Inhibition of tissue plasminogen activator inhibitor by defibrotide in atherosclerotic patients

Defibrotide, an antithrombotic drug, was previously shown to activate fibrinolysis. In order to elucidate the relationship between defibrotide treatment and fibrinolysis, ten atherosclerotic patients were given 1200 mg/day defibrotide intravenously for 7 days and then 400 mg/day intramuscularly for another 20 days. t-PA antigen assessed before and after venous occlusion was not affected by the treatment. Tissue PAI activity significantly decreased and t-PA activity, measured after venous occlusion, increased after 8 and 28 days of treatment; both these changes disappeared after defibrotide was discontinued. No particular side effects were detected throughout the investigation. The study suggests that defibrotide increases t-PA activity by reducing PAI activity.     

The correlation between the activity of tissue plasminogen activator (TPA), levels of tissue plasminogen activator inhibitor (PAI-1) antigen and serum lipids in healthy subjects

It is well known that abnormality of the coagulation-fibrinolytic system and serum lipids plays an important role in the development of thrombi and atherosclerotic disease. In the present study, the correlation between the activity of tissue plasminogen activator (TPA), the levels of tissue plasminogen activator inhibitor (PAI-1) and serum lipids (TC, TG, beta-Lp, HDLC, LDLC and apolipoproteins) were studied in 102 healthy subjects. The activity of TPA showed negative correlation with the obesity rate (r = 0.21, p less than 0.05) but levels of PAI-1 antigen showed a positive correlation with the obesity rate (r = 0.182, p less than 0.1). The activity of TPA showed no correlation with serum lipids including apolipoproteins, while the levels of PAI-1 antigen showed a positive correlation between TG and beta-Lp (r = 0.292, p less than 0.01, r = 0.211, p less than 0.05). The levels of PAI-1 antigen showed a negative correlation with HDLC (r = -0.286, p less than 0.01). These results indicate that TPA and PAI-1 play an important role in the development of thrombotic disease and atherosclerotic disease.     

Behaviour of tissue plasminogen activator, plasminogen activator inhibitor 1 and their complex in various disease states.

Plasma levels of tissue plasminogen activator (t-PA) antigen, plasminogen activator inhibitor 1 (PAI-1) antigen and t-PA/PAI-1 complex were measured in plasmas from 18 healthy subjects and 75 patients with various diseases (28 patients with haematological malignancies, 20 with thrombotic diseases, five with infectious diseases, four with liver diseases, ten with bleeding disorders and eight miscellaneous conditions). In addition, we studied ten patients with bleeding disorders after DDAVP infusion and 18 healthy subjects after venous occlusion. Plasma levels of t-PA antigen, PAI-1 antigen and t-PA/PAI-1 complex were increased in the patients compared with the healthy subjects. t-PA/PAI-1 complex levels correlated well with t-PA antigen levels and molar concentrations of t-PA antigen were similar to those of the t-PA/PAI-1 complex. Venous occlusion induced an increase in both t-PA antigen and PAI-1 antigen and the molar concentration of the t-PA/PAI-1 complex was equivalent to that of t-PA antigen. Following DDAVP infusion, the levels of t-PA antigen and t-PA/PAI-1 complex increased but PAI-1 antigen levels decreased, and the increase of t-PA antigen was greater than that of t-PA/PAI-1 complex. These findings indicate that PAI-1 antigen exceeds t-PA antigen in healthy subjects and in patients with various diseases. We conclude that part of the t-PA/PAI-1 complex is rapidly cleared from the circulation and that free t-PA increases after DDAVP infusion.     

肺动脉血栓栓塞与组织型纤溶酶原激活剂及纤溶酶原激活物抑制剂的关系

目的探讨组织型纤溶酶原激活剂及纤溶酶原激活物抑制剂1血浆水平、基因多态性与肺动脉血栓栓塞(肺栓塞)的关系。方法选择肺栓塞患者87例(肺栓塞组),另选健康体检者80例(对照组),采用酶联免疫吸附法检测2组组织型纤溶酶原激活剂及纤溶酶原激活物抑制剂1血浆水平。聚合酶链反应-限制性片段长度多态性技术检测纤溶酶原激活物抑制剂1基因多态性。结果肺栓塞组患者较对照组血浆组织型纤溶酶原激活剂明显下降、纤溶酶原激活物抑制剂1明显升高。肺栓塞组4G/4G基因型频率明显高于对照组(51.7% vs 25.0%,P0.05)。肺栓塞组和对照组均以4G/4G基因型个体的纤溶酶原激活物抑制剂1血浆水平最高,5G/5G基因型最低,4G/5G基因型居中。结论肺栓塞患者存在纤溶异常,4G/4G基因型患者呈明显低纤溶状态。     

Plasma plasminogen activator inhibitor activity and tissue plasminogen activator levels in patients with unstable angina and those with coronary spastic angina.

Plasminogen activator inhibitor (PAI) activity and tissue plasminogen activator (TPA) antigen were measured in venous samples in 14 patients with unstable angina consisting of eight patients with organic stenosed coronary arteries and six patients with coronary spastic angina (unstable angina group); in 14 patients with stable exertional angina (stable exertional angina group); and in 14 patients with chest pain syndrome (chest pain syndrome group). The plasma levels of PAI activity were higher (p less than 0.01) in the unstable angina group than in the stable exertional angina group and the chest pain syndrome group (12.3 +/- 1.0 versus 5.1 +/- 0.7 and 4.8 +/- 0.6 IU/ml). The plasma levels of TPA antigen were also higher (p less than 0.05) in the unstable angina group than in the stable exertional angina group and the chest pain syndrome group (10.2 +/- 1.3 versus 6.5 +/- 0.8 and 6.0 +/- 0.7 ng/ml). There were no significant differences in PAI activity and TPA antigen levels between the stable exertional angina group and the chest pain syndrome group. Furthermore, both PAI activity and TPA antigen levels in the unstable angina group decreased to the levels in the stable exertional angina group and the chest pain syndrome group after treatment (p less than 0.01). In conclusion, the increased plasma PAI activity in patients with unstable angina and in those with coronary spastic angina indicates that the fibrinolytic system is impaired in these patients.     

Determination of plasma tissue type plasminogen activator and plasminogen activator inhibitor activity in patients with ischemic stroke

Plasma tissue-type plasminogen activator and plasminogen activator inhibitor were determined during the acute, recovery and sequelae stages of patients with ischemic stroke by chromophoric substrate assay. The result showed that t-PA activity was elevated during the acute phase, remained elevated during the recovery stage and declined during the sequelae stage. Lowering of PAI activity was found during acute phase, which reversed during recovery phase and remained significantly elevated during sequelae stage. As a result, the ratio of PAI/t-PA fluctuated during different stages of the disease. Significant elevation of PAI and PAI/t-PA ratio during sequelae stage may be one of the risk factors of further thrombosis and contribute partly to the high relapsing rate of the disease. In addition, a positive correlation was found between PAI and serum cholesterol content.     

Relation of depressive mood to plasminogen activator inhibitor, tissue plasminogen activator, and fibrinogen levels in patients with versus without coronary heart disease

The increased risk of coronary heart disease (CHD) associated with depression is well documented. We hypothesized that impaired fibrinolysis is involved in this link. To explore the association of depressive mood and/or vital exhaustion with various measurements of fibrinolysis activity, 231 men (40 to 65 years old; 123 without CHD and taking no medication and 108 with documented CHD), completed the Center of Epidemiologic Studies Depression Scale and the Maastricht Questionnaire for vital exhaustion. Using classic cut-off points (Center of Epidemiologic Studies Depression Scale score >or=17, Maastricht Questionnaire score >or=8), 6.5% and 9.8% of subjects without CHD and 38% and 48.1% of those with CHD were classified as depressed and exhausted, respectively. Patients with CHD were older, had a higher body mass index, and higher levels of total cholesterol, glucose, plasminogen activator inhibitor 1 (PAI-1), tissue plasminogen activator (t-PA) antigen, and fibrinogen; 47% were treated for hypertension. Depressed subjects had higher levels of PAI-1 activity (p = 0.006) and exhausted patients had higher levels of PAI-1 activity (p = 0.011) and fibrinogen (p = 0.009). After adjusting for clinical condition (with or without CHD), smoking, hypertension, triglyceride concentration, and body mass index, PAI-1 activity remained higher in depressed subjects (p = 0.03). This association persisted after further adjustment for vital exhaustion or for t-PA antigen and fibrinogen levels. t-PA antigen and fibrinogen levels were not associated with depressive mood in multivariate analyses. No fibrinolytic variable was associated with vital exhaustion in multivariate analyses. In conclusion, depressive mood, but not vital exhaustion, is associated with higher levels of PAI-1 activity, suggesting a possible impairment of fibrinolysis and indicating a potential additional mechanism by which depressive mood may act as a cardiovascular risk factor.     

Activity of tissue plasminogen activator and plasminogen activator inhibitor in noninsulin-dependent diabetes mellitus

The activity of free tissue plasminogen activator (f-tPA) and plasminogen activator inhibitor (PAI) in the plasma of 82 noninsulin-dependent diabetics (NIDDM) was measured by bioimmunoassay of the euglobulin fraction obtained from the plasma, and the levels were compared with those of age- and gender-matched normal subjects. Comparison of these levels in both groups revealed that the f-tPA activity tended to be lower in NIDDM than in the controls, although the differences were not significant. Normal activity of PAI was seen, but f-tPA in NIDDM, when accompanied by macroangiopathy such as ischemic heart disease, was significantly depressed. When glycosylated hemoglobin levels were in excess of 10%, the f-tPA activity was significantly decreased, but no reduction was found in PAI activity as compared with controls. When NIDDM is associated with either macroangiopathy or high glycosylated hemoglobin levels, a decreased f-tPA activity, rather than an increased PAI activity, may contribute to the development of a defective fibrinolytic state.     

Identification and regulation of tissue plasminogen activator activity in rat cumulus-oocyte complexes

Plasminogen activators convert plasminogen into plasmin, a serine protease that initiates extracellular proteolysis. Two types of plasminogen activator activities have recently been demonstrated in granulosa cells, and the proteolysis-inducing enzymes are believed to be involved in ovulation. However, little attention has been paid to the presence of these enzymes in oocytes. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by a fibrin overlay technique, we studied plasminogen activator activity in oocytes. Denuded oocytes collected from ovaries of hypophysectomized, estrogen-treated immature rats contained a tissue-type plasminogen activator (tPA), but not urokinase (uPA). In contrast, oocyte-free granulosa cells in these preantral follicles contained uPA, but not tPA. The tPA activity found in oocytes was plasminogen-dependent; incubation with increasing numbers (25-200) of denuded oocytes resulted in a dose-dependent increase in fibrinolysis only in the presence of plasminogen. Cellular localization of tPA was studied in the preantral follicles using an immuno-cytochemical method. Positive tPA staining was detected in the cytoplasm, but not in the germinal vesicle or zona pellucida of the oocytes. Furthermore, analysis using a reverse fibrin-overlay method did not reveal the presence of a plasminogen activator inhibitor. Culturing of denuded oocytes for 24 h increased the cellular content of tPA, but the enzyme activity was not further enhanced by treatment with FSH or forskolin. Also, no tPA activity was detected in the medium. We further studied plasminogen activator activities in the cumulus-oocyte complexes. Although only tPA activity was detected in freshly obtained cumulus-oocyte complexes, incubation for 24 h increased both tPA and uPA activity. Furthermore, tPA, but not uPA, activity was stimulated by treatment with FSH or forskolin. This was accompanied by the secretion of tPA into the medium. The identity of tPA and uPA in the cumulus-oocyte complexes was further confirmed by immunoprecipitation with specific antibodies. Isolation of denuded oocytes and cumulus cells after hormonal stimulation of the cumulus-oocyte complexes suggested that tPA activity was stimulated in both cell types and that the cumulus cells may mediate the action of FSH and forskolin on oocytes. In conclusion, the detection and regulation of tPA activity in cumulus-oocyte complexes suggest possible involvement of this enzyme in ovulation or the process of cumulus cell expansion and dispersion. Changes in oocyte tPA content may also serve as an indicator of oocyte development.     

Tissue plasminogen activator release and plasminogen activator inhibitor levels in coronary artery disease

Unstable angina and Q wave myocardial infarction are associated with intraluminal coronary thrombosis, a process to which impaired fibrinolysis may contribute. The authors examined the extrinsic fibrinolytic system, including tissue plasminogen activator antigen, plasminogen activator inhibitor activity and antigen, and euglobulin clot lysis time before and after venous occlusion in 56 patients undergoing coronary angiography for chest pain syndromes and in 16 healthy controls. Fibrinolysis variables were similar (with greater than 95% confidence) in the patients with thrombus-associated coronary syndromes as compared with those with chest pain syndromes not due to coronary thrombosis. These fibrinolytic variables were also similar to those in patients without coronary artery disease and in healthy controls. Their data suggest that defective fibrinolysis is not involved, at least systemically, in the pathogenesis of thrombus-associated coronary artery syndromes.     

Release of tissue plasminogen activator and its fast-acting inhibitor in defective fibrinolysis

Releasable tissue plasminogen activator (t-PA) and the fast inhibitor of t-PA were measured in 18 controls and a pedigree with venous thrombosis. The functional assay was performed by a technique that destroys the t-PA inhibitor when blood is drawn. It was found that activator and inhibitor levels varied widely in the control group. One patient demonstrated inhibitor levels, on two different occasions, of 2.82 and 3.54 IU of t-PA per milliliter of plasma, as compared with a releasable activator level of 1.87 IU/mL. The t-PA antigen levels of this patient and the remainder of the pedigree were essentially normal for all seven subjects. Thus, it is suggested that the previously reported fibrinolytic disorder in this pedigree represents an imbalance between activator and inhibitor levels rather than an actual deficiency of t-PA.     

Increased plasminogen activator inhibitor antigen levels in diabetic patients with stable angina.

PAI-1 antigen, tPA antigen and thrombin - antithrombin III complexes (TAT) levels were measured in 10 males with stable angina and type-II diabetes mellitus and in 16 males with stable angina without diabetes or other risk factors (hyperfibrinogenaemia, hyperlipidaemia, diabetes, hypertension, smoking and obesity) known to increase PAI levels. Ten healthy men of equivalent age served as controls. Because only diabetics with coronary artery disease (CAD) showed a decreased fibrinolytic capacity, a second study was performed on the 16 non-diabetic CAD patients to determine whether submaximal workload induces significant changes of tPA and PAI levels. TAT levels were increased in CAD, and significantly so in the diabetic group. tPA levels were increased only in the CAD patients without diabetes. PAI levels were significantly increased in diabetic CAD patients (5.26 +/- 1.96 ng/ml) but not in the stable angina patients without diabetes (2.97 +/- 1.44 ng/ml). Immunologically-reactive tPA released after exercise was higher in the 16 CAD patients without diabetes than in controls. Our data could indicate that in stable angina without diabetes there is no chronic latent activation of the clotting system, with no impairment of fibrinolytic activity. On the other hand, the presence of diabetes mellitus seems to influence the fibrinolytic capacity in CAD, particularly increasing PAI levels.     

Regulation of endothelial tissue plasminogen activator and plasminogen activator inhibitor type 1 synthesis by diacylglycerol,phorbol ester,and thrombin

Summary The influence of diacylglycerols, which are physiological activators of protein kinase C, on the production of tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor type 1 (PAI-1) by human umbilical vein endothelial cells (HUVEC) was studied in order to gain insight into the regulation of fibrinolysis by these cells. 1,2-dioctanoyl-sn-glycerol (diC₈) stimulated tPA production in a dose- and time-dependent manner. The tPA antigen in cell supernatants increased from 0.9 ng/10⁶ cells in unstimulated cells to 12.4 ng (10⁶ cells after incubation with 400µM diC₈ for 24 hours. In contrast, PAI-1 production was not influenced by diC₈, whereas phorbol 12-myristrate 13-acetate (PMA) or thrombin stimulated both, tPA and PAI-1 production by HUVEC. Staurosporine and H7, which are inhibitors of protein kinase C, inhibited tPA synthesis by HUVEC. The degree of inhibition was dependent on the agonist used. While diC₈-induced tPA production was inhibited to more than 80% by H7 (10µM) and staurosporine (10 nM), higher doses of inhibitors were required to inhibit thrombin- and PMA-induced tPA production. Thrombin-induced PAI-1 production was inhibited to more than 80% by H7 (10µM) and to about 50% by staurosporine, whereas PMA-induced PAI-1 production was not inhibited by staurosporine, and only to about 50% by higher doses of H7 (30µM). These data suggest that activation of protein kinase C is a common intracellular trigger mechanism for the induction of tPA synthesis by HUVEC. Protein kinase C is most likely also involved in the regulation of PAI-1 synthesis by HUVEC.     

Stimulation in vivo of expression of intra-abdominal adipose tissue plasminogen activator inhibitor Type I by proinsulin

Aims/hypothesis: Impaired fibrinolytic system capacity secondary to increased plasminogen activator inhibitor type-1 expression has been suggested as a pathogenetic link between insulin resistance and increased cardiovascular risk in patients with Type II (non-insulin-dependent) diabetes mellitus, obesity, or both. In patients with syndromes of insulin resistance including those with Type II diabetes, precursors of insulin such as proinsulin can constitute more than 50 % of insulin-like molecules in blood. The aim of this study was to determine whether proinsulin can increase plasminogen activator inhibitor type-1 expression in intra-abdominal adipose tissue in vivo, potentially contributing to the increased PAI-1 seen with insulin resistance. Methods: Lightly sedated normal rabbits were given intravenous proinsulin, insulin, or vehicle alone under euglycaemic clamp conditions with serial sampling of blood and assessment of PAI-1 expression in visceral fat. Results: Both proinsulin and insulin increased expression of plasminogen activator inhibitor type-1 in intra-abdominal adipose tissue, 5.3-fold (p = 0.006 vs control) and 2.5-fold (p = 0.031 vs control) respectively. PAI-1 inhibitor activity in blood peaked 3 h after administration of each, 5.1-fold, p = 0.020, and 3.4-fold, p = 0.004, respectively but did not change under control conditions. Conclusion/interpretation: Hyperproinsulinaemia can contribute to increased expression of plasminogen activator inhibitor type-1 in intra-abdominal adipose tissue implicated in increasing PAI-1 activity in blood, impaired fibrinolysis, and accelerated atherogenesis typical of Type II diabetes. [Diabetologia (2001) 44: 1121–1124] Received: 21 March 2001 and in revised form: 21 May 2001     

Increased levels of tissue plasminogen activator antigen and factor VIII activity in nonvalvular atrial fibrillation: relation to predictors of thromboembolism

INTRODUCTION: Given that nonvalvular atrial fibrillation (AF)-associated stroke can be either cardioembolic or atherothrombotic, we investigated the relationships between nonvalvular AF and hemostatic factors reflecting intrinsic thrombogenic and atherogenic potentials (tissue plasminogen activator [t-PA] antigen, plasminogen activator inhibitor-1, and factor VIII activity). We also evaluated the clinical applicability of these hemostatic factors by examining whether AF subjects with established clinical or echocardiographic predictors of thromboembolism had higher levels of these factors. METHODS AND RESULTS: Of the 3,212 participants of a Chinese population-based study, 53 subjects (1.7%) with AF were identified. Among the hemostatic factors measured, t-PA antigen (median 12.8 vs 8.1 ng/mL; P < 0.01) and factor VIII activity (median 155% vs 133%; P < 0.05) were significantly higher in AF subjects after adjustment for age and sex. In multivariate analysis, features independently associated with t-PA antigen levels were AF, sex, body mass index, systolic blood pressure, total cholesterol, triglycerides, and left ventricular systolic dysfunction. Features independently associated with factor VIII activity levels included AF, age, and total cholesterol. Levels of both t-PA antigen and factor VIII activity were primarily elevated in AF subjects with predictors of thromboembolism (age > 75 years, hypertension, diabetes, and left ventricular systolic dysfunction), whereas in AF subjects with no thromboembolic predictors, plasma levels of hemostatic factors examined were similar to those without AF. CONCLUSION: We demonstrated that nonvalvular AF was independently associated with increased peripheral levels of t-PA antigen and factor VIII activity. Levels of both hemostatic factors were primarily elevated in AF subjects with predictors of thromboembolism. Whether these hemostatic factors are independently predictive of future thromboembolic events in AF patients requires further investigation.     

Thrombin activatable fibrinolysis inhibitor antigen levels are inversely correlated with plasminogen activator inhibitor-1 antigen levels in hyperthyroid patients

Both increased and decreased fibrinolytic activity have been reported in patients with hyperthyroidism. Elevated levels of plasma plasminogen activator inhibitor-1 (PAI-1) antigen have been found in hyperthyroid patients. Thrombin activatable fibrinolysis inhibitor (TAFI) is a novel plasma protein, which inhibits fibrinolysis through removal of C-terminal lysines from partially degraded fibrin. Previously, we showed that plasma TAFI antigen levels were increased in patients with overt and subclinical hypothyroidism. The aim of this study is to investigate plasma levels of TAFI and PAI-1 antigens in hyperthyroid patients. PAI-1 and TAFI antigen levels were measured in the plasma of 29 patients with hyperthyroidism (14 overt hyperthyroid and 15 subclinical hyperthyroid), and 26 healthy individuals. Although there were increased levels of PAI-1 antigen in hyperthyroid patients, plasma TAFI antigen levels were significantly lower compared to controls (80.79 ng/ml vs. 32.42 ng/ml, p = 0.000 for PAI-1; 10.42 microg/ml vs. 12.24 microg/ml, p = 0.009 for TAFI). Elevated PAI-1 antigen levels were positively correlated with free thyroid hormones, although TAFI antigen levels were in negative correlation with free thyroxine. Furthermore, an inverse correlation between PAI-1 and TAFI antigen levels was found. Our study demonstrated that TAFI antigen levels were decreased in patients with hyperthyroidism. Inverse correlation with PAI-1 suggests that the decrease in TAFI antigen levels may be due to activation of TAFI pathway. Further studies evaluating the underlying mechanisms of low TAFI antigen levels in hyperthyroidism should be undertaken.     

Evidence for increased levels of plasminogen activator inhibitor and tissue plasminogen activator in plasma of patients with angiographically verified coronary artery disease

We studied 234 consecutive patients who underwent coronary angiography because of severe angina pectoris. Tissue plasminogen activator (tPA), plasminogen activator inhibitor (PAI), and lipoprotein Lp(a) were measured in citrated plasma samples. The 214 patients showing significant coronary artery stenosis (greater than 50% reduction of luminal area in any of the great coronary arteries) had higher mean levels of tPA (P less than 0.001) and PAI (P less than 0.01) than a random population sample of similar age. PAI and tPA levels were higher in smokers than in either non-smokers or ex-smokers, and in patients with hypertension tPA was increased. Subjects with blood group A had a higher mean Lp(a) level than subjects with blood group O. There were positive correlations of PAI and tPA levels with serum triglycerides and with body mass index; Lp(a) correlated weakly with plasma fibrinogen concentrations. The findings suggest an impairment of the fibrinolytic system in patients with coronary artery disease, which offers a link between established risk factors and a plausible pathophysiological mechanism, namely thrombus turnover.     

t-PA、PAI活性测定在早期糖尿病肾病临床中的意义

目的 探讨2型糖尿病（T2DM）患者及糖尿病肾病（DN）患者中尿白蛋白排泄率（UAER）与血浆组织型纤溶酶原激活物（t-PA）、纤溶酶原激活物抑制物（PAI）活性的相关性。方法 选择60例T2DM病人，根据UAER分为单纯糖尿病（DMa）组、微量白蛋白尿组（DMb）和临床蛋白尿组（DMc）。此外，还选择了30例健康人作为对照组。采用发色底物显色法测定血浆t-PA和PAI的活性，并对其相关性进行统计分析。结果 （1）对照组、DMa组、DMb组和DMc组血浆卜PA活性递减，PAI的活性递增，各组比较有显著性差异（P〈0．01）。（2）t-PA与UAER呈负相关（r=0．615，P=0．000），PAI和UAER呈正相关（r=0．721，P=0．000）。结论 DN早期即有纤溶活性低下；t-PA和PAI可能作为DN肾脏损害程度的佐证，对指导临床用药以缓解或延迟DN的发生具有重要意义。