P2X1 purinoceptor in human platelets. Molecular cloning and functional characterization after heterologous expression.
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 P2X1 type. In the present study, we have cloned a cDNA encoding a P2X1 purinoceptor from human platelets using degenerate reverse transcription and polymerase chain reaction. Northern blotting with a P2X1-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 P2X1 cDNA exhibited both ATP- and ADP-stimulated Ca2+ 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-P2X1 fusion peptide reacted with a 70-kDa band on Western blot of human platelets. It is therefore concluded that functional P2X1 purinoceptors are present in human platelets.
Sun
B
B
Department of Thrombosis and Vascular Biology, Maryland Research Laboratories, Otsuka America Pharmaceutical, Inc., Rockville, Maryland 20850, USA. bings@mrl.oapi.com
Li
J
J
Okahara
K
K
Kambayashi
J
J
D000244:Adenosine Diphosphate / Q000502:physiology; D000255:Adenosine Triphosphate / Q000502:physiology; D001254:Astrocytoma; D001483:Base Sequence; D001792:Blood Platelets / Q000502:physiology*; D002118:Calcium / Q000502:physiology; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D015640:Ion Channel Gating; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018048:Receptors, Purinergic P2 / Q000235:genetics* / Q000502:physiology; D058469:Receptors, Purinergic P2X; D011994:Recombinant Proteins; D014162:Transfection
D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D002118:Calcium