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Related Experiment Videos

An extensive interaction interface between thrombin and factor V is required for factor V activation.

T Myles1, T H Yun, S W Hall

  • 1Division of Hematology, Stanford University School of Medicine, Stanford, CA 94305, USA. tmyles@stanford.edu

The Journal of Biological Chemistry
|April 20, 2001
PubMed
Summary
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This study reveals that thrombin

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Hematology

Background:

  • Thrombin plays a crucial role in blood coagulation by activating factor V (FV).
  • Understanding the interaction interface between thrombin and FV is essential for elucidating the mechanisms of coagulation.
  • Site-directed mutagenesis is a powerful tool for dissecting protein-protein interactions.

Purpose of the Study:

  • To investigate the specific residues and regions of human thrombin involved in the interaction with human factor V (FV).
  • To determine the contribution of anion-binding exosite I (ABE-I), anion-binding exosite II (ABE-II), and other regions to FV activation.
  • To elucidate the structural requirements for efficient thrombin-mediated cleavage of FV.

Main Methods:

  • Site-directed mutagenesis was used to generate 78 recombinant thrombin mutants.

Related Experiment Videos

  • A two-stage clotting assay was employed to measure the FV activation activity of thrombin mutants.
  • SDS-polyacrylamide gel electrophoresis was used to analyze the cleavage of FV by selected thrombin mutants.

    • Main Results:

    • Seventeen thrombin mutants exhibited significantly reduced FV activation (<50% of wild-type).
    • Key regions identified include anion-binding exosite I, anion-binding exosite II, the Leu(45)-Asn(57) insertion loop, and the Na(+) binding loop.
    • Mutants in ABE-I (e.g., R68A, R70A, Y71A) and ABE-II (e.g., R98A) showed substantial defects in FV activation (<30% of WT activity).
    • Mutations in the Na(+) binding loop (E229A, R233A) and the Leu(45)-Asn(57) insertion loop (W50A) also significantly impaired FV activation.
    • Efficient cleavage of FV at Arg(709) requires both ABEs and the Na(+)-bound form of thrombin.
    • Basic residues in both ABEs contribute to complex formation through electrostatic interactions with FV.

    Conclusions:

    • Thrombin activation of FV involves an extensive interaction interface, requiring both ABE-I and ABE-II.
    • The Na(+)-bound form of thrombin is critical for optimal procoagulant activity.
    • The S' subsite and specific loops also play important roles in the thrombin-FV interaction and efficient cleavage.
    • These findings provide detailed insights into the molecular basis of thrombin-mediated FV activation in hemostasis.