Regulation and secretion of plasminogen activators and their inhibitors in a human leukemic cell line (K562)

The secretion of tissue plasminogen activator (t-PA), urokinase (u-PA) and their inhibitors by the human leukemia cell line K562 was examined. K562 cells normally secrete both t-PA and u-PA in a ratio of 3:1. After addition of 10 or 1 ng/mL phorbol myristate acetate (PMA) to K562 cells, a marked decrease in enzymatic activity is observed in the medium. However, when t-PA antigen rather than activity is measured, an increased amount is found in the medium under these conditions. PMA also induces secretion of the two inhibitors of plasminogen activator: plasminogen activator inhibitor 1 (PAI-1) and plasminogen activator inhibitor 2 (PAI-2). This accounts for the decrease in total enzymatic activity under conditions when production of t-PA antigen is increased. A study of the time course of induction revealed that the synthesis of plasminogen activator occurred before that of its inhibitors. Low concentrations of PMA (0.1 ng/mL) induce t-PA antigen primarily and not the inhibitors. This results in an increase in total enzymatic activity, with 94% of the secreted activity being t-PA. Thus, the secretion of plasminogen activators and their inhibitors can be manipulated in certain leukemic cells by inducers such as PMA.     

Characterization and fibrinolytic properties of human omental tissue mesothelial cells. Comparison with endothelial cells

It has been reported that omental fat tissue is a good source of human microvascular endothelial cells. By characterization we demonstrate that the epitheloid cells isolated from omental tissue are not endothelial cells, but mesothelial cells. They contain abundant cytokeratins 8 and 18, which are absent in endothelial cells, and vimentin. No staining with the endothelial-specific antibodies EN-4 and PAL-E is observed. A faint and diffuse staining of von Willebrand factor (vWF) is seen in mesothelial cells, whereas microvascular endothelial cells from subcutaneous fat display vWF in distinct granular structures. Human peritoneal mesothelium produces plasminogen activator-dependent fibrinolytic activity, which is essential in the resolution of fibrous exudates and may therefore be important in preventing the formation of fibrous peritoneal adhesions. This fibrinolytic activity is plasminogen activator-dependent, but has not been fully characterized. We report here that human omental tissue mesothelial cells in vitro produce large amounts of tissue-type plasminogen activator (t-PA), together with type 1 and 2 plasminogen activator inhibitor (PAI-1 and PAI-2). PAI-1 is predominantly secreted into the culture medium, whereas the major part of PAI-2 is found in the cells. No urokinase-type plasminogen activator is detected. On stimulation with the inflammatory mediator tumor necrosis factor (TNF), at least a threefold decrease in t-PA antigen is observed, together with an increase in both PAI-1 and PAI-2. TNF also induces a marked change in cell shape. Whereas TNF and bacterial lipopolysaccharide (LPS) have similar effects on the production of PA inhibitor by human endothelial cells, LPS has no or only a relatively small effect on the fibrinolytic properties of mesothelial cells. The decreased fibrinolytic activity induced by the cytokine TNF may impair the natural dissolution of fibrin deposits at the peritoneum in the presence of an inflammatory reaction.     

Activated protein C stimulates the fibrinolytic activity of cultured endothelial cells and decreases antiactivator activity.

The effects of bovine activated protein C (APC) on the fibrinolytic activity of cultured bovine aortic endothelial cells were investigated. Confluent monolayers were incubated with purified APC under various conditions and changes in total fibrinolytic activity and in the level of plasminogen activator and plasminogen activator inhibitor (antiactivator) were monitored. The addition of APC to the cells in the absence of other blood or plasma components led to a rapid, dose-dependent increase of fibrinolytic activity both in the media and in cellular extracts. For example, 3.4 micrograms of APC per ml resulted in a 15-fold increase of fibrinolytic activity in the medium within 1 hour. The enhanced fibrinolytic activity reflected increases in both the urokinase-related and tissue-type plasminogen activators produced by these cells. Interestingly, treatment of cells with APC also caused a rapid, dose-dependent decrease in antiactivator activity. Diisopropyl fluorophosphate-inactivated APC did not decrease antiactivator or increase plasminogen activator. Although a small but significant direct (i.e., cell-independent) effect of APC on both fibrinolytic activity and antiactivator activity could be demonstrated, the major portion of these changes appeared to be cell-mediated. These observations indicate that the fibrinolytic potential of cultured endothelial cells is increased by APC and that the enzyme active site is essential for this change. Moreover, the results suggest that one of the primary mechanisms for this stimulation of endothelial cell fibrinolytic activity involves an APC-mediated decrease in antiactivator.     

Fibrinolytic properties of a human endothelial hybrid cell line (Ea.hy 926)

The fibrinolytic characteristics of the endothelial hybrid cell line EA.hy 926, established by fusing a human umbilical vein endothelial cell with a human carcinoma cell line, were studied. The hybrid cell line produced large amounts of tissue-type plasminogen activator (t- PA), plasminogen activator inhibitor type 1, and a small amount of urokinase. All plasminogen activator present in conditioned medium was complexed with inhibitor because the cells secreted plasminogen activator inhibitor in excess over plasminogen activator and no activator activity was detectable in conditioned media by direct activity assays. t-PA activator activity was, however, demonstrable in conditioned media after treatment with sodium dodecyl sulfate, in agreement with t-PA antigen determinations. Increased plasminogen activator inhibitor activity could be induced by incubating the cells in the presence of endotoxin or microtubule inhibitors, whereas increased t-PA activity could be induced by microtubule inhibitors. Interleukin-1 had no effect. The fibrinolytic characteristics of the hybrid cell line were stable for at least 30 passages. The perpetual human hybrid cell line EA.hy 926 therefore may be a useful tool for the study of fibrinolysis in cultured endothelial cells.     

Changes in plasminogen activator inhibitor 1 and tissue-type plasminogen activator during exercise in patients with coronary artery disease

As depressed fibrinolysis is implicated in the pathogenesis of coronary artery disease, we have studied the activation of fibrinolysis during maximal, symptom-limited exercise in a group of 68 men. After exercise they were divided, according to their coronary angiography and exercise 201Tl emission computed tomography results, into three groups. Group 1: persons with normal exercise 201Tl emission computed tomography results and no underlying diseases who served as controls; group 2: patients with coronary artery disease without exercise-induced myocardial ischemia, and group 3: patients with coronary artery disease with transient, exercise-induced myocardial ischemia. Before and at peak exercise we measured the plasminogen activator activity (PAA) in the euglobulin fraction of plasma by an amidolytic method and the concentrations of tissue plasminogen activator (t-PA), activator-inhibitor complex - plasminogen activator inhibitor 1 (PAI-1) complexed with t-PA - and total PAI-1 by enzyme immunoassay. The concentration of free PAI-1 in plasma was calculated by subtraction of the concentration of activator-inhibitor complex from that of total PAI-1. Under basal conditions, group 3 had significantly higher free and total PAI-1 levels than group 1. There were no statistically significant differences between the three groups in PAA, t-PA, and activator-inhibitor complex levels. At peak exercise, group 1 showed the highest release of t-PA accompanied with highest increases in PAA as well as in activator-inhibitor complex, the proportion of released t-PA antigen not bound to PAI-1 being highest in group 1. Free PAI-1 decreased significantly, but there were no differences between individual groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Tumor necrosis factor induces the production of urokinase-type plasminogen activator by human endothelial cells

Endothelial cells play an important role in the regulation of fibrinolysis by the production of several key regulatory proteins. The cytokines tumor necrosis factor (TNF), lymphotoxin, and interleukin-1 (IL-1), but not interleukin-6, increase the production of plasminogen activator inhibitor-1 (PAI-1) by endothelial cells, whereas they have no stimulatory effect on the production of tissue-type plasminogen activator (t-PA). Primary cultures of human endothelial cells release very little urokinase-type plasminogen activator (u-PA). We report here that TNF and lymphotoxin induce, in a concentration-dependent way, the production of both cellular and secreted u-PA antigen in primary and subcultured human endothelial cells. The TNF-induced increase was accompanied by a more than 10-fold increase in u-PA mRNA. Upon stimulation of early passage umbilical vein endothelial cells by TNF, u-PA was predominantly secreted at the basolateral side, whereas PAI activity and t-PA were found in more equal amounts at the apical and basolateral sides of the cell monolayers. TNF-stimulated u-PA secretion by subcultured human aorta endothelial cells showed only a marginal polarity. The u-PA antigen was present in a plasmin-activatable form (single chain u-PA) and in a nonactivatable form (probably u-PA: PAI-1 complex). During the induction of u-PA by TNF, the ratio between plasmin-activatable u-PA and total u-PA decreased markedly. This may indicate that TNF also increases the degree of u-PA activation. The parallel induction of the synthesis and secretion of both u-PA and PAI-1 by endothelial cells adds a new aspect to the alterations of the fibrinolytic system caused by inflammatory mediators. This aspect may be significant for the regulation of cell-associated and interstitial plasminogen activator activity.     

Endothelial stimulation by DDAVP in von Willebrand's disease and haemophilia

Desamino-D-arginine vasopressin (DDAVP) is known to stimulate factor VIII (FVIII) and plasminogen activator release from endothelial cells, and has been shown to stimulate prostacyclin (PGI2) production in normal and haemophilic subjects. In von Willebrand's disease (vWd) some patients have a dissociate response with regard to FVIII and plasminogen activator. The aim of our study was to compare the PGI2, FVIII and plasminogen activator response to DDAVP infusion in vWd with the response to DDAVP in normal and haemophilic subjects. PGI2 metabolites thromboxane B2 (TxB2), factor VIII coagulant activity, factor VIII-related antigen and plasminogen activator were measured before and after DDAVP infusion. There was a significant increase in PGI2 metabolites, factor VIII-related antigen and plasminogen activator in all groups following DDAVP, but no effect on TxB2 was found, and there was no evidence of any dissociate response to DDAVP in any of the groups. Basal levels of PGI2 metabolites, however, were significantly lower in vWd as compared to normal and haemophilic subjects. Post-DDAVP levels of PGI2 metabolites were also significantly lower in vWd as compared with normal subjects. This may be due to a reduced stimulus to PGI2 production in vWd secondary to defective platelet adhesion.     

Antithrombotic regulation in human endothelial cells by fibrinolytic factors

Vascular endothelial cells (ECs) modulate the blood fibrinolytic system by secreting tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), and their inhibitor, type-1 plasminogen activator inhibitor (PAI-1). ECs also express t-PA receptors (t-PAR) and u-PA receptors (u-PAR) on their cell surfaces, assembling both enzymes to regulate the cellular fibrinolytic activity. In addition, ECs modulate these factors in response to several stimuli. Fibrin clots on ECs induce the up- and downregulation of t-PA and PAI-1 production, respectively, thus causing an effective lysis of the fibrin clot. Heat shock (43 degrees C) increases the expression of u-PA, t-PA, PAI-1, and u-PAR by which ECs become more fibrinolytic around the cells. Furthermore, because ECs possess t-PAR and u-PAR on their cell surfaces, the binding of t-PA and u-PA is a critical event, which affords ECs the localized and condensed fibrinolytic potential. Therefore, ECs play a central role in antithrombotic activity by regulating the levels of these fibrinolytic factors.     

Absence of functional activity of tissue plasminogen activator in patients with severe forms of von Willebrand''s disease

Some patients with von Willebrand's disease do not respond to stimuli such as venous occlusion and infusion of a vasopressin analogue DDAVP. In these patients, fibrinolytic activity is not enhanced and von Willebrand's factor is not released into the blood. Skin biopsies and cryostat sections were used to study the fibrinolytic activity of skin vessels and localization of tissue plasminogen activator (t-PA) in three patients with severe form of von Willebrand's disease. On fibrin films, no fibrinolysis developed around the skin vessels of the patients; however, using specific polyclonal and monoclonal antibodies to t-PA, and peroxidase coupled specific IgG, presence of t-PA antigen was demonstrated in endothelial cells (EC) of all of them. In plasma no t-PA activity was detected either before or after venous occlusion although t-PA inhibitor activity was in a normal range. Small amounts of t-PA antigen was measured in blood by ELISA. From these results, it is concluded that in patients with severe forms of von Willebrand's disease, t-PA present in EC is not functional and can not transform plasminogen into plasmin.     

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.     

The putative mechanism of thrombosis in antiphospholipid syndrome: impairment of the protein C and the fibrinolytic systems by monoclonal anticardiolipin antibodies

The mechanism of thrombosis in patients with antiphospholipid syndrome is not clear. To investigate it, we examined the effect of monoclonal anticardiolipin (aCL) antibodies and beta2-glycoprotein I (beta2-GPI), which is required for formation of the aCL epitopes, on activated protein C (APC) and on fibrinolytic activity. First, APC activities were measured in the presence and absence of beta2-GPI or gamma M immunoglobulin (IgM) monoclonal aCLs (EY1C8 and EY2C9), or both, established from peripheral blood lymphocytes obtained from a patient with aCL. beta2-GPI exhibited a procoagulant activity by inhibiting APC activity as well as an anticoagulant activity by inhibiting thrombin generation. Any further inhibition of APC activity was caused by monoclonal aCL, and then only in the presence of beta2-GPI. The remaining tissue plasminogen activator (t-PA) of the sample consisting of beta2-GPI, two-chain recombinant t-PA, and plasminogen activator inhibitor (PAI)-1 was measured by a chromogenic assay using the synthetic substrate S-2251, Glu-plasminogen, and soluble fibrin monomer. beta2-GPI protected t-PA activity from inhibition by PAI-1. However, monoclonal aCLs (EY1C8 and EY2C9) inhibited the effect of beta2-GPI on fibrinolytic activity; that is, monoclonal aCLs inhibited fibrinolytic activity by elevating PAI-1 activity. Thrombosis in patients with aCL can be explained in part by both the inhibition of APC anticoagulant activity and the impairment of fibrinolytic activity by aCL.     

Endothelin-1 enhances plasminogen activator inhibitor-1 production by human brain endothelial cells via protein kinase C-dependent pathway.

The effects of endothelin-1 (ET-1) on the production of plasminogen activator inhibitor 1 (PAI-1) and tissue plasminogen activator (t-PA) by human brain-derived endothelial cells in culture were studied. At 100 nmol/L, ET-1 increased PAI-1 production by 88+/-6% within 72 hours, and increased PAI-1 mRNA expression within 1 hour of stimulation; there was no significant effect on t-PA production. PAI-1 activity was also examined and found to increase with ET-1 treatment. Suboptimal concentrations of ET-1 and tumor necrosis factor-alpha (TNF-alpha) acted synergistically to increase PAI-1 production. ET-1 activated protein kinase C and cAMP-dependent protein kinase pathways within 3 to 5 minutes of treatment, with the peak at 10 minutes. Activation of protein kinase C by phorbol-12-myristate-13-acetate (PMA) resulted in increased PAI-1 production, whereas activation of the cAMP-dependent protein kinase by forskolin or dibutyryl cAMP (dBu-cAMP) significantly decreased PAI-1 production. However, simultaneous activation of protein kinase C by PMA and cAMP-dependent protein kinase by dBu-cAMP only slightly attenuated PMA-induced PAI-1 increase. Inhibition of protein kinase C by GF-109213X abolished the effects of ET-1. These results demonstrate that ET-1 and TNF-alpha function synergistically to induce procoagulant activity of brain endothelial cells in a process that involves a protein kinase C-dependent pathway.     

Impaired fibrinolysis in coronary artery disease

We assessed endogenous endothelial-dependent fibrinolysis in 99 subjects with coronary artery disease (CAD) documented by angiography and in 28 control subjects with normal coronary arteries on angiography. We used specific, sensitive assays for plasma tissue-type plasminogen activator (t-PA) antigen, t-PA activity, plasminogen activator inhibitor (PAI) activity, plasminogen, and alpha-2 plasmin inhibitor (alpha-2 PI). Mean PAI activity was significantly higher, and mean t-PA activity after venous occlusion of the upper arm (a standard test of the capacity of vascular endothelium to release t-PA) was significantly lower in subjects with CAD than in subjects with normal coronary arteries. The mean increment in t-PA antigen after venous occlusion was significantly lower than normal in subjects with CAD with onset of symptoms before age 45 years. Subjects with CAD had a significantly increased mean plasma fibrinogen level compared with control subjects, and a significant positive correlation was observed between PAI activity and plasma fibrinogen in subjects with CAD. No significant abnormalities of plasminogen or alpha-2 PI were observed in any subset of subjects with CAD. These data support an association between impaired fibrinolysis and CAD, with contributions from both increased PAI activity and in younger subjects from reduced endothelial t-PA release.     

Fibrinolytic capacity following stimulation with desmopressin acetate in patients with diabetes mellitus

Tissue plasminogen activator (t-PA), tissue plasminogen activator inhibitor, (PAI), and von Willebrand factor (vWF) were measured in 30 diabetics and 17 control subjects. These studies were performed to clarify the role of obesity in causing abnormalities of the fibrinolytic system in diabetics. The t-PA antigen response measured after the infusion of desmopressin acetate (DDAVP) was similar in all groups. Peak responses to DDAVP for controls, type I diabetics, and type II diabetics were 21.2 +/- 9.5 ng/mL, 27.5 +/- 9.0 ng/mL, and 28.8 +/- 11.0 ng/mL (NS), respectively. These responses did not correlate with the body mass index (BMI) or any other of the indices examined. A significant decrease of t-PA activity as contrasted with t-PA antigen following DDAVP occurred in type II diabetics only. The decrease of t-PA activity strongly correlated with greater basal levels of plasminogen activator inhibitor in these same subjects. The plasma level of plasminogen activator inhibitor correlated with BMI but with no other index examined. In contrast to t-PA activity and PAI, vWF responses to DDAVP inversely correlated to basal vWF concentration in all groups. Basal concentrations of vWF were increased in both type I and II diabetics and showed no relationship to degree of obesity. In summary, these results suggest that type II diabetic subjects have decreased t-PA activity, which is best explained by increased levels of PAI. The increased PAI appears related to obesity and not diabetes per se.     

Effect of atorvastatin and fluvastatin on the expression of plasminogen activator inhibitor type-1 in cultured human endothelial cells

Inhibitors of HMG-CoA reductase, namely statins, improve endothelial function independently of their cholesterol-lowering effects. Plasminogen activator inhibitor type-1 (PAI-1) plays a critical role in vascular pathophysiology both at the intra- and extravascular levels. We therefore investigated the effects of atorvastatin (ATOR) and fluvastatin (FLU) on PAI-1 and also tissue-type plasminogen activator (t-PA) synthesis in 20% fetal calf serum-cultured human umbilical vein endothelial cells (HUVEC) stimulated or not by recombinant human pro-inflammatory cytokines, i.e. tumor necrosis factor alpha (TNFalpha) and interleukin 1alpha (IL-1alpha). In non-stimulated HUVEC, ATOR and FLU significantly diminished (-50% at 2.0 micromol/l) the constitutive production of PAI-1 (mRNA level and protein secretion). This effect was prevented by addition of mevalonate (100 micromol/l). In HUVEC cultivated in 20% fetal calf serum, the t-PA antigen accumulation was not significantly altered, whereas in low serum concentration (1%) a significant stimulatory effect of ATOR (+30%) and FLU (+76%) was observed. In TNFalpha-stimulated cells, ATOR and FLU had a modest down-modulating effect (-17 and -20%, respectively) on TNFalpha-induced increase in PAI-1 synthesis. No effect of statins was observed in IL-1alpha-stimulated HUVEC, suggesting that statins do not interfere with the up-regulation of PAI-1 synthesis by pro-inflammatory cytokines. However, ATOR and FLU inhibited the TNFalpha-induced decrease in t-PA release. In conclusion, these results show that statins favorably modulate the expression of fibrinolytic factors produced by human endothelial cells.     

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

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.     

Progress of fibrinolysis during tumor necrosis factor infusions in humans. Concomitant increase in tissue-type plasminogen activator, plasminogen activator inhibitor type-1, and fibrin(ogen) degradation products.

Several investigators have reported that tumor necrosis factor (TNF) can alter the production of plasminogen activator type-1 (PAI-1) and plasminogen activators (PAs) by endothelial cells in vitro. We have examined the in vivo effects of recombinant human TNF administration on fibrinolysis as assessed by parameters in plasma during a 24-hour period of continuous TNF infusion to 17 cancer patients with active disease. The plasma levels of PAI activity increased sevenfold after 3 and 24 hours of TNF infusion. This was the result of an increase of PAI-1 antigen; PAI-2 antigen was not detectable. Plasma concentrations of tissue-type PA (t-PA) antigen increased twofold to fivefold after 3 and 24 hours of TNF infusion, whereas urokinase-type PA antigen levels in plasma remained unaltered. After 3 hours of TNF infusion the plasma levels of alpha 2-antiplasmin were slightly decreased, 5% on average, suggesting that fibrinolysis continued. After 24 hours of TNF infusion a highly significant increase in fibrin- plus fibrinogen-degradation products, and separately of fibrin degradation products and fibrinogen degradation products, was found. This indicates that fibrinolysis persisted, at least partly, in the presence of high levels of PAI activity. Whereas PAI-1 production increased, t-PA production by human endothelial cells in vitro remains unaltered or even decreases on TNF addition. It has been shown previously that TNF infusion in our patients results in thrombin and fibrin generation. Therefore, it is possible that thrombin, not TNF, is the actual stimulus for t-PA production in our patients. We speculate that fibrin is formed during TNF infusions and that plasmin is generated by t-PA action immediately on the initial formation of (soluble) fibrin molecules. Such a process may explain the generation of degradation products of both fibrin and fibrinogen during infusion of TNF in patients.     

Effects of captopril therapy on endogenous fibrinolysis in men with recent,uncomplicated myocardial infarction

Objectives. This study investigated the effects of captopril therapy on endogenous fibrinolysis in men with recent, uncomplicated myocardial infarction.Background. Angiotensin-converting enzyme inhibitors reduce the incidence of acute coronary syndromes in patients with mild left ventricular dysfunction after myocardial infarction. Abnormal endogenous fibrinolysis, reflected in increased levels of endogenous tissue-type plasminogen activator (t-PA) antigen and plasminogen activator inhibitor type 1 activity, is associated with an increased risk of myocardial infarction in patients with ischemic heart disease.Methods. In a randomized, double-blind crossover study beginning 8 weeks after uncomplicated myocardial infarction, patients received 4 weeks of placebo and 4 weeks of captopril (75 mg daily) therapy. At the end of each treatment period, we measured t-PA antigen and plasminogen activator inhibitor type 1 antigen and activity.Results. Median values in the 15 patients after placebo and in 12 normal men matched for age and body mass index were, respectively, t-PA antigen 16.0 versus 9.5 ng/ml (p = 0.001), plasminogen activator inhibitor type 1 antigen 17.3 versus 8.6 ng/ml (p = 0.29) and plasminogen activator inhibitor type 1 activity 13.2 versus 6.3 AU/ml (p = 0.04). After 4 weeks of treatment with captopril in the 15 patients, the estimated (95% confidence interval) median reduction in t-PA antigen was 7.3 ng/ml (−4.6 to −10.3 ng/ml, p = 0.001), in plasminogen activator inhibitor type 1 antigen 3.1 ng/ml (+1.5 to −8.4 ng/ml, p = 0.17) and in plasminogen activator inhibitor type 1 activity −2.2 AU/ml (−1.0 to −4.3 AU/ml, p = 0.02).Conclusions. Treatment with captopril after uncomplicated myocardial infarction is associated with a significant decrease in elevated levels of t-PA antigen and plasminogen activator inhibitor type 1 activity. This may help to explain the reduction in risk of coronary thrombosis associated with the use of angiotensin-converting enzyme inhibitors.     

Activated protein C up-regulates procoagulant tissue factor activity on endothelial cells by shedding the TFPI Kunitz 1 domain

Thrombin and activated protein C (APC) signaling can mediate opposite biologic responses in endothelial cells. Given that thrombin induces procoagulant tissue factor (TF), we examined how TF activity is affected by APC. Exogenous or endogenously generated APC led to increased TF-dependent factor Xa activity. Induction required APC's proteolytic activity and binding to endothelial cell protein C receptor but not protease activated receptors. APC did not affect total TF antigen expression or the availability of anionic phospholipids on the apical cell membrane. Western blotting and cell surface immunoassays demonstrated that APC sheds the Kunitz 1 domain from tissue factor pathway inhibitor (TFPI). A TFPI Lys86Ala mutation between the Kunitz 1 and 2 domains eliminated both cleavage and the enhanced TF activity in response to APC in overexpression studies, indicating that APC up-regulates TF activity by endothelial cell protein C receptor-dependent shedding of the Kunitz 1 domain from membrane-associated TFPI. Our results demonstrate an unexpected procoagulant role of the protein C pathway that may have important implications for the regulation of TF- and TFPI-dependent biologic responses and for fine tuning of the hemostatic balance in the vascular system.