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 (Gi alpha) 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 Gi alpha protein and the minor soluble 40 kDa Gi alpha 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 pertussis-toxin-dependent ADP-ribosylation of the soluble Gi alpha protein was more pronounced and observed at lower agonist concentrations than the decrease of the membrane Gi alpha 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 Gi alpha 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 Gi alpha proteins. Treatment of platelets with the stable prostacyclin analog, iloprost, reduced the decrease of pertussis-toxin-dependent ADP-ribosylation of Gi alpha 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 Gi alpha proteins to a degree comparable to TRAP. It is concluded that the thrombin-induced activation of both the membrane and soluble Gi alpha 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/Gi protein is reduced by intracellular cAMP.
Wilhelm
B
B
Institut für Epidemiologie und Prophylaxe der Kreislaufkrankheiten, Universität München, Germany.
Siess
W
W
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects; D000242:Cyclic AMP / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D016285:Iloprost / Q000494:pharmacology*; D010446:Peptide Fragments / Q000494:pharmacology; D037342:Pertussis Toxin; D011450:Prostaglandin Endoperoxides, Synthetic / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018179:Receptors, Thrombin; D013917:Thrombin / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D010446:Peptide Fragments; D011450:Prostaglandin Endoperoxides, Synthetic; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D010566:Virulence Factors, Bordetella; C076901:thrombin receptor-activating peptide (P508-530); D000246:Adenosine Diphosphate Ribose; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D000242:Cyclic AMP; D037342:Pertussis Toxin; D013917:Thrombin; D019204:GTP-Binding Proteins; D016285:Iloprost