Tissue Factor is the central activator of the coagulation cascade and has emerged as a signaling receptor at the crossroads of G-protein coupled and integrin signaling pathways. Project 3 is based on our recent finding that reduction/oxidation of a critical disulfide bond in the extracellular domain of TF regulates prothrombotic activity. To elucidate how this novel regulatory mechanism separately controls signaling and prothrombotic activity of TF is the central theme of this project. Specifically, the applicant proposes to characterize the protein structural determinants that render TF susceptible to reduction in the context of enzyme and substrate interaction, the role of membrane composition on the stability of the critical disulfide bond, and the localization of reduced TF relative to cellular oxidative pathways, including protein disulfide isomerase implicated in oxidative activation of TF. The finding that TF can alternatively become S-nitrosylated is the basis for experiments to characterize cellular pathways that transfer NO to TF and to generate analytical tools to detect this posttranslation modification in the context of thrombus formation. The applicant proposes to test the hypothesis that thrombogenicity is under reciprocal control by signaling pathways with which TF is associated. Specifically, strategies are developed to demonstrate that integrins target TF to extracellular matrices and that the effects of integrin activation on local redox potential contribute to matrix thrombogenicty by oxidating TF. Mutagenesis is employed to unambiguously establish the signaling properties of the non-coagulant form of TF. Thrombogenic and signaling roles of TF are evaluated in atherosclerosis using an in vivo model of turbulent flow induced lesion development in atherosclerosis prone animals. The successful completion of these studies will provide valuable information on a novel regulatory pathway of TF-dependent thrombogenicity of particular relevance for the dynamics of platelet activation on extracellular matrices.
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