Interplay between P2Y1, P2Y12, and P2X1 receptors in the activation of megakaryocyte cation influx currents by ADP: evidence that the primary megakaryocyte represents a fully functional model of platelet P2 receptor signaling

Abstract

AbstractThe difficulty of conducting electrophysiologic recordings from the platelet has restricted investigations into the role of ion channels in thrombosis and hemostasis. We now demonstrate that the well-established synergy between P2Y1 and P2Y12 receptors during adenosine diphosphate (ADP)–dependent activation of the platelet αIIbβ3 integrin also exists in murine marrow megakaryocytes, further supporting the progenitor cell as a bona fide model of platelet P2 receptor signaling. In patch clamp recordings, ADP (30 μM) stimulated a transient inward current at –70 mV, which was carried by Na+ and Ca2+ and was amplified by phenylarsine oxide, a potentiator of certain transient receptor potential (TRP) ion channels by phosphatidylinositol 4,5-bisphosphate depletion. This initial current decayed to a sustained phase, upon which repetitive transient inward cation currents with pre-dominantly P2X1-like kinetics were super-imposed. Abolishing P2X1-receptor activity prevented most of the repetitive currents, consistent with their activation by secreted adenosine triphosphate (ATP). Recordings in P2Y1-receptor–deficient megakaryocytes demonstrated an essential requirement of this receptor for activation of all ADP-evoked inward currents. However, P2Y12 receptors, through the activation of PI3-kinase, played a synergistic role in both P2Y1 and P2X1-receptor–dependent currents. Thus, direct stimulation of P2Y1 and P2Y12 receptors, together with autocrine P2X1 activation, is responsible for the activation of nonselective cation currents by the platelet agonistADP.