An enzyme-linked immunosorbent assay (ELISA) for quantitation of plasminogen activator inhibitor 1 (PAI-1) was developed, based on two murine monoclonal antibodies (MA-7D4B7 and MA-7F5), raised against purified PAI-1 from HT-1080 fibrosarcoma cells, which react with non-overlapping epitopes. MA-7D4B7 was coated on microtiter plates and bound PAI-1 antigen was quantitated with MA-7F5 conjugated with horseradish peroxidase.In normal plasma, collected on citrate at pH 7.4, the PAI-1 level is 27 ± 16 ng/ml (mean ± SD, n=ll), with a corresponding value of 19 ± 11 ng/ml (n=12) in plasma collected on acid citrate pH 4.5, which inhibits the release of PAI-1 from platelets. The lower limit of the assay in plasma is 2 ng/ml; the intra-assay, inter-assay and inter-dilution coefficients of variation are 5.2%, 8.0% and 7.1% respectively.This ELISA was used to measure PAI-1 levels in plasma (collected on citrate, pH 7.4) of non-pregnant women and of women at different stages of pregnancy. A progressive increase is observed : before: 20±9 ng/ml, n=7; first trimester: 25 ± 12 ng/ml, n=5; second trimester: 40 ± 25 ng/ml, n=ll; third trimester: 98 ± 46 ng/ml, n=13. A correlation coefficient of 0.70 is found between the duration of pregnancy and the PAI-1 level.Preliminary data indicate that the PAI-1 antigen level is increased in several disease states, including myocardial infarction and deep vein thrombosis.Thus, this newly developed ELISA allows a direct measurement of the fast-acting inhibitor of plasminogen activator in plasma. Application of this assay to plasma of non-pregnant and pregnant women substantiates previous results obtained with the use of functional assays. In order to quantitate PAI-1 antigen circulating in plasma, blood should be collected under conditions that prevent platelet stimulation.
Summary Inflammation may promote malignant invasion by enhancing cancer cell-associated proteolysis. Here we present the effect of inflammatory cytokines on the plasminogen activation system of eight human colon carcinoma cell lines. Tumour necrosis factor a (TNF-a) and interleukin-l,B (IL-1,B) increased in several, but not all, cell lines the production of urokinase-type plasminogen activator (uPA), tissue-type PA (tPA) and plasminogen activator inhibitor type 1 (PAI-1) as analysed by zymography, enzyme immunoassays and Northern analysis. Interleukin 6 (IL-6) had no effect. uPA receptor (uPAR) mRNA levels were also upregulated. However, each individual cell line responded differently following exposure to TNF-a or IL-l,i. For example, there was a dose-dependent up-regulation of uPA and PAI-1 in SW 620 cells, whereas increased uPA production in SW 1116 cells was not accompanied by an increase in PAI-1. The TNF-a stimulatory effect was blocked by anti-TNF-a Fab fragments. All cell lines expressed both types of TNF receptor mRNAs, whereas no transcript for TNF-a, IL-1If, IL-6, IL-6 receptor or the IL-1 receptors was found. Our results demonstrate that TNF-a and IL-l, stimulate the plasminogen activation system in tumour cells but the responses differed even in cells derived from the same tissue origin.
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