INOSITOL 1,4,5-TRISPHOSPHATE-MEDIATED CA2+ RELEASE FROM PLATELET INTERNAL MEMBRANES IS REGULATED BY DIFFERENTIAL PHOSPHORYLATION

Platelets are activated by an increase in cytosolic Ca2+, and a portio n of this increase is derived from inositol 1,4,5-trisphosphate (InsP( 3))-mediated Ca2+ release from internal stores via the InsP(3) recepto r. Cytosolic cAMP inhibits platelet activation, and experiments were d esigned to determine if cAMP-dependent phosphorylation affects the rat e of InsP(3)-mediated Ca2+ release. Western blotting of platelet inter nal membranes with anti-InsP(3) receptor and anti-actin binding protei n antibodies revealed that the platelet contains type 1 InsP(3) recept or and that it is distinct from actin binding protein. The platelet In sP(3) receptor was shown to be phosphorylated by endogenous, membrane- bound kinases as well as by exogenous protein kinase A. Prior phosphor ylation of the InsP(3) receptor by endogenous kinases inhibited additi onal protein kinase A-dependent phosphorylation by 60%. Furthermore, e ndogenous phosphorylation resulted in a 2-fold increase in the InsP(3) -mediated Ca2+ release rate relative to dephosphorylated controls. Fol lowing endogenous phosphorylation, additional phosphorylation by prote in kinase A returned the Ca2+ release rate to control values, while pr otein kinase A-dependent phosphorylation of dephosphorylated membranes did not affect the release rate. These results suggest that the InsP( 3) receptor within intact platelets is phosphorylated by endogenous ki nases which results in a high InsP(3)-mediated Ca2+ release rate, and that increases in cAMP result in additional phosphorylation that inhib its Ca2+ release, thus contributing to inhibition of platelet activati on.