Background: One hemophilia treatment concept focuses on rebalancing coagulation and anticoagulation to restore normal blood clotting. Targeting the coagulation regulator, protein S (PS), in hemophilia shows promise to increase the generation of thrombin, a critical enzyme in the clotting process.

Objectives: This study aimed to: (1) assess whether inhibiting PS increases thrombin generation (TG) in plasma from individuals with hemophilia A (HA) and B (HB); and (2) develop a hepatocyte-targeted PS small interfering RNA (siRNA) therapy using N-acetylgalactosamine conjugation to restore hemostasis in hemophilia without increasing thromboembolic risks.

Methods: We assessed TG in plasma from patients with HA and HB. To target the liver specifically, we developed a PS-siRNA conjugated with N-acetylgalactosamine. This approach ensures that PS levels remain adequate in other cells, thereby minimizing the risk of thrombosis. Additionally, we evaluated the therapeutic potential of PS-siRNA in preclinical models.

Results: Inhibiting PS with a polyclonal antibody in plasma resulted in a 3- to 5-fold increase in TG in HA and a 4- to 9-fold increase in HB plasma, with a 70% reduction in plasma PS. In preclinical models, subcutaneous PS-siRNA therapy in HA mice and nonhuman primates successfully lowered PS levels and improved clot formation. It also prevented bleeding in both saphenous vein puncture and knee injury models in HA mice. Notably, it enhanced clotting without triggering widespread clot formation.

Conclusion: Reducing PS levels enhances TG in hemophilia models, and PS-siRNA therapy shows promise in improving hemostasis. This approach warrants further clinical investigation as a potential treatment for hemophilia.