Synthesis and mechanisms of action of nitric oxide in the human platelet

Nitric oxide (NO) is a gas produced by the nitric oxide synthase (NOS) family of enzymes. Constitutively expressed (c) and inducible (i) NOSs exist. NO has a number of physiologic and pathophysiologic functions. In particular, because of its antiplatelet effects, NO is thought to have an important role in hemostasis and thrombosis.;To assess whether platelets possess NOS isoforms, NO synthase assays were carried out in lysates of human platelets and immortalized human megakaryocytic cells (MEG-O1). Platelets were found to have only cNOS activity. In addition to cNOS activity, MEG-O1 cells also expressed iNOS activity and did so even in the absence of cytokine induction. Western, Northern and reverse transcriptase polymerase chain reaction analyses confirmed these results.;Novel activating signaling pathways in the platelet were investigated as possible targets of NO's inhibitory action. These pathways, which interact closely, are regulated by protein tyrosine kinases of the src family and by the enzyme phosphoinositide 3-kinase (PI3-K). Platelet incubation with NO donors prior to thrombin or thrombin receptor activating peptide (TRAP) activation resulted in the inhibition of PI3-K activity as assessed by lipid kinase assays of anti-phosphotyrosine immunoprecipitates. Inhibition of PI3-K activity by NO was also demonstrated in immunoprecipitates of the src family member, lyn. These effects were shown to be mediated by cGMP. Immunoprecipitation-Western analysis confirmed the physical association of lyn and PI3-K in a manner that was not regulated by TRAP. Assays of lyn-associated protein kinases demonstrated the differential regulation of these activities by TRAP and the ability of NO to antagonize the effects of TRAP. Western blots with anti-phosphotyrosine antibody revealed the inhibitory action of NO on tyrosine kinase activities in cytosolic and cytoskeletal fractions. Flow cytometry showed that NO and wortmannin (a specific PI3-K inhibitor) have at least additive inhibitory effects on the activation of surface molecules important in platelet aggregation, namely, the fibrinogen receptor glycoprotein IIb/IIIa and P-selectin. These studies provide novel clues to understanding the antiplatelet effects of endothelium/platelet-derived nitric oxide.