title: High-resolution epitope mapping by HX MS reveals the pathogenic mechanism and a possible therapy for autoimmune TTP syndrome.
abstract: Acquired thrombotic thrombocytopenic purpura (TTP), a thrombotic disorder that is fatal in almost all cases if not treated promptly, is primarily caused by IgG-type autoantibodies that inhibit the ability of the ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) metalloprotease to cleave von Willebrand factor (VWF). Because the mechanism of autoantibody-mediated inhibition of ADAMTS13 activity is not known, the only effective therapy so far is repeated whole-body plasma exchange. We used hydrogen-deuterium exchange mass spectrometry (HX MS) to determine the ADAMTS13 binding epitope for three representative human monoclonal autoantibodies, isolated from TTP patients by phage display as tethered single-chain fragments of the variable regions (scFvs). All three scFvs bind the same conformationally discontinuous epitopic region on five small solvent-exposed loops in the spacer domain of ADAMTS13. The same epitopic region is also bound by most polyclonal IgG autoantibodies in 23 TTP patients that we tested. The ability of ADAMTS13 to proteolyze VWF is impaired by the binding of autoantibodies at the epitopic loops in the spacer domain, by the deletion of individual epitopic loops, and by some local mutations. Structural considerations and HX MS results rule out any disruptive structure change effect in the distant ADAMTS13 metalloprotease domain. Instead, it appears that the same ADAMTS13 loop segments that bind the autoantibodies are also responsible for correct binding to the VWF substrate. If so, the autoantibodies must prevent VWF proteolysis simply by physically blocking normal ADAMTS13 to VWF interaction. These results point to the mechanism for autoantibody action and an avenue for therapeutic intervention.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104;
authors.forename: Veronica C
authors.initials: VC
authors.lastname: Casina
authors.affiliation: Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104; engl@mail.med.upenn.edu wenbing@mail.med.upenn.edu zhengl@uab.edu.
authors.forename: Wenbing
authors.initials: W
authors.lastname: Hu
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104;
authors.forename: Jian-Hua
authors.initials: JH
authors.lastname: Mao
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104;
authors.forename: Rui-Nan
authors.initials: RN
authors.lastname: Lu
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104;
authors.forename: Hayley A
authors.initials: HA
authors.lastname: Hanby
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104;
authors.forename: Brandy
authors.initials: B
authors.lastname: Pickens
authors.affiliation: Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104;
authors.forename: Zhong-Yuan
authors.initials: ZY
authors.lastname: Kan
authors.affiliation: Department of Molecular Biology, Pusan National University, Busan, South Korea 609-735;
authors.forename: Woon K
authors.initials: WK
authors.lastname: Lim
authors.affiliation: Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104;
authors.forename: Leland
authors.initials: L
authors.lastname: Mayne
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104;
authors.forename: Eric M
authors.initials: EM
authors.lastname: Ostertag
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104;
authors.forename: Stephen
authors.initials: S
authors.lastname: Kacir
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104;
authors.forename: Don L
authors.initials: DL
authors.lastname: Siegel
authors.affiliation: Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104; engl@mail.med.upenn.edu wenbing@mail.med.upenn.edu zhengl@uab.edu.
authors.forename: S Walter
authors.initials: SW
authors.lastname: Englander
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104; Division of Laboratory Medicine, Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35249 engl@mail.med.upenn.edu wenbing@mail.med.upenn.edu zhengl@uab.edu.
authors.forename: X Long
authors.initials: XL
authors.lastname: Zheng
mesh_terms: D051722:ADAM Proteins / Q000097:blood / Q000737:chemistry / Q000378:metabolism; D000071120:ADAMTS13 Protein; D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D000941:Antigens / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D002648:Child; D003710:Demography; D041961:Deuterium Exchange Measurement / Q000379:methods*; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D007700:Kinetics; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008875:Middle Aged; D008969:Molecular Sequence Data; D011485:Protein Binding; D059748:Proteolysis; D011697:Purpura, Thrombotic Thrombocytopenic / Q000473:pathology* / Q000628:therapy*; D016415:Sequence Alignment; D017384:Sequence Deletion; D057127:Single-Chain Antibodies / Q000378:metabolism; D055815:Young Adult
chemical_list: D000941:Antigens; D000939:Epitopes; D007074:Immunoglobulin G; D057127:Single-Chain Antibodies; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human
keywords: ADAMTS13; autoimmunity; hydrogen exchange; thrombotic thrombocytopenic purpura; von Willebrand factor