Extensive contacts between ADAMTS13 exosites and von Willebrand factor domain A2 contribute to substrate specificity

Gao, Wei‐Qiang; Anderson, Patricia J.; Sadler, J. Evan

doi:10.1182/blood-2008-04-148759

Cited by 95 publications

(152 citation statements)

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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%

“…To investigate further the VWF residues important for cleavage by ADAMTS13, we have considered the activities of previously reported short VWF A2 domain fragments that span the cleavage site. One, VWF64 (1605-1668), could not be cleaved by ADAMTS13 (13), but others, such as VWF73 (1596-1668), VWF76 (1593-1668), and VWF115 (1554-1668) are all proteolysed efficiently (14,18,19). The difference between the substrates that are and those that are not cleaved resides in the sequence N terminal to the scissile bond.…”

mentioning

confidence: 99%

“…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).…”

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confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…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%

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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“…We have previously reported a minimal functional substrate consisting of 73 amino-acid residues of the C-terminal region of the VWF A2 domain (Asp1596-Arg1668) and designated VWF73 (Kokame et al, 2004;Miyata et al, 2007). A recent study has shown that the VWF-binding exosites located in the T, C and S domains interact with different segments of VWF73 (Gao et al, 2008). These interactions increased the VWF-binding affinity and rate of substrate cleavage by 300-fold.…”

Section: Introductionmentioning

confidence: 99%

Production, crystallization and preliminary crystallographic analysis of an exosite-containing fragment of human von Willebrand factor-cleaving proteinase ADAMTS13

et al. 2009

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ADAMTS13 is a reprolysin-type metalloproteinase belonging to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin type 1 motif) family. It specifically cleaves plasma von Willebrand factor (VWF) and regulates platelet adhesion and aggregation. ADAMTS13 is a multi-domain enzyme. In addition to the N-terminal metalloproteinase domain, the ancillary domains, including a disintegrin-like domain, a thrombospondin-1 type 1 repeat, a Cysrich domain and a spacer domain, are required for VWF recognition and cleavage. In the present study, a fragment of the ADAMTS13 ancillary domains (ADAMTS13-DTCS; residues 287-685) was expressed using CHO Lec cells, purified and crystallized. Diffraction data sets were collected using the SPring-8 beamline. Two ADAMTS13-DTCS crystals with distinct unit-cell parameters generated data sets to 2.6 and 2.8 Å resolution, respectively.

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Extensive contacts between ADAMTS13 exosites and von Willebrand factor domain A2 contribute to substrate specificity

Cited by 95 publications

References 36 publications

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

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

Production, crystallization and preliminary crystallographic analysis of an exosite-containing fragment of human von Willebrand factor-cleaving proteinase ADAMTS13

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