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.
Highlights
In order to assess the requirement for the Arg-Gly- surface
Mutagenesis and Plasmid Construction-The RGDS sequence responsible for von Willebrand factor (vWF) binding to GpIIb/IIIa is located at amino acid residues 1744-1747 in the matureprotein, which corresponds to residues 2507-2510 in pro-vWF (5, 6, 14)
Multimers from RGDS-Mutant v WF Are Indistinguishable from wild type (WT)-vWF-RADS-vWF, RGES-vWF, andWT-vWF cDNAs were initially transfected into COS-1 cells to study the processing and multimerization of RGDS-mutant vWF
Summary
Mutagenesis and Plasmid Construction-The RGDS sequence responsible for vWF binding to GpIIb/IIIa is located at amino acid residues 1744-1747 in the matureprotein, which corresponds to residues 2507-2510 in pro-vWF (5, 6, 14). Transfections-To express vWF transiently, RGDS-mutant and wild type (WT) vWF cDNA in pMT2 was transfected into COS-1 cells using a DEAE-dextran protocol with a dimethyl sulfoxide shock (17).COS-1cells were rinsed twice with DMEM (Dulbecco'smodified Eagle's medium) prior to the addition of 10 pg of DNA in 4 ml of 10 mM Tris/DEAE-dextran; final concentratio2n50 pglml, pH 7.3. The sample was loaded onto a Sepharose CL-4B (Pharmacia) column from which vWF eluted in the void volume Both immunoaffinitypurified and chromatographically purified recombinant vWF were shown to be pure by silver stain analysis of SDS-PAGE gels, and each behaved identically in attachment assays. To assess HUVEC cell spreading, affinity-purified WT-vWF or RGDS-mutant vWwFas adsorbed to microtiter wells as described above, and cells were examined by phase contrast microscopy 1h after plating
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