Fig. 1. VWF binds to macrophages and triggers inflammatory signaling.

a Binding of plasma-derived VWF (pd-VWF) to primary human macrophages and b THP1 macrophages was assessed in vitro using confocal microscopy as detailed in Materials and Methods (VWF staining in green; nuclear DAPI staining in blue; cell membrane staining in red). c VWF internalization was assessed using THP1 macrophage and anti-VWF antibody staining in green, DAPI and anti-EEA1 (early endosomes antigen 1) antibody staining in red and co-localization in yellow. pd-VWF or recombinant VWF (300–600 nM) were incubated with macrophages for 30 min at 37 °C and cells analyzed by flow cytometry. Representative histograms are presented where red represents control cells not treated with VWF and blue cells treated with VWF. d pd-VWF and e recombinant VWF binding to primary human macrophages; f recombinant VWF binding to THP1 macrophages. Flow gating strategy is presented in Supplementary Fig. 14.Western blot analysis of phosphorylation of p38, JNK, IKBα, and p65 in g primary human and h primary murine macrophages incubated with VWF (10 µg/ml) or LPS (100 ng/ml) for 30 min. All scale bars are 5 μM. All experiments were performed in triplicate and source data for this figure are provided as a Source Data file.