Activated protein C cleavage of FVIII-WT and FVIII-QQ. (A) Western blot analysis of 10 nM of FVIII-WT and FVIII-QQ after 30-minute incubation with 6 nM of APC, 20 µM of PCPS, and 6 nM of hirudin. FVIII fragments were visualized with an anti-A2 antibody (GMA-012). (B) Western blot analysis of 10 nM of FVIII-WT, FVIII-QQ, and FVIII-R372Q after 30-minute incubation with 6 nM of APC, 20 µM of PCPS, and 6 nM of hirudin; 30 ng of purified protein was loaded on the gel, and FVIII fragments were visualized with GMA-012. (C) Inactivation of 10 nM of FVIII-WT (blue squares) and FVIII-QQ (red triangles) by 6 nM of APC in the presence of 20 µM of PCPS and 6 nM of hirudin over time in purified intrinsic Xase assay compared with inactivation of 10 nM of FVIII-WT (open blue squares) and FVIII-QQ (open red triangles) by 6 nM of APC with 100 nM of PS in the presence of 20 µM of PCPS and 6 nM of hirudin. Initial velocities of FXa generation throughout incubation were compared with 0-minute time point to determine residual FVIII activity. Representative plots of duplicate experiments are plotted. Data were fit to exponential decay or linear regression (FVIII-QQ with APC only). (D) Western blot analysis of 10 nM of FVIII-WT, FVIII-QQ, and FVIII-R372Q, 20 µM of PCPS and 6 nM of hirudin after 2- and 10-minute incubations with either 100 nM of PS or 6 nM of APC or 6 nM of APC and 100 nM of PS; 20 ng of purified protein was loaded on the gel, and FVIII fragments were visualized with GMA-012. FVIII-R372Q is resistant to cleavage at Arg372. SC, single chain.