Page 1996 - Williams Hematology ( PDFDrive )

1970 Part XII: Hemostasis and Thrombosis Chapter 115: Vascular Function In Hemostasis 1971

converted to citrulline and NO. The endothelial cell isoform of NO syn- and subsequent platelet aggregation. There is a broad spectrum of other
thase (eNOS or the NOS3 gene product) functions constitutively, and is effects of NO, including inhibition of leukocyte adhesion to endothelial
further activated by receptor-agonists that elevate intracellular calcium. cell surfaces, inhibition of smooth muscle migration, and reduction of
Major stimuli include ADP, thrombin, bradykinin, and shear stress. smooth muscle cell proliferation. These phenomena suggest that secre-
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Shear forces induce transcriptional activation of the eNOS gene because tion of NO into the microenvironment is a major component of the
its promoter contains a shear response consensus sequence (GAGACC). response to vascular injury 43
The NO that forms activates guanylate cyclase, thereby generating cyclic
GMP. NO becomes oxidized to nitrite and then to nitrate, which is mea- INHIBITION OF PLATELET ACTIVATION
surable in blood samples. NO in the circulation is rapidly inactivated
by erythrocytes. 11,47,48 NO has a vasodilatory effect on the pulmonary AND RECRUITMENT BY ECTO-ATP/
vasculature, and, in patients with congestive heart failure, its inhala-
tion decreases pulmonary hypertension and increases pulmonary ven- DASE1-CD39
tilation. 10,11,47–54 Acetylcholine released by activated nerve terminals in In addition to the platelet inhibition by PGI and NO, endothelial cells
2
the vessel wall activate the endothelial cell to produce and release NO. inhibit platelet function via the action of endothelial cell ecto-ATP/
This NO effect also explains the action of nitroglycerin, which has long Dase-1/CD39, an ecto-apyrase with ADPase and adenosine triphos-
been used to treat patients with angina resulting from coronary artery phatase (ATPase) activities. The cluster designation symbol for this
disease. 54 compound is CD39, the product of the ENTPD1, ectonucleotide tri-
Importantly, production of NO by endothelial cells is impaired in phosphate diphosphohydrolase gene. CD39 is localized mainly in
55
the presence of the thiol-containing amino acid, homocysteine. Cyn- endothelial cells and leukocytes. In endothelial cells, CD39 is located on
omolgus monkeys with diet-induced hyperhomocysteinemia demon- the cell surface with the major portion of the molecule facing the vessel
strated reduced blood flow in the lower extremity and an impaired lumen. 12,13,56 The enzyme has both N- and C-terminal transmembrane
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response to endothelial cell-dependent vasodilators. Similarly, pro- regions with small cytosolic portions anchoring the molecule. In addi-
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duction of NO by endothelial cells in vitro is significantly inhibited tion to CD39, CD73 (5’-nucleotidase) is present on vascular cells and
in the presence of homocysteine, possibly by a mechanism involving converts the adenosine monophosphate (AMP) generated from CD39
impairment of the enzyme glutathione peroxidase. 52,53 metabolism to adenosine (Fig. 115–5). In contrast to all other known
platelet inhibitors, acting in concert with CD73, CD39 can convert the
STRUCTURE AND BIOCHEMICAL PROPERTIES local environment from a prothrombotic ADP/ATP-rich entity to an
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OF NITRIC OXIDE SYNTHASE antithrombotic adenosine-rich environment. This phenomenon was
evident from observations that platelets became unresponsive to all ago-
There are two isoforms of NOS, the constitutive form (eNOS), syn- nists when in motion or in proximity to endothelial cells, even when
thesized by the endothelial cell and regulated by Ca and calmod- eicosanoid and NO production were blocked. Importantly, CD39 and
2+
2
ulin, and the cytokine-inducible, posttranscriptionally regulated form CD73 do not exert their action on the platelet per se but act in series to
47
(iNOS) . Both constitutive and inducible forms are mainly cytosolic, metabolize ATP and ADP secreted from activated platelets to AMP and
although a membrane-bound constitutive NOS isoform containing a hence to adenosine. 13,59 ADP released from activated platelets is metab-
myristoylation consensus sequence has been isolated from bovine aor- olized by CD39, thereby inhibiting ADP-induced platelet activation,
43
tic endothelial cells. eNOS has a molecular mass of 144 kDa and shares release and aggregation (Fig. 115–5).
57 percent amino acid sequence identity with neuronal NOS. The cofactor Most platelet agonists initiate secretion of dense granule contents
(6R-tetrahydro-L-biopterin [H B]) participates in inducible and con- within 15 to 20 seconds. The enhanced metabolism of ATP and ADP by
4
stitutive NOS isoform reactions. It is thought that H B stabilizes the therapeutically administered soluble CD39 would also reduce second-
4
enzyme in a manner allowing for maximum activity of the NOS subunit ary autoamplification and recruitment, and, consequently, thrombus
to which the pterin binds. 10,11,47,54 formation. 9,27,60 Because CD39 and CD73 are probably acting together,
they will theoretically increase levels of endogenous adenosine and
BLOCKADE OF PLATELET AGGREGATION AND elevate the threshold for platelet activation in the local microenviron-
SECRETION BY NITRIC OXIDE ment. In a murine model, soluble CD39 administration ameliorates
the extent of stroke and reverses excessive platelet reactivity without
Platelet activation and recruitment in response to all agonists, such as bleeding complications, even if administered 3 hours following stroke
ADP, collagen, epinephrine, and thrombin, is blocked by NO. Blockade induction. Therapeutic benefit of soluble CD39 has also been demon-
61
10
also occurs in vivo via formation of NO from endothelium. Impor- strated in animal models of cardiac ischemia, in the development of
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tantly, the inhibitory action of NO is not affected by aspirin either in atherosclerosis, regulation of leukocyte proinflammatory activity,
vivo or ex vivo. Therefore, NO production is not caused by participation inhibition of metastasis, and in transplantation medicine. That the
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of endothelial cell eicosanoids. preclinical therapeutic use of soluble CD39 could abrogate thrombosis
In addition to eNOS, the NOS3 gene product, endothelial cells without inducing the hemorrhage seen with the use of existing anti-
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stimulated by agonists such as cytokines express the inducible form of platelet therapies could provide a therapeutic advantage over existing
NO synthase, iNOS, the NOS2 gene product. Through this mechanism, therapies for thrombotic disorders, including those who are resistant to
9
NO can further inhibit platelet reactivity and reduce basal vessel tone by existing therapeutic paradigms. CD39 represents a major control sys-
inducing relaxation of vascular smooth muscle. The biochemical basis tem for blood fluidity. 68
for the reaction is that NO binds to the heme prosthetic group of gua-
nylyl cyclase. The inhibitory effect of NO on platelet activation can be THE PROTEIN C PATHWAY
monitored by measuring surface expression of P-selectin. The ability of
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NO to inhibit mobilization of intracellular platelet calcium results in The protein C pathway plays a critical role in the prevention of throm-
reduction of the conformational changes in platelet membrane glyco- bosis and is an integral part of the host inflammatory response. This
protein (GP)IIb/IIIa, an absolute requirement for fibrinogen binding pathway is initiated on the endothelial cell surface when thrombin

Kaushansky_chapter 115_p1967-1984.indd 1971 9/18/15 10:08 AM