Purified ADAMTS13 is cleaved by thrombin and FXa in a concentration- and time-dependent fashion. Purified ADAMTS13 (400 nM) was incubated with and without 9 nM thrombin (FIIa) in 20 mM Tris-HCl (pH 7.8) containing 150 mM NaCl and 5 mM Ca²⁺ at 37°C for 16 hours. Samples were electrophoresed on a 4% to 15% polyacrylamide gel under both reducing (A) and nonreducing (B) conditions, followed by Coomassie staining. Thrombin was detected as a band of approximately 36 kDa (white arrowhead); full-length ADAMTS13 and its proteolytic fragments are identified by arrows. (C) Western blot analysis of ADAMTS13 with and without complete thrombin proteolysis (under reducing and nonreducing conditions) using the anti-ADAMTS13 protease domain antibody. (D) Western blot analysis of ADAMTS13 with and without complete thrombin proteolysis (under reducing and nonreducing conditions) using the anti-myc epitope antibody. (E) Varying concentrations of thrombin (0 nM-90 nM) were incubated with 250 nM ADAMTS13 at 37°C for 0 to 6 hours in 20 mM Tris-HCl (pH 7.8) containing 150 mM NaCl and 5 mM Ca²⁺. Samples were analyzed under reducing conditions by Western blotting using the anti-ADAMTS13 protease domain antibody. (F) FXa (50 nM) was incubated with 250 nM ADAMTS13 at 37°C for 0 to 6 hours in 20 mM Tris-HCl (pH 7.8) containing 150 mM NaCl and 5 mM Ca²⁺, and analyzed as in panel E.