Evaluation of the Fibrin Plate Method for Estimating Plasminogen Activators

1972 ◽  
Vol 28 (01) ◽  
pp. 075-088 ◽  
Author(s):  
N. A Marsh ◽  
C. L Arocha-Pinango
Keyword(s):  
Tranexamic Acid ◽  
Plasminogen Activators ◽  
Heated Plate ◽  
Plasminogen Activation ◽  
Response Curves ◽  
Plate Method ◽  
Single Plate ◽  
Effect Of Additives ◽  
Threefold Increase ◽  
Fibrin Plate

SummaryA study was carried out in order to evaluate the Astrup and Mullertz fibrin plate method for estimating plasminogen activators.Choice of a suitable fibrinogen substrate was found to be the most important factor in setting up a workable assay. Many preparations contained a large proportion of non-clottable protein and plates made from these fibrinogens were usually unreliable. In addition, plasminogen content varied appreciably between preparations so that sensitivity of the method required careful calibration with each new batch of fibrinogen.The effect of additives in the fibrin plate was considered and it was found that calcium chloride alone was sufficient to ensure a stabilised plate which could be stored at 4° C for some time. The addition of tranexamic acid (AMCHA) was found to be a slightly more convenient way of estimating direct proteolytic activity, compared with the traditional heated plate. However neither method distinguished completely between proteolysis and plasminogen activation.In order to improve the precision of the method, the use of an analysis of variance technique has been studied. This technique provides information on the dose-response curves of test and unknown substances, and in addition produces an approximately threefold increase in precision over single plate tests.

scholarly journals Identification of lumbrokinase by fibrin-plate method, fibrin-zymography and liquid chromatography mass spectrometry

2019 ◽  
Author(s):  
Bingbing Ke’ ◽  
Ruiqing Xian ◽  
Hong Jiang ◽  
Jianghong Guo
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Eisenia Fetida ◽  
Plasminogen Activators ◽  
Liquid Chromatography Mass Spectrometry ◽  
Plate Method ◽  
Bovine Trypsin ◽  
Fibrin Plate ◽  
Human Plasminogen ◽  
Protein Components

AbstractLumbrokinase, extracted from the cultured earthworm of Eisenia fetida, has been widely used as biochemical medicine in China to prevent or treat thrombosis. In the present study, the mechanism of lumbrokinase was investigated using the fibrin plate method. The results revealed that lumbrokinase contained both fibrinolytic and kinase components. The method of fibrin-zymography was used to show the existence and activity of lumbrokinase, and we proved that the fibrin-zymogram gel could be adopted as the identification method of earthworm. Subsequently, the components were identified by mass spectrometry. According to the results, fibrinolytic related components existed in the drug. These proteins were further compared with other serine proteins. The result showed that the identified proteins were similar to human trypsin and bovine trypsin. Besides, some also exhibited similar characteristics with human plasminogen activators. The mentioned results demonstrated that lumbrokinase products contained two major groups of protein components, suggesting two different functions.

Interfering Factors in the Assay of Plasminogen Activators by the Fibrin Plate Method

1979 ◽  
Vol 41 (03) ◽  
pp. 590-600 ◽  
Author(s):  
G Wijngaards
Keyword(s):  
Special Reference ◽  
Plasminogen Activator ◽  
Inhibitory Action ◽  
Human Lung ◽  
Plasminogen Activators ◽  
Uterine Tissue ◽  
Plate Method ◽  
Tissue Extracts ◽  
Fibrin Plate ◽  
Tissue Plasminogen

SummaryThe assay of plasminogen activator activities on fibrin plates was re-evaluated with special reference to fibrinolysis inhibitors present in samples and in fibrin plates. The nature, action and stability of inhibiting material were studied in tissues with considerable differences in activator and inhibitor contents! human lung, liver and placenta. Extracts were tested for inhibitory capacity against purified human uterine tissue plasminogen activator, urokinase and plasmin on fibrin plates prepared from different grades of fibrinogens and fibrin.The tissue extracts inhibited fibrinolysis on fibrin plates to varying degrees, dependent on the sample medium, the type of fibrin plate and the kind of plasminogen activator. The influence of inhibitors in the sample and in the fibrin plate was partly abolished by the presence of 2 M KSCN in the sample. The procedure for preparing the samples as described by Astrup and Albrechtsen did not completely eliminate the inhibitory action against the added plasminogen activators.Comparison of urokinase inhibition with tissue activator inhibition by the tissue extracts as to the degree of denaturation in the Astrup and Albrechtsen procedure showed that they have much in common. Nevertheless, some differences were found which indicated the possible existence of separate urokinase and tissue activator inhibitors or of different inhibition mechanisms for these plasminogen activators.

Factors Involved In the Plasminogen Activation System in Human Breast Tumours

1994 ◽  
Vol 71 (05) ◽  
pp. 684-691 ◽  
Author(s):  
László Damjanovich ◽  
Csaba Turzó ◽  
Róza Ádány
Keyword(s):  
Epithelial Cells ◽  
Connective Tissue ◽  
Plasminogen Activators ◽  
Breast Tumour ◽  
Plasminogen Activation ◽  
Malignant Tumours ◽  
Plasminogen Activation System ◽  
Activation System ◽  
Malignant Breast ◽  
Pai 1

SummaryThe plasminogen activation system is a delicately balanced assembly of enzymes which seems to have primary influence on tumour progression. The conversion of plasminogen into serine protease plasmin with fibrinolytic activity depends on the actual balance between plasminogen activators (urokinase type; u-PA and tissue type; t-PA) and their inhibitors (type 1 and 2 plasminogen activator inhibitors; PAI-1 and PAI-2). The purpose of this study was to determine the exact histological localization of all the major factors involved in plasminogen activation, and activation inhibition (plasmin system) in benign and malignant breast tumour samples. Our results show that factors of the plasmin system are present both in benign and malignant tumours. Cancer cells strongly labelled for both u-PA and t-PA, but epithelial cells of fibroadenoma samples were also stained for plasminogen activators at least as intensively as tumour cells in cancerous tissues. In fibroadenomas, all the epithelial cells were labelled for PAM. Staining became sporadic in malignant tumours, cells located at the periphery of tumour cell clusters regularly did not show reaction for PAI-1. In the benign tumour samples the perialveolar connective tissue stroma contained a lot of PAI-1 positive cells, showing characteristics of fibroblasts; but their number was strongly decreased in the stroma of malignant tumours. These findings indicate that the higher level of u-PA antigen, detected in malignant breast tumour samples by biochemical techniques, does not necessarily indicate increased u-PA production by tumour cells but it might be owing to the increased number of cells producing u-PA as well. In malignant tumours PAI-1 seems to be decreased in the frontage of malignant cell invasion; i.e. malignant cells at the host/tumour interface do not express PAI-1 in morphologically detectable quantity and in the peritumoural connective tissue the number of fibroblasts containing PAI-1 is also decreased.

Dose-response Curves in the Fibrin Plate Assay Fibrinolytic Activity of Proteases

1974 ◽  
Vol 32 (02/03) ◽  
pp. 356-365 ◽  
Author(s):  
F Haverkate ◽  
D. W Traas
Keyword(s):  
Dose Response ◽  
Fibrinolytic Activity ◽  
Enzyme Concentration ◽  
Response Curves ◽  
Agar Gel ◽  
Porcine Tissue ◽  
Plate Assay ◽  
Fibrin Plate ◽  
Different Types ◽  
Dose Response Curves

SummaryIn the fibrin plate assay different types of relationships between the dose of applied proteolytic enzyme and the response have been previously reported. This study was undertaken to determine whether a generally valid relationship might exist.Trypsin, chymotrypsin, papain, the plasminogen activator urokinase and all of the microbial proteases investigated, including brinase gave a linear relationship between the logarithm of the enzyme concentration and the diameter of the circular lysed zone. A similar linearity of dose-response curves has frequently been found by investigators who used enzyme plate assays with substrates different from fibrin incorporated in an agar gel. Consequently, it seems that this linearity of dose-response curves is generally valid for the fibrin plate assay as well as for other enzyme plate bioassays.Both human plasmin and porcine tissue activator of plasminogen showed deviations from linearity of semi-logarithmic dose-response curves in the fibrin plate assay.

Fibrinolytic Activity of Cerebro-Spinal Fluid and the Development of Artificial Cerebral Haematomas in Dogs

1969 ◽  
Vol 21 (02) ◽  
pp. 294-303 ◽  
Author(s):  
H Mihara ◽  
T Fujii ◽  
S Okamoto
Keyword(s):  
Cerebrospinal Fluid ◽  
Carboxylic Acid ◽  
Fibrinolytic Activity ◽  
Clinical Implications ◽  
Trans Form ◽  
Plate Method ◽  
Blood Samples ◽  
Fibrin Plate ◽  
The Brain

SummaryBlood was injected into the brains of dogs to produce artificial haematomas, and paraffin injected to produce intracerebral paraffin masses. Cerebrospinal fluid (CSF) and peripheral blood samples were withdrawn at regular intervals and their fibrinolytic activities estimated by the fibrin plate method. Trans-form aminomethylcyclohexane-carboxylic acid (t-AMCHA) was administered to some individuals. Genera] relationships were found between changes in CSF fibrinolytic activity, area of tissue damage and survival time. t-AMCHA was clearly beneficial to those animals given a programme of administration. Tissue activator was extracted from the brain tissue after death or sacrifice for haematoma examination. The possible role of tissue activator in relation to haematoma development, and clinical implications of the results, are discussed.

Comparative Study of Proactivators of the Fibrinolysin System in Three Mammalian Species

1969 ◽  
Vol 21 (03) ◽  
pp. 594-603 ◽  
Author(s):  
Y Takada ◽  
A Takada ◽  
J. L Ambrus
Keyword(s):  
Guinea Pig ◽  
Comparative Study ◽  
Human Plasma ◽  
Mammalian Species ◽  
Gel Filtration ◽  
Plasminogen Activators ◽  
The Other ◽  
Rabbit Plasma ◽  
Plate Test ◽  
Fibrin Plate

SummarySephadex gel filtration of human plasma gave results suggesting the presence of two proactivators of plasminogen, termed proactivators A and B.Activity resembling that of proactivator A was found in rabbit plasma, but not in guinea pig plasma.Plasminogen activators produced by the interaction of proactivator A of human plasma with streptokinase had no caseinolytic or TAMe esterolytic effect.Proactivator A can be separated in a form apparently free from plasminogen, as shown by the heated fibrin plate test and by immunological analysis. On the other hand, proactivator B concentrates prepared so far are contamined with plasminogen.Human proactivators appear to be far more susceptible to streptokinase than are rabbit proactivators.Inhibitors of the fibrinolysin system were observed in the plasmas of all 3 species. These inhibitors are not present in the euglobulin fraction of plasma. Sephadex fractionation of euglobulin fractions results in proactivator preparations that do not contain inhibitors.

Plasminogen Activation by Invasive Human Pathogens

1997 ◽  
Vol 77 (01) ◽  
pp. 001-010 ◽  
Author(s):  
Michael D P Boyle ◽  
Richard Lottenberg
Keyword(s):  
Plasminogen Activators ◽  
Plasminogen Activation ◽  
Potential Importance ◽  
Human Pathogens

SummaryIn this review the interaction between invasive human pathogens expressing plasmin(ogen) receptors and/or producing plasminogen activators with the human plasmin(ogen) system is described. Evidence is presented for multiple mechanisms by which human pathogens can acquire a surface bound form of plasmin that cannot be regulated by host serpins. The potential importance of these pathways in providing the organisms with the ability to cross tissue barriers is discussed.

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