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有助于血管发病。纤维蛋白原与白细胞的结合增强了白细胞的附着，并促进其随后的渗出。白细胞粘附在血管损伤部位和固定形式的纤维蛋白（OGEN）的参与度，而不是其可溶性形式，是诱导的炎性分子的产生，包括氧自由基，蛋白水解酶和细胞因子。 白细胞表面上的整联蛋白alphambeta2和alphaxbeta2通过用作纤维蛋白原的受体来参与这些事件。 该建议的总体目的是了解白细胞β2整合素纤维蛋白原识别的分子基础。 我们已经确定了纤维蛋白原的伽马链中的序列，这些序列由alphambeta2和alphaxbeta2识别，以及α2的I域中的互补识别序列。 我们假设纤维蛋白原γ链和受体的I域内的离散区域分别构成整联蛋白结合和配体结合口袋。 伽马模块的高分辨率结构和I区域的高分辨率结构的可用性，以及表达纤维蛋白原模块和I域的有效系统，为解决了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。我们将通过将功能收益的突变方法验证鉴定残基的贡献，通过将它们引入没有整合蛋白结合功能的纤维蛋白原模块中，或者与不结合纤维蛋白原的相关α2整合蛋白的α-域的α-构域。 这项研究的结果将通过测试纤维蛋白原内复制受体结合位点的成分的肽的作用来证实体内，作为吞噬细胞募集到植入的生物材料的抑制剂。 最后，将研究构象改变形式的纤维蛋白原形式的促炎作用的机制。 我们假设纤维蛋白原向纤维蛋白转化，固定在表面上，与血小板整合蛋白alphaiibbeta3结合时，纤维蛋白原的构象变化以及其蛋白水解的结合将转化为与白细胞β2整合素相互作用的形式。此外，将评估金属蛋白酶的选择性诱导，即通过不同的纤维蛋白原序列的代表性信号事件。 总之，这些研究将阐明β2整合素识别纤维蛋白原识别的分子基础，并定义调节其促炎活性的机制。这些信息可能有助于设计新型治疗剂，以破坏纤维蛋白原 - 白细胞相互作用，并可能导致对整合素的配体识别基本原理的一般理解。