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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. Thrombomudlin 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 M{sub r} {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 K{sub d}'s for thrombin of 1.3 nM, 3.3 nM, and 4.7 nM, respectively. The K{sub d} 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 {sup 125}I-DIP-thrombin, while factor Va did not inhibit binding. Three synthetic peptides were tested for ability to inhibit 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 Hir{sup 53-64} and the thrombomodulin fifth-EGF-domain peptide Tm{sup 426-444} displaced DIP-thrombin from thrombomodulin, but the factor V peptide FacV{sup 30-43} which is similar in compositionmore » and charge to Hir{sup 53-64} 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.« less

Authors:
; ; ; ; ;  [1]
  1. Howard Hughes Medical Institute, St. Louis, MO (USA)
Publication Date:
OSTI Identifier:
5736518
Resource Type:
Journal Article
Journal Name:
Biochemistry; (USA)
Additional Journal Information:
Journal Volume: 29:47; Journal ID: ISSN 0006-2960
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; MEMBRANE PROTEINS; BIOCHEMICAL REACTION KINETICS; RECEPTORS; CROSS-LINKING; THROMBIN; ENDOTHELIUM; FIBRINOGEN; IODINE 125; PEPTIDES; TUMOR CELLS; ANIMAL CELLS; ANIMAL TISSUES; BETA DECAY RADIOISOTOPES; BLOOD COAGULATION FACTORS; BODY; CHEMICAL REACTIONS; COAGULANTS; DAYS LIVING RADIOISOTOPES; DRUGS; ELECTRON CAPTURE RADIOISOTOPES; ENZYMES; GLOBULINS; HEMATOLOGIC AGENTS; HEMOSTATICS; HYDROLASES; INTERMEDIATE MASS NUCLEI; INTERNAL CONVERSION RADIOISOTOPES; IODINE ISOTOPES; ISOTOPES; KINETICS; NUCLEI; ODD-EVEN NUCLEI; ORGANIC COMPOUNDS; PEPTIDE HYDROLASES; POLYMERIZATION; PROTEINS; RADIOISOTOPES; REACTION KINETICS; SERINE PROTEINASES; TISSUES; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Tsiang, Manuel, Lentz, S R, Dittman, W A, Wen, D, Scarpati, E M, and Sadler, J E. Equilibrium binding of thrombin to recombinant human thrombomodulin: Effect of hirudin, fibrinogen, factor Va, and peptide analogues. United States: N. p., 1990. Web. doi:10.1021/bi00499a005.
Tsiang, Manuel, Lentz, S R, Dittman, W A, Wen, D, Scarpati, E M, & Sadler, J E. Equilibrium binding of thrombin to recombinant human thrombomodulin: Effect of hirudin, fibrinogen, factor Va, and peptide analogues. United States. https://doi.org/10.1021/bi00499a005
Tsiang, Manuel, Lentz, S R, Dittman, W A, Wen, D, Scarpati, E M, and Sadler, J E. 1990. "Equilibrium binding of thrombin to recombinant human thrombomodulin: Effect of hirudin, fibrinogen, factor Va, and peptide analogues". United States. https://doi.org/10.1021/bi00499a005.
@article{osti_5736518,
title = {Equilibrium binding of thrombin to recombinant human thrombomodulin: Effect of hirudin, fibrinogen, factor Va, and peptide analogues},
author = {Tsiang, Manuel and Lentz, S R and Dittman, W A and Wen, D and Scarpati, E M and Sadler, J E},
abstractNote = {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. Thrombomudlin 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 M{sub r} {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 K{sub d}'s for thrombin of 1.3 nM, 3.3 nM, and 4.7 nM, respectively. The K{sub d} 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 {sup 125}I-DIP-thrombin, while factor Va did not inhibit binding. Three synthetic peptides were tested for ability to inhibit 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 Hir{sup 53-64} and the thrombomodulin fifth-EGF-domain peptide Tm{sup 426-444} displaced DIP-thrombin from thrombomodulin, but the factor V peptide FacV{sup 30-43} which is similar in composition and charge to Hir{sup 53-64} 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.},
doi = {10.1021/bi00499a005},
url = {https://www.osti.gov/biblio/5736518}, journal = {Biochemistry; (USA)},
issn = {0006-2960},
number = ,
volume = 29:47,
place = {United States},
year = {1990},
month = {11}
}