Factor Xa Variants as Novel Bypass Agents for the Treatment of Hemophilia in Murine Models

Hemophilia A and B are bleeding disorders caused by mutations in the genes encoding FVIII or FIX. Treatment involves protein replacement; however in some patients antibodies develop that neutralize FVIII or FIX. In these inhibitor patients, hemostasis can be achieved with activated prothrombin complex concentrates or recombinant FVIIa which bypass the intrinsic pathway generating thrombin to allow coagulation to occur. In principle, infusion of FXa should bypass deficiencies in the intrinsic pathway. However, this could trigger excessive coagulation as FXa can activate several clotting factors. Furthermore, FXa is rapidly inactivated by plasma inhibitors resulting in a short half-life ( in vivo experiments with FXa-I16L using age-matched male HB mice on the Balb/c strain to examine the effects on systemic coagulation as well as efficacy using three injury models. Using a modified aPTT clotting assay, hemostatically normal control (wt) animals had a clot time of 27 ± 3 sec (n = 4) while in untreated HB animals it was 69 ± 4 sec (n = 10). Administration of FXa-I16L (450 μg/kg) via tail vein almost completely corrected the aPTT 10 min post infusion (n = 5; 31 ± 5 sec) whereas PBS was without effect (n = 5; 63 ± 3 sec). The aPTT was shortened for more than 2 hours (50 ± 3 sec) and returned close to the starting value after 24 hr (62 ± 3 sec). In these experiments, infusion of the protein was well tolerated as all animals survived. Furthermore, using this high dose of FXa-I16L (450 μg/kg) platelet levels only marginally decreased over the course of the experiment while there was a transient increase in TAT levels at 10 min (68 ± 33 ng/mL versus 16 ± 8 ng/mL in HB controls) which returned back to baseline after 60 min (18 ± 7 ng/mL). Next we tested whether the improvement of the clotting times was associated with in vivo hemostatic performance. We used a tail clip assay in which blood loss was measured during a 10 min period after sectioning the distal part of the tail. Infusion of FXa-I16L in HB mice (n = 7; 450 μg/kg) significantly reduced total blood loss compared to PBS (n = 7) treated animals (p in vivo test of thrombosis, the FeCl 3 -carotid artery injury model, normal mice (n = 3) presented with vessel occlusion and interruption of blood flow within 9–12 min postvessel injury with 15% FeCl 3 while no evidence of clot formation was found for HB controls (n = 5). After injury of HB mice with FeCl 3 , followed by a 10 min observation period with stable blood flow, infusion of FXa-I16L (450 μg/kg) resulted in vessel occlusion and interruption of blood flow within 5–6 min in three out of five mice. In the last in vivo injury model, thrombus formation was assessed following a laser injury to a small vessel (arteriole) in the cremaster muscle by monitoring platelet accumulation at the site of injury. Untreated HB mice (n = 13, 30 sites) failed to form clots, while all HB animals treated with FXa-I16L (n= 10, >70 sites; 10, 30, or 90 μg/kg) formed a stable thrombus following injury that was comparable in size to wt mice (n = 3, 17 sites). Taken together our data show that FXa-I16L improves the hemophilic phenotype in HB mice following a series of hemostatic challenges and can restore thrombus formation upon injury at both micro and macrocirculation levels. Thus zymogen-like variants of FXa have properties that indicate their ability to serve as superior therapeutic procoagulants for bypassing deficiencies upstream of the common pathway.