12th Congress of the European Hematology ... - Haematologica

BRIC 126 and 4N1K) in cells lacking protein 4.1R compared with control
cells, but decreased in the GPC pathway (when challenged with
BRIC 10) in cells lacking protein 4.1R compared with control cells. 4.1R
(Madrid) cells were also shown to be resistant to TSP-1 mediated eryptosis.
This difference in PS exposure suggests an important regulatory
role for 4.1R in both of these apoptotic pathways, and may suggest that
CD47-induced PS exposure occurs independently of binding to 4.1R/p55.
Studies are ongoing with red cells obtained from a further 4.1R deficient
individual (4.1R -/- Lille) and initial analysis is confirms the alterations
seen in the 4.R -/- Madrid sample.
Zoe Plummer contributed equally to this work
0916
INHIBITION OF INCREASED SICKLE NEUTROPHIL ADHESION TO ICAM-1 BY NITRIC
OXIDE DONORS AND ACTIVATION OF CGMP SIGNALING
A. Canalli, N. Conran, S.T.O. Saad, F.F. Costa
Unicamp, CAMPINAS, Brazil
Background. Leukocytes play an important role in sickle cell disease
(SCD); increased numbers of leukocytes are associated with increased
morbidity and mortality in SCD and leukocytes participate significantly
in the vaso-occlusive process. Nitric oxide (NO) has recently been
implicated as important in SCD pathophysiology. NO plasma bioavailability
is thought to be decreased in SCD, and NO therapy has been proposed
for the treatment of SCD and vaso-occlusive crises. Aims. Since
NO is an important inflammatory mediator and may be important for
the inhibition of leukocyte adhesion and migration mechanisms, we
compared the adhesive properties of neutrophils from control subjects
and from SCD patients (SCD neutrophils)and looked at the effect of NO
donating agents on this adhesion. Nitric oxide metabolites and cGMP
levels (second messenger for NO) were also measured in these neutrophils.
Methods. Neutrophils were isolated from whole blood by separating
on a Ficoll gradient. Cell adhesion to recombinant ICAM-1 was
compared utilizing static adhesion assays. NO metabolites/cGMP levels
were measured in neutrophil extracts using specific assays. Results.
Neutrophils from SCD patients demonstrated a significantly greater
adhesion to ICAM-1-coated plates (10 µg/mL) than control neutrophils
(19.51±9.02%, n=13; 10.98±3.95%, respectively n=9; p=0.025). Coincubation
of SCD neutrophils with the nitric oxide donors, sodium
nitroprusside (SNP, 10 µM) and DEANO (1 µM) reduced their increased
adhesion to ICAM-1 to levels similar to those of control neutrophils
(19.12±3.00% reduced to 16.49±2.60, n=8; p=0.009 for SNP) and
(19.62±2.50%, reduced to 10.17±0.986%, n=4; p= 0.002 for DEANO).
In contrast, SNP and DEANO did not significantly affect normal neutrophil
adhesion to ICAM-1 (12.33±0.25% to 11.95±0.62%, n= 5; p<
0.05) and (13.21±1.26% to 10.89±1.91%, n=3; p
0.05; n=3). Levels of nitric oxide metabolites in SCD neutrophils did not
differ significantly from those in normal neutrophils (17.34±7.02%,
n=10; 16.30±7.00%, n=8; respectively). Furthermore, levels of cGMP
(0.11±0.02 pMol/1x107 neutrophils, n=9) in SCD neutrophils were not
significantly different to those observed in normal neutrophils
(0.142±0.036 pMol/1x107 neutrophils, n=7; p