Periodic Reporting for period 1 - VWF and NETs (Investigating the interplay between VWF, platelets, and neutrophil extracellular traps inpathologies involving thrombosis of the microvasculature) Reporting period: 2017-10-03 to 2019-10-02 Summary of the context and overall objectives of the project Thrombosis is a complex process involving activation of endothelial cells, and their release of Weibel-Palade body contents such as von Willebrand factor (VWF) and P-selectin. This in turn recruits platelets which form aggregates, and leukocytes such as neutrophils. The recent discovery of neutrophil extracellular traps (NETs) described a novel antimicrobial function of neutrophils. NETs are extracellular chromatin strands containing microbicidal proteins. The release of histones, serine proteases, and myeloperoxidase concentrated on DNA fibers into the extracellular space can contribute to many pathologies. NETs can bind platelets and red blood cells and therefore also contribute to thrombosis. Activated platelets can in turn stimulate neutrophils to make NETs, creating a vicious thrombo-inflammatory cycle.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. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far We isolated neutrophils from healthy volunteers, incubated them with VWF or bovine serum albumin (BSA, as a protein control), and stimulated them using stimuli representative of different triggers/pathways of NET formation (ionomycin, PMA, or bacterial lipopolysaccharides), under static conditions. We visualized NET production by fluorescence microscopy and measured production of reactive oxygen species (ROS) using flow cytometry. We also compared NET formation within blood circulation in VWF+/+ and VWF-/- animals using a mouse models of microvascular occlusion leading to multiorgan failure.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. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) In addition to normal hemostasis, there is increasing evidence that the VWF/ADAMTS13 axis is also involved in neutrophil-mediated inflammatory disease. VWF can directly interact with neutrophils via the same receptors that have been previously implicated in neutrophil extracellular trap (NET) formation. We hypothesized that VWF, at the interface of thrombosis and inflammation, could play a role in neutrophil activation leading to NETosis. The addition of VWF to neutrophils under static conditions reduced NET formation in vitro. In an in vivo mouse model involving microvascular occlusions, VWF-deficiency resulted in exacerbated NET formation. VWF may serve as an important regulator of neutrophil activation/NETosis, particularly during thrombo-inflammatory disease. This interesting new finding provides a basis for future research into how VWF can serve as a potential regulator of neutrophil activation, which could be broadly applicable in thrombotic disease. Representative image of NETs
