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Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay
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Effects of prothrombin on the individual activated protein C-mediated cleavages of coagulation factor Va

Sinh Tran1, Eva Norstrøm, Björn Dahlbäck

  • 1Department of Laboratory Medicine, Clinical Chemistry, Lund University, The Wallenberg Laboratory, University Hospital, Malmö, SE-205 02 Malmö, Sweden.

The Journal of Biological Chemistry
|January 17, 2008
PubMed

Insights

Prothrombin inhibits activated protein C (APC) from inactivating factor Va (FVa) by affecting both major cleavage sites. This protective effect, crucial for FVa function in the prothrombinase complex, is lost upon prothrombin activation.

Area of Science:

  • Biochemistry
  • Hematology
  • Molecular Biology

Background:

  • Factor Va (FVa) is a critical coagulation cofactor.
  • Activated protein C (APC) inactivates FVa through specific proteolytic cleavages.
  • Inhibition of APC-mediated FVa inactivation by factor Xa (FXa) and prothrombin is known, but prothrombin's precise mechanism is unclear.

Purpose of the Study:

  • To investigate the effect of prothrombin on the individual APC cleavage sites in FVa.
  • To elucidate the mechanism by which prothrombin inhibits APC-mediated FVa inactivation.

Main Methods:

  • Utilized recombinant FV variants (FV:R506Q/R679Q and FV:R306Q/R679Q).
  • Employed a prothrombinase-based FVa assay to monitor APC-mediated FVa inhibition.
  • Calculated apparent first-order rate constants for cleavage sites in the presence and absence of prothrombin.
  • Analyzed proteolytic products using Western blots.

Main Results:

  • Prothrombin significantly impaired APC-mediated cleavages at both Arg306 and Arg506 sites of FVa.
  • Prothrombin exhibited dose-dependent inhibition, with near-complete inhibition at 3 µM.
  • The inhibitory activity of prothrombin was lost after its cleavage by thrombin.
  • Prothrombin's inhibitory effect was more pronounced at the Arg306 site in the presence of protein S.

Conclusions:

  • Prothrombin protects FVa from APC inactivation by hindering both Arg306 and Arg506 cleavages.
  • Prothrombin's inhibition involves impaired APC function and, in the presence of protein S, decreased APC cofactor function.
  • FVa is protected within the prothrombinase complex by FXa and prothrombin, with this protection being alleviated upon prothrombin activation.

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