ADP is not an agonist at P2X(1) receptors: evidence for separate receptors stimulated by ATP and ADP on human platelets

Abstract

ADP, an important agonist in thrombosis and haemostasis, has been reported to activate platelets via three receptors, P2X(1), P2Y(1) and P2T(AC). Given the low potency of ADP at P2X(1) receptors and recognized contamination of commercial samples of adenosine nucleotides, we have re-examined the activation of P2X(1) receptors by ADP following HPLC and enzymatic purification. Native P2X(1) receptor currents in megakaryocytes were activated by alpha, beta-meATP (10 microM) and commercial samples of ADP (10 microM), but not by purified ADP (10 - 100 microM). Purified ADP (up to 1 mM) was also inactive at recombinant human P2X(1) receptors expressed in Xenopus oocytes. Purification did not modify the ability of ADP to activate P2Y receptors coupled to Ca(2+) mobilization in rat megakaryocytes. In human platelets, P2X(1) and P2Y receptor-mediated [Ca(2+)](i) responses were distinguished by their different kinetics at 13 degrees C. In 1 mM Ca(2+) saline, alpha,beta-meATP (10 microM) and commercial ADP (40 microM) activated a rapid [Ca(2+)](i) increase (lag time < or =0.5 s) through the activation of P2X(1) receptors. Hexokinase treatment of ADP shifted the lag time by approximately 2 s, indicating loss of the P2X(1) receptor-mediated response. A revised scheme is proposed for physiological activation of P2 receptors in human platelets. ATP stimulates P2X(1) receptors, whereas ADP is a selective agonist at metabotropic (P2Y(1) and P2T(AC)) receptors.

Figure 1

P2X₁ receptor currents in mouse megakaryocytes: responses to commercial grade and purified ADP. Whole-cell currents at a holding potential of −60 mV. Agonist applications (bar) were separated by 5 min to allow recovery of P2X₁ receptors from desensitization. (a) Representative traces during application of α,β-meATP, commercial ADP or HPLC-purified ADP, each at 10 μM. (b) Effect of commercially available and hexokinase-treated ADP (100 μM) compared to 10 μM α,β-meATP.

Figure 2

Human P2X₁ receptors expressed in Xenopus oocytes: responses to ATP, commercial grade ADP and purified ADP. P2X₁ receptor currents recorded by two-electrode voltage clamp at a holding potential of −60 mV. Agonist applications (bar) were separated by 5 min to allow recovery from desensitization. (a) Responses to ATP, commercial grade ADP, HPLC-purified ADP and hexokinase-treated ADP, each at 100 μM. (b) Average concentration:response relationships for ATP, commercial ADP and hexokinase-treated ADP, constructed from 4–6 oocytes.

Figure 3

P2Y receptor-mediated [Ca²⁺]_i responses to commercial grade ADP and hexokinase-treated ADP in a rat megakaryocyte. Ratiometric fluorescence measurements of fura-2 were used to monitor [Ca²⁺]_i following introduction of the dye under whole-cell patch clamp conditions. Hexokinase-treated (Hex-treated) ADP or commercial (comm.) ADP were applied for the periods indicated by the bars. Holding potential was −75 mV.

Figure 4

Effects of hexokinase purification on the ADP-evoked [Ca²⁺]_i responses of human platelets. [Ca²⁺]_i was monitored using the background-corrected 340/380 nm fluorescence ratio from stirred suspensions of fura-2-loaded human platelets at 13°C. The vertical arrow below the traces in (a) and (b), indicates injection of agonist. (a) Responses following selective activation of either P2X₁ receptors (α,β-meATP in 1 mM Ca²⁺-containing saline) or P2Y receptors (ADP in nominally Ca²⁺-free saline). (b) Responses to commercial grade ADP (comm. ADP) and hexokinase-treated ADP (hexADP) in the presence or nominal absence of external Ca²⁺ (1 mM). (c) Average lag times from 15–19 experiments for each of the conditions in (a) and (b). ^*P<0.05, ^**P<0.01, (Student's t-test) when compared to hexokinase-treated ADP in Ca²⁺-free saline.

Figure 5

Model for activation of platelets via P2 receptors. A/C: adenylyl cyclase; PLC: phospholipase-C; G: G-protein. Ap_nA, diadenosine polyphosphates. See text for full explanation.