Sialic acid prevents loss of large von Willebrand factor multimers by protecting against amino-terminal proteolytic cleavage.
Removal of sialic acid from the von Willebrand factor (vWF) subunit exposes additional cleavage sites in the amino-terminal region that are associated with loss of large multimers. The extent of large multimer loss was evaluated by examining the sites of subunit cleavage of native and carbohydrate-modified vWF after treatment with trypsin, chymotrypsin, or plasmin. In the presence of proteinase inhibitors, purified vWF was treated with neuraminidase alone to remove 90% to 95% of the sialic acid or with neuraminidase and beta-galactosidase to remove the sialic acid and 45% to 50% of the D-galactose, with little or no loss of large multimers observed. Digestion of native vWF with trypsin produced the greatest loss of large multimers, while chymotrypsin produced less and plasmin produced the least. Large multimer loss was more extensive with each enzyme after carbohydrate modification of vWF. The extent and approximate location of subunit cleavage was determined by immunoblotting and monoclonal antibody epitope mapping. Trypsin, chymotrypsin, and plasmin were shown to produce both amino- and carboxyl-terminal fragments. The number, location, and relative quantities of carboxyl-terminal fragments produced were unchanged after carbohydrate modification. However, digestion of the amino-terminal region was considerably more extensive after carbohydrate modification as judged by a marked decrease or absence of the larger fragments seen when native vWF was digested, and by the appearance of new smaller molecular mass species. Therefore, the greater loss of large multimers that occurs after carbohydrate modification is likely to be the result of cleavages in the amino-terminal region of the molecule. By protecting the vWF subunit against amino-terminal cleavage, sialic acid inhibits the loss of large multimers.
Berkowitz
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SD
Department of Basic and Clinical Research, Research Institute of Scripps Clinic, La Jolla, CA.
Federici
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AB
D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D000939:Epitopes; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D015394:Molecular Structure; D010446:Peptide Fragments / Q000276:immunology; D010447:Peptide Hydrolases / Q000378:metabolism*; D012794:Sialic Acids / Q000502:physiology*; D013329:Structure-Activity Relationship; D014841:von Willebrand Factor / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D012794:Sialic Acids; D014841:von Willebrand Factor; D010447:Peptide Hydrolases