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

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Chapter | 10 Hemostasis
The proteins stored in the α -granules are either synthesized
by megakaryocytes or are endocytosed by platelets
as they circulate around the body ( Reed, 2004 ). Like
other endocytotic cell types, platelets contain lysosomes
that release hydrolases. It has been suggested that these
hydrolases assist in the elimination of circulating platelet
aggregates to prevent inappropriate thrombus formation
( Rendu and Brohard-Bohn, 2001 ). Mitochondria and
cytoplasmic glycogen stores provide the energy to support
the granule exocytotic secretory process in activated
platelets ( Flaumenhaft, 2003 ). This exocytosis process
involves contraction of the actin-myosin cytoskeleton and
the apposition and fusion of each granule membrane with
the platelet plasma membrane ( Escolar and White, 1991 ;
Reed, 2004 ; Rendu and Brohard-Bohn, 2001 ). In addition
to the extrusion of granule contents, the alteration in
platelet morphology results in the exposure of both GPIIb-
IIIa integrin complexes and membrane markers, such as
CD62 and CD63, on the surface of the activated platelets
( Polasek, 2004 ). CD62, also referred to as P-selectin, has
been used as a marker of activated canine platelets ( Moritz
et al ., 2003a, 2003b ).
3. Platelet Activation
a. Immobilized Agonists
When the ECM is damaged, various macromolecules interact
with platelets as they roll along the exposed subendothelial
surface collagen. Of these, collagen-bound vWF is
considered to be the most important for the initial tethering
and adhesion of platelets to areas of vascular damage
( Nieswandt and Watson, 2003 ). This occurs through the
interaction of collagen-bound vWF with the GPIb-IX-V
integrin complex. However, at the medium and high shear
rates found in arteries and arterioles, this reaction is reversible
and must be followed by the more stable binding of
collagen to its specific platelet receptors α 2 β 1 and GPVI
( Table 10-2 ). The initial interaction between platelets and
the ECM through vWF-GPIb-IX-V binding is important
because it induces activation of intracellular tyrosine
kinases, causing increased affinity of α 2 β 1 for collagen
binding as well as an increase in the affinity of GPIIb-IIIa
for a variety of ligands including fibrinogen, vWF, fibronectin,
vitronectin, and thrombospondin ( Calvete, 2004 ).
The two collagen receptors, α 2 β 1 and GPVI, are thought
to have complementary functions in platelet adhesion
( Andrews and Berndt, 2004 ). In human platelets, α 2 β 1
binds collagen in an Mg 2 -dependent manner, tethering
platelets to the ECM before platelet activation. At the
same time, collagen- α 2 β 1 binding facilitates the interaction
between collagen and GPVI in a nontyrosine kinasedependent
reaction. GPVI is currently considered to be the
major collagen receptor because it initiates shape change,
activation, and secretion reactions following platelet adhesion
( Nieswandt and Watson, 2003 ). In mouse platelets,
it appears that GPVI provides the primary collagen signal
that activates and recruits α 2 β 1 integrins to the surface of
the platelet so that collagen signaling is amplified ( Chen
and Kahn, 2003 ). One of the important intracellular pathways
activated as a result of collagen binding to GPVI is
the PLC pathway, specifically the activation of the PLC γ 2
isomer ( Savage et al ., 2001 ).
As platelets adhere to collagen and receptor activation
occurs, they lose their resting discoid shape and extend
pseudopods as they spread over the endothelial surface
( Escolar and White, 1991 ; Gentry, 2000b ). This increases
the surface area of the platelets, which, coupled with the
clustering of integrin molecules on platelet membranes and
the increase in integrin affinity, results in the formation
of irreversible platelet-endothelial binding via vWF and
the GPIb-IX-V receptors and platelet-platelet interactions
via vWF and fibrinogen binding to GPIIb-IIIa receptors
( Boudreaux, 1996 ; Savage et al ., 2001 ). The GPIb-IX-V
receptor is a sialoglycoprotein-rich complex that contributes
to the net negative charge on the platelet surface. GPIb
consists of two disulfide-linked subunits, GPIb α and GPIb β ,
that are associated with GPIX in a 1:1 complex ( Lopez and
Dong, 1997 ). In mouse platelets, the activation function
of vWF binding to GPIb-IX-V is, like collagen binding to
the GPVI receptor, dependent on tyrosine phosphorylation
( Shattil and Newman, 2004 ). This phosphorylation activity
is dependent on the FcR, a molecule that associates noncovalently
with GPVI and GPIb-IX-V. FcR γ is a component
of the multisubunit high-affinity receptor for immunoglobulin
(IgE), and its tyrosine-phosphorylated active motif
helps to recruit tyrosine kinases to the glycoprotein receptors.
The binding site for vWF on the receptor complex is
localized to a site on the GPIb β chain, which can also serve
as a thrombin receptor. The outside-in signaling events
that ensue from vWF-GPIb β binding include activation
of protein kinase C (PKC), protein kinase G (PKG) and
phosphoinositol 3 kinase (PI3K), elevation of intracellular
Ca 2 , and rearrangement of the cytoskeleton ( Gibbins,
2004 ). These reactions complete the irreversible adhesion
of the activated platelet monolayer to the damaged endothelium.
As part of the defense mechanism against inappropriate
platelet adhesion and aggregation, GPIb-IX-V
receptors only bind vWF immobilized on collagen and not
soluble vWF, except under conditions of high shear flow
rates ( Lopez and Dong, 1997 ).
The continued growth of the platelet plug requires
platelet-platelet adhesion that is accomplished through a
process described as “ contact-dependent ” signaling and
involves the expression and activation of GPIIb-IIIa receptors
( Shattil and Newman, 2004 ). GPIIb-IIIa is the most
abundant integrin on the platelet surface and is not only
necessary for platelet-platelet aggregation but also for
platelet secretion, the development of procoagulant activity,
and clot retraction. This integrin is typical of type 1
membrane receptors in that it contains a relatively large