Abnormal thrombus formation and/or dissolution may result from an aberrant plasma fibrinolytic system. The long term objective of my laboratory is to understand the role of the fibrinolytic system in the pathogenesis of these disorders. It is clear that many of the fibrinolytic components of plasma originate from endothelium, and that endothelial cell-mediated fibrinolysis must therefore be precisely regulated. However, it is also apparent that the fibrinolytic system of these cells is too complex to study with existing enzymatic assays. For example, cultured bovine aortic endothelial cells (BAEs) produce not one, but multiple plasminogen activators (PAs) as well as a PA inhibitor (PAI) that can modulate their activities. Additional levels of regulation are implied since the urokinase-like PA (uPA) behaves as a proenzyme, and also binds to specific receptors on the cell surface. In additional levels of regulation are implies since the urokinase-like PA (uPA) behaves as a proenzyme, and also binds to specific receptors on the cell surface. In addition, most of the tissue-type PA (tPA) is in an inactive complex with PAI, but can be expressed under some conditions.
The specific aims of this proposal are to delineate the exact contribution of free and cell- associated uPA, and of free and PAI-bound tPA to the net fibrinolytic activity of BAEs under a variety of conditions. Bovine uPA, tPA, and tPA-PAI complexes will be purified, and polycolonal and monoclonal antibodies will be developed. These antibodies will be used in immunological assays to determine the concentration, cellular distribution, and biosynthetic rates of each of these fibrinolytic components under conditions which alter the net fibrinolytic activity of BAEs (e.g., interleukin 1, endotoxin, activated protein C, and thrombin). The tPA-PAI complexes will be characterized. Emphasis will be placed on defining the functional status of the individual domains of tPA (e.g., fibrin- binding domain) in complexes, on establishing conditions express the latent PA activity of complexes, and on delineating the process by which complexes are assembled by BAEs. The nature and significance of the binding of uPA to BAEs will be investigated. The PA activity and PAI sensitivity of receptor- bound UPA will be established, and the biochemical parameters of this interaction (e.g., rate constants, number of receptors, etc.) will be defined. The receptor itself will be characterized.
|Keeton, M; Eguchi, Y; Sawdey, M et al. (1993) Cellular localization of type 1 plasminogen activator inhibitor messenger RNA and protein in murine renal tissue. Am J Pathol 142:59-70|
|Schneiderman, J; Sawdey, M S; Keeton, M R et al. (1992) Increased type 1 plasminogen activator inhibitor gene expression in atherosclerotic human arteries. Proc Natl Acad Sci U S A 89:6998-7002|
|Seiffert, D; Keeton, M; Eguchi, Y et al. (1991) Detection of vitronectin mRNA in tissues and cells of the mouse. Proc Natl Acad Sci U S A 88:9402-6|
|Sawdey, M S; Loskutoff, D J (1991) Regulation of murine type 1 plasminogen activator inhibitor gene expression in vivo. Tissue specificity and induction by lipopolysaccharide, tumor necrosis factor-alpha, and transforming growth factor-beta. J Clin Invest 88:1346-53|
|Schleef, R R; Loskutoff, D J; Podor, T J (1991) Immunoelectron microscopic localization of type 1 plasminogen activator inhibitor on the surface of activated endothelial cells. J Cell Biol 113:1413-23|
|Sane, D C; Moser, T L; Parker, C J et al. (1990) Highly sulfated glycosaminoglycans augment the cross-linking of vitronectin by guinea pig liver transglutaminase. Functional studies of the cross-linked vitronectin multimers. J Biol Chem 265:3543-8|
|Schleef, R R; Podor, T J; Dunne, E et al. (1990) The majority of type 1 plasminogen activator inhibitor associated with cultured human endothelial cells is located under the cells and is accessible to solution-phase tissue-type plasminogen activator. J Cell Biol 110:155-63|
|Campochiaro, P A; Mimuro, J; Sugg, R et al. (1989) Retinal pigment epithelial cells produce a latent fibrinolytic inhibitor that is antigenically and biochemically related to type 1 plasminogen activator inhibitor produced by vascular endothelial cells. Exp Eye Res 49:195-203|
|Mimuro, J; Loskutoff, D J (1989) Binding of type 1 plasminogen activator inhibitor to the extracellular matrix of cultured bovine endothelial cells. J Biol Chem 264:5058-63|
|Mimuro, J; Loskutoff, D J (1989) Purification of a protein from bovine plasma that binds to type 1 plasminogen activator inhibitor and prevents its interaction with extracellular matrix. Evidence that the protein is vitronectin. J Biol Chem 264:936-9|
Showing the most recent 10 out of 23 publications