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

306
Chapter | 10 Hemostasis
is a relatively poor activator of TAFI, but in the presence
of TM its catalytic efficiency is increased over 1000-fold
( Nesheim, 2003 ). This enhancement of TAFI activation is
dependent on the formation of a ternary complex between
thrombin-TAFI and the EGF 3 through EGF 6 domains of
the TM molecule. Like the thrombin-TM-mediated activation
of PC, the activation of TAFI is calcium dependent
( Bajzar et al ., 1995 ). Plasmin is also a physiological activator
of TAFI with a catalytic efficiency eight times that
of thrombin. However, the ability of plasmin to activate
TAFI is reduced to only one-tenth that of thrombin-TM in
the presence of glycosaminoglycans, such as those found
in the extracellular matrix ( Mao et al ., 1999 ).
5. Regulation of Plasminogen Activation and Plasmin
Activity
Although it is important that fibrinolysis occurs after
wound healing has been initiated, it is equally important
that the degradation of the clot not occur prematurely. This
is achieved because, in the initial stages of thrombus formation,
the thrombin-induced release of tPA from endothelial
cells is effectively opposed by the localized activation
of TAFI. Through its affinity for and its ability to modify
partially degraded fibrin, TAFIa functions to block the ability
of tPA to activate plasminogen ( Fig. 10-3 ). Following
partial degradation by plasmin, the newly exposed lysine
residues of fibrin bind both tPA and plasmin with high
affinity promoting fibrinolysis (see Section II.D.3). TAFIa
catalyzes the release of both arginine and lysine residues
from the partially degraded fibrin thus reducing both tPA
and Glu-plasminogen binding, which has the effect of
reducing plasmin formation. The reduced binding of Gluplasminogen
to TAFIa modified fibrin has been correlated
with an increase in clot lysis time ( Sakharov et al ., 1997 ).
Both TAFI and TAFIa are cross-linked to fibrin by FXIIIa,
which localizes these molecules to the fibrin clot. TAFIa is
an unstable molecule, which decays rapidly at 37 ° C such
that its half-life is approximately 8 min ( Nesheim, 2003 ).
To date no physiological inhibitor of TAFI has been identified
( Bajzar, 2000 ).
PAI-1 is the major inhibitor of both tPA and uPA
( Tsikouris et al ., 2002 ). It inhibits plasminogen activation
by first forming a reversible complex with tPA that then
cleaves PAI-1 at its reactive site to release the C-terminal
peptide of the inhibitor. Further proteolytic activity of tPA
is blocked because the carboxyl group of one of the cleaved
PAI-1 arginine residues remains covalently bound to a serine
residue at the reactive center of tPA ( Dobrovolsky and
Titaeva, 2002 ). Because of the relatively high levels of PAI-
1 in the circulation, most of the tPA released by endothelial
cells is inactivated before it can interact with plasminogen.
Hence, in the absence of fibrin plasminogen is only slowly
converted to plasmin. Once tPA becomes bound to fibrin, it
is protected from the inhibitory effects of PAI-1 and plasmin
formation can proceed rapidly.
Antiplasmin is the major physiological inhibitor of plasmin
irrespective of whether the plasmin is bound to fibrin or
is free in the circulation. In plasma, the reaction between AP
and plasmin is rapid and the formation of a stable 1:1 bimolecular
complex between the two molecules results in the
irreversible inactivation of plasmin (Darien, 2000b) . Because
AP is partially degraded by plasmin, other circulating protease
inhibitors ( Table 10-6 ) can act as plasmin inhibitors,
particularly when the capacity of AP is exceeded by high
concentrations of free plasmin. These reactions are important
in preventing systemic lytic states from developing
such as occurs in certain disease states (see Section IV.B.4).
When fibrin is formed, it binds plasminogen and AP in
equimolar amounts. AP is bound via its N-terminal portion,
which leaves the C-terminal portion free to bind to one of
the kringle domains of the plasmin molecules that form in
response to tPA activity (Coughlin, 2005a) . This modulates
plasmin such that the proteolytic cleavages do not initially
destroy the fibrin network but rather open new affinity binding
sites for additional tPA and plasminogen. As this first
phase of fibrinolysis proceeds, more plasmin is generated on
the partially degraded fibrin, which can induce the second
phase that produces final degradation of the fibrin meshwork.
The existence of the two sequential phases of fibrin
degradation may be one of the mechanisms that provide
temporary stability of fibrin clots at sites of vascular damage
( Dobrovolsky and Titaeva, 2002 ).
III. LABORATORY ASSESSMENT OF
HEMOSTASIS
A. Quality Control and Reagent Variation
The common laboratory assays available to assess the
integrity of the hemostatic system can be divided into those
assessing primary hemostasis (platelet quantity and function)
and those assessing secondary hemostasis (clotting
factor activity and fibrin formation and dissolution).
Proper collection of plasma that is free from clots and
activated platelets is very important for all hemostasis testing.
To obtain the best sample, minimal pressure should be placed
on the vein and a single clean venipuncture performed, to
avoid release of excessive TF, which would activate the clotting
mechanism, consume coagulation factors, and therefore
artifactually prolong the clotting times. The stopper should
be removed from the Vacutainer and blood slowly expelled
from the syringe into the tube because excessive vacuum
turbulence may activate platelets. Ideally, the first few
drops of blood collected should be discarded because they
most likely would contain some TF. The blood should be
gently mixed with the citrate, to ensure anticoagulation, but
not too aggressively mixed so as to activate hemostasis.