Biochemical characterization of FVIII-BDD. (A) Canine (c) FVIII-BDD is predominantly synthesized as 160 000 single chain protein with a smaller proportion being processed as a heterodimer. Thrombin (IIa) cleaves cFVIII-BDD at the indicated sites to yield activated cFVIII. (B) Protein purity was assessed by loading 4 μg of human FVIII-BDD (H) and cFVIII-BDD (C) on a reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by staining with Coomassie blue (left; −IIa). FVIII-BDD (H or C; 800nM) was incubated with IIa (+IIa; 5nM) for 10 minutes, and the resulting activated FVIII was run on a reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (right, +IIa). The various domains of FVIII are indicated. SC indicates single chain; HC, heavy chain; LC, light chain; A3-C1-C2 (73 kDa), A1 (50 kDa), and A2 (43 kDa). (C) The specific activity of cFVIII-BDD and hFVIII-BDD was compared using a 1- or 2-stage aPTT in human-deficient plasma. For the 2-stage assay (+IIa), FVIII-BDD (human or canine; 20nM) in 20mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid/150mM NaCl/5mM CaCl₂/0.01% Tween 80, pH 7.4 (assay buffer) was intentionally activated with IIa (40nM) for 30 seconds at 25°C. Activated FVIII was immediately diluted into assay buffer with 0.1% albumin and then subsequently added to the aPTT clotting assay. In either the 1- (−IIa) or 2-stage aPTT (+IIa), the specific activity of cFVIII-BDD was 3-fold higher than hFVIII-BDD. The activation quotient was 22 for cFVIII and 28 for hFVIII. (D) A purified Xase assay was used to assess A2-domain stability. The Xase assay was performed by activating 20nM cFVIII-BDD or hFVIII-BDD with 40nM IIa for 30 seconds at 25°C. The reaction was stopped by adding 60nM hirudin. At various time points after activation, FVIIIa (0.2nM, final) was added to the Xase complex (hFIXa, 2nM; hFX, 300nM; and phospholipids, 20μM; phosphatidylcholine/phosphatidylserine, 75:25), and activation was measured by monitoring FXa generation using a chromogenic substrate.