Thrombopoietin Complements Gi- but Not Gq-dependent Pathways for Integrin αIIbβ3 Activation and Platelet Aggregation

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

doi:10.1074/jbc.m501174200

Abstract

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 alpha(IIb)beta(3) activation, as evaluated by binding of both fibrinogen and PAC-1 monoclonal antibody. However, ADP-induced activation of integrin alpha(IIb)beta(3) 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 alpha(IIb)beta(3) induced by TPO and G(i) stimulation occurred independently of thromboxane A(2) production and was not mediated by protein kinase C, MAP kinases, or Rho-dependent kinase. Importantly, TPO and G(i) activation of integrin alpha(IIb)beta(3) 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 alpha(IIb)beta(3) activation platelet aggregation by an alternative signaling pathway independent of phospholipase C but involving the phosphatidylinositol 3-kinase and the small GTPase Rap1B.

Highlights

Thrombopoietin (TPO)1 is the major regulator of megakaryopoiesis and platelet production [1]
Upon blockade of the P2Y1 receptor, platelet aggregation profiles very similar to those reported in Fig. 1B were recorded when ADP and TPO were added simultaneously or when treatment with ADP preceded the addition of TPO, indicating that the observed effects are independent of the order of agonists addition
We have demonstrated that co-stimulation of the Gi-coupled P2Y12 receptor for ADP and the cMpl receptor for TPO induces integrin ␣IIb␤3 activation and platelet aggregation in the absence of Gq-mediated signaling

Summary

EXPERIMENTAL PROCEDURES

Materials—Sepharose CL-2B, GSH-Sepharose, [32P]orthophosphate, and the enhanced chemiluminescence substrate were from Amersham Biosciences. Aliquots containing 50 ␮g of total platelet lysates were heated at 96 °C for 5 min in SDS sample buffer, separated on a 5–15% acrylamide gradient gel, and transferred to nitrocellulose. Platelet samples (50 ␮l) were incubated with 200 ␮g/ml biotin-fibrinogen and 10 ␮g/ml phycoerythrinstreptavidin in the absence or presence of 200 ␮M MRS2179 and treated with 10 ␮M ADP and/or 100 ng/ml TPO for 30 min at room temperature without stirring. For PAC-1 binding, platelet samples (50 ␮l, 0.5 ϫ 107 cells/ml) were incubated with 1 ␮g/ml PAC-1 Fab antibody and 10 ␮g/ml goat anti-mouse antibody conjugated with fluorescein and treated with agonists, as described for fibrinogen binding. Fibrinogen and PAC-1 Fab binding to platelets was quantified by flow cytometry

RESULTS

Platelet Activation by TPO and Gi Stimulation

DISCUSSION