title: 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 SUMMARY: Background Recent 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. Objectives To identify a novel substrate for a constitutively active gain of function (GoF) ADAMTS-13 variant (R568K/F592Y/R660K/Y661F/Y665F). Methods Fibrinogen 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. Conclusion The '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.
authors: Centre for Haematology, Imperial College London, London, UK. ksouth@imperial.ac.uk.
authors: K
authors: K
authors: South
authors: Centre for Haematology, Imperial College London, London, UK.
authors: M O
authors: MO
authors: Freitas
authors: Centre for Haematology, Imperial College London, London, UK.
authors: D A
authors: DA
authors: Lane
mesh_terms: D000071120:ADAMTS13 Protein / Q000737:chemistry* / Q000235:genetics; D001777:Blood Coagulation; D005337:Fibrin / Q000737:chemistry; D005340:Fibrinogen / Q000737:chemistry*; D005341:Fibrinolysin / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D018613:Microscopy, Confocal; D011487:Protein Conformation; D059748:Proteolysis; D011697:Purpura, Thrombotic Thrombocytopenic / Q000097:blood; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D013927:Thrombosis
chemical_list: D005337:Fibrin; D005340:Fibrinogen; D005341:Fibrinolysin; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human
keywords: ADAMTS-13 protein, human; allosteric regulation; fibrinogen; fibrinolysis; protein conformation; von Willebrand factor