title: Effects of replacement of factor VIII amino acids Asp519 and Glu665 with Val on plasma survival and efficacy in vivo.
abstract: Proteolytic cleavage of factor VIII (FVIII) to activated FVIIIa is required for participation in the coagulation cascade. The A2 domain is no longer covalently bound in the resulting activated heterotrimer and is highly unstable. Aspartic acid (D) 519 and glutamic acid (E) 665 at the A1-A2 and A2-A3 domain interfaces were identified as acidic residues in local hydrophobic pockets. Replacement with hydrophobic valine (V; D519V/E665V) improved the stability and activity of the mutant FVIII over the wild-type (WT) protein in several in vitro assays. In the current study, we examined the impact of mutations on secondary and tertiary structure as well as in vivo stability, pharmacokinetics (PK), efficacy, and immunogenicity in a murine model of Hemophilia A (HA). Biophysical characterization was performed with far-UV circular dichroism (CD) and fluorescence emission studies. PK and efficacy of FVIII was studied following i.v. bolus doses of 4, 10 and 40 IU/kg with chromogenic and tail clip assays. Immunogenicity was measured with the Bethesda assay and ELISA after a series of i.v. injections. Native secondary and tertiary structure was unaltered between variants. PK profiles were similar at higher doses, but at 4 IU/kg plasma survival of D519V/E665V was improved. Hemostasis at low concentrations was improved for the mutant. Immune response was similar between variants. Overall, these results demonstrate that stabilizing mutations in the A2 domain of FVIII can improve HA therapy in vivo.
authors.affiliation: Department of Pharmaceutical Sciences, University at Buffalo, The State University of New York, 359 Kapoor Hall, Buffalo, New York, 14214, USA.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Kosloski
authors.forename: Krithika A
authors.initials: KA
authors.lastname: Shetty
authors.forename: Hironao
authors.initials: H
authors.lastname: Wakabayashi
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Fay
authors.forename: Sathy V
authors.initials: SV
authors.lastname: Balu-Iyer
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D004195:Disease Models, Animal; D004355:Drug Stability; D005169:Factor VIII / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000493:pharmacokinetics / Q000494:pharmacology*; D006467:Hemophilia A / Q000097:blood / Q000188:drug therapy* / Q000235:genetics; D006487:Hemostasis / Q000187:drug effects; D006490:Hemostatics / Q000008:administration & dosage / Q000737:chemistry / Q000276:immunology / Q000493:pharmacokinetics / Q000494:pharmacology*; D057927:Hydrophobic and Hydrophilic Interactions; D007275:Injections, Intravenous; D008297:Male; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation; D015202:Protein Engineering; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
chemical_list: D006490:Hemostatics; C078147:F8 protein, human; D005169:Factor VIII