title: Kinetic analysis and binding studies of a new recombinant human factor VIIa for treatment of haemophilia.
abstract: OBJECTIVE
  LR769 is a new second-generation recombinant human Factor VIIa (rhFVIIa) developed for haemophilia treatment. We determined enzymatic properties of LR769 and its interaction with antithrombin, tissue factor, platelets and endothelial protein C receptor (EPCR), compared with NovoSevenRT.
  
  
  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 (Kd) for activated platelet binding was in the 1 μm range for both products. At saturation, more LR769 than NovoSevenRT was bound to the platelets. Binding to HUVEC was 25-50% higher for LR769 than for NovoSevenRT. Protein C, soluble EPCR, and anti-EPCR antibody all reduced the binding, indicating specificity for EPCR. LR769 was similar to NovoSevenRT in all kinetic assays. Active site titration demonstrated 0.7 mole of active site/mole of protein. The kcat/Kmvalues for activation of FX and FIX with purified recombinant tissue factor and phospholipids were 10.5 s-1/0.32 μm and 3.3 s-1/0.44 μm respectively. The apparent second-order rate constant for inactivation by human plasma AT was 5.9 ± 0.4 × 103m-1s-1. The Kdvalues for binding of LR769 to soluble tissue factor and full-length tissue factor were 8.1 nm and 0.9 nm, respectively, and the Kdfor binding to soluble EPCR was 41 nm.
  
  
  CONCLUSIONS
  Overall, LR769 exhibited characteristics similar to NovoSevenRT, but bound EPCR on HUVEC with somewhat higher affinity than NovoSevenRT.
authors.affiliation: LFB USA, Framingham, MA, USA.
authors.forename: J
authors.initials: J
authors.lastname: Grandoni
authors.affiliation: LFB France, Les Ulis, France.
authors.forename: G
authors.initials: G
authors.lastname: Perret
authors.affiliation: LFB France, Les Ulis, France.
authors.forename: C
authors.initials: C
authors.lastname: Forier
mesh_terms: D015942:Factor VIIa / Q000008:administration & dosage / Q000627:therapeutic use*; D006467:Hemophilia A / Q000188:drug therapy*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000627:therapeutic use; D015398:Signal Transduction
chemical_list: D011994:Recombinant Proteins; C103587:recombinant FVIIa; D015942:Factor VIIa
keywords: EPCR; FVIIa; antithrombin; haemophilia; inhibitors; platelets