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

Abstract

Platelets labeled with 2′,7′-dihydrodichlorofluorescein diacetate (DCF-DA) and stimulated with 50–400 nM peroxynitrite (ONOO −) produced a rapid increase of the fluorescence signal at 523 nm 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 μM 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 0.75 mM) 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 (1 U/mL) produced a rapid increase in fluorescence that corresponded to the formation of 20.5 nmol ONOO − per 10 7 cells, whereas stimulation with collagen and arachidonic acid was without effect. Hypoxia of platelets for 20 and 40 min followed by 5 min 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.