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, P2Y(1) and P2Y(12), to induce a range of functional responses. Here we have addressed the role and mechanism of P2Y(12) in modulating ADP-induced platelet shape change. Although the response depended upon activation of P2Y(1), it was potentiated by P2Y(12) as the P2Y(12)-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. P2Y(12) 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 P2Y(1) is required for activation of Rho kinase, P2Y(12) is able to potentiate its activity. We conclude that P2Y(12) 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 G(i)-coupled pathways.