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Thromb Haemost 2005; 94(06): 1138-1147
DOI: 10.1160/TH04-03-0201
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

An important role for the activation peptide domain in controlling factor IX levels in the blood of haemophilia B mice

Megan E. Begbie
1   Canadian Blood Services Research and Development
,
Asif Mamdani
1   Canadian Blood Services Research and Development
,
Sharon Gataiance
2   Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada
,
Louise J. Eltringham-Smith
2   Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada
,
Varsha Bhakta
1   Canadian Blood Services Research and Development
,
Gonzalo Hortelano
1   Canadian Blood Services Research and Development
2   Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada
,
William P. Sheffield
1   Canadian Blood Services Research and Development
2   Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada
› Author AffiliationsFinancial support: This work was supported by Canadian Blood Services Research and Development Grants XH00009 and XH00034 to WPS and GH.
Further Information

Publication History

Received 31 March 2005

Accepted after revision 13 October 2005

Publication Date:
07 December 2017 (online)

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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
 

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