Integrin αvβ3 Is Involved in the Stabilization of Ultra Long von Willebrand Factor Strings under Fluid Shear Stress on Human Endothelial Cells.

Von Willebrand factor (VWF) multimers attached to endothelial cells can provide a platform for thrombosis, especially when accompanied by ADAMTS13 deficiency. We characterized the structural features of ultralarge VWF (ULVWF) molecules acutely secreted from Weibel-Palade bodies and identified a receptor responsible for their binding to the surface of cultured human umbilical vein endothelial cells (HUVECs). Using fluorescence microscopy on live cells, VWF multimers formed extended strings within minutes after stimulation. String formation did not require exogenous platelets and occurred over a range of shear stress from 2.5 dyn/cm² to 40 dyn/cm². A subset of ULVWF strings spontaneously bound formalin-fixed platelets via platelet GPIb. Quick-freeze, deep-etch electron microscopy showed that ULVWF strings often merged to form bundles and networks. Each string was tethered to the endothelial membrane by a limited number of anchorage sites, many of them located on small membrane projections, suggesting a specific mode of interaction. Several independent approaches implicated integrin αvβ3 in anchoring ULVWF strings to the HUVEC surface. Either “RGDS” peptide or function blocking antibody (LM609) to integrin αvβ3 specifically and dose dependently inhibited ULVWF string formation 62 ± 0.7% and 53 ± 4%, respectively. Furthermore, integrin αv was seen decorating the extending ULVWF strings using a non-functional blocking antibody (LM142) in live-cell immunofluorescence. In addition, a lentiviral vector encoding shRNA against integrin αv resulted in approximately 70% reduction in cell surface αv expression by FACS analysis with antibody LM609. HUVEC infected with this lentivirus were significantly impaired in their ability to form ULVWF strings compared to cells infected with a control virus. In multiple experiments, shRNA knockdown of αv expression reduced ULVWF strings 75.3 ± 4.4% at 2.5dyn/cm² and 81.6 ± 2% at 7.5dyn/cm². These results indicated that ULVWF strings bind tightly to endothelial cells via very few anchorage sites and are relatively resistant to fluid shear stress. Although integrin αvβ3 does not appear to be required to stabilize ULVWF strings on mouse endothelium, these data suggest that integrin αvβ3 may participate in the stabilization of ULVWF strings on human endothelial cell surfaces.