Contribution of Streptokinase-Domains from Groups G and A (SK2a) Streptococci in Amidolytic/Proteolytic Activities and Fibrin-Dependent Plasminogen Activation: A Domain-Exchange Study

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.

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Effect of Lipoprotein(a) and LDL on Plasminogen Binding to Extracellular Matrix and on Matrix-dependent Plasminogen Activation by Tissue Plasminogen Activator

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650304 ◽

1996 ◽

Vol 75 (03) ◽

pp. 497-502 ◽

Cited By ~ 10

Author(s):

Hadewijch L M Pekelharing ◽

Henne A Kleinveld ◽

Pieter F C.C.M Duif ◽

Bonno N Bouma ◽

Herman J M van Rijn

Keyword(s):

Extracellular Matrix ◽

Endothelial Cells ◽

Plasminogen Activator ◽

Binding Sites ◽

Umbilical Vein ◽

Single Step ◽

Plasminogen Activation ◽

Structural Homology ◽

Tissue Plasminogen ◽

Umbilical Vein Endothelial Cells

SummaryLp(a) is an LDL-like lipoprotein plus an additional apolipoprotein apo(a). Based on the structural homology of apo(a) with plasminogen, it is hypothesized that Lp(a) interferes with fibrinolysis. Extracellular matrix (ECM) produced by human umbilical vein endothelial cells was used to study the effect of Lp(a) and LDL on plasminogen binding and activation. Both lipoproteins were isolated from the same plasma in a single step. Plasminogen bound to ECM via its lysine binding sites. Lp(a) as well as LDL were capable of competing with plasminogen binding. The degree of inhibition was dependent on the lipoprotein donor as well as the ECM donor. When Lp(a) and LDL obtained from one donor were compared, Lp(a) was always a much more potent competitor. The effect of both lipoproteins on plasminogen binding was reflected in their effect on plasminogen activation. It is speculated that Lp(a) interacts with ECM via its LDL-like lipoprotein moiety as well as via its apo(a) moiety.

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Studies on an Inhibitor of Plasminogen Activation in Human Serum

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649120 ◽

1973 ◽

Vol 30 (02) ◽

pp. 414-424 ◽

Cited By ~ 20

Author(s):

Ulla Hedner

Keyword(s):

Ion Exchange ◽

Human Serum ◽

Antithrombin Iii ◽

Gel Filtration ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Specific Adsorption ◽

Partial Purification ◽

Plasminogen Activation ◽

Preparative Electrophoresis

SummaryA procedure is described for partial purification of an inhibitor of the activation of plasminogen by urokinase and streptokinase. The method involves specific adsorption of contammants, ion-exchange chromatography on DEAE-Sephadex, gel filtration on Sephadex G-200 and preparative electrophoresis. The inhibitor fraction contained no antiplasmin, no plasminogen, no α1-antitrypsin, no antithrombin-III and was shown not to be α2 M or inter-α-inhibitor. It contained traces of prothrombin and cerulo-plasmin. An antiserum against the inhibitor fraction capable of neutralising the inhibitor in serum was raised in rabbits.

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Aggregated, Conformationally Changed Fibrinogen Exposes the Stimulating Sites for t-PA-Catalysed Plasminogen Activation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650269 ◽

1996 ◽

Vol 75 (02) ◽

pp. 326-331 ◽

Cited By ~ 7

Author(s):

Unni Haddeland ◽

Knut Sletten ◽

Anne Bennick ◽

Willem Nieuwenhuizen ◽

Frank Brosstad

Keyword(s):

Conformational Changes ◽

Gel Permeation Chromatography ◽

Tissue Type ◽

Plasminogen Activation ◽

Chromogenic Substrate ◽

Type Plasminogen Activator ◽

Gel Permeation ◽

Self Association ◽

Fibrin Monomers ◽

Stimulation Of

SummaryThe present paper shows that conformationally changed fibrinogen can expose the sites Aα-(148-160) and γ-(312-324) involved in stimulation of the tissue-type plasminogen activator (t-PA)-catalysed plasminogen activation. The exposure of the stimulating sites was determined by ELISA using mABs directed to these sites, and was shown to coincide with stimulation of t-PA-catalysed plasminogen activation as assessed in an assay using a chromogenic substrate for plasmin. Gel permeation chromatography of fibrinogen conformationally changed by heat (46.5° C for 25 min) demonstrated the presence of both aggregated and monomeric fibrinogen. The aggregated fibrinogen, but not the monomeric fibrinogen, had exposed the epitopes Aα-(148-160) and γ-(312-324) involved in t-PA-stimulation. Fibrinogen subjected to heat in the presence of 3 mM of the tetrapeptide GPRP neither aggregates nor exposes the rate-enhancing sites. Thus, aggregation and exposure of t-PA-stimulating sites in fibrinogen seem to be related phenomena, and it is tempting to believe that the exposure of stimulating sites is a consequence of the conformational changes that occur during aggregation, or self-association. Fibrin monomers kept in a monomeric state by a final GPRP concentration of 3 mM do not expose the epitopes Aα-(148-160) and γ-(312-324) involved in t-PA-stimulation, whereas dilution of GPRP to a concentration that is no longer anti-polymerizing, results in exposure of these sites. Consequently, the exposure of t-PA-stimulating sites in fibrin as well is due to the conformational changes that occur during selfassociation.

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Evaluation of the Fibrin Plate Method for Estimating Plasminogen Activators

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649043 ◽

1972 ◽

Vol 28 (01) ◽

pp. 075-088 ◽

Cited By ~ 27

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.

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Plasminogen Activation In Vivo upon Intravenous Infusion of DDAVP

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648390 ◽

1992 ◽

Vol 67 (01) ◽

pp. 111-116 ◽

Cited By ~ 64

Author(s):

Marcel Levi ◽

Jan Paul de Boer ◽

Dorina Roem ◽

Jan Wouter ten Cate ◽

C Erik Hack

Keyword(s):

Monoclonal Antibodies ◽

Plasminogen Activator ◽

Plasma Levels ◽

Fold Increase ◽

Fibrinolytic System ◽

Plasminogen Activation ◽

Plasminogen Activator Activity ◽

Insight Into

SummaryInfusion of desamino-d-arginine vasopressin (DDAVP) results in an increase in plasma plasminogen activator activity. Whether this increase results in the generation of plasmin in vivo has never been established.A novel sensitive radioimmunoassay (RIA) for the measurement of the complex between plasmin and its main inhibitor α2 antiplasmin (PAP complex) was developed using monoclonal antibodies preferentially reacting with complexed and inactivated α2-antiplasmin and monoclonal antibodies against plasmin. The assay was validated in healthy volunteers and in patients with an activated fibrinolytic system.Infusion of DDAVP in a randomized placebo controlled crossover study resulted in all volunteers in a 6.6-fold increase in PAP complex, which was maximal between 15 and 30 min after the start of the infusion. Hereafter, plasma levels of PAP complex decreased with an apparent half-life of disappearance of about 120 min. Infusion of DDAVP did not induce generation of thrombin, as measured by plasma levels of prothrombin fragment F1+2 and thrombin-antithrombin III (TAT) complex.We conclude that the increase in plasminogen activator activity upon the infusion of DDAVP results in the in vivo generation of plasmin, in the absence of coagulation activation. Studying the DDAVP induced increase in PAP complex of patients with thromboembolic disease and a defective plasminogen activator response upon DDAVP may provide more insight into the role of the fibrinolytic system in the pathogenesis of thrombosis.

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Assembly and Activation of the Intrinsic Fibrinolytic Pathway on the Surface of Human Endothelial Cells in Culture

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649800 ◽

1995 ◽

Vol 74 (02) ◽

pp. 698-703 ◽

Cited By ~ 29

Author(s):

Catherine Lenich ◽

Ralph Pannell ◽

Victor Gurewich

Keyword(s):

Endothelial Cells ◽

Umbilical Vein ◽

Factor Xii ◽

Plasminogen Activation ◽

High Molecular Weight Kininogen ◽

Human Endothelial Cells ◽

Intrinsic Pathway ◽

Local Fibrinolysis ◽

Umbilical Vein Endothelial Cells ◽

And Function

SummaryFactor XII has long been implicated in the intrinsic pathway of fibrinolysis, but the mechanism by which it triggers plasminogen activation and targets fibrinolysis has not been established. In the present study, the assembly and function of activated Factor XII (F.XIIa), prourokinase (pro-u-PA), high molecular weight kininogen (H-kininogen), and prekallikrein on human umbilical vein endothelial cells (HUVEC) was investigated. 125I-prekallikrein was shown to bind to HUVEC via receptor-bound H-kininogen in the presence of 50 μM ZnCl2. After the addition of F.XIIa, 78% of the 125I-prekallikrein initially bound to HUVEC was converted to 125I-kallikrein. However, only 6% of the HUVEC-bound 125I-pro-u-PA was thereby activated. This discrepancy was shown to be related to rapid dissociation (>50% within 15 min) of prekallikrein/kallikrein, but not pro-u-PA, from HUVEC. Increasing the level of cell-bound kallikrein increased the portion of cell-bound pro-u-PA activated, indicating that their co-localization was important for this pathway. Finally, F.XIIa was shown to trigger plasminogen activation on HUVEC via this pathway. This assembly of reactants on the endothelium suggests a mechanism whereby local fibrinolysis may be triggered by blood coagulation.

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Mechanism of Human Plasminogen Activation by Streptokinase and Human Plasmin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654808 ◽

1964 ◽

Vol 11 (01) ◽

pp. 085-093

Author(s):

W. F Blatt ◽

JL Gray ◽

H Jensen

Keyword(s):

Proteolytic Activity ◽

Low Ph ◽

Plasminogen Activation ◽

Sensitive Tool ◽

Human Plasminogen

SummaryA sensitive tool has been described for measuring fibrinolysis in reconstituted systems using thrombelastography. Activator mixtures with no appreciable proteolytic activity can similarly be tested in this system when the fibrinogen utilized has sufficient plasminogen present. Exposure of human plasminstreptokinase mixtures formed at pH 7.0 to acid conditions produced a striking loss of activator activity which could not be ascribed to low pH lability of the components, nor to plasmin action on the SK at pH 2.0. This is additional evidence for the hypothesis that human plasmin interacts with SK to form a complex capable of converting human and bovine plasminogen to plasmin.

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Interaction of Heparin with Fibrinolysis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654997 ◽

1963 ◽

Vol 09 (02) ◽

pp. 446-458 ◽

Cited By ~ 5

Author(s):

Rudolf Holemans ◽

Dionysios Adamis ◽

James F Horace

Keyword(s):

Human Plasma ◽

Fibrinolytic Activity ◽

Plasminogen Activation ◽

Fibrinolytic Agents ◽

High Concentration ◽

Enhancing Effect

SummaryHeparin in high concentration inhibits the fibrinolysis of human plasma clots or bovine fibrin by fibrinolytic agents which produce plasminogen activation. Heparin has no effect on the fibrinolytic activity of plasmin or Aspergillus protease.In order to produce inhibition of plasminogen activation heparin requires the presence of a co-factor which is present in citrated human plasma but absent from its euglobulin fraction.In none of the concentrations tested has heparin an enhancing effect on fibrinolysis.

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Plasminogen Activation in Diabetes Mellitus: Normalization of Blood Sugar Levels Improves Impaired Enzyme Kinetics In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657861 ◽

1985 ◽

Vol 54 (02) ◽

pp. 413-414 ◽

Cited By ~ 10

Author(s):

Margarethe Geiger ◽

Bernd R Binder

Keyword(s):

Plasminogen Activator ◽

Blood Sugar ◽

Metabolic Disorders ◽

Normal Values ◽

Diabetic Patients ◽

Type I ◽

Plasminogen Activation ◽

Lag Period ◽

Sugar Levels

SummaryWe have demonstrated previously that fibrin enhanced plasmin formation by the vascular plasminogen activator was significantly impaired, when components isolated from the plasma of three uncontrolled diabetic patients (type I) were used to study plasminogen activation in vitro. In the present study it can be demonstrated that functional properties of the vascular plasminogen activators as well as of the plasminogens from the same three diabetic patients are significantly improved after normalization of blood sugar levels and improvement of HbAlc values. Most pronounced the Km of diabetic vascular plasminogen activator in the presence of fibrin returned to normal values, and for diabetic plasminogen the prolonged lag period until maximal plasmin formation occurred was shortened to almost control values. From these data we conclude that the observed abnormalities of in vitro fibrinolysis are not primarily associated with the diabetic disease, but might be secondary to metabolic disorders caused by diabetes.

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