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

II. Mechanisms of Hemostasis
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globular external domain, formed by the association of
the N-terminal ends of both the α and β subunits, a single
pass transmembrane domain and short C-terminal cytoplasmic
tails composed of 20 to 60 amino acids ( Hynes,
2002 ). The ligand binding sites contained in the external
domain are converted to a high-affinity state through several
inside-out signaling mechanisms. Among these signals
are the interaction of the α and β subunit cytoplasmic
tails with cytoskeleton proteins, actin and talin, inducing
the formation of larger actin-based signaling complexes
that are essential for granule migration and the fusion of
granule and plasma membranes that precede the secretion
of granule contents ( Williams et al ., 1995 ). Fibrinogen is
the primary hemostatic ligand for GPIIb-IIIa, and fibrinogen
bridges between platelets are the backbone of stable
platelet aggregates. Other plasma proteins, including vWF,
fibronectin, vitronectin, and thrombospondin, that have an
RGD sequence similar to that present in fibrinogen can
also bind to the α subunit of the GPIIb-IIIa. Fibrinogen
has two RGD sequences per monomer, but it can also bind
to GPIIb-IIIa through a recognition site involving residues
400 to 411 on its γ chain ( Calvete, 2004 ; Williams et al .,
1995 ). Occupancy of GPIIb-IIIa binding sites with fibrinogen
up-regulates this integrin by inducing microclustering,
as well as initiating downstream signaling through activation
of Src and Syk protein kinases ( Tables 10-2 and 10-3 )
( Shattil and Newman, 2004 ).
It is now recognized that GPIIb-IIIa plays a continuing
role in platelet function after a thrombus has formed
through its role in clot retraction ( Osdoit and Rosa, 2001 ).
It has been postulated that one of the reasons platelet-rich
clots are more resistant to thrombolysis than platelet-poor
clots is related to the lower affinity of tissue plasminogen
activator (tPA) for the retracted fibrin fibers of a platelet-rich
clot compared to the less retracted fibrin fibers
of a platelet-poor clot ( Collet et al ., 2002 ) (see Section
II.D.2.b). Although the biochemical mechanisms involved
in clot retraction are poorly understood, studies with mouse
platelets have shown that after the initial wave of tyrosine
phosphorylation initiated by activation of the β 3 cytoplasmic
tail of the GPIIb-IIIa receptor, a sustained GPIIb-
IIIa-dependent tyrosine dephosphorylation of several
polypeptides occurs ( Osdoit and Rosa, 2001 ). This dephosphorylation
response causes actin-dependent changes in
the cytoskeleton that result in shrinkage in the size of the
platelet-fibrin mass.
b. Soluble Agonists
In addition to immobilized collagen and vWF on the surface
of damaged ECM, there are several soluble mediators
that act as potent platelet agonists in the aggregation of
mammalian platelets ( Gentry, 2000b ). The local accumulation
of thrombin, generated from TF expression on the surface
of damaged endothelial cells (see Section II.C.2), and
ADP, released from α -granule stores in activated platelets,
is essential for the growth of the primary hemostatic plug
on top of the initial monolayer of collagen-bound platelets.
Thrombin and ADP, and in some species TXA 2 , induce
similar platelet-platelet aggregation formation through activation
of the α IIb β 3 receptors ( Brass, 2003 ).
The major pathways of thrombin-induced activation
result from hydrolysis of specific thrombin substrates, protease-activated
receptors-1 and -4 (PAR-1, PAR-4), that are
members of the G-protein coupled seven transmembrane
domain receptor family ( Major et al ., 2003 ). To activate
these receptors, thrombin cleaves the N-terminus, exposing
a new N-terminus that serves as a “ tethered-ligand ” and
binds to the extracellular-2 domain of the cleaved receptor
( Dugina et al ., 2002 ). A highly effective local concentration
of this ligand is present, as it is not free to diffuse
away from the platelet surface ( Brass, 2003 ). Activation
of PAR-1 and PAR-4 causes a downstream activation
of G q , G 12 , and G i that, in turn, leads to the activation of
the β isomer of PLC, PI3-kinase, and the inhibition of
adenylyl cyclase, respectively ( Table 10-2 ) ( Grand et al .,
1996 ). Studies on PAR receptors to date have focused on
human and murine platelets, and results have revealed distinct
differences in PAR receptor expression. In human
platelets, activation of PAR-4 requires 10- to 100-fold
higher concentrations of thrombin than PAR-1. Hence,
PAR-1 is considered to be the more important thrombin
receptor ( Kahn et al ., 1998 ). This difference in receptor
sensitivity may be related to the hirudin-like sequences
in PAR-1, but not PAR-4, that facilitate receptor cleavage
by thrombin. In contrast, PAR-4 is the primary thrombin
receptor in mouse platelets ( Ishihara et al ., 1997 ). In
this species the cleavage of PAR-4 is facilitated by PAR-
3 receptors. PAR-3 is the only member of the PAR family
that does not possess an activating peptide, and, hence, it
cannot directly activate platelets. PAR-2, the fourth member
of the PAR family to be identified, is not activated by
thrombin but by other serine proteases such as trypsin, TF,
and FXa ( Camerer et al ., 2000 ). The PAR-2 receptor has
not been found on platelets but is present in a number of
other cell types, including endothelial cells. The potency
of thrombin as an agonist may also be related to its ability
to interact not only with PARs but also non-PARs, particularly
the GPIb α component of the GPIb-IX-V complex
( Soslau et al ., 2001 ). It has been suggested that, in human
platelets, binding of thrombin to GPIb α may facilitate
PAR-1 cleavage in a manner analogous to the role of PAR-
3 in mouse platelets ( DeCandia et al ., 2001 ).
ADP was the first low-molecular-weight platelet-aggregating
agent to be discovered and, like thrombin, is recognized
as a universal agonist. It is stored in dense granules,
in near molar amounts, and can be released not only from
this source but also from damaged endothelial cells and
red blood cells at sites of vascular damage ( Gachet, 2001 ).
ADP induces a broad range of biochemical changes in
platelets. By itself, it is a relatively weak agonist, but it