Monoclonal antibodies to human thrombomodulin whose binding is calcium dependent.
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.
Kimura
S
S
Tokyo Research Institute, Kowa Ltd.
Nagoya
T
T
Aoki
N
N
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
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