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Mutated factor X to correct hemophilias
amount of actiten generated was greater than that in nor- mal plasma (endogenous thrombin potential: 1819±202 vs. 1510±216 nM.min, respectively) although this was not a statistically significant difference. There was a slight increase in the lag time when actiten was used (10.2±1.1 min for actiten vs. 7.2±1.7 min for normal plasma; P<0.01). Actiten was then evaluated in hemophilia A plasma with or without a high titer of inhibitors (292 BU/mL) (Figure 6B, C). In this latter plasma, the addition of actiten, unlike that of FVIII, could restore thrombin generation in a dose- dependent manner. At a dose of 20 μg/mL, a total amount of 689±24 nM.min of thrombin was generated, providing significant correction of the plasma.
Actiten was next evaluated in FIX-deficient plasma, spiked or not with anti-FIX polyclonal antibodies, in which it behaved similarly as in the hemophilia A plasma or FVIII-deficient plasma (Figure 6D, E; Table 1). The potency of the molecule was then assessed in the absence of FX or FXI (Figure 6F, G). The behavior of the molecule was similar to that previously observed, with the amount
A
B
Figure 5. Evaluation of the stability of actiten in factor-deficient plasma sam- ples. (A) Evaluation in factor X (FX)-deficient plasma. Plasma-derived FX (pdFX) (10 μg/mL), pdFX (10 μg/mL) plus plasma-derived activated factor X (pdFXa) (0.1 μg/mL) and actiten (20 μg/mL) were incubated in FX-deficient plasma re- calcified by 7.5 mM CaCl2. Plasma clotting times were recorded manually (n=2- 4). (B) Evaluation in factor VIII (FVIII)-deficient plasma. FVIII (1 U/mL), pdFXa (0.1 μg/mL) and actiten (20 μg/mL) were incubated in FVIII-deficient plasma re-cal- cified by 7.5 mM CaCl2. Plasma clotting times were recorded manually (n=3).
of thrombin generated (1010±73 nM.min and 1285±119 nM.min, respectively) being close to that in normal plas- ma (1510±216 nM.min) but again with an increased lag time compared to that of normal plasma (12.1±1.8 min and 18.6±8.7 min vs. 7.2±1.7 min; P<0.001 and P<0.001, respectively).
It should be stressed that in all these thrombin genera- tion assays, the thrombin generated was efficiently con- trolled by the anticoagulant system and that no runaway of coagulation was detected at up to 120 μg/mL actiten. These data indicate that even following clotting induction, actiten did not overreact or escape the anticoagulation pathway.
In vivo evaluation of actiten potency
A rabbit model of hemophilia A was established to eval-
uate the potency of actiten in vivo. A cocktail of two anti- FVIII monoclonal antibodies severely impaired thrombin generation in rabbit plasma (Figure 7A). An in vivo assay was designed and is schematically represented in Figure 7B. The anti-FVIII antibodies or NaCl were infused into rabbits and the bleeding times were recorded. A statisti- cally significant difference was observed between rabbits administered NaCl or anti-FVIII (405±44 s vs. 2760.5±348 s; P<0.01), which validated the animal model (Figure 7C). The experiments were repeated but with the infusion of actiten, recombinant wild-type FX (recFX-WT) or NaCl following the infusion of anti-FVIII antibodies. RecFX-WT was produced from HEK293F cells and purified in the same way as actiten (Online Supplementary Figure S3). As a positive control, FVIIa (500 μg/kg) was shown to correct bleeding in this model (746±290 s; P<0.01) following its infusion. The bleeding times in anti-FVIII-treated rabbits (2861±2 s) were significantly longer than those in actiten- treated rabbits (P<0.05); bleeding times of the latter were similar to those in untreated wild-type rabbits (387±111 s vs. 347±51 s, respectively; P=ns) (Figure 7D). In contrast, the infusion of recFX-WT did not diminish the bleeding time with regard to those of anti-FVIII-treated rabbits (2847±668 s vs. 2861±2 s; P=ns) confirming the absence of contaminating FXa in the preparation and the specificity of actiten (Online Supplementary Figure S3). The hemoglo- bin losses were also compared and identical efficacy pro- files were found: rabbits treated with actiten lost the equivalent of 4±2 mg/dL hemoglobin; in contrast, the anti- FVIII-treated rabbits lost the equivalent of 268±169 mg/dL (P<0.05) (Figure 7E). Recombinant FVIIa diminished the loss of hemoglobin (61±38 mg/mL) in contrast to recFX- WT (420±222 mg/mL, P=ns). Markers of exaggerated coagulation (thrombin/antithrombin complexes, pro- thrombin fragment 1 and 2 and fibrinogen D-dimers) were also measured but none of them was significantly increased (Online Supplementary Table S1). These results demonstrate that actiten was able to restore coagulation in vivo, in a model of antibody-induced hemophilia A.
Discussion
Actiten as a NRF strategy has some specific features that are illustrated by the data presented here. First, actiten was able to normalize the amount of thrombin generated in vitro as well as in vivo during bleeding in a model of anti- body-induced hemophilia A. In contrast, most of the other NRF strategies partially restore coagulation but some
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