Partial characterization of a binding site for von Willebrand factor on glycocalicin.
The binding of von Willebrand factor (vWF) to platelet membrane glycoprotein Ib (GpIb) facilitates platelet adhesion to vascular subendothelium. In this study, we provide evidence that the vWF binding site is on glycocalicin (GC), a proteolytic fragment of GpIb, and we examine the role of the carbohydrate portion of GC on that binding. The binding to platelets of 6D1, a monoclonal antibody that recognizes an epitope on GpIb and blocks ristocetin-induced vWF binding to platelets, was inhibited by purified GC. In addition, purified GC inhibited ristocetin-dependent binding of 125I-labeled vWF to platelets. Since GC contains 60% carbohydrate by weight, we assessed the role of carbohydrate sequences on its interaction with antibody 6D1 and vWF. Based on the known sequence of the major oligosaccharide chain of GC--N-acetyl neuraminic acid, galactose, N-acetyl glucosamine, N-acetyl galactosamine--we treated GC sequentially with neuraminidase, beta-galactosidase, and beta-N-acetylglucosaminidase. Removal of sialic acid and galactose residues did not affect GC binding. Removal of N-acetyl glucosamine residues did not affect GC binding to 6D1 but did decrease the ability of GC to inhibit vWF binding to platelets, increasing the concentration needed to inhibit binding by 50% (IC50) 40-fold. This suggests that a portion of the oligosaccharide chains on GC contributes to the vWF binding activity of this molecule.
D000911:Antibodies, Monoclonal / Q000276:immunology; D002240:Carbohydrate Sequence; D006023:Glycoproteins / Q000032:analysis* / Q000276:immunology / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis* / Q000276:immunology / Q000494:pharmacology; D009005:Monosaccharides / Q000097:blood; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface / Q000032:analysis*
D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D008565:Membrane Proteins; D009005:Monosaccharides; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; C023592:glycocalicin; C028254:von Willebrand factor receptor; D006026:Glycoside Hydrolases