Platelet glycocalicin. Its membrane association and solubilization in aqueous media.
Glycocalicin has been extracted from human platelets by 3 M KCl and purified using affinity chromatography on columns of Sepharose-coupled wheat germ agglutinin as the most efficient step. Rabbit antiserum to the purified protein agglutinated human platelets and inhibited the agglutination induced by bovine Factor VIII-related protein. Crossed immunoelectrophoresis of Triton X-100 extracts of platelets in Triton X-100-containing agarose revealed the presence of two glycocalicin-related components of different electrophoretic mobilities giving a continuous double-peak immunoprecipitate with this antiserum. The fast-moving component, which represented the minor peak of the immunoprecipitate, corresponded to purified soluble glycocalicin. Crossed hydrophobic interaction immunoelectrophoresis did not demonstrate binding of the purified glycocalicin or the fast-moving component to phenyl-Sepharose CL-4B as hydrophobic matrix. The slow-moving component, which represented the major peak of the immunoprecipitate, showed a strong binding to the hydrophobic matrix. Immunoelectrophoretic quantitation of glycocalicin present in the aqueous media demonstrated that the presence of EDTA, N-ethylmaleimide and iodoacetamide during lysis of platelets significantly reduced the solubilization of glycocalicin. At the same concentrations these inhibitors strongly inhibited the calcium-activated protease of platelet sonicates. Sialic acid determination after acid hydrolysis of aliquots from the soluble fractions showed that their content of sialic acid was considerably higher when lysis was performed in the absence, rather than in the presence, of EDTA and that glycocalicin contributes significantly to the total platelet sialic acid.
D000371:Agglutination; D001792:Blood Platelets / Q000032:analysis*; D004492:Edetic Acid; D005033:Ethylmaleimide; D005615:Freezing; D006023:Glycoproteins / Q000097:blood*; D006801:Humans; D007106:Immune Sera; D007123:Immunoelectrophoresis, Two-Dimensional; D007460:Iodoacetamide; D008565:Membrane Proteins / Q000097:blood*; D010974:Platelet Aggregation; D019038:Platelet Glycoprotein GPIb-IX Complex; D012995:Solubility
D006023:Glycoproteins; D007106:Immune Sera; D008565:Membrane Proteins; D019038:Platelet Glycoprotein GPIb-IX Complex; C023592:glycocalicin; D004492:Edetic Acid; D005033:Ethylmaleimide; D007460:Iodoacetamide