Identification of glycoprotein Ib beta as one of the major proteins phosphorylated during exposure of intact platelets to agents that activate cyclic AMP-dependent protein kinase.

Platelet function is inhibited by prostaglandin E1, prostaglandin I2, or forskolin, agents that increase the intracellular concentration of cyclic AMP. The inhibition appears to result from cyclic AMP-stimulated phosphorylation of specific intracellular proteins. One of the major increases in phosphorylation occurs in a polypeptide of Mr = 24,000 (P24). In this study, an effort was made to identify P24. Platelets prelabeled with [32P]phosphate were incubated with prostaglandin E1, prostaglandin I2, or forskolin. Proteins that became phosphorylated were detected by autoradiography of sodium dodecyl sulfate-polyacrylamide gels. Several lines of evidence indicated that P24 was the beta-subunit of the plasma membrane glycoprotein (GP) Ib, a glycoprotein that is essential for the adhesion of platelets to damaged subendothelium, for the rapid response of platelets to thrombin, and for the attachment of the membrane skeleton to the cytoplasmic face of the plasma membrane. P24 co-migrated with GP Ib beta on reduced gels (Mr = 24,000) and also on nonreduced gels (when GP Ib beta is disulfide-linked to GP Ib alpha and migrates with Mr = 170,000). Like GP Ib beta, P24 was associated with actin filaments in Triton X-100 lysates. Like GP Ib beta, it was selectively associated with filaments of the membrane skeleton and was released from filaments when the Ca2+-dependent protease was active. Antibodies against GP Ib immunoprecipitated P24 from platelet lysates. Finally, exposure of Bernard-Soulier platelets (which lack GP Ib) to prostaglandin E1 resulted in phosphorylation of other polypeptides, but not of P24. These studies show that P24, one of the major polypeptides phosphorylated when platelets are exposed to agents that inhibit platelet function by increasing the concentration of cyclic AMP, is the beta-subunit of GP Ib.