GTP and cytosol stimulate phosphoinositide hydrolysis in isolated platelet membranes

Abstract

Hydrolysis of polyphosphoinositides by phospholipase C was examined in isolated membranes prepared from [32P]labelled platelets. In the presence of GTP[gamma]S, thrombin increased the release of inositol triphosphate and inositol biphosphate approximately 500%. GTP[gamma]S alone stimulated release 2 fold. Maximal activation of thrombin-induced phosphoinositide hydrolysis was observed at 10 uM GTP. Although addition of calcium had no effect, 2 mM EGTA completely inhibited inositolphosphate release. Addition of high speed supernatant to [32P]labelled membranes stimulated the release of inositolphosphates. This hydrolysis was further enhanced by the addition of GTP. These data demonstrate that the breakdown of polyphosphoinositides in isolated platelet membranes is dependent on GTP and stimulated by platelet cytosol.