Summary
The factors responsible for the removal of injected factor IX (fIX) from the blood
of individuals with haemophilia B are only partly understood, and may include binding
to endothelial or subendothelial sites, passive extravasation related to size or charge,
or interactions requiring fIX activation. To investigate these issues, we have produced
and characterised recombinant fIX proteins with amino acid changes: Δ155–177, an internal
deletion which removes most of the activation peptide while retaining the activation
cleavage sites; S365A, which inactivates the serine protease activity of fIXa; and
K5A, previously shown to eliminate fIX binding of endothelial/subendothelial collagen
IV. All proteins were expressed in stably transfected HEK 293 cells, purified by immunoaffinity
chromatography, and compared to the wild type HEK 293-derived protein (fIX (WT)).
Mutant fIX proteins K5A and Δ155–177 exhibited 72 and 202% of the specific activity
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 Δ155–177,
but were higher for K5A and S365A. The terminal catabolic halflife of Δ155–177, alone
among the mutants, was increased, by 45% versus fIX (WT). Nine hours post-injection,
the observed areas under the clearance curve (AUCs) of Δ155–177 and K5, but not S365A,
were elevated 2-fold. Δ155–177 was equally effective as fIX (WT) in reducing blood
loss following tail vein transection in haemophilia B mice. Our results suggest that
deletion of the multiple sites of fIX post-translational modification found within
the activation peptide eliminated important fIX clearance motifs.
Keywords
Factor IX - recombinant proteins - clearance - recovery - posttranslational Modifications