Conformational quiescence of ADAMTS13 prevents proteolytic promiscuity

Background Recent work has revealed that ADAMTS13 circulates in a ‘closed’ conformation, only fully interacting with VWF following a conformational change. We hypothesised that this conformational quiescence also maintains the substrate specificity of ADAMTS13 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) ADAMTS13 variant (R568K/F592Y/R660K/Y661F/Y665F) Methods Fibrinogen proteolysis was characterised using SDS PAGE and LC-MS/MS. Fibrin formation was monitored by turbidity measurements and fibrin structure visualised by confocal microscopy Results ADAMTS13 exhibits proteolytic activity against the Aα chain of human fibrinogen, but this is only manifest on its conformational activation. Accordingly, the GoF ADAMTS13 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 ADAMTS13 impairs fibrin formation in plasma based assays, alters clot structure and increases clot permeability. While GoF ADAMTS13 does not appear to proteolyse preformed cross-linked fibrin, its proteolytic activity against fibrinogen increases the susceptibility of fibrin to t-PA induced lysis by plasmin and increases the fibrin clearance rate more than 8 fold compared to WT ADAMTS13 (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 ADAMTS13 restricts its specificity and protects against fibrinogenolysis. Induced substrate promiscuity will be important as ADAMTS13 variants are developed as potential therapeutic agents against TTP and other cardiovascular diseases