Despite the prominent role for tissue factor (TF)in various physiological processes, primarily in those relatedto blood coagulation, there is a lack of information with respect to the structure and function of natural TFforms existing in vivo. Most experimental data related to TF have been derived using various forms ofrecombinant proteins. Our preliminary data indicate that monocytic TF present on the native membrane is150-400-fold more active than any TF, recombinant or natural, presented on an artificial membrane. Theprimary goal of this proposal is to study natural human TF from multiple sources by evaluating their structuraland functional properties and by identifying the cell membrane component(s) responsible for high TF activity.Substantial amounts of placental, monocyte and mtcroparticle TF proteins will be purified by immunoaffinitymethods. Characterization of purified TF species will utilize immunoassays, structural, biochemical andfunctional assays as well as structure-function analyses. Functional characterization of TF proteins bothpurified and presented on native membranes will include factor Vila-driven reactions in one enzyme-onesubstrate systems (fluorogenic assays, extrinsic factor Xase and factor IX activation) followed by morecomplex systems, such as TF-initiated thrombin generation in synthetic coagulation proteome and wholeblood. The posttranslational modifications of the purified TF species will be characterized usingdeglycosylation, tryptic digestion, mass-spectroscopy and sequencing. The influence of these modificationson TF activity will be analyzed. Purified human TF from placenta will be used as a standard in allevaluations, functional and structural. Three different species of recombinant human TF will be used forcomparison as well, i.e. TF^ea (full-length), TFi_242 (lacking the cytoplasmic domain), and TFi.2is (soluble;extracellular domain only). We will also attempt to understand the mechanism underlying high monocyte TFin situ activity by purifying, analyzing and evaluating the component(s) of the monocyte membrane, lipid raftsand monocyte-derived microparticles. The data accumulated during this study will expand our knowledgerelated to the structural and functional properties of natural human TF. New knowledge linking TF structureand environment with functional activity will be obtained.Relevance: This project will lead to a better understanding of the mechanisms regulating the activity of tissuefactor, a key protein in the initiation of blood clotting and an emerging link between inflammation andthrombosis.
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