Platelet Nitric Oxide Signaling Research Articles

P6003Coronary artery spasm: a consequence of impaired nitric oxide/hydrogen sulphide signalling?

Abstract Background Coronary artery spasm (CAS) represents a major cause of patient morbidity, with variable clinical response to prophylaxis with calcium antagonists and generally poor symptomatic relief with organic nitrates. The precipitation of CAS with acetylcholine may reflect impaired nitric oxide (NO) release and/or signalling. We have recently demonstrated that platelets from patients with CAS exhibit markedly impaired anti-aggregatory responses to the NO donor sodium nitroprusside (SNP) (“NO resistance”). Purpose In the current experiments we sought to determine whether N-acetylcysteine (NAC), which is known to potentiate haemodynamic responses to organic nitrates, reverses NO resistance in platelets from CAS patients. Methods Patients with CAS were studied during acute (n=11) and chronic (n=24) phases of symptoms. NAC (10 g/24 hours) was infused together with low dose NTG (2.5 μg/min) in patients presenting with acute exacerbations, and platelets were studied ex vivo. In blood samples taken from chronic CAS patients, in vitro studies were performed to evaluate the possible role of H2S release (via cysteine formation) from NAC in putative potentiation of NO effect. Results (1) In acute patients, NTG/NAC infusion resulted in increases in platelet response to SNP (p=0.003); (2) In vitro studies showed that incubation with NAC or the H2S donor NaHS potentiated SNP responses (Figure 1A); (3) Effects of NAC were reversed by co-incubation with aminooxyacetic acid (AOAA) and D, L-propargylglycine (PAG), inhibitors of enzymatic cysteine bioconversion to release H2S (Figure 1B). Figure 1 Conclusion CAS-associated impairment of platelet NO signaling reflects a deficiency of the H2S/NO interaction, and can be reversed using exogenous H2S donors, including NAC.

Impaired platelet nitric oxide response in patients with new onset atrial fibrillation

BackgroundClinical factors associated with thromboembolic risk in AF patients are well characterized and include new onset AF. Biochemically, AF is associated with inflammatory activation and impairment of nitric oxide (NO) signalling, which may also predispose to thromboembolism: the bases for variability in these anomalies have not been identified. We therefore sought to identify correlates of impaired platelet NO signalling in patients hospitalized with atrial fibrillation (AF), and to evaluate the impact of acuity of AF. Methods87 patients hospitalized with AF were evaluated. Platelet aggregation, and its inhibition by the NO donor sodium nitroprusside, was evaluated using whole blood impedance aggregometry. Correlates of impaired NO response were examined and repeated in a “validation” cohort of acute cardiac illnesses. ResultsWhilst clinical risk scores were not significantly correlated with integrity of NO signalling, new onset AF was associated with impaired NO response (6±5% inhibition versus 25±4% inhibition for chronic AF, p<0.01). New onset AF was a multivariate correlate (p<0.01) of impaired NO signalling, along with platelet ADP response (p<0.001), whereas the associated tachycardia was not. Platelet ADP response was predicted by elevation of plasma thrombospondin-1 concentrations (p<0.01). Validation cohort evaluations confirmed that acute AF was associated with significant (p<0.05) impairment of platelet NO response, and that neither acute heart failure nor acute coronary syndromes were associated with similar impairment. ConclusionRecent onset of AF is associated with marked impairment of platelet NO response. These findings may contribute to thromboembolic risk in such patients.

Aging of platelet nitric oxide signaling: pathogenesis, clinical implications, and therapeutics.

The nitric oxide (NO)/soluble guanylate cyclase (sGC) system is fundamental to endothelial control of vascular tone, but also plays a major role in the negative modulation of platelet aggregation. The phenomenon of platelet NO resistance, or decreased antiaggregatory response to NO, occurs increasingly with advanced age, as well as in the context of cardiovascular disease states such as heart failure, ischemic heart disease, and aortic valve disease. The central causes of NO resistance are "scavenging" of NO and dysfunction of sGC. In the current review, we discuss the roles of several modulators of NO synthesis and of the NO/sGC cascade on changes in platelet physiology with aging, together with potential therapeutic options to reduce associated thrombotic risk.

PT365 Is there a biochemical basis for differential thromboembolic risk in atrial fibrillation? Studies with platelet nitric oxide signalling

PT365 Is there a biochemical basis for differential thromboembolic risk in atrial fibrillation? Studies with platelet nitric oxide signalling

PM220 Hyperglycaemia, rather than diabetes per se, engenders impaired platelet nitric oxide signaling

PM220 Hyperglycaemia, rather than diabetes per se, engenders impaired platelet nitric oxide signaling

Platelet nitric oxide signalling in heart failure: role of oxidative stress

Heart failure is associated with deficient endothelial nitric oxide (NO) production as well as increased oxidative stress and accelerated NO degradation. The aim of this study was to evaluate platelet NO biosynthesis and superoxide anion (O(2)(-)) production in patients with heart failure. In platelets from patients with heart failure due to idiopathic dilated cardiomyopathy (n= 16) and healthy control subjects (n= 23), NO synthase (NOS) activity was evaluated by L-[(3)H]-arginine to l-[(3)H]-citrulline conversion, cGMP was determined by radioimmunoassay, vasodilator-stimulated phosphoprotein (VASP: total and serine-239-phosphorylated) was assessed by western blotting, and O(2)(-) production and O(2)(-) scavenging capacity were measured by pholasin-enhanced chemiluminescence. In platelets from patients with heart failure, basal NOS activity was higher than in those from controls; furthermore, whereas platelet NOS activity increased as expected in response to albuterol or collagen in controls, no increase occurred in platelets from heart failure subjects. Despite this, basal intraplatelet NO-attributable cGMP was lower in heart failure than in control subjects, as was serine-239 phosphorylation of VASP, suggesting a decrease in bioactive NO. Platelets from heart failure subjects exhibited higher basal and collagen-stimulated O(2)(-) production and impaired O(2)(-) scavenging capacity, resulting in higher oxidative stress, consistent with the observed decrease in bioactive NO. In heart failure, despite activation of NOS, platelets produce less bioactive NO, probably as a result of NO scavenging due to increased O(2)(-) production. This functional defect in the platelet l-arginine/NO/guanylyl cyclase pathway could contribute to the platelet activation observed in heart failure.

Platelet Nitric Oxide Signaling System in Patients with Coronary Artery Disease

Coronary artery disease (CAD) is the leading cause of death worldwide, and the major cause of hospital admissions in the Western countries. The pathogenesis of CAD is closely related to nitric oxide release and formation. The purpose of this study was to investigate the status of platelets nitric oxide in patients with coronary artery disease. We measured platelets aggregation, cGMP, NO (nitrite/nitrate level), NO synthase activity, plasma NO, and ionized Ca(2+) in 40 healthy volunteers and 120 patients with myocardial infarction, unstable and stable angina, with 40 subjects in each group. The subjects' age mean range was from 40-51 years. Platelets aggregation, NO, cGMP, NO synthase activity, plasma NO and ionized Ca(2+) have increased significantly (P <0.001) across the patients groups compared to controls. Platelets NO synthase activity (mean ± SD / U / 10(9) platelets) in healthy controls, MI, unstable angina and stable angina patients were 1.19 ± 0.56, 1.21 ± 0.64, 1.64 ± 0.98 and 1.57 ± 0.81 respectively. The cGMP (mean ± SD / pmole / 10(9) platelets) levels were 0.95 ± 0.41, 1.53 ± 0.64, 3.18 ± 0.77, and 5.12 ± 1.5 respectively. The present study demonstrated that platelets aggregation, NO, cGMP, NO synthase activity, plasma NO, and ionized Ca(2+) profoundly increased in CAD. The increases in NO-cGMP components may have resulted as a compensatory response to ameliorate platelet activity and Ca(2+) levels in CAD patients.

Pyridoxine Improves Platelet Nitric Oxide Synthase Dysfunction Induced by Advanced Glycation End Products in vitro

Advanced glycation end products (AGEs) increase platelet aggregation and suppress vascular nitric oxide (NO) synthase (NOS) activity, and these effects may contribute to the atherothrombotic disease seen in diabetes. The aims of this study were to determine in vitro whether pyridoxine can abrogate the impairment in platelet NOS activity caused by AGEs, and to determine the mechanism by which it does this. Platelet aggregation was measured by Born aggregometry. Intraplatelet cyclic guanosine-3',5'-monophosphate (cGMP, an index of bioactive NO) was measured by radioimmunoassay. Serine-1177-specific phosphorylation of NOS type 3 (NOS-3) and phosphorylation of protein kinase Akt were determined in platelets by Western blotting. Phosphatidylinositol 3-kinase (PI3K) activity in platelets was ascertained by homogeneous time-resolved fluorescence (HTRF) assay. We found that AGE-modified albumin (AGEs) 200 mg/L increased platelet aggregability and decreased intraplatelet cGMP; these effects were largely attenuated by pyridoxine. Western blotting studies revealed that AGEs decreased NOS-3 phosphorylation on serine-1177, increased NOS-3 O-glycosylation, and decreased serine phosphorylation of protein kinase Akt; all of these changes were abrogated by pyridoxine. Direct measurement of PI3K activity in platelets demonstrated that all of the above effects could be attributed to a suppression by AGEs of PI3K activity, which was prevented by co-incubation with pyridoxine. We conclude that pyridoxine is effective in ameliorating the dysfunction of platelet NO signaling in response to AGEs, through improving PI3K activity, and hence downstream Akt phosphorylation and in turn serine-1177 phosphorylation of NOS-3.

Decreased platelet nitric oxide contributes to increased circulating monocyte-platelet aggregates in hypertension

The aim of this study was to determine the effect of blood pressure (BP) on platelet nitric oxide (NO) signalling and on formation of circulating monocyte-platelet aggregates (MPA), as well as the role of platelet NO in modulating MPA in hypertension. We first examined platelet NO signalling in 23 untreated hypertensive (UH) and 23 normotensive (NT) subjects. Platelets from hypertensives exhibited reduced NO synthase activation by albuterol or collagen, as well as suppressed basal and stimulated NO-attributable cyclic guanosine-3',5'-monophosphate, compared with NT. In a second study, comprising 106 subjects with a wide BP range, circulating MPA showed a strong positive correlation with BP. On multiple regression analysis, using a model incorporating systolic BP (SBP), diastolic BP, age, lipids, gender, and smoking status, the only independent predictor of MPA was SBP. Nitric oxide synthase inhibition with N(G)-monomethyl-L-arginine increased MPA in NT but not in hypertensives, whereas the NO donor spermine NONOate (SNO) decreased MPA in NT but not in hypertensives. Platelet P-selectin expression was higher in hypertensives than in NT, and its expression was suppressed by SNO in NT only. Platelet NO production and responsiveness are suppressed with raised BP, and this may contribute to the increase in platelet P-selectin and hence in circulating MPA in hypertension.

Platelet-Derived Nitric Oxide Signaling and Regulation

Nitric oxide (NO) exerts important vasodilatory, antiplatelet, antioxidant, antiadhesive, and antiproliferative effects. Although endothelium derived NO has been shown to be of prime importance in cardio- and vasculoprotection, until recently little was known about the role of platelet-derived NO. New evidence suggests that NO synthesized by platelets regulates platelet functions, in particular suppressing platelet activation and intravascular thrombosis. Moreover, platelet NO biosynthesis may be decreased in patients with cardiovascular risk factors or with coronary heart disease, and this may contribute to arterial thrombotic disease in these patients. Here, we review the current state of knowledge as regards the role of platelet-derived NO, both in normal physiology and in cardiovascular disease states, and compare platelet NO signaling and regulation with that in endothelial cells.

Age decreases nitric oxide synthesis and responsiveness in human platelets and increases formation of monocyte–platelet aggregates☆

Ageing is associated with an increase in atherothrombotic disease. Platelet-derived nitric oxide (NO) inhibits platelet activation, but the effect of age on platelet NO signaling is unknown. We investigated platelet NO biosynthesis and responsiveness in older (> 45 years old) as compared with younger (< 30 years old) healthy human subjects. Platelet NO synthase (NOS) activity was evaluated by l-[3H]-arginine to l-[3H]-citrulline conversion, and cGMP was determined by radioimmunoassay. Platelet expression of NOS3, phosphoserine-1177-NOS3 and soluble guanylyl cyclase (sGC) were quantified by Western blotting. Circulating monocyte-platelet aggregates (MPA) were measured by flow cytometry. Basal NOS activity was similar in both groups. By contrast, whereas both albuterol and collagen stimulated platelet NOS in younger subjects, stimulation was absent in older subjects. Platelet NOS3 expression was similar in both age groups, but NOS3 serine-1177 phosphorylation was greater in younger subjects. Basal, albuterol- and collagen-stimulated cGMP, as well as sGC expression, were all greater in younger than older subjects, and within the younger group both cGMP (basal and stimulated) and sGC expression were greater in women than in men. Circulating MPA were greater in older subjects and, whilst NOS inhibition increased MPA further in both groups, it did so to a lesser extent in the older age bracket. These data suggest that platelet NO production and responsiveness decrease with age, and this is reflected in increased circulating MPA.