Selective inactivation of the Arg-Gly-Asp-Ser (RGDS) binding site in von Willebrand factor by site-directed mutagenesis.
In order to assess the requirement for the Arg-Gly-Asp-Ser (RGDS) consensus adhesion sequence in von Willebrand factor (vWF) for vWF binding to platelets and endothelial cells, point mutations were introduced into this sequence by site-directed mutagenesis. A glycine to alanine substitution yielded RADS-vWF, while an aspartate to glutamate substitution resulted in RGES-vWF. Recombinant RADS-vWF and RGES-vWF, purified from transformed Chinese hamster ovary cells, were compared with recombinant wild type vWF (WT-vWF) in functional assays with platelets and human umbilical vein endothelial cells (HU-VECs). High molecular weight RADS-vWF and RGES-vWF multimers inhibited binding of 125I-vWF to a mixture of insolubilized native type I and III collagen and competed effectively with 125I-vWF for binding to formalin-fixed platelets in the presence of ristocetin, indicating functional collagen and platelet glycoprotein Ib binding. However, RADS-vWF and RGES-vWF were unable to displace the binding of 125I-vWF to thrombin or ADP-activated platelets. The attachment of HUVECs to either RADS-vWF or RGES-vWF coated surfaces was reduced and spreading was almost completely inhibited, compared with WT-vWF. We conclude that point mutations of the RGDS sequence in vWF selectively impair binding to platelet glycoprotein IIb/IIIa and the HUVEC vitronectin receptor.
Beacham
D A
DA
Brigham and Women's Hospital Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115.
Wise
R J
RJ
Turci
S M
SM
Handin
R I
RI
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism; D002448:Cell Adhesion; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003094:Collagen / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides; D010957:Plasmids; D015539:Platelet Activation; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D012310:Ristocetin / Q000494:pharmacology; D014162:Transfection; D014841:von Willebrand Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides; D010980:Platelet Membrane Glycoproteins; D014841:von Willebrand Factor; D012310:Ristocetin; D003094:Collagen; C046617:arginyl-glycyl-aspartyl-serine