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.     

The role of urokinase and urokinase inhibitor in tumour cell metastasis

In this paper we summarize experiments in which murine melanoma B16-F1 cells of low metastatic potential were transfected with the human gene for prep rourokinase. B16-F1 cells selected for their secretion of high amounts of the human urokinase gene product (3- to 4-fold higher plasminogen activator activity than control cells) formed between 4- and 16-fold greater numbers of lung tumours in C57BL mice after tail vein injection. In correlative results, highly malignant B16-F10 melanoma cells transfected with a urokinase antisense sequence showed a significant inhibition of endogenous urokinase expression and a corresponding significant decrease in the number of tumours formed after tail vein injection.The human urokinase gene product expressed by the transfected cells does not bind to murine cell surface urokinase receptors, and the results may provide direct evidence for a role of secreted (non-surface bound) urokinase in the extravasation steps of tumour cell metastasis. These human urokinase secreting cells also showed a greater ability to spontaneously metastasize from a primary footpad tumour to the mouse lung.Extending the study of urokinase to the role of its natural inhibitor, plasminogen activator inhibitor 2 (PAI-2), we determined the constitutive and phorbol ester inducibility of the mRNA for PAI-2 and urokinase in human A375 melanoma cell lines and human prostate PC3 carcinoma cell lines of different metastatic potentials. Analysis of the changes in PAI-2 and urokinase mRNA levels indicate that variation in the levels of PAI-2 may be more important than urokinase in explaining the different metastatic abilities of A375 and PC3 sublines. Urokinase activity may promote metastasis by its initiation of a specific proteolysis pathway leading to the activation of metalloproteinases. We have previously shown that the metalloproteinase inhibitor, recombinant tissue inhibitor of metalloproteinases (rTIMP), inhibits B16 melanoma cell lung colonization.     

Immunological comparison between human and rat plasminogen activators in blood and the vessel wall.

To evaluate the rat as an experimental model for plasminogen activator research, the ability of antibodies specific for human tissue type plasminogen activator and urokinase to suppress the plasminogen activator activity in whole plasma and in the vessel wall was studied in both rat and man. Plasminogen activator activity in plasma was assayed on fibrin plates containing plasminogen. Plasminogen activator in the vessel wall was shown by the fibrin side technique. Antibodies against human tissue type melanoma cell activator and urokinase were raised in goats and mixed into the fibrin film or the fibrin plates. In both species antibodies to melanoma cell activator were able to suppress the plasminogen activator activity completely in plasma and in the vessel wall. Anti-urokinase, however, had no suppressing effect. In rat plasma the inhibitory effect on the fibrinolytic activity was seen only with high concentrations of antibodies against melanoma cell activator, which suggests that rat plasminogen activator in plasma and vessel walls is similar to, but not identical with, human tissue type plasminogen activator.     

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.     

Human lung cancer cell lines express cell membrane complement inhibitory proteins and are extremely resistant to complement-mediated lysis; a comparison with normal human respiratory epithelium in vitro,and an insight into mechanism(s) of resistance

Human lung cancer expresses cell membrane complement inhibitory proteins (CIP). We investigated whether human lung cancer cell lines also express cell-membrane CIP molecules and whether the biology of CIP molecules in these cell lines differs from that of CIP in normal human respiratory epithelium in culture. The cell lines ChaGo K-1 and NCI-H596 were compared with normal human nasal epithelium in primary cultures in respect to the level of cell membrane CIP expression of membrane cofactor protein (MCP; CD46), decay-accelerating factor (DAF; CD55) and CD59, in respect to the level of cell resistance to complement-mediated lysis, and in respect to the contribution of cell membrane CIP to cell resistance against complement-mediated lysis. We found, using flow cytometry, that both human lung cancer cell lines expressed MCP, DAF and CD59, as did normal nasal epithelial cells. However, normal cells showed a large subpopulation of low DAF-expressing cells (60% of all cells) and a smaller subpopulation of high DAF-expressing cells (40%), while the lung cancer cell lines showed only one cell population, of high DAF expression. In addition, both lung cancer cell lines expressed higher MCP levels, and NCI-H596 cells showed higher levels of CD59. Cell resistance to complement-mediated lysis of both lung cancer cell lines was much higher than that of normal cells. Fifty percent normal human serum, under the same concentrations of complement activators, induced lysis of less than a mean of 10% of lung cancer cells, while lysing up to a mean of 50% of nasal epithelial cells. Lung cancer cell resistance to complement was due to its ability to prevent significant activation of complement upon its cell membrane, as manifested by a failure of complement activators to increase cell membrane deposition of C3-related fragments. The exact mechanism for this resistance remains obscure. Unexpectedly, neutralizing antibodies, anti-MCP and anti-DAF were entirely ineffective and anti-CD59 was only slightly effective (18% mean cell lysis) in increasing the susceptibility of the lung cancer cell lines to complement, while the same antibodies were very effective in facilitating complement-mediated lysis of the normal nasal epithelial cells (50% mean cell lysis with CD59 MoAb). On the other hand, detachment of DAF and CD59 by phosphatidylinositol-specific phospholipase C (PIPLC) from the lung cancer cell lines abrogated their resistance to lysis. We suggest that the biology of cell membrane CIP molecules in human lung cancer cell lines is different from that of CIP in normal respiratory epithelial cells. Human lung cancer cell lines are able to prevent significant complement activation upon its cell membrane and are therefore especially resistant to complement-mediated lysis. Complement resistance may serve this common and highly lethal human cancer as an escape mechanism from the body's immunosurveillance and prevent effective immunotherapy with tumour-specific MoAbs.     

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.     

Quantification of plasminogen activator activity associated with herpesvirus-transformed cells

Herpesvirus-transformed cell lines were examined for plasminogen activator (PA) activity using a quantitative assay. Previous results of cold fibrin overlay assays indicated that herpesvirus-transformed hamster cell lines produce fibrinolytic activity. Quantification of this activity involved the use of an 125I-fibrin lysis assay in which medium previously incubated with transformed or normal cells was tested for its ability to lyse 125I-fibrin polymers. This assay indicated an enhanced production of plasminogen-dependent fibrinolytic activity by transformed hamster cells compared to hamster embryo fibroblasts. The kinetics of secretion failed to reveal a significant difference in plasminogen activator activity in cells transformed by either herpes simplex virus types 1 or 2 (HSV-1 or HSV-2); however, transformed cells exhibited a significant increase in activity over non-transformed cells. Further characterization of PA activity associated with cells transformed by HSV-1 or HSV-2 has revealed that the protease is secreted and can function extracellularly. Extracellular PA activity produced by HSV-transformed cells is detected more efficiently than the cell-associated enzyme. Extracellular PA can be induced in two different species of cells by infection with partially inactivated HSV-2. Lytic infection of human embryo lung cells by HSV-2 strain 333 did not enhance activity, but infection of these cells with virus inactivated by u.v. irradiation resulted in increased PA levels from the cells. Increased enzyme levels were also detected in hamster embryo fibroblasts infected with partially inactivated virus. Further investigation of this enzyme function may determine whether increased levels of protease will indicate oncogenic transformation in vitro by herpesviruses.     

Inhibition of urokinase activity and prevention of urokinase receptor binding by monoclonal antibodies

Two murine monoclonal antibodies produced against human urokinase-type plasminogen activator were characterized with respect to their antigen-binding specificity and their effects on urokinase activity and urokinase receptor binding. One of the antibodies binds to the protease domain of urokinase (Kass = 2.1 X 10(7) M-1). Antibody binding inhibits catalysis of plasminogen activation. It does not, however, affect amidolytic activity of urokinase towards the chromogenic substrate D-Val-Leu-Arg-p-nitroanilide. The antibody thus appears to interfere with plasminogen binding without directly affecting catalytically active amino acid residues of the enzyme. The other antibody binds to the aminoterminal fragment of urokinase (Kass = 1.0 X 10(7) M-1) and prevents binding of the enzyme to high affinity receptors on human granulocytes. Binding of this antibody neither influences plasminogen activation nor the amidolytic activity of urokinase. Both antibodies are potentially useful for the further analysis and manipulation of urokinase function.     

Increased synthesis of fibrinolytic inhibitor induced by muramyl dipeptide derivatives in human cultured endothelial cells

A decreased fibrinolytic activity induced by bacterial products and some muramyl peptides has been previously demonstrated in macrophages preparations. Since vascular endothelial cells are important for the fibrinolytic balance, we have studied the effects of MDP derivatives on cultured endothelial cells. The supernatant of MDP and murabutide treated cell cultures exhibited an increased fibrinolytic inhibitory activity when tested with urokinase. The MDP(D-D)-treatment had no effect. This increased inhibitory activity was detectable in the supernatant after a 6 h treatment and was suppressed by the addition of puromycin to the cell cultures. Furthermore, the endothelial cell culture supernatant also reduced the lytic activity of the human plasma plasminogen activator induced by venostasis. This was enhanced by MDP treatment of the cultures. These in vitro results suggested that adjuvant-active muramyl peptides may regulate the fibrinolytic balance at the vessel wall level. This could be of possible significance in the transendothelial cell migration where the role of plasminogen activator(s) has been involved.     

FATE OF INTRAVENOUSLY INJECTED CULTURED HUMAN CANCER CELLS IN THE LUNGS OF CONGENITALLY ATHYMIC NUDE MICE

Three cultured cell lines of human lung cancer with varied degrees of thromboplastic and fibrinolytic activities were injected subcutaneously or intravenously into congenitally athymice nude mice, and their fate was observed with light and electron microscopes. All the cell lines formed solid tumor masses at the sites of subcutaneous injection. When the cancer cells with high or moderate thromboplastic activity were injected intravenously, marked platelet aggregation and fibrin deposition, followed by neutrophilic infiltration, were seen around the cancer cells arrested in pulmonary arterioles or capillaries. Platelet aggregation and fibrin deposition were less conspicuous, when the cancer cells with low thromboplastic activity were injected. No metastatic foci were found 2 weeks after the intravenous injection of each cancer cell line. These results suggest that thromboplastic activity of human cancer cells is closely related to thrombus formation at the sites of lodgement and that thrombus develops without participation of immunological mechanisms.     

Immunohistochemical localization of coagulation, fibrinolytic and antifibrinolytic markers in adenocarcinoma of the lung.

Extravascular coagulation and fibrinolysis are intimately involved in and modulate cancer cell growth, invasion and metastasis. Samples from resection specimens of patients with primary lung cancer (adenocarcinomas) were tested with monoclonal (MAb) and polyclonal (PAb) antibodies against various factors of the coagulation or fibrinolysis systems, or against antigens of inflammatory or proliferating cells. MAb Ki-67 specific to nuclear antigens of proliferating cells showed a distinct but variable staining of cell nuclei throughout the tumor tissue. Nests of tumor tissue stained with cytokeratin-specific antibodies (PKK1), whereas other parts were negative. Fibrin(ogen) and fibronectin were found throughout the tumor tissue stroma and in the alveolar lining, and the most densely stained areas were at the transition zone between normal and tumor tissue. Fibrinolytic system components like tissue plasminogen activators (t-PA), and urokinase (u-PA), and their inhibitors PAI-1 and PAI-2 were all studied. All specimens were negative for t-PA (except endothelial linings), whereas urokinase-specific antibodies stained loosely packed tumor cells and macrophages within the tumor stromal tissue and alveolar septa. Both PAI-1 and PAI-2 were most prominently expressed within interstitial and alveolar macrophages. A weaker staining of tumor tissue cells was demonstrated. Inflammatory cells like macrophages and T lymphocytes were located in aggregates or diffusely spread within tumor stromal tissue. The inflammatory reaction was most intense at the border between normal lung and tumor tissue.     

Cells of the human myelomonocytic line RC-2A synthesize tissue factor-like procoagulant and urokinase-type plasminogen activator

Cells of the myelomonocytic leukemia cell line RC-2A were studied for their ability to synthesize clotting-promoting and fibrinolytic factors. The cells were observed to generate procoagulant activity (PCA) in readily measurable quantities. Incubation of RC-2A cells with phorbol myristate acetate (PMA; 3 ng/ml) or phytohemagglutinin (PHA, 10 micrograms/ml) for 18 h resulted in a 4-5-fold increase in PCA relative to unstimulated control. The PCA of RC-2A cells was tissue factor-like in that it was dependent on factor VII but not on factors VIII or IX. RC-2A cells also produced plasminogen activator (PA). Secreted PA was approximately 70% of the PA of an identical number of human monocyte-derived macrophages; fresh isolated monocytes synthesized virtually no PA. Compared to macrophages, RC-2A cells secreted less or no PA-inhibitors. Lysates of RC-2A cells contained over three times more PA than lysed macrophages. Stimulation of the cells with lectins (PHA, concanavalin A) or PMA was followed by a modest (2-3-fold) increase in PA. Enzyme immunoassay with antibodies to urokinase (u-PA) or tissue-type PA (t-PA) identified the RC-2A plasminogen activator as being of urokinase type.     

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.     

Host urokinase-type plasminogen activator participates in the release of malaria merozoites from infected erythrocytes

Malaria infection of red blood cells is associated with plasminogen activation. Surface immunofluorescence and immunoprecipitation experiments, using specific polyclonal and monoclonal antibodies raised against human urokinase, demonstrate that this activity is due to the binding of host urokinase-type plasminogen activator to the surface of erythrocytes infected by mature forms of Plasmodium falciparum malaria parasites. Depletion of urokinase from the culture medium leads to the inhibition of merozoite release and the accumulation of segmenter-infected erythrocytes; this inhibition is reversed by the addition of human single-chain or two-chain urokinase. These findings are consistent with host urokinase being involved in the process of merozoite release from the red blood cell.     

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.     

Regulation of fibrin deposition by malignant mesothelioma.

Malignant mesothelioma (MM) is a locally aggressive tumor that spreads by poorly understood mechanisms. Because neoplastic spread has been linked to altered fibrin turnover, we used immunohistochemistry of nine MM and three fibrous tumors of the pleura to confirm in vivo fibrin deposition and expression of selected coagulation and fibrinolytic reactants in MM. Tumor-associated fibrin was readily detectable at site of tissue invasion. Little fibrin was distributed within the tumor, but tissue factor and tissue factor pathway inhibitor, urokinase, urokinase receptor, and plasminogen activator inhibitors 1 and 2 were all detected in either epithelioid or sarcomatous areas of MM. We used the MS-1 human pleural mesothelioma cell line to determine how expression of these reactants is regulated. Fibrinolytic activity of MS-1 is mainly due to urokinase and is responsive to cytokine stimulation. Functional extrinsic activation and prothrombinase complexes assemble at the cell surface. MM express procoagulants as well as fibrinolytic reactants in vivo and in vitro that promote local fibrin formation and remodeling. Fibrin deposition occurs primarily at areas of tissue invasion and could promote local extension of this neoplasm. Sparsity of fibrin within the central portions of the tumor stroma suggests that local resorption of transitional fibrin occurs at sites of established MM.     

Localization of plasminogen activator activity within normal and injured lungs by in situ zymography

During inflammatory lung injury, the fibrinolytic activity that is normally present within bronchoalveolar lavage (BAL) fluid (BALF) is often suppressed due to increased levels of inhibitors, including plasminogen activator inhibitor (PAI)-1. Despite this suppression, BALF frequently contains fibrin degradation products, indicating persistence of fibrinolytic activity within the lung. To address this discrepancy and determine the sites where plasminogen activation is occurring, we developed an in situ zymographic technique for frozen sections of lung tissue that localizes plasminogen activator activity at the cellular level. After validating the method using enzyme inhibitors and mice with genetic manipulations of their plasminogen system genes, we applied the technique to lungs of normal and bleomycin-exposed mice. In normal mice, plasminogen activator activity was localized to bronchial epithelial cells, cells of the alveolar walls, and alveolar macrophages. After bleomycin exposure, in situ zymography showed that, despite loss of fibrinolytic activity within BALF, abundant enzymatic activity was associated with aggregates of inflammatory cells. PAI-1-deficient mice that are protected from bleomycin-induced fibrosis had preserved plasminogen activator activity in BALF and increased tissue activity, as determined by in situ zymography. We conclude that analysis of BALF does not adequately reflect the fibrinolytic activity that persists within microenvironments of the lung during inflammation.     

G-CSF increases secretion of urokinase-typeplasminogen activator by human lung cancer cells

We reported previously that granulocyte colony-stimulating factor (G-CSF) can promote the invasion ofhuman lung cancer cell lines in vitro. However, the exact mechanism of its stimulatory effect on invasionremains to be elucidated. In the present study we mainly focused our attention on the components of theplasminogen activation system in human lung cancer cell lines, because of the central role that plasminogenactivators play in regulating extracellular proteolysis. We showed that G-CSF induced a dose-dependentincrease in the urokinase-type plasminogen activator (uPA) activity in the conditioned medium of a PC-9lung cancer cell line. When the amounts of uPA activity were quantitated by densitometry, we found thateven at a concentration of 0.01 mg/ml, G-CSF had a stimulatory effect on the uPA release, while highconcentrations caused a 3.6-fold increase at a maximum concentration of 1 mg/ml. A Western blot analysisof the conditioned medium confirmed the findings observed in a zymographic analysis. The observed increasein uPA protein was paralleled by a significant increase in the uPA mRNA levels after treatment withG-CSF. However, our experiments failed to identify any alteration in the plasminogen activator inhibitor(PAI) secretion caused by G-CSF. In addition, we also found the expression of G-CSF receptor by PC-9cells, suggesting the possible pathway activated by G-CSF.©Kluwer Academic Publishers     

Modulation of urokinase receptors on human synovial cells and osteoarthritic chondrocytes by diacetylrhein

On the basis of both 125I-labelled urokinase-like plasminogen activator binding analysis and transmission electron microscopy of an urokinase-gold complex, we have shown the presence of specific receptors for human urokinase on the cell membrane of human synovial cells. By radio-ligand binding experiments we have shown the existence of similar receptors on the surface of human chondrocytes. In both cases the specific binding is attributable to interaction between the receptor and the A chain of the ligand, as previously shown in other cell model systems. Treatment of synoviocytes with 1,8-diacetoxy-anthraquinone-3-carboxylic acid (diacetylrhein) is able to reduce the number of surface urokinase receptors. At the same time the drug can reduce the fibrinolytic activity released into the culture medium of human synovial cells. Preliminary data indicate that chondrocytes from osteoarthritic patients have a larger number of urokinase receptors than chondrocytes of normal patients. Diacetylrhein can restore the receptor number to normal levels; the amount of urokinase in the synovial fluid of ostroarthritic patients is also reduced. We conclude that the use of this drug has the chance to significantly modify the natural history of osteoarthritis.