HDL3-MEDIATED INHIBITION OF THROMBIN-INDUCED PLATELET-AGGREGATION ANDFIBRINOGEN BINDING OCCURS VIA DECREASED PRODUCTION OF PHOSPHOINOSITIDE-DERIVED 2ND-MESSENGERS 1,2-DIACYLGLYCEROL AND INOSITOL 1,4,5-TRIS-PHOSPHATE

We demonstrate that physiological concentrations of HDL3 inhibit the t hrombin-induced platelet fibrinogen binding and aggregation in a time- and concentration-dependent fashion. The underlying mechanism includes HDL3-mediated inhibition of phosphatidylinositol 4,5-bis-phosphate tu rnover, 1,2-diacylglycerol and inositol 1,4,5-tris-phosphate formation , and intracellular calcium mobilization, The inhibitory effects of HD L3 on inositol 1,4,5-tris-phosphate formation and intracellular calciu m mobilization were abolished after covalent modification of HDL3 with dimethylsuberimidate. Furthermore, they could be blocked by calphosti n C and bis-indolylmaleimide, 2 highly selective and structurally unre lated protein kinase C inhibitors. However, the inhibitory effects of HDL3 were not blocked by H89, a protein kinase A inhibitor. In additio n, HDL3 failed to induce cAMP formation but stimulated the phosphoryla tion of the protein kinase C 40- to 47-kD major protein substrate. We observed a close temporal relationship between the HDL3-mediated inhib ition of thrombin-induced inositol 1,4,5-tris-phosphate formation, int racellular calcium mobilization, and fibrinogen binding and the phosph orylation of the protein kinase C 40- to 47-kD major protein substrate . Taken together, these findings indicate that the HDL3-mediated inhib ition of thrombin-induced fibrinogen binding and aggregation occurs vi a inhibition of phosphatidylinositol 4,5-bis-phosphate turnover and fo rmation of 1,2-diacylglycerol and inositol 1,4,5-tris-phosphate. Prote in kinase C may be involved in this process.