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Keywords:

  • platelet activation;
  • P2 receptors;
  • anti-platelet drug;
  • platelet secretion;
  • calcium

Adenosine diphosphate (ADP) is an important platelet agonist and ADP released from platelet dense granules amplifies responses to other agonists. There are three known subtypes of ADP receptor on platelets: P2X1, P2Y1 and P2T receptors. Sustained ADP-induced aggregation requires co-activation of P2Y1 and P2T receptors. AR-C69931MX, a selective P2T receptor antagonist and novel antithrombotic agent, was studied to characterize further the function of the P2T receptor. The roles of the P2Y1 receptor and thromboxane A2 were assessed using the selective P2Y1 antagonist A2P5P and aspirin respectively. Aggregation was measured by whole blood single-platelet counting and platelet-rich plasma turbidimetry, using hirudin anticoagulation. Dense granule release was estimated using [14C]-5-hydroxytryptamine (HT)-labelled platelets. Ca2+ mobilization, P-selectin expression, Annexin V binding and microparticle formation were determined by flow cytometry. P2T receptor activation amplified ADP-induced aggregation initiated by the P2Y1 receptor, as well as amplifying aggregation, secretion and procoagulant responses induced by other agonists, including U46619, thrombin receptor-activating peptide (TRAP) and collagen, independent of thromboxane A2 synthesis, which played a more peripheral role. P2T receptor activation sustained elevated cytosolic Ca2+ induced by other pathways. These studies indicate that the P2T receptor plays a central role in amplifying platelet responses and demonstrate the clinical potential of P2T receptor antagonists.