Relative Importance of the Glycoprotein Ib-Binding Domain and the RGD Sequence of von Willebrand Factor for Its Interaction With Endothelial Cells

Abstract

AbstractEndothelial cell adhesion to von Willebrand Factor is mainly mediated through an interaction between the αvβ3 integrin and the RGD sequence of von Willebrand factor (vWF ). To define the potential involvement of glycoprotein Ibα (GPIbα) as an endothelial vWF receptor, we compared cell adhesion to three recombinant vWF, the wild-type (WT-rvWF ) and two mutants, RGGS-rvWF (D1746G), defective for binding to platelet αIIbβ3, and ΔA1-rvWF with a deletion between amino-acids 478 and 716, which does not bind to platelet GPIbα. Adhesion of human umbilical vein endothelial cells to purified vWF recombinants was measured by automatized cell counting using an image analyzer. Whereas cell adhesion to ΔA1-rvWF was unchanged compared with WT-rvWF, reaching a plateau of 40% total cells at a concentration of 2.5 μg/mL rvWF, adhesion to RGGS-rvWF was only 10% of total cells. Cell stimulation by tumor necrosis factor-α (TNFα), reported to upregulate the expression of the putative endothelial GPIbα, did not modify adhesion to these rvWF. Monoclonal antibodies to vWF or GPIbα, blocking vWF interaction with platelet GPIbα, were unable to inhibit endothelial cell adhesion to rvWF. In contrast, antibody 9 to vWF, blocking the αvβ3-dependent endothelial cell adhesion to plasma vWF, inhibited adhesion to WT-rvWF as efficiently as to ΔA1-rvWF (50% inhibition at a concentration of 11 and 15 μg/mL, respectively). In agreement with the fact that endothelial cell adhesion to vWF appeared independent of the GPIbα-binding domain, we were unable to detect endothelial surface expression of GPIbα by flow cytometry or in cell lysates by immunoprecipitation followed by immunoblotting. Moreover, expression of GPIbα mRNA was undetectable in endothelial cells, even after stimulation by TNFα. These studies indicate that GPIbα is not expressed in human cultured endothelial cells and is not involved in adhesion to vWF-containing surfaces. Thus, in static conditions, cultured endothelial cells adhere to vWF through an αvβ3-dependent, GPIbα-independent mechanism.