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

II. Mechanisms of Hemostasis
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the extracellular matrix of endothelial cells bound to GAGs
such as heparan sulfate (Abildgaard, 1992). Consequently,
infusion of heparin can displace this surface-bound TFPI to
induce a two- to four-fold increase in circulating TFPI levels
(Golino et al ., 2002 ; Novotny et al ., 1991 ). The liver effectively
clears circulating TFPI by receptor-mediated endocytosis
resulting in a half-life for TFPI of less than 70 min
(Lindhal, 1997). The inhibitory activity of TFPI depends
on the activation of the TF pathway and the formation of
FXa. TFPI binds to FXa at, or near, its serine active site in a
reversible calcium-independent reaction to form a 1:1 complex
( Broze, 1992 ). This complex is formed most efficiently
when FXa is incorporated into the prothrombinase complex
(see Section II.C.3). The ability of TFPI to inhibit the activity
of the TF/FVIIa complex depends on the TFPI/FXa complex
forming an irreversible, calcium-dependent quaternary
complex consisting of TFPI-FXa-TF-FVIIa ( Lindahl, 1997 ).
Hence, TFPI is not only able to inhibit FXa activity, but it
is also able to suppress the generation of additional FXa by
blocking the TF/FVIIa-catalyzed conversion of FX to FXa.
TFPI is able to exert this bimodal inhibitory effect because
the inhibition of FXa depends on the first Kunitz-type
domain and an intact C-terminal region, whereas the second
Kunitz-type domain of TFPI molecule is responsible for the
binding of the TFPI/FXa complex to the TF/FVIIa complex
( Lindahl, 1997 ). Because TFPI does not interfere with
FXa activation by the tenase complex (FIXa-FVIIIa-Ca-PL,
see Section II.C.3), it does not completely block thrombin
formation. The importance of TFPI in regulating thrombin
generation and fibrin formation has been demonstrated in
animal models depleted of TFPI. These animals are sensitized
to TF and readily develop DIC (see Section IV.B.4) in
response to endotoxin, which causes the release of TF from
endothelial cells ( Huang et al ., 1997 ; Sandset et al ., 1991 ).
Hence, one of the factors involved in DIC is an imbalance
between the levels of TF and TFPI.
b. Serine Protease Inhibitors (Serpins)
Antithrombin (AT, also known as antithrombin III, ATIII)
is the principal member of the superfamily of serine protease
inhibitors, or serpins, that inhibit the action of several
serine proteases of the hemostatic system ( Pike et al .,
2005 ). Serpins have a common mechanism of action that
involves trapping and irreversibly inactivating the proteases
through the formation of a covalent bond followed by the
removal of the serpin-enzyme complex from the circulation
by the action of receptors, which specifically recognize the
inhibited conformation of the serpin ( Gettins, 2002 ). One
of the functions of serpins is to neutralize any of the procoagulant
enzymes that escape from the vicinity of a blood
clot and thus prevent inappropriate thrombus formation.
AT is a 58-kDa single-chain glycoprotein synthesized in
the liver and endothelial cells. Structural similarities exist
in the AT molecule across many species ( Frost et al ., 2002 ;
Johnstone, 2000 ). Although AT can inhibit several serine
protease of the coagulation system including thrombin,
FIXa, FXa, FXIa, and FXIIa, the principal targets are considered
to be thrombin and FXa ( Pike et al ., 2005 ). The AT
molecule has two functional sites: the reactive site for serpins
and a binding a site for glycosaminoglycans (GAGs),
such as heparin and heparan sulfate. GAGs serve as potent
cofactors that enhance the rate of thrombin inhibition from
one that is nonphysiological to a physiologically relevant
level. In its unactivated, or native state, the AT molecule
contains a reactive center loop that is partially inserted into
its A β -sheet. When thrombin comes in contact with AT,
it cleaves a specific bond in the reactive center loop causing
it to become fully inserted into the A β -sheet and at the
same time translocating thrombin from the top of the AT
molecule to the bottom ( Munoz and Linhardt, 2004 ). In
this way, a 1:1 stoichiometric complex is formed through
covalent bonding between the active site serine of thrombin
and the reactive site arginine of AT. The interaction
between GAGs, like heparin, and AT involves several steps.
First, a low-affinity interaction occurs forming ion pairs
between a cluster of positively charged amino acids, such
as arginine and lysine, in the D-helix of the AT molecule
and specific spatially defined negatively charged sulfo- and
carboxyl groups in the heparin pentasaccharide sequence
( Huntington et al ., 1996 ). This binding induces a conformation
change in AT that results in both stronger heparin
binding and the expulsion of the reactive center loop
of AT from the A β -sheet ( Munoz and Linhardt, 2004 ). In
effect, heparin facilitates the diffusion of thrombin and AT
toward each other through the formation of a tertiary complex
between heparin and AT. For example, in the presence
of heparin the inhibition of thrombin by AT is accelerated
1000-fold from 4.4 min to 0.27sec and the rate of inhibition
of FXa by AT is accelerated 10,000-fold ( Olson and
Shore, 1982 ; Pike et al ., 2005 ). The formation of the
FXa-AT-heparin complex requires the presence of calcium
ions to overcome the negative effects of the Gla-domains
in FXa ( Rezaie, 1998 ). Because clot-bound thrombin and
FXa are protected from inactivation by AT, the physiological
role of AT is not to cause the cessation of clotting but
rather to localize the clot and prevent it from spreading too
far beyond the site of damage ( Weitz et al ., 1990 ). It also
functions to prevent thrombin formation from occurring on
undamaged endothelium. Heparan sulfate is an extracellular
component of the extracellular matrix of all animal
cells, whereas heparin is localized to the granules of mast
cells and is only released locally in an allergic reaction. In
the vasculature, only heparan sulfate in the outer monolayer
of endothelial cells possesses the AT binding sites
that can accelerate the inhibitory action of AT, whereas
the heparan sulfate contained in the underlying layer of
smooth muscle cells lacks AT binding sites ( Munoz and
Linhardt, 2004 ). Thus, when thrombin comes in contact
with AT-heparan sulfate complex on the “ healthy ” intact
vascular wall, the T-AT complex irreversibly inactivates it.