Targeting Factor Xa Expression in Platelets As a Potential Gene Therapy Strategy for Hemophilia with Inhibitors

The development of inhibitory antibodies (inhibitors) is a critical issue in the replacement therapy of hemophilia. The effective therapy of hemophilia with inhibitors is still an urgent demand. In the coagulation cascade, factor X (FX) is the common point of both intrinsic and extrinsic pathway. FX is activated to factor Xa (FXa) by either factor IXa (FIXa)/factor VIIIa (FVIIIa) or factor VIIa (FVIIa)/tissue factor (TF) complexes. FXa forms a prothrombinase complex with factor Va (FVa), and activates prothrombin to thrombin. Since FXa activates prothrombin very efficiently and this process bypasses FIX and FVIII, it has been tried as a bypassing agent in the treatment of hemophilia with inhibitors for a long time. It was found that infusion of FXa in the plasma could alleviate the bleeding phenotype of hemophilia animals. However, studies disclosed that the half-life of FXa is too short to be an effective bypassing agent. Furthermore, improved plasma FXa concentration also increases the risk of disseminated intravascular coagulation (DIC). A protein ectopically expressed in platelets can be stored in platelets, and it can be released during the activation of platelets. Here, we hypothesize that if FXa can be targeted expressed and stored in platelets, the half-life of FXa could be increased, while at the same time the risk of DIC could be largely limited. When platelets are activated at the injury site, the stored FXa can be released and activate prothrombin on the surface of platelets, so as to initiate the blood coagulation. Thus, this platelet-stored FXa could be used in the treatment of hemophilia A (HA) and hemophilia B (HB) with inhibitors. To prove our hypothesis, we constructed a platelet-targeted FXa expression cassette (2bFXa) by putting FXa under the control of GPαIIb promoter. Then we made the corresponding lentiviral vector with this 2bFXa cassette. The 2bFXa lentiviral vector was used to transduce hematopoietic stem cells (HSCs) collected from HA and HB mice, the modified HSCs were then transplanted back into the lethally irradiated corresponding HA and HB recipient mice. We collected plasma and platelets from the recipients. FXa was successfully detected in the platelets but not in the plasma of both HA and HB recipients by either an FXa activity assay or FX ELISA. The level of platelet-stored FXa remains constant throughout the experiment. Platelet release assay indicates almost 85.7% stored FXa can be released after the stimulation of platelets. The storage of FXa in platelets was also demonstrated by confocal microscopy and flow cytometry analysis. We then carried out a tail clip survival test to evaluate the phenotypic correction of recipients, all HA and HB recipient mice survived the tail clip challenge, demonstrating the platelet-stored FXa is biologically functional. We also tested the same strategy on HA mice with inhibitors and acquired the same results, indicating that the strategy is effective for HA with inhibitors. During the whole period of experiment, the recipient mice were all in good health. We also kept on monitoring the complete blood cell counts for 48 weeks and the cell numbers remained in normal range, indicating that the blood cells were successfully recovered in the recipients. Taken together, our proof of principle studies suggest that targeting FXa expression in platelets could facilitate the storage of FXa in platelets and would not cause obvious FXa leakage. The platelet-stored FXa is active and can be released with the activation of platelets and corrects the bleeding phenotype of HA and HB mice. This new gene therapy method could eliminate the shortage of FXa and be functional in the treatment of hemophilia with inhibitors.