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Related Experiment Videos

Structural requirements for expression of factor Va activity.

Michael Kalafatis1, Daniel O Beck, Kenneth G Mann

  • 1Department of Chemistry, Cleveland State University, and The Lerner Research Institute, The Cleveland Clinic Foundation, Ohio, USA. m.kalafatis@csuohio.edu

The Journal of Biological Chemistry
|June 6, 2003
PubMed
Summary
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Cleavage of factor V at Arg1545 is crucial for factor Xa binding in prothrombinase. Residues 697-709 of factor V

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Hemostasis

Background:

  • Factor Va is a critical cofactor in the prothrombinase complex, essential for efficient thrombin generation.
  • The structure and function of factor Va are modulated by proteolytic cleavage events.
  • Understanding factor Va's interactions is key to comprehending coagulation disorders.

Purpose of the Study:

  • To investigate the role of specific cleavage sites in factor Va on its interaction with factor Xa.
  • To elucidate the contribution of factor Va domains to prothrombinase complex assembly and function.
  • To identify the binding site for prothrombin within the factor Va structure.

Main Methods:

  • Proteolytic cleavage of human factor V using snake venom proteases (Naja nigricollis nigricollis and Russell's viper venom) and alpha-thrombin.

Related Experiment Videos

  • Characterization of cleaved factor V variants (factor VNN, factor VRVV, factor VNN/IIa, factor VNN/RVV) using SDS-PAGE.
  • Determination of apparent dissociation constants (Kd,app) for factor Xa binding using purified components.
  • Inhibition studies using a synthetic peptide (D13R) representing residues 697-709 of factor Va.
  • Interaction studies of the D13R peptide with thrombin-agarose.

    • Main Results:

    • Cleavage of factor V by Naja nigricollis nigricollis protease (NN) at Asp697, Asp1509, and Asp1514 yielded factor VNN, which exhibited reduced affinity for factor Xa (Kd,app = 4 nM) and diminished clotting activity.
    • Cleavage by Russell's viper venom (RVV) at Arg1018 and Arg1545 produced factor VRVV with factor Xa affinity and clotting activity similar to thrombin-activated factor Va.
    • Further cleavage of factor VNN at Arg1545 by alpha-thrombin or RVV restored high affinity for factor Xa (Kd,app ≈ 0.5 nM).
    • A synthetic peptide (D13R) corresponding to residues 697-709 of factor Va competitively inhibited prothrombinase activity and specifically interacted with thrombin-agarose.

    Conclusions:

    • Cleavage at Arg1545 and the subsequent formation of the light chain of factor Va are essential for high-affinity binding and function of factor Xa within the prothrombinase complex.
    • Amino acid residues 697-709 in the heavy chain of factor Va contribute to the binding site for prothrombin.
    • These findings provide critical insights into the molecular mechanisms governing the assembly and function of the prothrombinase complex.