Mechanisms of Inhibition of Human Monoclonal Antibodies in Immune Thrombotic Thrombocytopenic Purpura

Abstract

Antibody binding to a plasma metalloprotease ADAMTS13 is necessary for the development of immune thrombotic thrombocytopenic purpura (iTTP). Inhibition of ADAMTS13-mediated VWF cleavage by such antibodies clearly plays a role in the pathophysiology of the disease, though the mechanisms by which they inhibit ADAMTS13 enzymatic function are not fully understood. At least some immunoglobulin G-type antibodies appear to affect the conformational accessibility of ADAMTS13 domains involved in both substrate recognition and inhibitory antibody binding. We utilized single chain fragments of the variable region (scFv's) previously identified via phage display from patients with iTTP to explore the mechanisms of action of inhibitory human monoclonal antibodies. Using recombinant full-length ADAMTS13, truncated ADAMTS13 variants, and native ADAMTS13 in normal human plasma, we found that, regardless of the conditions tested, all three inhibitory monoclonal antibodies affect enzyme turnover rate much more than substrate recognition of von Willebrand factor (VWF). Hydrogen-deuterium exchange plus mass spectrometry (HX-MS) experiments with each of these inhibitory antibodies demonstrated that residues in the active site of the catalytic domain of ADAMTS13 are differentially exposed to solvent in the presence and absence of monoclonal antibody binding. These results support the hypothesis that inhibition of ADAMTS13 in iTTP may not necessarily occur because the antibodies directly prevent VWF binding, but instead rely on allosteric effects that impair VWF cleavage, likely by affecting the conformation of the catalytic center in the protease domain of ADAMTS13. Our findings provide novel insight into the mechanism of autoantibody-mediated inhibition of ADAMTS13 and pathogenesis of iTTP.