Relative importance of the glycoprotein Ib-binding domain and the RGD sequence 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 vbeta3 integrin and the RGD sequence of von Willebrand factor (vWF). To define the potential involvement of glycoprotein Ib alpha (GPIb alpha) 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 alphaIIb beta3, and deltaA1-rvWF with a deletion between amino-acids 478 and 716, which does not bind to platelet GPIb alpha. 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 compared with WT-rvWF, reaching a plateau of 40% total cells at a concentration of 2.5 microg/mL rvWF, adhesion to RGGS-rvWF was only 10% of total cells. Cell stimulation by tumor necrosis factor-alpha (TNF alpha), reported to upregulate the expression of the putative endothelial GPIb alpha, did not modify adhesion to these rvWF. Monoclonal antibodies to vWF or GPIb alpha, blocking vWF interaction with platelet GPIb alpha, were unable to inhibit endothelial cell adhesion to rvWF. In contrast, antibody 9 to vWF, blocking the alpha vbeta3-dependent endothelial cell adhesion to plasma vWF, inhibited adhesion to WT-rvWF as efficiently as to deltaA1-rvWF (50% inhibition at a concentration of 11 and 15 microg/mL, respectively). In agreement with the fact that endothelial cell adhesion to vWF appeared independent of the GPIb alpha-binding domain, we were unable to detect endothelial surface expression of GPIb alpha by flow cytometry or in cell lysates by immunoprecipitation followed by immunoblotting. Moreover, expression of GPIb alpha mRNA was undetectable in endothelial cells, even after stimulation by TNF alpha. These studies indicate that GPIb alpha 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 alpha vbeta3-dependent, GPIb alpha-independent mechanism.
Perrault
C
C
INSERM U143, Hopital de Bicetre, France.
Lankhof
H
H
Pidard
D
D
Kerbiriou-Nabias
D
D
Sixma
J J
JJ
Meyer
D
D
Baruch
D
D
D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D002448:Cell Adhesion / Q000187:drug effects; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D009842:Oligopeptides; D010446:Peptide Fragments; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014841:von Willebrand Factor / Q000737:chemistry / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules; D009842:Oligopeptides; D010446:Peptide Fragments; D019038:Platelet Glycoprotein GPIb-IX Complex; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D014841:von Willebrand Factor; C047981:arginyl-glycyl-aspartic acid