FVIII-containing platelets modulate immune responses and attenuate inhibitor development in hemophilia A mice

Abstract

Background: Development of inhibitory antibodies (inhibitors) against FVIII is a significant complication of protein replacement therapy in hemophilia A (HA). Platelets, traditionally viewed as mediators of hemostasis, also modulate immune responses through cytokine release and interactions with immune cells. Harnessing these immunomodulatory properties may provide a novel strategy to prevent or suppress inhibitor formation. Objective: To investigate whether FVIII-engineered platelets and related platelet-based products modulate FVIII immune responses in HA mice. Methods: FVIII-containing platelets were isolated from 2bF8 transgenic mice. FVIII-deficient mice were infused with intact FVIII-containing platelets, desialylated FVIII-containing platelets (dPlts), or acidified platelet lysates in combination with or before recombinant human FVIII (rhF8) exposure. Anti-FVIII antibody titers were determined by the Bethesda assay and ELISA, and T cell responses were analyzed by flow cytometry and proliferation assays. Results: Co-infusion of FVIII platelets with rhF8 significantly reduced inhibitor titers compared with rhF8 alone. Acidified FVIII platelet lysates were potent, decreasing inhibitor titers by >20-fold when co-infused with rhF8. In contrast, co-infusion of dPlts with rhF8 did not suppress immune responses. However, repeated pre-sensitization with dPlts alone promoted immune tolerance to FVIII, evidenced by reduced inhibitor titers upon rhF8 immunization and attenuated CD4ô€€€ T cell proliferation upon subsequent rhF8 exposure. These findings reveal a hierarchy of immune modulation, with intact platelets providing partial protection, lysates strongly suppressing immune responses, and dPlts inducing immune tolerance. Conclusions: FVIII-engineered platelets and platelet-derived products are potent immune modulators. These strategies offer novel and translatable approaches to both restore hemostasis and prevent or eradicate inhibitors in HA.