Peroxynitrite can affect platelet responses by inhibiting energy production

Abstract

Peroxynitrite (ONOO-) strongly inhibits agonist-induced platelet responses. However, the mechanisms involved are not completely defined. Using porcine platelets, we tested the hypothesis that ONOO- reduces platelet aggregation and dense granule secretion by inhibiting energy production. It was found that ONOO- (25-300 μM) inhibited collagen-induced dense granule secretion (IC50 = 55±7 μM) more strongly than aggregation (IC50 = 124±16 μM). The antiaggregatory and antisecretory effects of ONOO- were only slightly (5-10%) reduced by 1H-[1,2,4]-oxadiazolo-[4,3-α]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase. In resting platelets ONOO- (50-300 μM) enhanced glycolysis rate and reduced oxygen consumption, in a dose dependent manner. The ONOO- effects on glycolysis rate and oxygen consumption were not abolished by ODQ. The extent of glycolysis stimulation exerted by ONOO- was similar to that produced by respiratory chain inhibitors (cyanide and antimycin A) or an uncoupler (2,4-dinitrophenol). Stimulation of platelets by collagen was associated with a rise in mitochondrial oxygen consumption, accelerated lactate production, and unchanged intracellular ATP content. In contrast to resting cells, in collagen-stimulated platelets, ONOO- (200 μM) distinctly decreased the cellular ATP content. The glycolytic activity and oxygen consumption of resting platelets were not affected by 8-bromoguanosine 3′,5′-cyclic monophosphate. Blocking of the mitochondrial ATP production by antimycin A slightly reduced collagen-induced aggregation and strongly inhibited dense granule secretion. Treatment of platelets with ONOO- (50-300 μM) resulted in decreased activities of NADH:ubiquinone oxidoreductase, succinate dehydrogenase and cytochrome oxidase. It is concluded that the inhibitory effect of ONOO- on platelet secretion and to a lesser extent on aggregation may be mediated, at least in part, by the reduction of mitochondrial energy production.