Vasodilator‐stimulated protein phosphorylation in platelets is mediated by cAMP‐ and cGMP‐dependent protein kinases

Abstract

Vasodilators such as sodium nitroprusside, nitroglycerin and various prostaglandins are capable of inhibiting platelet aggregation associated with an increase of either cGMP or cAMP. In our studies with intact platelets, prostaglandin E₁ and sodium nitroprusside stimulated the phosphorylation of several proteins which could be distinguished from proteins known to be phosphorylated by a calmodulin‐regulated protein kinase or by protein kinase C. Prostaglandin E₁ (10 μM) or dibutyryl cAMP (2 mM) stimulated the phosphorylation of proteins with apparent relative molecular masscs, M_r, of 240000, 68 000, 50 000, and 22 000 in intact platelets. These proteins were also phosphorylated in response to low concentrations (1–2 μM) of cAMP in a particulate fraction of platelets. In intact platelets, sodium nitroprusside (100 μM) and the 8‐bromo derivative of cGMP (2 mM) increased the phosphorylation of one protein of M_r 50000 which was also phosphorylated in response to low concentrations (1–2 μM) of cGMP in platelet membranes. An additional protein (M_r 24000) appeared to be phosphorylated to a lesser degree in intact platelets by prostaglandin E₁ and sodium nitroprusside. Since the phosphorylation of the protein of M_r 50000 was stimulated both in intact platelets by cyclic‐nucleotide‐elevating agents and cyclic nucleotide analogs, as well as in platelet membranes by cyclic nucleotides, this phosphoprotein was analyzed by limited proteolysis, tryptic fingerprinting and phosphoamino acid analysis. These experiments indicated that the 50‐kDa proteins phosphorylated by sodium nitroprusside and prostaglandin E₁ were identical, and that the peptide of the 50‐kDa protein phosphorylated by both agents was also the same as the peptide derived from the 50‐kDa protein phosphorylated in platelet membranes by cGMP‐ and cAMP‐dependent protein kinases, respectively. Regulation of protein phosphorylation mediated by cAMP‐ and cGMP‐dependent protein kinases may be the molecular mechanism by which those vasodilators, capable of increasing either cAMP or cGMP, inhibit platelet aggregation.