Identification of Key Coagulation Activity Determining Elements in Canine Factor VIII

Jenni Firrman; Qizhao Wang; Wenman Wu; Biao Dong; Wenjing Cao; Andrea Rossi Moore; Sean Roberts; Barbara A Konkle; Carol Miao; LinShu Liu; Dong Li; Weidong Xiao

doi:10.1016/j.omtm.2019.12.019

Identification of Key Coagulation Activity Determining Elements in Canine Factor VIII

Mol Ther Methods Clin Dev. 2020 Jan 15:17:328-336. doi: 10.1016/j.omtm.2019.12.019. eCollection 2020 Jun 12.

Authors

Jenni Firrman^{1

2}, Qizhao Wang³, Wenman Wu³, Biao Dong³, Wenjing Cao³, Andrea Rossi Moore³, Sean Roberts³, Barbara A Konkle^{4

5}, Carol Miao⁵, LinShu Liu², Dong Li⁶, Weidong Xiao^{1

2

3}

Affiliations

¹ Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, PA, USA.
² Dairy and Functional Foods Research Unit, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA, USA.
³ Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, USA.
⁴ Bloodworks Northwest, Seattle, WA, USA.
⁵ Department of Medicine, University of Washington, Seattle, WA, USA.
⁶ Shanghai Tongji Hospital, Tongji University, Shanghai, P.R. China.

Abstract

It is well known that canine factor VIII (cFVIII) has a higher specific activity than does human FVIII (hFVIII), and it has been previously demonstrated that cFVIII light chain is able to enhance hFVIII activity. The goal of this study was to first determine which amino acids in cFVIII light chain were responsible for enhancing hFVIII activity, and second to use these amino acids to develop a hFVIII variant with enhanced functional activity. We systemically screened segments of cFVIII light chain by testing an array of human-canine light chain hybrids and found that canine amino acids 1857-2147 were key to this enhancement. Each canine amino acid within this span was screened individually using a negative selection method, which led to the identification of 12 aa (JF12) in the FVIII light chain that could enhance activity. Substitution of the corresponding 12 aa into hFVIII (hFVIIIJF12BDD) elevated the specific activity profile in vitro. Furthermore, hFVIIIJF12BDD expressed an in vivo-displayed increased coagulation activity compared to wild-type, while maintaining normal secretion efficiency. In conclusion, we identified the amino acids in cFVIII that are the key determinants for higher specific activity and may be the basis for future development of therapeutic treatments for hemophilia A.

Keywords: FVIII protein; coagulation factor VIII; factor VIII; genetic engineering; hemophilia A.

Abstract

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