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

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... 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 P2Y-like separate components. This hypothesis is also supported by recent demonstration of a P2Y 1 -type purinoceptor in human platelets (14). In the present study, P2X 1 cDNA was cloned from human platelets, and its function was further characterized after heterologous expression in mammalian cells. MATERIALS AND METHODS Reagents and Solutions-ATP, ADP, ␣,␤-methylene-ATP, aspirin, and apyrase were purchased from Sigma. ATP and ADP were further purified by high performance liquid chromatography using a Partisil Sax 10 m, 250 ϫ 4.6-mm column and a gradual elution with 50 -750 mM ammonium phosphate buffer, pH 3.5. Fura-2 acetoxymethyl ester was from Molecular Probes (Eugene, OR); pyridoxalphosphate-6-azophenyl-2Ј,4Ј-disulfonic acid (PPADS) 1 was from Calbiochem; and suramin was from RBI (Natick, MA). HEPES-Tyrode buffer for calcium measurement contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 2 mM CaCl 2 , 10 mM glucose, and 20 mM HEPES, pH 7.4. In the case of calcium-free solution, CaCl 2 was replaced with 1 mM EGTA. RNA Isolation, cDNA Synthesis, and Polymerase Chain Reaction (PCR) Amplification-Blood was drawn from healthy volunteers and mixed with 0.1 volume of 3.8% sodium citrate followed by centrifugation at 150 ϫ g for 20 min. Supernatant (platelet-rich plasma) was collected without disturbing buffy coat and packed red blood cells. Aspirin (1 mM) and apyrase (20 g/ml, final concentration) were then added to prevent platelet activation by spontaneously released thromboxane and ADP, respectively (3). Contaminating leukocytes and red blood cells were removed by an additional centrifugation for 10 min at 150 ϫ g. The pH of resultant platelet-rich plasma was lowered to 6.5 with citric acid (4 l/ml from 1 M stock) before sedimentation of platelets at 800 ϫ g for 15 min (4). The pellet was lysed, and RNA was extracted with RNA STAT-60 (TEL-TEST B, Inc.). Possible contamination of platelet RNA specimens with leukocyte RNA was ruled out by reverse transcription-PCR amplification (negative) of ␤2 integrin, which presents in leukocytes but not in platelets, using two specific primers. First strand complementary DNA (cDNA) was synthesized utilizing a random hexamer primer using Superscript TM (Life Technologies, Inc.). Degenerate primers for P2X were designed and synthesized based on the conserved regions of rat P2X 1 , P2X 2 , and P2X 3 . The sequence for 5Ј primer used was 5Ј-TTCACCMTYYTCATCAARAACAGCATC-3Ј, and the 3Ј primer was 5Ј-ATRGTRGGRATGAKRYYRAAYTTSCC-3Ј