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J. Biol. Chem., Vol. 279, Issue 24, 25299-25306, June 11, 2004

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Contribution of Protease-activated Receptors 1 and 4 and Glycoprotein Ib-IX-V in the G_i-independent Activation of Platelet Rap1B by Thrombin^*

Paolo Lova, Francesca Campus, Rossana Lombardi¶||, Marco Cattaneo¶, Fabiola Sinigaglia**, Cesare Balduini, and Mauro Torti

From the Center of Excellence in Applied Biology, Department of Biochemistry, University of Pavia, via Bassi 21, 27100 Pavia, the ^¶Unit of Hematology and Thrombosis, Department of Surgery, Medicine, and Dentistry, Ospedale San Paolo, University of Milan, via di Rudini 8, 20142 Milan, the ^||A. Bianchi Bonomi Hemophilia and Thrombosis Center, Istituto di Ricovero e Cura a Carattere Scientifico Ospedale Maggiore, University of Milan, via Pace 9, 20122 Milan, and the ^**Department of Medical Sciences, University "A. Avogadro," via Solaroli 17, 28100 Novara, Italy

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 Ca²⁺ 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 Ca²⁺ 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-GPIb 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 GPIb 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.

Received for publication, December 3, 2003 , and in revised form, March 31, 2004.

^* This work was supported by grants from the Ministero dell'Istruzione, Università e Ricerca Scientifica: FIRB 2001 (to C. B.), Progetti di Ricerca di Interesse Nazionale 2001 (to M. C.), 2002 (to M. T.), and 2003 (to M. T.) and from the Consiglio Nazionale delle Ricerche: Target Project Biotechnology (to C. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

To whom correspondence should be addressed: Dept. of Biochemistry, University of Pavia, via Bassi 21, 27100 Pavia, Italy. Tel.: 39-0382-507238; Fax: 39-0382-507240; E-mail: mtorti{at}unipv.it.

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