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

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 (rFIX_K5R) or reduced (rFIX_K5A) 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 (rFIX_WT) 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 rFIX_K5A displayed the highest plasma exposure compared to rFIX_WT and rFIX_K5R. Immunofluorescence evaluation of liver tissue showed that rFIX_K5R was detectable up to 24 hours, whereas rFIX_WT and rFIX_K5A were detectable only up to 15 minutes. In the tail clip bleeding model, rFIX_K5R displayed significant hemostatic protection against bleeding incidence for up to 72 hours postintravenous administration of 50 IU/kg, whereas the efficacy of rFIX_K5A was already reduced at 24 hours. Similarly, in the mesenteric artery thrombus model, rFIX_K5R and rFIX_WT demonstrated prolonged efficacy compared to rFIX_K5A.

Conclusion: Using two different in vivo models of hemostasis and thrombosis, we demonstrate that mutated rFIX protein with enhanced binding (rFIX_K5R) to extravascular space confers prolonged hemostatic efficacy in vivo despite lower plasma exposure, whereas rFIX_K5A rapidly lost its efficacy despite higher plasma exposure.