Human Platelet Sialic Acid Content and Tritium Incorporation After ADP-induced Shape Change and Aggregation

Abstract

The distribution of surface sialic acid and its possible alteration after ADP-induced shape change and aggregation were studied. Gel-filtered platelets that were aggregated, disaggregated, and centrifuged showed decreased total protein and a significant decrease in sialic acid/mg protein. Platelet surface sialic acid was labeled by periodate oxidation followed by borotritiide reduction. Tritium was apparently incorporated only into sialic acid, since the label was removed by mild acid hydrolysis, had a mobility similar to that of the seven-carbon derivative of sialic acid, and was not taken up when periodate was omitted. When reduced platelet proteins were examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis, at least three radioactive peaks (1, 2, and 3) were consistently observed, with apparent molecular weights of about 200,000, 150,000, and 100,000 daltons, respectively. Peak 2 had the greatest radioactivity. A shoulder of slightly lower molecular weight than peak 2, or a small peak between peaks 2 and 3, was also seen. Peaks 2 and 3 corresponded to glycoproteins I and III identified with periodic acid-Schiff (PAS) staining, and the material between them to glycoprotein II. Peak 1, and two to four smaller radioactive peaks with apparent molecular weights between 25,000 and 60,000 daltons, were not usually associated with PAS staining. All radioactive peaks were much smaller in platelets labeled after treatment with neuraminidase, indicating that much of the labeled sialic acid was on the platelet surface. The amount of tritium incorporated and its labeling pattern were unaffected when shape change alone was induced with ADP. However, platelets that were aggregated and disaggregated with ADP had reduced tritium uptake with a uniformly decreased labeling pattern, suggesting that the decrease in sialic acid content/mg protein resulted from a generalized loss of surface sialic acid or glycoprotein.