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

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 (2bF8^Tg) 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 FVIII^null 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 2bF8^Tg 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 2bF8^Tg 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 2bF8^Tg-dPlts. Infusion of these dPlts expanded Treg populations in HA mice without inducing inhibitors. Moreover, prior sensitization with 2bF8^Tg-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