Antibody-mediated VWF targeting reconstitutes ADAMTS13 metalloproteinase activity and prevents thrombocytopenia in mice

Abstract

ADAMTS13 (a multidomain metalloproteinase) cleaves von Willebrand factor (VWF), thereby preventing the accumulation of ultra-large VWF multimers that promote platelet-rich thrombus formation. Severe ADAMTS13 deficiency causes thrombotic thrombocytopenic purpura (TTP). In immune-mediated TTP (iTTP), anti-ADAMTS13 autoantibodies inhibit ADAMTS13 activity, often by targeting non-catalytic domains that mediate substrate recognition. We developed ADAMTS13 mimetics by fusing the ADAMTS13 metalloproteinase domain to anti-human VWF A2 domain antibodies, aiming to replace native substrate-recognition domains with antibody-mediated substrate targeting while removing major autoantibody-targeted ADAMTS13 domains. The resulting mimetics bound to the recombinant VWF A2 domain and showed proteolytic activity toward both the recombinant VWF A2 domain and FRETS-VWF73 substrate. Kinetic analysis showed that the mimetics had not only lower kcat values than ADAMTS13 but also lower Km values, thereby maintaining overall catalytic efficiency. Importantly, ADAMTS13 mimetics retained VWF A2-cleaving activity in the presence of a spacer domain-directed anti-ADAMTS13 autoantibody both in vitro and in vivo. In an iTTP mouse model, ADAMTS13 mimetics prevented recombinant VWF-induced thrombocytopenia, whereas the parental anti-VWF A2 domain antibody did not. Platelet-preserving activity was also observed in ADAMTS13-deficient mice. In further analyses, some mimetics showed reduced binding to a conformationally restricted VWF A2 domain, resulting in reduced cleavage activity. Antibody binding correlated with platelet recovery in the iTTP mouse model, suggesting that epitope accessibility and antibody-mediated positioning contribute to in vivo activity. These findings demonstrate that antibody-mediated substrate targeting can reconstitute the VWF-cleaving activity of the ADAMTS13 metalloproteinase domain and suggest that ADAMTS13 mimetics represent a potential therapeutic approach for iTTP.