Electrostatic dependence of the thrombin-thrombomodulin interaction.

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Thrombin is a key enzyme in blood coagulation.
• Thrombomodulin acts as an anticoagulant cofactor by binding to thrombin.
• Understanding the kinetics of this interaction is crucial for anticoagulation therapies.

Purpose of the Study:

• To determine the rate constants for the binding interaction between thrombin and a functional fragment of thrombomodulin (TM456).
• To investigate the influence of ionic strength on the binding kinetics.
• To elucidate the role of electrostatic forces in steering the association process.

Main Methods:

• Surface plasmon resonance (SPR) was used to measure binding kinetics.
• Experiments were conducted at varying ionic strengths (100 mM to 250 mM NaCl).
• The effect of glycerol on association rates was also examined.

Main Results:

• The association rate constant (k(a)) was 6.7x10^6 M^-1 s^-1 and the dissociation rate constant was 0.033 s^-1 at physiological ionic strength.
• The binding equilibrium constant was determined to be 4.9 nM.
• A tenfold decrease in association rate was observed with increased ionic strength, while dissociation rate remained stable.
• Debye-Hückel analysis indicated a significant role for electrostatic interactions.

Conclusions:

• The thrombin-TM456 interaction is exceptionally fast and primarily governed by electrostatic steering.
• TM456 likely approaches thrombin along the direction of the thrombin molecular dipole.
• These findings provide a deeper understanding of the molecular mechanisms underlying anticoagulation.