Mechanistic insight into multiple antibody binding to ADAMTS13 in immune thrombotic thrombocytopenic purpura

• Published in: Research and practice in thrombosis and haemostasis 2024-10, Vol.8 (7), p.102603, Article 102603
• Main Authors: Halkidis, Konstantine, Meng, Chan, Pillai, Vikram G., Shay, Madison, Liu, Szumam, Zheng, X. Long
• Format: Article
• Language: English
• Subjects: ADAMTS-13; autoantibody; immune thrombotic thrombocytopenic purpura; mechanism of inhibition; Original
• ISSN: 2475-0379; 2475-0379
• DOI: 10.1016/j.rpth.2024.102603

Antibody-mediated inhibition of von Willebrand factor (VWF) cleavage by ADAMTS-13 results in immune thrombotic thrombocytopenic purpura (iTTP). However, the effects of multiple antibody binding to ADAMTS-13 are not fully understood. To determine how multiple antibodies affect ADAMTS13 activity under various conditions. Single-chain fragments of the variable region isolated via phage display from patients with iTTP, FRETS-VWF73, native ADAMTS-13 in normal human plasma, and hydrogen-deuterium exchange plus mass spectrometry were used. We found that 2 stimulatory antibodies affect ADAMTS-13 turnover rate more than its substrate recognition. Hydrogen-deuterium exchange plus mass spectrometry revealed that 1 of these 2 stimulatory antibodies bound to the CUB2 domain that presumably interacts with the spacer domain of ADAMTS-13. Spacer domain is targeted by most inhibitory antibodies in iTTP. Both inhibitory and stimulating antibodies could bind ADAMTS-13 simultaneously but when both were present the inhibitory activity predominates. The antibody-mediated stimulation was lost, but the inhibition persisted when a modified substrate with the amino acid residue leucine at position 1603 of VWF was replaced by an alanine (VWF73-L1603A), interfering with active site binding. These results support the hypothesis that the mechanism of action of both stimulatory and inhibitory anti-ADAMTS-13 antibodies in iTTP is through allosteric modification of the catalytic domain and that inhibition of ADAMTS-13 dominates when both are present. Our findings may provide a new avenue of exploration to develop targeted diagnostic and therapeutic approaches in the management of iTTP. •Antibody effects on ADAMTS-13 in immune thrombotic thrombocytopenic purpura are complex.•Single-chain fragments of the variable loop were used to explore antibody mechanisms.•Both stimulatory and inhibitory antibodies appear to work allosterically.•Inhibitory antibody activity supersedes stimulatory antibody effects when both are present.