title: Monoclonal antibodies to human thrombomodulin whose binding is calcium dependent.
abstract: Four monoclonal antibodies to human thrombomodulin were characterized. Binding of two of these antibodies was dependent on the presence of calcium ions, and approximately 5 mM calcium was required for their maximum binding. These two antibodies inhibited the binding of thrombin to thrombomodulin, thereby inhibiting activation of protein C catalyzed by thrombin-thrombomodulin complex. These two antibodies bind to a major active fragment formed by limited proteolytic digestions of thrombomodulin with elastase and trypsin, suggesting that the antibodies bind to the thrombin-binding site (or its vicinity) located in the epidermal growth factor (EGF)-homology domain. One of the other calcium-independent antibodies also inhibited the binding of thrombin and the activation of protein C, but the inhibition was very weak and was observed only when the antibody was present in a molar excess over thrombomodulin. This antibody did not bind to the protease digests of thrombomodulin. Another calcium-independent antibody did not inhibit either thrombin binding or protein C activation, but bound to the active fragment of protease digests, suggesting that the antibody binds to a region other than the thrombin-binding site in the EGF-homology domain. These observations suggest that thrombomodulin undergoes a calcium-dependent conformational change which may occur in proximity to a thrombin-binding site located in the EGF-homology domain.
authors.affiliation: Tokyo Research Institute, Kowa Ltd.
authors.forename: S
authors.initials: S
authors.lastname: Kimura
authors.forename: T
authors.initials: T
authors.lastname: Nagoya
authors.forename: N
authors.initials: N
authors.lastname: Aoki
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006735:Horseradish Peroxidase / Q000276:immunology; D006801:Humans; D015151:Immunoblotting; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D011486:Protein C / Q000032:analysis; D011487:Protein Conformation; D011863:Radioimmunoassay; D011956:Receptors, Cell Surface / Q000276:immunology*; D018179:Receptors, Thrombin; D013917:Thrombin / Q000494:pharmacology; D014914:Whole Blood Coagulation Time
chemical_list: D000911:Antibodies, Monoclonal; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D011486:Protein C; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D006735:Horseradish Peroxidase; D013917:Thrombin; D002118:Calcium