scholarly journals Activation of pro-urokinase and plasminogen on human sarcoma cells: a proteolytic system with surface-bound reactants.

1989 ◽  
Vol 108 (5) ◽  
pp. 1987-1995 ◽  
Author(s):  
R W Stephens ◽  
J Pöllänen ◽  
H Tapiovaara ◽  
K C Leung ◽  
P S Sim ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Surface ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Plasmin Activity ◽  
Exponential Process ◽  
Diisopropyl Fluorophosphate ◽  
Pai 1

Human HT-1080 fibrosarcoma cells produce urokinase-type plasminogen activator (u-PA) and type 1 plasminogen activator inhibitor (PAI-1). We found that after incubation of monolayer cultures with purified native human plasminogen in serum-containing medium, bound plasmin activity could be eluted from the cells with tranexamic acid, an analogue of lysine. The bound plasmin was the result of plasminogen activation on the cell surface; plasmin activity was not taken up onto cells after deliberate addition of plasmin to the serum-containing medium. The cell surface plasmin formation was inhibited by an anticatalytic monoclonal antibody to u-PA, indicating that this enzyme was responsible for the activation. Preincubation of the cells with diisopropyl fluorophosphate-inhibited u-PA led to a decrease in surface-bound plasmin, indicating that a large part, if not all, of the cell surface plasminogen activation was catalyzed by surface-bound u-PA. In the absence of plasminogen, most of the cell surface u-PA was present in its single-chain proenzyme form, while addition of plasminogen led to formation of cell-bound two-chain u-PA. The latter reaction was catalyzed by cell-bound plasmin. Cell-bound u-PA was accessible to inhibition by endogenous PAI-1 and by added PAI-2, while the cell-bound plasmin was inaccessible to serum inhibitors, but accessible to added aprotinin and an anticatalytic monoclonal antibody. A model for cell surface plasminogen activation is proposed in which plasminogen binding to cells from serum medium is followed by plasminogen activation by trace amounts of bound active u-PA, to form bound plasmin, which in turn serves to produce more active u-PA from bound pro-u-PA. This exponential process is subject to regulation by endogenous PAI-1 and limited to the pericellular space.

PURIFICATION AND CHARACTERIZATION OF REACTIVE AND NON-REACTIVE PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1) FROM HUMAN PLACENTA

1987 ◽  
Author(s):  
M Philips ◽  
A G Juul ◽  
S Thorsen ◽  
J Selmer ◽  
L Thim
Keyword(s):  
Monoclonal Antibody ◽  
Plasminogen Activator ◽  
Human Placenta ◽  
Solid Phase ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Single Chain ◽  
Plasminogen Activator Inhibitor 1 ◽  
Sds Page ◽  
Pai 1

Reactive and non-reactive forms of PAI-1 have been identified in various biological materials. The structural differences between these forms remain to be determined.A monoclonal antibody specific for a non-reactive PAI-1 and a monoclonal antibody reacting with both the reactive and nonreactive form of the inhibitor were obtained by immunization with a tissue-type plasminogen activator (t-PA)-PAI-1 complex (Philips et al., Thromb Haemostas 1986; 55:213-7). These antibodies were used for the isolation of reactive and non-reactive PAI-1 by solid-phase immunoadsorption from extracts of human placenta. The inhibitor preparations were further purified by HPLC. Reactive and non-reactive PAI-1 both migrated with a Mr ∼ 52,000 when analyzed by SDS-PAGE. Furthermore, the two inhibitor forms were indistinguishable by N-terminal sequence analysis. Two N-terminal sequences were found in about equal ammounts for both the reactive and non-reactive PAI-1. They were Ser-Ala-Val-His-His-Pro-Pro- and a two residues shorter sequence (Val-His-His-Pro-Pro-). These sequences are in agreement with the published cDNA sequence of PAI-1 and shows that the inhibitor is N-terminally heterogeneously processed. The second order rate constant (ki) for the reaction between reactive PAI-1 and single-chain t-PA was about 6 106 M-1s-1. Treatment with 4 M guanidinium-HCl partially converted the non-reactive PAI-1 to a reactive form exhibiting a similar k1 for inhibition of single-chain t-PA. SDS-PAGE showed that the t-PA-PAI-1 complex could be dissociated by 1,5 M NH4OH/ 39 mM SDS resulting in the release of a PAI-1 with approximately the same Mr as native PAI-1. This indicates either that t-PA does not cleave the inhibitor or that it cleaves a peptide bond close to the C-terminus.In conclusion a non-reactive and a reactive form of PAI-1 can be purified from placenta. The two forms are distinguishable by monoclonal antibodies but they show similar Mr′ls and the same N-terminal sequences.

scholarly journals High levels of plasminogen activator inhibitor-1, tissue plasminogen activator and fibrinogen in patients with severe COVID-19

2021 ◽  
Author(s):  
David Cabrera-Garcia ◽  
Andrea Miltiades ◽  
Samantha M Parsons ◽  
Katerina Elisman ◽  
Mohammad Taghi Mansouri ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Plasminogen Activation ◽  
Plasmin Activity ◽  
Plasminogen Activator Inhibitor 1 ◽  
Partial Explanation ◽  
Tissue Plasminogen ◽  
Activator Inhibitor ◽  
Pai 1

We measured plasma levels of fibrinogen, plasminogen, tissue plasminogen activator (t-PA) and plasminogen activation inhibitor 1 (PAI-1) in blood from 37 patients with severe coronavirus disease-19 (COVID-19) and 23 controls. PAI-1, t-PA and fibrinogen levels were significantly higher in the COVID-19 group. Increased levels of PAI-1 likely result in lower plasmin activity and hence decreased fibrinolysis. These observations provide a partial explanation for the fibrin-mediated increase in blood viscosity and hypercoagulability that has previously been observed in COVID-19. Our data suggest that t-PA administration may be problematic, but that other interventions designed to enhance fibrinolysis might prove useful in the treatment of the coagulopathy that is often associated with severe COVID-19.

Purification and characterization of the plasminogen activator inhibitors PAI-1, PAI-2, and PN-1 from the human glioblastoma U138

1993 ◽  
Vol 71 (5-6) ◽  
pp. 248-254 ◽  
Author(s):  
Patricia G. Murphy ◽  
Steven P. Lenz ◽  
Mark Dobson ◽  
Allan D. Arndt ◽  
David A. Hart
Keyword(s):  
Monoclonal Antibody ◽  
Plasminogen Activator ◽  
Concanavalin A ◽  
Northern Blot ◽  
Northern Blot Analysis ◽  
Plasminogen Activator Inhibitor ◽  
Immunoaffinity Column ◽  
Positive Hybridization ◽  
Activator Inhibitor ◽  
Pai 1

This investigation presents data which indicate that the plasminogen activator inhibitor (PAI) activity secreted from U138 cells is composed of three separate PAIs: PAI-1, PAI-2, and PN-1. It was demonstrated that the U138 PAI-1-like protein had an apparent molecular mass of 50 kilodaltons (kDa) and was purified to apparent homogeneity by elution from an anti-PAI-1 immunoaffinity column. These fractions were also reactive with a second anti-PAI-1 monoclonal antibody using immunoblotting techniques. Northern blot analysis of RNA isolated from unstimulated U138 cells demonstrated positive hybridization with the cDNA specific for human PAI-1. The U138 PAI-2-like protein was adherent to an anti-PAI-2 immunoaffinity column and was demonstrated to be nonadherent to concanavalin A – agarose, heparin–Sepharose, and the anti-PAI-1 immunoaffinity column. The eluted U138 PAI-2-like protein was demonstrated to have an apparent molecular mass of 60 kDa and was also reactive with a second anti-PAI-2 monoclonal antibody using immunoblotting techniques. Further, the cDNA specific for PAI-2 was demonstrated to hybridize to a 2.5-kilobase message from RNA isolated from U138 cells. A third PAI was detected that was nonadherent to concanavalin A – agarose and both of the anti-PAI columns. This 50-kDa PAI was adherent to heparin–Sepharose and thrombin–agarose columns, and was not reactive with any antibodies for either PAI-1 or PAI-2. Northern blot analysis of U138 RNA demonstrated positive hybridization with an oligodeoxynucleotide specific for PN-1. This investigation demonstrates with biochemical, immunological, and molecular data that the U138 glioblastoma constitutively produces three PAIs.Key words: plasminogen activator inhibitor, U138 glioblastoma, PAI purification, human tumor cell line, proteinase inhibitors.

Glutathione restores collagen degradation in TGF-β-treated fibroblasts by blocking plasminogen activator inhibitor-1 expression and activating plasminogen

2005 ◽  
Vol 289 (6) ◽  
pp. L937-L945 ◽  
Author(s):  
Praveen K. Vayalil ◽  
Mitchell Olman ◽  
Joanne E. Murphy-Ullrich ◽  
Edward M. Postlethwait ◽  
Rui-Ming Liu
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Collagen Degradation ◽  
Tissue Type ◽  
3T3 Cells ◽  
Plasmin Activity ◽  
Nih 3T3 ◽  
Activator Inhibitor ◽  
Pai 1 ◽  
Nih 3T3 Cells

Transforming growth factor (TGF)-β plays an important role in tissue fibrogenesis. We previously demonstrated that reduced glutathione (GSH) supplementation blocked collagen accumulation induced by TGF-β in NIH-3T3 cells. In the present study, we show that supplementation of GSH restores the collagen degradation rate in TGF-β-treated NIH-3T3 cells. Restoration of collagen degradation by GSH is associated with a reduction of type I plasminogen activator inhibitor (PAI)-1 expression/activity as well as recovery of the activities of cell/extracellular matrix-associated tissue-type plasminogen activator and plasmin. Furthermore, we find that NIH-3T3 cells constitutively express plasminogen mRNA and possess plasmin activity. Blockade of cell surface binding of plasminogen/plasminogen activation with tranexamic acid (TXA) or inhibition of plasmin activity with aprotinin significantly reduces the basal level of collagen degradation both in the presence or absence of exogenous plasminogen. Most importantly, addition of TXA or active PAI-1 almost completely eliminates the restorative effects of GSH on collagen degradation in TGF-β treated cells. Together, our results suggest that the major mechanism by which GSH restores collagen degradation in TGF-β-treated cells is through blocking PAI-1 expression, leading to increased PA/plasmin activity and consequent proteolytic degradation of collagens. This study provides mechanistic evidence for GSH's putative therapeutic effect in the treatment of fibrotic disorders.

Inducible lung-specific urokinase expression reduces fibrosis and mortality after lung injury in mice

2002 ◽  
Vol 283 (5) ◽  
pp. L1023-L1032 ◽  
Author(s):  
Thomas H. Sisson ◽  
Kerstin E. Hanson ◽  
Natalya Subbotina ◽  
Anjali Patwardhan ◽  
Noboru Hattori ◽  
...  
Keyword(s):  
Lung Injury ◽  
Plasminogen Activator ◽  
Regulatory System ◽  
Plasminogen Activator Inhibitor ◽  
Transgenic Animals ◽  
Plasminogen Activation ◽  
Plasminogen Activator Inhibitor 1 ◽  
Similar Reduction ◽  
Fibrotic Process ◽  
Pai 1

Plasminogen activator inhibitor-1 (PAI-1)-deficient transgenic mice have improved survival and less fibrosis after intratracheal bleomycin instillation. We hypothesize that PAI-1 deficiency limits scarring through unopposed plasminogen activation. If this is indeed true, then we would expect increased urokinase-type plasminogen activator (uPA) expression to result in a similar reduction in scarring and improvement in mortality. To test our hypothesis, using the tetracycline gene regulatory system, we have generated a transgenic mouse model with the features of inducible, lung-specific uPA production. After doxycycline administration, these transgenic animals expressed increased levels of uPA in their bronchoalveolar lavage (BAL) fluid that accelerated intrapulmonary fibrin clearance. Importantly, this increased plasminogen activator production led to a reduction in both lung collagen accumulation and mortality after bleomycin-induced injury. These results suggest that PAI-1 deficiency does protect against the effects of bleomycin-induced lung injury through unopposed plasmin generation. By allowing the manipulation of plasminogen activation at different phases of the fibrotic process, this model will serve as a powerful tool in further investigations into the pathogenesis of pulmonary fibrosis.

Plasminogen activator inhibitor-1 modulates adipocyte differentiation

2006 ◽  
Vol 290 (1) ◽  
pp. E103-E113 ◽  
Author(s):  
Xiubin Liang ◽  
Talerngsak Kanjanabuch ◽  
Su-Li Mao ◽  
Chuan-Ming Hao ◽  
Yi-Wei Tang ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Mrna Expression ◽  
Glucose Uptake ◽  
Plasminogen Activator ◽  
Adipocyte Differentiation ◽  
Plasminogen Activator Inhibitor ◽  
Plasmin Activity ◽  
Plasminogen Activator Inhibitor 1 ◽  
Activator Inhibitor ◽  
Pai 1

Increased plasminogen activator inhibitor-1 (PAI-1) is linked to obesity and insulin resistance. However, the functional role of PAI-1 in adipocytes is unknown. This study was designed to investigate effects and underlying mechanisms of PAI-1 on glucose uptake in adipocytes and on adipocyte differentiation. Using primary cultured adipocytes from PAI-1+/+ and PAI-1−/− mice, we found that PAI-1 deficiency promoted adipocyte differentiation, enhanced basal and insulin-stimulated glucose uptake, and protected against tumor necrosis factor-α-induced adipocyte dedifferentiation and insulin resistance. These beneficial effects were associated with upregulated glucose transporter 4 at basal and insulin-stimulated states and upregulated peroxisome proliferator-activated receptor-γ (PPARγ) and adiponectin along with downregulated resistin mRNA in differentiated PAI-1−/− vs. PAI-1+/+ adipocytes. Similarly, inhibition of PAI-1 with a neutralizing anti-PAI-1 antibody in differentiated 3T3-L1 adipocytes further promoted adipocyte differentiation and glucose uptake, which was associated with increased expression of transcription factors PPARγ, CCAAT enhancer-binding protein-α (C/EBPα), and the adipocyte-selective fatty acid-binding protein aP2, thus mimicking the phenotype in PAI-1−/− primary adipocytes. Conversely, overexpression of PAI-1 by adenovirus-mediated gene transfer in 3T3-L1 adipocytes inhibited differentiation and reduced PPARγ, C/EBPα, and aP2 expression. This was also associated with a decrease in urokinase-type plasminogen activator mRNA expression, decreased plasmin activity, and increased collagen I mRNA expression. Collectively, these results indicate that absence or inhibition of PAI-1 in adipocytes protects against insulin resistance by promoting glucose uptake and adipocyte differentiation via increased PPARγ expression. We postulate that these PAI-1 effects on adipocytes may, at least in part, be mediated via modulation of plasmin activity and extracellular matrix components.

Demonstration of a Functionally Active tPA-Like Plasminogen Activator in Human Platelets

1994 ◽  
Vol 71 (04) ◽  
pp. 493-498 ◽  
Author(s):  
D L Wang ◽  
Y T Pan ◽  
J J Wang ◽  
C H Cheng ◽  
C Y Liu
Keyword(s):  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Protein Band ◽  
Human Platelets ◽  
Free Form ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Western Blots ◽  
A Minor ◽  
Pai 1

SummaryThe mechanism of platelet-enhanced fibrinolysis is unclear. We therefore investigated the fibrinolytic activity of human platelets and demonstrated that they contain a tissue plasminogen activator (tPA)- like plasminogen activator, abbreviated as tPA-like-PA. This activator was detected by ELISA in platelet incubation medium and in platelet Triton extracts. Plasminogen activation assays showed that this tPA- like-PA could induce plasminogen activation to form plasmin. Western blots of Triton extracts incubated with anti-tPA antibody demonstrated a major 64-kD protein band, compared to a 70-kD band for standard single chain tPA, plus a minor 118-kD band corresponding to a complex of tPA-like-PA and plasminogen activator inhibitor (PAI-1). Western blots of Triton extracts incubated with anti-PAI-1 antibody produced an approximately similar high-molecular-weight (118 kD) protein band. Fibrin zymographic analysis of affinity-purified tPA-like- PA demonstrated a major and a minor fibrin lysis zone, which approximately corresponded to the tPA-like-PA and its complex with PAI-1 observed by Western blots. Immunogold labelling and electron microscopy demonstrated that platelet activator, either as the free form or co-localized with PAI-1, was present in granules and in channels of the open canalicular system. We conclude that platelets contain a functionally active tPA-like-PA, whose low fibrinolytic activity might be due to its readily forming a complex with PAI-1. This functionally active tPA-like-PA might contribute to the enhanced fibrinolytic activity of platelets observed in platelet-rich thrombi.

Plasminogen activator inhibitor-1 deficiency protects against atherosclerosis progression in the mouse carotid artery

Blood ◽  
2000 ◽  
Vol 96 (13) ◽  
pp. 4212-4215 ◽  
Author(s):  
Daniel T. Eitzman ◽  
Randal J. Westrick ◽  
Zuojun Xu ◽  
Julia Tyson ◽  
David Ginsburg
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Carotid Bifurcation ◽  
Plasminogen Activation ◽  
Plasminogen Activator Inhibitor 1 ◽  
Atherosclerosis Progression ◽  
Plasminogen Activation System ◽  
Activation System ◽  
Activator Inhibitor ◽  
Pai 1

Abstract Dissolution of the fibrin blood clot is regulated in large part by plasminogen activator inhibitor-1 (PAI-1). Elevated levels of plasma PAI-1 may be an important risk factor for atherosclerotic vascular disease and are associated with premature myocardial infarction. The role of the endogenous plasminogen activation system in limiting thrombus formation following atherosclerotic plaque disruption is unknown. This study found that genetic deficiency for PAI-1, the primary physiologic regulator of tissue-type plasminogen activator (tPA), prolonged the time to occlusive thrombosis following photochemical injury to carotid atherosclerotic plaque in apolipoprotein E-deficient (apoE−/−) mice. However, anatomic analysis revealed a striking difference in the extent of atherosclerosis at the carotid artery bifurcation between apoE−/− mice and mice doubly deficient for apoE and PAI-1 (PAI-1−/−/apoE−/−). Consistent with a previous report, PAI-1+/+/apoE−/−and PAI-1−/−/apoE−/− mice developed similar atherosclerosis in the aortic arch. The marked protection from atherosclerosis progression at the carotid bifurcation conferred by PAI-1 deficiency suggests a critical role for PAI-1 in the pathogenesis of atherosclerosis at sites of turbulent flow, potentially through the inhibition of fibrin clearance. Consistent with this hypothesis, intense fibrinogen/fibrin staining was observed in atherosclerotic lesions at the carotid bifurcation compared to the aortic arch. These observations identify significant differences in the pathogenesis of atherosclerosis at varying sites in the vascular tree and suggest a previously unappreciated role for the plasminogen activation system in atherosclerosis progression at sites of turbulent flow.

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