Plasminogen activators in alcoholic cirrhosis: demonstration of increased tissue type and urokinase type activator

Plasma samples from patients with alcoholic cirrhosis were analysed for plasminogen activators and for inhibitors of the fibrinolytic system. Plasminogen activator activity was considerably increased in patients' plasma compared with normal. Immunochemical characterisation of these plasminogen activators showed that they included both tissue type and urokinase type plasminogen activator. The major inhibitor of plasmin, alpha 2-antiplasmin, was decreased in the patients, but no evidence for the generation of plasmin was found.     

Immunological characterization of plasminogen activators in human mixed saliva

In mixed saliva obtained from six healthy volunteers, the plasminogen activators were characterized immunologically using antibodies specific for human tissue plasminogen activator and urokinase, which were raised in goats immunized with low molecular weight urokinase and tissue-type activator from melanoma cells. The fibrinolytic activity in mixed saliva upon stimulation was assayed on fibrin plates containing plasminogen after preincubation with immunoglobulins with and without specific antibodies. In both centrifuged and uncentrifuged saliva, antibodies against tissue plasminogen activator completely quenched the fibrinolytic activity. By contrast, antibodies against urokinase had no suppressive effect, neither did non-immunized goat serum influence the fibrinolytic activity in mixed saliva. In conclusion, during physiological conditions tissue plasminogen activator appears to regulate fibrinolytic activity in mixed saliva, in which no activity of urokinase-like plasminogen activators could be demonstrated.     

Regulation of plasminogen activators in connective tissues: Potential for thrombolytic therapy in collagen-vascular diseases

Cells in connective tissues such as skin, lung, ligaments, tendons, cartilage, synovium and intravertebral discs express plasminogen activators (PA) and plasminogen activator inhibitors (PAI) in a highly regulated fashion. Such PA and PAI very likely play a role in normal tissue maturation, tissue remodelling, and tissue repair. Dysregulation of this system can contribute to tissue degradation and loss of function. Alterations in regulation have been shown to occur locally in tissues such as joints (rheumatoid arthritis and osteoarthritis) or in lungs (acute respiratory distress syndrome and fibrotic lung disease), as well as systemically in inflammatory diseases such as scleroderma, systemic lupus erythematosus or rheumatoid arthritis. The dysfunction of the PA/PAI system in such diseases raises the possibility that thrombolytics such as urokinase or tissue plasminogen activator may have efficacy in inhibiting disease progression and reversing some disease associated alterations.     

Chromosomal assignments of genes for tissue plasminogen activator and urokinase in mouse

The genes encoding the two plasminogen activators, tissue plasminogen activator and urokinase, were mapped to mouse chromosomes using probes derived from the respective mouse cDNAs. DNA from mouse-Chinese hamster and mouse-rat somatic cell hybrids was digested with BamHI and EcoRI, respectively, and analyzed by Southern blot hybridization for the segregation of the two genes. Tissue plasminogen activator and urokinase cosegregated with mouse chromosomes 8 and 14, respectively. The plasminogen activator genes thus fall into two syntenic groups that are conserved in human and mouse.     

Protein engineering and comparative pharmacokinetic analysis of a family of novel recombinant hybrid and mutant plasminogen activators

We have created a number of new recombinant plasminogen activators based on tissue plasminogen activator (t-PA) including: (1) a t-PA mutant with a duplicated finger (F) domain, (2) a hybrid containing a Factor XII growth factor (G) domain in the place of the native G domain and (3) a hybrid containing the kringle (K) domain from urokinase inserted between the G and K1 domains of t-PA. We have also made two hybrids containing the five K domains from plasminogen: (1) linked to the urokinase B-chain or (2) linked to t-PA K2 and B-chain. The latter molecule contains six K domains. All the molecules were expressed in HeLa cells, purified and the rates of plasma clearance examined in a guinea pig model. Our results show that a wide range of strategies may be used to retard plasma clearance of plasminogen activators. Molecules containing the five plasminogen K domains were by far the most slowly cleared, up to approximately 100-fold more slowly than t-PA itself.     

Tissue- and Urokinase-Type Plasminogen Activators and Type 1 Plasminogen Activator Inhibitor in Melanomas and Benign Skin Pigment Neoplasms

The content of urokinase- and tissue-type plasminogen activators and plasminogen activator inhibitor PAI-1 in the cytosol of primary and metastatic melanomas and benign skin pigment neoplasms was estimated by enzyme immunoassay. It was shown that local growth and invasion of melanomas are related to suppressed expression of tissue plasminogen activator. The content of urokinase plasminogen activator increases in patients with distant metastases and large thickness of the primary tumor.     

Fibrinolytic behaviour during experimental haemorrhagic shock

The plasma levels of functional plasminogen activators and functional plasminogen activator inhibitors were evaluated in the initial period (90 min) of experimental haemorrhagic shock in the anaesthetised dog.Soon after bleeding (5 min) a hyperfibrinolytic state was found: euglobulin lysis time decreased and remained so for 1h, while there was an initial, although transient, increase in plasminogen activators. At 90 min after bleeding, a hypofibrinolytic balance was found, associated with a significant increase of plasminogen activator inhibitors relative to pre-bleeding values. This hypofibrinolysis coincides with a severe depletion of vascular plasminogen activator pool, as evaluated by an histochemical method, on femoral vein segments obtained at 90 min post-bleeding.We conclude that experimental haemorrhagic shock defines a situation of fast transition from hyperfibrinolysis to an intense hypofibrinolytic stage that may be important to explain the pathological heterogeneity found in haemorrhagic events.     

Enzyme-linked immunosorbent assay for human urokinase-type plasminogen activator and its proenzyme using a combination of monoclonal and polyclonal antibodies

A sensitive and specific enzyme-linked immunosorbent assay (ELISA) for human urokinase-type plasminogen activator (u-PA) and its inactive proenzyme (pro-u-PA) was developed. A monoclonal antibody was used as solid-phase antibody, while rabbit antibodies against human u-PA followed by peroxidase-conjugated third antibody were used for detection of bound u-PA. No reaction was observed with tissue-type plasminogen activator or with a variety of other human proteins. The assay was used for quantitation of u-PA in human urine and in culture fluid from human tumor cells. The recovery of added pro-u-PA was greater than 95%. A good agreement with the results obtained by enzymatic assays was found. The detection limit was less than 0.1 ng per ml, both for u-PA and pro-u-PA. The advantages of the use of ELISA compared with enzymatic assays and radioimmunoassays for quantitation of u-PA and pro-u-PA in biological samples are discussed.     

Plasminogen activator system, vascular endothelial growth factor, and colorectal cancer progression.

AIMS: To plasminogen activator system (PAS) consists of the plasminogen activators (urokinase (uPA) and tissue-type (tPA) plasminogen activators), the uPA receptor (uPAR), and the plasminogen activator inhibitors (PAI-1 and PAI-2). Plasminogen activators activate plasminogen to plasmin, which can break down extracellular matrix (ECM) components. Vascular endothelial growth factor (VEGF) is a mitogen for endothelial cells and is involved in angiogenesis. VEGF has been shown to upregulate uPA and this may facilitate tumour angiogenesis further. METHODS: PAS components and VEGF were determined by enzyme linked immunosorbent assay (ELISA) in paired colorectal tumour and normal tissue (n = 50) and correlated with pathological staging. RESULTS: uPA, uPAR, PAI-1, and VEGF values were significantly higher in tumour tissue (for example, tumour uPA: median, 2.3 (range, 0.1-6.7) ng/mg protein v normal uPA: median, 0.2 (range, 0-2.6) ng/mg protein). tPA was significantly higher in normal mucosa and there was no difference in PAI-2. uPA, uPAR, PAI-1, and VEGF values significantly correlated with each other and with Dukes's staging (uPA in adenomas: median, 0.42 (range, 0.1-1.2) ng/mg protein; upA in Dukes's B tumours: median, 2.1 (range, 0.4-4.3) ng/mg protein; and uPA in Dukes's D tumours: median, 4.0 (range, 3.7-4.2) ng/mg protein) and lymphatic invasion. In addition PAI-1 also correlated with tumour size and differentiation. CONCLUSION: The involvement of the PAS and VEGF in colorectal cancer appears to be complex. uPA, uPAR, PAI-1, and VEGF were upregulated in tumour tissue and this correlated with Dukes's staging and lymphatic invasion.     

Urokinase and Tissue Plasminogen Activators and Their Inhibitor PAI-1 in Human Tumors

The content of urokinase and tissue plasminogen activators and their inhibitor PAI-1 in tumors and histologically intact tissues from patients with breast, ovarian, and non-small-cell lung cancer was measured by enzyme immunoassay. The content of urokinase plasminogen activator and PAI-1 considerably increased in all malignant tumors, however the correlation between the expression of components of the plasminogen activation system and clinical morphological feature and prognosis of the disease depends on the type of tumor.     

Enzyme-Linked Immunosorbent Assay for Human Urokinase-Type Plasminogen Activator and its Proenzyme Using a Combination of Monoclonal and Polyclonal Antibodies

Abstract

A sensitive and specific enzyme-linked immunosorbent assay (ELISA) for human urokinase-type plasminogen activator (u-PA) and its inactive proenzyme (pro-u-PA) was developed. A monoclonal antibody was used as solid-phase antibody, while rabbit antibodies against human u-PA followed by peroxidase-conjugated third antibody were used for detection of bound u-PA. No reaction was observed with tissue-type plasminogen activator or with a variety of other human proteins. The assay was used for quantitation of u-PA in human urine and in culture fluid from human tumor cells. The recovery of added pro-u-PA was > 95%. A good agreement with the results obtained by enzymatic assays was found. The detection limit was less than 0.1 ng per ml, both for u-PA and pro-u-PA. The advantages of the use of ELISA compared with enzymatic assays and radioimmunoassays for quantitation of u-PA and pro-u-PA in biological samples are discussed.     

Immunological analysis of plasminogen activators from cultured human cancer cells

Summary Immunological similarities or differences between urokinase and plasminogen activators from 9 lines of cultured human caner cells with varying degrees of fibrinolytic activity were examined with antibodies against human urokinase.The antibodies completely inhibited the fibrinolytic activity of 4 lines of gastric cancer, 2 lines of lung cancer, 1 line of urinary bladder cancer and 1 line of renal cancer, indicating that the plasminogen activators from these cell lines were immunologically identical to urokinase. In 5 out of these cell lines, immunological identity was also confirmed by double diffusion analysis.The plasminogen activator from 1 line of lung cancer was found to be immunologically dissimilar to urokinase by a neutralization experiment and double diffusion analysis.These findings indicate that there are at least two immunologically distinguishable forms of plasminogen activators from human cancer cells.     

Platelet release protein which inhibits plasminogen activators.

An inhibitor of plasminogen activator has been identified in human platelets by the technique of sodium dodecyl sulphate polyacrylamide gel electrophoresis and zymography. The inhibitor has a molecular weight of about 40 000 and is distinct from known plasma protease inhibitors. It is associated almost exclusively with platelets, with only trace amounts in platelet free plasma. The inhibitor is released during platelet aggregation or in vitro coagulation. This inhibitor inhibits both tissue type plasminogen activator and urokinase but has no effect on plasmin. It forms a 1:1 complex with tissue type plasminogen activator, which retains activity detectable under the analytical conditions used. A similar complex with urokinase either forms less readily or retains less activity.     

Characterization of plasmin digested peptide maps for recombinant high molecular weight urokinase and pro-Urokinase . Its use for the estimation of low molecular weight urokinase in pro-UK/HMW-UK

The generation of active two chain urokinase from the proenzyme pro-urokinase by catalytic amounts of plasmin is an important regulatory step in plasminogen activation. The plasminogen activator, urokinase, converts plasminogen to plasmin which in turn dissolves fibrin blood clots. We have studied the conversion of pro-urokinase (Pro-UK) to high molecular weight urokinase (HMW-UK) by plasmin. This paper describes a peptide map that separates various plasmin cleavage products of Pro-UK/HMW-UK. A partially purified sample of HMW-UK containing trace amounts of plasmin was incubated at room temperature for 12 days. The digested peptide mixture after reduction and S-carbamide methylation was separated using reversed phase high performance liquid chromatography. The peptides were collected and analyzed for their N-terminal amino acid sequences, and in some cases by plasma desorption mass spectrometry (PDMS). The plasmin map so generated is useful in investigating the integrity of any HMW-UK produced via plasmin cleavage step.     

Immunohistochemical localisation of tissue plasminogen activator and urokinase in the vessel wall.

Immunoreactive plasminogen activators were studied in tissue sections using a peroxidase method and monospecific antibodies to tissue plasminogen activator produced by a melanoma. Tissue plasminogen activator reactivity was found in skin melanomas and in endothelial and smooth muscle cells of arteries and veins. Vessels of the umbilical cord showed higher reactivity than peripheral vessels. Only faint antiurokinase reactivity was found. By means of the fibrin slide technique, fibrinolytic activity could be shown in peripheral vessel walls but not in the umbilical cord, which suggests that immunoreactivity of tissue plasminogen activator bound to an inhibitor can also be demonstrated. This method may be a useful tool in further studies of tissue plasminogen activator in physiological as well as pathological processes.     

Secretion of plasminogen activators by human colorectal and gastric tumor explants

Conditioned media from explants of human colorectal and gastric tumors in short-term organ culture were analysed for plasminogen activator activity, activity toward the synthetic urokinase substrate, Spectrozyme-UK, and for the presence of urokinase antigen using monospecific goat antibody, by enzymelinked immunosorbent assay. Comparisons were made between primary tumors, adjacent normal mucosa and metastatic lesions. These analyses were carried out on unfractionated culture fluids and on fractions obtained by fast protein liquid chromatography separation using Superose 6 gels. Plasminogen activator activity, tested by azocaseinolysis in the presence of added plasminogen, was restricted to peaks of 55 kD and 155 kD. These were of the urokinase type as shown by specific immunoinhibition and by absorption by an antiurokinase antibody Affigel 10 column. Spectrozyme-UK, in addition to these peaks, detected a series of higher molecular weight activities, the largest of which appeared in the void volume, and were therefore of >10⁶ molecular weight. These activities were greatly increased by inclusion of trace plasmin indicating that these components were mostly in their proenzyme forms. The characteristics of these very large enzymes were similar to those isolated earlier from a human lung cancer cell line [10].Comparison of the primary and metastatic tumors confirmed earlier observations showing that urokinase secretion by the metastatic tumors was greatly reduced in comparison with the primary tumors: in the colon carcinomas it was 10 per cent of the value for the primary, in the gastric tumors 3 per cent, whether means or medians were compared (P<0·0001). This large difference was characteristic only of plasminogen activator secretion assayable by azocaseinolysis; activities toward Spectrozyme-UK, and antigen reacting with anti-urokinase antibody, were considerably less different in the two groups. In individual tissues, no correlation was found between the amount of extractable plasminogen activator and amounts secreted, or between the latter and the amount of lactic acid released. It is postulated that the greatly reduced plasminogen activator secretion by explants of metastatic tumors may be a phenotypic characteristic of distinct advantage for cancer cells destined to initiate metastatic foci, and may contribute to the ability of circulating cancer cells to lodge in the blood vessels of the target organ.This study is dedicated to the memory of Judith Madeja, who died on 2 November 1986.     

Substrate specificity of tissue plasminogen activator and urokinase as determined with synthetic chromogenic substrates

Three different synthetic chromogenic substrates )H-glutamylglycyl-L-arginine-p-nitroanilide (S-2227), pyro-glutamyl-glycyl-L-arginine-p-nitroanilide (S-2444), and H-D-isoleucyl-L-prolyl-L-arginine-p-nitroanilide (S-2288] were investigated for use in the measurement of plasminogen activator activity with high molecular weight urokinase (H-UK), low molecular weight urokinase (L-UK), and tissue plasminogen activator (TPA). The three substrates were hydrolyzed by both TPA-type and UK-type plasminogen activator. As regards the amidolytic activity of S-2227, TPA exhibited a weaker amidolytic activity, and L-UK a stronger activity. In the case of the amidolytic activity of S-2444, no great difference between the three activators was observed in terms of Vmax. As regards the amidolytic activity of S-2288, L-UK exhibited a stronger activity, and TPA a weaker activity. It is suggested that the molecular size of the synthetic chromogenic substrate was too small when compared to natural substrate (fibrin), and therefore that fibrin-binding sites around the catalytic site in TPA are not recognized.     

Factors of the plasminogen activator system in human testis, as demonstrated by in-situ hybridization and immunohistochemistry.

A growing body of evidence suggests that the plasminogen activator (PA) system is crucially involved in reproductive physiology in both sexes. Certain factors of the PA system have been found to be present in organs of the male reproductive tract in various species, and the presence of several of the factors was recently demonstrated in monkey testis. The present morphological study was therefore designed to investigate the occurrence and distribution of the tissue and urokinase plasminogen activators (t-PA and u-PA), the u-PA receptor (u-PAR) and the plasminogen activator inhibitors (PAI-1 and PAI-2) in normal human testis. Intraoperative specimens from seven patients undergoing orchidectomy or testicular biopsy were studied using in-situ hybridization (ISH) and immunohistochemistry (IHC). All five factors studied could be detected with both the ISH and IHC procedures. The ISH signals were localized to sites in the testicular tubules in a stage-specific manner. There was good correlation between results obtained with the two methods, though in IHC the tubules were stained more uniformly. The findings provide support for the involvement of the PA system in human male reproductive physiology.     

Plasminogen activation in human leukemia and in normal hematopoietic cells

The active process of pericellular proteolysis is central in tumor invasion, and in particular the essential role of the urokinase-type plasminogen activator (uPA) is well established. uPA-mediated plasminogen activation facilitates cell migration and invasion through extracellular matrices by dissolving connective tissue components. uPA, its receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1) are enriched in several types of tumors. The importance of proteolysis and especially plasminogen activation is less clear in hematopoietic malignancies than in solid tumors. However, patients with leukemia have an increased tendency to bleeding, not always attributable to thrombocytopenia, and tissue infiltration by leukemic cells, processes in which plasminogen activation may be involved. Several studies have indicated that plasminogen activators (PAs) are highly expressed by cultured leukemia cells. Furthermore, differing from adherent tumor cells, leukemic cells have an enhanced capacity to activate pro-uPA and mainly the active form of uPA is released to culture medium. Ex vivo studies have shown that uPAR, uPA and its inhibitors can be found on the surface of normal blood cells and on the blast cell surfaces from patients with acute leukemia as well as from plasma samples. Elevated levels of PAs and their inhibitors have been detected in leukemic cell lysates. Few studies have tried to demonstrate a correlation between prognosis of leukemia and levels of plasminogen activators. More in vivo studies are needed to show, if any of the factors of the plasminogen activation process can be used as tools in subclassification or as markers for prognosis in leukemia. This review article will focus on the in vivo studies of plasminogen activation in leukemia and will present several in vitro findings on PAs in normal leukocytes and leukemic cell lines.     

The haemostatic balance in groups of thrombosis-prone patients. With particular reference to fibrinolysis in patients with myocardial infarction

The concept of the haemostatic balance was reviewed, and its potential role in the regulation of tissue repair and the pathogenesis of thrombotic processes was surveyed. Physiological activation of coagulation appears to be dominated by effects of degenerated and injured cells of the vascular wall causing local release of thromboplastin and exposition of activating surfaces. Inhibition of coagulation impairs its progression and the non-thrombogenic nature of the normal endothelium is chiefly caused by the binding of inhibitory components (antithrombin-III, protein C) to specific receptor sites. Physiological activation of fibrinolysis appears to be triggered by and limited to the fibrin because of a specific affinity to fibrin of plasminogen and plasminogen activators. Systemic activation of fibrinolysis is prevented by primary (alpha 2-antiplasmin) and secondary (alpha 2-macroglobulin, alpha 1-antitrypsin) plasmin inhibitors. A plasminogen binding protein (histidine-rich glycoprotein), plasmin inhibitors and activator inhibitors appear to contribute to the regulation of the initial phase of fibrinolysis. A deviation from normal of the dynamic balance, regulating fibrin formation and resolution, may lead to a haemorrhagic and/or a thrombophilic state. Described were the optimization of selected methods for assessment of variables involved in the haemostatic balance. An overestimation of plasminogen concentrations in plasma may occur in patients with elevated levels of fibrinogen or fibrin degradation products, when using assays based on the activation of plasminogen by streptokinase followed by the hydrolysis of a synthetic chromogenic substrate. This source of error could be eliminated by presence of fibrinogen in excess in the plasminogen assay, thereby securing maximum stimulation of the plasminogen-streptokinase complex. The presence of cryoglobulin in plasma interferes with the assessment in euglobulins of plasminogen activator activities. Experiments indicate that tissue-type plasminogen activator adsorb cryoglobulins and that a cold-promoted activation of the factor XII-dependent proactivator system of fibrinolysis is related to the presence of cryoglobulins. Experiments supported the existence of an as yet not characterized factor XII-dependent proactivator. Strictly optimized procedures for the preparation of euglobulins for the accurate determination of plasminogen activators were recommended. The determination of plasminogen activator inhibition in plasma was optimized and simplified. The amidolytic assay of antithrombin-III was shown to be influenced by adsorption to laboratory utensils and aggregation of thrombin. This error could be corrected by protection with additives (Tween 80, polyethyleneglycol 6,000), which also improved the solubility of the chromogenic substrates in aqueous media. The role of thrombosis in myocardial infarction was reviewed.(ABSTRACT TRUNCATED AT 400 WORDS)