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.
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