STRUCTURE AND FUNCTION OF THROMBOSPONDIN

血小板反应蛋白的结构和功能

• 批准号: 2218392
• 负责人: DAVID W ESSEX
• 金额: $ 16.46万
• 依托单位: SUNY DOWNSTATE MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-12-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2218392
• 关键词: antibody formation; calcium; cell adhesion molecules; cell cycle; cell migration; disulfide bond; enzyme activity; enzyme complex; enzyme mechanism; extracellular matrix proteins; human tissue; immunoprecipitation; isomerase; oxidation reduction reaction; platelet activation; platelets; protease inhibitor; protein purification; protein structure function; thiols; thrombin; thrombospondins; wound healing

Platelet activation occurs at the sites of injury, where extensive tissue remodeling takes place as part of the subsequent wound healing. Activated platelets secrete adhesive proteins that participate in formation of the extracellular matrix, and the nature of the matrix alters the adhesion, migration and proliferation of cells. This proposal is based on the hypothesis that protein disulfide isomerase is released by activated platelets and catalyzes the isomerization of disulfide bonds, leading to changes in protein conformation and to formation of disulfide-linked protein-protein complexes, thereby modifying the composition and the nature of the extracellular matrix. The hypothesis is based on the observations that when material secreted by activated platelets is incubated at 37 degree, thrombospondin shows isomerization of disulfide bonds, reduction of disulfide bonds, and formation f disulfide-linked multimers and disulfide- linked complexes with thrombin-serpin complexes. Each of these is similar to reactions catalyzed by protein disulfide isomerase. Preliminary experiments confirmed the presence of disulfide isomerase activity in the supernatant solution of activated platelets. Additional experiments will establish the disulfide isomerase specificity, the requirement for cofactors and the approximate mass of the enzyme. The disulfide isomerase will be purified, and antibodies will be prepared. The role of the disulfide isomerase will be tested in three ways: i) it will be added to combinations of purified proteins to determine which become disulfide linked, ii) antibodies will be used to immunoprecipitate the disulfide isomerase from the platelet supernatant solution to determine which reactions are inhibited, and iii) the ability of cells to adhere, spread and migrate on the matrix formed in the presence or absence of the disulfide isomerase will be tested. The specific conditions for disulfide linking of thrombin-serpin complexes to thrombospondin will be determined, and the thiols of thrombospondin will be characterized further.

血小板激活发生在损伤部位，那里有广泛的组织 重塑是随后伤口愈合的一部分。 活性 血小板分泌粘附蛋白，参与血小板的形成 细胞外基质，基质的性质改变粘附， 细胞的迁移和增殖。 该提案基于 假设蛋白质二硫键异构酶是由激活的 血小板并催化二硫键异构化，导致 蛋白质构象的变化和二硫键连接的形成 蛋白质-蛋白质复合物，从而改变其组成和性质 细胞外基质。 该假设基于观察 当活化血小板分泌的物质在37℃下孵育时 程度，血小板反应蛋白显示二硫键异构化，减少 二硫键，以及二硫键连接的多聚体和二硫键的形成 与凝血酶-丝氨酸蛋白酶抑制剂复合物连接的复合物。 每一个都是相似的 蛋白质二硫键异构酶催化的反应。 初步的 实验证实二硫键异构酶活性的存在 活化血小板的上清液。 额外的实验将 建立二硫键异构酶特异性，要求 辅因子和酶的近似质量。 二硫键异构酶 将被纯化，并且将制备抗体。 的作用 二硫键异构酶将以三种方式进行测试：i) 将其添加到 纯化蛋白质的组合以确定哪些会变成二硫键 连接，ii) 抗体将用于免疫沉淀二硫键 从血小板上清液中测定异构酶 反应被抑制，以及 iii) 细胞粘附、扩散的能力 并在存在或不存在的情况下形成的基质上迁移 将测试二硫键异构酶。 二硫键的具体条件 将确定凝血酶-丝氨酸蛋白酶抑制剂复合物与血小板反应蛋白的连接， 并且将进一步表征血小板反应蛋白的硫醇。