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)

Pharmacol Res. 2010 Oct;62(4):344-51. doi: 10.1016/j.phrs.2010.05.007. Epub 2010 Jun 4.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blood Platelets / metabolism*
  • Cell Line
  • Deoxyadenine Nucleotides / chemical synthesis
  • Deoxyadenine Nucleotides / chemistry
  • Deoxyadenine Nucleotides / metabolism*
  • Humans
  • Iodine Radioisotopes / chemistry
  • Iodine Radioisotopes / metabolism
  • Mice
  • Protein Binding
  • Purinergic P2Y Receptor Antagonists / chemical synthesis
  • Purinergic P2Y Receptor Antagonists / chemistry
  • Purinergic P2Y Receptor Antagonists / metabolism*
  • Radiopharmaceuticals / metabolism*
  • Receptors, Purinergic P2Y1 / genetics
  • Receptors, Purinergic P2Y1 / metabolism*
  • Recombinant Proteins / metabolism


  • 2-iodo-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate
  • Deoxyadenine Nucleotides
  • Iodine Radioisotopes
  • Purinergic P2Y Receptor Antagonists
  • Radiopharmaceuticals
  • Receptors, Purinergic P2Y1
  • Recombinant Proteins