Factor IX oligomerization underlies reduced activity upon disruption of physiological conditions.
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.
['Simhadri|V L|VL|', 'Hamasaki-Katagiri|N|N|', 'Tseng|S C|SC|', 'Bentley|A A|AA|', 'Zichel|R|R|', 'Hershko|A Y|AY|', 'Komar|A A|AA|', 'Kimchi-Sarfaty|C|C|']
D000818:Animals D002417:Cattle D004591:Electrophoresis, Polyacrylamide Gel D005164:Factor IX D006801:Humans D006863:Hydrogen-Ion Concentration D008958:Models, Molecular D009994:Osmolar Concentration D011487:Protein Conformation D055503:Protein Multimerization D011994:Recombinant Proteins D012710:Serum Albumin, Bovine
factor IX oligomer BeneFIX® Mononine® clotting factor