Thrombotic thrombocytopenic purpura (TTP) is primarily caused by immunoglobulin G (IgG) autoantibodies against A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats, 13 (ADAMTS13). Nearly all adult idiopathic TTP patients harbor IgGs, which bind the spacer domain of ADAMTS13, a region critical for recognition and proteolysis of von Willebrand factor (VWF). We hypothesize that a modification of an exosite in the spacer domain may generate ADAMTS13 variants with reduced autoantibody binding while preserving or enhancing specific activity. Site-directed mutagenesis was used to generate a series of ADAMTS13 variants, and their functional properties were assessed. Of 24 novel ADAMTS13 variants, 2 (ie, M4, R660K/F592Y/R568K/Y661F and M5, R660K/F592Y/R568K/Y661F/Y665F) exhibited increased specific activity approximately 4- to 5-fold and approximately 10- to 12-fold cleaving a peptide VWF73 substrate and multimeric VWF, respectively. More interestingly, the gain-of-function ADAMTS13 variants were more resistant to inhibition by anti-ADAMTS13 autoantibodies from patients with acquired idiopathic TTP because of reduced binding by anti-ADAMTS13 IgGs. These results shed more light on the critical role of the exosite in the spacer domain in substrate recognition. Our findings also help understand the pathogenesis of acquired autoimmune TTP. The autoantibody-resistant ADAMTS13 variants may be further developed as a novel therapeutic for acquired TTP with inhibitors.

Previous studies have demonstrated that factor VIII (FVIII) or platelets alone increase cleavage of von Willebrand factor (VWF) by ADAMTS13 under mechanically induced shear stresses. We show in this study that the combination of FVIII and platelets at the physiological concentrations is more effective than either one alone. In the absence of FVIII, lyophilized platelets increase the formation of cleavage product by 2-3-fold. However, in the presence of physiological concentration of FVIII (1 nm), the formation of VWF cleavage product increases dramatically as a function of increasing platelets with the maximal rate enhancement of approximately 8-fold. Conversely, in the presence of a physiological concentration of lyophilized platelets (150 x 10(3)/microl), the half-maximal concentration of FVIII required to accelerate VWF proteolysis by ADAMTS13 reduces by approximately 10-fold (to approximately 0.3 nm) compared with that in the absence of platelets ( approximately 3.0 nm). Further studies using the FVIII derivative that lacks an acidic region (a3), an antiplatelet glycoprotein 1balpha IgG, and a purified recombinant VWF-A1 domain or glycoprotein 1balpha-stripped platelets demonstrate that the synergistic rate-enhancing effect of FVIII and platelets depends on their specific binding interactions with VWF. Our findings suggest that FVIII and platelets are cofactors that regulate proteolysis of multimeric VWF by ADAMTS13 under physiological conditions.

Background Immunoglobulin Gs (IgGs) against ADAMTS13 is the primary cause of acquired (idiopathic) thrombotic thrombocytopenic purpura (TTP). However, the domains of ADAMTS13 against which anti-ADAMT13 IgGs target have not been investigated in a large cohort of TTP patients. DESIGN AND METHODS: 67 acquired idiopathic TTP patients were prospectively collected from three major U.S. centers. An enzyme-linked immunosorbent assay determined plasma concentration of anti-ADAMTS13 IgGs, whereas an immunoprecipitation plus Western blot determined the binding domains of anti-ADAMTS13 IgGs. RESULTS: Plasma anti-ADAMTS13 IgGs from 67 patients all bound full-length ADAMTS13 and a variant truncated after the 8(th) TSP1 repeat (delCUB). Approximately 97%(65/67) of patients harbored IgGs targeted against a variant truncated after the spacer domain (MDTCS). However, only 12% of patients reacted with a variant lacking the Cys-rich and spacer domains (MDT). In addition, approximately 37%, 31%, and 46% of patients' IgG interacted with the ADAMTS13 fragment containing TSP1 2-8 repeats (T2-8), CUB domains, and TSP1 5-8 repeats plus CUB domains (T5-8CUB), respectively. The presence of IgGs targeted against the T2-8 and/or CUB domains was inversely correlated with the patient platelet counts on admission. Conclusion Our results from the multicenter study further demonstrate that the multiple domains of ADAMTS13, particularly the Cys-rich and spacer domains, are frequently targeted by anti-ADAMTS13 IgGs in patients with acquired (idiopathic) TTP. Our data shed more light on the pathogenesis of acquired TTP and provide further rationales for adjunctive immunotherapy.

Previous studies have shown that ADAMTS13 spacer domain is required for cleavage of von Willebrand factor (vWF). However, the exact amino acid residues within this domain critical for substrate recognition are not known. Epitope mapping of anti-ADAMTS13 IgG from patients with thrombotic thrombocytopenic purpura (TTP) and sequence alignment of the ADAMTS13 spacer domains of human, mouse and zebrafish with these of human and murine ADAMTS1, a closely related member of ADAMTS family, have provided hints to investigate the role of the amino acid residues between Arg(659) and Glu(664) of the ADAMTS13 spacer domain in substrate recognition. A deletion of all these six amino acid residues (i.e. Arg(659)-Glu(664)) from the ADAMTS13 spacer domain resulted in dramatically reduced proteolytic activity toward vWF73 peptides, guanidine-HCl denatured vWF, and native vWF under fluid shear stress, as well as ultra large vWF on endothelial cells. Site-directed mutagenesis, kinetic analyses, and peptide inhibition assays have further identified a role for amino acid residues Arg(659), Arg(660), and Tyr(661) in proteolytic cleavage of various substrates under static and fluid shear stress conditions. These findings may provide novel insight into the structural-function relationship of ADAMTS13, and help understand pathogenesis of TTP and other arterial thromboses associated with compromised vWF proteolysis.

Deficiency of A Disintegrin And Metalloprotease with ThromboSpondin (ADAMTS13) results in thrombotic thrombocytopenic purpura (TTP). Plasma infusion or exchange is the only effective treatment to date. We show in this study that an administration of a self-inactivating lentiviral vector encoding human full-length ADAMTS13 and a variant truncated after the spacer domain (MDTCS) in mice by in utero injection at embryonic days 8 and 14 resulted in detectable plasma proteolytic activity (~5-70%), which persisted for the length of the study (up to 24 weeks). Intravascular injection via a vitelline vein at E14 was associated with significantly lower rate of fetal loss than intra-amniotic injection, suggesting that the administration of vector at E14 may be a preferred gestational age for vector delivery. The mice expressing ADAMTS13 and MDTCS exhibited reduced sizes of von Willebrand factor (vWF) compared to the Adamts13(-/-) mice expressing enhanced green fluorescent protein (eGFP). Moreover, the mice expressing both ADAMTS13 and MDTCS showed a significant prolongation of ferric chloride-induced carotid arterial occlusion time as compared to the Adamts13(-/-) expressing eGFP. The data demonstrate the successful correction of the prothrombotic phenotypes in Adamts13(-/-) mice by a single in utero injection of lentiviral vectors encoding human ADAMTS13 genes, providing the basis for developing a gene therapy for hereditary TTP in humans.Molecular Therapy (2008); doi:10.1038/mt.2008.223.