Antibody-induced enhancement of factor VIIa activity through distinct allosteric pathways

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  • Lisbeth Moreau Andersen, Danmark
  • Peter Andreasen, Danmark
  • Ivan Svendsen, Novo Nordisk A/S, Danmark
  • Janneke Keemink
  • ,
  • Henrik Østergaard, Novo Nordisk A/S, Danmark
  • Egon Persson, Novo Nordisk A/S, Danmark
In the absence of its cofactor tissue factor (TF), coagulation factor VIIa (FVIIa) predominantly exists in a zymogen-like, catalytically incompetent state. Here we demonstrate that conformation-specific monoclonal antibodies (mAbs) can be used to characterize structural features determining the activity of FVIIa. We isolated two classes of mAbs, which both increased the catalytic efficiency of FVIIa more than 150-fold. The effects of the antibodies were retained with a FVIIa variant, which has been shown to be inert to allosteric activation by the natural activator TF, suggesting that the antibodies and TF employ distinct mechanisms of activation. The antibodies could be classified into two groups based on their patterns of affinities for different conformations of FVIIa. Whereas one class of antibodies affected both the K(m) and k(cat), the other class mainly affected the K(m). The antibody-induced activity enhancement could be traced to maturation of the S1 substrate binding pocket and the oxyanion hole, evident by an increased affinity for p-aminobenzamidine, an increased rate of antithrombin inhibition, an increased rate of incorporation of diisopropylfluorophosphate, and an enhanced fraction of molecules with a buried N terminus of the catalytic domain in the presence of antibodies. As demonstrated by site-directed mutagenesis, the two groups of antibodies appear to have overlapping, although clearly different, epitopes in the 170-loop. Our findings suggest that binding of ligands to specific residues in the 170-loop or its spatial vicinity may stabilize the S1 pocket and the oxyanion hole, and they may have general implications for the molecular understanding of FVIIa regulatory mechanisms.
OriginalsprogEngelsk
TidsskriftJournal of Biological Chemistry
Vol/bind287
Nummer12
Sider (fra-til)8994-9001
Antal sider8
ISSN0021-9258
DOI
StatusUdgivet - 16 mar. 2012

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