IMMUNOCHEMICAL LOCALIZATION OF MEGAKARYOCYTE PROTEINS

巨核细胞蛋白的免疫化学定位

• 批准号: 3342852
• 负责人: DOROTHY F BAINTON
• 金额: $ 13.27万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-09-30 至 1986-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3342852
• 关键词: Golgi apparatus; autoradiography; bone marrow; cell differentiation; electron microscopy; endoplasmic reticulum; fibrinogen; histochemistry /cytochemistry; human subject; immunochemistry; megakaryocytes; monoclonal antibody; platelet disorder; platelet factor 4; radiotracer; surface antigens; thrombin; thrombocytopenia

The goal of this project is to characterize the pathway of synthesis and distribution of Alpha-granule (fibrinogen, Beta-thromboglobulin and platelet factor 4) and plasma membrane (GPIIb-IIIa) proteins by megakaryocytes. These proteins will be identified in human and baboon megakaryocytes from bone marrow aspirates using electron microscopic and immunocytochemical techniques previously developed by the investigators to study these substances in platelets. Well-characterized mono and polyclonal antibodies will be used to localize the antigens in subcellular organelles: presence of antigens in the rough endoplasmic reticulum and Golgi complex will be used as evidence of their synthesis by megakaryocytes. The stage of maturation at which each of these substances is synthesized as well as the mechanisms by which they are distributed into cellular organelles will be determined. Identification of antigens in endocytotic organelles (coated vesicles, multivesicular bodies or lysosomes) alone, would indicate the presence of these substances in the cell is due to internalization. Incubation of megakaryocytes with (3H) antigens followed by autoradiographic localization will provide additional evidence for endocytosis. We also plan to characterize thrombin binding and internalization by megakaryocytes as well as the effects of thrombin on cellular organelles such as Alpha-granules and their contents. In order to facilitate these studies immune-affinity techniques for isolating megakaryocytes will be developed using anti-GPIIb-IIIa antibodies in conjunction with established methods. Additional monoclonal antibodies against cell surface antigens will be characterized and used to extend our studies of the megakaryocyte plasma membrane. These experiments may also provide new information on formation of the demarcation membrane during megakaryocyte maturation. In addition, synthesis and distribution of Alpha-granules and plasma membrane proteins will be studied in conditions in which these cellular functions may be altered. These processes will be studied in megakaryocytes obtained from 1) baboons made thrombocytopenic with anti-platelet antisera as well as humans with consumptive platelet disorders to determine the effects of stimulation of megakaryocytopoiesis, and 2) patients with hereditary platelet disorders in which there is deficiency of substances found in plasma membranes (Glanzmann's thrombasthenia) or Alpha-granules (Gray platelet syndrome). These studies may provide information which will advance our understanding of the pathophysiology of these diseases.

该项目的目标是表征合成途径和 α-颗粒（纤维蛋白原、β-血栓球蛋白和 血小板因子 4) 和质膜 (GPIIb-IIIa) 蛋白 巨核细胞。 这些蛋白质将在人类和狒狒中被鉴定 使用电子显微镜从骨髓中抽吸巨核细胞并 研究人员之前开发的免疫细胞化学技术 研究血小板中的这些物质。 特性良好的单声道和 多克隆抗体将用于在亚细胞中定位抗原 细胞器：粗面内质网中存在抗原 高尔基复合体将被用作其合成的证据 巨核细胞。 这些物质的成熟阶段 被合成以及它们被分配到的机制 细胞器将被确定。 抗原的鉴定 内吞细胞器（包被囊泡、多泡体或 溶酶体）单独存在，就表明这些物质存在于 细胞是由于内化。 巨核细胞与 (3H) 的孵育 抗原随后放射自显影定位将提供额外的 内吞作用的证据。 我们还计划表征凝血酶结合 和巨核细胞的内化以及凝血酶对 细胞器，例如阿尔法颗粒及其内容物。 为了 促进这些研究免疫亲和技术分离 将使用抗 GPIIb-IIIa 抗体开发巨核细胞 与既定方法相结合。 其他单克隆抗体 针对细胞表面抗原的特征将被表征并用于扩展我们的 巨核细胞质膜的研究。 这些实验也可能 提供关于分界膜形成的新信息 巨核细胞成熟。 此外，合成和分布 α-颗粒和质膜蛋白将在一定条件下进行研究 其中这些细胞功能可能会改变。 这些过程将 在从 1) 狒狒中获得的巨核细胞中进行了血小板减少症的研究 使用抗血小板抗血清以及患有消耗性血小板的人 确定刺激巨核细胞生成的影响的疾病， 2) 患有遗传性血小板疾病的患者，其中存在 质膜中发现的物质缺乏（Glanzmann's 血小板无力症）或阿尔法颗粒（灰色血小板综合征）。 这些研究 可能提供有助于加深我们理解的信息 这些疾病的病理生理学。