An autoantibody epitope comprising residues R660, Y661, and Y665 in the ADAMTS13 spacer domain identifies a binding site for the A2 domain of VWF

Pos, Wouter; Crawley, James T. B.; Fijnheer, Rob; Voorberg, Jan; Lane, David A.; Luken, Brenda M.

doi:10.1182/blood-2009-06-229203

Cited by 118 publications

(165 citation statements)

References 52 publications

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“…It seems clear that the same ADAMTS13 antigenic epitope identified by HX MS is a common binding site for many polyclonal autoantibodies produced by TTP patients. These results are consistent with those reported previously (8)(9)(10)35). The role of autoantibodies reported to bind elsewhere (4, 6, 14) will require further investigation.…”

Section: Resultssupporting

confidence: 83%

High-resolution epitope mapping by HX MS reveals the pathogenic mechanism and a possible therapy for autoimmune TTP syndrome

Casina

Mao

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Resultssupporting

confidence: 83%

High-resolution epitope mapping by HX MS reveals the pathogenic mechanism and a possible therapy for autoimmune TTP syndrome

Casina

Mao

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…The reduction in cleavage constant for the VWF115 Leu1603Ser variant is appreciably greater than the 18-fold reduction for the VWF115 Met1606Ala variant in which the P1′ residue was substituted (10), the 6.5-fold reduction for the VWF115 Asp1614Ala that disrupts the disintegrin-like domain binding exosite (11), and the 12.5-to 15-fold reduction for the Δ1660-1668 C-terminal deletion variant that abolishes the spacer domain binding exosite (9,13). Together, these results clearly demonstrate an essential role for VWF residue Leu1603, which is N terminal to the scissile bond, acting with VWF P1 residue, Tyr1605, in the cleavage process.…”

Section: Resultsmentioning

confidence: 99%

“…Once the A2 domain has unraveled, however, multiple exosite interactions contribute to the approximation of ADAMTS13 metalloprotease domain with the VWF Tyr1605-Met1606 scissile bond (9-16). The characterized functionally important interactions include the binding of the ADAMTS13 spacer domain with amino acids in the C-terminal region of the VWF A2 domain (9,12,14), and the interaction of the ADAMTS13 disintegrin-like domain with Asp1614 in VWF (11). Remarkably, little is known of the molecular interactions between the substrate and the metalloprotease domain of ADAMTS13.…”

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confidence: 99%

Mechanism of von Willebrand factor scissile bond cleavage by a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13)

Xiang

Groot²,

Crawley³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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The platelet-tethering function of von Willebrand factor (VWF) is proteolytically regulated by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), which cleaves the Tyr1605-Met1606 (P1-P1′) bond in the VWF A2 domain. To date, most of the functional interactions between ADAMTS13 and VWF that have been characterized involve VWF residues that are C terminal to the scissile bond. We now demonstrate that the substrate P3 position in VWF, Leu1603, is a critical determinant of VWF proteolysis. When VWF Leu1603 was substituted with Ser, Ala, Asn, or Lys in a short VWF substrate, VWF115, proteolysis was either greatly reduced or ablated (up to 400-fold reduction in k cat /K m ). As Leu1603 must interact with residues proximate to the Zn 2+ ion coordinated in the active center of ADAMTS13, we sought the corresponding S3 interacting residues. Substitution of 10 candidate residues in the metalloprotease domain of ADAMTS13 identified two spatially separated clusters centered on Leu198 or Val195 (acting with Leu232 and Leu274, or with Leu151, respectively), as possible subsites interacting with VWF. These experimental findings using the short VWF115 substrate were replicated using full-length VWF. It is hypothesized that VWF Leu1603 interacts with ADAMTS13 Leu198/Leu232/Leu274 and that Val195/Leu151 may form part of a S1 subsite. The recognition of VWF Leu1603 by ADAMTS13, in conjunction with previously reported remote exosites C terminal of the cleavage site, suggests a mechanism whereby the VWF P1-P1′ scissile bond is brought into position over the active site for cleavage. Together with recently characterized remote exosite interactions, these findings provide a general framework for understanding the ADAMTS family substrate interactions. microvascular thrombosis | thrombotic thrombocytopenia purpura V on Willebrand factor (VWF) is a large multimeric glycoprotein that is essential for normal hemostasis (1). Following vessel injury, VWF binds to exposed subendothelial collagen. Thereafter, in response to the shear forces exerted by the flowing blood, it unfolds from its inactive globular conformation into an active string-like form that can specifically recruit platelets (2-4). VWF multimeric size is a primary determinant of its platelettethering function and is proteolytically controlled by the plasma metalloprotease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) (5-8). The physiological importance of this system is highlighted by the clinical sequelae associated with dysfunction of the VWF/ADAMTS13 axis. Whereas ADAMTS13 deficiency can cause fatal microvascular thrombosis-thrombotic thrombocytopenic purpura (7), excessive VWF proteolysis causes bleeding (i.e., type 2A von Willebrand disease) (1).ADAMTS13 circulates in plasma as a constitutively active enzyme, which is unusual. Despite this behavior, plasma VWF remains essentially resistant to ADAMTS13 proteolysis in free circulation. This resistance is because shear-dependent unfolding...

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“…Multiple VWF‐binding exosites have been identified across a number of ADAMTS‐13 domains 5, which have informed the development of a so‐called ‘molecular zipper’ model of interaction and proteolysis 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16. An interaction occurs between ADAMTS‐13 and globular VWF, in which the distal C‐terminal tail of ADAMTS‐13 and the C‐terminal D4‐CK domains of VWF make contact 17.…”

Section: Introductionmentioning

confidence: 99%

“…This moderate affinity binding (K D of ~80–120 n m ) interaction has been described as a positioning one, allowing a small proportion of ADAMTS‐13 to circulate in complex with VWF 5, 18. Much tighter binding occurs following A2 domain unfolding, between the ADAMTS‐13 spacer domain exosites (Arg659, Arg660, Tyr661 and Tyr665) and the newly exposed VWF A2 residues Asn1651‐Arg1668 13. Next, exosites in the cysteine‐rich domain (Gly471‐Val474) and the disintegrin‐like domain (Arg349 and Leu350) of ADAMTS‐13 interact with complementary binding sites in the A2 domain, progressively closer to the cleavage site 9, 12.…”

Section: Introductionmentioning

confidence: 99%

Conformational quiescence of ADAMTS‐13 prevents proteolytic promiscuity

South

Freitas

Lane

2016

Journal of Thrombosis and Haemostasis

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Essentials Recently, ADAMTS‐13 has been shown to undergo substrate induced conformation activation.Conformational quiescence of ADAMTS‐13 may serve to prevent off‐target proteolysis in plasma.Conformationally active ADAMTS‐13 variants are capable of proteolysing the Aα chain of fibrinogen.This should be considered as ADAMTS‐13 variants are developed as potential therapeutic agents. Click to hear Dr Zheng's presentation on structure function and cofactor-dependent regulation of ADAMTS‐13 SummaryBackgroundRecent work has revealed that ADAMTS‐13 circulates in a ‘closed’ conformation, only fully interacting with von Willebrand factor (VWF) following a conformational change. We hypothesized that this conformational quiescence also maintains the substrate specificity of ADAMTS‐13 and that the ‘open’ conformation of the protease might facilitate proteolytic promiscuity.ObjectivesTo identify a novel substrate for a constitutively active gain of function (GoF) ADAMTS‐13 variant (R568K/F592Y/R660K/Y661F/Y665F).MethodsFibrinogen proteolysis was characterized using SDS PAGE and liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Fibrin formation was monitored by turbidity measurements and fibrin structure visualized by confocal microscopy.Results ADAMTS‐13 exhibits proteolytic activity against the Aα chain of human fibrinogen, but this is only manifest on its conformational activation. Accordingly, the GoF ADAMTS‐13 variant and truncated variants such as MDTCS exhibit this activity. The cleavage site has been determined by LC‐MS/MS to be Aα chain Lys225‐Met226. Proteolysis of fibrinogen by GoF ADAMTS‐13 impairs fibrin formation in plasma‐based assays, alters clot structure and increases clot permeability. Although GoF ADAMTS‐13 does not appear to proteolyse preformed cross‐linked fibrin, its proteolytic activity against fibrinogen increases the susceptibility of fibrin to tissue‐type plasminogen activator (t‐PA)‐induced lysis by plasmin and increases the fibrin clearance rate more than 8‐fold compared with wild‐type (WT) ADAMTS‐13 (EC50 values of 3.0 ± 1.7 nm and 25.2 ± 9.7 nm, respectively) in in vitro thrombosis models.ConclusionThe ‘closed’ conformation of ADAMTS‐13 restricts its specificity and protects against fibrinogenolysis. Induced substrate promiscuity will be important as ADAMTS‐13 variants are developed as potential therapeutic agents against thrombotic thrombocytopenic purpura (TTP) and other cardiovascular diseases.

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An autoantibody epitope comprising residues R660, Y661, and Y665 in the ADAMTS13 spacer domain identifies a binding site for the A2 domain of VWF

Cited by 118 publications

References 52 publications

High-resolution epitope mapping by HX MS reveals the pathogenic mechanism and a possible therapy for autoimmune TTP syndrome

High-resolution epitope mapping by HX MS reveals the pathogenic mechanism and a possible therapy for autoimmune TTP syndrome

Mechanism of von Willebrand factor scissile bond cleavage by a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13)

Conformational quiescence of ADAMTS‐13 prevents proteolytic promiscuity

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