Hayward C P, Bainton D F, Smith J W, Horsewood P, Stead R H, Podor T J, Warkentin T E, Kelton J G
Department of Medicine, McMaster University Medical Centre, Hamilton, Canada.
J Clin Invest. 1993 Jun;91(6):2630-9. doi: 10.1172/JCI116502.
In this report, we describe the intracellular localization of multimerin in platelets and its biosynthesis by Dami cells, a megakaryocytic cell line. Immunoelectron microscopy was used to examine frozen thin sections of resting and activated platelets. Multimerin was localized within the platelet alpha-granule in an eccentric position. Within activated platelets, multimerin was found in the surface-connected open cannalicular system and on the external plasma membrane. Light microscopic immunocytochemistry demonstrated multimerin in normal megakaryocytes and in Dami cells after stimulation with PMA. Confirmation of multimerin biosynthesis by Dami cells was obtained by metabolic labeling studies. Both platelet and Dami cell multimerin demonstrated several subunit sizes on reduced SDS-PAGE. However, peptide mapping confirmed structural homology between the different multimerin subunits. Glycosidase digestion demonstrated that multimerin is heavily glycosylated with mainly complex, N-linked carbohydrate. In contrast to the multimerin isolated from platelets, cultured Dami cells secreted mainly smaller multimers of the protein. Biosynthesis of multimerin by a megakaryocytic cell line supports endogenous biosynthesis by megakaryocytes as the origin of this platelet alpha-granule protein.
在本报告中，我们描述了多聚体蛋白在血小板中的细胞内定位及其由巨核细胞系Dami细胞进行的生物合成。免疫电子显微镜用于检查静息和活化血小板的冷冻薄片。多聚体蛋白定位于血小板α颗粒内的偏心位置。在活化血小板中，多聚体蛋白存在于表面连接的开放小管系统和外部质膜上。光学显微镜免疫细胞化学显示正常巨核细胞和经佛波酯（PMA）刺激后的Dami细胞中有多聚体蛋白。通过代谢标记研究证实了Dami细胞合成多聚体蛋白。在还原型十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上，血小板和Dami细胞的多聚体蛋白均显示出几种亚基大小。然而，肽图谱分析证实了不同多聚体蛋白亚基之间的结构同源性。糖苷酶消化表明多聚体蛋白高度糖基化，主要是复杂的N-连接碳水化合物。与从血小板中分离的多聚体蛋白不同，培养的Dami细胞主要分泌该蛋白的较小多聚体。巨核细胞系合成多聚体蛋白支持巨核细胞内源性合成作为这种血小板α颗粒蛋白的来源。
