Tissue plasminogen activator and plasminogen activator inhibitor-1 levels in patients with acute paraquat intoxication

To investigate the effects of reactive oxygen species (ROS) on tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) plasma levels, and their possible implications on clinical outcome, we measured tPA and PAI-1 levels in 101 patients with acute paraquat (PQ) intoxication. The control group consisted of patients who ingested non-PQ pesticides during the same period. tPA and PAI-1 levels were higher in the PQ group than in the controls. PQ levels were significantly correlated with ingested amount, timelag to hospital, tPA level, and hospitalization duration. tPA levels were correlated with PAI-1, fibrin degradation product (FDP), and D-dimer. D-dimer levels were lower in the PQ group than in the controls. Univariate analysis indicated the following significant determinants of death: age, ingested amount, PQ level, timelag to hospital, serum creatinine, lipase, pH, pCO(2), HCO(3) (-), WBC, FDP, PAI-1, and tPA. However, multivariate analysis indicated that only PQ level was significant independent factor predicting death. In conclusion, tPA and PAI-1 levels were higher, while D-dimer levels were lower in the PQ group than in the controls, implying that ROS stimulate tPA and PAI-1, but PAI-1 activity overrides tPA activity in this setting. Decreased fibrinolytic activity appears to be one of the clinical characteristics of acute PQ intoxication.     

Increased expression of plasminogen activator inhibitor-1 in cardiomyocytes contributes to cardiac fibrosis after myocardial infarction

Plasminogen activator inhibitor-1 (PAI-1) plays a critical role in tissue fibrosis by inactivating matrix metalloproteinases, which might effect on the progression of left ventricular dysfunction. However, little has been known about the expression of PAI-1 during cardiac remodeling. We used a mouse model of myocardial infarction (MI) by coronary ligation, in which the progression of left ventricular remodeling was confirmed by echocardiography. Histological examination showed that interstitial and perivascular fibrosis progressed in the post-MI (PMI) heart at 4 weeks after the procedure. We observed the dramatic induction of cardiac PAI-1 mRNA and PAI-1 antigen in plasma in the PMI mice, as compared with the sham-operated (sham) mice. In situ hybridization analysis demonstrated that strong signals for PAI-1 mRNA were localized to cardiomyocytes in the border of infarct area and around fibrous lesions, and to perivascular mononuclear cells, which seemed to be mast cells, only in hearts of the PMI mice. Importantly, less development of cardiac fibrosis after MI was observed in mice deficient in PAI-1 as compared to wild-type mice. The mRNA expression of cytokines, transforming growth factor-beta, and tumor necrosis factor-alpha, was also increased in hearts of the PMI mice, but not in the sham mice. These observations suggest that cardiomyocytes and mast cells contribute to the increased PAI-1 expression, resulting in the development of interstitial and perivascular fibrosis in the PMI heart, and that the regional induction of cytokines may be involved in this process.     

Tissue plasminogen activator and plasminogen activator inhibitor-1 in human choledochal bile

Fibrinolytic properties have been detected in animal and human gallbladder (GB) bile. Plasminogen activator inhibitor-1 (PAI-1) has been reported in greater concentration in GB stone bile and may be a nucleating factor in the pathogenesis of GB stone formation. It is unknown whether or not human choledochal bile has similar properties, which could have a role in choledocholithiasis. The aims of this study were to determine the presence of fibrinolytic properties of human choledochal bile and to compare those properties among normal, acalculous, and calculous-infected choledochal bile. Tissue plasminogen activator (t-PA) and PAI-1 of choledochal bile were measured by enzyme linked immunosorbent assay in patients with cholangitis due to acalculous bile duct obstructions (n = 9), choledocholithiasis with cholangitis (n = 20), and normal bile (n = 7). The t-PA concentration of choledochal bile was no different among the three groups (acalculous-infected bile, median 4.61 ng/ml, and calculous-infected bile, 4.61 ng/ml, versus normal bile, 7.33 ng/ml). PAI-1 was detected in choledochal bile in significantly greater concentrations in patients with acalculous cholangitis due to bile duct obstructions and choledocholithiasis with cholangitis (acalculous-infected bile, median 0.36 ng/ml, and calculous-infected bile, 0.1 ng/ml, versus normal bile, 0.02 ng/ml, p < 0.05), but the bile concentration of PAI-1 was no different between the acalculous and calculous-infected choledochal bile. Human choledochal bile possesses t-PA and PAI-1. PAI-1 was present in greater concentrations in both acalculous and calculous-infected choledochal bile. Increased levels of PAI-1 may be an epiphenomenon of cholangitis rather than a factor in the pathogenesis of choledocholithiasis.     

Acute psychological stress decreases plasma tissue plasminogen activator (tPA) and tissue plasminogen activator/plasminogen activator inhibitor-1 (tPA/PAI-1) complexes in cardiac patients

Tissue plasminogen activator (tPA) promotes fibrinolysis, and impaired fibrinolysis is associated with atherosclerosis and thrombosis. Plasminogen activator inhibitor-1 (PAI-1) inhibits t-PA expression. The effects of acute laboratory stressors on tPA and tPA/PAI-1 complexes were assessed in a sample of 11 cardiac patients. Participants were randomly assigned to either a stress or relaxation condition at time 1, and the alternative condition at time 2. Blood samples were taken before (pre) and after (post) each session and participants completed a battery of psychological questionnaires. Two-way repeated-measures analysis of variance revealed a statistically significant decrease in tPA (P=0.01) and tPA-PAI-1 complexes (P=0.04) during the mental stress condition. Anger-in had a strong relationship to decreases in tPA/PAI-1 levels in the stress condition (r=0.68, P?<?0.05). Relaxation had no significant effect on tPA and tPA/PAI-1 levels. These data suggest that decreased fibrinolysis mediates the relationship between mental stress and atherosclerosis.     

Treatment of acute myocardial infarction with recombinant tissue-type plasminogen activator

In Ireland, to date, coronary thrombolytic therapy has been confined almost exclusively to the use of streptokinase. However, a large body of evidence suggests that, in comparison to streptokinase, the agent recombinant tissue-type plasminogen activator (rt-PA) may be more effective in lysing coronary thrombi and achieving coronary reperfusion and causes fewer disturbances of the coagulation system. With these considerations in mind, we undertook a study to explore the future potential role of rt-PA in our particular clinical practice. Sixteen patients presenting to our centre with clinical and ECG features suggestive of acute myocardial infarction were treated with rt-PA and heparin infusion within 3.8 +/- 1.3 (mean +/- SD) [range 0.6 - 5.3] hours of the onset of their symptoms. Reperfusion, as assessed by clinical, electrocardiographic and biochemical criteria, was achieved in 15 of these 16 patients. One patient developed reocclusion that was successfully treated with repeat thrombolytic therapy. Follow up coronary angiography, performed in eight patients, confirmed successful reperfusion in seven. One patient developed an intracranial haemorrhage. The result of this pilot study highlight the importance of considering thrombolytic therapy in all patients presenting with suspected acute myocardial infarction (AMI). Our observations also suggest that rt-PA is very effective in restoring myocardial perfusion in patients with AMI who present at an early stage. As with all thrombolytic agents, it may be associated with haemorrhagic complications. Determination of the precise role of rt-PA, as opposed to other thrombolytic agents, awaits the results of ongoing clinical trials.     

Acute psychological stress decreases plasma tissue plasminogen activator (tPA) and tissue plasminogen activator/plasminogen activator inhibitor-1 (tPA/PAI-1) complexes in cardiac patients

Tissue plasminogen activator (tPA) promotes fibrinolysis, and impaired fibrinolysis is associated with atherosclerosis and thrombosis. Plasminogen activator inhibitor-1 (PAI-1) inhibits t-PA expression. The effects of acute laboratory stressors on tPA and tPA/PAI-1 complexes were assessed in a sample of 11 cardiac patients. Participants were randomly assigned to either a stress or relaxation condition at time 1, and the alternative condition at time 2. Blood samples were taken before (pre) and after (post) each session and participants completed a battery of psychological questionnaires. Two-way repeated-measures analysis of variance revealed a statistically significant decrease in tPA (P = 0.01) and tPA-PAI-1 complexes (P = 0.04) during the mental stress condition. Anger-in had a strong relationship to decreases in tPA/PAI-1 levels in the stress condition (r = 0.68, P < 0.05). Relaxation had no significant effect on tPA and tPA/PAI-1 levels. These data suggest that decreased fibrinolysis mediates the relationship between mental stress and atherosclerosis.     

Increased plasma levels of a rapid inhibitor of tissue plasminogen activator in young survivors of myocardial infarction

Certain risk factors for myocardial infarction have been linked with disturbances in fibrinolytic activity. The recent development in our laboratory of new sensitive and specific methods for determination of tissue plasminogen activator (t-PA) activity and antigen, as well as the discovery of a new rapid inhibitor of this enzyme, enabled us to study fibrinolytic function in detail in a representative population of postinfarction patients. Seventy-one patients (62 men and 9 women) who had survived a myocardial infarction before the age of 45 were compared with 50 healthy subjects of similar age, three years after the infarction. Low t-PA activity after venous occlusion, mostly explained by high plasma levels of the t-PA inhibitor and to some extent by impaired release of t-PA from the vessel wall, was a frequent finding in the patients. The level of t-PA inhibitor was positively and significantly correlated with levels of serum triglycerides. Our data suggest that reduced fibrinolytic capacity due to increased plasma levels of a rapid inhibitor of t-PA may have pathogenetic importance in myocardial infarction, particularly in patients with hypertriglyceridemia.     

Reduction in fibrotic tissue formation in mice genetically deficient in plasminogen activator inhibitor-1

Mice with homozygous deletion of the plasminogen activator inhibitor-1 gene (PAI-1(-/-)) are relatively protected from bleomycin-induced pulmonary fibrosis. At least part of the protective effect appears to occur during the latter stages of the pathological process when fibrotic tissue is being deposited. To investigate the effect of PAI-1 deficiency on fibrosis, we studied the accumulation of fibrotic tissue within subcutaneously implanted polyvinyl alcohol sponges. Similar to the effect of PAI-1 deficiency on bleomycin-induced pulmonary fibrosis, the accumulation of fibrotic tissue within implanted sponges occurred more slowly in PAI-1(-/-) compared to wild-type mice. Another striking difference observed in the PAI-1(-/-) mice was the rapid removal of a fibrin-rich matrix that formed within the sponges by 1 day after implantation in both wild-type and PAI-1(-/-) mice. The pattern of connective tissue invasion also differed: cells in wild-type mice infiltrated as individually penetrating cells whereas in PAI-1(-/-) mice they did so as a well-demarcated advancing front. Providing an alternative provisional matrix by impregnating sponges with a low concentration of collagen before implantation corrected the changes induced by PAI-1 deficiency. In conclusion, PAI-1 deficiency appears to affect fibrotic tissue formation in part by altering the provisional matrix that forms soon after tissue injury.     

Effect of heparin administration on fibrinogenolysis during thrombolytic therapy with tissue plasminogen activator for acute myocardial infarction

Heparin accelerates plasminogen activation by tissue type plasminogen activator (t-PA). Previous investigators have postulated that heparin administration during t-PA therapy might lead to enhanced fibrinogenolysis. In this paper, important coagulation parameters from a randomised trial of early versus delayed heparin administration, during t-PA therapy for acute myocardial infarction, were analysed. In contrast to the prediction, the late heparin group had significantly greater fibrinogen depletion (nadir fibrinogen 1.06±0.65 g/1 vs 1.46±0.61 g/l, p=0.0001) and higher fibrinogen degradation products (peak FBDP 675.9±785.3 μg/ml vs 299.7±543.5 μg/ml, p=0.0001). A possible mechanism for these findings is discussed. It has previously been demonstrated that heparin and FBDP are competitive activators of plasminogen activation by t-PA. It has also been shown that the stimulation of plasminogen activation by FBDP contributes substantially to fibrinogenolysis by t-PA. Thus, heparin may reduce fibrinogenolysis by interfering with the stimulation of plasminogen activation by FBDP.     

Expression of urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor, and plasminogen activator inhibitor-1 and -2 in hepatocellular carcinoma

It has become more and more clear in recent decades that the plasminogen activation system, which includes urokinase-type plasminogen activator (uPA), urokinase-type plasminogen activator receptor (uPAR), plasminogen activator inhibitor (PAI)-1 and PAI-2, plays a very important role in the aggressiveness of cancer. Using immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), the expression of these four components of the uPA system was analyzed in 19 cases of hepatocellular carcinoma (HCC) and 18 cases of the adjacent non-cancer tissues which all had chronic active hepatitis with liver fibrosis or liver cirrhosis. Four cases of normal liver tissues, as controls for immunohistochemical stains, were obtained from the hepatectomized liver of patients with metastatic cancer in the liver. The positive rates of uPA, uPAR, PAI-1 and PAI-2 for immunohistochemical stains in cancer tissues were 78.9, 68.4, 57.9 and 31.6%, respectively. Positive signals were mainly distributed in the cytoplasm of the cancer and in stromal cells. Moreover, the strong stains were chiefly located in the invasive front of the cancer cells. No specific stain was detected in four cases of normal liver tissues. In ELISA, there were significant differences between cancer and non-cancer tissues in concentration of uPA, uPAR and PAI-1 (P < 0.0003, 0.0024 and 0.01, respectively), but there was no significant difference in that of PAI-2 (P = 0.37). These results suggest that uPA, uPAR and PAI-1 are related to invasion of HCC.     

A randomized trial of immediate versus delayed elective angioplasty after intravenous tissue plasminogen activator in acute myocardial infarction

We compared the efficacy of immediate coronary angioplasty after acute myocardial infarction with that of elective angioplasty at 7 to 10 days in patients treated initially with intravenous tissue plasminogen activator. The plasminogen activator (150 mg) was administered 2.95 +/- 1.1 hours after the onset of symptoms, to 386 patients with acute myocardial infarction. Ninety minutes later, patency of the coronary artery serving the area of the infarct was demonstrated by coronary angiography in 288 patients (75 percent). Bleeding problems were frequently encountered, as evidenced by an average drop in hematocrit of 11.7 +/- 6.5 points from base line to nadir and by a need for transfusion not related to bypass surgery in 70 patients (18 percent). After successful thrombolysis, 197 patients with a patent but severely stenotic vessel suitable for angioplasty were randomly assigned to immediate angioplasty (n = 99) or, if indicated 7 to 10 days after infarction, to deferred (elective) angioplasty (n = 98). The incidence of reocclusion was similar in the two groups: 11 percent in the group assigned to immediate angioplasty and 13 percent in the group assigned to elective angioplasty. Neither group had a significant improvement in global left ventricular function, and regional wall motion in the infarct zone improved to a similar extent in the two groups. In the elective-angioplasty group, the rate of crossover to emergency angioplasty for recurrent ischemia was 16 percent (whereas 5 percent of the immediate-angioplasty group required emergency repeated angioplasty; P = 0.01). In 14 percent of the patients in the elective group, the stenosis was substantially reduced by the time of the seven-day follow-up angiography, obviating the need for angioplasty. We conclude that in patients with initially successful thrombolysis and suitable coronary-artery anatomy, immediate angioplasty offers no clear advantage over delayed elective angioplasty.     

Monoclonal antibodies against a recombinant form of plasminogen activator inhibitor-1: Effects on tissue plasminogen activator neutralizing and vitronectin binding properties

A panel of eight murine monoclonal antibodies was produced against a recombinant form of plasminogen activator inhibitor- 1 (rPAI-1). All the antibodies recognized active and latent forms of rPAI-1, and rPAI-1 complexed with tissue plasminogen activator (t-PA) as determined in enzyme-linked immunosorbent assays (ELISA). Three of the antibodies, FAG9, FAD3 and BBH2, were particularly effective at inhibiting the t-PA neutralizing activity of rPAI-1 as measured in a chromogenic assay. Three different antibodies, DD8E9, FEG7 and FGG7, inhibited the binding of [¹²⁵I]rPAI-1 to vitronectin immobilized on polystyrene wells. The combination of FGG7 and alkaline phosphatase-labeled BBH2 resulted in a sensitive sandwich ELISA for rPAI-I with detection limits in the range of 1–10 ng. Definition of epitopes recognized by these antibodies will be useful for identifying various domains on PAI-1 involved in its interaction with protease substrates and with its protein cofactor, vitronectin.     

Circadian fluctuations in plasma levels of tissue plasminogen activator antigen and plasminogen activator inhibitor activity

Daily fluctuations of t-PA antigen and PAM activity were measured in plasma samples of physically active young healthy volunteers (group 1, n = 11; age range 20–38 years) and compared to data obtained from resting patients (group 11, n = 23; age range 44–67 years) suffering from moderate valvular disease without evidence for inflammatory, neoplastic, or thrombosis-related diseases (e.g. deep vein thrombosis, coronary artery disease). t-PA antigen concentration showed a similar diurnal pattern in both study groups with the peak value at 06:00 but was significantly increased in the higher aged group at all collection times. PAM activity had its acrophase in both groups at or around 03:00 but no age-dependent differences could be demonstrated. t-PA antigen as well as PAI-1 activity fluctuations conserved their typical pattern despite differences in physical activity in the study groups.     

Simvastatin inhibits tissue factor and plasminogen activator inhibitor-1 expression of glomerular mesangial cells in hypercholesterolemic rabbits

Tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) activity and/or expression are upregulated in hypercholesterolemia. Despite extensive research on anti-thrombotic effect of statins, little is known about their effects on TF and PAI-1 expression in glomerular mesangial cells under hypercholesterolemic condition. Male rabbits were fed on either normal or high-cholesterol diet for 8 weeks. Then cholesterol-fed rabbits were randomly assigned to simvastatin or starch. At the end of 12 weeks, glomerular mesangial cells were collected. The concentrations of TF and PAI-1 mRNA were detected by RT-PCR. The plasma activities of TF and PAI-1 were determined with enzyme linked immunosorbent assay (ELISA) and chromogenic substrate method, respectively. The atherogenic diet caused a consistent increase in serum concentrations of total cholesterol (TC) and serum triglyceride (TG) (p < 0.05), increased TF and PAI-1 mRNA expression in glomerular mesangial cells and plasma activities as compared to the normal diet (p < 0.01). Four-week simvastatin treatment resulted in significant decrease of mesangial TF and PAI-1 mRNA (p < 0.01), and also of the plasma activities of TF (p < 0.05) and PAI-1 (p < 0.01). These results suggest that simvastatin might protect kidney from the formation of microthrombus under hypercholesterolemic condition and might be a possible pathogenesis of obesity-related glomerulopathy.     

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.     

Studies on the interaction between plasminogen activator inhibitor-1 and vitronectin

Recently, the functionally active form of plasminogen activator inhibitor type-1 (PAI-1) was found to bind to vitronectin both in plasma and in extracellular matrix. In the present study the formation of the complex between functionally active PAI-1 and vitronectin has been studied using vitronectin-coated microtitre plates. PAW bound to vitronectin in the microtitre plates was quantified using HRP-conjugated monoclonal antibodies towards PAI-1. Even at PAI-I concentrations of about I pmol/I the binding to the vitronectin-coated plates seem to be quantitative suggesting high affinity. In contrast, with ‘latent’ PAI-1 at similar molar concentrations, no binding was observed. The effects of pH, NaCl, KBr, KSCN, urea, guanidinium chloride and certain amino acids were studied on the interaction between active PAI-1 and vitronectin. The interaction was not affected at a wide pH range or by increasing the ionic strength by addition of NaCl, suggesting that the binding is more complicated than just an ionic binding. In addition, no effect was observed on the complex formation by 2 mol/I KBr. In contrast, 0.5 mol KSCN almost completely abolished complex formation. Furthermore, arginine and guanidinium chloride, both dissociated the complex readily. Half maximal binding was obtained at about 0.3 mol/I for both substances. In contrast hardly any effect was obtained with epsilon aminocaproic acid (EACA) or lysine in concentrations up to 1.6 mol/I. Our results suggest that guanidine groups and most likely also hydrophobic interactions are involved in the binding of the active form of PAI-1 to vitronectin.