Rusak Tomasz, Tomasiak Marian, Ciborowski Michal
Department of Physical Chemistry, Medical University of Bialystok, Białystok, Poland.
Acta Biochim Pol. 2006;53(4):769-76. Epub 2006 Oct 27.
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 microM) inhibited collagen-induced dense granule secretion (IC50 = 55 +/- 7 microM) more strongly than aggregation (IC(50) = 124 +/- 16 microM). The antiaggregatory and antisecretory effects of ONOO- were only slightly (5-10%) reduced by 1H-[1,2,4]-oxadiazolo-[4,3-alpha]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase. In resting platelets ONOO- (50-300 microM) 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 microM) 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 microM) 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.
过氧亚硝酸盐（ONOO-）强烈抑制激动剂诱导的血小板反应。然而，其中涉及的机制尚未完全明确。我们使用猪血小板来检验ONOO-通过抑制能量产生来降低血小板聚集和致密颗粒分泌的假说。结果发现，ONOO-（25 - 300微摩尔）对胶原诱导的致密颗粒分泌的抑制作用（IC50 = 55±7微摩尔）比对聚集的抑制作用（IC50 = 124±16微摩尔）更强。可溶性鸟苷酸环化酶抑制剂1H-[1,2,4]-恶二唑并-[4,3-α]喹喔啉-1-酮（ODQ）仅使ONOO-的抗聚集和抗分泌作用轻微降低（5 - 10%）。在静息血小板中，ONOO-（50 - 300微摩尔）以剂量依赖的方式提高糖酵解速率并降低耗氧量。ODQ并未消除ONOO-对糖酵解速率和耗氧量的影响。ONOO-对糖酵解的刺激程度与呼吸链抑制剂（氰化物和抗霉素A）或解偶联剂（2,4-二硝基苯酚）产生的刺激程度相似。胶原刺激血小板会导致线粒体耗氧量增加、乳酸生成加速以及细胞内ATP含量不变。与静息细胞不同，在胶原刺激的血小板中，ONOO-（200微摩尔）显著降低细胞ATP含量。静息血小板的糖酵解活性和耗氧量不受8-溴鸟苷3',5'-环磷酸的影响。抗霉素A阻断线粒体ATP产生会轻微降低胶原诱导的聚集并强烈抑制致密颗粒分泌。用ONOO-（50 - 300微摩尔）处理血小板会导致NADH：泛醌氧化还原酶、琥珀酸脱氢酶和细胞色素氧化酶的活性降低。得出的结论是，ONOO-对血小板分泌的抑制作用以及在较小程度上对聚集的抑制作用可能至少部分是由线粒体能量产生的减少介导的。
