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Selective inactivation of the Arg-Gly-Asp-Ser (RGDS) binding site in von Willebrand factor by site-directed mutagenesis.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 1992 Feb 15; Vol. 267 (5), pp. 3409-15. - Publication Year :
- 1992
-
Abstract
- 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.
- Subjects :
- Animals
Base Sequence
Binding Sites
Blood Platelets metabolism
Cell Adhesion
Cell Line
Cells, Cultured
Cloning, Molecular
Collagen metabolism
Endothelium, Vascular metabolism
Escherichia coli genetics
Humans
Kinetics
Molecular Sequence Data
Oligodeoxyribonucleotides
Plasmids
Platelet Activation
Platelet Membrane Glycoproteins metabolism
Ristocetin pharmacology
Transfection
von Willebrand Factor antagonists & inhibitors
von Willebrand Factor genetics
Mutagenesis, Site-Directed
Oligopeptides
von Willebrand Factor metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 267
- Issue :
- 5
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 1737795