Antithrombin (AT) is a major serpin inhibitor in plasma that regulates the proteolytic activity of coagulation proteases of intrinsic and extrinsic pathways. In addition to its anticoagulant function, AT also has a potent antiinflammatory function. The anticoagulant function of AT is mediated through its reactive center loop (RCL)-dependent inhibition of coagulation proteases, but the antiinflammatory signaling function of AT is mediated via its D-helix-dependent interaction with glycosaminoglycan (GAG) chains, attached to core proteins of heparan sulfate proteoglycans (HSPGs). Unlike the relatively well-understood anticoagulant function, the mechanism of the antiinflammatory function of AT is poorly understood. Moreover, because it is thought that a conformational change in the RCL of AT by D-helix-dependent interaction with vascular GAGs is required for the protease inhibitory function of the serpin, the contribution of the AT D-helix-GAG interaction to the physiological function of the serpin in two pathways remains unknown. The antiinflammatory function of AT requires its D- helix dependent interaction with 3-O-sulfate (3-OS) containing GAGs, the same type of vascular GAGs which are thought to be required for the conformational activation of AT in order for the serpin to optimally inhibit the activity of vitamin K-dependent coagulation proteases. The Syndecan-4 (Synd-4) sub-family of HSPGs has been identified as the primary receptor on which AT binds to elicit antiinflammatory responses in vascular endothelial cells. However, Synd-4 is also an essential co- receptor for receptor tyrosine kinases for signaling by basic fibroblast growth factor (FGF2 also called bFGF) in vascular endothelial cells. The mechanism by which AT and FGF2 utilize the same receptor to elicit paradoxical signaling responses is completely unknown. We have prepared several AT and receptor mutants and propose to investigate these questions, employing both cellular and animal models in 2 Specific Aims. We propose to investigate 1) the significance of AT D-helix interaction with vascular HSPGs to the physiological function of the serpin, in the alternative anticoagulant and antiinflammatory pathways by employing AT-deficient mice and 2) the mechanism by which AT elicits intracellular signaling responses in vascular endothelial cells. A sub-aim of Aim 2 will investigate the mechanism by which cleaved and/or latent AT elicit antiangiogenic responses in endothelial cells in response to FGF2. These studies will utilize multidisciplinary approaches including genetics, basic biochemistry, kinetics, cellular and molecular biology methods to provide key information related to the D-helix dependent antiinflammatory signaling mechanism of AT.
The studies of this application will employ multidisciplinary approaches to investigate the mechanisms through which antithrombin modulates procoagulant and proinflammatory pathways. Antithrombin binds to vascular heparan sulfate proteoglycans to elicit antiinflammatory signaling responses by an unknown mechanism and whether this interaction contributes to the anticoagulant function of antithrombin is not known. Understanding the mechanism of antithrombin-mediated regulation of coagulation proteases and inflammatory pathways can lead to development of new generation of therapeutic drugs that can potentially be useful for controlling thrombotic and inflammatory disorders.
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