407-3 Glutathione Peroxidase Prevents the Inactivation of Nitric Oxide and Restores the Inhibition of Platelet Function by S-Nitrosothiols

The interaction of nitric oxide (NO) with reactive oxygen species in the vasculature can inactivate NO leading to potentially adverse vascular consequences. Glutathione peroxidases (GSH-Px), a family of antioxidant enzymes present at reduced concentrations in plasma and platelets of patients with coronary artery disease, catalyze the reduction of hydrogen peroxide and lipid hydroperoxides (LOOH) by glutathione. Given the role of LOOH in platelet eicosanoid metabolism and their presence in atherosclerotic plaque, we investigated the effect of GSH-Px on the inhibition of platelet function by the naturally occurring NO donor, S-nitroso-glutathione (SNO-Glu). Subthreshold inhibitory concentrations of SNO-Glu were added to platelet-rich plasma, and aggregation was induced by arachidonate. The addition of GSHPx (0.2–20U/ml) to this system led to a dose-dependent inhibition of platelet aggregation with an IC50= 0.6U/ml GSH-Px (p < 0.05 by ANOVA). Superoxide dismutase (0.1–200U/ml), catalase (0.1–200U/ml), or GSH-Px without SNO-Glu did not alter platelet aggregation responses. The addi ion of GSH-Px to a subthreshold inhibitory concentration of sodium nitroprusside also did not affect platelet aggregation responses. LOOH increased platelet aggregation in the presence of SNO-Glu, an effect reversed by GSH-Px. Levels of cGMP were measured after platelets were incubated with SNO-Glu, exogenous LOOH, and GSH-Px. SNO-Glu alone increased cGMP levels, and this effect was attenuated by LOOH but restored by the addition of GSH-Px. GSH-Px activity was equivalent with either SNO-Glu or glutathione as cosubstrate. Incubation of SNO-Glu with GSH-Px led to a 48.5% decrease in the concentration of SNO-Glu as determined by HPLC-electrochemical detection. Incubation of SNO-Glu with albumin in the presence of GSH-Px led to increased formation of S-nitroso-albumin, a prevalent reservoir of EDRF in plasma. These results show that GSH-Px, at physiologically relevant concentrations, has a potent effect on NO-induced inhibition of platelet aggregation and that this enzyme may have two functions: (i) metabolism of LOOH, thereby preventing its inactivation of NO; and (ii) metabolism of SNO-Glu, thereby liberating NO and/or supporting further transnitrosation reactions These findings suggest that GSH-Px, in addition to its antioxidant functions, regulates the availability of NO in the vasculature and possibly alters plateletdependent thrombotic events