Hyperfunctional FIX expressed in platelets induced immune tolerance in FIX^nullmice that received 2bCoF9R338L-transduced HSCs (the noninhibitor model). (A) Flow cytometry analysis of platelets that were positive for hFIX protein expression in 2bCoF9R338L-transduced recipients. Platelets isolated from transduced recipients were permeabilized, stained with anti-hFIX–specific antibody (goat anti-hFIX primary antibody and donkey anti-goat IgG conjugated with Alexa-488 as a secondary antibody) and analyzed by flow cytometry. Platelets from FIX^null mice were used as a control in parallel. Shown are representative flow dot plots from recipients at 31 weeks after HSCT. (B) Summary data from flow cytometry analysis of hFIX⁺ platelets in 2bCoF9R338L-transduced FIX^null recipients. (C) Absolute levels of FIX expression in 2bCoF9R338L-transduced platelets. The absolute levels of FIX:C and FIX:Ag in 2bCoF9R338L-transduced platelets were calculated according to the platelet-FIX levels and the percentage of hFIX⁺ platelets in each recipient. (D) The incidence of anti-FIX inhibitor development in 2bCoF9R338L-transduced recipients after being challenged with rhFIX together with adjuvant. Eight months after transplantation, recipients were challenged with rhFIX at a dose of 200 U/kg in the presence of IFA twice with a 3-week interval. Two weeks after the second immunization, blood samples were collected, and plasma was isolated for a Bethesda assay to determine the inhibitor titers. FIX^null mice were immunized under the same protocol. (E) Anti-FIX inhibitor titers in 2bCoF9R338L-transduced recipients before and after challenge with rhFIX plus IFA. Data are presented as mean ± SD. **P < .01; ***P < .001. BU/mL, Bethesda units per mL; GPIIba, glycoprotein IIba; Post-Imm, post-immunization; Pre-Imm, pre-immunization.