Cleavage of N-terminal deletion and elongation substrates. (A) GST-VWFd5 (65 nM) was incubated at 37°C for the indicated time with 2 nM recombinant full-length ADAMTS13 (FL), truncated constructs MD or MDT, or 0.3 nM plasma ADAMTS13 (PL). No cleavage product () was detected by gel electrophoresis and Western blotting with anti-GST antibody. (B) GST-VWF64, GST-VWF73nl, and GST-VWF106nl (65 nM) were incubated for 2 hours with 2 nM recombinant ADAMTS13 (FL), or for 6 hours with 0.3 nM plasma ADAMTS13 (PL). Substrates and cleavage products () were detected by gel electrophoresis and Western blotting with anti-GST antibody. Asterisks (*) indicates nonspecific bands observed reproducibly for GST-VWFd5 and GST-VWF106nl. Results are representative of 3 independent experiments. (C) Time course for cleavage of 6.8 nM GST-VWF64 (○), GST-VWF73nl (●), and GST-VWF106nl (□) by 1 nM ADAMTS13 at 37°C. Cleavage products were quantitated by an ELISA method. Data points represent the mean values for 2 independent experiments; the range was 2% to 9% of the mean.