Open Access
CC BY-NC-ND 4.0 · Thromb Haemost 2023; 123(08): 751-762
DOI: 10.1055/a-2090-9739
Coagulation and Fibrinolysis

Modulation of Extravascular Binding of Recombinant Factor IX Impacts the Duration of Efficacy in Mouse Models

Steffi Knoll Machado
1   CSL Behring Innovation GmbH, Marburg, Germany
,
Hendrik Peil
1   CSL Behring Innovation GmbH, Marburg, Germany
,
Timo Kraushaar
1   CSL Behring Innovation GmbH, Marburg, Germany
,
Philipp Claar
1   CSL Behring Innovation GmbH, Marburg, Germany
,
Marcel Mischnik
1   CSL Behring Innovation GmbH, Marburg, Germany
,
Holger Lind
1   CSL Behring Innovation GmbH, Marburg, Germany
,
Eva Herzog
2   CSL Behring LLC, King of Prussia, Pennsylvania, United States
,
Michael Bacher
3   Institute of Immunology—Philipps University Marburg, Marburg, Germany
,
Marc Wolfgang Nolte
1   CSL Behring Innovation GmbH, Marburg, Germany
,
Maximilian Bielohuby
1   CSL Behring Innovation GmbH, Marburg, Germany
,
Sabine Pestel*
1   CSL Behring Innovation GmbH, Marburg, Germany
,
1   CSL Behring Innovation GmbH, Marburg, Germany
› Institutsangaben

Funding The work was funded by CSL Behring Innovation GmbH.


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Abstract

Background There is an emerging concept that in addition to circulating coagulation factor IX (FIX), extravascular FIX contributes to hemostasis.

Objective Our objective was to evaluate the efficacy of extravascular FIX using animal models of tail clip bleeding and ferric chloride-induced thrombosis.

Methods Mutant rFIX proteins with described enhanced (rFIXK5R) or reduced (rFIXK5A) binding to extracellular matrix were generated and characterized using in vitro aPTT, one-stage clotting, and modified FX assays. Using hemophilia B mice, pharmacokinetic (PK) parameters and in vivo efficacy of these proteins were compared against rFIX wild-type protein (rFIXWT) in a tail clip bleeding and FeCl3-induced thrombosis model. Respective tissue disposition of FIX was evaluated using immunofluorescence.

Results In vitro characterization demonstrated comparable clotting activity of rFIX proteins. The PK profile showed that rFIXK5A displayed the highest plasma exposure compared to rFIXWT and rFIXK5R. Immunofluorescence evaluation of liver tissue showed that rFIXK5R was detectable up to 24 hours, whereas rFIXWT and rFIXK5A were detectable only up to 15 minutes. In the tail clip bleeding model, rFIXK5R displayed significant hemostatic protection against bleeding incidence for up to 72 hours postintravenous administration of 50 IU/kg, whereas the efficacy of rFIXK5A was already reduced at 24 hours. Similarly, in the mesenteric artery thrombus model, rFIXK5R and rFIXWT demonstrated prolonged efficacy compared to rFIXK5A.

Conclusion Using two different in vivo models of hemostasis and thrombosis, we demonstrate that mutated rFIX protein with enhanced binding (rFIXK5R) to extravascular space confers prolonged hemostatic efficacy in vivo despite lower plasma exposure, whereas rFIXK5A rapidly lost its efficacy despite higher plasma exposure.

Authors' Contribution

The manuscript has been read and approved for submission by all authors. S.K.M., H.P., M.M., P.C., S.P., and P.P. contributed to the design and/or implementation of the research, to the analysis of the results and to the writing of the manuscript. M.M. performed the pharmacokinetic evaluations. T.K. planned and carried out SPR experiments. E.H., M.B., M.W.N., and M.B. provided critical feedback and helped shape the final manuscript.


* Authors contributed equally.


Supplementary Material



Publikationsverlauf

Eingereicht: 08. Juli 2022

Angenommen: 11. Januar 2023

Accepted Manuscript online:
10. Mai 2023

Artikel online veröffentlicht:
08. Juni 2023

© 2023. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/)

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