title: Activation, activity and inactivation of factor VIII in factor VIII products.
abstract: BACKGROUND
  Factor VIII (FVIII) products used in haemophilia A treatment show inter-and intra-product and inter-assay differences in specific activity. The mechanistic basis of these differences remains unclear.
  
  
  OBJECTIVE
  The aim of this study was to mechanistically compare the functional properties of an in-house excipient-free full-length FVIII standard and pharmacologic recombinant products containing full-length (products A and B) or B-domainless (C and D) FVIII.
  
  
  METHODS
  Factor VIII protein concentration was quantitated by ELISA. Product potency determinations (APTT, intrinsic tenase assays) and kinetic analyses detailing these products' activations by thrombin and FXa, their spontaneous and activated protein C (APC) catalysed inactivation and their performances in coagulation proteome reconstructions were studied +/- von Willebrand factor (VWF). Computational models were developed to facilitate interpretation of empirical data.
  
  
  RESULTS
  Factor VIII protein content per manufacturer activity unit was highest for product C with the other three products similar to the standard. Potency estimates, done five different ways, varied 20-30% in inter- and intra-assay comparisons, with product B consistently showing lower specific activity. Kinetic analyses showed the five FVIII species to differ somewhat in maximum rate of activation, the maximum level of activity achieved, the rate of spontaneous or APC catalysed inactivation and the magnitude of the effect of VWF on these parameters. When evaluated both computationally and empirically in the context of tissue factor initiated thrombin generation, product C appears the most dissimilar.
  
  
  CONCLUSIONS
  Assessments of FVIII activation/inactivation dynamics report larger differences between FVIII products than standard functional assays. However, all FVIII products promote a 'normal' thrombin generation response to TF.
authors.affiliation: Department of Biochemistry, College of Medicine University of Vermont, Burlington, VT, USA.
authors.forename: T
authors.initials: T
authors.lastname: Orfeo
authors.affiliation: Department of Biochemistry, College of Medicine University of Vermont, Burlington, VT, USA.
authors.forename: R
authors.initials: R
authors.lastname: Elsman
authors.affiliation: Department of Biochemistry, College of Medicine University of Vermont, Burlington, VT, USA.
authors.forename: M
authors.initials: M
authors.lastname: Gissel
authors.affiliation: Department of Biochemistry, College of Medicine University of Vermont, Burlington, VT, USA.
authors.forename: K G
authors.initials: KG
authors.lastname: Mann
authors.affiliation: Department of Biochemistry, College of Medicine University of Vermont, Burlington, VT, USA.
authors.forename: S
authors.initials: S
authors.lastname: Butenas
mesh_terms: D002384:Catalysis; D003029:Coagulants / Q000627:therapeutic use; D004797:Enzyme-Linked Immunosorbent Assay; D005169:Factor VIII / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use; D015951:Factor Xa / Q000378:metabolism; D006467:Hemophilia A / Q000188:drug therapy; D006801:Humans; D007700:Kinetics; D010314:Partial Thromboplastin Time; D011486:Protein C / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000627:therapeutic use; D013917:Thrombin / Q000378:metabolism; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D003029:Coagulants; D011486:Protein C; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D005169:Factor VIII; D013917:Thrombin; D015951:Factor Xa
keywords: computational modelling; factor VIII; factor VIII activation; factor VIII assays; factor VIII inactivation; factor VIII products