MEGAKARYOCYTE & PLATELET PROTEOGLYCANS

这项工作的目标是对 硫酸盐的结构、代谢和功能 大分子，即蛋白多糖和硫化蛋白， 巨核细胞和血小板。我们之前的工作证明了 三种蛋白多糖和几种硫酸化蛋白的存在 在豚鼠巨核细胞和血小板中，并存在 人类血小板中具有相同MR范围的蛋白多糖。我们会 将这些研究扩展如下。(1)每种植物的核心蛋白 N-端氨基酸将是蛋白多糖的特征 总氨基酸组成的序列分析和研究。 针对完整蛋白多糖、多肽制备的抗体 根据N-末端的选定序列合成的 核心蛋白的多肽，以及基于 这些肽序列将被用来筛选cDNA库 从HEL细胞制备以获得核心基因的c DNA 蛋白质；然后我们就可以确定总的氨基酸序列。(2) 血小板的硫化和非硫化低聚糖 蛋白多聚糖将被完全表征。(3)( 我们将探讨这三种蛋白多糖的亚细胞定位。 在光学和电子显微镜水平上的超微结构 确定它们的具体本地化。产生的抗体 针对完整的分子和N-末端序列将BC 用作探头。将Bc基因作为探针用于原位杂交 杂交研究以确定不同的核心是否 蛋白质在巨核细胞的不同阶段表达 成熟。(4)多聚糖合成在α-糖链中的作用 颗粒的形成将使用体外模型进行评估 巨核细胞在特定抑制物存在的情况下孵育 蛋白多糖的合成，并通过研究Gray患者 血小板综合症，看看他们是否缺乏蛋白多糖。(5)角色 将通过以下方法评估血小板功能中的表面蛋白多糖 用软骨素酶消化细胞表面 用抗膜蛋白多糖抗体孵育细胞，并 监测对血小板功能的影响。(6)硫酸化蛋白质 人类血小板的数量将被识别出来，而 表征了含有硫酸盐的部分。他们的职能作用 将通过用硫酸酶对血小板进行表面消化来评估。 这些研究将使我们了解 硫酸化大分子在血小板功能中的作用，以及 确定它们是否以及如何在以下异常中被更改 影响血小板功能。

The goals of this work are to obtain a complete understanding of the structure, metabolism and function of the sulfated macromolecules, i.e. the proteoglycans and sulfated proteins, of megakaryocytes and platelets. Our previous work has demonstrated the existence of three proteoglycans and several sulfated proteins in guinea pig megakaryocytes and platelets, and the existence of proteoglycans of the same Mr range in human platelets. We will extend these studies as follows. (1) The core proteins of each proteoglycan will be characterized by N-terminal amino acid sequence analysis and study of total amino acid composition. Antibodies prepared against the intact proteoglycans, peptides synthesized on the basis of selected sequences in the N-terminal peptides of the core proteins, and oligonucleotide probes based on the peptide sequences will be used to screen a cDNA library prepared from HEL cells in order to obtain cDNA for the core proteins; we can then determine total amino acid sequence. (2) The sulfated and non-sulfated oligosaccharides of platelet proteoglycans will be completely characterized. (3) (The subcellular localization of the three proteoglycans will be probed ultrastructurally at the light and electron microscopy level to determine their specific localization. Antibodies generated against the intact molecules and the N-terminal sequences will bc used as probes. The cDNA will bc used as a probe for in situ hybridization studies to determine whether the different core proteins are expressed at different stages of megakaryocyte maturation. (4) The role of poteoglycan synthesis in alpha granule formation will be assessed using an in vitro model in which megakaryocytes are incubated in the presence of specific inhibitors of proteoglycan synthesis, and by studying patients with the Gray Platelet Syndrome to see if they lack proteoglycans. (5) The role of surface proteoglycans in platelet function will be assessed by digesting the surface of the cells with chondroitinase or incubating the cells with anti-membrane proteoglycan antibody, and monitoring the effect on platelet function. (6) Sulfated proteins of human platelets will be identified and the nature of the sulfate-containing moieties characterized. Their functional role will be assessed by surface digestion of platelets with sulfatases. These studies will provide an understanding of the roles of the sulfated macromolecules in platelet function, and a basis for determining whether and how they are altered in abnormalities which affect platelet function.