RELATIVE IMPORTANCE OF THE GLYCOPROTEIN IB-BINDING DOMAIN AND THE RGDSEQUENCE OF VON-WILLEBRAND-FACTOR FOR ITS INTERACTION WITH ENDOTHELIAL-CELLS

Endothelial cell adhesion to von Willebrand Factor is mainly mediated through an interaction between the alpha v beta 3 integrin and the RGD sequence of von Willebrand factor (VWF). To define the potential invo lvement of glycoprotein Ib alpha (GPIb alpha) as an endothelial vWF re ceptor, we compared cell adhesion to three recombinant VWF, the wild-t ype (WT-rvWF) and two mutants, RGGS-rVWF (D1746G), defective for bindi ng to platelet alpha Ib beta 3, and Delta A1-rvWF with a deletion betw een amino-acids 478 and 716, which does not bind to platelet GPIb alph a. 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 Delta A1-rvWF was unchanged compare d with WT-rvWF, reaching a plateau of 40% total cells at a concentrati on of 2.5 mu g/ml. rvWF, adhesion to RGGS-rvWF was only 10% of total c ells. Cell stimulation by tumor necrosis factor-alpha (TNF alpha), rep orted to upregulate the expression of the putative endothelial GPIb al pha, did not modify adhesion to these rvWF. Monoclonal antibodies to V WF of GPIb alpha, blocking vWF interaction with platelet GPIb alpha, w ere unable to inhibit endothelial cell adhesion to rvWF. In contrast, antibody 9 to VWF, blocking the alpha v beta 3-dependent endothelial c ell adhesion to plasma vWF, inhibited adhesion to WT-rVWF as efficient ly as to Delta A1-rvWF (50% inhibition at a concentration of 11 and 15 mu g/mL, respectively). In agreement with the fact that endothelial c ell adhesion to vWF appeared independent of the GPIb alpha-binding dom ain, we were unable to detect endothelial surface expression of GPIb a lpha by flow cytometry or in cell lysates by immunoprecipitation follo wed by immunoblotting. Moreover, expression of GPIb alpha mRNA was und etectable in endothelial cells, even after stimulation by TNF alpha. T hese studies indicate that GPIb alpha is not expressed in human cultur ed endothelial cells and is not involved in adhesion to vWf-containing surfaces. Thus, in static conditions, cultured endothelial cells adhe re to VWF through an alpha v beta 3-dependent, GPIb alpha-independent mechanism. (C) 1997 by The American Society of Hematology.