Tissue factor (coagulation factor III) is a membrane glycoprotein and an essential cofactor for the """"""""extrinsic"""""""" initiation of coagulation. Only in the presence of tissue factor does factor VII (or VIIa) have significant proteolytic activity towards its substrates, factors IX and X. Tissue factor apoprotein has no procoagulant activity, but the activity can be restored by reassociation of the apoprotein with appropriate lipids. Procoagulant activity appears to be strictly dependent on association of the apoprotein with a phospholipid membrane. Existing information regarding the molecular mechanism of tissue factor's cofactor activity and the regulation of that activity is limited. The proposed research will investigate the reconstitution of tissue factor-membrane complexes, and will evaluate the influence of the lipid environment on the reconstituted activity. Assembly of tissue factor apoprotein into lipid vesicles will be studied using purified phospholipids to determine which variables in lipid structure (charge and acyl chains) influence physical association of tissue factor with lipids and which variables affect expression of procoagulant activity in the assembled complex. Plasma high density lipoprotein can alter the physical properties of phospholipid vesicles and has been shown to inhibit tissue factor activity. Individual apolipoproteins will be purified and tested for their effects on tissue factor activity. Possible lipoprotein effects to be specifically addressed include interference with factors X and VII access to tissue factor-vesicle complexes, and alterations of the tissue factor conformation in the lipid bilayer. These studies will contribute to understanding tissue factor function and its regulation, and provide information with which to evaluate the importance of plasma lipoprotein effects on the initiation of coagulation.
