title: Contribution of protease-activated receptors 1 and 4 and glycoprotein Ib-IX-V in the G(i)-independent activation of platelet Rap1B by thrombin.
abstract: Thrombin activates human platelets through three different membrane receptors, the protease-activated receptors PAR-1 and PAR-4 and the glycoprotein Ib (GPIb)-IX-V complex. We investigated the contribution of these three receptors to thrombin-induced activation of the small GTPase Rap1B. We found that, similarly to thrombin, selective stimulation of either PAR-1 or PAR-4 by specific activating peptides caused accumulation of GTP-bound Rap1B in a dose-dependent manner. By contrast, in PAR-1- and PAR-4-desensitized platelets, thrombin failed to activate Rap1B. Thrombin, PAR-1-, or PAR-4-activating peptides also induced the increase of intracellular Ca(2+) concentration and the release of serotonin in a dose-dependent manner. We found that activation of Rap1B by selected doses of agonists able to elicit comparable intracellular Ca(2+) increase and serotonin release was differently dependent on secreted ADP. In the presence of the ADP scavengers apyrase or phosphocreatine-phosphocreatine kinase, activation of Rap1B induced by stimulation of either PAR-1 or PAR-4 was totally inhibited. By contrast, thrombin-induced activation of Rap1B was only minimally affected by neutralization of secreted ADP. Concomitant stimulation of both PAR-1 and PAR-4 in the presence of ADP scavengers still resulted in a strongly reduced activation of Rap1B. A similar effect was also observed upon blockade of the P2Y12 receptor for ADP, as well as in P2Y12 receptor-deficient human platelets, but not after blockade of the P2Y1 receptor. Activation of Rap1B induced by thrombin was not affected by preincubation of platelets with the anti-GPIbalpha monoclonal antibody AK2 in the absence of ADP scavengers or a P2Y12 antagonist but was totally abolished when secreted ADP was neutralized or after blockade of the P2Y12 receptor. Similarly, cleavage of the extracellular portion of GPIbalpha by the cobra venom mocarhagin totally prevented Rap1B activation induced by thrombin in the presence of apyrase and in P2Y12 receptor-deficient platelets. By contrast, inhibition of MAP kinases or p160ROCK, which have been shown to be activated upon thrombin binding to GPIb-IX-V, did not affect agonist-induced activation of Rap1B in the presence of ADP scavengers. These results indicate that although both PAR-1 and PAR-4 signal Rap1B activation, the ability of thrombin to activate this GTPase independently of secreted ADP involves costimulation of both receptors as well as binding to GPIb-IX-V.
authors.affiliation: Center of Excellence in Applied Biology, Department of Biochemistry, University of Pavia, via Bassi 21, 27100 Pavia.
authors.forename: Paolo
authors.initials: P
authors.lastname: Lova
authors.forename: Francesca
authors.initials: F
authors.lastname: Campus
authors.forename: Rossana
authors.initials: R
authors.lastname: Lombardi
authors.forename: Marco
authors.initials: M
authors.lastname: Cattaneo
authors.forename: Fabiola
authors.initials: F
authors.lastname: Sinigaglia
authors.forename: Cesare
authors.initials: C
authors.lastname: Balduini
authors.forename: Mauro
authors.initials: M
authors.lastname: Torti
mesh_terms: D000244:Adenosine Diphosphate / Q000502:physiology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000502:physiology*; D018841:HSP90 Heat-Shock Proteins; D006801:Humans; D008565:Membrane Proteins / Q000502:physiology; D015539:Platelet Activation; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000502:physiology*; D044463:Receptor, PAR-1 / Q000502:physiology*; D018048:Receptors, Purinergic P2 / Q000502:physiology; D058925:Receptors, Purinergic P2Y12; D018179:Receptors, Thrombin / Q000502:physiology*; D013917:Thrombin / Q000494:pharmacology*; D020668:rap GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D018841:HSP90 Heat-Shock Proteins; D008565:Membrane Proteins; C548576:P2RY12 protein, human; D019038:Platelet Glycoprotein GPIb-IX Complex; D044463:Receptor, PAR-1; D018048:Receptors, Purinergic P2; D058925:Receptors, Purinergic P2Y12; D018179:Receptors, Thrombin; C092306:TRAP1 protein, human; D000244:Adenosine Diphosphate; D013917:Thrombin; C116753:RAP1B protein, human; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020668:rap GTP-Binding Proteins; C112908:protease-activated receptor 4