ADAMTS13 activity in aHUS (serum and recombinant). (A) Different concentrations of FRETS-VWF73 substrate were used to determine the kinetics of ADAMTS13 activity in serum samples of aHUS patients with reduced ADAMTS13 activity (patient identification numbers MA-8, MA-13, MA-15, MA-20, and MA-27) and in normal pooled serum. The results are shown as a linear graph summarizing 3 separate experiments. The generated fluorescence was correlated with the concentration of cleaved FRETS-VWF73, using data obtained through a calibration standard with 100% activity. (B) Activity of purified recombinant ADAMTS13 molecules (WT and mutants) was measured by monitoring fluorescence generated as a result of cleavage of FRETS-VWF73 substrate after incubation with 1 µg of recombinant ADAMTS13. (C) Comparison of secretion of recombinant ADAMTS13 variants to cell culture media. Forty-eight hours after transfection of HEK293 cells with cDNAs encoding ADAMTS13 (WT and mutants), supernatants and cell lysates were collected and used for western and immunoblotting using polyclonal ADAMTS13 antibody. ADAMTS13 in media is secreted from the cells, and ADAMTS13 in the cell lysates is synthesized inside the cells.