Proteolysis of ADAMTS13 by thrombin abolishes ADAMTS13 enzymatic activity toward purified human VWF. Human VWF was purified from human plasma by gel filtration. After gel filtration (A), the purity of VWF in each fraction was assessed by reducing SDS-PAGE on a 7.5% polyacrylamide gel, followed by silver staining (B). Fractions B8-B12 contained VWF, visualized as a band of approximately 250 kDa (arrow), and were free of major contaminating proteins. Only the highest purity VWF from fractions B8-B10 were used as a substrate for ADAMTS13. ADAMTS13 that had been pretreated with and without 9 nM thrombin in 20 mM Tris-HCl (pH 7.8) containing 150 mM NaCl and 5 mM Ca²⁺ for 16 hours at 37°C was assayed for activity. Subsamples were removed at 0, 1, 2, and 6 hours and assessed by Western blotting using an anti-VWF polyclonal antibody (C). ADAMTS13 generated characteristic VWF proteolytic fragments of 176 kDa and 140 kDa. Samples studied in panel C were also analyzed using a functional assay based on collagen binding (VWF-CBA) as outlined in “Materials and methods” (D). Results shown ± SEM, (n = 3).