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.

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.

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.

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.

Tissue plasminogen activator inhibition in diabetes mellitus.

Depression of fibrinolysis may be relevant to the vascular complications of diabetes mellitus. Plasminogen activator inhibitor (PAI) is an important inhibitor of fibrinolysis in humans, and we have found basal activities of PA inhibition to be elevated in patients with diabetes compared with a reference group of healthy subjects (mean +/- SD 268 +/- 268 vs. 105 +/- 48%; P less than .0001). With a monoclonal antibody, it was shown that high inhibition values were due to PAI-1. No differences in PA inhibition were noted in relation to type of diabetes, diabetic treatment, or presence or absence of vascular complications. Basal PA inhibition did not correlate with in vivo (B beta 15-42 antigen) or ex vivo (fibrin plate) fibrinolytic activity or HbA1. In patients with non-insulin-dependent diabetes mellitus, treatment with the anabolic steroid stanozolol significantly reduced PA inhibition. These findings suggest a further abnormality of fibrinolysis in diabetes, but the lack of a relationship among other measures of fibrinolysis renders its biologic significance uncertain.

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.

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.

Stability of plasminogen activator inhibitor 1 (PAI-1).

The stability of PAI-activity has been studied at different conditions. The inactivation followed first order kinetics. Lowering the temperature and decreasing the pH both, increased the stability of PAI-1 dramatically. Addition of the PAI-1 binding protein, vitronectin, to reactivated PAI-1, about doubled the half-life of PAI-1 at all conditions studied. In the presence of chloramine T, the inactivation of reactivated PAI-1 was very rapid. In this case the protective effect of purified vitronectin, human plasma or fetal calf serum, but not of bovine serum albumin, was pronounced. The stability of the spontaneously active high Mr form of PAI-1 (partially purified or in plasma), constituting a complex between PAI-1 and vitronectin, was quite similar to reactivated PAI-1 in the presence of vitronectin. Addition of pure vitronectin, human plasma or fetal calf serum to such material had no further stabilizing effect. Reactivated PAI-1, which was inactivated by incubation at physiological conditions could again be fully reactivated, in contrast to chloramine T-oxidized PAI-1, which was irreversibly inactivated.

Immunoreactivity of tissue plasminogen activator and of its inhibitor complexes. Biochemical and multicenter validation of a two site immunosorbent assay.

An enzyme linked immunosorbent assay (ELISA) based on goat polyclonal antibodies against human tissue plasminogen activator (tPA) was evaluated. The relative immunoreactivity of tPA in free form and tPA in complex with inhibitors was estimated by ELISA and found to be 100, 74, 94, 92 and 81% for free tPA and tPA in complex with PAI-1, PAI-2, alpha 2-antiplasmin and C1-inhibitor, respectively. Addition of tPA to PAI-1 rich plasma resulted in rapid and total loss of tPA activity without detectable loss of ELISA response, indicating an immunoreactivity of tPA in tPA/PAI-1 complex of about 100%. Three different treatments of citrated plasma samples (acidification/reneutralization, addition of 5 mM EDTA or of 0.5 M lysine) prior to determination by ELISA all resulted in increased tPA levels. The fact that the increase was equally large in all three cases along with good analytical recovery of tPA added to plasma, supported the notion that all tPA antigen present in plasma samples is measured by the ELISA. Analysis by ELISA of fractions obtained by gel filtration of plasma from a patient undergoing tPA treatment identified tPA/inhibitor complexes and free tPA but no low molecular weight degradation products of tPA. Determinations of tPA antigen were made at seven French clinical laboratories on coded and randomized plasma samples with known tPA antigen content. For undiluted samples there was no significant difference between the tPA levels found and those known to be present. The between-assay coefficient of variation was 7 to 10%. In conclusion, the ELISA appeared suited for determination of total tPA antigen in human plasma samples.

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.

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.

Characterization of monoclonal antibodies against human tissue plasminogen activator (tPA): quantitation of free tPA in human cell cultures by an ELISA.

Seven murine monoclonal antibodies produced against tissue plasminogen activator (tPA) were evaluated by means of enzyme-linked immunosorbent assays (ELISAs), and their effects on the enzymatic activities of tPA towards a synthetic substrate (S-2288) and plasminogen were investigated. One of the antibodies, TPA1-70, strongly inhibited the enzymatic activity of tPA in a fibrin agarose plate assay, while it did not affect the enzymatic activity towards the synthetic substrate or plasminogen. The antibody is directed to an epitope on the B-chain of tPA, which is necessary for the formation of a ternary complex of tPA, fibrin and plasminogen, but probably not to the active site. Another antibody, TPA2-14, partially inhibited the enzymatic activities of tPA towards the synthetic substrate and plasminogen, but it was not able to bind to the inactive tPA complexed with plasminogen activator inhibitor-1 (PAI-1). The antibody is directed to an epitope on the second kringle region, which is probably one of the PAI-1 binding sites. This property of the antibody enabled us to develop an ELISA for selective quantitation of free tPA in culture media conditioned with several human cell lines. The results indicate that tPA in these media exists either partially or almost entirely in a complex with PAI-1.

Fluctuation of TPA and PAI-1 antigen levels in plasma: difference of their fluctuation patterns between male and female.

The daytime fluctuation of the fibrinolytic activity and the antigen levels of tissue plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI-1) in plasma were analyzed in normal male and female volunteers. Samples were obtained at 8:30, 10:30, 12:30, 14:30 and 16:30 h. Euglobulin clot lysis time (ECLT) showed the longest time at 8:30 to 10:30 h and the shortest time at 16:30 h. The highest level of tPA was obtained at 8:30 h and the lowest at 14:30 h in men, whereas the highest value was at 8:30 h and the lowest was at 16:30 h in females. Active free PAI-1 showed highest value at 10:30 h in men and at 8:30 h in women. The lowest values obtained at 16:30 h in both men and women and were about one third of the highest values. ECLT was always shorter in females than in males and parameter such as tPA and PAI-1 were also lower in females than in males. The phase of the circadian rhythm of the fibrinolytic parameters may be advanced in females than in males.

Plasma levels of t-PA free PAI-1 and a complex of t-PA with PAI-1 in human males and females at various ages.

The concentration of tissue plasminogen activator (t-PA), plasminogen activator inhibitor 1 (PAI-1) and the complex of t-PA and PAI-1 (t-PA-PAI-1 complex) were measured using an enzyme immunoassay, where the first antibody was monoclonal antibody against PAI-1 and the second one was polyclonal anti-t-PA Fab' antibody. Plasma levels of t-PA and t-PA-PAI-1 complex increased with increase in age in the total population of males and females, but there was no age related change in free and total PAI-1. Plasma levels of t-PA antigen were higher in males than in females, and those in females gradually increased with increase in age, reaching the same levels as those of males at their 60s. Plasma levels of t-PA-PAI-1 complex were also higher in males than in females, but the difference was low at their 50s and no difference was observed at their 60s. Plasma levels of free PAI-1 were higher in males than in females, but those of males declined at age group higher than their 50s. At their 60s plasma levels of free PAI-1 were higher in females than in males. These results indicate that fibrinolytic parameters such as t-PA and PAI-1 were balanced at higher levels in plasma in males than in females.

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.

Dexamethasone increases the release of three 44 kD proteins immunologically related to plasminogen activator inhibitor-1 from human umbilical vein endothelial and rabbit coronary microvessel endothelial cells.

Endothelial cells synthesize and release different proteins involved in their function, and some of these proteins may play important roles in the cellular response to injury, infection, or glucocorticoids. We have examined the profile of proteins released from rabbit coronary microvascular endothelial (RCME) and human umbilical vein endothelial (HUVE) cells using two-dimensional polyacrylamide gel electrophoresis. The production of three anionic 44kD proteins was increased in RCME and HUVE-cell conditioned medium after treatment with dexamethasone, endotoxin or hypoxia-reoxygenation. The three 44 kD proteins were recognized by antisera raised against endothelial type plasminogen activator inhibitor (PAI-1). Dexamethasone treatment of HUVE and RCME cells reduced cellular and secreted plasminogen activator activity, but no significant effect of dexamethasone on PAI-1 activity in conditioned media could be demonstrated. These observations suggest that although the 44Kd proteins exhibit immunoreactivity with PAI-1 antisera, these proteins are most likely inactive forms of PAI-1.

Heparin cofactor II inhibits thrombin-stimulated release of tissue plasminogen activator from cultured human endothelial cells in the presence of dermatan sulfate.

To investigate the involvement of heparin cofactor II (HC II) in fibrinolytic system, endothelial cells from human umbilical vein were cultured in the presence of HC II or antithrombin III (AT III) combined with or without thrombin. Although AT III significantly inhibited thrombin-induced increase in tissue plasminogen activator antigen (t-PA:Ag) release, HC II did not exhibit such a suppressive effect. In contrast, in the presence of dermatan sulfate, HC II inhibited thrombin stimulation of t-PA:Ag release more strongly than AT III did. The release of plasminogen activator inhibitor-1 antigen (PAI-1:Ag) was also stimulated by thrombin; this stimulation was inhibited only by the combination of HC II and dermatan sulfate. Comparatively high concentrations of HC II significantly suppressed thrombin stimulation of t-PA:Ag and PAI-1:Ag release but did not cause an obvious change of both release in the absence of thrombin. Based on these results, it was suggested that HC II may inhibit an increase in fibrinolytic activity mediated by thrombin-stimulated endothelial cells in the liquid phase through a suppression of thrombin stimulation of t-PA:Ag release, when plasma is exposed to vascular smooth muscle cells or fibroblasts which synthesize a significant amount of dermatan sulfate.

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.

Dexamethasone-induced plasminogen activator inhibitor: characterization, purification, and preparation of monoclonal antibodies.

The effects of dexamethasone on protein synthesis were studied in human fibrosarcoma (HT-1080) cells. Dexamethasone induced a new protein of 46 kD which was rapidly secreted into the medium, while neither progesterone nor estradiol would induce the synthesis of this protein and only a small increase in its amount could be seen in the presence of testosterone. The 46 kD protein was partially purified by ammonium sulfate precipitation and gel filtration and mouse monoclonal antibodies to it were produced in mouse hybrid cells. Altogether 13 positive clones were found, of which six reacted only with native and seven reacted with the unreduced 46 kD protein in Western blotting. It was possible by using polyclonal antibodies to plasminogen activator inhibitor type I (PAI-1) and purified plasminogen activator inhibitor type I to confirm that the 46 kD protein purified and characterized here was PAI-1. In addition, the 46 kD protein clearly inhibited plasminogen activation, thus further confirming that protein isolated was an inhibitor of plasminogen activator. Since the induction of PAI-1 by dexamethasone was very extensive, it is possible that glucocorticoids regulate proteolysis and fibrinolysis in vivo by increasing the amount of the inhibitor of plasminogen activator and thus preventing the activation of plasminogen to plasmin. The reduction of activation of plasminogen to plasmin by glucocorticoid-induced inhibitor could be of great importance, e.g., in various blistering diseases, in metastases from malignant cells, and in the migration of inflammatory cells.