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Further localization of binding sites for thrombin and protein C in human thrombomodulin
T Hayashi1, M Zushi, S Yamamoto
1Division of Enzyme Cytology, University of Tokushima, Japan.
Insights
Researchers identified specific binding sites for thrombin and protein C on human thrombomodulin. Thrombin binds to the fifth epidermal growth factor (EGF) domain, while protein C interacts with the fourth EGF domain, crucial for its activation.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Interactions
Background:
- Human thrombomodulin is a key regulator of blood coagulation.
- It acts as a cofactor for thrombin-mediated protein C activation.
- Understanding the specific binding sites is crucial for elucidating its function.
Purpose of the Study:
- To map the binding sites of thrombin and protein C on the epidermal growth factor (EGF) domains of human thrombomodulin.
- To characterize the functional role of different EGF domains in protein C activation.
Main Methods:
- Expression of recombinant mutant thrombomodulin proteins in COS-1 cells.
- Assays for cofactor activity in thrombin-catalyzed protein C activation.
- Inhibition studies of thrombin binding using mutant proteins and synthetic peptides.
Main Results:
- Mutant EGF456 exhibited complete cofactor activity, while EGF56 showed none, and EGF45 showed partial activity.
- Thrombin binding was inhibited by both EGF45 and EGF56 domains.
- A synthetic peptide from the fifth EGF domain inhibited thrombin binding.
- Calcium ions were essential for protein C binding and activation.
Conclusions:
- Thrombin binds to the latter half of the fifth EGF domain of thrombomodulin.
- Protein C binds to the fourth EGF domain of thrombomodulin, mediated by calcium ions.
- These findings precisely map the interaction sites for thrombin and protein C on thrombomodulin.
Abstract:
To elucidate the binding sites for thrombin and protein C in the six epidermal growth factor (EGF) domains of human thrombomodulin, recombinant mutant proteins were expressed in COS-1 cells. Mutant protein EGF456, which contains the fourth, fifth, and sixth EGF domains from the NH2 terminus of thrombomodulin, showed complete cofactor activity in thrombin-catalyzed protein C activation, as did intact thrombomodulin or elastase-digested thrombomodulin. EGF56, containing the fifth and sixth EGF domains, did not have cofactor activity; but EGF45, containing the fourth and fifth EGF domains, had about one-tenth of the cofactor activity of EGF456. Thrombin binding to attached recombinant thrombomodulin (D123) was inhibited by EGF45 as well as by EGF56. A synthetic peptide (ECPEGYILDDGFICTDIDE), corresponding to Glu-408 to Glu-426 in the fifth EGF domain, inhibited thrombin binding to attached thrombomodulin (D123) with an apparent Ki of 95 microM. At Ca2+ concentrations of 0.25-0.3 mM, intact protein C was maximally activated by thrombin in the presence of EGF45, EGF456, or EGF1-6, which contains the first to sixth EGF domains; but such maximum cofactor activity was not observed when gamma-carboxyglutamic acid-domainless protein C was used. These findings suggest that: 1) thrombin binds to the latter half of the fifth EGF domain; and 2) protein C binds to the fourth EGF domain of thrombomodulin through Ca2+ ions.