Characterization Of Plasminogen Activator Research Articles

Characterization of plasminogen activators in unstimulated and stimulated human whole saliva.

The fibrinolytic activity of saliva from healthy males was studied on plasminogen-free and plasminogen-rich fibrin plates. A cell-bound plasminogen activator in human unstimulated and stimulated whole saliva was demonstrated. The assessed fibrinolytic activities could always be quenched by incorporation into the fibrin plates of IgG antibodies raised against human two-chain tissue-plasminogen activator (t-PA), while additional experiments indicated the absence in normal human saliva of urokinase-like and factor XII-dependent plasminogen activators as well as the absence of inhibitors of fibrinolysis. Thus, t-PA is the only type of plasminogen activator in normal human saliva. The present findings seem to support our recent clinical observations of a decrease in the incidence of bleeding complications and need for replacement therapy in hemophiliacs undergoing oral surgery during local antifibrinolytic therapy with tranexamic acid. Whether the findings might also be of importance in other pathological conditions of the oral cavity, such as impaired wound healing, remains to be elucidated.

Characterization of plasminogen activator from two human renal carcinoma cell lines

Plasminogen activator (PA) activity was identified in the conditioned medium of two human renal carcinoma cell lines, Cur and Caki-1. PA activity of medium, following chromatography on Con A-Sepharose, was divided into effluent and eluate fractions, the latter obtained after elution with methyl mannoside. The ratio of PA activity in effluent:eluate was 90:10 for Caki-1 and 60:40 for Cur. The PA of both effluent fractions and the Caki-1 eluate fraction was of the urokinase (UK) type. Identification rested on molecular weight determination by zymography (major component with Mr 52,000 and a less prominent component of 93,000), lack of binding to fibrin, inhibition by anti-UK antibodies, and lack of inhibitory effect of anti-tissue type PA (TPA) antibodies or the Erythrina trypsin inhibitor, which inhibits TPA but not UK. PA of the Cur eluate fraction gave a more complex pattern in that it bound significantly to fibrin (like TPA), was completely inhibited by both anti-UK and anti-TPA antibodies, but was unaffected by Erythrina trypsin inhibitor. These results raise the possibility of an unusual PA-like enzyme that immunologically cross reacts with anti-UK and anti-TPA. Most of the PA of both cell lines was secreted in a latent form that could be activated by trypsin treatment. The latency appears to result largely from secretion of urokinase proenzyme, which is consistent with the Mr 52,000 of the major PA species and the insensitivity to diisopropyl fluorophosphate inhibition prior to trypsin activation. However, in addition, a UK binding component was found in the conditioned medium, which produced an Mr 93,000 component by reaction with UK.

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.

Isolation and characterization of plasminogen activators from hyperlastic and malignant prostate tissue

Plasminogen activators from prostate tissue were purified to apparent homogeneity by a procedure involving reverse ammonium sulfate gradient solubilization, chromatography on gelatine-Sepharose, gel filtration on Sephadex G-150, and chromatography on Con A-Sepharose as a final step. Two activators were obtained. The predominant one exhibited physicochemical, immunochemical and functional properties indistinguishable from human urinary high molecular weight urokinase. The other one, which amounted to about 20% was immunochemically related to tissue type plasminogen activator and its plasminogen activator activity was enhanced by addition of CNBr-fibrinogen framents in a similar pattern as for the vascular plasminogen activator. The molecular weight, however, and enzymatic activities on the synthetic low molecular weight paranitroanilide substrates pyro-Glu-gly-Arg-p NA and H- D-Ile-Pro-Arg -p NA were different to vascular plasminogen activator but similar to high molecular weight urinary urokinase.

Localization and immunological characterization of plasminogen activators in human prostate tissue.

In tissue sections of human hyperplastic as well as carcinomatous prostate glands fibrinolytic activity could be attributed to collecting ducts and vessel walls. Inhibition studies with antibodies directed against human urokinase (u-PA) or tissue plasminogen activator (t-PA) revealed the presence of a plasminogen activator completely inhibitable by anti-t-PA IgG in the vessel walls, while in collecting ducts the plasminogen activator activity was inhibited by anti-u-PA IgG to only 80% and by anti-t-PA IgG to about 20%. A mixture of anti-u-PA and anti-t-PA IgG was able to inhibit fibrinolytic activity in collecting ducts completely. In carcinomatous prostate glands total fibrinolytic activity was significantly higher, but localization and relative contribution of anti-u-PA-inhibitable and anti-t-PA-inhibitable plasminogen activators remained the same.

Characterization of plasminogen activator in human cervical cells

Plasminogen activator activity was detected in human gynecologic specimens using a synthetic fluoregenic peptide substrate assay and confirmed by an 125I-labeled fibrin plate assay. Epithelial cells in these samples contain enzymatic activity that biochemically resembles both the well-characterized plasminogen activator, urokinase, and the less-specific plasminogen activator, trypsin. Inhibition of the cervical cell activity by diisopropylfluorophosphate and p-nitrophenyl- p′-guanidinobenzoate demonstrates that, like urokinase and trypsin, this plasminogen activator is also a serine protease. Polyacrylamide gel electrophoresis of plasminogen that had been incubated with cervical cells indicated the same mechanism of plasminogen activation as exhibited by urokinase. We attempted to correlate plasminogen activator activity of each sample with cytomorphologic diagnosis. Three of the four dysplastic samples analyzed showed higher plasminogen activator activity than did the normal samples.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

The Isolation and Characterization of Plasminogen Activators (Urokinase) from Human Urine*

The Isolation and Characterization of Plasminogen Activators (Urokinase) from Human Urine^*