Proteolytic inactivation of ADAMTS13 by plasmin. Recombinant ADAMTS13 (250 nM) was incubated with 9 nM human plasmin in 20 mM Tris-HCl (pH 7.8) containing 150 mM NaCl and 5 mM Ca2+ at 37°C for 30 minutes, causing partial proteolysis of ADAMTS13 (A). Samples were analyzed by Western blotting using either anti-ADAMTS13 protease domain (lane 1) or anti-myc epitope antibodies (lane 2). The rate of plasmin-dependent ADAMTS13 proteolysis was assessed by incubating varying concentrations of plasmin (0 nM-90 nM) with 250 nM ADAMTS13 at 37°C for 30 minutes to 6 hours (B). Samples were analyzed under reducing conditions by Western blotting using the anti-ADAMTS13 protease domain antibody. To assess the effect of plasmin proteolysis on ADAMTS13 function, ADAMTS13 was pretreated with or without 9 nM plasmin for 16 hours at 37°C and thereafter assayed for activity by 2 methods, as outlined in “Materials and methods.” Subsamples were removed from each assay at 0, 1, 2, 4, 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 that were visualized after 1 hour and increased in intensity over the 6-hour time course. There was also a concomitant decrease in the intensity of the 250-kDa VWF band. Analysis of VWF function as in Figure 6D using a VWF-CBA (D). Results shown ± SEM, (n = 3).
