Nitric oxide-dependent and independent effects on human platelets treated with peroxynitrite

Objective: Peroxynitrite (ONOO−) is an oxidant formed from the rapid reaction of superoxide and nitric oxide (NO) at sites of inflammation. The literature reports conflicting data on the effects of ONOO− in biological systems, with both NO- and oxidant-dependent effects having been demonstrated. The aim of this study was to investigate these distinct mechanisms through examining molecular aspects of the effects of ONOO− on human platelets, a system in which we have previously shown that ONOO− has both pro- and anti-aggregatory effects. Methods: Platelet function was assessed by measuring platelet P-selectin expression flow cytometrically, intraplatelet Ca2+ concentrations, and by light aggregometry. A colorimetric method was used to measure extracellular platelet membrane thiols. The contribution of NO and cGMP to the pharmacological effects of ONOO− was investigated using an inhibitor of the soluble guanylate cyclase (sGC), 1 H -[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ), and the NO scavenger oxy-haemoglobin. Results: Peroxynitrite (50–400 μM) caused a concentration-dependent increase in the number of platelets expressing P-selectin, an increase in intraplatelet Ca2+ concentrations and a decrease in platelet membrane thiols. Peroxynitrite-induced P-selectin expression was augmented by ODQ. In contrast, when P-selectin expression was elicited by collagen, ONOO− acted as an inhibitor of this process, an effect that was further enhanced by the addition of 1% plasma. ODQ or oxy-haemoglobin abolished this inhibitory effect. Finally, low concentrations (50–100 μM) of ONOO− inhibited collagen-induced platelet aggregation, an effect that was reversed by oxy-haemoglobin. Conclusions: Peroxynitrite exerts dual effects on platelets, which are either activating or inhibitory due to the conversion of ONOO− to NO or NO donors. Peroxynitrite-induced platelet activation seems to be due to thiol oxidation and an increase in intracellular Ca2+. It is important to note that inhibitory, NO-dependent effects occur at lower concentrations than the activating effects. These data are then consistent with the conflicting literature, showing both damaging and cytoprotective effects of ONOO− in biological systems. We hypothesize that the conversion of ONOO− to NO is the critical factor determining the outcome of ONOO− exposure in vivo.