FUNCTIONAL CHARACTERIZATION OF PLATELET THROMBOSPONDIN

凝血酶原蛋白(TSP)是一种重要的粘附性蛋白 来调节细胞的生长和迁移。该计划的长远目标 建议的研究是确定TSP在形成中的作用和 组织修复。这项提案的具体重点将指向 以下方面： (1)凝血酶敏感蛋白与钙高亲和力(KD)的相互作用 -100 nm)和低亲和力(KD-120微米)协同结合 利用蛋白质结构探针检测到了TSP。氨基酸 凝血酶反应蛋白的序列包括一个独特的结构基序 由多个钙结合位点组成，这些结合位点立即 相互毗邻。l提议的这一部分的具体目标 研究的目的是(1)定量测定45个钙与TSP和TO的直接结合。 基于类型3重复的合成肽以及(2)确定 构象变化可以在个体类型3的水平上检测到 重复一遍。 (2)凝血酶敏感蛋白TSP的功能域具有结合能力 肝素、纤维蛋白原、纤维连接蛋白、V型胶原、层粘连蛋白、纤溶酶原、 富含组氨酸的糖蛋白、硫酸糖脂和细胞表面 感受器。拟议研究的一个具体目标是确定这些地点 在TSP分子内通过产生 (1)保留功能活性的蛋白水解物片段；(2)融合 具有功能活性的蛋白质；(3)多克隆反融合蛋白 阻断功能的抗体，(4)阻断功能的单抗 功能，(5)TSP的变体形式，其中特定站点已被 突变或缺失；(6)模拟黄曲霉毒素活性的合成肽 TSP. (3)凝血酶敏感蛋白在生长和发育中的作用 细胞在体外对TSP合成的调节导致了这一假说 TSP参与了正常的形态发生过程，是一个组成部分 细胞对损伤的反应。这一假说将预测 凝血酶原蛋白将是正常发育的重要组成部分。一个 拟议研究的具体目的是确定TSP在人体内的功能 通过(1)TSP在发育中的小鼠胚胎中的定位和(2) TSP基因缺失或突变的一代小鼠。

Thrombospondin (TSP) is an adhesive protein which appears to be important in modulating cell growth and migration. The long-term objective of the proposed study is to determine the function of TSP in the formation and repair of tissue. Specific focus in this proposal will be directed toward the following areas: (1) The Interaction of Thrombospondin with Calcium High affinity (Kd - 100nM) and low affinity (Kd - 120 uM) cooperative binding of calcium to TSP has been detected using probes of protein structure. The amino acid sequence of thrombospondin includes a unique structural motif which appears to be composed of multiple calcium-binding sites that are immediately adjacent to each other.l The specific aims of this portion of the proposed study are to (1) quantitate the direct binding of 45 calcium to TSP and to synthetic peptides based on the type 3 repeats and (2) determine if a conformational change can be detected at the level of individual type 3 repeat. (2) Functional Domains of Thrombospondin TSP has the ability to bind to heparin, fibrinogen, fibronectin, type V collagen, laminin, plasminogen, histidine-rich glycoprotein, sulfated glycolipids and cell surface receptors. A specific aim of the proposed study is to identify the sites within the TSP molecule for these interactions through the production of (1) proteolytic fragments which retain the functional activity, (2) fusion proteins with functional activity, (3) polyclonal anti-fusion protein antibodies which block function, (4) monoclonal antibodies which block function, (5) variant forms of TSP in which specific sites have been mutated or deleted and (6) synthetic peptides which mimic the activity of TSP. (3) Thrombospondin in Growth and Development Recent data on the regulation of TSP synthesis by cells in vitro have led to the hypothesis that TSP is involved in the normal morphogenetic process and is a component of the cell's response to injury. This hypothesis would predict that thrombospondin would be an essential component of normal development. A specific aim of the proposed study is to determine the function of TSP in vivo by (1) the localization of TSP in the developing mouse embryo and (2) the generation of mice in which the TSP gene has been deleted or mutated.