Clinical Biochemistry of Domestic Animals (Sixth Edition) - UMK ...

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Chapter | 10 Hemostasis
2. Components of the Fibrinolytic System
a. Plasminogen
Plasminogen is secreted by the liver as a single-chain 92-
kDa glycoprotein with glutamine as the N-terminal amino
acid (Glu-plasminogen). It is composed of five kringle-like
domains containing “ lysine-binding sites ” and a C-terminal
domain homologous to other trypsin-like proteases
( Ponting et al ., 1992 ). The kringle domains mediate the
interaction of plasminogen, and its active form plasmin,
with substrates, inhibitors, and cell membranes.
Consequently the kringle domains are important in both
regulation of plasminogen activation and the localization
of the proteolytic activity of plasmin to the appropriate
physiological site ( Wu et al ., 1990 ). Plasminogen can be
cleaved by plasmin at several sites resulting in lysine as the
N-terminal amino acid (Lys-plasminogen) and the release
of an 8-kDa N-terminal polypeptide. Lys-plasminogen, in
the absence of fibrin, is converted to plasmin at a faster rate
than Glu-plasminogen. The cleavage of an Arg-Val peptide
bond in single-chain plasminogen converts it to plasmin,
which now consists of two polypeptide chains linked
by a disulfide bond ( Dobrovolsky and Titaeva, 2002 ). The
N-terminal heavy A chain contains the kringle domains
that convey substrate specificity to plasmin, whereas the
C-terminal light B chain contains the catalytic site.
b. Plasminogen Activators
tPA is synthesized and secreted by endothelial cells as a
70-kDa single-chain active enzyme. It is the only protease
of the hemostatic system secreted in an active form
( Dobrovolsky and Titaeva, 2002 ). The molecule is composed
of five domains: the N-terminal domain, which is
homologous to the finger-like domains of fibronectin, is
followed by a region homologous to the epidermal growth
factor (EGF) domain, two kringle-like domains similar to
those of plasminogen, and a C-terminal domain homologous
to other trypsin-like proteases ( van Zonneveld et al .,
1986 ). The finger-like kringle domains possess two distinct
fibrin-binding sites. Plasmin, kallikrein, and FXa can
all cleave tPA to form a two-chain form of the molecule
that, in the presence of fibrin, activates plasminogen at the
same rate as single-chain tPA (Dobrovolsky and Titaeva,
2002). Stimuli such as exercise, venous occlusion, and
intravenous injection of vasoactive drugs can increase the
release of tPA from endothelial cells, whereas inflammatory
mediators such as tissue necrosis factor α (TNF α ) and
interleukin-1 (IL-1) cause a decrease in free plasma tPA
levels. Recombinant tPA is currently being used to induce
thrombolysis in human patients with myocardial infarctions
and other thromboembolic problems (Dobrovolsky
and Titaeva, 2002).
A trypsin-like protease capable of activating plasminogen
was originally isolated from human urine and named
urokinase. It is now known that many cell types can synthesize
and secrete a single-chain 54-kDa glycoprotein, which
was initially referred to as prourokinase. Current nomenclature
designate these proteins as two forms: single-chain urokinase-type
plasminogen activator (scuPA) and two-chain
urokinase-type plasminogen activator (tcuPA). The scuPA
molecule is composed of three domains. The N-terminal
domain is homologous to the EGF domain, the middle protein
is homologous to the kringle domain of plasminogen,
and the C-terminal domain is homologous to trypsin-like
proteases (Dobrovolsky and Titaeva, 2002). Both plasmin
and kallikrein can cleave a Lys-Lys peptide bond in uPA to
form fully functional tcuPA. Thrombin cleaves an Arg-Phe
bond to produce a variant tcuPA molecule that appears incapable
of directly activating plasminogen until after it has
been further hydrolyzed by plasmin cleavage of a Lys-Ile
bond (Dobrovolsky and Titaeva, 2002). Although initially
considered to be a major component of the plasma fibrinolytic
system, it is now recognized that a more important
physiological function of uPA may be its ability to activate
metalloproteinases that degrade cellular matrix components
inducing pericellular proteolysis ( Lijnen, 2002 ). uPA mediates
this effect through its ability to bind to specific cellular
receptors (uPARs), which bring uPA into close contact with
cell-bound plasminogen and thus promote plasmin generation
on the cell surface. Thus, uPAR plays an important
role in cell migration and the tissue remodeling processes
that are required following vascular damage and thrombus
formation.
3. Mechanism of Fibrinolysis
Fibrinolysis, like coagulation, is organized on a surface
that facilitates the assembly and