Abstract
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. SummaryBackgroundADAMTS‐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.ObjectivesTo establish how the hyperactivity of GoF ADAMTS‐13 is manifested in experimental models mimicking the occlusive arterial thrombi present in acute ischemic stroke.MethodsThe 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.ResultsWild‐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 EC50 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 (EC50 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.ConclusionsThe 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.
Highlights
Evidence has been accumulating that implicates von Willebrand factor (VWF) as an important factor in the pathophysiology of acute ischemic stroke (AIS) [1]
When WT ADAMTS-13 was preincubated with 100 nM VWF D4-CK prior to being added to the assay, there was a significant decrease (P < 0.005) in the platelet coverage recorded at the end of the assay (Fig. 1A,B)
The extent of the decrease in platelet capture in the presence of conformationally activated WT ADAMTS-13 was similar to that observed in the presence of GoF ADAMTS-13 (Fig. 1A,B)
Summary
Evidence has been accumulating that implicates von Willebrand factor (VWF) as an important factor in the pathophysiology of acute ischemic stroke (AIS) [1]. VWF is an adhesive plasma glycoprotein (GP) that is secreted and circulates as large globular multimers [2,3,4,5]. In this globular conformation, VWF is quiescent and unable to capture platelets. At sites of vascular damage, VWF is tethered to the vessel wall through its constitutively exposed collagen-binding site in the A3 domain [6,7]. Under the shear force of flowing blood, the unfolding of its A2 domain [8,9,10] induces conformational changes at a macromolecular scale [11], and local conformational changes in the A1 domain expose the previously cryptic platelet GP)1b-binding site [11]. The ability of VWF to capture platelets and to initiate the formation of the platelet plug is dependent on its multimeric size, and is regulated by the plasma metalloprotease ADAMTS-13 [12,13]
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