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

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Chapter | 10 Hemostasis
exhibits synergy with the stronger agonists, such as collagen
and thrombin, which induce its secretion from dense
granules ( Mills, 1996 ). Further, low concentrations of ADP
potentiate and amplify the effects of other weak agonists
such as serotonin, epinephrine, and TXA2 ( Dangelmaier
et al ., 2001 ). So far, three platelet ADP receptors have been
identified: P2Y 1 , P2Y 12 , and P2X 1 ( Table 10-2 ) ( Gachet,
2001 ). The central role of ADP in platelet function may
be due, at least in part, to its ability to directly cause an
elevation in intracellular calcium levels while simultaneously
stimulating two biochemical pathways, one of which
is mediated through G q /PLC-mediated reactions ( Table 10-
3 ) and the other through G i2 -mediated inhibition of adenylyl
cyclase. Co-activation of both G q and G i2 pathways,
through P2Y 1 and P2Y 12 , respectively, are essential for
normal ADP-induced aggregation ( Gachet, 2001 ). P2Y 1
and P2Y 12 are GPCRs that exhibit the usual seven hydrophobic
domains and are sometimes referred to as “ metabotropic.
” In addition to activating the PLC β isomer of PLC,
the P2Y 1 receptor induces phosphorylation of myosin light
chain kinase (MLCK), which permits reversible incorporation
of proteins, such as actin and talin, into the cytoskeleton
to promote shape change ( Savage et al ., 2001 ). This
cytoskeletal reorganization is accompanied by an increase
in the levels of the regulatory subunit of P13 kinase analogous
to that observed following activation of platelets by
thrombin ( Rittenhouse, 1996 ). When ADP interacts with
the P2Y 12 receptor it causes the heterodimeric G i protein
to disassociate into its α i2 and β γ subunits, which in turn
causes the inhibition of adenylyl cyclase and a reduction
in intracellular cAMP levels. These biochemical reactions
slow the rate of calcium removal from the cytosol, which
helps sustain the aggregation response. ADP also facilitates
the development of stable platelet-platelet aggregates
because activation of both the P2Y 1 and P2Y 12 receptors
enhances the number and clustering of binding sites for
fibrinogen on α IIb β 3 receptors ( Gachet, 2001 ). The P2X 1
receptor resembles a ligand-gated ion channel protein,
exhibits two hydrophobic domains, and is referred to as
“ ionotropic ” ( Gachet, 2001 ; Mills, 1996 ). ADP interacts
with its receptors as the anion ADP 3 – , indicating that positively
charged groups on the receptors are involved in
ligand binding. The major advances in knowledge of ADP
receptors have come from studies with human and mouse
platelets. It appears that at least the P2Y 1 and P2Y 12 receptors
may be ubiquitous, although the relative number and
sensitivity of receptors may differ ( Coomber et al ., 2006 ).
For example, compared to human platelets, the P2Y 12
receptor may have a more important role in equine platelets
than the P2Y 1 receptor ( Mateos-Trigos et al ., 2002 ).
Platelet-activating factor (PAF) is also a potent soluble
inducer of platelet aggregation but, unlike the universal
agonists thrombin and ADP, not all mammalian platelets
exhibit specific PAF receptors. Like human platelets, cow,
sheep, horse, pig, dog, cat, elephant, guinea pig, and rabbit
platelets respond to PAF at concentrations between 10 10
and 10 7 M (Kulikov and Muzya, 1998 ). Platelets from
mice, rabbits, and hamsters are unresponsive to PAF. PAF
is a proinflammatory lipid mediator with a unique 1-Oalkyl-glycerophospholipid
backbone that exhibits multiple
biological properties, including activation of platelets, neutrophils,
monocytes, and macrophages ( Chao and Olson,
1993 ; Prescott et al ., 2000 ). In PAF activated endothelial
cells and platelets, cytoplasmic PLA 2 simultaneously
releases AA and lyso-PAF that is converted to additional
PAF molecules by acetyl CoA lyso-PAF transferase ( Chao
and Olson, 1993 ; Honda et al ., 2002 ). In endothelial cells,
PAF is translocated from its site of synthesis in the endoplasmic
reticulum to the plasma membrane where the polar
head of the molecule is inserted into the outer leaflet and
serves as a juxtacrine ligand ( Honda et al ., 2002 ). In contrast,
PAF formed in platelets, from lyso-PAF, is released
into the circulation. PAF is degraded in the plasma by
removal of an acetyl group at the sn-2 position, regenerating
lyso-PAF and acetate. This reaction is catalyzed by a
PAF acetylhydrolase that circulates in association with lowdensity
and high-density lipoproteins ( Prescott et al ., 2000 ).
The PAF receptor (PAFR) possesses the typical structure of
the GPCRs. Therefore, PAF can induce intracellular protein
phosphorylation in activated platelets indirectly through
DAG stimulated protein kinase C activation, as well as
through activation of tyrosine kinases ( Table 10-2 ).
It is well established that platelets from various species
exhibit different sensitivities when exposed in vitro to a single
dose of a weak agonist such as serotonin, epinephrine,
or arachidonic acid ( Dodds, 1978 ; Meyers and Wardrop,
1991 , Pelagalli et al ., 2002 ). Serotonin causes only a weak
aggregation response in human, rabbit, cat, cow, sheep, and
horse platelets, whereas canine platelets are unresponsive
(Bailey et al ., 2000 ; Mischke and Schulze, 2004 ; Ogawa
et al ., 1998 ). As in mammalian platelets, serotonin is present
in thrombocytes of birds and reptiles ( Maurer-Spurej, 2005 ).
In these species serotonin acts as a vasoconstrictor and plays
a role in endothermic body temperature regulation through
its ability to regulate skin blood flow. It has been postulated
that serotonin released from activated platelets may also have
a role in mammalian thermoregulation ( Maurer-Spurej, 2005 ).
Canine and equine platelets are relatively unresponsive to
epinephrine alone (Mischke and Shulze, 2004; Segura et al .,
2005 ). The ability of epinephrine to act synergistically with
collagen is used to advantage for the assessment of platelet
function in whole blood with the PFA analyzer (see Section
III.B.2.c) . However, the response of both canine and equine
platelets is lower when collagen-epinephrine is used as the
agonist compared to collagen-ADP (Mischke and Shulze,
2004; Segura et al ., 2005 ).
c. Intracellular Signaling Mechanisms
The ligand binding of integrins and agonist binding to
G-protein coupled receptors initiate intracellular signaling