The interaction of ADAMTS13 with PNG-VWF. (A) VWF (5 μg/mL) was incubated with 10 nM ADAMTS13 in 5 mM CaCl₂, 50 mM NaCl, and 20 mM Tris-HCl (pH 7.8) in the presence and absence of 1.5 M urea. At various time points, subsamples were taken and the reaction was stopped with EDTA, and the extent of proteolysis was determined by CBA. (B) MaxiSorp plates, coated with 7.5 μg/mL VWF, were incubated with decreasing concentration of ADAMTS13. Bound ADAMTS13 was detected with either pAb(-Tsp2-4) (main graph) or pAb Tsp2-4 (inset). (C) VWF was captured to the microtiter plate using a monoclonal anti-VWF antibody directed against the C-terminal SpII fragment. Captured VWF was incubated with decreasing concentration of ADAMTS13 and bound ADAMTS13 detected with pAb(-Tsp2-4). Error bars represent mean and SD.