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ADAMTS13 variants to escape autoantibodies in iTTP
ity, retaining only 22%. Single asparagine and alanine mutations of aromatic residues induced limited losses of activity. Cumulative double and triple asparagine or ala- nine mutations had more detrimental effects, with alanine mutations having less negative effects (RNRNN: 18%; RARAA: 35%). Mutation of the arginines by alanines had different impacts: R660A (RFAYY) had a higher reduction of activity (50% activity retained) compared to R568A (AFRYY) (83% activity retained). Mutation of both arginines (AFAYY) caused a large reduction of activity (24% that of the wild-type molecule). The classic GoF molecule (KYKFF) followed all trends observed for conser- vative mutations combined with arginine mutations and, surprisingly, also displayed reduced activity compared to the wild-type form (~37% activity retained). The full- length mutants AAAAA, AARAA, RAAAA, AAKAA, KAAAA, KAKAA, and KARAA all had similar activities (15-22%). The MDTCS variants displayed higher activi- ties than each of their full-length counterparts (wild-type MDTCS with 190% and MDTCS-AAAAA with 89% vs. 100% and 19%, respectively). In general, an inverse corre- lation was observed: a higher resistance to autoantibody binding attained through spacer exosite-3 mutations resulted, in general, in a lower residual FRETS-VWF73 activity (Figure 4).
The activities of five variants were tested in the pres- ence of patients’ autoantibodies in a FRETS-VWF73 assay format (Figure 5). Samples from patients TTP-007, TTP-
008 (exclusively anti-spacer autoantibodies) and patient TTP-085 (anti-spacer and anti-C-terminal autoantibodies) were used to provide a proof-of-concept. While the wild- type and the classic GoF were clearly inhibited, all other selected variants retained the same levels of activity.
Subsequently, several mutants with different levels of FRETS-VWF73 activities were tested in a static VWF mul- timer assay (Figure 6). The wild-type molecule showed activity after 30 min, with high molecular weight VWF multimers being cleaved and satellite bands accumulating in the lower molecular weight regions. After 24 h, these differences were further pronounced. All of the mutants tested in this assay showed reduced activity compared to the wild-type molecule. The classic GoF (KYKFF) demon- strated the highest activity among all the mutants tested. Among the autoantibody-resistant mutants, RARAA had the highest activity, with a reduction of high molecular weight VWF multimers and accumulation of satellite bands. While the truncated wild-type MDTCS variant had equal activity to full-length wild-type ADAMTS13, the MDTCS-AAAAA variant had very low activity, compara- ble to that of its full-length counterpart, AAAAA.
Discussion
In the current study we assessed the relevance of the spacer RFRYY epitope residues for autoantibody binding,
Figure 4. Inverse correlation observed between autoantibody reactivity and activity of ADAMTS13 variants. Only full-length ADAMTS13 variants are shown. The cyan- blue shading shows the area in which ADAMTS13 variants with reduced autoantibody binding are displayed. Blue dots represent conservative mutants; green dots represent semi-conservative mutants; red dots represent non-conservative mutants; gray dots represent alanine mutants (light-gray for only aromatic residues; dark- gray for arginine mutants); pink represents lysine/alanine hybrids.
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