Equilibrium binding of thrombin to recombinant human thrombomodulin: Effect of hirudin, fibrinogen, factor Va, and peptide analogues

Journal Article · Wed Oct 31 23:00:00 EST 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00499a005· OSTI ID:5736518

Tsiang, Manuel; Lentz, S R; Dittman, W A; Wen, D; Scarpati, E M; Sadler, J E ^[1]

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1. Howard Hughes Medical Institute, St. Louis, MO (USA)

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.

• OSTI ID: 5736518
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 29:47; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMAL CELLS
ANIMAL TISSUES
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BLOOD COAGULATION FACTORS
BODY
CHEMICAL REACTIONS
COAGULANTS
CROSS-LINKING
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
ENDOTHELIUM
ENZYMES
FIBRINOGEN
GLOBULINS
HEMATOLOGIC AGENTS
HEMOSTATICS
HYDROLASES
INTERMEDIATE MASS NUCLEI
INTERNAL CONVERSION RADIOISOTOPES
IODINE 125
IODINE ISOTOPES
ISOTOPES
KINETICS
MEMBRANE PROTEINS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PEPTIDE HYDROLASES
PEPTIDES
POLYMERIZATION
PROTEINS
RADIOISOTOPES
REACTION KINETICS
RECEPTORS
SERINE PROTEINASES
THROMBIN
TISSUES
TUMOR CELLS