Gain-of-Function ADAMTS13 Variants Resistant to Anti-ADAMTS13 Autoantibody Inhibition: A Potential Novel Therapy for Acquired Autoimmune Thrombotic Thrombocytopenic Purpura

Abstract 1188

Spacer domain of ADAMTS13 is essential for recognition and proteolytic cleavage of von Willebrand factor. It also contains major antigenic epitope targeted by anti-ADAMTS13 autoantibodies in patients with acquired (idiopathic) thrombotic thrombocytopenic purpura (TTP). Through site-directed mutagenesis and recombinant protein strategy, we identified two novel ADAMTS13 variants, which exhibited enhanced specific activity, but reduced inhibition by anti-ADAMTS13 autoantibodies. Among twenty-three ADAMTS13 variants tested, ADAMTS13-M4 (R660K/F592Y/R568K /Y661F) and ADAMTS13-M5 (R660K/F592Y/R568K /Y661F/Y665F) exhibited increased specific activity by 4∼5 fold and 12∼16 fold in cleavage of FRETS-VWF73 and plasma-derived multimeric VWF under denaturing conditions, respectively. Moreover, these two novel ADAMTS13 variants (i.e. M4 and M5) were resistant to inhibition by anti-ADAMTS13 IgG autoantibodies from 10 out of 12 acquired TTP patients tested. In contrast, proteolytic activity of wild-type ADAMTS13 and several other mutants (i.e. M1: R660K, M2: R660K/F592Y, and M3: R660K/F592Y/R568K) were significantly inhibited by same amount of anti-ADAMTS13 IgGs in all 12 patients' plasmas. The resistance to inhibition of these variants was the result of reduced binding interaction between the ADAMTS13 variants (M4 and M5) and the anti-ADAMTS13 IgGs. These findings demonstrate for the first time that an exosite modification in the spacer domain may be a viable strategy for identification of novel ADAMTS13 variants with more desirable enzymatic properties. The results provide a potential tool for treatment of acquired (autoimmune) TTP and perhaps other arterial thrombotic disorders.