Interactions of peroxynitrite and other nitrating substances with human platelets: the role of glutathione and peroxynitrite permeability.

Platelets labeled with 2',7'-dihydrodichlorofluorescein diacetate (DCF-DA) and stimulated with 50-400nM peroxynitrite (ONOO(-)) produced a rapid increase of the fluorescence signal at 523nm with good linearity and reproducibility. Platelet fluorescence was inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), suggesting that HCO(3)(-)/Cl(-) transporter mediated ONOO(-) transport into the platelets. Exposure of platelets to potassium superoxide, hydrogen peroxide, and sodium nitroprusside at concentrations of up to 100 microM did not generate a fluorescence signal. We also studied other nitrating compounds to establish the specificity of the DCF-DA-labeled platelet ONOO(-) assay. A rapid increase of fluorescence was observed when sodium hypochlorite (0.15 to 0.75mM) was added to platelets suspended in a buffered nitrite solution. Exposure of platelets to NO(2), nitroglycerin, and tetranitromethane produced a slow sustained increase of fluorescence. Endogenous glutathione appeared to be an essential factor in the generation of fluorescence by ONOO(-) and other nitrating compounds. We further studied other conditions that increased platelet fluorescence. Stimulation of platelets with thrombin (1U/mL) produced a rapid increase in fluorescence that corresponded to the formation of 20.5nmol ONOO(-) per 10(7) cells, whereas stimulation with collagen and arachidonic acid was without effect. Hypoxia of platelets for 20 and 40min followed by 5min of reoxygenation doubled the fluorescence from these platelets compared with control platelets. Thus, thrombin produced an effect that was likely due to the formation of ONOO(-) in platelets, whereas hypoxia-reoxygenation was likely to cause the formation of an active nitroglutathione-like molecule.