Engineered platelets as immune modulators: A novel strategy to prevent FVIII inhibitor formation in hemophilia A

Abstract Background: The development of inhibitory antibodies against factor VIII (FVIII) remains a major complication of replacement therapy in patients with hemophilia A (HA). Our previous work demonstrated that platelet-targeted FVIII gene therapy can induce immune tolerance. However, transfusion of FVIII-containing platelets alone neither triggers inhibitor formation nor leads to consistent immune tolerance induction. Objective: This study investigates the immunomodulatory potential of FVIII-containing platelets and their derivatives in shaping immune responses to FVIII in HA mice. Methods: Human B-domain deleted FVIII was expressed in platelets of 2bF8 transgenic (2bF8Tg) mice under the platelet-specific αIIb promoter. Platelets were harvested and used intact, enzymatically desialylated (dPlts), or lysed under acidic conditions. These products were infused into FVIIInull mice either alone or with recombinant human FVIII (rhFVIII), followed by immunization. Inhibitor titers were measured using the Bethesda assay. Regulatory T (Treg) induction and CD4⁺ T-cell proliferation were also evaluated. Results: Co-infusion of 2bF8Tg platelets with rhFVIII significantly reduced FVIII inhibitor titers by 8-fold compared to rhFVIII alone (32 ± 36 vs. 253 ± 125 BU/mL). Co-administration of acidified 2bF8Tg platelet lysates, which efficiently induce functional Treg cells, further decreased inhibitor titers by 21-fold. Desialylation of GPIbα removed 98% of terminal sialic acids, producing apoptotic-like 2bF8Tg-dPlts. Infusion of these dPlts expanded Treg populations in HA mice without inducing inhibitors. Moreover, prior sensitization with 2bF8Tg-dPlts before rhFVIII immunization markedly lowered both the incidence and titers of inhibitors. Splenic CD4⁺ T cells from dPlt-sensitized, rhFVIII-immunized mice failed to proliferate upon in vitro rhFVIII restimulation, whereas control animals showed a 5-fold increase in dividing CD4⁺ T cells under the same conditions. Conclusion: FVIII-containing platelets—especially in the form of desialylated platelets or acidified lysates—effectively suppress inhibitor formation and promote immune tolerance. This novel immune modulatory strategy may represent a translational approach to prevent or treat FVIII inhibitor development in hemophilia A. Keywords: Hemophilia A, Immune tolerance, Factor VIII, Platelets, Inhibitors