Plasminogen activator inhibitor-1 secretion of endothelial cells increases fibrinolytic resistance of an in vitro fibrin clot: evidence for a key role of endothelial cells in thrombolytic resistance

Time-dependent thrombolytic resistance is a critical problem in thrombolytic therapy for acute myocardial infarction. Platelets have been regarded as the main source of plasminogen activator inhibitor-1 (PAI-1) found in occlusive platelet-rich clots. However, endothelial cells are also known to influence the fibrinolytic capacity of blood vessels, but their ability to actively mediate time-dependent thrombolytic resistance has not been fully established. We will show that, in vitro, tumor necrosis factor-alpha-stimulated endothelial cells secrete large amounts of PAI-1 over a period of hours, which then binds to fibrin and protects the clot from tissue plasminogen activator- induced fibrinolysis. In vivo, endothelial cells covering atherosclerotic plaques are influenced by cytokines synthesized by plaque cells. Therefore, we propose that continuous activation of endothelial cells in atherosclerotic blood vessels, followed by elevated PAI-1 secretion and storage of active PAI-1 in the fibrin matrix, leads to clot stabilization. This scenario makes endothelial cells a major factor in time-dependent thrombolytic resistance.

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Abstract T P75: Temporal Profiles of Plasminogen Activator Inhibitor-1 Concentration and Activity Changes in Circulation after Focal Stroke and Tissue Type Plasminogen Activator Administration in Rats

Stroke ◽

10.1161/str.45.suppl_1.tp75 ◽

2014 ◽

Vol 45 (suppl_1) ◽

Author(s):

Qi Liu ◽

Xiang Fan ◽

Helen Brogren ◽

Ming-Ming Ning ◽

Eng H Lo ◽

...

Keyword(s):

Endothelial Cells ◽

Plasminogen Activator ◽

Plasminogen Activator Inhibitor ◽

Tissue Type ◽

Plasminogen Activator Inhibitor 1 ◽

Type Plasminogen Activator ◽

Temporal Profiles ◽

Activator Inhibitor ◽

Pai 1

Aims: Plasminogen activator inhibitor-1 (PAI-1) is the main and potent endogenous tissue-type plasminogen activator (tPA) inhibitor, but an important question on whether PAI-1 in blood stream responds and interferes with the exogenously administered tPA remains unexplored. We for the first time investigated temporal profiles of PAI-1 concentration and activity in circulation after stroke and tPA administration in rats. Methods: Permanent MCAO focal stroke of rats were treated with saline or 10mg/kg tPA at 3 hours after stroke (n=10 per group). Plasma (platelet free) PAI-1 antigen and activity levels were measured by ELISA at before stroke, 3, 4.5 (1.5 hours after saline or tPA treatments) and 24 hours after stroke. Since vascular endothelial cells and platelets are two major cellular sources for PAI-1 in circulation, we measured releases of PAI-1 from cultured endothelial cells and isolated platelets after direct tPA (4 μg/ml) exposures for 60 min in vitro by ELISA (n=4 per group). Results: At 3 hours after stroke, both plasma PAI-1 antigen and activity were significantly increased (3.09±0.67, and 3.42±0.57 fold of before stroke baseline, respectively, all data are expressed as mean±SE). At 4.5 hours after stroke, intravenous tPA administration significantly further elevated PAI-1 antigen levels (5.26±1.24), while as expected that tPA neutralized most elevated PAI-1 activity (0.33±0.05). At 24 hours after stroke, PAI-1 antigen levels returned to the before baseline level, however, there was a significantly higher PAI-1 activity (2.51±0.53) in tPA treated rats. In vitro tPA exposures significantly increased PAI-1 releases into culture medium in cultured endothelial cells (1.65±0.08) and platelets (2.02±0.17). Conclution: Our experimental results suggest that tPA administration may further elevate stroke-increased blood PAI-1 concentration, but also increase PAI-1 activity at late 24 hours after stroke. The increased PAI-1 releases after tPA exposures in vitro suggest tPA may directly stimulate PAI-1 secretions from vascular walls and circulation platelets, which partially contributes to the PAI-1 elevation observed in focal stroke rats. The underlying regulation mechanisms and pathological consequence need further investigation.

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The Roles of α2-Antiplasmin and Plasminogen Activator Inhibitor 1 (PAI-1) in the Inhibition of Clot Lysis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649570 ◽

1993 ◽

Vol 70 (02) ◽

pp. 301-306 ◽

Cited By ~ 51

Author(s):

Linda A Robbie ◽

Nuala A Booth ◽

Alison M Croll ◽

Bruce Bennett

Keyword(s):

Plasminogen Activator ◽

Plasminogen Activator Inhibitor ◽

Fibrin Clot ◽

Clot Lysis ◽

Plasminogen Activator Inhibitor 1 ◽

Dependent Inhibition ◽

Activator Inhibitor ◽

Pai 1

SummaryThe relative importance of the two major inhibitors of fibrinolysis, α2-antiplasmin (α2-AP) and plasminogen activator inhibitor (PAI-1), were investigated using a simple microtitre plate system to study fibrin clot lysis in vitro. Cross-linked fibrin clots contained plasminogen and tissue plasminogen activator (t-PA) at concentrations close to physiological. Purified α2-AP and PAI-1 caused dose-dependent inhibition. All the inhibition due to normal plasma, either platelet-rich or poor, was neutralised only by antibodies to α2-AP. Isolated platelets, at a final concentration similar to that in blood, 2.5 × 108/ml, markedly inhibited clot lysis. This inhibition was neutralised only by antibodies to PAI-1. At the normal circulating ratio of plasma to platelets, α2-AP was the dominant inhibitor. When the platelet:plasma ratio was raised some 20-fold, platelet PAI-1 provided a significant contribution. High local concentrations of PAI-1 do occur in thrombi in vivo, indicating a role for PAI-1, complementary to that of α2-AP, in such situations.

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Localization of a Vitronectin Binding Region of Plasminogen Activator Inhibitor-1

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653876 ◽

1995 ◽

Vol 73 (05) ◽

pp. 829-834 ◽

Cited By ~ 11

Author(s):

Jaya Padmanabhan ◽

David C Sane

Keyword(s):

Binding Site ◽

Plasminogen Activator ◽

Inhibitory Activity ◽

Plasminogen Activator Inhibitor ◽

Structural Homology ◽

Binding Region ◽

Plasminogen Activator Inhibitor 1 ◽

Activator Inhibitor ◽

Pai 1

SummaryThe PAI-1 binding site for VN was studied using two independent methods. PAI-1 was cleaved by Staph V8 protease, producing 8 fragments, only 2 of which bound to [125I]-VN. These fragments were predicted to overlap between residues 91-130. Since PAI-2 has structural homology to PAI-1, but does not bind to vitronectin, chimeras of PAI-1 and PAI-2 were constructed. Four chimeras, containing PAI-1 residues 1-70,1-105,1-114, and 1-167 were constructed and expressed in vitro. PAI-1, PAI-2, and all of the chimeras retained inhibitory activity for t-PA, but only the chimera containing PAI-1 residues 1-167 formed a complex with VN. Together, these results predict that the VN binding site of PAI-1 is between residues 115-130.

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Fibrate-modulated Expression of Fibrinogen, Plasminogen Activator Inhibitor-1 and Apolipoprotein A-I in Cultured Cynomolgus Monkey Hepatocytes

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615393 ◽

1998 ◽

Vol 80 (12) ◽

pp. 942-948 ◽

Cited By ~ 29

Author(s):

M. Kockx ◽

H. M. G. Princen ◽

T. Kooistra

Keyword(s):

Plasminogen Activator ◽

Cynomolgus Monkey ◽

Plasma Concentrations ◽

Plasminogen Activator Inhibitor ◽

Peroxisome Proliferator ◽

Plasminogen Activator Inhibitor 1 ◽

Apolipoprotein A ◽

Activator Inhibitor ◽

Pai 1

SummaryFibrates are used to lower plasma triglycerides and cholesterol levels in hyperlipidemic patients. In addition, fibrates have been found to alter the plasma concentrations of fibrinogen, plasminogen activator inhibitor-1 (PAI-1) and apolipoprotein A-I (apo A-I). We have investigated the in vitro effects of fibrates on fibrinogen, PAI-1 and apo A-I synthesis and the underlying regulatory mechanisms in primary monkey hepatocytes.We show that fibrates time- and dose-dependently increase fibrinogen and apo A-I expression and decrease PAI-1 expression in cultured cynomolgus monkey hepatocytes, the effects demonstrating different potency for different fibrates. After three consecutive periods of 24 h the most effective fibrate, ciprofibrate (at 1 mmol/l), increased fibrinogen and apo A-I synthesis to 356% and 322% of control levels, respectively. Maximum inhibition of PAI-1 synthesis was about 50% of control levels and was reached by 1 mmol/l gemfibrozil or ciprofibrate after 48 h. A ligand for the retinoid-X-receptor (RXR), 9-cis retinoic acid, and specific activators of the peroxisome proliferator-activated receptor-α (PPARα), Wy14,643 and ETYA, influenced fibrinogen, PAI-1 and apo A-I expression in a similar fashion, suggesting a role for the PPARα/RXRα heterodimer in the regulation of these genes. When comparing the effects of the various compounds on PPARα trans-activation activity as determined in a PPARα-sensitive reporter gene system and the ability of the compounds to affect fibrinogen, PAI-1 and apo A-I antigen production, a good correlation (r = 0.80; p <0.01) between PPARα transactivation and fibrinogen expression was found. Apo A-I expression correlated only weakly with PPARα transactivation activity (r = 0.47; p = 0.24), whereas such a correlation was absent for PAI-1 (r = 0.03; p = 0.95). These results strongly suggest an involvement of PPARα in the regulation of fibrinogen gene expression.

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Tumor necrosis factor increases the production of plasminogen activator inhibitor in human endothelial cells in vitro and in rats in vivo

Blood ◽

10.1182/blood.v72.5.1467.1467 ◽

1988 ◽

Vol 72 (5) ◽

pp. 1467-1473 ◽

Cited By ~ 213

Author(s):

VW van Hinsbergh ◽

T Kooistra ◽

EA van den Berg ◽

HM Princen ◽

W Fiers ◽

...

Keyword(s):

Endothelial Cells ◽

Plasminogen Activator ◽

Blood Platelets ◽

Interleukin 1 ◽

Plasminogen Activator Inhibitor ◽

Activator Inhibitor ◽

Necrosis Factor ◽

Pai 1

Abstract The vascular endothelium plays an important role in fibrinolysis by producing tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI). The monokine tumor necrosis factor (human recombinant TNF) increased the production of PAI by cultured human endothelial cells from umbilical vein (twofold) and from foreskin microvessles (four to eight fold). This was demonstrated by titration of endothelial cell-conditioned medium with t-PA, by reverse fibrin autography, and by immunoprecipitation of [35S]PAI-1 by anti-PAI-1 IgG. TNF also induced a marked increase of PAI-1 messenger RNA (mRNA) in the cells. The stimulation of PAI activity by TNF was seen at 4 U/mL and reached a maximum at 500 U/mL. Human recombinant lymphotoxin and interleukin-1 (alpha and beta) also stimulated the production of PAI activity, while interleukin-6 was ineffective. Separate additions of TNF or interleukin-1 (IL-1) at optimal concentrations (500 U/mL and 5 U/mL, respectively) resulted in a comparable stimulation of PAI production by endothelial cells. The simultaneous addition of both mediators resulted in an additive effect. The effect of TNF could not be prevented by the addition of polymyxin B or by anti-IL-1 antibodies. Therefore, it is unlikely that TNF acts through the induction of IL-1 secretion by endothelial cells. Two hours after a bolus injection of 250,000 U/kg TNF into rats, a fivefold increase in circulating PAI levels was found. In the next ten hours, the levels returned to normal. Blood platelets do not significantly contribute to the increase in circulating PAI, because the number of platelets did not change after TNF injection and the amount of PAI in blood platelets is not sufficient for several hours during an increase in PAI activity. The acute phase reactants, fibrinogen and alpha 2-antiplasmin in rat plasma, were altered little if any two to 24 hours after injection of 250,000 U/kg TNF. In vitro, TNF did not change PAI production by human and rat hepatocytes in primary monolayer culture. Therefore, it is most likely that vascular endothelial cells contribute to the increased amount of circulating PAI induced by TNF in vivo. This increase in PAI activity might decrease fibrinolysis.

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The biologic therapy of prostate cancer using plasminogen activator inhibitor-1 (PAI-1)

Journal of Clinical Oncology ◽

10.1200/jco.2006.24.18_suppl.14596 ◽

2006 ◽

Vol 24 (18_suppl) ◽

pp. 14596-14596

Author(s):

S. Chen ◽

D. O. Henry ◽

M. K. Wong

Keyword(s):

Prostate Cancer ◽

Plasminogen Activator ◽

Protease Activity ◽

Fluorescent Protein ◽

Tumor Regression ◽

Plasminogen Activator Inhibitor ◽

Plasminogen Activator Inhibitor 1 ◽

Activator Inhibitor ◽

Pai 1

14596 Background: Treating prostate cancer through the expression of intrinsic biologic modifiers is a relatively unexplored aspect of prostate cancer therapy. Plasminogen Activator Inhibitor-1 (PAI-1) is expressed at low levels in prostate cancer cells. PAI-1 is both an anti-angiogenesis agent, and also potently inhibits tumor proteases responsible for tumor invasion and metastases such as uPA and tPA. Thus we hypothesized that stimulation of tumor endogenous PAI-1 would result in a particularly powerful and profound prostate cancer regression. We present proof-of-concept from our experimental models that demonstrate significant tumor regression in experimental prostate tumors and supports this hypothesis. Methods and Results: Human prostate adenocarcinoma (PC3 cell line) xenograft tumors engineered to conditionally express either PAI-1 or Green Fluorescent Protein (GFP, control) were used to test our hypothesis. Stable cell lines were created that conditionally express either GFP or PAI-1 under the regulation of a doxycycline-responsive promoter (Tet-On). Thus gene expression is switched on in the presence of doxycycline. PC3 tumors were inoculated and allowed to reach at least 200 mm3 in size whereby the tumor-bearing mice were given doxycycline-doped drinking water. Genes were significantly turned on within 48 hours as monitored by the appearance of a GFP signal in control mice. The induction of PAI-1 results in significant inhibition of tumor growth as compared to GFP control. Importantly, in vitro induction of PAI-1 expression in PC-3 has no direct effects on cell growth as compared to any PC-3 control. Histological analysis of these tumors revealed a rich nexus of fine angiogenic vessels at the interface between control tumors and surrounding stroma. PAI-1 secreting tumors were significantly smaller and were pale, bland, and lacked peritumoral vessels. Protease activity measured by in-situ zymography directly on these tumors revealed that this was significantly reduced in PAI-1 expressing tumors as compared to GFP controls. Conclusion: PAI-1 expression results in tumor inhibition through direct anti-angiogenic effects and inhibition of tumor protease activity. No significant financial relationships to disclose.

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Thymosin β4 induces the synthesis of plasminogen activator inhibitor 1 in cultured endothelial cells and increases its extracellular expression

Blood ◽

10.1182/blood-2003-04-1015 ◽

2004 ◽

Vol 103 (4) ◽

pp. 1319-1324 ◽

Cited By ~ 32

Author(s):

Khalid N. I. Al-Nedawi ◽

Malgorzata Czyz ◽

Radoslaw Bednarek ◽

Janusz Szemraj ◽

Maria Swiatkowska ◽

...

Keyword(s):

Endothelial Cells ◽

Plasminogen Activator ◽

Human Fibroblasts ◽

Plasminogen Activator Inhibitor ◽

Binding Activity ◽

Mitogen Activated Protein Kinase ◽

Thymosin Β4 ◽

Plasminogen Activator Inhibitor 1 ◽

Activator Inhibitor ◽

Pai 1

Abstract Thymosin β4(Tβ4), a 4.9-kDa polypeptide primarily known as a main G-actin–sequestering peptide, is present in high concentrations in various cells and in the circulation. We have found that Tβ4 upregulates the expression of plasminogen activator inhibitor 1 (PAI-1) in endothelial cells measured both at the level of mRNA and protein synthesis. This effect seems to be cell specific and was not observed when other cells such as human fibroblasts, PC3, and U937 were tested. Tβ4 significantly activated the PAI-1 promoter in EA.hy 926 cells transiently transfected either with plasmid p800LUC containing PAI-1 promoter fragment (–800 to +71) or the PAI-1 promoter linked with green fluorescent protein. Tβ4 mediated up-regulation of PAI-1 involved activation of the mitogen-activated protein kinase cascade. Furthermore, Tβ4 enhanced c-Fos/c-Jun DNA-binding activity to the activator protein 1 (AP-1)–like element (–59 to –52). The specificity of this binding activity was demonstrated by competition electrophoretic mobility shift assay and after transfection of EA.hy 926 cells with the mutated PAI-1 promoter. Taken together, these data indicate that, in response to Tβ4 stimulation, AP-1 activity increases to enhance PAI-1 transcription through its unique AP-1–like element at –59 to –52 in the PAI-1 promoter.

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