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

II. Mechanisms of Hemostasis
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A short cytoplasmic tail at the C-terminus is followed by a
membrane-spanning region that contains serine/threoninerich
domains that serve as glycosylation sites, which support
its attachment to chondroitin sulfate (CS) in the extracellular
matrix. The membrane-spanning domain is followed
by a domain that consists of six epidermal growth factor
(EGF)-like repeats, which determine the biological activity
of TM. The N-terminal region of TM includes two amino
acid sequences that play no role in its hemostatic function.
Rather, the anticoagulant activity of TM is conferred
by EGF domains 4, 5, and 6, which are also the areas that
are involved in the ability of TM to enhance thrombin inhibition
by PCI ( Tsiang et al ., 1992 ). The EGF5 and EGF6
domains have been identified as thrombin binding sites ( Van
de Wouwer et al ., 2004 ). For APC to act efficiently as an
anticoagulant, the presence of the cofactor, protein S, at the
endothelial cell surface is also required. Protein S is a singlechain
polypeptide that is synthesized by hepatocytes, endothelial
cells, and megakaryocytes in forms that vary in size
from 69 to 84 kDa ( Johnstone, 2000 ). In plasma, the majority
of protein S circulates bound to the complement regulatory
protein C4b-binding protein and only about 30% is in the
free, more biologically active form ( Dahlback, 1995 ). Unlike
the other vitamin K-dependent hemostatic proteins, protein
S is not a zymogen and functions solely as a cofactor to promote
the formation of the inhibitory complex that consists of
APC-protein S-calcium on the negatively charged phospholipids
(PL) that are exposed on damaged endothelium and
activated platelets ( Walker, 1984 ). These PLs are also those
that are involved in the formation of the tenase complex and
the prothrombinase complex (see Section II.C.3). Hence,
protein S effectively acts as a mediator to increase the number
of APC molecules bound to the PL, to bring them in
closer contact with FVIIIa and FVa, and thus to accelerate
the rate at which APC can cleave these proteins to produce
their inactive forms, FVIIIai and FVai ( Dahlback, 2000 ).
The reduction in FVIIIa and FVa cofactor activity effectively
reduces the rate of thrombin formation. APC can only
inhibit FVIII when it is a component of the tenase complex
because it is protected from proteolysis as FVIII circulates
in plasma bound to vWF (see Section II.C.2). In contrast,
circulating FV, like FVa, can bind to membrane PLs where it
can be cleaved to generate an anticoagulant form of FV that
functions in synergy with protein S as an APC cofactor in
the inactivation of FVIIIa ( Dahlback, 2000 ). In this way, FV
can, like thrombin, function as both a procoagulant and an
anticoagulant cofactor in hemostasis. It is now recognized
that TM is not the only cofactor expressed on endothelial
cells that can function as a cofactor for thrombin-induced PC
activation. EPCR is constitutively expressed in endothelial
cells, particularly in large blood vessels, and is structurally
similar to the major histocompatibility class 1/CDI family
of molecules that are involved in immunity and inflammation
( Di Cera, 2003 ; Esmon, 2003 ; Van de Wouwer et al .,
2004 ). Not only does EPCR accelerate thrombin-mediated
activation of PC, but it also concentrates APC near the surface
of the vessel wall ( Stearns-Kurosawa et al ., 1996 ).
When APC is generated, it remains bound to EPCR for
only a short time before it becomes associated with protein
S on the surface of activated platelets or damaged endothelium
( Van de Wouwer et al ., 2004 ). After APC has caused
the inhibition of FVIIIa and FVa, it is itself inactivated by
several serpins including, α 1 -protease inhibitor, α 2 -M, and
protein C inhibitor ( Table 10-6 ). The APC pathway can also
be down-regulated by inflammatory cytokines such as IL-1 β
and TNF α , which reduce the expression of both TM and
EPCR ( Esmon, 2003 ). It would appear that TM can exert
multifunctions in the regulation of hemostasis. For example,
when TM is bound to CS on the extracellular matrix,
not only is the PC cofactor activity of TM enhanced but the
rate of neutralization of thrombin by both heparin-AT and
by PCI is accelerated ( Koyama et al ., 1991 ). TM also exhibits
antifibrinolytic activity through the ability of both EGF
domains 3 through 6 of the thrombin-TM complex to activate
TAFI (see Section II.D.3) ( Nesheim, 2003 ).
D. Fibrinolysis
1. Overview
It is essential that the coagulation pathways are counterbalanced
by a functional fibrinolytic cascade so that blood
fluidity can be maintained in the vascular system ( Welles,
1996 ). Like the coagulation pathway, the fibrinolytic system
is normally latent but can be fully activated within a minute
or two after stimulation ( Nesheim, 2003 ). The major
enzymatic component of this fibrin degradation system is
plasmin, which, like thrombin, is a serine protease but with
broader substrate specificity ( Lijnen, 2002 ). In addition to
cleaving specific sites on polymerized fibrin, under some
pathophysiological conditions plasmin can also degrade
fibrinogen, FV, and FVIII and activate metalloproteinases
(Darien, 2000b) . The fibrinolytic system shares several
biochemical characteristics with the coagulation system.
These include an initiation phase that involves the liberation
of tissue-type plasminogen activator (tPA) from damaged
endothelium; the conversion of the circulating zymogen,
plasminogen, to the active protease, plasmin; and positive
feedback reactions that increase the concentration of plasmin
in the area of a clot ( Fig. 10-3 ). The system is downregulated
by both protease inhibitors , such as plasminogen
activator inhibitor type 1 (PAI-1) and antiplasmin (AP), and
by a reaction mediated by the thrombin-TM complex ( Table
10.6 ) (Coughlin, 2005a ; Nesheim, 2003 ). The ability of
fibrin to act as an effective cofactor for enhanced plasmin
activity is an important factor in localizing the physiological
activity of this protease to the site of a fibrin clot ( Medved
and Nieuwenhuzen, 2003 ). Nonphysiological activators of
fibrinolysis, such as streptokinase, have only proved to be
effective in dissolving fibrin clots in human plasma.