BINDING OF A THROMBIN RECEPTOR TETHERED LIGAND ANALOG TO HUMAN PLATELET THROMBIN RECEPTOR

A thrombin receptor-radioligand binding assay was developed using [H-3 ]A(pF-F)R(ChA)(hR)Y-NH2 ([H-3]haTRAP), a high affinity thrombin recept or-activating peptide (TRAP), and human platelet membranes. Scatchard analysis of saturation binding data indicated that [H-3]haTRAP bound t o platelet membranes with a K-d of 15 nM and a B-max of 5.2 pmol/mg of protein. The binding was reduced by GPPNHP, a nonmetabolizable GTP an alogue. Various TRAPs and a TRAP antagonist, but not other receptor ag onists, displaced [H-3]haTRAP from the binding sites. SFLLRN-NH2, a th rombin receptor-tethered ligand analogue, and [H-3]haTRAP exhibited co mpetitive binding for the same binding sites. The relative affinity of these peptides for the binding site paralleled their EC(50) or IC50 v alues for platelet aggregation. These data indicate that [H-3]haTRAP b inds specifically and saturably to the functioning G protein-linked th rombin (tethered ligand) receptor in human platelet membranes.