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Related Experiment Videos

Activation of human factor V by meizothrombin

G Tans1, G A Nicolaes, M C Thomassen

  • 1Department of Biochemistry, University of Limburg, Maastricht, The Netherlands.

The Journal of Biological Chemistry
|June 10, 1994
PubMed
Summary

A modified prothrombin, R155A meizothrombin, potently activates factor V, especially on phospholipid surfaces. This suggests meizothrombin plays a key role in factor V activation during blood coagulation.

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Area of Science:

  • Biochemistry
  • Hematology
  • Molecular Biology

Background:

  • Prothrombin activation is a critical step in blood coagulation.
  • Meizothrombin, an intermediate in prothrombin activation, has been investigated for its enzymatic activities.
  • The role of meizothrombin in activating factor V, particularly in the presence of phospholipids, requires further elucidation.

Purpose of the Study:

  • To investigate the factor V activating potential of a modified meizothrombin derivative (R155A meizothrombin).
  • To compare the kinetics of factor V activation by R155A meizothrombin with that of native thrombin.
  • To determine the influence of negatively charged phospholipid vesicles on factor V activation by R155A meizothrombin.

Main Methods:

  • Preparation of recombinant human prothrombin with an Arg155 to Ala substitution.

Related Experiment Videos

  • Conversion of recombinant prothrombin to R155A meizothrombin.
  • Assay of factor V activation using R155A meizothrombin and thrombin in the presence and absence of phosphatidylserine/phosphatidylcholine vesicles.
  • Kinetic analysis using second-order rate constants.
  • Gel electrophoretic analysis of factor V cleavage products.

    • Main Results:

    • R155A meizothrombin demonstrated significantly enhanced factor V activation compared to thrombin, particularly on phospholipid vesicles (18 x 10^6 M^-1 S^-1 vs. 4.0 x 10^6 M^-1 S^-1).
    • Factor V activation by R155A meizothrombin was highly dependent on the concentration of phosphatidylserine/phosphatidylcholine vesicles, reaching a plateau at approximately 50 microM.
    • Both R155A meizothrombin and thrombin generated similar factor Va heavy and light chains, although cleavage rates differed.

    Conclusions:

    • R155A meizothrombin is a potent activator of factor V, with activity significantly enhanced by negatively charged phospholipids.
    • The findings suggest that meizothrombin, a major product of initial prothrombin activation, likely contributes significantly to in vivo factor V activation.
    • This study highlights a crucial role for meizothrombin in the hemostatic pathway beyond its role as a prothrombin intermediate.