An enzyme linked immunosorbent assay for determination of tissue plasminogen activator applied to patients with thromboembolic disease

Utilizing the immunoglobulin fraction from a goat antiserum against human uterine tissue plasminogen activator, an enzyme-linked immunoassay for tissue-type plasminogen activator in human plasma has been developed. With the new method, the concentration of t-PA in normal human acidified plasma is found to be 4.0 +/- 1.8 (SD) ng/ml. It increases to 12 ng/ml after a tourniquet test, and to 14 ng/ml after strenuous physical exercise. In a group of patients with idiopathic thromboembolic disease, the resting t-PA concentration was 5 ng/ml and the post-occlusion value 16 ng/ml. Furthermore, the patients also exhibited a normal post-occlusion rise in the concentration of plasmin-alpha 2-antiplasmin complex. However, in 37% of the post-occlusion patient plasmas, virtually no increase in t-PA could be detected by a specific activity assay. The results indicate that the reason for a defective post-occlusion fibrinolytic activity in a majority of cases may be the presence of increased concentrations of a fast-acting specific t-PA inhibitor.     

A sensitive assay, specific for endothelial cell type plasminogen activator inhibitor in blood plasma

A polyclonal antibody raised against plasminogen activator (PA-)inhibitor from endothelial cells fully precipitates the PA-inhibitor in endothelial cell conditioned medium but only a part of the PA-inhibitory activity in blood plasma. This indicates that the PA-inhibitory activity in blood plasma is not due to a single inhibitory component. Performing the assay for PA-inhibitory activity in plasma both in the presence and absence of saturating concentrations of anti-endothelial cell PA-inhibitor antibodies, allows the determination of endothelial cell type PA-inhibitor in plasma. The assay gives a linear dose-response curve of amount of plasma added versus t-PA neutralised. Values for endothelial cell type PA-inhibitor in plasma of a group of 20 healthy individuals are in the range of 0.0-16.8 IU/ml and are not normally distributed (median value 3.0 IU/ml). This method also reveals a second, so far unidentified, PA-inhibitory component in human plasma.     

Plasminogen activator inhibitor 1 and plasminogen activator inhibitor 2 in various disease states

The association of increased PA-inhibitor (PAI) activity and of PAI-1 and PAI-2 antigen levels with different pathological conditions was studied in a collective of over 300 patients. PAI-1 and PAI-2 levels were measured by specific radioimmunoassays. A good correlation was observed of PAI activity with PAI-1 antigen (r = 0.718; p less than 0.0001) but not with PAI-2 (r = 0.070; n.s.). Both in the controls and in the patients, PAI activity and PAI-1 antigen showed an extremely large range of values. PAI activity ranged from 0.5 to 68 U/ml and PAI-1 antigen from 6 to 600 ng/ml. Increased PAI activity and PAI-1 antigen was observed in patients with malignant tumors, cardiovascular or thromboembolic disease, in the postoperative phase, with hepatic insufficiency, after trauma and after extracorporeal circulation. The large spectrum of disease states with increased PAI activity and PAI-1 antigen reinforces previous suggestions that PAI-1 is an acute phase reactant. After extracorporeal circulation, PAI activity and PAI-1 concentrations strongly increased within one hour, remained elevated for at least one week and returned to preoperation values within 7 days. PAI-2 values ranged from below detection limit (15 ng/ml), observed in half of the plasmas, to 485 ng/ml in a pregnant woman. High values of PAI-2 were only observed in pregnancy.     

Adiponectin is inversely related to plasminogen activator inhibitor type 1 in patients with stable exertional angina

Adipose tissue is a secretory organ producing a variety of bioactive substances, such as adiponectin. Adiponectin has antiatherogenic properties while plasminogen activator inhibitor type 1 (PAI-1) is closely involved in the development of atherosclerosis.The relationship between adiponectin and PAI-1 in patients with coronary artery disease (CAD) has not been clarified. This study examined plasma levels of adiponectin and PAI-1 in 64 patients with stable exertional angina (SEA) and 65 patients with the chest pain syndrome (CPS). Plasma log-adiponectin levels were significantly lower in patients with SEA (0.62+/-0.08 micro g/dL) compared to those with CPS (0.86+/-0.05 micro g/dL) (p<0.0001). The plasma levels of log-PAI-1 were significantly higher in patients with SEA (1.23+/-0.18 ng/mL) compared to those with CPS (1.15+/-0.22 ng/mL) (p<0.05). Plasma log-adiponectin levels correlated negatively with diabetes mellitus (DM), body mass index (BMI), log-PAI-1 (r=-0.284, p<0.001), triglyceride (TG), and remnant-like particles cholesterol (RLP-C), and positively with high-density lipoprotein cholesterol (HDL-C) levels. Plasma levels of log-PAI-1 correlated positively with DM, BMI,TG and RLP-C levels, and negatively with HDL-C levels. Multiple logistic regression analysis identified sex, angina pectoris, and PAI-1 as independent determinants of hyperadiponectinemia (p<0.05). Adiponectin is inversely related to PAI-1. DM, BMI,TG, HDL-C, and RLP-C are common mediators between adiponectin and PAI-1, and treatment for common mediators may prevent the development of CAD by reducing PAI-1 and increasing adiponectin levels.     

Purification of a specific placental plasminogen activator inhibitor by monoclonal antibody and its complex formation with plasminogen activator

A monoclonal antibody of IgG2a-type was obtained against a specific fast acting plasminogen activator inhibitor found in placenta. The placental inhibitor was purified by affinity chromatography using the monoclonal antibody and additionally in a FPLC-system. A strong complex formation was found between the inhibitor and urokinase and also with the two-chain form of plasminogen activator of the tissue-type. A weaker complex was found between the placental inhibitor and the one-chain form of the tissue-type activator.     

Tissue-type plasminogen activator, type 1 plasminogen activator inhibitor and their complex in plasma with disseminated intravascular coagulation.

The levels of tissue-type plasminogen activator (t-PA), type 1 plasminogen activator inhibitor (PAI-1), and t-PA/PAI-1 complex antigens were analyzed in the plasma of disseminated intravascular coagulation (DIC) patients and healthy controls. Other fibrinolytic parameters such as the levels of plasminogen, alpha 2-antiplasmin (alpha 2-AP), plasmin/alpha 2-AP (PAP), and D-dimer were also estimated to clarify the fibrinolytic states in these plasmas. The antigens of t-PA, PAI-1, and t-PA/PAI-1 complex were found to increase from 8.5 +/- 4.3, 54.4 +/- 21.2, and 8.6 +/- 3.5 ng/ml in normal plasma to 36.4 +/- 25.1, 106.8 +/- 54.7, and 46.6 +/- 34.5 ng/ml in DIC plasma, respectively. The molar ratio of total t-PA to total PAI-1 was 1:6 and 1:3 in normal plasma and DIC plasma, respectively, indicating an enhanced fibrinolytic state in the DIC plasma. The DIC plasma revealed a significant consumption of plasminogen (62.1 +/- 27.8%), and alpha 2-AP (63.7 +/- 25.3%) and an increase in PAP (2.6 +/- 2.7 micrograms/ml) and D-dimer (3.9 +/- 10.7 micrograms/ml). These results suggest that the production and secretion of t-PA and PAI-1 from endothelial cells were enhanced in DIC, resulting in an increased t-PA/PAI-1 complex with dominant fibrinolytic activity.     

Secretion of tissue plasminogen activator and plasminogen activator inhibitor 1 during cardiopulmonary bypass

INTRODUCTION: Cardiopulmonary bypass (CPB) is associated with elevated tissue plasminogen activator (t-PA) levels during CPB and increased plasminogen activator inhibitor 1 (PAI-1) levels post-operatively. The goal of this study was to estimate the rate of t-PA and PAI-1 secretion in vivo, before, during and after CPB. MATERIALS AND METHODS: Estimated rates of t-PA and PAI-1 secretion were based on measured levels of active and total t-PA, and active and total PAI-1, obtained before, during and after CPB from nine males, combined with a computer model of each patient's vascular system that continuously accounted for secretion, clearance, hemodilution, blood loss and transfusion. RESULTS AND CONCLUSIONS: At baseline, the average t-PA and PAI-1 secretion rates were 0.74+/-0.33 and 1.28+/-0.74 pmol/s, respectively. Within 5 min of CPB initiation, t-PA secretion increased six-fold to 4.41+/-2.58 pmol/s, while PAI-1 secretion was unchanged, resulting in a six-fold increase in active t-PA levels. t-PA secretion remained elevated throughout CPB and into the early post-operative period. Average PAI-1 secretion did not start to increase until the end of CPB. By 2 h after surgery, average PAI-1 secretion had increased 15-fold to 19.60+/-17.10 pmol/s, resulting in reduced levels of active t-PA even though t-PA secretion was still elevated. We conclude that CPB induces an immediate sustained increase in t-PA secretion followed by a delayed progressive increase in PAI-1 production. Variations in the level of active t-PA are a function of the relative rates of t-PA versus PAI-1 secretion at different times during and after surgery.     

Circadian fluctuations of plasminogen activator inhibitor and tissue plasminogen activator levels in plasma of patients with unstable coronary artery disease and acute myocardial infarction

A decrease in the fibrinolytic potential, mainly due to an elevation of plasminogen activator inhibitor (PAI), has been described in patients with stable coronary artery disease and a previous myocardial infarction. We investigated plasma levels of PAI and tissue plasminogen activator (t-PA) and their possible circadian variations in patients with unstable coronary artery disease (CAD). Sixty-three patients were studied for at least 2 consecutive days during their stay at the coronary care unit (CCU). Diurnal plasma fluctuations in PAI and t-PA and onset of further myocardial ischemic episodes were monitored. As controls we used 22 age-matched patients submitted to the clinic because of non cardiac chest pain or valvular disease who revealed no evidence of CAD. PAI levels were significantly elevated in patients with unstable CAD (p less than 0.0001) but were not influenced by the extent of underlying CAD, history of previous myocardial infarction, known risk factors for CAD, or by extent of myocardial damage. The circadian variation of PAI levels with peak values between midnight and 6 A.M. found in controls was still present in patients but at a higher level. Preservation of circadian pattern in PAI plasma levels despite myocardial ischemic attacks indicates that elevation of PAI is rather not caused by a reactive phenomenon. On the other hand, elevated PAI levels and episodes of severe myocardial ischemia exhibiting a median time of onset at 10 A.M. seem to be closely related.     

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.     

Association of tissue plasminogen activator and plasminogen activator inhibitor polymorphism with myocardial infarction: A meta-analysis

Introduction

To investigate whether t-PA Alu repeat insertion/deletion (I/D) and PAI-1 4 G/5 G genetic variations are associated with the risk of MI.

Methods

We conducted a meta-analysis to assess the association between the t-PA I/D and PAI-1 4 G/5 G polymorphisms and risk of MI. We also performed subgroup analyses based on ethnicity (Caucasian, Asian, and African), gender and age. Forty one eligible studies including 12,461 cases and 14,993 controls were identified to evaluate the impact of PAI-1 4 G/5 G polymorphism on MI. Seven studies investigated the relationship between t-PA I/D and MI.

Results

This meta-analysis revealed that the PAI-1 4 G allele (4 G/4 G and 4 G/5 G genotype) was associated with an increased risk of MI compared with the 5 G allele in the overall population (OR = 1.094, 95% CI = 1.021 - 1.172, p = 0.011). The relative risks of MI for 4 G/4 G genotype was increased when compared to 5 G/5 G genotype and 5 G allele, with odds ratio at 1.157 (95% CI 1.015 - 1.320, p = 0.029) and 1.126 (95% CI = 1.015 - 1.249, p = 0.025). However, the results show that the 4 G/5 G polymorphism risk for MI was not associated with ethnicity stratification as Caucasian, Asian or African population. No substantial differences in the genotype distributions were observed in the MI group and control group along the lines of gender and age. After multivariable analysis t-PA I/D polymorphism showed no consistent association with MI.

Conclusions

This study suggests that the 4 G/5 G polymorphism of PAI-1 may be a risk factor for MI in overall populations.     

Plasminogen activators and plasminogen activator inhibitor 1 before and after venous occlusion of the upper limb in thromboangiitis obliterans (Buerger's disease).

Plasma levels of plasminogen activators (t-PA, u-PA) and their inhibitor (PAI-1) were studied in patients suffering from Buerger's disease and healthy volunteers before and after 15 minutes of venous occlusion test. The baseline levels of t-PA in group of patients did not differ from those of controls. On the contrary patients with Burger's disease showed a marked increase in u-PA antigen concentrations with concomitant decrease in PAI-1 antigen levels. During venous stasis t-PA antigen concentrations increased in all subjects, however it was much pronounced in controls. Venous occlusion resulted in significant decrease in free PAI-1 levels in the group of patients only. In conclusion, Buerger's disease is associated with the endothelial derangement with increased u-PA release and decreased PAI-1 release, which does not influence the function of fibrinolytic system. The fact that the reduced response of the endothelium to release t-PA after venous stasis goes in parallel with marked decrease in PAI-1 antigen levels seems to suggest that patients suffering from Buerger's disease are not at high risk of intravascular fibrin deposition.     

Types 1 and 2 plasminogen activator inhibitor and tumor necrosis factor alpha in patients with sepsis

We have determined the plasma concentrations of types 1 and 2 of plasminogen activator inhibitor (PAI-1 and PAI-2), tumor necrosis factor (TNF-alpha) and endotoxin in 47 patients with bacterial infection (22 patients presented with positive blood cultures). Results were compared with those observed in 30 healthy subjects. There was a significant increase in PAI-1 and TNF-alpha in patients as compared to controls (p less than 0.0001), whereas no differences for PAI-2 were observed. PAI-1 and TNF-alpha were significantly higher in 18 patients with gram-negative bacteremia as compared to all other patients (p less than 0.0001). However, no correlation between the analyzed parameters and either endotoxin or clinical outcome was observed. We conclude that there is an increase of PAI-1 and TNF-alpha in patients with sepsis, which is not related to the endotoxin concentration. Our results suggest that PAI-1, but not PAI-2, is the main plasminogen activator inhibitor in human sepsis.     

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.     

Induction of synthesis of plasminogen activator inhibitor type-1 by tissue-type plasminogen activator in human hepatic and endothelial cells

Plasminogen activator inhibitor type-1 (PAI-1) can modify fibrinolytic activity in vitro and in vivo. The present study was performed to determine whether pharmacologic concentrations of tissue-type plasminogen activator (t-PA) can initiate negative feedback by stimulating PAI-1 synthesis. In both human hepatoma cells (Hep G2) and human umbilical vein endothelial cells (HUVEC), t-PA increased the total concentrations and appearance of newly synthesized protein in conditioned media of free PAI-1 and PAI-1 complexed with t-PA in a dose and time dependent fashion judging from results after immunoprecipitation of metabolically labeled PAI-1. The t-PA effect was not attributable simply to release of stored or matrix-bound PAI-1. In HUVEC, Northern blot analyses indicated that t-PA increased steady-state levels of PAI-1 mRNA two-fold. In contrast PAI-1 mRNA expression was not increased in Hep G2 cells. Thus, mechanisms of stimulation appeared to differ in the two cell lines. The results obtained are consistent with the hypothesis that increased PAI-1 synthesis and secretion in response to t-PA may limit or attenuate fibrinolysis locally or systemically in vivo.     

Elevated plasminogen activator inhibitor (PAI), a cause of thrombophilia? A study in 203 patients with familial or sporadic venous thrombophilia

Two hundred and three patients with venous thrombophilia were investigated in order to find out whether an elevated plasma concentration of plasminogen activator inhibitor (PAI) could be a cause of their tendency to thrombosis. The patients were studied in an asymptomatic period about 3 months after their last thromboembolic episode. PAI activity was found to be elevated in 19 patients (9%), and a corresponding elevation of PAI-1 antigen was observed. In 16 out of the 19 patients with elevated PAI activity, follow-up could be performed after an additional asymptomatic period of about 1 year: in eight patients the elevation of PAI was transient and in eight it was persistent. Out of the eight patients with a persistent elevation of PAI, seven had a positive family history of thrombosis. Investigation of these families excluded a hereditary elevation of PAI activity in two families. In only two other families was elevated PAI activity also found among family members. The occurrence of elevated PAI activity, however, did not coincide with the occurrence of thrombosis in these individuals: except for the probands, all investigated family members who had a history of thrombosis had a normal PAI activity. We therefore conclude that, at least in our material, familial thrombophilia can not be attributed to an inherited, persistent elevation of the blood level of PAI.     

Plasminogen activator inhibitor-1 is the primary inhibitor of tissue-type plasminogen activator in pregnancy plasma

The aim of the present work was to clarify to what extent plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2) contribute to the increase in plasma inhibition of tissue-type plasminogen activator (t-PA) observed during pregnancy. It was demonstrated that a monoclonal antibody against PAI-1 almost completely quenched inhibition of single-chain t-PA and most of the inhibition of two-chain t-PA in plasma during the third trimester of pregnancy. The remaining inhibition of two-chain t-PA was to a great extent abolished by a PAI-2 antibody. The second order rate constant (k1) for inhibition of single-chain t-PA by the inhibitor neutralized by the PAI-1 antibody was about 4.8.10(6) M-1.s-1. The conversion of single-chain t-PA to the two-chain form increased the reaction rate with the inhibitor about 3-fold. These kinetic data are comparable with those obtained with PAI-1 in non-pregnancy plasma or with purified PAI-1. From the above results it is concluded that PAI-1 is the primary inhibitor of both single-chain and two-chain t-PA and that PAI-2 is the secondary inhibitor of two-chain t-PA in pregnancy plasma. The concentration of reactive PAI-1 versus gestation age was assayed in plasma from 6 women by binding of PAI-1 to 125I-labelled single-chain t-PA followed by quantitation of the labelled t-PA-PAI-1 complex after separation by SDS-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

A simple, sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator applicable to measurements in plasma

An indirect spectrophotometric assay for extrinsic plasminogen activator has been devised, which is based on the parabolic assay of Drapier et al. (5). The system contains activator, plasminogen, the synthetic plasmin substrate H-D-Val-Leu-Lys-pNA (S-2251, Kabi) and a mixture of soluble fibrinogen fragments prepared by treatment of fibrinogen with cyanogen bromide. The addition of these fibrinogen fragments considerably enhances the sensitivity and specificity of the method owing to specific stimulation of the plasminogen activation by extrinsic plasminogen activator. The assay conditions were optimized and the application for extrinsic plasminogen activator measurements in plasma euglobulin fractions is demonstrated.