Comparison the activity of rat FVIII (rFVIII) and human FVIII (hFVIII). (A) Expression plasmids of hFVIII-BDD-SQ and rFVIII-BDD-SQ were transfected into 293 T cells by Polyjet, and the FVIII activity was detected 48 h post-transfection by the activated partial thromboplastin time (aPTT) assay (n = 3, ***p =0.0001). (B) Expression plasmids hHC, hLC, rHC, and rLC were co-transfected into 293 T cells with combinations of hHC + hLC and rHC +rLC with the ratio of HC:LC at 1:1. FVIII activity was detected 48 h post-transfection by the aPTT assay (n = 3, ****p < 0.0001). The serial-diluted pooled normal human plasma (NHP) was employed as the standard. (C) Comparison of the activities of rFVIII and hFVIII by single-chain and dual-chain strategy (n =3, ***p =0.0001, ****p < 0.0001).