Factor IX oligomerization underlies reduced activity upon disruption of physiological conditions

Authors

  • V. L. Simhadri,

    1. Laboratory of Hemostasis, Division of Hematology, Center for Biologics Evaluation & Research, US FDA, Bethesda, MD, USA
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    • Equal contribution.
  • N. Hamasaki-Katagiri,

    1. Laboratory of Hemostasis, Division of Hematology, Center for Biologics Evaluation & Research, US FDA, Bethesda, MD, USA
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    • Equal contribution.
  • S. C. Tseng,

    1. Laboratory of Hemostasis, Division of Hematology, Center for Biologics Evaluation & Research, US FDA, Bethesda, MD, USA
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    • Equal contribution.
  • A. A. Bentley,

    1. Department of Biological, Geological & Environmental Sciences, Center for Gene Regulation in Health and Disease, Cleveland State University, Cleveland, OH, USA
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  • R. Zichel,

    1. Laboratory of Hemostasis, Division of Hematology, Center for Biologics Evaluation & Research, US FDA, Bethesda, MD, USA
    Current affiliation:
    1. Department of Biotechnology, Israel Institute for Biological Research, Ness-Ziona, Israel
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  • A. Y. Hershko,

    1. Department of Medicine, Meir Medical Center, Kfar Saba, Israel
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  • A. A. Komar,

    Corresponding author
    1. Department of Biological, Geological & Environmental Sciences, Center for Gene Regulation in Health and Disease, Cleveland State University, Cleveland, OH, USA
    • Correspondence: Chava Kimchi-Sarfaty, The Laboratory of Hemostasis, Division of Hematology, Center for Biologics Evaluation & Research, US FDA, Bethesda, MD 20892, USA.

      Tel.: 301 827 0039; fax: 301 402 2780;

      e-mail: chava.kimchi-sarfaty@fda.hhs.gov; and/or Anton A. Komar, The Center for Gene Regulation in Health and Disease, Department of Biological, Geological & Environmental Sciences, Cleveland State University, Cleveland, OH 44115, USA.

      Tel.: 216 687 2516; fax: 216 687 6972;

      e-mail: a.komar@csuohio.edu

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  • C. Kimchi-Sarfaty

    Corresponding author
    1. Laboratory of Hemostasis, Division of Hematology, Center for Biologics Evaluation & Research, US FDA, Bethesda, MD, USA
    • Correspondence: Chava Kimchi-Sarfaty, The Laboratory of Hemostasis, Division of Hematology, Center for Biologics Evaluation & Research, US FDA, Bethesda, MD 20892, USA.

      Tel.: 301 827 0039; fax: 301 402 2780;

      e-mail: chava.kimchi-sarfaty@fda.hhs.gov; and/or Anton A. Komar, The Center for Gene Regulation in Health and Disease, Department of Biological, Geological & Environmental Sciences, Cleveland State University, Cleveland, OH 44115, USA.

      Tel.: 216 687 2516; fax: 216 687 6972;

      e-mail: a.komar@csuohio.edu

    Search for more papers by this author

Summary

Coagulation factor IX (FIX) is a serine protease that plays a pivotal role in the blood coagulation cascade. FIX deficiency leads to a blood clotting disorder known as haemophilia B. FIX, synthesized as a prepro-peptide of 461 amino acids, is processed and secreted into plasma. The protein undergoes numerous modifications, including, but not limited to glycosylation, γ-carboxylation and disulphide bond formation. Upon processing and limited proteolysis, the protein is converted into an active protease. Under physiological conditions, the FIX zymogen is a monomer. The purpose of this work was to analyse the conditions that may affect FIX monomeric state and promote and/or reduce oligomerization. Using native gel electrophoresis and size exclusion chromatography, we found that under decreased pH and ionic strength conditions, the FIX zymogen can oligomerize, resulting in the formation of higher molecular weight species, with a concomitant reduction in specific activity. Similarly, FIX oligomers formed readily with low bovine serum albumin (BSA) concentrations; however, increased BSA concentrations impeded FIX oligomerization. We hypothesize that normal blood physiological conditions are critical for maintaining active FIX monomers. Under conditions of stress associated with acidosis, electrolyte imbalance and low albumin levels, FIX oligomerization is expected to take place thus leading to compromised activity. Furthermore, albumin, which is commonly used as a drug stabilizer, may enhance the efficacy of FIX biological drugs by reducing oligomerization.

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