Purified native HPNs inhibit proteolytic cleavage of rF-VWF73 and multimeric VWF by ADAMTS13. (A) Purified native HNPs (0-40 nM) were incubated with rF-VWF73 (2 µM) in the presence of rADAMTS13 (10 nM) and proteolytic cleavage of rF-VWF73 was determined by the rate of fluorescence generation per minute. Relative residual activity was plotted against concentrations of HNPs (means ± SEM, n = 3). (B) Purified native HNPs (0-150 µM) were incubated with plasma-derived multimeric VWF (25 µg/mL) in the presence of rADAMTS13 (10 nM) for 4 hours on a dialysis membrane floating on top of 1.5 M urea, 5 mM Tris-HCl, pH 8.0. In the last lane, EDTA (10 mM) was included as a control for complete inhibition of ADAMTS13 activity. The residual VWF was determined by 1% agarose electrophoresis, followed by western blotting with rabbit anti-VWF IgG. The results are representatives of 3 experiments. HMW, high molecular weight of VWF multimers; LMW, low molecular weight of VWF multimers; SEM, standard error of the mean.