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Relative Importance of the Glycoprotein Ib-Binding Domain and the RGD Sequence of von Willebrand Factor for Its Interaction With Endothelial Cells
Christelle Perrault,
Hanneke Lankhof,
Dominique Pidard,
Danièle Kerbiriou-Nabias,
Jan J. Sixma,
Dominique Meyer, and
Dominique Baruch
From INSERM U143, Hopital de Bicetre, Bicetre, France; the Department of Hematology, University Hospital, Utrecht, The Netherlands; and Unité Associée Institut Pasteur/INSERM U285, Institut Pasteur, Paris, France.
Endothelial cell adhesion to von Willebrand Factor is mainly mediated through an interaction between the  v 3 integrin and the RGD sequence of von Willebrand factor (vWF ). To define the potential involvement of glycoprotein Ib (GPIb) 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 IIb3, and  A1-rvWF with a deletion between amino-acids 478 and 716, which does not bind to platelet GPIb. 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 A1-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- (TNF), reported to upregulate the expression of the putative endothelial GPIb, did not modify adhesion to these rvWF. Monoclonal antibodies to vWF or GPIb, blocking vWF interaction with platelet GPIb, were unable to inhibit endothelial cell adhesion to rvWF. In contrast, antibody 9 to vWF, blocking the v3-dependent endothelial cell adhesion to plasma vWF, inhibited adhesion to WT-rvWF as efficiently as to A1-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 GPIb-binding domain, we were unable to detect endothelial surface expression of GPIb by flow cytometry or in cell lysates by immunoprecipitation followed by immunoblotting. Moreover, expression of GPIb mRNA was undetectable in endothelial cells, even after stimulation by TNF. These studies indicate that GPIb 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 v3-dependent, GPIb-independent mechanism.
Blood, Vol. 90 No. 6 (September 15), 1997:
pp. 2335-2344
© 1997 by The American Society of Hematology.
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