EVIDENCE FOR CAMP-DEPENDENT PLATELET ECTOPROTEIN KINASE-ACTIVITY THATPHOSPHORYLATES PLATELET GLYCOPROTEIN-IV (CD36)

The dephosphorylating enzyme alkaline phosphatase, by removing phospha te groups from the external platelet membrane proteins, modulates plat elet activation (Hatmi, M., Haye, B., Gavaret, J. M., Vargaftig, B. B. , and Jacquemin, C. (1991) Br. J. Pharmacol. 104, 554-558). This obser vation, together with findings reported by others (Ehrlich, Y. H., Dav is, T. B., Beck, E., Kornecki, E., and Lenox, R. H. (1986) Nature 320, 67-70; Dusenbery, K. E., Mendiola, J. R., and Skubitz, K. M. (1988) B iochem. Biophys. Res. Commun. 153, 7-13), indicate the existence of ec toprotein kinase activity on the blood platelet surface. In this study , we demonstrate that washed human platelets phosphorylate the synthet ic heptapeptide kemptide in a cAMP-dependent mode. The intensity of th e phosphorylation was concentration-dependent for kemptide. In additio n, incubation of platelets with [gamma-P-32]ATP resulted in a rapid in corporation of [P-32] phosphate into proteins at the outer membrane su rface that was sensitive to alkaline phosphatase treatment. When cAMP was added to the medium, major phosphorylation of an 88-kDa ectoprotei n occurred. Its isoelectric point determined by isoelectric focusing S DS-polyacrylamide gel electrophoresis was around pH 6.2. Phosphorylati ons of this 88-kDa polypeptide and of the exogenous kemptide substrate were both prevented by the specific protein kinase A inhibitor peptid e. When platelets were preincubated with [P-32]inorganic phosphate to label intracellular proteins, the protein phosphorylation pattern was different from that obtained with [gamma-P-32]ATP, indicating that the latter occurred at the outer surface of the cells. Prostacyclin, whic h induces the increase of intracellular cAMP levels and, consequently, its liberation into the extracellular medium, increased phosphorylati on of both kemptide and platelet 88-kDa polypeptide. The major protein of 88-kDa, which was phosphorylated in the presence of cAMP and exter nal [gamma-P-32]ATP, was identified by immunoprecipitation to GPIV (CD 36), one of thrombospondin and collagen binding sites on platelets. Th e phosphorylation of CD36 also occurred in platelet-rich plasma, sugge sting a physiological role for this ectoenzyme. In the present study, we clearly demonstrate the presence of an ectoprotein kinase A activit y at the surface of intact human platelets, and we revealed its princi pal endogenous substrate as being CD36.