一氧化氮依赖性和非依赖性对经过氧亚硝酸盐处理的人类血小板的影响。

Nitric oxide-dependent and independent effects on human platelets treated with peroxynitrite.

作者信息

Brown A S, Moro M A, Masse J M, Cramer E M, Radomski M, Darley-Usmar V

机构信息

Department of Cardiology, King's College Hospital, London, UK.

出版信息

Cardiovasc Res. 1998 Nov;40(2):380-8. doi: 10.1016/s0008-6363(98)00182-5.

DOI:10.1016/s0008-6363(98)00182-5

PMID:9893732

Abstract

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.

摘要

目的

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。

方法

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。

结果

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。

结论

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。