Amino acid residues Arg659, Arg660, and Tyr661 in the spacer domain of ADAMTS13 are critical for cleavage of von Willebrand factor

Jin, Sheng‐Yu; Skipwith, Christopher G.; Zheng, X. Long

doi:10.1182/blood-2009-07-235101

Cited by 72 publications

(46 citation statements)

References 45 publications

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“…1A) were expressed in stably transfected CHO- s cell lines and purified to homogeneity with a combination of Q-fast flow anion exchange and Ni-chelating affinity chromatography 15, 18 . As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…An extensive exosite interaction between the ADAMTS13-DTCS domains and the VWF-A2 domain 11, 17 appears to be necessary for productive VWF cleavage. A mutation or deletion in the DTCS domains 18-20 or an autoantibody that targets the spacer domain or others 19, 21-24 dramatically reduces or inhibits the ability of ADAMTS13 to cleave its VWF substrate. However, the role of more distal C-terminal domains of ADAMTS13 including the 2-8 TSP1 repeats and CUB domains is little known.…”

Section: Introductionmentioning

confidence: 99%

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Carboxyl Terminus of ADAMTS13 Directly Inhibits Platelet Aggregation and Ultra Large von Willebrand Factor String Formation Under Flow in a Free-Thiol–Dependent Manner

Bao

Mao

Zheng

2014

ATVB

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Summary Objective ADAMTS13 cleaves von Willebrand factor (VWF), thereby inhibiting thrombus formation. Proteolytic cleavage relies on the amino-terminal (MDTCS) domains, but the role of the more distal carboxyl-terminal domains of ADAMTS13 is not fully understood. A previous study demonstrated the presence of multiple surface- exposed free sulfhydryls on ADAMTS13 that appeared to interact with those on VWF under shear. Here, we determined the physiological relevance of such an interaction in antithrombotic responses under flow. Approaches and Results A microfluidic assay demonstrated that a carboxyl-terminal fragment of ADAMTS13, comprising either 2-8 thrombospondin type 1 (TSP1) repeats and CUB domains (T2C) or 5-8 TSP1 repeats and CUB domains (T5C), directly inhibited platelet adhesion/aggregation on a collagen surface under arterial shear. In addition, an intravital microscopic imaging analysis showed that the carboxyl-terminal fragment of ADAMTS13 (T2C or T5C) was capable of inhibiting the formation and elongation of platelet-decorated ultra large (UL) VWF strings and the adhesion of platelets/leukocytes on endothelium in mesenteric venules after oxidative injury. The inhibitory activity of T2C and T5C on platelet aggregation and ULVWF string formation was dependent on the presence of their surface free thiols; pretreatment of T2C and T5C or full-length ADAMTS13 with N-ethylmaleimide that reacts with free sulfhydryls abolished or significantly reduced its antithrombotic activity. Conclusion Our results demonstrate for the first time that the carboxyl-terminus of ADAMTS13 has direct antithrombotic activity in a free-thiol dependent manner. The free thiols in the carboxyl-terminal domains of ADAMTS13 may also contribute to the overall antithrombotic function of ADAMTS13 under pathophysiological conditions.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Carboxyl Terminus of ADAMTS13 Directly Inhibits Platelet Aggregation and Ultra Large von Willebrand Factor String Formation Under Flow in a Free-Thiol–Dependent Manner

Bao

Mao

Zheng

2014

ATVB

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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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“…Structural predictions of the arginine surrounded hydrophobic cluster have been confirmed by several functional studies. Arg660, Tyr661, and Tyr665 together are essential for VWF binding and cleavage [75,76]. These three residues are also very commonly found in the epitope site of ADAMTS13 antibodies [75,76].…”

Section: Structure and Functionmentioning

confidence: 99%

ADAMTS13 and von Willebrand factor interactions

Zander

Cao

Zheng

2015

Current Opinion in Hematology

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Purpose of review ADAMTS13 is a zinc-containing metalloprotease that cleaves von Willebrand factor (VWF). Deficiency of plasma ADAMTS13 activity is accountable for a potentially fatal blood disorder thrombotic thrombocytopenic purpura (TTP). Understanding of ADAMTS13–VWF interaction is essential for developing novel treatments to this disorder. Recent findings Despite the proteolytic activity of ADAMTS13 being restricted to the metalloprotease domain, the ancillary proximal C-terminal domains including the disintegrin domain, first TSP-1 repeat, cysteine-rich region, and spacer domain are all required for cleavage of VWF and its analogs. Recent studies have added to our understandings of the role of the specific regions in the disintegrin domain, the cysteine-rich domain, and the spacer domain responsible for its interaction with VWF. Additionally, regulative functions of the distal portion of ADAMTS13 including the TSP-1 2–8 repeats and the CUB domains have been proposed. Finally, fine mapping of anti-ADAMTS13 antibody epitopes have provided further insight into the essential structural elements in ADAMTS13 for VWF binding and the mechanism of autoantibody-mediated TTP. Summary Significant progress has been made in our understandings of the structure–function relationship of ADAMTS13 in the past decade. To further investigate ADAMTS13–VWF interactions for medical applications, these interactions must be studied under physiological conditions in vivo.

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Amino acid residues Arg659, Arg660, and Tyr661 in the spacer domain of ADAMTS13 are critical for cleavage of von Willebrand factor

Cited by 72 publications

References 45 publications

Carboxyl Terminus of ADAMTS13 Directly Inhibits Platelet Aggregation and Ultra Large von Willebrand Factor String Formation Under Flow in a Free-Thiol–Dependent Manner

Carboxyl Terminus of ADAMTS13 Directly Inhibits Platelet Aggregation and Ultra Large von Willebrand Factor String Formation Under Flow in a Free-Thiol–Dependent Manner

Conformational quiescence of ADAMTS‐13 prevents proteolytic promiscuity

ADAMTS13 and von Willebrand factor interactions

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