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Effects of Replacement of Factor VIII Amino Acids Asp519 and Glu665 with Val on Plasma Survival and Efficacy In Vivo

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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.

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ACKNOWLEDGMENTS

This work was supported by grants from the National Institutes of Health (R01 HL-70227 to SVB and RO1-HL38199 to PJF). MPK received pre-doctoral fellowships from Pfizer Inc. and the American Foundation for Pharmaceutical Education. Portions of the D519V/E665V protein used were supplied by Dr. John E. Murphy of Bayer Healthcare.

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Authors and Affiliations

  1. Department of Pharmaceutical Sciences, University at Buffalo, The State University of New York, 359 Kapoor Hall, Buffalo, New York, 14214, USA

    Matthew P. Kosloski, Krithika A. Shetty & Sathy V. Balu-Iyer

  2. Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York, USA

    Hironao Wakabayashi & Philip J. Fay

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  1. Matthew P. Kosloski
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  2. Krithika A. Shetty
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  3. Hironao Wakabayashi
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  5. Sathy V. Balu-Iyer
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Correspondence to Sathy V. Balu-Iyer.

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Kosloski, M.P., Shetty, K.A., Wakabayashi, H. et al. Effects of Replacement of Factor VIII Amino Acids Asp519 and Glu665 with Val on Plasma Survival and Efficacy In Vivo . AAPS J 16, 1038–1045 (2014). https://doi.org/10.1208/s12248-014-9627-2

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