卡托普利对家兔急性心肌梗塞早期血小板活化及纤溶活性的影响

本研究观察了家兔急性心肌梗塞（ＡＭＩ）早期血浆血小板α－颗粒膜蛋白（ＧＭＰ－１４０）、血栓素Ｂ２（ＴＸＢ２）、组织型纤溶酶酶原激活剂（Ｔ－ＰＡ）及其抑制物（ＰＡＩ－１）水平的变化及卡托利干预的ＰＡ活性显著降低；相关分析表明，血浆ＧＭＰ－１４０浓度与ＰＡＩ－１活性显著正相关。卡托普利干预后，血浆ＧＭＰ－１４０浓度、ＴＹＸＢ２浓度、Ｔ－ＰＡ浓度、ｔ－ＰＡ含量、ＰＡＩ－１活性均明显降低，而ｔ－ＰＡ活性显     

Tissue plasminogen activator, plasminogen activator inhibitors, and activator-inhibitor complex in liver disease.

AIMS--To identify the relative contribution of plasminogen activators, particularly tissue plasminogen activator (t-PA) and specific plasminogen activator inhibitors (PAI-1, PAI-2), to the fibrinolytic changes associated with various types of liver disease or severe chemical and physical damage to the liver. METHODS--Platelet rich (PRP) and platelet poor plasma (PFP) from patients with alcoholic cirrhosis, primary biliary cirrhosis, hepatic malignancy, or paracetamol overdose, or who were undergoing partial hepatectomy or liver transplantation, were assayed for t-PA, PAI-1, t-PA-PAI-1 complex and PAI-2 antigen values using specific enzyme linked immunosorbent assays (ELISAs) developed in this laboratory. RESULTS--Appreciable increases in the plasma concentration of t-PA, PAI-1, and t-PA-PAI-1 were seen in patients with alcoholic cirrhosis, primary biliary cirrhosis, and hepatic malignancy. Liver damage due to paracetamol overdose and partial hepatectomy both resulted in a striking increase in plasma PAI-1 concentration, although concentrations of t-PA and t-PA-PAI-1 complex were less affected. Concentrations of t-PA, PAI-1, and t-PA-PAI-1 complex returned to near normal values after successful liver transplantation in a patient with chronic active hepatitis. PAI-2 was also detected in several patients with chronic liver disorders. CONCLUSIONS--Haemorrhage due to fibrinolytic bleeding is commonly associated with liver disease. The patients studied here all had appreciable increases in circulating t-PA antigen concentrations. This was associated with increased concentrations of PAI-1 antigen and t-PA-PAI-1 complex and the balance between activator and inhibitor did not result in systemic plasmin generation. Reduced PAI-1 activity in cirrhosis or a critical difference in the ratio of t-PA to PAI-1 concentrations may explain the enhanced plasminogen activator activity previously noted in cirrhosis but not metastatic disease. Reduced hepatic clearance of t-PA and t-PA-PAI-1 complex due to impaired liver function may account for increased concentrations of free and complexed t-PA.     

Levels of tissue-type plasminogen activator and plasminogen activator inhibitor 1 in disseminated intravascular coagulation syndrome

Since disseminated intravascular coagulation (DIC) may directly reflect the abnormal regulation of the fibrinolytic system by endothelial cells, we have measured the levels of tissue-type plasminogen activator (t-PA), type 1 PA inhibitor (PAI-1) and t-PA . PAI-1 complex which is formed as a result of interaction on the two factors, in the plasma of patients with DIC (n = 51) and healthy controls (n = 42). Antigens of t-PA, PAI-1 and t-PA . PAI-1 complex were significantly increased in the DIC plasma (36.4 +/- 25.1, 106.8 +/- 54.7 and 46.6 +/- 34.5 ng/ml, respectively) compared with those in normal plasma (8.5 +/- 4.3, 54.4 +/- 21.2 and 8.6 +/- 3.5 ng/ml, respectively). The molar ratio of t-PA to PAI-1 was much higher in the DIC plasma (1:3) than in normal plasma (1:6), which caused enhancement of the whole fibrinolytic activity in the DIC plasma. These changes resulted in significant consumption of plasminogen, alpha 2-plasmin inhibitor (alpha 2-PI) and a significant increase of plasmin . alpha 2-PI complex (PPI) and D-dimer. These results suggest that t-PA and its specific inhibitor PAI-1 both of which are secreted from endothelial cells into blood, play an important role on the progress of DIC.     

抗PAI抑制作用的组织纤溶酶原激活剂在大肠杆菌中的表达

目的:构建t-PA活性不被PAI-1抑制的新一代t-PA突变体。方法:根据t-PA的结构特点,去除t-PA分子中的指状区、表皮生长因子和Kringle1区,以含全长t-PA编码区序列的pUC18质粒为模板,经PCR扩增编码氨基酸1~3和176~527位的截短式t-PADNA序列;并将该t-PA分子中的PAI-1结合位点,即第373~384位核苷酸(AAGCACAGGAGG)突变为(GCGGCCGCGGCG),相应的氨基酸KHRR则变为AAAA。结果:测序证实,t-PA突变体的DNA序列正确,将其克隆于大肠杆菌表达载体中,并在大肠杆菌中得到高效表达。表达蛋白占总菌体蛋白的30%,以包涵体形式存在。经蛋白质变性、复性,得到有活性的t-PA突变体。t-PA突变体与PAI-1反应后t-PA的活性未受到抑制。结论:t-PA突变体可能是一种用于治疗心肌梗死和脑血栓等血栓性疾病的强效且剂量要求低的新型生物工程药物。     

Tissue plasminogen activator and plasminogen activator inhibitor type 1 gene polymorphism in patients with gastric ulcer complicated with bleeding

Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) may be involved in the pathogenesis of peptic ulcers through suppression of fibrinolysis. This study was designed to investigate associations of t-PA and PAI-1 genes with clinical features of the patients with bleeding gastric ulcers. Eighty-four patients with peptic ulcers and 100 controls were studied between January 1998 and April 2000. We used polymerase chain reaction and endonuclease digestion to genotype for 4G/5G polymorphism in the promoter region of the PAI-1 gene and the Alurepeat insertion/deletion (I/D) polymorphism in intron h of the t-PA gene. Various clinical features, including lesion site, bleeding event, recurrence of ulcer, and rebleeding, were assessed using a multiple logistic regression model. The genotype distributions of both the t-PA and PAI-1 genes did not differ between the patient and control groups. The occurrence of the I/D or D/D genotype of t-PA was significantly higher in cases of duodenal ulcer (adjusted OR=4.39, 95% CI=1.12-17.21). When a dominant effect (i.e., 4G/4G or 4G/5G versus 5G/5G) of the 4G allele was assumed, the PAI-1 4G/4G genotype was independently associated with rebleeding after hemostasis (adjusted OR=5.07, 95% CI=1.03-24.87). Our data suggest that t-PA gene polymorphism is associated with duodenal ulcers, and that the PAI-1 gene may be a risk factor leading to recurrent bleeding after initial hemostasis.     

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.     

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.     

Tissue plasminogen activator and plasminogen activator inhibitor in patients with liver cirrhosis

The behaviour of tissue plasminogen activator (t-PA) and t-PA inhibitor (PAI) was studied in patients with decompensated liver cirrhosis. t-PA antigen showed a 3-fold significant increase with respect to healthy volunteers. t-PA activity in these patients did not significantly differ from that found in the controls, but the specific activity of t-PA was significantly lowered by 63%. PAI activity was significantly reduced in cirrhosis (−67%), while PAI-1 antigen was increased by 24%. Venous occlusion of the arm for 20min induced similar increases of t-PA antigen both in normal subjects and cirrhotic patients. These findings show that the total amount of t-PA is enhanced in liver cirrhosis, with no increase of t-PA activity due to the capacity of PAI to bind increased circulating t-PA antigen. The increased total amount of t-PA in cirrhotic patients could be explained in terms of a reduced clearance by the hepato-endothelial system.     

Increase of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) in plasma after thrombolytic therapy of patients with myocardial infarction. A randomised,placebo-controlled study

Based on recent studies we hypothesised that the marked generation of thrombin in patients who undergo coronary thrombolysis was associated with substantial deviations of endogenous tissue-type plasminogen activator (t-PA) and the plasminogen activator inhibitor type 1 (PAI-1) in plasma.In the present placebo-controlled study we observe in 20 patients treated with recombinant t-PA (rt-PA) a marked increase (p<0.001) of antigen concentrations in plasma of endogenous t-PA and PAI-1 during the first 12h after initiation of treatment. The concentrations of endogenous t-PA and PAI-1 in plasma correlated significantly (p<0.05) with an estimate of generated thrombin in vivo, determined as plasma concentrations of thrombin-antithrombin III complexes. We conclude that the generation of coagulant activity following coronary thrombolysis is associated with an increased synthesis and/or release of endogenous t-PA and PAI-1, which suggests that the procoagulant condition involves an altered functional state of the vascular endothelium.     

Tissue plasminogen activator and placental plasminogen activator inhibitor in human gingival fluid

Gingival crevicular fluid (GCF) is an extracellular exudate protecting periodontal tissue. Pathological changes in the periodontium are reflected in the composition of GCF. Crevicular fluid was collected from healthy volunteers on Millipore^® filter disks, and eluted at 100-fold dilution. The samples were tested for fibrinolytic activity and the presence of the plasminogen activators, t-PA and u-PA, and the specific plasminogen activator inhibitors, PAI-1 and PAI-2. In the diluted samples, t-PA was found at concentrations of 4–33 gmg/l, and PAI-2 at concentrations of 19–84 μg/l, whereas u-PA and PAI-1 were hardly detectable. Analyses of parotid and whole saliva yielded no evidence of gingival fluid contamination from these sources. The fibrinolytic activity of gingival fluid was completely quenched both by antibodies against t-PA and by PAI-2, indicating the presence of t-PA in its two chain form which is more susceptible to inhibition. This inhibition by PAI-2 may serve a regulatory purpose and prevent excessive proteolysis and tissue destruction.     

Plasminogen activator and plasminogen activator inhibitor activities in a reference population

The influence of age, gender, and aspirin ingestion on plasma levels of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) activities was studied in a reference population of 35 men and 35 women between the ages of 20 and 65 years. The t-PA values (mean +/- SD) in the women before and after 5 minutes of venous occlusion were 3.8 +/- 1.4 and 7.8 +/- 4.4 micrograms/L, respectively; in men these values were 3.3 +/- 1.2 and 8.8 +/- 8.9 micrograms/L. Men had higher mean PAI levels than did women (5.0 vs. 2.5 kU/L). T-PA showed an inverse relationship to PAI in both sexes. There was a negative correlation of t-PA levels with age, whereas PAI levels were positively correlated. The ingestion of a single dose of aspirin (650 mg) did not alter PAI or t-PA activities. This study indicates that factors such as age and sex may need to be considered when reference populations are developed for clinical studies of fibrinolysis.     

Quantification of a specific inhibitor (PAI-1) of tissue-type plasminogen activator (t-PA) in the plasma

In human plasma, the activation of plasminogen by tissue plasminogen activator (t-PA) is a fibrin localized process which allows the specific dissolution of thrombi. Most of the t-PA circulates as a complex with its inhibitor, PAI-1, which thereby regulates its activity. In the present work the authors have studied the kinetics of inhibition of t-PA by PAI-1 and have developed an assay for its specific detection. The assay is performed in microtitration plates containing a solid-phase fibrin network, as follows: the source of inhibitor is mixed with solutions containing increasing amounts of t-PA, then the residual t-PA is separated by means of a solid-phase fibrin support and detected with a coupled reaction using a plasmin selective chromogenic substrate. The change in absorbance is measured in a microtiter plate reader and converted to t-PA activity by reference to a standard curve. The residual t-PA activity is inversely proportional to the concentration of PAI-1. The quantitation of PAI-1 is based on the variation of the dissociation constant of the fibrin/t-PA interaction obtained in the presence of the inhibitor. Since other serine-protease inhibitors do not interfere with the assay, the method is specific for PAI-1 and can be safely used in other biological fluids.     

Relationship between tissue plasminogen activator,plasminogen activator inhibitor and CT image in chronic subdural hematoma.

The present study was performed to investigate the relationship between the concentrations of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI) and the CT images in 23 cases of chronic subdural hematomas (SDHs). The concentrations of t-PA and PAI-1 were quantified by enzyme-linked immunosorbent assay (ELISA). Chronic SDHs were divided into five groups according to their appearance on computed tomography: high-density (n = 4), isodensity (n = 8), low-density (n = 5), mixed-density (n = 3), layering (n = 3) types. The volume of hematoma was measured with an image analyzing software program. The concentrations of t-PA were higher in layering (41.2 +/- 0.3 ng/ml, mean +/- standard error of the mean) and high-density (40.0 +/- 1.1 ng/ml) types compared to those of low-density (23.3 +/- 4.1 ng/ml) and iso-density (25.1 +/- 3.7 ng/ml) types. The concentrations of PAI-1 were lower in layering (95.9 +/- 1.0 ng/ml) and high-density (103.4 +/- 34.5 ng/ml) types compared to that of low-density (192.5 +/- 2.6 ng/ml) type. So the ratio between t-PA and PAI-1 (t-PA/PAI) was greater in layering and high-density types. The volume of hematoma was larger in mixed-density and layering types but statistically insignificant. These results presumably suggest that the ratio between t-PA and PAI concentration may contribute to the pathogenesis of the chronic SDH.     

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.     

The activation of plasminogen by tissue plasminogen activator is not enhanced by different heparin species as assessed in vitro with a solid-phase fibrin method

The aim of this study was to evaluate the effect of several heparin species (standard heparin, heparin of low molecular weight: IC 831422, heparin of high affinity for antithrombin III: IC 831435, and heparin of low affinity for antithrombin III: IC 831436) on the different steps of the fibrinolytic mechanism i.e., interaction of tissue-type plasminogen activator (t-PA) with plasminogen activator inhibitor-1 (PAI-1), binding of t-PA and plasmin(ogen) to fibrin and activation of plasminogen on the fibrin surface, in the presence of plasma proteins and factors that modulate fibrinolysis. Fibrinolytic and plasmin amidolytic activities were measured in the presence and absence of heparin. The spectrophotometric assays were performed in the presence and absence of solid-phase fibrin using selective chromogenic substrates for plasmin and saturating concentrations of plasminogen.The overall fibrinolytic activity, the amidolytic activity of mixtures of t-PA or urokinase with plasminogen in the absence of fibrin, the binding of t-PA and plasmin(ogen) to fibrin and the t-PA/PAI-1 interaction were not modified by heparin. In contrast, the activation of plasminogen by fibrin-bound t-PA decreased as a function of the concentration of heparin. Although this effect was not significant at concentrations usually used in routine heparin therapy (0.1 to 1 iu/ml) it might have some relevance when heparin is injected in bolus doses.In conclusion, by using a solid-phase fibrin method which allows the analysis of t-PA/PAI-1 balance, data were obtained indicating that the heparin species tested here neither competed with fibrin for the binding of t-PA, nor potentiated the activation of plasminogen at the fibrin surface.     

Characterization of a panel of monoclonal antibodies against human tissue-type plasminogen activator

Five murine monoclonal antibodies have been produced against the 1-chain form of human tissue plasminogen activator (t-PA). Affinity constants, calculated from data generated in a solid phase radioimmunoassay, ranged from 4.9 x 10(8)M-1 to 2.3 x 10(9) M-1 for these antibodies. This panel was classified into three groups based on the antibodies' effects on both the plasminogen activation and amidolytic activities of t-PA: complete inhibitors (CD2), partial inhibitors (DB10), and those with limited effects (AE5, BA10 and EG2). This same pattern for the five antibodies was observed in an assay measuring the binding of 125I-t-PA to its fast-acting inhibitor, PAI-1. It is concluded that this panel of antibodies defines at least three domains on the t-PA molecule, including its catalytic site.     

Effects of black cohosh on the plasminogen activator system in vascular smooth muscle cells

Objective

The rhizome of the Cimicifuga racemosa plant (commonly known as black cohosh) has been used for menopausal complaints. Studies regarding the cardiovascular effects of black cohosh are lacking. We investigated the effect of black cohosh on the plasminogen activator system in cultured vascular smooth muscle cells (VSMCs).

Methods

VSMCs were isolated from rat aortae. Expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) proteins were evaluated by Western blot analysis and enzyme-linked immunosorbent assay, respectively. The activities of PAI-1 and t-PA in the conditioned media were assessed by fibrin overlay zymography. A 40% 2-propanol extract of black cohosh was used.

Results

Black cohosh extract (BcEx) stimulated the protein expression of PAI-1, but it did not affect that of t-PA. Vitamin E, a potent antioxidant, inhibited the BcEx-induced increase in PAI-1 expression, while ICI 182,780, an estrogen receptor antagonist, had no effect. Fibrin overlay zymography revealed that BcEx increased the activity of PAI-1 in the conditioned media, while concurrently decreasing that of free t-PA by inducing a binding to PAI-1.

Conclusions

BcEx induces PAI-1 protein expression in the VSMCs likely via an oxidant mechanism. It also stimulates the enzyme activity of PAI-1 and reduces that of free t-PA. These findings suggest that black cohosh might exert a negative influence on fibrinolysis.     

Effects of the Magenstrasse and Mill operation for obesity on plasma plasminogen activator inhibitor type 1, tissue plasminogen activator,fibrinogen and insulin

Plasminogen activator inhibitor 1 (PAI-1), tissue plasminogen activator (t-PA), fibrinogen and insulin were measured in 43 patients 3 years after they had undergone the Magenstrasse and Mill (MM) procedure and in 43 morbidly obese (MO) patients. Mean plasma PAI-1 was 61 ng/ml in the MO group compared to 30 ng/ml in the MM group (p < 0.0001); mean plasma t-PA was 10 ng/ml in the MO group compared to 7 ng/ml in the MM group (p < 0.001). Mean fibrinogen was 3.6 g/l in the MO group compared to 3.2 g/l in the MM group (p < 0.05). Mean plasma insulin levels were 32 U/ml in the MO group compared to 15 U/ml in the MM group. These changes suggest that use of the MM procedure may reduce mortality and morbidity from coronary heart disease in these high-risk obese patients.