Evidence that the purinergic receptor P2Y₁₂ potentiates platelet shape change by a Rho kinase-dependent mechanism

Abstract

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 G_i-coupled pathways.

• ADP
• Rho kinase
• shape change
• actin polymerisation
• P2Y12