Enzymatically-Active ADAMTS13 Variants Are Not Inhibited by Anti-ADAMTS13 Autoantibodies - A Potential Novel Therapeutic Strategy.

ADAMTS13, a reprolysin-type zinc metalloprotease consisting of a metalloprotease (MP), a disintegrin, a thrombospondin-1 (TSP), a cysteine-rich region (Cys), a spacer, seven additional TSP, and two CUB domains, is critical for preventing unwarranted VWF-platelet interaction in the circulation. Profound deficiency of ADAMTS13, due to genetic mutations of the ADAMTS13 gene or autoimmune inhibitors of the protease, causes microvascular thrombosis characteristic of TTP. Plasma exchange is a conventional but complex therapy of TTP that may be ineffective for patients with high inhibitor titers. In this study we produce recombinant ADAMTS13 variant proteins and analyze their binding with and suppression by TTP inhibitors. TTP IgG binds the ADAMTS13 variants ending downstream of the spacer domain but not those truncated upstream, consistent with recent reports that the spacer domain is essential for interaction between TTP IgG and ADAMTS13. Nevertheless, the spacer domain sequence alone may be insufficient since TTP IgG does not bind a fragment consisting solely of the spacer domain sequence but binds a fragment encompassing the sequence between the distal 2/3 of the MP and the spacer. ADAMTS13 ending at the TSP#1 or downstream are proteolytically active in cleaving either VWF multimers or a 73-amino acid fragment of the VWF A2 domain. Mixing studies show that each of ten TTP plasma samples tested suppresses the protease activity of ADAMTS13 ending downstream of the spacer domain, but not the activity of the variants truncated at the TSP#1 or Cys domain. The variant proteins truncated at TSP#1, when added to a series of plasma samples from a TTP patient with rising inhibitor titers, consistently yield the expected protease activity levels, even in those samples whose high inhibitor titers abolish the activity of the full-length ADAMTS13 at 15x of its concentration. These findings indiate that it is feasible to design ADAMTS13 variant proteins that are proteolytically active yet refractory to suppression by inhibitors of TTP. Such ADAMTS13 variants may be exploited to circumvent the inhibitors of TTP, potentially eliminating the need of performing plasma exchanges in the future.