Kinetic analysis and binding studies of a new recombinant human factor VIIa for treatment of haemophilia

Introduction/Aim LR 769 is a new second‐generation recombinant human Factor VII a (rh FVII a) developed for haemophilia treatment. We determined enzymatic properties of LR 769 and its interaction with antithrombin, tissue factor, platelets and endothelial protein C receptor ( EPCR ), compared with NovoSeven RT . Methods Kinetic enzyme assays and active site titration were used for enzymatic studies. Surface Plasmon Resonance ( SPR ) was used for determination of binding constants. Cellular binding was determined for platelets and cultured human umbilical vein endothelial cells ( HUVEC ). Results The dissociation constant ( K d ) for activated platelet binding was in the 1 μ m range for both products. At saturation, more LR 769 than NovoSeven RT was bound to the platelets. Binding to HUVEC was 25–50% higher for LR 769 than for NovoSeven RT . Protein C, soluble EPCR , and anti‐ EPCR antibody all reduced the binding, indicating specificity for EPCR . LR 769 was similar to NovoSeven RT in all kinetic assays. Active site titration demonstrated 0.7 mole of active site/mole of protein. The k cat / K m values for activation of FX and FIX with purified recombinant tissue factor and phospholipids were 10.5 s −1 /0.32 μ m and 3.3 s −1 /0.44 μ m respectively. The apparent second‐order rate constant for inactivation by human plasma AT was 5.9 ± 0.4 × 10 3 m −1 s −1 . The K d values for binding of LR 769 to soluble tissue factor and full‐length tissue factor were 8.1 n m and 0.9 n m , respectively, and the K d for binding to soluble EPCR was 41 n m . Conclusion Overall, LR 769 exhibited characteristics similar to NovoSeven RT , but bound EPCR on HUVEC with somewhat higher affinity than NovoSeven RT .