A selective role for phosphatidylinositol 3,4,5-trisphosphate in the Gi-dependent activation of platelet Rap1B.
The small GTP-binding protein Rap1B is activated in human platelets upon stimulation of a G(i)-dependent signaling pathway. In this work, we found that inhibition of platelet adenylyl cyclase by dideoxyadenosine or SQ22536 did not cause activation of Rap1B and did not restore Rap1B activation in platelets stimulated by cross-linking of Fcgamma receptor IIA (FcgammaRIIA) in the presence of ADP scavengers. Moreover, elevation of the intracellular cAMP concentration did not impair the G(i)-dependent activation of Rap1B. Two unrelated inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002, totally prevented Rap1B activation in platelets stimulated by cross-linking of FcgammaRIIA, by stimulation of the P2Y(12) receptor for ADP, or by epinephrine. However, in platelets from PI3Kgamma-deficient mice, both ADP and epinephrine were still able to normally stimulate Rap1B activation through a PI3K-dependent mechanism, suggesting the involvement of a different isoform of the enzyme. Moreover, the lack of PI3Kgamma did not prevent the ability of epinephrine to potentiate platelet aggregation through a G(i)-dependent pathway. The inhibitory effect of wortmannin on Rap1B activation was overcome by addition of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), but not PtdIns(3,4)P(2), although both lipids were found to support phosphorylation of Akt. Moreover, PtdIns(3,4,5)P(3) was able to relieve the inhibitory effect of apyrase on FcgammaRIIA-mediated platelet aggregation. We conclude that stimulation of a G(i)-dependent signaling pathway causes activation of the small GTPase Rap1B through the action of the PI3K product PtdIns(3,4,5)P(3), but not PtdIns(3,4)P(2), and that this process may contribute to potentiation of platelet aggregation.
Lova
Paolo
P
Department of Biochemistry, University of Pavia, via Bassi 21, 27100 Pavia, Italy.
Paganini
Simona
S
Hirsch
Emilio
E
Barberis
Laura
L
Wymann
Matthias
M
Sinigaglia
Fabiola
F
Balduini
Cesare
C
Torti
Mauro
M
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism; D000322:Adrenergic Agonists / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D001081:Apyrase / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002867:Chromones / Q000494:pharmacology; D016048:Dideoxyadenosine / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004837:Epinephrine / Q000494:pharmacology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D015539:Platelet Activation / Q000502:physiology; D010974:Platelet Aggregation / Q000502:physiology; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011961:Receptors, Fc / Q000378:metabolism; D017452:Receptors, IgG; D015290:Second Messenger Systems / Q000502:physiology*; D000077191:Wortmannin; D020668:rap GTP-Binding Proteins / Q000378:metabolism*
D000244:Adenosine Diphosphate; D000249:Adenosine Monophosphate; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases; D000322:Adrenergic Agonists; D000730:Androstadienes; D000963:Antimetabolites; D001081:Apyrase; D002867:Chromones; D016048:Dideoxyadenosine; D004791:Enzyme Inhibitors; D004837:Epinephrine; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D011961:Receptors, Fc; D017452:Receptors, IgG; D000077191:Wortmannin; D020668:rap GTP-Binding Proteins; C445323:FCGR2A protein, human; C499173:FCGR2B protein, human; C499172:Fcgr2b protein, mouse; C060974:phosphatidylinositol 3,4,5-triphosphate; C060980:phosphatidylinositol 3,4-diphosphate; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C117446:cangrelor; C494918:AKT1 protein, human; C116753:RAP1B protein, human; C492136:Rap1b protein, mouse