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

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 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.

Coordinate Activation of Human Platelet Protease-activated Receptor-1 and -4 in Response to Subnanomolar α-Thrombin

“…Expression of CD62 and CD63 on washed human platelets were quantified by flow cytometry using FITC-labeled CD63 antibody (Immunotech, Beckman Coulter Co., Fullerton, CA) and phycoerythrin-labeled CD62 antibody (BD Biosciences) as described previously (5). PAR-1 cleavage at Arg 41 -Ser 42 and release of PAR-1 activation peptide, PAR-1-(1-41), in the supernatants of washed platelets was quantified by an ELISA as described previously (16). PAR-4 cleavage at Arg 47 -Gly 48 was also measured by an ELISA as the release of PAR-4-(1-47) as described below.…”

“…Activation of human platelet PAR-1 arising from cleavage at Arg 41 -Ser 42 in response to ␣-thrombin and the simultaneous exposure of the previously cryptic tethered ligand domain (beginning with the sequence 42 SFLLRN), were first reported by Vu and colleagues (4). Two studies have confirmed cleavage at Arg 41 -Ser 42 by quantifying platelet PAR-1 activation peptide release (5,6). Monoclonal antibodies directed against the hirudin-like ␣-thrombin-binding domain of PAR-1 or against residues spanning the reported ␣-thrombin cleavage site (PAR-1 span antibodies) practically eliminated all responses of human platelets to Յ1 nM ␣-thrombin.…”

Substrate-Assisted Catalysis of the PAR1 Thrombin Receptor

“…DISCUSSION Thrombin has many substrates and inhibitors, including fibrinogen, prothrombin, protein C, antithrombin III, ␣ 2 -macroglobulin, and factors V, VII, XI, and XIII, that circulate at concentrations ranging from 30 nM to 10 M (26). By comparison, PAR1 is a rare substrate for thrombin with only 600 -1800 receptors present on the surface of each platelet (27,28). During the time period prior to clot formation, the prothrombinase complex on the surface of platelets generates 2-15 nM thrombin in whole blood (29).…”

Himbacine-Derived Thrombin Receptor Antagonists: C7-Spirocyclic Analogues of Vorapaxar

“…22 PAR-1 receptors isolated from human platelets were used for this study. The inhibition constants (K i ) for the spirocyclic analogues are presented in Table 1.…”