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S. Staessens et al.
Our observation that vWF is closely associated with fib- rin in platelet-rich regions further supports a potentially novel link between vWF and fibrin in thrombus forma- tion. It has been shown in vitro that fibrin and vWF can interact with each other via covalent crosslinking by FXIII or via thrombin-dependent incorporation, enhancing thrombus formation.32-34 Our histological data on vWF and fibrin indicate that fibrin degradation via plasmin alone might not be sufficient to achieve effective thrombolysis of platelet-rich thrombus material. Indeed, we and others have recently shown that targeting vWF, for example using the vWF-cleaving enzyme ADAMTS13, improves thrombolysis of rt-PA-resistant thrombi, reducing ischemic stroke brain injury in mice.19,35 Of note, plasmin can also cleave vWF, and plasmin activity was shown to regulate ADAMTS13 activity.36-38 It will be interesting to further elucidate the interplay between plasmin and ADAMTS13 in the degradation of fibrin/vWF structures that are present in ischemic stroke thrombi.
Taken together, our detailed histological analysis reveals different structural features in ischemic stroke thrombi that could be highly relevant for developing efficient phar- macological thrombolysis strategies. The presence of fib- rin, vWF and DNA could explain why the current ‘one size fits all’ therapy aiming only at fibrinolysis via rt-PA is not effective in all patients. Furthermore, we found that some thrombi consist of an RBC-rich core surrounded by a dense platelet-rich shell, which could further hamper rt- PA-mediated thrombolysis.
As far as thrombectomy is concerned, emerging evi- dence indicates that RBC-rich stroke thrombi are more easily retrieved via endovascular procedures in compari- son to more complex fibrin/platelet-rich thrombi.15,39 Even though it may seem intuitive that the retriever devices and techniques available today favor softer thrombi, the mechanisms that render platelet-rich thrombi more resist- ant to thrombectomy are not completely understood. Weafer et al. have recently shown that the degree of clot integration into the thrombectomy device is decreased in fibrin-rich thrombi, making these thrombi more resistant to mechanical removal.40 Our study reveals that platelet- rich thrombus material from ischemic stroke patients has particular features that include dense fibrin/vWF struc- tures, leukocytes and DNA. Thick fibrin strands have been shown to increase clot rigidity and fibrin was shown to influence the thrombus coefficient of friction and level of physical compression.10,41,42 Interestingly, whether or not the association of vWF with fibrin influences the mechan- ical properties of thrombi needs to be further investigated. Of note, DNA is able to modify the structure of fibrin, ren- dering it more resistant to mechanical forces.28,42 In fact, Ducroux et al. found a positive correlation between the amount of neutrophil extracellular DNA traps and the number of device passes needed to achieve successful
recanalization.15 Further studies are now needed to better understand how fibrin, DNA, vWF and platelets influence not only the mechanical properties of the occluding thrombus, but also its interaction with the thrombectomy device and the vessel wall.
This study has several limitations that are worth consid- ering. First, only thrombi from those patients in whom the thrombus did not dissolve spontaneously or during prior rt- PA treatment, and in whom the thrombus could be success- fully retrieved, were available for study. This impedes the assessment of thrombi that were rt-PA susceptible or thrombectomy resistant. Second, the scope of this study was focused on the description of common structural fea- tures of patient stroke thrombi, and did not include clinical and procedural parameters. Ongoing research on larger sets of stroke thrombi are needed to further elucidate the clini- cal impact of the described structural features. Our hypoth- esis is that the relative contribution of platelet-rich and RBC-rich areas will most likely determine the success rate of any given pharmacological and endovascular recanaliza- tion strategy. Interestingly, non-contrast computed tomog- raphy (NCCT) and magnetic resonance imaging are able to identify the presence of RBC-dominant thrombi via the presence of a hyperdense artery sign or a blooming artefact.43,44 Such information could guide treatment selec- tion in the future. Better understanding of thrombus com- position and its link with stroke etiology could also help to assess the likely thrombus origin in patients with embolic stroke of undetermined source, and to guide the develop- ment of targeted strategies for secondary stroke prevention.
As a final note, we emphasize that we describe typical features that are generally found in the majority of ischemic stroke thrombi, and that the heterogeneity between thrombi (Figure 2) does not allow for a single typical thrombus model.
In conclusion, we show that stroke thrombi consist of RBC-rich areas and platelet-rich areas. We found that RBC-rich areas have a limited complexity, while platelet- rich areas are characterized by dense fibrin structures aligned with vWF and abundant amounts of leukocytes and extracellular DNA. These findings are important to further improve acute ischemic stroke therapy, especially concerning platelet-rich thrombi that are rt-PA resistant and difficult to retrieve via thrombectomy.
Funding
This work was supported by research grants to SFDM from the Fonds voor Wetenschappelijk Onderzoek – Vlaanderen (FWO) (research grants G.0A86.13, G.0785.17 and 1509216N), the KU Leuven (OT/14/099 and ISP/14/02L2), the Queen Elisabeth Medical Foundation and by the European Union's Horizon 2020 Research and Innovation Program INSIST under grant agreement No 777072. FD is a postdoctoral fellow of the FWO (12U7818N).
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