Evidence that the purinergic receptor P2Y12 potentiates platelet shape change by a Rho kinase-dependent mechanism.
ADP activates human platelets through two G-protein coupled receptors, P2Y1 and P2Y12, to induce a range of functional responses. Here we have addressed the role and mechanism of P2Y12 in modulating ADP-induced platelet shape change. Although the response depended upon activation of P2Y1, it was potentiated by P2Y12 as the P2Y12-selective antagonists AR-C69931MX and 2MeSAMP partially inhibited shape change in the later phase of the response. This was paralleled by inhibition of pseudopod formation, platelet spheration, actin polymerisation and myosin light chain phosphorylation. P2Y12 is known to couple to activation of PI3 kinase and inhibition of adenylate cyclase, but we showed that neither of these signalling events couples to regulation of shape change by this receptor. However, by assessment of phosphorylation of its major substrate myosin light chain phosphatase, we provide direct evidence for activation of Rho kinase by ADP, and that although P2Y1 is required for activation of Rho kinase, P2Y12 is able to potentiate its activity. We conclude that P2Y12 plays a potentiatory role in ADP-induced shape change through regulation of the Rho kinase pathway, potentiating both myosin phosphorylation and actin polymerisation, and this forms part of an important signalling pathway additional to its well-established Gi-coupled pathways.
Hardy
Adam R
AR
Department of Pharmacology, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK.
Hill
Darryl J
DJ
Poole
Alastair W
AW
D000199:Actins / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism; D000730:Androstadienes / Q000378:metabolism; D001792:Blood Platelets / Q000166:cytology* / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D048430:Cell Shape; D002614:Chelating Agents / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D015539:Platelet Activation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011554:Pseudopodia / Q000378:metabolism; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058472:Receptors, Purinergic P2Y1; D058925:Receptors, Purinergic P2Y12; D015290:Second Messenger Systems / Q000502:physiology; D000077191:Wortmannin; D054460:rho-Associated Kinases
D000199:Actins; D000244:Adenosine Diphosphate; D000249:Adenosine Monophosphate; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases; D000730:Androstadienes; D002118:Calcium; D002614:Chelating Agents; D004533:Egtazic Acid; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009218:Myosins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D058925:Receptors, Purinergic P2Y12; D000077191:Wortmannin; D054460:rho-Associated Kinases; C546382:P2RY1 protein, human; C548576:P2RY12 protein, human; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; C117446:cangrelor