THE THROMBIN RECEPTOR EXTRACELLULAR DOMAIN CONTAINS SITES CRUCIAL FORPEPTIDE LIGAND INDUCED ACTIVATION

A thrombin receptor (TR) demonstrating a unique activation mechanism h as recently been isolated from a megakaryocytic (Dami) cell line. To f urther study determinants of peptide ligand-mediated activation phenom enon, we have isolated, cloned, and stably expressed the identical rec eptor from a human umbilical vein endothelial cell (HUVEC) library. Ch inese hamster ovary (CHO) cells expressing a functional TR (CHO-TR), p latelets, and HUVECs were then used to specifically characterize alpha -thrombin- and peptide ligand-induced activation responses using two d ifferent antibodies: anti-TR34-52 directed against a 20-amino acid pep tide spanning the thrombin cleavage site,and anti-TR1-160 generated ag ainst the NH2-terminal 160 amino acids of the TR expressed as a chimer ic protein in Escherichia coli. Activation-dependent responses to both alpha-thrombin (10 nM) and peptide ligand (20 muM) were studied using fura 2-loaded cells and microspectrofluorimetry. Whereas preincubatio n of CHO-TR with anti-TR34-52 abolished only a-thrombin-induced [Ca2+] i transients, preincubation with anti-TR1-160 abrogated both alpha-thr ombin- and peptide ligand-induced responses. This latter inhibitory ef fect was dose dependent and similar for both agonists, with an EC50 of approximately 90 mug/ml. Anti-TR1-160 similarly abolished peptide lig and-induced [Ca2+]i transients in platelets and HUVECs, whereas qualit atively different responses characterized by delayed but sustained ele vations in [Ca2+]i transients were evident using alpha-thrombin. Plate let aggregation to low concentrations of both ligands was nearly aboli shed by anti-TR1-160, although some shape change remained; anti-TR34-5 2 only inhibited alpha-thrombin-induced aggregation. These data establ ish that a critical recognition sequence for peptide ligand-mediated r eceptor activation is contained on the NH2-terminal portion of the rec eptor, upstream from the first transmembrane domain. Furthermore, alph a-thrombin-induced activation of HUVECs and platelets may be partially mediated by an alternative mechanism(s) or receptor(s).