Measuring the autoantibody reactivity and proteolytic activity of novel ADAMTS13 variants. (A) ADAMTS13 epitope expansion based on modeling. Known epitope residues are depicted in magenta; predicted residues are depicted in yellow. Native N-glycan residues are highlighted in green. Artificial N-glycans were introduced at positions 568 (NGLY1), 591 (NGLY2), 608 (NGLY3), 609 (NGLY4), 636 (NGLY5), and 637 (NGLY6). (B) Reactivity of ADAMTS13 variants against II-1 and I-9 monoclonal autoantibodies. (C) Reactivity of ADAMTS13 variants against TTP patient sera in heat map format. Numbers represent relative binding compared with WT (in percentage; 100% is red, 50% is white, and 0% is green). (D) Relative activity of ADAMTS13 variants in FRETS-VWF73 assay (WT-ADAMTS13 is 100%). (E) Activity of NGLY-ADAMTS13 variants in VWF multimer assay. (F) Relative activity of ADAMTS13 variants measured with FRETS-VWF73 in the presence of TTP patient sera (WT-ADAMTS13 is 100%; all values compared with WT-ADAMTS13 without any patient antibodies). (G) Mass spectrometry reveals presence of an N-glycan at amino acid position 608 in NGLY3 (K608N). The presence of the N-glycan at this position is derived from the detection of a K608N containing deamidated peptide of NGLY3 upon treatment with PNGaseF (for further details see supplemental Materials). OD, optical density.