Fig. 2

SZ-179 inhibits rADAMTS13-mediated cleavage of the minimal substrate GSH-VWF73-H and VWF-R1597W under native conditions. a, b GST-VWF73-H (2.8 μg) was pre-incubated with SZ-179 or isotype IgG1 for 2 h at 37°C and then incubated with 50 nM rADAMTS13 for 1 h. The cleavage products were analyzed by 15% reducing SDS-PAGE and Western blotting with an HRP-conjugated mouse anti-GST antibody. c, d VWF-R1597W (150 nM) was incubated with SZ-179 or isotype control murine IgG1 at 4 °C for 30 min, followed by 3 h with 50 nM rADAMTS13 at 4 °C. The proteolytic products were separated by electrophoresis in a 1.3% agarose gel and detected by anti-VWF. e Dose–response curve for inhibition of rADAMTS13-mediated cleavage of VWF-R1597W. f Wild-type VWF (150 nM) was treated with rADAMTS13 at 4 °C for 3 h as a control. Results represented as mean ± SD of four independent experiments