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Bothrojaracin: a potent two-site-directed thrombin inhibitor
V Arocas1, R B Zingali, M C Guillin
1Laboratoire de Recherche sur l'Hémostase et la Thrombose, Faculté Xavier Bichat, Paris, France.
Biochemistry
|July 16, 1996
Summary
Bothrojaracin, a thrombin inhibitor from snake venom, binds to two sites on thrombin, efficiently inhibiting both soluble and clot-bound forms. This dual-site interaction explains its high affinity and potent anticoagulant activity.
Area of Science:
- Biochemistry
- Pharmacology
- Toxicology
Background:
- Bothrojaracin is a 27 kDa protein from Bothrops jararaca venom that inhibits thrombin functions.
- It blocks fibrinogen clotting, platelet activation, and binding to fibrin and thrombomodulin without interacting with the catalytic site.
Purpose of the Study:
- To elucidate the binding mechanism and interaction sites of bothrojaracin with alpha-thrombin.
- To understand how bothrojaracin affects thrombin's interaction with other molecules and its inhibition by antithrombin III-heparin.
Main Methods:
- Surface plasmon resonance (SPR) to determine binding affinities (Kd) and inhibition constants.
- Competition assays using hirudin peptide, heparin, and prothrombin fragment 2.
- Investigating binding to different thrombin forms (alpha-thrombin, gamma-thrombin) and prothrombin.
Main Results:
- High-affinity binding of bothrojaracin to immobilized alpha-thrombin (Kd = 0.6 nM).
- Binding is dependent on exosite 1 and involves exosite 2 or nearby regions, confirmed by competition studies.
- Bothrojaracin inhibits platelet activation by clot-bound thrombin and slows thrombin dissociation from fibrin clots.
Conclusions:
- Bothrojaracin exhibits high-affinity binding to thrombin through a dual-site interaction involving exosite 1 and exosite 2.
- This interaction leads to efficient inhibition of both soluble and clot-bound thrombin, highlighting its potential as an anticoagulant.