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

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  • Authors:
  • K. South
  • F. Denorme
  • I. I. Salles-crawley
  • S. F. De Meyer
  • D. A. Lane

Abstract

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 nm and 3.81 ± 0.97 nm, 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 nm and 10.2 ± 5.6 nm, 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.

Bibliographical metadata

Original languageEnglish
Pages (from-to)2289-2299
Number of pages11
JournalJournal of Thrombosis and Haemostasis
Volume16
Issue number11
Early online date28 Aug 2018
DOIs
Publication statusPublished - Nov 2018

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