Abstract Plasma ADAMTS13 circulates in a folded conformation that is stabilized by an interaction between the central Spacer domain and the C-terminal CUB (complement components C1r and C1s, sea urchin protein Uegf, and bone morphogenetic protein-1) domains. Binding of ADAMTS13 to the VWF D4(-CK) domains or to certain activating murine monoclonal antibodies (mAbs) induces a structural change that extends ADAMTS13 into an open conformation that enhances its function. The objective was to characterize the mechanism by which conformational activation enhances ADAMTS13-mediated proteolysis of VWF. The activating effects of a novel anti-Spacer (3E4) and the anti-CUB1 (17G2) mAbs on the kinetics of proteolysis of VWF A2 domain fragments by ADAMTS13 were analyzed. mAb-induced conformational changes in ADAMTS13 were investigated by enzyme-linked immunosorbent assay. Both mAbs enhanced ADAMTS13 catalytic efficiency (kcat/Km) by ∼twofold (3E4: 2.0-fold; 17G2: 1.8-fold). Contrary to previous hypotheses, ADAMTS13 activation was not mediated through exposure of the Spacer or cysteine-rich domain exosites. Kinetic analyses revealed that mAb-induced conformational extension of ADAMTS13 enhances the proteolytic function of the metalloprotease domain (kcat), rather than augmenting substrate binding (Km). A conformational effect on the metalloprotease domain was further corroborated by the finding that incubation of ADAMTS13 with either mAb exposed a cryptic epitope in the metalloprotease domain that is normally concealed when ADAMTS13 is in a closed conformation. We show for the first time that the primary mechanism of mAb-induced conformational activation of ADAMTS13 is not a consequence of functional exosite exposure. Rather, our data are consistent with an allosteric activation mechanism on the metalloprotease domain that augments active site function.