Characterization of inactive fVIII constructs. (A) SDS-PAGE of wt fVIII, R372A/R1689A fVIII, and V634M fVIII with and without exposure to thrombin (factor IIa). MW STDS indicates molecular weight standards; SC, single chain fVIII; HC, fVIII heavy chain (A1-A2 domains); LC, fVIII light chain; LC_IIa, thrombin-cleaved light chain; A1, A1 domain; and A2, A2 domain. (B) Thrombin generation in fVIII-deficient plasma reconstituted with 2 μg/mL wt fVIII, R372A/R1689A fVIII, or V634M fVIII. (C) Binding of wt fVIII, R372A/R1689A fVIII, or V634M fVIII to immobilized VWF in the presence or absence of exposure to thrombin detected by ELISA as described in “Binding of fVIII to VWF.” (D) ELISA of binding of wt fVIII, R372A/R1689A fVIII, or V634M fVIII to immobilized domain-specific anti-fVIII capture mAbs 2-116 (A1), 1D4 (A2), 2-54 (A2), 2-93 (A2), 4A4 (A2), 2-113 (A3), G38 (A3), 5G12, 2A9 (C1), I-109 (C2), and I14 (C2, negative control). Biotinylated I14 was used as the detection antibody. (E) Clearance of 1 μg of wt fVIII, R372A/R1689A fVIII, or V634M fVIII after tail-vein injection in FVIII^−/− mice. Errors represent sample SDs.