Oxidized LDL Decreases l -Arginine Uptake and Nitric Oxide Synthase Protein Expression in Human Platelets

Abstract

Background Oxidized LDL (ox-LDL) promotes vasoconstriction and platelet activation. The present study was undertaken to determine the involvement of the l -arginine–nitric oxide (NO) pathway in ox-LDL–mediated platelet activation. Methods and Results Washed human platelets were incubated with native LDL or ox-LDL for 1 hour at 37°C followed by measurement of platelet function and indexes of the l -arginine–NO pathway. Ox-LDL but not native LDL caused a concentration-dependent increase in thrombin-induced platelet aggregation and 14 C-serotonin release. These effects of ox-LDL were inhibited by pretreatment of platelets with l -arginine, the precursor of NO. Ox-LDL also caused a concentration-dependent reduction in the uptake of 3 H- l -arginine by platelets. In addition, NO synthase activity, measured as conversion of 3 H- l -arginine to 3 H- l -citrulline, decreased on incubation of platelet cytosol with ox-LDL. Nitrite production was also reduced by treatment of platelets with ox-LDL. These effects of ox-LDL on NO synthase activity and nitrite production were reversed by pretreatment of platelets with l -arginine. Concurrent with the decrease in NO production, cytosolic cGMP was inhibited in ox-LDL–treated platelets. The inhibitory effects of ox-LDL were dependent in part on the increase of cholesterol in the platelets. Western blot analysis demonstrated ≈50% reduction in the expression of NO synthase protein in platelets treated with ox-LDL. Conclusions These observations indicate that the l -arginine–NO pathway is involved in the effects of ox-LDL on platelet function and that ox-LDL stimulates platelet function primarily by diminishing NO synthase expression as well as decreasing the uptake of l -arginine.