Mechanisms for Thrombopoietin-Induced Potentiation of Platelet Aggregation.

Abstract

Thrombopoietin (TPO) is the major cytokine regulating megakaryocytes proliferation and thrombopoiesis. Binding of TPO to the c-Mpl receptor expressed on human platelets promotes tyrosine phosphorylation of a number of proteins and potentiates platelet aggregation induced by low concentrations of a variety of agonists including ADP. Nevertheless, TPO alone is not able to induce any platelet functional response, such as shape change, granule secretion, or aggregation. It is now clear that platelet aggregation induced by many different agonists results from the concomitant activation of both Gi-and Gq-dependent signaling pathways. In particular, activation of a Gi-dependent pathway is essential, albeit not sufficient, to elicit complete platelet aggregation. Recently, it has been proposed that the small GTPase Rap1B is an important element in the Gi-dependent pathway for platelet aggregation. ADP activates Gq and Gi-dependent pathways by binding to the P2Y1 and the P2Y12 receptors, respectively. In this study we investigated the contribution of TPO in platelet activation induced by ADP. We found that TPO could restore ADP-induced platelet aggregation when the Gq-coupled receptor was blocked, but failed to restore ADP-induced aggregation in absence of Gi stimulation. Similarly, TPO was unable to restore U46619-induced platelet aggregation, when the Gi-coupled P2Y12 receptor for ADP was blocked, but caused irreversible platelet aggregation when added in combination with epinephrine, which binds to a Gi-coupled receptor. TPO did not cause any detectable calcium movements or pleckstrin phosphorylation in human platelets. Moreover, TPO failed to restore ADP-induced cytosolic calcium incresases and pleckstrin phosphorylation when the Gq-coupled P2Y1 receptor was blocked, although aggregation was completely restored. Moreover, we found that TPO induced activation of Rap1B in platelets, and could completely restore ADP-induced Rap1B activation, which was partially reduced upon blockade of the P2Y1 receptor. Finally, ADP-induced binding of both fibrinogen and PAC-1 monoclonal antibody to integrin αIIbβ3 was prevented by the blockade of the Gq-coupled P2Y1 receptor, but completely restored by the simultaneous addition of TPO. These findings indicate that TPO can complement Gi-, but not Gq-dependent pathways for integrin αIIbβ3 activation and platelet aggregation, through a new mechanism that does not involve activation of phospholipase C, but may involve the small GTPase Rap1B.