FUNCTIONAL CHARACTERIZATION OF PLATELET THROMBOSPONDIN

血小板血小板反应蛋白的功能特征

• 批准号: 2216347
• 负责人: John W LAWLER
• 金额: $ 38.5万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-09-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2216347
• 关键词: CD antigens; affinity chromatography; calcium binding protein; chimeric proteins; chondroitin sulfates; complementary DNA; fibrinogen; fibronectins; heparin; immunochemistry; integrins; laboratory mouse; molecular site; monoclonal antibody; osteonectin; phosphorylation; plasminogen; platelet aggregation; platelets; protein structure function; proteolysis; site directed mutagenesis; synthetic peptide; thrombospondins; tissue /cell culture

DESCRIPTION: Thrombospondin-1 (TSP-1) modulates cell adhesion, migration and growth by directing assembly of macromolecular complexes that integrate the signals produced by growth factors, cytokines and extracellular matrix at the cell surface. The long term goal of this project is to integrate in vitro and in vivo data obtained from protein biochemical, cell biological and genetic approaches. The proposal lists three specific aims. In Specific Aim 1, functional domains in TSP-1 will be mapped by a combination of approaches including (1) protease fagmentation and isolation of functional domains, (2) generation of fusion proteins with functional activity, (3) generation of polyclonal anti-fusion protein antibody that block function, (4) generation of monoclonal antibodies against fusion proteins that block function, (5) preparation of mutated or domain deleted forms of TSP-1, and (6) preparation of synthetic peptides that inhibit the activity of TSP-1. Specific Aim 2 will characterize the interaction of TSP-1 with bone and muscle cells with respect to its ability to support attachment, spreading, migration and proliferation. Receptors that interact with the carboxy-terminus of TSP-1 from bone and muscle cell lines will be identified by (1) expression cloning, (2) production of monoclonal antibodies to the cell surface that block binding, and (3) affinity chromatography. In Specific Aim 3, the applicant will determine the effect of TSP-1 deficiency on mice in two homogeneous genetic backgrounds, C57BL/6 and 129/5V. The phenotypic characterization in these studies will focus on (1) blood cell counts, (2) platelet aggregation, (3) phosphorylation of platelet proteins, (4) cartilage and bone formation, (5) histology of the major organ systems, and (6) angiogenesis. TSP-1-deficient mice will be crossed to mice that are deficient in other members of the TSP family. Finally, the role of TSP-1 in tumor vascularization will be evaluated by crossing the TSP-1-deficient mice to P53-deficient mice and mice carrying the MMTV/c-neu transgene.

描述：血小板传播1（TSP-1）调节细胞粘附， 通过指导大分子复合物的迁移和生长 整合了生长因子，细胞因子和 细胞表面的细胞外基质。这个长期目标 项目将整合从体外和体内数据。 蛋白质生化，细胞生物学和遗传方法。 这 提案列出了三个具体目标。 在特定目标1中，功能性 TSP-1中的域将通过方法组合来映射 包括（1）蛋白酶欺诈和功能的隔离 域，（2）具有功能活性的融合蛋白的产生， （3）封闭多克隆抗融合蛋白抗体的产生 功能，（4）抗融合的单克隆抗体产生 阻断功能的蛋白质，（5）突变或结构域的制备 删除的TSP-1形式，（6）制备合成肽 抑制TSP-1的活性。特定目标2将表征 TSP-1与骨骼和肌肉细胞相对于其相互作用 支持依恋，扩散，迁移和扩散的能力。 与TSP-1的羧基末端相互作用的受体 骨和肌肉细胞系将通过（1）表达克隆识别 （2）生产对细胞表面的单克隆抗体 块结合和（3）亲和色谱。在特定的目标3中 申请人将确定TSP-1缺乏症对小鼠的影响 两个均匀的遗传背景，C57BL/6和129/5V。 这 这些研究中的表型表征将集中于（1）血液 细胞计数，（2）血小板聚集，（3）血小板的磷酸化 蛋白质，（4）软骨和骨形成，（5）主要的组织学 器官系统和（6）血管生成。 TSP-1缺乏小鼠将是 越过缺乏TSP成员的小鼠 家庭。最后，TSP-1在肿瘤血管形成中的作用将 可以通过将TSP-1缺陷小鼠越过P53缺陷来评估 携带MMTV/C-NEU转基因的小鼠和小鼠。