Adenosine diphosphate (ADP) activates platelets by binding to purinoceptors on the platelet surface and, in contrast to purinoceptors on other cell types, adenosine triphosphate (ATP) is a competitive antagonist for this process ( Gachet et al, 1996 ). Current evidence suggests that there are three types of ADP receptor on platelet surfaces, classified as P2X1, P2Y1 and P2T (P2TAC or P2YADP) receptors ( MacKenzie et al, 1996 ; Daniel et al, 1998 ; Fagura et al, 1998 ; Geiger et al, 1998 ; Jin et al, 1998 ; Jantzen et al, 1999 Leon et al, 1999 ). Both the P2X1 and the P2Y1 receptors, but not the P2T receptor, have been cloned ( Jin et al, 1998 ; Sun et al, 1998 ). The P2T receptor has been characterized pharmacologically, using selective antagonists, as the receptor linked, via Gi, to inhibition of adenylate cyclase, mediating a fall in the cyclic AMP level in response to ADP ( Mills & Smith, 1972; Daniel et al, 1998 ; Jin et al, 1998 ; Savi et al, 1998 ; Jantzen et al, 1999 ). Studies of a patient with ADP receptor deficiency support the concept that the P2T receptor is a single, distinct receptor subtype ( Leon et al, 1999 ). Co-activation of both the P2Y1 and the P2T receptors (G-protein coupled receptors) is required for platelet aggregation to occur, as detected by turbidimetry ( Jin & Kunapuli, 1998). The P2X1 receptor (a ligand-gated ion channel), which is selectively activated by α,β-methylene ATP, mediates rapid transient Ca2+ influx, but has not been found to contribute to platelet aggregation ( MacKenzie et al, 1996 ; Jin & Kunapuli, 1998; Kunapuli, 1998). The P2Y1 receptor activates phospholipase C (PLC), via Gq, and this accounts for most of the elevation in cytosolic Ca2+ induced by ADP, via formation of IP3 and release of Ca2+ from intracellular stores ( Daniel et al, 1998 ; Leon et al, 1999 ).
ADP may be released from platelet dense granules, where it is stored in high concentration, or erythrocytes and endothelial cells ( Gachet et al, 1996 ). ADP released by platelets stimulated by other agonists, such as thrombin or collagen, amplifies aggregation and secretion responses induced by these agonists ( Cattaneo et al, 1991 , 1997; Colman et al, 1994 ).
Analogues of ATP that bind specifically to the P2T receptor, including AR-C66096 and AR-C67085 (formerly ARL or FPL 66096 and ARL or FPL 67085 respectively) allow in vitro study of the function of the P2T receptor ( Humphries et al, 1994, 1995a, 1995b; Daniel et al, 1998 ; Fagura et al, 1998 ; Jin & Kunapuli, 1998; Jin et al, 1998 ). Blockade of the P2T receptor with these agents abolishes the turbidimetric response to ADP ( Jin & Kunapuli, 1998) and also renders aggregation induced by the thrombin receptor (PAR1)-activating peptide, TRAP, reversible ( Trumel et al, 1999 ).
A more recent and related P2T receptor antagonist, AR-C69931MX ( Ingall et al, 1999 ), has been found to be a highly selective competitive antagonist at the P2T receptor (unpublished observations) and is currently being developed as an intravenous antithrombotic agent. We have used this agent to study the role of the P2T receptor in platelet aggregation and secretion, as well as its role in TRAP-induced procoagulant activity, as determined by platelet microparticle formation and Annexin V binding ( Dachary-Prigent et al, 1993 ). We have related our findings to the role of thromboxane A2 synthesis using aspirin. We have also assessed the effects of AR-C69931MX on agonist-induced rises in cytosolic Ca2+ to explore further the mechanism whereby P2T receptor activation achieves its effects.