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.     

Distribution of plasminogen activator inhibitor (PAI-1) in tissues.

Extracts of human tissue were analysed for plasminogen activator inhibitor (PAI-1) antigen and activity. PAI-1 was localised in tissues by an immunochemical method, using monoclonal antibodies. PAI-1 occurred throughout the body; its concentration and activity differed considerably from organ to organ. Extracts of liver and spleen had the greatest abundance of PAI-1, but the activity of the inhibitor was much higher in liver than in spleen: the liver may be a source of plasma PAI-1. Immunochemical staining for PAI-1 was observed in endothelium, platelets and their precursor cells, the megakaryocytes, and locations central to the process of haemostasis. PAI-1 also occurred in neutrophil polymorphs and macrophages, cells important in inflammatory and immune processes, but not in lymphocytes. Other cell types, in particular, vascular smooth muscle cells and mesangial cells, also stained positively for PAI-1 and such cells seem to represent an important reservoir of PAI-1.     

Estrogen regulates endothelial migration via plasminogen activator inhibitor (PAI-1)

Endothelial plasminogen activator inhibitor (PAI-1) controls vascular remodeling, angiogenesis and fibrinolysis. PAI-1 blood levels in women are related to estrogen. The aim of this study was to characterize the signaling pathways through which estrogen regulates PAI-1 in endothelial cells. Furthermore, we aimed to investigate whether PAI-1 is implicated in the control of endothelial migration by estrogen. Cultured human umbilical vein endothelial cells (HUVECs) and ovariectomized rats were used to test the effects of 17β-estradiol (E(2)) on PAI-1 expression and its role on endothelial migration. At physiological concentrations, E(2) increases the expression of PAI-1 in HUVEC within 6-12 h through activation of a signaling cascade initiated by estrogen receptor α and involving G proteins, phosphatidylinositol-3-OH kinase and Rho-associated kinase II. ROCK-II activation turns into an over-expression of c-Jun and c-Fos that is required for E(2)-induced expression of PAI-1. Estrogen-induced PAI-1 expression is implicated in HUVEC horizontal migration. PAI-1 regulation is found also in vivo, in female rats, where ovariectomy is associated with reduced PAI-1 expression, while estrogen replacement counteracts this change. In conclusion, E(2) increases PAI-1 synthesis in human endothelial cells and in rodent aorta through a G protein-initiated signaling that targets early-immediate gene expression. This regulatory pathway is implicated in endothelial cell migration. These findings describe new mechanisms of action of estrogens in the vessels, which may be important for vascular remodeling and hemostasis.     

Fibronectin, plasminogen activator inhibitor type 1 (PAI-1) and uterine artery Doppler velocimetry as markers of preeclampsia

Objective: The purpose of this study was to examine plasma levels of fibronectin and plasminogen inhibitor type 1 (PAI-1), and alterations in uterine artery (UtA) waveforms throughout normotensive and preeclamptic pregnancies and to analyze its predictive value for the detection of preeclampsia within the second trimester of pregnancy. Material and methods: Blood samples were collected from 102 healthy, nulliparous women between the 24th and 26th gestational week. Preeclampsia developed in 13 patients; 89 normotensive control subjects were matched from the same cohort. Plasma samples were assayed for fibronectin and PAI-1 by enzyme-linked immunosorbent assay. Color pulsed Doppler examinations of UtA were performed after blood sampling. Trends were compared between two groups. Results: Maternal plasma fibronectin and PAI-1 levels and average PI, RI and S/D ratios of patients with preeclampsia were significantly higher (p < 0.05). The best cut-off values for predicting preeclampsia of fibronectin, PAI-1, PI, RI, S/D ratio based on ROC curve analysis were 290 mg/ml, 77.3 ng/ml, 1,0615, 0.605 and 2,59 respectively. The areas under the curve equal to 0.705, 0.753, 0.689, 0.695 and 0.699 for fibronectin, PAI-1 and uterine artery Doppler PI, RI, S/D ratio were determined for the prediction of preeclampsia. Conclusions: Fibronectin, PAI-1 and UtA Doppler are potentially useful predictors of preeclampsia. Maternal plasma PAI-1 combinated with fibronectin had the highest predictive values in our study.     

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.     

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.     

Highly stable plasminogen activator inhibitor type one (VLHL PAI-1) protects fibrin clots from tissue plasminogen activator-mediated fibrinolysis

Plasminogen activator inhibitor-1 (PAI-1) is the major specific inhibitor of tissue-type plasminogen activator (tPA) which mediates fibrin clot lysis through activation of plasminogen. Wild-type-PAI-1 (wPAI-1) is rapidly converted to the latent form (half-life of approximately 2 h) and loses its ability to inhibit tPA. We developed a very long half-life PAI-1 (VLHL PAI-1), a recombinant protein with a half-life >700 h compared with wPAI-1. In this study, VLHL PAI-1 was assessed for its ability to inhibit clot lysis in vitro. Clot formation was initiated in normal plasma supplemented with tPA by the addition of either tissue factor or human recombinant FVIIa. Clot lysis time, monitored turbidimetrically in a microtiter plate reader, was determined at various concentrations of wPAI-1 and VLHL PAI-1. Both wPAI-1 and VLHL PAI-1 caused a significant increase in clot lysis time, although the latter was somewhat less effective at lower concentrations. The VLHL PAI-1, but not wPAI-1, maintained its anti-fibrinolytic activity after preincubation overnight at 37 degrees. These studies demonstrate that VLHL PAI-1 is an effective inhibitor of fibrin clot degradation. Due to the high stability of VLHL PAI-1 compared with wPAI-1, this novel inhibitor of tPA-mediated fibrinolysis may have therapeutic applications for treating surgical and trauma patients when used directly or in conjunction with the procoagulant recombinant FVIIa.     

A simplified estimation of tissue plasminogen activator (t-PA) inhibition in human plasma

A simplified estimation of the inhibition in plasma of the tissue-plasminogen activator (t-PA) is described. The assay is based on the addition to a plasma dilution of a fixed amount of t-PA, determination after incubation for a fixed period of time of the plasmin generated from added plasminogen by an amidolytic assay, estimation of residual t-PA activity and calculation of t-PA inhibition in plasma. Experiments performed to secure optimal conditions of the assay are reported. The recommended procedure gave no indication in normal plasma of an interaction between t-PA and secondary t-PA inhibitors (e.g. α₂-antiplasmin, α₂-macroglobulin). Precision studies yielded a day-to-day coefficient of variation of 7%.     

Production and characterisation of a set of monoclonal antibodies against tissue-type plasminogen activator (t-PA)

To generate bispecific monoclonal antibodies, reactive to both fibrin and tissue-type plasminogen activator (t-PA), we planned to generate anti-t-PA monoclonal antibodies (mAb) which eliminate negative aspects of t-PA such as the inhibition by plasminogen activator inhibitor-type 1 (PAI-1) and the rapid clearance of t-PA. Here we report on the isolation and characterisation of a set of 13 mAb against t-PA, some of which meet the above requirements. Apart from their potential in the production of bispecific antibodies, these and the other mAb can be useful in structure-function analysis and a variety of other applications.Experiments involving PAI-1 showed that one mAB (12-5-3) reacts only with free t-PA, and prevents the subsequent binding of PAI-1 to mAb-bound t-PA. In vitro studies on the receptor mediated uptake of t-PA by hepatic cells, showed that one mAb (1-3-1) specifically inhibited the association of t-PA with liver endothelial cells. Other tests showed that mAb 7-8-4 and 12-5-3, but not 1-3-1, inhibited in vitro the enzymatic activity of t-PA.On the basis of these and other observations, we conclude that especially mAb 1-3-1, and in vivo possibly 7-8-4 and 12-5-3 may be good candidates for incorporation in bispecific monoclonal antibodies.     

Definition of epitopes within plasminogen activator inhibitor type-1 (PAI-1) using multiple peptide synthesis

Three regions in PAI-1 were selected for epitope analysis on the basis of their proposed significance for the functional activity of PAI-1 and their surface-location. Antisera were raised against 3 peptides synthesized for the regions ¹¹¹P-V^{121 125}D-N¹³⁷ and ³³⁵A-E³⁵⁰ in PAI-1 and were evaluated by dot immunobinding assay. Confirmation of the antigenicity of the peptides was followed by synthesis of solid-phase sequential overlapping octapeptides for each region. Binding of the octapeptides to antisera against the 3 peptides and to a panel of monoclonal antibodies against PAI-1 was evaluated by ELISA.Antipeptide serum ¹²⁵D-N¹³⁷ was most reactive with the amino acid sequence ¹²⁶FSEVERAR¹³³ and antipeptide serum ³³⁵A-E³⁵⁰ reacted strongly with the 2 octapeptides in the sequence ³⁴²IVSARMAPE³⁵⁰. Two of 10 monoclonal antibodies recognized continuous epitopes within the synthesized peptides. ESPI-10 bound to ¹²⁵DFSEVERA¹³² and ESPI-12 bound to ³⁴²IVSARMAP³⁴⁹, a region spanning the reactive centre of PAI-1.     

Prognostic relevance of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitors PAI-1 and PAI-2 in gastric cancer

Expression of urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2) was evaluated in 125 surgically resected gastric cancers by immunohistochemical analysis. Tissue was stained immunohistochemically with a monoclonal antibody against human uPA and monoclonal antibodies against human PAI-1 and PAI-2. In addition, DNA ploidy patterns were determined by cytofluorometer after staining with propidium iodide. We found that 82 (66%) of the 125 gastric cancers expressed uPA as diffuse cytoplasmic staining, as intensely outlined luminal borders. PAI-1 expression was observed in 62 (50%) of 125 gastric cancer as a fine, diffuse and granular pattern in the cytoplasm. PAI-2 expression was observed in 65 (52%) of the 125 gastric cancers as a diffuse cytoplasmic staining. uPA-positive tumours showed a higher incidence of infiltration, lymph node metastasis and peritoneal dissemination than uPA-negative ones. Patients with uPA-positive tumours proved to have a significantly poorer prognosis than those with negative ones. PAI-1-negative tumours showed a higher incidence of liver metastasis and carried a poorer prognosis than PAI-1-positive ones. There was no significant correlation between uPA or PAI-1 expression and DNA ploidy patterns. Conversely, there was no significant relationship between PAI-2 expression and clinicopathological parameters and prognosis. According to the expression of uPA and PAI-1 status, groups of 19 uPA(–)/PAI-1(–), 44 uPA(+)/PAI-1(–), 23 uPA(–)/PAI-1(+) and 39 uPA(+)/PAI-1(+) were subdivided. Tumours with UPA(+)/PAI-1(–) had a significantly higher incidence of liver metastasis, lymph node metastasis and serosal invasion than the other groups of tumours. Patients with uPA(+)/PAI-1(–) tumours had a significantly poorer prognosis than those with uPA(–)/PAI-1(+) tumours. These results indicate that uPA expression is a useful biological prognostic indicator, and that uPA and PAI-1 may play an important part in the tumour progression and metastasis in gastric cancer.     

Effect of heparin administration on fibrinogenolysis during thrombolytic therapy with tissue plasminogen activator for acute myocardial infarction

Heparin accelerates plasminogen activation by tissue type plasminogen activator (t-PA). Previous investigators have postulated that heparin administration during t-PA therapy might lead to enhanced fibrinogenolysis. In this paper, important coagulation parameters from a randomised trial of early versus delayed heparin administration, during t-PA therapy for acute myocardial infarction, were analysed. In contrast to the prediction, the late heparin group had significantly greater fibrinogen depletion (nadir fibrinogen 1.06±0.65 g/1 vs 1.46±0.61 g/l, p=0.0001) and higher fibrinogen degradation products (peak FBDP 675.9±785.3 μg/ml vs 299.7±543.5 μg/ml, p=0.0001). A possible mechanism for these findings is discussed. It has previously been demonstrated that heparin and FBDP are competitive activators of plasminogen activation by t-PA. It has also been shown that the stimulation of plasminogen activation by FBDP contributes substantially to fibrinogenolysis by t-PA. Thus, heparin may reduce fibrinogenolysis by interfering with the stimulation of plasminogen activation by FBDP.     

Reduction of infarct size estimated enzymatically in patients with acute myocardial infarction after treatment with recombinant tissue type plasminogen activator or after spontaneous coronary recanalisation: A randomised,placebo controlled study

In a double-blind trial, 56 patients with suspected myocardial infarction (AMI) of a duration of less than 5 h were randomised to either 100 mg recombinant human tissue type plasminogen activator (rt PA) treatment or placebo intravenously over 3 h. We studied the possible influence of endogenous coronary recanalisation on the extent of myocardial damage in relation to the estimate of myocardial salvage by early treatment of AMI patients with rt PA. We used a computerised serum CK MB time activity curve method for estimation of reperfusion and infarct size. Patients with a first AMI had a significantly higher reperfusion rate than patients with a previous AMI (p=0.01). In the placebo group, the median enzymatic estimated infarct size was significantly lower in the patients with spontaneous reperfusion compared to patients with no endogenous reperfusion (p < 0.05). The median infarct size was significantly lower (33%) in patients treated with rt PA than in patients without spontaneous reperfusion (p < 0.05).We conclude that rt PA is efficient in inducing coronary reperfusion in the majority of patients with a first AMI. Our results confirm that intravenously, administered rt PA treatment can reduce the infarct size in patients with AMI. Our study extends previous observations by indicating that the extent of myocardial salvage induced by such treatment has been underestimated in the past, where patients with spontaneous coronary reperfusion have been included in the reference group.     

组织型纤溶酶原激活物在兔髂动脉损伤后平滑肌细胞内的表达

为进一步探讨动脉粥样硬化形成及血管成形术后再狭窄的发生机制，应用光镜、显微-微机图像分析、发色底物显色法活性测定、原位分子杂交等技术，研究兔血管损伤后不同时间组织型纤溶酶原激活物（t-PA）在平滑肌细胞（SMC）的表达。研究发现：血管损伤后4天t-PA活性明显增加[（7．50±0．05）×10-2IU／mg蛋白]，此时SMC开始从中膜向内膜迁移。损伤后7天，t-PA活性降至近于正常水平[（2．50±0．37）×10-2IU／mg蛋白]。正常血管壁t-PAmRNA杂交阴性，血管损伤后4天，内弹力板下及内膜SMC有大量t-PAmRNA表达，7天时仅表达少量。说明t-PA可能在血管损伤后的SMC增生和迁移过程中起重要作用。