Demonstration of cGMP‐dependent protein kinase and cGMP‐dependent phosphorylation in cell‐free extracts of platelets

Abstract

Homogenates, membranes and cytosol of rat and human platelets were found to contain cGMP‐dependent protein kinase immunoreactivity. Specific cGMP‐dependent protein kinase immunoreactivity was about 1.7 pmol protein kinase/mg protein for homogenates of human platelets and 0.7 pmol/mg for homogenates of rat platelets; the majority appeared to be associated with the membrane fraction. In membranes of platelets low concentrations of cAMP (0.5–2 μM) stimulated the phosphorylation of five major proteins with apparent relative molecular masses, M r , of 240000, 130000,50000,42000 and 22000 while low concentrations of cGMP (0.5–2 μM) stimulated the phosphorylation of three major proteins with apparent M r of 130000, 50000 and 46000. An affinity‐purified antibody against the cGMP‐dependent protein kinase was prepared which specifically inhibited the activity of cGMP‐dependent protein kinase. In membranes of human platelets this affinity‐purified antibody inhibited the cGMP‐stimulated phosphorlation of the three proteins with M r of 130000, 50000 and 46000 while it had no effect on the cAMP‐dependent and cyclic‐nucleotide‐independent protein phosphorylation. The results demonstrate that platelets contain a cGMP‐dependent protein kinase and at least three specific substrates for this enzyme. Two of these substrates, the proteins with apparent molecular M r of 130000 and 50000, are substrates for both cAMP‐ and cGMP‐dependent protein kinase. The protein with apparent M r of 130000 appears to be closely related to an intrinsic plasma membrane protein of vascular smooth muscle cells which is a substrate for a membrane‐associated cGMP‐dependent protein kinase. Therefore, cGMP‐dependent protein kinase and cGMP‐regulated phosphoproteins may mediate in platelets the intracellular effects of those hormones, vasodilators and drugs which elevate the level of cGMP and inhibit platelet aggregation.