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 extravascu-lar space confers prolonged hemostatic efficacy in vivo despite lower plasma exposure, whereas rFIX(K5A) rapidly lost its efficacy despite higher plasma exposure.