Human umbilical vein endothelial cells (HEC) in culture synthesize and release a plasminogen activator inhibitor which is found in the culture medium in two forms, an active form which binds to and inactives tissue plasminogen activator and urokinase and a latent form which lacks anti-activator activity in its native state but which becomes active upon treatment with SDS. Studies from this laboratory suggest that the inhibitor is synthesized and released from HEC in its active form and converts to the latent form in the extracellular environment. Using the human inhibitor from cultured HEC, I propose to investigate the mechanism by which the transition from active to latent inhibitor occurs and to determine whether the rate of this conversion can be regulated. The physiochemical properties of the two forms will be studied and compared employing isoelectric focusing, sucrose and cesium chloride centrifugation and molecular exclusion chromatography. A correlation between the active to latent inhibitor transition and changes in molecular properties will be sought. Studies will be performed with purified active inhibitor to determine whether this transition is catalyzed by a second component in the culture medium or if it is an intrinsic characteristic of the molecule occurring independently of its environment. The observed association between the inhibitor and the endothelial cell matrix will be further investigated by immunofluorescence and binding studies. These studies will further define a potentially important mechanism by which plasminogen activator inhibitor activity is controlled in vivo.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL030244-06
Application #
3341309
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1984-01-01
Project End
1990-12-31
Budget Start
1989-01-01
Budget End
1990-12-31
Support Year
6
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Madden, R M; Levin, E G; Marlar, R A (1991) Thrombin and the thrombin-thrombomodulin complex interaction with plasminogen activator inhibitor type-1. Blood Coagul Fibrinolysis 2:471-6
Levin, E G; Santell, L (1991) Thrombin- and histamine-induced signal transduction in human endothelial cells. Stimulation and agonist-dependent desensitization of protein phosphorylation. J Biol Chem 266:174-81
Idell, S; James, K K; Levin, E G et al. (1989) Local abnormalities in coagulation and fibrinolytic pathways predispose to alveolar fibrin deposition in the adult respiratory distress syndrome. J Clin Invest 84:695-705
Levin, E G; Marotti, K R; Santell, L (1989) Protein kinase C and the stimulation of tissue plasminogen activator release from human endothelial cells. Dependence on the elevation of messenger RNA. J Biol Chem 264:16030-6
Park, S; Harker, L A; Marzec, U M et al. (1989) Demonstration of single chain urokinase-type plasminogen activator on human platelet membrane. Blood 73:1421-5
Miles, L A; Dahlberg, C M; Levin, E G et al. (1989) Gangliosides interact directly with plasminogen and urokinase and may mediate binding of these fibrinolytic components to cells. Biochemistry 28:9337-43
Levin, E G; Santell, L (1988) Stimulation and desensitization of tissue plasminogen activator release from human endothelial cells. J Biol Chem 263:9360-5
Santell, L; Levin, E G (1988) Cyclic AMP potentiates phorbol ester stimulation of tissue plasminogen activator release and inhibits secretion of plasminogen activator inhibitor-1 from human endothelial cells. J Biol Chem 263:16802-8
Schwartz, B S; Monroe, M C; Levin, E G (1988) Increased release of plasminogen activator inhibitor type 2 accompanies the human mononuclear cell tissue factor response to lipopolysaccharide. Blood 71:734-41
Miles, L A; Levin, E G; Plescia, J et al. (1988) Plasminogen receptors, urokinase receptors, and their modulation on human endothelial cells. Blood 72:628-35

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