Inhibition of VWF proteolysis by ADAMTS13 under flow by the C-terminal fragments of ADAMTS13. (A) The C-terminal fragments block cleavage of VWF by ADAMTS13. Purified VWF (18.75 μg/mL or 50 nM) was incubated 10 mM EDTA (control) or 0 to 150 nM of CUB, T2-8, T5-8, T5-8CUB, and T2-8CUB (lanes 2-6) for 60 minutes. ADAMTS13 (50 nM) was then added into the reaction mixture in presence of 50 mM HEPES buffer containing 0.25% BSA, 5 mM CaCl₂, and 0.25 mM ZnCl₂ (total volume, 20 μL) in a 0.2 mL PCR tube with dome caps. The reaction mixture was subjected to vortexing at a fixed rotation rate of about 2500 rpm (set “8”) for 3 minutes on a mini vortexer. The cleavage of VWF was determined by Western blot with anti-VWF IgG, peroxidase conjugated anti–rabbit IgG and ECL reagents (arrowheads indicate the dimers of 176 kDa). (B) Quantitation of chemiluminescent signal. The signal on X-ray film within the 30 seconds to 1 minute was quantified by densitometry using NIH ImageJ software. The relative proteolytic activity of ADAMTS13 (%) after being inhibited by various C-terminal fragments was plotted against the concentrations of C-terminal fragments of ADAMTS13 added into the reaction.