Thrombotic thrombocytopenic purpura (TTP) is the prototypical microangiopathy characterized by disseminated microthromboses, hemolytic anemia, and ultimately organ dysfunction. A link with deficiency of the von Willebrand factorcleaving protease (ADAMTS13) has been demonstrated, but additional genetic and/or environmental triggers are thought to be required to incite acute illness. Here we report that 4 days of ADAMTS13 functional inhibition is sufficient to induce TTP in the baboon (Papio ursinus), in the absence of inciting triggers because injections with an inhibitory monoclonal antibody (mAb) consistently (n ‫؍‬ 6) induced severe thrombocytopenia (< 12 ؋ 10 9 /L), microangiopathic hemolytic anemia, and a rapid rise in serum lactate dehydrogenase. Immunohistochemical staining revealed the characteristic disseminated platelet-and von Willebrand factor-rich thrombi in kidney, heart, brain, and spleen but not lungs. Prolonged inhibition (14 days, n ‫؍‬ 1) caused myocardial ischemic damage and asplenia but not death. Control animals (n ‫؍‬ 5) receiving equal doses of a noninhibitory anti-ADAMTS13 mAb remained unaffected. Our results provide evidence for a direct link between TTP and ADAMTS13 inhibition and for a mild disease onset. Introductionvon Willebrand factor (VWF) is a multimeric glycoprotein that bridges platelets to injured arterial vessels through interactions with both subendothelial collagen and platelet membrane receptors. Unusually large VWF multimers (UL-VWFs) are released as VWF precursors into the bloodstream by stimulated endothelial cells. 1 These high-molecular-weight proteins are abnormally adhesive, being able to bind and cross-link platelets in circulation even in the absence of endothelial injury. 2 Normally, UL-VWFs are rapidly cleaved by circulating VWF-cleaving protease (AD-AMTS13), 3 which generates VWF multimers of sizes seen in normal plasma. 4 The inability to process UL-VWF in cases of ADAMTS13 deficiency can cause disseminated platelet-rich thrombi, which block terminal arterioles, 1,5 leading to hemolytic anemia with ischemic organ failure and ultimately death in patients with thrombotic thrombocytopenic purpura (TTP). Diagnosis is based on signs of concurrent thrombocytopenia with hemolytic anemia and fragmented red blood cells (schistocytes) in the absence of other identifiable primary causes. 6 ADAMTS13 deficiency can be hereditary by mutations in the ADAMTS13 gene 3 or acquired by inhibiting autoantibodies to ADAMTS13. 7 The former is currently treated by infusion of fresh frozen plasma, which contains donor ADAMTS13 to overcome the deficiency. The latter often requires plasma exchange to both replenish the diminished proteolytic activity and remove inhibitors.These plasma therapies could effectively reduce mortality to approximately 20%, 8 but morbidity still is considerable and not seldom as a consequence of the plasma therapy. 9,10 Safer therapeutic strategies are therefore required 11 and could focus on the inhibition of the platelet-VWF interaction 12 or on the reconst...

Soluble von Willebrand factor (VWF) has a low affinity for platelet glycoprotein (GP) Ib␣ and needs immobilization and/or high shear stress to enable binding of its A1 domain to the receptor. The previously described anti-VWF monoclonal antibody 1C1E7 enhances VWF/GPIb␣ binding and recognizes an epitope in the amino acids 764 -1035 region in the N-terminal DD3 domains. In this study we demonstrated that the DD3 region negatively modulates the VWF/GPIb-IX-V interaction; (i) deletion of the DD3 region in VWF augmented binding to GPIb␣, suggesting an inhibitory role for this region, (ii) the isolated DD3 region inhibited the GPIb␣ interaction of a VWF deletion mutant lacking this region, indicating that intramolecular interactions limit the accessibility of the A1 domain, (iii) using a panel of anti-VWF monoclonal antibodies, we next showed that the DD3 region is in close proximity with the A1 domain in soluble VWF but not when VWF was immobilized; (iv) destroying the epitope of 1C1E7 resulted in a mutant VWF with an increased affinity for GPIb␣. Our results support a model of domain translocation in VWF that allows interaction with GPIb␣. The suggested shielding interaction of the A1 domain by the DD3 region then becomes disrupted by VWF immobilization. The plasma protein von Willebrand factor (VWF)4 has a central role in normal primary hemostasis (1). The interaction of VWF with its platelet receptor glycoprotein (GP) Ib␣ in the GPIb-IX-V complex mediates platelet adhesion to extracellular matrices exposed at sites of vascular injury. This interaction is essential for thrombus formation at sites of high shear stress, as in microarterioles or in stenosed arteries.Mature VWF comprises a series of multimers that are composed of homodimers interlinked through disulfide bridges. The mature VWF subunit consists of four distinct types of internal homology present in two to three copies in the following order from the N terminus:

Background Recently, conformational activation of ADAMTS13 was identified. This mechanism showed the evolution from a condensed and inhibited conformation, in which the proximal MDTCS and distal T2-CUB2 domains are in close contact with each other, to an activated structure due to ding with the von Willebrand factor (VWF). Objectives Identification of cryptic epitope/exosite exposure after conformational activation and of sites of flexibility in ADAMTS13. Methods The activating effect of 25 anti-T2-CUB2 antibodies was studied in the FRETS-VWF73 and the vortex assay. Cryptic epitope/exosite exposure was determined in ELISA and VWF binding assay. The molecular basis for flexibility was hypothesized through RADAR analysis, tested in ELISA using deletion variants and visualized using electron microscopy. Results Eleven activating anti-ADAMTS13 antibodies, directed against the T5-CUB2 domains, were identified in the FRETS-VWF73 assay. RADAR analysis identified three linker regions in the distal domains. Interestingly, identification of an antibody recognizing a cryptic epitope in the metalloprotease domain confirmed the contribution of these linker regions to conformational activation of the enzyme. The proof of flexibility around both the T2 and metalloprotease domains by electron microscopy furthermore supported this contribution. In addition, cryptic epitope exposure was identified in the distal domains, as activating anti-T2-CUB2 antibodies increased the binding to folded VWF up to ~3-fold. Conclusion Conformational activation of ADAMTS13 leads to cryptic epitope/exosite exposure in both proximal and distal domains, subsequently inducing increased activity. Furthermore, three linker regions in the distal domains are responsible for flexibility and enable the interaction between the proximal and the T8-CUB2 domains.