Monoclonal antibodies to human thrombomodulin whose binding is calcium dependent

Area of Science:

• Biochemistry
• Immunology
• Molecular Biology

Background:

• Human thrombomodulin is a key regulator of the protein C anticoagulant pathway.
• Understanding thrombomodulin's structure and function is crucial for developing anticoagulant therapies.

Purpose of the Study:

• To characterize monoclonal antibodies binding to human thrombomodulin.
• To investigate the role of calcium ions in thrombomodulin's interaction with thrombin and protein C activation.

Main Methods:

• Characterization of four monoclonal antibodies against human thrombomodulin.
• Assessment of antibody binding in the presence and absence of calcium ions.
• Evaluation of antibody effects on thrombin binding and protein C activation.
• Analysis of antibody binding to thrombomodulin fragments generated by protease digestion.

Main Results:

• Two antibodies showed calcium-dependent binding and inhibited thrombin-thrombomodulin interaction and protein C activation.
• These calcium-dependent antibodies recognized a major active fragment, suggesting binding near the thrombin-binding site in the EGF-homology domain.
• One calcium-independent antibody weakly inhibited thrombin binding and protein C activation.
• Another calcium-independent antibody bound to a different region within the EGF-homology domain.

Conclusions:

• Thrombomodulin undergoes a calcium-dependent conformational change.
• This conformational change likely occurs near the thrombin-binding site within the EGF-homology domain.
• Antibody characterization provides insights into thrombomodulin's structure-function relationship.