Binding of a Thrombin Receptor Tethered Ligand Analogue to Human Platelet Thrombin Receptor

  1. Ho-Sam Ahn,
  2. Carolyn Foster,
  3. George Boykow,
  4. Leyla Arik,
  5. April Smith-Torhan,
  6. David Hesk and
  7. Meeta Chatterjee
  1. Schering-Plough Research Institute, Kenilworth, NJ 07033

    Abstract

    A thrombin receptor-radioligand binding assay was developed using [3H]A(pF-F)R(ChA)(hR)Y-NH2([3H]haTRAP), a high affinity thrombin receptor-activating peptide (TRAP), and human platelet membranes. Scatchard analysis of saturation binding data indicated that [3H]haTRAP bound to platelet membranes with aKd of 15 nm and a Bmax of 5.2 pmol/mg of protein. The binding was reduced by GPPNHP, a nonmetabolizable GTP analogue. Various TRAPs and a TRAP antagonist, but not other receptor agonists, displaced [3H]haTRAP from the binding sites. SFLLRN-NH2, a thrombin receptor-tethered ligand analogue, and [3H]haTRAP exhibited competitive binding for the same binding sites. The relative affinity of these peptides for the binding site paralleled their EC50 or IC50 values for platelet aggregation. These data indicate that [3H]haTRAP binds specifically and saturably to the functioning G protein-linked thrombin (tethered ligand) receptor in human platelet membranes.

    Footnotes

    • Send reprint requests to: Ho-Sam Ahn, Ph.D., Schering-Plough Research Institute, K-15-4-4600, Kenilworth, NJ 07033-0539. E-mail: ho-sam.ahn{at}spcorp.com

    • Abbreviations:
      TRAP
      thrombin receptor-activating peptide
      [3H]haTRAP
      alanine-parafluorophenylalanine-arginine-cyclohexylalanine-homoarginine-[3H]phenylalanine amide
      monoiodo-haTRAP
      alanine-parafluorophenylalanine-arginine-cyclohexylalanine-homoarginine-paraiodophenylalanine amide
      pF-F
      parafluorophenylalanine
      ChA
      cyclohexylalanine
      hR
      homoarginine
      Mpa
      3-mercaptopropionyl-phenylalanine-cyclohexylalanine-cyclohexylalanine-arginine-lysine-proline-asparagine-aspartate-lysine amide
      GPPNHP
      5′-guanylyl imidodiphosphate
      DMSO
      dimethylsulfoxide
      DMF
      dimethylformamide
      TFA
      trifluoroacetic acid
      EGTA
      ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
      HPLC
      high performance liquid chromatography
      • Received April 24, 1996.
      • Accepted September 25, 1996.
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    1. Molecular Pharmacology February 1, 1997 vol. 51 no. 2 350-356
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