Release Of Plasminogen Activator Inhibitor Type 1 Research Articles

Ghrelin attenuates plasminogen activator inhibitor‐1 production induced by tumor necrosis factor‐α in HepG2 cells via NF‐κB pathway

Plasminogen activator inhibitor type 1 (PAI-1), produced partly from liver is a risk factor for macrovascular and microvascular complications of diabetes. Ghrelin, a recently described orexigenic peptide hormone, attenuates PAI-1 induced by TNF-alpha in the human hepatoma cell line (HepG2). Exposure to TNF-alpha (1 ng/ml) for 24h caused a significant increase in PAI-1 mRNA expression and protein secretion, as evaluated by RT-PCR and ELISA, but pretreatment with ghrelin (1-100 ng/ml) inhibited both basal and TNF-alpha-induced PAI-1 release in a dose and time-dependent manner in HepG2. PDTC, selective NF-kappaB inhibitor, had no additive inhibitory effects with ghrelin. The results indicate that ghrelin inhibits both basal and TNF-alpha-induced PAI-1 production via NF-kappaB pathway in HepG2 cells, and suggest that the peptide plays a therapeutic role in atherosclerosis, especially in obese patients with insulin resistance, in whom ghrelin levels were reduced.

Repeated Thoracenteses Affect Proinflammatory Cytokines, Vascular Endothelial Growth Factor, and Fibrinolytic Activity in Pleural Transudates

Repeated thoracenteses is indicated in patients with refractory, symptomatic transudative effusions. However, their effect on cytokines and fibrinolytic activity in pleural transudates remains unclear. Twenty-one patients with symptomatic, large amount of free-flowing transudative effusions caused by heart failure were studied. Thoracentesis with drainage of 500 mL of pleural fluid per day was done for 3 consecutive days (days 1 to 3). Pleural fluid characteristics, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, IL-8, vascular endothelial growth factor (VEGF), tissue-type plasminogen activator (tPA), and plasminogen activator inhibitor type 1 (PAI-1) were measured during each tap. Chest ultrasonography was done on day 6 to detect the fibrin strands in pleural effusion and the outcome of effusion was evaluated within 7 days after repeated thoracenteses. Effusion levels of lactate dehydrogenase, neutrophils, TNF-alpha, IL-1 beta, IL-8, VEGF, and PAI-1 increased significantly during repeated thoracenteses. Furthermore, the values of PAI-1 and PAI-1/tPA obtained on days 2 and 3 were highly correlated with those of TNF-alpha, IL-1 beta, IL-8, and VEGF. On day 6, pleural fibrins were observed on chest ultrasonography in 6 patients (29%, fibrinous group) but were absent in the remaining 15 patients (nonfibrinous group). Compared with the nonfibrinous group, the effusion levels of TNF-alpha, IL-1 beta, VEGF, and PAI-1 on day 2 and day 3, and recurrence of symptomatic effusion after repeated thoracenteses were significantly higher in fibrinous group. Repeated thoracenteses may induce local release of proinflammatory cytokines, VEGF and PAI-1, which may result in fibrin deposition and impair resolution of pleural transudates.

Effect of acidosis on expression of mesothelial cell plasminogen activator inhibitor type-1

Abdominal mesothelial cells are exposed to carbon dioxide during laparoscopy. Previous data indicate that carbon dioxide increases release and expression of plasminogen activator inhibitor type-1 (PAI-1) and induces acidification. To assess the impact resulting from a range of pH, human mesothelial cells were exposed to culturing media balanced to pH levels of 6.0 to 8.0 for 90 min. Samples from cell media were withdrawn at several time points. Concentrations of PAI-1 and PAI-1 activity were measured using enzyme-linked immunoassay techniques. To focus on the effect of clinically relevant pH, cells were subjected to pH 6.4 and 7.4. Samples were withdrawn for PAI-1 assessments and for PAI-1 mRNA analyses. During exposure to various levels of pH, PAI-1 secretion and activity were variable. However, 5 h after exposure, greater concentration and activity of PAI-1 were observed in acidified cultures. More PAI-1 mRNA was isolated after exposure of cells to a pH of 6.4, apparently indicating transcriptional regulation. Mesothelial cells seem to respond to acidification by an increased release and production of PAI-1 in vitro.

Effect of Repeated Thoracenteses on Fluid Characteristics, Cytokines, and Fibrinolytic Activity in Malignant Pleural Effusion

To evaluate the effect of repeated thoracenteses on the fluid characteristics and the levels of various cytokines, including tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-5, IL-6, and IL-8, and of plasminogen activator inhibitor type 1 (PAI-1) and tissue type plasminogen activator in malignant pleural effusion and its clinical significance. A prospective study. Twenty-six patients with symptomatic and a large amount of free-flow malignant pleural effusions were studied. Thoracentesis with drainage of 500 mL of pleural fluid per day was performed for 3 continuous days (days 1 to 3). The effusion samples were collected to evaluate the changes of fluid characteristics, cytokine levels, and fibrinolytic activity. Chest ultrasonography was done on day 6 to observe the presence of fibrin strands. The result of pleurodesis was evaluated in the patients classified into groups based on chest ultrasonographic findings. The values of TNF-alpha, PAI-1, IL-8, and neutrophil count in pleural fluid increased significantly during repeated thoracenteses in 26 patients studied. A positive correlation was found between the concentrations of TNF-alpha and PAI-1 and between the values of IL-8 and neutrophils. On day 6, fibrin strands were observed in the pleural effusion on chest ultrasonography in 11 patients (42%, fibrinous group) but were absent in the remaining 15 patients (nonfibrinous group). During repeated thoracenteses, a significant increase of effusion PAI-1 and TNF-alpha was observed in the fibrinous group but not in the nonfibrinous group. In addition, the levels of effusion PAI-1 and TNF-alpha obtained from day 2 and day 3 were significantly higher in the fibrinous group than in the nonfibrinous group. The success rate of pleurodesis was significantly higher in the fibrinous group (11 of 11 patients, 100%) than in the nonfibrinous group (8 of 12 patients, 67%). Repeated thoracenteses may cause pleural inflammation and induce local release of proinflammatory cytokine as TNF-alpha, which may subsequently enhance the release of PAI-1 and lead to fibrin formation in malignant effusion. The presence of fibrin strands after repeated thoracenteses may be of considerable value in predicting the success of subsequent pleurodesis in patients with malignant pleural effusions.

Enhanced secretion of tissue plasminogen activator by simultaneous use of retinoic acid and ascorbic acid from tissue cultured gastroepiploic artery

Tissue-type plasminogen activator (tPA) is a key enzyme in the fibrinolysis system and the regulation of its expression has been extensively studied in cultured vascular endothelial cells. Many kinds of supplements including growth factors are needed, however, to keep endothelial cells viable, which leads the culture condition far from the physiological milieu. Using a new device of amorphous calcium phosphate coated culture plate, we succeeded in culturing ring-cut gastroepiploic artery in a basic medium of RPMI 1640 containing 10 % fetal calf serum. The overall normal vessel architecture and the antigenicity of von Willebrand factor, tPA and plasminogen activator inhibitor type 1 (PAI-1) were retained for at least 9 days. tPA was constantly secreted into the conditioned medium at least up to day 12. Employing this organ culture technique, we analyzed the effects of two well-known pro-fibrinolytic vitamins of retinoic acid (Vit. A) and ascorbic acid (Vit. C) on the release of tPA and PAI-1. The cultured artery responded well and the tPA secretion was enhanced by factors of 1.5 fold by Vit. A, 1.7 fold by Vit C and 3.2 fold by their combination, whereas none of these stimuli increased PAI-1 secretion. These results suggested that the cultured ring-cut artery retained functional endothelial cells for at least 9 days and was suitable in analyzing the regulatory mechanism of protein synthesis and secretion from the vascular wall. Using this method, vitamins A and C were shown to lead the intra-vascular condition to a profibrinolytic state.

Sodium hyaluronate increases the fibrinolytic response of human peritoneal mesothelial cells exposed to tumor necrosis factor alpha.

Sodium hyaluronate interferes with the fibrin degrading capacity of human peritoneal mesothelial cells exposed to tumor necrosis factor (TNF) alpha. Controlled laboratory experiment. Human peritoneal mesothelial cells were harvested from 5 patients undergoing laparotomy and cultured in vitro. Cells were treated with TNF-alpha, a cytokine typically involved in peritoneal inflammation, and sodium hyaluronate was added in a final concentration of 0.1%, 0.2%, or 0.4%. Controls received medium only. After 24 hours' incubation, tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), and plasminogen activator inhibitor type 1 (PAI-1) were measured in the medium and cell lysates using enzyme-linked immunosorbent assay techniques. Specific gene transcripts in cells treated with 0.4% sodium hyaluronate and controls were determined using a quantitative reverse transcription polymerase chain reaction. Concentrations of tPA, uPA, and PAI-1, and their specific gene transcripts. Sodium hyaluronate significantly increased tPA concentration in cell lysates without affecting its gene expression as determined after 24 hours (P =.02). The uPA concentration was significantly decreased by sodium hyaluronate in the medium but not in cell lysates (P<.0001). The uPA messenger RNA expression was 1000-fold increased compared with control. Sodium hyaluronate significantly decreased PAI-1 concentration in the medium and reduced its gene expression 500-fold (P =.04), while PAI-1 concentration in cell lysates did not change. Sodium hyaluronate affected the fibrinolytic response of TNF-alpha-stimulated human peritoneal mesothelial cells, most notably by decreasing PAI-1 transcription and release. This observation indicates that sodium hyaluronate counteracts the fibrinolytic decline induced by TNF-alpha and suggests a biological mechanism of action for sodium hyaluronate intra-abdominally.

Endothelin-1 does not contribute to the release of tissue plasminogen activator in vivo in man

Objectives: Endothelin-1 is a potent endothelium-derived vasoconstrictor peptide with autocrine and paracrine actions. Tissue plasminogen activator (t-PA) and its inhibitor, plasminogen activator inhibitor type 1 (PAI-1), are also released from the vascular endothelium and play a pivotal role in endogenous fibrinolysis. We, therefore, examined the effects of exogenous and endogenous endothelin-1 on t-PA and PAI-1 release in vivo in man. Design: Open investigative study. Setting: Clinical Research Centre, University of Edinburgh. Subjects: Fourteen healthy male volunteers. Interventions: Unilateral brachial artery infusions of endothelin-1 at 2.5 and 10 pmol/min, and the selective endothelin type B (ET B) receptor antagonist, BQ-788, at 1 nmol/min. Main outcome measures: Blood, flow and plasma fibrinolytic factors were measured in both forearms using venous occlusion plethysmography and venous blood samples withdrawn from the antecubital fossae. Results: Endothelin-1 caused a slow onset dose-dependent forearm vasoconstriction ( P<0.001) with a maximal reduction in blood flow of 40 ± 4% and 63 ± 3% at 2.5 and 10 pmol/min respectively. BQ-788 also caused a slow onset reduction in forearm blood flow ( P<0.001) reaching a maximum of 21 ± 3%. However, BQ-788 and endothelin-1 did not affect plasma concentrations of t-PA or PAI-1 in the venous effluent of the infused forearm. Conclusions: Despite sustaining significant vasoconstriction, neither endogenous nor exogenous endothelin-1 influences the release of t-PA or PAI-1 in the forearm vascular bed of man. This suggests that endothelin-1 does not provide a major contribution to the regulation of endogenous fibrinolysis in man.

Thrombin Upregulates Production of Plasminogen Activator Inhibitor Type 1 in Human Peritoneal Mesothelial Cells

To determine the influence of thrombin, which is generated intraperitoneally during peritoneal dialysis, on the synthesis of fibrinolytic system components in human peritoneal mesothelial cells (HMC). Confluently grown HMC, isolated from the omental tissue, were used in the experiments. Conditioned media were obtained by incubating cells with serum-free M199 containing the appropriate concentration of the test compound. Tissue type plasminogen activator (tPA) and plasminogen activator inhibitor type 1 (PAI-1) antigen concentrations were measured by ELISA. Northern blot analysis was conducted for mRNA expression experiments. To test thrombin specificity, we used the thrombin inhibitor hirudin. The protein kinase C (PKC) inhibitor Ro 31-8220 was inserted to examine whether the effect of thrombin depends on PKC activity. Thrombin increased PAI-1 antigen in the conditioned media of HMC in a time- and concentration-dependent manner. After 24 hours incubation, PAI-1 levels increased from 350+/-30 ng/10(5) cells in control conditions to 620+/-30 ng/10(5) cells in HMC exposed to 5 U/mL thrombin (n = 8, p < 0.05). In contrast, there was no effect of thrombin on tPA antigen levels. An increase of PAI-1 mRNA expression was also observed by Northern blot hybridization. Hirudin (10 U/mL) inhibited the thrombin-induced increase in PAI-1 synthesis. In addition, a complete inhibition of the stimulating effect of thrombin on PAI-1 synthesis was obtained by blocking PKC activity with Ro 31-8220 (3 micromol/L). Thrombin increases PAI-1 synthesis in HMC via a PKC-dependent mechanism. Thereby the synthesis of tPA is not affected. Thus, thrombin may not only promote fibrin formation in the peritoneal cavity, but may also inhibit fibrin degradation by release of free PAI-1 from HMC.

Pathogenetic implications of hyaluronan-induced modification of vascular smooth muscle cell fibrinolysis in diabetes.

Proteolysis, modulated in part by intramural fibrinolytic system proteins and their inhibitors, appears to influence vascular smooth muscle cell (SMC) migration and proliferation and remodeling of extracellular matrix (ECM). Alterations of fibrinolysis in circulating blood and of proteolysis within vessel walls in experimental animals and patients with diabetes have been associated with accelerated vascular disease. Hyaluronan, a prominent component of ECM in normal vessels, is increased in the tunica media of macroscopically normal arterial vessels from patients with type 2 diabetes. To determine whether hyaluronan alters the expression of the fibrinolytic system protein, plasminogen activator inhibitor type-1 (PAI-1), in human vascular SMCs, thereby potentially accelerating vascular disease in patients with type 2 diabetes. Urokinase-type and tissue-type plasminogen activators (uPA and tPA) and PAI-1 were assayed in vascular SMC conditioned media and in cell lysates, using enzyme-linked immunosorbent assay and western blotting. Hyaluronan increased the 24-h release of PAI-1 into conditioned media in a concentration-dependent and time-dependent manner (1.8-fold compared with control with 1 mg/ml hyaluronan; n = 9, P < 0.01). Although the accumulation of uPA in conditioned media tended to increase also, uPA content was reduced in cell lysates (64% of control with 0.1 mg/ml hyaluronan at 24 h; n = 9, P < 0.01) without any change in PAI-1. Concentrations of tPA in conditioned media and cell lysates were unchanged. Digestion of hyaluronan with hyaluronidase (50 turbidity reducing units (TRU)/ml) or exposure of the smooth muscle cells to antihuman CD44 antibody (1 microgram/ml) that binds to the hyaluronan cell surface receptor obviated the effects of hyaluronan. Our results indicate that increases in hyaluronan increase vascular SMC expression of PAI-1, a phenomenon that may alter the balance between proteolysis and its inhibition in vessels of patients with type 2 diabetes, thereby contributing to the acceleration of macroangiopathy.

Recycling of the urokinase receptor upon internalization of the uPA:serpin complexes.

The GPI-anchored urokinase plasminogen activator receptor (uPAR) does not internalize free urokinase (uPA) but readily internalizes and degrades uPA:serpin complexes in a process that requires the alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha2MR-LRP). This process is accompanied by the internalization of uPAR which renders it resistant to phosphatidylinositol-specific phospholipase C (PI-PLC). In this paper we show that during internalization of uPA:serpins at 37 degrees C, analysed by FACScan, immunofluorescence and immunoelectron microscopy, an initial decrease of cell surface uPAR was observed, followed by its reappearance at later times. This effect was not due to redistribution of previously intracellular receptors, nor to the surface expression of newly synthesized uPAR. Recycling was directly demonstrated in cell surface-biotinylated, uPA:PAI-1-exposed cells in which biotinylated uPAR was first internalized and subsequently recycled back to the surface upon incubation at 37 degrees C. In fact, uPAR was resistant to PI-PLC after the 4 degrees C binding of uPA:PAI-1 to biotinylated cells, but upon incubation at 37 degrees C PI-PLC-sensitive biotinylated uPAR reappeared at the cell surface. Binding of uPA:PAI-1 by uPAR, while essential to initiate the whole process, was, however, dispensable at later stages as both internalization and recycling of uPAR could be observed also after dissociation of the bound ligand from the cell surface.

Regulation of plasminogen activator inhibitor activity in endothelial cells by tissue-type plasminogen activator

Summary Tissue-type plasminogen activator (t-PA) may stimulate the expression of plasminogen activator inhibitor type 1 (PAI-1) mRNA in cultured human umbilical vein endothelial cells. The PAI-1 antigen in the conditioned medium of cells, pretreated with t-PA, was less than the control, probably due to the formation and degradation of the t-PA·PAI-1 complex. However, the PAI activity of the t-PA-pretreated cells reached the same level as control group, 24 h after the residual t-PA activity was rapidly neutralized by the newly synthesized PAI-1. To test the release of PAI-1 from the substratum of the endothelial cells, the cultured cells were metabolically prelabeled with 35S-methionine for 3h and then treated with t-PA. The 35S-PAI-1 of 46 kDa was found in the substratum and culture medium of cultured endothelial cells as analyzed by the SDS-PAGE after immunoprecipitation. During the treatment of endothelial cells with t-PA, the PAI-1 of 46 kDa in the cell substratum disappeared, and the 110kDa t-PA·PAI-1 complex, the 81 kDa degraded t-PA·PAI-1, and the 44 kDa degraded PAI-1 products concomitantly appeared in the conditioned medium instead. In summary, t-PA can regulate the fibrinolytic activity of endothelial cells by enhancing PAI-1 mRNA biosynthesis and release PAI-1 from the substratum to neutralize t-PA activity. The PAI-1 which released into the medium was immediately converted to the inactive latent form in the absence of active t-PA.

Suppression of plasminogen activator inhibitor type 1 release from cultured human umbilical vein endothelial cells by basic fibroblast growth factor

We investigated the release of plasminogen activator inhibitor type 1 (PAI-1) from cultured vascular endothelial cells after exposure to basic fibroblast growth factor (bFGF). Treatment with human recombinant bFGF of confluent cultures of endothelial cells derived from human umbilical vein resulted in a reduction of the accumulation of PAI-1 antigen (PAI-1:Ag) in the conditioned medium. The suppressive effect of bFGF completely disappeared in the presence of anti-bFGF antibody. The reduction of endothelial PAI-1:Ag release induced by bFGF was suggested to be independent of intracellular accumulation of cyclic AMP. On the other hand, nordihydroguaiaretic acid (NDGA), an inhibitor of the lipoxygenase pathway of arachidonic acid metabolism, suppressed the spontaneous release of PAI-1:Ag by itself; in the presence of NDGA, bFGF failed to further suppress the PAI-1:Ag release. The effect of bFGF and indomethacin, an inhibitor of the cyclooxygenase pathway, was additive on the PAI-1:Ag release. The present data suggest that bFGF reduces the endothelial PAI-1:Ag release via suppression of the putative lipoxygenase pathway which up-regulates a part of the spontaneous PAI-1:Ag release.

Attenuation of thrombolysis by release of plasminogen activator inhibitor type-1 from platelets

Although platelets contain approximately 90% of the total amount of plasminogen activator inhibitor type-1 (PAI-1) present in blood, the functional significance of PAI-1 in platelets has been controversial. Most assessments of platelet PAI-I have been performed with platelet lysates in which the PAI-I derived from platelets may have been inactivated during the course of lysis. This study was performed to determine whether elaboration of PAI-1 from platelets activated physiologically by thrombolysis of pre-formed clots inhibits activation of plasminogen by tissue-type plasminogen activator (t-PA). Human whole blood clots were formed in Chandler tubes, and release of PAI-1 from platelets was quantified during and after clot formation. Subsequently, clots were placed in different Chandler tubes, and the effects of platelet PAI-1 on lysis induced by t-PA were characterized. Both the activity and concentrations of PAI-1 elaborated from platelets peaked approximately 15 min after induction of clotting. Induction of clot lysis with t-PA, 1,000 to 5,000 ng/ml, was inhibited by platelet-rich compared with platelet-poor plasma. Platelets inhibited lysis of preformed clots by t-PA and plasminogen in buffer solutions as well. Both the inhibition of clot lysis and accumulation of PAI-1 released from platelets were prevented by attenuation of thrombin-mediated activation of platelets with hirudin. Furthermore, the PAI-I mediated inhibition was obviated by blockade of PAI-1 activity with a neutralizing monoclonal antibody to PAI-1. These results indicate that platelets inhibit clot lysis induced by t-PA by releasing functionally active PAI-1.

Increased synthesis of plasminogen activator inhibitor-1 by cultured human endothelial cells exposed to native and modified LDLs. An LDL receptor-independent phenomenon.

The effects of native and acetylated low density lipoproteins (LDLs and acetyl-LDLs, respectively) on the release of plasminogen activator inhibitor type 1 (PAI-1) by cultured human umbilical vein endothelial cells (ECs) were evaluated. LDL and acetyl-LDL incubated with ECs for 16-18 hours increased the PAI-1 antigen levels in conditioned medium. At a concentration of 100 micrograms/mL, LDL and acetyl-LDL increased PAI-1 by 10.8 and 12.0 ng/mL, respectively (p < 0.05 and p < 0.01 versus control). The increases in PAI-1 antigen levels exerted by the lipoproteins paralleled the changes in PAI-1 activity. The effect of LDL and acetyl-LDL was concentration dependent and specific for PAI-1 because tissue-type plasminogen activator and expression of procoagulant activity were not affected by either lipoprotein. In addition, total protein synthesis evaluated in [35S] methionine-labeled ECs was not affected, and studies with cycloheximide showed that the effect of LDL and acetyl-LDL on PAI-1 release was due to de novo protein synthesis. Experiments using the C7 monoclonal antibody against the LDL receptor and binding-defective LDL indicated that the effect of LDL on the synthesis of PAI-1 was not dependent on the interaction of the LDLs with their specific receptors. Finally, extensive oxidation of LDL prevented and even reversed the effect of LDL on PAI-1 release by ECs. It is concluded that LDL specifically increases the synthesis of PAI-1 by ECs with mechanisms that are not receptor mediated.

Mechanisms contributing to increased synthesis of plasminogen activator inhibitor type 1 in endothelial cells by constituents of platelets and their implications for thrombolysis.

We recently hypothesized that after pharmacologically induced coronary thrombolysis, increased activity of plasminogen activator inhibitor type 1 (PAI-1) retards recanalization, contributes to early reocclusion, or both. This hypothesis was based on the increased elaboration of PAI-1 that we observed in cultured liver cells exposed to growth factors releasable from platelets activated at sites of thrombosis in vivo. PAI-1 released locally is particularly likely to attenuate lysis of thrombi that are targets of thrombolytic drugs. Accordingly, the present study was performed to determine whether synthesis of PAI-1 by endothelial cells is augmented by products of platelets. Lysates from platelets (0.5-8.0 x 10(4)/mm3 media, i.e. less than 10% of the concentration of platelets in blood) increased synthesis and release of PAI-1 into both the extracellular matrix and conditioned media (by 2.8-fold and 3.3-fold within 6 and 24 hours, respectively). Synthesis of neither tissue-type plasminogen activator nor overall protein increased. Increased synthesis of PAI-1 was confirmed by immunoprecipitation of [35S]PAI-1 after metabolic labeling of cells. The increased elaboration of PAI-1 was consistent with increased transcription as reflected by the observed increase in PAI-1 mRNA of 2.2-fold in 4 hours. Effects of platelet lysates were simulated by transforming growth factor beta (TGF-beta), known to be present in platelet alpha-granules and released with platelet activation. Antibody to TGF-beta reduced the stimulation of PAI-1 synthesis by TGF-beta, as expected, by 82%.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of Synthesis of Plasminogen Activator Inhibitor Type-1 byTissue-Type Plasminogen Activator in Human Hepatic and Endothelial Cells

Plasminogen activator inhibitor type-1 (PAI-1) can modify fibrinolytic activity in vitro and in vivo. The present study was performed to determine whether pharmacologic concentrations of tissue-type plasminogen activator (t-PA) can initiate negative feedback by stimulating PAI-1 synthesis. In both human hepatoma cells (Hep G2) and human umbilical vein endothelial cells (HUVEC), t-PA increased the total concentrations and appearance of newly synthesized protein in conditioned media of free PAI-1 and PAI-1 complexed with t-PA in a dose and time dependent fashion judging from results after immunoprecipitation of metabolically labeled PAI-1. The t-PA effect was not attributable simply to release of stored or matrix-bound PAI-1. In HUVEC, Northern blot analyses indicated that t-PA increased steady-state levels of PAI-1 mRNA two-fold. In contrast PAI-1 mRNA expression was not increased in Hep G2 cells. Thus, mechanisms of stimulation appeared to differ in the two cell lines. The results obtained are consistent with the hypothesis that increased PAI-1 synthesis and secretion in response to t-PA may limit or attenuate fibrinolysis locally or systemically in vivo.