A novel binding site for ADAMTS13 constitutively exposed on the surface of globular VWF

Zanardelli, Sara; Chion, Alain; Groot, Evelyn; Lenting, Peter J.; McKinnon, Thomas A. J.; Laffan, Michael; Tseng, Michelle; Lane, David A.

doi:10.1182/blood-2009-05-224915

Cited by 99 publications

(128 citation statements)

References 40 publications

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“…Several studies have shown that the carboxy-terminal TSP2-8 and CUB1-2 domains are important for the processing of VWF by ADAMTS13. 22,30,31 Zheng and coworkers showed that platelet counts on admission were lower in patients with anti-TSP2-8 and/or anti-CUB1-2 IgG. 19 This observation suggests that antibodies directed towards the carboxy-terminal domains inhibit ADAMTS13 activity or alternatively enhance its clearance from the circulation.…”

Section: Discussionmentioning

confidence: 99%

Residues Arg568 and Phe592 contribute to an antigenic surface for anti-ADAMTS13 antibodies in the spacer domain

Pos¹,

Sorvillo²,

Fijnheer³

et al. 2011

Haematologica

118

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The online version of this article has a Supplementary Appendix. BackgroundThe majority of patients diagnosed with thrombotic thrombocytopenic purpura have autoantibodies directed towards the spacer domain of ADAMTS13. Design and MethodsIn this study we explored the epitope specificity and immunoglobulin class and immunoglobulin G subclass distribution of anti-ADAMTS13 antibodies. The epitope specificity of antispacer domain antibodies was examined using plasma from 48 patients with acute acquired thrombotic thrombocytopenic purpura by means of immunoprecipitation of ADAMTS13 variants containing single or multiple alanine substitutions. Using similar methods, we also determined the presence of anti-TSP2-8 and CUB1-2 domain antibodies in this cohort of patients.

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Section: Discussionmentioning

confidence: 99%

Residues Arg568 and Phe592 contribute to an antigenic surface for anti-ADAMTS13 antibodies in the spacer domain

Pos¹,

Sorvillo²,

Fijnheer³

et al. 2011

Haematologica

118

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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. 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.…”

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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“…This resistance is because shear-dependent unfolding of the VWF A2 domain is first required to expose both ADA-MTS13 binding sites and the Tyr1605-Met1606 scissile bond before cleavage can occur. 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)(10)(11)(12)(13)(14)(15)(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).…”

mentioning

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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A novel binding site for ADAMTS13 constitutively exposed on the surface of globular VWF

Cited by 99 publications

References 40 publications

Residues Arg568 and Phe592 contribute to an antigenic surface for anti-ADAMTS13 antibodies in the spacer domain

Residues Arg568 and Phe592 contribute to an antigenic surface for anti-ADAMTS13 antibodies in the spacer domain

Conformational quiescence of ADAMTS‐13 prevents proteolytic promiscuity

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

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