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 portion of this increase is derived from inositol 1,4,5-trisphosphate (InsP3)-mediated Ca2+ release from internal stores via the InsP3 receptor. Cytosolic cAMP inhibits platelet activation, and experiments were designed to determine if cAMP-dependent phosphorylation affects the rate of InsP3-mediated Ca2+ release. Western blotting of platelet internal membranes with anti-InsP3 receptor and anti-actin binding protein antibodies revealed that the platelet contains type 1 InsP3 receptor and that it is distinct from actin binding protein. The platelet InsP3 receptor was shown to be phosphorylated by endogenous, membrane-bound kinases as well as by exogenous protein kinase A. Prior phosphorylation of the insP3 receptor by endogenous kinases inhibited additional protein kinase A-dependent phosphorylation by 60%. Furthermore, endogenous phosphorylation resulted in a 2-fold increase in the InsP3-mediated Ca2+ release rate relative to dephosphorylated controls. Following endogenous phosphorylation, additional phosphorylation by protein kinase A returned the Ca2+ release rate to control values, while protein kinase A-dependent phosphorylation of dephosphorylated membranes did not affect the release rate. These results suggest that the InsP3 receptor within intact platelets is phosphorylated by endogenous kinases which results in a high InsP3-mediated Ca2+ release rate, and that increases in cAMP result in additional phosphorylation that inhibits Ca2+ release, thus contributing to inhibition of platelet activation.