FUNCTIONAL CHARACTERIZATION IN PLATELET THROMBOSPONDIN

凝血酶原蛋白是一种高相对分子质量的糖蛋白。 在血小板、内皮细胞、平滑肌细胞中被鉴定， 成纤维细胞，以及最近在大鼠上皮细胞系中 牙槽骨。拟议研究的目的是评估 凝血酶原蛋白是一种多功能蛋白的假说 细胞间和细胞间的大分子缔合 细胞与细胞外基质的相互作用。具体的焦点将被定向 到以下领域： (1)凝血酶敏感蛋白与钙离子的相互作用。沉积作用 系数、特性粘度、圆二色体、电子显微镜 凝血酶原蛋白的外观和对蛋白分解的抵抗力是深刻的 受EDTA去除二价阳离子的影响。两者的滴定 圆二色谱与有限胰酶产生的多肽图谱 消化意味着钙与凝血酶反应蛋白的相互作用 涉及多个站点之间的协作交互。在建议的 研究中，将进行钙和稀土元素的结合实验 离子直接测量结合位点数，以确认 合作相互作用的存在和确定解离 常量。 (2)凝血酶原蛋白的结构域。建议的一个具体目标 研究是为了建立一个详细的领域结构图 凝血酶敏感蛋白。纯化这些结构域的方法和 将开发针对这些结构域的单抗，并 用于下文所述的功能研究。电子显微镜， 反相高效液相色谱多肽图谱的初步研究 将使用N-末端氨基酸序列来定位结构 凝血酶原蛋白分子中的结构域。 (3)凝血酶原蛋白的功能结构域。凝血酶原蛋白有能力 结合钙、肝素、纤维蛋白(原)、纤维连接蛋白、富含组氨酸 糖蛋白、V型胶原、层粘连蛋白和细胞表面。一个明确的目标 拟议研究的目的是确定结构域 含有上述每种功能活性的凝血酶原蛋白。在……里面 此外，上述部分凝血酶原蛋白结合部分 化合物的鉴定和二价阳离子对这些化合物的影响 将对相互作用进行调查。 (4)细胞表面凝血酶敏感蛋白受体。几种不同的 将开发和使用固相结合分析来确定 血小板、内皮细胞、平滑肌表面的蛋白质 细胞、成纤维细胞、红细胞和巨噬细胞与 凝血酶敏感蛋白。潜在受体的功能意义将是 通过确定它们的亚细胞位置和能力进行评估 生理上相关的抑制剂，以抑制其与 凝血酶敏感蛋白。

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.