The vascular endothelium is recognized as the principle source of tissue plasminogen activator (tPA) in blood and therefore must play a major role in determining the fibrinolytic state of the vascular system. Cultured human endothelial cells (HEC) continue to produce a PA which is immunochemically similar to the tPA isolated from human plasma. Characterization of the HEC-tPA has shown that it has a molecular weight of 100,000, it is inactive in its native state, and it does not bind to fibrin clots. Preliminary studies on the molecular state of the HEC-tPA have suggested that it is a complex consisting of a 60,000 dalton anti-tPA immunoprecipitable component and an undefined 40,000 dalton inhibitor. Because endothelial cell cultures offer a useful model to define the contribution of the vascular endothelium in fibrinolysis it is proposed to initially characterize the tPA-containing complex released by primary cultures of HEC and then begin to define the mechanisms by which HEC-derived plasma PAs are regulated. Using cultured human umbilical vein endothelial cells I propose to purify the 100,000 dalton complex, determine the nature of the interaction between the tPA and its putative inhibitor, the site and timing of complex formation, and the identity and specificity of the inhibitor. Once the complex has been characterized studies will focus on the regulation of tPA production by plasma components. These studies will define the precise mechanisms by which the HEC-derived tPA and its modulated by inhibitors and the mechanisms by which cellular PA production is regulated. It is anticipated the proposed studies will have clinical application in determining potential therapeutic procedures for modulating the firbrinolytic state.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL030244-02
Application #
3341306
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1984-01-01
Project End
1986-12-31
Budget Start
1985-01-01
Budget End
1985-12-31
Support Year
2
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
San Diego
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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