Ahn H S, Foster C, Boykow G, Arik L, Smith-Torhan A, Hesk D, Chatterjee M
Schering-Plough Research Institute, Kenilworth, NJ 07033, USA.
Mol Pharmacol. 1997 Feb;51(2):350-6. doi: 10.1124/mol.51.2.350.
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 a Kd 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.
利用[3H]A(pF-F)R(ChA)(hR)Y-NH2（[3H]haTRAP），一种高亲和力的凝血酶受体激活肽（TRAP），以及人血小板膜，开发了一种凝血酶受体放射性配体结合测定法。对饱和结合数据的Scatchard分析表明，[3H]haTRAP与血小板膜结合，解离常数（Kd）为15 nM，最大结合容量（Bmax）为5.2 pmol/mg蛋白质。不可代谢的GTP类似物GPPNHP可降低这种结合。各种TRAP和一种TRAP拮抗剂，而非其他受体激动剂，能将[3H]haTRAP从结合位点上置换下来。凝血酶受体拴系配体类似物SFLLRN-NH2和[3H]haTRAP对相同的结合位点表现出竞争性结合。这些肽对结合位点的相对亲和力与其对血小板聚集的半数有效浓度（EC50）或半数抑制浓度（IC50）值平行。这些数据表明，[3H]haTRAP与人血小板膜中起作用的G蛋白偶联凝血酶（拴系配体）受体特异性且饱和性地结合。
