PROTEIN-KINASE C-DEPENDENT DESENSITIZATION OF HDL(3)-ACTIVATED PHOSPHOLIPASE-C IN HUMAN PLATELETS

In isolated human platelets, exposure of subfraction 3 high-density li poprotein (HDL(3)) binding sites to high concentrations of HDL(3) (1 m g/mL) causes rapid desensitization of HDL(3) (50 mu g/mL)-stimulated b reakdown of phosphatidylcholine, as shown in approximately a 70% depre ssion of the maximal 1,2-diacylglycerol release activity by phospholip ase C. This desensitization is HDL(3) dose dependent (IC50, 150+/-20 m u g/mL, n=6) and time dependent (t(1/2), <30 seconds). It requires the binding of HDL(3), as pretreatment of HDL(3) by tetranitromethane doe s not cause the desensitization of HDL(3)-induced phospholipase C acti vity. Permeabilization of human platelets with 10 mu g/mL digitonin, u sed to permit access of charged inhibitors to the cytosol, does not in terfere with the pattern of HDL(3) (1 mg/mL)-induced desensitization o f HDL(3) (50 mu g/mL)-stimulated phospholipase C. Inhibitors of protei n kinase C (100 mu mol/L H-7 and 10 mu mol/L staurosporine) markedly i nhibit desensitization of HDL(3)-induced phospholipase C activity, whe reas CAMP-dependent protein kinase inhibitor (1 mu mol/L), heparin (10 0 nmol/L), or concanavalin A (0.25 mg/mL) were ineffective. HDL(3)-ind uced desensitization is accompanied at least by the phosphorylation of the 94- and 110-kD proteins. Inhibition of HDL(3)-induced desensitiza tion by 100 mu mol/L H-7 or 10 mu mol/L staurosporine is characterized by a marked reduction of the phosphorylation state of these proteins in permeabilized platelets. Whereas protein kinase C inhibitors fully inhibited the phosphorylation of the 94- and 110-kD proteins, inhibito rs of protein kinase A were less effective. These data establish that phosphorylation by protein kinase C represent a step in the desensitiz ation of HDL(3) binding sites in human platelets.