THE SYNERGISM OF HYDROGEN-PEROXIDE WITH PLASMA S-NITROSOTHIOLS IN THEINHIBITION OF PLATELET ACTIVATION

Earlier studies have shown that inhibition of aggregation of washed pl atelets (WP) by NO was enhanced almost 100-fold by H2O2. In the presen t study, the interactions of H2O2 with nitrosothiols, the influence of the presence of plasma and the mechanism of the synergism were invest igated. H2O2 strongly enhanced the inhibitory effects of S-nitrosoglut athione (GSNO) on thrombin-induced aggregation of WP. S-Nitrosoalbumin also inhibited platelets, and this was similarly enhanced by H2O2. Th e synergism with H2O2 was demonstrable for both exogenous GSNO and NO in the presence of plasma when platelets were stimulated with collagen . The inhibition of platelets by GSNO and H2O2 was completely inhibite d by guanylate cyclase inhibitors. Synergism was also observed whether the H2O2 was added simultaneously or 1 min before or after the GSNO ( or NO). This suggests that the action of H2O2 follows the occupation b y NO of haem sites in guanylate cyclase and that a prior reaction betw een NO and H2O2 was not required. In the absence of exogenous GSNO or NO, H2O2 inhibited activation of platelets in plasma, an effect abolis hed by guanylate cyclase inhibitors. This suggested that endogenous NO donors in plasma or NO synthesized in platelets may interact with H2O 2. Addition of N-G-nitro-L-arginine methyl ester(hydrochloride) (L-NAM E) decreased the effects of the H2O2 by 25%, indicating that the major endogenous source of NO in platelet-rich plasma was not derived from platelet synthesis of NO but from NO donors in plasma, such as nitroso thiols. Inhibition by H2O2 was also enhanced by beta-mercaptosuccinate , a glutathione peroxidase inhibitor that protects the H2O2. These res ults suggest a potent synergism of H2O2 with endogenous plasma nitroso thiols that inhibit platelet function through an intracellular mechani sm involving guanylate cyclase.