Thrombopoietin complements G(i)- but not G(q)-dependent pathways for integrin {alpha}(IIb){beta}(3) 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 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.
Campus
Francesca
F
Department of Biochemistry, University of Pavia, via Bassi 21, 27100 Pavia.
Lova
Paolo
P
Bertoni
Alessandra
A
Sinigaglia
Fabiola
F
Balduini
Cesare
C
Torti
Mauro
M
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000737:chemistry; D000730:Androstadienes / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001792:Blood Platelets / Q000166:cytology*; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002867:Chromones / Q000494:pharmacology; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005340:Fibrinogen / Q000737:chemistry / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics* / Q000502:physiology; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics* / Q000502:physiology; D006801:Humans; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D015539:Platelet Activation; D010974:Platelet Aggregation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D018121:Receptors, Cytokine / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D058472:Receptors, Purinergic P2Y1; D053628:Receptors, Thrombopoietin; D013926:Thrombopoietin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D013928:Thromboxane A2 / Q000378:metabolism; D013931:Thromboxanes / Q000737:chemistry; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism; D000077191:Wortmannin; D020668:rap GTP-Binding Proteins / Q000378:metabolism
D000730:Androstadienes; D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D002867:Chromones; D016861:Cyclooxygenase Inhibitors; D004791:Enzyme Inhibitors; D009025:Morpholines; C546382:P2RY1 protein, human; D010750:Phosphoproteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D053628:Receptors, Thrombopoietin; D013931:Thromboxanes; C039077:platelet protein P47; C082218:MPL protein, human; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D013928:Thromboxane A2; D000244:Adenosine Diphosphate; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D005340:Fibrinogen; D013926:Thrombopoietin; D019869:Phosphatidylinositol 3-Kinases; D010738:Type C Phospholipases; C116753:RAP1B protein, human; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020668:rap GTP-Binding Proteins; D002118:Calcium; D000077191:Wortmannin