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PubMed1987

Microplate immunocapture assay for plasminogen activators and their specific inhibitors.

We report a convenient sensitive enzyme activity assay for urokinase and tissue-type plasminogen activators, based on a solid-phase microtitre plate method using readily available polyclonal antibodies. The sensitivities for urokinase (active and proenzyme) and tissue activator were better than 1 ng/ml. The specificity was very high, with no significant contribution of urokinase in tissue activator assays or vice versa. This method is particularly useful for the assay of urokinase proenzyme in samples containing inhibitors. We describe how this assay may also be used to measure specific inhibitors of plasminogen activators, making use of their rapid formation of stable complexes with solid-phase activator. Inhibitors may be assayed in samples containing proenzymes.

Binding, Competitive↗
PubMed1988

Measurements of the concentration of free plasminogen activator inhibitor (PAI-1) and its complex with tissue plasminogen activator in human plasma.

A new assay system to measure free plasminogen activator inhibitor (PAI-1) and its complex with tissue plasminogen activator (t-PA) was developed. A silicone piece coupled with anti-PAI-1 monoclonal antibody was incubated with human plasma or human plasma which had previously been incubated with t-PA in order to convert all the free PAI-1 to t-PA-PAI-1 complex. After incubation, the amounts of t-PA bound on each silicone piece were measured by enzyme immunoassay. The amounts of t-PA on a silicone piece incubated with plasma represent the amounts of t-PA-PAI-1 complex in the original plasma, and the amounts of t-PA on a silicone piece incubated with plasma previously added with t-PA represent total PAI-1 (the complex plus free PAI-1). Using the technique, the concentration of t-PA-PAI-1 complex seems to increase with age and there was a good correlation between the concentration of free PAI-1 and t-PA-PAI-1 complex in the plasma.

Antibodies, Monoclonal↗
PubMed1988

Measurement of tPA and tPA-PAI-1 complexes by ELISA, using monoclonal antibodies: clinical relevance.

Two ELISA methods using monoclonal antibodies, are described for the measurement of tPA:Ag and tPA-PAI-1 complexes. On normal population tPA:Ag was found with a mean value of 5 ng/ml. Furthermore, despite that tPA activity was very low, only 50% (mean value) was measured as stable complexes with PAI-1. Important increase of tPA:Ag was observed in various pathologies (cardiac infarction, septicemia, respiratory distress syndrome). In liver disease, tPA:Ag reached high levels up to 100 ng/ml. Impaired liver clearance can potentiate the increased concentration which results from endothelial release. In all patients with elevated tPA:Ag level, 70 to 100% of tPA was complexed to PAI-1. Excess release of PAI-1 accompanys the increased release of tPA as it is proved by presence of high residual PAI-1 activity. Addition of exogeneous tPA to these pathological plasmas induced a high increase in tPA-PAI-1 complexes. Venous stasis in normal population resulted in a parallel increase of tPA:Ag and tPA-PAI-1 complexes. Although about a two fold increase was obtained for both parameters, post venous stasis plasma presented a much higher fibrinolytic activity while PAI-1 activity was moderately elevated. tPA:Ag and tPA-PAI-1 complexes have diagnosis and prognosis value in various pathologies as indicators of stimulated release of fibrinolysis activator and inhibitor.

Animals↗
PubMed1989

Intraoperative evolution of coagulation parameters and t-PA/PAI balance in orthotopic liver transplantation.

The changes in coagulation and fibrinolysis were investigated in 10 patients undergoing orthotopic liver transplantation (OLT) which is known to be frequently associated with perturbations of haemostasis. The coagulation profile, already deteriorated before surgery in most patients, showed no appreciable further alteration. On the other hand, important modifications of fibrinolytic parameters occurred, essentially concerning tissue-type plasminogen activator (t-PA) and its specific inhibitor (PAI). t-PA activity constantly increased in the course of transplantation, reaching a maximum at the end of anhepaty. Large interindividual variations were noted in the level of t-PA activity (7.5 to 135 IU/ml). Free PAI activity followed a reverse kinetics, remaining low during the anhepatic stage, and dramatically increasing after allograft reperfusion. Despite the fibrinolytic potential related to high circulating t-PA levels, no biologic nor clinical evidence of systemic fibrinolysis was observed peroperatively. These findings suggest that PAI release could represent an early process making the use of antifibrinolytic drugs during OLT unnecessary.

Blood Coagulation Factors↗
PubMed1989

Fibrinolytic response in normal subjects to venous occlusion and DDAVP infusion.

A set of fibrinolytic parameters was measured in 40 healthy subjects before and after a venous occlusion (VO) test lasting 10 min. After VO, plasma levels of tissue-type plasminogen activator (t-PA) antigen increased in all subjects, t-PA activity increased only in 25 subjects who were considered responders and remained unchanged in 15 (non-responders). High levels of plasminogen activator inhibitor (PAI) in the non-responder group explain this discrepancy. The non-responders had basal levels of PAI activity and t-PA antigen higher than responders (p less than 0.0001) and their basal levels of t-PA activity were lower (p less than 0.001). DDAVP infusion elicited good responses in 7 of 9 non-responders to VO with a fall of PAI activity to 0. Our data indicate that a high proportion of healthy subjects do not have a fibrinolytic response after VO, that a lack of fibrinolytic response to VO is due to high plasma levels of PAI and that DDAVP infusion appear to be more selective than VO for detecting non-responders.

Adult↗
PubMed1989

Demonstration of plasminogen activator activity in the intima and media of the normal human aorta and other large arteries: immunological identification of the plasminogen activator(s).

The plasminogen activator activity (PAA) in extracts of the intima, media, and adventitia of the normal human aorta and other large arteries (carotid artery, renal artery and iliac artery) was studied with a sensitive, quantitative spectrophotometric assay using plasminogen and the chromogenic plasmin substrate S-2251. All layers of the arteries showed PAA which was highest in the adventitia, lowest in the media, while in the intima (aorta) PAA was intermediate, but much closer to that of the media. Plasminogen activator inhibition (PAI) was at the same level in all layers of the arteries studied. Plasmin inhibition (PI) was higher in adventitia than in intima (aorta), while in media the PI was intermediate. The PAA was due to the tissue-type plasminogen activator (t-PA), but not to the urokinase-type (u-PA), as judged by addition of respective antibodies. The relatively low PAA found in the intima of large arteries is therefore due to a low plasminogen activator and not a high plasminogen activator inhibitor activity or plasmin inhibitor level.

Adolescent↗
PubMed1990

Insulin, cortisol and catecholamines do not regulate circadian variations in fibrinolytic activity.

To evaluate possible hormonal regulators of the diurnal rhythm in fibrinolytic activity, we measured tissue plasminogen activator (t-PA) activity, plasminogen activator inhibitor activity (PAI-1), t-PA antigen, insulin, cortisol, and catecholamines in 6 healthy males (age 34 +/- 5) every 2 hours for 24 hours. Fibrinolysis was characterized by a peak in PAI-1 activity and a trough in t-PA activity at 0600 h. PAI-1 activity increased 92% and t-PA activity decreased 56% between 2400 h and 0600 h. t-PA antigen (principally a measure of t-PA/PAI-1 complex), peaked at 0800 h. In comparison, insulin levels were lowest at night when PAI-1 activity was rising. The weak inverse correlation between insulin and PAI-1 activity (r = -0.28, p less than 0.02), was not sufficient to explain the diurnal change in fibrinolysis. While cortisol demonstrated the expected circadian change, the increase in cortisol did not occur until 0400 h, 4-6 hours after the rise in PAI-1 and decrease in t-PA activity started. Supine resting plasma epinephrine and norepinephrine showed no circadian rhythm. From this data, we hypothesize that the increased level of PAI-1 in the morning consumes t-PA, leading to decreased t-PA activity and increased t-PA/PAI-1 complex. Further, we conclude that insulin, cortisol, and catecholamines are not responsible for the circadian rhythm of fibrinolysis.

Adult↗
PubMed1990

Hypofibrinolysis associated with vasculopathy in non insulin dependent diabetes mellitus.

The pathogenesis of diabetic vasculopathy has been related to modifications in hemostasis and fibrinolysis. 50 non insulin dependent diabetes mellitus patients have been studied. Euglobulin clot lysis time, fibrin plate, tissue plasminogen activator (t-PA) antigen, plasminogen activator inhibitor (PAI) activity, Protein C and S, cholesterol, triglycerides and Hb A1c were determined in blood samples. Diabetic patients showed decreased fibrinolytic activity, as measured by ECLT, with clearly increased PAI levels. Fibrinolytic response to venous occlusion was lower than normal. Vascular complications were associated both with an even higher PAI activity and with a decreased fibrinolytic response to venous occlusion. Elevated PAI activity and decreased fibrinolytic response to stimulus may contribute to vascular disease in diabetes.

Aged↗
PubMed1990

A modified quantitative whole blood clot lysis method for general laboratory analysis of fibrinolysis.

Because fibrinolysis is now recognized as an important factor in hypercoagulable states, we have developed and characterized an easily performed, rapid, and quantitative screening test that assesses a patient's fibrinolytic activity. This modification of the dilute whole blood clotting time (DWBCT) counts the number of intact erythrocytes released from the clot formed in samples obtained before and after the application of a venous occlusion cuff. Samples were corrected for the plasma volume changes that occurred during venous occlusion. This test was performed on nine healthy volunteers. Specimens were diluted 1:1 with PBS, rapidly clotted with thrombin and incubated at 37 C. Starting thirty minutes after the thrombin was added and then at twenty minute intervals until 110 minutes, the number of RBCs released from the clot were counted using a Coulter S Plus counter. There were consistently more RBCs released at each time period after venous occlusion (p less than 0.001). Aliquots were also obtained for measuring PAI activity and TPA levels. PAI activity was lower post-cuff at every point (p less than 0.001). TPA level was higher at every point (p less than 0.001) post-cuff. The addition of exogenous TPA, activated protein C, or anti-PAI antibodies increased the amount of clot lysis; while the addition of anti-TPA antibodies and EACA each prevented the post-cuff increase. Unlike the euglobulin lysis time (ELT) this modified DWBCT (mDWBCT) measures the patients intact fibrinolytic system, including PAI and erythrocytes, in a quantitative fashion. Unlike either the ELT or the DWBCT the mDWBCT can be performed within two hours, so results are rapidly available for clinical decisions. These studies have demonstrated an easily performed, inexpensive, quantitative screening test of a patient's overall fibrinolytic system that reacts appropriately to pharmacologic manipulations.

Aminocaproic Acid↗
PubMed1990

Suppression of plasminogen activator inhibitor 1 release by fibrin from human umbilical vein endothelial cells.

The effect of fibrin stimulation on the fibrinolytic potential in cultured human umbilical vein endothelial cells (HUVEC) was investigated in the normal state and aged state. The amount of antigen of the two fibrinolytic factors, tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1), was determined using ELISA and the ABC method, respectively. When a fibrin clot was overlayered on the normal HUVEC, the secretion of t-PA or PAI-1 from the HUVEC was greatly changed. That is, PAI antigen was decreased 3-fold and t-PA antigen increased slightly in the conditioned medium. On the other hand, when the aged HUVEC were stimulated by a fibrin clot, PAI antigen was increased 3-fold and t-PA antigen did not change in the conditioned medium. When the level of fibrinolytic activity in the conditioned medium was expressed as the molar ratio of PAI and t-PA (PAI/t-PA), the value in the fibrin-stimulated normal HUVEC was markedly reduced (a 3.5-fold decrease) when compared with that of the non-stimulated normal HUVEC, reflecting a profibrinolytic state. On the other hand, the value in the fibrin-stimulated aged HUVEC was markedly increased (a 5-fold increase) when compared with that of the non-stimulated aged HUVEC, reflecting an antifibrinolytic state. Actinomycin D- or cycloheximide-treated HUVEC showed no response to the fibrin stimulation. We conclude that the level of HUVEC-mediated fibrinolytic activity was regulated mainly by the production and secretion of PAI from the HUVEC to protect against the generation of thrombi. In the aged HUVEC, the regulatory mechanism acts in an opposite manner and a thrombotic process may be induced.

Cell Survival↗
PubMed1991

Molecular forms of plasminogen activator inhibitor-1 (PAI-1) and tissue-type plasminogen activator (t-PA) in human plasma.

Molecular forms of plasminogen activator inhibitor-1 (PAI-1) and tissue-type plasminogen activator (t-PA), identified by gel filtration and specific immunoassays, were studied in plasma from subjects with normal and elevated PAI-1 levels before and after in vitro or in vivo addition of t-PA. In normal plasma, PAI-1 occurs in three molecular forms, a Mr greater than 700 KDa inactive form of heterogeneous composition, an active 450 KDa form containing PAI-1/vitronectin complex and an inactive peak at Mr 50 KDa containing free PAI-1. Stimulation of platelets results in a significant increase of the 50 KDA form and a slight increase of the 450 KDa form. Patients with increased PAI activity levels have an increase of both the 450 KDa and the 50 KDa forms, whereas patients with thrombotic thrombocytopenic purpura have an increased 50 KDa form. In normal plasma, collected in the presence or absence of D-Phe-Pro-Arg-CH2Cl, t-PA occurs primarily as a Mr greater than 700 KDa form containing t-PA/PAI-1 complex. Addition of high concentrations of t-PA (70 ng/ml) to plasma in vitro or t-PA injection in vivo, results in t-PA inhibitor complexes, including t-PA/ alpha 2 antiplasmin. It is concluded that in subjects with increased PAI-1 levels in plasma, PAI-1 may occur as high molecular weight complexes with vitronectin of which 450 KDa was the most important part and as a 50 KDa inactive form; t-PA circulates primarily in complex with inhibitors. Thus, some of the molecular interactions between PAI-1, t-PA and vitronectin, previously demonstrated in purified systems in vitro, also occur in plasma.

Amino Acid Sequence↗
PubMed1991

Increase in levels of plasminogen activator and type-1 plasminogen activator inhibitor in human breast cancer: possible roles in tumor progression and metastasis.

We measured antigen levels of two kinds of plasminogen activators, tissue type plasminogen activator (t-PA) and urokinase type plasminogen activator (UK), as well as their primary inhibitor, type-1 plasminogen activator inhibitor (PAI-1) in the tissue extracts of benign and malignant breast tumors. Tumor tissues of 36 fibroadenomas and 39 breast cancers were examined. t-PA levels were not different in both groups. Malignant tumors contained the significantly higher levels of UK than benign tumors (p less than 0.001). Furthermore in breast cancer tissues, UK antigen levels of tumors with axillary lymph node involvements were significantly higher than those of tumors without lymph node involvements (p less than 0.05). PAI-1 antigen levels of breast cancer tissues were dramatically higher than those of fibroadenoma (p less than 0.001). PAI-1 levels of node positive carcinomas showed also values significantly higher than node negative ones (p less than 0.01). When we divided cancer tissues into three groups as node negative tumors, tumors with positive axillary nodes fewer than four and tumors with four or more positive nodes, PAI-1 levels increased corresponding to the progression of lymph node involvements (p less than 0.05). Immunohistochemical studies, using mouse monoclonal antibodies to human UK and PAI-1, showed that those immunoreactivities were diffusely distributed in the cytoplasm of human breast cancer cells. Their staining patterns were very similar to each other.

Adenofibroma↗
PubMed1991

The activation of plasminogen by T-PA-PAI-1 complex in the absence of fibrin.

The activity of tissue plasminogen activator-plasminogen activator inhibitor-1 (t-PA-PAI-1) complex to convert plasminogen to plasmin was examined by using fibrin autography and casein zymography where plasminogen was added or removed for casein layer. Fibrin autography was more sensitive to the activity of t-PA even in the complex with PAI-1, but casein zymography was more sensitive to plasmin. The inhibitory activity of PAI-1 toward t-PA diminished when plasma was clotted, thus in the presence of fibrin. These results indicate that t-PA-PAI-1 complex has a catalytic activity even in the absence of fibrin, but the activity is enhanced in its presence.

Fibrin↗
PubMed1991

Fibrin-dependent fibrinolytic activity during extracorporeal circulation.

Cardiopulmonary By-Pass (CPB) Surgery may at times induce a haemostatic defect, at present not too well understood, causing severe bleeding from the operative site and chest tube drain. We present here some data on antigen increase in tissue Plasminogen Activator (tPA) and D 2 Dimer (D2D) detected during CPB and apparently not compensated by enhanced Plasminogen Activator Inhibitor type 1 (PAI 1) activity. tPA concentration (antigenic) ranged around 6.15 ng/ml (SD 5.6) before thoracotomy and 5.8 g/ml (SD 4.74) 5-10 minutes after a heparin 250 IU/Kg bolus injection. During CPB, tPA increased to 20.34 ng/ml (SD 9.17) before protamine infusion, and 16.93 ng/ml (SD 8.13) after heparin neutralization. As the D2D went up to 2000-4000 ng/ml (before/after protamine) and it was not correlated by fibrinogen consumption or FDP production, we find these observations suggestive of fibrin-dependent fibrinolytic activity, as an acquired haemostatic defect developed during CPB.

Adult↗
PubMed1989

Inhibition of endothelial secretion of tissue-type plasminogen activator and its rapid inhibitor by agents which increase intracellular cyclic AMP.

We investigated the effect of agents which raise intracellular levels of cyclic AMP (cAMP) on the secretion of tissue-type plasminogen activator (t-PA) and type 1 plasminogen activator inhibitor (PAI-1) by cultured human umbilical-vein endothelial cells. Significant inhibition of baseline (unstimulated) t-PA and PAI-1 secretion was observed in response to several agents which, when added exogenously, cause increased intracellular cAMP: cholera toxin, 1-methyl-3-isobutylxanthine (MIX), dibutyryl-cAMP, and prostaglandin E1. These agents also significantly reduced or abolished the previously reported stimulatory effects of thrombin and histamine on t-PA secretion, and, with the exception of MIX, significantly reduced the previously reported stimulatory effect of thrombin on PAI-1 secretion. MIX at a concentration (10 microM) below that required to inhibit t-PA and PAI-1 secretion when tested alone, significantly increased the inhibitory effects of cholera toxin, dibutyryl-cAMP, and prostaglandin E1 on both t-PA and PAI-1 secretion. The data suggest that elevated intracellular levels of cAMP inhibit both spontaneous endothelial secretion of t-PA and PAI-1, and secretion induced by agents (thrombin and histamine) which stimulate endothelial phosphoinositide metabolism, consistent with bidirectional regulation of endothelial fibrinolytic protein secretion by the adenylate cyclase and phosphoinositide signal transduction pathways. The inhibitory effects of cAMP do not appear to be specific for t-PA and PAI-1, since cholera toxin and MIX also inhibited endothelial secretion of the adhesive protein, fibronectin. Significant inhibition of baseline endothelial t-PA and PAI-1 secretion was also caused by the stable prostacyclin analogue iloprost (ZK 36 374) and by arachidonic acid, which is converted by endothelial cells to prostacyclin, suggesting that prostacyclin produced endogenously by endothelial cells may inhibit secretion of fibrinolytic proteins by increasing intracellular cAMP.

Cyclic AMP↗