Inhibition of Platelet Function by the Endothelium

C H A P T E R

17 Inhibition of Platelet Function by the Endothelium Lea M. Beaulieu and Jane E. Freedman Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts

I. INTRODUCTION Cell
cell interactions, environmental factors, and cytokine/hormone concentrations play a major role in modulating the process of hemostasis and thrombus formation after vessel injury. A fine balance between prothrombotic and antithrombotic processes must be achieved to prevent either hemorrhage or thrombosis. During the activation, adhesion, and aggregation of platelets at the site of injury, the endothelium responds by limiting the size and growth of the hemostatic plug or thrombus or even reversing platelet reactivity. These responses are referred to as endothelial thromboregulation1
3 and require several forms of communication between endothelial cells and platelets. There are three primary (and functionally independent) pathways during the early stages of thromboregulation (Fig. 17-1 and Table 17-1):1
4 (1) nitric oxide (NO); (2) the eicosanoid prostacyclin; and (3) the ectonucleotidase CD39. In this chapter, these three thromboregulatory mechanisms are discussed in detail. In particular, this chapter highlights the enzymes, receptors, and signaling pathways involved and discusses animal models, clinical investigations, and the role of these pathways in disease development, including relevant genetic variants.

II. NITRIC OXIDE A. Biosynthesis of NO and Characteristics of Endothelial NO Synthase NO, a well-characterized platelet inhibitor and vasodilator, was first discovered in endothelial cells by Furchgott and Zawadzki in 1980.5 NO can exist in

Platelets, 3rd edition

three interrelated redox forms that expand the biological spectrum of NO: the free radical NO, the nitrosonium anion (NO1 ), and the nitrosyl anion (NO
).6,7 NO is inactivated by superoxide8 but stabilized by superoxide dismutase.9,10 It can also bind to heme-containing proteins, such as hemoglobin, to activate or inhibit their enzymatic activity.7 The biosynthesis of NO is carried out by a family of enzymes called nitric oxide synthases (NOS). The family consists of three members: endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS). Several recent reports also describe the existence of a distinct form of mitochondrial NOS (mtNOS).11
14 eNOS is expressed in endothelial cells,15,16 cardiomyocytes,17 bronchiolar epithelial cells,18,19 megakaryocytes, and platelets.20,21 The basal level of NO released from platelets has been reported to be similar to that from endothelial cells.22 eNOS is composed of an N-terminal oxygenase domain and a C-terminal reductase domain. The oxygenase domain—which contains binding sites for heme, tetrahydrobiopterin (BH4), and the substrate L-arginine—is linked by a calmodulin-recognition site to the reductase domain, which has binding sites for flavin adenine dinucleotide, flavin mononucleotide, and NADPH.23 The dimerization interface includes binding sites for BH4 and the Zn21-binding heme,24
26 which is mandatory for dimerization. An “N-terminal hook” domain,26 as well as the presence of L-arginine, further stabilize the dimerization process.23 eNOS catalyzes the multi-electron oxidation reaction of L-arginine with oxygen in a Ca21-dependent manner, forming L-citrulline and releasing NO.27 Intracellular localization, posttranslational modifications, and enzymatic activity of eNOS are tightly

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© 2013 Elsevier Inc. All rights reserved.

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17. INHIBITION OF PLATELET FUNCTION BY THE ENDOTHELIUM

Nitric Oxide

Prostacyclin

cGMP sGC cAMP

CD39

cAMP IP receptor

NO

PGI2 eNOS

cAMP A2 Receptor ADP

Adenosine AMP

CD39

eNOS

CD73

PGIS PGIS

ER

FIGURE 17-1 Overview of the 3 mechanisms utilized by endothelial cells to inhibit platelets. Nitric Oxide: Endothelial nitric oxide synthase (eNOS), located in the caveolae, releases nitric oxide (NO). Upon diffusion across the platelet plasma membrane, NO stimulates soluble guanylyl cyclase (sGC), which leads to the upregulation of cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP). Activated platelets will also produce NO through eNOS expressed on their cell surface. Prostacyclin: Prostacyclin synthase (PGIS), located on the endoplasmic reticulum (ER) and in the caveolae of the endothelial cells, releases prostacyclin (PGI2). On the platelet cell surface, prostacyclin receptor (IP receptor) binds PGI2, which leads to the increase in cellular cAMP. CD39: Adenosine diphosphate (ADP) that is released from activated platelets is hydrolyzed into adenosine monophosphate (AMP) on the endothelial cell surface by CD39. AMP is then converted into adenosine by CD73, also present on the endothelial cell surface. Adenosine will bind the platelet adenosine receptor (A2 receptor) and increase cAMP levels. All of these processes will lead to the inhibition of platelet aggregation and adhesion, controlling the size of the clot to allow for proper blood flow and healing of the endothelium.

regulated. In the endoplasmic reticulum, eNOS becomes irreversibly myristoylated at the N-terminal residue Gly2, whereas reversible palmitoylation at residues Cys15 and Cys26 takes place in the Golgi apparatus. Both acylation processes are crucial for the optimal targeting of eNOS to the plasma membrane.28
30 In the plasma membrane, eNOS is found to be highly enriched within caveolae,31 which are specialized membrane invaginations rich in signaling molecules and coated with the protein caveolin.32,33 The enzymatic activity is sevenfold higher in membrane-associated eNOS than cytosolic.33 In the plasma membrane, its activity is about tenfold greater in the caveolae fraction as compared to the whole plasma membrane.33 It has been reported that eNOS binds to the scaffolding domain of caveolin, which has an inhibitory effect on eNOS activity in vivo.34 The activity of eNOS is regulated by several other mechanisms, such as changes in intracellular calcium concentration, phosphorylation, and dephosphorylation at various tyrosine, serine and

threonine residues, as well as the association or dissociation of eNOS-interacting proteins (reviewed in Refs. 23, 30, and 35). Mechanical stimuli, such as shear flow in the blood vessel, can activate eNOS36 by Akt-mediated phosphorylation at Ser1179,37,38 leading to NO production and vasodilation. Estrogen also affects eNOS function. 17β-estradiol increases eNOS expression and activity (reviewed in Ref. 39), which can be inhibited by progestins40 and medroxyprogesterone acetate.41 Estrogen replacement therapy should therefore be beneficial by increasing eNOS levels and NO production. However, clinical studies have shown that there is an increased risk for DVT in postmenopausal women under hormone replacement therapy. A new aminoestrogen with anticoagulant properties, prolame, has been tested in vitro and in mouse models and has shown to induce NO production and reduced thrombi production in a thrombosis model.42 Conversely, under conditions such as the absence of L-arginine or BH4, or in the presence of the eNOS

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TABLE 17-1

Endothelial-derived Mediators of Platelet Activation

Class of Thromboregulator

Nitrovasodilator

Eicosanoid

Ecto-nucleotidase

Type of Thromboregulator

NO

Prostacyclin

CD39 and CD73

Site of Action

Released into blood stream

Released into blood stream

ADP/ATP removed from blood stream; adenosine released into blood stream

Levels

Basal levels, increased upon stimulation

Induced upon stimulation

Basal levels

Endothelial Thromboregulating Enzyme

eNOS

Prostacyclin synthase (PGIS)

CD39, CD73

Platelet Receptor for Endothelial Thromboregulator

NO diffusion across membrane, sGC

Prostacyclin receptor (IP receptor)

Adenosine receptor (A2)

Thromboregulatory Mechanism to Inhibit Platelet Reactivity

Upregulation of platelet cGMP production

Upregulation of platelet cAMP production

Enzymatic removal of ADP secreted from activated platelets; adenosine-induced upregulation of platelet cAMP

Platelet “Auto-regulation”

Platelet eNOS (NO production), superoxide, ROS

TXA2 synthesis

ADP/ATP purinergic receptors; CD39 (low expression)

Sensitivity to Aspirin

No

Yes

No

Abbreviations: ADP, adenosine diphosphate; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine 5’-monophosphate; eNOS, endothelial nitric oxide synthase; NO, nitric oxide; PGI2, prostaglandin I2; ROS, reactive oxygen species; sGC, soluble guanylyl cyclase; TXA2, thromboxane A2.

inhibitor NG-monomethyl-L-arginine (L-NMMA), eNOS can undergo a process called eNOS uncoupling whereby eNOS catalyzes an uncoupled NADPH oxidation leading to the formation of superoxide, instead of NO.43
45 Superoxide can react very rapidly with NO, forming the powerful oxidant peroxynitrite,46 which can then readily modify protein residues and lipid moieties.8,47

B. The Effect of Endothelial and Platelet NO on Platelet Reactivity NO has both antithrombotic and vasodilatory effects. In the nondiseased blood vessel, the intact endothelium releases NO to inhibit platelet adhesion to the endothelium48,49 and platelet aggregation.50
53 In addition, the vasodilatory effect of NO leads to smooth muscle relaxation. NO inhibits platelet activity primarily by binding to the heme-containing enzyme soluble guanylyl cyclase (sGC), which triggers a conformational change that increases its catalytic activity.54 Intracellular cyclic guanosine 50 -monophosphate (cGMP) levels are rapidly increased by sGC,55,56 affecting multiple signaling pathways,57,58 including cGMP-dependent receptor proteins, cGMP-regulated phosphodiesterases (PDE), and cGMP-dependent protein kinases.59 The increase in cGMP levels is accompanied by a decrease in intracellular Ca21 flux60,61 caused by activation of cGMP-dependent protein kinase G.7,62 Ca21 levels are

furthered lowered by a decreased rate of Ca21 entry from the extracellular environment and inhibition of receptor-mediated Ca21 release from the dense tubular system.63 The decrease in Ca21 levels inhibits the conformational change of GPIIb-IIIa into its active form64 and thus decreases platelet association with fibrinogen.65 The binding affinity of GPIIb-IIIa for fibrinogen is also decreased by cGMP-dependent inhibition of phosphoinositide 3-kinase activation (PI3K)66 and by cGMP-dependent phosphorylation of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) at Ser157.67 In addition, stimulation of cGMP-dependent protein kinases leads to the inhibition of phospholipase A2- and C-mediated responses,68 such as the inhibition of arachidonic acid release and inhibition of phospholipase C/G protein/receptor coupling.62 Thromboxane (TXA2) receptor becomes phosphorylated by cGMPdependent protein kinase and is unable to mediate platelet activation.69 Another cGMP-dependent mechanism preventing platelet adhesion to the endothelium is the downregulation of P-selectin expression by platelet NOS via an inhibitory effect on protein kinase C (PKC).70 NO-mediated platelet inhibition can also occur through cGMP-independent pathways. Such pathways include the modification of cellular or plasma proteins by S-nitrosylation of cysteine residues forming S-nitrosothiols.58,71,72 Transnitrosation reactions between protein thiol-bound NO and low-molecular-weight thiolbound NO occur in vivo.73,74 The bioavailability of albumin thiols, or low-molecular-weight thiols such as

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glutathione, was found to prolong the antiplatelet action of endothelial NO.75,76 Extracellular NO in the nanomolar range is required for cGMP-independent inhibition of platelet activation. Plasma components may play an important role in the activation of cGMP-independent signaling by S-nitrosothiols or peroxynitrite generators.77 NO is important not only for inhibiting platelet aggregation and adhesion but also for modulating platelet function itself. Platelet-derived NO is released both at rest22 and during aggregation78
80 and has been linked to important autoregulatory functions in platelet reactivity. The NO released from platelets in the resting state is in the nanomolar range.22 Shortly after activation, platelets release a large amount of NO (in the micromolar range) to prevent further platelet aggregation and adhesion in the growing thrombus.80,81 SHP-1 protein tyrosine phosphatase associates with eNOS in resting platelets. Upon activation, SHP-1 dephosphorylates eNOS, which leads to increased NO synthesis.82 Resveratrol, a main polyphenol in wine, has been shown to increase NO in platelets, in vitro, through protein kinase B.83 Estrogen has also been shown to regulate platelet NO release through the increase in cAMP and cGMP levels.84 The outcome of NO-mediated effects are concentration dependent, such that NO synthesized in a nanomolar range is cytoprotective to maintain vascular hemostasis, whereas NO released in the micromolar range is considered to produce cytotoxic effects promoting vascular pathology.7 One study showed that excessive NO production can inhibit Ca21 flux, prostacyclin production, and eNOS expression in endothelial cells.85 Thus, the bioavailability of NO is regulated in at least two ways: reaction with various reactive oxygen species (ROS)86,87 and desensitization of the NO/cGMP system.88 As discussed previously, under certain conditions eNOS can release the prothrombotic oxidant superoxide, which can react rapidly with NO to form peroxynitrite.46 NADPH oxidase has been identified as one primary source of superoxide in platelets87,89,90 and was found to play a role in platelet activation.90 Superoxide production by NADPH oxidase is decreased by NO-mediated PI3K inhibition, resulting in a reversal of platelet aggregation.91 This modulation of reactive oxygen and nitrogen species is a potential mechanism of regulating the bioavailability of NO at the site of platelet aggregation.87 Upon activation, platelets generate superoxide via the glutathione cycle and lipoxygenase pathway,92 leading to a reduction in NO levels. Hydrogen peroxide and lipid hydroperoxides are additional forms of ROS, which affect the bioavailability of NO. The plasma enzyme gluthathione peroxidase protects both endothelial cells and platelets93 from

oxidative damage by reduction of ROS.63 More recent work in vitro has shown that sphingosine-1-phosphate (S1P) released from platelets will increase eNOS activity and reverse the effects of hydrogen peroxide on the endothelium.94

C. NO-Mediated Endothelial
Platelet Interactions and Thrombotic Disease NO plays an important protective role in vascular hemostasis by suppressing thrombosis, atherosclerosis, and proliferation of vascular smooth muscle cells.95 Diverse animal and human in vivo studies examining eNOS activity and NO production have reported the level of NO bioavailability and eNOS function during thrombotic disease states, as well as studied the effect of NO-donating or NO-enhancing substances in various pathological settings. In atherosclerosis and hyperlipidemia, eNOS is dysfunctional and produces superoxide, which is implicated in endothelial dysfunction and impaired endothelium-dependent relaxations.96 Endothelial dysfunction or diminished NO bioavailability result in increased neutrophil adhesion to the endothelium and the initiation of atherosclerosis and thrombosis.97
99 During sepsis, there is increased interaction among immune cells, including neutrophils, the endothelium, and platelets. In vitro data has shown that increases in eNOS activity can decrease neutrophil activation and adhesion onto endothelial cells and platelet activation, aggregation, and adhesion to endothelial cells and neutrophils.100 Animal studies have helped elucidate the importance of NO in platelet
endothelial mediated homeostasis and vascular patency. Mice deficient in eNOS have decreased vascular reactivity and are hypertensive.101 Using platelets deficient in eNOS, it was shown that platelet-derived NO is important in regulating hemostasis, specifically by regulating platelet recruiment.81 eNOS deficiency is also associated with enhanced fibrinolysis due to lack of NO-dependent inhibition of the release of endothelial Weibel
Palade bodies,102 which contain tissue plasminogen activator.5 More recently, it was demonstrated that in eNOS
/
mice collateral circulation and angiogenesis was reduced due to impaired proliferation of vascular cells.103 Mice overexpressing the human eNOS gene were studied in a murine model of infarct-induced congestive heart failure.104 Both cardiac and pulmonary dysfunctions were attenuated and survival was drastically improved in the mice overexpressing human eNOS.104 The genetic overexpression of human eNOS in mice also attenuated myocardial infarction after ischemia/reperfusion injury but failed to significantly protect against post-ischemic myocardial contractile dysfunction.105

I. PLATELET BIOLOGY

II. NITRIC OXIDE

Overexpression of bovine eNOS was found to inhibit lesion formation in a mouse model of vascular remodeling,106 but when crossed with ApoE2/2 mice, atherosclerotic lesion formation was accelerated.107 In the human diseased blood vessel and during unstable coronary syndromes, release of both endothelial and platelet NO is impaired and thus contributes to thrombus formation. NO availability in the vascular system has been associated with various disease states and genetic variants. Risk factors for atherosclerosis— such as high cholesterol, male gender, family history, and age—have also been linked to this impaired endothelium-dependent vasodilation in coronary arteries.108 NO deficiency has also been linked to an increasing number of cardiac and noncardiac thrombotic disorders in humans. There is more platelet activation in unstable coronary disease,109 and these platelets produce significantly less NO compared to patients with stable coronary artery disease.110,111 Furthermore, in human atherosclerotic coronary arteries, endothelial NO-dependent vessel dilation is impaired.112 Endothelial dysfunction has also been associated with pulmonary hypertension, leading to eventual pulmonary vascular hypertrophy and thrombosis.113 Measurement of eNOS expression in the pulmonary vascular tissue of patients with pulmonary hypertension is decreased compared to healthy controls,114 and inhibition of eNOS leads to an increase in platelet deposition.115 Impaired bioavailability of NO due to a deficiency in plasma glutathione peroxidase was found to be the cause of thrombotic childhood stroke.116 Pharmacological agents may have direct beneficial effects by improving the function of endothelial cells and platelets.95,117 In a canine model of coronary artery stenosis, intravenous nitroglycerin infusion has been shown to inhibit platelet thrombus formation.118 In a rat study, inhaled NO resulted in a lower incidence of collagen-induced platelet aggregation in small pulmonary vessels compared to controls.119 The antithrombotic activity of NO was similar to the platelet GPIIb-IIIa antagonistic peptide G4120, suggesting that NO had a direct effect on inhibiting platelet activation.119 Chronic treatment with L-arginine, a substrate for eNOS, inhibits formation of atherosclerotic lesions in animal models of atherosclerosis.120,121 Human in vivo studies have shown that modulating NO clinically affects platelet function. Oral supplementation of L-arginine in healthy adults inhibits ADP-dependent platelet aggregation when compared to placebo,122 and inhibition of platelet eNOS enhances platelet aggregation and reduces bleeding times.123 Furthermore, infusion of L-arginine reversed the vasoconstrictive effect of eNOS inhibitor L-NMMA in coronary arteries.124 Some statins have also been used to upregulated eNOS. However, the

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results have been inconsistent. Clinical studies have shown that the presence of asymmetric dimethylarginine (ADMA) affects how statins work. Individuals with high levels of ADMA did not show any enhancement of NO synthesis upon treatment with simvastatin; those with low ADMA levels benefited from the treatment. Adding L-arginine to simvastatin treatment in patients with elevated ADMA levels improves endothelial function.125 Clinical studies have also looked at how dietary supplements can affect eNOS and NO levels. A peroxynitrite scavenger found in walnuts, γ-tocopherol, has shown in enhance platelet eNOS activity and endothelial-dependent vasodilation, potentially through upregulation of expression and phosphorylation of eNOS, as shown in rat models.126 In vitro, fish oil treatment of human endothelial cells increases eNOS gene expression and decreases NADPH oxidase expression.127 EGb 761, an extract of ginkgo biloba, increases eNOS expression and activation through phosphorylation at Ser478, resulting in the reduction of blood pressure and vasodilation in rat models.128 Folic acid has been shown to promote eNOS dimerization, which has been suggested as one way folic acid may improve endothelial function in patients with coronary artery disease.129 Finally, acetate has been shown to increase phosphorylation of eNOS by PKA and AMPK in HUVEC, which is suggested to explain the increase in forearm vasodilation in postmenopausal women given vinegar (acetic acid).130 The use of GPIIb-IIIa antagonists is known to reduce myocardial infarction and death in patients undergoing percutaneous coronary intervention and in patients with acute coronary syndromes (Chapter 55).131,132 Inhibition of GPIIb-IIIa in patients with known cardiovascular disease increases NO bioactivity.133 NO donors have also been found to directly reverse GPIIb-IIIa activation of platelets,134 although the therapeutic use of GPIIb-IIIa antagonists remains in flux.135,136 Angiotensin-converting enzyme (ACE) inhibitors have been largely used to treat hypertension. Experimental models and clinical studies have shown how some of these inhibitors can affect eNOS levels and activity. Some ACE inhibitors were shown to increase eNOS levels and activity in rat aortas; however, some were better than others.137 In patients with coronary artery disease or heart failure, ACE inhibitor treatment increased eNOS levels and activity in endothelial cells and cardiac tissue.138 S1P, which has been shown to augment eNOS levels in endothelial cells, has been studied as a potent immunosuppressor in kidney transplantation. Clinical trials, using its analogue FTY720, have shown that treatment traps lymphocytes in lymphoid organs, preventing migration to inflammatory sites. ApoE
/


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mice given FTY720 had a reduction in atherosclerotic plaque formation, suggested to occur through its immunosuppressive abilities and activation of eNOS.139 A PPARγ agonist, fenofibrate, has been studied for its role in reducing cardiovascular events, particularly in diabetic patients who have undergone limb amputation. Fenofibrate increases adiponectin as a way to protect the vasculature and increase revascularization. As previously mentioned, work in mice looking at collateral circulation has indicated a role of eNOS in angiogenesis and collateral formation.103 Fenofibrate treatment in mice was shown to stimulate the phosphorylation of AMPK and eNOS in ischemic tissue. Blockage of both pathways, individually, led to a reversal of fenofibrate effects on blood flow.140

D. Genetic Variants and Polymorphisms of Endothelial and Platelet eNOS Numerous investigations have elucidated eNOS polymorphisms and linked them to various thrombotic diseases in association studies, although many of these studies are now considered to be underpowered. The most common and thus extensively studied polymorphisms are the Glu298Asp polymorphism in exon 7 (also known as G894T), the T786C polymorphism in the promoter region of the eNOS gene, and the intron 4b/a. Table 17-2 provides an overview of these and other polymorphisms with regard to their association with thrombotic diseases. Genetic variability due to heritable factors has a major influence on platelet TABLE 17-2 Disease

aggregation,141 and the availability of plasma NO has been linked to several of these polymorphisms.142,143 In a recent meta-analysis, the effects of three of the most common eNOS genetic variations on the susceptibility to ischemic heart disease were examined:144 Glu298Asp, T786C, and intron 4. In this study,144 only homozygosity for Glu298Asp and intron 4a alleles were associated with respective 31% and 34% increased risks of ischemic heart disease. Studies attempting to demonstrate an association of clinical coronary artery disease or acute myocardial infarction with eNOS polymorphisms are inconsistent and vary between ethnic groups (Table 17-2). Japanese patients homozygous for the intron 4 polymorphism were associated with an increased risk of myocardial infarction.145 However, in a similar German population, there was no increased risk of cardiovascular disease,146 whereas a decreased risk of myocardial infarction was reported in Finnish men.147 In Koreans, both T768C and 4b/a polymorphisms are associated with coronary artery disease.148 In an study conducted in the United States, the Glu298Asp polymorphism is associated with the first myocardial infarction occurring in individuals younger than 50 years old.149 Part of the apparent variations in studies may lie in the various presentations of acute and chronic coronary artery disease, including coronary vasospasm, intraarterial thrombosis, and chronic plaque formation or plaque rupture. There are also numerous difficulties associated with reporting genetic associations linked to complex outcomes;150 for instance, many of the current trials involving eNOS polymorphisms are relatively

Polymorphisms of the eNOS Gene and Associated Disease States Cardiovascular Disease: MI, CAD

Hypertension

Stroke and Aneurysm

Diabetes

Other

T786C161,162,451

T786C152
155,158,157

T786C164,165

Retinopathy of PREMATURITY
T786C; G894T174

Intron 4b/a144
148,444,452,453,455

Intron 4b/a161,162,454

Intron 4b/a152,156,163

Intron 4b/a163,164,167

Lupus
T786C172,173

G894T 144,149,180,444,445,457
461

G894T161,162,453,461
465

G894T152
154,156,466

G894T164,165

Breast Cancer
T786C178

A922G444

Intron 13 CA VNTR468

A922G157

786/4b/ 894G163

Renal Disease
T786C; Intron 4b/a; G894T164,165,179,180

Intron 13 CA VNTR467,469

Intron 23 G10T463,465

298Asp/4b/-786T156

C Asp b166

ED
G894T175,176

C Glu b159

298Asp/4b/-786C156

C Glu a160

-786C/4b/894G163

Polymorphism/ T786C144,148,180,444
450 VNTR/SNP/ Haplotype

C Asp b166 Abbreviations: CAD, coronary artery disease; ED, erectile dysfunction; MI, myocardial infarction; SNP, single nucleotide polymorphisms; VNTR, variable number of tandem repeat polymorphisms.

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small and likely underpowered, in addition to the possible different distribution of eNOS polymorphisms among ethnic groups.151 eNOS polymorphisms are also associated with ischemic stroke and aneurysms. eNOS polymorphisms including G894T, T786C, and 4b/a, and haplotypes were associated with silent brain infarctions in Koreans.152 However, in a separate study, there was no link between these eNOS polymorphisms and intracranial aneurysms in Koreans153 and Indians.154 In a study conducted in the United States, there was an association between the T786C polymorphism and the increased risk for angiographic vasospasm after an aneurysm.155 Both eNOS polymorphisms and haplotypes have been associated with a susceptibility of ischemic stroke in Tunisians156 and Chinese.157 Conversely, intron 4aa polymorphism was shown to be protective of lacunar infarction, or silent strokes; T786C increased the chances in a Turkish population.158 In hypertension, various studies have found correlations between eNOS polymorphisms and the development or progression of the disease. In obese children and adolescents, eNOS haplotypes, but not polymorphisms, were associated with the development of hypertension.159 Haplotypes were also associated with gestation hypertension and preeclampsia.160 Interestingly, as seen with coronary artery disease and myocardial infarction, studies in Italians have shown no associations between eNOS variants and hypertension.161,162 In relationship to diabetes, eNOS haplotypes have been associated with both type 2 diabetes and the risk of ischemic stroke in individuals with diabetes in Koreans.163 There is no association of eNOS polymorphisms in Caucasian Brazilians with diabetes and the development of renal disease.164 However, in Asian Indians, there is an association of eNOS polymorphisms and haplotypes with the development of renal disease in diabetic patients, related to lower serum NO levels.165Additionally, another study done in Brazil, showed a relationship between eNOS haplotypes and NO levels in individuals with hypertension and type 2 diabetes.166 Polymorphisms in intron 4b/a in Germans are associated with lower eNOS-dependent vasodilation in individuals at an increased risk for type 2 diabetes.167 eNOS polymorphisms have been shown to alter treatment efficacy. T786C polymorphism has been shown to affect atorvastatin’s ability to modulate NO bioavailability and inflammation.168,169 The effect of exercise on lowering blood pressure in postmenopausal women has also shown to be more effective in individuals with the T786C polymorphism.170 Finally, eNOS haplotypes have been shown to determine the effectiveness of antihypertension therapy in women

with preeclampsia but not in gestational hypertension.171 eNOS polymorphisms have also been associated with various other diseases. eNOS haplotypes in combination with the T786C polymorphism is associated with susceptibility to systemic lupus erythematous in subjects from Kuwait172 but not in subjects from China.173 Retinopathy of prematurity in infants is associated with both T768C and G894T polymorphisms.174 G894T is associated with an increased risk of erectile dysfunction.175,176 eNOS polymorphisms are also associated with a reduced risk of radiation therapy toxicity used to treatment breast cancer.177 However, another polymorphism, T786C, is associated with sporadic breast cancer in women younger than 55 years old.178 Finally, in renal disease, eNOS polymorphisms are associated with functional deterioration179 and cardiovascular events.180

III. PROSTACYCLIN A. Prostacyclin Biosynthesis and Characteristics of Endothelial Prostacyclin Synthase Prostaglandin I2 (PGI2) or prostacyclin is a derivative of the C-20 unsaturated fatty acid arachidonic acid (5,8,11,14-eicosatetraenoic acid),181 which was discovered in 1976 as a substance inhibiting platelet secretion, platelet aggregation, and vasoconstriction.182,183 With a half-life of about 3 minutes,184,185 prostacyclin is extremely labile under physiological conditions, but this half-life is increased by binding to serum albumin to stabilize PGI2 activity and enhance its receptor binding.186 The biosynthesis of prostacyclin is part of the arachidonic acid metabolic pathway. Arachidonic acid is a member of the ω-6 series of essential fatty acids and is covalently bound at the sn-2-position in membrane phospholipids. It is released by phospholipase A2 upon activation of the enzyme by an increase in intracellular Ca21 concentration. Arachidonic acid is further metabolized by at least two major enzyme complexes: (1) prostaglandin endoperoxide H (PGH) synthase, which is commonly known as cyclooxygenase (COX), and (2) 5-lipoxygenase187 (see Chapter 53). COX is a bifunctional enzyme with two related catalytic functions, a bis-oxygenase (cyclooxygenase) that catalyzes first the formation of prostaglandin G2 (PGG2),188 and a hydroperoxidase that subsequently catalyzes the two-electron reduction of PGG2 to the unstable PGH2.189 PGH2 is further converted by several specific synthases into prostanoids such as PGE2, PGD2, PGF2α, prostacyclin, or TXA2, depending on the tissue in which this reaction occurs.187,190 Prostacyclin

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17. INHIBITION OF PLATELET FUNCTION BY THE ENDOTHELIUM

synthase (PGIS), in the endothelium, catalyzes the formation of prostacyclin from PGH2. PGIS is widely distributed, predominantly in endothelial and smooth muscle cells,191
196 but it is not expressed in platelets. PGIS, a membrane-bound enzyme of approximately 57 kDa, has one membrane anchor domain at the N-terminus197 and a large cytoplasmic domain containing a substrate channel, hemebinding sites, and two Couet-motifs.198,199 The enzyme is mainly localized in the membrane of the endoplasmic reticulum (ER)200 whereas the heme-containing domain faces the cytoplasmic site of the ER.198,201 The two isoforms of COX are also located in the ER membrane, though their active site is positioned toward the ER luminal side.202 In addition, recent studies demonstrated the presence of PGIS in caveolae of human endothelial cell membranes. It binds to caveolin-1, the primary coat protein of caveolae, which does not affect the enzymatic activity.203 Previous studies suggested that PGIS is subject to multilevel regulation in vivo. One proposed mechanism is “suicide” inactivation, by which PGIS undergoes a catalytic inactivation by its substrate PGH2.204 Another control mechanism has been described as selective inactivation of PGIS by tyrosine nitration at the active site at Tyrosine 430 due to exposure to micromolar peroxynitrite concentrations.205,206 At the gene expression level, it has been previously established that variations in the PGIS gene sequence can affect the enzymatic activity (see Section III.E).

B. The Platelet Prostacyclin Receptor: Biochemistry, Structure, and Function Prostacyclin is a potent vasodilator, antithrombotic, and antiplatelet agent that mediates its effects through a specific membrane-bound receptor, the prostacyclin receptor (IP receptor). The receptor belongs to the prostanoid family of G-protein-coupled receptors (GPCRs)190 and is expressed on platelets207,208 and smooth muscle cells.209 IP receptor expression within the cardiovascular system is most abundant in the aorta.210 It is also expressed in the atrium and ventricle of the heart, indicating possible roles for prostacyclin in cardiac tissue.211 There is no IP receptor expression found in veins.190 The human prostacyclin receptor has a molecular weight of 41 to 83 kDa,212,213 is located in the plasma membrane, seven transmembrane domains with a short extracellular N-terminal region and an extended intracellular C-terminal region.211 Prostaglandins have two structural features, a cyclopentane ring and side chains, that are recognized by their receptor to stabilize ligand binding. In the IP receptor, transmembrane

domains VI and VII bind with PGI2 side chains. Transmembrane domains I and II confer broader binding functions, including recognition and interaction with the cyclopentane ring of prostacyclin.181,214 The transmembrane domain I also contains a dimerization motif.211 The prostaglandins are not necessarily specific for an individual receptor. The binding pocket of the IP receptor can accommodate the cyclopentane rings of PGI2, PGE1, and PGE2.211 Prostacyclin analogs such as iloprost and cicaprost bind the receptor with the same affinity as prostacyclin. The rank order of agonist affinity for the cloned human prostacyclin receptor is: iloprost 5 cicaprost . PGE1 . carbacyclin PGE2 . PGD2, PGF2α.190,210,215,216 The cross-reactivity of IP ligands with several receptors for PGE2 (EP) has been reported.190,211 Further elucidation of substrate specificity of the IP receptor as well as the evaluation of the role of endogenous prostacyclin have been hampered by the lack of a potent, selective IP receptor antagonist and highly selective agonists.211,217,218 The human prostacyclin receptor contains two potential N-glycosylation sites, one in the extracellular N-terminal domain at Asn7 and one in the first extracellular loop at Asn78.211 A functional study of the glycosylation sites in HEK and COS-7 cells revealed a greater degree of glycosylation at Asn78 than at Asn7, which might play a role in membrane localization, ligand binding, and signal transduction of the IP receptor.219 The C-terminus of the IP receptor contains a consensus sequence for isoprenylation and is modified at Cys383 by a C-15 farnesyl isoprenoid chain.220 This modification has no effect on the ligand binding characteristics but is required for efficient coupling of the prostacyclin receptor to adenylyl cyclase as well as phospholipase C, both via the G proteins Gαs and Gαq, respectively.220 In addition, it was established that the isoprenyl modification is necessary for efficient agonist-mediated receptor internalization221 following receptor desensitization222 and G-protein uncoupling.223 Palmitoylation of the intracellular C-terminal domain at residues Cys308 and Cys311224 is not required for ligand binding but plays a crucial role in Gαs/adenylyl cyclase coupling. Cys308 is necessary for Gαq coupling and phospholipase C activation.224 Several sites in the intracellular loops and the C-terminal domain of the IP receptor represent consensus sequences for protein kinase A (PKA) and PKC phosphorylation.211,223 In particular, Ser328 was found to be a primary site for PKC phosphorylation.223 In addition to protein regulation via posttranslational modifications, the composition of the platelet membrane also plays a role in the expression of prostacyclin binding sites whereby an increase in cholesterol

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content lowers and an increase in phospholipids enhances the amount of bound prostacyclin.225 Furthermore, the expression of the prostacyclin receptor was shown to be upregulated by proinflammatory and megakaryocytopoietic cytokines.226

C. Prostacyclin: Signaling Mechanisms and Platelet Inhibition Prostacyclin is a very potent endogenous inhibitor of platelet aggregation, as well as a strong vasodilator that also inhibits the growth of vascular smooth muscle cells.182,191,193,215,227
231 It inhibits platelet activation induced by various stimulants such as thrombin, collagen, ADP, TXA2, or the calcium ionophore A23187.218 However, it shows selectivity towards TXA2-initiated platelet activation and its inhibitory potential decreases in the following order: TXA2 A23187 . thrombin . ADP.232 Besides inhibiting platelet activation and limiting thrombus size,191 prostacyclin can also prevent platelet56 and leukocyte adhesion to endothelial cells.233 After release from the vessel wall, prostacyclin mediates its inhibitory effects via the high-affinity prostacyclin receptors on the platelet surface, causing an increase in cAMP levels.215,234,235 The binding of prostacyclin to its receptor induces a signaling cascade through G-protein Gαs.181,236 This stimulates adenylate cyclase, assumed to be located in the dense tubular system,237 and leads to an increase in cAMP levels.234,235 Increased cAMP production activates PKA,238 which causes the phosphorylation of several key proteins239,240 such as myosin light chain kinase (MLCK),241 the platelet inositol 1,4,5-triphosphate receptor,242 and VASP.243 Activation of these proteins leads to the inhibition of several pathways: (1) Rhokinase and MLCK-activation and subsequent granule secretion, (2) GPIIb-IIIa activation, and (3) PKC activation and increase in intracellular calcium levels.218,244 Phosphorylated MLCK is inactive and has a reduced affinity for calmodulin, which then reduces the amount of phosphorylated myosin.245 The effect of this is a decreased platelet contractile activity, including secretion, and a decreased association of myosin with the platelet cytoskeleton.246 Through these mechanisms, prostacyclin is able to inhibit platelet activation and to cause disaggregation of existing platelet aggregates.247 Prostacyclin action is a dynamic process that is regulated on many levels. The binding of prostacyclin to its receptor and subsequent activation of adenylyl cyclase via Gαs is a concentration-dependent process. Similar to other GPCRs, the IP receptor may activate more than one G protein.211 At higher ligand

321

concentrations, the IP receptor activates phospholipase C, most likely through Gαq coupling,248 and causes calcium mobilization.249 Furthermore, it has been proposed that the net effect on cAMP levels by prostacyclin depends on its ability to stimulate as well as inactivate adenylyl cyclase.250 Prostacyclin receptor desensitization is another regulatory mechanism of PGI2 responsiveness. It is known that elevated prostacyclin levels are accompanied by reduced binding to the IP receptor and decreased responsiveness in human vascular disease, such as myocardial infarction251,252 and preeclampsia,253 as well as during administration of prostacyclin and its analogs.211,254 The desensitization process involves uncoupling of the receptor from G-proteins, inhibition of adenylyl cyclase activity, and subsequent receptor internalization. The result is a diminished response due to extended exposure to agonists or a high concentrations of agonist.222,223 Several studies have shown that desensitization and internalization of the human IP receptor takes place in platelets,222,255,256 in culture,249,257 as well as in vivo.258,259 PKC-mediated phosphorylation of the human IP receptor is a critical determinant of agonist-induced desensitization,223 whereas sequestration is independent of the PKC pathway and proceeds partially via an endocytotic pathway involving clathrin-coated vesicles.249 In platelets, the response to desensitization is augmented platelet
endothelial cell adhesion in the presence of thrombin, resulting in an enhanced tendency toward thrombus formation.251,256 The short-term desensitization of the platelet IP receptor is a reversible phenomenon whereby the receptor is not degraded but is rapidly recycled as a functionally active form after prostacyclin withdrawal.260 IP receptor responsiveness is also regulated by receptor co-expression and co-localization, receptor cross-regulation through shared G-proteins, as well as proposed heterodimerization with other prostanoid receptors.211,215,218,261,262 In platelets the IP receptor (a stimulatory receptor) co-localizes with the EP3 receptor (an inhibitory receptor) to regulate cAMP production and to act as a buffering mechanism against a local increase in prostacyclin levels.263,264 This observation emphasizes the functional advantage of nonspecific agonists. Maintenance of the balance between prostacyclin and TXA2 appears to be a critical regulator for the maintenance of vessel integrity,265 the interaction between platelets, and the endothelium193,266 and cardiovascular processes.218,267 Synthesized by platelets268 and released after platelet activation,269,270 TXA2 is a platelet agonist and vasoconstrictor participating in a positive feedback loop that mediates additional platelet activation and aggregation. The actions of PGI2

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counteract the actions of TXA2. Platelet activation and vasoconstriction induced by TXA2 can be inhibited by prostacyclin.215 Through increased platelet cAMP levels, prostacyclin blocks arachidonic acid formation271 and thus prevents synthesis of prothrombotic prostaglandins and thromboxanes by the human platelet,272,273 which further inhibits platelet activation.274 The formation of heterodimers between the prostacyclin receptor and the TXA2 receptor α (TPα) has been shown to regulate the prothrombotic role of TXA2.262 IP and TPα heterodimerization results in cAMP generation through TPα, changing the effect of this receptor to be antithrombotic.262 In addition, platelet-derived endoperoxides (like TXA2) can act as substrate and thus upregulate prostacyclin synthesis in endothelial cells.218,275,276 In general, there appear to be two mechanisms for prostacyclin synthesis by cultured endothelial cells: the first one involves prostacyclin synthesis from endogenous precursors, and the second one involves endoperoxides derived from stimulated platelets.275 As discussed previously, platelets release other antithrombotic substances, such as NO, that provide a negative feedback for thrombus formation. Indeed, low doses of NO and PGI2 synergize in their anti-aggregatory effect,277,278 although not at a level to inhibit platelet adhesion to the endothelium.56,279 Platelets also release factors that upregulate prostacyclin production. Bile salt-dependent lipase is released from platelets upon activation and has been shown to bind to HUVEC and increase prostacyclin synthesis.280

D. The Relevance of Prostacyclin In Vivo Prostacyclin as an antiplatelet and antithrombotic mediator plays an important role in the development of cardiovascular disease.281 Dysfunctional prostacyclin activity has been implicated in the development of various cardiovascular diseases including thrombosis, myocardial infarction, stroke, atherosclerosis, and hypertension.190 A reduction in the binding affinity of platelets for prostacyclin and cAMP synthesis has been observed in patients with acute myocardial infarction compared to healthy subjectss.252 Because PGIS is upregulated by various cytokines, prostacyclin biosynthesis has been reported to be increased in the presence of atherosclerosis and platelet activation.282 Mice deficient in the PGIS gene are hypertensive and develop vascular disorders with vascular wall thickening and interstitial fibrosis, particularly in the kidneys.283 Inhibition or deficiency of PGIS, in vivo, causes a marked enhancement of white platelet thrombus formation.284
286 Several investigators have reported that abnormal

prostacyclin synthesis or metabolism may be a risk factor for myocardial and cerebral infarction.287,288 Delivery of PGIS in vivo can prevent proliferation and migration of smooth muscle cells, key steps in the development of restenosis and atherosclerosis.289,290 Although prostacyclin reduces cerebral infarction in animal models,291,292 many therapeutic trials of prostacyclin have failed to show significant clinical improvement.293
295 A decrease in prostacyclin production has been implicated in the pathogenesis of severe pulmonary hypertension296 but not in human essential hypertension. However, in pregnancy-induced hypertension, a decrease in prostacyclin has been reported to precede the clinical manifestation.297 Mice deficient in the IP receptor have been studied in various disease settings.217,298
302 Murata et al. found that IP receptor knockout mice display an increased thrombotic tendency but have a reduction in inflammatory swelling and pain responses and are not susceptible to hypertension.298 Injury of the carotid artery leads to obstruction in the mice lacking the IP receptor, and this response was abolished in mice lacking TXA2.300 In the IP null mice, the production of TXA2 by platelets and components of the injured vessel wall was increased.300 Ovariectomized LDLR
/
female mice also lacking the IP receptor are no longer atheroprotective when estrogen is reintroduced. Estrogen, when bound to estrogen receptor alpha, upregulates prostacyclin through COX-2 activation.302 The opposing actions of prostacyclin and TXA2 on platelets and the vessel wall and their concentration levels at the site of injury are considered to be critical for thrombus formation183 and in various occlusive vascular diseases including coronary heart disease.262,303 During cardiac ischemia/reperfusion, the synthesis of PGI2 and TXA2 are significantly increased.304,305 It has been reported that PGI2 and its analogs attenuate cardiac ischemia/reperfusion injury when administered exogenously in animal models,306
309 possibly due to their inhibitory effect on platelets and neutrophils. Clinical studies suggested that loss of the IP receptor may contribute to atherogenesis in patients with chronic spinal cord injury.310 During spontaneous angina pectoris,311 severe atherosclerosis,312 and acute myocardial infarction,313 the prostacyclin binding capacity and the number of IP receptors has been reported to decrease, but not in other patients with angiographically proven coronary artery disease and stable angina.252,314 It has been shown that in myocardial infarction and unstable angina, biosynthesis of prostacyclin is considerably increased,109,313 which could lead to agonist-induced receptor changes such as desensitization of the PGI2 binding sites.314

I. PLATELET BIOLOGY

III. PROSTACYCLIN

Prostacyclin’s short half-life has led to the development and experimental application of several prostacyclin mimetics.215 The ones most commonly used are cicaprost,315 iloprost,316 and carbacyclin,215,317,318 which are all analogs of PGI2.319 Cicaprost is often the analog of choice as iloprost is less selective and partially acts as an agonist for prostaglandin E1 receptors.215 Administration of prostacyclin mimetics, such as epoprostenol, in the clinical setting has been used to limit platelet aggregation, but a major drawback is the profound vascular dilatation they induce.215 In pulmonary arterial hypertension (PAH), treatment with prostacyclin analogs, such as epoprostenol, treprostinil, iloprost and beraprost, continues to play an important role despite the complications induced by their generally short half-lives and complicated drug delivery systems.320 More recently, epoprostenol was shown to have no effect in patients with pulmonary embolism.321 This same drug has been shown to induce headaches in healthy patients322 and migraine-like events in migraine suffers323 because of the sensitization of sensory afferents around extracranial arteries. However, epoprostenol has been shown to increase the survival of patients with PAH324 and improve pulmonary function,325 even during thoracic transplantation.326 To improve the effects of beraprost on PAH, this drug has been used in conjuncture with other drugs, such as an angiotensin blocker,327 or has been reformulated to last longer to improve pulmonary pressure and quality of life.328 Iloprost is also being studied for its effectiveness in PAH, even during heart transplantations,329 and shows, alone or in conjuncture with other drugs,330 effectiveness in improving pulmonary arterial pressure and hemodynamics.331 Treprostinil has been extensively studied more recently, particularly as a replacement for epoprostenol because it is longer lasting.332,333 All studies report improved pulmonary vasodilation, exercise capacity, hemodynamics, and survival.334
338 Iloprost has been used for the treatment of systemic sclerosis, a connective tissue disease in which the immune system attacks body tissues, leading to a buildup of collagen in these tissues. Studies have looked at its effects on oxidative stress that occurs due to frequent ischemia/reperfusion events during the pathogenesis of this disease, but there has been no agreement if this drug has a role.339,340 However, iloprost has been shown to be effective in preventing pulmonary hypertension in patients with systemic sclerosis.341 Two prostacyclin analogues have been studied for the treatment of peripheral artery disease (PAD; arteriosclerosis obliterans)—iloprost and beraprost. Iloprost has been shown to be effective in reducing inflammation and oxidative stress in conjuncture with

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aspirin treatment;342 alone, it has not been effective to treat the disease.343 Beraprost has shown to be effective in the treatment of PAD by reducing renal anemia,344 decreasing endothelin-1 levels, and improving quality of life.345 Prostacyclin analogs are also being tested in renal diseases. Individuals with acute renal failure requiring continuous venovenous hemodiafiltration have an increased risk of thrombosis. Treatments to prevent this cause an increase in bleeding. A prostacyclin analog was shown to reduce platelet aggregation induced by ADP and collagen these patients.346,347 In another study, patients with renal failure undergoing coronary intervention were given iloprost prophylactically protected against contrast-induced nephropathy.348 Finally, individuals on chronic peritoneal dialysis experience endothelial injury and thrombosis. Patients treated with beraprost had a reduction in both Ddimer and von Willebrand factor markers, indicative of thrombosis and endothelial damage, respectively.349 Aspirin (acetylsalicylic acid) has an anti-thrombotic effect350 based on its preferential action on blocking COX in platelets and the endothelium by acetylating COX’s active center (Chapter 53).351 Aspirin has also been reported to increase NO production in neutrophils352,353 and in the arterial wall.354 There are two different cyclooxygenases, COX-1 and COX-2.187,188,355 COX-1 is involved in TXA2 synthesis in platelets while COX-2 is involved in the synthesis of prostacyclin in endothelial cells.187,356 Aspirin at low doses acetylates COX-1 in platelets and thus irreversibly blocks TXA2 synthesis for their lifetime in the circulation. At the same low doses, aspirin has little effect on the synthesis of PGI2. Thus, the overall effect of low dose aspirin is a reduced risk of thrombosis.187 The combined inhibition of both COX isoforms, but not the selective inhibition of COX-2, attenuates atherogenesis in LDLR
/
mice.357

E. Polymorphisms of the PGIS and Prostacyclin Receptor Genes The genomic organization of the PGIS gene has been investigated for several polymorphisms in association studies performed, primarily, in the Japanese population.358
369 Correlations between some of these polymorphisms, including single nucleotide polymorphisms (SNP), variable number of tandem repeat polymorphism (VNTR), and splicing mutations, and have found an increased risk for essential hypertension, myocardial infarction, and cerebral infarction.360,364,366
368 Two additional studies found polymorphisms in the French Caucasian population and two in African ethnic groups, but with no

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324 TABLE 17-3

17. INHIBITION OF PLATELET FUNCTION BY THE ENDOTHELIUM

Polymorphisms of the Prostacyclin Synthase (PGIS) Gene and Associated Disease States

Disease

Cardiovascular disease: MI, CAD, Thrombosis

Hypertension

Stroke and Aneurysm Other

Polymorphism/ VNTR/SNP/ Haplotype

SNP A1117C in Exon 8368,374

Nonsense Mutation in Exon 2, Codon 26365

Nonsense Mutation in Exon 2, Codon 26365

Renal Disease - Splicing Site Mutation in Intron 9359,360

M113T, L104R. R279C382

VNTR in Promoter
3-7 repeats of 9 bp362,363,369,372

VNTR in Promoter
3-7 repeats of 9 bp367

Respiratory Syncytial Virus Infection
VNTR378

V53V/S328S381

Splicing Site Mutation in Intron 9359,360

Lung Cancer
CpG Methylation in VNTRs in Promoter and Exon 1377

R212C381

SNP T192G366

Adenomatous Polyps
VNTR in Promoter376

SNP375

T192G/VNTR/A117C361 SNP373

Abbreviations: CAD, coronary artery disease; MI, myocardial infarction; SNP, single nucleotide polymorphisms; VNTR, variable number of tandem repeat polymorphism.

associated functional changes or disease-associations (Table 17-3).370,371 Other mutations have been identified, including a nonsense mutation in exon 2 at codon 26 (CGA/TGA) of the PGIS gene, which may be associated with hypertension. This polymorphism causes a large part of the PGIS mRNA to not be translated and is assumed to decrease the enzymatic activity of PGIS.365 A VNTR polymorphism in the 5’-upstream promoter region has also been found in Japanese subjects and is associated with cerebral infarction.367 This region contains binding sites for the transcription factors Sp1 and AP-2. The alleles vary in size from three to seven repeats of nine base pairs. In a study with almost 5000 Japanese subjects, the 3 and 4 repeats of the VNTR was found to be associated with increased systolic hypertension and higher pulse pressure.363,372 This repeat polymorphism was found to influence the promoter activity of the PGIS gene in human endothelial cells.363 Another mutation includes a splicing variant in intron 9 that results in a truncated protein lacking the heme-binding region, which is associated with hypertension.359,360 In the Croatian population, a SNP near PGIS and the potassium voltage gate channel genes is associated with a high prevalence of hypertension, particularly in inhabitants of the island of Vis, where 75% of the population has hypertension.373 The SNP, C1117A, which is a silent mutation, was identified in exon 8 and found to be associated with a risk for myocardial infarction, in a Japanese population368 and Chinese population.374 More recently, a study done in the United States showed that common SNPs in PGIS were associated with myocardial infarctions. One haplotype was associated with a higher risk of myocardial infarction and stroke. One SNP was associated with a lower risk for both diseases.375

Polymorphisms in PGIS that affect the promoter region and expression level have also been associated with cancer. Less than 6 repeats in the promoter region of PGIS was associated with an increased risk of adenomatous polyps, while more than 6 repeats was associated with a reduced risk. The presence of these repeats also determined the benefit of NSAID treatment for colorectal polps.376 Finally, CpG methylation within VNTRs in the promoter and in exon 1 and the first intron control PGIS expression level. Overexpression of PGIS was found to be protective in lung cancer. Human lung cancer cell lines were found to have lower levels of PGIS due to a high number of these CpG methylation sites.377 Finally, the number of VNTRs in the promoter of PGIS are inversely correlated with the severity of respiratory syncytial virus in Japanese infants. A low number of VNTRs, resulting in a lower level of urinary PGI2 metabolite levels correlated with the most severe respiratory syncytial virus infection in the lower respiratory tract. The higher number of VNTRs correlated with a less severe infection.378 A genetic analysis revealed the first two naturally occurring polymorphisms within the coding sequence of the human prostacyclin receptor, Val25Met in the transmembrane domain I and Arg212His in the third intracellular loop.379 Structure-function characterizations of these polymorphisms were performed at physiological pH (7.4) and an acidic pH (6.8) that would be encountered during stress such as renal, respiratory or heart failure. In particular, the Arg212His polymorphism showed significant reduction in receptor binding affinity at low pH and in signal transduction activity at both pH values, whereas the Val25Met polymorphism had no significant effects.379 A nuclear prostacyclin receptor has been identified which belongs

I. PLATELET BIOLOGY

IV. CD39 (NTPDase-1)

to the peroxisome proliferator-activated receptor (PPAR) family (PPARδ)380 and was suggested to be involved in angiogenesis.199 More recently, polymorphisms in the human prostacyclin receptor have been associated with thrombosis and cardiovascular disease. Two synonymous polymorphisms (V53V/ S328S) were associated with an increase in sP-selectin and 11-dehydro-thromboxane B2 levels in DVT patients compared to control.381 Another polymorphism, R212C, results in a dysfunctional receptor and is associated with intimal hyperplasia.381 Rare receptor polymorphisms that lead to impaired binding and activation of the prostacyclin receptor, including M113T, L104R, and R279C, were associated with coronary artery obstruction.382

IV. CD39 (NTPDase-1) A. Endothelial CD39—Biochemistry, Structure and Function Ecto-nucleoside triphosphate diphosphohydrolases (ENTPDases) is a family of membrane proteins that are ubiquitously expressed in eukaryotic cells and play a pivotal role in mediating platelet-endothelial interactions. These enzymes hydrolyze nucleoside 5’-di- and triphosphates in a Ca21 or Mg21-dependent manner.383,384 Endothelial CD39 (NTPDase-1), the major vascular ENTPDase, is a membrane-anchored glycoprotein with ecto-apyrase activity.385,386 As an integral component of the endothelial cell surface, it is activated by its extracellular substrates in the blood stream, ATP and ADP, which are rapidly hydrolyzed into AMP.383,387 An ecto-5’-nucleotidase (CD73) further converts AMP into adenosine.388,389 CD39 enzyme expression is found in a wide variety of vascular cells. The enzyme is highly expressed on human umbilical vein endothelial cells (HUVECs),390,391 which also has the highest enzymatic activity. It is also expressed on natural killer cells,392 a subset of T cells,390 activated B cells,393 Epstein
Barr virus-transformed B cells,385 megakaryocytes, and platelets.394 Structural analysis of CD39 showed that the protein is membrane-anchored at its N- and C- terminus, whereas each terminus is composed of one transmembrane domain and a short cytoplasmic tail.393 The middle of the protein forms a large extracellular loop containing a more central hydrophobic region.383,395 This large extracellular domain has four apyrase conserved regions (ACR) which were suggested to contain the sites of catalytic activities.396,397 The four ACR are highly conserved throughout the plant and animal kingdoms suggesting their importance in the biological function of CD39.396,398 A fifth ACR (termed ACR-5)

325

has been described in the C-terminal region of the extracellular domain.399,400 The ACR-4 in CD39 was suggested to contain the putative γ-phosphate binding motif, to be highly homologous with the actin-HSP70hexokinase superfamily,396 and to be involved in ATP hydrolysis.397 ACR-1 has been proposed to be the β-phosphate binding domain by analogy with the same superfamily396 and to play a role in ADP hydrolysis.397 The enzyme’s ADPase (but not ATPase) activity depends on the presence of divalent cations, with Ca21 preferred over Mg21.398 Heterologous interactions between both transmembrane domains of CD39 cause the tetramerization of the enzyme in the plasma membrane, which increases its activity over the monomeric form.401 Human CD39 has six potential N-linked glycosylation sites.383,393 The extent of glycosylation is different in endothelial cells, platelets, and leukocytes.394 The enzymatic activity of CD39 was reported to remain essentially unaltered by deglycosylation395 after the protein is properly folded and targeted to the membrane surface.402 CD39 also has several sites that may be modified by ectoprotein kinases,403,404 a few potential phosphorylation sites for intracellular PKC,403 as well as one N-terminal palmitoylation site.405 Oxidative modifications and proteolytic cleavages may modulate and regulate the enzymatic activity of CD39.406,407 CD39 is preferentially localized in caveolae of HUVECs and COS-7 cells,408 a process which is mediated by S-palmitoylation at the residue Cys13 of the intracytoplasmic N-terminal region.405 The activity of CD39 is cholesterol-dependent, whereby depletion or sequestering of membrane cholesterol results in inhibition of the enzymatic activity.409 The absence of caveolin-1 and the subsequent loss of caveolae formation does not affect the enzymatic activity or the targeting of CD39 to the membrane, as large aggregates of endogenous CD39 were found to colocalize with membrane-anchored CD73 and lipid rafts.409

B. The Effect of Endothelial CD39 on Platelet Reactivity Nucleotides serve as intracellular energy sources as well as extracellular signaling molecules, and they can be released upon cellular activation or injury and induce biological responses through specific receptors.384 Some of them can have pathological consequences, for example, the excessive ADP released from platelets and injured tissue have prothrombotic effects.2 Endothelial CD39 does not act on platelets per se; instead it reacts with the ADP released from activated

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platelets.391 Upon activation, platelet release ADP from their dense granules leads to further activation and aggregation of platelets. By hydrolyzing ADP to AMP, CD39 prevents further prothrombotic platelet activation387 with consequent restoration of platelets to the resting state.410 Activation of platelets with collagen or low levels of thrombin is inhibited by CD39.386 solCD39 strongly inhibits aggregation of human platelets induced by collagen, ADP, arachidonate, and thrombin receptor agonist peptide (TRAP).411,412 Platelet-released ADP is transiently hydrolyzed to AMP, which is further broken down to adenosine by endothelial CD73 (Figure 17-1).386,388 Adenosine, an antithrombotic and anti-inflammatory mediator,3 blocks ADP-induced platelet activation and aggregation413 by elevating intracellular cyclic AMP levels and blocking calcium mobilization,414 through the adenosine A2 receptor on platelets.415,416 A study of transgenic CD73
/
mice supports these findings by demonstrating a significant reduction in the occlusion time of the carotid artery, as well as a reduction in the tail bleeding time compared to wild-type mice.417 Platelet cAMP levels, but not cGMP levels, were also found to be significantly lower in these mice.417 Thus, endothelial CD39 and CD73 act in concert to prevent platelet activation and aggregation, while neither NO nor prostacyclin are involved in their inhibitory activity.387,413 ADP and ATP are also substrates for the purinergic type 2 receptors (P2) (see Chapter 14). Several P2 receptors have been localized on megakaryocytes, platelets,418
420 as well as on endothelial cells421,422 (see Chapter 14). ATP is a competitive antagonist of ADP for P2Y receptors on human platelets.423 The situation is further complicated by purinergic receptor desensitization where adenine nucleotides could also limit ADP-induced platelet activation423 and cause platelet dysfunction.424 CD39 appears to modulate the functional expression of P2Y receptors. The coexpression of CD39 and various P2 receptors in endothelial cells as well as platelets has been confirmed.394 A more recent study observed that P2Y1 and CD39 colocalize in the caveolae of endothelial cells.425 ADP and ATP released from activated platelets also affect the endothelium of the vessel wall. Through various G-protein-coupled P2 receptors both molecules stimulate the release of NO and prostacyclin from endothelial cells422,426 via a phospholipase C-mediated pathway that leads to increased intracellular Ca21 and subsequent activation of eNOS and phospholipase A2. This negative feedback mechanism can further limit the extent of platelet aggregation.422 The strong thromboregulatory potential of CD39 has been confirmed by experiments in which prostacyclin and NO production in the endothelium was

blocked by treatment with aspirin and hemoglobin, respectively. The inhibition of platelet activation and aggregation was maintained due to the presence of CD39 in the endothelial cell membrane.387,391,414 CD39 is therefore able to act independently of NO and prostacyclin; however, the presence of these thromboregulatory molecules enhances its activity.389 Interestingly, activation of endothelial cells by proinflammatory cytokines like tumor necrosis factor α (TNFα) or exposure to oxidative stress leads to loss of the enzymatic activity of CD39,386,406,407 but not the cell surface expression.406 Cholesterol depletion of CD39-containing membranes causes a significant delay in the inhibition of platelet aggregation and a substantial decrease in platelet disaggregation.409 Although the function of endothelial CD39 has been related to the inhibition of platelet activation and aggregation by ADP/ATP hydrolysis,386
387 the function of CD39 expressed on platelets and megakaryocytes has not yet been established. Interesting, vigorous exercise in men involves a reduction in CD39 expression in platelets but an increase in expression on Blymphocytes.427

C. CD39
In Vivo Studies The clinical relevance of CD39 has been primarily established by animal studies. Recombinant solCD39 is able to inhibit platelet aggregation,428 remaining active over an extended period of time. Following reperfusion injury429 or after exposure to oxidative stress during vascular inflammation,406 the enzymatic and antithrombotic properties of endothelial CD39 are rapidly lost. Locally administered soluble apyrase (a soluble enzyme with identical function to CD39) was found to be beneficial in inhibiting platelet reactivity within the vasculature of transplanted organs.430 Thus, targeted expression of a stable and active CD39 could be an effective therapeutic means to intervene in vascular inflammation395 and transplantation-associated diseases.431 Administration of solCD39 either before or 3 hours after arterial occlusion improves the neurological score and reduces the infarct size in a rat stroke model.432 SolCD39 may, therefore, have potential for the treatment of focal ischemic stroke in humans.432 In sympathetic nerve endings from guinea pig hearts, neuronal ATP enhances norepinephrine exocytosis. Treatment with solCD39 significantly attenuates norepinephrine release,433 suggesting that CD39 could provide cardioprotection by reducing ATP-mediated norepinephrine release. In a murine stroke model driven by an excessive platelet recruitment, solCD39 reduced the extent of stroke without increasing intracerebral hemorrhage.433

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IV. CD39 (NTPDase-1)

The transgenic expression of human CD39 (hCD39) in mice has been investigated in different disease settings. Mice that express hCD39, at base line, have no spontaneous bleeding tendencies; however, platelet aggregation is impaired, bleeding times are prolonged, and the mice are resistance to systemic thromboembolism.389 In denudated rat aortas adenovirally transfected with hCD39, neointimal formation was reduced compared to controls, which was associated with a decrease in vascular smooth muscle cell proliferation.434 In mice with dysfunctional CD39, bleeding times were normal, but they had increased cerebral infarct volumes and reduced postischemic perfusion. Treatment with solCD39 restored postischemic cerebral perfusion and rescued the mice from cerebral injury, suggesting a protective function of CD39 in stroke.435 WT mice that have undergone wire-induced femoral artery injury have a reduced platelet adhesion, leukocyte accumulation, and neointimal hyperplasia with treatment of human solCD39 compared to saline controls.436 Additionally, treating mice with solCD39 that is incorporated into lipid bilayer increases enzymatic activity and decreased mortality in mice that have undergone thromboplastin-induce thromboembolism.437 Studies with CD39 knockout mice have investigated the lack of this gene on cardiovascular properties and disease development.389,424,435,438
440 Although CD39deficient (CD39
/
) mice and wild-type mice displayed no differences in their phenotype, the CD39
/
mice had substantially prolonged bleeding times and prolonged platelet plug formation times with minimally perturbed coagulation parameters.424 This was due to a reduced response of the CD39
/
platelets to ADP, collagen, and low doses of thrombin. The platelet count was found to be 20% lower in CD39
/
mice compared to wild-type mice, but the CD39
/
platelets had no morphological abnormalities. The interaction between platelet and injured vasculature in vivo was considerably reduced.424 Platelet dysfunction was reversible and correlated with purinergic receptor P2Y1 desensitization, suggesting a dual role for CD39 in hemostasis and thrombotic reactions.424 The CD39
/
mice were also very sensitive to ischemia-reperfusion injuries as they were incapable of producing adenosine at the local endothelial vessel wall layer. Treatment with apyrase reversed the effect and protected the mice.424 Using endothelial cell cultures from the CD39
/
mice, it was shown both ADPase and ATPase activities were diminished.424 This suggests that other nucleotidases associated with endothelial cells, contribute only minimally to the hydrolysis of extracellular adenine nucleotides. Other disease models have been studied using CD39
/
mice, including intestinal ischemia and reperfusion injuries.438 In CD39-null or -deficient mice, 80% died due to intestinal injury as compared to wild-type

mice. Apyrase supplementation protected all wild-type mice from intestinal ischemia-related death, but did not fully protect CD39
/
mice. Adenosine treatment failed to improve the survival rate. Platelet adherence to postcapillary venules in wild-type mice was significantly decreased and vascular integrity was well preserved following apyrase treatment. The potential of CD39 to maintain vascular integrity suggests potential pharmacological benefit in mesenteric ischemic injury.438 Balloon injury in rabbit arteries decreases native CD39 activity, which was reversed by adenovirus-mediated gene transfer of CD39; however, platelet deposition was not altered.439 Studies of ApoE
/
mice showed that deletion of CD39 accelerates the development of atherosclerotic lesions, a process reversed by solCD39 administration.440 Furthermore, supplementation of solCD39 slows atherosclerosis in CD39 wild-type/ ApoE
/
mice.440 In conclusion, the deletion of CD39 renders the mice sensitive to vascular injury. Supplementation of CD39 by somatic gene transfer or administration of soluble CD39 has been shown to be advantageous in models of transplantation, inflammation, and atherosclerosis.

D. Polymorphisms of Endothelial CD39 No polymorphisms of CD39 have been associated with cardiovascular diseases. However, a few have been associated with diabetes and colitis (Table 17-4). A two-SNP haplotype is associated with an increased risk for type 2 diabetes in African Americans. This SNP results in a higher expression level of CD39. A four-SNP haplotype was found to be protective against diabetes. This SNP results in a lower expression level of CD39 compared to control subjects.441 Another CD39 polymorphism has been associated with an increased risk for Crohn’s disease. This SNP results in a lower expression level of CD39. Mouse studies using a CD39
/
model, showed that dextran sodium sulfate-induced colitis was more severe in the knockout mice compared to WT.442 However, in another study, CD39
/
mice given trinitrobenzene sulfonic acid-induced colitis had improved survival and a TABLE 17-4 Polymorphisms of the Ecto-nucleotidase CD39 Gene and Associated Disease States Disease Polymorphism/ VNTR/SNP/ Haplotype

Diabetes AG Haplotype

Other 441

Crohn’s Disease
Loss of Function Mutation442

GAGA Haplotype441 Abbreviations: SNP, single nucleotide polymorphisms; VNTR, variable number of tandem repeat polymorphisms.

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reduced inflammatory response compared to WT mice.443 Thus, although CD39 polymorphisms have been associated with colitis, the exact role of CD39 in the progression of this disease remains controversial.

V. SUMMARY Endothelial thromboregulation requires several forms of communication between endothelial cells and platelets in order to regulate clot formation, but still allow proper blood vessel repair. The three primary and functionally independent pathways involve NO, prostacyclin, and the ecto-nucleotidase CD39. Taken together, the endothelium utilizes these pathways to regulate and prevent uncontrolled platelet activation, aggregation, and adhesion to the vessel wall. In addition, the platelet itself possesses several protective and counter-regulatory mechanisms to balance its own prothrombotic actions and to stimulate endothelial antithrombotic factors and processes. As each of these pathways is defined, it is becoming more apparent that the underlying thromboregulatory mechanisms and enzymatic systems involved are interconnected. Disturbances of these balanced systems contribute to not only the pathophysiology of vascular thrombotic diseases, but other diseases including hemorrhage. Understanding how we can target these pathways to control platelet function without causing hemorrhage or thrombosis will be key in developing effective therapeutics for vascular diseases in the future.

Acknowledgements The authors thank Sybille Rex for her contribution to the writing of this chapter.

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