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

II. Mechanisms of Hemostasis
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through multiple, interrelated pathways. The major signal
transduction systems are summarized in Table 10-3 . These
biochemical pathways appear to be universal in platelets
from all species, although they have been primarily investigated
in human platelets. The limited information available
for nonhuman platelets indicates that variations occur in the
relative predominance of the individual enzymatic pathways
( Gentry, 1992, 2000b ). Protein phosphorylation by tyrosine
and serine/threonine kinases is known to be critical for the
modulation of the biological functions of platelets. Enzyme
systems that have been identified include the nonreceptor
tyrosine kinases, Src and Syk, that activate the cytoplasmic
tail of the GPIIb-IIIa integrin β 3 chain inducing an increase
in the affinity of the α IIb chain for fibrinogen ( Marshall et
al ., 2004 ). The fibrinogen binding reaction is also enhanced
by protein kinase C (PKC), following its activation through
a diacylglycerol (DAG)-dependent mechanism ( Buensuceso
et al ., 2005 ). α 2 β 1 activation of collagen-adherent platelets
also stimulates phosphorylation of proteins such as Src,
Syk, and the PLC γ 2 isomer of PLC, all of which are components
in the GPVI-FcR γ -chain cascade ( Inoue et al .,
2003 ). Activation of platelets by collagen-GPVI interaction
induces PKC-Ca 2 -mediated activation of the MLCK that,
in turn, induces the cytoskeletal changes necessary for granule
secretion. The phosphorylation of the myosin light chain
can also occur through a Ca 2 /calmodulin mechanism that is
mediated by an extracellular signal regulated kinase (Erk2)
(Toth-Zsamboki et al ., 2003 ).
The elevation of free cytosolic calcium, following
receptor-mediated influx of extracellular calcium and secondary
release of calcium from the dense tubular system, is
one of the critical biochemical events in platelet activation
in all species ( Gentry, 2000b ; Heemskerk and Sage, 1994 ).
In addition to mediating the activation of PKC, calcium is
directly involved in the regulation of phospholipid metabolism
through activation of the calcium-dependent enzymes
phospholipase C (PLC) and phospholipase A2 (PLA2). A
further response to increased intracellular calcium is the
lowering of intracellular cyclic adenosine monophosphate
(cAMP) levels through activation of cAMP phosphodiesterase.
The reduced cAMP levels exert a positive feedback
response to further increase intracellular calcium levels,
which, in turn, further enhance platelet reactivity and
aggregation ( Table 10-3 ).
PLC is one of the secondary messenger systems that is
universally activated by platelet agonists. It catalyses the
cleavage of membrane bound phosphatidyl 4,5 bisphosphate
(PIP2), one of the phosphorylated derivatives of PI,
to lipophilic membrane bound DAG and hydrophilic IP3
that is released into the cytoplasm ( Kroll and Schafer, 1989 ;
Nozawa et al ., 1991 ). DAG and IP3 act synergistically as
signal transducers to promote all aspects of the basic platelet
response, including shape change, increase in integrin and
receptor affinity, and secretion of granule contents ( Table
10-3 ). As noted previously, DAG-activated kinases, such
as PKC, cause protein phosphorylation to mediate cellular
changes such as increased affinity of GPIIb-IIIa integrins
and a rise in intracellular calcium. IP 3 is also instrumental
in elevating calcium levels through the activation of a
calcium transporting adenosine triphosphate (ATPase) system
that mobilizes calcium from the dense tubular system.
The continued functioning of the IP 3 -DAG system depends
on the continued agonist-receptor-mediated activation of
PLC, as both IP 3 and DAG are rapidly converted to inositol
and phosphatidic acid (PA), respectively. Both inositol and
PA can be utilized for the resynthesis of PI and therefore are
recycled within the platelet ( Nozawa et al ., 1991 ).
In platelets from most species of domestic animals,
collagen and thrombin are more potent activators of the
phospholipase A 2 (PLA 2 )-mediated secondary messenger
system than are ADP or PAF ( Gentry and Nyarko, 2000 ).
PLA 2 is an acyl hydrolase that cleaves the sn-2 acyl bond
of the platelet membrane phospholipids, PC and PE, to
release AA and lysophospholipids ( Puri, 1998 ). AA is the
major free fatty acid present in both platelet membranes
and granules. Following its release into the cytoplasm, it
is rapidly metabolized by the cyclooxygenase and lipoxygenase
enzyme systems into labile products that function
as both intracellular and extracellular messengers ( Gentry
and Nyarko, 2000 ). Unlike many nucleated cells, including
endothelial cells that contain two forms of cyclooxygenase
(COX), only the constitutively expressed form, COX-1, is
found in platelets. In platelets, COX-1 first oxidizes and
cyclizes AA to form prostaglandin PGG 2 , which it subsequently
hydrolyzes to prostaglandin PGH 2 . These cyclic
endoperoxides are rapidly metabolized to TXA 2 by thromboxane
synthetase; prostacyclin (PGI 2 ) by PGI synthetase;
or are converted to the eicosanoids, PGD 2 , PGE 2 , or PGF 2
by isomerase enzymes that have yet to be fully characterized
in platelets. In most mammalian platelets, TXA 2
is the major metabolite, and in human platelets, it exerts
a positive feedback effect on platelet aggregation through
its receptor. Variable responses to TXA 2 have been found
in canine platelets, and bovine platelets do not respond to
this agonist ( Gentry, 1992 , 2000b). It is possible that the
variable response of mammalian platelets to the agonist
effect of TXA 2 is related to different receptor populations
in different species. Because of the labile nature of TXA 2 ,
its stable metabolite, TXB 2 , is used to evaluate the extent
of AA metabolism in activated platelets. Comparable to
the difference in aggregation response to TXA 2 , the level
of TXB 2 released from thrombin-stimulated human, horse,
and cat platelets is 10-fold greater than the amount released
after similar activation of cow, pig, sheep, and mink platelets
( Gentry and Nyarko, 2000 ).
d. Negative Regulation of Aggregation
To balance the ability of platelets to be rapidly activated
when needed, a number of regulatory mechanisms exist