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Characterization of proexosite I on prothrombin.
P J Anderson1, A Nesset, K R Dharmawardana
1Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
The Journal of Biological Chemistry
|April 5, 2000
Summary
The study identifies and characterizes proexosite I on prothrombin, a key blood protein. This site shows significantly increased affinity for hirudin peptides upon thrombin activation, revealing insights into blood coagulation regulation.
Area of Science:
- Biochemistry
- Molecular Biology
- Hematology
Background:
- Activation of prothrombin to thrombin involves the expression of regulatory exosites I and II.
- These exosites play crucial roles in modulating thrombin's enzymatic activity and interactions.
- Understanding these regulatory sites is key to comprehending blood coagulation.
Purpose of the Study:
- To identify and characterize the exosite I binding site on human and bovine prothrombin.
- To investigate the expression and affinity changes of this site upon thrombin formation.
- To compare the binding characteristics of hirudin peptides to prothrombin and thrombin.
Main Methods:
- Quantitative affinity chromatography and equilibrium binding studies were performed.
- A fluorescein-labeled hirudin peptide derivative, [5F]Hir(54-65)(SO(3)(-)), was used as a specific ligand.
- Competitive binding assays with unlabeled hirudin peptides were conducted.
Main Results:
- Proexosite I was identified as a specific binding site for hirudin peptides on both prothrombin and thrombin.
- [5F]Hir(54-65)(SO(3)(-)) demonstrated distinct spectral differences and binding affinities for prothrombin versus thrombin.
- Human prothrombin and thrombin showed a 130-fold increase in affinity for the ligand upon thrombin activation; bovine proteins showed a 150-fold increase.
- Sulfated hirudin peptides exhibited higher affinity than nonsulfated analogs.
Conclusions:
- Proexosite I on prothrombin represents a low-affinity binding site in a distinct conformational state.
- Activation to thrombin results in a significant conformational change, increasing binding affinity by approximately 100-fold.
- These findings provide a detailed characterization of proexosite I and its role in thrombin's regulatory function during blood coagulation.