The P2X1 Receptor, an Adenosine Triphosphate–Gated Cation Channel, Is Expressed in Human Platelets but not in Human Blood Leukocytes

Abstract

Extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) activate multiple types of P2-nucleotide receptors expressed in platelets or leukocytes. Electrophysiological and biochemical studies have indicated expression of the P2X1 receptor, an ATP-gated cation channel, in human and rat platelets, rat basophilic leukemia (RBL) cells, and phorbol myristate acetate (PMA)-differentiated HL-60 myeloid cells. Although these findings suggest that P2X1 receptors are present in both blood leukocytes and blood platelets, the relative levels of P2X1receptor expression and function in human blood leukocytes and platelets have not been directly characterized. On the basis of both immunoblot analysis and functional assays of P2X1receptor-mediated ionic fluxes, we report that there is significant expression of P2X1 receptors in human platelets, but not in neutrophils, monocytes, or blood lymphocytes. Thus, unlike platelets and myeloid progenitor cell lines, fully differentiated human blood leukocytes do not express functionally significant numbers of P2X1 receptors, suggesting the downregulation of P2X1 receptor gene expression during the differentiation of phagocytic leukocytes. By contrast, P2X1 receptor expression is strongly maintained during megakaryocytic differentiation and platelet release. Immunoblot analysis indicated that the platelet P2X1 receptor migrates as an approximately 60-kD protein during SDS-electrophoresis under reducing or nonreducing conditions. Treatment of platelet membranes with endoglycosidase-F causes the P2X1 receptor band to migrate as a 46-kD protein, verifying the highly glycosylated nature of the mature receptor protein. Additional studies of nucleotide-induced changes in Ca2+influx/mobilization demonstrated that the platelet P2X1receptors are pharmacologically distinct from the well-characterized ADP receptors of these cells. This finding suggests a unique role for these ATP-gated ion channels during hemostasis or thrombosis.

ADP Receptor Defects/ADP-Rezeptor-Defekte

“…The other receptor, termed P2Y 12 , mediates a sustained ADP-induced aggregation not preceded by shape change. The combined action of P2Y 1 and P2Y 12 is necessary for the full platelet aggregation response to ADP. P2Y 12 is defective in some patients with congenital bleeding disorders.…”

“…P2Y 12 is defective in some patients with congenital bleeding disorders. Four patients with severe deficiency of P2Y 12 have been described so far. Sequence analysis of the P2Y 12 locus of three of these patients revealed homozygous mutations producing a frame shift mutation and premature truncation of the protein.…”

“…The fourth patient had an allele with a frame shift mutation and a normal allele, which could be silenced by an additional, as yet unknown mutation. Recently, we described a patient with a dysfunctional P2Y 12 . The patient is compound heterozygote: one allele contained a G to A transition resulting in an Arg 256 to Gln codon substitution (R256Q) and the other allele contained a C to T transition resulting in an Arg 265 to Trp codon substitution (R265W).…”

“…The patient is compound heterozygote: one allele contained a G to A transition resulting in an Arg 256 to Gln codon substitution (R256Q) and the other allele contained a C to T transition resulting in an Arg 265 to Trp codon substitution (R265W). Stable CHO cell lines were established expressing either wild-type P2Y 12 , P2Y 12 R256Q or P2Y 12 R265W. Neither mutation blocked the ability of the P2Y 12 receptor to translocate to the CHO cell surface.…”

“…Stable CHO cell lines were established expressing either wild-type P2Y 12 , P2Y 12 R256Q or P2Y 12 R265W. Neither mutation blocked the ability of the P2Y 12 receptor to translocate to the CHO cell surface. ADP at all tested concentrations (0.1±10 mM) greatly inhibited the forskolininduced increase of cAMP in CHO cells transfected with wild-type P2Y 12 , while CHO cells transfected with either mutant protein were only partially inhibited by ADP.…”