The central role of the P_2T receptor in amplification of human platelet activation, aggregation, secretion and procoagulant activity

Abstract

Summary. 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: P2X 1 , P2Y 1 and P 2T receptors. Sustained ADP-induced aggregation requires co-activation of P2Y 1 and P 2T receptors. AR-C69931MX, a selective P 2T receptor antagonist and novel antithrombotic agent, was studied to characterize further the function of the P 2T receptor. The roles of the P2Y 1 receptor and thromboxane A 2 were assessed using the selective P2Y 1 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 [14 C]-5-hydroxytryptamine (HT)-labelled platelets. Ca 21 mobilization, P-selectin expression, Annexin V binding and microparticle formation were determined by flow cytometry. P 2T receptor activation amplified ADPinduced aggregation initiated by the P2Y 1 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 A 2 synthesis, which played a more peripheral role. P 2T receptor activation sustained elevated cytosolic Ca 21 induced by other pathways. These studies indicate that the P 2T receptor plays a central role in amplifying platelet responses and demonstrate the clinical potential of P 2T receptor antagonists.

Novel uses for anti‐platelet agents as anti‐inflammatory drugs

“…Recently, a role for platelets has been reported for leukocyte recruitment in a murine model of chronic contact hypersensitivity, where clopidogrel administration reduced cell infiltration into skin tissue and the production of chemokines: MIP-1a, RANTES and TARC (Tamagawa-Mineoka et al, 2007). Specific P2Y 1 antagonism reduces platelet P-selectin expression and the occurrence of platelet-leukocyte complexes (Storey et al, 2000;Leon et al, 2003), which may represent a mechanism by which P2Y 1 antagonism has efficacy in reducing the extent of inflammation in vivo. Furthermore, the administration of clopidogrel also reduces platelet P-selectin expression, decreased platelet-PMN adhesion and plateletdependent ROS production in neutrophils (Evangelista et al, 2005), findings reciprocated with thienopyridine and AR-C69931MX (Storey et al, 2002a, b;Leon et al, 2003), but not aspirin (Storey et al, 2002a).…”

P2 receptors and platelet function

“…This is also the case in the procoagulant activity of platelets. While both receptors are indirectly involved through their role in platelet P-selectin exposure and in the formation of platelet-leukocyte conjugates leading to leukocyte tissue factor exposure [81,82], the P2Y 12 receptor is also directly implicated in the exposure of phosphatidylserine at the surface of platelets [81,83,84].…”

Cangrelor for the management and prevention of arterial thrombosis

“…This discovery supported the development of ATP analogues to be used as antagonists of ADPinduced platelet aggregation [14], one of these being cangrelor ( Figure 2); these ATP analogues were later shown to act selectively on the platelet P2Y 12 receptor without influencing P2Y 1 and P2X 1 [13,18]. Yet, several P2Y 12 antagonists, including cangrelor, were developed when a single ADP receptor on platelets was postulated and described as the P 2T receptor (T indicating 'thrombocyte') [14], designated so since its pharmacologic profile did not correspond to any cloned P2Y receptor at the time [10]. Subsequently there were demonstrated to be two distinct ADP receptors on platelets, eventually identified as P2Y 1 and P2Y 12 [10,13,18].…”

“…The other two platelet P2 receptors are selectively activated by ADP: P2Y 1 is coupled to Gq, which mediates mobilization of intracellular calcium and platelet shape change, whereas P2Y 12 is coupled to Gi, which mediates inhibition of adenylate cyclase and activation of PI3 kinase [10,12]. Activation of both P2Y 1 and P2Y 12 receptors is necessary for sustained ADP-induced platelet aggregation [13,14]. The success of the P2Y 12 receptor as a therapeutic target relates to its central role in the potentiation of dense granule secretion, platelet aggregation, pro-coagulant activity and overall thrombus growth and stability [14][15][16][17].…”