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 degrees 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 14C-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 3H-L-arginine by platelets. In addition, NO synthase activity, measured as conversion of 3H-L-arginine to 3H-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 approximately 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.