Tissue plasminogen activator and plasminogen activator inhibitor in patients with acute ischemic stroke: relation to stroke etiology

Recent studies suggest that high plasma levels of tissue-type plasminogen activator (tPA) and its inhibitor (plasminogen activator inhibitor-1, PAI-1) are markers of an increased risk of atherothrombotic ischemic events such as stroke and myocardial infarction. In this prospective study, we measured tPA antigen, PAI-1 antigen and activity, as well as tPA/PAI-1 complex in patients with acute stroke. Stroke subtypes were classified according to the TOAST criteria. From 132 consecutively screened patients, 89 (100%) were enrolled in this study, including 42 patients (47%) with large artery atherosclerosis (LAA), 32 (36%) with small vessel occlusion (SVO), and 15 (17%) with cardioembolism (CE). Nineteen age-matched neurologic patients without manifestations of cerebrovascular disease served as control subjects (CS). Patients with acute stroke had significantly higher plasma levels of tPA antigen (p < 0.001), PAI-1 antigen (p < 0.05) and PAI activity (p < 0.05) than patients in the control group. t-PA antigen, PAI activity and tPA/PAI-1 complex levels were similar regardless of stroke etiology. Only PAI-1 antigen was lower in patients with cardioembolic stroke than in stroke patients with LAA (p < 0.05). Plasma tPA antigen, PAI-1 antigen, and PAI activity are significantly increased in patients with acute ischemic stroke. Except for PAI-1 antigen, this increase appears not to be related to the underlying stroke etiology.     

Relationships between euglobulin clot lysis time and the plasma levels of tissue plasminogen activator and plasminogen activator inhibitor 1

The relationships between tissue plasminogen activator (tPA), its fast acting inhibitor (PAI-1) and euglobulin clot lysis time (ELT) were investigated with healthy volunteers' plasma. Turbidimetric clot lysis assay by the microtiter plate reader was utilized for ELT with a slight modification. Both tPA and PAI-1 showed the significant correlation with ELT. tPA had a significantly positive, not negative, correlation with ELT (R = 0.387, p less than 0.001). Higher correlation coefficients (R = 0.580, p less than 0.001 and R = 0.599, p less than 0.001) were obtained between ELT and total PAI-1 or free PAI-1 than tPA or tPA-PAI-1 complex (R = 0.427, p less than 0.001). The positive correlation was also obtained between tPA and PAI-1. These data suggest that PAI-1 is a highly important factor for ELT, especially, the amounts of free PAI-1 being the key factor to determine the ELT, which can represent the potential activity of the fibrinolytic system.     

血浆中PAI-1和TAFI水平与溶栓后出血性转化相关性研究

目的探索PAI及TAFI对急性脑梗死静脉溶栓后出血性转化的预警作用。方法前瞻性的将溶栓患者分为出血转化组和无出血转化组,对两组患者溶栓前及溶栓后静脉血浆PAI及TAFI浓度进行对比及统计学分析。结果溶栓前两组基线PAI-1水平差异有明显统计学意义(P=0.037)。溶栓后次日复查晨血PAI-1水平出血转化组明显较无出血转化组值更低(P=0.035)。溶栓前出血转化组的基线TAFI水平较无出血转化组比较TAFI值更低(P=0.024)。溶栓后次日复查晨血出血转化组TAFI值同样低于无出血转化组(P=0.042)。结论血浆中PAI-1和TAFI水平与溶栓后出血性转化相关性较高。当溶栓前及溶栓后PAI-1及TAFI的低水平表达均可增加溶栓后出血性转化的风险。     

Tissue plasminogen activator, plasminogen activator inhibitor-1, and tissue plasminogen activator/plasminogen activator inhibitor-1 complex as risk factors for the development of a first stroke

BACKGROUND NAD PURPOSE: Abnormalities in the fibrinolytic system have been associated with an increased risk for stroke in a few studies. This study was designed to test whether plasma levels of tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), and tPA/PAI-1 complex could predict a first-ever stroke. METHODS: The study was an incident case-control study nested within the V?sterbotten Intervention Program and the Northern Sweden Monitoring Trends and Determinants in Cardiovascular Disease (MONICA) cohorts. In this study 108 first-ever stroke cases were defined according to the MONICA classification, and 216 controls from the same cohort were randomly selected and matched for age, sex, sampling time, and geographic region. RESULTS: Stroke occurred on average 30 months after the blood sampling date. The mean plasma concentration of tPA/PAI-1 complex was higher for the stroke cases than for the controls (3.9 versus 3.0 microgram/L). In univariate regression analysis, significantly higher odds ratios were found for the tPA/PAI-1 complex as continuous variable. When divided into quartiles, the odds ratio was 2.74 for the highest quartile compared with the lowest. In the multivariate model, the tPA/PAI-1 complex remained an independent predictor for stroke. Additionally, tPA mass concentration quartiles 3 and 4 showed a significant association with all stroke as outcome. No association was found, however, for PAI-1. In subgroup analysis of cerebral hemorrhage (n=18), the mean tPA/PAI-1 complex level was higher for the cases than for the controls (4.8 versus 3.0 microgram/L), and in multivariate analysis including all controls (n=216), only tPA/PAI-1 complex remained significant. CONCLUSIONS: This prospective study shows that tPA/PAI-1 complex, a novel fibrinolytic marker, is independently associated with the development of a first-ever stroke, especially hemorrhagic stroke. This finding supports the hypothesis that disturbances in fibrinolysis precede a cerebrovascular event.     

Expression of type-1 plasminogen activator inhibitor in the kidney of diabetic rat models

INTRODUCTION: Intrarenal coagulation and fibrinolysis are thought to be involved in the pathogenesis of diabetic nephropathy. However, gene expression of fibrinolytic factors in diabetic nephropathy has not been clearly defined. Therefore we determined the gene expression of fibrinolytic factors in the kidneys of diabetic rats. MATERIALS AND METHODS: As a model of type1 diabetes male Sprague-Dawley rats were used. They were divided into three groups: control, streptozotocin (STZ)-induced diabetic, and insulin-treated diabetic. Otsuka Long-Evans Tokushima Fatty (OLETF) rats were used as a model of type 2 diabetes; and Long-Evans Tokushima Otsuka (LETO) rats, as the control. Renal gene expressions of type-1 plasminogen activator inhibitor (PAI-1), tissue-type PA (tPA), and urokinase-type PA (uPA) were examined by real-time PCR. Localization of PAI-1 mRNA was investigated by in situ hybridization. RESULTS: Renal PAI-1 mRNA levels (versus control) were increased by 60-80% in STZ-induced diabetic rats (10 days or 3 weeks post STZ injection); and insulin treatment reduced this increased expression to the control level. In OLETF rats (38 weeks old), the renal PAI-1 mRNA level was 2.5-fold higher than that in age-matched LETO rats. Both tPA and uPA mRNA levels were significantly lower than those in LETO rats. PAI-1 mRNA was observed in intraglomerular cells and tubular epithelial cells of both models. CONCLUSIONS: Renal PAI-1 gene expression is up-regulated in both type 1 and type 2 diabetic rats, and changes in gene expressions of fibrinolytic factors may play important roles in the development and pathogenesis of diabetic nephropathy.     

水蛭提取液对培养的大鼠脑皮质微血管内皮细胞分泌组织型纤溶酶原激活物和纤溶酶原激活剂抑制物1的影响

目的探讨水蛭提取液(HEL)对培养的大鼠脑皮质微血管内皮细胞分泌组织型纤溶酶原激活物(tPA)、纤溶酶原激活剂抑制物1(PAI-1)的影响。方法建立大鼠大脑皮质微血管内皮细胞培养实验模型。MTT法筛选HEL的有效浓度。检测培养上清液的tPA、PAI-1含量与活性变化,RT-PCR检测经HEL治疗组与生理盐水对照组处理后的微血管内皮细胞tPA与PAI-1的表达,免疫组化检测两组微血管内皮细胞tPA的表达。结果 HEL在一定浓度范围内(0.25～1mg/μl)可促进微血管内皮细胞的生长,有剂量依赖关系(P<0.05)。HEL治疗组较生理盐水对照组能促进培养的大鼠脑皮质微血管内皮细胞分泌tPA,同时提高其活性,促进tPA mRNA的表达及tPA免疫活性表达,且呈剂量依赖性表达增强(P<0.01)。结论 HEL在体外能激活内源性纤溶系统。     

Tissue plasminogen activator (tPA) activity is a novel and early marker of asymptomatic LEAD in type 2 diabetes

Introduction

Lower extremity arterial disease (LEAD) is often one of the first signs of a generalized atherosclerotic disease in type 1 and type 2 diabetic subjects.

Materials and methods

We studied 143 diabetic subjects at 30-70 years of age, M/F 69/74, 74 with type 1 and 69 with type 2 diabetes, without previously known or suspected lower extremity arterial disease. The relationship between early asymptomatic lower extremity arterial disease and blood levels of HbA1c, lipids and fibrinolysis markers (tPA-activity, tPA mass, PAI-1 activity, tPA-PAI-1 complex) was assessed. In parallel, a group with non-diabetic subjects (n = 80) was studied.

Results

35 (24%) diabetic subjects were classified as having sign(s) of LEAD, defined as having at least one reduced peripheral blood pressure measurement, 28% in type 1 vs 20% in type 2 diabetic subjects (p = NS). In univariate logistic regression analyses age, glycemic level (HbA1c), male gender (only in type 1 diabetic subjects), hypertension and tPA activity (only in type 2 diabetic subjects) were positively associated with LEAD. When markers of fibrinolysis were entered into a multivariate model adjusting for age, hypertension, and HbA1c, only tPA activity remained independently associated with LEAD (p = 0.01) and this was also found in type 2 diabetic subjects (p = 0.05). In type 1 diabetic subjects the increase in odds ratio was non-significant.

Conclusions

Tissue plasminogen activator (tPA) activity may be an independent and early marker for asymptomatic lower extremity arterial disease in diabetic subjects, particularly in type 2 diabetes. Thus an altered fibrinolytic activity could be an early marker of atherosclerosis development in the lower extremities but the cause-effect relationship remains unclear.     

Determinants of plasminogen activator inhibitor-1 in South Asians with ischaemic stroke

To investigate the relationship of circulating plasminogen activator inhibitor-1 (PAI-1) levels with features of insulin resistance and genotype at a single nucleotide insertion/deletion (4G/5G) polymorphism in the promoter region of the PAI-1 gene in 101 South Asian ischaemic stroke patients and 102 symptom-free reference subjects. The allele frequencies were 4G-0.51, 5G-0.49 and 4G-0.61, 5G-0.39 in patients and reference subjects, respectively. There was a significant association between PAI-1 promoter genotype and PAI-1 antigen levels in patients. Regression analysis with significant correlates in the model demonstrated age, gender and triglycerides in patients and fasting insulin and HDL cholesterol in reference subjects as independent predictors of PAI-1 antigen.     

Plasminogen activator inhibitor type 2 inhibits cell surface associated tissue plasminogen activator in vitro: potential receptor interactions

Regulation of cellular plasminogen activation is necessary for maintenance of tissue homeostasis. Despite increasing evidence for co-expression of tissue type plasminogen activator (tPA) and plasminogen activator inhibitor type-2 (PAI-2; SERPINB2) under patho/physiological conditions, the inhibition of cell-bound tPA mediated plasminogen activation by PAI-2 has not been addressed. Here we show that PAI-2 can inhibit cell-bound tPA activity in vitro and thus prevent plasmin formation. We also examined the potential involvement in this inhibition of the annexin II heterotetramer (AIIt), one of the many well characterized cell-surface co/receptors for tPA and plasminogen that efficiently promotes plasminogen activation. This receptor was of interest because AIIt has also been shown to directly bind PAI-2. Characterization of these potential interactions using purified protein systems revealed that PAI-2 directly bound AIIt via the p11 (S100A10) subunit. However, PAI-2 prevented AIIt/tPA-mediated plasminogen activation by its classic serpin inhibitory activity rather than through competition with tPA/plasminogen for binding. Further analysis showed that PAI-2 inhibited cell bound tPA-induced plasmin activity in both an AIIt-dependent and -independent manner. These data open new possibilities for further investigations regarding the regulation of cellular plasmin generation in vivo, especially in tissues where PAI-2 and tPA may be co-expressed.     

The acute effect of insulin on tissue plasminogen activator and plasminogen activator inhibitor in man

The present study was performed to elucidate the acute effect of insulin on levels of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor of endothelial cell type (PAI-1). Nine middle-aged, non-obese and non-smoking men were studied during a hyperinsulinemic, euglycemic glucose clamp for 2 h. Plasma insulin level during the clamp averaged 84 +/- 12 mU/l and euglycemia was maintained at 4.9 +/- 0.6 mmol/l. The t-PA activity gradually increased (75% mean increase after 2 h, p less than 0.001) and the PAI-1 activity decreased (49% mean decrease after 2 h, p less than 0.001) during the clamp. t-PA activity decreased and PAI-1 activity increased after the insulin infusion was ceased, but they were still 48% higher and 38% lower, respectively, after 60 min. PAI-1 and t-PA activities were not affected by saline infusion for 2 h. Thus, acute changes in the insulin levels lead to rapid alterations in the fibrinolytic system even when euglycemia is maintained. These effects may be induced by insulin itself or by the concomitant activation of the sympatho-adrenal system during the euglycemic clamp.     

Ageing and amyloid-beta peptide deposition contribute to an impaired brain tissue plasminogen activator activity by different mechanisms

Alzheimer's disease (AD) is the most common form of neurodegenerative disorder in the ageing population. It is characterized by the cerebral accumulation of toxic amyloid-beta peptide assemblies (Abeta). The serine protease plasmin, which is generated from the inactive zymogen plasminogen through its proteolytic cleavage by tissue- (tPA) or urokinase-type plasminogen activator, has been implicated in the catabolism of Abeta peptides. In this report, we studied the regulation of tPA activity in vivo during ageing in normal mice and in a mouse model of AD characterized by an exacerbated endogenous Abeta accumulation. We observed that cerebral tPA activity was decreased during ageing in normal mice and that this effect was worsened in mice overproducing Abeta peptides. These phenomena result, respectively, from a decrease in tPA expression and from an increase in the production of one of the tPA inhibitors, the plasminogen activator inhibitor type 1 (PAI-1). A similar study in sporadic AD and age-matched control brain tissues revealed that the tPA proteolytic activity was negatively correlated to Abeta peptides levels supporting the data observed in mice. Altogether, our data support a model in which amyloid deposition induces a decrease in tPA activity through the overproduction of PAI-1 by activated glial cells.     

Increased plasma levels of plasminogen activator inhibitor-1 and soluble vascular cell adhesion molecule after triacylglycerol infusion in man

Increased plasma plasminogen activator inhibitor-1 (PAI-1) has been implicated in the development of vascular disease. In type 2 diabetes mellitus high PAI-1 levels are associated with increased plasma concentrations of free fatty acids (FFA) and triacylglycerol indicating an association or a causal relationship. To answer that question, the effect of FFA/triacylglycerol on plasma PAI-1 was examined. Ten healthy male volunteers were studied for 6 h during infusion of triacylglycerol [1.5 ml/min]/heparin [0.2 IU/(kg.min)] (LIP; n=10), saline only (SAL; n=10), and saline/heparin (HEP; n=5). Plasma insulin concentrations were kept constant at approximately 35 pmol/l by intravenous somatostatin-insulin infusions and there was no significant change in plasma glucose levels during any of the study protocols. LIP increased plasma triacylglycerol and FFA approximately 3- (p < 0.001) and approximately 8- (p < 0.000001) fold, respectively, within 90 min. Baseline plasma PAI-1 measured by a bio-immunoassay was similar in HEP (11.4 +/- 2.8 ng/ml), SAL (16.6 +/- 3.6 ng/ml), and LIP studies (15.2 +/- 3.4 ng/ml). Since studies were initiated in the morning, PAI-1 decreased (p < 0.025) over time following its normal diurnal variation to 6.4 +/- 2.0 ng/ml and 4.0 +/- 2.4 ng/ml at 360 min in SAL and HEP, respectively. During LIP, however, PAI-1 increased to approximately 2.6 fold higher levels than during SAL at 360 min (16.4 +/- 4.0 ng/ml, p < 0.01). While tissue plasminogen activator (tPA) and adipsin, an adipocyte derived protease, were unaffected by LIP, changes in soluble vascular cell adhesion molecule-1 (sVCAM-1) were significantly correlated (p = 0.02) with those seen for PAI-1. This suggests that hyperlipidemia independent of insulin and plasma glucose levels stimulates vascular tissue and in turn might induce an increase in plasma PAI-1. PAI-1 then could contribute to the development of atherothrombotic vascular disease.     

Effect of peptides on the inactivation of tissue plasminogen activator by plasminogen activator inhibitor-1 and on the binding of tissue plasminogen activator to endothelial cells

The effectiveness of tissue plasminogen activator (tPA) in thrombolytic therapy is dependent upon the rate at which therapeutically administered tPA reaches the clot site and the proportion of that tPA which is enzymatically active. Interactions between tPA and its main plasma inhibitor (PAI-1) and between tPA and the endothelial cells lining blood vessels are two factors which may limit efficacy. In an attempt to identify the regions of the tPA molecule involved in these interactions, we have examined a series of synthetic peptides with amino acid sequences corresponding to different regions of the tPA molecule for their ability to protect tPA from inactivation by PAI-1 and for their ability to reduce the binding of tPA to endothelial cells. Three peptides were identified which were especially effective at maintaining tPA activity in the presence of PAI-1 and three others were found which had a lesser effect. These same peptides were also found to inhibit the binding of tPA to endothelial cells. This suggests that the same regions of the tPA molecule are involved in both processes. None of the peptides inhibited the binding of tPA to fibrin. These peptides may serve as models for the development of agents for enhancing the activity of both endogenous tPA and of tPA administered in thrombolytic therapy.     

Plasminogen activators and plasminogen activator inhibitors in liver deficiencies caused by chronic alcoholism or infectious hepatitis

Plasma concentrations of tissue-type plasminogen activator (t-PA), urokinase (u-PA), plasminogen activator inhibitor 1 (PAI-1) and PAI-2 were studied in 53 patients with liver deficiency caused by chronic alcoholism (n = 40), viral hepatitis (n = 10) or malignant disease of the liver (n = 3) and compared to that of a control group (n = 20) of healthy subjects. u-PA and PAI-1 levels were significantly increased in all patients with chronic alcoholism, whereas high t-PA was only observed in combination with disturbed liver function tests or with liver cirrhosis (two and six-fold above control values, respectively). A good correlation was observed between t-PA and gamma glutamyl transferase (r = 0.615; p less than 0.001). In patients with infectious hepatitis or with malignant disease of the liver t-PA was normal whereas u-PA and PAI-1 were increased. PAI-2 levels were close to or below the detection limit (15 ng/ml) in the control group and in most patients. However, in two patients with alcohol induced cirrhosis PAI-2 levels were approximately 45 ng/ml and in one patient with hepatocarcinoma even 66 ng/ml. Thus, in liver disease, marked elevations of t-PA, u-PA and PAI-1 levels may occur, with increased PAI-1 as an early marker of liver defects and t-PA a marker of severe liver defects.     

Urokinase-type plasminogen activator and metalloproteinase-2 are independently related to the carotid atherosclerosis in haemodialysis patients

INTRODUCTION: Matrix metalloproteinases (MMPs), their tissue inhibitors (TIMPs) system, and fibrinolytic system, have been implicated as important factors in atherosclerosis and vascular remodeling. However, no data are yet available on the associations between these two systems in relation to carotid atherosclerosis in hemodialysis (HD) patients. MATERIAL AND METHODS: We compared plasma levels of MMP-2, MMP-9, TIMP-1, TIMP-2; the parameters of fibrinolytic system: tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPAR) and its soluble receptor (suPAR), plasminogen activator inhibitor-1 (PAI-1), plasmin-alpha2-antiplasmin (PAP) complexes; high sensitivity C-reactive protein (hs CRP) as a marker of inflammation and a surrogate of atherosclerotic disease-intima media thickness (IMT) in HD patients and in healthy controls. RESULTS: The values of the uPA, suPAR, PAP, MMP-2, TIMPs, hs CRP and IMT in the patients significantly exceeded those in controls. The concentrations of MMP-9, tPA and PAI-1 were similar in both investigated groups. uPA, uPAR and PAP were positively associated with MMP-2/TIMPs system; all mentioned above parameters (except TIMP-2) and hsCRP were associated with IMT. Multivariate analysis showed that uPA, MMP-2 and age were the strong independent variables linked to IMT values in HD patients. CONCLUSIONS: The patients on haemodialysis treatment have evidence of disordered fibrinolysis/proteolysis balance in the plasma, independently associated with IMT on multivariate analysis. These data suggest the importance of uPA and MMP-2 levels in the developing of atherosclerosis in these patients.     

Cigarette smoking impairs bradykinin-stimulated tissue plasminogen activator release in human coronary circulation

BACKGROUND: Cigarette smoking is a major risk factor for acute coronary thrombosis. Bradykinin (BK) can induce the release of tissue plasminogen activator (tPA) from the coronary vasculature. The purpose of this study was to investigate whether smoking reduces BK-stimulated tPA release in human coronary circulation. MATERIALS AND METHODS: We examined two groups: 20 current smokers and 19 nonsmokers. By cardiac catheterization, graded doses of BK (0.2, 0.6 and 2.0 microg/min) and papaverine (PA) (12 mg) were administered into the coronary artery. Coronary blood flow (CBF) was measured using a Doppler flow wire. Blood samples from the aorta (Ao) and coronary sinus (CS) were assayed. RESULTS: BK increased both coronary artery diameter (CD) and CBF to a similar extent in the two groups. The net coronary tPA release was dose-dependently increased by BK in the two groups, but the degree of this increase in current smokers was significantly lower than that in nonsmokers. BK did not change plasminogen activator inhibitor type 1 (PAI 1) levels in either group. PA did not alter either tPA or PAI-1 levels in either group. CONCLUSIONS: These results suggest that cigarette smoking deteriorates coronary fibrolytic activity, independent of changes in CBF. These findings can at least partly explain the higher risk of coronary thrombosis in smokers.     

Imbalance of plasminogen activator inhibitor-I/ tissue plasminogen activator and tissue factor/tissue factor pathway inhibitor in young Japanese men with myocardial infarction

To evaluate the association between haemostatic parameters and increased risk of myocardial infarction (MI) at a young age, we measured fibrinogen, factor VII, antithrombin III, protein C, protein S, tissue factor (TF), free form tissue factor pathway inhibitor (TFPI), plasminogen, alpha2-antiplasmin, tissue plasminogen activator (tPA), plasminogen activator inhibitor-I (PAI-I), and lipoprotein (a) in 140 young men with MI before age 45 and 150 age-matched healthy men. TF, TF/TFPI ratio, PAI-I, PAI-I/tPA ratio, plasminogen, and lipoprotein (a) in young MI patients were all significantly higher than controls, while TFPI, antithrombin II, and tPA were significantly lower (P <0.001 of each). Significant determinants of MI risk were PAI-I/tPA ratio (R2 = 0.300, P <0.001), TF/TFPI ratio (R2 = 0.049, P <0.001), antithrombin III (R2 = 0.034, P <0.001), hyperlipidaemia (R2 = 0.019, P = 0.004), diabetes (R2 = 0.014, P = 0.015), lipoprotein (a) (R2 = 0.012, P = 0.023), alpha2-antiplasmin (R2= 0.014, P = 0.012), and protein C (R2= 0.012, P = 0.018). We conclude that the imbalances of PAI-I/tPA and TF/TFPI are significantly associated with MI at a young age, perhaps mediated via impaired fibrinolytic activity.     

Determination of plasminogen activator inhibitor (PAI) activity of human plasma after dilution in a PAI-depleted plasma

A simple and discriminating assay for the determination of the fast-acting plasminogen activator inhibitor (PAI) activity in human plasma is described. The method is based on the inhibition of purified tissue plasminogen activator (tPA) by plasma diluted with a PAI-depleted plasma and the subsequent measurement of residual tPA in the presence of CNBr-fibrinogen fragments, purified plasminogen and a plasmin sensitive chromogenic substrate CBS 10.65. This assay does not require any acidification step, and allows PAI determination directly on plasma. Since dilutions are made in PAI-depleted plasma, all the serine-protease inhibitors, except PAI, are kept constant in their effect on the assay. Thus, any detectable degree of inhibition can only be ascribed to PAI. Under these conditions, parallel titration curves of tPA are obtained in plasma and the values of PAI are reproducible when measured at different dilutions. The PAI levels of 31 normal volunteers ranged from 0.3 to 8.7 IU/ml (mean: 3.5 IU/ml). After venous occlusion, variations of PAI were associated with the release of tPA. A marked increase of PAI levels was observed in the post-operative period and in pregnancy. In this case both PAI-1 and PAI-2 related activities were measured. Due to its simplicity, the assay can be easily used for the screening of patients with thrombotic diseases.     

缺血性脑卒中患者血浆Hcy与PAI-1的变化及临床意义

目的观察缺血性脑卒中患者血浆总同型半胱氨酸(tHcys)与纤溶酶原激活物抑制剂-1(PAI-1)水平的变化并探索其临床意义。方法收集缺血性脑卒中患者110例为实验组,110名性别、年龄和种族等与实验组相匹配的无脑卒中者为对照组,采用荧光偏振免疫检测技术为基础的自动化免疫检测方法测定血浆tHcys。用酶联免疫吸附双抗体夹心法原理定量测定血浆中PAI-1含量。结果缺血性脑卒中组血浆tHcys水平显著高于对照组,P<0.01。PAI-1的平均血浆浓度实验组与对照组分别为(115.5±10.21)ng/ml和(38.2±5.53)ng/ml,P<0.01。结论血浆tHcys和PAI-1水平升高与缺血性脑卒中密切相关。血浆tHcys和PAI-1的检测可作为缺血性脑卒中治疗观察和愈后评价的参考指标。     

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.