Enhanced activity of an ADAMTS ADAMTS ‐13 variant (R568K/F592Y/R660K/Y661F/Y665F) against platelet agglutination in vitro in vitro and in a murine model of acute ischemic stroke

摘要

Summary Essentials ADAMTS13 requires a substrate‐induced conformational change to attain full activity in vitro . The efficacy of wild type ADAMTS13 in models of thrombosis/stroke may be enhanced by pre‐activation. A pre‐activated ADAMTS13 variant exhibits enhanced proteolysis of platelet agglutinates. This ADAMTS13 variant is protective in a murine model of stroke at a lower dose than WT ADAMTS13. Summary Background ADAMTS‐13 circulates in a closed conformation, only achieving full proteolytic activity against von Willebrand factor (VWF) following a substrate‐induced conformational change. A gain‐of‐function (GoF) ADAMTS‐13 variant (R568K/F592Y/R660K/Y661F/Y665F) is conformationally preactivated. Objectives To establish how the hyperactivity of GoF ADAMTS‐13 is manifested in experimental models mimicking the occlusive arterial thrombi present in acute ischemic stroke. Methods The ability of GoF ADAMTS‐13 to dissolve VWF–platelet agglutinates was examined with an assay of ristocetin‐induced platelet agglutination and in parallel‐flow models of arterial thrombosis. A murine model of focal ischemia was used to assess the thrombolytic potential of GoF ADAMTS‐13. Results Wild‐type (WT) ADAMTS‐13 required conformational activation to attain full activity against VWF‐mediated platelet capture under flow. In this assay, GoF ADAMTS‐13 had an EC 50 value more than five‐fold lower than that of WT ADAMTS‐13 (0.73 ± 0.21 n m and 3.81 ± 0.97 n m , respectively). The proteolytic activity of GoF ADAMTS‐13 against preformed platelet agglutinates under flow was enhanced more than four‐fold as compared with WT ADAMTS‐13 (EC 50 values of 2.5 ± 1.1 n m and 10.2 ± 5.6 n m , respectively). In a murine stroke model, GoF ADAMTS‐13 restored cerebral blood flow at a lower dose than WT ADAMTS‐13, and partially retained the ability to recanalize vessels when administration was delayed by 1 h. Conclusions The limited proteolytic activity of WT ADAMTS‐13 in in vitro models of arterial thrombosis suggests an in vivo requirement for conformational activation. The enhanced activity of the GoF ADAMTS‐13 variant translates to a more pronounced protective effect in experimental stroke.