https://orcid.org/0000-0002-1828-7957
Key Points
Anti-CUB domain antibodies target residues Trp1250, Lys1252 and Arg1367 in patients with immune TTP.
Binding of anti-CUB antibodies to ADAMTS13 disrupt spacer-CUB domain interactions thereby forcing ADAMTS13 in an open conformation.
Immune mediated thrombotic thrombocytopenic purpura (iTTP) is a rare life-threatening thrombotic disorder, which results from the development of autoantibodies targeting ADAMTS13. The majority of 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 towards the CUB-domains of ADAMTS13. Here, we explored if anti-CUB antibodies have a shared epitope located on CUB domains of ADAMTS13 that overlaps with spacer-CUB domain interface. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) 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 domain 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 towards either peptide substrates or VWF 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.
