Novel structurally altered P2X1 receptor is preferentially activated by adenosine diphosphate in platelets and megakaryocytic cells

Abstract

Experimental and clinical data suggest the presence of multiple types of adenosine diphosphate (ADP) receptors, one coupled to ligand-gated cation channels (P 2X ) and others coupled to G-proteincoupled (P 2Y ) receptors. This report identifies cDNA for a structurally altered P 2X1 -like receptor in megakaryocytic cell lines (Dami and CMK 11-5) and platelets that, when transfected into nonresponsive 1321 cells, confers a specific sensitivity to ADP with the pharmacologic rank order of ADP > > ATP > > > ␣,␤-methylene-ATP as measured by Ca ؉؉ influx. This receptor (P 2X1del ) contains a deletion of 17 amino acids (PALLREAENFTLFIKNS) that includes an NFT consensus sequence for N-linked glycosylation. Glycosylated forms of the P 2X1del and P 2X1wt receptors were indistinguishable electrophoretically by Western blot or by immunoprecipitation using available antihuman and antirat antibodies. These results indicate that the expression of the P 2X1del receptor results in an influx of Ca ؉؉ induced by ADP. Expression of P 2X1del receptor homomeric subunits is sufficient to express a receptor preferentially activated by ADP and suggests that this altered form, alone or in combination with P 2X1wt receptors, is a component of an ADP-activated ion channel. ( IntroductionAdenosine diphosphate (ADP) is known to play a key role in the development and extension of arterial thrombosis, the deposition of platelets onto collagen under flow conditions, collagen-induced aggregation, and the stabilization of thrombin-induced human platelet aggregates independent of fibrinogen binding to GPIIb/ IIIa, and it plays an important role in irreversible aggregation induced by PAR-1. [1][2][3][4][5] Reduced thrombus formation is observed when platelets from patients with storage pool deficiency are exposed to vascular subendothelium and when removal of ADP from blood-perfusing damaged arteries results in increased bleeding times. 4,6 Platelet aggregation is impaired in 2 patients with a congenital defect of platelet response to ADP, 1,7 further demonstrating that released ADP plays an important role in platelet activation though the mechanism of activation has not been elucidated. Significantly, inhibitors of ADP-induced aggregation are effective antithrombotic drugs. 8,9 Two subclasses of purinergic receptors have been described: P 2Y metabotropic receptors coupled to heterotrimeric G-proteins and P 2X ion channel receptors. Evidence has been provided for 2 distinct G-protein-coupled receptors in human platelets, one coupled to the inhibition of stimulated adenylate cyclase (the P 2YAC receptor) and the other a P 2Y1 receptor (the P2T PLC receptor) coupled to the activation of phospholipase C. [10][11][12] The P2T AC (or P2Y AC ) receptor, now termed the P2Y 12 receptor, has been recently identified. 13 AR-C69096, an adenosine triphosphate (ATP) analog, is a selective inhibitor of ADP-induced platelet aggregation that antagonizes ADP-induced activation through the P2Y 12 receptor. 14 ATP is a competitive inhibitor of ADP-induced aggregation...

Quantification of ADP and ATP receptor expression in human platelets

“…Some recent studies have been able to demonstrate P2X 1 -mediated amplification of the responses to both collagen and P2Y receptors [29,30]. Our findings of high P2X 1 expression in platelets together with reports of a patient with bleeding disorder with a dominant-negative mutation in the platelet P2X 1 receptor [31,32], suggests that a synergistic role for this receptor in platelet aggregation merits investigation in more physiological assays.…”

Heteromultimeric P2X1/2 Receptors Show a Novel Sensitivity to Extracellular pH

“…However, the molecular characterization of the guinea pig P2X 1 receptor is required before further conclusions can be drawn. The recent report of phenotypically altered rat P2X 1 receptors generated by alternative splicing (Greco et al, 2001) further complicates the comparison of native and recombinant P2X receptor responses. We are left with the conclusion that heteromeric assemblies of rP2X 1 and rP2X 2 subunits would best explain the unique pH sensitivity and unusual pharmacological activity of agonists at P2X 1 -like responses.…”

Molecular Physiology of P2X Receptors

“…A recent paper on the characterization of the platelet receptor drew attention to the dangers of using impure commercial preparations of nucleotides (296), showing that actions ascribed to ADP were not observed after it was purified. On the other hand, Greco et al (156) found a splice variant of the P2X 1 receptor to be abundant in human platelets. This variant lacks 17 amino acids at the beginning of exon 6, and the difference appears to have a large effect on the agonist selectivity of the receptor.…”

“…vI3). Finally, a spliced form of the hP2X 1 receptor that lacks most of exon 6 (including the conserved glycosylation site Asn-184) has been found in platelets and megakaryocyte cell line (156). When expressed in fibroblasts and studied by calcium imaging, this receptor showed a much reduced sensitivity to ␣␤meATP.…”