The P2Y(12) receptor induces platelet aggregation through weak activation of the alpha(IIb)beta(3) integrin--a phosphoinositide 3-kinase-dependent mechanism.
High concentrations of adenosine-5'-diphosphate ADP are able to induce partial aggregation without shape change of P2Y(1) receptor-deficient mouse platelets through activation of the P2Y(12) receptor. In the present work we studied the transduction pathways selectively involved in this phenomenon. Flow cytometric analyses using R-phycoerythrin-conjugated JON/A antibody (JON/A-PE), an antibody which recognizes activated mouse alpha(IIb)beta(3) integrin, revealed a low level activation of alpha(IIb)beta(3) in P2Y(1) receptor-deficient platelets in response to 100 microM ADP or 1 microM 2MeS-ADP. Adrenaline induced no such activation but strongly potentiated the effect of ADP in a dose-dependent manner. Global phosphorylation of (32)P-labeled platelets showed that P2Y(12)-mediated aggregation was not accompanied by an increase in the phosphorylation of myosin light chain (P(20)) or pleckstrin (P(47)) and was not affected by the protein kinase C (PKC) inhibitor staurosporine. On the other hand, two unrelated phosphoinositide 3-kinase inhibitors, wortmannin and LY294002, inhibited this aggregation. Our results indicate that (i) the P2Y(12) receptor is able to trigger a P2Y(1) receptor-independent inside-out signal leading to alpha(IIb)beta(3) integrin activation and platelet aggregation, (ii) ADP and adrenaline use different signaling pathways which synergize to activate the alpha(IIb)beta(3) integrin, and (iii) the transduction pathway triggered by the P2Y(12) receptor is independent of PKC but dependent on phosphoinositide 3-kinase.
Kauffenstein
G
G
INSERM U.311, Etablissement Français du Sang-Alsace, 10 rue Spielmann, 67065 Strasbourg Cedex, France.
Bergmeier
W
W
Eckly
A
A
Ohlmann
P
P
Léon
C
C
Cazenave
J P
JP
Nieswandt
B
B
Gachet
C
C
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001798:Blood Proteins / Q000378:metabolism; D002867:Chromones / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004837:Epinephrine / Q000494:pharmacology; D005340:Fibrinogen / Q000378:metabolism; D005434:Flow Cytometry; D007700:Kinetics; D008565:Membrane Proteins; D051379:Mice; D008855:Microscopy, Electron, Scanning; D009025:Morpholines / Q000494:pharmacology; D018994:Myosin Light Chains / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010974:Platelet Aggregation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D018048:Receptors, Purinergic P2 / Q000378:metabolism / Q000502:physiology*; D058925:Receptors, Purinergic P2Y12; D015398:Signal Transduction; D019311:Staurosporine / Q000494:pharmacology; D013997:Time Factors; D000077191:Wortmannin
D000730:Androstadienes; D001798:Blood Proteins; D002867:Chromones; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D009025:Morpholines; D018994:Myosin Light Chains; C548577:P2ry12 protein, mouse; D010750:Phosphoproteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D018048:Receptors, Purinergic P2; D058925:Receptors, Purinergic P2Y12; C039077:platelet protein P47; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D005340:Fibrinogen; D000242:Cyclic AMP; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D019311:Staurosporine; D000077191:Wortmannin; D004837:Epinephrine