RAS ACTIVATION IN PLATELETS AFTER STIMULATION OF THE THROMBIN RECEPTOR, THROMBOXANE A(2) RECEPTOR OR PROTEIN-KINASE-C

Several reports have indicated that the small G-protein Ras is not pre sent immunologically in platelets. However, here we report the identif ication of Ras in platelets by immunoprecipitation with the Ras-specif ic monoclonal antibodies Y13-259 or Y13-238, followed by Western blott ing. The presence of Pas was not due to contamination of samples with erythrocytes or leucocytes. Immunofluorescence studies indicated that Ras was present in a peripheral rim pattern in fixed, permeabilized pl atelets, suggesting an intracellular, plasma membrane location. Activa tion of platelets with the thrombin receptor peptide(42-50), the prost aglandin H-2/thromboxane A(2) mimetic U46619 or phorbol 12-myristate 1 3-acetate induced a rapid increase in GTP-bound, activated Ras. In eac h case, this increase was inhibited by the protein kinase C (PKC) inhi bitor bisindolylmaleimide GF 109203X, suggesting that Ras is activated downstream of PKC in platelets. Thus the activation of Ras in platele ts by agonists will now allow consideration of multiple potential Ras- dependent signal transduction pathways in platelet activation processe s.