This application is for a Program Project Grant from the National Heart, Lung and Blood Institute to support our continuing studies of the mechanism by which blood clotting is initiated. All of our studies to date suggest that the tissue factor pathway is the only significant pathway in a physiological sense. Accordingly, we are proposing a series of definitive experiments relating to the mechanism of action of tissue factor. In Project 1, a kinetic analysis of two tissue factor dependent reactions will be performed. This should shed light on the mechanism of action of this essential cofactor in addition to quantifying the newly described activation of Factor IX. In Project 2, we will be investigating the molecular consequences of the interaction of Factor VIIa and tissue factor. This will be accomplished mainly by active site mapping techniques utilizing competitive inhibitors and by systematically varying substrate composition. In Project 3, we will be investigating the way in which tissue factor is inserted into preformed liposomes, its interaction with Factor VIIa, and developing a radioimmunoassay in a bovine system. This is of particular interest insofar as we have described a plasma inhibitor of tissue factor. In Project 4, we will be attempting to confirm in a human system what we have learned from the bovine. In addition, we will directly compare the efficiency of glass activation of blood coagulation with that induced by tissue factor. In Project 5, which will be performed in a consortium arrangement with Yale University School of Medicine, we will continue our studies on the purification of human tissue factor. Once this is accomplished, radioimmunoassays will be developed for this substance.
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