Activation of pro-urokinase and plasminogen on human sarcoma cells: a proteolytic system with surface-bound reactants.

Human HT-1080 fibrosarcoma cells produce urokinase-type plasminogen activator (u-PA) and type 1 plasminogen activator inhibitor (PAI-1). We found that after incubation of monolayer cultures with purified native human plasminogen in serum-containing medium, bound plasmin activity could be eluted from the cells with tranexamic acid, an analogue of lysine. The bound plasmin was the result of plasminogen activation on the cell surface; plasmin activity was not taken up onto cells after deliberate addition of plasmin to the serum-containing medium. The cell surface plasmin formation was inhibited by an anticatalytic monoclonal antibody to u-PA, indicating that this enzyme was responsible for the activation. Preincubation of the cells with diisopropyl fluorophosphate-inhibited u-PA led to a decrease in surface-bound plasmin, indicating that a large part, if not all, of the cell surface plasminogen activation was catalyzed by surface-bound u-PA. In the absence of plasminogen, most of the cell surface u-PA was present in its single-chain proenzyme form, while addition of plasminogen led to formation of cell-bound two-chain u-PA. The latter reaction was catalyzed by cell-bound plasmin. Cell-bound u-PA was accessible to inhibition by endogenous PAI-1 and by added PAI-2, while the cell-bound plasmin was inaccessible to serum inhibitors, but accessible to added aprotinin and an anticatalytic monoclonal antibody. A model for cell surface plasminogen activation is proposed in which plasminogen binding to cells from serum medium is followed by plasminogen activation by trace amounts of bound active u-PA, to form bound plasmin, which in turn serves to produce more active u-PA from bound pro-u-PA. This exponential process is subject to regulation by endogenous PAI-1 and limited to the pericellular space.

Tissue-type plasminogen activator in subretinal fluid.

The plasminogen activation system is quantitatively the major mechanism of extracellular proteolysis. To evaluate its role in retinal detachment, plasmin and plasminogen activators were measured in subretinal fluid (SRF) from 12 eyes of twelve patients. Plasmin was detected in 5 eyes (mean 6.26 micrograms/ml, SD = 3.7 micrograms/ml). Tissue-type plasminogen activator was present in 5 eyes (mean activity 0.33 IU/ml, SD = 0.24 IU/ml) but the activity did not associate with the plasmin activity in all of the SRF samples. Urokinase-type plasminogen activator was not detected in SRF. We conclude that the plasmin system has been activated in SRF in some eyes with retinal detachment with tissue-type plasminogen activator as the predominant activator. Plasmin in SRF may enhance dispersion of pigment epithelial cells into the subretinal space and the vitreous, a phenomenon seen frequently in eyes with retinal detachment.

Epstein Barr virus (EBV) antibodies in the diagnosis of NPC--comparison between IFA and two commercial ELISA kits.

BACKGROUND: Antibodies to Epstein Barr Virus (EBV) antigens have been used for the diagnosis of nasopharyngeal carcinoma (NPC). While immunofluorescence assays (IFA) of IgA antiviral capsid and early antigens have been the mainstay of this diagnosis, enzyme immunoassays (ELISA) of various EBV antigens are now available. However in almost all of these assays, the sensitivities and specificities have been calculated using blood donors and normal hospital staff as controls, who may not be the most appropriate controls. We wanted to evaluate the usefulness of IFA and ELISA of various EBV antigens in a clinical setting to distinguish between patients with NPC and those suspected of NPC but being biopsy negative. METHODS: Between January 1987 and June 1988, 322 consecutive patients suspected of NPC and who had a post-nasal biopsy were studied. Blood was taken for EBV tests before diagnosis. Tests included IFA and ELISA IgA anti-VCA and anti-EA and ELISA IgA and IgG anti-ribonucleotide reductase, a cloned EA antigen. RESULTS: IFA IgA anti-VCA together with IFA IgA anti-EA both at a cut-off of 1:10 gave the best discrimination between patients with NPC and those suspected of NPC but were biopsy negative. CONCLUSION: The ELISA IgG anti-ribonucleotide reductase test is convenient to perform and looks very promising. An ELISA using a cocktail of cloned EA peptides may be even better.

Urokinase-type plasminogen activator in colorectal carcinomas and adenomatous polyps: quantitative expression of active and proenzyme.

Total urokinase-type plasminogen activator (u-PA) content (proenzyme plus active enzyme) was significantly higher in 20 colorectal carcinomas and in 27 adenomatous polyps than in metaplastic polyps and autologous normal mucosa. u-PA content was also markedly increased in adenomatous polyps and autologous colonic mucosa removed from familial polyposis coli patients. Using a new monoclonal antibody technique to distinguish the proenzyme of u-PA from the active enzyme, we found that 70% of the u-PA in polyp and cancer tissue was present in the proenzyme form compared to 47% in normal colonic mucosa. For colon cancers, there was a significant correlation between their stage of invasiveness and the levels of proenzyme. No correlation was observed between the u-PA content of adenomatous polyps and their size or degree of dysplasia. Study of the u-PA content of the colonic mucosa may offer a useful biochemical correlate of epithelial cell transformation in the colon.

Rapid appearance of plasmin in tear fluid after ocular allergen exposure.

Specific allergens were applied topically to the conjunctivae of 18 allergic patients. In cases with positive allergic reaction, proteolytic activity, identified as plasmin using zymographic analysis and a monoclonal anticatalytic antibody to plasmin, was found to appear within 3-5 min in the tear fluid, reaching concentrations of 2.2-28.6 micrograms/ml. No plasmin was detected, either in the tear fluid of 30 unchallenged non-atopic control persons, or atopic patients provoked with a test material that had given no reaction in a skin prick test. These findings suggest that generation of plasmin is a consequence of the conjunctival allergic reaction.

Isolation of plasma membrane from human blood monocytes. Subcellular fractionation and marker distribution.

The isolation of plasma membrane from human peripheral blood monocytes is described. Monocytes were isolated by centrifugal elutriation, to eliminate an adherence step, thus minimizing functional and surface antigenic alterations to the cells. Monocytes were surface-labelled with a radiolabelled monoclonal antibody, 125I-WVH-1, and then disrupted by nitrogen cavitation. Membranes were separated according to equilibrium buoyant density by isopycnic centrifugation on a sucrose gradient. The subcellular membranes were localized using marker enzymes for the plasma membrane, 5'-nucleotidase and leucine 2-naphthylamidase (leucine aminopeptidase), and for intracellular membranes: galactosyltransferase (Golgi), arylsulfatase C (endoplasmic reticulum), monoamine oxidase (mitochondria), catalase (peroxisomes), beta-hexosaminidase and beta-glucuronidase (lysosomal vesicles) and lactate dehydrogenase (cytosol). The monoclonal antibody 125I-WVH-1 was shown to label the plasma membrane, as judged by known markers, and represents a highly specific trace label, applicable to the use of plasma membrane as an immunogen for monoclonal antibody production. The NAD-splitting enzyme, NAD+ nucleosidase, was detected and its presence on the plasma membrane was demonstrated. The subcellular localization of non-specific esterase in human mononuclear phagocytes is controversial. No evidence was found for alpha-naphthyl acetate esterase activity on the plasma membrane or in lysosomal vesicles. However, a membrane-bound esterase in fractions with properties similar to the smooth endoplasmic reticulum was detected.

Monoclonal antibodies inhibitory to human plasmin. Definitive demonstration of a role for plasmin in activating the proenzyme of urokinase-type plasminogen activator.

Four monoclonal antibodies raised against purified human plasminogen were characterized for their effects on the activation of plasminogen and on three enzymic properties of plasmin: (a) thioesterolysis, (b) fibrinolysis, (c) conversion of high-Mr urokinase to its low-Mr form. None of the monoclonal antibodies inhibited plasminogen (plg) activation by urokinase. The monoclonal antibodies characterized in this study fell into three groups. Anti-plg 1 inhibited (a), (b) and (c), while anti-plg 2 inhibited activities (a), (b) and (c) to varying degrees but also formed complexes with plasmin that were stable to sodium dodecyl sulphate. Anti-plg 3 and anti-plg 4 inhibited only activity (c). Selective use of these monoclonal antibodies demonstrated unequivocally that plasmin mediates the activation of the proenzyme form of urokinase-type plasminogen activator. Besides their use in affinity chromatography, therefore, these antibodies are valuable for defining the role of plasmin in the mechanisms of extracellular matrix degradation.