Conformational quiescence of ADAMTS‐13 prevents proteolytic promiscuity

Abstract

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.

ADAMTS13 activity as a novel risk factor for incident type 2 diabetes mellitus: a population-based cohort study

“…After an initial interaction with globular VWF, ADAMTS13 undergoes a conformational change that increases its ability to break down VWF [33]. Recent research suggests that this conformational change increases the ability of ADAMTS13 to break down not only VWF but also other proteins such as fibrinogen [34]. ADAMTS13 activity, as measured in our study, may partly reflect this process.…”

Conformational quiescence of ADAMTS‐13 prevents proteolytic promiscuity: reply

“…However, in our experiments, using the same commercial fibrinogen preparation, we did not induce extensive plasminogen activation, and instead observed limited proteolysis of the Aa chain (South et al [1], fig. 1).…”

“…The findings of Mansfield et al [1] are consistent with the results from a subgroup analysis of patients receiving chemotherapy in the FRAGMATIC study, which were presented at the XXV ISTH Congress [3]. In brief, patients with primary bronchial carcinoma, non-small-cell lung cancer or small-cell lung cancer within 6 weeks of diagnosis and prior to definitive anticancer treatment were enrolled in the FRAGMATIC study: a phase III trial investigating the impact on overall survival of adding low molecular weight heparin to usual care in patients with lung cancer [4].…”

“…We thank Cao and Long Zheng for their interest in our article recently published in JTH [1], in which we reported our findings that C-terminally truncated variants of ADAMTS-13 directly cleave the Aa chain of fibrinogen. We postulated that conformationally activated ADAMTS-13 [2,3] may be able to cleave substrate(s) in addition to its usual substrate, the A2 domain of von Willebrand factor (VWF).…”

“…Predictors of active cancer thromboembolic outcomes: validation of the Khorana score among patients with lung cancer: comment We read with interest the article by Mansfield et al [1] in which the authors report their attempt to validate the Khorana Risk Score (KRS) [2] in the prediction of venous thromboembolism (VTE) among ambulant patients with lung cancer receiving chemotherapy. In their prospectively collected lung cancer database of 719 patients, there were 83 (11.5%) episodes of VTE.…”