Detection of proteolysis of FL-VWF variants by collagen-binding assay. (A-D) The VWF variants and ADAMTS13 were separately preincubated in 20 mM Tris (pH 7.8), 50 mM NaCl, 5 mM CaCl₂ ± 1.5 M urea at 37°C for 45 minutes. VWF (1 µg/mL) and ADAMTS13 (5 nM) were then combined, incubated at 37°C, and reactions stopped after 15 minutes and 180 minutes by the addition of EDTA. To analyze the extent of proteolysis, CBA was determined. For each FL-VWF variant the VWF:CBA/VWF:Ag ratio at both time points was normalized against the ratio determined at the start of the assay (100%). VWF concentrations in assays with urea (B,D) contained 7 μg/mL VWF, hence samples could be diluted further and urea no longer interfered with the assay. Results are means ± SEM from 3 independent analyses.