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.
