Localization of thrombomodulin-binding site within human thrombin.

Area of Science:

• Biochemistry
• Molecular Biology
• Hematology

Background:

• Thrombomodulin is a critical cofactor for thrombin in protein C activation.
• The precise binding site of thrombomodulin on human alpha-thrombin was previously undefined.
• Understanding this interaction is vital for regulating coagulation and thrombosis.

Purpose of the Study:

• To elucidate the specific binding site of thrombomodulin on human alpha-thrombin.
• To identify the molecular interactions mediating thrombin-thrombomodulin complex formation.
• To characterize the role of specific thrombin residues in thrombomodulin binding.

Main Methods:

• Epitope mapping using a monoclonal antibody (MT-6) that inhibits thrombin-thrombomodulin binding.
• Enzymatic digestion of thrombin (Staphylococcus aureus V8 protease, elastase) and peptide isolation via HPLC.
• Characterization of peptide fragments and synthetic peptides to determine binding activity.

Main Results:

• An 8.5-kDa thrombin fragment and a derived peptide (residues Thr-147 to Asp-175) bound to MT-6 and thrombomodulin.
• A synthetic peptide (Thr-147 to Ser-158) inhibited thrombin binding to thrombomodulin.
• Elastase cleavage between Ala-150 and Asn-151 abolished binding to both MT-6 and thrombomodulin.

Conclusions:

• The binding site for thrombomodulin on human alpha-thrombin is localized to the sequence between Thr-147 and Ser-158 in the B-chain.
• This region is crucial for the interaction between thrombin and thrombomodulin, impacting protein C activation.
• The findings provide a molecular basis for understanding thrombin-thrombomodulin complex formation and function.