Anti-cysteine/spacer antibodies that open ADAMTS13 are a common feature in iTTP

Abstract

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is caused by an autoantibody-mediated deficiency in ADAMTS13. In healthy individuals, ADAMTS13 has a folded conformation in which the central spacer (S) domain interacts with the C-terminal CUB domains. We recently showed that ADAMTS13 adopts an open conformation in iTTP and that patient immunoglobulin G antibodies (IgGs) can open ADAMTS13. Anti-ADAMTS13 autoantibodies in patients with iTTP are directed against the different ADAMTS13 domains, but almost all patients have autoantibodies binding to the cysteine/spacer (CS) domains. In this study, we investigated whether the autoantibodies against the CS and CUB domains can disrupt the S-CUB interaction of folded ADAMTS13, thereby opening ADAMTS13. To this end, we purified anti-CS and anti-CUB autoantibodies from 13 patients with acute iTTP by affinity chromatography. The successfully purified anti-CS (10/13 patients) and anti-CUB (4/13 patients) autoantibody fractions were tested further in our ADAMTS13 conformation enzyme-linked immunosorbent assay to study whether they could open ADAMTS13. Interestingly, all purified anti-CS fractions (10/10 patients) were able to open ADAMTS13. On the other hand, only half of the purified anti-CUB fractions (2/4 patients) opened ADAMTS13. Our finding highlights that anti-CS autoantibodies that open ADAMTS13 are a common feature of the autoimmune response in iTTP.

Figure 1.

The presence of anti-CS autoantibodies that open ADAMTS13 is a common feature of the autoimmune response in patients with iTTP. (A-B) IgG fractions from 13 patients with acute iTTP were purified against CS (A) and CUB (B) using affinity chromatography. The specificity of the purified autoantibody fractions against CS (A) or CUB (B) was tested, using ELISA, on coated CS (light green) and MDT (orange) (A) and CUB (lavender) and T2-T8 (red) (B). Bound autoantibodies were detected using HRP-labeled polyclonal goat anti-human (Fc-specific) IgGs. Data are shown as the residual OD value that was calculated by subtracting the OD value of the sample on the fragment of interest (CS, MDT, CUB, or T2-T8), with the mean OD + 3 SD value of the binding of purified IgGs of 9 healthy donors to the corresponding fragment. Anti-CS (A) or anti-CUB (B) autoantibody fractions were used for further analysis if the residual OD value of the sample on CS(A, light green) or CUB (B, lavender), respectively, was positive, whereas the residual OD of binding to the other ADAMTS13 domains (MDT (A, orange) or T2-T8 (B, red)) was <50% of the binding to CS (A, light green) or CUB (B, lavender). Hence, only the anti-CUB autoantibody fraction from patient F was not used further. The anti-CS autoantibody fractions isolated from 10 patients (C) and the anti-CUB autoantibody fractions isolated from 4 patients (D) were individually preincubated with NHP containing closed ADAMTS13 and tested, using 1C4-ELISA, to investigate which autoantibodies could open ADAMTS13. Data are expressed as the mean CI ± SD (n = 3). A mean CI ≤ 0.50 (filled circle) represents closed ADAMTS13, whereas a mean CI > 0.50 (open circle) represents open ADAMTS13.