This competing continuation application is based on three major findings that demonstrate that tissue factor (TF), the initiating protease receptor of the coagulation cascade, regulates integrin-dependent cell migration of relevance for angiogenesis. (1) A specific epitope in the TF extracellular domain is implicated in beta-1-integrin-dependent epithelial cell migration and TF interacts with a subset beta-1 integrin heterodimers as well as alpha-v-beta-3. (2) On the intracellular side, the p85 subunit of phosphatidylinositol-3 kinase, an adaptor involved in cell migration, binds to the TF cytoplasmic domain in a Ser 258 phosphorylation regulated manner. (3) TF cytoplasmic domain-deleted mice show enhanced angiogenesis and endothelial cell sprouting, providing evidence for a physiologically relevant regulatory role of TF in integrin function. We propose to characterize the interaction between TF and integrins in endothelial cells with the following specific aims.
Aim 1 is to map the integrin binding site on TF and to characterize with which integrins TF associates in endothelial cells. These studies will define novel molecular interactions of TF and provide tools to address integrin-specificity in the """"""""non-hemostatic"""""""" interactions of TF.
Aim 2 is to characterize the relationship of integrins and Ser 258 phosphorylation of the TF cytoplasmic domain in endothelial cells. In addition to regulation by palmitoylation of Cys 245, Ser 258 phosphorylation is influenced by interactions outside the cytoplasmic domain. The proposed experiments will establish whether integrin interactions regulate TF cytoplasmic domain phosphorylation, whether Ser 258 phosphorylated TF is associated with specific integrins, and how TF and integrin assembly into endothelial cell-derived microparticles is regulated by phosphorylation.
Aim 3 is to define the role of the TF cytoplasmic domain in tumor growth and angiogenesis. The goals of the proposed experiments are to characterize tumor development in TF cytoplasmic domain deleted mice and to define the mechanism by which the TF cytoplasmic domain suppresses angiogenesis in ex vivo and in vitro assays. Together, these experiments will characterize a novel pathway of integrin cooperation with TF of relevance for endothelial cell migration.
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