The Endothelial Glycocalyx Contributes to the Anchorage of von Willebrand Factor Fibers to the Vascular Endothelium

The von Willebrand factor (vWF) is part of the primary hemostasis and promotes the adhesion of circulating platelets to the activated or damaged endothelium. Increased levels of plasma vWF have been found in patients suffering from severe infections or malignant tumors suggesting that vWF contributes to diseases beyond coagulation disorders. Under quiescent conditions, vWF is stored within endothelial cells in large vesicles, the Weibel-Palade bodies. Upon stimulation, endothelial cells release the densely packed vWF to the blood. The shear force of the blood flow enables the mechanical elongation and thus exposure of the platelet binding domain A1. The required force that mediates the dynamic change from a globular conformation to an elongated fiber depends strongly on the anchorage of vWF to the endothelial cell surface. Previous studies suggested that P-selectin and αvβ3 integrins may bind vWF to the vessel wall, however their precise role is unclear and further binding partners have been proposed. In the present study we investigated whether the endothelial glycocalyx is able to anchor VWF fibers to the endothelium. The endothelial glycocalyx is composed of several carbohydrates while heparan sulfate (HS) is one of the major components. We used microfluidic devices to mimic the pathophysiological environment of malignant melanoma and we followed the binding of vWF fibers and platelets to the endothelial cell surface by fluorescence microscopy. Enzymatic removal of the HS or silencing of the exostosin glycosyltransferase 1, which contributes to the synthesis of HS, reduced the capability of endothelial cells to anchor vWF fibers and to trap platelets. Stabilization of the glycocalyx by chitosan oligosaccharides or overexpression of the HS decorated proteoglycan syndecan-1 (SDC-1) increased the retention of vWF at the cellular interface. The direct molecular interaction between red fluorescent protein tagged SDC-1 and green fluorescent vWF was further documented by live cell fluorescence microscopy and quantitative flow cytometry. In a murine melanoma model, we found that protection of the endothelial glycocalyx through the silencing of the HS degrading heparanase increased the number of vWF fibers anchored to the wall of tumor blood vessels. In conclusion, we identified HS chains exposed by proteoglycans such as SDC-1 as a relevant binding factor for vWF at the endothelial cell surface in vitro and in vivo.