Evidence that the purinergic receptor P2Y₁₂potentiates 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 Gi-coupled pathways.

The critical role of myosin IIA in platelet internal contraction

“…1C). Combined ADP receptor blockade with the P2Y 1 receptor antagonist A3P5P (200 μ m ) and the P2Y 12 receptor antagonist 2‐MeSAMP [35] (10 μ m ) did not inhibit shape change (Fig. 1D).…”

RhoA/ROCK pathway mediates p38 MAPK activation and morphological changes downstream of P2Y12/13 receptors in spinal microglia in neuropathic pain

“…Formerly, the P2Y12 and P2Y13 receptors were believed to activate Gi coupled signaling cascade that causes inhibition of stimulated adenylyl cyclase (Hollopeter et al, ; Zhang et al, ; Fumagalli et al, ). Two independent groups revealed the P2Y12‐mediated activation of RhoA and Rho‐associated coiled‐coil‐containing protein kinase (ROCK) that reorganized the actin cytoskeleton in a Gi‐independent manner in the platelet (Soulet et al, ; Hardy et al, ). In addition, Malaval et al () reported the activation of RhoA/ROCK1 under the P2Y13 signaling cascade in hepatocytes.…”

Canonical Wnt signaling negatively regulates platelet function

“…Indomethacin-treated (10 M) washed platelets (2 ϫ 10 8 /ml) were treated with the Rho kinase inhibitor, Y27632 (10 M) (Sigma), and/or the intracellular Ca 2ϩ chelator, BAPTA-AM (20 M) (Sigma), or vehicle control, for 10 min as before (38). Wnt3a (50 nM) or its carrier was added for 1 min before platelet stimulation with 5 M ADP or 1.5 M TRAP in a Chrono-log aggregometer under stirring in the presence of 1 mM EGTA.…”