Purified VWF (1.5 mM) was pre-denatured with 1.5 M guanidine-HCl in 20 mM Tris-HCl (pH 8.0) at 37uC for 2 h. After a 1:10 dilution with 20 mM Tris-HCl (pH 8.0), the denatured VWF was incubated with anti-VWF mAbs (0-200 mg/ml) at 37uC for 30 min and then treated with 25 nM rADAMTS13 that had been activated by incubation with 5 mM CaCl 2 at 37uC for 1 h. After incubation at 37uC for 1.5 h, the reaction was stopped by the addition of 20 mM EDTA.