Summary

The factors responsible for the removal of injected factor IX(fIX) from the blood of individuals with haemophilia B are onlypartly understood, and may include binding to endothelial orsubendothelial sites, passive extravasation related to size orcharge, or interactions requiring fIX activation. To investigatethese issues, we have produced and characterised recombinantfIX proteins with amino acid changes: δ 155–177, an internaldeletion which removes most of the activation peptide while retainingthe activation cleavage sites; S365A, which inactivates theserine protease activity of fIXa; and K5A, previously shown toeliminate fIX binding of endothelial/subendothelial collagen IV.All proteins were expressed in stably transfected HEK 293 cells,purified by immunoaffinity chromatography, and compared tothe wild type HEK 293-derived protein (fIX (WT)). Mutant fIX proteins K5A and ? 155–177 exhibited 72 and 202% of the specificactivity of fIX (WT), respectively; S365A was without activity.Following intravenous injection in haemophilia B (fIX knockout)mice, recoveries did not differ for fIX (WT) and δ 155–177,but were higher for K5A and S365A.The terminal catabolic halflifeof δ155–177, alone among the mutants, was increased, by45% versus fIX (WT). Nine hours post-injection, the observedareas under the clearance curve (AUCs) of δ 155–177 and K5,but not S365A, were elevated 2-fold. δ 155–177 was equally effectiveas fIX (WT) in reducing blood loss following tail veintransection in haemophilia B mice.Our results suggest that deletionof the multiple sites of fIX post-translational modificationfound within the activation peptide eliminated important fIXclearance motifs.