FUNCTIONAL CHARACTERIZATION IN PLATELET THROMBOSPONDIN

血小板反应蛋白是一种高分子量糖蛋白， 在血小板，内皮细胞，平滑肌细胞， 成纤维细胞，最近，在上皮细胞系，从大鼠 肺泡 拟议研究的目的是评估 假设血小板反应蛋白是一种多功能蛋白， 参与细胞与细胞之间的大分子结合， 细胞与细胞外基质的相互作用。 具体重点将放在 以下领域： (1)血小板反应蛋白与钙离子的相互作用。 沉降 系数，特性粘数，圆二色性，电镜 血小板反应蛋白的外观和对蛋白水解的抵抗性 受EDTA去除二价阳离子的影响。 两者的滴定 圆二色性和有限胰蛋白酶产生的肽模式 消化意味着钙与血小板反应蛋白的相互作用 涉及多个站点之间的协作交互。 拟议 研究，结合实验将与钙和镧系元素进行 离子直接测量结合位点的数量，以确认 合作相互作用的存在，并确定解离 constants. (2)血小板反应蛋白的结构域。 建议的具体目标 研究的目的是建立一个详细的域结构图， 血小板反应蛋白 用于纯化这些结构域的方法和 将开发针对这些结构域的单克隆抗体， 用于下述功能研究。 电子显微镜， 通过反相高压液相色谱法进行肽图谱分析， N-末端氨基酸测序将用于定位结构 凝血酶敏感蛋白分子内的结构域。 (3)血小板反应蛋白的功能域。 血小板反应蛋白有能力 与钙、肝素、纤维蛋白（原）、纤连蛋白、富组氨酸 糖蛋白、V型胶原、层粘连蛋白和细胞表面。 具体目标 的结构域， 血小板反应蛋白，其包含这些功能活性中的每一种。 在 此外，上述一些化合物的血小板反应蛋白结合部分可与血小板反应蛋白结合。 将识别化合物以及二价阳离子对这些化合物的影响 互动将被调查。 (4)血小板反应蛋白的细胞表面受体。 几种不同 固相结合分析将被开发和使用，以确定 血小板、内皮细胞、平滑肌表面蛋白 细胞、成纤维细胞、红细胞和巨噬细胞， 血小板反应蛋白 潜在受体的功能意义将是 通过测定它们的亚细胞位置和 生理学相关的抑制剂，以抑制它们与 血小板反应蛋白

Thrombospondin is a high molecular weight glycoprotein which has been identified in platelets, endothelial cells, smooth muscle cells, fibroblasts and, most recently, in an epithelial cell line from rat alveolar. The objective of the proposed study is to evaluate the hypothesis that thrombospondin is a multifunctional protein which mediates the macromolecular associations involved in cell-to-cell and cell-to-extracellular matrix interactions. Specific focus will be directed to the following areas: (1) The interaction of thrombospondin with calcium. The sedimentation coefficient, intrinsic viscosity, circular dichrosim, electron microscopic appearance and resistance to proteolysis of thrombospondin are profoundly affected by the removal of divalent cations with EDTA. Titration of both the circular dichroism and the peptide pattern produced by limited tryptic digestion imply that the interaction of calcium with thrombospondin involves cooperative interactions between multiple sites. In the proposed study, binding experiments will be performed with calcium and lanthanide ions to directly measure the number of binding sites, to confirm the presence of cooperative interactions and to determine the dissociation constants. (2) Structural domains of thrombospondin. A specific aim of the proposed study is to establish a detailed map of the domain structure of thrombospondin. Methods for the purification of these domains and monoclonal antibodies directed against these domains will be developed and used in the functional studies described below. Electron microscopy, peptide mapping by reverse phase high pressure liquid chromatography and N-terminal amino acid sequencing will be used to orient the structural domains within the thrombospondin molecule. (3) Functional domains of thrombospondin. Thrombospondin has the ability to bind to calcium, heparin, fibrin(ogen), fibronectin, histidine-rich glycoprotein, type V collagen, laminin and cell surfaces. A specific aim of the proposed study is to identify the structural domain of thrombospondin which contains each of these functional activities. In addition, the thrombospondin-binding portion of some of the above mentioned compounds will be identified and the effect of divalent cations on these interactions will be investigated. (4) Cell surface receptors for thrombospondin. Several different solid-phase binding assays will be developed and employed to identify proteins on the surface of platelets, endothelial cells, smooth muscle cells, fibroblasts, red cells and macrophages which bind to thrombospondin. The functional significance of potential receptors will be evaluated by determination of their subcellular location and the ability of physiologically relevant inhibitors to inhibit their binding to thrombospondin.