Abstract
The dephosphorylating enzyme alkaline phosphatase, by removing phosphate groups from the external platelet membrane proteins, modulates platelet activation (Hatmi, M., Haye, B., Gavaret, J. M., Vargaftig, B. B., and Jacquemin, C. (1991) Br. J. Pharmacol. 104, 554-558). This observation, together with findings reported by others (Ehrlich, Y. H., Davis, T. B., Bock, E., Kornecki, E., and Lenox, R. H. (1986) Nature 320, 67-70; Dusenbery, K. E., Mendiola, J. R., and Skubitz, K. M. (1988) Biochem. Biophys. Res. Commun. 153, 7-13), indicate the existence of ectoprotein kinase activity on the blood platelet surface. In this study, we demonstrate that washed human platelets phosphorylate the synthetic heptapeptide kemptide in a cAMP-dependent mode. The intensity of the phosphorylation was concentration-dependent for kemptide. In addition, incubation of platelets with [gamma-32P]ATP resulted in a rapid incorporation of [32P] phosphate into proteins at the outer membrane surface that was sensitive to alkaline phosphatase treatment. When cAMP was added to the medium, major phosphorylation of an 88-kDa ectoprotein occurred. Its isoelectric point determined by isoelectric focusing SDS-polyacrylamide gel electrophoresis was around pH 6.2. Phosphorylations of this 88-kDa polypeptide and of the exogenous kemptide substrate were both prevented by the specific protein kinase A inhibitor peptide. When platelets were preincubated with [32P]inorganic phosphate to label intracellular proteins, the protein phosphorylation pattern was different from that obtained with [gamma-32P]ATP, indicating that the latter occurred at the outer surface of the cells. Prostacyclin, which induces the increase of intracellular cAMP levels and, consequently, its liberation into the extracellular medium, increased phosphorylation of both kemptide and platelet 88-kDa polypeptide. The major protein of 88-kDa, which was phosphorylated in the presence of cAMP and external [gamma-32P]ATP, was identified by immunoprecipitation to GPIV (CD36), one of thrombospondin and collagen binding sites on platelets. The phosphorylation of CD36 also occurred in platelet-rich plasma, suggesting a physiological role for this ectoenzyme. In the present study, we clearly demonstrate the presence of an ectoprotein kinase A activity at the surface of intact human platelets, and we revealed its principal endogenous substrate as being CD36.
Highlights
In this study, we demonstrate that washed human platelets phosphorylate the synthetic heptapeptide kemptide in a cAMP-dependent mode
We clearly demonstrate the presence of an ectoprotein kinase A activity at the surface of intact human platelets, and we revealed its principal endogenous substrate as being CD36
It is well known that intracellular protein kinase A (PKA)1 is important in the regulation of various platelet functions [11,12,13], but ecto-PKA activity in the plasma membrane has not been demonstrated in blood platelets
Summary
We demonstrate that washed human platelets phosphorylate the synthetic heptapeptide kemptide in a cAMP-dependent mode. Its isoelectric point determined by isoelectric focusing SDS-polyacrylamide gel electrophoresis was around pH 6.2 Phosphorylations of this 88-kDa polypeptide and of the exogenous kemptide substrate were both prevented by the specific protein kinase A inhibitor peptide. Extracellular ATP is known to exert an inhibitory effect on platelet activation by competing with adenosine diphosphate, increasing intracellular cAMP levels and probably phosphorylating surface platelet proteins [9]. Naik et al [10] have reported protein kinase and phosphatase activities on the membrane surface of human platelets, which rapidly phosphorylated and dephosphorylated 39- and 42-kDa proteins, whose function was undetermined. It is well known that intracellular protein kinase A (PKA) is important in the regulation of various platelet functions [11,12,13], but ecto-PKA activity in the plasma membrane has not been demonstrated in blood platelets. Our results suggest that rephosphorylation of CD36 by ecto-PKA could restore the binding properties of the resting state
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