Brown A S, Moro M A, Masse J M, Cramer E M, Radomski M, Darley-Usmar V
Department of Cardiology, King's College Hospital, London, UK.
Cardiovasc Res. 1998 Nov;40(2):380-8. doi: 10.1016/s0008-6363(98)00182-5.
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.
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.
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.
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.
过氧亚硝酸盐（ONOO⁻）是超氧化物和一氧化氮（NO）在炎症部位快速反应形成的一种氧化剂。文献报道了关于ONOO⁻在生物系统中作用的相互矛盾的数据，已证实其具有依赖NO和依赖氧化剂的两种作用。本研究的目的是通过研究ONOO⁻对人血小板作用的分子层面，来探究这些不同的机制，我们之前已表明在该系统中ONOO⁻具有促聚集和抗聚集两种作用。
通过流式细胞术检测血小板P选择素表达、血小板内Ca²⁺浓度以及光散射比浊法来评估血小板功能。采用比色法测量细胞外血小板膜硫醇。使用可溶性鸟苷酸环化酶（sGC）抑制剂1H-[1,2,4]恶二唑并[4,3-α]喹喔啉-1-酮（ODQ）和NO清除剂氧合血红蛋白，研究NO和cGMP对ONOO⁻药理作用的贡献。
过氧亚硝酸盐（50 - 400微摩尔）导致表达P选择素的血小板数量呈浓度依赖性增加、血小板内Ca²⁺浓度升高以及血小板膜硫醇减少。ODQ增强了过氧亚硝酸盐诱导的P选择素表达。相反，当胶原诱导P选择素表达时，ONOO⁻起到该过程的抑制剂作用，加入1%血浆可进一步增强此作用，ODQ或氧合血红蛋白可消除这种抑制作用。最后，低浓度（50 - 100微摩尔）的ONOO⁻抑制胶原诱导的血小板聚集，氧合血红蛋白可逆转此作用。
过氧亚硝酸盐对血小板发挥双重作用，由于ONOO⁻转化为NO或NO供体，其作用可为激活或抑制。过氧亚硝酸盐诱导的血小板激活似乎是由于硫醇氧化和细胞内Ca²⁺增加。需要注意的是抑制性的、依赖NO的作用在比激活作用更低的浓度下发生。这些数据与相互矛盾的文献一致，表明ONOO⁻在生物系统中具有损伤和细胞保护两种作用。我们推测ONOO⁻向NO的转化是决定体内ONOO⁻暴露结果的关键因素。
