Disruption of ADAMTS13-Autoantibody Complexes with an ADAMTS13 Spacer Domain As a Potential Therapeutic Strategy for Immune Thrombotic Thrombocytopenic Purpura
2019
Background: Immune thrombotic thrombocytopenic purpura (iTTP) is primarily caused by acquired immunoglobulin (Ig) G against ADAMTS13, a member of the A Disintegrin and Metalloprotease with Thrombospondin Type 1 repeats family, which specifically cleaves von Willebrand factor (VWF). Such proteolytic cleavage is essential for normal hemostasis. An inability to cleave VWF due to IgG autoantibody-mediated inhibition of plasma ADAMTS13 activity results in an accumulation of ultra large VWF multimers and disseminated microvascular thromboses, a pathognomonic feature of TTP. Previous studies have demonstrated that the spacer domain of ADAMTS13 is the primary target of IgG autoantibody; also, spacer domain is important for recognition of VWF under various conditions. Objective: In the present study, we explored if an isolated spacer domain can compete or reverse IgG autoantibody-mediated inhibition of ADAMTS13 activity, an important question to be addressed for developing a new strategy to treat iTTP. Methods:A recombinant ADAMTS13-spacer domain was expressed and purified to homogeneity; additionally, a single-chain fragment of the variable region of anti-ADAMTS13 IgG (i.e. scFv4-20) was isolated via phage display from a patient with iTTP, which was shown to specifically target the spacer domain and potently inhibit plasma ADAMTS13 activity with sub-nanomolar affinity. A functional assay with a fluorescein-labeled VWF73 substrate was used to determine the ADAMTS13 activity. Results: First, when a purified recombinant spacer domain (0-1.0 µM) was pre-incubated with scFv4-20 at various concentrations, followed by an addition of normal human plasma (NHP) that provides wild-type ADAMTS13 and a fluorescein-labeled recombinant VWF73 (F-rVWF73) substrate, we demonstrated that the inhibitory activity of scFv4-20 towards plasma ADAMTS13 decreased in a concentration-dependent manner. As a control, recombinant Cys-rich domain had no effect on blocking the scFv4-20-mediated inhibition of plasma ADAMTS13 activity under the same conditions. When scFv4-20 was pre-incubated with plasma ADAMTS13, recombinant spacer domain could also reverse the scFv4-20-mediated inhibition of ADAMTS13 activity but to a lesser degree. At the final concentration of 1.0 µM, recombinant spacer domain was able to increase ADAMTS13 activity by 2.1-fold when the recombinant spacer domain was added prior to the formation of ADAMTS13-antibody complexes but only by 1.5-fold if the recombinant spacer domain was added after the ADAMTS13 and antibody complexes were formed. Conclusion: Our results demonstrate that autoantibody-mediated inhibition of plasma ADAMTS13 is reversible and can be blocked by a molecular mimic that is the primary antibody-binding region of the full-length ADAMTS13 protein. The data suggest that it may be feasible to explore the development of agents capable of displacing inhibitory antibodies against ADAMTS13 in patients with iTTP. Disclosures Zheng: Alexion: Speakers Bureau; Ablynx/Sanofi: Consultancy, Speakers Bureau; Shire/Takeda: Research Funding; Clotsolution: Other: Co-Founder.
Keywords:
- Correction
- Source
- Cite
- Save
- Machine Reading By IdeaReader
0
References
0
Citations
NaN
KQI