Proteolytic cleavage of VWF and VWF73 under flow or static condition by ADAMTS13 and C-terminal truncated variants. (A) Rotation speed–dependent cleavage of VWF by ADAMTS13. Native plasma VWF (37.5 μg/mL or 150 nM) was incubated with ADAMTS13 (about 60 nM) for 1 minute and then vortexed for 3 minutes at 22°C at rotation speeds from 0 to about 3200 rpm (set at “0-10”). (B) Dose-dependent cleavage of VWF by ADAMTS13. VWF (18.75 μg/mL or 75 nM) was vortexed for 3 minutes without (lanes 1 and 7) or with various concentrations of rADAMTS13 (lane 2-6) or 2.5 μL of normal human plasma with 30 μg/mL (lane 8) or 60 μg/mL (lane 9) heparin or TTP patient plasma (lane 10). (C) Cleavage of VWF by ADAMTS13 and variants. VWF (18.75 μg/mL or 75 nM) was incubated and vortexed for 3 minutes without (−) or with (+) about 60 nM of FL-A13, delCUB, and MDTCS in absence (−) or presence (+) of 10 mM EDTA. (D) Cleavage of guanidine-HCl–denatured VWF by ADAMTS13 and variants. Denatured VWF (37.5 μg/mL or about 150 nM) was incubated without (−) or with (+) about 60 nM of purified FL-A13, delCUB, and MDTCS in absence (−) or presence (+) of 10 mM EDTA for 1 hour. All the reactions above were quenched by addition of SDS sample buffer and heated at 100°C for 5 minutes. The cleavage product (dimer of 176 kDa) was determined by Western blot with peroxidase-conjugated rabbit anti-VWF IgG, followed by chemiluminescent ECL reagents. The signal was obtained by exposure to X-ray film within 5 to 30 seconds. (E) Cleavage of GST-VWF73-H by ADAMTS13 and variants. GST-VWF73-H at various concentrations (0 to 200 nM) was incubated with about 60 nM of FL-A13, delCUB, and MDTCS for 10 minutes at 37°C. The cleavage product (34.4 kDa, arrowheads indicated) was determined by Western blot with rabbit anti-GST IgG and Alexa Fluor680–conjugated antirabbit IgG. (F) The plot of the fluorescent signal; obtained by Odyssey infrared fluorescent image system against concentrations of GST-VWF73 substrate.