ACTIVATION OF THE CLONED PLATELET THROMBIN RECEPTOR DECREASES THE PERTUSSIS-TOXIN-DEPENDENT ADP-RIBOSYLATION OF THE MEMBRANE AND SOLUBLE INHIBITORY GUANINE-NUCLEOTIDE-BINDING-ALPHA PROTEINS - INHIBITION BY THE PROSTACYCLIN ANALOG, ILOPROST

Thrombin binds at least to two sites of the platelet surface; to the recently cloned thrombin receptor [Vu, T. K., Hung, D. T., Wheaton, V. I. & Coughlin, S. R. (1991) Cell 64, 1057-1068] and to glycoprotein Ib. In the present study, the decrease of pertussis-toxin-dependent ADP-ribosylation of membrane and soluble inhibitory guanine-nucleotide-binding alpha (G(ialpha)) proteins was measured after platelet stimulation with a thrombin-receptor-activating peptide (TRAP), and compared to stimulation with thrombin. Stimulation of intact platelets with TRAP decreased the pertussis-toxin-dependent ADP-ribosylation of the major membrane 41-kDa G(ialpha) protein and the minor soluble 40 kDa G(ialpha) protein recently described in platelets [Gennity, J. M. & Siess, W. (1991) Biochem. J. 279, 643-650]. The kinetics and extent of the decrease of pertussis-toxin-dependent ADP-ribosylation after stimulation of TRAP were similar to the effect of thrombin. The decrease of pertussistoxin-dependent ADP-ribosylation of the soluble G(ialpha) protein was more pronounced and observed at lower agonist concentrations than the decrease of the membrane G(ialpha) protein. Desensitization of the thrombin receptor by incubating platelets with a low concentration of TRAP reduced the subsequent decrease of pertussis-toxin-dependent ADP-ribosylation of G(ialpha) proteins, evoked by TRAP or thrombin. Platelet stimulation with gamma-thrombin that does not bind to glycoprotein Ib also showed a decrease in the pertussis-toxin-dependent ADP-ribosylation of the soluble and membrane G(ialpha) proteins. Treatment of platelets with the stable prostacyclin analog, iloprost, reduced the decrease of pertussis-toxin-dependent ADP-ribosylation of G(ialpha) proteins induced by TRAP or thrombin. Among other platelet stimuli tested (endoperoxide/thromboxane analog U44619, collagen, ADP, vasopressin), only U44619 decreased the pertussis-toxin-dependent ADP-ribosylation of the soluble and membrane G(ialpha) proteins to a degree comparable to TRAP. It is concluded that the thrombin-induced activation of both the membrane and soluble G(ialpha) proteins in platelets occurs via stimulation of the recently cloned thrombin receptor and is independent of the binding of thrombin to glycoprotein Ib. Furthermore, the coupling thrombin receptor/G(i) protein is reduced by intracellular cAMP.