MEGAKARYOCYTE & PLATELET PROTEOGLYCANS

这项工作的目标是获得一个完整的理解 硫酸盐的结构、代谢和功能 大分子，即蛋白聚糖和硫酸化蛋白， 巨核细胞和血小板。 我们之前的工作已经证明 存在三种蛋白聚糖和几种硫酸化蛋白 在豚鼠巨核细胞和血小板中， 在人血小板中具有相同Mr范围的蛋白聚糖。 我们将 将这些研究扩展如下。 (1)每种蛋白质的核心 蛋白聚糖将通过N-末端氨基酸来表征 序列分析和总氨基酸组成的研究。 针对完整蛋白聚糖、肽制备的抗体 基于N-末端中的选定序列合成 核心蛋白的肽，以及基于 肽序列将用于筛选cDNA文库 从HEL细胞制备，以获得核心的cDNA 蛋白质;然后我们可以确定总的氨基酸序列。 (2)的 硫酸化和非硫酸化血小板寡糖 蛋白聚糖将被完全表征。 (3)（请参阅 这三种蛋白聚糖的亚细胞定位将被探测 在光学和电子显微镜水平的超微结构， 确定其具体位置。 产生的抗体 与完整的分子相比，N-末端序列将 用作探针。 该cDNA将用作原位探针 杂交研究，以确定是否不同的核心， 蛋白质在巨核细胞的不同阶段表达 成熟 (4)低聚糖合成在α 使用体外模型评估颗粒形成，其中 巨核细胞在特异性抑制剂存在下孵育 蛋白多糖合成的研究，并通过研究患有灰色 血小板综合症来看看他们是否缺乏蛋白多糖。 (5)的作用 将通过以下方法评估血小板功能中的表面蛋白聚糖 用软骨素酶消化细胞表面，或 用抗膜蛋白聚糖抗体孵育细胞，和 监测对血小板功能的影响。 (6)硫酸化蛋白 人血小板的性质将被确定， 表征的含硫酸盐部分。 职能作用 将通过用硫酸酯酶表面消化血小板来评估。 这些研究将使人们了解 硫酸化大分子在血小板功能中的作用， 确定它们是否以及如何在异常中改变， 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.