Functional analysis of ADAMTS13 Cys-rich domain point mutants and engineered glycan mutants. (A) Activity assays were set up in which 0.5 nM ADAMTS13 or ADAMTS13 point mutant in concentrated conditioned media was incubated with 6 µM VWF115. Control reactions containing media without ADAMTS13 were set up in parallel. At time points from 0 to 90 minutes, aliquots were removed, stopped with EDTA, and analyzed by SDS-PAGE and Coomassie staining. Uncleaved VWF115 is a 16.9-kDa protein. Cleavage products of 10 kDa and 6.9 kDa arising from specific ADAMTS13-mediated proteolysis are marked by arrows. (B) ADAMTS13 variants containing novel engineered glycans at different positions on the surface of the Cys-rich domain were generated and their ability to proteolyze VWF115 assessed. The analysis was done as in panel A, except that 1 nM ADAMTS13 or variant was used and the third time point was taken at 60 minutes.