Partial Characterization of a Fibrinolytic Inhibitor Produced by Cultured Endothelial Cells Derived from Human Umbilical Vein

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

doi:10.1055/s-0038-1657146

Cited by 24 publications

(6 citation statements)

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“…This may be especially true of cultured endothelial cells because all endothelial cells tested, including those isolated from humans (8)(9)(10)(11)36), rabbits (6,7), and now cows have been shown to contain both activities. Obviously, then, special consideration should be given in designing any experimental approach to characterize and quantitate the fibrinolytic components of such cells.…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

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confidence: 99%

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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.

349

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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Section: Discussionmentioning

confidence: 99%

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.

349

170

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show abstract

“…When lesser quantities of UK (10 rulml, -2 nM) were added, the major inhibitor of UK was reported to be a2-antiplasmin (22). The concentration of free PA-inhibitor in the plasma of healthy persons is in general below 5 U per ml (0,15 nM) (7,8,11).Thus the addition of UK in a large molar excess compared to that of free PA-inhibitor explains why these studies did not identify the primary inhibitor of UK in human plasma Fast-acting inhibitors of UK have been found in extracts of human placenta (24,25) , and in the conditioned media of fibroblasts (26,27), endothelial cells (28,29), hepatoma cells (30), monocytes and macrophages (31)(32)(33). At present the relationship of these inhibitors with the plasma PA-inhibitor is not yet established.…”

Section: Discussionmentioning

confidence: 99%

The Fast-Acting Inhibitor of Tissue-Type Plasminogen Activator in Plasma Is also the Primary Plasma Inhibitor of Urokinase

1986

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SummaryWe have compared the ability of a plasminogen activator inhibitor (PA-inhibitor) in human plasma, to form complexes with radioiodinated tissue-type plasminogen activator (t-PA) and high molecular weight urokinase (HMr-UK). Addition of 125I-t-PA (final concentration 10 IU/ml) or of 125I-HMr-UK (2 IU/ml) to a plasma containing 33 U/ml of PA-inhibitor resulted in the rapid formation of a 110,000 Mr complex of 125I-t-PA or a 95,000 Mr complex of 125I-HMr-UK with PA-inhibitor. Upon prolonged incubation of the plasma with 125I-HMr-UK a secondary complex of a Mr of 88,000 was observed, which probably derives from limited degradation of the 95,000 complex. Preincubation of the plasma with unlabelled t-PA, HMr-UK or LMr-UK at higher concentrations prevented the subsequent formation of complexes between radiolabelled PAs and the PA-inhibitor. These results thus demonstrate that t-PA and UK form complexes with the same PA-inhibitor. The rate of complex formation of 125I-t-PA or of 125I-HMr-UK with the plasma PA-inhibitor was similar (second order rate constant of association with PA-inhibitor in the order of 107 M−1s−1).

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“…In this study we examine the effects of lipopolysaccharide (LPS) on the production of fibrinolytic inhibitor by cultured human endothelial cells. The ability of these cells to synthesize inhibitor(s) of plasminogen activators has been demonstrated previously (7,10,12,22,28). Recently, we have observed that muramyl dipeptide (MDP), the immunoadjuvant properties of which have been well documented (3,20), could stimulate this fibrinolytic inhibitor synthesis (6).…”

mentioning

confidence: 82%

Effect of polymyxin B and colimycin on induction of plasminogen antiactivator by lipopolysaccharide in human endothelial cell culture

Dubor¹,

Dosne²,

Chedid³

1986

Infect Immun

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The effect of lipopolysaccharide (LPS) on the production of fibrinolytic inhibitor by human endothelial cells was determined because results of previous experiments have shown us that it is possible to stimulate this synthesis with muramyl dipeptide. Treatment of these cells with LPS resulted in a marked enhancement of fibrinolytic inhibitor, as estimated in a urokinase-induced fibrinolysis assay. A dose-response curve was obtained for LPS concentrations ranging from 10 to 1,000 ng/ml, thus demonstrating the great sensitivity of these cells. This inhibitor did not reduce plasmin activity and formed complexes with highand low-molecularweight urokinase as visualized by fibrin enzymography on sodium dodecyl sulfate-polyacrylamide electrophoretic gels. The molecular weight of this inhibitor was estimated to be 54 to 58 kilodaltons. These findings led us to conclude that LPS stimulates formation of a plasminogen antiactivator. This LPS effect could be suppressed by polymyxin B and colimycin. The stimulatory effect of muramyl dipeptide required doses which were at least 1,000 times greater than those of LPS and was not decreased by polymyxin B. These results show the possibility of independent modulation of plasminogen antiactivator production at the endothelial level, which could be important in endotoxemia. Under these conditions colimycin might have an additional advantage for clinical use because of its ability to prevent fibrinolytic inhibition.

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Partial Characterization of a Fibrinolytic Inhibitor Produced by Cultured Endothelial Cells Derived from Human Umbilical Vein

Cited by 24 publications

References 4 publications

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

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

The Fast-Acting Inhibitor of Tissue-Type Plasminogen Activator in Plasma Is also the Primary Plasma Inhibitor of Urokinase

Effect of polymyxin B and colimycin on induction of plasminogen antiactivator by lipopolysaccharide in human endothelial cell culture

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