A mutated factor X activatable by thrombin corrects bleedings in vivo in a rabbit model of antibody-induced hemophilia A

Abstract

Rendering coagulation factor X sensitive to thrombin was proposed as a strategy to bypass the need for factor VIII. In this study, this nonreplacement strategy was evaluated in vitro and in vivo for its ability to correct factor VIII but also factor IX, X and XI deficiencies. A novel modified factor X, named actiten, was generated and produced in the HEK293F cell line. The molecule possesses the required post-translational modifications, partially maintaining its ability to be activated by RVV-X, factor VIIa/tissue factor, and factor VIIIa/factor IXa and acquires the ability to be activated by thrombin. The potency of the molecule was evaluated in plasma samples with deficiencies of the respective factors and in plasma samples from patients with hemophilia A, some of which contained inhibitors. Actiten dose-dependently corrected all the deficient plasmas that were assayed. It was able to normalize the thrombin generation at 20 μg/mL although the lag time was increased. It was then assayed in a rabbit antibody-induced model of hemophilia A in which, in contrast to recombinant wild-type factor X, it normalized the bleeding time and the loss of hemoglobin. No sign of thrombogenicity was observed and the generation of activated factor X was controlled by the anticoagulation pathway in all the coagulation assays performed. These data indicate that actiten may be considered as a possible non-replacement factor to treat hemophilia, with the advantage of being a zymogen that corrects bleeding only when needed.