STUDIES ON GLYCOPROTEINS .3. ISOLATION OF SIALYLGLYCOPEPTIDES FROM HUMAN PLATELET MEMBRANES

No direct evidence has been obtained for the presence of glycoproteins on platelet membranes despite the importance of membrane phenomena in platelet physiology and blood coagulation. In order to obtain such information a purified membrane fraction has been prepared from homogenized platelets and subjected to proteolytic digestion with trypsin and Pronase. Two major size classes of N-acetylneuraminic acid containing glycopeptides of molecular weight approximately 12,000 and 3,500, respectively, have been obtained in equimolar amounts and correspond to a total of about 107 such heterosaccharide residues per platelet. Purification by gradient elution from DEAE-Sephadex at pH 2.5 produced three major N-acetylneuraminic acid containing peaks in each class. Chemical analysis showed that aspartic acid and glucosamine were the major constituents of all six glycopeptide fractions other than hexose. Gas chromatography of the sugars as their alditol acetates showed that mannose and galactose were found in all six fractions, glucose in four out of six, and that fucose was present in trace amounts. The range of analytical data observed were hexoses, 15-30%; N-acetylglucosamine, 15-35%; tV-acetylneuraminic acid, 7-20%; and amino acids, 28-50%. Isoelectric focussing of partially purified, nondififusible glycopeptides indicated a range of pK from 1.8 to 2.2 for the bound N-acetylneuraminic acid which is significantly lower than the accepted value for the free acid; this was confirmed by paper electrophoresis of individual glycopeptides. No evidence was obtained for the presence of alkalilabile O-seryl glycosidic linkages in the platelet membrane. These results (i) confirm that glycoproteins are an integral part of the platelet membrane, (ii) demonstrate that they are composed of a variety of complex heterosaccharide units, and (iii) show that the heterosaccharide units of the platelet membrane are significantly different from those isolated from the erythrocyte membrane. © 1969, American Chemical Society. All rights reserved.