. Tissue factor is a glycoprotein present in the plasma membranes of many cell types. It serves as an essential cofactor for factor VII, enabling it to proteolytically activate factors IX and X. Healthy cells normally in contact with blood have little or no tissue factor, but other cells, such as fibroblasts, can contain tissue factor in accessible, inaccessible, high, or low activity states. While tissue factor expression can be regulated at the level of gene transcription in response to stimuli, tissue factor already present in cells may be actively regulated with regard to accessibility and specific activity. Expressed tissue factor may be further regulated by inhibitory proteins in blood. The biochemical processes which control tissue factor expression from the point of translational maturity to inactivation at the cell surface, are unknown. Experiments in this proposal will address these processes through study of tissue factor phosphorylation, lipid-dependent tissue factor activity, and inhibitory blood proteins. Recent experiments demonstrating that tissue factor can be phosphorylated, considered with other published data, strongly suggests that tissue factor expression may be modulated by protein kinases and possibly by complimentary phosphatases. Proposed experiments will test the hypothesis that phosphorylation of tissue factor is a regulatory modification. I will also examine the biochemistry of such regulation. Other experiments will study the reassociation of purified tissue factor with lipids and the influence of the lipid milieu on tissue factor activity. Lipid-binding proteins which are known to inhibit tissue factor function (c reactive protein, apolipoprotein A-11, lipoprotein associated coagulation inhibitor, placental anticoagulant protein) will be studied and compared with regard to tissue factor-dependent activation of factors IX and X when tissue factor is present in various lipid environments, including tissue factor expressed in the environment provided by the cell membrane. The proposed experiments will provide new information pertinent to control of tissue factor expression by living cells. Particularly, the characteristics of membrane lipid variability and lipid-binding proteins which are important in the initiation of coagulation, will be elucidated.
