COS-induced modification of the endothelial glycocalyx promotes VWF binding. (A) Surfaces of HUVECs, treated with COSs (2µg/mL) or untreated (CTL), imaged by AFM. Height images provide 3-dimensional topography of the cellular surface. The height is false color coded. Black corresponds to a height of 0 µm and white to a height of 2 µm. Overview images are further magnified as indicated by square regions (i-ii). Corresponding lateral deflection images show the interaction between the scanning tip and the cellular surface in the depicted areas (i-ii). Further magnification of the lateral deflection images indicate an increased lateral deflection (white circle) on HUVECs treated with COSs in comparison with nontreated cells (CTL) (iii-iv). Scale bars, 1 µm. (B) Fluorescence microscopic analysis of the endothelial glycocalyx (red) and anchored VWF fibers (green). Cell nuclei are labeled in blue. The endothelial glycocalyx staining was more pronounced in COS-treated HUVECs. The lack of the endothelial glycocalyx staining beneath VWF fibers, which were in close proximity to the HUVEC surface, indicates an occupation of the HS chains by VWF. Scale bars, 10 µm. (C) Quantitative evaluations of VWF fluorescence staining of at least 10 fields of view of 3 independent experiments. **P ≤ .01 (Student t test).