Inhibition of Mycobacterium tuberculosis-induced signalling by transforming growth factor-ß in human mononuclear phagocytes.

Tuberculosis (TB) is associated with excessive production and bioactivation of transforming growth factor bets (TGF-ß) in situ. Here, modification of expression of components of plasminogen/plasmin pathway in human monocytes (MN) by inhibitors of TGF-ß signalling was examined. Smad3 siRNA effectively inhibited TGF-ß-induced urokinase plasminogen activator receptor (uPAR). Agents known to interfere with TGF-ß signalling, including the Smad inhibitors SIS3 and erythromycin derivatives, and ALK5 receptor inhibitor (SB 431542) in inhibition of uPAR expression in response to Mycobacterium tuberculosis (MTB) were examined. Inhibition by SIS3 only inhibited uPAR mRNA significantly. SIS3 may prove to be an effective adjunct to TB therapy.

Inactivation of plasminogen activator inhibitor type 1 by activated factor XII plays a role in the enhancement of fibrinolysis by contact factors in-vitro.

AIMS: Several activated coagulation factors have been reported to enhance fibrinolysis by inactivating plasminogen activator inhibitor type 1 (PAI-1), a serine protease inhibitor. We analyzed the interaction between PAI-1 and the three serine proteases generated during contact activation of plasma, activated factor XII (FXIIa), FXIa, and kallikrein, and evaluated their effects on fibrinolysis in-vitro. MAIN METHODS: Effects of kaolin on euglobulin clot lysis time (ECLT) and behavior of PAI-1 in factor-depleted plasma were analyzed. KEY FINDINGS: The ECLT of pooled plasma obtained from normal volunteers (designated as 100%) was shortened to 62.1+/-3.1% by Ca(2+) (5 mM) and 29.9+/-3.1% by kaolin. Activated protein C reversed the ECLT shortened by Ca(2+)-supplementation (86.3+/-17.4%), but did not affect the ECLT shortened by kaolin (31.4+/-2.1%). Thus, in contrary to Ca(2+)-supplementation, kaolin appeared to shorten the ECLT by a mechanism independent of thrombin generation. In three kinds of contact factor-depleted plasma, kaolin did not shorten ECLT only in FXII-depleted plasma. PAI-1 was cleaved to its inactive form in the Ca(2+) as well as the kaolin-supplemented euglobulin fraction in normal plasma, the latter of which, however, was not observed in FXII-depleted plasma. Similarly, a high molecular weight complex between FXIIa and PAI-1, as well as a cleaved form of PAI-1, was observed in kaolin-supplemented normal plasma, but neither was found in kaolin-supplemented FXII-depleted plasma. SIGNIFICANCE: PAI-1 inactivation by FXIIa appears to be a mechanism by which contact phase coagulation factors enhance fibrinolysis independently of thrombin generation.

The concentration of u-PA and PAI-1 antigen in tissue extracts of nasopharyngeal carcinoma.

We measured the antigen levels of urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor-1 (PAI-1) in tissue extracts from nasopharyngeal carcinomas. An increase in u-PA antigen was observed with the advanced stages of disease. However, the levels of PAI-1 antigen decreased with each advanced stage. These results suggest that local administration of antiplasminic agents may be effective in suppressing tumor invasion.

Familial idiopathic osteonecrosis mediated by familial hypofibrinolysis with high levels of plasminogen activator inhibitor.

Familial hypofibrinolysis with 3 generation, autosomal dominant, very high levels of plasminogen activator inhibitor activity (PAI-Fx) and antigen (PAI-Ag) was etiologically associated with bilateral idiopathic osteonecrosis in 2 brothers. They, their mother, 2 brothers, sister, and all 4 of her children (none of whom had yet developed osteonecrosis), all had very high PAI and could not elevate tissue plasminogen activator after 10 minutes of venous occlusion at 100 mmHg. Familial high PAI levels with concurrent hypofibrinolysis co-segregated with familial combined hyperlipidemia, both being independent risk factors for premature coronary heart disease. If thrombi block venous drainage in the femur, familial hypofibrinolysis mediated by familial high PAI with inability to lyse thrombi would contribute to venous hypertension of bone, bone anoxia, and bone death characteristic of osteonecrosis.

Enhancement of spontaneous fibrinolytic activity in diabetic retinopathy.

The fibrinolytic system was studied in 96 patients with type I diabetes mellitus. Patients were grouped according to their degree of retinopathy; 38 patients with no evidence of retinopathy, 28 patients with background retinopathy and 30 patients with proliferative retinopathy. Thirty healthy individuals served as controls. The basal fibrinolytic activity as measured by clot lysis time and t-PA activity was increased in diabetic patients. This was associated with low levels of plasminogen activator inhibitor. Increased levels of D-dimer in diabetic patients further indicate enhanced in vivo fibrinolysis. The increase in fibrinolytic activity was highest in diabetics without retinopathy, and decreased with increasing retinopathy. Endothelial release of t-PA after venous occlusion was not different between controls and all diabetic groups. These findings suggest that in type I diabetics the fibrinolytic system is in an activated state. With worsening of retinopathy this increase in fibrinolytic activity diminishes.

Discovery of a genetic polymorphism of human plasma protein C inhibitor (PCI): genetic survey utilizing isoelectric focusing followed by immunoblotting, immunological and biochemical characterization.

The objectives of this study were to determine the genetic basis of the electrophoretic differences of human plasma protein C inhibitors (PCI) from 977 individuals. Three discrete antibodies were produced against the PCI purified from human plasma and peptides that corresponded to the N-terminal 15 amino acid residues and the C-terminal 15 residues of human PCI, the chemical structures of which were determined by cDNA sequence analysis. The combined techniques of polyacrylamide gel isoelectric focusing and immunoblotting with these three different antibodies resolved the plasma PCI into several isoprotein bands, with a pH range of 6-7. These PCI isoproteins, however, were not stained by anti-human kallikrein, anti-human protein C or anti-human urokinase antibodies. Therefore, each of the PCI bands, which were detected by immunoblotting with the anti-PCI antibody and the two different anti-peptide antibodies, were derived from free PCI, and not an inactive PCI species. Two common phenotypes, designated PCI 1 and 1-2, were recognized, and family studies showed that they represented homozygosity or heterozygosity for two autosomal codominant alleles, PCI*1 and PCI*2. A population study of plasma samples collected from 977 Japanese individuals indicated that the frequencies of the PCI*1 and PCI*2 alleles were 0.988 and 0.012, respectively.

Urokinase expression in mononuclear phagocytes: cytokine-specific modulation by interferon-gamma and tumor necrosis factor-alpha.

This study delineates the regulatory effects of inflammatory cytokines on mononuclear phagocyte plasminogen activator (PA) activity. The mechanisms by which mononuclear phagocytes modulate PA activity are described. Mononuclear phagocytes regulate net PA activity by the balanced expression of urokinase-type PA (uPA), in either secreted or membrane-associated forms, and a specific plasminogen activator inhibitor, PAI-2. Therefore, understanding how immunomodulators regulate macrophage PA activity requires that the comparative effects of uPA and PAI-2 be elucidated. We determine how recombinant interferon-gamma (IFN) and tumor necrosis factor-alpha (TNF) regulate plasminogen activation in monoblast-like U937 cells and normal human monocytes. In U937 cells, both IFN and TNF induced concurrent increases in secreted PA and PA inhibitor activities. These effects were accompanied by increased immunoreactive uPA and PAI-2 in conditioned media (enzyme-linked immunosorbent assay) and steady-state levels of cellular uPA and PAI-2 mRNA (Northern analysis). To determine the relative abilities of IFN and TNF to either promote or inhibit plasmin generation, we directly compared the effects IFN and TNF, using optimal stimulating concentrations. IFN induced PA activity to 180% of the level achieved by TNF. In contrast, IFN elicited only 78% of the PA inhibitor produced by TNF stimulation. These differences in secreted activity can be explained by the shift in balance between uPA and PAI-2 proteins. Immunoreactive uPA was induced equally by IFN and TNF, but TNF generated higher levels of PAI-2. The same overall pattern of results was seen in normal human monocytes. IFN and TNF differ greatly in the ability to augment receptor-bound PA activity in U937 cells, as IFN induced a twofold increase but TNF had no effect. We conclude that IFN and TNF modulate mononuclear phagocyte proteolytic activity through coordinate regulation of secreted and receptor-bound uPA, balanced against concurrent expression of PAI-2. These effects are cytokine specific, as IFN is superior to TNF in stimulating expression of both secreted and receptor-associated PA activities. These properties suggest mechanisms by which mononuclear phagocytes control proteolysis in cytokine-rich inflammatory foci.

Inhibition of plasminogen activator inhibitor-1 activity results in promotion of endogenous thrombolysis and inhibition of thrombus extension in models of experimental thrombosis.

BACKGROUND: We investigated the effect of inhibition of plasminogen activator inhibitor-1 (PAI-1) activity by a murine monoclonal anti-human PAI-1 antibody (MAI-12) on in vitro thrombolysis and on in vivo thrombolysis and thrombus extension in an experimental animal model for thrombosis. METHODS AND RESULTS: Thrombolysis, mediated by recombinant tissue-type plasminogen activator (rt-PA), was studied in an in vitro system consisting of fibrinogen, plasminogen, and thrombin. Addition of purified platelets during clot formation inhibited rt-PA-mediated thrombolysis in a dose-dependent manner. Platelet-dependent thrombolysis resistance could be completely neutralized by the monoclonal antibody MAI-12, supporting the notion that the observed resistance is due to PAI-1 released from alpha-granules of platelets. Subsequently, we monitored in vivo thrombolysis and thrombus extension of human whole blood thrombi that were allowed to form in rabbit jugular veins. During thrombus formation, either MAI-12 or an irrelevant antibody was incorporated. Thrombolysis and thrombus extension were simultaneously measured during endogenous thrombolysis or upon administration of different dosages of rt-PA. We observed that endogenous thrombolysis was enhanced in the presence of MAI-12 compared with the control antibody. Significantly, thrombus extension was partially prevented by MAI-12 and not by the control antibody irrespective of whether exogenous rt-PA was systematically administered. CONCLUSIONS: These observations further confirm the essential role of PAI-1 in the regulation of the thrombolytic system and support the exploration of adjunctive therapy based on inhibition of PAI-1 activity in thrombolytic strategies.

Effects of defibrotide on fibrinolytic activity in diabetic patients with stable angina pectoris.

18 type II diabetes mellitus patients with coronary artery disease (CAD) have been studied. Tissue plasminogen activator (t-PA) antigen and activity, plasminogen activator inhibitor (PAI) antigen and activity, thrombin-antithrombin III (TAT) complexes were determined in blood samples. Diabetic CAD patients showed higher TAT levels with clearly increased PAI levels whereas t-PA levels levels were similar in patients and controls. Long term defibrotide treatment induced marked changes in fibrinolytic parameters of these diabetic patients with CAD with increased t-PA activity, that could be related to an evident reduction of PAI antigen and activity. Drugs able to modulate PAI activity may be useful in clinical conditions at high risk of thrombotic vascular complications like diabetics with stable angina.

Imbalance of plasminogen activators and their inhibitors in human colorectal neoplasia. Implications of urokinase in colorectal carcinogenesis.

Neoplastic growth and metastatic spread of adenocarcinomas is characterized by a marked increase of urokinase-type plasminogen activator (u-PA) and a decrease of tissue-type plasminogen activator (t-PA). In this study, the authors determined the activity and antigen levels of u-PA and t-PA, and their inhibitors, plasminogen-activator inhibitors types 1 and 2 (PAI-1 and PAI-2), in normal mucosa, adenomatous polyps, and adenocarcinomas of the human colon. The decrease in t-PA activity in the neoplastic tissues, determined enzymatically and zymographically, was significantly correlated with an increase in PAI-1 and PAI-2, in particular in carcinomas. In spite of significantly higher inhibitor levels in the neoplastic tissues, u-PA was found to be increased as well, both in antigen level and in activity. The authors conclude that PAI-1 and PAI-2 are significantly increased in neoplastic tissue of the human colon and contribute considerably to the decrease of t-PA activity in carcinomas. However, the malignancy-associated increase in u-PA seems not to be affected by the plasminogen activator inhibitors. Thus, it appears that there is an imbalance between plasminogen activators and their inhibitors in colonic neoplasia in favor of u-PA, which may contribute to plasmin-mediated growth, invasiveness, and metastasis. This feature was also noticed in adenomatous polyps, supporting the malignant potency of adenomas.

Thrombin neutralizes plasminogen activator inhibitor 1 (PAI-1) that is complexed with vitronectin in the endothelial cell matrix.

Vitronectin endows plasminogen activator inhibitor 1 (PAI-1), the fast-acting inhibitor of both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), with additional thrombin inhibitory properties. In view of the apparent association between PAI-1 and vitronectin in the endothelial cell matrix (ECM), we analyzed the interaction between PAI-1 and thrombin in this environment. Upon incubating 125I-labeled alpha-thrombin with endothelial cell matrix (ECM), the protease formed SDS-stable complexes exclusively with PAI-1, with subsequent release of these complexes into the supernatant. Vitronectin was required as a cofactor for the association between PAI-1 and thrombin in ECM. Metabolic labeling of endothelial cell proteins, followed by incubation of ECM with t-PA, u-PA, or thrombin, indicated that all three proteases depleted PAI-1 from ECM by complex formation and proteolytic cleavage. Proteolytically inactive thrombin as well as anticoagulant thrombin, i.e., thrombin in complex with its endothelial cell surface receptor thrombomodulin, did not neutralize PAI-1, emphasizing that the procoagulant moiety of thrombin is required for a functional interaction with PAI-1. A physiological implication of our findings may be related to the mutual neutralization of both PAI-1 and thrombin, providing a new link between plasminogen activation and the coagulation system. Evidence is provided that in ECM, procoagulant thrombin may promote plasminogen activator activity by inactivating PAI-1.

Extracellular and cell-associated localizations of plasminogen activators and plasminogen activator inhibitor-1 in cultured endothelium.

The extracellular localizations of urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), and plasminogen activator inhibitor-1 (PAI-1) were examined in cultured bovine capillary endothelial cells (BCEs) by an immunofluorescence method using BCEs treated with or without saponin and focal contact preparations. The specific immunofluorescence of cell surface uPA showed a patchy or strand-like distribution and was colocalized with vinculin strands indicating that uPA secreted from BCEs was mainly deposited at the cell surface of focal contacts. BCEs at a subconfluent density showed a higher intensity of specific immunofluorescence for uPA than when they were at a confluent density. tPA was observed over the dorsal surface of cultured BCEs and accentuated at their margins, suggesting that tPA was diffusely distributed on the luminal surface of BCEs in vivo. PAI-1 was distributed in the extracellular matrix under cultured BCEs. These findings suggest that uPA and PAI-1 are located under BCEs participating in the regulation of proteolytic activities provoked by plasminogen-PAs-plasmin system in vivo. The localization of tPA appears to be consistent with its function, which is to maintain the fluidity of the blood and to initiate thrombolysis in vivo.

Deep vein thrombosis and fibrinolysis. Defective urokinase type plasminogen activator release.

In the present study 57 consecutive patients with a first episode of venographically proven deep vein thrombosis were investigated to evaluate the release of tissue-type plasminogen activator (t-PA) and of urokinase-type plasminogen activator (u-PA) in response to DDAVP stimulation as well as the resting plasminogen activator inhibitor (PAI) concentration, comparing this to the results obtained in 66 similar patients with a clinical suspicion of thrombosis but with a normal venogram. All assays were performed without knowledge of the patient's status. Four patients in the deep vein thrombosis-group (7%) had an absent u-PA antigen response upon DDAVP infusion, while a normal response was observed in all control subjects. Patients and controls showed similar increases in t-PA antigen level upon DDAVP. High resting PAI antigen levels were encountered in 5 patients in the deep vein thrombosis-group (9%) and in 6 subjects in the control group (9%). The results from this controlled study indicate that a defective release of u-PA may occur in patients with deep vein thrombosis and may have pathogenetic significance. Furthermore it is concluded that elevation of PAI levels cannot be considered as a specific risk factor for venous thrombosis.

PAI-1 plays an important role in the expression of t-PA activity in the euglobulin clot lysis by controlling the concentration of free t-PA.

The antigen levels of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) were assayed in the plasma and in the euglobulin fraction, and their contributions to the euglobulin clot lysis time (ECLT) and t-PA activity were analyzed. Total and free PAI-1 levels in both fractions showed significant positive correlation with ECLT (p less than 0.001), whereas t-PA antigen level did not have a high correlation coefficient with ECLT. t-PA activity showed significant negative correlation with ECLT (p less than 0.001) and positive correlation with free t-PA level (p less than 0.001), which was calculated by the ratio of the concentrations of t-PA-PAI-1 complex and the free PAI-1. Thus free t-PA seems to dissolve the euglobulin clot and its concentration seems to be controlled by the concentration of free PAI-1. These findings were confirmed by the analyses of the effects of C1-inactivator and antibody against t-PA to regular ECLT and kaolin activated ECLT, the latter of which was only inhibited by the addition of C1-inactivator whereas the former was inhibited by anti-t-PA antibody.

Inverse relationship between cortisol excretion rate and plasminogen activator inhibitor-1 (PAI-1) antigen and activity in premenopausal obese women.

The present study was undertaken in order to investigate the possibility of a relationship between plasminogen activator inhibitor-1 (PAI-1) and cortisol excretion rate in 15 obese women. We found a highly significant linear inverse correlation between cortisol excretion rate and both PAI-1 antigen (r = 0.79, P less than 0.001) and activity (r = 0.77, P less than 0.001). In addition, stepwise regression analysis showed that cortisol excretion rate maintained a strong negative relationship with PAI-1 antigen (significance level 0.03) and activity (significance level 0.003), when adjusted for other variables taken in examination (waist to hip ratio, body mass index, insulin, DHEAS, age). Even though this study only demonstrates a negative correlation, the possibility of a direct inhibitory effect of cortisol on PAI-1 production should be considered. In conclusion, the present study demonstrates an inverse correlation between cortisol excretion rate and PAI-1 antigen and activity, suggesting a possible role for cortisol in protecting obese women from reduced fibrinolytic activity.

A kinetic model of the circulatory regulation of tissue plasminogen activator.

A computer simulation was developed to study the regulation of active tissue plasminogen activator (t-PA) levels in plasma by kinetically modeling t-PA secretion, t-PA inhibition by plasminogen activator inhibitor type 1 (PAI-1), and hepatic clearance of t-PA, PAI-1 and t-PA/PAI-1 complex throughout a simplified human circulatory system. The model indicates that as the active PAI-1 concentration increases, the percent of t-PA in the active form decreases exponentially. Further, the reaction between t-PA and PAI-1 substantially reduces the half-lives of both active factors. By adjusting the t-PA and PAI-1 secretion rates to provide the best fit between simulated and measured circadian variations in t-PA, PAI-1 and complex, the model predicts that the diurnal rhythm in active t-PA levels is principally due to changes in the rate of PAI-1 secretion and not to variations in the t-PA secretion rate. In conclusion, the model predicts that PAI-1 is an important regulator of the concentration, half-life and circadian variation of active t-PA.

Standard heparin but not LMW heparin induces a concomitant increase of t-PA and PAI-1 without modification of global fibrinolysis: study after 60 days treatment.

The relationship between heparin and fibrinolysis is strongly suggested. We have studied the influence on fibrinolysis of standard heparin (SH), Calciparin and low molecular weight heparin (LMWH), Fraxiparin, given preventively in an elderly population. Patients were randomized into two groups (SH, LMWH). We investigated fibrinolytic parameters (ECLT, t-PA antigen, PAI-1 activity and antigen, t-PA/PAI-1 complexes) before treatment (D0) and at D30 and D60, before and after venous occlusion (VO). Values at D0, D30 and D60 were compared within each group. A significant and marked increase in t-PA and t-PA/PAI-1 complexes at D30 and D60 before and after VO was noted only in the SH group. The mechanism and clinical relevance of this increase remains to be established.

Immunological and molecular characterization of plasminogen activator inhibitors 1 and 2 in baboon (Papio anubis) placental tissues.

Two major plasminogen activator inhibitors (PAI-1 and PAI-2) increase in the peripheral circulation during pregnancy in humans. PAI-1 is of vascular endothelial origin whereas PAI-2 is produced primarily by human placental tissues. This study was undertaken to determine a) if PAI-1 and PAI-2 are also present in the baboon and b) their association with pregnancy. Citrated plasma was obtained from pregnant baboons sequentially at 15 +/- 3-day intervals between Days 30 and 140 of pregnancy. PAI activity increased significantly (p less than 0.05) at Day 120 (15.3 IU/ml) and 140 (21.8 IU/ml) of gestation and returned to baseline (2.6 IU/ml) 48 h post cesarean section. Placental tissues obtained at cesarean section during the third trimester were either placed in explant culture, fixed for immunocytochemistry, or frozen for RNA extraction. Western blot analysis of tissue culture media (TCM) indicated that the polyclonal antibody to PAI-1 reacted with a major band (Mr 47 000) in TCM from placental tissues while the PAI-2 antibody reacted primarily with a doublet (Mr 67 000 and 69 000) in these same media. PAI-1 was immunocytochemically localized primarily in the chorioamniotic tissue (CAM-D) and PAI-2 was found predominantly in placental villi. Slot blot hybridization with cDNAs to PAI-1 and PAI-2 indicated that the mRNA for PAI-2 was found primarily in placental villi, whereas the mRNA for PAI-1 was present in all three tissue compartments.(ABSTRACT TRUNCATED AT 250 WORDS)

Identity between the placental protein PP10 and the specific plasminogen activator inhibitor of placental type PAI-2.

The highly specific plasminogen activator inhibitor of placental type, PAI-2, occurs in the placenta in a low molecular mass form of 46.6 kDa, and in pregnancy plasma in a (possibly glycosylated) high molecular mass form of 60 kDa. Extensive knowledge is available about the functional properties of PAI-2 as a plasminogen activator inhibitor and about its molecular biology and regulation. Of the several placenta proteins (PP) isolated, one of them, PP10, has a molecular mass of 48 kDa and its occurrence in malignancy and in complications during pregnancy has been the topic of a number of studies, though its properties and physiological significance are unknown. The present findings constitute evidence of immunological identity between PP10 and PAI-2. The sections of the amino acid sequence of PP10 analysed here were found to have identical counterparts in the sequence of the low molecular mass form of PA1-2, but in several preparations PP10 was found to occur in an inactive two-chain form due to cleavage of an Arg-Thr bond, the two peptide chains being linked to each other by a disulphide bridge. The cleavage site is identical to that observed in the reaction between PAI-2 and urokinase. The results make it possible to coordinate and correlate the findings of many separate studies and our own observations on PP10 and PAI-2.

Plasminogen activator inhibitor-1 is induced in microvascular endothelial cells by a chondrocyte-derived transforming growth factor-beta.

We have previously demonstrated that a chondrocyte-derived TGF-beta inhibits spontaneous endothelial sprout formation in an in vitro model of angiogenesis (Pepper et al., J.Cell.Physiol. 146:170-179, 1991). We suggested that the inhibition might be mediated by an antiproteolytic effect. In this paper, we describe the induction of PAI-1 and PAI-1 mRNA in microvascular endothelial cells by chondrocyte conditioned medium. This effect can be significantly reduced by the addition of anti-TGF-beta antibodies to the conditioned medium, and can be mimicked by the addition of exogenous TGF-beta 1. Taken together with our previous observations, these results demonstrate that the inhibition of endothelial sprout formation occurs concomitantly with an increase in the production of PAI-1, a physiological plasminogen activator inhibitor. This suggests that TGF-beta-induced antiproteolysis is responsible for the inhibition of sprout formation.