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

II. Mechanisms of Hemostasis
291
TABLE 10-3 Intracellular Activation Signaling
Pathways
Effector
Tyrosine and serine/
threonine kinases
(e.g., Src, Syk, Erk, PKC)
↑ intracellular calcium
PLC α , PLC β
IP 3
DAG
PLA 2
Major Actions
Enzyme activation including
PLC, PLA 2 , MLCK
Calmodulin mediated activation
of MLCK
Activation of PLC, PLA 2 , PKC
Activation of
phosphodiesterase → ↓ cAMP
Hydrolysis of PI to IP 3 and DAG
Activation of calciumdependent
ATPase → release of
calcium from dense granules
Activation of PKC → integrin
conformation changes →
↑ affinity of GPIIb-IIIa for
fibrinogen
Hydrolysis of PC → release of
AA and lyso-PAF
Abbreviations: AA, arachidonic acid; DAG, diacyl glycerol; IP 3 , inositol 3-phosphate;
MLCK, myosin light chain kinase; PAF, platelet activating factor; PI, phosphoinositol;
PKC, protein kinase C; PLA 2 , phospholipase A 2 ; PLC, phospholipase C.
2004 ; Shattil and Newman, 2004 ). Other examples of integrins
involved in cross-membrane signaling are the ephrins
that activate specific Eph kinases to ephrins, enhancing
platelet adhesion through phosphatidylinositol 3 (PI3)
kinase and protein kinase C (PKC) mediated mechanisms
(see Table 10-3 ) ( Prevost et al ., 2004 ). CD40, a phosphorylated
transmembrane glycoprotein, a member of the TNF
receptor superfamily, is constitutively expressed in platelets,
but it is only functional following platelet activation
by other agonists. It has been shown that CD40 participates
in the release of P selectin (CD62P) and other α -granule
constituents, following agonist stimulation of human platelets
( Inwald et al ., 2003 ).
Several members of the GPCR superfamily have been
identified as integral components of platelet membranes
( Coughlin, 2005b ). Platelet agonists bind to the surfaceaccessible
N-terminal domain of these proteins causing information
to be transmitted through a seven-transmembrane
domain. This induces a conformational change in the intracellular
C-terminal domain which, in turn, activates GPCR
proteins associated with the intracellular surface of the
receptor ( Hamm, 2001 ). GPCR proteins are heterotrimeric,
being composed of single α , β , and γ subunits, and are
classified into families according to the structure of their α
subunit. Members of four families have been identified in
human platelets. Activation of G s and G i proteins induces
an increase and decrease in intracellular cAMP levels,
respectively, activation of G q stimulates the β isoforms of
phospholipase C (PLC, see Section II.B.3.c), and activation
of the G 12 contributes to the regulation of the platelet actin
cytoskeleton through protein phosphorylation ( Brass, 2003 ).
Whether a similar array of G protein receptors is expressed
on other types of mammalian platelets has yet to be determined.
However, based on studies with murine platelets, it is
evident that the abundance of G protein types is necessary to
support the similarity of platelet responses to multiple dissimilar
agonists ( Yang et al ., 2002 ).
The most important group of platelet membrane lipids
involved in hemostasis are the phospholipids (phosphoglycerides),
which constitute 63% and 57% of the
total lipid content of pig and human platelets, respectively
( Gentry and Nyarko, 2000 ). The major phospholipids
involved in platelet function are phosphatidylserine (PS),
phosphatidyl ethanolamine (PE), phosphatidylcholine
(PC), sphingosine (SP), and phosphatidyl inositol (PI). PI
is the parent molecule for the downstream, or secondary
messenger, signal transducers inositol triphosphate (IP3)
and 1,2 diacylglycerol (DAG) ( Table 10-3 ), whereas PC is
hydrolyzed to arachidonic acid (AA) in activated platelets
( Nozawa et al ., 1991 ). In all species, AA serves as the precursor
of thromboxane A 2 (TXA 2 ) and prostaglandins, such
as prostacyclin (PGI 2 ), and serves as an autocrine agonist
in some species ( Gentry and Nyarko, 2000 ). In unstimulated
platelets the negatively charged PS and PE are found
predominantly on the intracellular side of the plasma membrane,
whereas the neutral phospholipids, PC and SP, are
localized to the outer leaflet. In response to the elevated
intracellular calcium levels that occur following platelet
activation, PS and PE are translocated to the outer leaflet
of the membrane. Here they congregate to form the lipid
platform that is essential for the localized platelet membrane
binding and proteolytic activity of two key enzyme
complexes in thrombin formation, namely the factor
VIIIa-IXa-Ca-PS (tenase) complex and the factor Va-IXa-
Ca-PS (prothrombinase) complex (see Section II.C.3).
Platelets contain three types of organelles, also known
as granules, in which they transport various specific hemostatic
and wound healing compounds around the body
( Tablin, 2000 ). Dense granules contain small nonprotein
molecules such as ADP, ATP, serotonin, Ca 2 , and Mg 2 .
The release of ADP and serotonin, from the first layer of
activated platelets that adhere to a site of vascular damage,
is important in the recruitment of additional platelets and
the development of the platelet plug ( Fig. 10-1 ) α-granules,
the largest granule population, contain a variety of proteins
such as albumin, fibronectin, thrombospondin, and the
hemostatic proteins, fibrinogen, and FV ( Polasek, 2004 ).
They also contain platelet-specific proteins, including
β -thromboglobulin and platelet factor 4, platelet derived
growth factor (PDGF), epidermal growth factor (EGF),
transforming growth factor β (TGFβ ), and endothelial cell
growth factor (ECGF) ( Rendu and Brohard-Bohn, 2001 ).