RECOGNITION OF FIBRINOGEN BY LEUKOCYTE INTERGRINS

白细胞整合素对纤维蛋白原的识别

• 批准号: 6390461
• 负责人: Tatiana P Ugarova
• 金额: $ 22.7万
• 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/6390461
• 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而促成血管发病。纤维蛋白原与白细胞的结合增强了白细胞与内皮的附着并促进其随后的外渗。血管损伤部位的白细胞粘附和固定形式的纤维蛋白（原）（而不是其可溶形式）的接合诱导了包括氧自由基、蛋白水解酶和细胞因子在内的炎症分子的产生。 白细胞表面的整合素α M β 2和α X β 2通过充当纤维蛋白原的受体参与这些事件。 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。 我们已经鉴定了被α M β 2和α X β 2识别的纤维蛋白原γ链内的序列以及α M β 2的I结构域内的互补识别序列。 我们假设，纤维蛋白原的γ-链内和受体的I-结构域内的离散区域分别构成整合素结合口袋和配体结合口袋。 γ-模块和I-结构域的高分辨率结构的可用性，以及用于表达纤维蛋白原模块和I-结构域的有效系统，为解决它们相互识别的机制提供了前所未有的机会。 使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，通过将其引入无整联蛋白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α L β 2整联蛋白的α LI结构域，进一步验证所鉴定残基的贡献。 通过检测复制纤维蛋白原内受体结合位点组分的肽作为吞噬细胞募集至植入生物材料的抑制剂的作用，将在体内证实本研究的结果。 最后，将研究构象改变形式的纤维蛋白原的促炎作用的机制。 我们推测，纤维蛋白原的构象变化后，其转化为纤维蛋白，其固定到表面上，其结合血小板整合素α IIb β 3，其蛋白水解将转化成一种形式的分子能力与白细胞β 2整合素相互作用。此外，将评价不同纤维蛋白原序列对金属蛋白酶（代表性信号传导事件）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，并确定调节其促炎活性的机制。这些信息可能有助于设计新的治疗药物来破坏纤维蛋白原-白细胞相互作用，并可能导致对整合素识别配体的基本原理的普遍理解。