The central role of the P-2T receptor in amplification of human platelet activation, aggregation, secretion and procoagulant activity

Adenosine diphosphate (ADP) is an important platelet agonist and ADP releas ed from platelet dense granules amplifies responses to other agonists. Ther e are three known subtypes of ADP receptor on platelets: P2X(1), P2Y(1) and P-2T receptors. Sustained ADP-induced aggregation requires co-activation o f P2Y(1) and P-2T receptors. AR-C69931MX, a selective P-2T receptor antagon ist and novel antithrombotic agent, was studied to characterize further the function of the P-2T receptor. The roles of the P2Y(1) receptor and thromb oxane A(2) were assessed using the selective P2Y(1) antagonist A2P5P and as pirin respectively. Aggregation was measured by whole blood single-platelet counting and platelet-rich plasma turbidimetry, using hirudin anticoagulat ion. Dense granule release was estimated using [C-14]-5-hydroxytryptamine ( HT)-labelled platelets. Ca2+ mobilization, P-selectin expression, Annexin V binding and microparticle formation were determined by flow cytometry. P-2 T receptor activation amplified ADP-induced aggregation initiated by the P2 Y(1) receptor, as well as amplifying aggregation, secretion and procoagulan t responses induced by other agonists, including U46619, thrombin receptor- activating peptide (TRAP) and collagen, independent of thromboxane A(2) syn thesis, which played a more peripheral role. P-2T receptor activation susta ined elevated cytosolic Ca2+ induced by other pathways. These studies indic ate that the P-2T receptor plays a central role in amplifying platelet resp onses and demonstrate the clinical potential of P-2T receptor antagonists.