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Related Experiment Videos

Membrane-mediated assembly of the prothrombinase complex.

P L Giesen1, G M Willems, H C Hemker

  • 1Cardiovascular Research Institute Maastricht, University of Limburg, The Netherlands.

The Journal of Biological Chemistry
|October 5, 1991
PubMed
Summary

Prothrombinase assembly on phospholipid bilayers is significantly enhanced by factor Va, with factor Xa rapidly associating with membrane-bound factor Va. This process is faster than random collisions, indicating efficient surface-mediated complex formation.

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Area of Science:

  • Biochemistry
  • Biophysics
  • Membrane Biophysics

Background:

  • Prothrombinase is a key enzyme complex in the blood coagulation cascade.
  • Understanding its assembly on phospholipid surfaces is crucial for comprehending hemostasis and developing procoagulant therapies.

Purpose of the Study:

  • To investigate the mechanism and kinetics of prothrombinase assembly on defined phospholipid surfaces.
  • To quantify the role of factor Va in enhancing factor Xa binding and complex formation.

Main Methods:

  • Utilized macroscopic planar bilayers composed of dioleoyl-phosphatidylserine (DOPS) and dioleoyl-phosphatidylcholine (DOPC).
  • Employed ellipsometry to measure factor Xa binding constants.
  • Assessed steady-state thrombin production to determine binding affinities and assembly rates.

Related Experiment Videos

  • Investigated prothrombinase assembly in suspensions of phospholipid vesicles of varying diameters.

    • Main Results:

    • Factor Xa binding to DOPS/DOPC bilayers showed a dissociation constant (Kd) of 47 nM, reduced to 2.2 pM with preadsorbed factor Va.
    • Comparable Kd values were observed in phospholipid vesicle suspensions.
    • Prothrombinase assembly on surfaces with sparse factor Va was faster than diffusion-limited collisions, suggesting rapid lateral diffusion of factor Xa to factor Va.
    • A high bimolecular rate constant (kon) of 2.8 x 10^13 (mol/cm^2)^-1 s^-1 was estimated for surface complex formation.
    • In vesicle suspensions, kon values increased with vesicle diameter, consistent with collision-limited assembly.

    Conclusions:

    • Factor Va dramatically enhances prothrombinase assembly on phospholipid membranes by facilitating rapid, surface-diffusion-driven complex formation.
    • The assembly mechanism shifts from collision-limited in solution to highly efficient surface-mediated association.
    • These findings provide insights into the spatial regulation of coagulation factor assembly on membrane surfaces.