Hyperfunctional FIX-Padua expressed and stored in platelets was fully γ-carboxylated. Intracellular location of codon-optimized FIX-Padua in 2bCoF9R338L-transduced platelets of FIX-primed FIX^null mice was examined by immunofluorescent confocal microscopy. The percentage of platelets that were positive for hFIX protein was determined by flow cytometry. To evaluate whether ectopic expression of codon-optimized FIX-Padua in platelets is fully γ-carboxylated, platelet lysates from 2bCoF9R338L-transduced recipients at various time points with varying FIX:Ag levels (0.69 to 15.62 mU/10⁸ platelets) were treated with or without 10% BaSO₄. The remaining FIX in supernatants after BaSO₄ precipitation was determined by ELISA. Various amounts of rhFIX were treated with BaSO₄ as controls in parallel. (A-I) Immunofluorescent confocal microscopy. hFIX was stained with a primary goat anti-hFIX antibody and a secondary donkey anti-goat IgG conjugated with Alexa-488 (in green). Endogenous VWF protein was stained with a rabbit anti-human VWF antibody that crosses with mouse VWF and a secondary donkey anti-rabbit IgG conjugated with Alexa-594 (in red). Scale bars, 10 μm. (J) Representative dot plots from flow cytometry analysis of hFIX⁺ platelets. hFIX was stained with a primary goat anti-hFIX antibody and a secondary donkey anti-goat IgG conjugated with Alexa-488. Platelets were identified using anti-GPIIb antibody directly conjugated with phycoerythrin. (K) Summary data from flow cytometry analysis of hFIX⁺ platelets in 2bCoF9R338L-transduced rhFIX-primed FIX^null recipients at weeks 20 to 28 after transplantation. (L) Absolute levels of FIX expression in 2bCoF9R338L-transduced platelets in rhFIX-primed recipients. 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. (M) Platelet-FIX:Ag in the supernatants of platelet lysates from 2bCoF9R338L- or 2bF9-transduced recipients after BaSO₄ precipitation. Platelet-FIX:Ag levels in platelet lysates without BaSO₄ treatment were defined as 100%. (N) FIX:Ag in the supernatants of rhFIX after BaSO₄ precipitation. FIX:Ag levels in supernatants of rhFIX without BaSO₄ treatment were defined as 100%. Data are presented as mean ± SD.