Binding of antibodies present in plasma from 27 different patients with iTTP against CUB domain alanine variants. (A) Heat map of the average binding of antibodies present in plasma of patients with iTTP to various CUB domain and spacer domain alanine combination variants, expressed as a percentage relative to wtADAMTS13 binding (n = 3). (B) Average (± standard deviation) binding of patient samples to all tested ADAMTS13 variants, relative to wtADAMTS13. Significance was determined using a paired repeated-measures 1-way analysis of variance (ANOVA), with Geisser-Greenhouse correction, followed by a post hoc Tukey multiple comparisons test. (C) The relative reduction of antibody binding to CUB domain alanine variants combined with spacer 5ALA, as compared with binding to the 5ALA variants. (D) Linked scatterplot of binding of patient samples to 5ALA, 5ALA-1250A-1252A, and 5ALA-1250A-1252A-1367A, showing the effect per patient. Significance was determined using a paired repeated-measures 1-way ANOVA, with Geisser-Greenhouse correction, followed by a post hoc Tukey multiple comparisons test. ∗∗∗∗, P < .0001; ∗∗∗, P < .001; WT, wild-type.