STRUCTURE AND FUNCTION OF THROMBOSPONDIN

血小板反应蛋白的结构和功能

基本信息

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
项目类别：
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

项目摘要

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作为随后伤口愈合的一部分进行的。活性血小板分泌参与形成的粘附蛋白细胞外基质，并且基质的性质改变了粘附，细胞的迁移和增殖。该建议基于假设蛋白质二硫化物异构酶被激活释放血小板和催化二硫键的异构化，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，从而修改成分和性质细胞外基质。该假设基于观察结果当被激活的血小板分泌的材料在37处孵育程度，血小板传播显示二硫键的异构化，减少二硫键和形成f二硫键的多聚体和二硫键 - 将复合物与凝血酶 - 塞平蛋白复合物联系起来。这些都相似由蛋白质二硫异构酶催化的反应。初步的实验证实了二硫化物异构酶活性活化血小板的上清液溶液。其他实验将建立二硫异构酶特异性，要求辅因子和酶的近似质量。二硫异构酶将被纯化，并准备抗体。角色二硫异构酶将通过三种方式进行测试：i）将添加到纯化蛋白质的组合以确定哪种变为二硫化物链接，ii）抗体将用于免疫沉淀二硫化物来自血小板上清液溶液的异构酶，以确定哪个反应被抑制，iii）细胞粘附，扩散的能力并在存在或不存在的基质上迁移将测试二硫异构酶。二硫化物的特定条件将确定凝血酶 - 塞平蛋白复合物与血小板传播的联系，确定，血小板传播的硫醇将进一步表征。