Factor Xa-factor Va complex assembles in two dimensions with unexpectedly high affinity: an experimental and...

Area of Science:

• Biochemistry
• Molecular Biology
• Membrane Biophysics

Background:

• The prothrombin activation complex is crucial for blood coagulation.
• Understanding the assembly kinetics and binding affinity of this complex is vital.

Purpose of the Study:

• To investigate how phospholipid vesicle concentration and size affect the assembly and affinity of the prothrombin activation complex.
• To determine the binding kinetics and affinity between factors Va and Xa in the presence of vesicles.

Main Methods:

• Monitoring prethrombin 1 activation to measure complex formation between factors Va and Xa.
• Varying phospholipid vesicle concentrations and sizes to assess their impact on reaction rates.
• Investigating two-dimensional binding on membrane surfaces under various protein densities.

Main Results:

• Activation rates initially increased and then decreased with rising vesicle concentration.
• Larger vesicles showed inhibition only at higher phospholipid concentrations.
• Prolonged incubation reduced inhibition by high vesicle concentrations, suggesting slow redistribution.
• Dissociation constant (Kd) was determined to be ≤ 25 pM, indicating tighter binding than previously reported.
• Complex formation was independent of surface density when all proteins were membrane-bound.

Conclusions:

• The overall affinity of the factor Va-factor Xa interaction is higher than previously appreciated.
• Factor Va and factor Xa form a stable complex upon binding to the same vesicle.
• Phospholipid vesicle characteristics significantly influence the kinetics and affinity of prothrombin activation complex assembly.