Kinetic analysis of ADAMTS13 disintegrin-like domain mutants. Activity assays were set up using 10 nM ADAMTS13 (or ADAMTS13 mutants, as shown) and either 1 μM VWF115 (A) or VWF115(D1614A) (B). Samples were stopped with EDTA from 0 to 2 hours and percentage of VWF115 proteolysis was determined by HPLC. The reduced proteolytic function of ADAMTS13(R349A) seen in panel A was completely lost when VWF115(D1614A) was used as a substrate (B), suggesting that the influence of R349A is dependent on D1614 in VWF115. ADAMTS13 and ADAMTS13(R349A) were further analyzed by measuring the initial rate of VWF115 (C) or VWF115(D1614A) (D) as a function of substrate concentration. Initial rates of proteolysis (< 15% cleavage) were analyzed after 20 minutes by HPLC. Using VWF115 (C), a large difference between the V_max for ADAMTS13 and ADAMTS13(R349A) was evident, confirming the time-course results in panel A. When VWF115(D1614A) was used, this difference was completely lost (D), confirming the results in panel B. Graphs shown are single reactions, but representative of 3 separate experiments. The kinetic values derived from panels C and D are shown in Table 1 (n = 3).