Dysfunctional endogenous FIX impairs prophylaxis in a mouse hemophilia B model

Factor IX (FIX) binds to collagen IV (Col4) in the subendothelial basement membrane. In hemophilia B, this FIX-Col4 interaction reduces the plasma recovery of infused FIX and plays a role in hemostasis. Studies examining the recovery of infused BeneFix (FIX_WT) in null (cross-reactive material negative, CRM^-) hemophilia B mice suggest the concentration of Col4 readily available for binding FIX is ∼405 nM with a 95% confidence interval of 374 to 436 nM. Thus, the vascular cache of FIX bound to Col4 is several-fold the FIX level measured in plasma. In a mouse model of prophylactic therapy (testing hemostasis by saphenous vein bleeding 7 days after infusion of 150 IU/kg FIX), FIX_WT and the increased half-life FIXs Alprolix (FIX_FC) and Idelvion (FIX_Alb) produce comparable hemostatic results in CRM^- mice. In bleeding CRM^- hemophilia B mice, the times to first clot at a saphenous vein injury site after the infusions of the FIX agents are significantly different, at FIX_WT < FIX_FC < FIX_Alb Dysfunctional forms of FIX, however, circulate in the majority of patients with hemophilia B (CRM⁺). In the mouse prophylactic therapy model, none of the FIX products improves hemostasis in CRM⁺ mice expressing a dysfunctional FIX, FIX_R333Q, that nevertheless competes with infused FIX for Col4 binding and potentially other processes involving FIX. The results in this mouse model of CRM⁺ hemophilia B demonstrate that the endogenous expression of a dysfunctional FIX can deleteriously affect the hemostatic response to prophylactic therapy.