RECOGNITION OF FIBRINOGEN BY LEUKOCYTE INTERGRINS

白细胞整合素对纤维蛋白原的识别

• 批准号: 6537649
• 负责人: Tatiana P Ugarova
• 金额: $ 23.38万
• 依托单位: CLEVELAND CLINIC FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6537649
• 关键词: binding sites; biological signal transduction; biomaterial interface interaction; fibrinogen; gene mutation; human subject; inflammation; integrins; intermolecular interaction; laboratory mouse; leukocytes; metalloendopeptidases; protein binding; protein sequence; site directed mutagenesis

The interaction of leukocytes with fibrinogen contributes to vascular pathogenesis by mediating an inflammatory response. Fibrinogen binding to leukocytes enhances leukocyte attachment to endothelium and promotes their subsequent extravasation. Leukocyte adhesion at sites of vascular injury and engagement of immobilized form of fibrin(ogen), but not its soluble form, induced production of inflammatory molecules including oxygen radicals, proteolytic enzymes and cytokines. The integrins alphaMbeta2 and alphaXbeta2 on the surface of leukocytes participate in these events by serving as receptors for fibrinogen. The overall objective of this proposal is to understand the molecular basis for fibrinogen recognition by leukocyte beta2 integrins. We have identified sequences within the gamma-chain of fibrinogen which are recognized by alphaMbeta2 and alphaXbeta2 and the complementary recognition sequences within the I-domain of alphaMbeta2. We hypothesize that discrete regions within the gamma-chain of fibrinogen and within the I- domain of the receptor constitute the integrin-binding and ligand-binding pockets, respectively. The availability of the high-resolution structure of the gamma-module and the I-domain, and efficient systems for expression of fibrinogen modules and the I-domain, provide an unprecedented opportunity to solve the mechanism of their mutual recognition. Using peptide chemistry and mutational analyses we will determine critical amino acid residues involved in the formation of ligand-receptor contacts. We will further verify the contribution of identified residues using a gain-of-function mutational approach by introducing them into the modules of fibrinogen without integrin-binding function or into the alphaLI-domain of the related alphaLbeta2 integrin which does not bind fibrinogen. Results from this study will be substantiated in vivo by testing the effect of peptides duplicating the components of the receptor binding site within fibrinogen as inhibitors of phagocyte recruitment to implanted biomaterials. Finally, the mechanism underlying proinflammatory effect of conformationally altered form of fibrinogen will be studied. We hypothesize that conformational changes in fibrinogen upon its transformation to fibrin, its immobilization onto surfaces, its binding to platelet integrin alphaIIbbeta3, and its proteolysis will transform the molecule into a form competent to interact with leukocyte beta2 integrins. Furthermore, selective induction of metalloproteinases, a representative signaling event, by different fibrinogen sequences will be evaluated. Together, these studies will clarify the molecular basis of fibrinogen recognition by beta2 integrins and define the mechanism that regulates its proinflammatory activity. This information may assist in the design of novel therapeutic agents to disrupt fibrinogen-leukocyte interactions and may lead to general understanding of the basic principles of ligand recognition by integrins.

白细胞与纤维蛋白原的相互作用通过介导炎症反应参与血管发病。纤维蛋白原与白细胞的结合增强了白细胞对内皮的附着，促进了白细胞的外渗。白细胞在血管损伤部位的粘附和固定形式的纤维蛋白（原）的接合，而不是其可溶性形式，诱导炎症分子的产生，包括氧自由基、蛋白水解酶和细胞因子。白细胞表面的整合素alphabeta2和alphaXbeta2作为纤维蛋白原受体参与了这些事件。本提案的总体目标是了解白细胞β 2整合素识别纤维蛋白原的分子基础。我们在纤维蛋白原γ -链中发现了被alphabeta2和alphaXbeta2识别的序列，以及在alphabeta2的i结构域内发现了互补的识别序列。我们假设纤维蛋白原γ链和受体I-结构域内的离散区域分别构成整合素结合袋和配体结合袋。γ -模块和i结构域的高分辨率结构的可用性，以及纤维蛋白原模块和i结构域的高效表达系统，为解决它们相互识别的机制提供了前所未有的机会。利用肽化学和突变分析，我们将确定参与配体-受体接触形成的关键氨基酸残基。我们将使用功能获得突变方法进一步验证鉴定的残基的贡献，方法是将它们引入没有整合素结合功能的纤维蛋白原模块或引入不结合纤维蛋白原的相关alphaLbeta2整合素的alpha - i结构域。这项研究的结果将在体内得到证实，通过测试复制纤维蛋白原内受体结合位点成分的肽对吞噬细胞向植入生物材料募集的抑制作用。最后，探讨纤维蛋白原构象改变的促炎作用机制。我们假设纤维蛋白原在转化为纤维蛋白、固定在表面、与血小板整合素alphaIIbbeta3结合以及其蛋白水解过程中的构象变化将使该分子转化为能够与白细胞beta2整合素相互作用的形式。此外，我们将评估不同纤维蛋白原序列对金属蛋白酶（一种代表性信号事件）的选择性诱导。总之，这些研究将阐明β 2整合素识别纤维蛋白原的分子基础，并确定其促炎活性的调节机制。这一信息可能有助于设计新的治疗药物来破坏纤维蛋白原-白细胞的相互作用，并可能导致对整合素识别配体的基本原理的一般理解。