Page 138 - 2020_11-Haematologica-web

N.A.G. Graça et al.
and the types of mutations that confer highest resistance to binding of patients’ anti-ADAMTS13 autoantibodies. In our study, the spacer RFRYY epitope made a major con- tribution to total autoantibody reactivity in 89% of the patients with immune TTP screened. Other studies reached similar conclusions.17,18 In our study, 12/18 patients (67%), had autoantibody mixtures comprising almost exclusively anti-spacer RFRYY autoantibodies. We also had 6/18 patients (33%) who had C-terminal anti- TSP2-8 and/or anti-CUB1-2 domain autoantibodies, a prevalence slightly below the range reported in the litera- ture (Online Supplementary Table S1). One patient (TTP- 042) also had autoantibodies targeting other unknown areas in the N-terminal region (Figure 2).
We show that different types of mutations in ADAMTS13 spacer exosite-3 have different impacts on immune TTP patients’ autoantibody binding. Our data indicate that the two arginines are less important for autoantibody binding than the three aromatic residues
A
present in this epitope (AFAYY had a median reactivity of 64%, while RARAA had only 27%). Conservative muta- tions of aromatic residues did not reduce reactivity towards patients’ autoantibodies (RYRFF median reactivi- ty: 92%). Replacing the arginines in this mutant by lysines (KYKFF, classic GoF mutant) led to a decrease in median reactivity (in agreement with the original study26), albeit quantitatively limited (73% binding). Thus, introduction of highly conservative mutations of aromatic residues is not sufficient to reduce the binding of autoantibodies tar- geting the RFRYY epitope. Semi-conservative mutations of aromatic residues with leucine had an intermediate, yet limited efficacy at autoantibody escape. Non-conservative asparagine or classic alanine mutations of the aromatic residues had the greatest success. Individual mutations had a low impact, and, by themselves, Y661 and Y665 appeared to be the least important residues for overall autoantibody binding. A more pivotal contribution was observed for F592, as shown when individually or cumu-
B
Figure 5. Patients’ samples inhibit wild-type and gain-of-function (KYKFF) ADAMTS13 variants but not non-conservative or alanine ADAMTS13 variants. The activ- ities of selected ADAMTS13 variants were tested in the presence of pathogenic patients’ autoantibodies in FRETS-VWF73 assays. Three patients’ samples, repre- sentative of the patients’ autoantibody repertoire, were incubated directly with ADAMTS13 variants (each variant at 0.2 mg/mL). (A). samples from TTP-007 and TTP- 008 (only anti-spacer autoantibodies) were diluted 40x. (B) Samples from TTP-008 (anti-spacer autoantibodies) and TTP-085 (anti-spacer and anti-C-terminal autoan- tibodies) were diluted 20x. As expected, both wild-type and gain-of-function variants were inhibited. Variants RNRNN, AARAA and RARAA displayed full-resistance and retained their baseline proteolytic activity levels. WT: wild-type; CHO: Chinese hamster ovary; GoF: gain-of-function.
2626
haematologica | 2020; 105(11)