title: Equilibrium binding of thrombin to recombinant human thrombomodulin: effect of hirudin, fibrinogen, factor Va, and peptide analogues.
abstract: Thrombomodulin is an endothelial cell surface receptor for thrombin that acts as a physiological anticoagulant. The properties of recombinant human thrombomodulin were studied in COS-7, CHO, CV-1, and K562 cell lines. Thrombomodulin was expressed on the cell surface as shown by the acquisition of thrombin-dependent protein C activation. Like native thrombomodulin, recombinant thrombomodulin contained N-linked oligosaccharides, had Mr approximately 100,000, and was inhibited or immunoprecipitated by anti-thrombomodulin antibodies. Binding studies demonstrated that nonrecombinant thrombomodulin expressed by A549 carcinoma cells and recombinant thrombomodulin expressed by CV-1 and K562 cells had similar Kd's for thrombin of 1.3 nM, 3.3 nM, and 4.7 nM, respectively. The Kd for DIP-thrombin binding to recombinant thrombomodulin on CV-1(18A) cells was identical with that of thrombin. Increasing concentrations of hirudin or fibrinogen progressively inhibited the binding of 125I-DIP-thrombin, while factor Va did not inhibit binding. Three synthetic peptides were tested for ability to inhibit DIP-thrombin binding. Both the hirudin peptide Hir53-64 and the thrombomodulin fifth-EGF-domain peptide Tm426-444 displaced DIP-thrombin from thrombomodulin, but the factor V peptide FacV30-43 which is similar in composition and charge to Hir53-64 showed no binding inhibition. The data exclude the significant formation of a ternary complex consisting of thrombin, thrombomodulin, and hirudin. These studies are consistent with a model in which thrombomodulin, hirudin, and fibrinogen compete for binding to DIP-thrombin at the same site.
authors.affiliation: Howard Hughes Medical Institute, Washington University School of Medicine, Saint Louis 63110.
authors.forename: M
authors.initials: M
authors.lastname: Tsiang
authors.forename: S R
authors.initials: SR
authors.lastname: Lentz
authors.forename: W A
authors.initials: WA
authors.lastname: Dittman
authors.forename: D
authors.initials: D
authors.lastname: Wen
authors.forename: E M
authors.initials: EM
authors.lastname: Scarpati
authors.forename: J E
authors.initials: JE
authors.lastname: Sadler
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002460:Cell Line; D015943:Factor Va / Q000494:pharmacology; D005340:Fibrinogen / Q000494:pharmacology; D005456:Fluorescent Dyes; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D007531:Isoflurophate; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism*; D018179:Receptors, Thrombin; D011994:Recombinant Proteins / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D006629:Hirudins; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D011994:Recombinant Proteins; D007531:Isoflurophate; D015943:Factor Va; D005340:Fibrinogen; D013917:Thrombin