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), 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), and the NO scavenger oxy-haemoglobin.


RESULTS
Peroxynitrite (50-400 microM) 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 microM) 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.
Brown
A S
AS
Department of Cardiology, King's College Hospital, London, UK.
Moro
M A
MA
Masse
J M
JM
Cramer
E M
EM
Radomski
M
M
Darley-Usmar
V
V
D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D003094:Collagen / Q000494:pharmacology; D006152:Cyclic GMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D016166:Free Radical Scavengers; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors; D006801:Humans; D007150:Immunohistochemistry; D009566:Nitrates / Q000494:pharmacology*; D009569:Nitric Oxide / Q000378:metabolism*; D010069:Oxadiazoles / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology*; D010108:Oxyhemoglobins / Q000494:pharmacology; D019007:P-Selectin / Q000378:metabolism; D015539:Platelet Activation / Q000187:drug effects*; D011810:Quinoxalines / Q000494:pharmacology
C095284:1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one; D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D009566:Nitrates; D010069:Oxadiazoles; D016877:Oxidants; D010108:Oxyhemoglobins; D019007:P-Selectin; D011810:Quinoxalines; C039642:peroxynitric acid; D009569:Nitric Oxide; D003094:Collagen; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D002118:Calcium