Immunological Relationship Between Plasminogen Activator Inhibitors from Different Sources

1987 ◽  
Vol 57 (01) ◽  
pp. 029-034 ◽  
Author(s):  
Göran Urdén ◽  
Joanna Chmielewska ◽  
Tomas Carlsson ◽  
Björn Wiman
Keyword(s):  
Plasminogen Activator ◽  
Polyclonal Antibodies ◽  
Active Form ◽  
Polyclonal Antiserum ◽  
The Other ◽  
Hep G2 ◽  
Inhibitor Activity ◽  
Tissue Plasminogen ◽  
Immunological Relationship ◽  
Different Sources

SummaryPolyclonal antibodies have been raised against the inhibitor moiety in the purified complex between tissue plasminogen activator and its fast inhibitor (PA-inhibitor) in human plasma/ serum. A radioimmunoassay for quantitation of PA-inhibitor antigen was developed. The polyclonal antiserum and a previously described monoclonal antibody against the PA-inhibitor (14) have been used to study the immunological relationship between PA-inhibitors from plasma, serum, platelets, placenta extract and conditioned media from Hep G2 and HT 1080 cells. It was demonstrated that the ratio between PA-inhibitor activity and antigen varied considerably between the different sources. In the plasma samples studied, similar activity and antigen concentrations were found, suggesting that the PA-inhibitor in these samples mainly was in an active form. On the other hand the other sources seemed to contain variable amounts of inactive PA-inhibitor forms. Immunoadsorption experiments revealed that the PA-inhibitor (activity and antigen) from all the sources were specifically bound to the insolubilized antibodies (polyclonal and monoclonal). In no case, however, could active PA-inhibitor be eluted from the immunoadsorption columns. Also the competitive radioimmunoassays suggested that the PA-inhibitors from the different sources studied, were closely immunologically related.

scholarly journals Conversion of the active to latent plasminogen activator inhibitor from human endothelial cells

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 1090-1098
Author(s):  
EG Levin ◽  
L Santell
Keyword(s):  
Endothelial Cells ◽  
Conditioned Medium ◽  
Plasminogen Activator ◽  
Active Form ◽  
Plasminogen Activator Inhibitor ◽  
Human Endothelial Cells ◽  
Inhibitor Activity ◽  
Latent Form ◽  
Activator Inhibitor ◽  
Pai 1

The plasminogen activator inhibitor from human endothelial cells (PAI- 1) exists in two forms in the culture medium: an active form that binds to and inactivates plasminogen activators and a latent form that in its native state has no anti-activator activity. Inhibitor activity associated with the latent form can be generated by treatment with protein denaturants and makes up more than 98% of the total inhibitor activity in conditioned medium. Plasminogen activator inhibitor activity is also found in cell cytosol. This inhibitor activity is stable to SDS-treatment but is not enhanced by it. We investigated the relationship between this active cell-associated inhibitor and the latent PAI-1 found in the conditioned medium. Both intracellular and extracellular inhibitors were immunoprecipitated by a monoclonal antibody produced against the latent inhibitor from HT1080 fibrosarcoma cells and electrophoresis on SDS gels of various acrylamide concentrations demonstrated that both forms had the same Mr. Incubation of cytosol inhibitor at 37 degrees C resulted in a decline in inhibitor activity with a half-life of approximately 4 hours, a rate of decline similar to that of the active PAI-1 in conditioned medium, with less than 10% of the original activity present after eight hours. This decline is accelerated at higher temperatures and is not affected by the presence of a variety of protease inhibitors. Approximately 90% of the activity can be regenerated after SDS treatment suggesting that the cell associated inhibitor, during incubation at 37 degrees C, converts to a form similar to that found in conditioned medium. Despite these similarities, the apparent Stoke's radii of the active intracellular inhibitor and the latent inhibitor in conditioned medium were significantly different with values of 2.77 nm and 2.40 nm for active and latent PAI-1, respectively. Incubation of the active form at 37 degrees C resulted in the shift of the Stoke's radius to that similar to the latent PAI-1 (2.45 nm). Thus, the active and latent PAI-1, while being immunologically similar and of the same apparent Mr, can be differentiated by their behavior on gel permeation columns. This suggests that the intracellular inhibitor is a precursor to the latent form.

scholarly journals Quantitation and properties of the active and latent plasminogen activator inhibitors in cultures of human endothelial cells

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1309-1313 ◽  
Author(s):  
EG Levin
Keyword(s):  
Endothelial Cells ◽  
Conditioned Medium ◽  
Plasminogen Activator ◽  
Active Form ◽  
Specific Inhibitor ◽  
Human Endothelial Cells ◽  
Inhibitor Activity ◽  
Active Inhibitor ◽  
Latent Form ◽  
Cell Extracts

Abstract Human endothelial cells release two forms of a plasminogen activator- specific inhibitor: an active form that readily binds to and inhibits plasminogen activators and an inactive or latent form that has no anti- activator activity but which can be activated by denaturation. Latent and active forms of plasminogen activator-specific inhibitor were measured in cultures of human umbilical vein endothelial cells. Latent inhibitor was activated by treatment with 1% sodium dodecyl sulfate (SDS), and both forms were assayed by the 125I-fibrin plate method. After 16 hours, the conditioned medium contained 104.6 U/mL latent inhibitor activity and 2.6 U/mL active inhibitor. The level of each form in the culture medium increased with time although the activity associated with the latent form rose more rapidly: the ratio of latent to active inhibitor activity was 12 at four hours (10.3 U/mL v 0.86 U/mL) and reached 56 at 24 hours (155.3 U/mL v 2.80 U/mL). Intracellular inhibitor activity was associated with the active form only; no additional inhibitor activity was observed following SDS treatment of cell extracts. A decline in active inhibitor activity occurred during incubation at 37 degrees C with a 50% reduction in activity occurring in two hours. Treatment of conditioned medium with 10 U/mL thrombin also resulted in a loss of active inhibitor activity. The latent inhibitor, however, was not affected by either of these conditions. The inhibitor activity lost during incubation at 37 degrees C or thrombin treatment could be regenerated by SDS treatment, suggesting that the loss of the active inhibitor activity represented a conversion of this form to its latent counterpart. Thus, the concentration, stability, and regulation of these two forms of plasminogen activator inhibitor in human endothelial cell cultures differ significantly.

Effects of a Defibrotide—Heparin Combination on Some Measures of Haemostasis in Healthy Volunteers

1989 ◽  
Vol 17 (1) ◽  
pp. 36-40 ◽  
Author(s):  
E. M. Pogliani ◽  
M. Salvatore ◽  
C. Fowst ◽  
R. Girardello ◽  
C. Marelli
Keyword(s):  
Healthy Volunteers ◽  
Prothrombin Time ◽  
Plasminogen Activator ◽  
Open Study ◽  
Antithrombin Iii ◽  
The Other ◽  
Sodium Heparin ◽  
Tissue Plasminogen ◽  
Clinically Significant ◽  
Plasma Heparin

In an open-study design five healthy volunteers were first given 2500 IU sodium heparin intravenously and then, after 72 h, another injection of the same dosage of sodium heparin followed immediately by 400 mg defibrotide intravenously. In two separate experiments, prothrombin time, activated partial prothrombin time, antithrombin III, tissue plasminogen activator, its inhibitor and plasma heparin levels were measured at baseline and after 15 min in one experiment, and at baseline and after 2 h in the other experiment. The most important finding was that an interaction exists between heparin and defibrotide on haemostatic activity: activated partial prothrombin time was increased three-fold in volunteers given the defibrotide—heparin combination compared with volunteers given heparin alone. This statistically and clinically significant effect was evident 15 min after administration of defibrotide–heparin and was still present after 2 h. Possible explanations for this effect are discussed briefly.

scholarly journals Secretion of Active Recombinant Human Tissue Plasminogen Activator Derivatives in Escherichia coli

2001 ◽  
Vol 67 (6) ◽  
pp. 2657-2664 ◽  
Author(s):  
Jiradej Manosroi ◽  
Chatchai Tayapiwatana ◽  
Friedrich Götz ◽  
Rolf G. Werner ◽  
Aranya Manosroi
Keyword(s):  
Escherichia Coli ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Specific Activity ◽  
Stop Codon ◽  
Active Form ◽  
Site Directed Mutagenesis ◽  
Amidolytic Activity ◽  
Tissue Plasminogen ◽  
Kringle 2

ABSTRACT The DNA fragment coding for kringle 2 plus serine protease domains (K2S) of tissue plasminogen activator (tPA) was inserted into a phagemid vector, pComb3HSS. In the recombinant vector, pComb3H-K2S, the K2S gene was fused togpIII of ΦM13 and linked to the OmpA signal sequence. The resulting gene, rK2S-gpIII, was inducibly expressed in Escherichia coli XL-1 Blue. The protein was presented on the phage particle. To stop the expression of gpIII,a stop codon between K2S and the gpIIIgene was inserted by site-directed mutagenesis. This mutated vector, MpComb3H-K2S, was transformed in XL-1 Blue. After induction with IPTG (isopropyl-β-d-thiogalactopyranoside), rK2S was found both in the periplasm as an inactive form of approximately 32% and in the culture supernatant as an active form of approximately 68%. The secreted form of rK2S was partially purified by ammonium sulfate (55%) precipitation. The periplasmic form was isolated from whole cells by chloroform extraction. The fibrin binding site of kringle 2 was demonstrated in all expressed versions (phage-bound, periplasmic, and secreted forms) using the monoclonal anti-kringle 2 antibody (16/B). Only the secreted form of rK2S revealed a fibrinogen-dependent amidolytic activity with the specific activity of 236 IU/μg. No amidolytic activity of rK2S was observed in either the periplasmic or the phage-bound form. The secretion of rK2S as an active enzyme offers a novel approach for the production of the active-domain deletion mutant tPA, rK2S, without any requirements for bacterial compartment preparation and in vitro refolding processes. This finding is an important technological advance in the development of large-scale, bacterium-based tPA production systems.

scholarly journals Conversion of the active to latent plasminogen activator inhibitor from human endothelial cells

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 1090-1098 ◽  
Author(s):  
EG Levin ◽  
L Santell
Keyword(s):  
Endothelial Cells ◽  
Conditioned Medium ◽  
Plasminogen Activator ◽  
Active Form ◽  
Plasminogen Activator Inhibitor ◽  
Human Endothelial Cells ◽  
Inhibitor Activity ◽  
Latent Form ◽  
Activator Inhibitor ◽  
Pai 1

Abstract The plasminogen activator inhibitor from human endothelial cells (PAI- 1) exists in two forms in the culture medium: an active form that binds to and inactivates plasminogen activators and a latent form that in its native state has no anti-activator activity. Inhibitor activity associated with the latent form can be generated by treatment with protein denaturants and makes up more than 98% of the total inhibitor activity in conditioned medium. Plasminogen activator inhibitor activity is also found in cell cytosol. This inhibitor activity is stable to SDS-treatment but is not enhanced by it. We investigated the relationship between this active cell-associated inhibitor and the latent PAI-1 found in the conditioned medium. Both intracellular and extracellular inhibitors were immunoprecipitated by a monoclonal antibody produced against the latent inhibitor from HT1080 fibrosarcoma cells and electrophoresis on SDS gels of various acrylamide concentrations demonstrated that both forms had the same Mr. Incubation of cytosol inhibitor at 37 degrees C resulted in a decline in inhibitor activity with a half-life of approximately 4 hours, a rate of decline similar to that of the active PAI-1 in conditioned medium, with less than 10% of the original activity present after eight hours. This decline is accelerated at higher temperatures and is not affected by the presence of a variety of protease inhibitors. Approximately 90% of the activity can be regenerated after SDS treatment suggesting that the cell associated inhibitor, during incubation at 37 degrees C, converts to a form similar to that found in conditioned medium. Despite these similarities, the apparent Stoke's radii of the active intracellular inhibitor and the latent inhibitor in conditioned medium were significantly different with values of 2.77 nm and 2.40 nm for active and latent PAI-1, respectively. Incubation of the active form at 37 degrees C resulted in the shift of the Stoke's radius to that similar to the latent PAI-1 (2.45 nm). Thus, the active and latent PAI-1, while being immunologically similar and of the same apparent Mr, can be differentiated by their behavior on gel permeation columns. This suggests that the intracellular inhibitor is a precursor to the latent form.

BOVINE PULMONARY ARTERY ENDOTHELIAL CELLS (BPAECs) STIMULATE TISSUE TYPE PLASMINOGEN ACTIVATOR (tPA) RELEASE BY HUMAN MELANOMA CELL LINES (HMCLs)

1987 ◽  
Author(s):  
J Wojta ◽  
M Vetterlein ◽  
R N Eoover
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Active Form ◽  
Plasminogen Activator Inhibitor ◽  
Activity Level ◽  
The Other ◽  
Tissue Type ◽  
Malignant Cells ◽  
Other Hand ◽  
Type Plasminogen Activator

It is known that treatment of endothelial cells (ECs) with extracts of malignant cells results in an increased level of tPA release by such ECs. On the other hand, no data are available concerning an influence of ECs on the fibrinolytic activity (PA) of malignant cells. Therefore it was the aim of this investigation to study whether the tPA production of malignant cells is influenced by ECs. Eighteen different HMCLs were screened for their ability to produce tPA using a combined assay system for tPA activity and tPA antigen employing monoclonal anti tPA antibodies. All 18 HMCLs produced tPA antigen ranging from 0.2 to 15.5ng/105 cells/24 hours. Twelve of these cells produced also tPA activity from 0.2 to 5.0 IU/105 cells/24 hours. To determine the influence of ECs on the PA of HMCLs, BPAECs were selected because their PA consists mainly of urokinase type plasminogen activator (uPA). All 18 HMCLs were cultivated on monolayers of BPAECs and as a control on plastic. After 24 hours the average tPA antigen level in the supernatant for the HMCLs grown on monolayers of BPAECs was 4.2ng/105 cells as compared to 2.9ng/105 cells of the control. However, the tPA activity average level was < 0.1 IU/105 cells for the HMCLs grown on BPAECs and 0.9 IU/105 cells for the HMCLs grown on plastic. To further investigate these effects two HMCLs were grown on plastic and incubated with conditioned media (CM) of BPAECs and compared to non conditioned medium (NCM). After 24 hours the levels of tPA antigen in the supernatant were 194 and 28.5ng/l05 cells after incubation with CM and compared to 14.0 and 14.9ng/105 cells for the control. In contrast to the first experiment the tPA activity level was also increased after incubation with CM. The respective values were 9.5 and 11.6 IU/105 cells and 3.8 and 4.8 IU/105 cells for the control. Our data suggest that BPAECs release a "factor" which is stimulating both the release of tPA activity and tPA antigen. This "factor" remains to be further characterized. On the other hand our results also suggest that BPAECs might release a plasminogen activator inhibitor (PAI) in its active form inhibiting the tPA simultanously released by cocultivated HMCLs.

scholarly journals PRODUCTION AND CHARACTERIZATION OF ANTIBODIES TO TISSUE PLASMINOGEN ACTIVATOR: APPLICATION FOR THE PLATELET FLOW CYTOMETRY ASSAY

2020 ◽  
Vol 13 (5) ◽  
pp. 62-72
Author(s):  
E. I. Yusova ◽  
Keyword(s):  
Flow Cytometry ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Binding Sites ◽  
Polyclonal Antibodies ◽  
Immune Serum ◽  
Flow Cytometry Assay ◽  
Tissue Plasminogen

Tissue plasminogen activator (tPA) is one of the key protein of plasminogen/plasmin system that converts plasminogen in the active proteinase plasmin. Platelets are able to bind both tPA and plasminogen on their surface, thus providing stimulatory effects on activation of zymogen. The present study was aimed to produce polyclonal antibodies against tPA and characterize their immunochemical capacities for further application in flow cytometry assay to study interaction between tPA and platelets. The experimental methods involved immunization of rabbit with tPA, collection of immune serum, synthesis of tPA-containing immunoaffine sorbent, ELISA, and flow cytometry. Polyclonal monospecific antibodies against tPA with high affinity to the antigen (Кd = 4.05・10–9 М) were obtained. Flow cytometry assay based on the use of the produced antibodies showed the presence of binding sites for tPA on the plasma membrane of inactive platelets. Moreover, agonist-stimulated platelets were revealed to expose more binding sites than their resting counterparts. Certain subpopulations of platelets that differ in the ability to bind tPA on their surface were also identified. Obtained data are of significant importance for further investigation of mechanisms underlying the role of platelets to regulate fibrinolytic rates.

A Kinetic Model of the Circulatory Regulation of Tissue Plasminogen Activator

1991 ◽  
Vol 66 (03) ◽  
pp. 321-328 ◽  
Author(s):  
Wayne L Chandler
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Active Form ◽  
Circadian Variation ◽  
Circulatory System ◽  
Hepatic Clearance ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Plasminogen ◽  
Activator Inhibitor Type ◽  
Pai 1

SummaryA computer simulation was developed to study the regulation of active tissue plasminogen activator (t-PA) levels in plasma by kinetically modeling t-PA secretion, t-PA inhibition by plasminogen activator inhibitor type 1 (PAI-1), and hepatic clearance of t-PA, PAI-1 and t-PA/PAI-1 complex throughout a simplified human circulatory system. The model indicates that as the active PAI-1 concentration increases, the percent of t-PA in the active form decreases exponentially. Further, the reaction between t-PA and PAI-1 substantially reduces the half-lives of both active factors. By adjusting the t-PA and PAI-1 secretion rates to provide the best fit between simulated and measured circadian variations in t-PA, PAI-1 and complex, the model predicts that the diurnal rhythm in active t-PA levels is principally due to changes in the rate of PAI-1 secretion and not to variations in the t-PA secretion rate. In conclusion, the model predicts that PAI-1 is an important regulator of the concentration, half-life and circadian variation of active t-PA.

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