Tissue factor (TF), the primary triggering agent of the blood clotting cascade in normal hemostasis, is also implicated as a key initiator in the pathogenesis of a variety of life-threatening thrombohemorrhagic disorders. TF has two known functions: (1) TF greatly accelerates the activation of zymogen factor VII (fVII to the catalytically activate form, fVIIa; and (2) TF is the protein cofactor for fVIIa enzymatic activity. Neither function of TF is well understood in molecular terms. The goal of this study is to understand how TF interacts with fVII and fVIIa to assemble the functional TF:fVIIa complex, which is responsible for initiating the clotting cascade. The interactive sites on TF and fVIIa will be identified using mutagenesis, peptides, and antibodies. A battery of assays will be used that will permit measurement of specific aspects of TF/fVII function. Importantly, results from the activity assays will be compared to direct biophysical measures of protein-protein and protein-phospholipid interactions. The demonstration that the two major functions of TF can be separated by mutagenesis underscores the feasibility of this approach. This new insight into the structure/function relationship of TF has led to the formulation of several hypotheses, the testing of which will now enable the direct investigation of the poorly understood mechanism by which TF accelerates fVII activation. An important development from these studies will be the generation of novel anti-thrombotic agents with extremely high specificity (since they will inhibit only one specific aspect of TF or fVII function).
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