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

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Chapter | 10 Hemostasis
TABLE 10-4 Inhibitory Intracellular Signaling
Mechanisms
Effector
PGI 2
cAMP
NO/cGMP/cGKI
PECAM-1
Major Actions
↑ adenylyl cyclase activity → ↑ cAMP
Inhibition of PI hydrolysis → ↓ IP 3 and ↓ DAG
↓ free cytosolic calcium
Inhibition of PI hydrolysis
Suppression of adhesion and aggregation
responses
Inhibition of ADP2Y 12 receptor activation
Inhibition of GPIIb-IIIa receptor activity
Inhibition of collagen stimulated calcium
Mobilization, granule secretion, and
aggregation
Abbreviations: DAG, diacyl glycerol; GKI, GMP-dependent protein kinase; IP 3 ,
inositol 3-phosphate; NO, nitric oxide; PECAM, platelet endothelial cell adhesion
molecule; PGI 2 , prostacyclin; PI, phosphoinositol.
that prevent unwarranted platelet activation and limit the
extent of aggregation. Most of the important physiological
platelet inhibitors are endothelial-derived factors and their
mediators ( Table 10-4 ). The PGI 2 /cAMP and nitric oxide
(NO)/cGMP inhibitory pathways have multiple synergistic
interactions with respect to cyclic nucleotide generation/degeneration
and protein phosphorylation in platelets
( Schwarz et al ., 2001 ).
Eicosanoids, such as PGI 2 and TXA 2 , are autocoids that
only influence the activity of cells in the proximity of the
producing cell. Both compounds are metabolites of AA,
and whereas TXA 2 is the major product in platelets, PGI 2
is the major end product in endothelial cells after stimulation
by agonists such as thrombin, ADP, and inflammatory
mediators. PGI 2 acts as a potent platelet inhibitor,
whereas enhancement of aggregation occurs in response to
TXA 2 ( deGroot and Sixma, 1996 ). To a great extent, platelet
function is regulated by the intracellular concentration
of cAMP. In platelets, as in other cells, cAMP formation
is regulated bimodally by GPCRs that stimulate or inhibit
adenylyl cyclase ( Tables 10-2 and 10-3 ). PGI 2 suppresses
intracellular signaling in activated platelets by stimulating
adenylyl cyclase formation via G s -coupled IP receptors
( Moncada and Vane, 1976 ). This results in the activation
of a cAMP-dependent protein kinase (PKA) that stimulates
the phosphorylation of a low-molecular-weight GTPbinding
protein, rap 1B, which subsequently dissociates
from the platelet membrane and inhibits PLC membrane
binding. The net effect of this response is to suppress PI
hydrolysis and reduce intracellular levels of IP 3 and DAG
( Table 10-4 ). In addition to these effects on phospholipid
metabolism, elevated cAMP levels reduce platelet reactivity
through a number of mechanisms. These include a
reduction in GPIIb-IIIa expression and a reduction in the
magnitude and duration of elevated cytosolic-free calcium
after platelet activation ( Gentry, 2000b ).
Human platelets generate cGMP through the soluble
NO-activated guanylyl cyclase enzyme and degrade it
through the phosphodiesterase (PDE 2 and 5) ( Feil et al .,
2003 ). The inhibitor effects of cGMP on platelet adhesion/activation
are mediated through a cGMP-dependent
protein kinase, cGKI ( Table 10-4 ). The isomer present
in platelets is cGKI β , a homodimer consisting of two
75-kDa subunits. Each subunit is composed of an N-terminal
domain that suppresses kinase activity in the absence of
cGMP, a regulatory domain that contains two nonidentical
cGMP-binding sites, and a kinase domain that catalyzes
the transfer of the γ phosphate of ATP to the hyroxyl group
of a serine/threonine side chain of the target protein ( Feil
et al ., 2003 ). One of the ways in which cGKI contributes
to a negative feedback response is through the inhibition
of the G q /G i -coupled receptor responses, particularly in
response to activation of the ADP-P2Y 12 receptor ( Atkas
et al ., 2002 ). Another negative feedback response is initiated
by cGKI-mediated phosphorylation of platelet proteins,
such as vasodilator-stimulated phosphoprotein (VASP),
and is correlated with inhibition of the GPIIb-IIIa-fibrinogen
receptor ( Feil et al ., 2003 ). Studies with cGKI-deficient
mice have shown that platelet cGKI, but not endothelial or
smooth muscle cGKI, plays an important role in the prevention
of platelet adhesion and aggregation in the microcirculation
after ischemia ( Massberg et al ., 1999 ).
Platelet endothelial cell adhesion molecule (PECAM-1),
also known as CD31, is a 130-kDa type 1 transmembrane
glycoprotein whose function is regulated by phosphorylation
of serine residues in the cytoplasmic domain ( Newman
and Newman, 2003 ). Based on cDNA sequencing, there
appears to be considerable homology in PECAM-1 across
species. Phosphorylated PECAM-1 serine residues have
been detected in resting platelets, and their level of phosphorylation
increases two- to three-fold in collagen and
thrombin-activated platelets. PKC is thought to play a primary
role in this phosphorylation process, although Src
kinases may also be involved ( Edmead et al ., 1999 ). The
presence of two distinct immunoreceptor tyrosine-based
inhibitory motifs (ITIMs) in the cytoplasmic domain characterize
PECAM-1 as belonging to the Ig-ITIM family of
inhibitory receptors ( Newman, 1999 ). ITIM-containing
inhibitory receptors are thought to function primarily by
counteracting signal transduction pathways initiated by
receptors via their immunoreceptor tyrosine-based activation
motifs, such as the FcR γ chain that is associated with
the GPVI collagen receptor ( Ravetch and Lanier, 2000 ).
PECAM-1 is capable of inhibiting calcium mobilization,
granule secretion, and aggregation following collagen activation
of the GPVI/FcR γ chain complexes ( Table 10-4 )
( Newman and Newman, 2003 ). It is important to note, however,
that PECAM-1 inhibitory signaling can be overcome