Nitric oxide-dependent and independent effects on human platelets treated

Objective: Peroxynitrite (ONOO ) is an oxidant formed from the rapid reaction of superoxide and nitric oxide (NO) at sites of 2 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 2 2 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, 21 intraplatelet Ca concentrations, and by light aggregometry. A colorimetric method was used to measure extracellular platelet membrane 2 thiols. The contribution of NO and cGMP to the pharmacological effects of ONOO was investigated using an inhibitor of the soluble guanylate cyclase (sGC), 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), and the NO scavenger oxy-haemoglobin. Results: Peroxynitrite (50-400 mM) caused a concentration-dependent increase in the number of platelets expressing P-selectin, an increase in 21 intraplatelet Ca concentrations and a decrease in platelet membrane thiols. Peroxynitrite-induced P-selectin expression was augmented 2 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 2 (50-100 mM) of ONOO inhibited collagen-induced platelet aggregation, an effect that was reversed by oxy-haemoglobin. Conclusions: 2 Peroxynitrite exerts dual effects on platelets, which are either activating or inhibitory due to the conversion of ONOO to NO or NO 21 donors. Peroxynitrite-induced platelet activation seems to be due to thiol oxidation and an increase in intracellular Ca . It is important to note that inhibitory, NO-dependent effects occur at lower concentrations than the activating effects. These data are then consistent with 2 the conflicting literature, showing both damaging and cytoprotective effects of ONOO in biological systems. We hypothesize that the 2 2 conversion of ONOO to NO is the critical factor determining the outcome of ONOO exposure in vivo. © 1998 Elsevier Science B.V. All rights reserved.