RECOGNITION OF FIBRINOGEN BY LEUKOCYTE INTERGRINS

白细胞与纤维蛋白原的相互作用通过介导炎症反应参与了血管的发病。纤维蛋白原与白细胞结合可增强白细胞与内皮细胞的粘附性，并促进其随后的外渗。白细胞在血管损伤部位的黏附和固定化形式的纤维蛋白(原)的接触，而不是其可溶性形式，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αMbeta2和αXbeta2作为纤维蛋白原的受体参与了这些事件。这项建议的总体目标是了解白细胞β2整合素识别纤维蛋白原的分子基础。我们已经鉴定了纤维蛋白原的伽马链中被αMbeta2和αXbeta2识别的序列以及αMbeta2的I-结构域中的互补识别序列。我们假设纤维蛋白原的伽马链内的离散区域和受体的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。伽马模块和I结构域的高分辨率结构的可获得性，以及高效的纤维蛋白原模块和I结构域表达系统的出现，为解决它们的相互识别机制提供了前所未有的机会。利用多肽化学和突变分析，我们将确定参与形成配体-受体接触的关键氨基酸残基。我们将使用功能增益突变方法进一步验证已识别残基的贡献，方法是将它们引入不具有整合素结合功能的纤维蛋白原模块，或引入相关的不与纤维蛋白原结合的αLbeta2整合素的αLI结构域。这项研究的结果将通过测试复制纤维蛋白原内受体结合部位成分的多肽作为抑制吞噬细胞向植入的生物材料募集的效果来在体内得到证实。最后，我们将研究构象改变形式的纤维蛋白原的促炎作用的机制。我们假设，当纤维蛋白原转变为纤维蛋白时，它的构象变化，它固定在表面，它与血小板整合素αIIbbeta3结合，它的蛋白分解将使该分子转变为能够与白细胞β2整合素相互作用的形式。此外，还将评估不同的纤维蛋白原序列对金属蛋白酶的选择性诱导，这是一种典型的信号事件。总之，这些研究将阐明β2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与白细胞的相互作用，并可能导致对整合素识别配体的基本原理的总体理解。

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.