A phosphatidylserine binding site in factor Va C1 domain regulates both assembly and activity of the prothrombinase complex

Blood. 2008 Oct 1;112(7):2795-802. doi: 10.1182/blood-2008-02-138941. Epub 2008 Jun 27.


Tightly associated factor V(a) (FVa) and factor X(a) (FXa) serve as the essential prothrombin-activating complex that assembles on phosphatidylserine (PS)-containing platelet membranes during blood coagulation. We have previously shown that (1) a soluble form of PS (C6PS) triggers assembly of a fully active FVa-FXa complex in solution and (2) that 2 molecules of C6PS bind to FVa light chain with one occupying a site in the C2 domain. We expressed human factor V(a) (rFVa) with mutations in either the C1 domain (Y1956,L1957)A, the C2 domain (W2063,W2064)A, or both C domains (Y1956,L1957,W2063,W2064)A. Mutations in the C1 and C1-C2 domains of rFVa reduced the rate of activation of prothrombin to thrombin by FXa in the presence of 400 muM C6PS by 14 000- to 15 000-fold relative to either wild-type or C2 mutant factor rFVa. The K(d')s of FXa binding with rFVa (wild-type, C2 mutant, C1 mutant, and C1-C2 mutant) were 3, 4, 564, and 624 nM, respectively. Equilibrium dialysis experiments detected binding of 4, 3, and 2 molecules of C6PS to wild-type rFVa, C1-mutated, and C1,C2-mutated rFVa, respectively. Because FVa heavy chain binds 2 molecules of C6PS, we conclude that both C2 and C1 domains bind one C6PS, with binding to the C1 domain regulating prothrombinase complex assembly.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acids / metabolism
  • Animals
  • Binding Sites
  • COS Cells
  • Chlorocebus aethiops
  • DNA Mutational Analysis
  • Factor V / metabolism*
  • Factor Va / chemistry*
  • Factor Va / metabolism*
  • Factor Xa / metabolism*
  • Humans
  • Models, Biological
  • Mutant Proteins / metabolism
  • Phosphatidylserines / metabolism*
  • Protein Structure, Tertiary
  • Prothrombin / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Solubility
  • Structure-Activity Relationship


  • Amino Acids
  • Mutant Proteins
  • Phosphatidylserines
  • Recombinant Proteins
  • prothrombinase complex
  • Factor Va
  • Factor V
  • Prothrombin
  • Factor Xa