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

Development of inhibitory antibodies (inhibitors) against factor VIII (FVIII) is a significant complication of protein replacement therapy in hemophilia A (HA). Platelets (Plt), 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. The object was to investigate whether FVIII-engineered Plt and related Plt-based products modulate FVIII immune responses in HA mice. FVIII-containing Plt were isolated from 2bF8 transgenic mice. FVIII-deficient mice were infused with intact FVIII-containing Plt, desialylated FVIII-containing Plt (dPlt), or acidified Plt lysates in combination with or before recombinant human FVIII (rhF8) exposure. Anti-FVIII antibody titers were determined by the Bethesda assay and enzyme-linked immunosorbent assay, and T-cell responses were analyzed by flow cytometry and proliferation assays. Co-infusion of FVIII Plt with rhF8 significantly reduced inhibitor titers compared with rhF8 alone. Acidified FVIII Plt lysates were potent, decreasing inhibitor titers by >20-fold when co-infused with rhF8. In contrast, co-infusion of dPlt with rhF8 did not suppress immune responses. However, repeated pre-sensitization with dPlt alone promoted immune tolerance to FVIII, evidenced by reduced inhibitor titers upon rhF8 immunization and attenuated CD4⁺ T-cell proliferation upon subsequent rhF8 exposure. These findings reveal a hierarchy of immune modulation, with intact Plt providing partial protection, lysates strongly suppressing immune responses, and dPlt inducing immune tolerance. FVIII-engineered Plt and Plt-derived products are potent immune modulators. These strategies offer novel and translatable approaches to both restore hemostasis and prevent or eradicate inhibitors in HA.