title: Quantification of recombinant and platelet P2Y(1) receptors utilizing a [(125)I]-labeled high-affinity antagonist 2-iodo-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate ([(125)I]MRS2500).
abstract: The ADP-activated P2Y(1) receptor is broadly expressed and plays a crucial role in ADP-promoted platelet aggregation. We previously synthesized 2-iodo-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate (MRS2500), as a selective, high-affinity, competitive antagonist of this receptor. Here we report utilization of a trimethylstannyl precursor molecule for the multi-step radiochemical synthesis of a [(125)I]-labeled form of MRS2500. [(125)I]MRS2500 bound selectively to Sf9 insect cell membranes expressing the human P2Y(1) receptor but did not specifically bind to membranes isolated from empty vector-infected cells. Binding of [(125)I]MRS2500 to P2Y(1) receptors was saturable with a Kd of 1.2nM. Known agonists and antagonists of the P2Y(1) receptor inhibited [(125)I]MRS2500 binding to P2Y(1) receptor-expressing membranes with potencies in agreement with those previously observed in functional assays of this receptor. A high-affinity binding site for [(125)I]MRS2500 also was observed on intact human platelets (Kd=0.61nM) and mouse platelets (Kd=1.20nM) that exhibited the pharmacological selectivity of the P2Y(1) receptor. The densities of sites observed were 151 sites/platelet and 229 sites/platelet in human and mouse platelets, respectively. In contrast, specific binding was not observed in platelets isolated from P2Y(1) receptor(-/-) mice. Taken together, these data illustrate the synthesis and characterization of a novel P2Y(1) receptor radioligand and its utility for examining P2Y(1) receptors natively expressed on human and mouse platelets.
authors.affiliation: Inserm UMR-S949 and Université Louis Pasteur, 67087 Strasbourg, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Ohlmann
authors.forename: Sonia
authors.initials: S
authors.lastname: de Castro
authors.forename: Garth G
authors.initials: GG
authors.lastname: Brown
authors.forename: Christian
authors.initials: C
authors.lastname: Gachet
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Jacobson
authors.forename: T Kendall
authors.initials: TK
authors.lastname: Harden
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000378:metabolism*; D002460:Cell Line; D003838:Deoxyadenine Nucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D011485:Protein Binding; D058921:Purinergic P2Y Receptor Antagonists / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D019275:Radiopharmaceuticals / Q000378:metabolism*; D058472:Receptors, Purinergic P2Y1 / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: C495475:2-iodo-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate; D003838:Deoxyadenine Nucleotides; D007457:Iodine Radioisotopes; D058921:Purinergic P2Y Receptor Antagonists; D019275:Radiopharmaceuticals; D058472:Receptors, Purinergic P2Y1; D011994:Recombinant Proteins