Thrombopoietin complements G_i- but not G_q-dependent pathways for integrin α_IIbβ₃ activation and platelet aggregation

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 α_IIbβ ₃ activation, as evaluated by binding of both fibrinogen and PAC-1 monoclonal antibody. However, ADP-induced activation of integrin α_IIbβ₃ 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 α_IIbβ₃ 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 α_IIbβ₃ 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 α_IIbβ₃ activation platelet aggregation by an alternative signaling pathway independent of phospholipase C but involving the phosphatidylinositol 3-kinase and the small GTPase Rap1B.