Kinetic inhibition of recombinant full-length ADAMTS13 and truncated variants by an antispacer antibody (scFv20). (A) Cartoon representation of the structure of ADAMTS13 and truncated variants. Each recombinant protein has a 6-His tag and a V5 tag at the C-terminus. Rectangles: red, metalloprotease domain; light blue, disintegrin domain; dark blue, cysteine-rich domain; yellow, spacer domain; and green, 2 complement C1r/C1s, Uegf, Bmp1 (CUB) domains. Circles: thrombospondin type-1 domains (T2-8). Spacer domain, the binding site of scFv4-20 to ADAMTS13, is labeled. Assays were done at 25°C and pH 6.0. (B) The relative inhibitory activity of scFv4-20 incubated with full-length ADAMTS13 for different lengths of time (0, 15, 30, and 60 minutes). Activity was normalized to velocity (ΔFU/s) in the absence of scFv4-20 (buffer control) to derive relative velocity. (C-E) Titration of recombinant full-length ADAMTS13, truncated ADAMTS13 MT4, and MDTCS, respectively, with FRETS-VWF73 in the presence of various concentrations of scFv4-20 (n ≥ 3). Data in the plots are shown as mean ± standard error of the mean (SEM).