Binding of single alanine variants in region Y658-Y665 of the spacer domain to antibodies from patients with acquired TTP. (A) Immunoprecipitation of single alanine variants in region Y658-Y665 of MDTCS with protein G Sepharose coupled with human monoclonal antibodies I-9 and II-1. (B) Reactivity of IgG present in 6 patient samples (I-VI) with recombinant ADAMTS13 proteins MDTCS13, MDTCS in which the spacer domain of ADAMTS13 was replaced by the spacer domain of ADAMTS1 (MDTCS1), TSP2-8, and CUB1-2. (C) Binding of patient-derived IgG (samples I-VI) to single alanine variants in region Y658-Y665 of MDTCS. (D) Reactivity of patient-derived IgG (samples I-VI) toward truncated ADAMTS13 variants containing multiple amino acid substitutions: MDTCS-RY1, MDTCS-RY2, MDTCS-YY, and MDTCS-RYY. Pull-down of ADAMTS13 proteins with anti-V5 antibody was included in all experiments as positive control (+), whereas in a monoclonal antipneumococcal antibody (subclass IgG1; panel A), or IgG derived from normal plasma was used as a negative control (−; panels B-D).