Enhanced activity of ADAMTS13 variant (R568K/F592Y/R660K/Y661F/Y665F) against platelet agglutination in vitro and in a murine model of acute ischaemic stroke
File(s)
Author(s)
South, Kieron
Denorme, Frederik
Salles, Isabelle
De Meyer, Simon
Lane, David
Type
Journal Article
Abstract
Background
ADAMTS13 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) ADAMTS13 variant (R568K/F592Y/R660K/Y661F/Y665F) is conformationally pre‐activated.
Objectives
To establish how the hyperactivity of GoF ADAMTS13 is manifest in experimental models mimicking the occlusive arterial thrombi present in acute ischaemic stroke.
Methods
The ability of GoF ADAMTS13 to dissolve VWF‐platelet agglutinates was examined using an assay of ristocetin‐induced platelet agglutination and in parallel flow models of arterial thrombosis. A murine model of focal ischaemia was used to assess the thrombolytic potential of GoF ADAMTS13.
Results
WT ADAMTS13 required conformational activation to attain full activity against VWF‐mediated platelet capture under flow. In this assay GoF ADAMTS13 had an EC50 value >5‐fold lower than wild type (WT) (0.73±0.21 nM and 3.81±0.97 nM, respectively). The proteolytic activity of GoF ADAMTS13 against pre‐formed platelet agglutinates under flow was enhanced >4‐fold compared to WT (EC50 values of 2.5±1.1 nM and 10.2±5.6 nM, respectively). In a murine stroke model GoF ADAMTS13 restored cerebral blood flow at a lower dose than WT ADAMTS13 and partially retained the ability to recanalise vessels when administration was delayed by 1 hour.
Conclusion
The limited proteolytic activity of WT ADAMTS13 in in vitro models of arterial thrombosis suggests an in vivo requirement for conformational activation. The enhanced activity of the GoF ADAMTS13 variant translates to a more pronounced protective effect in experimental stroke.
ADAMTS13 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) ADAMTS13 variant (R568K/F592Y/R660K/Y661F/Y665F) is conformationally pre‐activated.
Objectives
To establish how the hyperactivity of GoF ADAMTS13 is manifest in experimental models mimicking the occlusive arterial thrombi present in acute ischaemic stroke.
Methods
The ability of GoF ADAMTS13 to dissolve VWF‐platelet agglutinates was examined using an assay of ristocetin‐induced platelet agglutination and in parallel flow models of arterial thrombosis. A murine model of focal ischaemia was used to assess the thrombolytic potential of GoF ADAMTS13.
Results
WT ADAMTS13 required conformational activation to attain full activity against VWF‐mediated platelet capture under flow. In this assay GoF ADAMTS13 had an EC50 value >5‐fold lower than wild type (WT) (0.73±0.21 nM and 3.81±0.97 nM, respectively). The proteolytic activity of GoF ADAMTS13 against pre‐formed platelet agglutinates under flow was enhanced >4‐fold compared to WT (EC50 values of 2.5±1.1 nM and 10.2±5.6 nM, respectively). In a murine stroke model GoF ADAMTS13 restored cerebral blood flow at a lower dose than WT ADAMTS13 and partially retained the ability to recanalise vessels when administration was delayed by 1 hour.
Conclusion
The limited proteolytic activity of WT ADAMTS13 in in vitro models of arterial thrombosis suggests an in vivo requirement for conformational activation. The enhanced activity of the GoF ADAMTS13 variant translates to a more pronounced protective effect in experimental stroke.
Date Issued
2018-11
Date Acceptance
2018-08-03
Citation
Journal of Thrombosis and Haemostasis, 2018, 16 (11), pp.2289-2299
ISSN
1538-7836
Publisher
Wiley
Start Page
2289
End Page
2299
Journal / Book Title
Journal of Thrombosis and Haemostasis
Volume
16
Issue
11
Copyright Statement
© 2018 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Sponsor
British Heart Foundation
British Heart Foundation
British Heart Foundation
British Heart Foundation
Identifier
https://onlinelibrary.wiley.com/doi/full/10.1111/jth.14275
Grant Number
PG/12/55/29740
PG/14/87/3118
FS/10/004/28165
PG/14/90/31219
Subjects
Science & Technology
Life Sciences & Biomedicine
Hematology
Peripheral Vascular Disease
Cardiovascular System & Cardiology
ADAMTS-13 protein
conformational activation
hemostasis
stroke
von Willebrand factor
VON-WILLEBRAND-FACTOR
MYOCARDIAL ISCHEMIA/REPERFUSION INJURY
CONFORMATIONAL ACTIVATION
VICINAL CYSTEINES
RESISTANT
VWF
DEFICIENCY
PROTEASE
ADAMTS-13 protein
conformational activation
hemostasis
stroke
von Willebrand factor
Cardiovascular System & Hematology
1102 Cardiorespiratory Medicine and Haematology
1103 Clinical Sciences
Publication Status
Published
Date Publish Online
2018-08-28