title: Factor IX oligomerization underlies reduced activity upon disruption of physiological conditions.
abstract: 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.
authors.affiliation: Laboratory of Hemostasis, Division of Hematology, Center for Biologics Evaluation & Research, US FDA, Bethesda, MD, USA.
authors.forename: V L
authors.initials: VL
authors.lastname: Simhadri
authors.forename: N
authors.initials: N
authors.lastname: Hamasaki-Katagiri
authors.forename: S C
authors.initials: SC
authors.lastname: Tseng
authors.forename: A A
authors.initials: AA
authors.lastname: Bentley
authors.forename: R
authors.initials: R
authors.lastname: Zichel
authors.forename: A Y
authors.initials: AY
authors.lastname: Hershko
authors.forename: A A
authors.initials: AA
authors.lastname: Komar
authors.forename: C
authors.initials: C
authors.lastname: Kimchi-Sarfaty
mesh_terms: D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D005164:Factor IX / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009994:Osmolar Concentration; D011487:Protein Conformation; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D012710:Serum Albumin, Bovine; D005164:Factor IX
keywords: BeneFIX®; Mononine®; clotting factor; factor IX; oligomer