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J. Biol. Chem., Vol. 280, Issue 26, 24386-24395, July 1, 2005

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Thrombopoietin Complements G_i- but Not G_q-dependent Pathways for Integrin _IIb3 Activation and Platelet Aggregation^*

Francesca Campus, Paolo Lova, Alessandra Bertoni¶, Fabiola Sinigaglia¶, Cesare Balduini, and Mauro Torti||

From the Department of Biochemistry, University of Pavia, via Bassi 21, 27100 Pavia and the ^¶Department of Medical Sciences, University "A. Avogadro," via Solaroli 17, 28100 Novara, Italy

Binding of thrombopoietin (TPO) to the cMpl receptor on human platelets potentiates aggregation induced by a number of agonists, including ADP. In this work, we found that TPO was able to restore ADP-induced platelet aggregation upon blockade of the G_q-coupled P2Y1 purinergic receptor but not upon inhibition of the G_i-coupled P2Y12 receptor. Moreover, TPO triggered platelet aggregation upon co-stimulation of G_z by epinephrine but not upon co-stimulation of G_q by the thromboxane analogue U46619. Platelet aggregation induced by TPO and G_i stimulation was biphasic, and cyclooxygenase inhibitors prevented the second but not the first phase. In contrast to ADP, TPO was unable to induce integrin _IIb3 activation, as evaluated by binding of both fibrinogen and PAC-1 monoclonal antibody. However, ADP-induced activation of integrin _IIb3 was blocked by antagonists of the G_q-coupled P2Y1 receptor but was completely restored by the simultaneous co-stimulation of cMpl receptor by TPO. Inside-out activation of integrin _IIb3 induced by TPO and G_i stimulation occurred independently of thromboxane A₂ production and was not mediated by protein kinase C, MAP kinases, or Rho-dependent kinase. Importantly, TPO and G_i activation of integrin _IIb3 was suppressed by wortmannin and Ly294002, suggesting a critical regulation by phosphatidylinositol 3-kinase. We found that TPO did not activate phospholipase C in human platelets and was unable to restore ADP-induced phospholipase C activation upon blockade of the G_q-coupled P2Y1 receptor. TPO induced a rapid and sustained activation of the small GTPase Rap1B through a pathway dependent on phosphatidylinositol 3-kinase. In ADP-stimulated platelets, Rap1B activation was reduced, although not abolished, upon blockade of the P2Y1 receptor. However, accumulation of GTP-bound Rap1B in platelets activated by co-stimulation of cMpl and P2Y12 receptor was identical to that induced by the simultaneous ligation of P2Y1 and P2Y12 receptor by ADP. These results indicate that TPO can integrate G_i, but not G_q, stimulation and can efficiently support integrin _IIb3 activation platelet aggregation by an alternative signaling pathway independent of phospholipase C but involving the phosphatidylinositol 3-kinase and the small GTPase Rap1B.

Received for publication, February 1, 2005 , and in revised form, April 28, 2005.

^* This work was supported by Grant PRIN 2003 from Ministero dell'Istruzione, Università e Ricerca Scientifica (to M. T.) and from Grant CIB 2004 from Consorzio Interuniversitario Biotecnologie (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 equally contributed to the work.

^|| To whom correspondence should be addressed. Tel.: 39-0382-987238; Fax: 39-0382-987240; E-mail: mtorti{at}unipv.it.

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