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.ADAMTS13 | von Willebrand factor | thrombotic thrombocytopenic purpura | hydrogen exchange | autoimmunity A cquired thrombotic thrombocytopenic purpura (TTP) is characterized by severe thrombocytopenia and microangiopathic hemolytic anemia, resulting from disseminated microvascular thrombosis. Patients with TTP may suffer from widespread organ damage, resulting in death if not aggressively treated (1, 2). In most patients, thrombotic microangiopathy is caused by IgG-type autoantibodies against the plasma metalloprotease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) (3-5). ADAMTS13 regulates the function of the multidomain von Willebrand factor (VWF) (∼600 to ∼20,000 kDa) by cleaving it at the central A2 domain to regulate platelet-induced blood clot formation (3, 4).Immunological studies show that many acquired TTP patients with low plasma ADAMTS13 activity (less than 10%) harbor IgG autoantibodies that bind ADAMTS13 especially at the Cysrich and/or the spacer domain (4, 6). Deletion of these domains or grouped mutations therein can eliminate the binding of polyclonal anti-ADAMTS13 IgGs derived from many patients (4, 7-11). However, the exact ADAMTS13 binding sites of these autoantibodies are not known. Current treatment...

Human neutrophil peptides inhibit cleavage of von Willebrand factor by ADAMTS13: a potential link of inflammation to TTP

“…Our finemapping study of anti-ADAMTS13 antibodies demonstrated that the RRY motif in the spacer domain of ADAMTS13 is the frequent target of anti-ADAMTS13 IgGs in patients with acquired TTP. 40 HNPs share the same motif that is present in the ADAMTS13-spacer domain; therefore, it is possible that the released HNPs may boost the formation of a specific pathogenic autoantibody that binds the exosite 3 (RRYGEE) in the spacer domain of ADAMTS13. A monoclonal anti-human IgG (scFv4-20) isolated by phage display from a patient with acquired TTP appears to bind HNPs (not shown).…”

Human neutrophil peptides and complement factor Bb in pathogenesis of acquired thrombotic thrombocytopenic purpura

“…23,24 The autoantibodies bind to the spacer domain of ADAMTS13, which may physically block its substrate recognition. 7 The inability to cleave newly released ultralarge (UL) VWF results in the accumulation of ULVWF polymers on endothelial cells 25 and in the circulation. 26 Consequently, the ULVWF multimers serve as templates for rapidly recruiting platelets, 27,28 neutrophils, 28,29 and complement components 30 from the circulation to the sites of vascular injury.…”

“…Treatment with plasma infusion, aimed at increasing plasma ADAMTS13 activity to greater than 5%, is clinically effective in hereditary TTP. 6 In patients with acquired TTP, the immunoglobulin (IgG) type of autoantibodies, which bind primarily to the spacer domain of ADAMTS13, 7 result in competitive inhibition of plasma ADAMTS13 activity. Therapeutic plasma exchange, often used in combination with immunosuppression, including corticosteroids, vincristine, cyclophosphamide, and rituximab, etc., remains the treatment of choice for acquired TTP with inhibitors.…”

Rapid Fine Conformational Epitope Mapping Using Comprehensive Mutagenesis and Deep Sequencing

“…Co-crystallization provides unambiguous epitope identification but can require considerable effort and generation of many antigen variants to identify one that is compatible with crystallization (2). Mass spectrometry-based methods utilizing hydrogen/ deuterium exchange identify epitopes to a ϳ5-amino acid resolution only under rigorous control experiments that limit throughput (16,17). Competing display-based methods use many sorts (18), identify only partial epitopes (19,20), or are limited by restricting mutations to alanine (21).…”