Trp1250, Lys1252, and Arg1367 of ADAMTS13 comprise a hot spot for anti-CUB domain antibodies in patients with iTTP

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare life-threatening thrombotic disorder, which results from the development of autoantibodies targeting ADAMTS13. Most patients (>90%) with iTTP display antibodies against a shared epitope in the spacer domain of ADAMTS13. Nevertheless, a smaller population of patients (20%-40%) also has antibodies directed toward the CUB (complement C1r/C1s, Uegf, Bmp1) domains of ADAMTS13. Here, we explored whether anti-CUB antibodies have a shared epitope located on CUB domains of ADAMTS13 that overlaps with the spacer-CUB domain interface. Hydrogen-deuterium exchange mass spectrometry revealed that a panel of patient-derived human monoclonal anti-CUB domain antibodies specifically targeted peptides 1248-1253 and 1359-1377 in the CUB1 and CUB2 domains, respectively. A parallel alanine screen showed that residues W1250, K1252, and R1367 are crucially involved in binding of anti-CUB domain antibodies. A triple alanine variant containing W1250A/K1252A/R1367A ameliorated the binding of all patient-derived monoclonal antibodies. This triple-alanine variant also showed greatly reduced binding of anti-CUB antibodies upon analysis of plasma samples of a panel of 27 patients with iTTP. Functional analysis of the anti-CUB antibodies showed that all antibodies were able to induce an open conformation but did not inhibit activity toward either peptide substrates or von Willebrand factor multimers under flow conditions. Collectively, our findings show that anti-CUB antibodies target residues W1250/K1252 and R1367. Binding of pathogenic antibodies disrupt the spacer-CUB domain interface, thereby inducing an open conformation in ADAMTS13.