Periodic Reporting for period 1 - VWF and NETs (Investigating the interplay between VWF, platelets, and neutrophil extracellular traps inpathologies involving thrombosis of the microvasculature)
Periodo di rendicontazione: 2017-10-03 al 2019-10-02
Our first objective was to study VWF involvement in the induction of NETosis. We aimed elucidate the role of VWF as an important mediator of NET formation by incubating neutrophils with various sources of VWF (plasma and recombinant). Fluorescence imaging and flow cytometry was used to unravel the kinetics of VWF/platelet-mediated NET formation. Our second objective was to assess the physiological relevance of (inhibiting) platelet/VWF involvement in NETs-related pathology. We investigated the role of VWF/NET interactions in two critical clinical settings where microvascular thrombosis contributes to multiorgan failure and/or mortality: sudden inflammatory response syndrome (SIRS) and severe malaria. Mouse models of disease were used and combined with strategies for inhibition of VWF/platelet/NETs interactions to assess their effect on disease progression.
In summary, with this research project we aimed to better understand how platelets and VWF can drive NET formation in thrombo-inflammation, as this may also lead to more targeted therapeutic approaches in diseases which currently have limited treatment options.
VWF could be visualized within punctate structures upon neutrophil stimulation, and consistently reduced the percentage of neutrophils releasing NETs. To be able to compare multiple donors, the percentage of NET formation was normalized to a condition containing only stimulation medium (defined as 100%). This effect was dose-dependent, and specific to VWF as other plasma proteins did not have the same effect. Factor VIII was dispensible for the reduction in NET release. We obtained mechanistic insight as to how this process is taking place using flow cytometric analysis to investigate signaling pathways, identifying a key player in NETosis as being downregulated upon exposure of neutrophils to VWF.
We next investigated the effect of VWF-deficiency on NET formation in vivo using a mouse model of involving thrombotic occlusion of the microvasculature. Interestingly, VWF-/- animals had a higher percentage of NETs detectable in their blood circulation compared to VWF+/+ mice in this animal model. NET biomarkers were also higher in plasma collected from VWF-/- compared to VWF+/+ mice. However, no difference in NETs was detected in the affected tissues of these mice. Furthermore, no difference was found between disease progression or outcome in VWF+/+ compared to VWF-/-, indicating that the presence of increased NETs did not contribute to an exacerbated disease progression.