FUNCTIONAL CHARACTERIZATION OF PLATELET THROMBOSPONDIN

血小板反应蛋白（TSP）是一种重要的粘附蛋白， 调节细胞生长和迁移。 的长期目标 建议的研究是确定TSP在形成中的作用， 组织修复。 本提案的具体重点将针对 以下领域： (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 uM）的钙与 TSP已使用蛋白质结构的探针检测。 的氨基酸 血小板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， 建议的这一部分的具体目标 研究是（1）定量45钙与TSP的直接结合， 基于3型重复序列的合成肽，和（2）确定 可以在个体3型水平上检测到构象变化 重复. (2)血小板反应蛋白TSP的功能结构域具有结合 肝素、纤维蛋白原、纤连蛋白、V型胶原、层粘连蛋白、纤溶酶原， 富组氨酸糖蛋白、硫酸化糖脂和细胞表面 受体。 拟议研究的一个具体目标是确定 在TSP分子内，通过产生 (1)保留功能活性的蛋白水解片段，（2）融合 具有功能活性的蛋白，（3）多克隆抗融合蛋白 阻断功能的抗体，（4）阻断 功能，（5）TSP的变体形式，其中特定位点已被 突变或缺失的肽和（6）模拟 茶匙 (3)血小板反应蛋白与生长发育 体外细胞对TSP合成的调节导致了以下假设 TSP参与了正常的形态发生过程， 细胞对损伤的反应。 这一假设预测， 血小板反应蛋白将是正常发育的重要组成部分。 一 拟议研究的具体目标是确定TSP在 通过（1）TSP在发育中的小鼠胚胎中的定位和（2） TSP基因缺失或突变的小鼠的产生。

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.