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Objective: 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 (N45 years old) as compared with younger (b30 years old) healthy human subjects. Methods: Platelet NO synthase (NOS) activity was evaluated by l-[ 3 H]-arginine to l-[ 3 H]-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. Results: 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. Conclusions: These data suggest that platelet NO production and responsiveness decrease with age, and this is reflected in increased circulating MPA.

Aims 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 (O2−) production in patients with heart failure. Methods and results 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-[3H]-arginine to l-[3H]-citrulline conversion, cGMP was determined by radioimmunoassay, vasodilator-stimulated phosphoprotein (VASP: total and serine-239-phosphorylated) was assessed by western blotting, and O2− production and O2− 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 O2− production and impaired O2− scavenging capacity, resulting in higher oxidative stress, consistent with the observed decrease in bioactive NO. Conclusion In heart failure, despite activation of NOS, platelets produce less bioactive NO, probably as a result of NO scavenging due to increased O2− production. This functional defect in the platelet l-arginine/NO/guanylyl cyclase pathway could contribute to the platelet activation observed in heart failure.