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Localization of the thrombin-binding domain on prothrombin fragment 2
P C Liaw1, J C Fredenburgh, A R Stafford
1McMaster University and the Hamilton Civic Hospitals Research Centre, Hamilton, Ontario L8V 1C3, Canada.
The Journal of Biological Chemistry
|May 16, 1998
Summary
Human prothrombin fragment 2 (F2) interacts with thrombin via its kringle inner loop and COOH-terminal peptide. This binding induces conformational changes, affecting thrombin
Area of Science:
- Biochemistry
- Molecular Biology
- Hematology
Background:
- Human prothrombin fragment 2 (F2) is known to interact with thrombin.
- Previous co-crystallographic studies suggested salt bridge formation between F2's kringle inner loop and thrombin's anion-binding exosite II.
- F2 binding to thrombin can induce conformational changes in thrombin's active site and exosite I.
Purpose of the Study:
- To precisely localize the thrombin-binding domain on human prothrombin fragment 2 (F2).
- To investigate the specific regions of F2 responsible for its interaction with thrombin and the resulting functional consequences.
Main Methods:
- Utilized plasma, recombinant, and synthetic F2 peptides for binding studies.
- Employed thrombin-agarose affinity chromatography to assess binding.
- Performed kinetic assays using chromogenic substrates and antithrombin inhibition to evaluate functional effects.
- Used fluorescently labeled active site-blocked thrombin and hirudin to probe binding interactions and allosteric changes.
Main Results:
- F2, rF2-(1-116), rF2-(55-116), and sF2-(63-116), all containing the kringle inner loop and COOH-terminal peptide, bound to thrombin.
- Analogues lacking either the kringle inner loop or the COOH-terminal peptide did not bind, indicating both regions are crucial for interaction.
- F2 and sF2-(63-116) bound to exosite II, reduced heparin-catalyzed antithrombin inhibition, and induced allosteric changes in thrombin's active site and exosite I.
- A specific mutation (V78E) in sF2-(63-116) significantly increased its affinity for thrombin.
Conclusions:
- Contrary to prior structural predictions, both the kringle inner loop and the COOH-terminal connecting peptide of F2 are essential for thrombin binding.
- F2 binding to thrombin induces significant allosteric effects, impacting thrombin's enzymatic activity and interaction with inhibitors.
- These findings enhance the understanding of F2-thrombin interactions and their functional implications in hemostasis.