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Quantitative characterization of the thrombin-heparin interaction. Discrimination between specific and nonspecific
S T Olson1, H R Halvorson, I Björk
1Henry Ford Hospital, Division of Biochemical Research, Detroit, Michigan 48202.
The Journal of Biological Chemistry
|April 5, 1991
Summary
Human alpha-thrombin binds to heparin via nonspecific electrostatic interactions, primarily involving 5-6 anionic residues. A second weaker binding site explains deviations with longer heparin chains.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Heparin is a crucial anticoagulant.
- Thrombin is a key enzyme in the coagulation cascade.
- Understanding thrombin-heparin interactions is vital for anticoagulant drug development.
Purpose of the Study:
- To investigate the binding mechanism between human alpha-thrombin and heparin.
- To differentiate between sequence-specific and nonspecific binding modes.
- To characterize the affinity and stoichiometry of the interaction.
Main Methods:
- Extrinsic fluorescence probe (p-aminobenzamidine) measurements.
- Quantitative affinity chromatography.
- Analysis using nonspecific and discrete binding site models.
- Sedimentation equilibrium ultracentrifugation.
Main Results:
- Binding is primarily a nonspecific electrostatic association, not sequence-specific.
- Apparent binding affinity increases with heparin chain length.
- An intrinsic dissociation constant (KD,obs) of 6-10 microM was determined at physiological ionic strength.
- A second weaker binding site on thrombin for heparin was identified, explaining deviations with longer chains and complex insolubility.
Conclusions:
- Thrombin-heparin interaction is predominantly governed by nonspecific electrostatic forces.
- Heparin chain length influences binding affinity, with longer chains showing increased affinity.
- The findings suggest a complex binding mechanism involving at least two sites on thrombin.