Background: 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.
Objectives: To determine how multiple antibodies affect ADAMTS13 activity under various conditions.
Methods: 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.
Results: 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.
Conclusion: 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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC11609530 | PMC |
http://dx.doi.org/10.1016/j.rpth.2024.102603 | DOI Listing |
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