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

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

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

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.

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.

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.