Sialic acid in mature megakaryocytes: detection by wheat germ agglutinin

Abstract

Abstract The characteristics of the surface of guinea pig megakaryocytes were investigated with wheat germ agglutinin (WGA). Purified guinea pig megakaryocytes and platelets were incubated with WGA conjugated with rhodamine, cytocentrifuged, and then exposed to Chromomycin A3 for the assessment of ploidy. The fluorescence emission of the DNA-Chromomycin complex was similar to that of fluorescein, and thus both rhodamine-WGA and Chromomycin A3 fluorescence could be analyzed in the same cell. Ploidy was assessed by microdensitometry of Chromomycin A3 fluorescence. Eight hundred megakaryocytes were analyzed by four parameters: (1) labeling by WGA, (2) ploidy, (3) morphological stage, and (4) size. The results were analyzed by a computer-assisted program. Although all platelets had reacted with WGA, only about half of the isolated megakaryocytes had been labeled by the lectin. The analysis of the megakaryocytes that had reacted revealed that 72% of stage III and 77% of stage IV megakaryocytes as compared with 35% of stage I and 29% of stage II cells had been labeled by the lectin. WGA reacted with 44% of 8N megakaryocytes and 60% and 59% of 16N and 32N cells. However, WGA labeling was independent of megakaryocyte size. The digestion of 15% and 48% of megakaryocyte sialic acid with neuraminidase from Vibrio cholera resulted in a 67% and 89% decrease in the binding of rhodamine- WGA, respectively, as determined by microdensitometry. The study indicated that sialic acid serves as a receptor for WGA and that sialoglycoproteins and possibly gangliosides become exposed on the surface of mature megakaryocytes. WGA can recognize mature megakaryocytes by biochemical criteria and the assessment of lectin binding could complement the morphological staging of megakaryocytes.