Hemodynamic forces modulate the effects of cytokines on fibrinolytic activity of endothelial cells

We investigated the effects of hemodynamic force on fibrinolytic activity of cultured human umbilical vein endothelial cells stimulated by cytokines, using a modified cone-plate viscometer in which well- controlled and -defined shear forces were generated. Treatment of the cells with interleukin (IL)-beta or tumor necrosis factor alpha (TNFalpha) under static conditions had no effect on tissue plasminogen activator (t-PA) secretion, while release of plasminogen activator inhibitor 1 (PAI-1) increased. When cells were exposed to increasing shear stress up to 24 dynes/cm2, levels of t-PA and t-PA/PAI-1 complex significantly increased relative to shear stress, while total PAI-1 and active PAI-1 secretion decreased gradually. In the presence of IL-1beta or TNFalpha, the increase in production of t-PA and the t-PA/PAI-1 complex was further augmented. Dot blot hybridization analysis of cultured cells in similar experimental conditions using t-PA and PAI-1 cDNA probes revealed no t-PA mRNA in 3 microg total RNA from static endothelial cells under resting or cytokine-stimulated conditions, but abundant t-PA mRNA was detected in cells subjected to a shear force of 18 dynes/cm2, and the increase was further augmented by addition of cytokines. In contrast, PAI-1 mRNA was detected in resting and cytokine- stimulated, nonsheared endothelial cells, but levels decreased after exposure to shear stress, even in the presence of cytokines. These results indicate a role for hemodynamic forces in regulating fibrinolytic activity with or without cytokine stimulation.     

Plasma from patients with cirrhosis increases tissue plasminogen activator release from vascular endothelial cells in vitro

ABSTRACT— Aims/Background: In patients with severe liver disease, blood levels of many coagulation and fibrinolytic factors are lowered due to a diminished synthetic capability in the liver. Tissue plasminogen activator (t-PA) is synthesized by the vascular endothelial cells, however, and is increased in such patients. Methods: Amounts of t-PA secreted were determined by immunosorbent assay after plasma from patients with cirrhosis was added to cultured human umbilical vein endothelial cells to determine whether a plasma factor directly enhanced t-PA secretion from vascular endothelial cells. Results: Release of t-PA was significantly higher with exposure to plasma from patients with decompensated cirrhosis than when plasma from patients with compensated cirrhosis or normal subjects was used (p<0.01 and p<0.05, respectively). Plasminogen activator inhibitor 1(PAI-1) concentrations were measured similarly but did not differ among the three groups. Conclusions: Our results indicate that factors in plasma from patients with decompensated cirrhosis directly stimulate t-PA release from the vascular endothelial cells, while any increased PAI-1 release observed in comparable in vivo situations is probably an indirect response to an increase of t-PA or a result of impaired hepatic clearance.     

Increased plasma tissue-type plasminogen activator levels in patients with chronic thrombocytopenia

Plasma fibrinolytic factors were measured in 14 patients with chronic idiopathic thrombocytopenic purpura (ITP), in 5 patients with chronic central thrombocytopenia and in 16 healthy volunteers. The von Willebrand factor (vWF), tissue-type plasminogen activator (t-PA) and D-dimer (DD) antigens were found to be significantly higher in both patient groups than in the control group. No difference appeared in euglobulin fibrinolytic activity and plasminogen activator inhibitor activity. The increases in both t-PA and vWF suggest the occurrence of an endothelial cell stimulation, associated with the reduction of circulating platelet number. The correlation of increased DD and t-PA levels during ITP can be the proof of a fibrinolysis activation and suggest an antifibrinolytic role of platelets at physiological concentrations. These results can justify antifibrinolytic therapy in bleeding thrombocytopenic patients.     

Experimental endotoxemia in humans: analysis of cytokine release and coagulation, fibrinolytic, and complement pathways

Endotoxemia was evoked by bolus injection of Escherichia coli endotoxin (2 ng/kg body weight) in six healthy subjects to investigate the early kinetics of cytokine release in relation to the development of clinical and hematologic abnormalities frequently seen in gram-negative septicemia. The plasma concentration of tumor necrosis factor (TNF) increased markedly after 30 to 45 minutes, and reached a maximal level after 60 to 90 minutes. In each volunteer, the initial increase of plasma interleukin 6 (IL-6) concentrations occurred 15 minutes after the initial TNF increase, and maximal IL-6 concentrations were reached at 120 to 150 minutes. A transient increase in body temperature and pulse rate occurred simultaneously with the initial TNF and IL-6 increases, whereas a significant decrease in blood pressure occurred after 120 minutes. These changes were proportional to the changes in TNF and IL-6 concentrations. Coagulation activation, as assessed by a rise of prothrombin fragments and thrombin-antithrombin III complexes, was noted after 120 minutes, in the absence of activation of the contact system. A two- to sixfold increase in the concentrations of tissue plasminogen activator (t-PA) and von Willebrand factor antigen indicated endothelial cell activation. This increase started at 120 and 90 minutes, respectively. The release of t-PA coincided with activation of the fibrinolytic pathway, as measured by plasmin-alpha 2-antiplasmin complexes. The fibrinolytic activity of t-PA was subsequently offset by release of plasminogen activator inhibitor, observed 150 minutes after the endotoxin injection, and reaching a peak at 240 minutes. No complement activation was detected. These results show that in humans endotoxin induces an early, rapidly counteracted fibrinolytic response, and a more long-lasting activation of thrombin by a mechanism other than contact system activation. In addition, our data suggest that endotoxin-induced leukopenia and endothelial cell activation are mediated by TNF.     

Short-term effects of transdermal nicotine on acute tissue plasminogen activator release in vivo in man.

OBJECTIVE: Cigarette smoking impairs peripheral endothelium-dependent vasodilatation and acute tissue plasminogen activator (t-PA) release in man. The aim of the study was to determine if this endothelial dysfunction is, in part, mediated by the effects of nicotine. METHODS: Blood flow and plasma fibrinolytic factors were measured in both forearms of eight healthy male non-smokers during unilateral brachial artery infusion of the endothelium-dependent vasodilator, substance P (2 to 8 pmol/min). Endothelium-independent vasodilatation was assessed using intra-arterial infusion of sodium nitroprusside (2 to 8 microg/min). Subjects attended after 7 days treatment with transdermal nicotine or placebo in a double blind randomised crossover design. RESULTS: Plasma cotinine concentrations rose from 0.4+/-0.1 (placebo) to 125+/-25 ng/ml during nicotine administration (P<0.001). On both treatment days, substance P caused dose-dependent increases in blood flow and plasma t-PA antigen and activity concentrations (P<0.001 for all) but had no effect on plasma plasminogen activator inhibitor type 1 (PAI-1) concentrations. Compared with placebo, nicotine administration increased the substance-P-induced release of t-PA antigen and activity (P<0.05 for both) without an effect on endothelium-dependent or -independent vasodilatation. CONCLUSIONS: Short-term transdermal nicotine treatment does not affect endothelium-dependent vasomotion but does increase substance-P-induced t-PA release in vivo in man. This suggests that nicotine administration alters specific aspects of endothelial function and enhances the acute endogenous fibrinolytic capacity in vivo. The long-term effects of nicotine exposure, including the potential to cause depletion of endothelial t-PA stores, now needs to be assessed.     

Thromboembolic disease and anomalies of fibrinolysis

Quantitative abnormalities of fibrinolytic system factors (tissue plasminogen activator (t-PA), plasminogen activator inhibitor (PA-inhibitor) are often found in the patients exhibiting an idiopathic thromboembolic pathology. Exploration of fibrinolytic system is performed by taking blood samples prior and after stimulation (after venous occlusion or DDAVP injection). Patients can be "good responders" (that is presenting with an increase of fibrinolytic activity after stimulation) or "bad responders". Among these latter (that is 30 p. 100 of cases), there are two groups: patients exhibiting an increase of PA inhibitor level, this concealing fibrinolytic role of t-PA released by stimulation (20 p. 100 of cases), patients failing to present any t-PA release through endothelial cells stimulation (10 p. 100 of cases). Furthermore, an hypofibrinolysis was demonstrated a long time ago, in certain thrombogenic conditions (post-operative period, obesity, elderly patients). Hypofibrinolysis was recently demonstrated, according to such conditions, as liable to an increase in the PA inhibitor levels. As pathogenic role of hypofibrinolysis is then demonstrated, therapeutic studies reducing the PA inhibitor level or increasing the t-PA rate produced and released by endothelial cells are to be developed.     

Induction of plasminogen activator inhibitor by products released from platelets

Activation of platelets and augmentation of plasma plasminogen activator inhibitor (PAI) type I activity accompany acute myocardial infarction. To determine whether the two may be related, platelet compounds including epidermal growth factor and transforming growth factor beta as well as platelet lysates were studied in rabbits in vivo. After intravenous infusion of epidermal growth factor (1 and 5 micrograms/kg), plasma PAI activity increased sevenfold and 20-fold, peaking at 2 hours. After infusions of transforming growth factor beta (0.2 and 0.5 microgram/kg), plasma PAI activity increased sevenfold and 12-fold but peaked more slowly (at 5 hours). After infusion of platelet lysates (lysates from 2.8 and 5.6 X 10(8) platelets/kg), the increase was 19-fold and 35-fold, with a peak at 4 hours. Platelet lysates induced a pronounced increase of plasma PAI type 1 messenger RNA (Northern blots) in aorta, liver, and myocardium. Anti-transforming growth factor beta neutralizing antibody markedly attenuated the plasma PAI increase. Concentrations in plasma of fibrinogen and alpha 2-antiplasmin were virtually unaffected under all conditions. Thus, platelet-associated growth factors and platelet lysates, shown previously to increase plasma PAI type 1 messenger RNA expression and protein production in cultured hepatocytes and vascular endothelial cells in vitro, augment plasma PAI in vivo as well. Accordingly, activation of platelets and release of platelet-associated growth factors appear to contribute to the increased plasma PAI seen after myocardial infarction.     

The renin angiotensin system and endothelial dysfunction in chronic heart failure: role of endogenous fibrinolysis

Regulation of vascular tone by the endothelium is abnormal in patients with heart failure and contributes to the characteristic peripheral vasoconstriction and increased afterload. This endothelial dysfunction is mediated through several endothelium-derived factors, including nitric oxide; there is an important interplay between the endothelium and the renin angiotensin system. The benefits of ACE inhibition in heart failure relate, in part, to a reduction in ischemic events which may be mediated by improvements in endothelial function and the endothelium derived fibrinolytic parameters: tissue plasminogen activator (t-PA) and its inhibitor, plasminogen activator inhibitor type 1 (PAI-1). In addition to potential improvements in the regulation of vasomotion, ACE inhibitor therapy may increase bradykinin induced t-PA release and/or reduce angiotensin II mediated PAI-1 release. Recent evidence suggests that both angiotensin II type 1 receptor (AT(1)) antagonism and ACE inhibition improve basal fibrinolytic parameters in patients with heart failure which may facilitate the acute endogenous fibrinolytic response. 1999 by CHF, Inc.     

Fibrinolytic actions of intra-arterial angiotensin II and bradykinin in vivo in man

OBJECTIVES: Angiotensin II and bradykinin are potent endogenous vasoactive peptides which may play a role in the regulation of endogenous fibrinolysis and, thereby, contribute to the beneficial actions of ACE inhibitors. The aims of the study were to determine the acute effect of angiotensin II and bradykinin on the local vascular release of tissue plasminogen activator (t-PA) and its inhibitor, plasminogen activator inhibitor type 1 (PAI-1), and the endothelium-derived haemostatic factor, von Willebrand factor (vWf) from the forearm. METHODS: Blood flow, and plasma haemostatic and fibrinolytic factors, were measured in both forearms of sixteen healthy men: eight subjects received intra-arterial angiotensin II (5, 50 and 500 pmol/min) which was coinfused with sodium nitroprusside (SNP; 0.3, 1.5 and 7.5 microg/min, respectively), and eight received intra-arterial bradykinin at 10-3000 pmol/min. RESULTS: Despite substantial rises in plasma angiotensin II concentrations (P<0.001) which caused pressor effects (P<0.003) at the highest dose, angiotensin II infusion did not affect local plasma t-PA, PAI-1 or vWf concentrations. In contrast, bradykinin caused substantial dose-dependent increases in blood flow and t-PA release (>100 ng/100 ml of tissue/min) in the infused forearm (P<0. 001 for both) without affecting plasma PAI-1 or vWf concentrations. CONCLUSIONS: Despite high local concentrations with breakthrough of significant systemic effects, angiotensin II did not affect acute endothelial cell t-PA, PAI-1 or vWf release in healthy men. In contrast, bradykinin is a potent vasodilator and selective stimulus for acute local t-PA release. This may, at least in part, explain the fibrinolytic actions of ACE inhibitors in heart failure and ischaemic heart disease.     

Marked bradykinin-induced tissue plasminogen activator release in patients with heart failure maintained on long-term angiotensin-converting enzyme inhibitor therapy

OBJECTIVES: The aim of the present study was to assess the contribution of angiotensin-converting enzyme (ACE) inhibitor therapy to bradykinin-induced tissue-type plasminogen activator (t-PA) release in patients with heart failure (HF) secondary to ischemic heart disease. BACKGROUND: Bradykinin is a potent endothelial cell stimulant that causes vasodilatation and t-PA release. In large-scale clinical trials, ACE inhibitor therapy prevents ischemic events. METHODS: Nine patients with symptomatic HF were evaluated on two occasions: during and following seven-day withdrawal of long-term ACE inhibitor therapy. Forearm blood flow was measured using bilateral venous occlusion plethysmography. Intrabrachial bradykinin (30 to 300 pmol/min), substance P (2 to 8 pmol/min), and sodium nitroprusside (1 to 4 pmol/min) were infused, and venous blood samples were withdrawn from both forearms for estimation of fibrinolytic variables. RESULTS: On both study days, bradykinin and substance P caused dose-dependent vasodilatation and release of t-PA from the infused forearm (p < 0.05 by analysis of variance [ANOVA]). Long-term ACE inhibitor therapy caused an increase in forearm vasodilatation (p < 0.05 by ANOVA) and t-PA release (p < 0.001 by ANOVA) during bradykinin, but not substance P, infusion. Maximal local plasma t-PA activity concentrations approached 100 IU/ml, and maximal forearm protein release was approximately 4.5 microg/min. CONCLUSIONS: Long-term ACE inhibitor therapy augments bradykinin-induced peripheral vasodilatation and local t-PA release in patients with HF due to ischemic heart disease. Local plasma t-PA activity concentrations approached those seen during systemic thrombolytic therapy for acute myocardial infarction. This may contribute to the primary mechanism of the anti-ischemic effects associated with long-term ACE inhibitor therapy.     

Inhibition of clot lysis and decreased binding of tissue-type plasminogen activator as a consequence of clot retraction.

Tissue-type plasminogen activator (t-PA) is less active in vivo and in vitro against clots that are enriched in platelets, even at therapeutic concentrations. The release of radioactivity from 125I-fibrin-labeled clots was decreased by 47% 6 hours after the addition of t-PA 400 U/mL when formed in platelet-rich versus platelet-poor plasma. This difference was not due to the release of plasminogen activator inhibitor-1 (PAI-1) by platelets. Thus, the fibrinolytic activity of t-PA in the supernatant was similar in the two preparations and fibrin autography demonstrated only a minor degree of t-PA-PAI-1 complex formation. Furthermore, a similar platelet-dependent reduction in clot lysis was seen with a t-PA mutant resistant to inhibition by PAI-1. The reduction in t-PA activity correlated with a decrease in t-PA binding to platelet-enriched clot (60% +/- 3% v platelet-poor clot, n = 5). This reduction in binding was also shown using t-PA treated with the chloromethylketone, D-Phe-Pro-Arg-CH2Cl (PPACK) (36% +/- 13%, n = 3), and with S478A, a mutant t-PA in which the active site serine at position 478 has been substituted by alanine (43% +/- 6%, n = 3). In contrast, fixed platelets and platelet supernatants had no effect on the binding or lytic activity of t-PA. Pretreatment with cytochalasin D 1 mumol/L, which inhibits clot retraction, also abolished the platelet-induced inhibition of lysis and t-PA binding by platelets. These data suggest that platelets inhibit clot lysis at therapeutic concentrations of t-PA as a consequence of clot retraction and decreased access of fibrinolytic proteins.     

Histone deacetylase inhibitors stimulate tissue-type plasminogen activator production in vascular endothelial cells

A reduced capacity for acute tissue-type plasminogen activator (t-PA) release is likely to be associated with an impaired endogenous defense against intravascular thrombosis. Efficient approaches to pharmacologically restore a defective t-PA release have been lacking, but recent observations suggest that histone deacetylase inhibitors (HDACis) enhance t-PA production in vitro. HDACis have diverse chemical structures and different HDAC-enzyme sub-class targeting. We here compared the effects of several clinically used HDACis on t-PA production in endothelial cells. Human umbilical vein endothelial cells were exposed to a panel of 11 different HDACis and t-PA mRNA and protein levels were quantified. All HDACis dose-dependently stimulated t-PA mRNA and protein expression with similar maximal efficacy but with different potencies. Already at low concentrations, the majority of inhibitors caused significant and sustained effects on t-PA production. In addition, selected HDACis were capable of normalizing t-PA production when suppressed by the inflammatory cytokine TNF-α. We conclude that HDACis targeting classical HDAC enzymes are powerful inducers of t-PA expression in cultured endothelial cells and could be promising candidates for pharmacological modulation of endogenous fibrinolysis in man.     

Relation of C-reactive protein to endothelial fibrinolytic function in healthy adults

Increased plasma concentrations of C-reactive protein (CRP) independently predict future atherothrombotic events in healthy asymptomatic adults. CRP may promote atherothrombosis by altering fibrinolytic balance; however, the influence of increased plasma CRP concentrations on endothelial fibrinolysis in healthy adults is unclear. We tested the hypothesis that endothelial release of tissue-type plasminogen activator (t-PA) is impaired in adults with increased plasma CRP concentrations independent of other cardiometabolic risk factors. Fifty-four healthy adults were studied: 24 with CRP <1.0 mg/L (low CRP; 18 men and 6 women, 55 ± 2 years old), 18 with CRP 1.0 to 3.0 mg/L (moderate CRP; 8 men and 10 women, 58 ± 2 years old), and 12 with CRP >3.0 mg/L (high CRP; 7 men and 5 women, 56 ± 2 years old). Net endothelial release of t-PA was determined in vivo in response to intrabrachial infusions of bradykinin (125 to 500 ng/min) and sodium nitroprusside (2.0 to 8.0 μg/min). Capacity of the endothelium to release t-PA was significantly lower (～30%) in the high (0.32 ± 0.5 to 38.9 ± 6.0 ng · 100 ml tissue(-1) · min(-1)) and moderate (-0.05 ± 0.4 to 39.3 ± 5.2 ng · 100 ml tissue(-1) · min(-1)) compared to the low (0.42 ± 0.9 to 61.8 ± 5.2 ng · 100 ml tissue(-1) · min(-1)) CRP group. There was no significant difference in t-PA release between the high and moderate CRP groups. Plasma CRP concentrations were inversely related to t-PA release (r = -0.38, p <0.05). In conclusion, these results indicate that the capacity of the endothelium to release t-PA is decreased in adults with plasma CRP ≥1.0 mg/L. Endothelial fibrinolytic dysfunction may underlie the increased atherothrombotic risk associated with increases in plasma CRP concentrations in otherwise healthy adults.     

Thrombin regulation of mRNA levels of tissue plasminogen activator and plasminogen activator inhibitor-1 in cultured human umbilical vein endothelial cells

Cultured human umbilical vein endothelial cells release tissue plasminogen activator (t-PA) and type 1 plasminogen activator inhibitor (PAI-1) in response to alpha thrombin stimulation. In order to study the mechanisms of thrombin stimulation, we measured changes in levels of mRNA for t-PA and PAI-1 following exposure of endothelial cells to 3 U/mL alpha thrombin. Alpha thrombin causes a significant and time- dependent increase in the mRNA levels of both t-PA and PAI-1. Catalytically inactivated diisofluorophosphate (DIP) treated thrombin and alpha thrombin pretreated with hirudin do not alter t-PA and PAI-1 mRNA levels. We conclude that the increased secretion of t-PA and PAI-1 by human umbilical vein endothelial cells in response to alpha thrombin is mediated at least partially through an increase in mRNA levels. In addition, an active thrombin catalytic site is required for these increases in mRNA to occur.     

Serotonin induces the expression of tissue factor and plasminogen activator inhibitor-1 in cultured rat aortic endothelial cells

Serotonin (5-hydroxytryptamine, or 5-HT), released from activated platelets, not only accelerates aggregation of platelets but also is known to promote mitosis, migration, and contraction of vascular smooth muscle cells (VSMCs). These effects are considered to contribute to thrombus formation and atherosclerosis. The aim of this study was to investigate the effects of 5-HT on the expressions of coagulative and fibrinolytic factors in rat aortic endothelial cells. Endothelial cells were stimulated with various concentrations of 5-HT (0.1 approximately 10 microM), and the expressions of tissue factor (TF), tissue factor pathway inhibitor (TFPI), plasminogen activator inhibitor-1 (PAI-1), and tissue-type plasminogen activator (TPA) messenger RNAs (mRNAs) were evaluated by Northern blot analysis. The activities of TF and PAI-1 were also measured. TF and PAI-1 mRNA were increased significantly in a concentration- and time-dependent manner. However, TFPI and TPA mRNA expression did not change. The inductions of TF and PAI-1 mRNAs were inhibited by a 5-HT1/5-HT2 receptor antagonist (methiothepin) and a selective 5-HT2A receptor antagonist (MCI-9042). These results indicate that 5-HT increases procoagulant activity and reduces fibrinolytic activities of endothelial cells through the 5-HT2A receptor. It was concluded that the modulation of procoagulant and hypofibrinolytic activities of endothelial cells by 5-HT synergistically promotes thrombus formation at the site of vessel injury with the platelet aggregation, VSMC contraction, and VSMC proliferation.     

Activated protein C decreases plasminogen activator-inhibitor activity in endothelial cell-conditioned medium

Confluent cultures of endothelial cells from human umbilical cord were used to study the effect of activated human protein C (APC) on the production of plasminogen activators, plasminogen activator-inhibitor, and factor VIII-related antigen. Addition of APC to the cells in a serum-free medium did not affect the production of tissue-type plasminogen activator (t-PA) or factor VIII-related antigen; under all measured conditions, no urokinase activity was found. However, less plasminogen activator-inhibitor activity accumulated in the conditioned medium in the presence of APC. This decrease was dose dependent and could be prevented by specific anti-protein C antibodies. No decrease was observed with the zymogen protein C or with diisopropylfluorophosphate-inactivated APC. APC also decreased the t-PA inhibitor activity in endothelial cell-conditioned medium in the absence of cells, which suggests that the effect of APC is at least partly due to a direct effect of APC on the plasminogen activator- inhibitor. High concentrations of thrombin-but not of factor Xa or IXa-- had a similar effect on the t-PA inhibitor activity. The effect of APC on the plasminogen activator-inhibitor provides a new mechanism by which APC may enhance fibrinolysis. The data suggest that activation of the coagulation system may lead to a secondary increase of the fibrinolytic activity by changing the balance between plasminogen activator(s) and its (their) fast-acting inhibitor.     

Induction of endothelial cell expression of the plasminogen activator inhibitor type 1 gene by thrombosis in vivo.

BACKGROUND. We have shown previously that products from activated platelets can augment synthesis of plasminogen activator inhibitor type 1 (PAI-1) in cultured endothelial and hepatoma (Hep G2) cells in vitro and increase plasma PAI-1 activity in vivo in rabbits. Accordingly, the effects of activation of platelets associated with thrombosis and thrombolysis in vivo on plasma PAI-1 activity and expression of the PAI-1 gene in endothelium, liver, and other organs were characterized. METHODS AND RESULTS. Endothelial injury giving rise to platelet-rich thrombi was induced with electrical stimulation in carotid arteries in rabbits. Clot lysis and recanalization were induced subsequently with intravenous tissue-type plasminogen activator (t-PA) and verified with Doppler flow probes. Plasma PAI-1 activity (mean +/- SD) increased from 6 +/- 2 arbitrary units (AU)/ml to 129 +/- 48 AU/ml (n = 15) within several hours after recanalization. When t-PA had failed to induce recanalization, the increase was much less (from 7 +/- 2 to 42 +/- 23 AU/ml, n = 11). To define mechanisms responsible for these changes, PAI-1 messenger RNA (mRNA) was evaluated by Northern blot analysis and localized in tissues by in situ hybridization. Strong and consistent induction of PAI-1 mRNA was evident in aorta, heart, and liver of animals subjected to thrombosis (twofold to threefold increases compared with values in controls), particularly in those in which thrombolysis had been induced (fourfold to sixfold). After thrombolysis, an intense, PAI-1 mRNA-specific signal was detected in endothelium of aorta, liver, and heart, with less intense signals in endothelium of lung, adrenals, and kidneys. CONCLUSIONS. The increases in plasma PAI-1 activity follow a preceding increase in endothelial cell expression of the PAI-1 gene as reflected by PAI-1 mRNA levels. Thus, increased synthesis of endothelial cell PAI-1 after thrombosis and thrombolysis may attenuate endogenous fibrinolysis early after coronary thrombolysis, thereby potentiating early, thrombotic reocclusion.     

Endothelin modulation of tissue plasminogen activator release from human vascular endothelial cells in culture.

To clarify a possible involvement of the vasoconstrictive peptide endothelin in the regulation of endothelial cell-mediated fibrinolytic system, confluent cultures of vascular endothelial cells from human umbilical vein were incubated in serum-free medium in the presence of endothelin-1 at 100 nM and below, and tissue plasminogen activator antigen (t-PA:Ag) in the medium was determined by enzyme immunoassay. Endothelin-1 at 1 nM and above significantly decreased the release of t-PA:Ag from the endothelial cells after a 24 h incubation. The t-PA:Ag release was also decreased by either endothelin-2 or endothelin-3 at 10 nM. The activity of lactate dehydrogenase in the medium was not changed by endothelin-1 at 100 nM and below, suggesting that the peptide did not cause nonspecific cell damage. The decrease in the t-PA:Ag release induced by endothelin-1 occurred in the presence or absence of 8-bromo cyclic AMP, which is an active congener of cyclic AMP; 3-isobutyl-1-methylxanthine, which is an inhibitor of phosphodiesterase; and forskolin, which is a stimulator of adenylate cyclase. These results strongly indicated that cyclic AMP which is known to down-regulate t-PA:Ag release was not involved in the endothelin-1 effect. However, endothelin-1 failed to decrease the t-PA:Ag release in the presence of either calcium ionophore A23187 or EGTA; the ionophore itself markedly decreased the release. The cytosolic calcium accumulation was significantly increased by endothelin-1. These results suggest that endothelin-1 decreases the release of t-PA:Ag from human endothelial cells through an excess accumulation of intracellular, especially cytosolic which would be mediated by an extracellular, calcium-dependent mechanism.