Molecular and Cellular Biology of Plasminogen Activation

X I t h I n t e r n a t I o n a l w o r k S h o p o n
Molecular and Cellular Biology
of Plasminogen Activation
International Scientific
Committee
T. Antalis, Rockville, USA
N. Behrendt, Copenhagen, Denmark
F. Blasi, Milan, Italy
P. Bock, Nashville, USA
N. Brünner, Copenhagen, Denmark
T. Bugge, Bethesda, USA
P. DeClerk, Leuven, Belgium
V. Ellis, Norwich, United Kingdom
M. Gyetko, Ann Arbor, USA
K. Hajjar, New York, USA
M. Huang, Fujian, China
E.K.O. Kruithof, Geneva, Switzerland
K. Liu, Umeå, Sweden
D. Loskutoff, La Jolla, USA
V. Magdolen, München, Germany
R. Medcalf, Box Hill, Australia
D. Monard, Basel, Switzerland
Y. Nagamine, Basel, Switzerland
L. Ossowski, New York, USA
L. Pathy, Budapest, Hungary
16–20 June 2007
C. Peterson, Knoxville, USA
M. Ploug, Copenhagen, Denmark
M. Ranson, Wollongong, Australia
J. Rømer, Copenhagen, Denmark
M.P. Stoppelli, Naples, Italy
D. Strickland, Rockville, USA
A. Vaheri, Helsinki, Finland
M. Wilczynska, Umeå, Sweden
Organizing Committee
P. Andreasen, Aarhus, Denmark
T. Ny, Umeå, Sweden
Workshop Coordinator
E. Wolff, Urbana, USA
Participants
ConferenCe Centre Vår Gård
SaltSJöbaden,Sweden
130 participants from Australia, Austria,
Belgium, Canada, China, Denmark,
Germany, Italy, Japan, Mexico, Norway,
Poland, Russia, Spain, Sweden,
Switzerland, United Kingdom, and USA.

ii X I t h I n t e r n a t i o n a l W o r k s h o p o n

The XIth International Workshop on Molecular
and Cellular Biology of Plasminogen Activation
is sponsored by
Nobel Committee for Chemistry
Swedish Cancer Society
Swedish Research Council
American Diagnostica Inc.
The organizing committee would like to thank the organizers of the
previous International Workshops of Plasminogen Activation for
generously contributing unspent funds.
Molecular & Cellular Biology of Plasminogen Activation iii

Housing Grant Recipients
Nina Ahlskog, Umeå, Sweden
Daniela Alfano, Naples, Italy
Lisbeth Andersen, Aarhus, Denmark
Annapaola Andolfo, Milan, Italy
Esther Ardite, Barcelona, Spain
Rashna Balsara, Notre Dame, USA
Nathalie Beaufort, Munich, Germany
Julie Bødker, Aarhus, Denmark
Patrick Brunner, Vienna, Austria
Katharaina Bruno, Chicago, USA
Blake Cochran, Wollongong, Australia
David Croucher, Sydney, Australia
Angels Diaz-Ramos, Barcelona, Spain
Daniel Dupont, Aarhus, Denmark
Monika Ehnman, Stockholm, Sweden
Monika Ehart, Vienna, Austria
Paola Franco, Naples, Italy
Yongzhi Guo, Umeå, Sweden
Peter Hägglöf, Cambridge, United Kingdom
Jakob Harslund, Frederiksberg, Denmark
Emir Henic, Lund, Sweden
Karin Hultman, Gothenburg, Sweden
Benedikte Jacobsen, Copenhagen, Denmark
Lotte Jensen, Frederiksberg, Denmark
Anna Juncker-Jensen, Copenhagen, Denmark
Jodi Lee, Wollongong, Australia
Shih-Hon Li, Urbana, USA
Anna Lillis, Baltimore, USA
Sergei Lobov, Wollongong, Australia
Immacolata Longanesi Cattani, Naples, Italy
Ida Katrine Lund, Copenhagen, Denmark
Chris Madsen, Milan, Italy
Rajani Maiya, New York, USA
Lester Meissenheimer, Leuven, Belgium
Judit Mihaly, Vienna, Austria
Evelyn Nieves-Li, Urbana, USA
Bjorn Olausson, Umeå, Sweden
Mohan Pabba, Umeå, Sweden
Justin Paul, New York, USA
Valentina Pirazzoli, Milan, Italy
Boris Pliyev, Moscow, Russia
Gerald Prager, Vienna, Austria
Patrycja Przygodzka, Umeå, Sweden
Tomasz Przygodzki, Umeå, Sweden
Alessandro Salvi, Brescia, Italy
Morten Rasch, Copenhagen, Denmark
Birgitte Rønø, Copenhagen, Denmark
Gian Maria Sarra Ferraris, Milan, Italy
Rima Sulniute, Umeå, Sweden
Berta Vidal, Barcelona, Spain
Patrik Wahlberg, Umeå, Sweden
Ying Wei, San Francisco, USA
Malgorzata Wygrecka, Giessen, Germany
Wendy Xolalpa, Mexico, Mexico
Aiwu Zhou, New York, USA
iv X I t h I n t e r n a t i o n a l W o r k s h o p o n

Table of Contents
Saturday, 16 June 2007 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
Sunday, 17 June 2007 ..............................................................2
Session 1—Vitronectin and the Plasminogen Activation System ............................2
Session 2—Proteases, Inhibitors, Receptors, and Substrates in Vascular Biology . . . . . . . . . . . . . .3
Session 3—Cell Signalling Upstream and Downstream of the
Plasminogen Activation System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
Monday, 18 June 2007 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
Session 4—Proteases, Inhibitors, Receptors, and Substrates in Neurobiology
and Neuronal Pathology ..............................................................4
Session 5—Proteases, Inhibitors, Receptors, and Substrates in Normal Development ..........5
Session 6—The Plasminogen Activation System and Programmed Cell Death ................5
Tuesday, 19 June 2007 .............................................................6
Session 7—Proteases, Inhibitors, Receptors, and
Substrates in Tissue Repair and Tissue Remodelling ......................................6
Session 8—Proteases, Inhibitors, Receptors, and
Substrates in Inflammation and Infectious Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
Wednesday, 20 June 2007 ..........................................................7
Session 9—Proteases, Inhibitors, Receptors, and Substrates in Cancer .......................7
Session 10—Proteases, Inhibitors, and Receptors as Therapeutic Targets and
Therapeutic Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
Poster Presentations ................................................................9
Abstracts for Oral Presentations ...................................................13
Abstracts for Poster Presentations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .63
Author Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .129
Attendee List .....................................................................132
Molecular & Cellular Biology of Plasminogen Activation 1

Saturday, 16 June 2007
14:00–18:00 Registration
17:30–17:40 Welcome and Meeting Overview
Peter Andreasen and Tor Ny
17:40–18:25 Popular lecture: “Carl Linnaeus—The man who gave all of us our names”
Anna Rask-Andersen
18:30–20:00 Dinner
20:15–21:00 Lecture: “Control of invasive growth by plasminogen-related growth factors
(HGF and MSP)”
Paolo Michieli and Paolo Comoglio
Sunday, 17 June 2007
7:00–8:30 Breakfast
X I t h I n t e r n a t I o n a l w o r k S h o p o n
Molecular and Cellular Biology
of Plasminogen Activation
8:30–10:15 Session 1—Vitronectin and the Plasminogen Activation System
Session Chairs: Michael Ploug and Mingdong Huang
8:30–8:35 Introduction by Session Chairs
8:35–8:55 001 • Defining the Native Disulfide Topology in the SMB Domain of Human
Vitronectin • Li X, Zou G, Yuan W, Lu W*
8:55–9:15 002 • The Vitronectin Binding Site on the Urokinase Receptor Comprises Residues
from Both Domain I and the Flanking Interdomain Linker Region • Gårdsvoll H and
Ploug M*
9:15–9:35 003 • uPAR-induced Cell Adhesion and Migration: Vitronectin Provides the Key •
Madsen CD*, Sarra Ferraris GM, Andolfo A, Cunningham O, Sidenius N
9:35–9:55 004 • Interactions between PAI-1 and Vitronectin: Two Proteins, Two Sites, and Two
Phases • Schar CR, Jensen JK, Blouse GE, Minor KH, Andreasen PA, Peterson CB*
9:55–10:15 005 • How Does Vitronectin Accelerate PAI-1’s Protease Inhibition? • Zhou A* and
Wei Z
Relevant Posters—051–056
2 X I t h I n t e r n a t i o n a l W o r k s h o p o n

10:15–10:35 Break
10:35–12:20 Session 2—Proteases, Inhibitors, Receptors, and Substrates in Vascular Biology
Session Chairs: Marie Ranson and Thomas Bugge
10:35–10:40 Introduction by Session Chairs
10:40–11:00 006 • The Interaction between Tissue-type Plasminogen Activator and the Low
Density Lipoprotein Receptor-Related Protein Induces Activation of the NF-kB
Pathway during Cerebral Ischemia • Yepes M*, Brotzge XH, Polavarapu R
11:00–11:20 007 • Unique Secretory Dynamics of Tissue Plasminogen Activator (tPA) Is
Beneficial to Maintain Fibrinolytic Activity on Cell Surface • Suzuki Y*, Ihara H,
Mogami H, Urano T
11:20–11:40 008 • The Macrophage Low-Density Lipoprotein Receptor Related Protein (LRP)
Modulates Murine Lipoprotein Metabolism • Lillis AP*, Mikhailenko I, Robinson S,
Migliorini M, Battey F, Pizzo SV, Strickland DK
11:40–12:00 009 • Annexin 2 Mediates Plasminogen-Dependent Recruitment of Neovascular
Mural Cells in Lymphoma Angiogenesis • Ling Q, Ruan J, Yan L, Sui G-Z, Deora AB,
Church S, Cohen-Gould L, Rafii S, Lyden D, Hajjar KA*
12:00–12:20 010 • Activation of Latent PDGF-CC by Tissue Plasminogen Activator Impairs
Blood Brain Barrier Integrity during Ischemic Stroke • Su EJ, Fredriksson L,
Geyer M, Folestad E, Cale J, Mann K, Gao Y, Pietras K, Andreé J, Yepes M, Strickland DK,
Betsholtz C, Eriksson U, Lawrence DA*
12:20–12:30 Group photo
Relevant Posters—057–060
12:30–15:00 Lunch and free time
15:00–17:30 Poster Session A—Posters 051–084
17:30–19:00 Dinner
19:00–21:10 Session 3—Cell Signalling Upstream and Downstream of the Plasminogen
Activation System
Session Chairs: Francesco Blasi and Egbert K.O. Kruithof
19:00–19:05 Introduction by Session Chairs
19:05–19:25 011 • Plasminogen Activator Inhibitor-1 Gene Regulation: Cross Talk between
Hypoxia and Insulin Signalling • Flugel D and Kietzmann T*
19:25–19:45 012 • Role of Rho GTPases and p38 MAP Kinase in the Regulation of t-PA and
PAI-1 Expression in Cultured Human Endothelial Cells • Fish RJ*, Dunoyer-Geindre
S, Kruithof EKO
19:45–20:05 013 • Urokinase Receptor/a5b1 Integrin Interaction and Signaling in Cancer Cells •
Wei Y*, Tang CH, Kim Y, Robillard L, Kugler MC, Hill M, Brumwell A, Chapman HA
20:05–20:10 Break
Molecular & Cellular Biology of Plasminogen Activation 3

20:10–20:30 014 • Activated Human Neutrophils Rapidly Release the Chemotactically Active
D2D3 Form of the Urokinase-type Plasminogen Activator Receptor (uPAR/CD87) •
Pliyev BK* and Tkachuk VA
20:30–20:50 015 • PDGF-DD Bioavailability Is Regulated by the uPA/uPAR System:
Implications for Tumor Growth • Ehnman M*, Li H, Fredriksson L, Eriksson U
20:50–21:10 064 • Detection and Prevention of Hepatic Fibrosis Targeting Proteolytic TGF-b
Activation Reaction • Kojima S
Relevant Posters—061–076
Monday, 18 June 2007
7:00–8:30 Breakfast
8:30–11:00 Session 4—Proteases, Inhibitors, Receptors, and Substrates in Neurobiology and
Neuronal Pathology
Session Chairs: Denis Monard and Dan Lawrence
8:30–8:35 Introduction by Session Chairs
8:35–8:55 017 • A Novel Neuronal Death Pathway Triggered By Excess Tissue Plasminogen
Activator • Li J*, Snyder EY, Sidman RL
8:55–9:15 018 • The Inhibitor of Serine Proteases Protease Nexin-1 and its Receptor LRP
Modulate SHH Signalling during Cerebellar Development • Vaillant C, Michos O,
Orolicki S, Brellier F, Taieb S, Moreno E, Té H, Zeller R, Monard D*
9:15–9:35 019 • Fibrin Deposition Accelerates Neurovascular Damage and
Neuroinflammation in Mouse Models of Alzheimer’s Disease • Paul J* and
Strickland S
9:35–9:55 020 • Anxiety-like Behavior and Impaired Fear Extinction in Mice with Altered
Control of Extracellular Brain Proteolytic Activity • Meins M*, Herry C, Moreno E,
Fischer C, Lüthi A, Monard D
9:55–10:00 Break
10:00–10:20 021 • Tissue Plasminogen Activator Is Co-Packaged and Co-Transported to
Synaptic Sites with a Key Neuromodulator Associated with Synaptic Plasticity •
Lochner JE*, Spangler E, Schuttner LC, Scalettar BA
10:20–10:40 022 • Tissue Plasminogen Activator Modulates Cellular and Behavioral Response to
Cocaine • Maiya R*, Zhou Y, Norris EH, Kreek MJ, Strickland S
10:40–11:00 023 • Characterisation of the Pathway of Polymerisation of Wildtype Neuroserpin
and the Ser49Pro Mutant that Underlies the Dementia FENIB • Hägglöf P*,
Belorgey D, Karlsson-Li S, Sharp LK, Lomas DA
Relevant Poster—077
4 X I t h I n t e r n a t i o n a l W o r k s h o p o n

11:00–11:20 Break
11:20–12:25 Session 5—Proteases, Inhibitors, Receptors, and Substrates in Normal
Development
Session Chairs: Kui Liu and Leif Lund
11:20–11:25 Introduction by Session Chairs
11:25–11:45 024 • Matriptase Is an Essential Inhibitory Target for Hepatocyte Growth Factor
Activator Inhibitor-1 during both Embryonic Development and Postnatal Life •
Szabo R*, Molinolo A, List K, Bugge TH
11:45–12:05 025 • Mice with very low Matriptase Are Viable and Phenocopy Human Autosomal
Ichthyosis with Hypotrichosis Syndrome • List K*, Currie B, Scharschmidt T, Szabo R,
Molinolo A, Shireman J, Segre J, Bugge TH
12:05–12:25 026 • Role of Urokinase-Receptor in Hematopoietic Stem Cell Trafficking •
Montuori N, Selleri C, Ricci P, Visconte V,Carriero MV, Rotoli B, Rossi G, Ragno P*
Relevant Posters—078–084
12:25–17:30 Lunch and Free Time
17:30–18:30 Dinner
18:30–20:15 Session 6—The Plasminogen Activation System and Programmed Cell Death
Session Chairs: Patrizia Stoppelli and Bernd Binder
18:30–18:35 Introduction by Session Chairs
18:35–18:55 027 • Plasminogen Activator Inhibitor-1, PAI-1, Regulates the Akt Survival Pathway
• Rømer MU, Larsen L, Offenberg H, Brünner N, Lademann U*
18:55–19:15 028 • A Host Plasminogen Activator Inhibitor-1 Deficiency Promotes Proliferation
and Resistance to Apoptosis by Activation of the PI3-K/Akt Pathway in Endothelial
Cells • Balsara RD*, Castellino FJ, Ploplis VA
19:15–19:35 029 • Bomapin Is a Redox-Regulated Serpin which Stabilizes Retinoblastoma
Protein during Apoptosis and Increases Proliferation of Leukemia Cells •
Przygodzka P, Olausson B, Tengel Y, Larsson G, Wilczynska M*
19:35–19:55 030 • Hepsin as a Cell Survival Factor • Qiu D, Owen K, Edwards DR, Ellis V*
19:55–20:15 031 • Urokinase (uPA) Protects Endothelial Cell against Apoptosis by Upregulating
the X-Linked Inhibitor of Apoptosis Protein (XIAP) •Prager GW*, Koschelnick Y,
Mihaly J, Brunner P, Binder BR
Relevant Posters—085–087
Molecular & Cellular Biology of Plasminogen Activation 5

Tuesday, 19 June 2007
7:00–8:30 Breakfast
8:30–9:55 Session 7—Proteases, Inhibitors, Receptors, and Substrates in Tissue Repair and
Tissue Remodelling
Session Chairs: Katherine Hajjar and Dudley Strickland
8:30–8:35 Introduction by Session Chairs
8:35–8:55 032 • Pro-fibrinolytic Effects of Metalloproteinases during Skin Wound Healing in
the Absence of Plasminogen • Lund LR*, Green KA, Almholt K, Ploug M, Bugge TH,
Rømer J
8:55–9:15 033 • Complementary Roles of Intracellular and Pericellular Collagen Degradation
Pathways in Mesenchymal Cell Survival and Proliferation • Wagenaar-Miller RA,
Engelholm LH, Gavard J, Yamada S, Gutkind JS, Behrendt N, Holmbeck K, Bugge TH*
9:15–9:35 034 • Interplay between MMPs and the Endocytic Collagen Receptor, uPARAP/
Endo180, in Collagen Degradation • Behrendt N*, Madsen DH, Ingvarsen S, Hillig T,
Wagenaar-Miller R, Kjøller L, Gårdsvoll H, Høyer-Hansen G, Bugge TH, Engelholm LH
9:35–9:55 035 • The Urokinase Receptor Ko Mice Have Reduced Keratinocytes Proliferation
and Migration during Wound Healing and Are Protected in a Skin Carcinogenesis
Protocol • D’Alessio S, Mazzieri, R, Gerasi L, Blasi F*
9:55–10:15 Break
Relevant Poster—088
10:15–12:00 Session 8—Proteases, Inhibitors, Receptors, and Substrates in Inflammation and
Infectious Diseases
Session Chairs: Toni Antalis and Vincent Ellis
10:15–10:20 Introduction by Session Chairs
10:20–10:40 036 • Distinct Roles of Plasmin in Staphylococcus aureus-induced Sepsis and
Infection Models • Guo Y*, Li J, Hagström E, Ny T
10:40–11:00 037 • Proteolytic Activation of the Human Urokinase/Plasmin System by
Staphylococcus aureus • Beaufort N*, Wojciechowski P, Sommerhoff CP, Schmitt M,
Potempa J, Magdolen V
11:00–11:20 038 • The Maintenance of High Affinity Plasminogen Binding by PAM Variants
from Group A Streptococci Is Mediated by Conserved Arg and His Residues in Both
the A1 and A2 Repeat Domains • Ranson M*, Sanderson-Smith ML, Walker MJ, Fu Q,
Castellino FJ, Prorok M
11:20–11:40 039 • Plasminogen Activator Inhibitor-1 (PAI-1) Is an Inhibitor of Factor VII-
Activating Protease in Patients with Acute Respiratory Distress Syndrome •
Wygrecka M*, Morty RE, Markart P, Kanse SM, Andreasen PA, Wind T, Guenther A,
Preissner KT
6 X I t h I n t e r n a t i o n a l W o r k s h o p o n

11:40–12:00 040 • Improved Muscle Regeneration in PAI-1-deficient Mice Is Associated with an
Enhanced Inflammatory Response and Reduced Fibrin Deposition after Injury •
Ardite E*, Vidal B, Jardí M, González B, Muñoz-Cánoves P
Relevant Posters—089–095
12:00–12:20 Presentation of the 2009 Plasminogen Activation Meeting
12:20–14:30 Lunch and Free Time
14:30–17:00 Poster Session B—Posters 085–116
17:00 Reception and Gala Banquet and Boat Trip
Wednesday, 20 June 2007
7:00–8:30 Breakfast
8:30–9:55 Session 9—Proteases, Inhibitors, Receptors, and Substrates in Cancer
Session Chairs: Keld Danø and Thomas Kietzmann
8:30–8:35 Introduction by Session Chairs
8:35–8:55 041 • Invasion and Metastasis of Carcinoma Cells Is Prevented by Urokinase-
Derived Antagonists of avb5 Integrin Activation • Franco P, Vocca I, Alfano D,
Votta G, Carriero MV, Estrada Y, Netti PA, Ossowski L, Stoppelli MP*
8:55–9:15 042 • Urokinase Receptor/Integrin Interactions in Lung Tumor Development •
Tang CH, Hill M, Kim Y, Wei Y, Chapman HA*
9:15–9:35 043 • uPA and uPAR Expressing Stromal Cells Accompany the Transition to
Invasive Breast Cancer • Nielsen BS*, Rank F, Illemann M, Lund LR, Danø K
9:35–9:55 044 • Generation of the Malignant Phenotype in HT-1080 Tumor Cells by
PAI-1 Involves Modulation of Proteasomal Activity and Phosphatases • Mihaly J*,
Carroll VA, Breuss JM, Prager GW, Binder BR
9:55–10:15 Break
Relevant Posters—096–106
10:15–12: 20 Session 10—Proteases, Inhibitors, and Receptors as Therapeutic Targets and
Therapeutic Proteins
Session Chairs: Gunilla Høyer-Hansen and Ann Gils
10:15–10:20 Introduction by Session Chairs
10:20–10:40 045 • PEGylated DX-1000: Pharmacokinetics, Anti-Tumor and Anti-Metastatic
Effects of a Specific Plasmin Inhibitor • Devy L*, Rabbani SA, Stochl M, Ruskowski M,
Mackie I, Naa L, Toews M, van Gool R, Chen J, Ley A, Ladner RC, Dransfield DT,
Henderikx P
Molecular & Cellular Biology of Plasminogen Activation 7

10:40–11:00 046 • Cytotoxic Potential of a Novel uPA-activity Dependent and EGF Receptor
Targeting Pro-drug • Rønø B*, Kim GB, Liu S, Kristjansen PEG, Neville DM, Leppla SH,
Bugge TH, Rømer J
11:00–11:20 047 • Inhibition of Mouse uPA Activity by Mouse Monoclonal Antibodies in vitro
and in vivo • Lund IK*, Jögi A, Behrendt N, Ploug M, Gårdsvoll H, Lund LR, Rømer J,
Høyer-Hansen G
11:20–11:40 048 • Crystal Structure of Human Urokinase Complexed with a Cyclic Peptidyl
Inhibitor, uPAin-1 • Zhao G, Yuan C, Bian C, Wind T, Andreasen PA, Huang M*
11:40–12:00 049 • Discovery of a Novel Zymogen Targeting Inhibitor of Urokinase-type
Plasminogen Activator: Evidence for Structural Flexibility of the Protease Domain •
Blouse GE, Bøtkjær KA, Deryugina EI, Kjelgaard S, Byszuk O, Mortensen KK, Quigley JP,
Andreasen PA*
12:00–12:20 050 • A Novel Type of Agent Blocking the Association of uPA to its Receptor
uPAR: uPA-binding Aptamers • Dupont DM*, Madsen JB, Kjems J, Andreasen PA
Relevant Posters—107–116
12:20 Lunch and departure
8 X I t h I n t e r n a t i o n a l W o r k s h o p o n

Poster Presentations
• Poster numbers 51–84 will be
displayed from Saturday evening
through Monday morning.
• Presenters of poster numbers 51–84
will present in Poster Session A on
Sunday, 17 June from 15:00–17:30.
• Poster numbers 85–116 will be
displayed from Monday afternoon
through Wednesday morning.
• Presenters of poster numbers 85–116
will present in Poster Session B on
Tuesday, 19 June from 14:30–17:00.
• Presenters should be present at their
posters during the first 2 hours of the
poster session.
051 • Identification and Analysis of the Vn
Binding Site in Mouse uPAR • Pirazzoli V*,
Andolfo AP, Madsen CD, Sidenius N
052 • Novel uPAR Binding Site in Vitronectin
• Andolfo A* and Sidenius N
053 • Dissecting Serpin-protease Reaction
Pathways by the Use of Monoclonal
Antibodies • Bødker JS*, Blouse GE,
Dupont DM, Andreasen PA
054 • PAI-1-vitronectin Interactions Involve
an Extended Binding Surface and Mutual
Conformational Rearrangements • Blouse GE,
Peterson CB, Dupont DM, Ploug M, Gårdsvoll H,
Schar CR, Perron MJ, Minor KH, Shore JD,
Andreasen PA*
055 • Intact (non-cleavable) Cell-surface
u-PAR Accelerates Clearance of
tcu-PA:PAI-1:u-PAR Complexes and
Subsequent Re-surfacing of Intact and
Functional u-PAR • Nieves-Li EC* and
Manchanda N
056 • The Central b-sheet of PAI-1
Demonstrates Two Dynamically Distinct
Regions • Li S*, Lawrence DA, Schwartz BS
057 • Regulation of Cancer Cell Plasmin
Generation by Annexin A2-S100A10
Heterotetramer (AIIt) • Waisman DM*
058 • Plasminogen Activator Inhibitor Type 2
Binds to S100A10 in Annexin 2 Heterotetramer
and Prevents Annexin 2-dependent Plasmin
In-site Formation by Inhibiting tPA • Lobov S*,
Croucher D, Ranson M
059 • Understanding the Structural Basis
of the Differential-Receptor-Mediated
Endocytosis Mechanisms of PAI-1 and PAI-2
in Cancer • Cochran BJ*, Lobov S, Croucher D,
Ranson M
060 • A Low-glycemic-index Diet Reduces
Plasma PAI-1 Activity in Overweight Women
• Jensen L*, Krog-Mikkelsen I, Sloth B, Flint A,
Astrup A, Raben A, Tholstrup T, Brünner N
061 • Urokinase Receptor-independent
Signalling of the Urokinase-type Plasminogen
Activator via Phosporylation of STAT1 • Ehart
M* and Binder BR
062 • Urokinase Receptor Promotes Neo-
Angiogenesis through its Ser88-Arg-Ser-Arg-
Tyr92 Chemotactic Sequence • Longanesi-
Cattani I*, Bifulco K, Cantelmo AR, Di Carluccio
G, Spina R, Liguori E, Stoppelli MP, Carriero MV
063 • The Density Enhanced Phosphatase 1
(DEP-1) Down-Modulates Urokinase Receptor
(uPAR) Surface Expression in Confluent
Endothelial Cells • Brunner PM*, Heier PC,
Prager GW, Mihaly J, Priglinger U, Binder BR
064 • Detection and Prevention of Hepatic
Fibrosis Targeting Proteolytic TGF-b
Activation Reaction • Kojima S*
Molecular & Cellular Biology of Plasminogen Activation 9

065 • Domain 1 of uPAR Is Required for its
Morphological and Functional b2 Integrinmediated
Connection with Actin Cytoskeleton
in Human Endothelial Cells • Del Rosso M*,
Fibbi G, Margheri F, Serratì S, Pucci M,
Manetti M, Ibba-Manneschi L
066 • The uPA/uPAR/Vn Pathway of
Signaling to MAPK-activation • Sarra
Ferraris GM*, Madsen C, Sidenius N
067 • Regulation of tPA and PAI-1 Gene
Expression in Astrocytes • Hultman K*,
Tjärnlund-Wolf A, Blomstrand F, Nilsson M,
Medcalf R, Jern C
068 • Identification of a Mitotic Epitope in the
Domain 2 of the Urokinase Receptor (uPAR)
• Degryse B*, Eden G, Arnaudova R, Furlan F,
Blasi F
069 • Vitronectin Inhibits Plasminogen
Activator Inhibitor-1 (PAI-1)-Induced
Chemotaxis by Blocking PAI-1 Binding to
the LDL Receptor-Related Protein • Neels JG,
Kamikubo Y, Degryse B*
070 • Estradiol Inhibits EGF-induced Cell
Migration and uPAR Expression in Estrogen
Receptor-a Negative, GPR30 Positive Ovarian
Cancer Cells • Henic E*, Noskova V, Høyer-
Hansen G, Hansson S, Casslén B
071 • Methylation of the PAI-1 Gene in Oral
Squamous Cell Carcinomas and Normal Oral
Mucosa • Gao S, Krogdahl A, Sørensen JA,
Dabelsteen E, Andreasen PA*
072 • Urokinase Signaling through Its
Receptor Promotes Invasiveness and
Metastasis of Pancreatic Cancer Cells •
Xue A*, Xue M, Jackson C, Song E, Allen BJ,
Smith RC
073 • Tissue Plasminogen Activator Induces
Cell Proliferation in Pancreatic Cancer by
a Non-catalytic Mechanism that Requires
ERK1/2 Activation through Epidermal
Growth Factor Receptor and Annexin A2 •
Ortiz-Zapater E, Peiró S, Roda O, Corominas JM ,
Aguilar S, Ampurdanés C, Real FX, Navarro P*
074 • Modulation of Lung Carcinoma Cell
Lines and Primary Cultures Migration by
uPA-Derived and EGFR Inhibitors • Franco P*,
Mancini A, Votta G, Caputi M, Stoppelli MP
075 • A Novel Role of Ku80 in Regulation
of PAI-1 Gene Expression in Migrating
Endothelial Cells Induced by Thymosin b4
• Bednarek R*, Boncela J, Smolarczyk K,
Cierniewski CS
076 • Signaling Pathway Involved in
Inhibition of PAI-1 Expression by CNP in
Endothelial Cells • Jerczynska H*, Cierniewski
CS, Pawlowska Z
077 • Interaction of Alzheimer’s Amyloid
b-peptide (Ab) 1-40 with PAI-2 • Pabba M*,
Przygodzki T, Malisauskas M, Olofsson A,
Morozova-Roche L, Wilczynska M, Ny T
078 • The Subcellular Itinerary of Hepatocyte
Growth Factor Activator Inhibitor-1 in MDCK
Cells • Godiksen S, Selzer-Plon J, Pedersen EDK,
Borger Rasmussen H, Bugge TH, Vogel LK*
079 • Evidence for a Matriptase-Prostasin
(CAP1/PRSS8) Serine Protease Zymogen-
Cascade-Regulating Epithelial Differentiation
• List K*, Netzel-Arnett S, Currie B, Szabo R,
Molinolo A, Antalis TM, Bugge TH
080 • The Plasminogen Activation System
in Monocytic Cell Differentiation and
Proliferation: Potential Target for Plasminogen
Activation Inhibitor Type- 2-Based
Therapeutics • Lee JA*, Croucher DR, Ranson M
081 • Identification and Localization of
Novel Serine Proteases in the Mouse Ovary •
Wahlberg P*, Nylander Å, Kui L, Ny T
082 • Alpha-enolase/Plasminogen Binding Is
Required during Myogenesis in vitro and in
vivo • Diaz-Ramos A*, Llorens A, Luque T,
López-Alemany R
083 • The Serpinb8 Is Alternatively Spliced
to the Known Long Form and a Novel Short
Form • Olausson B*, Przygodzka P, Dahl L,
Carlsson L, Wilczynska M
10 X I t h I n t e r n a t i o n a l W o r k s h o p o n

084 • Characterization of a Combined PAI-1
and TIMP-1 Gene-deficient Mouse Model •
Harslund J*, Nielsen OL, Brünner N, Offenberg H
085 • Dual Role of the uPA/uPAR System in
Apoptosis of Mesangial Cells and Diabetic
Nephropathy • Tkachuk N, Tkachuk S*, Kiyan J,
Shushakova N, Haller H, Dumler I
086 • Phenotypic Consequences of
Plasminogen Activator Inhibitor-1 Gene
Ablation on STAT1 Activation and Cell Cycle
Progression in Proliferating Endothelial
Cells • Balsara RD*, Morin SJ, Meyer CA,
Castellino FJ, Ploplis VA
087 • Urokinase and its Receptor as Novel
C-Myc Target Genes Affecting Cell Migration
and Apoptosis • Alfano D*, Iaccarino I,
Stoppelli MP
088 • Effect of Plasminogen on Cell Migration
Using an in vitro Wound Model • Sulniute R*,
Li J, Ny T
089 • uPA, but not its Receptor uPAR, Is
Necessary for Experimentally-induced and
Pathological Muscle Regeneration • Vidal B*,
Serrano AL, Jardí M, Suelves M, Muñoz-
Cánoves P
090 • Urokinase-type Plasminogen Activator
Deficiency Strongly Attenuates Ischemia
Reperfusion Injury and Acute Kidney
Allograft Rejection • Gueler F, Rong S,
Mengel M, Park J-K, Kirsch T, Haller H,
Dumler I, Shushakova N*
091 • a1-antitrypsin Polymerization Studies
using Gas-phase Electrophoretic Mobility
Molecular Analysis (GEMMA) • Przygodzki T*,
Mallya M, Phillips RL, Belorgey D, Hägglöf P,
Lomas DA, Ny T
092 • The Plasminogen Interaction of
Antigen 85B Protein from Mycobacterium
Tuberculosis: Role of Lys89 • Xolalpa W*,
Vallecillo AJ, Rosales L, Ruiz BH, Espitia C
093 • Plasminogen as a Factor in Innate
Immunity • Ahlskog N*, Guo Y, Ny T
094 • The Inflammatory Cytokine Oncostatin
M Induces Plasminogen Activator Inhibitor-
1 in Human Vascular Smooth Muscle Cells
in vitro via PI3 kinase and MAP-kinase
Dependent Pathways • Demyanets S, Kaun C,
Rychli K, Rega G, Pfaffenberger S, Maurer G,
Huber K, Wojta J*
095 • Crohn’s Disease but not Chronic
Ulcerative Colitis Induces the Expression
of PAI-1 in Enteric Neurons • Laerum OD*,
Illemann M, Skarstein A, Helgeland L, Øvrebø K,
Danø K, Nielsen BS
096 • The Effect of Matrix Metalloprotease
3 Deficiency in Spontaneous Metastasis •
Juncker-Jensen A*, Rømer J, Almholt K
097 • Tumor Cell Expression of C4.4A, a
Structural Homologue of the Urokinase
Receptor, Correlates with Poor Prognosis
in Non-Small Cell Lung Cancer • Skov BG,
Hansen LV, Ploug M, Pappot H*
098 • Urokinase Receptor Splice Variant
uPAR-del4/5 and rab31 mRNA Expression
in Breast Cancer • Magdolen V*, Kotzsch M,
Sieuwerts A, Grosser M, Meye A, Smid M,
Schmitt M, Luther T, Foekens JA
099 • PN-1, a Serine Protease Inhibitor,
Increases MMP-9 Activity in Breast Cancer
Cell Line • Fayard B* and Monard D
100 • Cleavage of uPAR: Mechanism and
Prognostic Significance • Høyer-Hansen G*,
Almasi CE, Pappot H
101 • Expression of Urokinase Receptor
(uPAR) and Plasminogen Activator
Inhibitor-1 (PAI-1) in Human Colon Cancer
and their Matched Liver Metastases •
Illemann M*, Bird N, Majeed A, Laerum OD,
Lund LR, Danø K, Nielsen BS
102 • A Structural Basis for Differential Cell
Signaling Initiated by PAI-1 and
PAI-2: Implications for Metastatic Potential •
Croucher D*, Saunders D, Ranson M
Molecular & Cellular Biology of Plasminogen Activation 11

103 • Thrombin Induces Tumor Invasion
through the Induction and Association of
Matrix Metalloproteinase-9 and b-1 Integrin
on the Cell Surface • Bruno K*, Radjabi R,
Sawada K, Montag A, Kossiakoff A, Lengyel E
104 • Overexpression of Protease Nexin-1
mRNA in Oral Squamous Cell Carcinomas •
Gao S, Krogdahl A, Sørensen JA, Dabelsteen E,
Andreasen PA*
105 • The Matrix Metalloprotease (MMP)
Inhibitor Galardin Increases Collagen
Deposition and Reduces Spontaneous
Metastasis in the MMTV-PymT Transgenic
Breast Cancer Model • Almholt K*, Lærum OD,
Lund LR, Danø K, Johnsen M, Rømer J
106 • Proteomics of uPAR Protein: Protein
Interactions in Cancer Metastasis • Saldanha R,
Molloy M, Xu N, Baker MS*
107 • A New Tagging System for Production
of Recombinant Proteins in Drosophila
S2 Cells Using the Third Domain of the
Urokinase Receptor • Gårdsvoll H*, Hansen LV,
Jørgensen TJD, Ploug M
108 • Photoaffinity Labeling of uPAR with
Cyclic Peptides • Jacobsen B*, Gårdsvoll H,
Barkholt V, Østergaard S, Ploug M
109 • In Vivo Inhibition of the Murine
uPA-uPAR Interaction using Monoclonal
Antibodies Raised in uPAR Deficient Mice •
Rasch MG*, Pass J, Jögi A, Rønø B, Gårdsvoll H,
Lund LR, Høyer-Hansen G, Lund IK
110 • RNA Interference for Urokinase-
Targeting Limits Growth of Hepatocellular
Carcinoma Xenografts in Nude Mice •
Salvi A*, Arici B, Barlati S, De Petro G
111 • Potent and Broad Anti-tumor Activity
of an Engineered Matrix Metalloproteinaseactivated
Anthrax Lethal Toxin that Targets
Tumor Vasculature • Liu S, Wang H,
Currie BM, Molinolo A, Leung HJ, Moayeri M,
Alfano RW, Frankel AE, Leppla SH, Bugge TH*
112 • Elucidation of the Epitope of MA-
31C9, a Non-inhibitory Anti-human PAI-1
Antibody • Meissenheimer LM*, Dewilde M,
Compernolle G, Declerck PJ, Gils A
113 • Residues outside the Epitope Determine
the Function of MA-159M12, an Inhibitory
Anti-rat PAI-1 Antibody • Meissenheimer LM*,
Compernolle G, Declerck PJ, Gils A
114 • Conformational Probes and Activity
Regulators of Plasminogen Activator
Inhibitor-1, Isolated from Phage-displayed
Disulphide Bridge-constrained Peptide
Libraries • Dupont DM, Jensen JK, Mathiasen L,
Blouse GE, Wind T, Andreasen PA*
115 • Urokinase-type Plasminogen Activatorinhibiting
Cyclic Peptides Demonstrate New
Modalities for Inhibition of Serine Proteases •
Andersen LM*, Wind T, Hansen HD, Blouse GE,
Christensen A, Jensen JK, Malmendal A,
Nielsen NC, Andreasen PA
116 • In vivo Treatment with Monoclonal
Antibodies against Mouse Urokinase-type
Plasminogen Activator in Cancer Models •
Jögi A*, Lund IK, Høyer-Hansen G, Lund LR,
Danø K, Rømer J
12 X I t h I n t e r n a t i o n a l W o r k s h o p o n

Abstracts for Oral Presentations
i001i
Defining the Native Disulfide Topology in the SMB Domain of
Human Vitronectin
Li X, Zou G, Yuan W, Lu W*
Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland, USA
Presenting author e-mail: luw@umbi.umd.edu
The N-terminal 44 amino acid residues of the human plasma glycoprotein vitronectin, known as
the somatomedin B (SMB) domain, mediates the interaction between vitronectin and plasminogen
activator inhibitor 1 (PAI-1) in a variety of important biological processes. Several laboratories
have published conflicting reports on the native disulfide topology in the SMB domain with no
consensus reached thus far. Using native chemical ligation and orthogonal protection of selected
Cys residues, we chemically synthesized three topological analogs of SMB with predefined
disulfide connectivities corresponding to those previously published. In addition, we oxidatively
folded a fully reduced SMB in aqueous solution, and prepared, by CNBr cleavage, the N-terminal
segment of 51 amino acid residues of intact vitronectin purified from human blood. Biochemical
and functional characterizations allowed us to conclude that (1) only the Cys5-Cys21, Cys9-Cys39,
Cys19-Cys32 and Cys25-Cys31 connectivity is present in native vitronectin; (2) only the native
disulfide connectivity is functional; (3) the native disulfide pairings can be readily formed during
spontaneous (oxidative) folding of the SMB domain in vitro. Our results unequivocally define the
native disulfide topology in the SMB domain of human vitronectin, and provide important clues
as to how the controversy arose in the first place.
Molecular & Cellular Biology of Plasminogen Activation 13

i002i
The Vitronectin-Binding Site on the Urokinase Receptor
Comprises Residues from Both Domain I and the Flanking Interdomain
Linker Region
Gårdsvoll H and Ploug M*
Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark
Presenting author e-mail: m-ploug@finsenlab.dk
The urokinase-type plasminogen activator receptor (uPAR) has been implicated as a modulator
of several biochemical processes that are active during tumor invasion and metastasis e.g.
extracellular proteolysis, cell adhesion and cell motility. The structural basis for the high-affinity
interaction between the urokinase-type plasminogen activator (uPA) and uPAR, which focuses
cell surface associated plasminogen activation in vivo, is now thoroughly characterized by sitedirected
mutagenesis studies and X-ray crystallography. In contrast, the structural basis for the
interaction between uPAR and the extracellular matrix protein vitronectin, which is involved
in the regulation of cell adhesion and motility, remains to be clarified. In the present study, we
have identified the functional epitope on uPAR that is responsible for its interaction with the
full-length, extended form of vitronectin using a comprehensive alanine-scanning library of
purified single-site uPAR mutants (244 positions tested). Interestingly, the 5 residues identified
as ‘’hot spots’’ for vitronectin binding form a contiguous epitope comprising two exposed loops
connecting the central 4-stranded b-sheet in uPAR domain I (Trp32, Arg58 and Ile63) as well
as a proximal region of the flexible linker peptide connecting uPAR domains I and II (Arg91
and Tyr92). This binding topology provides the molecular basis for the observation that uPAR
can form a ternary complex with uPA and vitronectin. We also show that the affinity for the
small SMB domain of vitronectin is increased approximately 4-fold for uPAR-uPA complexes as
compared to unoccupied uPAR.
14 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i003i
uPAR-induced Cell Adhesion and Migration:
Vitronectin Provides the Key
Madsen CD* 1 , Sarra Ferraris GM1 , Andolfo A1 , Cunningham O1 1, 2
, Sidenius N
1 The FIRC Institute of Molecular Oncology (IFOM), Milan, Italy;
2 Molecular Genetics Unit, DIBIT, Università Vita-Salute San Raffaele, Milan, Italy
Presenting author e-mail: chris.madsen@ifom-ieo-campus.it
Expression of the GPI-anchored membrane receptor uPAR induces profound changes in cell
adhesion and migration, and its expression correlates with the malignant phenotype of cancers. To
identify the key molecular interactions essential for uPAR function in these processes; we carried
out a complete functional alanine-scan of uPAR in HEK293 cells. Of the 255 mutant receptors
characterized, 34 failed to induce changes in cell adhesion, cell morphology accompanied by
actin-rearrangement and focal adhesion turn-over, and cell migration. Remarkably, the molecular
defect of all of these mutants was a specific reduction in integrin-independent cell binding to the
somatomedin-B domain of the extracellular matrix component, vitronectin. In order to mimic the
membrane-ECM interaction induced by uPAR-Vn, we generated a GPI-anchored plasminogen
activator inhibitor-1 (PAI-1). This, like uPAR, binds specifically and with high affinity to the SMB
domain of Vn but shares no other similarity with uPAR. Surprisingly, the chimeric PAI-1/GPI
recapitulated the biological effects of uPAR expression. A direct uPAR-Vn interaction is thus both
required and sufficient to initiate downstream signalling leading to changes in cell morphology
and migration. Together these data demonstrate a novel mechanism by which a cell adhesion
molecule lacking inherent signalling capability evokes complex cellular responses, independently
of lateral interactions with signalling receptors, by modulating the contact between the cell and
the matrix. The importance of the uPAR/Vn-interaction was not cell-type specific as all mutants
identified were subsequently confirmed in CHO cells. Finally we have mapped the direct
vitronectin binding epitope (W32, R58, I63, R91, Y92) of uPAR.
Molecular & Cellular Biology of Plasminogen Activation 15

i004i
Interactions between PAI-1 and Vitronectin: Two Proteins,
Two Sites, and Two Phases
Schar CR 1 , Jensen JK 2 , Blouse GE 2 , Minor KH 1 , Andreasen PA 2 , Peterson CB* 1
1Department of Biochemistry, Cellular, and Molecular Biology, University of Tennessee, Knoxville,
Tennessee, USA;
2Laboratory of Cellular Protein Science, Department of Molecular and Structural Biology, University of
Aarhus, Aarhus, Denmark
Presenting author e-mail: cbpeters@utk.edu
We have generated a mutant form of vitronectin that lacks the well-characterized N-terminal
somatomedin B domain, known to house the primary high-affinity site for binding of PAI-1 to
vitronectin. Residual binding of PAI-1 to this deletion mutant of vitronectin is observed. Also, we
have used a large battery of mutant forms of PAI-1 to evaluate the specific interactions required
for binding. With these reagents, the second binding site for vitronectin on PAI-1 was mapped
to a region around helix D rich in charged amino acids. We have used kinetic and equilibrium
measurements with surface plasmon resonance to study PAI-1 to full-length and the truncated
mutant of vitronectin that is missing the somatomedin B domain. Clearly, the interaction of
vitronectin and PAI-1 at the second site is weaker than the primary interaction between the
somatomedin B domain and the flexible joint region that lies between helices D, E and F on
PAI-1. Most notably, the off rate for binding is much faster, comparable to that for latent PAI-1
dissociation from vitronectin. Interestingly, latent PAI-1 binds nearly as well at the second site
as does active PAI-1, consistent with less dramatic structural changes in the helix D region upon
conversion to the latent structure compared to those that occur with expansion of the central beta
sheet that affect the primary binding site interactions with the somatomedin B domain. These
characteristic features of binding at the two sites are consistent with FRET experiments and
stopped-flow fluorescence measurements that reveal biphasic binding between vitronectin and
PAI-1.
16 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i005i
How Does Vitronectin Accelerate PAI-1’s Protease Inhibition?
Zhou A* and Wei Z
Department of Haematology, University of Cambridge, CIMR, Cambridge, United Kingdom
Presenting author e-mail: awz20@cam.ac.uk
Vitronectin binds and stabilises PAI-1’s activity through its somatomedin B (SMB) domain.
Vitronectin can also accelerate PAI-1’s protease (thrombin and activated protein C) inhibition
by more than 100-fold. To investigate the mechanism underlying this acceleration, firstly we
prepared SMB domain alone and various SMB containing fragments of vitronectin. Kinetics
studies showed that these fragments of vitronectin, like urea-treated vitronectin, had little effect
on PAI-1’s protease inhibition. Secondly cross-linking experiment with inactive thrombin variant
(S195A) showed that thrombin could only be cross-linked to native VN (not urea-treated VN) in
the presence of active PAI-1. Thirdly, mutagenasis studies of thrombin showed that substitution
of the surface exposed residue Asp100 with Arg attenuated vitronectin PAI-1’s inhibition
of thrombin by more than 50%, while other mutations of thrombin such as Glu97Arg and
Asp178Arg etc had no effect. Altogether, these data indicate that native vitronectin has a cryptic
thrombin binding site, which is exposed upon PAI-1 binding (likely masked in urea treated VN)
and interacts with thrombin surface near Asp100, and vitronectin accelerates PAI-1’s protease
inhibition by bridging PAI-1 and thrombin together.
Molecular & Cellular Biology of Plasminogen Activation 17

i006i
The Interaction between Tissue-type Plasminogen Activator and
the Low Density Lipoprotein Receptor-Related Protein Induces
Activation of the NF-kB Pathway during Cerebral Ischemia
Yepes M*, Brotzge XH, Polavarapu R
Department of Neurology and Center for Neurodegenerative Disease, Emory University School of
Medicine, Atlanta, Georgia, USA
Presenting author e-mail: myepes@emory.edu
We previously demonstrated that the interaction between tPA and the low density lipoprotein
receptor-related protein (LRP) following middle cerebral artery occlusion (MCAO) increases
the permeability of the blood brain barrier (BBB). Here we studied the relation between tPA and
NF-kB activation following MCAO. Wild-type (WT), tPA (tPA–/–), and plasminogen (Plg–/–)
deficient mice underwent MCAO and analysis of NF-kB activation by immunohistochemistry,
Western blot (p65-phosphorylation) and electrophoretic mobility shift assay (EMSA). We observed
a rapid activation of NF-kB in WT and Plg–/– mice that was abolished in tPA–/– animals. The
effect of MCAO on LRP expression was studied by immunohistochemistry and quantitative
real-time PCR (qRT-PCR). We found that MCAO induces a rapid increase in LRP expression
in WT mice that is significantly attenuated in tPA–/– mice. Treatment of WT mice with either
the receptor associated protein (RAP) or anti-LRP antibodies inhibited MCAO-induced NF-kB
activation. The intracerebral injection of tPA into tPA–/– mice following MCAO resulted in NF-kB
activation that was not observed when tPA was co-administered with either RAP or LRP-blocking
antibodies. Immunohistochemistry and qRT-PCR demonstrated an increase in the expression of
inducible nitric oxide synthase (NF-kB-dependent gene) in the ischemic area in WT mice that
was attenuated in tPA–/– mice and in WT animals treated with RAP or anti-LRP antibodies.
We conclude that during cerebral ischemia tPA has a plasminogen-independent ‘’cytokine-like’’
function and that the interaction between tPA and LRP results in NF-kB pathway activation and
induction of NF-kB-dependent genes with known effect on cell death and BBB permeability.
18 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i007i
Unique Secretory Dynamics of Tissue Plasminogen Activator (tPA)
Is Beneficial to Maintain Fibrinolytic Activity on Cell Surface
Suzuki Y*, Ihara H, Mogami H, Urano T
Department of Physiology, Hamamatsu University School of Medicine, Hamamatsu, Japan
Presenting author e-mail: seigan@hama-med.ac.jp
Introduction: Vascular endothelial cells (VECs) express and secrete various anti-thrombotic
molecules to keep the vascular patency. tPA, the primary fibrinolytic enzyme in vasculature, is
one of them. After secretion as an active form, tPA initiates fibrinolysis both on VECs and in blood
where its specific inhibitor of PAI-1 exists. Here, we analyzed the dynamics of tPA secretion from
the containing granules and its modulation by PAI-1 using total internal reflection fluorescence
microscopy (TIRF-M). Method: An established cell-line of VECs was cultured and transfected
with green fluorescent protein (GFP)-tagged either wild type tPA, tPA (S478A)(Ser478 at active
center is replaced by Ala, no ability to complex with PAI-1) or tPA (catalytic domain; CD)(deleted
in finger-, EGF-like-, kringle1- and 2- domains). The exocytotic dynamics of these tPAs-GFP near
the plasma membrane (PM) were analyzed by TIRF-M. Results: (1) tPA-GFP showed unique
dynamics of slow disappearance from PM after opening of its containing granules (fluorescence
half life: TF1/2=10sec). (2) Supplemented PAI-1 shortened TF1/2 and increased tPA-PAI-1
complex but not free tPA in supernatant. TF1/2 of tPA(S478A)-GFP was slower than tPA-GFP,
indicating that the complex formation with PAI-1 is essential for rapid dissociation of tPA from
the opened granular membrane. (3) TF1/2 of tPA(CD)-GFP was faster than tPA-GFP, indicating
that FEK domains are responsible for slow disappearance. Conclusion: tPA has unique, slow
secretory dynamics which is beneficial to maintain fibrinolytic activity on VECs. PAI-1 facilitates
tPA dissociation and suppresses fibrinolytic activity on VECs.
Molecular & Cellular Biology of Plasminogen Activation 19

i008i
The Macrophage Low-density Lipoprotein-Receptor-Related
Protein (LRP) Modulates Murine Lipoprotein Metabolism
Lillis AP* 1,2 , Mikhailenko I 1 , Robinson S 1 , Migliorini M 1 , Battey F 1 , Pizzo SV 2 , Strickland DK 1
1Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore,
Maryland, USA;
2 Department of Pathology, Duke University Medical Center, Durham, North Carolina, USA
Presenting author e-mail: alillis@som.umaryland.edu
Very low-density lipoproteins (VLDL) and chylomicrons transport cholesterol and triglycerides
(TG) to muscle, adipose and other extra-hepatic tissues. Lipoprotein lipase selectively removes
and hydrolyzes TGs, transferring free fatty acids to these tissues. The resulting remnant
lipoproteins bind to two members of the low-density lipoprotein receptor (LDLR) family, LDLR
itself and the LDLR-related protein (LRP). LRP is a large endocytic receptor that recognizes more
than 30 different ligands. Accumulating evidence supports a role for hepatic LRP in the clearance
of remnant lipoproteins. However, LRP is also abundant in resident liver macrophages (Kupffer
cells) which reside in the sinusoids of the liver in close proximity to the space between endothelial
cells and hepatocytes where lipoprotein remnants are sequestered and modified before being
recognized by LRP and the LDL receptor. To investigate a possible role of macrophage LRP in
remnant lipoprotein metabolism, we developed tissue-specific knockouts using the LoxP/Cremediated
recombination system on mice genetically deficient in the LDLR. By crossing mice
carrying loxP-tagged LRP with a LysMCre transgenic mouse line expressing the Cre recombinase
at the mouse M lysozyme locus, we have generated LDLR-deficient mice in which the LRP gene
is also selectively and efficiently deleted in macrophages (macLRP- mice). After placing macLRP-
mice (and their sibling controls which are deficient in LDLR but express LRP normally) on a high
fat, high cholesterol (Western) diet for 3 weeks, we observed elevated serum triglycerides (5.0 vs
2.8 mg/ml, p

i009i
Annexin 2 Mediates Plasminogen-Dependent Recruitment of
Neovascular Mural Cells in Lymphoma Angiogenesis
Ling Q 1 , Ruan J 2 , Yan L 1 , Sui G-Z 1 , Deora AB 1 , Church S 2 , Cohen-Gould L 1 , Rafii S 2 , Lyden D 3 ,
Hajjar KA* 1
Departments of 1 Cell and Developmental Biology, 2 Medicine, 3 Pediatrics, Weill Cornell Medical College,
New York, New York, USA
Presenting author e-mail: khajjar@med.cornell.edu
Malignant tumor progression depends upon the development of tumor-associated blood vessels,
either via co-option of nearby host vascular cells, or through recruitment of marrow-derived
progenitor cells. Annexin 2 (A2) is a cell surface co-receptor for plasminogen (Plg) and tPA that
augments the catalytic efficiency of Plg activation. We showed previously that A2–/– mice display
impaired growth factor-induced postnatal angiogenesis in the corneal pocket, oxygen-induced
retinopathy, and Matrigel plug assays. Here, we examined neovascularization of experimental
lymphoma in mice with fibrinolytic deficiency states. For EL4, a T cell lymphoma, and B6RV2, a
B cell lymphpma, tumor growth was rapid in wildtype and tPA–/– mice, but severely retarded
in both A2–/– and Plg–/– mice. Immunohistochemical staining of EL4 tissue on day 8 revealed
reduced vascular density, frequent intravascular fibrin thrombi, and dilated microvessels in
A2–/–, but not A2+/+, mice. Electron microscopy and immunofluorescence revealed a paucity
of pericytes and secondary dropout of endothelial cells within A2–/– tumor microvessels. Flow
cytometric analysis of circulating marrow-derived progenitor cells showed reduced populations
of VEGFR1+/CD11b+ hematopoietic precursor cells in A2–/– versus A2+/+ tumor-bearing mice.
In lethally irradiated A2–/– mice, tumor growth was rescued completely upon engraftment
with A2+/+ marrow. Transplantation of green fluorescent protein (GFP)-positive bone marrow
led to the recruitment of abundant GFP+/CD11b+/CD68+ cells to locations surrounding
tumor neovessels. In addition, transplantation of normal marrow restored the investment of
tumor microvessels with a-smooth muscle actin-positive pericytes. These data indicate that A2
contributes critically to tumor angiogenesis in experimental lymphoma, by enabling recruitment
of A2+ myelomonocytic cells that instruct pericyte recruitment and neovascular stabilization.
Molecular & Cellular Biology of Plasminogen Activation 21

i010i
Activation of Latent PDGF-CC by Tissue Plasminogen Activator
Impairs Blood Brain Barrier Integrity during Ischemic Stroke
Su EJ 1 , Fredriksson L 2 , Geyer M 1 , Folestad E 2 , Cale J 1 , Mann K 1 , Gao Y 3 , Pietras K 2 , Andreé J 4 , Yepes M 5 ,
Strickland D 3 , Betsholtz C 4 , Eriksson U 2 , Lawrence DA* 1
1Department of Internal Medicine, Division of Cardiovascular Medicine, University of Michigan
Medical School, Ann Arbor, Michigan, USA;
2 Ludwig Institute for Cancer Research, Stockholm Branch, Karolinska Institutet, Stockholm, Sweden;
3Center for Vascular and Inflammatory Disease and Departments of Surgery and Physiology, School of
Medicine, University of Maryland, Baltimore, Maryland, USA;
4Laboratory of Vascular Biology, Division of Matrix Biology, Department of Medical Biochemistry and
Biophysics, Karolinska Institutet, Stockholm, Sweden;
5Department of Neurology and Center for Neurodegenerative Disease, Emory University School of
Medicine, Atlanta, Georgia, USA
Presenting author e-mail: dlawrenc@umich.edu
The current treatment for ischemic stroke, intravenous tPA, only benefits a limited number of
patients. This is due in part to the requirement that tPA be administered within 3 hours of the
onset of symptoms. The reasons for this time constraint are not known but may be due to the
unique activities that tPA has in the brain beyond its well established role as a fibrinolytic enzyme.
For example, tPA has been shown to play a role in regulating cerebrovascular permeability
after stroke; however, the specific substrate for tPA in brain is not known. The recent discovery
that tPA cleaves latent PDGF-CC lead us to hypothesize that tPA may mediate cerebrovascular
permeability via activation of latent PDGF-CC. To test this possibility, active PDGF-CC was
injected directly into the CSF in the absence of ischemia and the extravasation of Evans Blue
dye in the brain was examined. These studies showed a significant increase in Evans Blue
extravasation after active PDGF-CC injection. This effect was similar to that seen with tPA
injection, suggesting that tPA and PDGF-CC may be acting on the same pathway. To test this
neutralizing antibodies against PDGF-CC were co-injected with tPA and were found to block
the effect of tPA. To evaluate whether PDGF-CC plays a role in regulating cerebrovascular
permeability during stroke, the PDGF receptor inhibitor Gleevec was used in a photothrombotic
stroke model. Stroke is induced specifically in the middle cerebral artery by the local activation
of intravenous Rose Bengal with a cold laser. Mice treated with Gleevec showed significant
reductions in Evans Blue extravasation at 24-hours and stroke volumes at 72-hours, indicating a
strong effect of PDGF signaling on cerebrovascular integrity. Taken together, these data suggest
that PDGF-CC is a downstream substrate of tPA in the CNS. These studies may provide important
insights into new therapeutic strategies for treating stroke patients.
22 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i011i
Plasminogen Activator Inhibitor-1 Gene Regulation:
Cross Talk between Hypoxia and Insulin Signalling
Flugel D and Kietzmann T*
Faculty of Chemistry, Department Biochemistry, University of Kaiserslautern, Kaiserslautern, Germany
Presenting author e-mail: tkietzm@gwdg.de
A number of pathological conditions like cancer, diabetes or the metabolic syndrome are
associated with enhanced plasminogen activator inhibitor-1 (PAI-1) levels. In addition, these
diseases are often combined with hypoxia and an impaired insulin signalling. Therefore we asked
whether hypoxia and insulin can exert direct effects on PAI-1 expression. We found that hypoxia
induced PAI-1 expression via the hypoxia-inducible transcription factor-1 (HIF-1) which binds
to hypoxia responsive elements in the PAI-1 promoter. HIF-1 is a heterodimer from which HIF-
1alpha becomes hydroxylated under normoxia. This enables binding of the von Hippel-Lindau
(VHL) protein which targets HIF-1alpha for proteasomal degradation.
Interestingly, HIF-1a can also respond to non-hypoxic stimuli like insulin which acts via
phosphatidylinositol 3-kinase and protein kinase B. Although PKB induces HIF-1alpha
stabilization, HIF-1a is not a direct substrate for PKB/Akt. Therefore, we aimed to investigate
whether glycogen synthase kinase-3 (GSK3) may have an impact on the VHL-dependent HIF-
1alpha degradation.
We found that inhibition of GSK3 with LiCl and insulin as well as depletion with siRNA induced
HIF-1alpha and PAI-1 expression whereas overexpression of GSK3alpha and GSK3beta reduced it.
These effects were mediated posttranslationally via the oxygen-dependent degradation domain of
HIF-1alpha. Mutation of the proline residues critical for the VHL-dependent degradation as well
as usage of VHL-deficient cells did not prevent GSK3-mediated ubiquitylation and degradation of
HIF-1alpha. However, inhibition of the proteasome by MG132 partially reversed the GSK3 effects
indicating that GSK3 could target HIF-1alpha to the proteasome by phosphorylation. Further,
we identified three putative target sites for GSK3 within HIF-1alpha and mutation of these sites
increased HIF-1alpha transactivity, protein stability and PAI-1 expression.
Thus, the present data show that hypoxia and insulin signalling merge at HIF-1alpha which
appears to have a prominent role for the regulation of PAI-1 expression.
Molecular & Cellular Biology of Plasminogen Activation 23

i012i
Role of Rho GTPases and p38 MAP Kinase in the Regulation of
t-PA and PAI-1 Expression in Cultured Human Endothelial Cells
Fish RJ*, Dunoyer-Geindre S, Kruithof EKO
Service of Angiology and Haemostasis, Department of Internal Medicine, Geneva University Hospital
and Medical School, Geneva, Switzerland
Presenting author e-mail: Richard.Fish@medecine.unige.ch
HMG-CoA reductase inhibitors, or statins, are used as cholesterol-lowering drugs for
cardiovascular disease (CVD). Clinical evidence suggests that statins are also beneficial for CVD
patients independently from their effects on cholesterol levels. Statins increase endothelial cell
expression of t-PA while lowering levels of PAI-1. This could increase the fibrinolytic potential
of the endothelium and be beneficial in CVD. We investigated the mechanism of statin-induced
changes in t-PA and PAI-1 expression in HUVEC. t-PA and PAI-1 mRNA were measured by
quantitative RT-PCR. A time- and dose-dependent increase in t-PA, and decrease in PAI-1,
were measured in fluvastatin-treated HUVEC. These changes were reversed by mevalonate
or geranylgeranyl pyrophosphate, and mimicked by a geranylgeranyl transferase inhibitor -
demonstrating that the statins effects were mediated by inhibition of protein geranylgeranylation.
Rho family GTPases are geranylgeranylated and therefore potential targets for the effects of
fluvastatin. Dominant negative (DN) RhoA, Rac1 and Cdc42 were expressed in HUVEC. A
minor increase in t-PA expression was measured in cells expressing DNRhoA, while DNRac1
or DNCdc42 expression each lead to a three-fold increase. PAI-1 expression decreased in cells
expressing DNRhoA or DNRac1, but not in cells expressing DNCdc42. Fluvastatin-induced t-PA
expression is most likely mediated by p38 MAP kinase (p38) because the p38 inhibitor, SB202190,
reversed fluvastatin-induced t-PA expression but not the decrease in PAI-1. Our results show
a role for p38 in statin-induced t-PA, with geranylgeranylated Rho GTPases as potential statin
targets. Endothelial t-PA and PAI-1 expression appear to be differentially regulated by Rho
GTPases.
24 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i013i
Urokinase Receptor/Alpha5Beta1 Integrin Interaction and
Signaling in Cancer Cells
Wei Y*, Tang CH, Kim Y, Robillard L, Kugler MC, Hill M, Brumwell A, Chapman HA
Pulmonary and Critical Care Division, Department of Medicine, Cardiovascular Research Institute,
University of California, San Francisco, California, USA
Presenting author e-mail: ying.wei@ucsf.edu
The urokinase receptor (uPAR) is commonly upregulated in tumor cells and thought to promote
cancer invasion and metastasis. Our previous studies show that uPAR binds fibronectin (Fn)
receptor a5b1 integrin, the complex shifting the integrin binding site on Fn from the canonical
RGD motif to a nearby Fn heparin binding domain (HepII). We hypothesized that uPAR
interactions with a5b1 modulate Fn signaling and promote tumor progression in vivo. To study the
effects of uPAR/a5b1 interactions on signaling, endogenous uPAR was stably silenced by RNAi in
malignant tumor cell lines HT1080 and H1299 and reconstituted with WT uPAR or a uPAR point
mutant (H249A) discovered to be defective in a5b1-binding. Fn attachment initiated src/FAK,
Rac1, and ERK activation followed by MMP-9 expression. In both uPAR knockdown and H249A
uPAR bearing tumor cells Fn adhesion initiated src/FAK activation but was unable to activate
Rac1 or ERK or induce MMP-9. Thus MMP-9 induction by Fn depends on two signals: one from
a5b1 leading to active src/FAK and one from a5b1/uPAR leading to active Rac1. Both are required
from ERK activation and MMP-9 induction. In vivo, knockdown of uPAR in tumor cells injected
intravenously or into the lung orthotopically resulted in markedly reduced tumor incidence and
size. UPAR knockdown cells maintained low levels of MMP-9 after at least two days in the mouse
lungs. Collectively, these data demonstrate that uPAR/a5b1 complexes are required for MMP-9
induction through an ERK-dependent and that this signaling pathway may be relevant to tumor
development in vivo.
Molecular & Cellular Biology of Plasminogen Activation 25

i014i
Activated Human Neutrophils Rapidly Release the Chemotactically
Active D2D3 Form of the Urokinase-type Plasminogen Activator
Receptor (uPAR/CD87)
Pliyev BK* 1,2 and Tkachuk VA 1,2
1Department of Biological and Medical Chemistry, School of Basic Medicine, Moscow State University,
Moscow, Russia;
2Molecular Endocrinology Laboratory, Institute of Experimental Cardiology, Cardiology Research
Center, Moscow, Russia
Presenting author e-mail: bpliyev@cardio.ru
The urokinase-type plasminogen activator receptor (uPAR/CD87) exists both in cell-bound and
soluble forms. Soluble uPAR (suPAR) is readily detected in blood and is markedly increased
during inflammation and cancer. Neutrophils contain extensive intracellular pools of uPAR that
are translocated to the plasma membrane upon activation. In the present study, we investigated
the ability of human neutrophils to shed uPAR from cell surface following activation and
addressed the possible involvement of the released receptor in inflammatory response. We
first observed that resting neutrophils spontaneously release suPAR. This release was strongly
and rapidly (within minutes) enhanced by calcium ionophore ionomycin and to a lesser extent
when cells were primed with TNF-alpha or LPS and then stimulated with fMLP or IL-8. We
demonstrate that suPAR is produced by resting and activated neutrophils predominantly as a
truncated form devoid of N-terminal D1 domain (D2D3 form) that lacks GPI anchor. Migration
of human embryonic kidney (HEK) 293 cells stably transfected with the human formyl peptide
receptor FPRL1 towards the supernatants harvested from activated neutrophils was significantly
diminished when suPAR was immunodepleted from the supernatants. We conclude that activated
neutrophils release the chemotactically active D2D3 form of suPAR that acts as a ligand of
FPRL1. The release of suPAR by activated cells was significanty but not completely inhibited by
alpha 1-antichymotrypsin, a specific inhibitor of cathepsin G, indicating that cathepsin G acts as
a shedding protease for membrane-bound uPAR. Neutrophils isolated from synovial fluids of
rheumatoid arthritis patients released significantly (p < 0.01) higher amounts of suPAR compared
with cells isolated from pared peripheral blood samples suggesting that the release of suPAR by
neutrophils is increased in vivo in the sites of acute inflammation. We suggest that production of
the chemotactically active D2D3 form of suPAR by activated human neutrophils in vivo could
contribute to the recruitment of monocytes and other formyl peptide receptors-expressing cells to
the sites of acute inflammation where neutrophils accumulation and activation occur.
26 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i015i
PDGF-DD Bioavailability Is Regulated by the uPA/uPAR System:
Implications for Tumor Growth
Ehnman M*, Li H, Fredriksson L, Eriksson U
Ludwig Institute for Cancer Research, Stockholm Branch, Karolinska Institutet, Stockholm, Sweden
Presenting author e-mail: monika.ehnman@licr.ki.se
Members of the Platelet-derived growth factor (PDGF) family are involved in many physiological
and pathological events such as embryonic development, blood vessel maturation, fibrotic
disease and cancer. In contrast to the classical PDGFs, the novel and closely related PDGF-CC
and PDGF-DD are latent factors that need to be extracellularly processed before they can initiate
PDGF receptor activation. We have previously identified tissue plasminogen activator (tPA) as
an activator of PDGF-CC and we are now elucidating the biological relevance of the urokinase
plasminogen activator (uPA) and its receptor for the proteolytic activation of PDGF-DD.
Both tPA and uPA are plasminogen activators accounting for the generation of plasmin in
different tissues, including blood, thereby initializing a proteolytic cascade leading to breakdown
of extracellular matrix components, activation of other proteases and growth factors. The uPA/
uPAR system has been extensively studied during the years and the components have been
shown to play a role as prognostic markers in various cancers and in the metastatic process.
Here we have elucidated the structural requirements for uPA-mediated activation of PDGF-DD.
To further explore if this activation process may play a role in NIH/3T3 cell transformation and
subsequent tumor growth in nude mice, we generated stable NIH/3T3 cell lines expressing either
activated PDGF-DD or latent PDGF-DD and compared their in vitro growth capacity and in vivo
tumorigenicity. Our data suggest that regulated PDGF-DD activation is important for NIH/3T3
tumor development in vivo and indicate that the uPA/uPAR system may be of importance for
localized and regulated PDGF-DD activation.
Molecular & Cellular Biology of Plasminogen Activation 27

i016i
TGF-b1-Induced Plasminogen Activator Inhibitor-
1 Expression in Vascular Smooth Muscle Cells Requires
Cooperative Rho/ROCK and EGFR Signaling
Higgins PJ* and Samarakoon R
Center for Cell Biology & Cancer Research, Albany Medical College, Albany, New York, USA
Presenting author e-mail: higginp@mail.amc.edu
TGF-b1 and its target gene encoding plasminogen activator inhibitor-1 (PAI-1) are major causative
factors in the pathophysiology of arteriosclerosis and perivascular fibrosis. The definition of TGFb1-activated
signaling networks that impact PAI-1 transcription in vascular smooth muscle cells
(VSMC) may identify novel, therapeutically-relevant, opportunities to manage PAI-1-associated
cardiovascular disease. TGF-b1-induced PAI-1 expression in VSMC required cooperative EGFR/
MEK-ERK and Rho/ROCK signaling. Transient transfection of a dominant negative RhoA (DN-
RhoA) expression construct or pretreatment of VSMC with C3 transferase (a Rho inhibitor) and
Y-27632 (an inhibitor of p160ROCK, a downstream effector of Rho) dramatically attenuated the
TGF-b1-initiated PAI-1 inductive response. Genetic EGFR1 deficiency or pharmacologic inhibition
of EGFR activity (with AG1478) virtually ablated TGF-b1-stimulated ERK1/2 activation as well
as PAI-1 expression but not SMAD2 phosphorylation. Interference with Rho/ROCK signaling,
in contrast, prevented SMAD2 activation and nuclear accumulation. Infection of VSMC with
an adenovirus encoding a mutant Y845F-EGFR effectively decreased TGF-b1-induced PAI-1
expression implicating the pp60c-src phosphorylation site (Y845) of the EGFR in the inductive
response. This is consistent with previous findings that pp60c-src is activated by TGF-b1 in
VSMC and that dominant negative pp60c-src (DN-Src) expression vectors significantly block
TGF-b1 induced PAI-1 expression. SMAD2 activation, moreover, is not sufficient to induce PAI-
1 expression by TGF-b1 in the absence of EGFR signaling both in VSMC and mouse embryonic
fibroblasts. Thus, two distinct but cooperative pathways involving EGFR/MEK-ERK signaling
and Rho-dependent SMAD2 activation are required for TGF-b1-induced PAI-1 expression in
VSMC.
28 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i017i
A Novel Neuronal Death Pathway Triggered by
Excess Tissue Plasminogen Activator
Li J* 1 , Snyder EY 2 , Sidman RL 1
1Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston,
Massachusetts, USA;
2 Stem Cell and Regeneration Program, The Burnham Institute for Medical Research, La Jolla, California,
USA
Presenting author e-mail: jli7@caregroup.harvard.edu
We have explored molecular features of the cell death pathway in nervous (nr) mutant mice
that show mitochondrial abnormalities and dendritic growth retardation in cerebellar Purkinje
neurons (PNs) in postnatal weeks 2-3, and massive PN degeneration and motor incoordination
thereafter. We found a mutation-induced dramatic 10-fold excess of tissue plasminogen activator
(tPA) in nr cerebellum, affecting two types of downstream targets: voltage-dependent anion
channels (VDACs) and neurotrophins. VDACs affect synaptic communication and apoptotic
cell death, as well as mitochondrial metabolite fluxes. The tPA/plasmin system contains five
‘’kringle’’ binding segments, of which kringle 5 can specifically bind brain mitochondrial VDACs
to induce partial closure of these channels and interfere with mitochondria-related regulation of
intracellular calcium and pH. Neurotrophins participate in regulation of neuronal morphogenesis
and maintenance. Degradation of particular neurotrophins by tPA/plasmin may account for
tPA-mediated decreases in synaptic organization and neuronal survival. We have established
that neural stem cells (NSCs) transplanted into neonatal nr cerebellum rescued PNs from cell
death weeks later by restoring molecular homeostasis of tPA and its downstream targets. Two
weeks after NSC injection, and a week before onset of PN degeneration, correction of tPA was
accompanied by 1) normal amount and distribution of VDACs and normal mitochondrial shape,
and 2) neurotrophin normalcy and PN dendritic growth. Likewise, NSCs contacting cerebellar
slices in vitro rectified tPA levels and rescued host PNs, as did pharmacological decrease of tPA to
normal but not subnormal levels. Neurotoxic action of tPA is open to further study in this system.
Molecular & Cellular Biology of Plasminogen Activation 29

i018i
The Inhibitor of Serine Proteases Protease Nexin-1 and its Receptor
LRP Modulate SHH Signalling during Cerebellar Development
Vaillant C 1 , Michos O 2 , Orolicki S 1 , Brellier F 1 , Taieb S 1 , Moreno E 1 , Té H 1 , Zeller R 2 , Monard D* 1
1 Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland;
2 Developmental Genetics, DKBW Center for Biomedicine, University of Basel Medical School, Basel,
Switzerland
Presenting author e-mail: denis.monard@fmi.ch
Development of the postnatal cerebellum relies on tight regulation of the cell number by
morphogens that control the balance between cell proliferation, survival and differentiation. Here
we analyze the role of the serine protease inhibitor Protease Nexin-1 (PN-1) in the proliferation
and differentiation of Cerebellar Granular Neuron Precursors (CGNPs) via modulation of their
main mitogenic factor, Sonic Hedgehog (SHH). Our studies show that PN-1 interacts with the
Low-density lipoprotein receptor-Related Proteins (LRPs) to antagonize SHH-induced CGNP
proliferation and inhibits the activity of the SHH transcriptional target Gli1. The binding of
PN-1 to LRPs interferes with SHH-induced Cyclin D1 expression. CGNPs isolated from PN-
1 deficient mice exhibit enhanced basal proliferation rates due to over-activation of the SHH
pathway and show higher sensitivity to exogenous SHH. In vivo, the PN-1 deficiency alters the
expression of SHH target genes. In addition, the onset of CGNP differentiation is delayed, which
results in an enlarged outer External Granular Layer. Furthermore, the PN-1 deficiency leads
to an overproduction of CGNPs and enlargement of the Internal Granular Layer in a subset of
cerebellar lobes during late development and adulthood. We propose that PN-1 contributes to
shaping of the cerebellum by promoting cell cycle exit.
30 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i019i
Fibrin Deposition Accelerates Neurovascular Damage and
Neuroinflammation in Mouse Models of Alzheimer’s Disease
Paul J* and Strickland S
Laboratory of Neurobiology and Genetics, The Rockefeller University, New York, New York, USA
Presenting author e-mail: jpaul@rockefeller.edu
Cerebrovascular dysfunction contributes to the pathology and progression of Alzheimer’s disease
(AD) but the mechanisms are not completely understood. Using transgenic mouse models of
Alzheimer’s disease (TgCRND8 and Tg2576), we evaluated blood-brain barrier damage and
the role of fibrin and fibrinolysis in the progression of b-amyloid pathology. These mouse
models showed age-dependent fibrin deposition coincident with areas of blood-brain barrier
permeability as demonstrated by Evans blue extravasation. Three lines of evidence suggest that
fibrin contributes to the pathology: 1, AD mice with only one functional plasminogen gene and
therefore with reduced fibrinolysis have increased neurovascular damage relative to AD mice;
2 Conversely, AD mice with only one functional fibrinogen gene have decreased blood-brain
barrier damage; 3, Treatment of AD mice with the plasmin inhibitor tranexamic acid aggravated
pathology, while removal of fibrinogen from the circulation of AD mice with ancrod treatment
attenuated measures of neuroinflammation and vascular pathology. These results suggest fibrin
is a mediator of inflammation and may impede the reparative process for neurovascular damage
in AD. Fibrin and the mechanisms involved in its accumulation and clearance may present novel
therapeutic targets in slowing the progression of Alzheimer’s disease.
Molecular & Cellular Biology of Plasminogen Activation 31

i020i
Anxiety-like Behavior and Impaired Fear Extinction in Mice with
Altered Control of Extracellular Brain Proteolytic Activity
Meins M*, Herry C, Moreno E, Fischer C, Lüthi A, Monard D
Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland
Presenting author e-mail: mmeins@fmi.ch
Regulation of serine proteases has been implicated in modulating synaptic plasticity. Protease
nexin-1 (PN-1), an endogenous serine protease inhibitor, is expressed in specific neuronal
subpopulations in the adult central nervous system. The in vivo expression of PN-1 is regulated
by neuronal activity. In mice lacking PN-1 (PN-1 –/–), the brain homogenate proteolytic profile
is altered and NMDA receptor-mediated transmission is reduced. To examine the in vivo
consequences of these changes, we compared cognitive and emotional responses of PN-1 –/– mice
to their wildtype littermates. No memory impairments were detected. In contrast, PN-1 –/– mice
displayed enhanced anxiety-like and avoidance behavior. Differences between wildtype and
PN-1 –/– littermates were also detected in cued fear conditioning. Reduced extinction of freezing
behavior was detected in the PN-1 –/– mice. This was reflected at the cellular level by decreased
immunoreactivity of c-FOS, an indicator of neuronal activity, in the PN-1 –/– basolateral
amygdala. Surprisingly, increased immunoreactivity was detected in the same area in the PN-1
–/– mice which underwent fear conditioning without extinction training. This may suggest that
the increased reaction displayed by PN-1 –/– mice to some stressful situations may be the result
of increased neuronal activation of the relevant neurons. The increased anxiety-like behavior and
the reduced fear extinction in the PN-1 –/– mice could be explained by an impaired modulation
of NDMA receptor mediated activity in some regions of the amygdala. To investigate this
hypothesis, we are analyzing downstream effectors of NMDA receptor activation in these areas.
These results not only corroborate findings supporting the importance of the control of proteolytic
activity in synaptic plasticity but they also suggest the PN-1 –/– mice as an interesting model to
study the brain structures and the molecular mechanisms involved in fear extinction.
32 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i021i
Tissue Plasminogen Activator Is Co-Packaged and
Co-Transported to Synaptic Sites with a Key
Neuromodulator Associated with Synaptic Plasticity
Lochner JE* 1 , Spangler E 1 , Schuttner LC 1 , Scalettar BA 2
1 Biochemistry & Molecular Biology, 2 Physics, Lewis & Clark College, Portland, Oregon, USA
Presenting author e-mail: lochner@lclark.edu
Tissue plasminogen activator (tPA) is highly expressed in the hippocampus and is implicated
in modifying synaptic efficacy during learning and memory. tPA is postulated to exert its
influence on synaptic plasticity by initiating an extracellular proteolytic cascade that promotes the
conversion of precursor brain-derived neurotrophic factor (proBDNF) to mature BDNF (mBDNF)
extracellularly, at synapses. mBDNF is a potent neuromodulator that is known to augment
synapse density and elicit long-lasting enhancement of synaptic transmission. Motivated by
recent interest in possible interactions between tPA and proBDNF, we have developed a system
that facilitates study of the packaging, transport, and secretion of these two neuromodulatory
proteins in cultured hippocampal neurons. We find 89% colocalization of tPA and proBDNF
chimeras within dense-core granules (DCGs) of mature hippocampal neurons, indicating efficient
co-packaging of these neuromodulators. Additionally, we find that these two neuromodulators
undergo rapid co-transport (at speeds of ~1 µm/s) along neuronal processes, suggesting that tPA
and pro-BDNF are co-delivered to synaptic sites in DCGs. Evaluation of the colocalization of these
two neuromodulators at the organelle level in post-synaptic dendritic spines reveals that tPA
and proBDNF chimeras colocalize in individual DCGs in 27% of spines, and that 80% of spines
which contain DCGs contain both chimeras in the same DCG. Our results suggest that efficient
co-packaging and co-transport produces a pool of DCGs containing both tPA and proBDNF at
post-synaptic sites, where these neuromodulators can undergo activity-dependent co-release from
DCGs and then interact and/or mediate changes that influence learning and memory.
Molecular & Cellular Biology of Plasminogen Activation 33

i022i
Tissue Plasminogen Activator Modulates Cellular
and Behavioral Response to Cocaine
Maiya R* 1 , Zhou Y 2 , Norris EH 1 , Kreek MJ 2 , Strickland S 1
1 2 Laboratory of Neurobiology and Genetics, Laboratory of the Biology of Addictive Diseases, The
Rockefeller University, New York, New York, USA
*Presenting author e-mail: rmaiya@mail.rockefeller.edu
Cocaine exposure induces long lasting molecular and cellular adaptations in the brain. Tissue
plasminogen activator (tPA), an extracellular protease implicated in regulating neuronal plasticity,
may orchestrate some of these cocaine-induced neuro-adaptations. In this study, the effects
of acute and chronic cocaine administration on tPA activity in the brain were examined. Mice
were injected with cocaine in the acute (3 injections of 15 mg/kg) or chronic (3x15mg/kg for 14
days) ‘’binge’’ paradigm. In situ and in-gel zymography revealed that in the amygdala of WT
animals, acute cocaine exposure increased tPA activity whereas chronic cocaine administration
decreased tPA activity. Acute cocaine also elevated levels of plasma corticosterone, corticotropin
releasing factor (CRF), and CRF receptor 1 (CRF-R1) mRNAs in the amygdala of both WT and
tPA–/– animals. Phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2), a marker
for post-synaptic plasticity events, was observed in the amygdala of WT but not tPA–/– mice
after acute cocaine exposure. In comparison to WT animals, tPA–/– animals showed attenuated
c-fos expression in the amygdala after acute cocaine administration. These results suggest altered
cocaine-induced neuro-adaptation in the amygdala of tPA–/– mice. Cocaine-induced anxiety
was also examined in both WT and tPA–/– animals using the elevated plus maze. In contrast to
WT animals, tPA–/– animals were resistant to the anxiogenic effects of acute cocaine. After acute
cocaine, tPA–/– mice displayed increased total arm entries in comparison to WT mice, suggesting
enhanced cocaine-induced locomotor stimulation. In summary, these results suggest a significant
role for tPA in the modulation of cocaine-induced behaviors and neuronal plasticity.
34 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i023i
Characterisation of the Pathway of Polymerisation
of Wildtype Neuroserpin and the Ser49Pro Mutant
that Underlies the Dementia FENIB
Hägglöf P*, Belorgey D, Karlsson-Li S, Sharp LK, Lomas DA
Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research,
Cambridge, United Kingdom
Presenting author e-mail: pmh43@cam.ac.uk
Neuroserpin is expressed during the late stage of development in neurons of the central and
peripheral nervous system and in the adult brain. The target proteinase of neuroserpin is tissue
plasminogen activator (tPA) and it is likely to be important in the control of synaptic plasticity,
in learning, memory and can act as a neuroprotectant. It can decrease the toxicity of the Ab1-
42 peptide that is central to the pathogenesis of Alzheimer’s disease. Point mutations in the
neuroserpin gene underlie the autosomal dominant dementia, familial encephalopathy with
neuroserpin inclusion bodies, (FENIB). This is characterised by the retention of ordered polymers
of neuroserpin within the endoplasmic reticulum of neurons. We have previously shown that
polymers result from the sequential linkage between the reactive centre loop of one neuroserpin
molecule and b-sheet A of another. We show here that the polymerisation of both wildtype and
the Ser49Pro mutant of neuroserpin that causes FENIB is dependent of pH demonstrating that
the His-338 residue in the shutter region is important for in the opening of b-sheet A. Tryptophan,
extrinsic fluorescence and fluorphore labelled cysteine mutants suggest that the RCL may be
preinserted in b-sheet A and then expelled during in an at least two steps in the formation of
polymers: a fast unimolecular process that is likely to reflect opening of b-sheet A followed by
the slower process of polymer formation. Taken together these data suggest a two-step kinetic
mechanism for the formation of the polymers of neuroserpin that underlie the dementia FENIB.
Molecular & Cellular Biology of Plasminogen Activation 35

i024i
Matriptase Is an Essential Inhibitory Target for
Hepatocyte Growth Factor Activator Inhibitor-1 during
both Embryonic Development and Postnatal Life
Szabo R*, Molinolo A, List K, Bugge TH
Oral and Pharyngeal Cancer Branch, NIDCR, NIH, Bethesda, Maryland, USA
Presenting author e-mail: rszabo@nidcr.nih.gov
Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a Kunitz-type transmembrane serine
protease inhibitor proposed to inhibit the activity of several trypsin-like serine proteases,
including hepsin, hepatocyte growth factor activator, prostasin (CAP1/PRSS8), and matriptase.
Here we generated HAI-1-deficient mice to determine the biological function of HAI-1 and
identify physiological HAI-1 inhibitory targets during development and postnatal life. HAI-
1-deficient mice died at mid-gestation due to placental insufficiency caused by a disruption of
the epithelial integrity of placental chorionic trophoblasts associated with chorionic basement
membrane dissolution, loss of E-cadherin, and loss of membrane-associated b-catenin.
Interestingly, however, matriptase gene ablation in HAI-1-deficient embryos restored the integrity
of chorionic trophoblasts and enabled both normal placentation and development to term. HAI-
1 was recently reported to also be required for mouse postnatal survival beyond two weeks. To
determine the inhibitory targets for HAI-1 during postnatal life, we generated HAI-1 null mice
in a matriptase hypomorphic background, which display 82 - >99 % reduction in matriptase
mRNA levels in epithelial tissues. Low matriptase levels enabled both embryonic development
and normal postnatal survival of HAI-1-deficient mice when followed for up to six months. Taken
together, this study identifies matriptase as an essential inhibitory target for HAI-1 during both
embryonic development and postnatal life.
36 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i025i
Mice with very low Matriptase Are Viable and Phenocopy
Human Autosomal Ichthyosis with Hypotrichosis Syndrome
List K* 1 , Currie B 1 , Scharschmidt T 2 , Szabo R 1 , Molinolo A 1 , Shireman J 1 , Segre J 2 , Bugge TH 1
1 Oral and Pharyngeal Cancer Branch, NIDCR, NIH, Bethesda, Maryland, USA;
2 National Human Genome Research Institute, NIH, Bethesda, Maryland, USA
Presenting author e-mail: klist@nidcr.nih.gov
Complete deficiency of the type II transmembrane serine protease matriptase or its candidate
downstream target, the GPI-anchored serine protease prostasin, causes epidermal barrier
disruption and neonatal death of mice. Here we report the surprising observation that mice with a
one-hundred-fold reduction in epidermal matriptase mRNA levels, generated by inserting a leaky
splice acceptor site into intron 1 of the matriptase gene, are viable and fertile. Interestingly, these
matriptase hypomorphic mice display skin and hair defects that are identical to those of humans
with Autosomal Recessive Ichthyosis with Hypotrichosis (ARIH): an inherited disorder recently
linked to homozygosity for a G827R substitution in the matriptase serine protease domain. Thus,
matriptase hypomorphic mice developed marked hyperkeratosis with impaired desquamation
and focal dermal inflammation, as well as hypotrichosis with brittle, thin, uneven, and sparse
hair. Proteomic analysis of matriptase hypomorphic epidermis revealed markedly reduced
prostasin activation and profilaggrin proteolytic processing. This work strongly supports reduced
matriptase activity as one etiological origin of human ARIH and provides a mouse model for the
exploration of matriptase in other physiological and pathological processes, including epithelial
carcinogenesis.
Molecular & Cellular Biology of Plasminogen Activation 37

i026i
Role of Urokinase-Receptor in Hematopoietic Stem Cell Trafficking
Montuori N 1 , Selleri C 2 , Ricci P 2 , Visconte V 1 , Carriero MV 3 , Rotoli B 2 , Rossi G 1 , Ragno P* 4
1 Department of Cellular and Molecular Biology and Pathology, 2 Department of Biochemistry and
Medical Biotechnology, ‘’Federico II’’ University, Naples, Italy;
3 Department of Experimental Oncology, National Cancer Institute, Naples, Italy;
4 Department of Chemistry, University of Salerno, Salerno, Italy
Presenting author e-mail: pragno@unisa.it
Cleaved forms of soluble urokinase-receptor (c-suPAR) have been detected in body fluids from
patients affected by various tumors. We reported increased c-suPAR levels in sera of healthy
donors during granulocyte colony-stimulating factor (G-CSF)-induced mobilization of CD34+
hematopoietic stem cells (HSCs). In vitro, c-suPAR or its derived chemotactic peptide (uPAR84-
95) stimulated migration of human CD34+ HSCs and inactivated CXCR4, the chemokine receptor
primarily responsible for HSC retention in bone marrow (BM). These results suggested that
c-suPAR could potentially contribute to regulate HSC trafficking from and to BM. Therefore, we
investigated uPAR84-95 effects on mobilization of mouse CD34+ hematopoietic stem/progenitor
cells (HSCs/HPCs). We first demonstrated that uPAR84-95 stimulated in vitro dose-dependent
migration of mouse CD34+ M1 leukemia cells and inactivated murine CXCR4. uPAR84-95
capability to induce mouse HSC/HPC release from bone marrow and migration into the
circulation was then investigated in vivo. uPAR84-95 intraperitoneal administration induced rapid
leukocytosis, which was associated with an increase in peripheral blood CD34+ HSCs/HPCs. In
vitro colony assays confirmed that uPAR84-95 mobilized hematopoietic progenitors, showing an
increase in circulating colony-forming cells.
We are now studying suPAR and c-suPAR effects on HSCs, to investigate their role in HSC
retention in BM, and, therefore, their potential contribution also to the homing and the
engraftment of HSCs to the BM. In vitro effects of anti-uPAR antibodies and of suPAR and csuPAR
on adhesion and proliferation of human G-CSF-mobilized and BM HSCs have been
examined in long-term cultures (LTC), which represent the best surrogate in vitro of BM
hematopoiesis.
38 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i027i
Plasminogen Activator Inhibitor-1, PAI-1, Regulates
the Akt Survival Pathway
Rømer MU, Larsen L, Offenberg H, Brünner N, Lademann U*
Department of Veterinary Pathobiology, Faculty of Life Science, University of Copenhagen, Frederiksberg,
Denmark
Presenting auther e-mail: ul@life.ku.dk
Plasminogen Activator Inhibitor-1, PAI-1, inhibits the activation of the plasminogen activation
system, which is involved in cancer growth and dissemination. Increased tumor tissue levels of
PAI-1 would therefore be expected to inhibit cancer cell progression. However, PAI-1 is elevated
in many solid tumors and this elevation has consistently been shown to be associated with shorter
patient survival. The reason for this has remained unclear.
The phosphatidylinositol-3 kinase (PI3K)/Akt pathway is overactivated in a wide range of tumor
types, and this triggers a cascade of responses, including inhibition of apoptosis. PAI-1 has been
reported to modulate the PI3 kinase /Akt pathway, thus it is possible that PAI-1 inhibits apoptosis
of tumor cells via activation of this pathway.
We have previously shown that PAI-1–/– fibrosarcoma cells are significantly more sensitive than
wild-type fibrosarcoma cells to etoposide-induced apoptosis. To further study the signalling
pathways involved in the anti-apoptotic function of PAI-1 we asked the question whether Akt
activation is involved in PAI-1 mediated inhibition of apoptosis. We demonstrate that Akt is
hyperfosforylated in wild type fibrosarcoma cells (endogenous PAI-1 expression) compared
with PAI-1–/– fibrosarcoma cells. This hyperphosphorylation is accompanied by increased
phosphorylation of downstream targets of Akt. When wild type fibrosarcoma cells were treated
with a specific inhibitor against Akt1/2 (Akt inhibitor VIII) and PI3K (LY294002) it induced a
sensitizing effect on etoposide induced cell death, further supporting an apoptosis regulating role
of the PI3K/Akt survival pathway in our cell model.
Altogether, our data suggests that PAI-1 inhibits etoposide induced cell death via activation of the
Akt survival pathway.
Molecular & Cellular Biology of Plasminogen Activation 39

i028i
A Host Plasminogen Activator Inhibitor-1 Deficiency Promotes
Proliferation and Resistance to Apoptosis by Activation
of the PI3-K/Akt Pathway in Endothelial Cells
Balsara RD* 1,2,3 , Castellino FJ 1,2,3 , Ploplis VA 1,2,3
1 2 3 W. M. Keck Center for Transgene Research, Department of Chemistry and Biochemistry, Notre Dame
Cancer Institute, University of Notre Dame, Indiana, USA
Presenting author e-mail: rbalsara@nd.edu
Angiogenic function of plasminogen activator inhibitor-1 (PAI-1) and its underlying mechanism
were studied utilizing endothelial cells (EC) isolated from the aortas of wild-type (WT) and PAI-
1-deficient mice. The enhanced proliferation of PAI-1–/– EC was associated with hyperactivation
of Akt(P-Ser473) and accompanied by resistance to apoptosis. Disruption of VEGF/VEGFR-1
interaction attenuated activation of Akt(P-Ser473) in PAI-1–/– EC. Involvement of the PI3k/Akt
survival axis in both WT and PAI-1–/– EC was discerned by treating cells with the
pharmacological inhibitor wortmannin, which also diminished cell proliferation. Furthermore,
the higher levels of Akt(P-Ser473) in PAI-1–/– EC were regulated by higher levels of inactive
phosphatase PTEN. In PAI-1–/– EC, increased levels of Akt(P-Ser473) were associated with higher
levels of inactive caspase-9, which is a direct phosphorylation target of Akt. Subsequently lower
protein levels and activity of pro- and active-caspase-3 were detected in PAI-1–/– EC. Treatment
of PAI-1–/– EC with exogenous r-PAI-1 controlled the increased rate of cell proliferation with
concomitant modulation of the components of the Akt pathway. In the presence of r-PAI-1, levels
of inactive PTEN(P-Ser380) and Akt(P-Ser473) were decreased, and the levels of pro- and activecaspase-3
were increased. A r-PAI-1 mutant that had compromised binding ability to low density
lipoprotein receptor-related protein (LRP) failed to alter cell proliferation of PAI-1–/– cells. These
results indicate that PAI-1 mediates its anti-proliferative activity in an LRP-dependent manner.
Additionally, PAI-1 plays a pivotal role in modulating the Akt pathway, which is an important
participant in transducing signaling events during angiogenesis.
40 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i029i
Bomapin Is a Redox-regulated Serpin which Stabilizes Retinoblastoma
Protein during Apoptosis and Increases Proliferation of Leukemia Cells
Przygodzka P, Olausson B, Tengel T, Larsson G, Wilczynska M*
Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden
Presenting author e-mail: Malgorzata.Wilczynska@medchem.umu.se
Bomapin is a hematopoietic- and leukemia-specific intracellular serine protease inhibitor.
Its expression is restricted to bone marrow and to peripheral blood leukocytes of patients
with myeloid leukemias. Consistent with this, bomapin is also constitutively expressed in
promyelocytic and monocytic cell lines (HL60, THP1, and AML-193). Herein we show that
naturally expressed bomapin is localized in the nucleus, where it is stabilized by a disulfide
bond linking the only two bomapin cysteines. Computer modeling has shown that the cysteines
are distant in the reduced bomapin, but can easily be disulfide-linked without distortion of
the overall bomapin structure. Stable, ectopic expression of wild-type bomapin in K562 cells
increased cell proliferation by about 50%. Importantly, the enhancement of cell proliferation was
not observed for a bomapin mutant lacking the disulfide bond. We show also that in proliferating
leukemia cells, bomapin existed in a multi-protein complex including lamin-A/C, retinoblastoma
(Rb) protein, beta-actin, and non-muscle myosin IIA. Bomapin expression resulted in stabilization
of Rb protein during serum deprivation-induced apoptosis. We propose that bomapin is a
redox-regulated serpin which enhances proliferation potential of leukemia cells via assembly
into a multi-protein complex including also the Rb protein, and stabilizes Rb protein against
degradation during apoptosis.
Molecular & Cellular Biology of Plasminogen Activation 41

i030i
Hespin as a Cell Survival Factor
Qiu D, Owen K, Edwards DR, Ellis V*
Biomedical Research Centre, School of Biological Sciences, University of East Anglia, Norwich, United
Kingdom
Presenting author e-mail: v.ellis@uea.ac.uk
Hepsin is a type II transmembrane serine protease upregulated in a variety of cancers and may
be functionally involved in disease progression. How hepsin might mediate these effects and
via which substrates is currently unknown. Our qRT-PCR data show that hepsin expression
in prostate cancer tissue is positively correlated with both malignancy (p

i031i
Urokinase (uPA) Protects Endothelial Cell against Apoptosis by
Upregulating the X-linked Inhibitor of Apoptosis Protein (XIAP)
Prager GW* 1,2 , Koschelnick Y 1 , Mihaly J 1 , Brunner P 1 , Binder BR 1
1 Department of Vascular Biology and Thrombosis Research, Centre for Bio-Molecular Medicine and
Pharmacology, 2 Clinical Division of Oncology, Department of Medicine I and Cancer Center, Medical
University Vienna, Austria
Presenting author e-mail: gerald.prager@meduniwien.ac.at
uPA has originally been implicated to assist the angiogenic process by it’s proteolytic properties.
It is now becoming increasingly evident that uPA additionally elicits many pro-angiogenic
responses like differentiation, proliferation and cell migration in a non-proteolytic fashion via
induction of intracellular signal transduction.
We now demonstrate that in endothelial cells uPA protects against apoptosis by transcriptional
upregulation of inhibitor of apoptosis proteins (IAPs), among them most prominently the Xlinked
inhibitor of apoptosis protein (XIAP). In contrast to canonical growth factors, like vascular
endothelial growth factor (VEGF), uPA elicits anti-apoptosis independently of the PI3-kinase
pathway. uPA-induced cell survival is dependent on the type of extracellular matrix used
indicating the involvement of integrin adhesion receptors. Thereby, uPA induces phosphorylation
of the CDC42 downstream effector p21-activated kinase 1 (PAK1) and phosphorylation of
IkappaB kinase alpha (IKKa), which itself induces NFkappaB activation. Blocking NFkappaB by
the use of the specific NFkappaB inhibitor BAY 11-7082 or by adenoviral-mediated overexpression
of its inhibitor IkB, inhibits uPA-induced XIAP expression as well as uPA-induced cell survival.
Furthermore, silencing XIAP expression by siRNA significantly reduces cell survival efficiencies
of uPA in endothelial cells.
From these data we conclude that uPA, which is a main player in endothelial cell migration and
invasion, provides an additional, PI3-kinase independent autocrine or paracrine cell survival
mechanism.
Molecular & Cellular Biology of Plasminogen Activation 43

i032i
Pro-fibrinolytic Effects of Metalloproteinases during Skin
Wound Healing in the Absence of Plasminogen
Lund LR* 1 , Green KA 1 , Almholt K 1 , Ploug M 1 , Bugge TH 2 , Rømer J 1
1 Finsen Laboratory, Rigshospitalet, Copenhagen Biocenter, Copenhagen, Denmark;
2Proteases and Tissue Remodeling Unit, National Institute of Dental and Craniofacial Research, National
Institutes of Health, Bethesda, Maryland, USA
Presenting author e-mail: lund@inet.uni2.dk
Genetic ablation of plasminogen as well as pharmacological inhibition of metalloproteinase
activity delays skin wound healing in mice, while the combined inhibition of these two
enzyme systems completely prevents healing. In the present study the impact of plasmin and
metalloproteinases as pro-fibrinolytic enzymes has been investigated by comparing skin wound
healing in the absence and presence of fibrin. Plasminogen-deficiency impairs skin wound
healing kinetics, but this delay is only partially restored in the absence of fibrin. This suggests that
plasmin-mediated fibrinolysis is the primary, but not the exclusive, requirement for healing of
wounds in these mice. In addition, we observe that lack of fibrin reduces plasminogen activation
significantly during wound healing. The pro-fibrinolytic role of metalloproteinases is revealed
by the finding that lack of fibrin partially restores the otherwise arrested healing of plasminogendeficient
wounds after metalloproteinase-inhibition. In conclusion, the residual impairment
of skin wound healing in the absence of fibrin suggests the existence of a fibrin-independent
substrate(s) for plasmin and metalloproteinases. Furthermore, these in vivo data reveal that
galardin-sensitive metalloproteinases mediate compensatory fibrinolysis to facilitate wound
healing in the absence of plasmin.
44 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i033i
Complementary Roles of Intracellular and Pericellular Collagen
Degradation Pathways in Mesenchymal Cell Survival and Proliferation
Wagenaar-Miller RA 1 , Engelholm LH 2 , Gavard J 1 , Yamada S 3 , Gutkind JS 1 , Behrendt N 2 , Holmbeck K 3 ,
Bugge TH* 1
1 Oral and Pharyngeal Cancer Branch, NIDCR, NIH, Bethesda, Maryland, USA;
2 Finsen Laboratory, Copenhagen, Denmark;
3 Skeletal Morphogenesis Branch, NIDCR, NIH, Bethesda, Maryland, USA
Presenting author e-mail: thomas.bugge@nih.gov
Mesenchymal cells must be capable of proteolytically remodeling their collagen-rich pericellular
environments to survive, proliferate, and properly differentiate. Two key turnover pathways
have been described for collagen: intracellular cathepsin-mediated degradation and pericellular
collagenase-mediated degradation. Here we show that intracellular and pericellular collagen
turnover pathways have complementary functions in collagen remodeling. Combined, but
not individual, deficits in intracellular collagen degradation (uPARAP/Endo180 ablation) and
pericellular collagen degradation (MT1-MMP ablation) caused proliferative failure and poor
survival of osteoblasts and chondrocytes within their respective collagen type I- and collagen
type II-rich osteogenic and chondrogenic niches. This severely impaired overall bone formation,
leading to uniform postnatal death of combined uPARAP/Endo180;MT1-MMP-deficient
mice. These findings have important implications for the use of pharmacological inhibitors of
collagenase activity aimed at preventing connective tissue destruction in a variety of diseases.
Molecular & Cellular Biology of Plasminogen Activation 45

i034i
Interplay between MMPs and the Endocytic Collagen
Receptor, uPARAP/Endo180, in Collagen Degradation
Behrendt N* 1 , Madsen DH 1 , Ingvarsen S 1 , Hillig T 1 , Wagenaar-Miller R 2 , Kjøller L 1 , Gårdsvoll H 1 ,
Høyer-Hansen G 1 , Bugge TH 2 , Engelholm LH 1
1 The Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark;
2Oral & Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, NIH,
Bethesda, Maryland, USA
Presenting author e-mail: niels.behrendt@finsenlab.dk
The uPAR-associated protein (uPARAP/Endo180) is an endocytic cell surface receptor on
mesenchymal cells that is centrally engaged in the turnover of collagens of several subtypes. It
has recently become clear that uPARAP/Endo180-mediated collagen degradation takes place
both during bone development and in the invasive growth of malignant tumors. In this work, we
have focused on the interplay between this new mechanism and the more extensively studied,
extracellular routes of collagenolysis. In collagen internalization assays, collagen that has been
pre-cleaved by a mammalian collagenase is taken up much more efficiently than intact, native
collagen by uPARAP/Endo180 positive cells. This preference is governed by the acquisition of
a gelatin like structure of collagen, occurring upon collagenase mediated cleavage under native
conditions. Furthermore, the growth of uPARAP/Endo180 deficient fibroblasts on a native
collagen matrix leads to a dramatic accumulation of large collagen fragments in the culture
supernatant. In contrast, wildtype fibroblasts possess the ability to direct a complete collagen
breakdown sequence, including both initial, extracellular cleavage, endocytic (uPARAP/Endo180
mediated) uptake of large, defined fragments and a final, intracellular degradation step. Thus,
our work shows that extracellular collagenolysis and endocytic collagen turnover can occur as
an integrated mechanism in cultured fibroblasts and fibroblast-like cells. Most likely, a similar
mechanism is operative in the stromal cell types that are responsible for collagenolysis during
tumor invasion.
46 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i035i
The Urokinase Receptor Ko Mice Have Reduced Keratinocytes
Proliferation and Migration during Wound Healing and
Are Protected in a Skin Carcinogenesis Protocol
D’Alessio S, Mazzieri R, Gerasi L, Blasi F*
H. San Raffaele and IFOM (FIRC Institute of Molecular Oncology), Milan, Italy
Presenting author e-mail: Blasi.Francesco@hsr.it
We have found that the urokinase receptor (uPAR) is implicated in multistage mouse skin
carcinogenesis, since uPAR Ko mice show a reduced susceptibility to tumor formation after a
DMBA-TPA protocol, compared to wt-mice. The uPAR Ko mice have 70% less and much smaller
papillomas. However, the rate of conversion of papillomas to carcinomas, is not different between
wt and Ko mice, indicating that the role of uPAR is in the first stage of tumorigenesis. We also
have noticed that uPAR is also involved in the regulation of wound healing, as uPAR Ko mice
display a slight delay in wound closure compared to wt-skin. In order to directly understand
the role of endogenous uPAR in these two in vivo events, we examined uPAR null-and wildtype
keratinocytes behaviour in vitro. Under non–permissive conditions uPAR wt grow faster
than uPAR Ko keratinocytes (and differentiate faster). After a strong growth stimulation, such
as addition of EGF or serum, the difference becomes even larger as the uPAR Ko keratinocytes
do not respond to EGF. Moreover EGF induces cell growth and activates ERK/MAP kinase
in murine keratinocytes only when these cells express uPAR even though the EGF receptor
(EGFR) expression level was not different uPAR Ko and wt cells, both in the absence and in the
presence of EGF. Moreover EGFR tyrosine phosphorylation, which is involved in cell growth, was
specifically decreased in uPAR Ko expressing cells both in the absence and in the presence of EGF.
In terms of cell proliferation, the rescue of the uPAR +/+ phenotype was achieved by expressing
murine, but not human, uPAR cDNA, in uPAR Ko cells. This suggests a role for also for uPA in
EGFR activation, possibly through an autocrine signalling, as murine uPA cannot bind the human
uPAR.
Besides a reduced growth rate in vitro, uPAR-deficient keratinocytes are also unable to migrate,
adhere and spread on a glass substrate because they fail to produce and secrete their own LN-5
substrate, which is an important requirement for reepithelialization of cutaneous wounds. In fact,
when we generated full-thickness excision wounds in the skin, immunological analysis of frozen
sections 3 days after injury, revealed that LN5 was highly expressed in uPAR wt but was almost
absent in uPAR-deficient mice, meaning that also in vivo LN5 deposition is controlled by the uPAR
genotype. This effect can be observed at both RNA and protein level.
In summary, our data provide evidence that uPAR controls cell migration and proliferation both
in vivo and in vitro under stressed conditions such as tumor growth and wound healing.
Molecular & Cellular Biology of Plasminogen Activation 47

i036i
Distinct Roles of Plasmin in Staphylococcus aureus-induced
Sepsis and Infection Models
Guo Y*, Li J, Hagström E, Ny T
Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden
Presenting author e-mail: yong-zhi.guo@medchem.umu.se
Plasmin is an important mediator in inflammatory cell migration and activation during host
defense against bacterial infection. However, the exact functional role of plasmin/ogen during
infection and sepsis is unclear. We have investigated the role of plasmin in murine Staphylococcus
aureus-induced sepsis and infection models by using wild-type (plg +/+ ) and plasminogen-deficient
(plg –/– ) mice. Our results showed that when mice were injected intravenously with 1 × 10 7 CFU of
S. aureus to induce systemic infection, the plg +/+ control mice had a 21-day survival rate of 86.7%.
The 21-day survival rate in the plg –/– group was 50%. However, when 16 times more bacteria were
injected to the mice, the average survival day for the plg –/– mice was 3 days longer than for the
plg +/+ mice. 24 hours after the induction of sepsis, serum IL-6 and IL-10 levels and the bacterial
counts in all detected organs were significantly higher in plg +/+ mice than in plg –/– mice. In plg +/+
mice, blockade of IL-6 by intravenous injection of anti-IL-6 antibodies significantly prolonged
the onset of mortality and improved the survival rate during sepsis. During sepsis, both the
phosphorylated and total STAT3 protein levels were dramatically lower in the neutrophils of
plg –/– mice as compared with plg +/+ mice. These data indicate that plasmin plays different roles
during infection and sepsis. In the infectious model plasmin has beneficial effects on host antibacterial
immune responses. However, in the septic model, plasmin seems to excessively promote
the production of inflammatory cytokines and tissue destruction, cripples neutrophil function, as
well as impairs bacterial killing ability in plg +/+ mice.
48 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i037i
Proteolytic Activation of the Human Urokinase/Plasmin System
by Staphylococcus aureus
Beaufort N* 1 , Wojciechowski P 1,2 , Sommerhoff CP 3 , Schmitt M 1 , Potempa J 2 , Magdolen V 1
1 Department of Obstetrics and Gynecology, Technical University of Munich, Munich, Germany;
2Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian
University, Krakow, Poland;
3Department of Clinical Chemistry and Clinical Biochemistry, Ludwig-Maximilians-University,
Munich, Germany
Presenting author e-mail: nbeaufortgbb@yahoo.fr
The major opportunistic human pathogen Staphylococcus aureus is known to interact with and
engage the human plasminogen activation system through plasmin(ogen) binding and nonproteolytic
activation, a capacity which is thought to participate to bacterial spread and invasion.
We here report that staphylococcal reference and clinical strains efficiently convert the zymogen
pro-urokinase (pro-uPA) into its active counterpart uPA. Using selective protease inhibitors,
purified bacterial proteases, and/or protease-deficient bacterial strains, we establish that this
processing involves the secreted thermolysin-like metalloprotease aureolysin, and displays a
2.6 x 10-3 M-1 s-1 catalytic efficiency. Additionally, aureolysin not only targets pro-uPA, but also
cleaves and disables the serpins plasminogen activator inhibitor-1 and a2-antiplasmin, whereas it
converts plasminogen into angiostatin and mini-plasminogen.
Altogether, we propose that, in parallel to the staphylokinase-dependent activation of
plasminogen, aureolysin contributes significantly to the activation of the fibrinolytic system by S.
aureus, and thus may promote bacterial spread and invasion.
Molecular & Cellular Biology of Plasminogen Activation 49

i038i
The Maintenance of High Affinity Plasminogen Binding by PAM
Variants from Group A Streptococci Is Mediated by Conserved
Arg and His Residues in Both the a1 and a2 Repeat Domains
Ranson M* 1 , Sanderson-Smith ML 1 , Walker MJ 1 , Fu Q 2 , Castellino FJ 2 , Prorok M 2
1 School of Biological Sciences, University of Wollongong, Australia;
2W.M. Keck Center for Transgene Research and Department of Chemistry and Biochemistry, University
of Notre Dame, Notre Dame, Indiana, USA
Presenting author e-mail: mranson@uow.edu.au
Subversion of the plasminogen activation system is implicated in the virulence of Group A
streptococci (GAS). GAS display several receptors for human plasminogen on the cell surface
including the high affinity plasminogen-binding group A streptococcal M protein (PAM),
which mediates an important virulence mechanism for a subset of GAS isolates. The major
plasmin(ogen)-binding domain of PAM is comprised of two characteristic tandem repeats
designated a1 and a2. We have recently shown that mutation of previously identified key
residues Lys98, Arg101, His102 and Lys111 within the a1 and a2 repeats reduced, but did not
abrogate plasminogen binding by full-length PAM using solid-phase binding assays. Loss of
plasminogen binding was only observed following simultaneous mutation of Arg101, Arg114,
His102 and His115 in both the a1 and a2 repeats regardless of the presence of residues Lys98
and Lys111. The on- and off-rate constants underlying the Kd values obtained from these assays
were further examined using PAM and select variants by surface plasmon resonance (SPR). The
relative affinity of PAM for streptokinase-resistant murine plasminogen compared to human
plasminogen was also investigated by SPR and will be reported. This demonstration of a nonlysine-dependent,
high affinity interaction between plasminogen and a receptor is unique. We
suggest that the highly conserved Arg and His residues from either the a1 or a2 repeats in PAMlike
proteins compensates for variation elsewhere in the binding repeats, or indeed for loss of one
of the repeats, and explains the maintenance of high affinity plasminogen binding by naturally
occurring PAM-like variants.
50 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i039i
Plasminogen Activator Inhibitor-1 (PAI-1) Is an
Inhibitor of Factor VII-Activating Protease in Patients
with Acute Respiratory Distress Syndrome
Wygrecka M* 1 , Morty RE 2 , Markart P 2 , Kanse SM 1 , Andreasen PA 3 , Wind T 3 , Guenther A 2 , Preissner KT 1
1 2 Department of Biochemistry, Department of Internal Medicine, Faculty of Medicine, Justus-Liebig-
University Giessen, Giessen, Germany;
3 Department of Molecular and Structural Biology, University of Aarhus, Aarhus, Denmark
Presenting author e-mail:malgorzata.wygrecka@innere.med.uni-giessen.de
Factor VII-activating protease (FSAP) is a novel plasma-derived serine protease structurally
homologous to tissue-type (tPA) and urokinase-type (uPA) plasminogen activators. We
demonstrate that plasminogen activator inhibitor-1 (PAI-1), the predominant inhibitor of tPA and
uPA in plasma and tissues, is an inhibitor of FSAP as well. We detected PAI-1-FSAP complexes
in addition to high levels of extracellular RNA, an important FSAP cofactor, in bronchoalveolar
lavage fluids from patients with acute respiratory distress syndrome (ARDS). Hydrolytic activity
of FSAP was inhibited by PAI-1 with a second-order inhibition rate constant (Ka) of 3.38 ± 1.12 x
10 5 M-1.s-1. Residue Arg346 was a critical recognition element on PAI-1 for interaction with FSAP.
RNA, but not DNA, fragments (>400 nucleotides in length) dramatically enhanced the reactivity
of PAI-1 with FSAP, and 4 µg.ml-1 RNA increased the Ka to 1.61 ± 0.94 x 10 6 M-1.s-1. RNA also
stabilized the active conformation of PAI-1, increasing the half-life for spontaneous conversion of
active to latent PAI-1 from 48.4 ± 8 min to 114.6 ± 5 min. In contrast, little effect of DNA on PAI-1
stability was apparent. Residues Arg76 and Lys80 in PAI-1 were key elements mediating binding
of nucleic acids to PAI-1. FSAP-driven inhibition of vascular smooth muscle cell proliferation
was antagonized by PAI-1, suggesting functional consequences for the FSAP-PAI-1 interaction.
These data indicate that extracellular RNA and PAI-1 can regulate FSAP activity, thereby playing
a potentially important role in hemostasis and cell functions under various pathophysiological
conditions such as ARDS.
Molecular & Cellular Biology of Plasminogen Activation 51

i040i
Improved Muscle Regeneration in PAI-1-deficient Mice
Is Associated with an Enhanced Inflammatory Response
and Reduced Fibrin Deposition after Injury
Ardite E*, Vidal B, Jardí M, González B, Muñoz-Cánoves P
Center for Genomic Regulation (CRG), Program on Differentiation and Cancer, Barcelona, Spain
Presenting author e-mail: esther.ardite@crg.es
We have previously shown that uPA activity is required for muscle regeneration in vivo, since
muscle regeneration was impaired in uPA-deficient mice (uPA–/–) after injury, correlating with
persistent accumulation of fibrin. In contrast, mice lacking the uPA inhibitor PAI-1 (PAI-1–/–)
showed an improved and accelerated muscle regeneration with respect to wild-type (WT) mice
after injury, which correlated with decreased fibrin deposition in PAI-1–/– muscles. These data
suggest that the regulation of fibrinolysis by the uPA/PAI-1 balance is important for efficient
muscle regeneration. Moreover, while the recruitment of inflammatory cells to regenerating
muscle was reduced in uPA–/– mice, it was increased in PAI-1–/– mice with respect to WT
mice, suggesting that uPA/PAI-1 activities may play a role in the inflammatory response at the
site of injury. To test this hypothesis, we analyzed whether transplantation of WT bone marrow
would rescue the defective regeneration of uPA–/– mice. Transplantation of WT bone marrow
into uPA–/– mice restored the inflammatory cell recruitment to damaged uPA–/– muscle to the
levels normally observed in WT mice; more importantly, this normalized inflammatory response
was accompanied by a rescued muscle regeneration in uPA–/– mice. Additional transplantation
experiments of WT bone marrow into PAI-1 –/– mice are ongoing. Altogether, these results
indicate that the uPA/PAI-1 balance may be important for efficient inflammation and muscle
regeneration, with important implications for human muscular dystrophies.
52 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i041i
Invasion and Metastasis of Carcinoma Cells Is Prevented by
Urokinase-Derived Antagonists of avb5 Integrin Activation
Franco P 1 , Vocca I 1 , Alfano D 1 , Votta G 1 , Carriero MV 2 , Estrada Y 4 , Netti PA 3 , Ossowski L 4 ,
Stoppelli MP* 1
1 Institute of Genetics and Biophysics ‘’Adriano Buzzati-Traverso’’, National Research Council, Naples,
Italy;
2 National Cancer Institute of Naples, Via M. Semmola, Naples, Italy;
3 Department of Materials and Production Engineering, University ‘’Federico II’’, Piazzale Tecchio,
Naples, Italy;
4 Department of Medicine, Mount Sinai School of Medicine, New York, New York, USA
Presenting author e-mail: stoppell@igb.cnr.it
We have previously shown that phosphorylation of urokinase (uPA) as well as substitution of
the critical serine with glutamic acid residues (His-uPA138E/303E) impairs its motogen ability.
Phospho-mimicking uPA strongly inhibited carcinoma cell invasion, as shown by the reduced
metastatic ability of HEp3 cell clones stably expressing His-uPA138E/303E in the chick embryo
chorioallantoic membrane. The relationship between this inhibitory effect and urokinase receptor
(uPAR) expression was analyzed by using several uPA variants lacking the uPAR binding
domain (residues 9-45 of the human sequence) and carrying the relevant Ser to Glu substitutions
(dGFa138E/303E) in HEK-293 cells. A strong inhibitory effect of cell migration toward different
matrix chemoattractants resulted, in a growth factor domain- and uPAR-independent manner.
The inhibitory uPA reduces the speed of cell translocation by 5 fold and alters the pattern of Factin
formation in HEK-293 cells. His-uPA138E/303E and dGFa138E/303E specifically bind to
avb5 integrin, as binding is inhibited by specific anti-integrin antibodies and by pre-incubation
of ligand with purified avb5. Finally, cell exposure to picomolar concentrations of His-uPA138E/
303E or dGFa138E/303E decreases the affinity of avb5 integrin for 125I-vitronectin and impairs
cell chemotactic response to vitronectin. Also, the extent of talin associated to h5 is markedly
decreased. Although the uPA-derived antagonists do not bind directly to a5b1, a4b1, a1b1,
they prevent cell response to fibronectin, laminin and collagen. Similarly, a general inhibition is
observed in the presence of the natural antagonist endostatin, which binds to avb5 and in the
presence of anti- avb5 blocking antibodies. These data, taken together, show that avb5 can convey
a strong signal inhibiting cell migration and suggest that this inhibition extends to other integrins.
Molecular & Cellular Biology of Plasminogen Activation 53

i042i
Urokinase Receptor/Integrin Interactions in Lung Tumor Development
Tang CH, Hill M, Kim Y, Wei Y, Chapman HA*
Pulmonary and Critical Care Division, Department of Medicine, Cardiovascular Research Institute,
University of California, San Francisco, California, USA
Presenting author e-mail: hal.chapman@ucsf.edu
Upregulation of urokinase receptors (uPAR) is common during cancer progression and thought to
promote invasion and metastasis. Urokinase receptors bind urokinase and a set of beta1 integrins,
but it remains unclear whether or how uPAR/integrin binding contributes to tumor progression.
Using site-directed mutagenesis, single amino acid mutants of the urokinase receptor were
identified that fail to associate with either integrins alpha3beta1 (D262A) or alpha5beta1 (H249A).
A recombinant uPAR bearing both D262A and H249A mutations (H/D uPAR) was found to be
expressible, to bind urokinase normally, but to no longer co-precipitate with beta1 integrins. To
study the functional effects of these mutations, endogenous uPAR was first stably silenced in
H1299 lung cancer cells and then wild type or mutant uPARs expressed. Orthotopic implantation
into nude mice of H1299 cells with uPAR knockdown resulted in lower incidence and markedly
suppressed lung tumor area (8 mm2 vs 1.5 mm2 , n = 14) compared to parent cells at 32 days,
and this was reversed by wild type but not H/D uPAR re-constitution. H1299 cells expressing
wt and H/D mutant uPAR were compared for matrix-induced ERK activation, MMP expression,
and invasion through matrigel-coated transwells in vitro. Compared with wt uPAR reconstituted
H1299 cells, H/D uPAR cells showed virtually no fibronectin or laminin-induced ERK activation,
MMP upregulation, or matrix-dependent migration. Collectively, these data indicate that uPAR/
integrin complexes are required for matrix-dependent induction of MMP through an ERKdependent
pathway in H1299 cells and that this signaling pathway appears important to lung
tumor development in vivo.
54 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i043i
uPA- and uPAR-Expressing Stromal Cells Accompany
the Transition to Invasive Breast Cancer
Nielsen BS* 1 , Rank F 2 , Illemann M 1 , Lund LR 1 , Danø K 1
1 The Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark;
2 Department of Pathology, Rigshospitalet, Copenhagen, Denmark
Presenting author e-mail: schnack@finsenlab.dk
The transition from ductal carcinoma in situ (DCIS) of the breast to invasive ductal carcinoma is
facilitated by proteolytic degradation of the basement membrane. The transition can be identified
as microinvasive foci in a small proportion of DCIS lesions. We have previously reported that
MMP-13 is frequently expressed in such foci. To establish if plasmin-directed proteolysis is
likely to be involved in early invasion we have studied the expression of urokinase plasminogen
activator (uPA) and its receptor (uPAR) in human DCIS lesions with and without microinvasion.
uPA mRNA was detected in periductal stromal cells in all of 9 DCIS lesions with microinvasion
and in 2 of 9 DCIS lesions without microinvasion by in situ hybridization. The uPA mRNA
signal was seen in numerous stromal cells in microinvasive areas together with MMP-13 mRNA
expressing cells. Double immunofluorescence analyses, using emission fingerprinting, showed
that the uPA expressing stromal cells included both macrophages and myofibroblasts. uPAR was
focally upregulated in periductal stromal cells in all of the 9 DCIS lesions with microinvasion
and in only 2 of the 9 DCIS lesions without microinvasion. uPAR was seen in both macrophages
and myofibroblasts in microinvasive areas and it was evident that uPA and uPAR co-localized
in both fibroblast-like cells and macrophage-like cells. The early invasive carcinoma cells were
negative for both uPA and uPAR. We conclude that periductal macrophages and myofibroblasts
are strongly involved in the initial steps of breast cancer invasion by focally upregulating the
expression of the plasminogen activation system and MMP-13.
Molecular & Cellular Biology of Plasminogen Activation 55

i044i
Generation of the Malignant Phenotype in HT-1080 Tumor Cells by
PAI-1 Involves Modulation of Proteasomal Activity and Phosphatases
Mihaly J* 1 , Carroll VA 2 , Breuss JM 1 , Prager GW 1,3 , Binder BR 1
1 Department of Vascular Biology and Thrombosis Research, Medical University Vienna, Austria;
2 Imperial College of Medicine, Oxford, United Kingdom;
3 First Clinic for Internal Medicine, Oncology, Medical University Vienna, Austria
Presenting author e-mail: judit.mihaly@meduniwien.ac.at
Elevated levels of PAI-1 are often found in several types of cancer and increased PAI-1 levels
positively correlate with poor outcome and prognosis. We described that prolonged exposure of
‘’non-malignant’’, low PAI-1 secreting HT-1080 cells to exogenous PAI-1 increased cell adhesion
via redistribution of integrins of the broad substrate specificity alpha-v heterodimer type to
the cell surface. This PAI-1 induced integrin redistribution was dependent on the presence of
uPA, uPAR and a member of the LDLR-family as shown by the use of firboblasts derived from
respective gene deficient mice and involved sustained activation of the ERK/MAPK pathway.
The involvement of ERK1 in that process was also demonstrated by overexpression of EGFP
tagged ERK1 that mimicked integrin redistribution and cell adhesion. Furthermore, exposure
to exogenous PAI-1 also influences proteasomal activity: at early timepoints (2 hours) a PAI-1/
uPA/LDLR dependent increase of proteasomal activity was found, whereas after 18 hours the
proteasomal activity in PAI-1 stimulated cells dropped below levels measured in unstimulated
control. The activity of ERK1/2 is tightly controlled by dual specificity phosphatases, whereby
one of the major players is the MKP1/DUSP1 and exposure of cells to exogenous PAI-1 also
modulated DUSP-1 on the mRNA and protein levels, while DUSP4 remained unaffected. Active
ERK1/2 controls proteasomal degradation of its regulator MKP1/DUSP1 by phosphorylation.
From these data we propose a model in which PAI-1 triggered sustained ERK activation is
maintained by a fine tuned regulatory mechanism involving modulation of the proteasomal
activity providing the optimal kinase-phosphatase balance required for survival of detached/
metastasizing and proliferation of the lodged cells.
56 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i045i
PEGylated DX-1000: Pharmacokinetics: Anti-Tumor and
Anti-Metastatic Effects of a Specific Plasmin Inhibitor
Devy L* 1 , Rabbani SA 2 , Stochl M 3 , Ruskowski M 4 , Mackie I 5 , Naa L 1 , Toews M 3 , van Gool R 1 , Chen J 3 ,
Ley A 3 , Ladner RC 3 , Dransfield DT 3 , Henderikx P 1
1 Dyax s.a. Building 22, Sart-Tilman, Liege, Belgium;
2 Department of Medicine and Oncology, McGill University Health Centre, Montreal, QC, Canada;
3 Dyax Corporation, Cambridge, Massachusetts, USA;
4Department of Radiology, Division of Nuclear Medicine, University of Massachusetts Medical School,
Worcester, Massachusetts, USA;
5 Haematology Department, University College London, London, United Kingdom
Presenting author e-mail: ldevy@dyax.com
Using phage display, we identified a TFPI-derived Kunitz domain protein which is a specific high
affinity inhibitor of plasmin (Ki=99±15pM) referred to as DX-1000. DX-1000 blocked plasminmediated
proMMP-9 activation, invasiveness (44-66%) of uPA-expressing HT-1080 cells and
tube formation of HUVECs (IC 50 =1.4±0.3nM) and mouse endothelial cells (IC 50 =16.6± 0.1nM).
DX-1000 did not significantly affect haemostasis and coagulation in vitro. However, due to its
low molecular weight (~7 kDa), the protein exhibited a rapid plasma clearance rate in vivo (b
phase half-life=27 minutes in mice and 1 hour in rabbits). After site-specific PEGylation, DX-1000
retained its activity and exhibited a decreased plasma clearance (b phase half-life=13 hours in
mice and 59 hours in rabbits). 4PEG-DX-1000 was effective in vitro, and inhibited growth of MDA-
MB231 tumor cells in nude mice (45% TGI). 4PEG-DX-1000 treatment caused a marked inhibition
of angiogenesis, a significant reduction of uPA expression, and inhibition of tumor proliferation
and MAPK phosphorylation. 4PEG-DX-1000 treatment significantly reduced the number of
metastatic foci in the lung (46%) and the liver (57%) and decreased the level of active FAK in the
primary tumors. Together, the results from these studies provide compelling evidence for the role
of plasmin inhibitors as therapeutic agents for blocking breast cancer growth and metastasis.
Molecular & Cellular Biology of Plasminogen Activation 57

i046i
Cytotoxic Potential of a Novel uPA-activity Dependent
and EGF Receptor-targeting Pro-drug
Rønø B* 1, 5 , Bae Kim G 2 , Liu S 3 , Kristjansen PEG 4 , Neville DM 2 , Leppla SH 3 , Bugge TH 5 , Rømer J 1
1 Finsen Laboratory, Rigshospitalet, Copenhagen, Copenhagen, Denmark;
2 National Institutes of Mental Health, NIH, Bethesda, Maryland, USA;
3 National Institutes of Allergy and Infectious Diseases, NIH, Bethesda, Maryland, USA;
4 Institute of Molecular Pathology, University of Copenhagen, Copenhagen, Denmark;
5 National Institutes of Dental and Craniofacial Research, NIH, Bethesda, Maryland, USA
Presenting author e-mail: brono@finsenlab.dk
To exploit this preferential expression of uPA and uPAR in cancer we have constructed a novel
pro-drug whose activation is dependent on proteolytic processing by uPA. To further improve
the cancer specificity, the pro-drug targets epidermal growth factor receptor (EGFR) expressing
cells solely. The pro-drug, termed DT-U2-TGFa, was generated by replacing the native proteolytic
activation sequence in the diphtheria toxin with an amino acid sequence recognised by uPA and
in addition by exchanging the diphtheria receptor-binding domain with transforming growth
factor alpha (TGFa).
We have examined the activation, specificity, and cytotoxic potential of the pro-drug. By
incubating the pro-drug with recombinant uPA DTU2TGFa was cleavaged by uPA in a
time dependent manner. In a cell culture based assay, the uPA/uPAR and EGFR expressing
human head and neck cancer cell line, HN6, bound, activated, and internalised DT-U2-TGFa.
Furthermore, a colorimetric cytotoxicity assay showed that the cells were killed by DT-U2-TGFa
in a dose dependent manner. Incubation with different uPA inhibitors prior to treatment with
DT-U2-TGFa efficiently reduced activation of the pro-drug, substantiating the requirement for
active uPA. EGFR transduced murine fibroblasts, NR6W, were sensitive to DT-U2-TGFa, while
the parental EGFR negative NR6 cells were not affected, indicating that the cytotoxicity of DTU2-
TGFa is restricted to EGFR expressing cells. In support of these data HN6 cells pre-incubated with
TGFa were completely rescued from DT-U2-TGFa induced cell death.
In conclusion, we have constructed, produced, and tested a novel pro-drug that targets uPA/
uPAR and EGFR expressing cells. Our data provides evidence of a highly specific pro-drug with
promising cytotoxic efficacy in cancer cells.
58 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i047i
Inhibition of Mouse uPA Activity by Mouse
Monoclonal Antibodies in vitro and in vivo
Lund IK*, Jögi A, Behrendt N, Ploug M, Gårdsvoll H, Lund LR, Rømer J, Høyer-Hansen G
Finsenlaboratoriet, Copenhagen BioCenter, Copenhagen, Denmark
Presenting author e-mail: ikl@finsenlab.dk
The potential of monoclonal antibodies (mAbs) as anti-cancer therapeutics has lately been
demonstrated for a number of antigens. To enable in vivo therapy experiments using mAbs in
murine cancer models, we immunized mice deficient in murine urokinase plasminogen activator
(muPA) with recombinant muPA, thus developing murine mAbs directed against muPA.
We have selected 5 mAbs (mU1-mU5) reacting with muPA in ELISA, Surface Plasmon Resonance
(SPR) analysis, and Western blotting. Analysing the epitope location on muPA using the
recombinant amino-terminal fragment of muPA (mATF) and the B-chain revealed that only
mU1 recognized an epitope exclusively located in the B-chain, encompassing the catalytic site of
muPA. SPR analyses demonstrated that mU2 was unable to bind receptor (muPAR)-bound muPA
and prevented binding of muPAR to muPA. In vitro cell binding experiments using 125I-mATF
illustrated an efficient mU2-induced interference of muPA-muPAR interaction on the cell surface.
Using an enzyme kinetic assay measuring muPA-dependent plasminogen activation, four mAbs
were found to inhibit muPA catalytically activity to various extend, with mU1 having the most
pronounced effect. Application of mU1 or mU2 in an anthrax toxin assay dependent on muPA
activity yielded a very high cell rescue.
Importantly, treatment of tissue-type plasminogen (tPA) deficient mice with mU1 resulted in
significantly delayed wound healing mimicking the phenotype observed in uPA;tPA doubledeficient
mice. Moreover, data concerning the in vivo effect of mU1 on liver fibrin deposits of tPA
deficient mice as compared to uPA;tPA double-deficient mice will be provided.
Molecular & Cellular Biology of Plasminogen Activation 59

i048i
Crystal Structure of Human Urokinase Complexed
with a Cyclic Peptidyl Inhibitor, uPAin-1
Zhao G 1 , Yuan C 1 , Bian C 1 , Wind T 2 , Andreasen PA 2 , Huang M* 1
1State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter,
Chinese Academy of Sciences, Fuzhou, Fujian, China;
2 Department of Molecular Biology, University of Aarhus, Aarhus, Denmark
Presenting author e-mail: mhuang@fjirsm.ac.cn
Urokinase-type plasminogen activator (uPA) plays a crucial role in the regulation of tumor
cell adhesion and migration. The inhibition of uPA activity is a promising mechanism for anticancer
therapy. A cyclic peptidyl inhibitor, upain-1, CSWRGLENHRMC, was identified recently
as a competitive and highly specific uPA inhibitor (Hansen et al. J Biol Chem 280, 38424-37). We
determined the crystal structure of uPA in complex with upain-1 at 2.15 Å. The structure reveals
that the cyclic peptide adopts a rigid conformation stabilized by two tight beta turns formed by
Leu6-His9 and His9-Cys12 segments, respectively. The Glu7 residue of upain-1 forms hydrogen
bonds with the main chain nitrogen atoms of residues 4, 5, and 6 of upain-1, and is also critical
for maintaining the active conformation of upain-1. The Arg4 of upain-1 is inserted into the uPA’s
specific S1 pocket. The Ser2 residue of upain-1 locates close to the S1b pocket of uPA. The Gly5
and Glu7 residues of upain-1 occupy the S2 pocket and the oxyanion hole of uPA, respectively.
Furthermore, the Asn8 residue of upain-1 binds to the 37-loop and 60-loop of uPA and renders
the specificity of upain-1 for uPA. The steric hindrance of the side chain of Glu7 and the indole
ring of Trp3 residue of upain-1 prevents the carboxyl group of Arg4 residue at the cleavage site to
be accessible by the catalytic Ser195 residue of uPA, thus making upain-1 behave as an inhibitor
rather than a substrate. Based on this structure, we propose a new pharmacophore for the design
of highly specific uPA inhibitors.
60 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i049i
Discovery of a Novel Zymogen Targeting Inhibitor of
Urokinase-type Plasminogen Activator: Evidence for
Structural Flexibility of the Protease Domain
Blouse GE 1 , Bøtkjær KA 1 , Deryugina EI 3 , Kjelgaard S 1 , Byszuk O 1 , Mortensen KK 1 , Quigley JP 3 ,
Andreasen PA* 1
1 2 Laboratory of Cellular Protein Science, BioDesign Laboratory, Department of Molecular Biology,
University of Aarhus, Aarhus, Denmark;
3 Department of Cell Biology, Scripps Research Institute, La Jolla, California, USA
Presenting author e-mail: pa@mb.au.dk
Considerable interest is emerging for pharmacologic interference with proteolytic enzyme targets.
One such validated target is urokinase-type plasminogen activator (uPA), which has received
much attention due to a suspected causal role in the clinical progression of cancer and other
processes of pathological tissue remodeling. Nonetheless, development of specific small molecule
inhibitors has proven a daunting task. In nature, a key mechanism of protease regulation is the
point of zymogen activation. Thus far, controlling protease activity by targeting the zymogen
activation step is an underexploited strategy. We have now designed a specific monoclonal
antibody inhibitor (Mab-112) with sub-nanomolar affinity to pro-uPA. A detailed mechanistic
evaluation with several biophysical methods elucidated a novel multifunctional mechanism
whereby Mab-112 retards the protelytic conversion of single-chain pro-uPA into the two-chain
form and subsequently averts the conformational transition to a mature protease by sequestering
the two-chain form in a zymogen-like state. Furthermore, Mab-112 is a non-competitive inhibitor
of two-chain uPA, stabilising the protease in a non-catalytic conformation. Functional studies
employing high intravasating (HT-hi/diss) and low intravasating (HT-lo/diss) variants of the
human HT-1080 cell line demonstrate that Mab-112 is an effective inhibitor of intravasation in the
chorioallantoic membrane assay. These findings show the utility of pharmacological interference
of zymogen activation as a plausible and robust means to regulate uPA activity and the
downstream effects of plasminogen activation. Furthermore, a strategy that targets regions related
to pro-enzyme activation likely provide a unique inhibitor-protease interaction surface and is thus
expected to enhance the chances of engineering high inhibitor specificity.
Molecular & Cellular Biology of Plasminogen Activation 61

i050i
A Novel Type of Agent Blocking the Association of uPA
to its Receptor uPAR: uPA-binding Aptamers
Dupont DM* 1,2 , Madsen JB 1 , Kjems J 1,2 , Andreasen PA 1,2
1 Department of Molecular Biology, 2 iNANO Centre, University of Aarhus, Aarhus, Denmark
Presenting author e-mail: dmd@mb.au.dk
Systematic evolution of ligands by exponential enrichment (SELEX) is a relatively new approach
for generating agents of potential therapeutic and diagnostic interest. The technique combines
the ability of RNA or DNA oligonucleotides to fold into a variety of three-dimensional structures,
with the possibility of selecting, from very large pools of random sequences (~10 15 ), the ones
capable of binding to a target of interest. We have generated a library of serum-stable 2´-fluoropyrimidine
modified RNA oligonucletides and used it in a SELEX experiment to select sequences,
or so-called aptamers, binding to human urokinase-type plasminogen activator (uPA). As
analysed by surface plasmon resonance (SPR), the aptamers bind to the amino terminal fragment
of human, but not murine, uPA, with K D -values in the low nanomolar range. SPR analyses and
cell binding assays have shown that the aptamers block the association of uPA with its receptor
uPAR. In consistency with their binding area in the amino terminal fragment, they do not inhibit
the uPA proteolytic activity directly, but inhibit uPAR-dependent plasminogen activation on cell
surfaces. By RNA sequence alignments and computerised secondary structure predictions, we
were able to identify the uPA binding regions of the aptamers and delete regions unnecessary
for uPA binding. uPA-binding aptamers represent a promising principle for interfering with the
pathophysiological functions of the plasminogen activation system. Targeting the receptor-ligand
interaction through uPA may be advantageous as compared to uPAR-targeting agents which may
not be antagonists of all functions of uPAR. Aptamers are also potential tools for analytical and
imaging purposes.
62 X I t h I n t e r n a t i o n a l W o r k s h o p o n

Abstracts for Poster Presentations
i051i
Identification and Analysis of the Vn Binding Site in Mouse uPAR
Pirazzoli V* 1 , Andolfo AP 1 , Madsen CD 1 , Sidenius N 1,2
1 IFOM Fondation, FIRC Institute of Molecular Oncology, Milan, Italy;
2 Molecular Genetics Unit, DIBIT, Università Vita-Salute San Raffaele, Milan, Italy
Presenting author e-mail: valentina.pirazzoli@ifom-ieo-campus.it
A complete functional alanine scan of human uPAR conducted recently in our laboratory
identified the interaction with Vn as being the only essential direct uPAR-interaction required for
the receptor to induce changes in cell adhesion, morphology, migration and signaling. To address
the importance of the Vn-interaction in vivo it is our goal to generate a knock-in mouse where the
uPAR gene has been modified to encode a receptor deficient in Vn-binding but otherwise normal
(i.e. with normal uPA-binding). With such a goal in mind, we now report the analysis of the
Vn-binding epitope in mouse uPAR. When over-expressed in CHO cells, wild-type mouse uPAR
(muPAR) was found to induce changes in cell morphology and colony formation very similar
to that of the human receptor. These changes include the formation of actin-rich lamellipodia,
loss of stress fibers, reduced cell-cell contact as well as a complete failure to form colonies when
seeded a low density. We had previously identified 5 residues in human uPAR which appear to
engage Vn directly and which when changed into alanine disrupt the Vn-interaction completely
or partially. To establish if the corresponding, fully conserved, residues in muPAR (W32, R58, I63,
R92 and Y93) are also responsible for Vn-binding we mutated these into alanine and tested their
ability to induce RGD-independent Vn-adhesion as well as changes in cell morphology. The result
of this analysis documented that a substitution of any of these residues completely impaired
muPAR binding to Vn. Additional experiments using recombinant soluble muPAR confirmed the
impaired Vn-binding and a normal uPA-binding.
Molecular & Cellular Biology of Plasminogen Activation 63

i052i
Novel uPAR Binding Site in Vitronectin
Andolfo A* 1 and Sidenius N 1,2
1 FIRC Institute of Molecular Oncology Foundation, Milan, Italy;
2 Dibit, Fondazione Vita-salute San Raffaele, Milan, Italy
Presenting author e-mail: annapaola.andolfo@ifom-ieo-campus.it
The ability of the serum protein vitronectin (Vn) to modulate cell adhesion requires the
aminoterminal somatomedin B (SMB) domain (amino acids 1-44) which binds both plasminogen
activator inhibitor-1 (PAI-1) and urokinase-type plasminogen activator receptor (uPAR) as well
as the flanking ArgGlyAsp sequence (RGD, amino acids 45-47) responsible for the interaction
with Vn-receptors of the integrin family. Here we report our data on uPAR-Vn interaction site
mapping by a complete alanine scan of the SMB domain. We expressed and purified the mutants
from mammalian cell cultures and assayed their biochemical and biological activity. In contrast
to previously published data using SMB domains expressed in E. coli, we find that single alanine
substitution only has marginal effects on PAI-1 binding (< 2-fold reduction in affinity) and no
detectable effects on integrin mediated RGD-dependent cell adhesion. We confirm the previously
published data on uPAR binding site in Vn, that is overlapping to the known PAI-1 binding site
(F13, D22, L24, Y27, Y28). Most importantly, we find additional residues in the SMB domain that
are exclusively required for uPAR binding (S4, G7, R8), thus defining a composite site with two
distinct epitopes located on opposite sides of the SMB-structure.
In conclusion, the presence of two separate uPAR binding sites in the SMB, which are both critical
for binding, raises important questions towards how and if a single molecule of uPAR can engage
these two sites contemporarily.
64 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i053i
Dissecting Serpin-protease Reaction Pathways
by the Use of Monoclonal Antibodies
Bødker JS*, Blouse GE, Dupont DM, Andreasen PA
Department of Molecular Biology, University of Aarhus, Aarhus, Denmark
Presenting author e-mail: jsb@mb.au.dk
The formation of a stable PAI-1-protease complex is believed to progress through several
separate steps. The steps include exosite interactions between the protease 37-loop and the serpin
reactive centre loop (RCL); formation of a reversible Michaelis complex; formation of a stable
acyl-enzyme intermediate; release of exosite interactions; insertion of the N-terminal side of the
RCL as b-strand 4A which translocates the protease to the opposite pole of the serpin. We have
now obtained further information about the PAI-1-protease mechanism by several approaches
including the use of monoclonal anti-PAI-1 antibodies having epitopes near the path believed
to be followed by the protease during translocation, stopped-flow analyses with fluorescently
labeled PAI-1, BIACORE, and site-directed mutagenesis. Mab-5, which has an epitope near the
region where the RCL first inserts into b-sheet A, caused a slight reduction in the rate of Michaelis
complex formation, but promoted an 100-fold reduction in the rate of RCL insertion, an effect
reduced by mutations known to affect the release from the protease-serpin exosite interactions.
Another antibody, Mab-2, with an epitope in a-helix F, caused a 5–10-fold reduction in the rate of
loop insertion, induction of PAI-1 substrate behaviour, and accumulation of a rapidly dissociating
complex on BIACORE, presumably an acyl-enzyme complex with partial loop insertion and
lacking exosite interactions. These results lend further support to the existence of two forms for
acyl-enzyme intermediates in the serpin-protease reaction mechanism, one with intact exosite
interactions and one in which release of exosite interactions is coupled to the initial phase of RCL
insertion.
Molecular & Cellular Biology of Plasminogen Activation 65

i054i
PAI-1-vitronectin Interactions Involve an Extended Binding
Surface and Mutual Conformational Rearrangements
Blouse GE 1 , Peterson CB 2 , Dupont DM 1 , Ploug M 3 , Gårdsvoll H 3 , Schar CR 2 , Perron MJ 4 , Minor KH 2 ,
Shore JD 4 , Andreasen PA* 1
1 Department of Molecular Biology, University of Aarhus, Aarhus, Denmark;
2Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville,
Tennessee, USA;
3 Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark;
4 Department of Pathology, Henry Ford Health System, Detroit, Michigan, USA
Presenting author e-mail: pa@mb.au.dk
It has been proposed that binding of PAI-1 to native vitronectin induces a reorganisation of
vitronectin from a closed to an open conformation allowing assembly of vitronectin oligomers
with changed cell and matrix binding functions. In order to elucidate early molecular events
during interactions between PAI-1 and vitronectin, we have applied a robust strategy that
combines protein engineering, fluorescence spectroscopy and rapid reaction kinetics. Fluorescence
stopped-flow experiments indicated a fast, concentration dependent, biphasic binding of PAI-
1 to native monomeric vitronectin, but a monophasic association with vitronectins N-terminal
somatomedin B domain (SMB), suggesting that multiple phases of the binding interaction
occur only when full-length vitronectin is present. Nonetheless, in all cases, the initial fast
interaction was followed by slower fluorescence changes, which we, by the use of an engineered,
fluorescently silent PAI-1 with non-natural amino acids, demonstrated to be caused by reciprocal
structural changes within the PAI-1 structure as well as in native vitronectin. Furthermore,
measuring the effect of vitronectin on the rate of insertion of the reactive centre loop into b-sheet A
of PAI-1 during reaction with target proteases by the use of fluorescently labelled PAI-1 variants,
we observed that both full-length vitronectin and SMB had protease-specific effects on the rate
of loop insertion, but that the two had clearly different effects. We interpret these results as
support for a model of PAI-1-vitronectin binding in which there is an extended interaction surface
implicating parts of vitronectin other than the somatomedin B domain.
66 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i055i
Intact (non-cleavable) Cell-surface u-PAR Accelerates
Clearance of tcu-PA:PAI-1:u-PAR Complexes and Subsequent
Re-surfacing of Intact and Functional u-PAR
Nieves-Li EC* and Manchanda N
Department of Biochemistry and Internal Medicine, University of Illinois at Urbana-Champaign,
Urbana, Illinois, USA
Presenting author e-mail: enieves@uiuc.edu
Urokinase plasminogen activator receptor (u-PAR) has been well-defined as a promoter of
plasminogen activation and modulates several cellular processes such as adhesion, proliferation,
and migration. U-PAR has been shown to be susceptible to proteolytic cleavage by several
proteases, one of them being its ligand two-chain u-PA (tcu-PA). This dissociates D1 from
the rest of the anchored receptor and leads to loss of ligand binding. However, exposure of
the chemotactic epitope after D1 removal gives u-PAR a new role in signaling and migration.
We found that cell-surface plasminogen activation is impaired upon D1 removal. We have
investigated the role of receptor cleavage in the regulation of cell-surface plaminogen activation.
We engineered and expressed wt u-PAR as well as tcu-PA cleavage resistant u-PAR (tcr-uPAR)
in HEK293 cells. Using three different approaches to follow u-PAR internalization and recycling,
i.e. biotin labeled receptor, biotin-labeled tcu-PA-PAI-1 complexes, and assays for plasminogen
activation, we found that intact receptor better promotes the internalization of cell-surface u-
PAR:u-PA-PAI-1 complexes and promotes subsequent re-surfacing of unoccupied and functional
receptor. We hypothesize that intact cell-surface u-PAR may accelerate removal of inhibited
tcu-PA from the cell-surface environment and that its re-expression may promote cell-surface
plasminogen activation.
Molecular & Cellular Biology of Plasminogen Activation 67

i056i
The Central b-sheet of PAI-1 Demonstrates
Two Dynamically Distinct Regions
Li S* 1 , Lawrence DA 2 , Schwartz BS 1,3
1 Department of Biochemistry, University of Illinois at Urbana-Chanpaign, Urbana, Illinois, USA;
2 Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA;
3 Department of Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA
Presenting author e-mail: SLI3@uiuc.edu
PAI-1 is an inhibitory serpin that can spontaneously transit with a half-life of 1-2 hours at 37°C
to a latent form whereby the RCL is intact but fully inserted into b-sheet A. The labile nature of
native PAI-1 is thought to be due to the ability of its central sheet to exist in equilibrium between
open and closed conformations. The stable variant, 14-1B, contains 4 amino acid substitutions that
convey a 70-fold enhancement in the inhibitory half-life compared to wild-type PAI-1 (wtPAI-
1). X-ray crystal structures of latent PAI-1 and active 14-1B suggest that some of the mutations
in the stable variant keep b-sheet A closed, preventing incorporation of the RCL. We tested this
hypothesis using RCL-mimicking peptides, which have been shown to insert into b-sheet A
as s4A. We found that 14-1B is less susceptible than wtPAI-1 to inactivation by such peptides.
The PAI-1 cofactor, vitronectin (Vn), did not affect the binding of a pentamer, Ac-TVASS, to
14-1B, yet enhanced the binding of an octamer, Ac-TVASSSTA. The binding of Vn to wtPAI-1
modestly delays the transition to latency. Yet we found that that Vn did not protect wtPAI-1
from inactivation by the peptides, rather slightly enhancing such inactivation. These results are
inconsistent with the currently held model on how active PAI-1 is stabilized by its physiologic
cofactor. We propose that sheet A breathes in a pigeon-toed fashion in which the region
underneath a-helix F remains closed and the region near the RCL is dynamic.
68 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i057i
Regulation of Cancer-Cell Plasmin Generation by
Annexin A2-S100A10 Heterotetramer (AIIt)
Waisman DM*
Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia,
Canada
Presenting author e-mail: david.waisman@dal.ca
Many findings over several decades strongly suggest an important role for the plasminogen
activation system in cancer cell invasion and metastasis. We have investigated the role of the
S100A10, annexin A2 and the heterotetrameric complex formed by these subunits (AIIt) in
plasminogen regulation. Surface plasmon resonance studies established that S100A10 bound
directly to tPA, plasminogen and plasmin whereas the annexin A2 bound only plasmin. AIIt and
S100A10, but not annexin A2, dramatically stimulated the plasminogen activator-dependent
conversion of plasminogen to plasmin in vitro. Immunofluorescence microscopy demonstrated
the colocalization of S100A10 with the uPA/uPAR complex. We have also utilized both antisense
and siRNA methodologies to knockdown S100A10. Loss of extracellular S100A10 resulted in a
70-90% loss in cellular plasmin generation. Under these conditions the levels of annexin A2 were
unchanged. The S100A10 knockdown cells were also less invasive and showed a dramatic loss in
tumor formation and metastatic potential. We also observed that the addition of plasminogen to
cancer cells resulted in the oxidation of annexin A2 and S100A10. AIIt stimulated autoproteolysis
of the plasmin Lys468-Gly469 bond and also catalyzed the reduction of the plasmin
Cys462Cys541disulfide. These two reactions resulted in the release of angiostatin (Lys78-Lys468)
from plasmin. However, oxidation of annexin A2 was not observed after knockdown of S100A10.
Thus, AIIt acts as a plasmin reductase, catalyzing the release of angiostatin from plasmin. Results
form these studies have established that S100A10 is a key regulatory protein of cell surface
plasmin and angiostatin generation. Funded by the CIHR and NCIC.
Molecular & Cellular Biology of Plasminogen Activation 69

i058i
Plasminogen Activator Inhibitor Type 2 Binds to S100A10 in
Annexin 2 Heterotetramer and Prevents Annexin 2-dependent
Plasmin In-site Formation by Inhibiting tPA
Lobov S*, Croucher D, Ranson M
School of Biological Sciences, University of Wollongong, NSW, Australia
Presenting author e-mail: sergei@uow.edu.au
Plasminogen Activator Inhibitor type 2 is a well known member of the serpin superfamily.
Extracellular PAI-2 efficiently inhibits uPA and tPA in solution but there seem to be no
physiological role for PAI-2 as tPA inhibitor. Interestingly however, PAI-2 as well as tPA and
plasminogen are capable of binding to annexin 2 heterotetramer (AIIt). AIIt acts as a receptor
for tPA and plasminogen on the cell surface thereby promoting tPA-dependent plasmin in-site
formation. The fact that PAI-2 is capable of binding to AIIt is known for more then a decade.
However, neither the mechanism nor the physiological outcomes of the binding are known.
Here we show that PAI-2 binds to AIIt via the S100A10 subunit and that the CD-loop of PAI-2
plays a minor role in the binding. Furthermore, the interaction of PAI-2 with AIIt, unlike tPA and
plasminogen, is AIIt C-terminal lysine independent, which might involve novel binding sites in
S100A10 and AIIt. In spite of seemingly different sites of binding to AIIt, PAI-2 and plasminogen
could not simultaneously bind to AIIt. Furthermore, simultaneous binding of PAI-2 and tPA
seemed to be able to exist only for the PAI-2/tPA complex. Enzyme kinetic analyses confirmed
that PAI-2 inhibits AIIt-bound tPA in vitro and thus prevents plasmin in-site formation. We
conclude that there is a complex relationship between PAI-2/plasminogen/tPA when AIIt is
present. We propose that extracellular PAI-2 may have an important physiological role as a tPA
inhibitor on the cell surface.
70 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i059i
Understanding the Structural Basis of the Differential-Receptor-
Mediated Endocytosis Mechanisms of PAI-1 and PAI-2 in Cancer
Cochran BJ* 1 , Lobov S 1 , Croucher D 2 , Ranson M 1
1 Cancer Research Laboratory, School of Biological Sciences, University of Wollongong, Australia;
2 Cancer Research Program, Garvan Institute of Medical Research, Australia
Presenting author e-mail: blake@uow.edu.au
The urokinase-type plasminogen activator (uPA) and its inhibitors, plasminogen activator
inhibitor type-1 (PAI-1) and type-2 (PAI-2) play an important role in the progression of a number
of forms of cancer. Whilst both PAI-1 and PAI-2 clear uPA activity from the cell surface through
irreversible complex formation, uPA:PAI-1 initiates cell signalling events most likely associated
with poor cancer prognosis. This activation is dependent on interactions between PAI-1 and
the very low density lipoprotein receptor (VLDLr). Upon internalisation, uPA:PAI-2 does not
activate these cell signalling pathways, possibly accounting for the association between PAI-2
expression and good prognosis in some cancer types. The mechanisms underlying this differential
functionality are poorly understood and is believed to be due to the absence of LDLR binding
sites in the PAI-2 moiety of the uPA:PAI-2 complex.
This study aims to reconstitute the LDLR binding ability of PAI-1 in PAI-2 via the replacement
of structural elements and residues of PAI-2 with those corresponding to PAI-1. To date, we
have constructed an ahelix D domain swap which retained uPA inhibitory activity. We intend
to compare the LDLR binding characteristics of this protein chimera to PAI-1. The findings of
this research may help explain the underlying reasons for the distinct biological roles of PAI-1
and PAI-2 in cancer. A more complete understanding of the biological roles of PAI-2 will provide
important information relating to the function of the plasminogen activation system in cancer and
may aid in the continuing development of highly specific and effective anti-uPA treatments.
Molecular & Cellular Biology of Plasminogen Activation 71

i060i
A Low-glycemic-index Diet Reduces Plasma PAI-1 Activity
in Overweight Women
Jensen L* 1 , Krog-Mikkelsen I 2 , Sloth B 2 , Flint A 2 , Astrup A 2 , Raben A 2 , Tholstrup T 2 , Brünner N 1
1 Department of Veterinary Pathobiology, 2 Department of Human Nutrition, Centre for Advanced Food
Studies, Faculty of Life Sciences, University of Copenhagen, Frederiksberg, Denmark
Presenting author e-mail: lje@life.ku.dk
An elevated level of plasminogen activator inhibitor-1 (PAI-1) in plasma is a core feature of the
metabolic syndrome. PAI-1 has been shown to decrease during weight loss. However, effects of
healthy diets on PAI-1 levels may not solely depend on weight loss. The relevance of glycemic
index in preventing the metabolic syndrome is controversial.
The purpose of the present study was to investigate the effect of 10 weeks intake of a low
glycemic index (LGI) vs. a high glycemic index (HGI) high-carbohydrate, low fat ad libitum diet
on plasma PAI-1 activity and antigen levels in overweight women.
44 healthy overweight women (BMI 27.5 ± 0.2 kg/m2) were randomly assigned to a parallel 10
week intervention with a LGI (n=22) or HGI (n=22) diet. To study the postprandial effect of LGI
vs. HGI diets a subgroup of 29 subjects (LGI, n=14; HGI n=15) were assigned to participate in an
additional 4-h meal test on the last day of the intervention. Fasting blood samples were obtained
before and after the 10 weeks, and postprandially at 10 weeks.
PAI-1 activity in plasma decreased (P = 0.02) after the LGI diet, a decrease that was significantly
different (P < 0.01) from the change after a HGI diet. Changes in PAI-1 antigen levels were
not significantly different between any groups. Postprandial PAI-1 concentrations showed no
significant differences between groups.
A LGI diet reduces plasma PAI-1 activity, suggesting that a beneficial role of a LGI diet in
overweight adults could be related to PAI-1.
72 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i061i
Urokinase Receptor-independent Signalling of the Urokinase-type
Plasminogen Activator via Phosporylation of STAT1
Ehart M* and Binder BR
Department of Vascular Biology and Thrombosis Research, Medical University of Vienna, Austria
Presenting author e-mail: monika.ehart@meduniwien.ac.at
Phosphorylation of STAT-proteins is one possible way of signalling induced by the urokinase-
type plasminogen activator (uPA). We analyzed the dependency of STAT1 phosphorylation on the
presence of the urokinase receptor in mouse skin fibroblasts. We found that human uPA, which
is assumed not to bind to the mouse urokinase receptor, induces STAT1 phosphorylation. Much
to our surprise this phosphorylation was induced only in cells derived from urokinase receptor
knock out mice, but not in cells derived from wild type mice. Since we and others have shown
that gp130 is involved in uPA induced STAT signalling, we analyzed association of uPA with
gp130. We could precipitate gp130 together with uPA from cells derived from urokinase receptor
knock out mice but not from cells derived from wild type mice. We further analyzed urokinase
receptor independent uPA signalling in the human kidney epithelial cell line HEK-293 and the
human adenocarcinoma cell line MCF-7 and obtained data supporting the notion that gp130
transuces uPA signals only in the absence of urokinase receptor.
Molecular & Cellular Biology of Plasminogen Activation 73

i062i
Urokinase Receptor Promotes Neo-Angiogenesis through
its Ser88-Arg-Ser-Arg-Tyr92 Chemotactic Sequence
Longanesi-Cattani I* 1 , Bifulco K 1 , Cantelmo AR 1 , Di Carluccio G 1 , Spina R 1 , Liguori E 1 , Stoppelli MP 2 ,
Carriero MV 1
1 Department of Experimental Oncology, National Cancer Institute of Naples, Italy;
2 Institute of Genetics and Biophysics ‘’Adriano Buzzati-Traverso,’ Naples, Italy
*Presenting author e-mail: immalonganesi@libero.it
Angiogenesis is a highly coordinated process required for normal development, in response
to injury and for tumor growth. This process is sustained by a tightly regulated motility of
endothelial cells. Angiogenesis is regulated by chemotactic stimuli and requires the activation of
several signalling pathways that converge on cytoskeletal remodelling. The Ser88-Arg-Ser-Arg-
Tyr92 chemotactic sequence of the urokinase receptor binds to formyl peptide receptor-like-1
(FPRL-1), a G protein-coupled cell receptor (Resnati et al. 2002). Furthermore, we have found
that the Ser-Arg-Ser-Arg-Tyr (SRSRY) peptide binds to the high affinity formyl peptide receptor
(FPR), and specifically promotes cytoskeletal rearrangements and directional cell migration in
a vitronectin receptor-dependent manner (Gargiulo et al. 2005). Since Human Umbelical Vein
Endothelial Cells (HUVEC) express FPRL-1 as well as avb3 vitronectin receptors, we investigated
on the possibility that SRSRY may triggers neo-angiogenesis. By conventional Boyden chamber
assays, we found that SRSRY promotes directional cell migration of HUVEC in a dose-dependent
manner, with an extent similar to VEGF. Maximal chemotactic effect is reached at 10 nM
SRSRY concentration. In a tube-formation assay, SRSRY shows a remarkable ability to promote
angiogenesis of endothelial cells. According to cell migration data, pro-angiogenic effect is dosedependent
and peaks at 10 nM SRSRY. Both endothelial cell migration and in vitro angiogenesis
promoted by SRSRY are inhibited by cell pre-incubation with blocking anti-avb3 monoclonal
antibodies, indicating that SRSRY exhibits an avb3-dependent pro-angiogenetic effect. Although
preliminary, these data suggest the development of SRSRY-derived pharmacological compounds
to be employed in tissue repairing and cardiovascular diseases.
74 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i063i
The Density Enhanced Phosphatase 1 (DEP-1) Down-Modulates
Urokinase Receptor (uPAR) Surface Expression in Confluent
Endothelial Cells
Brunner PM* 1 , Heier PC 1 , Prager GW 1,2 , Mihaly J 1 , Priglinger U 3 , Binder BR 1
1Department of Vascular Biology and Thrombosis Research, Center for Biomolecular Medicine and
Pharmacology, Medical University Vienna, Austria;
2 Clinical Division of Oncology, Department of Medicine I, Medical University Vienna, Austria;
3 Department of Emergency Medicine, Medical University Vienna, Austria
Presenting author e-mail: patricia.heier@univie.ac.at
It is known that a proteolytic machinery is indispensable for ordered endothelial cell migration
and tissue invasion. Such machinery is largely provided by the prourokinase-urokinase-plasmin
system. Endothelial cells (ECs) in vessels are forming a confluent layer that can be mimicked
by confluent ECs in cell culture. Compared to sparse cultures we could show that the surface
expression of uPAR is decreased by 40%. To examine the cause of this decrease we investigated
the MAP-Kinase-pathway being the major regulatory system for uPAR-expression.
We found that between sparse (50,000 cells/cm2), subconfluent (100,000 cells/cm2) and confluent
cells (150,000 cells/cm2) there was no change in ERK1/2 expression or its phosphorylation.
Therefore we analyzed phosphatases as possible candidates for density dependent uPARexpression.
While Dual-Specificity Phosphatase 1 (DUSP1) was not and DUSP4 was only
borderline upregulated with density, Density Enhanced Phosphatase 1 (DEP-1) significantly
increased with cell density. Transfection of ECs with expression plasmids for wild-type
and mutated forms of DEP-1 revealed that wild-type DEP-1 but not forms mutated in the
phosphatase-domain decreased uPAR-mRNA and cell surface expression. To analyse in detail
how DEP-1 interferes with uPAR-expression we applied reporter assays using plasmids encoding
ERK1 and different DEP-1 forms. We found that DEP-1 influenced the MAP-Kinase pathway
downstream of ERK1/2 in addition to its tyrosine kinase dephosphorylating activity. From this
data we conclude that the quiescent state of contact inhibited ECs is at least partially mediated by
DEP-1 (and possibly also by DUSP4) that inhibits the MAP-Kinase pathway also downstream of
ERK1/2.
Molecular & Cellular Biology of Plasminogen Activation 75

i064i
Detection and Prevention of Hepatic Fibrosis Targeting
Proteolytic TGF-b Activation Reaction
Kojima S*
Molecular Cellular Pathology Research Unit, RIKEN, Japan
Presenting author e-mail: skojima@postman.riken.go.jp
Transforming growth factor (TGF)-b, the most fibrogenic cytokine participating in the
pathogenesis of liver diseases, is produced as a high molecular weight latent form, and thus must
be activated before exerting its biological activities. TGF-b activation is the reaction, by which 25
kD active TGF-b molecule is released from the latent complex. We showed that TGF-b is activated
by plasmin (PLN) and plasma kallikrein (PLK) during pathogenesis of liver fibrosis and impaired
liver regeneration, respectively, and that blockage of these activation reactions with low molecular
weight protease inhibitors prevented the development of the diseases in animal models.
PLN and PLK cleaved between K56-L57 and R58-L59 in LAP portion of human latent TGF-b1,
respectively. We produced antibodies that specifically recognize the neo-epitopes formed by
protease degradation, namely the cut ends of each cleavage site. The anti-R58 antibodies strongly
stained liver sections from patients with fulminant hepatitis compared to normal liver sections
from patients that died from pulmonary embolism. On the other hand, the anti-L59 antibodies
were successively used to establish ELISA to detect LAP degradation fragments in mouse serum.
These results suggest a key role for PLN/PLK in the generation of active TGF-b, namely that a
PLN/PLK-dependent activation reaction occurs around hepatic stellate cells to produce active
TGF-b, which may induce fibrosis and inhibit the proliferation of hepatocytes, thereby addressing
a potential use for PLN/PLK inhibitors in hepatitis therapy. These results also suggest a potential
usage of a LAP degradate as a biomarker for liver fibrosis.
76 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i065i
Domain 1 of uPAR Is Required for its Morphological
and Functional b2 Integrin-mediated Connection with
Actin Cytoskeleton in Human Endothelial Cells
Del Rosso M* 1 , Fibbi G 1 , Margheri F 1 , Serratì S 1 , Pucci M 1 , Manetti M 2 , Ibba-Manneschi L 2
1 Department of Experimental Pathology and Oncology, University of Florence, Italy;
2 Department of Anatomy, Histology and Forensic Medicine, University of Florence, Italy
Presenting author e-mail: delrosso@unifi.it
We have previously shown that MMP12-dependent cleavage of uPAR domain 1 blocks
angiogenesis in Systemic Sclerosis (SSc) endothelial cell. Since integrin association accounts
for uPAR invasion required in angiogenesis, this study was undertaken to show whether fullsize
and truncated uPAR differentially associated to integrins and with motor components
of the cytoskeleton. Truncated and full-size uPAR and its association with integrins and actin
cytoskeleton in SSc and normal (N) microvascular endothelial cells (MVEC), isolated from skin
biopsies, were studied by Confocal Laser Scanning Microscopy and immuno-precipitation.
Integrin composition of endothelial cells was studied by RT-PCR. Cell migration and capillary
morphogenesis were studied on fibrin(ogen) substrates. Involvement of Rac and Cdc42 was
showed by Western blot.
Only full-size uPAR is connected with actin cytoskeleton in endothelial cells. Such connection
is mediated by uPAR-associated alphaM-beta2 and alphaX-beta2 integrins and is absent in
endothelial cells of SSc patients. The cleaved receptor does not associate with beta2 integrins
and with actin. Beta3 integrins associated with both full-size and cleaved uPAR at focal contacts.
uPAR-beta2 integrins uncoupling in N-MVEC impaired activation of Rac and Cdc42, which
mediate uPAR-dependent cytoskeletal rearrangements and cell motility, as well as integrin
engagement-delivered signals to actin cytoskeleton. Invasion and capillary morphogenesis on
fibrin(ogen)-coated substrates indicated that uPAR ligation by uPA empowers beta2 and beta3
integrin-dependent fibrin(ogen) invasion and that such system is impaired in SSc endothelial cell.
Conclusions. uPAR truncation and the following loss of beta2 integrin-mediated uPAR connection
with actin cytoskeleton account for reduced angiogenic properties of SSc endothelial cells.
Molecular & Cellular Biology of Plasminogen Activation 77

i066i
The uPA/uPAR/Vn Pathway of Signaling to MAPK-activation
Sarra Ferraris GM* 1 , Madsen C 1 , Sidenius N 1,2
1 IFOM Foundation, FIRC Institute of Molecular Oncology, Milan Italy;
2 Molecular Genetics Unit, DIBIT, Università Vita-Salute San Raffaele, Milan Italy
Presenting author e-mail: gianmaria.sarraferraris@ifom-ieo-campus.it
The urokinase-type plasminogen activator receptor (uPA(R)), despite the lack of a transmembrane
domain, behaves as a signaling molecule resulting in increased ERK1/2-activation when
overexpressed or upon uPA-binding. In accordance, we find that CHO and 293 cells overexpressing
uPAR display increased basal ERK1/2-activation.
Two different alanine substitution in uPAR (W32A and T54A), which impair Vn-binding, both
failed to cause increased basal ERK1/2-activation. The addition of uPA to cells expressing the
T54A receptor induced rapid binding to vitronectin and ERK1/2 activation, while uPA binding
to cells expressing the W32A mutant failed to induce both vitronectin binding and ERK1/2
activation. Both mutants bind uPA equally well and these data thus allow us to conclude
that uPA-induced ERK1/2 activation is mediated by the induction of the uPAR/vitronectininteraction.
Using cells expressing theT54A receptor we compared the kinetics of uPAR/Vn-induced ERK1/2
activation with the ones triggered by other signalling receptors.
uPA and LPA treatments caused a sustained ERK1/2 activation while EGF an Mn2+ displayed a
peek at 5 minutes followed by a rapid decrease of phosphorylated ERK1/2 levels.
Moreover EGF receptor inhibitors, LPA1 receptor inhibitor and Pertussis Toxin did not affect uPAuPAR
signalling pathway while PI3 kinase inhibitors decreased partially but significantly ERK1/2
activation in response to uPA and LPA.
These data underline the importance of Vn binding in the uPA-uPAR signalling, suggesting a new
way, which is independent of the EGFR and pertussis toxin sensitive GPCR’s, by which uPAR
may signal to ERK1/2.
78 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i067i
Regulation of tPA and PAI-1 Gene Expression in Astrocytes
Hultman K* 1 , Tjärnlund-Wolf A 1 , Blomstrand F 1 , Nilsson M 1 , Medcalf R 2 , Jern C 1
1 Institute of Neuroscience and Physiology, the Sahlgrenska Academy at Göteborg University, Sweden;
2 Australian Centre for Blood Diseases, Monash University, Alfred Medical Research and Education
Precinct (AMREP), Australia
Presenting author e-mail: karin.hultman@neuro.gu.se
We have shown that PKC activation acts in synergy with retinoic acid (RA) to induce tissue-type
plasminogen activator (tPA) gene expression in human endothelial cells. The aim of the present
study was to investigate whether tPA is regulated in a similar manner in astrocytes, as well as to
characterize plasminogen activator inhibitor type 1 (PAI-1) gene expression in these cells.
Native human astrocytes were treated with RA, PKC activator (PMA), PKA activator (forskolin),
cytokines or growth factors for 3, 6, 14 and 20 hours. RA or PMA alone induced a 3-fold upregulation
of tPA at 20 hours. Combined treatment resulted in a 9-fold induction and increased
intracellular storage pools of tPA as visualized by immunocytochemistry. Pretreatment with
actinomycin D or cycloheximide completely blocked both RA and PMA mediated tPA mRNA
increase. Cytokines and growth factors caused a general weak up-regulation of tPA and PAI-1 at
3 and 6 hours followed by a slight down-regulation at 20 hours. Forskolin induced a strong timedependent
down-regulation of both proteins. For all treatments, similar inductions were observed
at the mRNA (RT-PCR) and protein level (ELISA).
We show for the first time that in astrocytes, RA and PKC activation induce a strong up-regulation
of tPA gene expression and that this response is dependent on both gene transcription and de
novo protein synthesis. As we and others have identified functional variants in the tPA and PAI-1
promoters, we will now proceed by investigating whether there is an allele-specific regulation in
human astrocytes.
Molecular & Cellular Biology of Plasminogen Activation 79

i068i
Identification of a Mitotic Epitope in the Domain 2 of the
Urokinase Receptor (uPAR)
Degryse B* 1 , Eden G 2 , Arnaudova R 1 , Furlan F 1 , Blasi F 1,2
1Department of Molecular Biology and Functional Genomics, DIBIT, University Vita Salute San
Raffaele, Milan, Italy;
2 IFOM, FIRC Institute of Molecular Oncology, Milan, Italy
Presenting author e-mail: degryse.bernard@hsr.it
The first characterized chemotactic sequence of uPAR (SRSRY) is located in the linker region
between domain 1 and 2. SRSRY peptide promotes cell migration through binding to receptors
such as FPRL1. Recently, we identified in the domain 2 another chemotactic epitope that we
named D2A, 130IQEGEEGRPKDDR142 (Degryse et al., 2005, J. Biol. Chem. 280, 24792-24803). D2A
synthetic peptide dose-dependently stimulates cell migration with a maximum at 1 pM. We have
also shown that D2A promotes cell migration through binding to avb3 and a5b1 integrins and
activation of integrin-dependent signaling.
Since uPAR can regulate cell proliferation, we have started investigating the effects of D2A on
cell growth. D2A stimulates cell growth in a dose-dependent manner in various cell types and
species, whereas a scrambled version of D2A has no effect. In rat smooth muscle cells, the optimal
dose is 1-10 pM while in human HT29 and HT1080 cells it is 100 pM. These results demonstrate
that D2A is a mitogen at doses that are slightly higher compared to the optimal dose required for
chemotaxis.
We have identified a mitotic/chemotactic epitope in the domain 2 of uPAR showing that this
region is particularly important for uPAR-induced signaling. These data suggest that uPAR is a
membrane-bound mitogen acting through binding to other membrane receptor(s).
80 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i069i
Vitronectin Inhibits Plasminogen Activator Inhibitor-1
(PAI-1)-Induced Chemotaxis by Blocking PAI-1
Binding to the LDL Receptor-Related Protein
Neels JG 1,2 , Kamikubo Y 1,3 , Degryse B* 1,4
1Division of Vascular Biology, Department of Cell Biology, The Scripps Research Institute, La Jolla,
California, USA;
Present addresses:
2 Department of Medicine, University of California San Diego, La Jolla, California, USA;
3Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla,
California, USA;
4Department of Molecular Biology and Functional Genomics, DIBIT, University Vita-Salute San
Raffaele, Milan, Italy
Presenting author e-mail: degryse.bernard@hsr.it
We have previously shown (Degryse et al., 2004, J. Biol. Chem. 279, 22595-22604) that PAI-1
activates the Jak/Stat signaling pathway and stimulates cell migration by binding to the LDL
receptor-related protein (LRP), a large endocytic receptor. Since vitronectin (VN) influences most
of the biological functions of PAI-1, we explored the effects of VN on PAI-1-induced signaling and
cell migration. In fact, VN inhibits PAI-1-promoted signaling and chemotaxis. VN acts by binding
to PAI-1 inhibiting the activation of the Jak/Stat pathway and cell migration. VN exerts these
inhibitory effects by blocking PAI-1 binding to LRP, its motogenic receptor.
We have unveiled a new inhibitory role of VN which has all the hallmarks of a molecule that can
regulate every steps of the cell migration cycle by providing ‘’Stop’’ (under the form of VN/PAI-
1 complex) and ‘’Go’’ signals to the cell. VN shares striking similarities with urokinase, which is
the other main ligand of uPAR and can also generate ‘’Stop’’ and ‘’Go’’ signals. Furthermore, it is
intriguing that urokinase and VN induce ‘’Stop’’ signals by creating uPA/PAI-1 and VN/PAI-1
complexes that block uPAR-, integrin- and LRP-dependent cell migration.
Molecular & Cellular Biology of Plasminogen Activation 81

i070i
Estradiol Inhibits EGF-induced Cell Migration and uPAR Expression in
Estrogen Receptor-a Negative, GPR30 Positive Ovarian Cancer Cells
Henic E* 1 , Noskova V 1 , Høyer-Hansen G 2 , Hansson S 1 , Casslén B 1
1 Department of Gynecology & Obstetrics, University Hospital, Lund, Sweden;
2 Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark
Presenting author e-mail: emir.henic@med.lu.se
EGF is a stimulator of proliferation as well as migration in ovarian cancer cells. In contrast, the
effects of estrogen are incompletely explored and some results are contradictory. In addition to
genomic effects via classical nuclear estrogen receptors (ER), accumulating evidence suggest that
estrogens initiate rapid non-genomic signaling through cell membrane receptors. GPR30 is a
newly identified receptor with all characteristics of a membrane ER.
In this study we show that estradiol attenuates the stimulatory effect of EGF on both cell
migration and uPAR expression in ovarian cancer cell lines. We have previously shown that EGF
up-regulates uPAR expression via three distinct mechanisms. Studying each of these mechanisms
in the presence of estradiol, we found no inhibition of neither the production of uPAR, i.e.
the level of uPAR mRNA, nor the elimination of cell surface uPAR, i.e. by internalization for
lysosomal degradation or by shedding to the medium. Instead estradiol inhibited the very rapid
increase of detergent extractable uPAR, which occurs within minutes of EGF stimulation, and is
likely to represent mobilization of uPAR from detergent resistant domains, like lipid rafts. ICI
182,780, which is a nuclear ER antagonist, has agonistic properties with GPR30. This compound,
like estradiol, inhibited the immediate effect of EGF on uPAR expression, and also did not inhibit
that effect of estradiol. OVCAR-3 cells are ERa negative, but express mRNA for GPR30.
Thus, our observations suggest that modulation of the EGF response by estradiol is mediated by
GPR30.
82 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i071i
Methylation of the PAI-1 Gene in Oral Squamous
Cell Carcinomas and Normal Oral Mucosa
Gao S 1 , Krogdahl A 2 , Sørensen JA 3 , Dabelsteen E 4 , Andreasen PA* 1
1 Department of Molecular Biology, University of Aarhus, Aarhus, Denmark;
2 Department of Pathology, Odense University Hospital, Odense, Denmark;
3 Department of Plastic Surgery, Odense University Hospital, Odense, Denmark;
4 School of Dentistry, University of Copenhagen, Copenhagen, Denmark
Presenting author e-mail: pa@mb.au.dk
It has become clear that CpG methylation plays an important role in development and
progression of cancer and the associated changes in gene expression. We have become interested
in the possibility that decreased CpG methylation of specific genes may lead to over-expression
in tumours and have studied the plasminogen activator inhibitor-1 (PAI-1) gene in this respect.
The PAI-1 gene has 25 CpG sites within 960 bp around the transcription initiation site. We studied
CpG methylation of the PAI-1 gene by bisulfite sequencing and PAI-1 mRNA levels by real-time
RT-PCR. In a series of 20 cases of oral carcinomas and matched samples of adjacent histologically
normal tissue from the same patients, we observed that the methylation frequency in tumours
was lower than in the adjacent non-tumour tissue. By real time RT-PCR analysis, 17 of 20 patients
with oral carcinoma were found to have between 2.5 and 50 fold increased PAI-1 mRNA levels,
as compared with the adjacent non-tumour tissue. The mRNA level in the tumours was inversely
correlated with decreased methylation frequency in the tumours. We conclude that demethylation
of the PAI-1 gene promoter region may contribute to the higher expression of PAI-1 in the tumour
than in surrounding histologically normal tissue in some oral carcinoma patients. The methylation
at some specific CpG sites may play an important role in the regulation of PAI-1 gene expression.
Molecular & Cellular Biology of Plasminogen Activation 83

i072i
Urokinase Signaling through Its Receptor Promotes
Invasiveness and Metastasis of Pancreatic Cancer Cells
Xue A*, Xue M, Jackson C, Song E, Allen BJ, Smith RC
The University of Sydney, Department of Surgery, Royal North Shore Hospital, NSW, Australia
Presenting author e-mail: aiqunn@med.usyd.edu.au
Urokinase-type plasminogen activator (uPA) system plays an important role in tumour
pathogenesis. However, non-specific inhibition of all uPA system members causes side-effects,
so it is necessary to clarify the specific function of uPA system members to provide the basis for
selective blockade and reduction of therapeutic side-effects. In this study, we explored the role of
uPA, uPA receptor (uPAR) and PA inhibitor (PAI) in pancreatic ductal adenocarcinoma (PDAC)
pathogenesis in vitro. Cox regression analysis showed that uPAR was independently associated
with shorter overall patient survival, whereas increased levels of tumour-associated PAI-2 is a
potential indicator of good outcome for patients. Receiver-operating characteristic area under the
curve (ROC AUC) further confirmed that uPAR mRNA levels, as a signer factor, had achieved
the highest AUC (AUC=0.84, p < 0.0001 ) in PDAC. When analysis of uPAR was combined with
uPA, AUC was significantly increased up to 0.93, with sensitivity of 88.2% and specificity of 87.5%
(p < 0.000). We also found that silencing uPAR by the specific small interfering RNA (siRNA)
significantly inhibits cell proliferation, induces G0-G1 arrest and cell apoptosis, and suppresses
cell migration in pancreatic cancer cells. Moreover, suppression of uPAR or uPA expression
differentially regulates the activation of MAP kinases. However, specific blocking PAI-2 has not
been observed significant impact on pancreatic cancer cell proliferation and migration. Taken
together, our data indicate that uPAR may be important biomarker for human pancreatic cancer
progression and prognosis as well as potentially selective therapeutic target.
84 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i073i
Tissue Plasminogen Activator Induces Cell Proliferation in Pancreatic
Cancer by a Non-catalytic Mechanism that Requires ERK1/2 Activation
through Epidermal Growth Factor Receptor and Annexin A2
Ortiz-Zapater E 1,2 , Peiró S 1,2 , Roda O 1,2 , Corominas JM 3 , Aguilar S 1 , Ampurdanés C 1 , Real FX 1,2 ,
Navarro P* 1
1 Unitat de Biologia Cel-lular i Molecular, IMIM-prbb, Barcelona, Spain;
2 Departament de Ciències Experimentals i de la Salut, Facultat de Ciències de la Salut i de la Vida,
Universitat Pompeu Fabra, Barcelona, Spain;
3 Departament de Patologia, Hospital del Mar, UAB, Barcelona, Spain
Presenting author e-mail: pnavarro@imim.es
Pancreatic cancer is one of the most aggressive human tumors, being the fifth-leading cause of
cancer death in the developed countries. Our previous data have shown that tissue plasminogen
activator (tPA) is overexpressed in pancreatic ductal adenocarcinomas, playing a critical role
in several events associated to tumor progression such as cell proliferation, invasion and
angiogenesis. Prior work supports the notion that the effects of tPA on cell invasion require its
proteolytic activity. Here, we identify the molecular mechanism responsible for the proliferative
effects of tPA on pancreatic tumor cells. tPA activates the ERK1/2 signalling pathway in a
manner that is independent of its catalytic activity. We also show that at least two membrane
receptors, EGFR and AnxA2, which are overexpressed in pancreatic cancer, are involved in
the transduction of tPA signalling in pancreatic tumors, suggesting the establishment of an
amplification loop involved in tumor cell proliferation. These results add novel insights into the
non-catalytic functions of tPA in cancer and the molecular mechanisms involved in its effects on
cell proliferation.
Molecular & Cellular Biology of Plasminogen Activation 85

i074i
Modulation of Lung Carcinoma Cell Lines and Primary
Cultures Migration by uPA-Derived and EGFR Inhibitors
Franco P* 1 , Mancini A 2 , Votta G 1 , Caputi M 2 , Stoppelli MP 1
1 Institute of Genetics and Biophysics ‘’Adriano Buzzati-Traverso’’, National Research Council, Naples,
Italy;
2 Department of Cardiothoracic and Respiratory Sciences, Second University of Naples, Naples,Italy
Presenting author e-mail: franco@igb.cnr.it
The detachment of malignant cells from primary tumors and their subsequent migration to
distant sites, including intravasation and extravasation, leads to tumor dissemination. The
uPA/uPAR system plays a crucial role in tumor progression and invasion. Over-expression of
uPAR correlates with a poor prognosis in many neoplastic conditions, including lung tumors,
suggesting that the specific inhibition of the uPA/uPAR system or/and the relative interactors
may be a useful strategy to prevent metastasis. Among the functional partners of uPAR is the
EGFR, which is over-expressed in NSCL (non-small-cell-lung) cancers and correlates with an high
metastatic ability of the tumor. In particular, the EGFR is a widely recognised molecular target for
anti-neoplastic therapies.
In this study, we investigated the functional cross-talk between the two systems and the
possibility to simultaneously inhibit both receptors by the aid of uPAR or EGFR inhibitors.
In particular, we demostrated that both epidermoid carcinoma Calu-1 and squamous
mucoepidermoid NCI-H-292 cell lines pre-treated with anti-EGFR antibodies, fail to migrate not
only toward EGF but also toward uPA. Similarly, pre-treatment of the same cells with anti-uPAR
antibodies, induce a strong inhibition of both uPA- and EGF-dependent migration.
We also tested whether the anti-proliferative pharmacological inhibitors of EGFR kinase, namely
IRESSA or gefitinib (ZD1839) and tyrphostin (AG-1478) may interfere with the Calu-1 and the
NCI-H-292 cell migration. These inhibitors prevent not only EGF-dependent but also uPAdependent
migration, suggesting that EGFR tyrosine kinase activity is required for uPA-induced
directional migration.
To further investigate the relationship between the two systems, we employed non-transformed
human retinal pigment epithelial (RPE) and breast carcinoma MCF-7 cell lines in which uPAR
expression level is stably reduced by the RNA-interference technology. In these clones, EGFR
levels are not modified. However, in RPE and MCF-7 clones EGF-dependent migration is strongly
impaired, confirming that the uPAR is required to the EGF/EGFR-dependent migration.
These results show that the EGF/EGFR and uPA/uPAR systems are functionally interdependent
in lung carcinoma cell migration and suggest that targeting either one may affect both systems.
86 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i075i
A Novel Role of Ku80 in Regulation of PAI-1 Gene Expression
in Migrating Endothelial Cells Induced by Thymosin b4
Bednarek R* 1 , Boncela J 1 , Smolarczyk K 1 , Cierniewski CS 1,2
1 Center of Medical Biology, Polish Academy of Science, Lodz, Poland;
2 Department of Medical and Molecular Biophysics, Medical University, Lodz, Poland
Presenting author e-mail: rbednarek@cbm.pan.pl
Our data demonstrate that increased intracellular expression of thymosin b4 (Tb4) is
necessary and sufficient to induce PAI-1 gene expression in endothelial cells. To describe the
mechanism of this effect, we produced Tb4 mutants with impaired functional motifs and tested
their intracellular location and activity. Cytoplasmic distributions of Tb4(AcSDKPT/4A),
Tb4(KLKKTET/7A), and Tb4(K16A) mutants fused with GFP did not differ significantly
from those of wild Tb4. Overexpression of Tb4, Tb4(AcSDKPT/4A) and Tb4(K16A) affected
intracellular formation of actin filaments. As expected, Tb4(K16A) uptake by nuclei was impaired.
On the other hand, overexpression of Tb4(KLKKTET/7A) resulted in developing the actin
filament network typical of adhering cells, indicating that the mutant lacked the actin binding
site. The mechanism by which intracellular Tb4 induced the PAI-1 gene did not depend upon
the N-terminal tetrapeptide AcSDKP, and depended only partially on its ability to bind G actin
or enter the nucleus. Both Tb4 and Tb4(AcSDKPT/4A) induced the PAI-1 gene to the same
extent, while mutants Tb4(KLKKTET/7A) and Tb4(K16A) retained about 60% of the original
activity. By proteomic analysis, the Ku80 subunit of ATP-dependent DNA helicase II was found
to be associated with Tb4. Ku80 and Tb4 consistently co-immunoprecipitated in a complex from
endothelial cells. Furthermore, downregulation of Ku80 by specific siRNA resulted in dramatic
reduction in PAI-1 expression, both at the level of mRNA and protein synthesis. These data
suggest that Ku80 functions as a novel receptor for Tb4, initiates signaling leading to activation of
PAI-1 expression by an as yet unknown mechanism, and mediates the intracellular activity of Tb4.
Molecular & Cellular Biology of Plasminogen Activation 87

i076i
Signaling Pathway Involved in Inhibition of
PAI-1 Expression by CNP in Endothelial Cells
Jerczynska H*, Cierniewski CS, Pawlowska Z
Department of Molecular and Medical Biophysics, Medical University in Lodz, Lodz, Poland
Presenting author e-mail: hanuka@zdn.am.lodz.pl
We have reported previously that C-type natriuretic peptide (CNP) was an effective inhibitor of
PAI-1 synthesis and release from human endothelial cells. This inhibitory effect was stronger in
Tumor Necrosis Factor alpha TNFa - stimulated cells.
The signaling pathways required for this effect have not been elucidated. In the current study we
investigated the signal transduction pathway involved in the inhibitory CNP action in human
endothelial cells. We examined if CNP can modulate the known signaling pathways involved
in the regulation of PAI-1 expression as well as a potential cGMP role. To characterize the signal
transduction pathway, we used selective inhibitors of MAP kinase, PI3K/Akt, and 8-bromocGMP,
a soluble analog of cGMP. The activation level of ERK1/2, JNK and NFaB was determined
by ELISA with the use of specific antibodies. Our results imply that CNP inhibition of TNFaactivated
PAI-1 gene expression takes place via regulation of signal transduction pathways
involving directly PI3K/Akt and cGMP-dependent protein kinase, possibly by inhibiting NFkBdependent
pathways. Thus, our results explain at least in part the mechanism involved in PAI-1
expression inhibition by CNP in TNF-stimulated endothelial cells.
88 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i077i
Interaction of Alzheimer’s Amyloid b-peptide (Ab) 1-40 with PAI-2
Pabba M* 1 , Przygodzki T 1 , Malisauskas M 1 , Olofsson A 2 , Morozova-Roche L 1 , Wilczynska M 1 , Ny T 1
1 Department of Medical Biochemistry and Biophysics, 2 Umeå Center for Molecular Pathogenesis, Umeå
University, Umeå, Sweden
Presenting author e-mail: pabba.mohan@medchem.umu.se
Alzheimer’s disease (AD) is one of the most known dementia in elderly. The disease is
characterized by the presence of senile plaques that are almost entirely composed of an amyloid
b-peptide. Amyloid b-peptide which is a proteolytic fragment produced from amyloid precursor
protein, is also found in intracellular compartments, and recently several physiological roles of the
peptide have been proposed. Acceleration as well as inhibition of Ab-peptide protein aggregation
during Alzheimer’s disease is accomplished by various factors, including serpins. Serpins are
the largest and broadly distributed superfamily of protease inhibitors. Plasminogen activator
inhibitor type 2 (PAI-2) belongs to ovalbumin sub-clade of the serpin superfamily. Although it
has been extensively studied, its biological functions remain unknown. It has been shown that
expression of PAI-2 is high in microglia cells surrounding senile plaques in the brain of patients
with Alzheimer’s disease, suggesting that PAI-2 might have a role in disease. Our western
blot and sucrose cushion sedimentation experiments show that Ab-peptide interacted and coaggregate
with PAI-2. In addition, atomic force microscopy and thioflavin-T analyses reveal that
PAI-2 inhibit protofibril formation by the Ab-peptide. Taken together these data suggest that
Ab-peptide 1-40 interacts with PAI-2 in vitro. Studies on interaction of Ab-peptide with PAI-2 in
neuronal cells are in progress.
Molecular & Cellular Biology of Plasminogen Activation 89

i078i
The Subcellular Itinerary of Hepatocyte Growth
Factor Activator Inhibitor-1 in MDCK Cells
Godiksen S 1 , Selzer-Plon J 1 , Pedersen EDK 1 , Borger Rasmussen H 1 , Bugge TH 2 , Vogel LK* 1
1 Department of Cellular and Molecular Medicine, University of Copenhagen, Denmark;
2 Oral and Pharyngeal Cancer Branch, NIDCR, NIH, Bethesda, Maryland, USA
Presenting author e-mail: vogel@imbg.ku.dk
Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a Kunitz-type transmembrane serine
protease inhibitor that forms inhibitor complexes with several trypsin-like serine proteases that
is required for mouse placental development and embryo survival and is a key regulator of
carcinogenesis. HAI-1 is expressed in polarized epithelial cells, where the plasma membrane
is divided by tight juntions into an apical and a basolateral domain. Here we show that HAI-
1 at steady state is mainly located on the basolateral membrane in both MDCK cells and
mammary gland epithelial cells. After biosynthesis HAI-1 is exocytosed to the basolateral plasma
membrane from where 20% of the HAI-1 molecules are proteolytically cleaved and released
into the basolateral media. The remaining membrane associated HAI-1 is endocytosed and
recycles between the basolateral plama membrane and endosomes. However, a minor fraction
of basolaterally located HAI-1 is trancytosed to the apical plasma membrane, from where it
is probably cleaved and released into the media. HAI-1 is raft associated during most of its
intracellular transport in a methyl-b-cyclodextrin dependent manner.
90 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i079i
Evidence for a Matriptase-Prostasin (CAP1/PRSS8) Serine Protease
Zymogen-Cascade-Regulating Epithelial Differentiation
List K* 1 , Netzel-Arnett S 2 , Currie B 1 , Szabo R 1 , Molinolo A 1 , Antalis TM 2 , Bugge TH 1
1 Oral and Pharyngeal Cancer Branch, NIDCR, NIH, Bethesda, Maryland, USA;
2 Center for Vascular and Inflammatory Disease, University of Maryland, Baltimore, Maryland, USA
Presenting author e-mail: klist@nidcr.nih.gov
Epidermal ablation of matriptase and prostasin has identical deleterious effects on terminal
differentiation, suggesting a functional interrelationship between the two membrane serine
proteases. Here we present histological, biochemical, and genetic evidence for a matriptaseprostasin
zymogen cascade regulating epithelial differentiation. Enzymatic gene trapping of
matriptase combined with prostasin immunohistochemistry revealed that matriptase was colocalized
with prostasin in transitional layer cells of the epidermis, and that the developmental
onset of expression of the two membrane proteases correlated with acquisition of epidermal
barrier function. Purified soluble matriptase efficiently converted soluble prostasin zymogen to
an active two-chain form that formed SDS-stable complexes with the serpin protease nexin-1.
Whereas two forms of prostasin with molecular weights corresponding to the prostasin zymogen
and active prostasin were present in wildtype epidermis, prostasin was exclusively found in
the zymogen form in matriptase-deficient epidermis. Moreover, we found that matriptase and
prostasin displayed a near-ubiquitous co-localization in simple, stratified, and pseudo-stratified
epithelia of the integumentary system, digestive tract, respiratory tract, and urogenital tract,
suggesting that the proposed matriptase-prostasin zymogen cascade may have additional roles in
epithelial biology besides regulating terminal epidermal differentiation. Because matriptase is an
auto-activating protease, it may serve as the initiator of zymogen cascades, similar to the closely
related protease, enteropeptidase.
Molecular & Cellular Biology of Plasminogen Activation 91

i080i
The Plasminogen Activation System in Monocytic Cell
Differentiation and Proliferation: Potential Target for
Plasminogen Activation Inhibitor Type-2-Based Therapeutics
Lee JA* 1 , Croucher DR 2 , Ranson M 1
1 School of Biological Sciences, University of Wollongong, NSW Australia;
2 Cancer Research Program, Garvan Institute of Medical Research, Sydney, Australia
Presenting author e-mail: jal31@uow.edu.au
The progression of tumours to malignancy is aided by the surrounding stroma and infiltrating
leukocytes, of which tumour associated macrophages (TAMs) contribute up to 70% of the tumour
mass in some cancers. TAMs potentiate malignancy by contributing to the production of the
extracellular protease, plasmin. Originating from peripheral blood monocytes, TAMs have been
found to over express components of the u-PA system and this behaviour identifies them as
potential targets in the development of anti-u-PA targeted cancer therapies. One such potential
therapy utilises the accelerated internalisation mechanisms of cell surface u-PA upon inhibition
by its specific inhibitor, plasminogen activation inhibitor (PAI) type-2. This research shows for
the first time that the internalisation of PAI-2 by differentiated U-937 and THP-1 monocytic cell
lines is mediated in part by the low density lipoprotein receptor (LDLr) family of endocytosis
receptors, in a u-PA dependent manner. Importantly, this shows that TAMs expressing high levels
of u-PA may be targeted using PAI-2 therapeutics. Assessment of physiological consequences
associated with exogenous PAI interactions at the cell surface revealed a reduction in proliferation
for both cell lines when cultured in the presence of PAI-2 and PAI type-1, suggesting that u-PA:
PAI complex formation at the cell surface disrupts mitogenic signalling cascades mediated via
receptor bound u-PA. Based on these findings current research is being directed at the role of
exogenous PAI-2 in proliferation and differentiation of monocytes.
92 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i081i
Identification and Localization of Novel Serine
Proteases in the Mouse Ovary
Wahlberg P*, Nylander Å, Kui L, Ny T
Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden
Presenting author e-mail: patrik.wahlberg@medchem.umu.se
Proteolytic degradation of extracellular matrix components is believed to play an essential role
for ovulation to occur. Recent studies in our laboratory have suggested that the plasminogen (PA)
and matrix metalloproteinase (MMP) systems are not required in this process. In this study, we
have used a microarray approach to identify new proteases that are involved in ovulation. We
discovered three new serine proteases that were relatively highly expressed during ovulation:
HtrA1, which was not regulated during ovulation; PRSS23, which was downregulated by
gonadotropins; and PRSS35, which was upregulated by gonadotropins. We have investigated
the expression patterns of these proteases during gonadotropin-induced ovulation in immature
mice and in the corpus luteum (CL) of pseudopregnant mice. We found that HtrA1 was highly
expressed in granulosa cells throughout follicular development and ovulation. It was also highly
expressed in the forming and regressing CL. PRSS23 was highly expressed in atretic follicles and
it was expressed in the ovarian stroma and theca tissues after ovulation was induced. PRSS35 was
expressed in the theca layers of developing follicles. It was also highly induced in granulosa cells
of preovulatory follicles. PRSS35 was also expressed in the forming and regressing CL. These data
suggest that HtrA1 and PRSS35 are involved in ovulation and CL formation and regression and
that PRSS23 may play a role in follicular atre.
Molecular & Cellular Biology of Plasminogen Activation 93

i082i
Alpha-enolase/Plasminogen Binding Is Required
during Myogenesis in vitro and in vivo
Diaz-Ramos A*, Llorens A, Luque T, López-Alemany R
Institute of Biomedical Investigation of Bellvitge (IDIBELL), Molecular Oncology Center (COM),
L’Hospitalet de Llobregat, Barcelona, Spain
Presenting author e-mail: madiaz@idibell.org
During myogenesis, an important proteolyitc activity and extensive extracellular matrix (ECM)
remodelation takes place. Plasmin, generated by activation of plasminogen, is an extracellular
protease specialized in the degradation of the ECM components. Alpha-enolase constitutes a
receptor for plasminogen in several cell lines, serving to focalize proteolytic activity on the cell
surface.
Previous studies show a role for components of the plasminogen activation system during
myogenesis in vitro and in vivo. Preliminary results of our laboratory have shown that alphaenolase
expression was up-regulated in a myogenic cell line upon differentiation, but the role of
alpha-enolase as a plasminogen receptor in myogenesis deserves further analysis.
In the presence of inhibitors of plasminogen/alpha-enolase binding, myogenic differentiation,
fusion and migration were abrograted, using primary cultures of myogenic cells or Muscle
Precursor Cells (MPCs).
The effect of inhibitors of plasminogen/alpha-enolase binding were evaluated in a regeneration
model in mice after a muscle. Regeneration parameters were blocked by inhibitors of
plasminogen/alpha-enolase. The mdx mice (the animal model for Duchenne Muscular Dystrophy,
DMD) presented a more severe dystrophinopathy with the same treatment. Inflammatory cells
infiltration and fibrin deposition in injured tissues are currently being evaluated.
Since inhibitors of plasminogen/alpha-enolase binding have an inhibitory effect on MPCs
differentiation and muscle regeneration in vivo, our results demonstrate that plasmin activity
is necessary for myogenesis to take place correctly, in an alpha-enolase dependent way.
Plasminogen/alpha-enolase binding therefore could be an important target in the development of
treatments for DMD.
94 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i083i
The Serpinb8 Is Alternatively Spliced to the Known
Long Form and a Novel Short Form
Olausson B* 1 , Przygodzka P 1 , Dahl L 2 , Carlsson L 2 , Wilczynska M 1
1 2 Department of Medical Biochemistry and Biophysics, Umeå Center for Molecular Medicine, Umeå
University, Umeå, Sweden
Presenting author e-mail: bjorn.olausson@medchem.umu.se
Serpinb8 belongs to the ovoalbumin-serpin subfamily, and is known as an intracellular protease
inhibitor with a unique distribution pattern in human tissues. Here we show that serpinb8 can
be alternatively spliced to the already known long form and to a novel short form. Compared to
the long form, the short form of serpinb8 is missing exon 7, and instead it has a short nucleotide
extension. Exon 7 in the long serpinb8 encodes part of beta-sheet A and the reactive center loop.
Therefore, the short form of serpinb8 can be predicted not to function as protease inhibitor. By
using real-time PCR, we found that in myelomonocytic leukemia cell lines, as well as in mouse
hematopoietic stem cells, the long form of serpinb8 is expressed at very low levels, but the
expression increases during monocytic, granulocytic, and megakaryocytic differentiations to
reach maximum in finally differentiated blood cells. In contrast, the short form of serpinb8 is
temporarily expressed during monocytic differentiation of HL-60 cells and is not detectable in the
differentiated monocytes. Our preliminary real-time PCR data on the RNA from 18 human tissues
suggest that the short form of serpinb8 is expressed only in few tissues as pancreas, oesophagus,
and kidney. Studies on the cellular localization of the spliced forms of serpinb8 are in progress.
Molecular & Cellular Biology of Plasminogen Activation 95

i084i
Characterization of a Combined PAI-1 and TIMP-1
Gene-deficient Mouse Model
Harslund J* 1 , Nielsen OL 2 , Brünner N 1 , Offenberg H 1
1 2 Section of Biomedicine, Section of Pathology, Department of Veterinary Pathobiology, Faculty of Life
Sciences, University of Copenhagen, Frederiksberg, Denmark
Presenting author e-mail: jhar@life.ku.dk
The endogenous proteinase inhibitors PAI-1 and TIMP-1 are two distinct proteins with separate
molecular pathways. However, a much closer relationship between PAI-1 and TIMP-1 has been
proposed indicating some degree of functional overlap due to their involvement in ECM turnover,
tissue remodelling and cellular migration and signalling. To study the physiological implications
of PAI-1 and TIMP-1 we have generated a combined PAI-1 and TIMP-1 gene deficient mouse
model. We present the results on generating this specific mouse model with particular emphasis
on phenotypical characteristics, haematological parameters, a histological evaluation and gene
expression studies of PAI-1 and TIMP-1 in various organs. We observed a significant deviation in
segregation of offspring only in male mice (p

i085i
Dual Role of the uPA/uPAR System in Apoptosis of
Mesangial Cells and Diabetic Nephropathy
Tkachuk N, Tkachuk S*, Kiyan J, Shushakova N, Haller H, Dumler I
Nephrology Department, Hannover Medical School, Hannover, Germany
Presenting author e-mail: Tkatchouk.Sergei@mh-hannover.de
In diabetic nephropathy apoptosis has been considered as a major mechanism for regulation
of cell amount in the places of mesangial hypercellularity. Given these functions for apoptosis
together with the observation that uPAR and uPA may be upregulated during renal injury, the
present study was designed to investigate the role of the uPA/uPAR system in apoptosis of
mesangial cells (MC) under high glucose and growth factors withdrawal conditions. Our data
show that uPA can elicit both pro-apoptotic and anti-apoptotic effects in human MC depending
on the apoptosis-inducing stimulus. Thus, uPA abrogated MC apoptosis induced by growth
factors withdrawal conditions, whereas apoptosis initiated in MC by high glucose was enhanced
in presence of uPA. Amino-terminal fragment (ATF) of uPA completely retained effects of uPA.
uPAR was shown to be involved in both pro-apoptotic and anti-apoptotic effects of uPA. Cellular
redistribution of uPAR in response to high glucose was observed. Members of MAPK and PI3kinase
pathways and Bad protein were found to be downstream molecules mediating the effects
of uPA. The opposite effects of uPA on MC apoptosis are directed via different uPAR-interacting
transmembrane partners. Thus, whereas the anti-apoptotic effect of uPA was mediated by
integrins, its pro-apoptotic effect required uPAR interaction with cation-independent mannose-
6-phosphate receptor (M6PR). In vivo, uPAR and M6PR were upregulated in the kidney of mice
in streptozotocin (STZ)-induced diabetes that confirmed an important role for these receptors in
mediating functional response of mesangial cells to high glucose.
Molecular & Cellular Biology of Plasminogen Activation 97

i086i
Phenotypic Consequences of Plasminogen Activator
Inhibitor-1 Gene Ablation on STAT1 Activation and Cell
Cycle Progression in Proliferating Endothelial Cells
Balsara RD* 1,2,3 , Morin SJ 1 , Meyer CA 1 , Castellino FJ 1,2,3 , Ploplis VA 1,2,3
1 2 3 W. M. Keck Center for Transgene Research, Department of Chemistry and Biochemistry, Notre Dame
Cancer Institute, University of Notre Dame, Indiana, USA
Presenting author e-mail: rbalsara@nd.edu
The enhanced proliferation observed in PAI-1-deficient endothelial cells (EC) is associated with
Akt hyperactivation resulting in loss of growth control and resistance to apoptosis. Additionally,
involvement of other signaling pathways that regulate cell proliferation was investigated.
Evaluation of the STAT protein profile revealed that PAI-1–/– EC exhibited lower levels of
STAT1, which is a negative regulator of cellular proliferation. The pro-proliferative STAT3 and
STAT5 protein levels were similar in WT and PAI-1–/– cells. Furthermore, the activation status of
STAT1 between WT and PAI-1–/– EC differs. The extent of phosphorylation of Ser727 within the
STAT1 transcriptional activation domain is lower in PAI-1–/– EC. Immunofluorescent analyses
of proliferating WT and PAI-1–/– EC demonstrate that the cellular distribution of STAT1(P-
Tyr701), which is crucial for STAT1 dimerization and nuclear translocation, is different between
the two cell types. In WT cells, STAT1(P-Tyr701) appears to be diffuse throughout the whole
cell, including the nucleus. In contrast, in PAI-1–/– cells, STAT1(P-Tyr701) seems to be localized
in the cytoplasm and plasma membrane. Additionally, it was observed that in WT cells STAT1
preferentially binds to Jak1, whereas in the PAI-1–/– EC, STAT1 is preferentially bound to Jak2.
This differential binding may be responsible for the different activation states of STAT1. Cell cycle
progression analysis demonstrated that a higher percentage of PAI-1–/– EC are in the S-phase
compared to WT cells, which is likely a consequence of diminished STAT1 levels observed in the
PAI-1-deficient cells.
98 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i087i
Urokinase and its Receptor as Novel C-Myc Target
Genes Affecting Cell Migration and Apoptosis
Alfano D*, Iaccarino I, Stoppelli MP
Institute of Genetics and Biophysics ‘’Adriano Buzzati-Traverso’’, National Research Council, Naples,
Italy
Presenting author e-mail: alfano@igb.cnr.it
The binding of the serine protease urokinase (uPA) to its receptor (uPAR) plays a central role
in cell adhesion, migration and invasion. According to our recently published data, uPAR
overexpression and signalling prevents DNA damage-induced apoptosis as well as anoikis of
retinal pigment epithelial cells (RPE). Among the genes regulating cell proliferation and survival
is the oncogene c-Myc. In an attempt to characterise the effect of c-Myc activation on uPA and
uPAR expression as well as on the cell survival/apoptosis balance, we employed an RPE cell line
carrying an inducible c-MycER. As previously reported in other cell systems, induction of c-Myc
in RPE cells leads to an increased cell sensitivity to different pro-apototic stimuli, like uv-light
and serum starvation. To investigate the genes regulated in these conditions, the gene expression
profile following c-Myc activation for 4-16 hrs was determined. Surprisingly, a marked downregulation
of the mRNAs coding for uPA and uPAR was observed. Furthermore, addition of
recombinant uPA to the culture medium improuves cell survival, suggesting that
c-Myc-induced apoptosis is, at least in part, due to uPA down-regulation. As expected, following
c-Myc activation, we observed a remarkable decrease in cell migration ability. In conclusion, the
uPA/uPAR system is subjected to a c-Myc-dependent downregulation under conditions in which
a concomitant increase in the sensitivity to apoptosis and a decrease in cell motility is observed.
We propose that during oncogenic transformation the uPA/uPAR system might restrain c-Myc
proliferative and invasive potential.
Molecular & Cellular Biology of Plasminogen Activation 99

i088i
Effect of Plasminogen on Cell Migration Using an in vitro Wound Model
Sulniute R*, Li J, Ny T
Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden
Presenting author e-mail: rima.sulniute@medchem.umu.se
In vivo studies on incisional wounds have shown that plasminogen (plg) plays an important role
in wound healing. However, due to the complexity of the in vivo system, the exact functional roles
that plg plays in wound healing at the molecular level remain obscure.
We therefore established a simplified in vitro model in order to directly investigate the molecular
mechanism by which plg affects keratinocyte migration in wound healing. In this model, a
scratch wound is made on a monolayer of adherent DOK (early neoplastic/dysplastic human oral
keratinocytes) cell line and the wound closure process is monitored thereafter. Our data reveal
that at 24 hours after scratch wounding, DOK cells had migrated about 65% of the original wound
area in the presence of 2 µM plg (mimicking the plasma level of plg), whereas these cells only
migrated about 45% when incubated in the plg-depleted medium. Furthermore, wound closure
was accelerated up to 80% of the original wound when the level of plg was doubled
(4 µM). Interestingly, when PMSF, a serine protease inhibitor, was added in the presence of plg,
the cells had the same migration rate as those in the plg-depleted medium. Altogether, our data
suggest that plg, probably through its proteolytic activity, is essential in keratinocyte migration in
an in vitro wound healing model. These findings are in consistence with our in vivo studies in plg
deficient mice.
100 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i089i
uPA, but not its Receptor uPAR, Is Necessary for Experimentallyinduced
and Pathological Muscle Regeneration
Vidal B*, Serrano AL, Jardí M, Suelves M, Muñoz-Cánoves P
Center for Genomic Regulation (CRG), Differentiation and Cancer Program, Barcelona, Spain
Presenting author e-mail: berta.vidal@crg.es
Extracellular proteolysis takes place during skeletal muscle regeneration. We have previously
shown that uPA is required for muscle regeneration in vivo, since regeneration was impaired
in uPA–/– mice after muscle injury. Moreover, the double mutant uPA–/–mdx mice showed
exacerbated dystrophinopathy compared to uPA+/+mdx mice, the animal model for Duchenne
muscular dystrophy. uPA was critical for a correct inflammatory response, as demonstrated by
the reduced number of infiltrated macrophages and T-cells in uPA–/–mdx mice, as compared
to uPA+/+mdx mice. Consistent herewith, loss of uPA also reduced the number of infiltrated
inflammatory cells in experimentally-injured muscle. In contrast, the muscular compartment
did not seem to be affected by loss of uPA, since isolated uPA–/– satellite cells showed normal
myogenesis in vitro. Since several cellular functions of uPA do not require its proteolytical activity,
but rather its ability to bind uPAR, and because uPAR expression was induced in regenerating
muscle of WT and mdx mice, we hypothesized that the role of uPA in muscle regeneration might
be dependent, at least in part, on its binding to uPAR. Thus, we analyzed the consequences of
uPAR deficiency (uPAR–/– mice) in experimentally-induced muscle regeneration and mdx
dystrophinopathy (uPAR–/–mdx mice). Experimentally-induced muscle regeneration was
indistinguishable between WT and uPAR–/– mice after histological analyses at 2, 10 and 25
days post-injury. Muscular dystrophy was also similar in uPAR+/+mdx and uPAR–/–mdx
mice, indicating that uPAR is dispensable for muscle tissue remodeling during regeneration.
These results suggest that uPA regulates key processes during muscle regeneration in a uPARindependent
manner.
Molecular & Cellular Biology of Plasminogen Activation 101

i090i
Urokinase-type Plasminogen Activator Deficiency Strongly Attenuates
Ischemia Reperfusion Injury and Acute Kidney Allograft Rejection
Gueler F 1 , Rong S 1 , Mengel M 2 , Park J-K 1 , Kirsch T 1 , Haller H 1 , Dumler I 1 , Shushakova N* 1
1 Department of Nephrology, 2 Department of Pathology, Medical School Hanover, Hanover, Germany
Presenting author e-mail: nshushakova@phenos.com
Central mechanisms leading to ischemia induced allograft rejection are apoptosis and
inflammation, processes highly regulated by the urokinase-type plasminogen activator (uPA)
and its specific receptor (uPAR). Recently, up-regulation of uPA and uPAR has been shown to
correlate with allograft rejection in human biopsies. However, the causal connection of uPA/
uPAR in mediating of transplant rejection and underlying molecular mechanisms remain poorly
understood. In this study, we evaluated the role of uPA/uPAR in a mice model for IR injury
and for acute kidney allograft rejection. uPAR but not uPA deficiency strongly protected kidney
from IR injury. For the transplant model, allografts from H2b uPA–/–, uPAR–/– and wildtype
(WT) mice were transplanted into MHC-incompatible H2d WT recipients. Again uPAR but not
uPA deficiency of the allograft caused superior recipient survival and strongly attenuated loss of
renal function. We could demonstrate that uPAR-deficient allografts showed reduced generation
of reactive oxygen species (ROS) and apoptosis. Moreover, transplant-induced up-regulation
of adhesion molecule ICAM-1 was completely abrogated in uPAR-deficient allografts due to
inadequate C5a and TNFa signalling, leading to reduced leukocyte infiltration. Our results
demonstrate that the local renal uPAR plays an important role in the apoptotic and inflammatory
responses mediating IR-injury and transplant rejection.
102 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i091i
a1-antitrypsin Polymerization Studies using Gas-phase
Electrophoretic Mobility Molecular Analysis (GEMMA)
Przygodzki T* 1 , Mallya M 2 , Phillips RL 2 , Belorgey D 2 , Hägglöf P 2 , Lomas DA 2 , Ny T 1
1 Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden;
2 Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research,
Cambridge, United Kingdom
Presenting author e-mail: tomasz.przygodzki@medchem.umu.se
Several proteins from the serpin superfamily are known to have capability of forming polymers.
One of them is the elastase inhibitor-a1-antitrypsin (AT). Beside wild type AT which does not
polymerize in physiological conditions, a highly polymerizing, natural mutant, Z AT, has been
described.
The kinetics of serpin polymerization is still a matter of debate. We followed this process by a
novel approach: gas-phase electrophoretic mobility molecular analysis (GEMMA). This technique
allows the analysis of the distribution of macromolecules in solution with respect to their size. A
relatively short time required for sample analyses, a broad range of macromolecule size that can
be separated as well as the fact that non-covalent complexes remain intact upon measurement
make this technique very useful for the study of protein assembly.
We followed the polymerization process of both wild type and Z AT over time at three
temperatures. We observed no oligomers formation of wild type AT at temperatures of 36°C and
45°C whereas Z AT was polymerizing in all conditions. Velocity of wt AT oligomers formation
at 55°C was approximately 10 times lower than that for the Z mutant. We have observed
dimeric, trimeric, tetrameric and pentameric species in the case of both proteins indicating that
polymerization proceeds, by sequential addition of monomers, however the fast formation of
dimers suggests that the dimer can also be a building block in formation of the polymers.
Molecular & Cellular Biology of Plasminogen Activation 103

i092i
The Plasminogen Interaction of Antigen 85B Protein
from Mycobacterium Tuberculosis: Role of Lys89
Xolalpa W* 1 , Vallecillo AJ 1 , Rosales L 2 , Ruiz BH 2 , Espitia C 1
1 Departamento de Inmunología y, 2 Departamento de Biología Molecular, Instituto de Investigaciones
Biomédicas, Universidad Nacional Autónoma de México, México, México
Presenting author e-mail: wendyxv@hotmail.com
Interactions of microorganims with the Plasminogen (Plg) system molecules have been
recognized in numerous bacteria species. Our group has recently identified several Plg binding
proteins in Mycobacterium tuberculosis extracts combining 2D-PAGE and Ligand Blotting tools.
Interestingly, proteins belonging to the antigen 85 (Ag85) complex, which have a role on cell wall
synthesis and bind the extracellular matrix protein fibronectin, were identified as Plg binding
proteins. This binding was inhibited by the lysine analogue e-aminocaproic acid, involving
the participation of lysine binding sites (LBSs) of Plg kringles. In order to characterize the Plg-
Ag85 interaction, we took advantage of molecular modeling tools. Using the theoretical model
of the lysine-LBS interaction of the homodimeric complex of kringle 2 from tPA as reference of
interactions between a kringle domain and a non C-terminal lysine, the Lys89 from Ag85A and
Ag85B was recognized as a better candidate to mediate Plg interaction. To assess the role of this
aa, site-specific mutation was introduced in the M. tuberculosis Ag85B recombinant protein
replacing the lysine by arginine. Recombinant Ag85B and Ag85BArg89 proteins were evaluated
in their capability to bind to Plg by a solid-phase binding assay on ELISA plates. The results
demonstrate that Plg binding on Ag85BArg89 protein is reduced until 90 percent in comparison
with the wild type Ag85B protein. Binding to fibronectin was also evaluated in both proteins but
no significant difference was found. These results confirm the specific role of Lys89 on Ag85B-Plg
interaction giving to Ag85B a new role as putative Plg receptor.
104 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i093i
Plasminogen as a Factor in Innate Immunity
Ahlskog N*, Guo Y, Ny T
Department of Medical Chemistry and Biophysics, Umeå University, Umeå, Sweden
Presenting author e-mail: nina.ahlskog@medchem.umu.se
Recent results in our group strongly indicate a link between plasmin/plasminogen and the innate
immune system, the body’s initial response to infection. Plasminogen-deficient mice were shown
to have a higher mortality during S. aureus-induced infection (i. v. injection of low dose viable
bacteria), but had a better survival rate during the initial stages of sepsis (high dose bacteria) as
compared to their wild-type littermates. This phenotypic switch was shown to be specific to the
active plasmin, as well as dependent on plasmin levels.
Using a human mast cell line (HMC-1.2) and a mouse intra-peritoneal macrophage cell line (J774),
we have found that the phenotypic switch seen in vivo, is not due to the phagocytizing capability
of the macrophages or the anti-microbial activity of the mast cells in response to infection and
sepsis. The cells were exposed to viable bacteria in doses mimicking infection and sepsis for 20
hours, after which the bacterial number, as well as the cell number, was ascertained. The antimicrobial
response of both the cell types was comparable, regardless of the presence or absence
of plasminogen in the medium. The migrating capability of the cells was tested using Boyden
Micro Chemotaxis Chamber, where the percentage of cells migrating through the membrane of
the chamber was found to be comparable for both cell types, with or without plasminogen in the
medium. The cellular response, e.g. release of cytokines and proteases, of the cells upon exposure
to bacteria will be studied further as well as other leukocytic cell types.
Molecular & Cellular Biology of Plasminogen Activation 105

i094i
The Inflammatory Cytokine Oncostatin M Induces Plasminogen
Activator Inhibitor-1 in Human Vascular Smooth Muscle Cells in
vitro via PI3 kinase and MAP-kinase Dependent Pathways
Demyanets S 1 , Kaun C 1 , Rychli K 1 , Rega G 1 , Pfaffenberger S 1 , Maurer G 1 , Huber K 2 , Wojta J* 1
1Department of Internal Medicine II, Medical University of Vienna, Ludwig Boltzmann Foundation for
Cardiovascular Research, Vienna, Austria;
2 3rd Medical Department for Cardiology and Emergency Medicine, Wilhelminenspital, Vienna, Austria
Presenting author e-mail: johann.wojta@meduniwien.ac.at
Plasminogen activator inhibitor-1 (PAI-1) plays a pivotal role in the regulation of the fibrinolytic
system and in the modulation of extracellular proteolysis. Increased expression of PAI-1 was
found in atherosclerotic lesions and high PAI-1 plasma levels were shown to be associated
with coronary heart disease. Smooth muscle cells seem to be a major source of PAI-1 within
the vascular wall and PAI-1 was implicated in smooth muscle cell migration, proliferation and
apoptosis. We treated human coronary artery smooth muscle cells (HCASMC) and human aortic
SMC (HASMC) with the glycoprotein 130 (gp130) ligands cardiotrophin-1 (CT-1), interleukin-
6 (IL-6), leukemia inhibitory factor (LIF) or oncostatin M (OSM). OSM, but not CT-1, IL-6 or
LIF increased PAI-1 production significantly in both HCASMC and HASMC up to 20-fold as
determined by a specific ELISA. OSM upregulated also mRNA specific for PAI-1 up to 4.5-fold
after 24 hours in these cells and HCASMC and HASMC were shown to express gp130, OSM
receptor, IL-6 receptor and LIF receptor using RealTime-polymerase chain reaction (RealTime-
PCR). PD98059, a MEK inhibitor and LY294002, a PI3K inhibitor, but not AG-490, a JAK/STAT
inhibitor, abolished the OSM-dependent PAI-1 induction almost completely. We hypothesize that
if the effect of OSM on PAI-1 expression in smooth muscle cells is operative in vivo it could—via
modulation of fibrinolysis and extracellular proteolysis—be involved in the development of
vascular pathologies such as plaque progression, destabilization and subsequent thrombus
formation, and restenosis and neointima formation.
106 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i095i
Crohn’s Disease but not Chronic Ulcerative Colitis
Induces the Expression of PAI-1 in Enteric Neurons
Laerum OD* 1,2 , Illemann M 1 , Skarstein A 3 , Helgeland L 2 , Øvrebø K 3 , Danø K 1 , Nielsen BS 1
1 The Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark;
2The Gade Institute, Section of Pathology, University of Bergen and Department of Pathology, Haukeland
University Hospital, Bergen, Norway;
3Department of Surgical Sciences, University of Bergen, and Department of Surgery, Haukeland
University Hospital, Bergen, Norway
Presenting author e-mail: ole.laerum@gades.uib.no
Chronic inflammation of the intestinal wall is the common characteristic of Crohn’s disease
and ulcerative colitis; disorders, which in some cases can be difficult to distinguish. The
inflammation also affects the local neuronal plexuses of the enteric nervous system. It is known
that plasminogen activator inhibitor-1 (PAI-1) and urokinase receptor (uPAR) are upregulated in
neurons after experimental peripheral nerve injury and have been linked to nerve regeneration.
The expression of PAI-1 and uPAR in neuronal cells in lesions of the gastrointestinal tract was
analyzed by immunohistochemical techniques. PAI-1 was found in a subset of neurons primarily
located in the submucosal plexus of the small and large intestine in 24 of 28 cases (86%) with
Crohn’s disease, but in none of 17 cases with chronic ulcerative colitis and not in normal colon
(n=5), acute appendicitis (n=9) or colon adenocarcinomas (n=17). The PAI-1 was seen in the
perikarya of the neurons and a few proximal axons, whereas nerves were negative. uPAR was
seen in nerves in all types of lesion varying from 21-88% of the cases, most frequent in colon
adenocarcinomas. No uPAR-positive nerves were detected in normal colon. PAI-1-positive
neurons in inflammatory bowel disease are linked to chronic inflammation in Crohn’s disease,
implying PAI-1 as a potential parameter for the differential diagnosis between Crohn’s disease
and ulcerative colitis. The findings also suggest that PAI-1 in neurons is related to pain and that
both PAI-1 and uPAR are involved in neuronal repair in the inflamed tissue.
Molecular & Cellular Biology of Plasminogen Activation 107

i096i
The Effect of Matrix Metalloprotease 3 Deficiency
in Spontaneous Metastasis
Juncker-Jensen A*, Rømer J, Almholt K
Finsenlaboratoriet, Rigshospitalet, København, Denmark
Presenting author e-mail: ajjensen@finsenlab.dk
Cancer metastasis is dependent on proteolytic degradation of extracellular matrix barriers,
making extracellular proteolysis an attractive target for anti-cancer therapy. The matrix
metalloprotease (MMP) family consists of 23 (human) MMPs that are involved in physiological
and pathological degradation of extracellular matrix. Several MMPs are expressed in human
breast cancer, and we have demonstrated that a number of MMPs are expressed in tumors in
the MMTV-PyMT transgenic breast cancer model in mice. MMP-3 (stromelysin 1) is expressed
in many parts of the stroma of the tumors, resembling the expression of MMP-3 in fibroblasts of
human breast cancers.
We have now studied the effect of MMP-3 deficiency in MMTV-PyMT mice, in a cohort of 63
mice consisting of 32 MMTV-PyMT,MMP3 wild-type mice and 31 MMTV-PyMT,MMP3 knockout
mice. Primary tumor growth was monitored by weekly measurements of the tumor volume
and lung metastasis was quantitated using unbiased, stereological methods. All mice were
sacrificed at age 87 +/– 3 days. There was no difference in tumor onset or final tumor burden
when comparing wild-type and MMP-3 deficient mice. The median lung metastasis volume was
increased from 0,04 mm3 in wild-type mice to 0,13 mm3 in MMP-3 deficient mice. Due to large
variations in the lung metastasis volumes, this increase is not significant (Mann-Whitney U test,
p=0,40).
We are currently analyzing lymph node metastasis in the same mice, and we are analyzing the
tumor activity of potential MMP-3 targets including the metastasis-associated MMP-2 and -9.
108 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i097i
Tumor-Cell Expression of C4.4A, a Structural Homologue
of the Urokinase Receptor, Correlates with Poor
Prognosis in Non-Small Cell Lung Cancer
Skov BG 1 , Hansen LV 2 , Ploug M 2 , Pappot H* 2
1 2 KAS Herlev, Department of Pathology, div. Gentofte, The Finsen Laboratory, Copenhagen University
Hospital, Copenhagen, Denmark
Presenting author e-mail: pappot@rh.regionh.dk
C4.4A is a metastasis associated protein. This glycolipid-anchored protein is a structural
homologue of the urokinase receptor, uPAR, which is a well-established prognostic marker in
various human cancers. Although, several pilot studies demonstrate C4.4A expression in various
human tumors, little is known about its prognostic significance. We therefore aim to explore
the possible association between C4.4A expression and clinicopathological features and disease
prognosis in patients with non-small cell lung cancer (NSCLC).
Tissue sections from 109 NSCLC patients were subjected to immunohistochemical staining using
a polyclonal antibody that specifically recognizes human C4.4A. Staining frequency and intensity
was scored semiquantitatively and grouped into cancers with high and low expression of C4.4A.
Kaplan-Meier survival curves were generated to evaluate the significance of C4.4A expression in
prognosis of NSCLC patients.
High C4.4A expression was observed in 44% of the specimens analyzed, and it correlates with
overall survival (P=0.012). Intriguingly, a very strong correlation was noted between high
expression of C4.4A in pulmonary adenocarcinoma and survival (P

i098i
Urokinase Receptor Splice Variant uPAR-del4/5 and
rab31 mRNA Expression in Breast Cancer
Magdolen V* 1 , Kotzsch M 2 , Sieuwerts A 3 , Grosser M 2 , Meye A 4 , Smid M 3 , Schmitt M 1 , Luther T 4 ,
Foekens JA 3
1 Institute of Pathology, Technical University Dresden, Germany;
2 Clinical Research Unit, Department Obstetrics and Gynecology, Technical University München,
Germany;
3 Department of Medical Oncology, Erasmus Medical Center Rotterdam, Netherlands;
4 Medizinisches Labor Ostsachsen, Bautzen, Germany
Presenting author e-mail: viktor.magdolen@lrz.tum.de
In the present study, we evaluated the impact of mRNA expression of the uPAR splice variant
uPAR-del4/5 (lacking exons 4 and 5) on metastasis-free survival (MFS) in 280 node-negative
breast cancer patients. Furthermore, microarray techniques were applied to identify differentially
expressed genes associated with high uPAR-del4/5 mRNA levels in tumor tissue and tumor cell
lines.
High uPAR-del4/5 mRNA values were strongly associated with shorter MFS of breast cancer
patients (P=0.001). In multivariate analysis, uPAR-del4/5 significantly contributed to the base
model of traditional prognostic factors for MFS (HR=3.29). By microarray analyses, the gene
encoding rab31, a member of the Ras oncogene superfamily, was identified as one of seven
genes to be more than 2-fold upregulated in tumor samples and cell lines with high uPARdel4/5
mRNA expression. rab31 was selected for analysis of mRNA expression in the same set
of 280 patients. High rab31 mRNA values were significantly associated with shorter DMFS in
multivariate analysis (HR=2.27, P

i099i
PN-1, a Serine Protease Inhibitor, Increases MMP-9 Activity
in Breast Cancer Cell Line
Fayard B* and Monard D
Friedrich Miesher Institute, Basel, Switzerland
Presenting author e-mail: berengere.fayard@fmi.ch
Protease Nexin-1 (PN-1) belongs to the serpin family (serine protease inhibitor), acting on a
broad range of protease including tPA, uPA, FactorXIa and thrombin. PN-1 is overexpressed in
many malignant tumors, its role in cancer remains however unknown. To study its involvement
in tumor cells and particularly in invasion, we used four cell lines isolated from the same mouse
mammary tumor named as 67NR, 168FARN, 4T07 and 4T1 but displaying different metastatic
properties once reinjected in the mouse mammary fat pad. Here we show that, in these cell lines,
the level of Matrix Metalloproteinase-9 (MMP-9) activity, known to be one of the key molecules in
invasion, correlates with PN-1 expression. Indeed, the less invasive cell line, 168FARN, presents
the lowest MMP-9 activity but also does not express PN-1. In contrast, the three other cell lines
showing high levels of MMP-9 activity express PN-1.
In 168FARN, addition of PN-1 leads to an increase of secreted MMP-9. This effect is mediated
through its binding to LRP (lipoprotein receptor-related protein) as indicated by the inhibitory
effect of a peptide known to antagonize binding of PN-1 to LRP and by assays performed in LRP–
/– MEF cells. PN-1 does not lead to an increase in MMP-9 mRNA but an increased intracellular
level of the metalloprotease is detected when secretion is blocked by Brefeldin.
Unexpectedly, PN-1 showed ability to increase MMP-9 activity in cancer cell lines by a mechanism
dependent of its binding to LRP receptor. These results provide evidence for a new function for
PN-1, which as protease inhibitor, regulates the levels of secreted MMP-9 proteinas a mean to
affect the cellular invasive behavior of tumor cells.
Molecular & Cellular Biology of Plasminogen Activation 111

i100i
Cleavage of uPAR: Mechanism and Prognostic Significance
Høyer-Hansen G*, Almasi CE, Pappot H
Finsen Laboratory, Copenhagen Biocenter, Copenhagen Denmark
Presenting author e-mail: gunilla@finsenlab.dk
On the cell surface uPA cleaves uPAR(I-III) in the linker region between domains I and II of uPAR.
Physiological concentrations of uPA cleave glycolipid-anchored uPAR but not soluble uPAR
(suPAR). This is due to a difference in the conformation of the linker region between domains I
and II. In media from PMA-stimulated U937 cells intact suPAR, but not cleaved, suPAR(II-III), is
present even though on the cell surface both uPAR(I-III) and uPAR(II-III) are present in similar
amounts. Treatment of the cells with Pi-PLC releases both uPAR forms from the cell surface.
Biotinylation of cell surface proteins verified the localization of both uPAR forms, whereas nonbiotinylated
uPAR(I-III) and uPAR(II-III) were found in the water-phase indicating intracellular
location. When uPA mediated uPAR cleavage was inhibited by incubation with a neutralizing
anti-uPA antibody, uPAR(II-III) was not found on the cell surface. The intracellular pattern of
uPAR(I-III) and uPAR(II-III) was not affected by inhibiting the cell surface cleavage. However, in
the media from cells grown in the presence of the neutralizing anti-uPA antibody, both suPAR(I-
III) and suPAR(II-III) were present. This indicates that uPAR(II-III) is shed from the cell surface
when uPA-mediated cleavage of uPAR is blocked.
The total amount of all uPAR forms in tumor lysates or blood correlates with prognosis in several
forms of cancer. However, the amounts of the cleaved forms may be directly related to the uPA
activity and in lung cancer uPAR(I) is an even stronger prognostic marker than the total amount
of all uPAR forms.
112 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i101i
Expression of Urokinase Receptor (uPAR) and
Plasminogen Activator Inhibitor-1 (PAI-1) in Human
Colon Cancer and their Matched Liver Metastases
Illemann M* 1 , Bird N 2 , Majeed A 2 , Laerum OD 1,3 , Lund LR 1 , Danø K 1 , Nielsen BS 1
1 The Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark;
2 Academic Surgical Unit, University of Sheffield, Sheffield, England;
3 The Gade Institute, Haukeland University Hospital, Bergen, Norway
Presenting author e-mail: millemann@finsenlab.dk
In primary colon adenocarcinomas, expression of uPAR and PAI-1 is focally upregulated in
the invasive front, primarily in recruited and activated inflammatory cells and myofibroblasts.
To compare the expression of uPAR and PAI-1 in primary colon adenocarcinomas with that
in colon cancer liver metastases, we have analyzed matched samples from 14 patients. In the
primary tumors, we found uPAR-immunoreactivity primarily in macrophages and PAI-1immunoreactivity
primarily in myofibroblasts located in the invasive front in agreement with
previous findings. The growth pattern of colon cancer liver metastases can be divided into
two major groups, those with desmoplastic encapsulation (desmoplastic growth pattern) and
those without (solid growth pattern). In the current material we had 8 and 6 cases, respectively.
In all liver metastases with desmoplastic and one with solid growth pattern we found uPARimmunoreactivity
primarily in macrophages and neutrophils located in the tumor edge,
whereas the remaining 5 cases showing solid growth had uPAR-immunoreactivity almost only
in neutrophils. PAI-1 was in cases with desmoplastic growth seen in myofibroblasts within
the desmoplastic capsule, while metastases with solid growth had only a few PAI-1 positive
myofibroblasts. Our findings suggest that metastasis-induced desmoplasia with recruitment of
macrophages upregulate the expression of uPAR and PAI-1 in a pattern similar to that in the
primary tumors. The metastases with solid growth, in contrast, do not induce uPAR expression in
the recruited macrophages and PAI-1 is limited to the sporadically appearing myofibroblasts.
Molecular & Cellular Biology of Plasminogen Activation 113

i102i
A Structural Basis for Differential Cell Signaling Initiated by
PAI-1 and PAI-2: Implications for Metastatic Potential
Croucher D* 1 , Saunders D 1 , Ranson M 2
1 Cancer Research Program, Garvan Institute of Medical Research, Sydney, Australia;
2 School of Biological Sciences, University of Wollongong, Wollongong, Australia
Presenting author e-mail: d.croucher@garvan.org.au
Tumour expression of the uPA inhibitor PAI-1 strongly correlates with poor patient prognosis.
However, tumour expression of the related uPA inhibitor PAI-2 generally correlates with good
patient prognosis. Currently, there are no adequate biochemical/functional data to explain this
discrepancy.
One theory for the tumour-promoting effects of PAI-1 is based on the signaling events initiated
upon PAI-1 inhibition of cell surface uPA. On MCF-7 cells, uPA binding to uPAR induces a
transient pulse of ERK activation. Inhibition by PAI-1 unveils a cryptic high affinity site within the
PAI-1 moiety that binds to members of the LDLR endocytosis receptor family. Binding of this high
affinity site in PAI-1 to VLDLr sustains ERK phosphorylation and initiates cell proliferation via an
as yet unknown mechanism.
Through biochemical analysis we have shown that unlike PAI-1, the PAI-2 moiety of uPA:PAI-2
does not contain a high affinity site for LDLR members, although the uPA:PAI-2 complex is still
efficiently endocytosed by these receptors. Therefore, upon inhibition of uPA, PAI-2 does not
induce the sustained ERK phosphorylation and proliferation associated with PAI-1 inhibition.
This differential binding and subsequent signal propagation is possibly due to an incomplete
LDLR binding motif within the helix-D of PAI-2, which is complete in the helix-D PAI-1.
These data present a possible mechanism by which PAI-2 is able to clear cell surface uPA, and
hence proteolytic activity, via VLDLr without initiating the cell signaling events and increased
metastatic potential associated with high PAI-1. This may partially explain why high tumour
levels of PAI-1 correlate with poor prognosis, whereas high tumour levels of PAI-2 correlate with
good prognosis.
114 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i103i
Thrombin Induces Tumor Invasion through the Induction
and Association of Matrix Metalloproteinase-9 and b-1 Integrin
on the Cell Surface
Bruno K* 1 , Radjabi R 2 , Sawada K 2 , Montag A 3,4 , Kossiakoff A 1 , Lengyel E 2
1 Biochemistry and Molecular Biology, 2 Departments of Obstetrics and Gynecology/Section of
Gynecologic Oncology, 3 Pathology, 4 Committee on Cancer Biology, University of Chicago, Chicago,
Illinois, USA
Presenting author e-mail: kbruno@uchicago.edu
The procoagulatory serine protease thrombin is known to induce invasion and metastasis in
various cancers. However, the mechanisms by which it promotes tumorigenesis are poorly
understood. Since the 92 kDa gelatinase (MMP-9) is a known mediator of tumor cell invasion,
we sought to determine if and how thrombin regulates MMP-9. The thrombin receptor,PAR-1, is
expressed in osteosarcomas, as determined by immunohistochemistry and RT-PCR. Stimulation
of U2-OS osteosarcoma cells with thrombin and a thrombin receptor activating peptide (TRAP)
induced pro-MMP-9 secretion as well as cell surface associated pro-MMP-9 expression and
proteolytic activity. This was paralleled by an increase in MMP-9 mRNA and MMP-9 promoter
activity. Thrombin induced invasion of U2-OS cells through matrigel could be inhibited with
a MMP-9 antibody and was mediated by the PI3-kinase signaling pathway. The stimulation
of MMP-9 by thrombin was paralleled by an increase in b1-integrin mRNA and b1-integrin
expression on the cell surface, which was also mediated by PI3-kinase and was also required for
invasion. Thrombin activation induced and co-localized both b1-integrin and pro-MMP-9 on the
cell membrane, as evidenced by coprecipitation and confocal microscopy. The thrombin mediated
association of these two proteins, as well as thrombin mediated invasion of U2-OS cells could be
blocked with a cyclic peptide and with an antibody preventing binding of the MMP-9 hemopexin
domain to the b1-integrin I-like domain. These results suggest that thrombin induces expression
and association of b1-integrin with MMP-9 and that the cell surface localization of the protease by
the integrin promotes tumor cell invasion.
Molecular & Cellular Biology of Plasminogen Activation 115

i104i
Overexpression of Protease Nexin-1 mRNA in
Oral Squamous Cell Carcinomas
Gao S 1 , Krogdahl A 2 , Sørensen JA 3 , Dabelsteen E 4 , Andreasen PA* 1
1 Department of Molecular Biology, University of Aarhus, Aarhus, Denmark;
2 Department of Pathology, Odense University Hospital, Odense, Denmark;
3 Department of Plastic Surgery, Odense University Hospital, Odense, Denmark;
4 School of Dentistry, University of Copenhagen, Copenhagen, Denmark
Presenting author e-mail: pa@mb.au.dk
Protease nexin-1 (PN-1) belongs to the serpin family of serine protease inhibitors. It is the
phylogenetically closest relative of plasminogen activator inhibitor-1 (PAI-1). PN-1 is less specific
than PAI-1, inhibiting, besides uPA, also plasmin and thrombin at physiologically relevant rates.
While there are numerous studies of the occurrence and functions of PAI-1 in cancer, a possible
tumour biological role of PN-1 has been almost totally neglected. We have now initiated studies
of the occurrence of PN-1 in tumours, also with the presumption that investigations of the tumour
biological functions of PN-1 may provide clues to the uncertainty about the molecular and
cellular mechanisms underlying the correlation between a high PAI-1 level in tumours and a poor
prognosis for the patient. We compared the level of PN-1 mRNA in 20 cases of oral squamous cell
carcinomas and in matched samples of the corresponding normal oral tissues. We found that the
average PN-1 levels in tumours and normal tissues were significantly different, being increased
up to 13 fold in tumour samples compared with the average level in normal tissues. The PN-1
mRNA level was significantly higher in tumours from patients with lymph node metastasis than
in tumours from patients without. We could conclude that PN-1 is frequently overexpressed in
oral squamous cell carcinoma and may correlate with lymph node metastasis. Development of an
anti-PN-1 antibody suitable for immunohistochemical identification of PN-1 expressing cell types
in different carcinoma types may help us to understand its tumour biological functions.
116 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i105i
The Matrix Metalloprotease (MMP) Inhibitor Galardin Increases
Collagen Deposition and Reduces Spontaneous Metastasis
in the MMTV-PymT Transgenic Breast Cancer Model
Almholt K* 1 , Lærum OD 2 , Lund LR 1 , Danø K 1 , Johnsen M 3 , Rømer J 1
1 Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark;
2 The Gade Institute, Section of Pathology, Haukeland University Hospital, Bergen, Norway;
3 Department of Molecular Biology, University of Copenhagen, Copenhagen, Denmark
Presenting author e-mail: kasper@finsenlab.dk
Matrix metalloproteases (MMPs) and other extracellular proteases are recognized as critical
factors in cancer progression. MMP inhibitors (MMPIs) have proven effective primarily by
reducing growth of xenograft tumors in mice. However, small molecule MMPIs have not been
tested in any transgenic/spontaneous tumor model. We chose the MMTV-PymT transgenic breast
cancer model to analyze the effect of galardin/GM6001, a potent MMPI that reacts with most
MMPs. Prior to the experiment on cancer-bearing mice we determined a treatment regimen based
on the ability to inhibit skin wound healing in mice. We then followed a cohort of 54 MMTV-
PymT-transgenic mice that received either galardin or placebo. Treatment was started at age 6
weeks with galardin at 100 mg/kg/day in the form of s.c. implanted slow-release tablets. The
growth of the primary tumors was reduced but not severely retarded by galardin treatment. All
mice were killed at age 13½ weeks, at which time the average primary tumor burden was reduced
to 1.69 cm3 in galardin-treated mice compared to 3.29 cm3 in control mice (t-test, p=0.0014). It is
likely that some MMPs promote while others inhibit tumor growth, and the net effect of inhibiting
a whole range of MMPs was not sufficient to prevent tumor growth. However, the tumors
from galardin-treated mice often had a lower tumor grade as determined by histopathological
evaluation. The tumors from galardin-treated mice also showed a much higher degree of collagen
deposition compared to tumors from placebo-treated mice. We then quantified the total lung
metastases volume in the same mice. The median metastasis volume was reduced to 0.003 mm3 in
galardin-treated mice compared to 0.56 mm3 in control mice (t-test, p

i106i
Proteomics of uPAR Protein: Protein Interactions in Cancer Metastasis
Saldanha R, Molloy M, Xu N, Baker MS*
Australian Proteome Analysis Facility Ltd and CORE in Biomolecular Frontiers, Macquarie University,
Sydney, Australia
Presenting author e-mail: mbaker@proteome.org.au
uPAR is a key player in cancer metastasis. Our past studies show that when metastatic HCT116
cells express a 5’ uPAR antisense cDNA fragment this results in (i) reduced uPAR cell surface
expression; (ii) reduced cell-surface uPA binding, (iii) reduced adhesion and plasminogendependant
matrix degradation, (iv) reduced Erk MAP kinase activity, Src kinase activity and
MMP-9 secretion; (v)reduced metastasis in Nu/Nu mice and (vi) new data that shows statistically
significant expression/phosphorylation changes in only a small percentage (~4%) of the ~850
proteins reproducibly detected by 2DE (e.g., stathmin, C-Myc binding protein, translation
initiation factor 3, tropomyosin 3, histone H2B type 12, profilin-2, importin, HSP90 to name but
a few). To further characterize how uPAR operates we have now identified (and are quantifying
using IP-iTRAQ) uPAR protein binding complexes from malignant OVCA 429 cells. Proteins
that specifically co-purified with anti-uPAR mAbs were validated using orthogonal approaches
(e.g., cross-over IP Westerns). Of particular interest were a range of signal transduction proteins,
proteins previously associated with plasminogen activation (phospholipase C, LRP, and annexin
A2, enolase, thrombospondin) and some novel proteins (TGF-bR2 and only the integrin, av-b6
(although these cells express other integrins). Physical sites of interaction between uPAR and avb6
were definitively identified by peptide mapping strategies as was direct interaction of TGF-bR2
with uPAR. Functional consequences of uPAR interactions are being studied and preliminary data
show that uPAR mediates many biological effects through protein:protein interactions.
118 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i107i
A New Tagging System for Production of Recombinant Proteins in
Drosophila S2 Cells Using the Third Domain of the Urokinase Receptor
Gårdsvoll H* 1 , Hansen LV 1 , Jørgensen TJD 2 , Ploug M 1
1 Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark;
2 Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense,
Denmark
Presenting author e-mail: gvoll@finsenlab.dk
The use of protein fusion tag technology greatly facilitates detection, expression and purification
of recombinant proteins, and the demands for new and more effective systems are therefore
expanding. We have used a soluble truncated form of the third domain of the urokinase
receptor as a convenient C-terminal fusion partner for various recombinant extracellular human
proteins used in basic cancer research. The stability of this cystein-rich domain, which structure
adopts a three-finger fold, provides an important asset for its applicability as a fusion tag for
expression of recombinant proteins. Up to 20 mg of intact fusion protein were expressed by
stably transfected Drosophila S2 cells per liter of culture using this strategy. Purification of these
secreted fusion proteins from the conditioned serum free medium of S2 cells was accompanied
by an efficient one-step immunoaffinity chromatography procedure using the immobilized
anti-uPAR monoclonal antibody R2. An optional enterokinase cleavage site is included between
the various recombinant proteins and the linker region of the tag, which enables generation of
highly pure preparations of tag-free recombinant proteins. Using this system we successfully
produced soluble and intact recombinant forms of extracellular proteins such as CD59, C4.4A and
vitronectin, as well as a number of truncated domain constructs of these proteins. In conclusion,
the present tagging system offers a convenient general method for the robust expression and
efficient purification of a variety of recombinant proteins.
Molecular & Cellular Biology of Plasminogen Activation 119

i108i
Photoaffinity Labeling of uPAR with Cyclic Peptides
Jacobsen B* 1 , Gårdsvoll H 1 , Barkholt V 2 , Østergaard S 3 , Ploug M 1
1 Finsen Laboratory, Rigshospitalet, Copenhagen Biocenter, Denmark;
2 Enzyme and Protein Chemistry, BioCentrum-DTU, The Technical University of Denmark, Denmark;
3 Novo Nordisk A/S, Novo Research Park, Denmark
Presenting author e-mail: bjacobsen@finsenlab.dk
In view of its involvement in cancer invasion and metastasis, the high-affinity uPA-uPAR
interaction represents an attractive target for rational drug design, thus requiring detailed
knowledge of the receptor-ligand interface. In this study, we use photoaffinity labeling as a tool
for delineation of structural aspects of this interaction. The technique enables the covalent crosslinking
of two molecules by excitation of a photoactivatable probe, which thereby can abstract a
hydrogen atom from a C-H-bond in its vicinity. This has been used successfully to identify the
uPAR-binding sites of linear phage-display peptides. We have now synthesized a range of cyclic
peptides mimicking the long b-hairpin of the growth factor-like domain of uPA, which includes
residues essential for its binding to uPAR (Tyr24, Phe25, Ile28 and Trp30). In the peptides AE03
and AE04, the photoprobe p-benzoyl-L-phenylalanine has been inserted at positions equivalent to
Trp30 and Tyr24 in uPA, respectively, and biotin has been included as a detection and purification
tag. Photolysis of uPAR-peptide complexes causes a covalent incorporation of the cyclic peptides
into uPAR. The specificity of this insertion reaction was certified by the inhibition obtained when
preincubating with receptor-binding derivatives of uPA. By cleavage with chymotrypsin and
western blotting with streptavidin, AE03 is shown to incorporate into uPAR domain I, while AE04
is domains II + III-specific. These results are supported by the recently solved crystal structure
of uPAR in complex with the amino-terminal fragment of uPA, revealing a hydrophobic ligandbinding
cavity formed by the assembly of all three domains of uPAR.
120 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i109i
In Vivo Inhibition of the Murine uPA-uPAR Interaction using
Monoclonal Antibodies Raised in uPAR Deficient Mice
Rasch MG*, Pass J, Jögi A, Rønø B, Gårdsvoll H, Lund LR, Høyer-Hansen G, Lund IK
Finsen Laboratory, Copenhagen Biocenter, Copenhagen, Denmark
Presenting author e-mail: mrasch@finsenlab.dk
The interaction between uPA and uPAR is a potential target for anti-invasive cancer therapy.
Cancer invasion and metastasis is dependent on the interaction between cancer and stromal cells,
which can be studied in genetically induced murine cancer models. To enable in vivo studies of
the effect on inhibition of uPA-uPAR binding in genetically induced murine cancer models, we
have generated murine mAbs against murine uPAR (muPAR) by immunizing uPAR–/– mice with
recombinant muPAR.
We have selected five mAbs (mR1-mR5) of which mR1, mR3, and mR5 recognize epitopes in
domain I of muPAR, while mR2 and mR4 recognize epitopes in domain II-III of muPAR. In cell
binding experiments mR1 and mR4 antagonized the binding of 125I-mATF with IC50 values of
0.67 nM for mR1 and 0.40 nM for mR4 compared to 0.14 nM for unlabeled mATF. Additionally,
mR1 could rescue 50% P388D.1 cells in an anthrax-toxin based assay, which requires cell-bound
uPA to kill the cells. In contrast to all previously generated uPAR specific mAbs, mR3 binds
domain I without interfering with uPA binding.
In vivo efficacy of mR1 was demonstrated by the ability of mR1 to rescue mice treated with a lethal
dose of uPA-activatable anthrax toxins. Another in vivo approach showed that mR1 treated
tPA–/– mice mimicked the hepatic fibrin deposits of uPAR–/–/tPA–/– mice. Thus, mR1 is
an efficient in vivo antagonist of the uPA-uPAR interaction and well suited for therapeutic
intervention.
Molecular & Cellular Biology of Plasminogen Activation 121

i110i
RNA Interference for Urokinase-Targeting Limits Growth
of Hepatocellular Carcinoma Xenografts in Nude Mice
Salvi A*, Arici B, Barlati S, De Petro G
Division of Biology and Genetics, Department of Biomedical Sciences and Biotechnology, IDET Centre
of Excellence, University of Brescia, Brescia, Italy
Presenting author e-mail: asalvi@med.unibs.it
Gene expression of urokinase-type plasminogen activator (u-PA) is up-regulated in human
hepatocellular carcinoma (HCC) and the levels of u-PA mRNA are inversely related to HCC
patients’ survival. The purpose of this study was to examine the effects of vector-based RNA
interference (RNAi) of u-PA on the growth of human HCC xenografts in nude mice in order to
investigate the u-PA role in human HCC.
Our results showed that the s.c. injection of siRNA u-PA SKHep1C3 stable transfected cells
(pSsiRNAu-PA) led to a growth delay in xenograft development. The molecular characterization
of nodules (carried out by PCR, RT-PCR/Agilent technology and immunoistochemical analysis)
revealed the presence of plasmid DNA, the u-PA gene expression knock-down, at both mRNA
and protein levels, giving evidences of a long-term and target-specific inhibition by vectorbased
RNAi 11 weeks after cell inoculation. Furthermore the immunohistochemical and
immunofluorescence evaluation of fibronectin (FN) expression and organization revealed FN
fibrils in pSsiRNAu-PA xenografts and in pSsiRNA u-PA-cells. These results represent the first
experimental approach to inhibit u-PA in HCC xenografts using an ablative strategy, supporting
the notion that u-PA is involved in HCC development, with a major role for u-PA in the initial
phases of HCC growth; providing evidence that u-PA siRNA construct can be maintained in each
xenograft for up to 11 weeks of tumour development; also identifying the ability to organize FN
fibrils by SKHep1C3 cells as a downstream effect of u-PA knock-down. These data could open
the possibility for experimental HCC gene therapy by vector-based RNAi against u-PA in animal
models.
122 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i111i
Potent and Broad Anti-tumor Activity of an Engineered
Matrix Metalloproteinase-activated Anthrax Lethal
Toxin that Targets Tumor Vasculature
Liu S 1 , Wang H 1 , Currie BM 2 , Molinolo A 2 , Leung HJ 1 , Moayeri M 1 , Alfano RW 3 , Frankel AE 3 ,
Leppla SH 1 , Bugge TH* 2
1 Laboratory of Bacterial Diseases, NIAID, NIH, Bethesda, Maryland, USA;
2 Oral and Pharyngeal Cancer Branch, NIDCR, NIH, Bethesda, Maryland, USA;
3 Cancer Research Institute of Scott and White Memorial Hospital, Temple, Texas, USA
Presenting author e-mail: thomas.bugge@nih.gov
Anthrax lethal toxin (LT), which specifically inactivates MEKs, can treat human melanomas with
BRAF V600E mutations, because of their addiction to the MEK-ERK pathway. To more selectively
target tumors, we have developed a matrix-metalloproteinase (MMP)-activated anthrax LT, which
specifically targets tumors overexpressing MMPs. Surprisingly, the MMP-activated LT has potent
anti-tumor activity against a wide range of other tumor types, regardless of their BRAF mutation
status. Moreover, the engineered toxin could be curative to established malignant solid tumors
when used systemically, despite having very low tissue toxicity. This is largely due to the indirect
targeting of tumor angiogenesis. Moreover, the engineered toxin not only exhibits much lower
toxicity than wild-type LT to mice, but also shows higher toxicity to tumors because of its greater
bioavailability. Thus, in theory, all tumor types are expected to respond to the MMP-activated LT
therapy, and patients with tumors containing the BRAF mutation may derive additional benefits
due to the direct toxicity of the toxin to the cancer cells. We are currently preparing a clinical trial
to test the MMP-activated LT in cancer patients.
Molecular & Cellular Biology of Plasminogen Activation 123

i112i
Elucidation of the Epitope of MA-31C9, a Non-inhibitory
Anti-human PAI-1 Antibody
Meissenheimer LM*, Dewilde M, Compernolle G, Declerck PJ, Gils A
Laboratory for Pharmaceutical Biology, Katholieke Universiteit Leuven, Leuven, Belgium
Presenting author e-mail: lester.meissenheimer@pharm.kuleuven.be
Plasminogen Activator Inhibitor 1 (PAI-1) is the most important physiological inhibitor of
plasminogen activators in vivo. MA-31C9 is a non-inhibitory monoclonal antibody raised towards
human PAI-1, which is often used as a control antibody in studies on the evaluation of PAI-1
inhibitory antibodies. The goal of this study was to elucidate the epitope of MA-31C9.
Wild-type human PAI-1 has a high affinity for MA-31C9 (i.e. KA=6.8±1.9x109M-1 using surface
plasmon resonance (SPR)) whereas rat PAI-1 does not bind to MA-31C9 (KA

i113i
Residues outside the Epitope Determine the Function of
MA-159M12, an Inhibitory Anti-rat PAI-1 Antibody
Meissenheimer LM*, Compernolle G, Declerck PJ, Gils A
Laboratory for Pharmaceutical Biology, Katholieke Universiteit Leuven, Leuven, Belgium
Presenting author e-mail: lester.meissenheimer@pharm.kuleuven.be
Rat models have been shown to be suitable for in vivo investigations on thrombolysis and
fibrinolysis. MA-159M12 is a monoclonal antibody raised towards rat PAI-1 that exerts an
inhibitory effect by accelerating the active to latent conversion. The goal of this study was to study
the mechanism of action of MA-159M12.
MA-159M12 has a high affinity for rat PAI-1 (KA=3.4±1.1x109M-1) but does not bind to human
PAI-1 (KA

i114i
Conformational Probes and Activity Regulators of Plasminogen
Activator Inhibitor-1, Isolated from Phage-displayed
Disulphide Bridge-constrained Peptide Libraries
Dupont DM, Jensen JK, Mathiasen L, Blouse GE, Wind T, Andreasen PA*
Department of Molecular Biology, University of Aarhus, Aarhus, Denmark
Presenting author e-mail: pa@mb.au.dk
To find new probes and inhibitors of PAI-1s molecular interactions and conformational
changes, we screened a phage-displayed peptide library containing 109 different sequences
with the formats X7, CX7C, CX10C, and CX3CX3CX3C with human PAI-1 as bait. We isolated
phages harbouring three families of peptides. One family of peptides had a consensus core
motif of CFGaC, referred to a paionin-1, in which a is an aromatic residue. As determined by
site-directed mutagenesis, paionin-1 bound to in a hydrophobic pocket under a-helix D and
inhibited the binding of uPA-PAI-1 complex to the endocytosis receptors LRP and VLDLR. A
second family of peptide sequences with the formats CX7C and CX10C and the common core
sequence WPRY bound to the area of PAI-1 becoming exposed upon detachment of b-strand 1C
during latency transition. The access of paionin-3 to its binding site was blocked by the glycans
attached to Asn267 in PAI-1 expressed in mammalian cells. A third peptide was of the format
CX3CX3CX3CX5CX3CX3CX3C, will be referred to as paionin-4, and probably resulted from
an unplanned cloning artefact in the library. Paionin-4 accelerated the rate of conversion of
PAI-1 to the latent state and had a binding area on PAI-1 overlapping with that of the latencyinducing
monoclonal antibody MA33B8. Binding analyses showed that paionin-4 has a stronger
affinity to conformational forms of PAI-1 with an inserted RCL than to active PAI-1. Thus, some
of the isolated peptides are conformational probes, while others represent novel approaches to
pharmacological interference with pathophysiological functions of PAI-1.
126 X I t h I n t e r n a t i o n a l W o r k s h o p o n

i115i
Urokinase-type Plasminogen Activator-inhibiting Cyclic Peptides
Demonstrate New Modalities for Inhibition of Serine Proteases
Andersen LM* 1 , Wind T 1 , Hansen HD 1 , Blouse GE 1 , Christensen A 1 , Jensen JK 1 , Malmendal A 2 ,
Nielsen NC 2 , Andreasen PA 1
1 Department of Molecular Biology, 2 Department of Chemistry, University of Aarhus, Aarhus, Denmark
Presenting author e-mail: lma@mb.au.dk
In order to find new principles for inhibition of the enzymatic activity of urokinase-type
plasminogen activator (uPA), we screened phage-displayed random, disulphide bridgeconstrained
peptide repertoires with human and murine uPA as baits. With human uPA, the most
frequently isolated sequence was CSWRGLENHRMC, referred to as upain-1. With murine uPA,
the most frequent isolated sequence was CPAYSRYLDC, referred to as mupain-1. The selected
peptide sequences inhibited human or murine uPA competitively, with Ki values around 500 nM.
The Ki values were in excellent agreement with the KD values determined by BIACORE analysis.
By an inhibitory screen against other murine and human serine proteases, including trypsin, both
upain-1 and mupain-1 were found to be highly selective for human and murine uPA, respectively.
However, upain-1 did not measurably inhibit murine uPA and mupain-1 did not measurably
inhibit human uPA. Site-directed mutagenesis of the peptides as well as of the enzymes identified
Arg4 of upain-1 and Arg6 of mupain-1 as the P1 residues and indicated that binding specificity
depends on extended binding interactions involving specific surface loops of the enzymes and
several residues of the peptides. Comparison of the solution structure of upain-1, as determined
by NMR, and the results of the site-directed mutagenesis indicated that conformational changes of
the peptide are a prerequisite for upain-1-uPA binding. Peptide-derived inhibitors such as upain-
1 and mupain-1 provide novel mechanistic information about enzyme-inhibitor and enzymesubstrate
interactions, important tools for dissecting structural determinants of substrate and
inhibitor recognition, and alternative methodologies for designing effective protease inhibitors.
Molecular & Cellular Biology of Plasminogen Activation 127

i116i
In vivo Treatment with Monoclonal Antibodies against Mouse
Urokinase-type Plasminogen Activator in Cancer Models
Jögi A*, Lund IK, Høyer-Hansen G, Lund LR, Danø K, Rømer J
Finsen Laboratory, Rigshospitalet, Copenhagen Biocenter, København, Denmark
Presenting author e-mail: ajogi@finsenlab.dk
Degradation of extra cellular matrix is pivotal to tumor metastasis and invasive growth and
plasmin has a well-documented role in both processes. In a transgenic breast cancer model uPAdeficiency
was shown to confer reduced frequency of metastasis (Almholt et al (2005) Int J Cancer).
Inhibition of the activity of uPA is hence a potential approach to anti-invasive cancer therapy. In
order to evaluate this strategy preclinically in mice, reagents are needed that inhibit the activity of
murine uPA. This protein and its associated factors is highly species specific and although several
strong inhibitors of human uPA activity are known, none of these are efficient inhibitors in the
mouse. In order to develop such inhibitors, we have recently, by immunization of uPA-deficient
mice with mouse uPA, generated anti-catalytic monoclonal antibodies (mAbs) against murine
uPA. Here we report the first biological effects from systemic administration of such an inhibitory
anti-mouse uPA mAb. In a wound healing model, where migrating leading edge keratinocytes
model the invasive tumor cells, i.p. injections of anti-uPA mAb in tPA-deficient mice shifted the
healing time toward that seen in tPA;uPA double-deficient mice. Increasing dose of the mAb led
to increasingly delayed healing time in a dose dependent manner, approaching that of the genedeficient
mice. Immunoblotting of protein extracts from the wounds reveal that less plasmin is
generated in wounds of anti-uPA treated mice, compared to none-treated or mock treated mice.
We are currently testing this mAb in a transgenic breast cancer model.
128 X I t h I n t e r n a t i o n a l W o r k s h o p o n

Author Index
(The authors present at the meeting are indicated in bold characters.)
Aguilar, S, 073
Ahlskog, N, 093
Alfano, D, 041, 087, 111
Alfano, RW, 111
Allen, BJ, 072
Almasi, CE, 100
Almholt, K, 032, 096, 105
Ampurdanés, C, 073
Andersen, LM, 115
Andolfo, A, 003, 051, 052
Andreasen, PA, 004, 039, 048, 049,
050, 053, 054, 071, 104, 114, 115
Andreé, J, 010
Antalis, TM, 079
Ardite, E, 040
Arici, B, 110
Arnaudova, R, 068
Astrup, A, 060
Bae Kim, G, 046
Baker, MS, 105
Balsara, RD, 028, 086
Barkholt, V, 108
Barlati, S, 110
Battey, F, 008
Beaufort, N, 037
Bednarek, R, 075
Behrendt, N, 033, 034, 047
Belorgey, D, 023, 091
Betsholtz, C, 010
Bian, C, 048
Bifulco, K, 062
Binder, BR, 031, 044, 061, 063
Bird, N, 101
Blasi, F, 035, 068
Blomstrand, F, 067
Blouse, GE, 004, 049, 053, 054, 114,
115
Bødker, JS, 053
Boncela, J, 075
Borger Rasmussen, H, 078
Bøtkjær, KA, 049
Brellier, F, 018
Breuss, JM, 044
Brotzge, XH, 006
Brumwell, A, 013
Brunner, PM, 031, 063
Brünner, N, 027, 060, 084
Bruno, K, 103
Bugge, TH, 024, 025, 032, 033, 034,
046, 078, 079, 111
Byszuk, O, 049
Cale, J, 010
Cantelmo, AR, 062
Caputi, M, 074
Carlsson, L, 083
Carriero, MV, 026, 041, 062
Carroll, VA, 044
Casslén, B, 070
Castellino, FJ, 028, 038, 086
Chapman, HA, 013, 042
Chen, J, 045
Christensen, A, 115
Church, S, 009
Cierniewski, CS, 075, 076
Cochran, BJ, 059
Cohen-Gould, L, 009
Compernolle, G, 112, 113
Corominas, JM, 073
Croucher, D, 058, 059, 080, 102
Cunningham, O, 003
Currie, B, 025, 079, 111
Dabelsteen, E, 071, 104
Dahl, L, 083
D’Alessio, S, 035
Danø, K, 043, 095, 101, 105, 116
De Petro, G, 110
Declerck, PJ, 112, 113
Degryse, B, 068, 069
Del Rosso, M, 065
Demyanets, S, 094
Deora, AB, 009
Deryugina, EI, 049
Devy, L, 045
Dewilde, M, 112
Di Carluccio, G, 062
Diaz-Ramos, A, 082
Dransfield, DT, 045
Dumler, I, 085, 090
Dunoyer-Geindre, S, 012
Dupont, DM, 050, 053, 054, 114
Eden, G, 068
Edwards, DR, 030
Ehart, M, 061
Ehnman, M, 015
Ellis, V, 030
Engelholm, LH, 033, 034
Eriksson, U, 010, 015
Espitia, C, 092
Estrada, Y, 041
Fayard, B, 099
Fibbi, G, 065
Fischer, C, 020
Fish, RJ, 012
Flint, A, 060
Flugel, D, 011
Foekens, JA, 098
Folestad, E, 010
Franco, P, 041, 074
Frankel, AE, 111
Fredriksson, L, 010, 015
Fu, Q, 038
Furlan, F, 068
Gao, S, 071, 104
Gao, Y, 010
Gårdsvoll, H, 002, 034, 047, 054,
107, 108, 109
Gavard, J, 033
Gerasi, L, 035
Geyer, M, 010
Gils, A, 112, 113
Godiksen, S, 078
González, B, 040
Green, KA, 032
Grosser, M, 098
Gueler, F, 090
Guenther, A, 039
Guo, Y, 036, 093
Gutkind, JS, 033
Hägglöf, P, 023, 091
Hagström, E, 036
Hajjar, KA, 009
Haller, H, 085, 090
Hansen, HD, 115
Hansen, LV, 097, 107
Hansson, G, 070
Harslund, J, 084
Heier, PC, 063
Helgeland, L, 095
Henderikx, P, 045
Henic, E, 070
Herry, C, 020
Higgins, PJ, 016
Hill, M, 013, 042
Hillig, T, 034
Holmbeck, K, 033
Høyer-Hansen, G, 034, 047, 070,
100, 109, 116
Molecular & Cellular Biology of Plasminogen Activation 129

Huang, M, 048
Huber, K, 094
Hultman, K, 067
Iaccarino, I, 087
Ibba-Manneschi, L, 065
Ihara, H, 007
Illemann, M, 043, 095, 101
Ingvarsen, S, 034
Jackson, C, 072
Jacobsen, B, 108
Jardí, M, 040, 089
Jensen, JK, 004, 114, 115
Jensen, L, 060
Jerczynska, H, 076
Jern, C, 067
Jögi, A, 047, 109, 116
Johnsen, M, 105
Jørgensen, TJD, 107
Juncker-Jensen, A, 096
Kamikubo, Y, 069
Kanse, SM, 039
Karlsson-Li, S, 023
Kaun, C, 094
Kietzmann, T, 011
Kim, Y, 013, 042
Kirsch, T, 090
Kiyan, J, 085
Kjelgaard, S, 049
Kjems, J, 050
Kjøller, L, 034
Kojima, S, 064
Koschelnick, Y, 031
Kossiakoff, A, 103
Kotzsch, M, 098
Kreek, MJ, 022
Kristjansen, PEG, 046
Krogdahl, A, 071, 104
Krog-Mikkelsen, I, 060
Kruithof, EKO, 012
Kugler, MC, 013
Kui, L, 081
Lademann, U, 027
Ladner, RC, 045
Laerum, OD, 095, 101, 105
Larsen, L, 027
Larsson, G, 029
Lawrence, DA, 010, 056
Lee, JA, 080
Lengyel, E, 103
Leppla, SH, 046, 111
Leung, HJ, 111
Ley, A, 045
Li, H, 015
Li, J, 036, 088
Li, J, 017
Li, S, 056
Li, X, 001
Liguori, E, 062
Lillis, AP, 008
Ling, Q, 009
List, K, 024, 025, 079
Liu, S, 046, 111
Llorens, A, 082
Lobov, S, 058, 059
Lochner, JE, 021
Lomas, DA, 023, 091
Longanesi-Cattani, I, 062
López-Alemany, R, 082
Lu, W, 001
Lund, IK, 047, 109, 116
Lund, LR, 032, 043, 047,
101,105,109,116
Luque, T, 082
Luther, T, 098
Lüthi, A, 020
Lyden, D, 009
Mackie, I, 045
Madsen, CD, 003, 051, 066
Madsen, JB, 050
Madsen, DH, 034
Magdolen, V, 037, 098
Maiya, R, 022
Majeed, A, 101
Malisauskas, M, 077
Mallya, M, 091
Malmendal, A, 115
Manchanda, N, 055
Mancini, A, 074
Manetti, M, 065
Mann, K, 010
Margheri, F, 065
Markart, P, 039
Mathiasen, L, 114
Maurer, G, 094
Mazzieri, R, 035
Medcalf, R, 067
Meins, M, 020
Meissenheimer, LM, 112, 113
Mengel, M, 090
Meye, A, 098
Meyer, CA, 086
Michos, O, 018
Migliorini, M, 008
Mihaly, J, 031, 044, 063
Mikhailenko, I, 008
Minor, KH, 004, 054
Moayeri, M, 111
Mogami, H, 007
Molinolo, A, 024, 025, 079, 111
Molloy, M, 105
Monard, D, 018, 020, 099
Montag, A, 103
Montuori, N, 026
Moreno, E, 018, 020
Morin, SJ, 086
Morozova-Roche, L, 077
Mortensen, KK, 049
Morty, RE, 039
Muñoz-Cánoves, P, 040, 089
Naa, L, 045
Navarro, P, 073
Neels, JG, 069
Netti, PA, 041
Netzel-Arnett, S, 079
Neville, DM, 046
Nielsen, BS, 043, 095, 101
Nielsen, NC, 115
Nielsen, OL, 084
Nieves-Li, EC, 055
Nilsson, M, 067
Norris, EH, 022
Noskova, V, 070
Ny, T, 036, 077, 081, 088, 091, 093
Nylander, A, 081
Offenberg, H, 020, 084
Olausson, B, 029, 083
Olofsson, A, 077
Orolicki, S, 018
Ortiz-Zapater, E, 073
Ossowski, L, 041
Østergaard, S, 108
Øvrebø, K, 095
Owen, K, 030
Pabba, M, 077
Pappot, H, 097, 100
Park, J-K, 090
Pass, J, 109
Paul, J, 019
Pawlowska, Z, 076
Pedersen, EDK, 078
Peiró, S, 073
Perron, MJ, 054
Peterson, CB, 004, 054
Pfaffenberger, S, 094
Phillips, RL, 091
Pietras, K, 010
Pirazzoli, V, 051
Pizzo, SV, 008
Pliyev, BK, 014
Ploplis, VA, 028,086
Ploug, M, 002, 032, 047, 054, 097,
107, 108
Polavarapu, R, 006
Potempa, J, 037
Prager, GW, 031, 044, 063
130 X I t h I n t e r n a t i o n a l W o r k s h o p o n

Preissner, KT, 039
Priglinger, U, 063
Prorok, M, 038
Przygodzka, P, 029, 083
Przygodzki, T, 077, 091
Pucci, M, 065
Qiu, D, 030
Quigley, JP, 049
Rabbani, SA, 045
Raben, A, 060
Radjabi, R, 103
Rafii, S, 009
Ragno, P, 026
Rank, F, 043
Ranson, M, 038, 058, 059, 080, 102
Rasch, MG, 109
Real, FX, 073
Rega, G, 094
Ricci, P, 026
Robillard, L, 013
Robinson, S, 008
Roda, O, 073
Rømer, J, 032, 046, 047, 096, 105, 116
Rømer, MU, 027
Rong, S, 090
Rønø, B, 046, 109
Rosales, L, 092
Rossi, G, 026
Rotoli, B, 026
Ruan, J, 009
Ruiz, BH, 092
Ruskowski, M, 045
Rychli, K, 094
Saldanha, R, 105
Salvi, A, 110
Samarakoon, R, 016
Sanderson-Smith, ML, 038
Sarra Ferraris, GM, 003, 066
Saunders, D, 102
Sawada, K, 103
Scalettar, BA, 021
Schar, CR, 004, 054
Scharschmidt, T, 025
Schmitt, M, 037, 098
Schuttner, LC, 021
Schwartz, BS, 056
Segre, J, 025
Selleri, C, 026
Selzer-Plon, J, 078
Serrano, AL, 089
Serrati, S, 065
Sharp, LK, 023
Shireman, J, 025
Shore, JD, 054
Shushakova, N, 085, 090
Sidenius, N, 003, 051, 052, 066
Sidman, RL, 017
Sieuwerts, A, 098
Skarstein, A, 095
Skov, BG, 097
Sloth, B, 060
Smid, M, 098
Smith, RC, 072
Smolarczyk, K, 075
Snyder, EY, 017
Sommerhoff, CP, 037
Song, E, 072
Sørensen, JA, 071, 104
Spangler, E, 021
Spina, R, 062
Stochl, M, 045
Stoppelli, MP, 041, 062, 074, 087
Strickland, DK, 008, 010
Strickland, S, 019, 022
Su, EJ, 010
Suelves, M, 089
Sui, G-Z, 009
Sulniute, R, 088
Suzuki, Y, 007
Szabo, R, 024, 025, 079
Taieb, S, 018
Tang, CH, 013, 042
Té, H, 018
Tengel, T, 029
Tholstrup, T, 060
Tjärnlund-Wolf, A, 067
Tkachuk, N, 085
Tkachuk, S, 085
Tkachuk, VA, 014
Toews, M, 045
Urano, T, 007
Vaillant, C, 018
Vallecillo, AJ, 092
van Gool, R, 045
Vidal, B, 040, 089
Visconte, V, 026
Vocca, I, 041
Vogel, LK, 078
Votta, G, 041, 074
Wagenaar-Miller, RA, 033, 034
Wahlberg, P, 081
Waisman, DM, 057
Walker, MJ, 038
Wang, H, 111
Wei, Y, 013, 042
Wei, Z, 005
Wilczynska, M, 029, 077, 083
Wind, T, 039, 048, 114, 115
Wojciechowski, P, 037
Wojta, J, 094
Wygrecka, M, 039
Xolalpa, W, 092
Xu, N, 105
Xue, A, 072
Xue, M, 072
Yamada, S, 033
Yan, L, 009
Yepes, M, 006, 010
Yuan, C, 048
Yuan, W, 001
Zeller, R, 018
Zhao, G, 048
Zhou, A, 005
Zhou, Y, 022
Zou, G, 001
Molecular & Cellular Biology of Plasminogen Activation 131

Attendees
Ahlskog, Nina
Umeå University
nina.ahlskog@medchem.umu.se
Alfano, Daniela
Institute of Genetics & Biophysics
alfano@igb.cnr.it
Almholt, Kasper
Finsen Laboratory
kasper@finsenlab.dk
Alpízar Alpízar, Warner
The Gades Institute - Haukeland University
Hospital
awarnercr@yahoo.com
Andersen, Lisbeth Moreau
University of Aarhus
lma@mb.au.dk
Andolfo, Annapaola
Fondazione Ifom
annapaola.andolfo@ifom-ieo-campus.it
Andreasen, Peter A.
University of Aarhus
pa@mb.au.dk
Antalis, Toni
University of Maryland School of Medicine
tantalis@som.umaryland.edu
Ardite, Esther
Center for Genomic Regulation (CRG)
esther.ardite@crg.es
Baker, Mark
Australian Proteome Analysis Facility
mbaker@proteome.org.au
Balsara, Rashna
University of Notre Dame
rbalsara@nd.edu
Beaufort, Nathalie
KliFo der Frauenklinik, TU München
nbeaufortgbb@yahoo.fr
Bednarek, Radoslaw
Polish Academy of Science - Lodz
rbednarek@cbm.pan.pl
Behrendt, Niels
Finsen Laboratory, Rigshospitalet
niels.behrendt@finsenlab.dk
Binder, Bernd R.
Medical University of Vienna
bernd.binder@univie.ac.at
Blasi, Francesco
Fondazione Ifom
francesco.blasi@ifom-ieo-campus.it
Bødker, Julie Støve
University of Aarhus
jsb@mb.au.dk
Brunner, Patrick
Medical University of Vienna
patrick.brunner@meduniwien.ac.at
Bruno, Katharina
University of Chicago
kbruno@uchicago.edu
Bugge, Thomas
National Institutes of Health
tbugge@mail.nih.gov
Chapman, Hal
University of California San Francisco
hal.chapman@ucsf.edu
Cierniewski, Czeslaw
Polish Academy of Sciences
cciern@zdn.am.lodz.pl
Cochran, Blake
University of Wollongong
blake@uow.edu.au
Croucher, David
University of Wollongong
d.croucher@garvan.org.au
Dano, Keld
Finsen Laboratory
kelddano@dadlnet.dk
Degryse, Bernard
Fondazione Ifom
degryse.bernard@hsr.it
Del Rosso, Mario
University of Florence
delrosso@unifi.it
Devy, Laetitia
DYAX SA
ldevy@dyax.com
Diaz Ramos, Mª Angeles
Institut Investigacio BiomedicaBellvitge
madiaz@idibell.org
Dumler, Inna
Hannover Medical School
dumler.inna@mh-hannover.de
Dupont, Daniel
University of Aarhus
dmd@mb.au.dk
Eden, Gabriele
Fondazione Ifom
gabriele.eden@ifom-ieo-campus.it
132 X I t h I n t e r n a t i o n a l W o r k s h o p o n

Ehart, Monika
Medical University of Vienna
monika.ehart@meduniwien.ac.at
Ehnman, Monika
Ludwig Institute for Cancer Research
monika.ehnman@licr.ki.se
Ellis, Vincent
University of East Anglia
v.ellis@uea.ac.uk
Fayard, Berengere
Friedrich Miescher Institute for Biomedical
Research
berengere.fayard@fmi.ch
Fibbi, Gabriella
University of Florence
fibbi@unifi.it
Fish, Richard
Geneva University Medical Faculty and University
Hospital
Richard.Fish@medecine.unige.ch
Franco, Paola
Institute of Genetics & Biophysics
franco@igb.cnr.it
Friberger, Petfer
DiA-Service/American Diagnostica
petter.friberger@dia-service.se
Gårdsvoll, Henrik
Finsen Laboratory, Rigshospitalet
gvoll@finsenlab.dk
Gils, Ann
Laboratory for Pharmaceutical Biology, KULeuven
ann.gils@pharm.kuleuven.be
Godiksen, Sine
University of Copenhagen
sinego@imbg.ku.dk
Guo, YongZhi
Umeå University
Yong-zhi.guo@medchem.umu.se
Hägglöf, Peter
University of Cambridge
pmh43@cam.ac.uk
Hajjar, Katherine
Weill Cornell Medical College
khajjar@med.cornell.edu
Harslund, Jakob
University of Copenhagen
jhar@life.ku.dk
Henic, Emir
University Hospital Lund
emir.henic@med.lu.se
Høyer-Hansen, Gunilla
Finsen Laboratory
gunilla@finsenlab.dk
Huang, Mingdong
Chinese Academy of Sciences
mhuang@fjirsm.ac.cn
Hultman, Karin
Institution of Neurosciences and Physiology
karin.hultman@neuro.gu.se
Illemann, Martin
Finsen Laboratory
millemann@finsenlab.dk
Jacobsen, Benedikte
Finsen Laboratory, Rigshospitalet
bjacobsen@finsenlab.dk
Jensen, Lotte
University of Copenhagen
lje@life.ku.dk
Jerczynska, Hanna
Medical University of Lodz - Poland
hanuka@zdn.am.lodz.pl
Jerke, Uwe
Franz-Volhard-Klink, Charite-Buch Berlin
uwe.jerke@charite.de
Jern, Christina
Institution of Neurosciences and Physiology
christina.jern@neuro.gu.se
Juncker-Jensen, Anna
Finsen Laboratory
ajjensen@finsenlab.dk
Kietzmann, Thomas
University of Kaiserslautern
tkietzm@gwdg.de
Kinnby, Bertil
Malmö University College
Bertil.Kinnby@od.mah.se
Kojima, Soichi
Molecular Cellular Pathology Research Unit,
RIKEN
skojima@postman.riken.go.jp
Kruithof, Egbert
University Hospital of Geneva
egbert.kruithof@hcuge.ch
Kusch, Angelika
Charite-Campus Buch, Dept of Molecular and
Clinical Cardiology
angelika.kusch@charite.de
Lademann, Ulrik
University of Copenhagen
ul@life.ku.dk
Laerum, Ole Didrik
The Gade Institute, University of Bergen
ole.laerum@gades.uib.no
Lawrence, Daniel
University of Michigan Medical School
dlawrenc@med.umich.edu
Lee, Jodi
University of Wollongong
jal31@uow.edu.au
Li, Jianxue
BIDMC, Harvard Medical School
jli7@caregroup.harvard.edu
Molecular & Cellular Biology of Plasminogen Activation 133

Li, Jinan
Umeå University
jinan.li@medchem.umu.se
Li, Shih-Hon
University of Illinois at Urbana-Champaign
sli3@uiuc.edu
Lillis, Anna
University of Maryland School of Medicine
alillis@som.umaryland.edu
Liu, Kui
Umeå University
kui.liu@medchem.umu.se
Lobov, Sergei
University of Wollongong
sergei@uow.edu.au
Lochner, Janis
Lewis & Clark College
lochner@lclark.edu
Longanesi Cattani, Immacolata
National Cancer Institute of Naples - Italy
immalonganesi@libero.it
Lu, Wuyuan
University of Maryland
luw@umbi.umd.edu
Lund, Ida K.
Finsen Laboratory
ikl@finsenlab.dk
Lund, Leif R.
Finsen Laboratory
lund@inet.uni2.dk
Madsen, Chris
Fondazione Ifom
chris.madsen@ifom-ieo-campus.it
Magdolen, Viktor
Clinical Research Unit, TU München
viktor-magdolen@lrz.tum.de
Maiya, Rajani
The Rockefeller University
rmaiya@mail.rockefeller.edu
Manchanda, Naveen
University of Illinois at Urbana-Champaign
manchand@uiuc.edu
Markart, Philipp
University of Giessen Lung Center
philipp.markart@innere.med.uni-giessen.de
Meins, Marita
Friedrich Miescher Institute for Biomedical
Research
mmeins@fmi.ch
Meissenheimer, Lester
KULeuven: Lab Pharmaceutical Biology
lester.meissenheimer@pharm.kuleuven.be
Mihaly, Judit
Medical University of Vienna
judit.mihaly@meduniwien.ac.at
Monard, Denis
Friedrich Miescher Institute for Biomedical
Research
monard@fmi.ch
Navarro, Pilar
Institut Municipal dInvestigació Mèdica
pnavarro@imim.es
Nielsen, Boye Schnack
Finsen Laboratory, Rigshospitalet
schnack@finsenlab.dk
Nieves-Li, Evelyn
University of Illinois at Urbana-Champaign
enieves@uiuc.edu
Nuttall, Robert
Dalhousie University
r.nuttall@dal.ca
Ny, Tor
Umeå University
tor.ny@medchem.umu.se
Olausson, Björn
Umeå University
bjorn.olausson@medchem.umu.se
Pabba, Mohan
Umeå University
pabba.mohan@medchem.umu.se
Pappot, Helle
Rigshospitalet 5073
pappot@rh.regionh.dk
Paul, Justin
The Rockefeller University
jpaul@rockefeller.edu
Peterson, Cynthia
University of Tennessee
cbpeters@utk.edu
Pirazzoli, Valentina
Fondazione Ifom
valentina.pirazzoli@ifom-ieo-campus.it
Pliyev, Boris
Moscow State University
bpliyev@cardio.ru
Ploug, Michael
Finsen Laboratory
m-ploug@finsenlab.dk
Prager, Gerald
Medical University of Vienna
gerald.prager@meduniwien.ac.at
Przygodzka, Patrycja
Umeå University
patrycja.przygodzka@medchem.umu.se
Przygodzki, Tomasz
Umeå University
tomasz.przygodzki@medchem.umu.se
Ragno, Pia
University of Salerno
pragno@unisa.it
134 X I t h I n t e r n a t i o n a l W o r k s h o p o n

Ranson, Marie
University of Wollongong
mranson@uow.edu.au
Rasch, Morten
Finsen Laboratory
mrasch@finsenlab.dk
Resnati, Massimo
S.Raffaele Hospital- DIBIT
resnati.massimo@hsr.it
Rønø, Birgitte
Finsen Laboratory
brono@finsenlab.dk
Salvi, Alessandro
University of Brescia
asalvi@med.unibs.it
Sarra Ferraris, Gian Maria
Fondazione Ifom
gianmaria.sarraferraris@ifom-ieo-campus.it
Schmitt, Manfred
Clinical Research Unit, Dept. Obstetrics and
Gynecology
manfred.schmitt@LRZ.tum.de
Schwartz, Brad
University of Illinois at Urbana-Champaign
comuc@med.uiuc.edu
Sidenius, Nicolai
Fondazione Ifom
nicolai.sidenius@ifom-ieo-campus.it
Stoppelli, Maria Patrizia
Institute of Genetics & Biophysics
stoppell@igb.cnr.it
Strickland, Dudley
University of Maryland School of Medicine
dstrickland@som.umaryland.edu
Sulniute, Rima
Umeå University
rima.sulniute@medchem.umu.se
Suzuki, Yuko
Hamamatsu University School of Medicine
seigan@hama-med.ac.jp
Szabo, Roman
National Institutes of Health
rszabo@nidcr.nih.gov
Tkachuk, Sergey
Hannover Medical School
Tkatchouk.Sergei@mh-hannover.de
Urano, Tetsumei
Hamamatsu University School of Medicine
uranot@hama-med.ac.jp
Vidal, Berta
Center for Genomic Regulation (CRG)
berta.vidal@crg.es
Vogel, Lotte Katrine
University of Copenhagen
vogel@imbg.ku.dk
Wahlberg, Patrik
Umeå University
patrik.wahlberg@medchem.umu.se
Wei, Ying
University of California San Francisco
ying.wei@ucsf.edu
Wikström, Clas
Umeå University
clas.wikstrom@medchem.umu.se
Wilczynska, Malgorzata
Umeå University
Malgorzata.Wilczynska@medchem.umu.se
Wojta, Johann
Medical University of Vienna
johann.wojta@meduniwien.ac.at
Wygrecka, Malgorzata
University of Giessen Lung Center
malgorzata.wygrecka@innere.med.uni-giessen.de
Xolalpa, Wendy
Instituto de Investigaciones Biomedicas UNAM
wendyxolalpa@yahoo.com.mx
Xue, Aiqun
University of Sydney Royal North Shore Hospital
aiqunn@med.usyd.edu.au
Yepes, Manuel
Emory University
myepes@emory.edu
Zhou, Aiwu
University of Cambridge
awz20@cam.ac.uk
Molecular & Cellular Biology of Plasminogen Activation 135

Notes
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136 X I t h I n t e r n a t i o n a l W o r k s h o p o n