Quantification of recombinant and platelet P2Y1 receptors utilizing a [125I]-labeled high-affinity antagonist 2-iodo-N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate ([125I]MRS2500)

• Published in: Pharmacological research 2010-10, Vol.62 (4), p.344-351
• Main Authors: Ohlmann, Philippe, de Castro, Sonia, Brown Jr, Garth G., Gachet, Christian, Jacobson, Kenneth A., Harden, T. Kendall
• Format: Article
• Language: English
• ISSN: 1043-6618; 1096-1186
• DOI: 10.1016/j.phrs.2010.05.007

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 multistep 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 1.2 nM. 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 = 0.61 nM) and mouse platelets (Kd = 1.20 nM) 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.