No requirement of P2X1 purinoceptors for platelet aggregation

Abstract

ADP produces a series of responses in rabbit platelets such as shape changes, aggregation and intracellular Ca²⁺ mobilization. In human platelets, the P2X1 receptor mediates a rapid increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) upon stimulation with ADP. We investigated whether this phenomenon is also present in rabbit platelets. We found that the P2X1 receptor-mediated response was absent because there was (1) no elevation of [Ca²⁺]_i in response to α,β-methylene-ATP, a selective P2X1 receptor agonist, in fura-2-loaded platelets; (2) no change in the ADP-induced [Ca²⁺]_i increase and platelet aggregation after P2X1 receptor desensitization with α,β-methylene-ATP; (3) complete inhibition of the ADP-induced [Ca²⁺]_i elevation by the P2Y1 receptor specific antagonist, A3′P5′PS, with a similar ID₅₀ value both in the presence and absence of external Ca²⁺. These results indicate that ADP-induced [Ca²⁺]_i elevation is mainly mediated by P2Y1 receptors in rabbit platelets. We conclude that the P2X1 receptor does not play a significant role in the ADP-induced platelet shape changes and aggregation.

Introduction

ADP produces a series of responses in rabbit platelets similar to those in human platelets. The first event is a change in platelet shape, namely, a rapid disc–sphere transformation. The shape change is followed by platelet aggregation. Unlike human platelets, ADP does not induce the release of arachidonic acid in rabbit platelets. ADP causes a numbers of intracellular events in rabbit platelets, including phospholipase C activation, intracellular Ca²⁺ mobilization and adenylate cyclase inhibition (Matsuoka and Suzuki, 1983; Vickers et al., 1986; Nishio et al., 1992).

The ADP receptor on platelets is a unique ADP-selective purinocepter, termed P2T, which is recognized as a member of P2 purinoceptor family (Gordon, 1986). At P2T purinoceptor sites, ADP acts as an agonist and ATP as an antagonist. Hourani and Hall (1994)proposed a two-receptor model to explain the actions of ADP on platelets: the rapid Ca²⁺ influx is mediated by a receptor-operated Ca²⁺ channel, while the intracellular Ca²⁺ mobilization and adenylate cyclase inhibition are modulated by the P2T receptor through G-proteins. Two subtypes of purinoceptors have been recently cloned in human platelets: ionotropic (P2X types) (Clifford et al., 1998) and G-protein-coupled (P2Y types) purinoceptors (Leon et al., 1997). In 1998, Kunapuli et al. proposed a three-receptor model for the effects of ADP on platelets (Daniel et al., 1998): P2Y1 purinoceptors coupled to G_q, another receptor coupled to G_i protein-adenylate cyclase inhibition, and P2X1 purinoceptors, which induce a rapid increase in Ca²⁺ entry (Mahaut-Smith et al., 1992; MacKenzie et al., 1996). However, the role of ADP-evoked Ca²⁺ entry via P2X1 receptors in the function of blood platelets remains unclear.

This paper shows, using α,β-methylene-ATP (α,β-Me-ATP) as an agonist for P2X1 receptors (MacKenzie et al., 1996), that the P2X1 receptor is not involved in ADP-induced platelet shape changes and aggregation and that the [Ca²⁺]_i elevation in response to ADP is due to activation of P2Y1 purinoceptors in rabbit platelets.