Production of a fibrinolytic inhibitor by cultured endothelial cells derived from human umbilical vein

Dosne, A.M.; Dupuy, Évelyne; Bodevin, E

doi:10.1016/0049-3848(78)90309-2

Cited by 113 publications

(34 citation statements)

References 14 publications

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“…Furthermore, these inhibitors appear to differ between different cell types. The secretion of Uk inhibitors by murine macrophages, which also synthesize a Uk-type PA (15,16, manuscript in preparation), as well as by hepatoma tissue culture cells (36), endothelial cells (37)(38)(39), and other cell types (40)(41)(42), further underlines the possible generality of these phenomena.…”

Section: Discussionmentioning

confidence: 99%

Concomitant secretion of prourokinase and of a plasminogen activator-specific inhibitor by cultured human monocytes-macrophages.

Vassalli¹,

Dayer²,

Wohlwend³

et al. 1984

The Journal of Experimental Medicine

410

195

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The plasminogen activator (PA) produced by freshly purified human monocytes-macrophages and histiocytic, lymphoma-derived U 937 cells was analyzed by zymography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and found to migrate with an apparent Mr of 55,000, identical to that of urokinase (Uk). By immunoprecipitation with antibodies specific for the two different types of PA, the enzyme was shown to be immunologically related to urokinase, and not to tissue PA. Urokinase was secreted in the form of the inactive Mr 55,000 zymogen prourokinase , and could be converted to the active Mr 55,000 enzyme by limited proteolysis with plasmin. Conditioned media from cultures of U 937 cells and monocytes-macrophages inhibited the fibrinolytic activity of exogenously added urokinase. Using [125I]-labeled urokinase we observed the formation of an enzyme-ligand complex, which was not dissociated by boiling in SDS and migrated with an apparent Mr 40,000 daltons higher than the free enzyme; since complexed urokinase was functionally inactivated as a PA, the ligand is an inhibitor of urokinase. This inhibitor is different from fibroblast-produced protease- nexin , in that it did not interact with thrombin. These results suggest that plasminogen activation by mononuclear phagocytes can be modulated through the secretion of both (pro)enzyme and a specific inhibitor.

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Section: Discussionmentioning

confidence: 99%

Concomitant secretion of prourokinase and of a plasminogen activator-specific inhibitor by cultured human monocytes-macrophages.

Vassalli¹,

Dayer²,

Wohlwend³

et al. 1984

The Journal of Experimental Medicine

410

195

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“…Endothelial cells secrete several proteins involved in different aspects of the hemostatic response including vWf, which promotes platelet adhesion and aggregation (8,12), protein S, which potentiates the anticoagulant action of protein C (40,41), and both plasminogen activators (42,43) and plasminogen activator inhibitor (44), which together regulate activation of fibrinolysis. Therefore, control of endothelial cell synthesis and secretion of these proteins is of central importance in coordinating the local hemostatic response to vessel injury.…”

Section: Resultsmentioning

confidence: 99%

Fibrin induces release of von Willebrand factor from endothelial cells.

Ribes¹,

Francis²

1987

J. Clin. Invest.

160

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Addition of fibrinogen to human umbilical vein endothelial cells in culture resulted in release of von Willebrand factor (vWf) from Weibel-Palade bodies that was temporally related to formation of fibrin in the medium. Whereas no release occurred before gelation, the formation of fibrin was associated with disappearance of Weibel-Palade bodies and development of extracellular patches of immunofluorescence typical of vWf release. Release also occurred within 10 min of exposure to preformed fibrin but did not occur after exposure to washed red cells, clot liquor, or structurally different fibrin prepared with reptilase. Metabolically labeled vWf was immunopurified from the medium after release by fibrin and shown to consist of highly processed protein lacking pro-vWf subunits. The contribution of residual thrombin to release stimulated by fibrin was minimized by preparing fibrin clots with nonstimulatory concentrations of thrombin and by inhibiting residual thrombin with hirudin or heating.We conclude that fibrin formed at sites of vessel injury may function as a physiologic secretagogue for endothelial cells causing rapid release of stored vWf.

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“…In addition, cultured rabbit (6,7) and human (8)(9)(10)(11) endothelial cells were found to be associated with an antifibrinolytic activity, suggesting that endothelium itself also may produce molecules that modulate the fibrinolytic activity of the vessel wall. However, no such inhibitor activity has been detected in confluent bovine aortic endothelial cells (BAEs) (9,12).…”

mentioning

confidence: 99%

Detection of an unusually stable fibrinolytic inhibitor produced by bovine endothelial cells.

Loskutoff¹,

Mourik²,

Erickson³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

347

170

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Fibrin/agar films were prepared and used to detect plasminogen activators produced by cultured bovine aortic endothelial cells (fibrin autography). One preparation of fibrin underwent spontaneous lysis upon incubation at 37TC. This lysis was prevented by antibodies to tissue-type plasminogen activator but not by antibodies to urokinase. Conditioned medium from the confluent endothelial cells was fractionated by polyacrylamide gel electrophoresis in the presence of NaDodSO4. The gels were analyzed on indicator films prepared with the spontaneously lysing fibrin (reverse fibrin autography). Unexpectedly, as the opaque fibrin film cleared, a distinct lysis-resistant zone appeared in the indicator gel at a region corresponding to Mr 55,000. Experiments were devised to determine whether the lysis-resistant zone in the indicator film reflected the presence of a cellular inhibitor in the polyacrylamide gel. The corresponding region was excised from a polyacrylamide gel, extracted with buffer, and tested directly for antifibrinolytic activity by the 'lS-labeled fibrin plate method. Urokinase-mediated fibrinolytic activity was inhibited by the gel extract in a dose-dependent manner indicating the presence of such an inhibitor. Inhibitor activity was detected in Triton X-100 extracts of washed monolayers and in conditioned medium, where it accumulated with time. The endothelial cell inhibitor not only survived exposure to NaDodSO4 but also was active after incubation at pH 12 or treatment with 5% (vol/vol) 2-mercaptoethanol, 6 M urea, 4 M guanidine hydrochloride, or 1 M acetic acid. Considerable activity also remained after heating at 1000C for 30 min. These results indicate that cultured bovine aortic endothelial cells synthesize and secrete a previously undetected, unusually stable fibrinolytic inhibitor of Mr 55,000. Reverse fibrin autography offers a convenient approach for studying such molecules.The production of both urokinase-like and tissue-type plasminogen activators (PAs) by cultured endothelial cells (1) emphasizes the potential role of endothelium in the specific catabolism of locally deposited fibrin and in the general maintenance of blood vessel patency. Expression of these cellular activities should be regulated precisely to ensure that the normal hemostatic role of endothelium (2) is not compromised.Plasma contains a number of molecules that may function in this way by either promoting (3, 4) or inhibiting (5) endothelial cell-mediated fibrinolysis. In addition, cultured rabbit (6, 7) and human (8-11) endothelial cells were found to be associated with an antifibrinolytic activity, suggesting that endothelium itself also may produce molecules that modulate the fibrinolytic activity of the vessel wall. However, no such inhibitor activity has been detected in confluent bovine aortic endothelial cells (BAEs) (9,12).We have used cultured i3AEs as a model to identify factors that influence the overall fibrinolytic activity of endothelium (13). Cellular fibrinolytic activity was found to change with the...

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Production of a fibrinolytic inhibitor by cultured endothelial cells derived from human umbilical vein

Cited by 113 publications

References 14 publications

Concomitant secretion of prourokinase and of a plasminogen activator-specific inhibitor by cultured human monocytes-macrophages.

Concomitant secretion of prourokinase and of a plasminogen activator-specific inhibitor by cultured human monocytes-macrophages.

Fibrin induces release of von Willebrand factor from endothelial cells.

Detection of an unusually stable fibrinolytic inhibitor produced by bovine endothelial cells.

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