Abstract Number: PB0296
Meeting: ISTH 2022 Congress
Background: We recently identified a novel p.R102S mutation in the metalloprotease (MP) domain of ADAMTS13 in a congenital thrombotic thrombocytopenic purpura (cTTP) patient with decreased ADAMTS13 antigen (32 IU/dL) and undetectable ADAMTS13 activity. The in vitro effect of the mutation on ADAMTS13 secretion and activity has not been studied yet. Whether the mutation, which eliminates the H-bond of R102 with P297 in the loop that connects the MP with the disintegrin-like (D) domain, thereby affects preactivation of the MP domain is also unknown.
Aims: We aimed to investigate the in vitro effect of the p.R102S mutation on ADAMTS13 secretion, activity and preactivation.
Methods: CHO K1 or HEK293-T cells were transfected with wild-type (WT) or p.R102S mutant expression plasmids. ADAMTS13 antigen levels in expression medium were measured in ELISA. WT and p.R102S mutant were further purified by Zn2+-affinity chromatography. Preactivation of the MP domain was studied using anti-CUB1 monoclonal antibody (mAb) 17G2 which induces a 2-fold increase in ADAMTS13 activity in the FRETS-VWF73 assay, and exposes a cryptic epitope in the MP domain which is recognized by mAb 6A6 in ELISA.
Results: Secretion of the p.R102S mutant was impaired to 35% of WT secretion. ADAMTS13 activity of purified p.R102S mutant was severely reduced to 12% of WT activity. Although the activity of the p.R102S mutant was low, it could still be 2-fold activated by 17G2. In line with this, the 6A6 epitope was accessible when the p.R102S mutant was preincubated with 17G2.
Conclusion(s): The p.R102S mutation impairs ADAMTS13 secretion and severely reduces ADAMTS13 activity explaining the patient’s phenotype. However, the mutation did not impair preactivation of the MP domain. Molecular modelling studies are ongoing to further understand how the mutation affects ADAMTS13 activity.
To cite this abstract in AMA style:De Waele L, Vermeersch L, Tersteeg C, De Meyer S, Pavenski K, Vanhoorelbeke K. In vitro characterization of a novel Arg102 mutation in the metalloprotease domain of ADAMTS13 [abstract]. https://abstracts.isth.org/abstract/in-vitro-characterization-of-a-novel-arg102-mutation-in-the-metalloprotease-domain-of-adamts13/. Accessed September 22, 2023.
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