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.

Fig. 1

FVIII structure. Native structure of B-domainless FVIII generated with Polyview-3D (PDB code: 2R7E), with heavy (green) and light (blue) chains. Residues Asp519 and Glu665 (red) were replaced with Val in the stabilized mutant D519V/E665V

Fig. 2

a Circular dichroism of WT and D519V/E665V. Secondary structural analysis by far-UV CD of WT (dashed black line) and D519V/E665V (solid gray line). No changes were observed in the profiles characterized by the large negative 215 nm band corresponding to beta-sheet structures. b Intrinsic fluorescence of WT and D519V/E665V. Tertiary structural analysis following fluorescent excitation at 265 nm of WT (dashed black line) and D519V/E665V (solid gray line). Overlapping peak position and shape indicates similar microenvironments of aromatic amino acids

Fig. 3

Pharmacokinetics of D519V/E665V FVIII. PK profiles of D519V/E665V FVIII in HA mice after i.v. doses of 4, 10, and 40 IU/kg. Profiles for the two variants at 10 and 40 IU/kg were similar, but D519V/E665V substantially extended plasma survival at 4 IU/kg (no samples containing measurable WT were recovered following the 4 IU/kg dose)

Fig. 4

Efficacy of WT and D519V/E665V FVIII. Blood loss in a tail clip assay was measured in HA mice that received no treatment (open squares), WT FVIII (closed circles), or D519V/E665V FVIII (open circles). Normal mice (open triangles) are shown for reference. FVIII variant type was statistically significant (p < 0.005) by two-way ANOVA at the 10 IU/kg dose

Fig. 5

Immunogenicity of WT and D519V/E665V FVIII. Inhibitory titers (a) and total titers (b) were determined by a Bethesda assay and ELISA, respectively in HA mice treated intravenously for 4 weeks with either WT or D519V/E665V FVIII (n = 8/group). Mean and standard deviation are shown. No significant differences were observed