The activity of constitutive nitric oxide synthase is increased by the pathway cAMP/cAMP-activated protein kinase in human platelets. New insights into the antiaggregating effects of cAMP-elevating agents

Human platelets synthesize nitric oxide (NO) through an endothelial-type NO synthase (ecNOS) activated also by substances enhancing 3′,5′- cyclic adenosine monophosphate (cAMP) concentrations, such as catecholamines, β-adrenoceptor agonists and adenosine. To verify whether cAMP directly activates ecNOS through the cAMP-dependent protein kinase A (PKA), we evaluated (i) the influence of 8-Br-cAMP, adenosine and forskolin on ecNOS activity and phosphorylation at Ser ¹¹⁷⁷ and (ii) the effect of PKA inhibition on ecNOS activity. Platelets from 10 healthy male volunteers were used for aggregation studies and measurement of NOS activity (conversion of l-[ ³H]-arginine to l-[ ³H]-citrulline) following exposure to 8-Br-cAMP, adenosine and forskolin, both in the absence and in the presence of the PKA inhibitor Rp-cAMPS (100 μmol/l). The phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein (VASP) at Ser ¹⁵⁷ and Ser ²³⁹ and of ecNOS at Ser ¹¹⁷⁷ was evaluated by Western blot. NOS activity (pmol l-citrulline/10 ⁸ platelets) increased from 0.090±0.002 to 0.148±0.013 with 500 μmol/l 8-Br-cAMP (p1177. The study shows that NOS activity of human platelets is increased by the cAMP/PKA pathway which is involved in NO synthesis induced by adenosine, forskolin and potentially by every antiaggregating substance enhancing intraplatelet cAMP via receptor-dependent and -independent mechanisms.