P2X1 Purinoceptor in Human Platelets

Abstract

ADP is an important physiological platelet agonist. The molecular identity of the ADP receptor(s) in human platelets, however, is still unclear. Although P2T purinoceptor was believed to be the ligand-gated cation channel for ADP in human platelets, recent patch clamp studies now suggest it is P2X 1 type. In the present study, we have cloned a cDNA encoding a P2X 1 purinoceptor from human platelets using degenerate reverse transcription and polymerase chain reaction. Northern blotting with a P2X 1 -specific probe revealed a band of 1.8 kilobases in human platelets as well as in several megakaryoblastic cell lines. 1321N1 human astrocytoma cells expressing the cloned P2X 1 cDNA exhibited both ATP-and ADP-stimulated Ca 2؉ influx that could be blocked by the purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid and suramin. Additionally, a polyclonal antibody raised against glutathione-S-transferase-P2X 1 fusion peptide reacted with a 70-kDa band on Western blot of human platelets. It is therefore concluded that functional P2X 1 purinoceptors are present in human platelets.Platelet activation is known to be involved in the development of atherosclerosis and restenosis after angioplasty. ADP is an important endogenous platelet agonist. Stimulation of platelets with ADP has been shown to mediate platelet shape change, aggregation, and further release of ADP and ATP from activated platelets. The ADP receptor in platelets is reported to possess a unique, pharmacologically characteristic P2T-type purinoceptor (1). Activation of the ADP receptor causes immediate activation of a non-selective cation channel that mediates calcium influx and mobilization of calcium from intracellular stores (2, 3). Membrane binding experiments have indicated the presence of both high and low affinity binding sites for ADP in platelets (4, 5). Furthermore, pharmacological and clinical studies have also suggested the presence of two types of ADP receptors (6, 7). Several platelet membrane ADP-binding proteins have been proposed as putative ADP receptor (8 -10), but no peptide sequence is available for the cloning purposes. Recently, it has been shown by patch clamp techniques and intracellular calcium measurements that both ADP and ATP can activate a channel and mediate an increase in intracellular calcium concentration in human platelets. It has also been suggested that the ADP/ATP-gated non-selective cation channel resembles a P2X 1 purinoceptor (11).Extracellular ATP and ADP interact with two subgroups of P2 purinoceptors, P2X and P2Y (12, 13). Molecular cloning has identified seven members of P2X, a non-selective cation channel with two transmembrane domains; P2Y, which belongs to the seven-transmembrane domain G-protein-coupled receptor family, consists of more than seven members identified to date. Based on the effects of ADP on adenylyl cyclase, activation of phospholipase C, and intracellular calcium mobilization, we hypothesize that the P2T receptor in human platelets may consist of one P2X-like and one or more P...

Discovery and recognition of purine receptor subtypes on platelets

“…In electrophysiological studies ADP and ATP have been shown to directly open cation channels, with characteristics similar to those of the P2X 1 receptors [Mahaut-Smith et al, 1990, 1992MacKenzie et al, 1996;Sage et al, 1997]. The presence of P2X 1 receptors has now been demonstrated on platelets using molecular techniques Clifford et al, 1998;Scase et al, 1998;Sun et al, 1998], although the significance of this response is not clear. The influx of Ca 2+ could enhance the effect of Ca 2+ mobilised from stores, but as platelets lack voltage-sensitive Ca 2+ channels, the depolarisation will not trigger a greater influx of Ca 2+ through these, as happens in excitable cells such as smooth muscle [see Sage et al, 1992Sage et al, , 1997.…”

“…A genetically modified mouse has also been generated lacking the P2X 1 receptor [Mulryan et al, 2000], but although so far no data have been reported on their haemostatic function, presumably there were no obvious abnormalities. The presence of this receptor raises the question of the true role of ATP in platelet function: although it has always been regarded as an ADP antagonist [Macfarlane and Mills, 1975], it is considerably more potent than ADP at the cloned P2X 1 receptor [Valera et al, 1994;Sun et al, 1998]. …”

ATP-induced apoptosis of human granulosa luteal cells cultured in vitro

“…Molecular cloning has led to the identification of several subtypes in each family (22). ATP-sensitive receptors have been identified in various cell types, including smooth-muscle cells (23), vascular endothelial cells (24), mast cells (25), and platelets (26). Human granulosa cells are also known to have P 2y -purinoceptors (formerly known as P 2u type), and its downstream signal cascade appears to be coupled to [Ca 2ϩ ] i (27,28).…”

P2X1-mediated ERK2 Activation Amplifies the Collagen-induced Platelet Secretion by Enhancing Myosin Light Chain Kinase Activation

“…Both the Rho/Rho kinase and Ca 2ϩ /CaM-regulated MLC kinase pathways mediate MLC phosphorylation independently of each other in human platelets (11,12) as a function of the agonist applied. P2X 1 is highly expressed in human platelets and megakaryocytes (13,14); because of the role of Ca 2ϩ /CaM in platelet MLC kinase activation, we have presently investigated the P2X 1 -mediated Ca 2ϩ influx in relation to MLC phosphorylation and platelet shape change. We have previously shown that the P2X 1 -mediated Ca 2ϩ influx triggers phosphorylation of the extracellular signal-regulated kinase (ERK2), a reaction found to contribute to platelet activation and secretion induced by low concentrations of collagen (15,16).…”