Science.gov

Sample records for activator plasminogen activator

  1. [Plasminogen activator from Agkistrodon halys halys venom].

    PubMed

    Karbovs'kyĭ, V L; Levkiv, M Iu; Savchuk, O M; Hornyts'ka, O V; Volkov, H L; Bukhan, Ts

    2006-01-01

    Plasminogen activator "Ahh-32" from Agkistrodon halys halys venom has been isolated and purified using affinity and ion-exchange chromatography. The purified enzyme consists of the single peptide-chain with molecular weigth of 32 kDa. It can convert free plasminogen into active form--plasmin. "Ahh-32" was inhibited by DFP and benzamidine. Besides, the enzyme influences significantly the activation of plasminogen by streptokinase without having effect on analogical process in case of usage of tissue tipe plasminogen activator. The obtained protein can be used as an instrument under investigation of protein-protein interactions in haemostasis system.

  2. The plasminogen activator system: biology and regulation.

    PubMed

    Irigoyen, J P; Muñoz-Cánoves, P; Montero, L; Koziczak, M; Nagamine, Y

    1999-10-01

    The regulation of plasminogen activation involves genes for two plasminogen activators (tissue type and urokinase type), two specific inhibitors (type 1 and type 2), and a membrane-anchored urokinase-type plasminogen-activator-specific receptor. This system plays an important role in various biological processes involving extracellular proteolysis. Recent studies have revealed that the system, through interplay with integrins and the extracellular matrix protein vitronectin, is also involved in the regulation of cell migration and proliferation in a manner independent of proteolytic activity. The genes are expressed in many different cell types and their expression is under the control of diverse extracellular signals. Gene expression reflects the levels of the corresponding mRNA, which should be the net result of synthesis and degradation. Thus, modulation of mRNA stability is an important factor in overall regulation. This review summarizes current understanding of the biology and regulation of genes involved in plasminogen activation at different levels.

  3. The effects of residual platelets in plasma on plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays

    PubMed Central

    Barnard, Sunelle A.; Loots, Du Toit; Rijken, Dingeman C.

    2017-01-01

    Due to controversial evidence in the literature pertaining to the activity of plasminogen activator inhibitor-1 in platelets, we examined the effects of residual platelets present in plasma (a potential pre-analytical variable) on various plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays. Blood samples were collected from 151 individuals and centrifuged at 352 and 1500 g to obtain plasma with varying numbers of platelet. In a follow-up study, blood samples were collected from an additional 23 individuals, from whom platelet-poor (2000 g), platelet-containing (352 g) and platelet-rich plasma (200 g) were prepared and analysed as fresh-frozen and after five defrost-refreeze cycles (to determine the contribution of in vitro platelet degradation). Plasminogen activator inhibitor-1 activity, plasminogen activator inhibitor-1 antigen, tissue plasminogen activator/plasminogen activator inhibitor-1 complex, plasma clot lysis time, β-thromboglobulin and plasma platelet count were analysed. Platelet α-granule release (plasma β-thromboglobulin) showed a significant association with plasminogen activator inhibitor-1 antigen levels but weak associations with plasminogen activator inhibitor-1 activity and a functional marker of fibrinolysis, clot lysis time. Upon dividing the study population into quartiles based on β-thromboglobulin levels, plasminogen activator inhibitor-1 antigen increased significantly across the quartiles while plasminogen activator inhibitor-1 activity and clot lysis time tended to increase in the 4th quartile only. In the follow-up study, plasma plasminogen activator inhibitor-1 antigen was also significantly influenced by platelet count in a concentration-dependent manner. Plasma plasminogen activator inhibitor-1 antigen levels increased further after complete platelet degradation. Residual platelets in plasma significantly influence plasma plasminogen activator inhibitor-1 antigen levels mainly through release of

  4. Design of a novel chimeric tissue plasminogen activator with favorable Vampire bat plasminogen activator properties.

    PubMed

    Kazemali, MohammadReza; Majidzadeh-A, Keivan; Sardari, Soroush; Saadatirad, Amir Hossein; Khalaj, Vahid; Zarei, Najmeh; Barkhordari, Farzaneh; Adeli, Ahmad; Mahboudi, Fereidoun

    2014-12-01

    Fibrinolytic agents are widely used in treatment of the thromboembolic disorders. The new generations like recombinant tissue plasminogen activator (t-PA, alteplase) are not showing promising results in clinical practice in spite of displaying specific binding to fibrin in vitro. Vampire bat plasminogen activator (b-PA) is a plasminogen activator with higher fibrin affinity and specificity in comparison to t-PA resulting in reduced probability of hemorrhage. b-PA is also resistant to plasminogen activator inhibitor-1 (PAI-1) showing higher half-life compared to other variants of t-PA. However, its non-human origin was a driving force to design a human t-PA with favorable properties of b-PA. In the present study, we designed a chimeric t-PA with desirable b-PA properties and this new molecule was called as CT-b. The construct was prepared through kringle 2 domain removal and replacement of t-PA finger domain with b-PA one. In addition, the KHRR sequence at the initial part of protease domain was replaced by four alanine residues. The novel construct was integrated in Pichia pastoris genome by electroporation. Catalytic activity was investigated in the presence and absence of fibrin. The purified protein was analyzed by western blot. Fibrin binding and PAI resistance assays were also conducted. The activity of the recombinant protein in the presence of fibrin was 1560 times more than its activity in the absence of fibrin, showing its higher specificity to fibrin. The fibrin binding of CT-b was 1.2 fold more than t-PA. In addition, it was inhibited by PAI enzyme 44% less than t-PA. Although the presented data demonstrate a promising in vitro activity, more in vivo studies are needed to confirm the therapeutic advantage of this novel plasminogen activator.

  5. An immunohistochemical study of the distribution of plasminogen and plasminogen activators in bullous pemphigoid.

    PubMed

    Venning, V A; Wojnarowska, F; Cederholm-Williams, S

    1993-03-01

    Abnormalities of the cutaneous plasminogen/plasminogen activator system have been associated with acantholytic disorders, psoriasis, keratinocytes in culture, and epidermis in healing wounds. The present study was undertaken to investigate the possible role of the plasmin/plasminogen protease system in lesion development in bullous pemphigoid (BP). Using polyclonal antibodies and a fluorescent technique, the immunohistochemical distribution of plasmin/plasminogen, fibrinogen and the plasminogen activators, urokinase (uPA) and tissue plasminogen activator (tPA), were studied in lesional and non-lesional skin from nine BP patients, one with linear IgA disease (LAD) and one with pemphigoid gestationis (PG). The distribution of the proteases was compared with that in normal skin (n = 4) and in suction blisters (n = 2). In normal skin, fibrinogen, tPA and uPA were absent from the epidermis and plasminogen was confined to the basal layer. Uninvolved BP skin was identical to controls. Focal areas of suprabasal plasminogen expression in the region of a blister was seen in 3/9 BP lesions and in 1/2 suction blisters. In 6/9 BP lesions and both uninvolved and lesional LAD and PG skin were identical to controls, and no suprabasal expression of plasminogen was present. These findings suggest that suprabasal plasminogen expression is unlikely to play a fundamental role in the pathogenesis of blister formation in BP as enhanced expression was not present in every case and the finding was not specific to BP, also occurring in a suction blister. Enhanced plasminogen expression rather may be a reflection of the processes of tissue repair.

  6. Fibrin and plasminogen structures essential to stimulation of plasmin formation by tissue-type plasminogen activator.

    PubMed

    Suenson, E; Petersen, L C

    1986-04-22

    Plasminogen activation catalysed by tissue-type plasminogen activator (t-PA) has been examined in the course of concomitant fibrin formation and degradation. Plasmin generation has been measured by the spectrophotometric method of Petersen et al. (Biochem. J. 225 (1985) 149-158), modified so as to allow for light scattering caused by polymerized fibrin. Glu1-, Lys77- and Val442-plasminogen are activated in the presence of fibrinogen, des A- and des AB-fibrin and the rate of plasmin formation is found to be greatly enhanced by both des A- and des AB-fibrin polymer. Plasmin formation from Glu1- and Lys77-plasminogen yields a sigmoidal curve, whereas a linear increase is obtained with Val442-plasminogen. The rate of plasmin formation from Glu1- and Lys77-plasminogen declines in parallel with decreasing turbidity of the fibrin polymer effector. In order to study the effect of polymerization, this has been inhibited by the synthetic polymerization site analogue Gly-Pro-Arg-Pro, by fibrinogen fragment D1 or by prior methylene blue-dependent photooxidation of the fibrinogen used. Inhibition of polymerization by Gly-Pro-Arg-Pro reduces plasmin generation to the low rate observed in the presence of fibrinogen. Antipolymerization with fragment D1 or photooxidation has the same effect on Glu1-plasminogen activation, but only partially reduces and delays the stimulatory effect on Lys77- and Val442-plasminogen activation. The results suggest that protofibril formation (and probably also gelation) of fibrin following fibrinopeptide release is essential to its stimulatory effect. The gradual increase and subsequent decline in the rate of plasmin formation from Glu1- or Lys77-plasminogen during fibrinolysis may be explained by sequential exposure, modification and destruction of different t-PA and plasminogen binding sites in fibrin polymer.

  7. A miniaturized fibrinolytic assay for plasminogen activators

    NASA Technical Reports Server (NTRS)

    Lewis, M. L.; Nachtwey, D. S.; Damron, K. L.

    1991-01-01

    This report describes a micro-clot lysis assay (MCLA) for evaluating fibrinolytic activity of plasminogen activators (PA). Fibrin clots were formed in wells of microtiter plates. Lysis of the clots by PA, indicated by change in turbidity (optical density, OD), was monitored with a microplate reader at five minutes intervals. Log-log plots of PA dilution versus endpoint, the time at which the OD value was halfway between the maximum and minimum value for each well, were linear over a broad range of PA concentrations (2-200 International units/ml). The MCLA is a modification and miniaturization of well established fibrinolytic methods. The significant practical advantages of the MCLA are that it is a simple, relatively sensitive, non-radioactive, quantitative, kinetic, fibrinolytic micro-technique which can be automated.

  8. Inactivation of plasminogen activator inhibitor by oxidants

    SciTech Connect

    Lawrence, D.A.; Loskutoff, D.J.

    1986-10-21

    The rapidly acting plasminogen activator inhibitor (PAI) purified from cultured bovine endothelial cells (BAEs) was inactivated during iodination with chloramine T and other oxidizing iodination systems. Inactivation was observed in the absence of iodine, suggesting that the loss of activity resulted from the oxidizing conditions employed. In an attempt to further study the nature of this inactivation, the PAI was treated with chloramine T under conditions that specifically oxidize methionine and cystein residues. Both PAI inhibitory activity and the ability of the PAI to form complexes with tissue-type PA were decreased in a dose-dependent manner by such treatment. PAI activity was measured with the lysis of /sup 125/I-labelled fibrin. The reductase is a DTT-dependent enzyme that specifically converts methionine sulfoxide to methionine. Little activity was restored by either the reductase or DTT alone. These results indicate that the oxidation of at least one critical methionine residue is responsible for the loss of PAI activity upon iodination. In this respect, the BAE PAI resembles ..cap alpha../sub 1/-protease inhibitor, a well-characterized elastase inhibitor that also is inactivated by oxidants. Both inhibitors are members of the serine protease inhibitor superfamily (Serpins), and both have a methionine residue in their reactive center.

  9. Tissue Plasminogen Activator Reduces Neurological Damage after Cerebral Embolism

    NASA Astrophysics Data System (ADS)

    Zivin, Justin A.; Fisher, Marc; Degirolami, Umberto; Hemenway, Carl C.; Stashak, Joan A.

    1985-12-01

    Intravenous administration of tissue plasminogen activator immediately after the injection of numerous small blood clots into the carotid circulation in rabbit embolic stroke model animals caused a significant reduction in neurological damage. In vitro studies indicate that tissue plasminogen activator produced substantial lysis of clots at concentrations comparable to those expected in vivo, suggesting that this may be the mechanism of action of this drug. Drug-induced hemorrhages were not demonstrable. Tissue plasminogen activator may be of value for the immediate treatment of embolic stroke.

  10. Involvement of the plasminogen activation system in cow endometritis.

    PubMed

    Moraitis, S; Taitzoglou, I A; Tsantarliotou, M P; Boscos, C M; Kaldrimidou, E; Saratsis, Ph

    2004-01-15

    The objectives of this study were to investigate the: (a) presence and activity of components of the "plasminogen activators/plasmin" system in dairy cows with or without endometritis; (b) variations in enzyme activity according to the degree of endometritis; and (c) associations between these enzymes and changes in endometrial histology after intrauterine antibiotic treatment. Endometrial biopsies were collected from anestrus (no palpable ovarian structures and milk progesterone <1 ng/ml) Holstein cows, 30-40 days postpartum. On the basis of a vaginoscopic examination, rectal palpation of the cervix and uterus, and endometrial histology, there were 92 cows with endometritis and 20 cows without endometritis. After biopsy collection, each cow was given an intrauterine infusion of 1.5x10(6) IU of procaine penicillin G. In cows with endometritis, genital tract examinations and biopsies were repeated 2 weeks later. Both plasminogen activators (PAs), tissue type (t-PA) and urokinase (u-PA), were immunologically identified in all uterine biopsies. Plasminogen activator activity (PAA) increased, whereas plasminogen activator inhibition (PAI) and plasmin inhibition (PI) decreased in proportion to the degree of inflammation. Two weeks after intrauterine treatment, PAA had decreased significantly in all cows that had reduced severity of endometrial inflammation and had increased significantly in all cows with increased severity of inflammation. The change in the degree of inflammation depended upon plasminogen activator activity; cows with higher PAA were more likely to improve. In conclusion, there was evidence for a role of the plasminogen activation proteolytic system in bovine endometritis.

  11. Characterization of the murine plasminogen/urokinase-type plasminogen-activator system.

    PubMed

    Lijnen, H R; Van Hoef, B; Collen, D

    1996-11-01

    The murine plasminogen/urokinase-type plasminogen-activator (u-PA) system was studied using purified proteins, plasma and endothelioma cells. Recombinant murine u-PA was obtained as a single-chain molecule of 45 kDa which was converted to two-chain u-PA with plasmin by cleavage of the Lys159-Ile160 peptide bond. Murine plasminogen, purified from plasma as a single-chain protein of 95 kDa, was resistant to quantitative activation with murine recombinant two-chain u-PA: only 15% activation within 1 h at 37 degrees C was obtained in mixtures of 1 microM plasminogen and 5 nM recombinant two-chain u-PA, whereas quantitative activation was observed in the autologous human system. Addition of 6-aminohexanoic acid to native murine plasminogen resulted in quantitative activation within 1 h. In murine plasma in vitro, plasminogen was also resistant to quantitative activation with u-PA (50% activation within 1 h at 37 degrees C with 50 nM recombinant two-chain u-PA, whereas in the human system nearly quantitative activation was obtained). Murine plasma clots submerged in murine plasma were resistant to lysis with u-PA; < or = 2% clot lysis in 2 h was obtained with 80 nM recombinant two-chain u-PA in the autologous murine system whereas 50% clot lysis in 2 h required only 15 nM recombinant two-chain u-PA in the autologous human system. Saturable binding of murine recombinant two-chain u-PA was observed to murine endothelioma cells that are genetically deficient in u-PA (u-PA-/- End cells). Binding was characterized by a Kd of 5.5 nM and 800000 binding sites/cell. However, u-PA-/- End cells did not significantly stimulate the activation rate of murine plasminogen by murine recombinant two-chain u-PA and did not enhance the plasmin-mediated conversion rate of murine recombinant single-chain u-PA to its two-chain derivative. Murine recombinant two-chain u-PA bound to murine endothelioma cells was quantitatively inhibited by murine plasminogen-activator inhibitor-1 (PAI-1). Thus

  12. Polyphosphate colocalizes with factor XII on platelet-bound fibrin and augments its plasminogen activator activity

    PubMed Central

    Lionikiene, Ausra S.; Georgiev, Georgi; Klemmer, Anja; Brain, Chelsea; Kim, Paul Y.

    2016-01-01

    Activated factor XII (FXIIa) has plasminogen activator capacity but its relative contribution to fibrinolysis is considered marginal compared with urokinase and tissue plasminogen activator. Polyphosphate (polyP) is released from activated platelets and mediates FXII activation. Here, we investigate the contribution of polyP to the plasminogen activator function of αFXIIa. We show that both polyP70, of the chain length found in platelets (60-100 mer), and platelet-derived polyP significantly augment the plasminogen activation capacity of αFXIIa. PolyP70 stimulated the autoactivation of FXII and subsequent plasminogen activation, indicating that once activated, αFXIIa remains bound to polyP70. Indeed, complex formation between polyP70 and αFXIIa provides protection against autodegradation. Plasminogen activation by βFXIIa was minimal and not enhanced by polyP70, highlighting the importance of the anion binding site. PolyP70 did not modulate plasmin activity but stimulated activation of Glu and Lys forms of plasminogen by αFXIIa. Accordingly, polyP70 was found to bind to FXII, αFXIIa, and plasminogen, but not βFXIIa. Fibrin and polyP70 acted synergistically to enhance αFXIIa-mediated plasminogen activation. The plasminogen activator activity of the αFXIIa-polyP70 complex was modulated by C1 inhibitor and histidine-rich glycoprotein, but not plasminogen activator inhibitors 1 and 2. Platelet polyP and FXII were found to colocalize on the activated platelet membrane in a fibrin-dependent manner and decorated fibrin strands extending from platelet aggregates. We show that in the presence of platelet polyP and the downstream substrate fibrin, αFXIIa is a highly efficient and favorable plasminogen activator. Our data are the first to document a profibrinolytic function of platelet polyP. PMID:27694320

  13. Minor Role of Plasminogen in Complement Activation on Cell Surfaces

    PubMed Central

    Hyvärinen, Satu; Jokiranta, T. Sakari

    2015-01-01

    Atypical hemolytic uremic syndrome (aHUS) is a rare, but severe thrombotic microangiopathy. In roughly two thirds of the patients, mutations in complement genes lead to uncontrolled activation of the complement system against self cells. Recently, aHUS patients were described with deficiency of the fibrinolytic protein plasminogen. This zymogen and its protease form plasmin have both been shown to interact with complement proteins in the fluid phase. In this work we studied the potential of plasminogen to restrict complement propagation. In hemolytic assays, plasminogen inhibited complement activation, but only when it had been exogenously activated to plasmin and when it was used at disproportionately high concentrations compared to serum. Addition of only the zymogen plasminogen into serum did not hinder complement-mediated lysis of erythrocytes. Plasminogen could not restrict deposition of complement activation products on endothelial cells either, as was shown with flow cytometry. With platelets, a very weak inhibitory effect on deposition of C3 fragments was observed, but it was considered too weak to be significant for disease pathogenesis. Thus it was concluded that plasminogen is not an important regulator of complement on self cells. Instead, addition of plasminogen was shown to clearly hinder platelet aggregation in serum. This was attributed to plasmin causing disintegration of formed platelet aggregates. We propose that reduced proteolytic activity of plasmin on structures of growing thrombi, rather than on complement activation fragments, explains the association of plasminogen deficiency with aHUS. This adds to the emerging view that factors unrelated to the complement system can also be central to aHUS pathogenesis and suggests that future research on the mechanism of the disease should expand beyond complement dysregulation. PMID:26637181

  14. Photonic activation of plasminogen induced by low dose UVB.

    PubMed

    Correia, Manuel; Snabe, Torben; Thiagarajan, Viruthachalam; Petersen, Steffen Bjørn; Campos, Sara R R; Baptista, António M; Neves-Petersen, Maria Teresa

    2015-01-01

    Activation of plasminogen to its active form plasmin is essential for several key mechanisms, including the dissolution of blood clots. Activation occurs naturally via enzymatic proteolysis. We report that activation can be achieved with 280 nm light. A 2.6 fold increase in proteolytic activity was observed after 10 min illumination of human plasminogen. Irradiance levels used are in the same order of magnitude of the UVB solar irradiance. Activation is correlated with light induced disruption of disulphide bridges upon UVB excitation of the aromatic residues and with the formation of photochemical products, e.g. dityrosine and N-formylkynurenine. Most of the protein fold is maintained after 10 min illumination since no major changes are observed in the near-UV CD spectrum. Far-UV CD shows loss of secondary structure after illumination (33.4% signal loss at 206 nm). Thermal unfolding CD studies show that plasminogen retains a native like cooperative transition at ~70 ºC after UV-illumination. We propose that UVB activation of plasminogen occurs upon photo-cleavage of a functional allosteric disulphide bond, Cys737-Cys765, located in the catalytic domain and in van der Waals contact with Trp761 (4.3 Å). Such proximity makes its disruption very likely, which may occur upon electron transfer from excited Trp761. Reduction of Cys737-Cys765 will result in likely conformational changes in the catalytic site. Molecular dynamics simulations reveal that reduction of Cys737-Cys765 in plasminogen leads to an increase of the fluctuations of loop 760-765, the S1-entrance frame located close to the active site. These fluctuations affect the range of solvent exposure of the catalytic triad, particularly of Asp646 and Ser74, which acquire an exposure profile similar to the values in plasmin. The presented photonic mechanism of plasminogen activation has the potential to be used in clinical applications, possibly together with other enzymatic treatments for the elimination of

  15. Vampire bat salivary plasminogen activator is quiescent in human plasma in the absence of fibrin unlike human tissue plasminogen activator.

    PubMed

    Gardell, S J; Hare, T R; Bergum, P W; Cuca, G C; O'Neill-Palladino, L; Zavodny, S M

    1990-12-15

    The vampire bat salivary plasminogen activator (Bat-PA) is a potent PA that exhibits remarkable selectivity toward fibrin-bound plasminogen (Gardell et al, J Biol Chem 256: 3568, 1989). Herein, we describe the activity of recombinant DNA-derived Bat-PA (rBat-PA) in a human plasma milieu. rBat-PA and recombinant human single-chain tissue plasminogen activator (rt-PA) are similarly efficacious at lysing plasma clots. In stark contrast to rt-PA, the addition of 250 nmol/L rBat-PA to plasma in the absence of a clot failed to deplete plasminogen, alpha 2-antiplasmin and fibrinogen. The lytic activities exhibited by finger-domain minus Bat-PA (F- rBat-PA) and finger and epidermal growth factor-like domains minus Bat-PA (FG- rBat-PA) were less than rBat-PA, especially at low concentrations of PA; nevertheless, these truncated forms also possessed a strict requirement for a fibrin cofactor. The loss of PA activity following the addition of rBat-PA to plasma was slower than that observed when either rt-PA or two-chain rt-PA was added. The efficacy, fibrin selectivity, and decreased susceptibility to inactivation exhibited by rBat-PA in vitro in a human plasma milieu suggests that rBat-PA may be superior to rt-PA for the treatment of thrombotic complications.

  16. Biochemical assays on plasminogen activators and hormones from kidney sources

    NASA Technical Reports Server (NTRS)

    Barlow, Grant H.; Lewis, Marian L.; Morrison, Dennis R.

    1988-01-01

    Investigations were established for the purpose of analyzing the conditioned media from human embryonic kidney cell subpopulations separated in space by electrophoresis. This data is based on the experiments performed on STS-8 on the continuous flow electrophoresis system. The primary biological activity that was analyzed was plasminogen activator activity, but some assays for erythropoeitin and human granulocyte colony stimulating activity were also performed. It is concluded that a battery of assays are required to completely define the plasminogen activator profile of a conditioned media from cell culture. Each type of assay measures different parts of the mixture and are influenced by different parameters. The functional role of each assay is given along with an indication of which combination of assays are required to answer specific questions. With this type of information it is possible by combinations of assays with mathematical analysis to pinpoint a specific component of the system.

  17. Photonic Activation of Plasminogen Induced by Low Dose UVB

    PubMed Central

    Correia, Manuel; Snabe, Torben; Thiagarajan, Viruthachalam; Petersen, Steffen Bjørn; Campos, Sara R. R.; Baptista, António M.; Neves-Petersen, Maria Teresa

    2015-01-01

    Activation of plasminogen to its active form plasmin is essential for several key mechanisms, including the dissolution of blood clots. Activation occurs naturally via enzymatic proteolysis. We report that activation can be achieved with 280 nm light. A 2.6 fold increase in proteolytic activity was observed after 10 min illumination of human plasminogen. Irradiance levels used are in the same order of magnitude of the UVB solar irradiance. Activation is correlated with light induced disruption of disulphide bridges upon UVB excitation of the aromatic residues and with the formation of photochemical products, e.g. dityrosine and N-formylkynurenine. Most of the protein fold is maintained after 10 min illumination since no major changes are observed in the near-UV CD spectrum. Far-UV CD shows loss of secondary structure after illumination (33.4% signal loss at 206 nm). Thermal unfolding CD studies show that plasminogen retains a native like cooperative transition at ~70 ºC after UV-illumination. We propose that UVB activation of plasminogen occurs upon photo-cleavage of a functional allosteric disulphide bond, Cys737-Cys765, located in the catalytic domain and in van der Waals contact with Trp761 (4.3 Å). Such proximity makes its disruption very likely, which may occur upon electron transfer from excited Trp761. Reduction of Cys737-Cys765 will result in likely conformational changes in the catalytic site. Molecular dynamics simulations reveal that reduction of Cys737-Cys765 in plasminogen leads to an increase of the fluctuations of loop 760–765, the S1-entrance frame located close to the active site. These fluctuations affect the range of solvent exposure of the catalytic triad, particularly of Asp646 and Ser74, which acquire an exposure profile similar to the values in plasmin. The presented photonic mechanism of plasminogen activation has the potential to be used in clinical applications, possibly together with other enzymatic treatments for the elimination of

  18. The tissue-type plasminogen activator-plasminogen activator inhibitor 1 complex promotes neurovascular injury in brain trauma: evidence from mice and humans.

    PubMed

    Sashindranath, Maithili; Sales, Eunice; Daglas, Maria; Freeman, Roxann; Samson, Andre L; Cops, Elisa J; Beckham, Simone; Galle, Adam; McLean, Catriona; Morganti-Kossmann, Cristina; Rosenfeld, Jeffrey V; Madani, Rime; Vassalli, Jean-Dominique; Su, Enming J; Lawrence, Daniel A; Medcalf, Robert L

    2012-11-01

    The neurovascular unit provides a dynamic interface between the circulation and central nervous system. Disruption of neurovascular integrity occurs in numerous brain pathologies including neurotrauma and ischaemic stroke. Tissue plasminogen activator is a serine protease that converts plasminogen to plasmin, a protease that dissolves blood clots. Besides its role in fibrinolysis, tissue plasminogen activator is abundantly expressed in the brain where it mediates extracellular proteolysis. However, proteolytically active tissue plasminogen activator also promotes neurovascular disruption after ischaemic stroke; the molecular mechanisms of this process are still unclear. Tissue plasminogen activator is naturally inhibited by serine protease inhibitors (serpins): plasminogen activator inhibitor-1, neuroserpin or protease nexin-1 that results in the formation of serpin:protease complexes. Proteases and serpin:protease complexes are cleared through high-affinity binding to low-density lipoprotein receptors, but their binding to these receptors can also transmit extracellular signals across the plasma membrane. The matrix metalloproteinases are the second major proteolytic system in the mammalian brain, and like tissue plasminogen activators are pivotal to neurological function but can also degrade structures of the neurovascular unit after injury. Herein, we show that tissue plasminogen activator potentiates neurovascular damage in a dose-dependent manner in a mouse model of neurotrauma. Surprisingly, inhibition of activity following administration of plasminogen activator inhibitor-1 significantly increased cerebrovascular permeability. This led to our finding that formation of complexes between tissue plasminogen activator and plasminogen activator inhibitor-1 in the brain parenchyma facilitates post-traumatic cerebrovascular damage. We demonstrate that following trauma, the complex binds to low-density lipoprotein receptors, triggering the induction of matrix

  19. A Novel Interaction between Complement Inhibitor C4b-binding Protein and Plasminogen That Enhances Plasminogen Activation*

    PubMed Central

    Agarwal, Vaibhav; Talens, Simone; Grandits, Alexander M.; Blom, Anna M.

    2015-01-01

    The complement, coagulation, and fibrinolytic systems are crucial for the maintenance of tissue homeostasis. To date numerous interactions and cross-talks have been identified between these cascades. In line with this, here we propose a novel, hitherto unknown interaction between the complement inhibitor C4b-binding protein (C4BP) and plasminogen of the fibrinolytic pathway. Binding of C4BP to Streptococcus pneumoniae is a known virulence mechanism of this pathogen and it was increased in the presence of plasminogen. Interestingly, the acute phase variant of C4BP lacking the β-chain and protein S binds plasminogen much stronger than the main isoform containing the β-chain and protein S. Indeed, the complement control protein (CCP) 8 domain of C4BP, which would otherwise be sterically hindered by the β-chain, primarily mediates this interaction. Moreover, the lysine-binding sites in plasminogen kringle domains facilitate the C4BP-plasminogen interaction. Furthermore, C4BP readily forms complexes with plasminogen in fluid phase and such complexes are present in human serum and plasma. Importantly, whereas the presence of plasminogen did not affect the factor I cofactor activity of C4BP, the activation of plasminogen by urokinase-type plasminogen activator to active plasmin was significantly augmented in the presence of C4BP. Taken together, our data demonstrate a novel interaction between two proteins of the complement and fibrinolytic system. Most complexes might be formed during the acute phase of inflammation and have an effect on the homeostasis at the site of injury or acute inflammation. PMID:26067271

  20. Evaluation of Prognostic Values of Tissue Plasminogen Activator and Plasminogen Activator Inhibitor-1 in Crimean-Congo Hemorrhagic Fever Patients

    PubMed Central

    Gurbuz, Yunus; Ozturk, Baris; Tutuncu, Emin Ediz; Sencan, Irfan; Cicek Senturk, Gonul; Altay, Fatma Aybala

    2015-01-01

    Background: Crimean-Congo hemorrhagic fever (CCHF) is a widespread disease in Turkey, and was responsible for many deaths in endemic regions during the last decade. The pathogenesis of the disease is not fully understood yet. Objectives: In this study we aimed to determine the levels of tissue plasminogen activator (tPA) and Plasminogen activator inhibitor-1 (PAI-1) as predictors of prognosis in CCHF. Patients and Methods: Patients who were diagnosed by the polymerase chain reaction (PCR) and IgM positivity in the reference laboratory were included in this study. Tissue Plasminogen activator and PAI-1 levels were measured by the enzyme linked immunosorbent assay (ELISA) using a commercial kit (human t-PA ELISA and human PAL-1 ELISA; BioVendor research and diagnostic products, BioVendor-Laboratorni medicina a.s., Brno, Czech Republic). Results: A total of 46 patients participated in this study. The significant differences between recovering patients and the patients who died, regarding Aspartate aminotransferase (AST), Creatine Phosphokinase (CPK), Lactate Dehydrogenase (LDH), Prothrombin Time (PT), activated Partial Thromboplastin time (aPTT), and thrombocyte and fibrinogen levels, were consistent with many clinical studies in the literature. The fatal cases were found to have higher tPA and PAI-1 levels in contrast to the patients who completely recovered. Conclusions: We think that these findings may help the progress of understanding of CCHF pathogenesis. PMID:26587219

  1. Effects of tiaprofenic acid on plasminogen activators and inhibitors in human OA and RA synovium.

    PubMed

    Pelletier, J P; McCollum, R; Cloutier, J M; Martel-Pelletier, J

    1992-01-01

    The effect of therapeutic and pharmacological concentrations of tiaprofenic acid, a non-steroidal anti-inflammatory drug (NSAID), on the synthesis of the plasminogen activators, urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA), and the plasminogen activator inhibitors 1 and 2 (PAI-1 and PAI-2), by human synovial membranes isolated from osteoarthritis (OA) and rheumatoid arthritis (RA) sufferers was evaluated. Both forms of plasminogen activator (PA) and PA inhibitor (PAI) were synthesized by the arthritic synovium. PAI-1 and PAI-2 were both synthesized in greater amounts than the plasminogen activators. Tiaprofenic acid induced a dose-dependent decrease in uPA synthesis in both OA and RA, particularly in OA synovium, but had no true effect on tPA. Tiaprofenic acid also exerted a suppressive effect on the synthesis of PAI-1 in both OA and RA synovial membranes, and on the release of PAI-2 in RA synovium. The results of this study indicate that a decrease in uPA synthesis may be one of the mechanisms by which tiaprofenic acid could exert its effects on the arthritic process. The suppressive action of tiaprofenic acid on PAI is not likely to have a significant impact on the balance of plasminogen activators and plasminogen activator inhibitors, as plasminogen activator inhibitors are synthesized in greater amounts than plasminogen activators.

  2. Plasminogen activator activity in cortical granules of bovine oocytes during in vitro maturation.

    PubMed

    Rekkas, Constantinos A; Besenfelder, Urban; Havlicek, Vitezslav; Vainas, Emmanuel; Brem, Gottfried

    2002-04-15

    In this study, we provide evidence that plasminogen activator of tissue-type (t-PA), at least, is present in extracts of bovine oocyte cortical granules, and that its activity varies significantly with the duration of oocyte in vitro maturation. Cortical granules were collected from bovine oocytes by means of micromanipulation, after 0, 12, or 24 h of IVM. Our results show that plasminogen activator activity of cortical granule extracts was significantly higher after 24 h of IVM than after 12 h of IVM or before IVM. This activity was apparently due, at least partly, to tissue-type plasminogen activator as shown immunologically. No evidence was found for the presence of urokinase-type plasminogen activator, plasminogen activator inhibitors or plasmin inhibitors in bovine oocyte cortical granule extracts. Our findings further support the hypothesis that t-PA activity of oocyte origin may have a role in oocyte maturation or fertilization, as well as in post-fertilization events, such as cortical reaction and formation of the zona block to polyspermy.

  3. Altered expression of urokinase-type plasminogen activator and plasminogen activator inhibitor in high-risk soft tissue sarcomas.

    PubMed

    Benassi, M S; Ponticelli, F; Azzoni, E; Gamberi, G; Pazzaglia, L; Chiechi, A; Conti, A; Spessotto, P; Scapolan, M; Pignotti, E; Bacchini, P; Picci, P

    2007-09-01

    In recent years, classification of soft-tissue sarcomas (STS) has improved with cytogenetic analyses, but their clinical behavior is still not easily predictable. The aim of this study was to detect alterations in the urokinase-type plasminogen system, involved in tumor growth and invasion, by comparing mRNA levels of its components with those of paired normal tissues, and relating them with patient clinical course. Real-time PCR was performed on human STS cell lines and tissues from highly malignant STS, including leiomyosarcomas and malignant fibrous histiocytomas, to evaluate the expression of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1). Immunohistochemistry of gene products was also performed. Median mRNA values of all genes studied were higher in tumors than in paired normal tissues. In agreement with data on STS cell lines, significant up-regulation for uPA and PAI-1 genes compared to reference values was seen. Moreover, different levels of expression were related to histotype and metastatic phenotype. There was accordance between uPA mRNA and protein expression, while immunodetection of PAI-1 product was weak and scattered. Clearly, the controversial role of PAI-1 protein requires further biological analyses, but evident involvement of uPA/PAI-1 gene overexpression in STS malignancy may highlight a molecular defect useful in discriminating STS high-risk patients.

  4. Mechanism of the action of SMTP-7, a novel small-molecule modulator of plasminogen activation.

    PubMed

    Koyanagi, Keiji; Narasaki, Ritsuko; Yamamichi, Shingo; Suzuki, Eriko; Hasumi, Keiji

    2014-06-01

    SMTP-7 is a small molecule that promotes the proteolytic activation of plasminogen by relaxing its conformation. SMTP-7 has excellent therapeutic activities against thrombotic stroke in several rodent models. The objective of this study was to elucidate detailed mechanism of the action of SMTP-7 in vitro. We report here that the action of SMTP-7 requires a cofactor with a long-chain alkyl or alkenyl group, and that the fifth kringle domain (kringle 5) of plasminogen is involved in the SMTP-7 action. In this study, we found that the SMTP-7 action to enhance plasminogen activation depended on the presence of a certain type of surfactant, and we screened biologically relevant molecules for their cofactor activity for the SMTP action. As a result, phospholipids, sphingolipids, and oleic acid were found to be active in assisting the SMTP-7 action. On the contrary, stearic acid and bile acids were inactive. Thus, a certain structural element, not only the surface-activating potential, is required for a compound to act as a cofactor for the SMTP-7 action. The plasminogen molecule consists of a PAN domain, five kringle domains, and a serine protease domain. The cofactor-dependent effects of SMTP-7 was observed with plasminogen species including kringle 5 such as intact plasminogen (Glu-plasminogen), des-PAN plasminogen (Lys-plasminogen), and des-[PAN - (kringles 1-4)] plasminogen (mini-plasminogen). However, SMTP-7 effect was not observed with the smallest plasminogen species des-[PAN - (kringles 1-4) and a half of kringle 5)] plasminogen (micro-plasminogen). Thus, kringle 5 is crucial for the action of SMTP-7.

  5. Pivotal role of tissue plasminogen activator in the mechanism of action of electroconvulsive therapy.

    PubMed

    Hoirisch-Clapauch, Silvia; Mezzasalma, Marco A U; Nardi, Antonio E

    2014-02-01

    Electroconvulsive therapy is an important treatment option for major depressive disorders, acute mania, mood disorders with psychotic features, and catatonia. Several hypotheses have been proposed as electroconvulsive therapy's mechanism of action. Our hypothesis involves many converging pathways facilitated by increased synthesis and release of tissue-plasminogen activator. Human and animal experiments have shown that tissue-plasminogen activator participates in many mechanisms of action of electroconvulsive therapy or its animal variant, electroconvulsive stimulus, including improved N-methyl-D-aspartate receptor-mediated signaling, activation of both brain-derived neurotrophic factor and vascular endothelial growth factor, increased bioavailability of zinc, purinergic release, and increased mobility of dendritic spines. As a result, tissue-plasminogen activator helps promote neurogenesis in limbic structures, modulates synaptic transmission and plasticity, improves cognitive function, and mediates antidepressant effects. Notably, electroconvulsive therapy seems to influence tissue-plasminogen activator metabolism. For example, electroconvulsive stimulus increases the expression of glutamate decarboxylase 65 isoform in γ-aminobutyric acid-releasing neurons, which enhances the release of tissue-plasminogen activator, and the expression of p11, a protein involved in plasminogen and tissue-plasminogen activator assembling. This paper reviews how electroconvulsive therapy correlates with tissue-plasminogen activator. We suggest that interventions aiming at increasing tissue-plasminogen activator levels or its bioavailability - such as daily aerobic exercises together with a carbohydrate-restricted diet, or normalization of homocysteine levels - be evaluated in controlled studies assessing response and remission duration in patients who undergo electroconvulsive therapy.

  6. The tissue plasminogen activator-plasminogen proteolytic cascade accelerates amyloid-beta (Abeta) degradation and inhibits Abeta-induced neurodegeneration.

    PubMed

    Melchor, Jerry P; Pawlak, Robert; Strickland, Sidney

    2003-10-01

    Accumulation of the amyloid-beta (Abeta) peptide depends on both its generation and clearance. To better define clearance pathways, we have evaluated the role of the tissue plasminogen activator (tPA)-plasmin system in Abeta degradation in vivo. In two different mouse models of Alzheimer's disease, chronically elevated Abeta peptide in the brain correlates with the upregulation of plasminogen activator inhibitor-1 (PAI-1) and inhibition of the tPA-plasmin system. In addition, Abeta injected into the hippocampus of mice lacking either tPA or plasminogen persists, inducing PAI-1 expression and causing activation of microglial cells and neuronal damage. Conversely, Abeta injected into wild-type mice is rapidly cleared and does not cause neuronal degeneration. Thus, the tPA-plasmin proteolytic cascade aids in the clearance of Abeta, and reduced activity of this system may contribute to the progression of Alzheimer's disease.

  7. Schistosomes Enhance Plasminogen Activation: The Role of Tegumental Enolase

    PubMed Central

    Figueiredo, Barbara C.; Da'dara, Akram A.; Oliveira, Sergio C.; Skelly, Patrick J.

    2015-01-01

    Schistosoma mansoni is a blood fluke parasite that causes schistosomiasis, a debilitating disease of global public health importance. These relatively large parasites are able to survive prolonged periods in the human vasculature without inducing stable blood clots around them. We show here that the intravascular life stages (schistosomula and adult males and females) can all promote significant plasminogen (PLMG) activation in the presence of tissue plasminogen activator (tPA). This results in the generation of the potent fibrinolytic agent plasmin which could degrade blood clots forming around the worms in vivo. We demonstrate that S. mansoni enolase (SmEno) is a host-interactive tegumental enzyme that, in recombinant form, can bind PLMG and promote its activation. Like classical members of the enolase protein family, SmEno can catalyze the interconversion of 2-phospho-D-glycerate (2-PGA) and phosphoenolpyruvate (PEP). The enzyme has maximal activity at pH 7.5, requires Mg2+ for optimal activity and can be inhibited by NaF but not mefloquin. Suppressing expression of the SmEno gene significantly diminishes enolase mRNA levels, protein levels and surface enzyme activity but, surprisingly, does not affect the ability of the worms to promote PLMG activation. Thus, while SmEno can enhance PLMG activation, our analysis suggests that it is not the only contributor to the parasite’s ability to perform this function. We show that the worms possess several other PLMG-binding proteins in addition to SmEno and these may have a greater importance in schistosome-driven PLMG activation. PMID:26658895

  8. Distribution of plasminogen activator inhibitor (PAI-1) in tissues.

    PubMed Central

    Simpson, A J; Booth, N A; Moore, N R; Bennett, B

    1991-01-01

    Extracts of human tissue were analysed for plasminogen activator inhibitor (PAI-1) antigen and activity. PAI-1 was localised in tissues by an immunochemical method, using monoclonal antibodies. PAI-1 occurred throughout the body; its concentration and activity differed considerably from organ to organ. Extracts of liver and spleen had the greatest abundance of PAI-1, but the activity of the inhibitor was much higher in liver than in spleen: the liver may be a source of plasma PAI-1. Immunochemical staining for PAI-1 was observed in endothelium, platelets and their precursor cells, the megakaryocytes, and locations central to the process of haemostasis. PAI-1 also occurred in neutrophil polymorphs and macrophages, cells important in inflammatory and immune processes, but not in lymphocytes. Other cell types, in particular, vascular smooth muscle cells and mesangial cells, also stained positively for PAI-1 and such cells seem to represent an important reservoir of PAI-1. Images PMID:1864986

  9. Arrhenius temperature dependence of in vitro tissue plasminogen activator thrombolysis

    NASA Astrophysics Data System (ADS)

    Shaw, George J.; Dhamija, Ashima; Bavani, Nazli; Wagner, Kenneth R.; Holland, Christy K.

    2007-06-01

    Stroke is a devastating disease and a leading cause of death and disability. Currently, the only FDA approved therapy for acute ischemic stroke is the intravenous administration of the thrombolytic medication, recombinant tissue plasminogen activator (tPA). However, this treatment has many contraindications and can have dangerous side effects such as intra-cerebral hemorrhage. These treatment limitations have led to much interest in potential adjunctive therapies, such as therapeutic hypothermia (T <= 35 °C) and ultrasound enhanced thrombolysis. Such interest may lead to combining these therapies with tPA to treat stroke, however little is known about the effects of temperature on the thrombolytic efficacy of tPA. In this work, we measure the temperature dependence of the fractional clot mass loss Δm(T) resulting from tPA exposure in an in vitro human clot model. We find that the temperature dependence is well described by an Arrhenius temperature dependence with an effective activation energy Eeff of 42.0 ± 0.9 kJ mole-1. Eeff approximates the activation energy of the plasminogen-to-plasmin reaction of 48.9 kJ mole-1. A model to explain this temperature dependence is proposed. These results will be useful in predicting the effects of temperature in future lytic therapies.

  10. Tissue Plasminogen Activator (tPA) Mediates Neurotoxin-Induced Cell Death and Microglial Activation

    DTIC Science & Technology

    2001-07-01

    Alzheimer’s disease and stroke. Tissue plasminogen activator (tPA), a protease converting plasminogen to plasmin, is necessary for neurodegeneration. In mice lacking tPA (tPA-/1), neurons are resistant to neurotoxic death. Delivery of tPA into tpA-/- mice restores susceptibility to neuronal death, indicating that tPA is neurotoxic in the context of excitotoxic injury. Although tPA is synthesized by neurons, the increase in tPA upon injury derives primarily from activated microglia, the immune cells of the brain. Microglia in tPA-/- mice demonstrate reduced activation.

  11. Feasibility of Tissue Plasminogen Activator Formulated for Pulmonary Delivery

    PubMed Central

    Dunn, John S.; Nayar, Rajiv; Campos, Jackie; Hybertson, Brooks M.; Zhou, Yue; Manning, Mark Cornell; Repine, John E.; Stringer, Kathleen A.

    2007-01-01

    Purpose This study was conducted to assess the feasibility of a pulmonary formulation of tissue plasminogen activator (tPA) for nebulization into the airway by measuring protein stability, biologic activity, particle size, and estimating human lung distribution. Methods Formulations were derived by varying the surfactant and protein concentrations. Protein stability and recovery of each nebulized tPA formulation were assessed by ultraviolet spectroscopy. Formulations that met protein stability feasibility criteria were assessed for biologic and fibrinolytic activities. Biologic activity was determined by their ability to inhibit superoxide anion production by human neutrophils. Fibrinolytic activity was assessed by the cleavage of plasminogen to plasmin. Aerodynamic properties were assessed using a cascade impactor, and an estimation of human airway deposition was made via a human lung replica. Results Twenty-seven tPA formulations were initially assessed, 15 of which met protein stability criteria. Subsequently, three of these formulations maintained biologic and fibrinolytic activities. These formulations exhibited particle sizes of 2.4–3.1 μm, and had respirable doses ≥65%. A formulation of 1 mg mL−1 tPA and 0.1% Tween 80 exhibited a 45% deposition in the lower airways of a human lung replica. Conclusions A suitable pulmonary tPA formulation was identified that, following nebulization, maintained protein stability as well as biologic and fibrinolytic activities, and resulted in an optimal respirable dose and human airway deposition. This formulation may be applicable in the treatment of lung diseases, such as acute respiratory distress syndrome by permitting targeted pulmonary delivery of a therapeutic protein to the lungs. PMID:16180128

  12. Plasminogen activator inhibitor-1 4G/5G polymorphism is associated with metabolic syndrome parameters in Malaysian subjects.

    PubMed

    Al-Hamodi, Zaid H; Saif-Ali, Riyadh; Ismail, Ikram S; Ahmed, Khaled A; Muniandy, Sekaran

    2012-05-01

    The plasminogen activator inhibitor-1 4G/5G and tissue plasminogen activator Alu-repeat insertion/deletion polymorphisms might be genetic determinations of increased or decreased of their plasma activities. The aim of this study was to investigate the association of plasminogen activator inhibitor-1 4G/5G and tissue plasminogen activator Alu-repeat I/D polymorphisms with metabolic syndrome parameters in normal Malaysian subjects and to assess the impact of these polymorphisms on their plasma activities and antigens. The genetic polymorphisms were genotyped in 130 normal subjects. In addition, the plasma activities and antigens of plasminogen activator inhibitor-1 and tissue plasminogen activator as well as levels of insulin, glucose, and lipid profile at fasting state were investigated. The subjects with homozygous 4G/4G showed association with an increased triglyceride (p = 0.007), body mass index (p = 0.01) and diastolic blood pressure (p = 0.03). In addition, the plasminogen activator inhibitor-1 4G/5G polymorphism modulates plasma plasminogen activator inhibitor-1 activity and antigen and tissue plasminogen activator activity (p = 0.002, 0.014, 0.003) respectively. These results showed that, the plasminogen activator inhibitor-1 4G/5G polymorphism is associated with metabolic syndrome parameters, plasminogen activator inhibitor-1 and tissue plasminogen activator activities in Malaysian subjects, and may serve to increase the risk of type 2 diabetes and cardiovascular disease in Malaysian subjects.

  13. Tissue-type plasminogen activator and plasminogen embedded in casein rule its degradation under physiological situations: manipulation with casein hydrolysate.

    PubMed

    Silanikove, Nissim; Shapiro, Fira; Merin, Uzi; Leitner, Gabriel

    2013-05-01

    The aims of this study were to test the assumption that tissue-type plasminogen activator (t-PA) and plasminogen (PG) are closely associated with the casein micelle and form a functional complex that rules casein degradation. This assumption was essentially verified for bovine milk under conditions wherein the plasmin system was activated by treatment with casein hydrolysate. It was also shown that urokinase-type PA (u-PA), the second type of plasminogen activator present in milk, was not involved in casein degradation. In agreement with previous studies, we show that treatment with casein hydrolysate precipitously reduced mammary secretion, disrupted the tight junction integrity (increase in Na+ and decrease in K+ concentrations), induced hydrolysis of casein, and activated various elements of the innate and acquired immune system. In the present study, we have identified t-PA as the principal PA, which is responsible for the conversion of PG to plasmin. It was found that t-PA and plasminogen are present in freshly secreted milk (less than 10 min from its secretion), suggesting that they are secreted as a complex by the mammary gland epithelial cells. Further research is needed to provide the direct evidence to verify this concept.

  14. An Active Site Water Network in the Plasminogen Activator Pla from Yersinia pestis

    SciTech Connect

    Eren, Elif; Murphy, Megan; Goguen, Jon; van den Berg, Bert

    2010-08-13

    The plasminogen activator Pla from Yersinia pestis is an outer membrane protease (omptin) that is important for the virulence of plague. Here, we present the high-resolution crystal structure of wild-type, enzymatically active Pla at 1.9 {angstrom}. The structure shows a water molecule located between active site residues D84 and H208, which likely corresponds to the nucleophilic water. A number of other water molecules are present in the active site, linking residues important for enzymatic activity. The R211 sidechain in loop L4 is close to the nucleophilic water and possibly involved in the stabilization of the oxyanion intermediate. Subtle conformational changes of H208 result from the binding of lipopolysaccharide to the outside of the barrel, explaining the unusual dependence of omptins on lipopolysaccharide for activity. The Pla structure suggests a model for the interaction with plasminogen substrate and provides a more detailed understanding of the catalytic mechanism of omptin proteases.

  15. An active site water network in the plasminogen activator pla from Yersinia pestis.

    PubMed

    Eren, Elif; Murphy, Megan; Goguen, Jon; van den Berg, Bert

    2010-07-14

    The plasminogen activator Pla from Yersinia pestis is an outer membrane protease (omptin) that is important for the virulence of plague. Here, we present the high-resolution crystal structure of wild-type, enzymatically active Pla at 1.9 A. The structure shows a water molecule located between active site residues D84 and H208, which likely corresponds to the nucleophilic water. A number of other water molecules are present in the active site, linking residues important for enzymatic activity. The R211 sidechain in loop L4 is close to the nucleophilic water and possibly involved in the stabilization of the oxyanion intermediate. Subtle conformational changes of H208 result from the binding of lipopolysaccharide to the outside of the barrel, explaining the unusual dependence of omptins on lipopolysaccharide for activity. The Pla structure suggests a model for the interaction with plasminogen substrate and provides a more detailed understanding of the catalytic mechanism of omptin proteases.

  16. Thrombolytic Therapy by Tissue Plasminogen Activator for Pulmonary Embolism.

    PubMed

    Islam, Md Shahidul

    2017-01-01

    Clinicians need to make decisions about the use of thrombolytic (fibrinolytic) therapy for pulmonary embolism (PE) after carefully considering the risks of major complications from bleeding, and the benefits of treatment, for each individual patient. They should probably not use systemic thrombolysis for PE patients with normal blood pressure. Treatment by human recombinant tissue plasminogen activator (rt-PA), alteplase, saves the lives of high-risk PE patients, that is, those with hypotension or in shock. Even in the absence of strong evidence, clinicians need to choose the most appropriate regimen for administering alteplase for individual patients, based on assessment of the urgency of the situation, risks for major complications from bleeding, and patient's body weight. In addition, invasive strategies should be considered when absolute contraindications for thrombolytic therapy exist, serious complications arise, or thrombolytic therapy fails.

  17. [Thrombolysis by tissue plasminogen activator in acute myocardial infarct].

    PubMed

    Keltai, M; Dékány, P; Németh, J; Palik, I; Sitkei, E; Szente, A; Arvay, A

    1991-09-15

    The authors participated in the European multicenter investigation, ESPRIT, organized by the Wellcome Research Laboratories. Thrombolytic treatment by intravenous tissue plasminogen activator was performed in 25 patients with early (less than 6h) myocardial infarction. The efficacy of the treatment was controlled by repeat coronary arteriography at 60 minutes, at 90 minutes and at 24 hours of the tpA treatment. The infarct related artery was reperfused in 9/25 patients at 60 minutes, in 16/25 at 90 minutes and 17/18 at 24 hours. Four patients died after unsuccessful treatment or reocclusion. In two patients significant bleeding occurred at the puncture site but no transfusion was required. No other untoward effect was registered. The left ventricular function did not change significantly during the first day of infarction. It is concluded, that tpA is a safe thrombolytic agent in myocardial infarction. Its thrombolytic efficacy is similar to that of streptokinase.

  18. Urokinase type plasminogen activator receptor expression in colorectal neoplasms

    PubMed Central

    Suzuki, S; Hayashi, Y; Wang, Y; Nakamura, T; Morita, Y; Kawasaki, K; Ohta, K; Aoyama, N; Kim, S; Itoh, H; Kuroda, Y; Doe, W

    1998-01-01

    Background—The urokinase type plasminogen activator receptor (uPAR) may play a critical role in cancer invasion and metastasis. 
Aims—To study the involvement of uPAR in colorectal carcinogenesis. 
Methods—The cellular expression and localisation of uPAR were investigated in colorectal adenomas and invasive carcinomas by in situ hybridisation, immunohistochemistry, and northern and western blot analyses. 
Results—uPAR mRNA expression was found mainly in the cytoplasm of dysplastic epithelial cells of 30% of adenomas with mild (19%), moderate (21%), and severe (47%) dysplasia, and in that of carcinomatous cells of 85% of invasive carcinomas: Dukes' stages A (72%), B (93%), and C (91%). Some stromal cells in the adjacent neoplastic epithelium were faintly positive. Immunoreactivity for uPAR was detected in dysplastic epithelial cells of 14% of adenomas and in carcinomatous cells of 49% of invasive carcinomas. uPAR mRNA and protein concentrations were significantly higher in severe than in mild or moderate dysplasia (p<0.05); they were notably higher in Dukes' stage A than in severe dysplasia (p<0.05), and significantly higher in Dukes' stage B than in stage A (p<0.05), but those in stage B were not different from those in stage C or in metastatic colorectal carcinomas of the liver. 
Conclusions—Colorectal adenoma uPAR, expressed essentially in dysplastic epithelial cells, was upregulated with increasing severity of atypia, and increased notably during the critical transition from severe dysplasic adenoma to invasive carcinoma. These findings implicate uPAR expression in the invasive and metastatic processes of colorectal cancer. 

 Keywords: urokinase type plasminogen activator receptor; colorectal adenoma; colorectal cancer; adenoma-carcinoma sequence PMID:9824607

  19. Nattokinase-promoted tissue plasminogen activator release from human cells.

    PubMed

    Yatagai, Chieko; Maruyama, Masugi; Kawahara, Tomoko; Sumi, Hiroyuki

    2008-01-01

    When heated to a temperature of 70 degrees C or higher, the strong fibrinolytic activity of nattokinase in a solution was deactivated. Similar results were observed in the case of using Suc-Ala-Ala-Pro-Phe-pNA and H-D-Val-Leu-Lys-pNA, which are synthetic substrates of nattokinase. In the current study, tests were conducted on the indirect fibrinolytic effects of the substances containing nattokinase that had been deactivated through heating at 121 degrees C for 15 min. Bacillus subtilis natto culture solutions made from three types of bacteria strain were heat-treated and deactivated, and it was found that these culture solutions had the ability to generate tissue plasminogen activators (tPA) from vascular endothelial cells and HeLa cells at certain concentration levels. For example, it was found that the addition of heat-treated culture solution of the Naruse strain (undiluted solution) raises the tPA activity of HeLa cells to about 20 times that of the control. Under the same conditions, tPA activity was raised to a level about 5 times higher for human vascular endothelial cells (HUVEC), and to a level about 24 times higher for nattokinase sold on the market. No change in cell count was observed for HeLa cells and HUVEC in the culture solution at these concentrations, and the level of activity was found to vary with concentration.

  20. Sensitive, coupled assay for plasminogen activator using a thiol ester substrate for plasmin

    SciTech Connect

    Coleman, P L; Green, G D.J.

    1980-01-01

    Several assays for plasminogen activator employ a direct assay method. These are remarkably sensitive methods, yet they suffer in comparison to the sensitivity of coupled methods. Coupling the assay with plasminogen not only amplifies the sensitivity by the multiplicative effect of plasmin, but insures that only those proteases specific for plasminogen are assayed. The choice of substrate for plasmin is critical. A thiol ester substrate, thiobenzyl benzyloxy-carbonyl-L-lysinate (Z-Lys-SBzl), has been synthesized which combines high k/sub cat/ with alkaline stability. In an effort to characterize the plasminogen activator from hepatoma tissue culture (HTC) and its hormonally-controlled inhibitor, Z-Lys-SBzl was used in a coupled approach providing an assay which is superior to the /sup 125/I-fibrinolytic assay. It is also extremely sensitive to plasminogen activator and can be used for routine analysis of purification as well as kinetic and binding studies. (ERB)

  1. Tissue plasminogen activator and plasminogen activator inhibitor type 1 gene polymorphism in patients with gastric ulcer complicated with bleeding.

    PubMed Central

    Kim, Hong-Soo; Hwang, Kyu-Yoon; Chung, Il-Kwon; Park, Sang-Heum; Lee, Moon-Ho; Kim, Sun-Joo; Hong, Sae-Yong

    2003-01-01

    Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) may be involved in the pathogenesis of peptic ulcers through suppression of fibrinolysis. This study was designed to investigate associations of t-PA and PAI-1 genes with clinical features of the patients with bleeding gastric ulcers. Eighty-four patients with peptic ulcers and 100 controls were studied between January 1998 and April 2000. We used polymerase chain reaction and endonuclease digestion to genotype for 4G/5G polymorphism in the promoter region of the PAI-1 gene and the Alurepeat insertion/deletion (I/D) polymorphism in intron h of the t-PA gene. Various clinical features, including lesion site, bleeding event, recurrence of ulcer, and rebleeding, were assessed using a multiple logistic regression model. The genotype distributions of both the t-PA and PAI-1 genes did not differ between the patient and control groups. The occurrence of the I/D or D/D genotype of t-PA was significantly higher in cases of duodenal ulcer (adjusted OR=4.39, 95% CI=1.12-17.21). When a dominant effect (i.e., 4G/4G or 4G/5G versus 5G/5G) of the 4G allele was assumed, the PAI-1 4G/4G genotype was independently associated with rebleeding after hemostasis (adjusted OR=5.07, 95% CI=1.03-24.87). Our data suggest that t-PA gene polymorphism is associated with duodenal ulcers, and that the PAI-1 gene may be a risk factor leading to recurrent bleeding after initial hemostasis. PMID:12589088

  2. Keeping the blood flowing-plasminogen activator genes and feeding behavior in vampire bats.

    PubMed

    Tellgren-Roth, Asa; Dittmar, Katharina; Massey, Steven E; Kemi, Cecilia; Tellgren-Roth, Christian; Savolainen, Peter; Lyons, Leslie A; Liberles, David A

    2009-01-01

    The blood feeding vampire bats emerged from New World leaf-nosed bats that fed on fruit and insects. Plasminogen activator, a serine protease that regulates blood coagulation, is known to be expressed in the saliva of Desmodus rotundus (common vampire bat) and is thought to be a key enzyme for the emergence of blood feeding in vampire bats. To better understand the evolution of this biological function, we studied the plasminogen activator (PA) genes from all vampire bat species in light of their feeding transition to bird and subsequently mammalian blood. We include the rare species Diphylla ecaudata and Diaemus youngi, where plasminogen activator had not previously been studied and demonstrate that PA gene duplication observed in Desmodus is not essential to the vampire phenotype, but relates to the emergence of predominant mammalian blood feeding in this species. Plasminogen activator has evolved through gene duplication, domain loss, and sequence evolution leading to change in fibrin-specificity and susceptibility to plasminogen activator inhibitor-1. Before undertaking this study, only the four plasminogen activator isoforms from Desmodus were known. The evolution of vampire bat plasminogen activators can now be linked phylogenetically to the transition in feeding behavior among vampire bat species from bird to mammalian blood.

  3. Keeping the blood flowing—plasminogen activator genes and feeding behavior in vampire bats

    NASA Astrophysics Data System (ADS)

    Tellgren-Roth, Åsa; Dittmar, Katharina; Massey, Steven E.; Kemi, Cecilia; Tellgren-Roth, Christian; Savolainen, Peter; Lyons, Leslie A.; Liberles, David A.

    2009-01-01

    The blood feeding vampire bats emerged from New World leaf-nosed bats that fed on fruit and insects. Plasminogen activator, a serine protease that regulates blood coagulation, is known to be expressed in the saliva of Desmodus rotundus (common vampire bat) and is thought to be a key enzyme for the emergence of blood feeding in vampire bats. To better understand the evolution of this biological function, we studied the plasminogen activator (PA) genes from all vampire bat species in light of their feeding transition to bird and subsequently mammalian blood. We include the rare species Diphylla ecaudata and Diaemus youngi, where plasminogen activator had not previously been studied and demonstrate that PA gene duplication observed in Desmodus is not essential to the vampire phenotype, but relates to the emergence of predominant mammalian blood feeding in this species. Plasminogen activator has evolved through gene duplication, domain loss, and sequence evolution leading to change in fibrin-specificity and susceptibility to plasminogen activator inhibitor-1. Before undertaking this study, only the four plasminogen activator isoforms from Desmodus were known. The evolution of vampire bat plasminogen activators can now be linked phylogenetically to the transition in feeding behavior among vampire bat species from bird to mammalian blood.

  4. Plasminogen activator inhibitor-2 (PAI-2) in eosinophilic leukocytes.

    PubMed

    Swartz, Jonathan M; Byström, Jonas; Dyer, Kimberly D; Nitto, Takeaki; Wynn, Thomas A; Rosenberg, Helene F

    2004-10-01

    Plasminogen activator inhibitor-2 (PAI-2) as a potential eosinophil protein was inferred from our gene microarray study of mouse eosinophilopoiesis. Here, we detect 47 kDa intracellular and approximately 60 kDa secretory forms of PAI-2 in purified human eosinophil extracts. PAI-2 is present at variable concentrations in eosinophil lysates, ranging from 30 to 444 ng/10(6) cells, with a mean of 182 ng/10(6) cells from 10 normal donors, which is the highest per-cell concentration among all leukocyte subtypes evaluated. Enzymatic assay confirmed that eosinophil-derived PAI-2 is biologically active and inhibits activation of its preferred substrate, urokinase. Immunohistochemical and immunogold staining demonstrated PAI-2 localization in eosinophil-specific granules. Immunoreactive PAI-2 was detected in extracellular deposits in and around the eosinophil-enriched granuloma tissue encapsulating the parasitic egg in livers of wild-type mice infected with the helminthic parasite Schistosoma mansoni. Among the possibilities, we consider a role for eosinophil-derived PAI-2 in inflammation and remodeling associated with parasitic infection as well as allergic airways disease, respiratory virus infection, and host responses to tumors and metastasis in vivo.

  5. Transforming growth factor-beta modulates plasminogen activator activity and plasminogen activator inhibitor type-1 expression in human keratinocytes in vitro.

    PubMed

    Wikner, N E; Elder, J T; Persichitte, K A; Mink, P; Clark, R A

    1990-11-01

    Transforming growth factor beta (TGF-beta) is a multifunctional mediator with effects on cellular growth, differentiation, and extracellular matrix (ECM) metabolism. Because TGF-beta stimulates fibronectin expression in cultured human keratinocytes, we wished to determine whether it might also affect ECM degradation through the plasminogen activator (PA)-plasminogen activator inhibitor (PAI) system. Immunofluorescence of human keratinocytes using a monospecific antiserum to type 1 PAI (PAI-1) showed enhanced cellular and ECM staining when they were cultured in the presence of TGF-beta. The antiserum also identified an Mr 50,000 protein in conditioned media that was markedly enhanced by TGF-beta. A corresponding stimulation of PAI-1 mRNA was demonstrated by quantitative RNA blot analysis. Total plasminogen activating activity of conditioned medium was markedly decreased by TGF-beta. Zymography showed this to be at least partially due to decreased secreted urokinase activity. TGF-beta may play an important role in stabilizing the provisional matrix synthesized by keratinocytes in healing wounds.

  6. Inhibition of cell surface mediated plasminogen activation by a monoclonal antibody against alpha-Enolase.

    PubMed

    López-Alemany, Roser; Longstaff, Colin; Hawley, Stephen; Mirshahi, Massoud; Fábregas, Pere; Jardí, Merce; Merton, Elizabeth; Miles, Lindsey A; Félez, Jordi

    2003-04-01

    Localization of plasmin activity on leukocyte surfaces plays a critical role in fibrinolysis as well as in pathological and physiological processes in which cells must degrade the extracellular matrix in order to migrate. The binding of plasminogen to leukocytic cell lines induces a 30- to 80-fold increase in the rate of plasminogen activation by tissue-type (tPA) and urokinase-type (uPA) plasminogen activators. In the present study we have examined the role of alpha-enolase in plasminogen activation on the cell surface. We produced and characterized a monoclonal antibody (MAb) 11G1 against purified alpha-enolase, which abrogated about 90% of cell-dependent plasminogen activation by either uPA or tPA on leukocytoid cell lines of different lineages: B-lymphocytic, T-lymphocytic, granulocytic, and monocytic cells. In addition, MAb 11G1 also blocked enhancement of plasmin formation by peripheral blood neutrophils and monocytes. In contrast, MAb 11G1 did not affect plasmin generation in the presence of fibrin, indicating that this antibody did not interact with fibrinolytic components in the absence of cells. These data suggest that, although leukocytic cells display several molecules that bind plasminogen, alpha-enolase is responsible for the majority of the promotion of plasminogen activation on the surfaces of leukocytic cells.

  7. Transgenic chickens expressing human urokinase-type plasminogen activator.

    PubMed

    Lee, Sung Ho; Gupta, Mukesh Kumar; Ho, Young Tae; Kim, Teoan; Lee, Hoon Taek

    2013-09-01

    Urokinase-type plasminogen activator is a serine protease that is clinically used in humans for the treatment of thrombolytic disorders and vascular diseases such as acute ischemic stroke and acute peripheral arterial occlusion. This study explored the feasibility of using chickens as a bioreactor for producing human urokinase-type plasminogen activator (huPA). Recombinant huPA gene, under the control of a ubiquitous Rous sarcoma virus promoter, was injected into the subgerminal cavity of freshly laid chicken eggs at stage X using the replication-defective Moloney murine leukemia virus (MoMLV)-based retrovirus vectors encapsidated with VSV-G (vesicular stomatitis virus G) glycoprotein. A total of 38 chicks, out of 573 virus-injected eggs, hatched and contained the huPA gene in their various body parts. The mRNA transcript of the huPA gene was present in various organs, including blood and egg, and was germ-line transmitted to the next generation. The level of active huPA protein was 16-fold higher in the blood of the transgenic chicken than in the nontransgenic chicken (P < 0.05). The expression of huPA protein in eggs increased from 7.82 IU/egg in the G0 generation to 17.02 IU/egg in the G1 generation. However, huPA-expressing embryos had reduced survival and hatchability at d 18 and 21 of incubation, respectively, and the blood clotting time was significantly higher in transgenic chickens than their nontransgenic counterparts (P < 0.05). Furthermore, adult transgenic rooster showed reduced (P < 0.05) fertility, as revealed by reduced volume of semen ejaculate, sperm concentration, and sperm viability. Taken together, our data suggest that huPA transgenic chickens could be successfully produced by the retroviral vector system. Transgenic chickens, expressing the huPA under the control of a ubiquitous promoter, may not only be used as a bioreactor for pharming of the huPA drug but also be useful for studying huPA-induced bleeding and other disorders.

  8. Human epidermal plasminogen activator. Characterization, localization, and modulation.

    PubMed

    Morioka, S; Jensen, P J; Lazarus, G S

    1985-12-01

    Using biochemical and immunocytochemical approaches, we have investigated the plasminogen activator (PA) of primary human epidermal cell cultures. A rabbit antibody raised against human urinary PA (urokinase) inhibited greater than or equal to 96% of the PA activity in the keratinocyte cultures. Immunoblot and double immunodiffusion analyses of keratinocyte PA with anti-urokinase antibody confirmed that epidermal PA was of the urokinase type. Immunocytochemical investigation of human keratinocyte cultures with anti-urokinase antibody revealed two characteristic staining patterns for PA. First, cells at the advancing edge of subconfluent colonies were cytoplasmically stained in a granular pattern. Similar staining was observed at the migrating edges of confluent epidermal cell cultures that had been wounded by cutting with a blade. This induction of PA staining was independent of cell division. Secondly, differentiated epidermal cells located on the surface of colonies were stained either at the plasma membrane or homogeneously throughout the cell. The highly differentiated, spontaneously shed cells were usually very heavily stained by anti-urokinase antibody. These immunocytochemical experiments suggest that PA expression is highly regulated in human epidermal cells. Specifically, PA expression appears to be related to cellular differentiation and to cell movement in expanding or wounded keratinocyte colonies.

  9. Tissue Plasminogen Activator Neurotoxicity is Neutralized by Recombinant ADAMTS 13

    PubMed Central

    Fan, Mengchen; Xu, Haochen; Wang, Lixiang; Luo, Haiyu; Zhu, Ximin; Cai, Ping; Wei, Lixiang; Lu, Lu; Cao, Yongliang; Ye, Rong; Fan, Wenying; Zhao, Bing-Qiao

    2016-01-01

    Tissue plasminogen activator (tPA) is an effective treatment for ischemic stroke, but its neurotoxicity is a significant problem. Here we tested the hypothesis that recombinant ADAMTS 13 (rADAMTS 13) would reduce tPA neurotoxicity in a mouse model of stroke. We show that treatment with rADAMTS 13 in combination with tPA significantly reduced infarct volume compared with mice treated with tPA alone 48 hours after stroke. The combination treatment significantly improved neurological deficits compared with mice treated with tPA or vehicle alone. These neuroprotective effects were associated with significant reductions in fibrin deposits in ischemic vessels and less severe cell death in ischemic brain. The effect of rADAMTS13 on tPA neurotoxicity was mimicked by the N-methyl-D-aspartate (NMDA) receptor antagonist M-801, and was abolished by injection of NMDA. Moreover, rADAMTS 13 prevents the neurotoxicity effect of tPA, by blocking its interaction with the NMDA receptor NR2B and the attendant phosphorylation of NR2B and activation of ERK1/2. Finally, the NR2B-specific NMDA receptor antagonist ifenprodil abolished tPA neurotoxicity and rADAMTS 13 treatment had no further beneficial effect. Our data suggest that the combination of rADAMTS 13 and tPA may provide a novel treatment of ischemic stroke by diminishing the neurotoxic effects of exogenous tPA. PMID:27181025

  10. Tissue plasminogen activator prevents white matter damage following stroke

    PubMed Central

    Correa, Fernando; Gauberti, Maxime; Parcq, Jérôme; Macrez, Richard; Hommet, Yannick; Obiang, Pauline; Hernangómez, Miriam; Montagne, Axel; Liot, Géraldine; Guaza, Carmen; Maubert, Eric; Ali, Carine; Vivien, Denis

    2011-01-01

    Tissue plasminogen activator (tPA) is the only available treatment for acute stroke. In addition to its vascular fibrinolytic action, tPA exerts various effects within the brain, ranging from synaptic plasticity to control of cell fate. To date, the influence of tPA in the ischemic brain has only been investigated on neuronal, microglial, and endothelial fate. We addressed the mechanism of action of tPA on oligodendrocyte (OL) survival and on the extent of white matter lesions in stroke. We also investigated the impact of aging on these processes. We observed that, in parallel to reduced levels of tPA in OLs, white matter gets more susceptible to ischemia in old mice. Interestingly, tPA protects murine and human OLs from apoptosis through an unexpected cytokine-like effect by the virtue of its epidermal growth factor–like domain. When injected into aged animals, tPA, although toxic to the gray matter, rescues white matter from ischemia independently of its proteolytic activity. These studies reveal a novel mechanism of action of tPA and unveil OL as a target cell for cytokine effects of tPA in brain diseases. They show overall that tPA protects white matter from stroke-induced lesions, an effect which may contribute to the global benefit of tPA-based stroke treatment. PMID:21576385

  11. Plasminogen activator inhibitor-1 in acute hyperoxic mouse lung injury.

    PubMed Central

    Barazzone, C; Belin, D; Piguet, P F; Vassalli, J D; Sappino, A P

    1996-01-01

    Hyperoxia-induced lung disease is associated with prominent intraalveolar fibrin deposition. Fibrin turnover is tightly regulated by the concerted action of proteases and antiproteases, and inhibition of plasmin-mediated proteolysis could account for fibrin accumulation in lung alveoli. We show here that lungs of mice exposed to hyperoxia overproduce plasminogen activator inhibitor-1 (PAI-1), and that PAI-1 upregulation impairs fibrinolytic activity in the alveolar compartment. To explore whether increased PAI-1 production is a causal or only a correlative event for impaired intraalveolar fibrinolysis and the development of hyaline membrane disease, we studied mice genetically deficient in PAI-1. We found that these mice fail to develop intraalveolar fibrin deposits in response to hyperoxia and that they are more resistant to the lethal effects of hyperoxic stress. These observations provide clear and novel evidence for the pathogenic contribution of PAI-1 in the development of hyaline membrane disease. They identify PAI-1 as a major deleterious mediator of hyperoxic lung injury. PMID:8981909

  12. Tissue plasminogen activator in the bed nucleus of stria terminalis regulates acoustic startle.

    PubMed

    Matys, T; Pawlak, R; Strickland, S

    2005-01-01

    The bed nucleus of stria terminalis is a basal forebrain region involved in regulation of hormonal and behavioral responses to stress. In this report we demonstrate that bed nucleus of stria terminalis has a high and localized expression of tissue plasminogen activator, a serine protease with neuromodulatory properties and implicated in neuronal plasticity. Tissue plasminogen activator activity in the bed nucleus of stria terminalis is transiently increased in response to acute restraint stress or i.c.v. administration of a major stress mediator, corticotropin-releasing factor. We show that tissue plasminogen activator is important in bed nucleus of stria terminalis function using two criteria: 1, Neuronal activation in this region as measured by c-fos induction is reduced in tissue plasminogen activator-deficient mice; and 2, a bed nucleus of stria terminalis-dependent behavior, potentiation of acoustic startle by corticotropin-releasing factor, is attenuated in tissue plasminogen activator-deficient mice. These studies identify a novel site of tissue plasminogen activator expression in the mouse brain and demonstrate a functional role for this protease in the bed nucleus of stria terminalis.

  13. Bacterial pathogens activate plasminogen to breach tissue barriers and escape from innate immunity.

    PubMed

    Peetermans, Marijke; Vanassche, Thomas; Liesenborghs, Laurens; Lijnen, Roger H; Verhamme, Peter

    2016-11-01

    Both coagulation and fibrinolysis are tightly connected with the innate immune system. Infection and inflammation cause profound alterations in the otherwise well-controlled balance between coagulation and fibrinolysis. Many pathogenic bacteria directly exploit the host's hemostatic system to increase their virulence. Here, we review the capacity of bacteria to activate plasminogen. The resulting proteolytic activity allows them to breach tissue barriers and evade innate immune defense, thus promoting bacterial spreading. Yersinia pestis, streptococci of group A, C and G and Staphylococcus aureus produce a specific bacterial plasminogen activator. Moreover, surface plasminogen receptors play an established role in pneumococcal, borrelial and group B streptococcal infections. This review summarizes the mechanisms of bacterial activation of host plasminogen and the role of the fibrinolytic system in infections caused by these pathogens.

  14. A cleavage-resistant urokinase plasminogen activator receptor exhibits dysregulated cell-surface clearance.

    PubMed

    Nieves, Evelyn C; Manchanda, Naveen

    2010-04-23

    Urokinase plasminogen activator receptor (u-PAR) binds urokinase plasminogen activator (u-PA) and participates in plasminogen activation in addition to modulating several cellular processes such as adhesion, proliferation, and migration. u-PAR is susceptible to proteolysis by its cognate ligand and several other proteases. To elucidate the biological significance of receptor cleavage by u-PA, we engineered and expressed a two-chain urokinase plasminogen activator (tcu-PA) cleavage-resistant u-PAR (cr-u-PAR). This mutated receptor was similar to wild-type u-PAR in binding u-PA and initiating plasminogen activation. However, cr-u-PAR exhibited accelerated internalization and resurfacing due to direct association with the endocytic receptor alpha(2)-macroglobulin receptor/low density lipoprotein receptor-related protein in the absence of the enzyme x inhibitor complex of tcu-PA and plasminogen activator inhibitor-1 (tcu-PA.PAI-1). cr-u-PAR-expressing cells had enhanced migration compared with wild-type u-PAR-expressing cells, and cr-u-PAR was less sensitive to chymotrypsin cleavage as compared with wt u-PAR. Our studies suggest that these mutations in the linker region result in a rearrangement within the cr-u-PAR structure that makes it resemble its ligand-bound form. This constitutively active variant may mimic highly glycosylated cleavage-resistant u-PAR expressed in certain highly malignant cancer-cells.

  15. Site-restricted plasminogen activation mediated by group A streptococcal streptokinase variants.

    PubMed

    Cook, Simon M; Skora, Amanda; Walker, Mark J; Sanderson-Smith, Martina L; McArthur, Jason D

    2014-02-15

    SK (streptokinase) is a secreted plasminogen activator and virulence factor of GAS (group A Streptococcus). Among GAS isolates, SK gene sequences are polymorphic and are grouped into two sequence clusters (cluster type-1 and cluster type-2) with cluster type-2 being further classified into subclusters (type-2a and type-2b). In the present study, we examined the role of bacterial and host-derived cofactors in SK-mediated plasminogen activation. All SK variants, apart from type-2b, can form an activator complex with Glu-Plg (Glu-plasminogen). Specific ligand-binding-induced conformational changes in Glu-Plg mediated by fibrinogen, PAM (plasminogen-binding group A streptococcal M protein), fibrinogen fragment D or fibrin, were required for type-2b SK to form a functional activator complex with Glu-Plg. In contrast with type-1 and type-2a SK, type-2b SK activator complexes were inhibited by α2-antiplasmin unless bound to fibrin or to the GAS cell-surface via PAM in combination with fibrinogen. Taken together, these data suggest that type-2b SK plasminogen activation may be restricted to specific microenvironments within the host such as fibrin deposits or the bacterial cell surface through the action of α2-antiplasmin. We conclude that phenotypic SK variation functionally underpins a pathogenic mechanism whereby SK variants differentially focus plasminogen activation, leading to specific niche adaption within the host.

  16. [Diabetic nephropathy and plasminogen activator inhibitor 1 in urine samples].

    PubMed

    Torii, Kunio; Kimura, Hideki; Li, Xuan; Okada, Toshiharu; Imura, Toshio; Oida, Koji; Miyamori, Isamu; Furusaki, Fumio; Ono, Tomoko; Yoshida, Haruyoshi

    2004-06-01

    Plasminogen activator inhibitor-1 (PAI-1) may contribute to renal fibrosis because of its involvement in matrix (ECM) accumulation through inhibition of plasmin-dependent ECM degradation. The aim of this study is to determine urinary PAI-1 concentrations and its intrarenal localization in patients with various renal diseases and to identify inducers for PAI-1 expression in human cultured proximal renal tubular cells (HRCs). Urinary PAI-1 concentrations were significantly higher in patients with overt diabetic nephropathy (DN, n=36) than in proliferative glomerulonephritis (PGN, n=8), nephrotic syndrome (NS, n=10) and healthy controls (n=12). Urinary PAI-1 concentrations (ng/gCr) were directly correlated with urinary N-acetyl glucosaminidase (NAG) levels (r=0.58, p<0.05). As for intrarenal localization of PAI-1 antigen, strong stainings for PAI-1 were observed in proximal tubular cells of renal biopsy samples from patients with DN, while no stainings for PAI-1 were found in renal tissues of PGN or NS. Immunoblot analysis revealed the presence of PAI-1 protein in whole cell lyzates from HRCs grown to semiconfluency. Exposure of growth-arrested HRCs with hypoxia (1% O2) or TNF-alpha (10 ng/ml) for 24 hours increased the secretion rate of PAI-1 protein by about 2.0-fold, while 24-hour treatment with high glucose (450 mg/dl) did not increase PAI-1 secretion at all, compared with that of the control cells under normal glucose (100 mg/dl) and normoxia (18% O2). These findings suggest that PAI-1 expression is upregulated especially in the proximal renal tubular cells of DN, which may be explained partially by hypoxia and inflammatory cytokines but not high glucose.

  17. Zinc-triggered induction of tissue plasminogen activator by brain-derived neurotrophic factor and metalloproteinases.

    PubMed

    Hwang, Ih-Yeon; Sun, Eun-Sun; An, Ji Hak; Im, Hana; Lee, Sun-Ho; Lee, Joo-Yong; Han, Pyung-Lim; Koh, Jae-Young; Kim, Yang-Hee

    2011-09-01

    Tissue plasminogen activator (tPA) is necessary for hippocampal long-term potentiation. Synaptically released zinc also contributes to long-term potentiation, especially in the hippocampal CA3 region. Using cortical cultures, we examined whether zinc increased the concentration and/or activity of tPA. Two hours after a 10-min exposure to 300 μM zinc, expression of tPA and its substrate, plasminogen, were significantly increased, as was the proteolytic activity of tPA. In contrast, increasing extracellular or intracellular calcium levels did not affect the expression or secretion of tPA. Changing zinc influx or chelating intracellular zinc also failed to alter tPA/plasminogen induction by zinc, indicating that zinc acts extracellularly. Zinc-mediated extracellular activation of matrix metalloproteinase (MMP) underlies the up-regulation of brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase (Trk) signaling. Consistent with these findings, co-treatment with a neutralizing antibody against BDNF or specific inhibitors of MMPs or Trk largely reversed tPA/plasminogen induction by zinc. Treatment of cortical cultures with p-aminophenylmercuric acetate, an MMP activator, MMP-2, or BDNF alone induced tPA/plasminogen expression. BDNF mRNA and protein expression was also increased by zinc and mediated by MMPs. Thus, an extracellular zinc-dependent, MMP- and BDNF-mediated synaptic mechanism may regulate the levels and activity of tPA.

  18. Streptokinase variants from Streptococcus pyogenes isolates display altered plasminogen activation characteristics - implications for pathogenesis.

    PubMed

    Cook, Simon M; Skora, Amanda; Gillen, Christine M; Walker, Mark J; McArthur, Jason D

    2012-12-01

    Streptococcus pyogenes (group A streptococcus, GAS) secretes streptokinase, a potent plasminogen activating protein. Among GAS isolates, streptokinase gene sequences (ska) are polymorphic and can be grouped into two distinct sequence clusters (termed cluster type-1 and cluster type-2) with cluster type-2 being further divided into sub-clusters type-2a and type-2b. In this study, far-UV circular dichroism spectroscopy indicated that purified streptokinase variants of each type displayed similar secondary structure. Type-2b streptokinase variants could not generate an active site in Glu-plasminogen through non-proteolytic mechanisms while all other variants had this capability. Furthermore, when compared with other streptokinase variants, type-2b variants displayed a 29- to 35-fold reduction in affinity for Glu-plasminogen. All SK variants could activate Glu-plasminogen when an activator complex was preformed with plasmin; however, type-2b and type-1 complexes were inhibited by α(2) -antiplasmin. Exchanging ska(type-2a) in the M1T1 GAS strain 5448 with ska(type-2b) caused a reduction in virulence while exchanging ska(type-2a) with ska(type-1) into 5448 produced an increase in virulence when using a mouse model of invasive disease. These findings suggest that streptokinase variants produced by GAS isolates utilize distinct plasminogen activation pathways, which directly affects the pathogenesis of this organism.

  19. Enzymatic vitreolysis with recombinant tissue plasminogen activator for vitreomacular traction

    PubMed Central

    Raczyńska, Dorota; Lipowski, Paweł; Zorena, Katarzyna; Skorek, Andrzej; Glasner, Paulina

    2015-01-01

    Aims The aim of our research was to gain data about the efficacy of intravitreal injections of a recombinant tissue plasminogen activator (rTPA) in dissolving vitreoretinal tractions (VRTs). Materials and methods The study group consisted of patients of our Ophthalmology Clinic who had received an injection of rTPA (TPA Group) for an existent vitreomacular traction confirmed by optical coherence tomography and stereoscopic examinations. The control group consisted of patients who had declined treatment despite the existence of a vitreomacular traction confirmed by the same diagnostic methods. Each group consisted of 30 people (30 eyes). The observation period was 6 months. Conclusion In both groups some of the VRTs had dissolved. In the TPA group the traction dissolved in 10 patients (33.33%) and in the control group only in 5 (16.67%). It is also important to point out that the mean baseline membrane thickness was higher in the TPA group than in the control group. Observing patients in both groups we noticed that the dissolution of vitreoretinal membrane occurred most frequently in those cases where the membrane was thin. In the TPA group, the mean membrane thickness after 6 months decreased considerably. At the same time, no significant change in the membrane thickness could be observed in the control group. Observation of the retinal thickness allows us to draw the following conclusion: in the TPA group, the retinal thickness in the macular area (edema) had decreased over the study period, whereas in the control group it had increased. In those cases where the traction had dissolved, the edema of the retina decreased by the end of the 6-month period in both groups. In the TPA group, the dissolution of the membrane occurred most often within 3 months from the primary injection. Based on statistics, we can confirm that in the control group there was a decrease in visual acuity during the 6 months of the study period. At the same time, visual acuity in the TPA

  20. [Development and optimization of the methods for determining activity of plasminogen activator inhibitor-1 in plasma].

    PubMed

    Roka-Moĭia, Ia M; Zhernosiekov, D D; Kondratiuk, A S; Hrynenko, T V

    2013-01-01

    The activity and content of plasminogen activator inhibitor-1 (PAI-1) are important indicators of pathological processes, because its content in plasma increases at acute myocardium infarction, tumor, diabetes mellitus, etc. The present work is dedicated to the development and optimization of the methods of PAI-1 activity definition, which can be used in clinical practice. We have proposed the modification of the method COATEST PAI with the usage of chromogenic substrate S2251. According to our modification, the cyanogen bromide fragments of human fibrinogen have been changed into bovine desAB-fibrin. We have also developed the method with the usage of fibrin films. In this method fibrin is used as a stimulator of activation reaction and as a substrate at the same time. Using fibrin, the native substrate of plasmin, we provide high specificity of the reaction and exclude the cross-reaction with other plasma enzymes.

  1. Endotoxin induction of an inhibitor of plasminogen activator in bovine pulmonary artery endothelial cells

    SciTech Connect

    Not Available

    1986-01-05

    The effects of bacterial lipopolysaccharide (endotoxin) on the fibrinolytic activity of bovine pulmonary artery endothelial cells were examined. Endotoxin suppressed the net fibrinolytic activity of cell extracts and conditioned media in a dose-dependent manner. The effects of endotoxin required at least 6 h for expression. Cell extracts and conditioned media contained a 44-kDa urokinase-like plasminogen activator. Media also contained multiple plasminogen activators with molecular masses of 65-75 and 80-100 kDa. Plasminogen activators in extracts and media were unchanged by treatment of cells with endotoxin. Diisopropyl fluorophosphate (DFP)-abolished fibrinolytic activity of extracts and conditioned media. DFP-treated samples from endotoxin-treated but not untreated cells inhibited urokinase and tissue plasminogen activator, but not plasmin. Inhibitory activity was lost by incubation at pH 3 or heating to 56/sup 0/C for 10 min. These treatments did not affect inhibitory activity of fetal bovine serum. Incubation of /sup 125/I-urokinase with DFP-treated medium from endotoxin-treated cells produced an inactive complex with an apparent molecular mass of 80-85 kDa.

  2. Inhibitors of Urokinase Type Plasminogen Activator and Cytostatic Activity from Crude Plants Extracts

    PubMed Central

    Zha, Xueqiang; Diaz, Ricardo; Franco, Jose Javier Rosado; Sanchez, Veronica Forbes; Fasoli, Ezio; Barletta, Gabriel; Carvajal, Augusto; Bansal, Vibha

    2014-01-01

    In view of the clear evidence that urokinase type plasminogen activator (uPA) plays an important role in the processes of tumor cell metastasis, aortic aneurysm, and multiple sclerosis, it has become a target of choice for pharmacological intervention. The goal of this study was thus to determine the presence of inhibitors of uPA in plants known traditionally for their anti-tumor properties. Crude methanol extracts were prepared from the leaves of plants (14) collected from the subtropical dry forest (Guanica, Puerto Rico), and tested for the presence of inhibitors of uPA using the fibrin plate assay. The extracts that tested positive (6) were then partitioned with petroleum ether, chloroform, ethyl acetate and n-butanol, in a sequential manner. The resulting fractions were then tested again using the fibrin plate assay. Extracts from leaves of Croton lucidus (C. lucidus) showed the presence of a strong uPA inhibitory activity. Serial dilutions of these C. lucidus partitions were performed to determine the uPA inhibition IC50 values. The chloroform extract showed the lowest IC50 value (3.52 μg/mL) and hence contained the most potent uPA inhibitor. Further investigations revealed that the crude methanol extract and its chloroform and n-butanol partitions did not significantly inhibit closely related proteases such as the tissue type plasminogen activator (tPA) and plasmin, indicating their selectivity for uPA, and hence superior potential for medicinal use with fewer side effects. In a further evaluation of their therapeutic potential for prevention of cancer metastasis, the C. lucidus extracts displayed cytostatic activity against human pancreatic carcinoma (PaCa-2) cells, as determined through an MTS assay. The cytostatic activities recorded for each of the partitions correlated with their relative uPA inhibitory activities. There are no existing reports of uPA inhibitors being present in any of the plants reported in this study. PMID:23896619

  3. Therapeutic administration of plasminogen activator inhibitor-1 prevents hypoxic-ischemic brain injury in newborns.

    PubMed

    Yang, Dianer; Nemkul, Niza; Shereen, Ahmed; Jone, Alice; Dunn, R Scott; Lawrence, Daniel A; Lindquist, Diana; Kuan, Chia-Yi

    2009-07-08

    Disruption of the integrity of the blood-brain barrier (BBB) is an important mechanism of cerebrovascular diseases, including neonatal cerebral hypoxia-ischemia (HI). Although both tissue-type plasminogen activator (tPA) and matrix metalloproteinase-9 (MMP-9) can produce BBB damage, their relationship in neonatal cerebral HI is unclear. Here we use a rodent model to test whether the plasminogen activator (PA) system is critical for MMP-9 activation and HI-induced brain injury in newborns. To test this hypothesis, we examined the therapeutic effect of intracerebroventricular injection of plasminogen activator inhibitor-1 (PAI-1) in rat pups subjected to unilateral carotid artery occlusion and systemic hypoxia. We found that the injection of PAI-1 greatly reduced the activity of both tPA and urokinase-type plasminogen activator after HI. It also blocked HI-induced MMP-9 activation and BBB permeability at 24 h of recovery. Furthermore, magnetic resonance imaging and histological analysis showed the PAI-1 treatment reduced brain edema, axonal degeneration, and cortical cell death at 24-48 h of recovery. Finally, the PAI-1 therapy provided a dose-dependent decrease of brain tissue loss at 7 d of recovery, with the therapeutic window at 4 h after the HI insult. Together, these results suggest that the brain PA system plays a pivotal role in neonatal cerebral HI and may be a promising therapeutic target in infants suffering hypoxic-ischemic encephalopathy.

  4. Modular design of a novel chimeric protein with combined thrombin inhibitory activity and plasminogen-activating potential.

    PubMed

    Wirsching, Frank; Luge, Cornelia; Schwienhorst, Andreas

    2002-03-01

    In order to design plasminogen activators with improved thrombolytic properties we sought to construct the bifunctional protein HLS-2 which combines both a plasminogen-activating and an anticoagulative activity. The chimeric protein comprises four elements: a derivative of thrombin inhibitor hirudin, a 6-amino acid spacer, the sequence of plasminogen-activator staphylokinase (Sak), and a 13-amino acid expression tag at the C-terminus. The gene of the fusion protein was obtained by SOE-PCR, cloned into pCANTAB5E, and expressed in E. coli BL21. HLS-2 was purified from periplasmatic extracts and characterized by Western blotting. Plasminogen-activation of HLS-2 and of Sak in equimolar mixtures with plasminogen showed near equivalence as measured by plasmin-mediated cleavage of chromogenic substrate S-2403. For catalytic amounts of plasminogen-activator, however, HLS-2 was less effective by a factor of 1.7. HLS-2 also inhibited both the amidolytic and the fibrinolytic activities of thrombin. Similar concentrations of either commercial HV1 (42 pmol/L) or HLS-2 (250 pmol/L) were required to halve the initial rate of thrombin reaction with fluorogenic substrate Tos-Gly-Pro-Arg-AMC, suggesting the retention of high-affinity inhibition of thrombin by the fusion protein sufficiently strong to substitute anticoagulative comedication during fibrinolytic treatment. The results provide a rationale for further testing the efficacy of HLS-2 for the lysis of platelet-rich arterial blood clots and for the prevention of reocclusion after thrombolysis.

  5. Prevotella intermedia stimulates tissue-type plasminogen activator and plasminogen activator inhibitor-2 expression via multiple signaling pathways in human periodontal ligament cells.

    PubMed

    Guan, Su-Min; He, Jian-Jun; Zhang, Ming; Shu, Lei

    2011-06-01

    Prevotella intermedia is an important periodontal pathogen that induces various inflammatory and immune responses. In this study, we investigated the effects of P. intermedia on the plasminogen system in human periodontal ligament (hPDL) cells and explored the signaling pathways involved. Using semi-quantitative reverse transcription (RT)-PCR and quantitative real-time RT-qPCR, we demonstrated that P. intermedia challenge increased tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor (PAI)-2 expression in a concentration- and time-dependent manner, but exerted no influence on urokinase-type plasminogen activator and PAI-1mRNA expression in hPDL cells. Prevotella intermedia stimulation also enhanced tPA protein secretion as confirmed by enzyme-linked immunosorbent assay. Western blot results revealed that P. intermedia treatment increased phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 kinase (p38). ERK, JNK and protein kinase C inhibitors significantly attenuated the P. intermedia-induced tPA and PAI-2 expression. Furthermore, p38 and phosphatidylinositol 3-kinase inhibitors markedly decreased PAI-2 expression, whereas they showed no or little inhibition on tPA expression. In contrast, inhibition of protein kinase A greatly enhanced the upregulatory effect of P. intermedia on tPA and PAI-2 expression. Our results suggest that P. intermedia may contribute to periodontal tissue destruction by upregulating tPA and PAI-2 expression in hPDL cells via multiple signaling pathways.

  6. The construction and expression of chimeric urokinase-type plasminogen activator genes containing kringle domains of human plasminogen.

    PubMed

    Boutaud, A; Castellino, F J

    1993-06-01

    A series of chimeric urokinase-type plasminogen activator (uPA) genes, which contain combinations of kringle domains of human plasminogen (HPg) in place of the uPA kringle (KuPA), has been constructed and expressed. Some of the resulting recombinant (r) variant uPA chimeras contain modules that potentially mediate the macroscopic binding of HPg to its activation effectors, fibrin(ogen) and 6-aminohexanoic acid (EACA). Such binding sites are not possessed by KuPA, but are present in certain of the HPg kringles, viz., kringle 1 (K1HPg), kringle 4 (K4HPg), and kringle 5 (K5HPg). The recombinant (r) chimeras constructed included molecules with replacements of KuPA with K1HPg (r-[KuPA-->K1HPg]uPA), and with KuPA replaced by double kringle combinations of K1HPgK4HPg (r-[KuPA-->K1HPgK4HPg]uPA), K2HPgK3HPg (r-[KuPA-->K2HPgK3HPg]uPA), and K4HPgK5HPg (r-[KuPA-->K4HPgK5HPg]uPA). All of these variant genes, along with their wild-type (wt) r-uPA counterparts, were expressed in human kidney 293 cells. In cases wherein EACA-binding kringles from HPg have been placed in uPA, this property has been retained in the chimeric molecule and employed as an essential part of the purification procedures for the variants. The steady state amidolytic activity of two-chain (tc) wtr-uPA toward the chromogenic substrate, H-D-pyroglutamyl-Gly-L-Arg-p-nitroanilide (S2444), is characterized by a kcat/KM (pH 7.4, 37 degrees C) of 120 s-1 mM-1. This value ranges from 92 s-1 mM-1 (tcr-[KuPA-->K1HPg]uPA) to 166 s-1 mM-1 (tcr-[KuPA-->K1HPgK4HPg]uPA) for each of the variants, demonstrating that the catalytic efficiency of the active site is altered only in a small way by changes in the noncatalytic domain of uPA. Small differences are also observed in the abilities of these tcr variants to interact with the fast-acting plasma inhibitor of uPA, viz., plasminogen activator inhibitor-1 (PAI-1). The second-order rate constant for the interaction of PAI-1 with tcr-uPA, 0.46 x 10(7) M-1s-1 (pH 7.4, 10 degrees

  7. Expression of Active Human Tissue-Type Plasminogen Activator in Escherichia coli

    PubMed Central

    Qiu, Ji; Swartz, James R.; Georgiou, George

    1998-01-01

    The formation of native disulfide bonds in complex eukaryotic proteins expressed in Escherichia coli is extremely inefficient. Tissue plasminogen activator (tPA) is a very important thrombolytic agent with 17 disulfides, and despite numerous attempts, its expression in an active form in bacteria has not been reported. To achieve the production of active tPA in E. coli, we have investigated the effect of cooverexpressing native (DsbA and DsbC) or heterologous (rat and yeast protein disulfide isomerases) cysteine oxidoreductases in the bacterial periplasm. Coexpression of DsbC, an enzyme which catalyzes disulfide bond isomerization in the periplasm, was found to dramatically increase the formation of active tPA both in shake flasks and in fermentors. The active protein was purified with an overall yield of 25% by using three affinity steps with, in sequence, lysine-Sepharose, immobilized Erythrina caffra inhibitor, and Zn-Sepharose resins. After purification, approximately 180 μg of tPA with a specific activity nearly identical to that of the authentic protein can be obtained per liter of culture in a high-cell-density fermentation. Thus, heterologous proteins as complex as tPA may be produced in an active form in bacteria in amounts suitable for structure-function studies. In addition, these results suggest the feasibility of commercial production of extremely complex proteins in E. coli without the need for in vitro refolding. PMID:9835579

  8. Changes in levels of plasminogen activator activity in normal and germ-cell-depleted testes during development.

    PubMed

    Lacroix, M; Smith, F E; Fritz, I B

    1982-05-01

    Levels of plasminogen activator activity were determined in testes obtained from normal and irradiated rats in various ages. During normal development, plasminogen activator activity per g testis increased rapidly between 40 and 60 days of age, but a comparable rise did not occur in germ-cell depleted testes of irradiated rats. Levels of enzyme in various populations of testicular cells were highest in Sertoli (varying between 1800 and 6300 units/mg protein in cell maintained under different culture conditions), and lowest in peritubular myoid cells (about 1 unit/mg protein), with intermediate levels in germinal cells (ranging between 147 and 560 units/Mg protein in residual bodies, spermatocytes and spermatids). No protease inhibitor could be detected in germ-cell extracts. The addition to the medium in which Sertoli cells were in culture of particles which can be phagocytosed (autoclaved E. coli) resulted in an increased formation of plasminogen activator activity by Sertoli cells. A synergistic enhancement of enzyme production resulted following the addition of submaximal quantities of dibutyryl cyclic AMP and autoclaved bacteria to sertoli cells in culture. On the basis of these data, we suggest that the presence of advanced germinal cells during gonadal development may stimulate the synthesis of plasminogen activator by Sertoli cells, mediated in part by the phagocytosis of residual bodies by sertoli cells which occurs prior to spermiation.

  9. Urokinase-type plasminogen activator is induced in migrating capillary endothelial cells

    PubMed Central

    1987-01-01

    Cellular migration is an essential component of invasive biological processes, many of which have been correlated with an increase in plasminogen activator production. Endothelial cell migration occurs in vivo during repair of vascular lesions and angiogenesis, and can be induced in vitro by wounding a confluent monolayer of cells. By combining the wounded monolayer model with a substrate overlay technique, we show that cells migrating from the edges of an experimental wound display an increase in urokinase-type plasminogen activator (uPA) activity, and that this activity reverts to background levels upon cessation of movement, when the wound has closed. Our results demonstrate a direct temporal relationship between endothelial cell migration and uPA activity, and suggest that induction of uPA activity is a component of the migratory process. PMID:3121633

  10. Metabolic factors, adipose tissue, and plasminogen activator inhibitor-1 levels in Type 2 diabetes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plasminogen activator inhibitor-1 (PAI-1) production by adipose tissue is increased in obesity, and its circulating levels are high in type 2 diabetes. PAI-1 increases cardiovascular risk by favoring clot stability, interfering with vascular remodeling, or both. We investigated in obese diabetic per...

  11. Production of Plasminogen Activator in Cultures of Superior Cervical Ganglia and Isolated Schwann Cells

    NASA Astrophysics Data System (ADS)

    Alvarez-Buylla, Arturo; Valinsky, Jay E.

    1985-05-01

    Plasminogen activator has been implicated in tissue remodeling and cell migration during embryogenesis. In the developing nervous system, these processes are evident in the migration of neurons, axonal extension, Schwann cell migration, and the ensheathment and myelination of nerves. We have studied the production of plasminogen activator in cultures of superior cervical ganglia under conditions in which both neurons and glia are present. We have found that a principal source of the enzyme in these cultures is the glial cells and that the enzyme could not be detected at the growing tips of neurites. Plasminogen activator is also produced by Schwann cells isolated from neonatal rat sciatic nerve. The production of the enzyme by these cells is stimulated 6- to 10-fold by cholera toxin. Isolated Schwann cells and glial cells in the ganglion explant cultures produce the tissue form of plasminogen activator, a form of the enzyme not often found in nonmalignant cells. Preliminary experiments suggest that neuronal-glial interactions may regulate enzyme production by Schwann cells.

  12. Plasminogen activation in synovial tissues: differences between normal, osteoarthritis, and rheumatoid arthritis joints

    PubMed Central

    Busso, N.; Peclat, V.; So, A.; Sappino, A.

    1997-01-01

    OBJECTIVE—To analyse the functional activity of the plasminogen activators urokinase (uPA) and tissue type plasminogen activator (tPA) in human synovial membrane, and to compare the pattern of expression between normal, osteoarthritic, and rheumatoid synovium. The molecular mechanisms underlying differences in PA activities between normal and pathological synovial tissues have been further examined.
METHODS—Synovial membranes from seven normal (N) subjects, 14 osteoarthritis (OA), and 10 rheumatoid arthritis (RA) patients were analysed for plasminogen activator activity by conventional zymography and in situ zymography on tissue sections. The tissue distribution of uPA, tPA, uPA receptor (uPAR), and plasminogen activator inhibitor type-1 (PAI-1) was studied by immunohistochemistry. uPA, tPA, uPAR, and PAI-1 mRNA values and mRNA distribution were assessed by northern blot and in situ hybridisations respectively.
RESULTS—All normal and most OA synovial tissues expressed predominantly tPA catalysed proteolytic activity mainly associated to the synovial vasculature. In some OA, tPA activity was expressed together with variable amounts of uPA mediated activity. By contrast, most RA synovial tissues exhibited considerably increased uPA activity over the proliferative lining areas, while tPA activity was reduced when compared with N and OA synovial tissues. This increase in uPA activity was associated with increased levels of uPA antigen and its corresponding mRNA, which were localised over the synovial proliferative lining areas. In addition, in RA tissues, expression of the specific uPA receptor (uPAR) and of the plasminogen activator inhibitor-type 1 (PAI-1) were also increased.
CONCLUSION—Taken together, these results show an alteration of the PA/plasmin system in RA synovial tissues, resulting in increased uPA catalytic activity that may play a part in tissue destruction in RA.

 PMID:9370880

  13. Importance of viability and attachment to an ascites tumor in the release of plasminogen activator.

    PubMed Central

    Dong, Q.; Zhou, M.; Subbarao, V.; Ts'ao, C.

    1991-01-01

    Tumor plasminogen activator (PA) has been alleged to play a role in the growth and metastasis of tumors. Before such a role can be realized, PA first must be released from tumor cells. Having determined intra- and extracellular PA and PA-inhibitor activities in an experimental pancreatic ascites tumor grown in hamsters, the release of PA from these cells was investigated. No PA activity was detected in the suspension medium of freshly isolated tumor cells; inclusion of plasminogen, fibrinogen, or collagen in the medium yielded similar negative results. On the other hand, PA activity was demonstrated to be released in a time-dependent manner from these tumor cells embedded in fibrin clots. Plasminogen activator activity also was not found in the suspension medium of frozen-thawed tumor cells, despite the fact that most of them had breaks on their cell membrane. Unlike freshly isolated tumor cells, PA was not released from frozen-thawed cells embedded in fibrin clots. Full PA activity was demonstrated in frozen-thawed cells treated with Triton X-100, however. Frozen-thawed cells exhibited signs of severe damage, and more than 80% of them failed to exclude trypan blue. Obviously PA is released from viable tumor cells embedded in fibrin clots but not suspended in artificial medium. The PA-release mechanism, not PA itself, is destroyed in cells rendered nonviable by freeze thawing. Images Figure 1 Figure 5 Figure 6 Figure 7 PMID:1902626

  14. Dietary omega-3 polyunsaturated fatty acids induce plasminogen activator activity and DNA damage in rabbit spermatozoa.

    PubMed

    Kokoli, A N; Lavrentiadou, S N; Zervos, I A; Tsantarliotou, M P; Georgiadis, M P; Nikolaidis, E A; Botsoglou, N; Boscos, C M; Taitzoglou, I A

    2017-02-20

    The aim of this study was to determine the effect(s) of dietary omega-3 polyunsaturated fatty acids (ω-3 PUFA) on rabbit semen. Adult rabbit bucks were assigned to two groups that were given two diets, a standard diet (control) and a diet supplemented with ω-3 PUFA. Sperm samples were collected from all bucks with the use of an artificial vagina in 20-day intervals, for a total period of 120 days. The enrichment of membranes in ω-3 PUFA was manifested by the elevation of the 22:5 ω-3 (docosapentaenoic acid [DPA]) levels within 40 days. This increase in DPA content did not affect semen characteristics (i.e., concentration, motility and viability). However, it was associated with the induction of lipid peroxidation in spermatozoa, as determined on the basis of the malondialdehyde content. Lipid peroxidation was associated with DNA fragmentation in ω-3 PUFA-enriched spermatozoa and a concomitant increase in plasminogen activator (PA) activity. The effects of ω-3 PUFA on sperm cells were evident within 40 days of ω-3 PUFA dietary intake and exhibited peack values on day 120. Our findings suggest that an ω-3 PUFA-rich diet may not affect semen characteristics; however, it may have a negative impact on the oxidative status and DNA integrity of the spermatozoa, which was associated with an induction of PAs activity.

  15. Mouse ovarian granulosa cells produce urokinase-type plasminogen activator, whereas the corresponding rat cells produce tissue-type plasminogen activator

    PubMed Central

    1987-01-01

    It is well established that rat ovarian granulosa cells produce tissue plasminogen activator (tPA). The synthesis and secretion of the enzyme are induced by gonadotropins, and correlate well with the time of follicular rupture in vivo. We have found that in contrast, mouse granulosa cells produce a different form of plasminogen activator, the urokinase-type (uPA). As with tPA synthesis in the rat, uPA production by mouse granulosa cells is induced by gonadotropins, dibutyryl cAMP, and prostaglandin E2. However, dexamethasone, a drug which has no effect on tPA synthesis in rat cells inhibits uPA synthesis in the mouse. Results of these determinations made in cell culture were corroborated by examining follicular fluid, which is secreted in vivo predominantly by granulosa cells, from stimulated rat and mouse ovarian follicles. Rat follicular fluid contained only tPA, and mouse follicular fluid only uPA, indicating that in vivo, granulosa cells from the two species are secreting different enzymes. The difference in the type of plasminogen activator produced by the rat and mouse granulosa cells was confirmed at the messenger RNA level. After hormone stimulation, only tPA mRNA was present in rat cells, whereas only uPA mRNA was found in mouse cells. Furthermore, the regulation of uPA levels in mouse cells occurs via transient modulation of steady-state levels of mRNA, a pattern similar to that seen with tPA in rat cells. PMID:3040774

  16. Soluble fibrin degradation products potentiate tissue plasminogen activator-induced fibrinogen proteolysis.

    PubMed Central

    Weitz, J I; Leslie, B; Ginsberg, J

    1991-01-01

    Despite its affinity for fibrin, tissue plasminogen activator (t-PA) administration causes systemic fibrinogenolysis. To investigate the mechanism, t-PA was incubated with plasma in the presence or absence of a fibrin clot, and the extent of fibrinogenolysis was determined by measuring B beta 1-42. In the presence of fibrin, there is a 21-fold increase in B beta 1-42 levels. The potentiation of fibrinogenolysis in the presence of fibrin is mediated by soluble fibrin degradation products because (a) the extent of t-PA induced fibrinogenolysis and clot lysis are directly related, (b) once clot lysis has been initiated, fibrinogenolysis continues even after the clot is removed, and (c) lysates of cross-linked fibrin clots potentiate t-PA-mediated fibrinogenolysis. Fibrin degradation products stimulate fibrinogenolysis by binding t-PA and plasminogen because approximately 70% of the labeled material in the clot lysates binds to both t-PA- and plasminogen-Sepharose, and only the bound fractions have potentiating activity. The binding site for t-PA and plasminogen is on the E domain because characterization of the potentiating fragments using gel filtration followed by PAGE and immunoblotting indicates that the major species is (DD)E complex, whereas minor components include high-molecular weight derivatives containing the (DD)E complex and fragment E. In contrast, D-dimer is the predominant species found in the fractions that do not bind to the adsorbants, and it has no potentiating activity. Thus, soluble products of t-PA-induced lysis of cross-linked fibrin potentiate t-PA-mediated fibrinogenolysis by providing a surface for t-PA and plasminogen binding thereby promoting plasmin generation. The occurrence of this phenomenon after therapeutic thrombolysis may explain the limited clot selectivity of t-PA. Images PMID:1900308

  17. Trypanosoma cruzi: the immunological induction of macrophage plasminogen activator requires thymus-derived lymphocytes

    PubMed Central

    1977-01-01

    In this article we describe methods in which unstimulated mouse peritoneal macrophages were induced to secrete high livels of plasminogen activator under in vitro conditions. The exposure of sensitized peritoneal or spleen cell populations from Trypanosoma cruzi- infected animals to either viable or heat-killed trypanosomes lead to the release of an inducing factor(s). Maximal levels of plasminogen activator secretion are achieved by the incubation of such factors (s) with unstimulated macrophages for 48 h. A significant increase in enzyme secretion was already observed after a 24 h incubation. The production of the inducing factor(s) by sensitized cells was immunologically specific and unrelated antigens did not stimulate the production of the factor(s) by sensitized peritoneal or spleen cell populations. The inducing factor(s) was produced by nylon-wool- fractionated spleen and peritoneal cells which had been depleted of marcrophages. Pretreatment of sensitized spleen cells with anti-theta serum and C abolished the production of the activating factor(s). The active supernatant fluids were able to induce secretion of macrophage plasminogen activator across H-2 barriers. Attempts to induce trypanocidal activity in unstimulated macrophages have not been successful. PMID:327013

  18. Characterization of the Annonaceous acetogenin, annonacinone, a natural product inhibitor of plasminogen activator inhibitor-1

    PubMed Central

    Pautus, Stéphane; Alami, Mouad; Adam, Fréderic; Bernadat, Guillaume; Lawrence, Daniel A.; De Carvalho, Allan; Ferry, Gilles; Rupin, Alain; Hamze, Abdallah; Champy, Pierre; Bonneau, Natacha; Gloanec, Philippe; Peglion, Jean-Louis; Brion, Jean-Daniel; Bianchini, Elsa P.; Borgel, Delphine

    2016-01-01

    Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of the tissue type and urokinase type plasminogen activators. High levels of PAI-1 are correlated with an increased risk of thrombotic events and several other pathologies. Despite several compounds with in vitro activity being developed, none of them are currently in clinical use. In this study, we evaluated a novel PAI-1 inhibitor, annonacinone, a natural product from the Annonaceous acetogenins group. Annonacinone was identified in a chromogenic screening assay and was more potent than tiplaxtinin. Annonacinone showed high potency ex vivo on thromboelastography and was able to potentiate the thrombolytic effect of tPA in vivo in a murine model. SDS-PAGE showed that annonacinone inhibited formation of PAI-1/tPA complex via enhancement of the substrate pathway. Mutagenesis and molecular dynamics allowed us to identify annonacinone binding site close to helix D and E and β-sheets 2A. PMID:27876785

  19. Characterization of the Annonaceous acetogenin, annonacinone, a natural product inhibitor of plasminogen activator inhibitor-1

    NASA Astrophysics Data System (ADS)

    Pautus, Stéphane; Alami, Mouad; Adam, Fréderic; Bernadat, Guillaume; Lawrence, Daniel A.; de Carvalho, Allan; Ferry, Gilles; Rupin, Alain; Hamze, Abdallah; Champy, Pierre; Bonneau, Natacha; Gloanec, Philippe; Peglion, Jean-Louis; Brion, Jean-Daniel; Bianchini, Elsa P.; Borgel, Delphine

    2016-11-01

    Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of the tissue type and urokinase type plasminogen activators. High levels of PAI-1 are correlated with an increased risk of thrombotic events and several other pathologies. Despite several compounds with in vitro activity being developed, none of them are currently in clinical use. In this study, we evaluated a novel PAI-1 inhibitor, annonacinone, a natural product from the Annonaceous acetogenins group. Annonacinone was identified in a chromogenic screening assay and was more potent than tiplaxtinin. Annonacinone showed high potency ex vivo on thromboelastography and was able to potentiate the thrombolytic effect of tPA in vivo in a murine model. SDS-PAGE showed that annonacinone inhibited formation of PAI-1/tPA complex via enhancement of the substrate pathway. Mutagenesis and molecular dynamics allowed us to identify annonacinone binding site close to helix D and E and β-sheets 2A.

  20. Structural Differences between Active Forms of Plasminogen Activator Inhibitor Type 1 Revealed by Conformationally Sensitive Ligands*

    PubMed Central

    Li, Shih-Hon; Gorlatova, Natalia V.; Lawrence, Daniel A.; Schwartz, Bradford S.

    2008-01-01

    Plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor (serpin) in which the reactive center loop (RCL) spontaneously inserts into a central β-sheet, β-sheet A, resulting in inactive inhibitor. Available x-ray crystallographic studies of PAI-1 in an active conformation relied on the use of stabilizing mutations. Recently it has become evident that these structural models do not adequately explain the behavior of wild-type PAI-1 (wtPAI-1) in solution. To probe the structure of native wtPAI-1, we used three conformationally sensitive ligands: the physiologic cofactor, vitronectin; a monoclonal antibody, 33B8, that binds preferentially to RCL-inserted forms of PAI-1; and RCL-mimicking peptides that insert into β-sheet A. From patterns of interaction with wtPAI-1 and the stable mutant, 14-1B, we propose a model of the native conformation of wtPAI-1 in which the bottom of the central sheet is closed, whereas the top of the β-sheet A is open to allow partial insertion of the RCL. Because the incorporation of RCL-mimicking peptides into wtPAI-1 is accelerated by vitronectin, we further propose that vitronectin alters the conformation of the RCL to allow increased accessibility to β-sheet A, yielding a structural hypothesis that is contradictory to the current structural model of PAI-1 in solution and its interaction with vitronectin. PMID:18436534

  1. Regulation of plasminogen activation by TGF-β in cultured human retinal endothelial cells

    PubMed Central

    Wileman, S.; Booth, N.; Moore, N.; Redmill, B.; Forrester, J.; Knott, R.

    2000-01-01

    BACKGROUND/AIMS—Regulation of plasmin mediated extracellular matrix degradation by vascular endothelial cells is important in the development of angiogenesis. The aim was to determine whether transforming growth factor β (TGF-β) affected the regulation of components of the plasminogen system by human retinal endothelial cells, in order to define more clearly the role of TGF-β in retinal angiogenesis in the context of diabetes mellitus.
METHODS—Human retinal endothelial cells (HREC) were isolated from donor eyes and used between passages 4-8. The cells were cultured in medium supplemented with 2, 5, 15, or 25 mM glucose, plus or minus TGF-β (1 ng/ml). The concentrations of tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), and plasminogen activator inhibitor type 1 (PAI-1) in cell conditioned medium were determined by ELISA and the level of PAI-1 mRNA was determined using northern hybridisation. Cell associated plasminogen activity was determined using a clot lysis assay and a chromogenic assay.
RESULTS—Under basal conditions (5 mM glucose), HREC produced PAI-1, t-PA, and trace amounts of u-PA. Cell surface plasminogen activation observed by lysis of fibrin or by cleavage of chromogenic substrate, was mediated by t-PA. Glucose at varying concentrations (2-25 mM) had no significant effect on t-PA mediated clot lysis. In contrast, treatment with TGF-β resulted in increased synthesis of PAI-1 protein and mRNA. The increased expression of the PAI-1 mRNAs by TGF-β did not occur uniformly, the 2.3 kb mRNA transcript was preferentially increased in comparison with the 3.2 kb mRNA (p<0.05).
CONCLUSIONS—These data demonstrate that TGF-β increases PAI-1 and decreases cell associated lysis. This is sufficient to decrease the normal lytic potential of HREC.

 PMID:10729302

  2. Structural Basis of Interaction Between Urokinase-type Plasminogen Activator and its Receptor

    SciTech Connect

    Barinka,C.; Parry, G.; Callahan, J.; Shaw, D.; Kuo, A.; Cines, B.; Mazar, A.; Lubkowski, J.

    2006-01-01

    Recent studies indicate that binding of the urokinase-type plasminogen activator (uPA) to its high-affinity receptor (uPAR) orchestrates uPAR interactions with other cellular components that play a pivotal role in diverse (patho-)physiological processes, including wound healing, angiogenesis, inflammation, and cancer metastasis. However, notwithstanding the wealth of biochemical data available describing the activities of uPAR, little is known about the exact mode of uPAR/uPA interactions or the presumed conformational changes that accompany uPA/uPAR engagement. Here, we report the crystal structure of soluble urokinase plasminogen activator receptor (suPAR), which contains the three domains of the wild-type receptor but lacks the cell-surface anchoring sequence, in complex with the amino-terminal fragment of urokinase-type plasminogen activator (ATF), at the resolution of 2.8 {angstrom}. We report the 1.9 {angstrom} crystal structure of free ATF. Our results provide a structural basis, represented by conformational changes induced in uPAR, for several published biochemical observations describing the nature of uPAR/uPA interactions and provide insight into mechanisms that may be responsible for the cellular responses induced by uPA binding.

  3. Distortion of the catalytic domain of tissue-type plasminogen activator by plasminogen activator inhibitor-1 coincides with the formation of stable serpin-proteinase complexes.

    PubMed

    Perron, Michel J; Blouse, Grant E; Shore, Joseph D

    2003-11-28

    Plasminogen activator inhibitor-1 (PAI-1) is a typical member of the serpin family that kinetically traps its target proteinase as a covalent complex by distortion of the proteinase domain. Incorporation of the fluorescently silent 4-fluorotryptophan analog into PAI-1 permitted us to observe changes in the intrinsic tryptophan fluorescence of two-chain tissue-type plasminogen activator (tPA) and the proteinase domain of tPA during the inhibition reaction. We demonstrated three distinct conformational changes of the proteinase that occur during complex formation and distortion. A conformational change occurred during the initial formation of the non-covalent Michaelis complex followed by a large conformational change associated with the distortion of the proteinase catalytic domain that occurs concurrently with the formation of stable proteinase-inhibitor complexes. Following distortion, a very slow structural change occurs that may be involved in the stabilization or regulation of the trapped complex. Furthermore, by comparing the inhibition rates of two-chain tPA and the proteinase domain of tPA by PAI-1, we demonstrate that the accessory domains of tPA play a prominent role in the initial formation of the non-covalent Michaelis complex.

  4. Binding of Tissue-type Plasminogen Activator to the Glucose-regulated Protein 78 (GRP78) Modulates Plasminogen Activation and Promotes Human Neuroblastoma Cell Proliferation in Vitro*

    PubMed Central

    Gonzalez-Gronow, Mario; Gomez, Cristian Farias; de Ridder, Gustaaf G.; Ray, Rupa; Pizzo, Salvatore V.

    2014-01-01

    The glucose-regulated protein 78 (GRP78) is a plasminogen (Pg) receptor on the cell surface. In this study, we demonstrate that GRP78 also binds the tissue-type plasminogen activator (t-PA), which results in a decrease in Km and an increase in the Vmax for both its amidolytic activity and activation of its substrate, Pg. This results in accelerated Pg activation when GRP78, t-PA, and Pg are bound together. The increase in t-PA activity is the result of a mechanism involving a t-PA lysine-dependent binding site in the GRP78 amino acid sequence 98LIGRTWNDPSVQQDIKFL115. We found that GRP78 is expressed on the surface of neuroblastoma SK-N-SH cells where it is co-localized with the voltage-dependent anion channel (VDAC), which is also a t-PA-binding protein in these cells. We demonstrate that both Pg and t-PA serve as a bridge between GRP78 and VDAC bringing them together to facilitate Pg activation. t-PA induces SK-N-SH cell proliferation via binding to GRP78 on the cell surface. Furthermore, Pg binding to the COOH-terminal region of GRP78 stimulates cell proliferation via its microplasminogen domain. This study confirms previous findings from our laboratory showing that GRP78 acts as a growth factor-like receptor and that its association with t-PA, Pg, and VDAC on the cell surface may be part of a system controlling cell growth. PMID:25059665

  5. Acute ischemic stroke after cardiac catheterization: the protamine low-dose recombinant tissue plasminogen activator pathway.

    PubMed

    Guevara, Carlos; Quijada, Alonso; Rosas, Carolina; Bulatova, Katya; Lara, Hugo; Nieto, Elena; Morales, Marcelo

    2016-05-20

    Intravenous thrombolysis is the preferred treatment for acute ischemic stroke; however, it remains unestablished in the area of cardiac catheterization. We report three patients with acute ischemic stroke after cardiac catheterization. After reversing the anticoagulant effect of unfractionated heparin with protamine, all of the patients were successfully off-label thrombolyzed with reduced doses of intravenous recombinant tissue plasminogen activator (0.6 mg/kg). This dose was preferred to reduce the risk of symptomatic cerebral or systemic bleeding. The sequential pathway of protamine recombinant tissue plasminogen activator at reduced doses may be safer for reducing intracranial or systemic bleeding events, whereas remaining efficacious for the treatment of acute ischemic stroke after cardiac catheterization.

  6. Clinical significance of plasminogen activator inhibitor activity in patients with exercise-induced ischemia

    SciTech Connect

    Sakata, K.; Kurata, C.; Taguchi, T.; Suzuki, S.; Kobayashi, A.; Yamazaki, N.; Rydzewski, A.; Takada, Y.; Takada, A. )

    1990-10-01

    To assess the fibrinolytic system in patients with exercise-induced ischemia and its relation to ischemia and severity of coronary artery disease (CAD), 47 patients with CAD confirmed by results of coronary angiography underwent symptom-limited multistage exercise thallium-201 emission computed tomography. All patients with CAD had exercise-induced ischemia as assessed from thallium-201 images. Pre- and peak exercise blood samples from each patient and preexercise blood samples from control subjects were assayed for several fibrinolytic components and were also assayed for plasma adrenaline. The extent of ischemia was defined as delta visual uptake score (total visual uptake score in delayed images minus total visual uptake score in initial images) and the severity of CAD as the number of diseased vessels. In the basal condition, plasminogen activator inhibitor (PAI) activity was significantly higher in patients with exercise-induced ischemia as compared to control subjects (p less than 0.01), although there were no significant differences in other fibrinolytic variables between the two groups. Moreover, PAI activity in the basal condition displayed a significantly positive correlation with the extent of ischemia (r = 0.47, p less than 0.01). Patients with exercise-induced ischemia were divided into two groups (24 with single-vessel disease and 23 with multivessel disease). There were no significant differences in coronary risk factors, hemodynamics, or plasma adrenaline levels during exercise between single-vessel and multivessel disease except that delta visual uptake score was significantly higher in multivessel disease (p less than 0.01).

  7. PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1): A KEY FACTOR LINKING FIBRINOLYSIS AND AGE-RELATED SUBCLINICAL AND CLINICAL CONDITIONS

    PubMed Central

    Cesari, Matteo; Pahor, Marco; Incalzi, Raffaele Antonelli

    2010-01-01

    The close relationship existing between aging and thrombosis has growingly been studied in this last decade. The age-related development of a pro-thrombotic imbalance in the fibrinolysis homeostasis has been hypothesized at the basis of this increased cardiovascular and cerebrovascular risk. Fibrinolysis is the resulting of the interactions among multiple plasminogen activators and inhibitors constituing the enzymatic cascade, and ultimately leading to the degradation of fibrin. The plasminogen activator system plays a key role in a wide range of physiological and pathological processes. Plasminogen activator inhibitor-1 (PAI-1) is a member of the superfamily of serine-protease inhibitors (or serpins), and the principal inhibitor of both the tissue-type and the urinary-type plasminogen activator, the two plasminogen activators able to activate plasminogen. In this review, current evidence describing the central role played by PAI-1 in a number of age-related subclinical (i.e., inflammation, atherosclerosis, insulin resistance) and clinical (i.e., obesity, comorbidities, Werner syndrome) conditions is presented. Despite some controversial and unclear issues, PAI-1 represents an extremely promising marker which may become a biological parameter to be growingly considered in the prognostic evaluation, in the disease monitoring, and as treatment target of age-related conditions in the next future. PMID:20626406

  8. [Plasminogen activator inhibitor type 1 activity in women with unexplained very early recurrent pregnancy loss].

    PubMed

    Ivanov, P; Komsa-Penkova, R; Ivanov, I; Konova, E; Kovacheva, K; Simeonova, M; Tanchev, S

    2010-01-01

    The aim of the study was to assess the independent role of polymorphism 4G/5G (PL 4G/5G)--genotype 4G/4G in plasminogen activator inhibitor type 1 (PAI-1) in the development of very early recurrent pregnancy loss (RPL)--before 10 weeks of gestation of pregnancy. The polymorphism 4G/5G as well as Factor V Leiden (FVL), prothrombin (FII) gene mutation 20210 G > A and polymorphism 677 C > T in methylentetrahydrofolat reductase (MTHFR) gene was investigated in 110 women with recurrent pregnancy loss before 10 weeks of gestation and in 97 healthy women with at least one uncomplicated full-term pregnancy. A significant prevalence of PL 4G/5G in women with RPL was found in comparison to prevalence of the polymorphism in controls (41.8% versus 26.8% respectively in patients and controls, OR: 1.96, 95% CI: 1.05 3.69, p = 0.034). The difference in prevalence of the polymorphism remains still significant after exclusion of patients and control carriers of FVL, FII 202010 G > A and 677 C > T in MTHFR (the prevalence of PL 4G/5G alone was 44.1% and 24% respectively in patients and controls, OR: 2,5, 95% CI: 1,15 5, 45, p = 0.018). The found association of PL 4G/5G in PAI-1 with early recurrent pregnancy loss encourage an extension of the list of inherited thrombophilic factors with this one. This result also could have had an implication for adjustment of further prophylactic low-molecular weight heparin implication in further pregnancy to prevent a poor foetal outcome.

  9. Circadian fluctuations in circulating plasminogen activator inhibitor-1 are independent of feeding cycles in mice.

    PubMed

    Oishi, Katsutaka; Ohkura, Naoki; Yasumoto, Yuki; Yamamoto, Saori

    2017-01-01

    To evaluate the involvement of the day-night feeding cycle in the circadian regulation of circulating plasminogen activator inhibitor-1 (PAI-1) concentrations, mice were fed with a diet for eight hours during either daytime (DF) or nighttime (NF) for one week. The reversed feeding cycle did not affect the circadian phases of plasma PAI-1 levels as well as the nocturnal wheel-running activity, although the phase of Pai-1 mRNA expression was significantly advanced for 8.6 hours in the livers of DF, compared with NF mice. The day-night feeding cycle is not a critical Zeitgeber for circadian rhythm of circulating PAI-1.

  10. Vampire bat salivary plasminogen activator promotes rapid and sustained reperfusion without concomitant systemic plasminogen activation in a canine model of arterial thrombosis.

    PubMed

    Mellott, M J; Stabilito, I I; Holahan, M A; Cuca, G C; Wang, S; Li, P; Barrett, J S; Lynch, J J; Gardell, S J

    1992-02-01

    The efficacy of recombinant vampire bat salivary plasminogen activator (bat-PA) as a thrombolytic agent was compared with that of human tissue-type plasminogen activator (t-PA) in a canine model of arterial thrombosis. An occlusive thrombus was formed in the femoral artery by insertion of a thrombogenic copper coil; femoral arterial blood flow was monitored with a Doppler flow meter. Bat-PA and t-PA, when administered by 5-minute intravenous infusion (14 nmol/kg), reperfused seven out of eight and four out of eight dogs, respectively. The median reperfusion times in the bat-PA and t-PA groups were 24 and greater than or equal to 131 minutes, respectively. The mean reperfusion times (+/- SEM) in the recanalized bat-PA- and t-PA-treated dogs were similar (20 +/- 5 and 11 +/- 2 minutes, respectively, p = NS). Maximal blood flow after reperfusion was greater with bat-PA than with t-PA (80 +/- 10% and 41 +/- 15% of control flow, respectively, p less than 0.05). Furthermore, the median reocclusion time was markedly delayed in the bat-PA group relative to the t-PA group (131 versus 34 minutes, respectively, p less than 0.05). Plasma fibrinogen and plasminogen were not significantly depleted by the administration of t-PA or bat-PA. However, plasma alpha 2-antiplasmin activity was moderately depressed in the t-PA group relative to the bat-PA group (p less than 0.05). The clearance profile for t-PA was monoexponential, with a half-life (t1/2) of 2.4 +/- 0.3 minutes and a mean residence time of 3.5 +/- 0.4 minutes. The clearance profile for bat-PA was biexponential, with a t1/2 alpha of 0.9 +/- 0.2 minutes, a t1/2 beta of 20.2 +/- 2.7 minutes, and a mean residence time of 21.3 +/- 4.3 minutes. The steady-state volume of distribution displayed by bat-PA was 16-fold greater than that of t-PA. Zymography of serial plasma samples from the bat-PA-treated dogs failed to demonstrate the apparent generation of a complex between bat-PA and plasminogen activator inhibitor-1; the

  11. Decrease in plasminogen activator correlates with synapse elimination during neonatal development of mouse skeletal muscle.

    PubMed Central

    Hantaï, D; Rao, J S; Kahler, C; Festoff, B W

    1989-01-01

    Previous studies have implicated proteases, acting extracellularly, in the mechanism of polyneuronal synapse elimination. Most studies have focused on mammalian, especially rodent, skeletal muscle, where retraction of subordinate nerve terminals occurs during a narrow time window 2-3 weeks after birth. To date no specific protease(s) has been detected that (i) coincides in time with maximal synapse elimination and (ii) is known to act extracellularly on specific extracellular matrix proteins. In previous studies of denervation in adult mouse muscle, rapid activation of urokinase-type plasminogen activator, a neutral serine protease, was detected. This enzyme, by activation of plasminogen to plasmin, specifically degrades matrix components such as fibronectin, type IV collagen, and laminin in muscle. We now present evidence for an initial increase and subsequent decrease in soluble urokinase-type PA--and, to a lesser extent, tissue PA--in developing muscle, suggesting postnatal developmental regulation of these enzymes during the period of maximal synapse elimination. Although considerably higher in specific activity, membrane-bound PA activity followed the wave of synapse elimination, possibly indicating a longer half-life of membrane-bound enzyme(s). Images PMID:2492103

  12. Immunohistochemical localization of urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor and α2-antiplasmin in human corneal perforation: a case report

    PubMed Central

    2012-01-01

    Background Corneal ulceration leading to perforation is associated with infectious and non-infectious destructive conditions in the cornea. The fibrinolytic (plasminogen/plasmin) system is considered to contribute to tissue remodeling in the wound healing process and it is believed to play an important role in proteolysis and fibrosis. To determine the localization of urokinase-type plasminogen activator (u-PA), u-PA receptor (u-PAR) and α2-antiplasmin (α2AP) in the tissue of a corneal perforation, we investigated immunohistochemical expressions of u-PA, u-PAR, α2AP, CD68, and α-smooth muscle actin (α-SMA) in a patient with corneal perforation that developed from an ulcer of no clear cause. Case presentation The patient was a 77-year-old woman who presented with a perforated corneal ulcer in her right eye. The cause of her corneal ulcer was unknown. Double immunohistochemistry was performed for the combinations of u-PA with u-PAR, CD68 or α-SMA and α2AP with CD68 or α-SMA to detect the localization of u-PA and α2AP. u-PA and u-PAR co-localization was seen in the corneal ulceration area. u-PA was mainly observed in CD68-positive cells and in some α-SMA positive cells. On the other hand, α2AP was not expressed in CD68-positive cells, but was expressed in α-SMA positive cells. Conclusion We identified expression of the u-PA/u-PAR complex and α2AP in a patient with a corneal ulcer. These two molecules are believed to play a crucial role in inflammatory cell recruitment, ECM synthesis and degradation during corneal wound healing. PMID:23190581

  13. Expression and localization of urokinase-type plasminogen activator receptor in bovine cumulus-oocyte complexes.

    PubMed

    García, Daniela C; Miceli, Dora C; Rizo, Gabriela; García, Elina V; Valdecantos, Pablo A; Roldán-Olarte, Mariela

    2016-04-01

    Urokinase-type plasminogen activator (uPA) is a serine protease involved in extracellular matrix remodeling through plasmin generation. uPA usually binds to its receptor, uPAR, which is anchored to the plasma membrane through a glycosylphosphatidylinositol anchor. uPA/uPAR binding increases proteolytic activity in the neighborhood of the cells containing uPAR and activates intracellular signaling pathways involved in extracellular matrix remodeling, cell migration and proliferation. The aim of this work was to study the expression of uPA, uPAR and plasminogen activator inhibitor-1 (PAI-1) in immature and in vitro matured bovine cumulus-oocyte complexes (COCs). uPA is only expressed in the cumulus cells of immature and in vitro matured COCs, while uPAR and PAI-1 are expressed in both the cumulus cells and the immature and in vitro matured oocytes. In addition, uPAR protein was localized by confocal microscopy in the plasma membrane of oocytes and cumulus cells of immature COCs. Results from this research led us to hypothesize that the uPA/uPAR interaction could cause the local production of uPA-mediated plasmin over oocyte and cumulus cell surface; plasmin formation could also be regulated by PAI-1.

  14. Plasminogen activators, their inhibitors, and urokinase receptor emerge in late stages of melanocytic tumor progression.

    PubMed Central

    de Vries, T. J.; Quax, P. H.; Denijn, M.; Verrijp, K. N.; Verheijen, J. H.; Verspaget, H. W.; Weidle, U. H.; Ruiter, D. J.; van Muijen, G. N.

    1994-01-01

    Degradation of the extracellular matrix and other tissue barriers by proteases like plasminogen activators (PAs) is a prerequisite for neoplastic growth and metastasis. Recently, we reported that highly metastatic behavior of human melanoma cells in nude mice correlates with urokinase-type PA (u-PA) expression and activity and with PA inhibitor type 1 and 2 (PAI-1, PAI-2) expression. Here we report on the occurrence of components of the PA system in the various stages of human melanoma tumor progression in situ. We studied the protein distribution on freshly frozen lesions of common nevocellular nevi (n = 25), dysplastic (= atypical) nevi (n = 16), early primary melanomas (n = 8), advanced primary melanomas (n = 11), and melanoma metastases (n = 17). Tissue-type PA was present in endothelial cells in all lesions, whereas in metastases it could be detected in tumor cells in a minority of the lesions. u-PA, its receptor, PAI-1, and PAI-2 could not be detected in benign and in early stages but appeared frequently in advanced primary melanoma and melanoma metastasis lesions. u-PA was detected in stromal cells and in tumor cells at the invasive front, the u-PA receptor and PAI-2 in tumor cells, and PAI-1 in the extracellular matrix surrounding tumor cells. Localization of the corresponding messenger RNAs and enzyme activities revealed a similar distribution. We conclude that plasminogen activation is a late event in melanoma tumor progression. Images Figure 1 Figure 3 Figure 4 Figure 5 PMID:8291613

  15. Enhanced production of prostaglandins and plasminogen activator during activation of human articular chondrocytes by products of mononuclear cells.

    PubMed

    Meats, J E; McGuire, M K; Ebsworth, N M; Englis, D J; Russell, R G

    1984-01-01

    We have examined the way in which products of cultured human blood mononuclear cells activate human articular chondrocytes. Conditioned medium from mononuclear cells enhanced the production of prostaglandin E by cultured human chondrocytes and also stimulated fibrinolytic activity in these cultures. These two effects may be interrelated, since the increased fibrinolysis in response to products of mononuclear cells was partially inhibited by indomethacin, an inhibitor of prostaglandin biosynthesis. The increased fibrinolysis is probably attributable to plasminogen activator, since it was strongly dependent on the presence of plasminogen. Increased amounts of PGE and chondroitin sulphate were also released from intact fragments of cartilage exposed to medium from cultured mononuclear cells. The time course and dose dependence of these effects were studied. The addition of exogenous arachidonic acid markedly enhanced production of PGE2. Ultrogel AcA54 was used to fractionate medium from cultured mononuclear cells and the chondrocyte-stimulating activity eluted with an apparent molecular weight between 12 000 and 25 000 daltons. Adherent and non-adherent mononuclear blood cells were also partially separated and conditioned medium from each was assayed for chondrocyte-stimulating factors. Both populations released factor(s) which increased the production of prostaglandin E by chondrocytes, but more activity came from the adherent mononuclear cells. The possible interrelationship between the chondrocyte activating factor studied here and others described in the literature is discussed.

  16. The role of plasminogen activator inhibitor-1 in gastric mucosal protection

    PubMed Central

    Kenny, Susan; Steele, Islay; Lyons, Suzanne; Moore, Andrew R.; Murugesan, Senthil V.; Tiszlavicz, Laszlo; Dimaline, Rod; Pritchard, D. Mark; Varro, Andrea

    2013-01-01

    Gastric mucosal health is maintained in response to potentially damaging luminal factors. Aspirin and nonsteroidal anti-inflammatory drugs (NSAIDs) disrupt protective mechanisms leading to bleeding and ulceration. The plasminogen activator system has been implicated in fibrinolysis following gastric ulceration, and an inhibitor of this system, plasminogen activator inhibitor (PAI)-1, is expressed in gastric epithelial cells. In Helicobacter pylori-negative patients with normal gastric histology taking aspirin or NSAIDs, we found elevated gastric PAI-1 mRNA abundance compared with controls; the increase in patients on aspirin was independent of whether they were also taking proton pump inhibitors. In the same patients, aspirin tended to lower urokinase plasminogen activator mRNA. Immunohistochemistry indicated PAI-1 localization to epithelial cells. In a model system using MKN45 or AGS-GR cells transfected with a PAI-1 promoter-luciferase reporter construct, we found no evidence for upregulation of PAI-1 expression by indomethacin, and, in fact, cyclooxygenase products such as PGE2 and PGI2 weakly stimulated expression. Increased gastric PAI-1 mRNA was also found in mice following gavage with ethanol or indomethacin, but plasma PAI-1 was unaffected. In PAI-1−/− mice, gastric hemorrhagic lesions in response to ethanol or indomethacin were increased compared with C57BL/6 mice. In contrast, in PAI-1-H/Kβ mice in which PAI-1 is overexpressed in parietal cells, there were decreased lesions in response to ethanol and indomethacin. Thus, PAI-1 expression is increased in gastric epithelial cells in response to mucosal irritants such as aspirin and NSAIDs probably via an indirect mechanism, and PAI-1 acts as a local autoregulator to minimize mucosal damage. PMID:23494120

  17. Carotid Artery Stenting for Acute Ischemic Stroke Patients after Intravenous Recombinant Tissue Plasminogen Activator Treatment

    PubMed Central

    Deguchi, Ichiro; Hayashi, Takeshi; Neki, Hiroaki; Yamane, Fumitaka; Ishihara, Shoichiro; Tanahashi, Norio; Takao, Masaki

    2016-01-01

    We herein report three ischemic stroke patients who underwent emergency carotid artery stenting after receiving intravenous tissue plasminogen activator (t-PA) treatment. All patients received antiplatelet medications immediately before stent placement for loading as well as dual antiplatelet therapy after stenting. Under high-dose and dual antiplatelet therapy, none of the three patients showed symptomatic intracranial hemorrhaging. However, one case showed reocclusion of the placed stent after acute thrombosis. As a result, new treatment strategies for the use of antiplatelet agents during emergency stent placement must be developed, particularly for patients who have received intravenous t-PA therapy. PMID:27725550

  18. Hematologic and surgical management of the dental patient with plasminogen activator deficiency.

    PubMed

    Scheitler, L E; Hart, N; Phillips, G; Weinberg, J B

    1988-12-01

    Anticoagulation therapy is used to treat patients with a variety of hemostatic disorders in an attempt to prevent thrombus formation. A thorough understanding of the patient's medical history is essential before dental treatment that may require alteration of this anticoagulation therapy. Alteration of anticoagulation therapy should be undertaken only after consultation with the patient's physician because some patients are at greater risk than others for thrombus formation or hemorrhage. This case of a 29-year-old man with plasminogen activator deficiency illustrates how consultation can result in a coordinated treatment plan for medical and dental management formulated to help ensure safe surgical treatment for these medically compromised patients.

  19. Management of plastic bronchitis with nebulized tissue plasminogen activator: another brick in the wall.

    PubMed

    Colaneri, Massimo; Quarti, Andrea; Pozzi, Marco; Gasparini, Stefano; Carloni, Ines; de Benedictis, Fernando Maria

    2014-02-13

    Plastic bronchitis is a rare complication of a variety of respiratory diseases and congenital heart disease surgery, particularly Fontan procedure. Bronchial casts with rubber-like consistency develop acutely and may cause severe life-threatening respiratory distress. The management of plastic bronchitis is yet not well defined. Early intermittent, self-administered nebulization of tissue plasminogen activator was found to be effective in preventing deterioration of acute respiratory symptoms in a patient with primary ciliary dyskinesia and recurrent cast formation. Further investigation into new therapeutic strategies for this devastating disease is advocated.

  20. Geometry of GPPE binding to picrate and to the urokinase type plasminogen activator.

    PubMed

    Zesławska, Ewa; Stürzebecher, Jörg; Oleksyn, Barbara J

    2007-11-15

    Crystal structure of 2-(4-guanidynephenyl)-1-phenyl-ethanone (GPPE) in two different environments was determined in order to compare the binding geometry of these compound to a simple picrate anion and to protein, urokinase-type plasminogen activator (uPA), which may be treated as a target for anti-cancer drugs. It was shown that the conformation and the hydrogen-bonding formation by GPPE molecule are similar in both environments, but several important differences were discovered and described.

  1. Depletion of tissue plasminogen activator attenuates lung ischemia-reperfusion injury via inhibition of neutrophil extravasation

    PubMed Central

    Zhao, Yunge; Sharma, Ashish K.; LaPar, Damien J.; Kron, Irving L.; Ailawadi, Gorav; Liu, Yuan; Jones, David R.; Laubach, Victor E.

    2011-01-01

    Ischemia-reperfusion (IR) injury following lung transplantation remains a major source of early morbidity and mortality. Histologically, this inflammatory process is characterized by neutrophil infiltration and activation. We previously reported that lung IR injury was significantly attenuated in plasminogen activator inhibitor-1-deficient mice. In this study, we explored the potential role of tissue plasminogen activator (tPA) in a mouse lung IR injury model. As a result, tPA knockout (KO) mice were significantly protected from lung IR injury through several mechanisms. At the cellular level, tPA KO specifically blocked neutrophil extravasation into the interstitium, and abundant homotypic neutrophil aggregation (HNA) was detected in the lung microvasculature of tPA KO mice after IR. At the molecular level, inhibition of neutrophil extravasation was associated with reduced expression of platelet endothelial cell adhesion molecule-1 mediated through the tPA/ LDL receptor-related protein/NF-κB signaling pathway, whereas increased P-selectin triggered HNA. At the functional level, tPA KO mice incurred significantly decreased vascular permeability and improved lung function following IR. Protection from lung IR injury in tPA KO mice occurs through a fibrinolysis-independent mechanism. These results suggest that tPA could serve as an important therapeutic target for the prevention and treatment of acute IR injury after lung transplantation. PMID:21378024

  2. Involvement of urokinase-type plasminogen activator system in cancer: an overview.

    PubMed

    Mekkawy, Ahmed H; Pourgholami, Mohammad H; Morris, David L

    2014-09-01

    Currently, there are several studies supporting the role of urokinase-type plasminogen activator (uPA) system in cancer. The association of uPA to its receptor triggers the conversion of plasminogen into plasmin. This process is regulated by the uPA inhibitors (PAI-1 and PAI-2). Plasmin promotes degradation of basement membrane and extracellular matrix (ECM) components as well as activation of ECM latent matrix metalloproteases. Degradation and remodeling of the surrounding tissues is crucial in the early steps of tumor progression by facilitating expansion of the tumor mass, release of tumor growth factors, activation of cytokines as well as induction of tumor cell proliferation, migration, and invasion. Hence, many tumors showed a correlation between uPA system component levels and tumor aggressiveness and survival. Therefore, this review summarizes the structure of the uPA system, its contribution to cancer progression, and the clinical relevance of uPA family members in cancer diagnosis. In addition, the review evaluates the significance of uPA system in the development of cancer-targeted therapies.

  3. Prevention of adult respiratory distress syndrome with plasminogen activator in pigs.

    PubMed

    Hardaway, R M; Williams, C H; Marvasti, M; Farias, M; Tseng, A; Pinon, I; Yanez, D; Martinez, M; Navar, J

    1990-12-01

    Death from traumatic shock has been associated with loss of blood externally or internally. However, many patients die after trauma, even though blood volume restoration is adequate. Death is often due to pulmonary failure (adult respiratory distress syndrome [ARDS]). Death and ARDS have been associated with disseminated intravascular coagulation (DIC) and microclots in the lungs. Dissolution of the microclots after trauma can be achieved by activation of endogenous plasmin. Nine pigs were anesthetized for 48 h. Trauma was administered by 60 standard blows to each thigh resulting in a bruise of muscle but no skin, bone, or major vessel injury. Nutrition and respiration were maintained at normal levels. All nine pigs died with severe lung pathology and low PaO2. Ten other traumatized pigs were treated with a plasminogen activator iv 4 h after trauma. Five of these were treated with tissue plasminogen activator (tPA) and five with urokinase. All treated pigs survived 48 h and maintained a normal PaO2. Autopsy showed minimal lung pathology.

  4. The nature of interactions between tissue-type plasminogen activator and platelets

    SciTech Connect

    Torr, S.R.; Winters, K.J.; Santoro, S.A.; Sobel, B.E. )

    1990-07-15

    To elucidate interactions responsible for inhibition of aggregation of platelets in platelet-rich plasma (PRP) harvested from whole blood preincubated with t-PA, experiments were performed with PRP and washed platelets under diverse conditions of preincubation. Both ADP and collagen induced aggregation were inhibited in PRP unless aprotinin had been added to the preincubated whole blood concomitantly with t-PA. However, in washed platelets prepared after the same exposure aggregation was intact. When washed platelets were supplemented with fibrinogen degradation products (FDPs) in concentrations simulating those in whole blood preincubated with t-PA, aggregation induced with either ADP or collagen was inhibited. Thus, the inhibition in PRP depended on generation of FDPs by activated plasminogen. The functional integrity of surface glycoprotein (GP) IIb/IIIa receptors in washed platelets was documented by autoradiography after SDS-PAGE of surface labeled GPs and by fibrinogen binding despite preincubation of the whole blood or washed platelets themselves with t-PA and plasminogen as long as exogenous calcium (greater than or equal to 0.1 microM) was present. In contrast, when calcium was absent, the platelet GP IIb/IIIa receptor was rendered susceptible to degradation by plasmin, and aggregation was inhibited by preincubation at 37 degrees C even if aprotinin was present when aggregation was being assayed. These observations reconcile disparate results in the literature from studies in vivo and in vitro by demonstrating that inhibition of aggregation of platelets in PRP and in whole blood reflects indirect effects of plasminogen activation rather than direct effects of t-PA or plasmin on the platelets themselves.

  5. Interaction between plasminogen activator inhibitor type 1 (PAI-1) bound to fibrin and either tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). Binding of t-PA/PAI-1 complexes to fibrin mediated by both the finger and the kringle-2 domain of t-PA.

    PubMed Central

    Wagner, O F; de Vries, C; Hohmann, C; Veerman, H; Pannekoek, H

    1989-01-01

    Plasminogen activation is catalyzed both by tissue-type-(t-PA) and by urokinase-type plasminogen activator (u-PA). This reaction is controlled by plasminogen activator inhibitor type 1 (PAI-1) that is either present in plasma or bound to fibrin, present in a thrombus. We studied the mechanism of in vitro inhibition of both t-PA and u-PA activity by PAI-1 bound to fibrin. It is shown that activation of latent PAI-1 unmasks a specific fibrin-binding site that is distinct from its reactive site. This reactive site of activated PAI-1 bound to fibrin is fully exposed to form complexes with t-PA and u-PA, that are unable to activate plasminogen. Upon complex formation with either one of the plasminogen activators, PAI-1 apparently undergoes a conformational change and loses its affinity for fibrin. Consequently, complexes of u-PA and PAI-1 dissociate from the fibrin matrix and are encountered in the fluid phase. In contrast, t-PA/PAI-1 complexes remain bound to fibrin. By employing recombinant t-PA deletion-mutant proteins, that precisely lack domains involved in fibrin binding, we demonstrate that binding of t-PA/PAI-1 complexes is mediated by both the "finger" (F) and the "kringle-2" (K2) domain of t-PA. A model is proposed that explains inhibition of the fibrinolytic process, at the level of plasminogen activation by t-PA, directed by PAI-1 bound to fibrin. An implication of the proposed model is that t-PA/PAI-1 complexes and free t-PA compete for the same binding sites on fibrin. Images PMID:2503541

  6. Physiological and pathological roles of tissue plasminogen activator and its inhibitor neuroserpin in the nervous system

    PubMed Central

    Lee, Tet Woo; Tsang, Vicky W. K.; Birch, Nigel P.

    2015-01-01

    Although its roles in the vascular space are most well-known, tissue plasminogen activator (tPA) is widely expressed in the developing and adult nervous system, where its activity is believed to be regulated by neuroserpin, a predominantly brain-specific member of the serpin family of protease inhibitors. In the normal physiological state, tPA has been shown to play roles in the development and plasticity of the nervous system. Ischemic damage, however, may lead to excess tPA activity in the brain and this is believed to contribute to neurodegeneration. In this article, we briefly review the physiological and pathological roles of tPA in the nervous system, which includes neuronal migration, axonal growth, synaptic plasticity, neuroprotection and neurodegeneration, as well as a contribution to neurological disease. We summarize tPA's multiple mechanisms of action and also highlight the contributions of the inhibitor neuroserpin to these processes. PMID:26528129

  7. Unimpeded skin carcinogenesis in K14-HPV16 transgenic mice deficient for plasminogen activator inhibitor

    PubMed Central

    Masset, A.; Maillard, C.; Sounni, NE.; Jacobs, N.; Bruyère, F.; Delvenne, P.; Tacke, M.; Reinheckel, T.; Foidart, J-M.; Coussens, LM.; Noël, A.

    2011-01-01

    Angiogenesis, extracellular matrix remodeling and cell migration are associated with cancer progression and involve at least, the plasminogen activating system and its main physiological inhibitor, the plasminogen activator inhibitor-1 (PAI-1). Considering the recognized importance of PAI-1 in the regulation of tumor angiogenesis and invasion in murine models of skin tumor transplantation, we explored the functional significance of PAI-1 during early stages of neoplastic progression in the transgenic mouse model of multistage epithelial carcinogenesis (K14-HPV16 mice). We have studied the effect of genetic deletion of PAI-1 on inflammation, angiogenesis, lymphangiogenesis, as well as tumor progression. In this model, PAI-1 deficiency neither impaired keratinocyte hyperproliferation or tumor development, nor affected the infiltration of inflammatory cells and development of angiogenic or lymphangiogenic vasculature. We are reporting evidence for concomitant lymphangiogenic and angiogenic switches independent to PAI-1 status. Taken together, these data indicate that PAI-1 is not rate limiting for neoplastic progression and vascularization during premalignant progression, or that there is a functional redundancy between PAI-1 and other tumor regulators, masking the effect of PAI-1 deficiency in this long-term model of multi-stage epithelial carcinogenesis. PMID:20232379

  8. Staphylokinase has distinct modes of interaction with antimicrobial peptides, modulating its plasminogen-activation properties

    PubMed Central

    Nguyen, Leonard T.; Vogel, Hans J.

    2016-01-01

    Staphylokinase (Sak) is a plasminogen activator protein that is secreted by many Staphylococcus aureus strains. Sak also offers protection by binding and inhibiting specific antimicrobial peptides (AMPs). Here, we evaluate Sak as a more general interaction partner for AMPs. Studies with melittin, mCRAMP, tritrpticin and bovine lactoferricin indicate that the truncation of the first ten residues of Sak (SakΔN10), which occurs in vivo and uncovers important residues in a bulge region, improves its affinity for AMPs. Melittin and mCRAMP have a lower affinity for SakΔN10, and in docking studies, they bind to the N-terminal segment and bulge region of SakΔN10. By comparison, lactoferricin and tritrpticin form moderately high affinity 1:1 complexes with SakΔN10 and their cationic residues form several electrostatic interactions with the protein’s α-helix. Overall, our work identifies two distinct AMP binding surfaces on SakΔN10 whose occupation would lead to either inhibition or promotion of its plasminogen activating properties. PMID:27554435

  9. Characterization and biological activities of recombinant human plasminogen kringle 1-3 produced in Escherichia coli.

    PubMed

    You, Weon-Kyoo; So, Seung-Ho; Sohn, Young-Doug; Lee, Hyosil; Park, Doo-Hong; Chung, Soo-Il; Chung, Kwang-Hoe

    2004-07-01

    Angiogenesis, the formation of new capillaries from preexisting blood vessels, is involved in many pathological conditions, for example, tumorigenesis, diabetic retinopathy, and rheumatoid arthritis. Angiostatin, which contains the kringle 1-4 domains of plasminogen, is known to be a potent inhibitor of angiogenesis and a strong suppressor of various solid tumors. In this study, we expressed recombinant protein containing the kringle 1-3 domains of human plasminogen in Escherichia coli and investigated its biological activities. The protein was successfully refolded from inclusion bodies and purified at a 30% overall yield, as a single peak by HPLC. The purified recombinant protein had biochemical properties that were similar to those of the native form, which included molecular size, lysine-binding capacity, and immunoreactivity with a specific antibody. The recombinant protein was also found to strongly inhibit the proliferation of bovine capillary endothelial cells in vitro, and the formation of new capillaries on chick embryos. In addition, it suppressed the growth of primary Lewis lung carcinoma and B16 melanoma in an in vivo mouse model. Our findings suggest that the recombinant kringle 1-3 domains in a prokaryote expression system have anti-angiogenic activities, which may be useful in clinical and basic research in the field of angiogenesis.

  10. Purified plasminogen activating factor produced by malignant lymphoid cells abrogates lymphocyte cytotoxicity.

    PubMed Central

    Sundar, S K; Bergeron, J; Menezes, J

    1984-01-01

    Immunosuppression is a generally observed phenomenon in patients with malignancies. Here we report that plasminogen activating factor (PAF) produced by human (P3HR-1) and simian (B95-8) lymphoid cells of malignant origin abrogates lymphocyte cytotoxicity. PAF has been purified from Epstein-Barr (EB) virus genome carrying lymphocyte cytotoxicity. PAF has been purified from Epstein-Barr (EB) virus genome carrying lymphoid lines by affinity chromatography using lysine-Sepharose columns. Purified PAF consistently inhibited Killer cell activity against the following targets: K-562, EB virus superinfected Raji cells and in vitro EB virus transformed autologous B lymphocytes. Furthermore PAF also inhibited the antibody-dependent cellular cytotoxicity. The results presented also indicate that PAF affects the effector lymphocytes and not the target cells. Taken together, these observations emphasize the importance of factors such as PAF, released by malignant cells, as inhibitors/modulators of immune mechanisms effective against tumour cells. PMID:6430612

  11. Acceleration of Tissue Plasminogen Activator-Mediated Thrombolysis by Magnetically Powered Nanomotors

    PubMed Central

    2015-01-01

    Dose control and effectiveness promotion of tissue plasminogen activator (t-PA) for thrombolysis are vitally important to alleviate serious side effects such as hemorrhage in stroke treatments. In order to increase the effectiveness and reduce the risk of stroke treatment, we use rotating magnetic nanomotors to enhance the mass transport of t-PA molecules at the blood clot interface for local ischemic stroke therapy. The in vitro experiments demonstrate that, when combined with magnetically activated nanomotors, the thrombolysis speed of low-concentration t-PA (50 μg mL–1) can be enhanced up to 2-fold, to the maximum lysis speed at high t-PA concentration. Based on the convection enhanced transport theory due to rotating magnetic nanomotors, a theoretical model is proposed and predicts the experimental results reasonably well. The validity and efficiency of this enhanced treatment has been demonstrated in a rat embolic model. PMID:25006696

  12. Tissue plasminogen activator is required for the development of fetal alcohol syndrome in mice.

    PubMed

    Noel, Melissa; Norris, Erin H; Strickland, Sidney

    2011-03-22

    Ethanol exposure during developmental synaptogenesis can lead to brain defects referred to as fetal alcohol syndrome (FAS), which can include mental health problems such as cognitive deficits and mental retardation. In FAS, widespread neuronal death and brain mass loss precedes behavioral and cognitive impairments in adulthood. Because tissue plasminogen activator (tPA) has been implicated in neurodegeneration, we examined whether it mediates FAS. Neonatal WT and tPA-/- mice were injected with ethanol to mimic FAS in humans. In WT mice, ethanol elicited caspase-3 activation, significant forebrain neurodegeneration, and decreased contextual fear conditioning in adults. However, tPA-deficient mice were protected from these neurotoxicities, and this protection could be abrogated by exogenous tPA. Selective pharmacological modulators of NMDA and GABAA receptor pathways revealed that the effects of tPA were mediated by the NR2B subunit of the NMDA receptor. This study identifies tPA as a critical signaling component in FAS.

  13. Cardiovascular-renal complications and the possible role of plasminogen activator inhibitor: a review

    PubMed Central

    D'Elia, John A.; Bayliss, George; Gleason, Ray E.; Weinrauch, Larry A.

    2016-01-01

    Since angiotensin increases the expression of plasminogen activator inhibitor (PAI), mechanisms associated with an actively functioning renin–angiotensin–aldosterone system can be expected to be associated with increased PAI-1 expression. These mechanisms are present not only in common conditions resulting in glomerulosclerosis associated with aging, diabetes or genetic mutations, but also in autoimmune disease (like scleroderma and lupus), radiation injury, cyclosporine toxicity, allograft nephropathy and ureteral obstruction. While the renin–angiotensin–aldosterone system and growth factors, such as transforming growth factor-beta (TGF-β), are almost always part of the process, there are rare experimental observations of PAI-1 expression without their interaction. Here we review the literature on PAI-1 and its role in vascular, fibrotic and oxidative injury as well as work suggesting potential areas of intervention in the pathogenesis of multiple disorders. PMID:27679717

  14. Participation of the urokinase-type plasminogen activator receptor (uPAR) in neutrophil transendothelial migration.

    PubMed

    Pliyev, Boris K; Antonova, Olga A; Menshikov, Mikhail

    2011-05-01

    The mechanisms underlying migration of neutrophils across endothelium are not completely understood. The urokinase-type plasminogen activator receptor (uPAR) plays a key role in neutrophil adhesion and migration. In the present study, we addressed whether uPAR regulates neutrophil transendothelial migration. We first showed that siRNA-mediated knockdown of uPAR in human umbilical vein endothelial cells (HUVECs) did not affect neutrophil migration across HUVEC monolayers indicating that endothelial uPAR does not regulate neutrophil transmigration. In contrast, the transmigration was significantly inhibited by Fab' fragment of anti-uPAR monoclonal antibody and proteolytically inactive urokinase (uPA), whereas inhibition of proteolytical activity of endogenous uPA (with amiloride or plasminogen activator inhibitor-1) did not affect the transmigration. Both the anti-uPAR Fab' fragment and proteolytically inactive uPA did not exert significant effects upon the transmigration conducted in the presence of F(ab')(2) fragment of blocking antibody to integrin Mac-1 indicating that uPAR regulates Mac-1-dependent transmigration. Mac-1-dependent, but not Mac-1-independent, transmigration was significantly reduced in the presence of N-acetyl-d-glucosamine and d-mannose, the saccharides that disrupt uPAR/Mac-1 association, but was unaffected in the presence of control saccharides (d-sorbitol and sucrose). We conclude that physical association of uPAR with Mac-1 mediates the regulatory effect of uPAR over the transmigration. Finally, we provide evidence that the functional cooperation between uPAR and Mac-1 is essential at both adhesion and diapedesis steps of neutrophil migration across endothelium. Thus, uPAR expressed on neutrophil plasma membrane regulates transendothelial migration independently of uPA proteolytical activity and acting as a cofactor for integrin Mac-1.

  15. Tissue-type plasminogen activator induces synaptic vesicle endocytosis in cerebral cortical neurons.

    PubMed

    Yepes, M; Wu, F; Torre, E; Cuellar-Giraldo, D; Jia, D; Cheng, L

    2016-04-05

    The release of the serine proteinase tissue-type plasminogen activator (tPA) from the presynaptic terminal of cerebral cortical neurons plays a central role in the development of synaptic plasticity, adaptation to metabolic stress and neuronal survival. Our earlier studies indicate that by inducing the recruitment of the cytoskeletal protein βII-spectrin and voltage-gated calcium channels to the active zone, tPA promotes Ca(2+)-dependent translocation of synaptic vesicles (SVs) to the synaptic release site where they release their load of neurotransmitters into the synaptic cleft. Here we used a combination of in vivo and in vitro experiments to investigate whether this effect leads to depletion of SVs in the presynaptic terminal. Our data indicate that tPA promotes SV endocytosis via a mechanism that does not require the conversion of plasminogen into plasmin. Instead, we show that tPA induces calcineurin-mediated dynamin I dephosphorylation, which is followed by dynamin I-induced recruitment of the actin-binding protein profilin II to the presynaptic membrane, and profilin II-induced F-actin formation. We report that this tPA-induced sequence of events leads to the association of newly formed SVs with F-actin clusters in the endocytic zone. In summary, the data presented here indicate that following the exocytotic release of neurotransmitters tPA activates the mechanism whereby SVs are retrieved from the presynaptic membrane and endocytosed to replenish the pool of vesicles available for a new cycle of exocytosis. Together, these results indicate that in murine cerebral cortical neurons tPA plays a central role coupling SVs exocytosis and endocytosis.

  16. Binding site of amiloride to urokinase plasminogen activator depends on species.

    PubMed

    Jankun, J; Skrzypczak-Jankun, E

    2001-10-01

    A novel drug candidate is checked on its potency on animal models before it can advance to human phase of the research. Usually negative results on animal phase disqualify it. Targeting specific enzymes by small chemicals raises the question about the appropriateness of this approach. As an example, the urokinase (uPA) is recognized as an important enzyme responsible for cancer metastasis and angiogenesis. It is therefore important to ask the question if a small chemical will inhibit uPA of different species with the same or different potency. Using DNA sequence and known structure of uPA we have modeled 3D structures of uPAs for several different species. By theoretical calculations we have determined most probable structure of amiloride/uPAs complexes. Catalytic triad (B57, B102, B195) and specificity pocket (B187-B197, B212-B229) are highly conserved in all cases, and are the regions responsible for proteolytic activity and recognition of the substrate. Significant differences were observed in a different region (loop B93-B101), that we identified as binding site of amiloride to the tissue plasminogen activator (tPA). Although tPA shares the same function of activating plasminogen and it is structurally similar to uPA. Amiloride is a specific inhibitor of uPA but does not inhibit tPA. Our study shows that predicted position of amiloride depends on species and in some cases was located, as expected, in the specificity pocket, but in the other cases close to the loop B93-B101. This location could weaken affinity of binding or prevent inhibition of uPA. Therefore, drug screening and elimination process based solely on animal study, without careful structural analysis, could lead to the elimination of potential drugs for humans.

  17. New insights into the size and stoichiometry of the plasminogen activator inhibitor type-1.vitronectin complex.

    PubMed

    Podor, T J; Shaughnessy, S G; Blackburn, M N; Peterson, C B

    2000-08-18

    Plasminogen activator inhibitor-type 1 (PAI-1) is the primary inhibitor of endogenous plasminogen activators that generate plasmin in the vicinity of a thrombus to initiate thrombolysis, or in the pericellular region of cells to facilitate migration and/or tissue remodeling. It has been shown that the physiologically relevant form of PAI-1 is in a complex with the abundant plasma glycoprotein, vitronectin. The interaction between vitronectin and PAI-1 is important for stabilizing the inhibitor in a reactive conformation. Although the complex is clearly significant, information is vague regarding the composition of the complex and consequences of its formation on the distribution and activity of vitronectin in vivo. Most studies have assumed a 1:1 interaction between the two proteins, but this has not been demonstrated experimentally and is a matter of some controversy since more than one PAI-1-binding site has been proposed within the sequence of vitronectin. To address this issue, competition studies using monoclonal antibodies specific for separate epitopes confirmed that the two distinct PAI-1-binding sites present on vitronectin can be occupied simultaneously. Analytical ultracentrifugation was used also for a rigorous analysis of the composition and sizes of complexes formed from purified vitronectin and PAI-1. The predominant associating species observed was high in molecular weight (M(r) approximately 320,000), demonstrating that self-association of vitronectin occurs upon interaction with PAI-1. Moreover, the size of this higher order complex indicates that two molecules of PAI-1 bind per vitronectin molecule. Binding of PAI-1 to vitronectin and association into higher order complexes is proposed to facilitate interaction with macromolecules on surfaces.

  18. Butyrate stimulates tissue-type plasminogen-activator synthesis in cultured human endothelial cells.

    PubMed Central

    Kooistra, T; van den Berg, J; Töns, A; Platenburg, G; Rijken, D C; van den Berg, E

    1987-01-01

    Incubation of cultured human endothelial cells with 5 mM-dibutyryl cyclic AMP led to an approx. 2-fold increase in tissue-type plasminogen-activator (t-PA) production over a 24 h incubation period. The stimulating effect of dibutyryl cyclic AMP could be explained by the slow liberation of butyrate, as the effect could be reproduced by addition of free butyrate to the medium, but not by addition of 8-bromo cyclic AMP or forskolin, agents known to raise intracellular cyclic AMP levels. With butyrate, an accelerated accumulation of t-PA antigen in the conditioned medium (CM) was observed after a lag period of about 6 h. Increasing amounts of butyrate caused an increasingly stimulatory effect, reaching a plateau at 5 mM-butyrate. The relative enhancement of t-PA production in the presence of 5 mM-butyrate varied among different endothelial cell cultures from 6- to 25-fold in 24 h CM. Such an increase in t-PA production was observed with both arterial and venous endothelial cells. The butyrate-induced increases in t-PA production were accompanied by increased t-PA mRNA levels. Analysis of radiolabelled CM and cell extracts by SDS/polyacrylamide-gel electrophoresis indicated that the potent action of butyrate is probably restricted to a small number of proteins. The accumulation of plasminogen activator inhibitor type 1 (PAI-1) in CM from butyrate-treated cells varied only moderately. In our study of the relationship between structure and stimulatory activity, we found that a straight-chain C4 monocarboxylate structure with a methyl group at one end and a carboxy moiety at the other seems to be required for the optimal induction of t-PA in cultured endothelial cells. Images Fig. 2. Fig. 3. Fig. 5. Fig. 7. PMID:2827633

  19. Design, synthesis and in vitro evaluation of potent, novel, small molecule inhibitors of plasminogen activator inhibitor-1.

    PubMed

    Folkes, Adrian; Brown, S David; Canne, Lynne E; Chan, Jocelyn; Engelhardt, Erin; Epshteyn, Sergey; Faint, Richard; Golec, Julian; Hanel, Art; Kearney, Patrick; Leahy, James W; Mac, Morrison; Matthews, David; Prisbylla, Michael P; Sanderson, Jason; Simon, Reyna J; Tesfai, Zerom; Vicker, Nigel; Wang, Shouming; Webb, Robert R; Charlton, Peter

    2002-04-08

    We have synthesized and evaluated a series of tetramic acid-based and hydroxyquinolinone-based inhibitors of plasminogen activator inhibitor-1 (PAI-1). These studies resulted in the identification of several compounds which showed excellent potency against PAI-1. The design, synthesis and SAR of these compounds are described.

  20. Recombinant tissue plasminogen activator as a novel treatment option for infective endocarditis: a retrospective clinical study in 32 children.

    PubMed

    Levitas, Aviva; Krymko, Hanna; Richardson, Justin; Zalzstein, Eli; Ioffe, Viktoriya

    2016-01-01

    Infective endocarditis is a life-threatening infectious syndrome, with high morbidity and mortality. Current treatments for infective endocarditis include intravenous antibiotics, surgery, and involve a lengthy hospital stay. We hypothesised that adjunctive recombinant tissue plasminogen activator treatment for infective endocarditis may facilitate faster resolution of vegetations and clearance of positive blood cultures, and therefore decrease morbidity and mortality. This retrospective study included follow-up of patients, from 1997 through 2014, including clinical presentation, causative organism, length of treatment, morbidity, and mortality. We identified 32 patients, all of whom were diagnosed with endocarditis and were treated by recombinant tissue plasminogen activator. Among all, 27 patients (93%) had positive blood cultures, with the most frequent organisms being Staphylococcus epidermis (nine patients), Staphylococcus aureus (six patients), and Candida (nine patients). Upon treatment, in 31 patients (97%), resolution of vegetations and clearance of blood cultures occurred within hours to few days. Out of 32 patients, one patient (3%) died and three patients (9%) suffered embolic or haemorrhagic events, possibly related to the recombinant tissue plasminogen activator. None of the patients required surgical intervention to assist vegetation resolution. In conclusion, it appears that recombinant tissue plasminogen activator may become an adjunctive treatment for infective endocarditis and may decrease morbidity as compared with current guidelines. Prospective multi-centre studies are required to validate our findings.

  1. High-fat diet enhances and plasminogen activator inhibitor-1 deficiency attenuates bone loss in mice with Lewis Lung carcinoma

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study determined the effects of a high-fat diet and plasminogen activator inhibitor-1 deficiency (PAI-1-/-) on bone structure in mice bearing Lewis lung carcinoma (LLC) in lungs. Reduction in bone volume fraction (BV/TV) by 22% and 21%, trabecular number (Tb.N) by 8% and 4% and bone mineral de...

  2. Components of the plasminogen activation system promote engraftment of porous polyethylene biomaterial via common and distinct effects.

    PubMed

    Reichel, Christoph A; Hessenauer, Maximilian E T; Pflieger, Kerstin; Rehberg, Markus; Kanse, Sandip M; Zahler, Stefan; Krombach, Fritz; Berghaus, Alexander; Strieth, Sebastian

    2015-01-01

    Rapid fibrovascularization is a prerequisite for successful biomaterial engraftment. In addition to their well-known roles in fibrinolysis, urokinase-type plasminogen activator (uPA) and tissue plasminogen activator (tPA) or their inhibitor plasminogen activator inhibitor-1 (PAI-1) have recently been implicated as individual mediators in non-fibrinolytic processes, including cell adhesion, migration, and proliferation. Since these events are critical for fibrovascularization of biomaterial, we hypothesized that the components of the plasminogen activation system contribute to biomaterial engraftment. Employing in vivo and ex vivo microscopy techniques, vessel and collagen network formation within porous polyethylene (PPE) implants engrafted into dorsal skinfold chambers were found to be significantly impaired in uPA-, tPA-, or PAI-1-deficient mice. Consequently, the force required for mechanical disintegration of the implants out of the host tissue was significantly lower in the mutant mice than in wild-type controls. Conversely, surface coating with recombinant uPA, tPA, non-catalytic uPA, or PAI-1, but not with non-catalytic tPA, accelerated implant vascularization in wild-type mice. Thus, uPA, tPA, and PAI-1 contribute to the fibrovascularization of PPE implants through common and distinct effects. As clinical perspective, surface coating with recombinant uPA, tPA, or PAI-1 might provide a novel strategy for accelerating the vascularization of this biomaterial.

  3. Components of the Plasminogen Activation System Promote Engraftment of Porous Polyethylene Biomaterial via Common and Distinct Effects

    PubMed Central

    Reichel, Christoph A.; Hessenauer, Maximilian E. T.; Pflieger, Kerstin; Rehberg, Markus; Kanse, Sandip M.; Zahler, Stefan; Krombach, Fritz; Berghaus, Alexander; Strieth, Sebastian

    2015-01-01

    Rapid fibrovascularization is a prerequisite for successful biomaterial engraftment. In addition to their well-known roles in fibrinolysis, urokinase-type plasminogen activator (uPA) and tissue plasminogen activator (tPA) or their inhibitor plasminogen activator inhibitor-1 (PAI-1) have recently been implicated as individual mediators in non-fibrinolytic processes, including cell adhesion, migration, and proliferation. Since these events are critical for fibrovascularization of biomaterial, we hypothesized that the components of the plasminogen activation system contribute to biomaterial engraftment. Employing in vivo and ex vivo microscopy techniques, vessel and collagen network formation within porous polyethylene (PPE) implants engrafted into dorsal skinfold chambers were found to be significantly impaired in uPA-, tPA-, or PAI-1-deficient mice. Consequently, the force required for mechanical disintegration of the implants out of the host tissue was significantly lower in the mutant mice than in wild-type controls. Conversely, surface coating with recombinant uPA, tPA, non-catalytic uPA, or PAI-1, but not with non-catalytic tPA, accelerated implant vascularization in wild-type mice. Thus, uPA, tPA, and PAI-1 contribute to the fibrovascularization of PPE implants through common and distinct effects. As clinical perspective, surface coating with recombinant uPA, tPA, or PAI-1 might provide a novel strategy for accelerating the vascularization of this biomaterial. PMID:25658820

  4. Imaging of Prostate Cancer Using Urokinase-Type Plasminogen Activator Receptor PET.

    PubMed

    Skovgaard, Dorthe; Persson, Morten; Kjaer, Andreas

    2017-04-01

    Urokinase-type plasminogen activator receptor (uPAR) overexpression is an important biomarker for aggressiveness in cancer including prostate cancer (PC) and provides independent clinical information in addition to prostate-specific antigen and Gleason score. This article focuses on uPAR PET as a new diagnostic and prognostic imaging biomarker in PC. Many preclinical uPAR-targeted PET imaging studies using AE105 in cancer models have been undertaken with promising results. A major breakthrough was obtained with the recent human translation of uPAR PET in using (64)Cu- and (68)Ga-labelled versions of AE105, respectively. Clinical results from patients with PC included in these studies are encouraging and support continuation with large-scale clinical trials.

  5. Recombinant tissue plasminogen activator in two patients with basilar artery occlusion.

    PubMed Central

    Herderscheê, D; Limburg, M; Hijdra, A; Koster, P A

    1991-01-01

    Two patients with angiographically proved basilar artery occlusion were treated with systemic recombinant tissue plasminogen activator (rtPA) according to protocol. The first patient was in a locked-in state and gradually deteriorated. On repeat angiography the basilar artery remained occluded. He died and necropsy revealed a pontine haemorrhagic infarction. The second patient, who was comatose and with decerebrate posturing, made a remarkable recovery. Angiography showed reperfusion. Therapy was initiated in the first patient after six hours and in the second after two hours. Treatment with rtPA is promising but probably not feasible for every patient. Success may depend on duration of occlusion and composition of occluding thrombus. Images PMID:1901349

  6. Hemorrhagic Transformation after Tissue Plasminogen Activator Reperfusion Therapy for Ischemic Stroke: Mechanisms, Models, and Biomarkers.

    PubMed

    Wang, Wei; Li, Mingchang; Chen, Qianxue; Wang, Jian

    2015-12-01

    Intracerebral hemorrhagic transformation (HT) is well recognized as a common cause of hemorrhage in patients with ischemic stroke. HT after acute ischemic stroke contributes to early mortality and adversely affects functional recovery. The risk of HT is especially high when patients receive thrombolytic reperfusion therapy with tissue plasminogen activator, the only available treatment for ischemic stroke. Although many important publications address preclinical models of ischemic stroke, there are no current recommendations regarding the conduct of research aimed at understanding the mechanisms and prediction of HT. In this review, we discuss the underlying mechanisms for HT after ischemic stroke, provide an overview of the models commonly used for the study of HT, and discuss biomarkers that might be used for the early detection of this challenging clinical problem.

  7. Hemorrhagic Transformation After Tissue Plasminogen Activator Reperfusion Therapy for Ischemic Stroke: Mechanisms, Models, and Biomarkers

    PubMed Central

    Wang, Wei; Li, Mingchang; Chen, Qianxue; Wang, Jian

    2014-01-01

    Summary Intracerebral hemorrhagic transformation (HT) is well recognized as a common cause of hemorrhage in patients with ischemic stroke. HT after acute ischemic stroke contributes to early mortality and adversely affects functional recovery. The risk of HT is especially high when patients receive thrombolytic reperfusion therapy with tissue plasminogen activator, the only available treatment for ischemic stroke. Although many important publications address preclinical models of ischemic stroke, there are no current recommendations regarding the conduct of research aimed at understanding the mechanisms and prediction of HT. In this review, we discuss the underlying mechanisms for HT after ischemic stroke, provide an overview of the models commonly used for the study of HT, and discuss biomarkers that might be used for early detection of this challenging clinical problem. PMID:25367883

  8. Gastrin stimulates expression of plasminogen activator inhibitor-1 in gastric epithelial cells.

    PubMed

    Nørsett, Kristin G; Steele, Islay; Duval, Cedric; Sammut, Stephen J; Murugesan, Senthil V M; Kenny, Susan; Rainbow, Lucille; Dimaline, Rod; Dockray, Graham J; Pritchard, D Mark; Varro, Andrea

    2011-09-01

    Plasminogen activator inhibitor (PAI)-1 is associated with cancer progression, fibrosis and thrombosis. It is expressed in the stomach but the mechanisms controlling its expression there, and its biological role, are uncertain. We sought to define the role of gastrin in regulating PAI-1 expression and to determine the relevance for gastrin-stimulated cell migration and invasion. In gastric biopsies from subjects with elevated plasma gastrin, the abundances of PAI-1, urokinase plasminogen activator (uPA), and uPA receptor (uPAR) mRNAs measured by quantitative PCR were increased compared with subjects with plasma concentrations in the reference range. In patients with hypergastrinemia due to autoimmune chronic atrophic gastritis, there was increased abundance of PAI-1, uPA, and uPAR mRNAs that was reduced by octreotide or antrectomy. Immunohistochemistry revealed localization of PAI-1 to parietal cells and enterochromaffin-like cells in micronodular neuroendocrine tumors in hypergastrinemic subjects. Transcriptional mechanisms were studied by using a PAI-1-luciferase promoter-reporter construct transfected into AGS-G(R) cells. There was time- and concentration-dependent increase of PAI-1-luciferase expression in response to gastrin that was reversed by inhibitors of the PKC and MAPK pathways. In Boyden chamber assays, recombinant PAI-1 inhibited gastrin-stimulated AGS-G(R) cell migration and invasion, and small interfering RNA treatment increased responses to gastrin. We conclude that elevated plasma gastrin concentrations are associated with increased expression of gastric PAI-1, which may act to restrain gastrin-stimulated cell migration and invasion.

  9. Urokinase-Type Plasminogen Activator Promotes Dendritic Spine Recovery and Improves Neurological Outcome Following Ischemic Stroke

    PubMed Central

    Wu, Fang; Catano, Marcela; Echeverry, Ramiro; Torre, Enrique; Haile, Woldeab B.; An, Jie; Chen, Changhua; Cheng, Lihong; Nicholson, Andrew; Tong, Frank C.; Park, Jaekeun

    2014-01-01

    Spines are dendritic protrusions that receive most of the excitatory input in the brain. Early after the onset of cerebral ischemia dendritic spines in the peri-infarct cortex are replaced by areas of focal swelling, and their re-emergence from these varicosities is associated with neurological recovery after acute ischemic stroke (AIS). Urokinase-type plasminogen activator (uPA) is a serine proteinase that plays a central role in tissue remodeling via binding to the urokinase plasminogen activator receptor (uPAR). We report that cerebral cortical neurons release uPA during the recovery phase from ischemic stroke in vivo or hypoxia in vitro. Although uPA does not have an effect on ischemia- or hypoxia-induced neuronal death, genetic deficiency of uPA (uPA−/−) or uPAR (uPAR−/−) abrogates functional recovery after AIS. Treatment with recombinant uPA after ischemic stroke induces neurological recovery in wild-type and uPA−/− but not in uPAR−/− mice. Diffusion tensor imaging studies indicate that uPA−/− mice have increased water diffusivity and decreased anisotropy associated with impaired dendritic spine recovery and decreased length of distal neurites in the peri-infarct cortex. We found that the excitotoxic injury induces the clustering of uPAR in dendritic varicosities, and that the binding of uPA to uPAR promotes the reorganization of the actin cytoskeleton and re-emergence of dendritic filopodia from uPAR-enriched varicosities. This effect is independent of uPA's proteolytic properties and instead is mediated by Rac-regulated profilin expression and cofilin phosphorylation. Our data indicate that binding of uPA to uPAR promotes dendritic spine recovery and improves functional outcome following AIS. PMID:25339736

  10. NR2D-containing NMDA receptors mediate tissue plasminogen activator-promoted neuronal excitotoxicity.

    PubMed

    Baron, A; Montagne, A; Cassé, F; Launay, S; Maubert, E; Ali, C; Vivien, D

    2010-05-01

    Although the molecular bases of its actions remain debated, tissue-type plasminogen activator (tPA) is a paradoxical brain protease, as it favours some learning/memory processes, but increases excitotoxic neuronal death. Here, we show that, in cultured cortical neurons, tPA selectively promotes NR2D-containing N-methyl-D-aspartate receptor (NMDAR)-dependent activation. We show that tPA-mediated signalling and neurotoxicity through the NMDAR are blocked by co-application of an NR2D antagonist (phenanthrene derivative (2S(*), 3R(*))-1-(phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid, PPDA) or knockdown of neuronal NR2D expression. In sharp contrast with cortical neurons, hippocampal neurons do not exhibit NR2D both in vitro and in vivo and are consequently resistant to tPA-promoted NMDAR-mediated neurotoxicity. Moreover, we have shown that activation of synaptic NMDAR prevents further tPA-dependent NMDAR-mediated neurotoxicity and sensitivity to PPDA. This study shows that the earlier described pro-neurotoxic effect of tPA is mediated by NR2D-containing NMDAR-dependent extracellular signal-regulated kinase activation, a deleterious effect prevented by synaptic pre-activation.

  11. A novel plasminogen activator from Agkistrodon blomhoffii Ussurensis venom (ABUSV-PA): purification and characterization.

    PubMed

    Liu, Shuqing; Sun, Ming-Zhong; Greenaway, Frederick T

    2006-10-06

    A plasminogen activator with arginine ester hydrolysis activity (ABUSV-PA) has been identified and purified to homogeneity from Chinese Agkistrodon blomhoffii Ussurensis snake venom. ABUSV-PA, a monomeric protein with molecular mass of 27815.2 Da, was purified 180-fold with 0.02% recovery for protein and 3.6% recovery for esterase activity. ABUSV-PA reacts optimally with its substrate N(alpha)-tosyl-l-arginine-methyl ester (TAME) at approximately pH 7.5 and at 51 degrees C. Measurement from inductively coupled plasma-atomic emission spectroscopy (ICP-AES) reveals that ABUSV-PA is a Zn(2+)-containing protein with a stoichiometry of 1:1 [Zn(2+)]:[ABUSV-PA]. Analyses of esterase hydrolysis and UV absorption and CD spectra indicate that Zn(2+) plays an important role in maintaining the structural integrity rather than the esterase activity of ABUSV-PA. Divalent metal ions, including Ca(2+), Mg(2+), Cu(2+), Ni(2+), Mn(2+), and Co(2+), increase the TAME hydrolysis activity of ABUSV-PA. A red-shift of the emission wavelengths of the synchronous fluorescence of ABUSV-PA, compared to those of free Tyr and Trp, indicates a conformation where the Tyr and Trp residues are in exposed hydrophilic environments. The presence of zinc increases the hydrophobicity of the conformational environments surrounding the Trp residues of ABUSV-PA and affects the secondary structure of ABUSV-PA, as proved by UV absorption and CD spectroscopy.

  12. Tissue plasminogen activator inhibits NMDA-receptor-mediated increases in calcium levels in cultured hippocampal neurons

    PubMed Central

    Robinson, Samuel D.; Lee, Tet Woo; Christie, David L.; Birch, Nigel P.

    2015-01-01

    NMDA receptors (NMDARs) play a critical role in neurotransmission, acting as essential mediators of many forms of synaptic plasticity, and also modulating aspects of development, synaptic transmission and cell death. NMDAR-induced responses are dependent on a range of factors including subunit composition and receptor location. Tissue-type plasminogen activator (tPA) is a serine protease that has been reported to interact with NMDARs and modulate NMDAR activity. In this study we report that tPA inhibits NMDAR-mediated changes in intracellular calcium levels in cultures of primary hippocampal neurons stimulated by low (5 μM) but not high (50 μM) concentrations of NMDA. tPA also inhibited changes in calcium levels stimulated by presynaptic release of glutamate following treatment with bicucculine/4-aminopyridine (4-AP). Inhibition was dependent on the proteolytic activity of tPA but was unaffected by α2-antiplasmin, an inhibitor of the tPA substrate plasmin, and receptor-associated protein (RAP), a pan-ligand blocker of the low-density lipoprotein receptor, two proteins previously reported to modulate NMDAR activity. These findings suggest that tPA can modulate changes in intracellular calcium levels in a subset of NMDARs expressed in cultured embryonic hippocampal neurons through a mechanism that involves the proteolytic activity of tPA and synaptic NMDARs. PMID:26500501

  13. Plasminogen activator inhibitor type-1 determines plasmin formation in patients with ischaemic heart disease.

    PubMed

    Pedersen, O D; Gram, J; Jespersen, J

    1995-05-01

    The aim of the present study was to find out whether plasminogen activator inhibitor type-1 (PAI-1) controls the formation of plasmin in patients with ischaemic heart disease. We examined PAI activity, PAI-1 antigen, tissue type plasminogen activator (t-PA) activity, t-PA antigen, plasmin-alpha2-antiplasmin complex (PAP-complex) and fibrin degradation products D-dimer in 62 patients before (unstimulated) and after infusion of 1-desamino-8-D-arginine vasopressin (DDAVP; stimulated). DDAVP was used in a standardized dose to trigger the release of t-PA from the vascular endothelium. We observed that under basal conditions (unstimulated) median plasma t-PA activity for the whole group of patients was 86.5 mIU/ml (0-900), and after stimulation 2550 mIU/ml (0-6800), P < 0.0001; median plasma concentration of t-PA antigen was 14.7 ng/ml (7.0-115.5) under basal conditions, and after stimulation 34.1 ng/ml (15.8-58.6), P < 0.0001; median plasma PAI activity was 16.9 IU/ml (1.5-144.8) under basal conditions, and after stimulation 3.1 IU/ml (0-118.5), P < 0.0001; median plasma concentration of PAI-1 antigen was 21.5 ng/ml (8.1-132.2) under basal conditions, and after stimulation 14.9 ng/ml (4.8-149.0), P < 0.0001; the median plasma concentration of PAP-complex was 469.5 ng/ml (185.0-1802.0) under basal conditions, and after stimulation 695.5 (243.0-2292.0), P < 0.0001; median plasma concentration of D-dimer was 298.0 ng/ml (103.0-948.0) under basal conditions, and after stimulation 296.5 ng/ml (97.0-917.0), P < 0.0008.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. The plasminogen activator system in the ovine placentome during late gestation and stage-two of parturition.

    PubMed

    McNeel, Anthony K; Cushman, Robert A; Vallet, Jeffrey L

    2013-06-01

    The process of placental separation is not completely understood. In domestic animals, especially cattle, it is important that expulsion of the fetal membranes takes place in a timely manner in order to achieve maximal reproductive efficiency. The activity of the matrix-metalloprotease (MMP) family of proteases is known to be reduced in placentomes from cases of retained placenta. Members of the MMP family are known to be activated by the plasminogen activator (PA) family of proteases. We hypothesized that the expression and activity of the PA family increase in the cotyledon and/or caruncle as parturition approaches, with maximal expression and activity at parturition. To test this hypothesis, we performed reverse-transcriptase quantitative PCR and plasminogen-casein zymography to detect the presence and activity of PA family members in the placentome leading up to and during parturition in spontaneous and dexamethasone-induced parturient ewes. The results from our experiments indicated that serine proteases inhibitor E1 (SERPINE1) mRNA abundance in the cotyledon was different between treatment groups (P = 0.0002). In the caruncle, gene expression for plasminogen activator urokinase-type (PLAU) was different (P = 0.0154), and there was a strong trend for differences in SERPINE1 expression (P = 0.0565). These results demonstrate that expression of the PA system in the placentome changes from late pregnancy to parturition, and the presence or activity of these enzymes may occur after fetal expulsion.

  15. Urokinase type plasminogen activator receptor (uPAR) as a new therapeutic target in cancer

    PubMed Central

    Montuori, Nunzia; Pesapane, Ada; Rossi, Francesca W; Giudice, Valentina; De Paulis, Amato; Selleri, Carmine; Ragno, Pia

    2016-01-01

    The urokinase (uPA)-type plasminogen activator receptor (uPAR) is a GPI-anchored receptor that focuses urokinase (uPA) proteolytic activity on the cell surface. uPAR also regulates cell adhesion, migration and proliferation, protects from apoptosis and contributes to epithelial mesenchymal transition (EMT), independently of uPA enzymatic activity. Indeed, uPAR interacts with beta1, beta2 and beta3 integrins, thus regulating their activities. uPAR cross-talks with receptor tyrosine kinases through integrins and regulates cancer cell dormancy, proliferation and angiogenesis. Moreover, uPAR mediates uPA-dependent cell migration and chemotaxis induced by fMet-Leu-Phe (fMLF), through its association with fMLF-receptors (fMLF-Rs). Further, uPAR is an adhesion receptor because it binds vitronectin (VN), a component of provisional extracellular matrix. High uPAR expression predicts for more aggressive disease in several cancer types for its ability to increase invasion and metastasis. In fact, uPAR has been hypothesized to be the link between tumor cell dormancy and proliferation that usually precedes the onset of metastasis. Thus, inhibiting uPAR could be a feasible approach to affect tumor growth and metastasis. Here, we review the more recent advances in the development of uPAR-targeted anti-cancer therapeutic agents suitable for further optimization or ready for the evaluation in early clinical trials. PMID:27896223

  16. Tissue-type plasminogen activator is a neuroprotectant in the central nervous system

    PubMed Central

    Yepes, Manuel

    2015-01-01

    Tissue-type plasminogen activator (tPA) is a serine proteinase found not only in the intravascular space but also in a well-defined sub-set of neurons in the brain. tPA is rapidly released from neurons after either exposure to hypoxia or hypoglycemia in vitro, or the induction of cerebral ischemia in vivo. It has been proposed that tPA has a neurotoxic effect in the ischemic brain. However, recent evidence indicate that once released into the synaptic cleft tPA activates specific cell signaling pathways that promote the detection and adaptation to metabolic stress. More specifically, the non-proteolytic interaction of tPA with N-methyl-D-aspartate receptors (NMDARs) and a member of the low-density lipoprotein receptor (LDLR) family in dendritic spines activates the mammalian target of rapamycin (mTOR) pathway that adapts cellular processes to the availability of energy and metabolic resources. TPA-induced mTOR activation in neurons leads to hypoxia-inducible factor 1α (HIF-1α) accumulation, HIF-1α-induced expression and membrane recruitment of the neuronal transporter of glucose GLUT3, and GLUT3-mediated uptake of glucose. These and other data discussed in this Review suggest that the postulated neurotoxic effect of tPA needs to be reconsidered and instead indicate the emergence of a new paradigm: that tPA is an endogenous neuroprotectant in the central nervous system (CNS). PMID:26347605

  17. In vitro thrombolytic efficacy of echogenic liposomes loaded with tissue plasminogen activator and octafluoropropane gas

    NASA Astrophysics Data System (ADS)

    Shekhar, Himanshu; Bader, Kenneth B.; Huang, Shenwen; Peng, Tao; Huang, Shaoling; McPherson, David D.; Holland, Christy K.

    2017-01-01

    Echogenic liposomes loaded with the thrombolytic recombinant tissue-type plasminogen activator (rt-PA) are under development for the treatment of ischemic stroke. These agents are designed to co-encapsulate cavitation nuclei to promote bubble activity in response to ultrasound exposure, and to enable localized delivery of thrombolytic. Stable cavitation improves the efficacy of the thrombolytic through enhanced fluid mixing. Echogenic liposomes that encapsulate air-filled microbubbles nucleate scant stable cavitation activity in response to 120 kHz intermittent ultrasound exposure, and have demonstrated thrombolytic efficacy equivalent to rt-PA alone. It was hypothesized that encapsulating octafluoropropane (OFP) gas within rt-PA-loaded liposomes instead of air will enhance ultrasound-mediated stable cavitation activity and increase thrombolytic efficacy compared to previous studies. The thrombolytic efficacy and cavitation activity nucleated from liposomes that encapsulate OFP microbubbles and rt-PA (OFP t-ELIP) was evaluated in vitro. Human whole blood clots were exposed to human fresh-frozen plasma alone, rt-PA (0, 0.32, 1.58, and 3.15 µg ml-1), or OFP t-ELIP at equivalent enzymatic activity, with and without exposure to intermittent ultrasound. Further, numerical simulations were performed to gain insight into the mechanisms of cavitation nucleation. Sustained ultraharmonic activity was nucleated from OFP t-ELIP when exposed to ultrasound. Furthermore, the thrombolytic efficacy was enhanced compared to rt-PA alone at concentrations of 1.58 µg ml-1 and 3.15 µg ml-1 (p  <  0.05). These results indicate that OFP t-ELIP can nucleate sustained stable cavitation activity and enhance the efficacy of thrombolysis.

  18. Expression of tissue type and urokinase type plasminogen activators as well as plasminogen activator inhibitor type-1 and type-2 in human and rhesus monkey placenta

    PubMed Central

    HU, ZHAO-YUAN; LIU, YI-XUN; LIU, KUI; BYRNE, SIMON; NY, TOR; FENG, QIANG; OCKLEFORD, COLIN D.

    1999-01-01

    The distribution of mRNAs and antigens of tissue type (t) and urokinase type (u) plasminogen activators (PA) plus their corresponding inhibitors, type-1 (PAI-1) and type-2 (PAI-2) were studied in human and rhesus monkey placentae by in situ hybridisation and immunocytochemistry. Specific monkey cRNA and antibodies against human tPA, uPA, PAI-1 and PAI-2 were used as probes. The following results were obtained. (1) All the molecules tPA, uPA, PAI-1 and PAI-2 and their mRNAs were identified in the majority of the extravillous cytotrophoblast cells of the decidual layer between Rohr's and Nitabuch's striae and in cytotrophoblast cells of the chorionic plate, basal plate, intercotyledonary septae and cytotrophoblast cells of the chorionic villous tree. (2) Expression of uPA and PAI-2 was noted in villous trophoblast whereas tPA and PAI-1 were mainly concentrated where detachment from maternal tissue occurs. (3) No expression of tPA, uPA, PAI-1 and PAI-2 was observed in the basal plate endometrial stromal cells, chorionic plate connective tissue cells, septal endometrial stromal cells or villous core mesenchyme. (4) The distribution of probes observed following in situ hybridisation is generally consistent with the immunofluorescence pattern of the corresponding antigens and no significant interspecies differences were noted. It is possible that both decidual and extravillous trophoblast cells of placentae of human and rhesus monkey are capable of producing tPA, uPA, PAI-1 and PAI-2 to differing extents. Coordinated expression of these genes in the tissue may play an essential role in the maintenance of normal placentation and parturition. The differences in distribution we observed are consistent with the suggestion that coordinated expression of tPA and its inhibitor PAI-1 may play a key role in fibrinolytic activity in the early stages of placentation and separation of placenta from maternal tissue at term. On the other hand, uPA with its inhibitor PAI-2 appears

  19. Soluble urokinase plasminogen activator receptor levels reflect organ damage in systemic lupus erythematosus.

    PubMed

    Enocsson, Helena; Wetterö, Jonas; Skogh, Thomas; Sjöwall, Christopher

    2013-11-01

    Assessments of disease activity and organ damage in systemic lupus erythematosus (SLE) remain challenging because of the lack of reliable biomarkers and disease heterogeneity. Ongoing inflammation can be difficult to distinguish from permanent organ damage caused by previous flare-ups or medication side effects. Circulating soluble urokinase plasminogen activator receptor (suPAR) has emerged as a potential marker of inflammation and disease severity, and an outcome predictor in several disparate conditions. This study was done to evaluate suPAR as a marker of disease activity and organ damage in SLE. Sera from 100 healthy donors and 198 patients with SLE fulfilling the 1982 American College of Rheumatology classification criteria and/or the Fries criteria were analyzed for suPAR by enzyme immunoassay. Eighteen patients with varying degree of disease activity were monitored longitudinally. Disease activity was assessed by the SLE disease activity index 2000 and the physician's global assessment. Organ damage was evaluated by the Systemic Lupus International Collaborating Clinics/American College of Rheumatology damage index (SDI). Compared with healthy control subjects, serum suPAR levels were elevated significantly in patients with SLE. No association was recorded regarding suPAR levels and SLE disease activity in cross-sectional or consecutive samples. However, a strong association was observed between suPAR and SDI (P < 0.0005). Considering distinct SDI domains, renal, neuropsychiatric, ocular, skin, and peripheral vascular damage had a significant effect on suPAR levels. This study is the first to demonstrate an association between serum suPAR and irreversible organ damage in SLE. Further studies are warranted to evaluate suPAR and other biomarkers as predictors of evolving organ damage.

  20. Tissue-type plasminogen activator triggers the synaptic vesicle cycle in cerebral cortical neurons

    PubMed Central

    Wu, Fang; Torre, Enrique; Cuellar-Giraldo, David; Cheng, Lihong; Yi, Hong; Bichler, Edyta K; García, Paul S; Yepes, Manuel

    2015-01-01

    The active zone (AZ) is a thickening of the presynaptic membrane where exocytosis takes place. Chemical synapses contain neurotransmitter-loaded synaptic vesicles (SVs) that at rest are tethered away from the synaptic release site, but after the presynaptic inflow of Ca+2 elicited by an action potential translocate to the AZ to release their neurotransmitter load. We report that tissue-type plasminogen activator (tPA) is stored outside the AZ of cerebral cortical neurons, either intermixed with small clear-core vesicles or in direct contact with the presynaptic membrane. We found that cerebral ischemia-induced release of neuronal tPA, or treatment with recombinant tPA, recruits the cytoskeletal protein βII-spectrin to the AZ and promotes the binding of SVs to βII-spectrin, enlarging the population of SVs in proximity to the synaptic release site. This effect does not require the generation of plasmin and is followed by the recruitment of voltage gated calcium channels (VGCC) to the presynaptic terminal that leads to Ca+2-dependent synapsin I phosphorylation, freeing SVs to translocate to the AZ to deliver their neurotransmitter load. Our studies indicate that tPA activates the SV cycle and induces the structural and functional changes in the synapse that are required for successful neurotransmission. PMID:26126868

  1. Tissue Plasminogen Activator Alters Intracellular Sequestration of Zinc through Interaction with the Transporter ZIP4

    SciTech Connect

    Emmetsberger, Jaime; Mirrione, Martine M.; Zhou, Chun; Fernandez-Monreal, Monica; Siddiq, Mustafa M.; Ji, Kyungmin; Tsirka, Stella E.

    2010-09-17

    Glutamatergic neurons contain free zinc packaged into neurotransmitter-loaded synaptic vesicles. Upon neuronal activation, the vesicular contents are released into the synaptic space, whereby the zinc modulates activity of postsynaptic neurons though interactions with receptors, transporters and exchangers. However, high extracellular concentrations of zinc trigger seizures and are neurotoxic if substantial amounts of zinc reenter the cells via ion channels and accumulate in the cytoplasm. Tissue plasminogen activator (tPA), a secreted serine protease, is also proepileptic and excitotoxic. However, tPA counters zinc toxicity by promoting zinc import back into the neurons in a sequestered form that is nontoxic. Here, we identify the zinc influx transporter, ZIP4, as the pathway through which tPA mediates the zinc uptake. We show that ZIP4 is upregulated after excitotoxin stimulation of the mouse, male and female, hippocampus. ZIP4 physically interacts with tPA, correlating with an increased intracellular zinc influx and lysosomal sequestration. Changes in prosurvival signals support the idea that this sequestration results in neuroprotection. These experiments identify a mechanism via which neurons use tPA to efficiently neutralize the toxic effects of excessive concentrations of free zinc.

  2. Human retinal pigment epithelial lysis of extracellular matrix: functional urokinase plasminogen activator receptor, collagenase, and elastase.

    PubMed Central

    Elner, Susan G

    2002-01-01

    PURPOSE: To show (1) human retinal pigment epithelial (HRPE) expression of functional urokinase plasminogen activator receptor (uPAR; CD87), (2) HRPE secretion of collagenase and elastase, (3) uPAR-dependent HRPE migration, and (4) uPAR expression in diseased human retinal tissue. METHODS: Immunohistochemistry for uPAR was performed on cultured HRPE cells and in sections of human retina. Double-immunofluorescent staining of live human RPE cells with anti-CR3 antibody (CD11b) was performed to demonstrate the physical proximity of this beta 2 integrin with uPAR and determine whether associations were dependent on RPE confluence and polarity. Extracellular proteolysis by HRPE uPAR was evaluated using fluorescent bodipy-BSA and assessed for specificity by plasminogen activator inhibitor-1 (PAI-1) inhibition. The effect of interleukin-1 beta (IL-1 beta) on uPAR expression was assessed. Collagenase and elastase secretion by unstimulated and IL-1-stimulated HRPE cells was measured by 3H-labelled collagen and elastin cleavage. HRPE-associated collagenase was also assessed by cleavage of fluorescent DQ-collagen and inhibited by phenanthroline. Using an extracellular matrix assay, the roles of uPAR and collagenase in HRPE migration were assessed. RESULTS: Immunoreactive uPAR was detected on cultured HRPE cells and increased by IL-1. On elongated, live HRPE cells, uPAR dissociated from CD11b (CR3) and translocated to anterior poles of migrating cells. Extracellular proteolysis was concentrated at sites of uPAR expression and specifically inhibited by PAI-1. Cultured HRPE cells secreted substantial, functional collagenase and elastase. IL-1 upregulated uPAR, collagenase, and elastase activities. Specific inhibition of uPAR, and to a lesser degree collagenase, reduced HRPE migration in matrix/gel assays. Immunoreactive uPAR was present along the HRPE basolateral membrane in retinal sections and in sections of diseased retinal tissue. CONCLUSIONS: HRPE cells express functional u

  3. Increased production of plasminogen activator inhibitor in vitro by pleural leukocytes from rats intratracheally instilled with crocidolite asbestos

    SciTech Connect

    Xiao Yang Li; Brown, G.M.; Donaldson, K. ); Lamb, D. )

    1991-08-01

    The authors have previously reported that normal pleural leukocytes secrete a urokinase-type plasminogen activator inhibitor (PAI) in culture. In view of the pathogenic effects of asbestos on the pleura, in particular pleural fibrosis, they have extended these observation to crocidolite asbestos-exposed rats. Pleural leukocytes from rats exposed to crocidolite asbestos were found to secrete more PAI in culture than controls. The activity of PAI in pleural leukocyte-conditioned medium increased in a dose-dependent manner in relation to the quantity of asbestos injected into the lung. However, with increasing time post asbestos instillation, there was no significant change in the secretion of PAI by pleural leukocytes in culture compared with earlier time points of crocidolite-exposed rats. Plasminogen activator was not detectable in the conditioned medium at any time point. The data derived from this study may help to elucidate the pathogenesis of some pleural disorders caused by exposure to fibrous dusts in the lungs.

  4. Transforming Growth Factor-Beta and Urokinase-Type Plasminogen Activator: Dangerous Partners in Tumorigenesis—Implications in Skin Cancer

    PubMed Central

    Santibanez, Juan F.

    2013-01-01

    Transforming growth factor-beta (TGF-β) is a pleiotropic factor, with several different roles in health and disease. TGF-β has been postulated as a dual factor in tumor progression, since it represses epithelial tumor development in early stages, whereas it stimulates tumor progression in advanced stages. During tumorigenesis, cancer cells acquire the capacity to migrate and invade surrounding tissues and to metastasize different organs. The urokinase-type plasminogen activator (uPA) system, comprising uPA, the uPA cell surface receptor, and plasminogen-plasmin, is involved in the proteolytic degradation of the extracellular matrix and regulates key cellular events by activating intracellular signal pathways, which together allow cancer cells to survive, thus, enhancing cell malignance during tumor progression. Due to their importance, uPA and its receptor are tightly transcriptionally regulated in normal development, but are deregulated in cancer, when their activity and expression are related to further development of cancer. TGF-β regulates uPA expression in cancer cells, while uPA, by plasminogen activation, may activate the secreted latent TGF-β, thus, producing a pernicious cycle which contributes to the enhancement of tumor progression. Here we review the specific roles and the interplay between TGF-β and uPA system in cancer cells and their implication in skin cancer. PMID:23984088

  5. Promotion of Wound Healing by an Agonist of Adenosine A2A Receptor Is Dependent on Tissue Plasminogen Activator.

    PubMed

    Montesinos, M Carmen; Desai-Merchant, Avani; Cronstein, Bruce N

    2015-12-01

    Impaired wound healing, as it occurs in diabetes mellitus or long-term corticoid treatment, is commonly associated with disability, diminished quality of life, and high economic costs. Selective agonists of the A2A receptor subtype of adenosine, an endogenous regulator of inflammation, promote tissue repair in animal models, both healthy and with impaired healing. Plasmin-mediated proteolysis of fibrin and other matrix proteins is essential for cell migration at sites of injury. Since adenosine A2A receptor activation increases plasminogen activator release from macrophages and mast cells, we studied the effect of a selective agonist, CGS-21680, on full-thickness excisional wound closure in wild-type, urokinase plasminogen activator (uPA)-deficient, and tissue plasminogen activator (tPA)-deficient mice. Wound closure was impaired in tPA- and uPA-deficient mice as compared with wild-type mice, and topical application of CGS-21680 significantly increased the rate at which wounds closed in wild-type mice and uPA-deficient mice, but not in tPA-deficient mice. Immunostaining of tissue sections showed that tPA was present in endothelial cells and histiocytes by day 3 post-wound and also by day 6. In contrast, uPA was more prominent in these cell types only by day 6 post-wound. Our results confirm that plasminogen activation contributes to wound repair and are consistent with the hypothesis that adenosine A2A receptor activation promotes wound closure by a mechanism that depends upon tPA, but not uPA. Moreover, our results suggest that topical adenosine A2A receptor agonists may be useful in promotion of wound closure in patients with impaired wound healing.

  6. A novel plasminogen activator from Agkistrodon blomhoffii Ussurensis venom (ABUSV-PA): Purification and characterization

    SciTech Connect

    Liu Shuqing; Sun Mingzhong . E-mail: ming-zhong.sun@case.edu; Greenaway, Frederick T.

    2006-10-06

    A plasminogen activator with arginine ester hydrolysis activity (ABUSV-PA) has been identified and purified to homogeneity from Chinese Agkistrodon blomhoffii Ussurensis snake venom. ABUSV-PA, a monomeric protein with molecular mass of 27815.2 Da, was purified 180-fold with 0.02% recovery for protein and 3.6% recovery for esterase activity. ABUSV-PA reacts optimally with its substrate N {sub {alpha}}-tosyl-L-arginine-methyl ester (TAME) at {approx}pH 7.5 and at 51 {sup o}C. Measurement from inductively coupled plasma-atomic emission spectroscopy (ICP-AES) reveals that ABUSV-PA is a Zn{sup 2+}-containing protein with a stoichiometry of 1:1 [Zn{sup 2+}]:[ABUSV-PA]. Analyses of esterase hydrolysis and UV absorption and CD spectra indicate that Zn{sup 2+} plays an important role in maintaining the structural integrity rather than the esterase activity of ABUSV-PA. Divalent metal ions, including Ca{sup 2+}, Mg{sup 2+}, Cu{sup 2+}, Ni{sup 2+}, Mn{sup 2+}, and Co{sup 2+}, increase the TAME hydrolysis activity of ABUSV-PA. A red-shift of the emission wavelengths of the synchronous fluorescence of ABUSV-PA, compared to those of free Tyr and Trp, indicates a conformation where the Tyr and Trp residues are in exposed hydrophilic environments. The presence of zinc increases the hydrophobicity of the conformational environments surrounding the Trp residues of ABUSV-PA and affects the secondary structure of ABUSV-PA, as proved by UV absorption and CD spectroscopy.

  7. Statins Increase Plasminogen Activator Inhibitor Type 1 Gene Transcription through a Pregnane X Receptor Regulated Element.

    PubMed

    Stanley, Frederick M; Linder, Kathryn M; Cardozo, Timothy J

    2015-01-01

    Plasminogen activator inhibitor type 1 (PAI-1) is a multifunctional protein that has important roles in inflammation and wound healing. Its aberrant regulation may contribute to many disease processes such as heart disease. The PAI-1 promoter is responsive to multiple inputs including cytokines, growth factors, steroids and oxidative stress. The statin drugs, atorvastatin, mevastatin and rosuvastatin, increased basal and stimulated expression of the PAI-1 promoter 3-fold. A statin-responsive, nuclear hormone response element was previously identified in the PAI-1 promoter, but it was incompletely characterized. We characterized this direct repeat (DR) of AGGTCA with a 3-nucleotide spacer at -269/-255 using deletion and directed mutagenesis. Deletion or mutation of this element increased basal transcription from the promoter suggesting that it repressed PAI-1 transcription in the unliganded state. The half-site spacing and the ligand specificity suggested that this might be a pregnane X receptor (PXR) responsive element. Computational molecular docking showed that atorvastatin, mevastatin and rosuvastatin were structurally compatible with the PXR ligand-binding pocket in its agonist conformation. Experiments with Gal4 DNA binding domain fusion proteins showed that Gal4-PXR was activated by statins while other DR + 3 binding nuclear receptor fusions were not. Overexpression of PXR further enhanced PAI-1 transcription in response to statins. Finally, ChIP experiments using Halo-tagged PXR and RXR demonstrated that both components of the PXR-RXR heterodimer bound to this region of the PAI-1 promoter.

  8. Plasminogen Activator Inhibitor-1 Is Involved in Streptozotocin-Induced Bone Loss in Female Mice

    PubMed Central

    Tamura, Yukinori; Kawao, Naoyuki; Okada, Kiyotaka; Yano, Masato; Okumoto, Katsumi; Matsuo, Osamu; Kaji, Hiroshi

    2013-01-01

    In diabetic patients, the risk of fracture is high because of impaired bone formation. However, the details of the mechanisms in the development of diabetic osteoporosis remain unclear. In the current study, we investigated the role of plasminogen activator inhibitor (PAI)-1 in the pathogenesis of type 1 diabetic osteoporosis by using PAI-1–deficient mice. Quantitative computed tomography analysis showed that PAI-1 deficiency protected against streptozotocin-induced bone loss in female mice but not in male mice. PAI-1 deficiency blunted the changes in the levels of Runx2, osterix, and alkaline phosphatase in tibia as well as serum osteocalcin levels suppressed by the diabetic state in female mice only. Furthermore, the osteoclast levels in tibia, suppressed in diabetes, were also blunted by PAI-1 deficiency in female mice. Streptozotocin markedly elevated the levels of PAI-1 mRNA in liver in female mice only. In vitro study demonstrated that treatment with active PAI-1 suppressed the levels of osteogenic genes and mineralization in primary osteoblasts from female mouse calvaria. In conclusion, the current study indicates that PAI-1 is involved in the pathogenesis of type 1 diabetic osteoporosis in females. The expression of PAI-1 in the liver and the sensitivity of bone cells to PAI-1 may be an underlying mechanism. PMID:23715621

  9. Role of plasminogen activator inhibitor-1 in glucocorticoid-induced diabetes and osteopenia in mice.

    PubMed

    Tamura, Yukinori; Kawao, Naoyuki; Yano, Masato; Okada, Kiyotaka; Okumoto, Katsumi; Chiba, Yasutaka; Matsuo, Osamu; Kaji, Hiroshi

    2015-06-01

    Long-term use of glucocorticoids (GCs) causes numerous adverse effects, including glucose/lipid abnormalities, osteoporosis, and muscle wasting. The pathogenic mechanism, however, is not completely understood. In this study, we used plasminogen activator inhibitor-1 (PAI-1)-deficient mice to explore the role of PAI-1 in GC-induced glucose/lipid abnormalities, osteoporosis, and muscle wasting. Corticosterone markedly increased the levels of circulating PAI-1 and the PAI-1 mRNA level in the white adipose tissue of wild-type mice. PAI-1 deficiency significantly reduced insulin resistance and glucose intolerance but not hyperlipidemia induced by GC. An in vitro experiment revealed that active PAI-1 treatment inhibits insulin-induced phosphorylation of Akt and glucose uptake in HepG2 hepatocytes. However, this was not observed in 3T3-L1 adipocytes and C2C12 myotubes, indicating that PAI-1 suppressed insulin signaling in hepatocytes. PAI-1 deficiency attenuated the GC-induced bone loss presumably via inhibition of apoptosis of osteoblasts. Moreover, the PAI-1 deficiency also protected from GC-induced muscle loss. In conclusion, the current study indicated that PAI-1 is involved in GC-induced glucose metabolism abnormality, osteopenia, and muscle wasting in mice. PAI-1 may be a novel therapeutic target to mitigate the adverse effects of GC.

  10. Mechanistic characterization and crystal structure of a small molecule inactivator bound to plasminogen activator inhibitor-1

    PubMed Central

    Li, Shih-Hon; Reinke, Ashley A.; Sanders, Karen L.; Emal, Cory D.; Whisstock, James C.; Stuckey, Jeanne A.; Lawrence, Daniel A.

    2013-01-01

    Plasminogen activator inhibitor type-1 (PAI-1) is a member of the serine protease inhibitor (serpin) family. Excessive PAI-1 activity is associated with human disease, making it an attractive pharmaceutical target. However, like other serpins, PAI-1 has a labile structure, making it a difficult target for the development of small molecule inhibitors, and to date, there are no US Food and Drug Administration–approved small molecule inactivators of any serpins. Here we describe the mechanistic and structural characterization of a high affinity inactivator of PAI-1. This molecule binds to PAI-1 reversibly and acts through an allosteric mechanism that inhibits PAI-1 binding to proteases and to its cofactor vitronectin. The binding site is identified by X-ray crystallography and mutagenesis as a pocket at the interface of β-sheets B and C and α-helix H. A similar pocket is present on other serpins, suggesting that this site could be a common target in this structurally conserved protein family. PMID:24297881

  11. Statins Increase Plasminogen Activator Inhibitor Type 1 Gene Transcription through a Pregnane X Receptor Regulated Element

    PubMed Central

    Stanley, Frederick M.; Linder, Kathryn M.; Cardozo, Timothy J.

    2015-01-01

    Plasminogen activator inhibitor type 1 (PAI-1) is a multifunctional protein that has important roles in inflammation and wound healing. Its aberrant regulation may contribute to many disease processes such as heart disease. The PAI-1 promoter is responsive to multiple inputs including cytokines, growth factors, steroids and oxidative stress. The statin drugs, atorvastatin, mevastatin and rosuvastatin, increased basal and stimulated expression of the PAI-1 promoter 3-fold. A statin-responsive, nuclear hormone response element was previously identified in the PAI-1 promoter, but it was incompletely characterized. We characterized this direct repeat (DR) of AGGTCA with a 3-nucleotide spacer at -269/-255 using deletion and directed mutagenesis. Deletion or mutation of this element increased basal transcription from the promoter suggesting that it repressed PAI-1 transcription in the unliganded state. The half-site spacing and the ligand specificity suggested that this might be a pregnane X receptor (PXR) responsive element. Computational molecular docking showed that atorvastatin, mevastatin and rosuvastatin were structurally compatible with the PXR ligand-binding pocket in its agonist conformation. Experiments with Gal4 DNA binding domain fusion proteins showed that Gal4-PXR was activated by statins while other DR + 3 binding nuclear receptor fusions were not. Overexpression of PXR further enhanced PAI-1 transcription in response to statins. Finally, ChIP experiments using Halo-tagged PXR and RXR demonstrated that both components of the PXR-RXR heterodimer bound to this region of the PAI-1 promoter. PMID:26379245

  12. Targeting of peptide conjugated magnetic nanoparticles to urokinase plasminogen activator receptor (uPAR) expressing cells

    NASA Astrophysics Data System (ADS)

    Hansen, Line; Unmack Larsen, Esben Kjær; Nielsen, Erik Holm; Iversen, Frank; Liu, Zhuo; Thomsen, Karen; Pedersen, Michael; Skrydstrup, Troels; Nielsen, Niels Chr.; Ploug, Michael; Kjems, Jørgen

    2013-08-01

    Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are currently being used as a magnetic resonance imaging (MRI) contrast agent in vivo, mainly by their passive accumulation in tissues of interest. However, a higher specificity can ideally be achieved when the nanoparticles are targeted towards cell specific receptors and this may also facilitate specific drug delivery by an enhanced target-mediated endocytosis. We report efficient peptide-mediated targeting of magnetic nanoparticles to cells expressing the urokinase plasminogen activator receptor (uPAR), a surface biomarker for poor patient prognosis shared by several cancers including breast, colorectal, and gastric cancers. Conjugation of a uPAR specific targeting peptide onto polyethylene glycol (PEG) coated USPIO nanoparticles by click chemistry resulted in a five times higher uptake in vitro in a uPAR positive cell line compared to nanoparticles carrying a non-binding control peptide. In accordance with specific receptor-mediated recognition, a low uptake was observed in the presence of an excess of ATF, a natural ligand for uPAR. The uPAR specific magnetic nanoparticles can potentially provide a useful supplement for tumor patient management when combined with MRI and drug delivery.Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are currently being used as a magnetic resonance imaging (MRI) contrast agent in vivo, mainly by their passive accumulation in tissues of interest. However, a higher specificity can ideally be achieved when the nanoparticles are targeted towards cell specific receptors and this may also facilitate specific drug delivery by an enhanced target-mediated endocytosis. We report efficient peptide-mediated targeting of magnetic nanoparticles to cells expressing the urokinase plasminogen activator receptor (uPAR), a surface biomarker for poor patient prognosis shared by several cancers including breast, colorectal, and gastric cancers. Conjugation of a uPAR specific

  13. Tissue-type plasminogen activator suppresses activated stellate cells through low-density lipoprotein receptor-related protein 1

    PubMed Central

    Kang, Liang-I; Isse, Kumiko; Koral, Kelly; Bowen, William C; Muratoglu, Selen; Strickland, Dudley K; Michalopoulos, George K; Mars, Wendy M

    2015-01-01

    Hepatic stellate cell (HSC) activation and trans-differentiation into myofibroblast (MFB)-like cells is key for fibrogenesis after liver injury and a potential therapeutic target. Recent studies demonstrated that low-density lipoprotein receptor-related protein 1 (LRP1)-dependent signaling by tissue-type plasminogen activator (t-PA) is a pro-fibrotic regulator of the MFB phenotype in kidney. This study investigated whether LRP1 signaling by t-PA is also relevant to HSC activation following injury. Primary and immortalized rat HSCs were treated with t-PA and assayed by western blot, MTT, and TUNEL. In vitro results were then verified using an in vivo, acute carbon tetrachloride (CCl4) injury model that examined the phenotype and recovery kinetics of MFBs from wild-type animals vs mice with a global (t-PA) or HSC-targeted (LRP1) deletion. In vitro, in contrast to kidney MFBs, exogenous, proteolytically inactive t-PA suppressed, rather than induced, activation markers in HSCs following phosphorylation of LRP1. This process was mediated by LRP1 as inhibition of t-PA binding to LRP1 blocked the effects of t-PA. In vivo, following acute injury, phosphorylation of LRP1 on activated HSCs occurred immediately prior to their disappearance. Mice lacking t-PA or LRP1 retained higher densities of activated HSCs for a longer time period compared with control mice after injury cessation. Hence, t-PA, an FDA-approved drug, contributes to the suppression of activated HSCs following injury repair via signaling through LRP1. This renders t-PA a potential target for exploitation in treating patients with fibrosis. PMID:26237273

  14. Effects of addition of tissue-type plasminogen activator in in vitro fertilization medium on bovine embryo development and quality.

    PubMed

    Krania, F; Dovolou, E; Rekkas, C A; Theodosiadou, E K; Pappas, I; Amiridis, G S

    2015-02-01

    Plasminogen activators/Plasmin system plays pivotal role in regulating reproductive functions of mammals. Here, we examined the effects of modification of in vitro fertilization medium (IVF medium) with the addition of tissue-type plasminogen activator (t-PA), on bovine embryo development and quality, assessed by quantification of expression of various genes related to metabolism, oxidation, implantation and apoptosis. In addition, plasminogen activator activity (PAA) and plasminogen activator inhibition (PAI) were measured in the spent media. After conventional IVM, 2016 cumulus-oocyte complexes (COCs) were divided into four groups with modified composition of the IVF medium containing t-PA and/or its inhibitor epsilon-aminocaproic acid (control, t-PA, t-PA+ε-ACA, ε-ACA). Presumptive zygotes were cultured for 8 days in synthetic oviductal fluid (SOF) medium; gene expression studies were carried out on morulae and blastocysts. t-PA alone significantly suppressed cleavage and blastocyst formation rates, but this effect was neutralized by the addition of ε-ACA. PAA in the treated group was significantly reduced by ε-ACA, but without total elimination. Significant differences were detected in the expression of genes related to apoptosis and/or cell cycle arrest (BAX, BCL2L1, KAT2B) between embryos produced in t-PA-modified media and controls, giving an overall notion that the inferior developmental competence of treated embryos may be attributed to apoptotic phenomena induced by t-PA. In conclusion, it appears that excessive t-PA content in the IVF media, suppresses blastocyst formation rate, possibly due to induction of apoptotic phenomena.

  15. A collaborative study to establish the 3rd International Standard for tissue plasminogen activator.

    PubMed

    Sands, Dawn; Whitton, Colin M; Merton, R Elizabeth; Longstaff, Colin

    2002-08-01

    An international collaborative study was organised to replace the 2nd International Standard (IS) for tissue plasminogen activator (tPA). The 2nd IS for tPA (86/670) was used to calibrate the replacement Standard, which was selected from two candidate materials included in the collaborative study. Participants were provided with five sets of four samples (A, B, C, D) and asked to use sample A (2nd IS, 86/670, 850 IU/ml) to determine the activity of B (86/624, approximately 850 IU/ml), C and D (coded duplicates of the same material, 98/714 approximately 11,000 IU/ml). A total of 14 laboratories returned results from Europe, USA, Japan and Australia, providing data from 60 independent assays. Four laboratories used a reference method based on a published monograph from the European Pharmacopoeia for Alteplase for Injection, 1998, and the remaining 10 used their own method. Fibrin was used as promoter of tPA activity by 12 out of the 14 laboratories, the remaining two used kits where fibrinogen fragments were the promoter. Data from this collaborative study and the previous study to establish the 2nd IS for tPA show that tPA from melanoma cells and recombinant tPA from CHO cells are both suitable materials as International Standards. It was agreed that sample C, D, recombinant tPA, 98/714, be established as the 3rd International Standard for tPA with a potency of 10,000 IU per ampoule, calculated as the mean value from laboratories using fibrin as a promoter of tPA activity. The standard was established by WHO in November 2000.

  16. Association of Protein S Deficiency with Thrombosis in a Kindred with Increased Levels of Plasminogen Activator Inhibitor-1

    DTIC Science & Technology

    1993-10-15

    family with assay. Clin Chim Acts. 1983;127:279-88. hereditary thrombophilia . Blood. 1989;73:479-83. 22. Griffn JH, Gruber A, Fernandez JA. Reevaluation of...SMe E. Elevated plasminogen 25 Boiseol C, David H. Quantitative determination of serum triglycer- activator inhibitor (PAl), a cause of thrombophilia ...A study in 203 ides by the use of enzymes. Cliii Chem. 1973;19:476-82. patients with familial or sporadic venous thrombophilia . Thromb 26. Remnilgton

  17. Enablers of the Implementation of Tissue Plasminogen Activator in Acute Stroke Care: A Cross-Sectional Survey

    PubMed Central

    Grady, Alice; Bryant, Jamie; Carey, Mariko; Paul, Chris; Sanson-Fisher, Rob

    2014-01-01

    Objective To assess emergency physicians’ perceptions of individual and system enablers to the use of tissue Plasminogen Activator in acute stroke. Method Australian fellows and trainees of Australasian College for Emergency Medicine completed a 57-item online survey assessing enablers to implementation of evidence-based practice across six domains: knowledge, skills, modelling, monitoring, feedback, and maintenance. Demographic and workplace characteristics were obtained. Descriptive statistics were calculated to describe demographic and workplace characteristics of responders, and survey responses. Each domain received an overall score (%) based on the number of responders agreeing with all items within the domain. Results A total of 429 (13%) Australasian College for Emergency Medicine members responded. 17.7% of respondents reported they and/or their workplace met all knowledge-related enablers, however only 2.3% had all skill-related enablers in place. Of respondents who decide which patients receive tissue Plasminogen Activator treatment, 18.1% agreed that all maintenance-related enablers are in place at their hospital, compared to 6.6% for those who do not decide which patients receive tissue Plasminogen Activator treatment. None of the respondents had all items in place cross all domains. Conclusions Even when allowing for the low response rate, it seems likely there is a lack of individual and system enablers supporting the implementation of best-practice stroke care in a number of Australian hospitals. Quality improvement programs could target all domains, particularly the skills-training and feedback emergency physicians receive, to aid implementation of tissue Plasminogen Activator treatment for acute stroke. PMID:25490546

  18. Plasminogen Activator Inhibitor-1 in depression: Results from Animal and Clinical Studies

    PubMed Central

    Jiang, Haitang; Li, Xiaoli; Chen, Suzhen; Lu, Na; Yue, Yingying; Liang, Jinfeng; Zhang, Zhijun; Yuan, Yonggui

    2016-01-01

    Evidence suggests that plasminogen activator inhibitor-1 (PAI-1) is a stress-related factor, and serum PAI-1 levels are increased in patients with major depressive disorders (MDD). Herein, we analysed PAI-1 protein levels in the brain, cerebrospinal fluid (CSF) and serum of rodents exposed to chronic unpredictable mild stress or treated with escitalopram. In addition, we examined PAI-1 concentrations in serum obtained from 17 drug-free depressed patients before and after escitalopram treatment. We found that PAI-1 expression was increased in area 1 of the cingulate cortex and prelimbic cortex of the medial prefrontal cortex as well as in the hippocampal cornu ammonis 3 and dentate gyrus in stressed rats. A downregulation of PAI-1 following chronic escitalopram treatment was also found. PAI-1 levels were higher in the CSF and serum in stressed rats than in controls, although the difference did not reach statistical significance in the serum. Escitalopram treatment significantly decreased PAI-1 levels in the serum, but not in the CSF. MDD patients had significantly greater serum PAI-1 concentrations than controls. Our results suggest that PAI-1 is implicated in the pathophysiology of depression. PMID:27456456

  19. Plasminogen activator inhibitor I 4G/5G polymorphism in neonatal respiratory distress syndrome.

    PubMed

    Armangil, Didem; Yurdakök, Murat; Okur, Hamza; Gürgey, Aytemiz

    2011-08-01

    Fibrin monomers inhibit surfactant function. 4G/5G insertion/deletion polymorphism plays an important role in the regulation of plasminogen activator inhibitor 1 (PAI-1) gene expression. To examine the genotype distribution of PAI-1 polymorphism in 60 infants with respiratory distress syndrome (RDS) and 53 controls, an allele-specific polymerase chain reaction (PCR) was used. The proportion of 4G/4G, 4G/5G, and 5G/5G genotypes did not differ statistically between the RDS and control groups (P > .05). Having PAI-1 4G/4G genotype polymorphism appears to increase the risk of RDS (odds ratio [OR] =1.5; 95% confidence interval [CI], 0.5-4.3), although it was not statistically significant. No relation was found between the PAI-1 4G/5G polymorphisms and RDS, but there was an increased risk associated with the 4G variant of the PAI-1 gene. We believe that our findings of increased 4G allele of the PAI-1 gene in infants with RDS would also help to clarify the pathogenesis of RDS.

  20. Early intracardiac thrombosis in preterm infants and thrombolysis with recombinant tissue type plasminogen activator

    PubMed Central

    Ferrari, F; Vagnarelli, F; Gargano, G; Roversi, M; Biagioni, O; Ranzi, A; Cavazzuti, G

    2001-01-01

    OBJECTIVES—To determine the incidence of catheter related thrombosis and to test the efficacy of recombinant tissue type plasminogen activator (rt-PA) in preterm infants.
STUDY DESIGN—From January 1995 to December 1998, echocardiography was performed in the first few days of life in 76 very low birthweight (⩽ 1500 g) infants out of a total of 147 having an umbilical catheter placed. When intracardiac thrombosis was diagnosed, rt-PA infusion was performed.
RESULTS—Four infants (5%) developed an intracardiac thrombosis during the first few days of life. In three of them, rt-PA at a dose of 0.4-0.5 mg/kg in a 20-30 minute bolus led to dissolution of the clot. One patient received a three hour infusion after the bolus, at a dose of 0.1 mg/kg/h, with resolution of the thrombus. No systemic effects were observed after rt-PA infusion.
CONCLUSIONS—Early thrombosis may occur as a complication of umbilical catheterisation in preterm infants; early echocardiographic detection of this disorder allows complete, safe, and rapid lysis with rt-PA.

 PMID:11420328

  1. Impacts of tissue-type plasminogen activator (tPA) on neuronal survival

    PubMed Central

    Chevilley, Arnaud; Lesept, Flavie; Lenoir, Sophie; Ali, Carine; Parcq, Jérôme; Vivien, Denis

    2015-01-01

    Tissue-type plasminogen activator (tPA) a serine protease is constituted of five functional domains through which it interacts with different substrates, binding proteins, and receptors. In the last years, great interest has been given to the clinical relevance of targeting tPA in different diseases of the central nervous system, in particular stroke. Among its reported functions in the central nervous system, tPA displays both neurotrophic and neurotoxic effects. How can the protease mediate such opposite functions remain unclear but several hypotheses have been proposed. These include an influence of the degree of maturity and/or the type of neurons, of the level of tPA, of its origin (endogenous or exogenous) or of its form (single chain tPA versus two chain tPA). In this review, we will provide a synthetic snapshot of our current knowledge regarding the natural history of tPA and discuss how it sustains its pleiotropic functions with focus on excitotoxic/ischemic neuronal death and neuronal survival. PMID:26528141

  2. Induction of plasminogen activator by UV light in normal and xeroderma pigmentosum fibroblasts

    SciTech Connect

    Miskin, R.; Ben-Ishai, R.

    1981-10-01

    Normal and DNA repair-deficient human fibroblasts have been used to study induction of plasminogen activator (PA) by DNA damage. UV light induced the synthesis of PA in skin fibroblasts of all types of xeroderma pigmentosum (XP) in XP heterozygotes and in human amniotic cells. Enzyme induction was, however, not observed in fibroblasts of normal adults. In classical XP, which are deficient in excision repair, PA synthesis occurred in a narrow range of low-UV fluences. In such strains, the level of enzyme produced was correlated with the extent of repair deficiency. UV fluences required for PA induction in XP variants and XP heteozygotes were at least 10 times those inducing enzyme synthesis in excision-deficient XP. Maximum enzyme induction occurred 48 hr after irradiation, and the highest levels of enzyme produced were 15-20 times those of PA baseline levels. Electrophoretic analysis showed that UV irradiation enhances the synthesis of the M/sub r/ 60,000 human urokinase-type PA, which is present in low amounts in untreated cells. Our results suggest that PA induction in human cells is caused by unrepaired DNA damage and represents a eukaryotic SOS-like function. In addition, PA induction may provide a sensitive assay for detection of cellular DNA repair deficiencies and identification of XP heterozygotes.

  3. ADAM9 promotes lung cancer metastases to brain by a plasminogen activator-based pathway.

    PubMed

    Lin, Chen-Yuan; Chen, Hung-Jen; Huang, Cheng-Chung; Lai, Liang-Chuan; Lu, Tzu-Pin; Tseng, Guan-Chin; Kuo, Ting-Ting; Kuok, Qian-Yu; Hsu, Jennifer L; Sung, Shian-Ying; Hung, Mien-Chie; Sher, Yuh-Pyng

    2014-09-15

    The transmembrane cell adhesion protein ADAM9 has been implicated in cancer cell migration and lung cancer metastasis to the brain, but the underpinning mechanisms are unclear and clinical support has been lacking. Here, we demonstrate that ADAM9 enhances the ability of tissue plasminogen activator (tPA) to cleave and stimulate the function of the promigratory protein CDCP1 to promote lung metastasis. Blocking this mechanism of cancer cell migration prolonged survival in tumor-bearing mice and cooperated with dexamethasone and dasatinib (a dual Src/Abl kinase inhibitor) treatment to enhance cytotoxic treatment. In clinical specimens, high levels of ADAM9 and CDCP1 correlated with poor prognosis and high risk of mortality in patients with lung cancer. Moreover, ADAM9 levels in brain metastases derived from lung tumors were relatively higher than the levels observed in primary lung tumors. Our results show how ADAM9 regulates lung cancer metastasis to the brain by facilitating the tPA-mediated cleavage of CDCP1, with potential implications to target this network as a strategy to prevent or treat brain metastatic disease.

  4. Hypothermia Increases Tissue Plasminogen Activator Expression and Decreases Post-Operative Intra-Abdominal Adhesion

    PubMed Central

    Lee, Chien-Chang; Wang, Hsuan-Mao; Chou, Tzung-Hsin; Wu, Meng-Che; Hsueh, Kuang-Lung; Chen, Shyr-Chyr

    2016-01-01

    Background Therapeutic hypothermia during operation decreases postoperative intra-abdominal adhesion formation. We sought to determine the most appropriate duration of hypothermia, and whether hypothermia affects the expression of tissue plasminogen activator (tPA). Methods 80 male BALB/c mice weighing 25–30 g are randomized into one of five groups: adhesion model with infusion of 15°C saline for 15 minutes (A); 30 minutes (B); 45 minute (C); adhesion model without infusion of cold saline (D); and sham operation without infusion of cold saline (E). Adhesion scores and tPA levels in the peritoneum fluid levels were analyzed on postoperative days 1, 7, and 14. Results On day 14, the cold saline infusion groups (A, B, and C) had lower adhesion scores than the without infusion of cold saline group (D). However, only group B (cold saline infusion for 30 minutes) had a significantly lower adhesion scores than group D. Also, group B was found to have 3.4 fold, 2.3 fold, and 2.2 fold higher levels of tPA than group D on days 1, 7, and 14 respectively. Conclusions Our results suggest that cold saline infusion for 30 minutes was the optimum duration to decrease postoperative intra-abdominal adhesion formation. The decrease in the adhesion formations could be partly due to an increase in the level of tPA. PMID:27583464

  5. Elevation of serum urokinase plasminogen activator receptor and liver stiffness in postoperative biliary atresia

    PubMed Central

    Udomsinprasert, Wanvisa; Honsawek, Sittisak; Jirathanathornnukul, Napaphat; Chongsrisawat, Voranush; Poovorawan, Yong

    2016-01-01

    AIM To investigate serum urokinase-type plasminogen activator receptor (uPAR) and liver stiffness in biliary atresia (BA) and examine the correlation of circulating uPAR, liver stiffness, and clinical outcomes in postoperative BA children. METHODS Eighty-five postKasai BA children and 24 control subjects were registered. Circulating uPAR was measured using enzyme-linked immunosorbent essay. Liver stiffness was analyzed using transient elastography. RESULTS BA children had significantly greater circulating uPAR and liver stiffness scores than control subjects (P < 0.001). Circulating uPAR and liver stiffness were substantially higher in jaundiced BA children than non-jaundiced BA children (P < 0.001). In addition, circulating uPAR was positively associated with serum aspartate aminotransferase (r = 0.507, P < 0.001), alanine aminotransferase (r = 0.364, P < 0.001), total bilirubin (r = 0.559, P < 0.001), alkaline phosphatase (r = 0.325, P < 0.001), and liver stiffness scores (r = 0.508, P < 0.001). CONCLUSION Circulating uPAR and liver stiffness values were greater in BA children than healthy controls. The increased circulating uPAR was associated with liver dysfunction in BA. As a consequence, serum uPAR and liver stiffness may be used as noninvasive biomarkers indicating the progression of liver fibrosis in postKasai BA. PMID:27957246

  6. Prospective, longitudinal study of plastic bronchitis cast pathology and responsiveness to tissue plasminogen activator.

    PubMed

    Heath, Lauren; Ling, Shelley; Racz, Jennifer; Mane, Gerta; Schmidt, Lindsay; Myers, Jeffrey L; Tsai, Wan C; Caruthers, Regine L; Hirsch, Jennifer C; Stringer, Kathleen A

    2011-12-01

    Plastic bronchitis (PB) is a rare disease that often occurs in patients with congenital heart disease (CHD) who have undergone staged single-ventricle palliation. It is characterized by the formation of rubbery "casts" in the airways. PB treatment frequently includes inhaled tissue plasminogen activator (tPA). However, the efficacy of tPA to reduce cast burden is unknown. This is further complicated by our lack of knowledge of cast composition. We obtained spontaneously expectorated PB casts from children (n = 4) with CHD and one adult patient with idiopathic PB. Pathological assessment was made from paraffin-preserved samples. Casts were treated with phosphate-buffered saline (PBS) or tPA. Cast response to tPA was assessed by changes in cast weight and the production of fibrin D-dimer. Independent of dose, tPA reduced cast weight compared with PBS-treatment (P = 0.001) and increased D-dimer levels. Histological staining showed that PB casts from all patients were composed of fibrin and contained notable numbers of lymphocytes. Cast composition did not change over time. Collectively, these data support that in our PB patients, casts are composed of fibrin and are responsive to tPA treatment. This makes inhaled tPA a potentially viable option for symptomatic relief of PB while we work to unravel the complexity of PB pathogenesis.

  7. Management of plastic bronchitis with topical tissue-type plasminogen activator.

    PubMed

    Gibb, Elizabeth; Blount, Robert; Lewis, Nancy; Nielson, Dennis; Church, Gwynne; Jones, Kirk; Ly, Ngoc

    2012-08-01

    Plastic bronchitis or cast bronchitis is a rare disease of unclear etiology characterized by formation of airway casts that can lead to life-threatening airway obstruction. There is currently limited data regarding optimal treatment of plastic bronchitis. Several therapies have been suggested, but recurrences are common and mortality remains high. We report the case of a 6-year-old boy with refractory eosinophilic bronchial casts, unresponsive to low-dose systemic corticosteroids, inhaled corticosteroids, azithromycin, and dornase alfa, who was treated successfully and safely with direct instillation of tissue-type plasminogen activator (tPA) to the obstructing casts during flexible bronchoscopy and inhaled tPA. Our case illustrates that the current therapy for plastic bronchitis remains inadequate. To our knowledge, this case is the first to show that direct instillation of tPA can be used safely for treatment of this disease. The use of tPA via direct administration into the airways during bronchoscopy and via a nebulizer appeared to be a safe and effective therapy for plastic bronchitis and should be considered early in the course of the disease to prevent complications of severe airway obstruction.

  8. Urokinase-type plasminogen activator: a new target for male contraception?

    PubMed Central

    Qin, Ying; Han, Yan; Xiong, Cheng-Liang; Li, Hong-Gang; Hu, Lian; Zhang, Ling

    2015-01-01

    Urokinase-type plasminogen activator (uPA) is closely related to male reproduction. With the aim of investigating the possibility for uPA as a potential contraceptive target, in the present work, Kunming male mice were immunized by human uPA subcutaneous injection at three separate doses for 3 times. Then the potency of the anti-human uPA antibody in serum was analyzed, and mouse fertility was evaluated. Serum antibody titers for human uPA in immunized groups all reached 1:10,240 or higher levels by enzyme linked immunosorbent assay, and mating experiments revealed that pregnancy rates and the mean number of embryos implanted after mating declined obviously (P < 0.05) when compared with control groups. However, the mating capacity and reproductive organ weights had no obvious change, and histological analysis of the testes and epididymides also showed normal morphology for immunized male mice. Sperm function tests suggested that the sperm concentration, sperm viability, sperm motility, and in vitro fertilization rate for the cauda epididymis sperm in uPA-immunized groups were lower than those in the controls (P < 0.05). Together, these observations indicated that subcutaneous injection human uPA to the male mice could effectively reduce their fertility, and uPA could become a new target for immunocontraception in male contraceptive development. PMID:25578931

  9. Plasminogen activator inhibitor-1 gene-deficient mice. II. Effects on hemostasis, thrombosis, and thrombolysis.

    PubMed Central

    Carmeliet, P; Stassen, J M; Schoonjans, L; Ream, B; van den Oord, J J; De Mol, M; Mulligan, R C; Collen, D

    1993-01-01

    The effects of plasminogen activator inhibitor-1 (PAI-1) gene inactivation on hemostasis, thrombosis and thrombolysis were studied in homozygous PAI-1-deficient (PAI-1-/-) mice, generated by homologous recombination in D3 embryonic stem cells. Diluted (10-fold) whole blood clots from PAI-1-/- and from PAI-1 wild type (PAI-1+/+) mice underwent limited but significantly different (P < 0.001) spontaneous lysis within 3 h (6 +/- 1 vs 3 +/- 1%, respectively). A 25-microliters 125I-fibrin-labeled normal murine plasma clot, injected into a jugular vein, was lysed for 47 +/- 5, 66 +/- 3, and 87 +/- 7% within 8 h in PAI-1+/+, heterozygous PAI-1-deficient (PAI-1+/-), and PAI-1-/- mice, respectively (P = 0.002 for PAI-1+/+ vs PAI-1-/- mice). Corresponding values after pretreatment with 0.5 mg/kg endotoxin in PAI-1+/+ and PAI-1-/- mice, were 35 +/- 5 and 91 +/- 3% within 4 h, respectively (P < 0.001). 11 out of 26 PAI-1+/+ but only 1 out of 25 PAI-1-/- mice developed venous thrombosis (P = 0.004) within 6 d after injection of 10 or 50 micrograms endotoxin in the footpad. Spontaneous bleeding or delayed rebleeding could not be documented in PAI-1-/- mice after partial amputation of the tail or of the caecum. Thus, disruption of the PAI-1 gene in mice appears to induce a mild hyperfibrinolytic state and a greater resistance to venous thrombosis but not to impair hemostasis. Images PMID:8254029

  10. Is plasminogen activator inhibitor-1 a physiological bottleneck bridging major depressive disorder and cardiovascular disease?

    PubMed

    Savoy, C; Van Lieshout, R J; Steiner, M

    2016-06-01

    Major depressive disorder (MDD) is estimated to affect one in twenty people worldwide. MDD is highly comorbid with cardiovascular disease (CVD), itself one of the single largest causes of mortality worldwide. A number of pathological changes observed in MDD are believed to contribute to the development of cardiovascular disease, although no single mechanism has been identified. There are also no biological markers capable of predicting the future risk of developing heart disease in depressed individuals. Plasminogen activator inhibitor-1 (PAI-1) is a prothrombotic plasma protein secreted by endothelial tissue and has long been implicated in CVD. An expanding body of literature has recently implicated it in the pathogenesis of major depressive disorder as well. In this study, we review candidate pathways implicating MDD in CVD and consider how PAI-1 might act as a mediator by which MDD induces CVD development: chiefly through sleep disruption, adiposity, brain-derived neurotrophic factor (BDNF) metabolism, systemic inflammation and hypothalamic-pituitary-adrenal (HPA)-axis dysregulation. As both MDD and CVD are more prevalent in women than in men, and incidence of either condition is dramatically increased during reproductive milestones, we also explore hormonal and sex-specific associations between MDD, PAI-1 and CVD. Of special interest is the role PAI-1 plays in perinatal depression and in cardiovascular complications of pregnancy. Finally, we propose a theoretical model whereby PAI-1 might serve as a useful biomarker for CVD risk in those with depression, and as a potential target for future treatments.

  11. Systemic thrombolysis with recombinant tissue plasminogen activator in acute ischemic stroke: first Croatian experiences.

    PubMed

    Matijević, Vesna; Alvir, Domagoj; Malojčić, Branko; Unušić, Lea; Supe, Svjetlana; Boban, Marina; Bujan-Kovač, Andrea; Habek, Mario; Poljaković, Zdravka

    2010-12-01

    In September 2003, recombinant tissue plasminogen activator (rt-PA) for acute treatment of ischemic stroke was finally approved by the Croatian Ministry of Health. For the next 5 years, only three stroke units in the country implemented this therapy in their routine practice until summer 2008, when neurological wards in most Croatian hospitals started to treat acute stroke patients with systemic thrombolysis. We present a 2-year experience of thrombolytic therapy (2006-2008) in the stroke unit of the University Hospital in Zagreb, Croatian largest hospital, serving nearly one-fifth of the citizens of Croatia. Obtained data (vitals at admission and before administration of rt-PA; NIHSS and MRS scores at admission, 2 h and 7th day after rt-PA treatment, "time to door" and "door to needle" intervals, duration of hospital treatment as well as outcomes and complications of our 66 thrombolysed patients) are presented and discussed. We also present our results regarding benefits of this therapy as well as possible reasons for complications noticed.

  12. Successful treatment of mobile right atrial thrombus and acute pulmonary embolism with intravenous tissue plasminogen activator

    PubMed Central

    Bajaj, R; Ramanakumar, Ajay; Mamidala, Suresh; Kumar, Deepti

    2013-01-01

    An 89-year-old woman came with symptoms of progressively worsening dyspnoea at rest over the preceding week. She was normotensive, had elevated jugular venous pressure and clear lungs. ECG revealed atrial fibrillation with the rapid ventricular rate. Labs were significant for markedly elevated pro-brain natriuretic peptide of 43 000 pg/mL and troponin-T of 1 ng/mL. An urgent 2D echocardiogram was obtained, which revealed the severely dilated right atrium and a large linear mobile mass in the right atrium consistent with a thrombus. An emergent CT scan revealed multiple bilateral pulmonary emboli. She received intravenous tissue plasminogen activator. Repeat echocardiogram performed 6 h later showed no evidence of the right atrial thrombus. She was subsequently maintained on intravenous heparin and transitioned to Coumadin. Early recognition of this rare but potentially fatal complication is important as prompt treatment measures can help in preventing life-threatening complications of the right atrial thrombus. PMID:23892824

  13. Thrombolysis with intravenous recombinant tissue plasminogen activator during early postpartum period: a review of the literature.

    PubMed

    Akazawa, Munetoshi; Nishida, Makoto

    2017-02-21

    Thromboembolic events are one of the leading causes of maternal death during the postpartum period. Postpartum thrombolytic therapy with recombinant tissue plasminogen activator (rt-PA) is controversial because the treatment may lead to massive bleeding. Data centralization may be beneficial for analyzing the safety and effectiveness of systemic thrombolysis during the early postpartum period. We performed a computerized MEDLINE and EMBASE search. We collected data for 13 cases of systemic thrombolytic therapy during the early postpartum period, when limiting the early postpartum period to 48 hours after delivery. Blood transfusion was necessary in all cases except for one (12/13; 92%). In seven cases (7/13; 54%), a large amount of blood was required for transfusion. Subsequent laparotomy to control bleeding was required in five cases (5/13; 38%), including three cases of hysterectomy and two cases of hematoma removal, all of which involved cesarean delivery. In cases of transvaginal delivery, there was no report of laparotomy. The occurrence of severe bleeding was high in relation to cesarean section, compared with vaginal deliveries. Using rt-PA in relation to cesarean section might be worth avoiding. However, the paucity of data in the literature makes it difficult to assess the ultimate outcomes and safety of this treatment.

  14. Neurotoxic effects of exogenous recombinant tissue-type plasminogen activator on the normal rat brain.

    PubMed

    Goto, Hisaharu; Fujisawa, Hirosuke; Oka, Fumiaki; Nomura, Sadahiro; Kajiwara, Koji; Kato, Shoichi; Fujii, Masami; Maekawa, Tsuyoshi; Suzuki, Michiyasu

    2007-04-01

    Thrombolytic therapy with intravenous and intra-arterial recombinant tissue-type plasminogen activator (rtPA) has been established for the treatment of acute ischemic stroke. However, tPA has also been suggested to have neurotoxic effects. The purpose of this study was to examine direct neurotoxicity of rtPA in vivo. The animals (Wistar rats) were divided to the following three groups: low-dose (15 micromol/L) rtPA group (n = 6); high-dose (30 micromol/L) rtPA group (n = 6); and control (physiological saline) group (n = 6). The rtPA solution was perfused into the cortex via a microdialysis probe. The volume of the lesion was quantified histologically by image analysis of the lesions. Blood-brain barrier (BBB) disruption was evaluated by intravenous injection of Evans blue, and injury to the basal lamina was evaluated by immunohistochemistry using an anti-laminin antibody. In the rtPA-perfused animals, a pale lesion was produced around the probe, and microscopically, neurons showed necrotic changes. The volume of the lesions increased significantly as the concentration of perfused rtPA was increased. Marked extravasation of Evans blue was observed, and laminin immunoreactivity of blood vessels in the rtPA-induced lesions was lost. These results suggest that rtPA promotes acute direct neurotoxicity and participates in disruption of the microvascular basal lamina to cause BBB disruption, thereby increasing edema formation.

  15. Plasminogen activator inhibitor (PAI)-1 suppresses inhibition of gastric emptying by cholecystokinin (CCK) in mice.

    PubMed

    Gamble, Joanne; Kenny, Susan; Dockray, Graham J

    2013-08-10

    The intestinal hormone cholecystokinin (CCK) delays gastric emptying and inhibits food intake by actions on vagal afferent neurons. Recent studies suggest plasminogen activator inhibitor (PAI)-1 suppresses the effect of CCK on food intake. In this study we asked whether PAI-1 also modulated CCK effects on gastric emptying. Five minute gastric emptying of liquid test meals was studied in conscious wild type mice (C57BL/6) and in transgenic mice over-expressing PAI-1 in gastric parietal cells (PAI-1H/Kβ mice), or null for PAI-1. The effects of exogenous PAI-1 and CCK8s on gastric emptying were studied after ip administration. Intragastric peptone delayed gastric emptying in C57BL/6 mice by a mechanism sensitive to the CCK-1 receptor antagonist lorglumide. Peptone did not delay gastric emptying in PAI-1-H/Kβ mice. Exogenous CCK delayed gastric emptying of a control test meal in C57BL/6 mice and this was attenuated by administration of PAI-1; exogenous CCK had no effect on emptying in PAI-1-H/Kβ mice. Prior administration of gastrin to increase gastric PAI-1 inhibited CCK-dependent effects on gastric emptying in C57BL/6 mice but not in PAI-1 null mice. Thus, both endogenous and exogenous PAI-1 inhibit the effects of CCK (whether exogenous or endogenous) on gastric emptying. The data are compatible with emerging evidence that gastric PAI-1 modulates vagal effects of CCK.

  16. Preparation of thermosensitive magnetic liposome encapsulated recombinant tissue plasminogen activator for targeted thrombolysis

    NASA Astrophysics Data System (ADS)

    Hsu, Hao-Lung; Chen, Jyh-Ping

    2017-04-01

    Recombinant tissue plasminogen activator (rtPA) was encapsulated in thermosensitive magnetic liposome (TML) prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, distearolyphosphatidyl ethanolamine-N-poly(ethylene glycol) 2000, cholesterol and Fe3O4 magnetic nanoparticles by solvent evaporation/sonication and freeze-thaw cycles method. Response surface methodology was proved to be a powerful tool to predict the drug encapsulation efficiency and temperature-sensitive drug release. Validation experiments verified the accuracy of the model that provides a simple and effective method for fabricating TML with controllable encapsulation efficiency and predictable temperature-sensitive drug release behavior. The prepared samples were characterized for physico-chemical properties by dynamic light scattering, transmission electron microscopy, X-ray diffraction and differential scanning calorimetry. Temperature-sensitive release of rtPA could be confirmed from in vitro thrombolysis experiments. A thrombolytic drug delivery system using TML could be proposed for magnetic targeted delivery of rtPA to the site of thrombus followed by temperature-triggered controlled drug release in an alternating magnetic field.

  17. Effect of Regulatory Element DNA Methylation on Tissue-Type Plasminogen Activator Gene Expression

    PubMed Central

    Rivier-Cordey, Anne-Sophie; Caetano, Carlos; Fish, Richard J.; Kruithof, Egbert K. O.

    2016-01-01

    Expression of the tissue-type plasminogen activator gene (t-PA; gene name PLAT) is regulated, in part, by epigenetic mechanisms. We investigated the relationship between PLAT methylation and PLAT expression in five primary human cell types and six transformed cell lines. CpG methylation was analyzed in the proximal PLAT gene promoter and near the multihormone responsive enhancer (MHRE) -7.3 kilobase pairs upstream of the PLAT transcriptional start site (TSS, -7.3 kb). In Bowes melanoma cells, the PLAT promoter and the MHRE were fully unmethylated and t-PA secretion was extremely high. In other cell types the region from -647 to -366 was fully methylated, whereas an unmethylated stretch of DNA from -121 to +94 was required but not sufficient for detectable t-PA mRNA and t-PA secretion. DNA methylation near the MHRE was not correlated with t-PA secretion. Specific methylation of the PLAT promoter region -151 to +151, inserted into a firefly luciferase reporter gene, abolished reporter gene activity. The region -121 to + 94 contains two well-described regulatory elements, a PMA-responsive element (CRE) near -106 and a GC-rich region containing an Sp1 binding site near +59. Methylation of double-stranded DNA oligonucleotides containing the CRE or the GC-rich region had little or no effect on transcription factor binding. Methylated CpGs may attract co-repressor complexes that contain histone deacetylases (HDAC). However, reporter gene activity of methylated plasmids was not restored by the HDAC inhibitor trichostatin. In conclusion, efficient PLAT gene expression requires a short stretch of unmethylated CpG sites in the proximal promoter. PMID:27973546

  18. Wood Bark Smoke Induces Lung and Pleural Plasminogen Activator Inhibitor 1 and Stabilizes Its mRNA in Porcine Lung Cells

    DTIC Science & Technology

    2011-08-01

    in situ. Plasminogen activator inhibitor 1 was measured in bronchoalveolar lavage fluids by Western blotting. Induction of PAI-1 was determined at the...As measured in bronchoalveolar lavage (BAL) fluid, this defect in fibrinolysis is mainly attributable to overexpression of plasminogen activator...monitoring Pigs were monitored for 48 h. The following variables were measured : number of smoke breaths received, volume of smoke received, peak carboxy

  19. The omptins of Yersinia pestis and Salmonella enterica cleave the reactive center loop of plasminogen activator inhibitor 1.

    PubMed

    Haiko, Johanna; Laakkonen, Liisa; Juuti, Katri; Kalkkinen, Nisse; Korhonen, Timo K

    2010-09-01

    Plasminogen activator inhibitor 1 (PAI-1) is a serine protease inhibitor (serpin) and a key molecule that regulates fibrinolysis by inactivating human plasminogen activators. Here we show that two important human pathogens, the plague bacterium Yersinia pestis and the enteropathogen Salmonella enterica serovar Typhimurium, inactivate PAI-1 by cleaving the R346-M347 bait peptide bond in the reactive center loop. No cleavage of PAI-1 was detected with Yersinia pseudotuberculosis, an oral/fecal pathogen from which Y. pestis has evolved, or with Escherichia coli. The cleavage and inactivation of PAI-1 were mediated by the outer membrane proteases plasminogen activator Pla of Y. pestis and PgtE protease of S. enterica, which belong to the omptin family of transmembrane endopeptidases identified in Gram-negative bacteria. Cleavage of PAI-1 was also detected with the omptins Epo of Erwinia pyrifoliae and Kop of Klebsiella pneumoniae, which both belong to the same omptin subfamily as Pla and PgtE, whereas no cleavage of PAI-1 was detected with omptins of Shigella flexneri or E. coli or the Yersinia chromosomal omptins, which belong to other omptin subfamilies. The results reveal a novel serpinolytic mechanism by which enterobacterial species expressing omptins of the Pla subfamily bypass normal control of host proteolysis.

  20. Tissue plasminogen activator promotes the effects of corticotropin-releasing factor on the amygdala and anxiety-like behavior.

    PubMed

    Matys, Tomasz; Pawlak, Robert; Matys, Elzbieta; Pavlides, Constantine; McEwen, Bruce S; Strickland, Sidney

    2004-11-16

    Stress-induced plasticity in the brain requires a precisely orchestrated sequence of cellular events involving novel as well as well known mediators. We have previously demonstrated that tissue plasminogen activator (tPA) in the amygdala promotes stress-induced synaptic plasticity and anxiety-like behavior. Here, we show that tPA activity in the amygdala is up-regulated by a major stress neuromodulator, corticotropin-releasing factor (CRF), acting on CRF type-1 receptors. Compared with WT, tPA-deficient mice responded to CRF treatment with attenuated expression of c-fos (an indicator of neuronal activation) in the central and medial amygdala but had normal c-fos responses in paraventricular nuclei. They exhibited reduced anxiety-like behavior to CRF but had a sustained corticosterone response after CRF administration. This effect of tPA deficiency was not mediated by plasminogen, because plasminogen-deficient mice demonstrated normal behavioral and hormonal changes to CRF. These studies establish tPA as an important mediator of cellular, behavioral, and hormonal responses to CRF.

  1. Tissue-type plasminogen activator deficiency delays bone repair: roles of osteoblastic proliferation and vascular endothelial growth factor.

    PubMed

    Kawao, Naoyuki; Tamura, Yukinori; Okumoto, Katsumi; Yano, Masato; Okada, Kiyotaka; Matsuo, Osamu; Kaji, Hiroshi

    2014-08-01

    Further development in research of bone regeneration is necessary to meet the clinical demand for bone reconstruction. Recently, we reported that plasminogen is crucial for bone repair through enhancement of vessel formation. However, the details of the role of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) in the bone repair process still remain unknown. Herein, we examined the effects of plasminogen activators on bone repair after a femoral bone defect using tPA-deficient (tPA(-/-)) and uPA-deficient (uPA(-/-)) mice. Bone repair of the femur was delayed in tPA(-/-) mice, unlike that in wild-type (tPA(+/+)) mice. Conversely, the bone repair was comparable between wild-type (uPA(+/+)) and uPA(-/-) mice. The number of proliferative osteoblasts was decreased at the site of bone damage in tPA(-/-) mice. Moreover, the proliferation of primary calvarial osteoblasts was reduced in tPA(-/-) mice. Recombinant tPA facilitated the proliferation of mouse osteoblastic MC3T3-E1 cells. The proliferation enhanced by tPA was antagonized by the inhibition of endogenous annexin 2 by siRNA and by the inhibition of extracellular signal-regulated kinase (ERK)1/2 phosphorylation in MC3T3-E1 cells. Vessel formation as well as the levels of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) were decreased at the damaged site in tPA(-/-) mice. Our results provide novel evidence that tPA is crucial for bone repair through the facilitation of osteoblast proliferation related to annexin 2 and ERK1/2 as well as enhancement of vessel formation related to VEGF and HIF-1α at the site of bone damage.

  2. Plasminogen activator inhibitor-1 mitigates brain injury in a rat model of infection-sensitized neonatal hypoxia-ischemia.

    PubMed

    Yang, Dianer; Sun, Yu-Yo; Nemkul, Niza; Baumann, Jessica M; Shereen, Ahmed; Dunn, R Scott; Wills-Karp, Marsha; Lawrence, Daniel A; Lindquist, Diana M; Kuan, Chia-Yi

    2013-05-01

    Intrauterine infection exacerbates neonatal hypoxic-ischemic (HI) brain injury and impairs the development of cerebral cortex. Here we used low-dose lipopolysaccharide (LPS) pre-exposure followed by unilateral cerebral HI insult in 7-day-old rats to study the pathogenic mechanisms. We found that LPS pre-exposure blocked the HI-induced proteolytic activity of tissue-type plasminogen activator (tPA), but significantly enhanced NF-κB signaling, microglia activation, and the production of pro-inflammatory cytokines in newborn brains. Remarkably, these pathogenic responses were all blocked by intracerebroventricular injection of a stable-mutant form of plasminogen activator protein-1 called CPAI. Similarly, LPS pre-exposure amplified, while CPAI therapy mitigated HI-induced blood-brain-barrier damage and the brain tissue loss with a therapeutic window at 4 h after the LPS/HI insult. The CPAI also blocks microglia activation following a brain injection of LPS, which requires the contribution by tPA, but not the urinary-type plasminogen activator (uPA), as shown by experiments in tPA-null and uPA-null mice. These results implicate the nonproteolytic tPA activity in LPS/HI-induced brain damage and microglia activation. Finally, the CPAI treatment protects near-normal motor and white matter development despite neonatal LPS/HI insult. Together, because CPAI blocks both proteolytic and nonproteolytic tPA neurotoxicity, it is a promising therapeutics of neonatal HI injury either with or without infection.

  3. Expression of the Plague Plasminogen Activator in Yersinia pseudotuberculosis and Escherichia coli

    PubMed Central

    Kutyrev, V.; Mehigh, R. J.; Motin, V. L.; Pokrovskaya, M. S.; Smirnov, G. B.; Brubaker, R. R.

    1999-01-01

    Enteropathogenic yersiniae (Yersinia pseudotuberculosis and Yersinia enterocolitica) typically cause chronic disease as opposed to the closely related Yersinia pestis, the causative agent of bubonic plague. It is established that this difference reflects, in part, carriage by Y. pestis of a unique 9.6-kb pesticin or Pst plasmid (pPCP) encoding plasminogen activator (Pla) rather than distinctions between shared ∼70-kb low-calcium-response, or Lcr, plasmids (pCD in Y. pestis and pYV in enteropathogenic yersiniae) encoding cytotoxic Yops and anti-inflammatory V antigen. Pla is known to exist as a combination of 32.6-kDa (α-Pla) and slightly smaller (β-Pla) outer membrane proteins, of which at least one promotes bacterial dissemination in vivo and degradation of Yops in vitro. We show here that only α-Pla accumulates in Escherichia coli LE392/pPCP1 cultivated in enriched medium and that either autolysis or extraction of this isolate with 1.0 M NaCl results in release of soluble α and β forms possessing biological activity. This process also converted cell-bound α-Pla to β-Pla and smaller forms in Y. pestis KIM/pPCP1 and Y. pseudotuberculosis PB1/+/pPCP1 but did not promote solubilization. Pla-mediated posttranslational hydrolysis of pulse-labeled Yops in Y. pseudotuberculosis PB1/+/pPCP1 occurred more slowly than that in Y. pestis but was otherwise similar except for accumulation of stable degradation products of YadA, a pYV-mediated fibrillar adhesin not encoded in frame by pCD. Carriage of pPCP by Y. pseudotuberculosis did not significantly influence virulence in mice. PMID:10024583

  4. Link between plasminogen activator inhibitor-1 and cardiovascular risk in chronic hepatitis C after viral clearance.

    PubMed

    Chang, Ming-Ling; Lin, Yu-Sheng; Pao, Li-Heng; Huang, Hsin-Chih; Chiu, Cheng-Tang

    2017-02-13

    The pathophysiological implications of plasminogen activator inhibitor-1 (PAI-1) in HCV infection remain obscure. This prospective study evaluated 669 HCV patients, of whom 536 had completed a course of anti-HCV therapy and had pre-, peri- and post-therapy measurements of various profiles, including PAI-1 levels. Multivariate analysis demonstrated, before anti-HCV-therapy, platelet count and PAI-1-rs1799889 genotype were associated with PAI-1 levels. Among patients with a sustained virological response (SVR, n = 445), platelet count was associated with PAI-1 level at 24 weeks post-therapy. GEE analysis showed that PAI-1-rs-1799889 and interferon-λ3-rs12979860 genotypes affected PAI-1 levels early and late in therapy, respectively. At 24 weeks post-therapy, higher lipid, brain natriuretic peptide, homocysteine and PAI-1 levels and PAI-1 activity were noted only in SVR patients compared with pre-therapy levels. Within 24 weeks post-therapy, 2.2% of the SVR (mean age: 57.8 yr; 8 smoking males; the 2 females had pre-therapy hypercholesteremia or cardiovascular family history of disease) and 0% of the non-SVR patients experienced a new cardiovascular event. Platelet counts consistently correlated with PAI-1 levels regardless of HCV infection. PAI-1-rs-1799889 and interferon-λ3-rs12979860 genotypes mainly affected PAI-1 levels longitudinally. Within 24 weeks post-anti-HCV therapy, the SVR patients showed increasing PAI-1 levels with accelerating cardiovascular risk, especially the vulnerable cases.

  5. Link between plasminogen activator inhibitor-1 and cardiovascular risk in chronic hepatitis C after viral clearance

    PubMed Central

    Chang, Ming-Ling; Lin, Yu-sheng; Pao, Li-Heng; Huang, Hsin-Chih; Chiu, Cheng-Tang

    2017-01-01

    The pathophysiological implications of plasminogen activator inhibitor-1 (PAI-1) in HCV infection remain obscure. This prospective study evaluated 669 HCV patients, of whom 536 had completed a course of anti-HCV therapy and had pre-, peri- and post-therapy measurements of various profiles, including PAI-1 levels. Multivariate analysis demonstrated, before anti-HCV-therapy, platelet count and PAI-1-rs1799889 genotype were associated with PAI-1 levels. Among patients with a sustained virological response (SVR, n = 445), platelet count was associated with PAI-1 level at 24 weeks post-therapy. GEE analysis showed that PAI-1-rs-1799889 and interferon-λ3-rs12979860 genotypes affected PAI-1 levels early and late in therapy, respectively. At 24 weeks post-therapy, higher lipid, brain natriuretic peptide, homocysteine and PAI-1 levels and PAI-1 activity were noted only in SVR patients compared with pre-therapy levels. Within 24 weeks post-therapy, 2.2% of the SVR (mean age: 57.8 yr; 8 smoking males; the 2 females had pre-therapy hypercholesteremia or cardiovascular family history of disease) and 0% of the non-SVR patients experienced a new cardiovascular event. Platelet counts consistently correlated with PAI-1 levels regardless of HCV infection. PAI-1-rs-1799889 and interferon-λ3-rs12979860 genotypes mainly affected PAI-1 levels longitudinally. Within 24 weeks post-anti-HCV therapy, the SVR patients showed increasing PAI-1 levels with accelerating cardiovascular risk, especially the vulnerable cases. PMID:28211910

  6. The Role of Urokinase Plasminogen Activator and Plasmin Activator Inhibitor-1 on Vein Wall Remodeling in Experimental Deep Vein Thrombosis

    PubMed Central

    Baldwin, Joe F.; Sood, Vikram; Elfline, Megan A.; Luke, Cathy E.; Dewyer, Nicholas A.; Diaz, Jose A.; Myers, Dan D.; Wakefield, Thomas; Henke, Peter K.

    2012-01-01

    OBJECTIVE Deep vein thrombosis (DVT) resolution instigates an inflammatory response, resulting in vessel wall damage and scarring. Urokinase-plasminogen activator (uPA) and its inhibitor, plasminogen activator inhibitor-1 (PAI-1), are integral components of the fibrinolytic system, essential for VT resolution. This study determined the vein wall response when exposed to increased and decreased plasmin activity. Methods A mouse inferior vena cava (IVC) ligation model in uPA −/− or PAI-1 −/− and their genetic wild types (B6/SvEv and C57/BL6, respectively) was used to create stasis thrombi, with tissue harvest at either 8 or 21d. Tissue analysis included gene expression of vascular smooth muscle cells (alpha SMA [αSMA], SM22) and endothelial marker (CD31), by real time PCR, ELISA, matrix metalloproteinase (MMP) -2 and 9 activity by zymography and vein wall collagen by picrosirius red histological analysis. A P < .05 was considered significant. RESULTS Thrombi were significantly larger in both 8d and 21d uPA −/− as compared to WT, and were significantly smaller in both 8 and 21d PAI-1 −/− as compared to WT. Correspondingly, 8d plasmin levels were reduced in half in uPA −/− and increased 3 fold in PAI-1 −/− when compared to respective WT thrombi (P < .05, N = 5 – 6). The endothelial marker CD31 was elevated 2 fold in PAI-1 −/− mice at 8d, but reduced 2.5 fold at 21d in uPA −/− as compared with WT (P = .02, N = 5 – 6), suggesting less endothelial preservation. Vein wall VSMC gene expression showed that 8d and 21d PAI-1 −/− mice had 2.3 and 3.8 fold more SM22 and 1.8 and 2.3 fold more αSMA expression than respective WT (P < .05, N = 5 – 7), as well as 1.8 fold increased αSMA (+) cells (N = 3 – 5, P ≤ .05). No significant difference in MMP2 or 9 activity was found in the PAI-1 −/− mice compared with WT, while 5.4 fold more MMP9 was present in 21d WT than 21d uPA −/− (P = .03, N = 5). Lastly, collagen was ~2 fold

  7. Monocyte procoagulant activity and plasminogen activator. Role in human renal allograft rejection

    SciTech Connect

    Cole, E.H.; Cardella, C.J.; Schulman, J.; Levy, G.A.

    1985-10-01

    Currently the mechanism of renal allograft rejection is not well understood. This study was designed to determine whether induction of monocyte procoagulant activity (MCPA) is important in the pathogenesis of renal allograft rejection. The MPCA assay was performed utilizing a one stage clotting assay both in normal and in factor-VII-deficient plasma. There was no increase in spontaneous MPCA in 20 patients with endstage renal failure and in 10 patients following abdominal or orthopedic operation, as compared with 20 normal controls. MPCA was assessed daily in 18 patients who had received renal allografts. Rejection episodes (RE) were predicted on the basis of persistent elevation in MPCA as compared with pretransplant levels. Rejection was diagnosed clinically and treated on the basis of standard criteria. Treated RE were compared with those predicted by elevated MPCA, and 3 patients were assessed as having no RE by MPCA and by standard criteria. In 8 RE, MPCA correlated temporally with RE (same day) when compared with standard criteria. In 12 RE, MPCA was predictive of rejection preceding standard criteria by at least 24 hr. There were 7 false-positive predictions on the basis of MPCA; however, there was only 1 false negative. MPCA was shown to be a prothrombinase by its dependence only on prothrombin and fibrinogen for full activity. MPCA may be important in the pathogenesis of allograft rejection, and additionally it may be a useful adjunct in the clinical management of this disease.

  8. Activation of tissue plasminogen activator gene transcription by Neovastat, a multifunctional antiangiogenic agent.

    PubMed

    Gingras, Denis; Nyalendo, Carine; Di Tomasso, Geneviève; Annabi, Borhane; Béliveau, Richard

    2004-07-16

    We recently reported that Neovastat, an antiangiogenic drug that is currently undergoing Phase III clinical trials for the treatment of non-small cell lung cancer, may inhibit angiogenesis through an increase in tPA activity. Here, we show that Neovastat also stimulates tPA gene transcription in endothelial cells, in a TNFalpha-like manner. RT-PCR analysis and gene reporter assays using the human tPA promoter indicated that upregulation of the tPA gene transcription by both Neovastat and TNFalpha was correlated with the phosphorylation of JNK1/2 and of IkappaB and that SP600125 and BAY11-7082, inhibitors of JNK and IkappaK, respectively, inhibit the increase of tPA gene transcription induced by Neovastat and TNFalpha. These results suggest that Neovastat induces tPA gene transcription through activation of the JNK and NFkappaB signaling pathways, leading to an increase of tPA secretion by endothelial cells. This may lead to the localized destruction of the fibrin provisional matrix that is necessary for neovessel formation and thus contribute to the reported antiangiogenic properties of this compound.

  9. Tissue and urokinase plasminogen activators instigate the degeneration of retinal ganglion cells in a mouse model of glaucoma

    PubMed Central

    Chintala, Shravan K

    2015-01-01

    Elevated intraocular pressure (IOP) promotes the degeneration of retinal ganglion cells (RGCs) during the progression of Primary Open-Angle Glaucoma (POAG). However, the molecular mechanisms underpinning IOP-mediated degeneration of RGCs remain unclear. Therefore, by employing a mouse model of POAG, this study examined whether elevated IOP promotes the degeneration of RGCs by up-regulating tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) in the retina. IOP was elevated in mouse eyes by injecting fluorescent-microbeads into the anterior chamber. Once a week, for eight weeks, IOP in mouse eyes was measured by using Tono-Pen XL. At various time periods after injecting microbeads, proteolytic activity of tPA and uPA in retinal protein extracts was determined by fibrinogen/plasminogen zymography assays. Localization of tPA and uPA, and their receptor LRP-1 (low-density receptor-related protein-1) in the retina was determined by immunohistochemistry. RGCs’ degeneration was assessed by immunostaining with antibodies against Brn3a. Injection of microbeads into the anterior chamber led to a progressive elevation in IOP, increased the proteolytic activity of tPA and uPA in the retina, activated plasminogen into plasmin, and promoted a significant degeneration of RGCs. Elevated IOP up-regulated tPA and LRP-1 in RGCs, and uPA in astrocytes. At four weeks after injecting microbeads, RAP (receptor associated protein; 0.5 and 1.0 μM) or tPA-Stop (1.0 and 4.0 μM) was injected into the vitreous humor. Treatment of IOP-elevated eyes with RAP led to a significant decrease in proteolytic activity of both tPA and uPA, and a significant decrease in IOP-mediated degeneration of RGCs. Also, treatment of IOP-elevated eyes with tPA-Stop decreased the proteolytic activity of both tPA and uPA, and, in turn, significantly attenuated IOP-mediated degeneration of RGCs. Results presented in this study provide evidence that elevated IOP promotes the degeneration of

  10. Treatment of recent onset central retinal vein occlusion with intravitreal tissue plasminogen activator: a pilot study

    PubMed Central

    Glacet-Bernard, A.; Kuhn, D.; Vine, A.; Oubraham, H.; Coscas, G.; Soubrane, G.

    2000-01-01

    AIMS—To study the effects of intravitreal tissue plasminogen activator (tPA) in recent onset central retinal vein occlusion (CRVO).
METHODS—15 patients with recent onset CRVO (from 1-21 days' duration, mean 8 days) were given 75-100 µg of tPA intravitreally associate with low dose low molecular weight heparin. CRVO was perfused in nine patients and with mild ischaemia not exceeding 100 disc diameters in six patients. Follow up ranged from 5 to 21 months for 14 patients (mean 8 months). Visual acuity measurement, macular threshold (Humphrey perimeter), fluorescein angiography with the scanning laser ophthalmoscope with special emphasis on retinal circulation times, and retinal perfusion were performed at days 0, 1, and 8 and months 1, 3, and 6.
RESULTS—Visual acuity was significantly improved on the first day after treatment in only one eye, and decreased transiently in six eyes (40%). Retinal blood velocity was not significantly modified by tPA injection. Retinal ischaemia developed in six eyes (43%), leading to panretinal photocoagulation in five eyes including one with rubeosis iridis. At the end of follow up, visual acuity had improved to 20/30 or better in five eyes (36%), including two with complete recovery; visual acuity was worse than 20/200 in three eyes (28%). No complication of tPA injection was observed.
CONCLUSION—Intravitreal tPA treatment for CRVO appears to be simple and safe, but did not significantly modify the course of the occlusion in our patients immediately after treatment. Final visual outcome did not differ significantly from that observed in the natural course of the disease, but final visual acuity seemed to be slightly better. A randomised study is required to determine if intravitreal tPA actually improves visual outcome in CRVO.

 PMID:10837386

  11. Passenger mutations and aberrant gene expression in congenic tissue plasminogen activator-deficient mouse strains

    PubMed Central

    Szabo, R.; Samson, A. L.; Lawrence, D. A.; Medcalf, R. L.; Bugge, T. H.

    2017-01-01

    Summary Background The ability to generate defined null mutations in mice revolutionized the analysis of gene function in mammals. However, gene-deficient mice generated by using 129-derived embryonic stem cells may carry large segments of 129 DNA, even when extensively backcrossed to reference strains, such as C57BL/6J, and this may confound interpretation of experiments performed in these mice. Tissue plasminogen activator (tPA), encoded by the PLAT gene, is a fibrinolytic serine protease that is widely expressed in the brain. A number of neurological abnormalities have been reported in tPA-deficient mice. Objectives To study genetic contamination of tPA-deficient mice. Materials and methods Whole genome expression array analysis, RNAseq expression profiling, low- and high-density SNP analysis, bioinformatics, and genome editing was used to analyze gene expression in tPA-deficient mouse brains. Results and conclusions Genes differentially expressed in the brain of Plat−/− mice from two independent colonies highly backcrossed onto the C57BL/6J strain clustered near Plat on chromosome 8. SNP analysis attributed this anomaly to about 20 Mbp of DNA flanking Plat being of 129 origin in both strains. Bioinformatic analysis of these 129-derived chromosomal segments identified a significant number of mutations in genes co-segregating with the targeted Plat allele, including several potential null mutations. Using zinc finger nuclease technology, we generated novel “passenger mutation”-free isogenic C57BL/6J-Plat−/− and FVB/NJ-Plat−/− mouse strains by introducing an 11 bp deletion in the exon encoding the signal peptide. These novel mouse strains will be a useful community resource for further exploration of tPA function in physiological and pathological processes. PMID:27079292

  12. Tissue plasminogen activator in trabecular meshwork attenuates steroid induced outflow resistance in mice.

    PubMed

    Kumar, Sandeep; Shah, Shaily; Tang, Hai Michael; Smith, Matthew; Borrás, Teresa; Danias, John

    2013-01-01

    Tissue plasminogen activator, a serine protease encoded by the PLAT gene is present in the trabecular meshwork (TM) and other ocular tissues and has been reported to be downregulated by treatment with steroids in vitro. Steroids are known to cause changes in outflow facility of aqueous humor in many species. In the present study, we tested whether overexpression of PLAT can prevent and/or reverse the outflow facility of mouse eyes treated with steroids. Animals received bilateral injection with 20 µl of triamcinolone acetonide (TA) (40 mg/ml) suspension subconjunctivally to induce outflow facility changes. Some animals received unilateral intracameral injection with 2 µl of adenoviral suspension [3-4 x 10(12) virus genomes per milliliter (vg/ml)] carrying sheep PLAT cDNA (AdPLAT) either concurrently with TA injection or one week after TA injection, whereas others received bilateral intracameral injection with 2 µl of adenoviral suspension (9 x 10(12) vg/ml) carrying no transgene (AdNull) concurrently with TA injection. Animals were sacrificed one week after AdPLAT or AdNull treatment. Endogenous mRNA expression levels of mouse PAI-1 and MMP-2, -9 and -13 were also measured using qRT-PCR. Outflow facility one week after AdPLAT administration was increased by 60% and 63% respectively for animals that had not or had been pretreated with steroids. Overexpression of PLAT significantly upregulated expression of PAI-1, MMP-2, -9 and -13 compared to the levels found in TA only treated eyes. These findings suggest that overexpression of PLAT in TM of mouse eyes can both prevent and reverse the decrease in outflow facility caused by steroid treatment and is associated with upregulation of MMPs.

  13. Endothelium-Derived Hyperpolarizing Factor Mediates Bradykinin Stimulated Tissue Plasminogen Activator Release In Humans

    PubMed Central

    Rahman, Ayaz M.; Murrow, Jonathan R.; Ozkor, Muhiddin A.; Kavtaradze, Nino; Lin, Ji; De Staercke, Christine; Hooper, W. Craig; Manatunga, Amita; Hayek, Salim; Quyyumi, Arshed A.

    2014-01-01

    Aims Bradykinin stimulates tissue plasminogen activator (t-PA) release from human endothelium. Although bradykinin stimulates both nitric oxide and endothelium-derived hyperpolarizing factor (EDHF) release, the role of EDHF in t-PA release remains unexplored. This study sought to determine the mechanisms of bradykinin-stimulated t-PA release in the forearm vasculature of healthy human subjects. Methods In 33 healthy subjects (age 40.3±1.9 years) forearm blood flow (FBF) and t-PA release were measured at rest, and after intra-arterial infusions of bradykinin (400ng/min) and sodium nitroprusside (3.2 mg/min). Measurements were repeated after intra-arterial infusion of TEA (1 μmol/min), fluconazole (0.4 μmol.min-1.L-1), and NG-monomethyl-L-arginine (L-NMMA, 8 μmol/min) to block nitric oxide, and their combination in separate studies. Results Bradykinin significantly increased net t-PA release across the forearm (P<0.0001). Fluconazole attenuated both bradykinin-mediated vasodilation (-23.3±2.7% FBF, P<0.0001) and t-PA release (from 50.9±9.0 to 21.3±8.9 ng/min/100ml, P=0.02). TEA attenuated FBF (-14.7±3.2%, P=0.002) and abolished bradykinin-stimulated t-PA release (from 22.9+5.7 to - 0.8±3.6 ng/min/100ml, P=0.0002). L-NMMA attenuated FBF (P<0.0001), but did not inhibit bradykinin-induced t-PA release (P=NS). Conclusion Bradykinin-stimulated t-PA release is partly due to cytochrome P450-derived epoxides, and is inhibited by K+ca channel blockade. Thus, bradykinin stimulates both EDHF-dependent vasodilation and t-PA release. PMID:24925526

  14. Preclinical evaluation of a urokinase plasminogen activator receptor-targeted nanoprobe in rhesus monkeys

    PubMed Central

    Chen, Yushu; Gong, Li; Gao, Ning; Liao, Jichun; Sun, Jiayu; Wang, Yuqing; Wang, Lei; Zhu, Pengjin; Fan, Qing; Wang, Yongqiang Andrew; Zeng, Wen; Mao, Hui; Yang, Lily; Gao, Fabao

    2015-01-01

    Purpose To translate a recombinant peptide containing the amino-terminal fragment (ATF) of urokinase plasminogen activator receptor-targeted magnetic iron oxide (IO) nanoparticles (uPAR-targeted human ATF-IONPs) into clinical applications, we conducted a pilot study to evaluate the toxicity and pharmacokinetics of this nanoparticle in normal rhesus monkeys. Methods We assessed the changes in the following: magnetic resonance imaging (MRI) signals from pretreatment stage to 14 days posttreatment, serum iron concentrations from 5 minutes posttreatment to 12 weeks posttreatment, routine blood examination and serum chemistry analysis results from pretreatment stage to 12 weeks after administration, and results of staining of the liver with Perls’ Prussian Blue and hematoxylin–eosin at 24 hours and 3 months posttreatment in two rhesus monkeys following an intravenous administration of the targeted nanoparticles either with a polyethylene glycol (ATF-PEG-IONP) or without a PEG (ATF-IONP) coating. Results The levels of alkaline phosphatase, alanine transaminase, and direct bilirubin in the two monkeys increased immediately after the administration of the IONPs but returned to normal within 20 days and stayed within the normal reference range 3 months after the injection. The creatinine levels of the two monkeys stayed within the normal range during the study. In addition, red blood cells, white blood cells, hemoglobin level, and platelets remained normal during the 3 months of the study. Conclusion All of the results suggest that a transient injury in terms of normal organ functions, but no microscopic necrotic lesions, was observed at a systemic delivery dose of 5 mg/kg of iron equivalent concentration in the acute phase, and that no chronic toxicity was found 3 months after the injection. Therefore, we conclude that uPAR-targeted IONPs have the potential to be used as receptor-targeted MRI contrasts as well as theranostic agents for the detection and treatment of

  15. Recombinant Human Plasminogen Activator Inhibitor-1 Promotes Cementogenic Differentiation of Human Periodontal Ligament Stem Cells.

    PubMed

    Jin, Hexiu; Choung, Han-Wool; Lim, Ki-Taek; Jin, Bin; Jin, Chengbiao; Chung, Jong-Hoon; Choung, Pill-Hoon

    2015-12-01

    The periodontium, consisting of gingiva, periodontal ligament (PDL), cementum, and alveolar bone, is necessary for the maintenance of tooth function. Specifically, the regenerative abilities of cementum with inserted PDL are important for the prevention of tooth loss. Periodontal ligament stem cells (PDLSCs), which are located in the connective tissue PDL between the cementum and alveolar bone, are an attractive candidate for hard tissue formation. We investigated the effects of recombinant human plasminogen activator inhibitor-1 (rhPAI-1) on cementogenic differentiation of human PDLSCs (hPDLSCs) in vitro and in vivo. Untreated and rhPAI-1-treated hPDLSCs mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) and dentin matrix were transplanted subcutaneously into the dorsal surface of immunocompromised mice to assess their capacity for hard tissue formation at 8 and 10 weeks posttransplantation. rhPAI-1 accelerated mineral nodule formation and increased the mRNA expression of cementoblast-associated markers in hPDLSCs. We also observed that rhPAI-1 upregulated the levels of osterix (OSX) and cementum protein 1 (CEMP1) through Smad2/3 and p38 pathways, whereas specific inhibitors of Smad3 and p38 inhibited the enhancement of mineralization of hPDLSCs by rhPAI-1. Furthermore, transplantation of hPDLSCs with rhPAI-1 showed a great ability to promote cementogenic differentiation. Notably, rhPAI-1 induced hPDLSCs to regenerate cementum-like tissue with PDL fibers inserted into newly formed cementum-like tissue. These results suggest that rhPAI-1 may play a key role in cementogenic differentiation of hPDLSCs. rhPAI-1 with hPDLSCs may be a good candidate for future clinical applications in periodontal tissue regeneration and possibly in tooth root bioengineering.

  16. Saturated fatty acid intake can influence increase in plasminogen activator inhibitor-1 in obese adolescents.

    PubMed

    Masquio, D C L; de Piano, A; Campos, R M S; Sanches, P L; Corgosinho, F C; Carnier, J; Oyama, L M; do Nascimento, C M P O; de Mello, M T; Tufik, S; Dâmaso, A R

    2014-04-01

    The aim of this study was to verify if saturated fatty acid intake adjusted by tertiles can influence metabolic, inflammation, and plasminogen activator inhibitor-1 (PAI-1) in obese adolescents. Body mass, height, body mass index, waist circumference, blood pressure, and body composition of 108 obese adolescents were obtained. Fasting glucose, insulin, PAI-1, and CRP were determined. Insulin resistance was assessed by Homeostasis Model Assessment (HOMA-IR) and insulin sensitivity by Quantitative Insulin Sensitivity Check Index (QUICKI). Dietetic intake was estimated by a 3-day dietary record, and volunteers were divided according to consumption of saturated fatty acids: tertile 1 [Low Saturated Fatty Acid Intake (Low-SFA): ≤12.14 g], tertile 2 [Moderate Saturated Fatty Intake (Moderate SFA intake): 12.15-20.48 g], and tertile 3 [High Saturated Fatty Acid Intake (High-SFA Intake); >20.48 g]. Statistical analysis was performed using STATISTICA 7.0 software and the significance level was set at p<0.05. The most important finding in the present study is that Moderate and High-SFA intakes presented significantly higher values of PAI-1 than Low-SFA Intake. PAI-1 was positively associated with saturated fatty intake, waist circumference, mean blood pressure, and HOMA-IR. SFA intake was predictor of PAI-1 independent of body fat, HOMA-IR and total-cholesterol. In addition, PAI-1 was an independent predictor of blood pressure. HOMA-IR and QUICKI presented significantly higher and lower, respectively, in High-SFA compared to Moderate-SFA intake. High-SFA influenced cardiovascular disease risks, since it increased PAI-1 and insulin resistance, and decreased insulin sensibility, leading to vicious cycle among food ingestion, pro-thrombotic state, and cardiovascular risks in obese adolescents.

  17. Hepatic Microenvironment Affects Oval Cell Localization in Albumin-Urokinase-Type Plasminogen Activator Transgenic Mice

    PubMed Central

    Braun, Kristin M.; Thompson, Anne W.; Sandgren, Eric P.

    2003-01-01

    Mice carrying an albumin-urokinase type plasminogen activator transgene (AL-uPA) develop liver disease secondary to uPA expression in hepatocytes. Transgene-expressing parenchyma is replaced gradually by clones of cells that have deleted transgene DNA and therefore are not subject to uPA-mediated damage. Diseased liver displays several abnormalities, including hepatocyte vacuolation and changes in nonparenchymal tissue. The latter includes increases in laminin protein within parenchyma and the appearance of cytokeratin 19-positive bile ductule-like cells (oval cells) both in portal regions and extending into the hepatic parenchyma. In this study, we subjected AL-uPA mice to two-thirds partial hepatectomy to identify the response of these livers to additional growth stimulation. We observed several changes in hepatic morphology. First, the oval cells increased in number and often formed ductules in the parenchyma. Second, this cellular change was accompanied by a further increase in laminin associated with single or clusters of oval cells. Third, desmin-positive Ito cells increased in number and maintained close association with oval cells. Fourth, these changes were localized precisely to uPA-expressing areas of liver. Regenerating clones of uPA-deficient cells appeared to be unaffected both by stromal and cellular alterations. Thus, additional growth stimulation of diseased uPA-expressing liver induces an oval cell-like response, as observed in other models of severe hepatic injury, but the localization of this response seems to be highly regulated by the hepatic microenvironment. PMID:12507902

  18. Plasminogen Activator Inhibitor-1 Is Involved in Impaired Bone Repair Associated with Diabetes in Female Mice

    PubMed Central

    Mao, Li; Kawao, Naoyuki; Tamura, Yukinori; Okumoto, Katsumi; Okada, Kiyotaka; Yano, Masato; Matsuo, Osamu; Kaji, Hiroshi

    2014-01-01

    Previous studies suggest that fracture healing is impaired in diabetes; however, the underlying mechanism remains unclear. Here, we investigated the roles of plasminogen activator inhibitor-1 (PAI-1) in the impaired bone repair process by using streptozotocin (STZ)-induced diabetic female wild-type (PAI-1+/+) and PAI-1-deficient (PAI-1−/−) mice. Bone repair and the number of alkaline phosphatase (ALP)-positive cells at the site of a femoral bone damage were comparable in PAI-1+/+ and PAI-1−/− mice without STZ treatment. Although the bone repair process was delayed by STZ treatment in PAI-1+/+ mice, this delayed bone repair was blunted in PAI-1−/− mice. The reduction in the number of ALP-positive cells at the site of bone damage induced by STZ treatment was attenuated in PAI-1−/− mice compared to PAI-1+/+ mice. On the other hand, PAI-1 deficiency increased the levels of ALP and type I collagen mRNA in female mice with or without STZ treatment, and the levels of Osterix and osteocalcin mRNA, suppressed by diabetic state in PAI-1+/+ mice, were partially protected in PAI-1−/− mice. PAI-1 deficiency did not affect formation of the cartilage matrix and the levels of types II and X collagen and aggrecan mRNA suppressed by STZ treatment, although PAI-1 deficiency increased the expression of chondrogenic markers in mice without STZ treatment. The present study indicates that PAI-1 is involved in the impaired bone repair process induced by the diabetic state in part through a decrease in the number of ALP-positive cells. PMID:24651693

  19. Recombinant Human Plasminogen Activator Inhibitor-1 Promotes Cementogenic Differentiation of Human Periodontal Ligament Stem Cells

    PubMed Central

    Jin, Hexiu; Choung, Han-Wool; Lim, Ki-Taek; Jin, Bin; Jin, Chengbiao; Chung, Jong-Hoon

    2015-01-01

    The periodontium, consisting of gingiva, periodontal ligament (PDL), cementum, and alveolar bone, is necessary for the maintenance of tooth function. Specifically, the regenerative abilities of cementum with inserted PDL are important for the prevention of tooth loss. Periodontal ligament stem cells (PDLSCs), which are located in the connective tissue PDL between the cementum and alveolar bone, are an attractive candidate for hard tissue formation. We investigated the effects of recombinant human plasminogen activator inhibitor-1 (rhPAI-1) on cementogenic differentiation of human PDLSCs (hPDLSCs) in vitro and in vivo. Untreated and rhPAI-1-treated hPDLSCs mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) and dentin matrix were transplanted subcutaneously into the dorsal surface of immunocompromised mice to assess their capacity for hard tissue formation at 8 and 10 weeks posttransplantation. rhPAI-1 accelerated mineral nodule formation and increased the mRNA expression of cementoblast-associated markers in hPDLSCs. We also observed that rhPAI-1 upregulated the levels of osterix (OSX) and cementum protein 1 (CEMP1) through Smad2/3 and p38 pathways, whereas specific inhibitors of Smad3 and p38 inhibited the enhancement of mineralization of hPDLSCs by rhPAI-1. Furthermore, transplantation of hPDLSCs with rhPAI-1 showed a great ability to promote cementogenic differentiation. Notably, rhPAI-1 induced hPDLSCs to regenerate cementum-like tissue with PDL fibers inserted into newly formed cementum-like tissue. These results suggest that rhPAI-1 may play a key role in cementogenic differentiation of hPDLSCs. rhPAI-1 with hPDLSCs may be a good candidate for future clinical applications in periodontal tissue regeneration and possibly in tooth root bioengineering. PMID:25808697

  20. Tissue-type plasminogen activator controls neuronal death by raising surface dynamics of extrasynaptic NMDA receptors

    PubMed Central

    Lesept, Flavie; Chevilley, Arnaud; Jezequel, Julie; Ladépêche, Laurent; Macrez, Richard; Aimable, Margaux; Lenoir, Sophie; Bertrand, Thomas; Rubrecht, Laëtitia; Galea, Pascale; Lebouvier, Laurent; Petersen, Karl-Uwe; Hommet, Yannick; Maubert, Eric; Ali, Carine; Groc, Laurent; Vivien, Denis

    2016-01-01

    N-methyl-d-aspartate receptors (NMDARs) are ion channels whose synaptic versus extrasynaptic localization critically influences their functions. This distribution of NMDARs is highly dependent on their lateral diffusion at the cell membrane. Each obligatory subunit of NMDARs (GluN1 and GluN2) contains two extracellular clamshell-like domains with an agonist-binding domain and a distal N-terminal domain (NTD). To date, the roles and dynamics of the NTD of the GluN1 subunit in NMDAR allosteric signaling remain poorly understood. Using single nanoparticle tracking in mouse neurons, we demonstrate that the extracellular neuronal protease tissue-type plasminogen activator (tPA), well known to have a role in the synaptic plasticity and neuronal survival, leads to a selective increase of the surface dynamics and subsequent diffusion of extrasynaptic NMDARs. This process explains the previously reported ability of tPA to promote NMDAR-mediated calcium influx. In parallel, we developed a monoclonal antibody capable of specifically blocking the interaction of tPA with the NTD of the GluN1 subunit of NMDAR. Using this original approach, we demonstrate that the tPA binds the NTD of the GluN1 subunit at a lysine in position 178. Accordingly, when applied to mouse neurons, our selected antibody (named Glunomab) leads to a selective reduction of the tPA-mediated surface dynamics of extrasynaptic NMDARs, subsequent signaling and neurotoxicity, both in vitro and in vivo. Altogether, we demonstrate that the tPA is a ligand of the NTD of the obligatory GluN1 subunit of NMDAR acting as a modulator of their dynamic distribution at the neuronal surface and subsequent signaling. PMID:27831563

  1. Validation of an immunoassay to measure plasminogen-activator inhibitor-1 concentrations in human saliva

    PubMed Central

    Zhang, Xi; Dimeski, Goce; Punyadeera, Chamindie

    2014-01-01

    Introduction: We have previously shown that the concentrations of D-dimer are significantly elevated in saliva compared with plasma. Saliva offers several advantages compared with blood analysis. We hypothesised that human saliva contains plasminogen activator inhibitor-1 (PAI-1) and that the concentrations are not affected by the time of saliva collection. The aim was to adopt and validate an immunoassay to quantify PAI-1 concentrations in saliva and to determine whether saliva collection time has an influence in the measurement. Materials and methods: Two saliva samples (morning and afternoon) from the same day were collected from healthy subjects (N = 40) who have had no underlying heart conditions. A customized AlphaLISA® immunoassay (PerkinElmer®, MA, USA) was adopted and used to quantify PAI-1 concentrations. We validated the analytical performance of the customized immunoassay by calculating recovery of known amount of analyte spiked in saliva. Results: The recovery (95.03%), intra- (8.59%) and inter-assay (7.52%) variations were within the acceptable ranges. The median salivary PAI-1 concentrations were 394 pg/mL (interquartile ranges (IQR) 243.4–833.1 pg/mL) in the morning and 376 (129.1–615.4) pg/mL in the afternoon and the plasma concentration was 59,000 (24,000–110,000) pg/mL. Salivary PAI-1 did not correlate with plasma (P = 0.812). Conclusions: The adopted immunoassay produced acceptable assay sensitivity and specificity. The data demonstrated that saliva contains PAI-1 and that its concentration is not affected by the time of saliva collection. There is no correlation between salivary and plasma PAI-1 concentrations. Further studies are required to demonstrate the utility of salivary PAI-1 in CVD risk factor studies. PMID:24969919

  2. Modulation by the noble gas argon of the catalytic and thrombolytic efficiency of tissue plasminogen activator.

    PubMed

    David, Hélène N; Haelewyn, Benoît; Risso, Jean-Jacques; Abraini, Jacques H

    2013-01-01

    Argon has been shown to provide cortical as well as, under certain conditions, subcortical neuroprotection in all models so far (middle cerebral artery occlusion, trauma, neonatal asphyxia, etc.). This has led to the suggestion that argon could be a cost-efficient alternative to xenon, a metabolically inert gas thought to be gold standard in gas pharmacology but whose clinical development suffers its little availability and excessive cost of production. However, whether argon interacts with the thrombolytic agent tissue plasminogen activator, which is the only approved therapy of acute ischemic stroke to date, still remains unknown. This latter point is not trivial since previous data have clearly demonstrated the inhibiting effect of xenon on tPA enzymatic and thrombolytic efficiency and the critical importance of the time at which xenon is administered, during or after ischemia, in order not to block thrombolysis and to obtain neuroprotection. Here, we investigated the effect of argon on tPA enzymatic and thrombolytic efficiency using in vitro methods shown to provide reliable prediction of the in vivo effects of both oxygen and the noble inert gases on tPA-induced thrombolysis. We found that argon has a concentration-dependent dual effect on tPA enzymatic and thrombolytic efficiency. Low and high concentrations of argon of 25 and 75 vol% respectively block and increase tPA enzymatic and thrombolytic efficiency. The possible use of argon at low and high concentrations in the treatment of acute ischemic stroke if given during ischemia or after tPA-induced reperfusion is discussed as regards to its neuroprotectant action and its inhibiting and facilitating effects on tPA-induced thrombolysis. The mechanisms of argon-tPA interactions are also discussed.

  3. Plasminogen activator inhibitor-1 is elevated in patients with COPD independent of metabolic and cardiovascular function

    PubMed Central

    Waschki, Benjamin; Watz, Henrik; Holz, Olaf; Magnussen, Helgo; Olejnicka, Beata; Welte, Tobias; Rabe, Klaus F; Janciauskiene, Sabina

    2017-01-01

    Introduction Plasminogen activator inhibitor-1 (PAI-1), a major inhibitor of fibrinolysis, is associated with thrombosis, obesity, insulin resistance, dyslipidemia, and premature aging, which all are coexisting conditions of chronic obstructive pulmonary disease (COPD). The role of PAI-1 in COPD with respect to metabolic and cardiovascular functions is unclear. Methods In this study, which was nested within a prospective cohort study, the serum levels of PAI-1 were cross-sectionally measured in 74 stable COPD patients (Global Initiative for Chronic Obstructive Lung Disease [GOLD] Stages I–IV) and 18 controls without lung disease. In addition, triglycerides, high-density lipoprotein cholesterol, fasting plasma glucose, waist circumference, blood pressure, smoking status, high-sensitive C-reactive protein (hs-CRP), adiponectin, ankle–brachial index, N-terminal pro-B-type natriuretic peptide, and history of comorbidities were also determined. Results The serum levels of PAI-1 were significantly higher in COPD patients than in controls, independent of a broad spectrum of possible confounders including metabolic and cardiovascular dysfunction. A multivariate regression analysis revealed triglyceride and hs-CRP levels to be the best predictors of PAI-1 within COPD. GOLD Stages II and III remained independently associated with higher PAI-1 levels in a final regression analysis. Conclusion The data from the present study showed that the serum levels of PAI-1 are higher in patients with COPD and that moderate-to-severe airflow limitation, hypertriglyceridemia, and systemic inflammation are independent predictors of an elevated PAI-1 level. PAI-1 may be a potential biomarker candidate for COPD-specific and extra-pulmonary manifestations. PMID:28356730

  4. Association of Geographical Factors With Administration of Tissue Plasminogen Activator for Acute Ischemic Stroke

    PubMed Central

    Kunisawa, Susumu; Morishima, Toshitaka; Ukawa, Naoto; Ikai, Hiroshi; Otsubo, Tetsuya; Ishikawa, Koichi B.; Yokota, Chiaki; Minematsu, Kazuo; Fushimi, Kiyohide; Imanaka, Yuichi

    2013-01-01

    Background Intravenous tissue plasminogen activator (tPA) is an effective treatment for acute ischemic stroke if administered within a few hours of stroke onset. Because of this time restriction, tPA administration remains infrequent. Ambulance use is an effective strategy for increasing tPA administration but may be influenced by geographical factors. The objectives of this study are to investigate the relationship between tPA administration and ambulance use and to examine how patient travel distance and population density affect tPA utilization. Methods and Results We analyzed administrative claims data from 114 194 acute ischemic stroke cases admitted to 603 hospitals between July 2010 and March 2012. Mixed‐effects logistic regression models of patients nested within hospitals with a random intercept were generated to analyze possible predictive factors (including patient characteristics, ambulance use, and driving time from home to hospital) of tPA administration for different population density categories to investigate differences in these factors in various regional backgrounds. Approximately 5.1% (5797/114 194) of patients received tPA. The composition of baseline characteristics varied among the population density categories, but adjustment for covariates resulted in all factors having similar associations with tPA administration in every category. The administration of tPA was associated with patient age and severity of stroke symptoms, but driving time showed no association. Ambulance use was significantly associated with tPA administration even after adjustment for covariates. Conclusion The association between ambulance use and tPA administration suggests the importance of calling an ambulance for suspected stroke. Promoting ambulance use for acute ischemic stroke patients may increase tPA use. PMID:24045119

  5. Angiotensinogen and Plasminogen Activator Inhibitor-1 Gene Polymorphism in Relation to Renovascular Disease

    SciTech Connect

    Reis, Kadriye Altok Onal, Baran; Gonen, Sevim; Arinsoy, Turgay; Erten, Yasemin; Ilgit, Erhan; Soylemezoglu, Oguz; Derici, Ulver; Guz, Galip; Bali, Musa; Sindel, Sukru

    2006-02-15

    The present study was designed to evaluate angiotensinogen (AGT) M235T and plasminogen activator inhibitor-1 (PAI-1) (4G/5G) polymorphisims in relation to the occurrence of atherosclerotic renal artery stenosis (ARAS) and recurrent stenosis. In this study, 30 patients were enrolled after angiographic demonstration of ARAS; 100 healthy subjects for AGT polymorphism and 80 healthy subjects for PAI-1 polymorphism were considered the control group. The patients were followed for a mean 46.1 {+-} 9.2 months. The patients had significantly higher frequencies of the MT genotype and the T allele than control group ({chi}{sup 2} = 18.2, p < 0.001 and {chi}{sup 2} = 11.5 p < 0.001). There were no significant differences in the PAI-1 genotype and allele findings when the data for all patients were compared with that for the controls ({chi}{sup 2}= 2.45, p = 0.29 and {chi}{sup 2} = 0.019, p = 0.89). There were no significant differences in the genotype and allele findings for the patients with and without restenosis (p > 0.05). The C-reactive protein (CRP) level was higher in the patients with restenosis than in the patients without restenosis (7.694 {+-} 0.39 mg/L and 1.56 {+-} 1.08 mg/L) (p = 0.001). Our results suggest that the M235T MT genotype and T allele might be associated with increased risk of atherosclerotic renal artery stenosis. The CRP level might be an independent predictor for recurrent stenosis.

  6. Abnormal expression of plasminogen activator inhibitors in patients with gestational trophoblastic disease.

    PubMed Central

    Estellés, A.; Grancha, S.; Gilabert, J.; Thinnes, T.; Chirivella, M.; España, F.; Aznar, J.; Loskutoff, D. J.

    1996-01-01

    We previously reported significantly elevated levels of plasminogen activator inhibitor type 1 (PAI-1) in plasma and placenta from pregnant women with severe pre-eclampsia, and pre-eclampsia is a frequent problem in molar pregnancies. As increases in PAI-1 may contribute to the placental alterations that occur in pre-eclampsia, we have begun to investigate changes in PAI-1 as well as PAI-2 and several other components of the fibrinolytic system in patients with trophoblastic disease. Significant increases in plasma PAI-1 and decreases in plasma PAI-2 levels were observed in molar pregnancies when compared with the levels in normal pregnant women of similar gestational age. PAI-1 antigen levels also were increased, and PAI-2 levels were decreased in placenta from women with molar pregnancies compared with placenta obtained by spontaneous abortion. Immunohistochemical analysis revealed strong positive and specific staining of PAI-1 in trophoblastic epithelium in molar pregnancies and relatively weak staining of PAI-2. No association between the distribution of PAI-1 and vitronectin was found, and no specific signal for tissue type PA, urokinase type PA, tumor necrosis factor-alpha, or interleukin-1 was detected. In situ hybridization revealed an increase in PAI-1 but not PAI-2 mRNAs in placenta from molar pregnancies in comparison with placenta from abortions. These results demonstrate increased PAI-1 protein and mRNA in trophoblastic disease and suggest that localized elevated levels of PAI-1 may contribute to the hemostatic problems associated with this disorder. Images Figure 1 Figure 2 Figure 3 PMID:8863672

  7. Circadian Variation of Plasminogen-Activator-Inhibitor-1 Levels in Children with Meningococcal Sepsis

    PubMed Central

    Boeddha, Navin P.; Driessen, Gertjan J.; Cnossen, Marjon H.; Hazelzet, Jan A.; Emonts, Marieke

    2016-01-01

    Objective To study whether the circadian variation of plasminogen-activator-inhibitor-1 (PAI-1) levels, with high morning levels, is associated with poor outcome of children with meningococcal sepsis presenting in the morning hours. Design Retrospective analysis of prospectively collected clinical and laboratory data. Setting Single center study at Erasmus MC-Sophia Children’s Hospital, Rotterdam, the Netherlands. Subjects 184 patients aged 3 weeks to 18 years with meningococcal sepsis. In 36 of these children, PAI-1 levels at admission to the PICU were measured in plasma by ELISA. Interventions None. Measurements and main results Circadian variation was studied by dividing one day in blocks of 6 hours. Patients admitted between 6:00 am and 12:00 am had increased illness severity scores and higher PAI-1 levels (n = 9, median 6912 ng/mL, IQR 5808–15600) compared to patients admitted at night (P = 0.019, n = 9, median 3546 ng/mL, IQR 1668–6118) or in the afternoon (P = 0.007, n = 7, median 4224 ng/mL, IQR 1804–5790). In 184 patients, analysis of circadian variation in relation to outcome showed more deaths, amputations and need for skin grafts in patients admitted to the PICU between 6:00 am and 12:00 am than patients admitted during the rest of the day (P = 0.009). Conclusions Circadian variation of PAI-1 levels is present in children with meningococcal sepsis and is associated with illness severity, with a peak level in the morning. Whether circadian variation is an independent risk factor for morbidity and mortality in meningococcal sepsis needs to be explored in future studies. PMID:27893784

  8. Plasminogen activator inhibitor-1 is involved in impaired bone repair associated with diabetes in female mice.

    PubMed

    Mao, Li; Kawao, Naoyuki; Tamura, Yukinori; Okumoto, Katsumi; Okada, Kiyotaka; Yano, Masato; Matsuo, Osamu; Kaji, Hiroshi

    2014-01-01

    Previous studies suggest that fracture healing is impaired in diabetes; however, the underlying mechanism remains unclear. Here, we investigated the roles of plasminogen activator inhibitor-1 (PAI-1) in the impaired bone repair process by using streptozotocin (STZ)-induced diabetic female wild-type (PAI-1+/+) and PAI-1-deficient (PAI-1-/-) mice. Bone repair and the number of alkaline phosphatase (ALP)-positive cells at the site of a femoral bone damage were comparable in PAI-1+/+ and PAI-1-/- mice without STZ treatment. Although the bone repair process was delayed by STZ treatment in PAI-1+/+ mice, this delayed bone repair was blunted in PAI-1-/- mice. The reduction in the number of ALP-positive cells at the site of bone damage induced by STZ treatment was attenuated in PAI-1-/- mice compared to PAI-1+/+ mice. On the other hand, PAI-1 deficiency increased the levels of ALP and type I collagen mRNA in female mice with or without STZ treatment, and the levels of Osterix and osteocalcin mRNA, suppressed by diabetic state in PAI-1+/+ mice, were partially protected in PAI-1-/- mice. PAI-1 deficiency did not affect formation of the cartilage matrix and the levels of types II and X collagen and aggrecan mRNA suppressed by STZ treatment, although PAI-1 deficiency increased the expression of chondrogenic markers in mice without STZ treatment. The present study indicates that PAI-1 is involved in the impaired bone repair process induced by the diabetic state in part through a decrease in the number of ALP-positive cells.

  9. Dissociation of severity of stroke and aphasia recovery early after intravenous recombinant tissue plasminogen activator thrombolysis.

    PubMed

    Kremer, Christine; Kappelin, Johan; Perren, Fabienne

    2014-10-01

    Clinical observation suggested to us that aphasia recovers relatively better than other deficits early after intravenous recombinant tissue plasminogen activator (IV-rtPA) treatment in stroke patients with minor deficits, while the reverse seemed the case in those with severe deficits. Retrospective analysis of acute ischemic stroke patients with aphasia admitted within 3 hours from symptom onset and treated with IV-rtPA was carried out. Stroke severity, aphasia and global neurological impairment were assessed at admission and 24 hours after thrombolysis. Improvement of aphasia (gain of ⩾ 1 point on the National Institutes of Health Stroke Scale [NIHSS] aphasia score) and global neurological improvement (gain of ⩾ 4 points on the NIHSS) were compared in minor strokes (NIHSS ⩽ 7), moderate strokes (NIHSS 8-15), and major strokes (NIH ⩾ 16). Sixty-nine of 243 stroke patients suffered from aphasia. Improvement of aphasia occurred in 7/16 minor strokes, 11/25 moderate strokes, and 7/28 severe strokes. Improvement of ⩾ 4 points on the NIHSS occurred in 3/16 minor strokes, 17/25 moderate strokes and 15/28 severe strokes. There is a significant (X(2)=4.073, p<0.05) dissociation of recovery of aphasia and that of other neurological deficits between minor versus severe strokes. This confirms the clinically suspected dissociation between a good early recovery from aphasia in minor strokes relative to recovery of other neurological deficits, as opposed to a better recovery from other neurological deficits than from aphasia in patients with severe strokes.

  10. Differential regulation of protease activated receptor-1 and tissue plasminogen activator expression by shear stress in vascular smooth muscle cells

    NASA Technical Reports Server (NTRS)

    Papadaki, M.; Ruef, J.; Nguyen, K. T.; Li, F.; Patterson, C.; Eskin, S. G.; McIntire, L. V.; Runge, M. S.

    1998-01-01

    Recent studies have demonstrated that vascular smooth muscle cells are responsive to changes in their local hemodynamic environment. The effects of shear stress on the expression of human protease activated receptor-1 (PAR-1) and tissue plasminogen activator (tPA) mRNA and protein were investigated in human aortic smooth muscle cells (HASMCs). Under conditions of low shear stress (5 dyn/cm2), PAR-1 mRNA expression was increased transiently at 2 hours compared with stationary control values, whereas at high shear stress (25 dyn/cm2), mRNA expression was decreased (to 29% of stationary control; P<0.05) at all examined time points (2 to 24 hours). mRNA half-life studies showed that this response was not due to increased mRNA instability. tPA mRNA expression was decreased (to 10% of stationary control; P<0.05) by low shear stress after 12 hours of exposure and was increased (to 250% of stationary control; P<0.05) after 24 hours at high shear stress. The same trends in PAR-1 mRNA levels were observed in rat smooth muscle cells, indicating that the effects of shear stress on human PAR-1 were not species-specific. Flow cytometry and ELISA techniques using rat smooth muscle cells and HASMCs, respectively, provided evidence that shear stress exerted similar effects on cell surface-associated PAR-1 and tPA protein released into the conditioned media. The decrease in PAR-1 mRNA and protein had functional consequences for HASMCs, such as inhibition of [Ca2+] mobilization in response to thrombin stimulation. These data indicate that human PAR-1 and tPA gene expression are regulated differentially by shear stress, in a pattern consistent with their putative roles in several arterial vascular pathologies.

  11. Studies on a complex mechanism for the activation of plasminogen by kaolin and by chloroform: the participation of Hageman factor and additional cofactors

    PubMed Central

    Ogston, Derek; Ogston, C. Marie; Ratnoff, Oscar D.; Forbes, Charles D.

    1969-01-01

    As demonstrated by others, fibrinolytic activity was generated in diluted, acidified normal plasma exposed to kaolin, a process requiring Hageman factor (Factor XII). Generation was impaired by adsorbing plasma with glass or similar agents under conditions which did not deplete its content of Hageman factor or plasminogen. The defect could be repaired by addition of a noneuglobulin fraction of plasma or an agent or agents eluted from diatomaceous earth which had been exposed to normal plasma. The restorative agent, tentatively called Hageman factor-cofactor, was partially purified by chromatography and had an apparent molecular weight of approximately 165,000. It could be distinguished from plasma thromboplastin antecedent (Factor XI) and plasma kallikrein, other substrates of Hageman factor, and from the streptokinase-activated pro-activator of plasminogen. Evidence is presented that an additional component may be needed for the generation of fibrinolytic activity in mixtures containing Hageman factor, HF-cofactor, and plasminogen. The long-recognized generation of plasmin activity in chloroform-treated euglobulin fractions of plasma was found to be dependent upon the presence of Hageman factor. Whether chloroform activation of plasminogen requires Hageman factor-cofactor was not determined, but glass-adsorbed plasma, containing Hageman factor and plasminogen, did not generate appreciable fibrinolytic or caseinolytic activity. These studies emphasize the complex nature of the mechanisms which lead to the generation of plasmin in human plasma. PMID:4241814

  12. Involvement of the serine protease inhibitor, SERPINE2, and the urokinase plasminogen activator in cumulus expansion and oocyte maturation.

    PubMed

    Lu, Chung-Hao; Lee, Robert Kuo-Kuang; Hwu, Yuh-Ming; Lin, Ming-Huei; Yeh, Ling-Yu; Chen, Ying-Jie; Lin, Shau-Ping; Li, Sheng-Hsiang

    2013-01-01

    The serpin peptidase inhibitor, clade E, member 2 (SERPINE2) inhibits urokinase-type plasminogen activator (PLAU) and tissue-type plasminogen activator. Higher SERPINE2 expression levels were detected in cumulus cells of human immature oocytes than in those of mature oocytes. The objective of this study was to evaluate whether high SERPINE2 levels in cumulus cells are associated with oocyte immaturity. Using the mouse cumulus-oocyte complex as an experimental model, the effects of elimination and overexpression of SERPINE2 in cumulus cells on cumulus expansion and oocyte maturation were assayed by in vitro maturation. Serpine2 and PLAU transcripts were the most highly expressed serpins and plasminogen activators, respectively. Their expression was coordinately regulated in cumulus cells during gonadotropin-induced oocyte maturation. Silencing of Serpine2 expression using small interfering RNAs or blockage of SERPINE2 protein using a specific antibody had no effect on oocyte maturation. However, overexpression of Serpine2 or exogenous supplementation with high levels of SERPINE2 impaired cumulus expansion and oocyte maturation, probably by decreasing hyaluronan synthase 2 (Has2) and versican (Vcan) mRNA expression. Amiloride, a specific PLAU inhibitor, also suppressed these processes. PLAU supplementation of the oocyte in vitro maturation medium caused earlier and more extensive expansion of cumulus cells and oocyte maturation that may be mediated by increased Has2 mRNA expression. However, these effects were neutralized by coincubation with SERPINE2 or amiloride and PLAU. In conclusion, SERPINE2 and PLAU are involved in cumulus expansion and oocyte maturation. High SERPINE2 levels impair these processes, probably by decreasing cumulus matrix gene expression as well as reducing cumulus hyaluronan contents and inhibiting PLAU activity. These findings may explain why cumulus cells surrounding immature human oocytes express high SERPINE2 levels.

  13. The interplay between tissue plasminogen activator domains and fibrin structures in the regulation of fibrinolysis: kinetic and microscopic studies

    PubMed Central

    Thelwell, Craig; Williams, Stella C.; Silva, Marta M. C. G.; Szabó, László; Kolev, Krasimir

    2011-01-01

    Regulation of tissue-type plasminogen activator (tPA) depends on fibrin binding and fibrin structure. tPA structure/function relationships were investigated in fibrin formed by high or low thrombin concentrations to produce a fine mesh and small pores, or thick fibers and coarse structure, respectively. Kinetics studies were performed to investigate plasminogen activation and fibrinolysis in the 2 types of fibrin, using wild-type tPA (F-G-K1-K2-P, F and K2 binding), K1K1-tPA (F-G-K1-K1-P, F binding), and delF-tPA (G-K1-K2-P, K2 binding). There was a trend of enzyme potency of tPA > K1K1-tPA > delF-tPA, highlighting the importance of the finger domain in regulating activity, but the differences were less apparent in fine fibrin. Fine fibrin was a better surface for plasminogen activation but more resistant to lysis. Scanning electron and confocal microscopy using orange fluorescent fibrin with green fluorescent protein-labeled tPA variants showed that tPA was strongly associated with agglomerates in coarse but not in fine fibrin. In later lytic stages, delF-tPA-green fluorescent protein diffused more rapidly through fibrin in contrast to full-length tPA, highlighting the importance of finger domain-agglomerate interactions. Thus, the regulation of fibrinolysis depends on the starting nature of fibrin fibers and complex dynamic interaction between tPA and fibrin structures that vary over time. PMID:20966169

  14. The interplay between tissue plasminogen activator domains and fibrin structures in the regulation of fibrinolysis: kinetic and microscopic studies.

    PubMed

    Longstaff, Colin; Thelwell, Craig; Williams, Stella C; Silva, Marta M C G; Szabó, László; Kolev, Krasimir

    2011-01-13

    Regulation of tissue-type plasminogen activator (tPA) depends on fibrin binding and fibrin structure. tPA structure/function relationships were investigated in fibrin formed by high or low thrombin concentrations to produce a fine mesh and small pores, or thick fibers and coarse structure, respectively. Kinetics studies were performed to investigate plasminogen activation and fibrinolysis in the 2 types of fibrin, using wild-type tPA (F-G-K1-K2-P, F and K2 binding), K1K1-tPA (F-G-K1-K1-P, F binding), and delF-tPA (G-K1-K2-P, K2 binding). There was a trend of enzyme potency of tPA > K1K1-tPA > delF-tPA, highlighting the importance of the finger domain in regulating activity, but the differences were less apparent in fine fibrin. Fine fibrin was a better surface for plasminogen activation but more resistant to lysis. Scanning electron and confocal microscopy using orange fluorescent fibrin with green fluorescent protein-labeled tPA variants showed that tPA was strongly associated with agglomerates in coarse but not in fine fibrin. In later lytic stages, delF-tPA-green fluorescent protein diffused more rapidly through fibrin in contrast to full-length tPA, highlighting the importance of finger domain-agglomerate interactions. Thus, the regulation of fibrinolysis depends on the starting nature of fibrin fibers and complex dynamic interaction between tPA and fibrin structures that vary over time.

  15. Inhibition of urokinase plasminogen activator “uPA” activity alters ethanol consumption and conditioned place preference in mice

    PubMed Central

    Al Maamari, Elyazia; Al Ameri, Mouza; Al Mansouri, Shamma; Bahi, Amine

    2014-01-01

    Urokinase plasminogen activator, uPA, is a serine protease implicated in addiction to drugs of abuse. Using its specific inhibitor, B428, we and others have characterized the role of uPA in the rewarding properties of psychostimulants, including cocaine and amphetamine, but none have examined the role of uPA in ethanol use disorders. Therefore, in the current study, we extended our observations to the role of uPA in ethanol consumption and ethanol-induced conditioned place preference. The general aim of the present series of experiments was to investigate the effects of the administration of the B428 on voluntary alcohol intake and ethanol conditioned reward. A two-bottle choice, unlimited-access paradigm was used to compare ethanol intake between vehicle- and 3, 10, and 30 mg/kg B428-administered mice. For this purpose, the mice were presented with an ethanol solution (2.5%–20%) and water, at each concentration for 4 days, and their consumption was measured daily. Consumption of saccharin and quinine solutions was also measured. Systemic administration of B428 dose-dependently decreased ethanol intake and preference. Additionally, B428 mice did not differ from vehicle mice in their intake of graded solutions of tastants, suggesting that the uPA inhibition did not alter taste function. Also, ethanol metabolism was not affected following B428 injection. More importantly, 1.5 g/kg ethanol-induced conditioned place preference acquisition was blocked following B428 administration. Taken together, our results are the first to implicate uPA inhibition in the regulation of ethanol consumption and preference, and suggest that uPA may be considered as a possible therapeutic drug target for alcoholism and abstinence. PMID:25258509

  16. Interaction of Leptospira elongation factor Tu with plasminogen and complement factor H: a metabolic leptospiral protein with moonlighting activities.

    PubMed

    Wolff, Danielly G; Castiblanco-Valencia, Mónica M; Abe, Cecília M; Monaris, Denize; Morais, Zenaide M; Souza, Gisele O; Vasconcellos, Sílvio A; Isaac, Lourdes; Abreu, Patrícia A E; Barbosa, Angela S

    2013-01-01

    The elongation factor Tu (EF-Tu), an abundant bacterial protein involved in protein synthesis, has been shown to display moonlighting activities. Known to perform more than one function at different times or in different places, it is found in several subcellular locations in a single organism, and may serve as a virulence factor in a range of important human pathogens. Here we demonstrate that Leptospira EF-Tu is surface-exposed and performs additional roles as a cell-surface receptor for host plasma proteins. It binds plasminogen in a dose-dependent manner, and lysine residues are critical for this interaction. Bound plasminogen is converted to active plasmin, which, in turn, is able to cleave the natural substrates C3b and fibrinogen. Leptospira EF-Tu also acquires the complement regulator Factor H (FH). FH bound to immobilized EF-Tu displays cofactor activity, mediating C3b degradation by Factor I (FI). In this manner, EF-Tu may contribute to leptospiral tissue invasion and complement inactivation. To our knowledge, this is the first description of a leptospiral protein exhibiting moonlighting activities.

  17. CXCL12-mediated induction of plasminogen activator inhibitor-1 expression in human CXCR4 positive astroglioma cells.

    PubMed

    Oh, Jae-Wook; Olman, Mitchell; Benveniste, Etty Nadia

    2009-04-01

    Glioblastoma is the most malignant and common brain tumor. To promote their growth, these glioma cells secrete a variety of soluble factors including plasminogen activator inhibitor-1 (PAI-1), which functions as an inhibitor of plasminogen activators. We report here with the basis of microarray gene expression analysis that CXCR4 expressing glioma cells are capable of expressing PAI-1 mRNA and protein upon CXCL12 stimulation. Pretreatment with U0126, an inhibitor of mitogen activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) 1/2, abrogated CXCL12-induced PAI-1 expression. Pertussis toxin (PTX), an inhibitor of Galpha(i) proteins, also had inhibitory effects, indicating that the activation of Galpha(i) and ERK MAPK are required for this response. Interestingly, CXCL12 showed additive effects with another PAI-1 inducers, tumor necrosis factor (TNF)-alpha and/or tumor growth factor (TGF)-beta1, in increasing PAI-1 expression. These results indicate that CXCL12/CXCR4 signaling in glioma cells may be another mechanism for these cells to express PAI-1, which may be involved in angiogenesis and tumor invasion in brain tumors.

  18. Beta-catenin up-regulates the expression of the urokinase plasminogen activator in human colorectal tumors.

    PubMed

    Hiendlmeyer, Elke; Regus, Susanne; Wassermann, Stella; Hlubek, Falk; Haynl, Angela; Dimmler, Arno; Koch, Claudia; Knoll, Claudia; van Beest, Moniek; Reuning, Ute; Brabletz, Thomas; Kirchner, Thomas; Jung, Andreas

    2004-02-15

    Expression of the urokinase plasminogen activator (uPA) increases during the progression of colorectal tumors from adenomas to carcinomas. The highest amounts of uPA are found at the invasion front of carcinomas, which also displays a strong expression of nuclear beta-catenin and is therefore a region expressing beta-catenin target genes at high levels. Here we show that beta-catenin contributes to the transactivation of uPA. Therefore, beta-catenin might have an impact on the capacity of colorectal tumors for invasion and metastasis, as well as dormancy, which are hallmarks of cancer.

  19. The retinal tolerance to bevacizumab in co‐application with a recombinant tissue plasminogen activator

    PubMed Central

    Lüke, Matthias; Januschowski, Kai; Warga, Max; Beutel, Julia; Leitritz, Martin; Gelisken, Faik; Grisanti, Salvatore; Schneider, Toni; Lüke, Christoph; Bartz‐Schmidt, Karl Ulrich; Szurman, Peter

    2007-01-01

    Aim To investigate the retinal toxicity of bevacizumab in co‐application with a commercially available recombinant tissue plasminogen activator (rt‐PA), and to facilitate a new therapeutic concept in the treatment of massive subretinal haemorrhage caused by neovascular age‐related macular degeneration (AMD). Methods Isolated bovine retinas were perfused with an oxygen‐preincubated nutrient solution. The electroretinogram (ERG) was recorded as a transretinal potential using Ag/AgCl electrodes. Bevacizumab (0.25 mg/ml) and rt‐PA (20 μg/ml) were added to the nutrient solution for 45 min. Thereafter, the retina was reperfused for 60 min with normal nutrient solution. Similarly, the effects of rt‐PA (20 μg/ml, 60 μg/ml and 200 μg/ml) on the a‐ and b‐wave amplitudes were investigated. The percentages of a‐ and b‐wave reduction during application and at washout were calculated. Results During application of bevacizumab (0.25 mg/ml) in co‐application with 20 μg/ml (rt‐PA), the ERG amplitudes remained stable. The concentrations of rt‐PA alone (20 μg/ml and 60 μg/ml) did not induce significant reduction of the b‐wave amplitude. In addition, 20 μg/ml rt‐PA did not alter the a‐wave amplitude. However, 60 μg/ml rt‐PA caused a slight but significant reduction of the a‐wave amplitude. A full recovery was detected for both concentrations during the washout. At the highest tested concentration of 200 μg/ml rt‐PA, a significant reduction of the a‐ and b‐wave amplitudes was provoked during the exposure. The reduction of ERG amplitudes remained irreversible during the washout. Conclusion The present study suggests that a subretinal injection of 20 µg/ml rt‐PA in co‐application with bevacizumab (0.25 mg/ml) for the treatment of massive subretinal haemorrhage seems possible. This is a safety study. Therefore, we did not test the clinical effectiveness of this combined treatment. PMID:17383998

  20. Inhibition of establishment of primary and micrometastatic tumors by a urokinase plasminogen activator receptor antagonist.

    PubMed

    Ignar, D M; Andrews, J L; Witherspoon, S M; Leray, J D; Clay, W C; Kilpatrick, K; Onori, J; Kost, T; Emerson, D L

    1998-01-01

    Tumor establishment and metastasis are dependent on extracellular matrix proteolysis, tumor cell migration, and angiogenesis. Urokinase plasminogen activator (uPA) and its receptor are essential mediators of these processes. The purpose of this study was to investigate the effect of a recombinant human uPAR antagonist on growth, establishment, and metastasis of tumors derived from human cancer cell lines. A noncatalytic recombinant protein, consisting of amino acids 1-137 of human uPA and the CH2 and CH3 regions of mouse IgG1 (uPA-IgG), was expressed, purified, and shown to bind specifically to human uPAR and to saturate the surface of human tumor cells which express uPAR. Daily i.p. administration of uPA-IgG to nude mice extended latencies of unstaged tumors derived from Lox melanoma and SW48 colon carcinoma cells by 7.7 and 5.5 days, respectively. uPA-IgG treatment did not affect the growth of Lox or KB tumors staged to 200 mg before antagonist treatment commenced. The effect of uPA-IgG on the establishment of micrometastases was assessed in SCID mice. KB head/neck tumor cells were injected in the tail vein and allowed to seed for 48 h before initiation of daily i.p. injections of uPA-IgG for 24 days. The number of lung colonies ranged between 5 and 30% of vehicle-treated mice in two separate experiments. Furthermore, a single 800 microg dose of uPA-IgG administered 1 h prior to tail vein injection of KB cells reduced lung colony formation to just 3.5% of vehicle-treated SCID mice. These data demonstrate that antagonism of uPAR arrested metastasis and inhibited the establishment of primary tumors and micrometastases. Thus, small molecule uPAR antagonists may serve as useful adjuvant agents in combination with existing cancer chemotherapy.

  1. Novel inhibitors of urokinase-type plasminogen activator and matrix metalloproteinase expression in metastatic cancer cell lines.

    PubMed

    Cakarovski, Kristina; Leung, Jenny Y; Restall, Christina; Carin-Carlson, Anna; Yang, Eunice; Perlmutter, Patrick; Anderson, Robin; Medcalf, Robert; Dear, Anthony E

    2004-07-01

    The plasminogen-activating (PA) and matrix metalloproteinase (MMP) enzyme systems are implicated in proteolytic turnover of the extracellular matrix (ECM) associated with biologic processes including wound healing, inflammation and angiogenesis. Aberrant expression of components of the PA and MMP enzyme systems occurs in the pathogenesis of metastatic cancer. Oxamflatin (Ox), a novel hydroxamic acid derivative, inhibits u-PA mRNA expression and proteolytic activity while simultaneously upregulating the expression of the natural inhibitor of u-PA, plasminogen activator inhibitor type 2 (PAI-2) in metastatic cancer cells. We have characterized the effects of Ox and a novel derivative, Metacept-1 (MCT-1), on PA and MMP-mediated proteolysis and invasion in several metastatic tumor lines. Both compounds are able to inhibit u-PA-, MMP-2- and MMP-9-mediated gene expression at low micromolar concentrations as well as u-PA- and MMP-mediated proteolysis as assessed by zymography, with MCT-1 being the more effective of the 2 agents in some assays. Cellular invasion assays correlate with gene expression and zymography experiments identifying both Ox and MCT-1 as able to inhibit invasion of metastatic cancer cell lines through matrigel at nanomolar concentrations, with MCT-1 more effective than Ox in 2 of the 3 cancer cell lines assessed.

  2. Lack of association between plasminogen activator inhibitor type-1 (PAI-1) gene 4G/5G polymorphism and osteoarthritis.

    PubMed

    Bayram, Banu; Sayin, Emrah; Erkasap, Nilüfer; Onlü, Harun; Ozkurt, Mete; Sahin, Fezan; Türkoğlu, Züleyha

    2012-01-01

    This study was conducted in Turkish osteoarthritis patients to determine the frequency of 4G/5G polymorphism genotypes of plasminogen activator inhibitor type-1 gene and to examine the role of this polymorphism in osteoarthritis development. Genomic DNA obtained from 200 persons (140 patients with osteoarthritis and 60 healthy controls) was used in the study. DNA was amplified by polymerase chain reaction using 4G allele- and 5G allele-specific primers. Polymerase chain reaction products were assessed with CCD camera by being exposed to 2% agarose gel electrophoresis. No statistically significant difference between the groups with respect to genotype distribution was found (P > 0.05) in the study. The 4G allele frequency was indicated as 44% and 5G allele was as 56% in patients, whereas this was 45-55% in the control group. This study has established that 4G/5G polymorphism genotypes of plasminogen activator inhibitor type-1 gene do not play a role in the development of osteoarthritis in the Turkish population.

  3. Urokinase-type plasminogen activator receptor (uPAR) as a promising new imaging target: potential clinical applications

    PubMed Central

    Persson, Morten; Kjaer, Andreas

    2013-01-01

    Urokinase-type plasminogen activator receptor (uPAR) has been shown to be of special importance during cancer invasion and metastasis. However, currently, tissue samples are needed for measurement of uPAR expression limiting the potential as a clinical routine. Therefore, non-invasive methods are needed. In line with this, uPAR has recently been identified as a very promising imaging target candidate. uPAR consists of three domains attached to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor and binds it natural ligand uPA with high affinity to localize plasminogen activation at the cell surface. Due to the importance of uPAR in cancer invasion and metastasis, a number of high-affinity ligands have been identified during the last decades. These ligands have recently been used as starting point for the development of a number of ligands for imaging of uPAR using various imaging modalities such as optical imaging, magnetic resonance imaging, single photon emission computer tomography (SPECT) and positron emission topography (PET). In this review, we will discuss recent advances in the development of uPAR-targeted imaging ligands according to imaging modality. In addition, we will discuss the potential future clinical application for uPAR imaging as a new imaging biomarker. PMID:23701192

  4. Expression of plasminogen activator-related genes in the adipose tissue of lactating dairy sheep in the early post-weaning period.

    PubMed

    Theodorou, G; Lampidonis, A D; Laliotis, G P; Bizelis, I; Politis, I

    2012-06-01

    There is growing evidence that plasminogen activator inhibitor type 1 (PAI-1) is expressed in adipose tissue and its expression is implicated in inflammation that accompanies obesity-associated diseases. The physiological role of other genes implicated in the plasminogen-activating cascade such as urokinase-type plasminogen activator (u-PA), u-PA receptor (u-PAR) and plasminogen activator inhibitor type 2 (PAI-2) in ovine adipose tissue remains unknown. The objective of this study was to examine the changes in the expression of four plasminogen activator (PA)-related genes during the early post-weaning period in dairy ewes. A total of 21 subcutaneous adipose tissue samples were obtained from seven lactating dairy ewes of the Chios breed at weeks 1, 2 and 4 after weaning. Results indicated that expression of all PA-related genes was detected in most of the samples examined. Greatest expression of u-PAR corresponded to highest (week 1), while greatest expression of PAI-2 corresponded to lowest (week 4) rate of lipolysis, as indicated by the expression of hormone-sensitive lipase, in the ovine adipose tissue. There were no significant differences in the expression of the other two PA-related genes (u-PA, PAI-1) throughout the experimental period. Plasminogen activator-related genes are not expressed in a coordinated manner in the adipose tissue of lactating dairy sheep in the early post-weaning period. In conclusion, adipose tissue mobilization is correlated with highest expression of u-PAR and lowest expression of PAI-2.

  5. Intravenous tissue plasminogen activator and size of infarct, left ventricular function, and survival in acute myocardial infarction.

    PubMed Central

    Van de Werf, F.; Arnold, A. E.

    1988-01-01

    STUDY OBJECTIVE--To assess effect of intravenous recombinant tissue type plasminogen activator on size of infarct, left ventricular function, and survival in acute myocardial infarction. DESIGN--Double blind, randomised, placebo controlled prospective trial of patients with acute myocardial infarction within five hours after onset of symptoms. SETTING--Twenty six referral centres participating in European cooperative study for recombinant tissue type plasminogen activator. PATIENTS--Treatment group of 355 patients with acute myocardial infarction allocated to receive intravenous recombinant plasminogen activator. Controls comprised 366 similar patients allocated to receive placebo. INTERVENTION--All patients were given aspirin 250 mg and bolus injection of 5000 IU heparin immediately before start of trial. Patients in treatment group were given 100 mg recombinant tissue plasminogen activator over three hours (10 mg intravenous bolus, 50 mg during one hour, and 40 mg during next two hours) by infusion. Controls were given placebo by same method. Full anticoagulation treatment and aspirin were given to both groups until angiography (10-22 days after admission). beta Blockers were given at discharge. END POINT--Left ventricular function at 10-22 days, enzymatic infarct size, clinical course, and survival to three month follow up. MEASUREMENTS AND MAIN RESULTS--Mortality was reduced by 51% (95% confidence interval -76 to 1) in treated patients at 14 days after start of treatment and by 36% (-63 to 13) at three months. For treatment within three hours after myocardial infarction mortality was reduced by 82% (-95 to -31) at 14 days and by 59% (-83 to -2) at three months. During 14 days in hospital incidence of cardiac complications was lower in treated patients than controls (cardiogenic shock, 2.5% v 6.0%; ventricular fibrillation, 3.4% v 6.3%; and pericarditis, 6.2% v 11.0% respectively), but that of angioplasty or artery bypass, or both was higher (15.8% v 9

  6. The profibrinolytic enzyme subtilisin NAT purified from Bacillus subtilis Cleaves and inactivates plasminogen activator inhibitor type 1.

    PubMed

    Urano, T; Ihara, H; Umemura, K; Suzuki, Y; Oike, M; Akita, S; Tsukamoto, Y; Suzuki, I; Takada, A

    2001-07-06

    In this report, we demonstrate an interaction between subtilisin NAT (formerly designated BSP, or nattokinase), a profibrinolytic serine proteinase from Bacillus subtilis, and plasminogen activator inhibitor 1 (PAI-1). Subtilisin NAT was purified to homogeneity (molecular mass, 27.7 kDa) from a saline extract of B. subtilis (natto). Subtilisin NAT appeared to cleave active recombinant prokaryotic PAI-1 (rpPAI-1) into low molecular weight fragments. Matrix-assisted laser desorption/ionization in combination with time-of-flight mass spectroscopy and peptide sequence analysis revealed that rpPAI-1 was cleaved at its reactive site (P1-P1': Arg(346)-Met(347)). rpPAI-1 lost its specific activity after subtilisin NAT treatment in a dose-dependent manner (0.02-1.0 nm; half-maximal effect at approximately 0.1 nm). Subtilisin NAT dose dependently (0.06-1 nm) enhanced tissue-type plasminogen activator-induced fibrin clot lysis both in the absence of rpPAI-1 (48 +/- 1.4% at 1 nm) and especially in the presence of rpPAI-1 (78 +/- 2.0% at 1 nm). The enhancement observed in the absence of PAI-1 seems to be induced through direct fibrin dissolution by subtilisin NAT. The stronger enhancement by subtilisin NAT of rpPAI-1-enriched fibrin clot lysis seems to involve the cleavage and inactivation of active rpPAI-1. This mechanism is suggested to be important for subtilisin NAT to potentiate fibrinolysis.

  7. Clearance of the heavy and light polypeptide chains of human tissue-type plasminogen activator in rats.

    PubMed Central

    Rijken, D C; Emeis, J J

    1986-01-01

    In order to assess which part of the tissue-type plasminogen activator (t-PA) molecule should be (genetically) modified to obtain more-slowly-clearing mutants, two-chain t-PA and its isolated heavy and light chains were radiolabelled and injected into rats. The vast majority of t-PA and the heavy chain disappeared from the blood circulation with half-lives of 2.3 and 1.0 min respectively. The clearance of the light chain was biphasic, owing to complex-formation with plasma proteinase inhibitors. The disappearance of di-isopropylphospho-light chain, which has a blocked active site, was nearly monophasic, with a half-life of 5.7 min. Organ distribution studies showed that hepatic clearance constituted the major pathway in all cases. These results strongly suggest that t-PA is recognized by the liver primarily through the heavy chain. PMID:3099771

  8. Role of plasminogen activator inhibitor in the reciprocal regulation of bovine aortic endothelial and smooth muscle cell migration by TGF-beta 1.

    PubMed Central

    Petzelbauer, E.; Springhorn, J. P.; Tucker, A. M.; Madri, J. A.

    1996-01-01

    Vascular endothelial and smooth muscle cells exhibit reciprocal migratory responses after transforming growth factor (TGF)-beta 1 treatment. Endothelial cells exhibit a decreased migratory rate and smooth muscle cells exhibit an increased migratory rate. Previous studies have demonstrated increases in extracellular matrix and integrin synthesis and expression in response to TGF-beta 1. In this report, we illustrate the roles of plasminogen activator inhibitor in modulating the migratory rates in these two cell types. Endothelial cells appear to require a proteolytic phenotype for rapid migration, whereas vascular smooth muscle cells appear to require an anti-proteolytic phenotype. Modulation of proteinase/anti-proteinase activity ratios was accomplished via TGF-beta 1 induction, addition of exogenous plasminogen activator inhibitor, addition of anti-catalytic antibodies directed against urokinase plasminogen activator, overexpression of plasminogen activator inhibitor utilizing stable transfectants, and the use of vitronectin as a substratum. The reciprocal migratory behaviors exhibited by these two vascular cell types in response to TGF-beta 1 is discussed in the context that these two vascular cell types utilize distinct adhesive and signaling pathways in their interactions with extracellular matrix components and responsiveness to proteolytic activity. Images Figure 1 Figure 2 Figure 3 PMID:8780396

  9. Regulation of proteinases during mouse peri-implantation development: urokinase-type plasminogen activator expression and cross talk with matrix metalloproteinase 9.

    PubMed

    Martínez-Hernández, M G; Baiza-Gutman, L A; Castillo-Trápala, A; Armant, D Randall

    2011-02-01

    Trophoblast cells express urokinase-type plasminogen activator (PLAU) and may depend on its activity for endometrial invasion and tissue remodeling during peri-implantation development. However, the developmental regulation, tissue distribution, and function of PLAU are not completely understood. In this study, the expression of PLAU and its regulation by extracellular matrix proteins was examined by RT-PCR, immunocytochemistry, and plasminogen-casein zymography in cultured mouse embryos. There was a progressive increase in Plau mRNA expression in blastocysts cultured on gestation days 4-8. Tissue-type plasminogen activator (55 kDa) and PLAU (a triplet of 40, 37, and 31 kDa) were present in conditioned medium and embryo lysates, and were adsorbed to the culture plate surface. The temporal expression pattern of PLAU, according to semi-quantitative gel zymography, was similar in non-adhering embryos and embryos cultured on fibronectin, laminin, or type IV collagen, although type IV collagen and laminin upregulated Plau mRNA expression. Immunofluorescence revealed PLAU on the surface of the mural trophectoderm and in non-spreading giant trophoblast cells. Exogenous human plasminogen was transformed to plasmin by cultured embryos and activated endogenous matrix metalloproteinase 9 (MMP9). Indeed, the developmental expression profile of MMP9 was similar to that of PLAU. Our data suggest that the intrinsic developmental program predominantly regulates PLAU expression during implantation, and that PLAU could be responsible for activation of MMP9, leading to localized matrix proteolysis as trophoblast invasion commences.

  10. Challenging delivery of VLHL NS plasminogen activator inhibitor-1 by osmotic pumps in diabetic mouse: A case report.

    PubMed

    Jankun, Jerzy

    2012-10-01

    ALZET(®) osmotic pumps are implantable devices used in animals for the continuous infusion of drugs or proteins at controlled rates from 1 day to 4 weeks. Pumps have been used successfully in a number of studies on the effects of controlled delivery of a wide range of experimental agents, independent of their properties. In the present study, use of these pumps was made in mice with diabetic nephropathy. Plasminogen activator inhibitor-1 (PAI-1) mediates diabetic nephropathy, which is characterized by the excessive accumulation of extracellular matrix (ECM) in the kidney. Disproportionate PAI-1 inactivates tissue plasminogen activator, which is one of the proteolytic enzymes in a cascade responsible for ECM remodeling in the kidney. The decrease of PAI-1 in the kidney has been shown to arrest the progression of nephropathy in experimental animals. This was achieved using inactive PAI-1R which increased the clearance of wild-type PAI-1 in order to protect net proteolytic activity and ECM clearance. However, this protein has a brief half-life in vivo, therefore, high and frequent doses are required. Thus, VLHL NS PAI-1 protein with a long half-life of over 700 h (Gln197Cys, Gly355Cys) inactivated by single point mutation (Arg369Ala) was used. Following the sacrifice of animals the tips of the flow moderators of the osmotic pumps in the treated animals were found to be clogged. In addition, from each pump from the treatment group, but not controls, we collected 50-150 μl of clear liquid containing VLHL NS PAI-1, cellular and serum proteins suggesting early pump sealing by cellular material. In conclusion, despite encouraging results obtained for the PAI-1R protein, the method of VLHL PAI-1 delivery should be ameliorated.

  11. Immunoradiometric quantitation of tissue plasminogen activator-related antigen in human plasma: crypticity phenomenon and relationship to plasma fibrinolysis

    SciTech Connect

    Wun, T.C.; Capuano, A.

    1987-05-01

    A two-site immunoradiometric assay for tissue plasminogen activator (tPA) antigen has been developed using immunoaffinity purified antibody. Various treatments enhanced the detection of tPA antigen in the plasma samples. Maximum detection was obtained by acidification of plasma to pH 4.8 to 6.5 or addition of 0.5 mol/L of L-lysine or L-arginine. Acidification or addition of lysine to plasma is also required for maximum immunoadsorption of plasma tPA antigen on anti-tPA-Ig-sepharose. These results indicate that plasma tPA antigen is partially cryptic to antibody in untreated plasma. The plasma tPA antigen isolated by immunoadsorption of either untreated plasma or acidified plasma on anti-tPA-Ig-sepharose consists mainly of a 100-kd plasminogen activator species as determined by fibrin-agar zymography. The 100-kd activity is possibly a tPA:inhibitor complex. A standardized sample preparation method was conveniently adopted by mixing 3 vol of plasma and 1 vol of 2 mol/L of L-lysine for the assay. Reconstitution and recovery studies showed that the method is specific and permits full detection of both free tPA and tPA:inhibitor complex. The validity of the assay is further supported by the finding that the spontaneous plasma fibrinolysis previously demonstrated to be dependent on plasma tPA antigen is correlated with tPA antigen content. Using the standardized assay, we found that tPA antigen concentrations in 16 blood bank plasmas are equivalent to 3.7 to 20 ng of 60 kd tPA/mL. In all the plasma tested, more than half of the antigen is undetected unless the plasma is treated as described above.

  12. Effects of Lewis lung carcinoma on trabecular microstructural changes in wild-type and plasminogen activator inhibitor-1 deficient mice fed a high-fat diet

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bone is a major target organ of metastasis. The present study investigated the effects of Lewis lung carcinoma (LLC) on trabecular microstructural changes, using tomographic analysis, in distal femur and lumbar 4 vertebra from LLC-bearing wild-type and plasminogen activator inhibitor-1 (PAI-1) defi...

  13. PULMONARY LOCALIZATION AND EXPRESSION OF PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1) IN HEALTHY OR HYPERTENSIVE RATS EXPOSED TO PARTICULATE MATTER (PM)

    EPA Science Inventory

    PULMONARY LOCALIZATION AND EXPRESSION OF PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1) IN HEALTHY OR HYPERTENSIVE RATS EXPOSED TO PARTICULATE MATTER (PM). GS Backus1, R Vincent2, UP Kodavanti2, 1Curriculum in Toxicology, UNC, Chapel Hill; 2NHEERL, ORD, US EPA, Research Triangle Park,...

  14. Effects of a high-fat diet on spontaneous metastasis of Lewis lung carcinoma in plasminogen activator inhibitor-1 deficient and wild-type mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We investigated the effects of plasminogen activator inhibitor-1 (PAI-1) deficiency on spontaneous metastasis of Lewis lung carcinoma (LLC) in PAI-1 deficient (PAI-1-/-) and wildtype mice (C57BL/6J background) fed the AIN93G diet or that diet modified with 45% calories from fat. The high-fat diet i...

  15. Fiber intake and plasminogen activator inhibitor-1 in type 2 diabetes: Look AHEAD (Action for Health in Diabetes) Trial findings at baseline and 1 year

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plasminogen activator inhibitor 1 (PAI-1) is elevated in obese individuals with type 2 diabetes and may contribute, independently of traditional factors, to increased cardiovascular disease risk. Fiber intake may decrease PAI-1 levels. We examined the associations of fiber intake and its changes wit...

  16. Fontan patient with plastic bronchitis treated successfully using aerosolized tissue plasminogen activator: a case report and review of the literature.

    PubMed

    Do, Thomas B; Chu, James M; Berdjis, Farhouch; Anas, Nick G

    2009-04-01

    Plastic bronchitis is an uncommon condition characterized by the production of large pale bronchial casts that obstruct the tracheobronchial tree. The cellular content, cohesiveness, and often rubber-like consistency distinguish bronchial casts from the usual mucus plugs found with such disease states as asthma. Plastic bronchitis can be found secondary to many conditions, and a simplified classification scheme organizes it into two groups: an inflammatory type consisting of casts with an eosinophilic inflammatory infiltrate and an acellular type with a predominance of fibrin distinguished by its relative lack of cellular infiltrate, its mucin predominance, and its appearance only in children with congenital cyanotic heart disease. This report describes a 5-year-old girl who experienced plastic bronchitis 3 months after a Fontan procedure for hypoplastic left heart syndrome that was treated successfully with aerosolized tissue plasminogen activator.

  17. Effect of Vitreoscilla hemoglobin expression on growth and specific tissue plasminogen activator productivity in recombinant Chinese hamster ovary cells

    SciTech Connect

    Pendse, G.J.; Bailey, J.E. . Dept. of Chemical Engineering)

    1994-12-01

    Previous studies suggest that secretion of cloned proteins synthesized by recombinant Chinese hamster ovary (CHO) cells can be adenosine triphosphate (ATP) limited. Other research indicates that the presence of cloned Vitreoscilla hemoglobin (VHb) enhances ATP production in oxygen-limited Escherichia coli. To evaluate the influence of VHb expression on recombinant CHO cell productivity, the vhb gene has been fused to the mouse mammary tumor virus (MMTV) promoter and cloned in a CHO cell line previously engineered to express human tissue plasminogen activator (tPA). Western blot analysis confirms dexamethasone-inducible VHb expression in all of the clones tested. Batch cultivation experiments with one VHb-expressing clone and the parental CHO-tPA cells show a reduced specific growth rate in the VHb-expressing cells. The VHb-expressing clone exhibits specific tPA production 40 to 100% greater than the parental CHO-tPA culture.

  18. Ethanol-withdrawal seizures are controlled by tissue plasminogen activator via modulation of NR2B-containing NMDA receptors.

    PubMed

    Pawlak, Robert; Melchor, Jerry P; Matys, Tomasz; Skrzypiec, Anna E; Strickland, Sidney

    2005-01-11

    Chronic ethanol abuse causes up-regulation of NMDA receptors, which underlies seizures and brain damage upon ethanol withdrawal (EW). Here we show that tissue-plasminogen activator (tPA), a protease implicated in neuronal plasticity and seizures, is induced in the limbic system by chronic ethanol consumption, temporally coinciding with up-regulation of NMDA receptors. tPA interacts with NR2B-containing NMDA receptors and is required for up-regulation of the NR2B subunit in response to ethanol. As a consequence, tPA-deficient mice have reduced NR2B, extracellular signal-regulated kinase 1/2 phosphorylation, and seizures after EW. tPA-mediated facilitation of EW seizures is abolished by NR2B-specific NMDA antagonist ifenprodil. These results indicate that tPA mediates the development of physical dependence on ethanol by regulating NR2B-containing NMDA receptors.

  19. The crystal structures of 3-TAPAP in complexes with the urokinase-type plasminogen activator and picrate.

    PubMed

    Zesławska, Ewa; Jacob, Uwe; Stürzebecher, Jörg; Oleksyn, Barbara J

    2006-01-01

    The urokinase-type plasminogen activator (uPA) is a protein involved in tissue remodeling and other biological processes. The inhibitors of uPA have been shown to prevent the spread of metastasis and tumor growth, and accordingly this enzyme is widely accepted as a promising anticancer target. In this work, we have investigated the conformation of the uPA inhibitor 3-TAPAP in two different crystalline environments of a picrate and a uPA complex. These structures were compared to the known structure of the 3-TAPAP in the complex with trypsin. In the complexes with the proteins, trypsin, and uPA, the binding mode of 3-TAPAP is similar. A larger difference in the conformation, in the comparison to these structures, has been observed by us in the 3-TAPAP picrate crystal. This observation contradicts the hypothesis that 3-TAPAP derivatives inhibit serine proteinases in preformed stable conformations.

  20. Scale up and pharmacokinetic study of a novel mutated chimeric tissue plasminogen activator (mt-PA) in rats

    PubMed Central

    Raigani, Mozhgan; Rouini, Mohammad-Reza; Golabchifar, Ali-Akbar; Mirabzadeh, Esmat; Vaziri, Behrouz; Barkhordari, Farzaneh; Davami, Fatemeh; Mahboudi, Fereidoun

    2017-01-01

    Because of high mortality caused by cardiovascular diseases, various fibrinolytic agents with diverse pharmacokinetic and pharmacodynamic properties have been developed. A novel mutated chimeric tissue plasminogen activator (mt-PA) was developed by the removal of first three domains of t-PA, insertion of GHRP sequence and mutation towards resistance to plasminogen activator inhibitor-1 (PAI-1). Mt-PA protein was expressed in Expi293F cells. The expression level of mt-PA was found to be 5000 IU/mL. Following purification, the pharmacokinetic properties of mt-PA were evaluated in three doses in rats. Data related to mt-PA were best fitted to two compartment model. With the increase in dose, the Area Under the plasma concentration-time Curve (AUC0→∞) increased. The elimination half-life (t1/2) of mt-PA was in the range of 19.1–26.1 min in three doses while that of Alteplase was 8.3 min. The plasma clearance (CLp) of mt-PA ranged from 3.8 to 5.9 mL/min in three doses, which was several times lower than that of Alteplase (142.6 mL/min). The mean residence time (MRT) of mt-PA ranged from 23.3–31.8 min in three doses, which was 4–5 times greater than that of Alteplase (6 min). Mt-PA showed extended half-life and mean residence time and is a good candidate for further clinical studies. PMID:28223717

  1. The Clinical Value of Soluble Urokinase Plasminogen Activator Receptor (suPAR) Levels in Autoimmune Connective Tissue Disorders

    PubMed Central

    Toldi, Gergely; Balog, Attila

    2016-01-01

    The assessment of the general inflammatory condition of patients with autoimmune connective tissue disorders (ACTD) is a major challenge. The use of traditional inflammatory markers including CRP-levels and erythrocyte sedimentation rate (ESR) is limited by several preanalytical factors and their low specificities. Soluble urokinase plasminogen activator receptor (suPAR) is one of the novel candidate markers that is increasingly used in immune mediated disorders. In our studies we compared suPAR levels of patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), systemic sclerosis (SSc) and ankylosing spondylitis with those of healthy controls. suPAR provided valuable clinical information on disease activity in RA, SLE and SSc. We identified a subgroup of remitted RA patients, who presented still clinical symptoms of inflammatory activity which correlated to high plasma suPAR (while ESR and CRP were normal). In SLE we established specific suPAR cut-off values that support the discrimination between patients with high and those with moderate SLE activity. In patients with SSc suPAR correlated with objective measures of lung and other complications. In the majority of ACTDs including SLE, SSc or RA, suPAR is seemingly a good biomarker that would provide valuable clinical information. However, before the introduction of this novel parameter in laboratory repertoire important issues should be elucidated. These include the establishment of appropriate and disease specific cutoff values, clarification of interfering preanalytical values and underlying conditions and declaration of age- and gender-specific reference ranges. PMID:27683525

  2. Expression and large-scale production of the biochemically active human tissue-plasminogen activator in hairy roots of Oriental melon (Cucumis melo).

    PubMed

    Kim, Sung-Ryong; Sim, Joon-Soo; Ajjappala, Hemavathi; Kim, Yong-Hwan; Hahn, Bum-Soo

    2012-01-01

    Human tissue-plasminogen activator (t-PA) is a thrombolytic protein that plays an active role in dissolving fibrin clots by fibrinolysis and in activating plasminogen to plasmin in blood vessels. t-PA and synthetic t-PA (st-PA) genes were expressed as enzymatically active form in hairy roots of Oriental melon (Cucumis melo L. cv. Geumssaragi-euncheon) infected by Agrobacterium rhizogenes. The insertion of the t-PA genes in genomic DNA of transgenic hairy roots was verified by PCR. The presence and expression of t-PA-specific transcripts in the total RNAs of transgenic hairy roots were confirmed by RT-PCR. Western blot analysis of the transgenic hairy roots showed a single major band of 59-kDa recombinant t-PAs. ELISA demonstrated that the highest level of recombinant t-PA (798 ng mg⁻¹) was detected in hairy roots expressing t-PA. Similarly, the maximum fibrinolysis of recombinant t-PAs was observed in hairy roots transformed with t-PA. WPM medium was found to be more suitable for rapid growth of hairy roots among all the seven media types tested. The hairy root production was 5.8 times higher than that of White medium. The total yield of hairy roots grown on WPM medium was 621.8±8.7 g L⁻¹ at pH 7.0. These studies demonstrate that the hairy roots could be employed for the mass production of enzymatically active t-PA.

  3. Protease-Activated Receptor 2 (PAR2) Is Upregulated by Acanthamoeba Plasminogen Activator (aPA) and Induces Proinflammatory Cytokine in Human Corneal Epithelial Cells

    PubMed Central

    Tripathi, Trivendra; Abdi, Mahshid; Alizadeh, Hassan

    2014-01-01

    Purpose. Acanthamoeba plasminogen activator (aPA) is a serine protease elaborated by Acanthamoeba trophozoites that facilitates the invasion of trophozoites to the host and contributes to the pathogenesis of Acanthamoeba keratitis (AK). The aim of this study was to explore if aPA stimulates proinflammatory cytokine in human corneal epithelial (HCE) cells via the protease-activated receptors (PARs) pathway. Methods. Acanthamoeba castellanii trophozoites were grown in peptone-yeast extract glucose for 7 days, and the supernatants were collected and centrifuged. The aPA was purified using the fast protein liquid chromatography system, and aPA activity was determined by zymography assays. Human corneal epithelial cells were incubated with or without aPA (100 μg/mL), PAR1 agonists (thrombin, 10 μM; TRAP-6, 10 μM), and PAR2 agonists (SLIGRL-NH2, 100 μM; AC 55541, 10 μM) for 24 and 48 hours. Inhibition of PAR1 and PAR2 involved preincubating the HCE cells for 1 hour with the antagonist of PAR1 (SCH 79797, 60 μM) and PAR2 (FSLLRY-NH2, 100 μM) with or without aPA. Human corneal epithelial cells also were preincubated with PAR1 and PAR2 antagonists and then incubated with or without PAR1 agonists (thrombin and TRAP-6) and PAR2 agonists (SLIGRL-NH2 and AC 55541). Expression of PAR1 and PAR2 was examined by quantitative RT-PCR (qRT-PCR), flow cytometry, and immunocytochemistry. Interleukin-8 expression was quantified by qRT-PCR and ELISA. Results. Human corneal epithelial cells constitutively expressed PAR1 and PAR2 mRNA. Acanthamoeba plasminogen activator and PAR2 agonists significantly upregulated PAR2 mRNA expression (1- and 2-fold, respectively) (P < 0.05). Protease-activated receptor 2 antagonist significantly inhibited aPA, and PAR2 agonists induced PAR2 mRNA expression in HCE cells (P < 0.05). Protease-activated receptor 1 agonists, but not aPA, significantly upregulated PAR1 mRNA expression, which was significantly inhibited by PAR1 antagonist in HCE cells

  4. Caveolin-1 mediates tissue plasminogen activator-induced MMP-9 up-regulation in cultured brain microvascular endothelial cells.

    PubMed

    Jin, Xinchun; Sun, Yanyun; Xu, Ji; Liu, Wenlan

    2015-03-01

    Thrombolysis with tissue plasminogen activator (tPA) increases matrix metalloproteinase-9 (MMP-9) activity in the ischemic brain, which exacerbates blood-brain barrier injury and increases the risk of symptomatic cerebral hemorrhage. The mechanism through which tPA enhances MMP-9 activity is not well understood. Here we report an important role of caveolin-1 in mediating tPA-induced MMP-9 synthesis. Brain microvascular endothelial cell line bEnd3 cells were incubated with 5 or 20 μg/ml tPA for 24 hrs before analyzing MMP-9 levels in the conditioned media and cellular extracts by gelatin zymography. tPA at a dose of 20 μg/mL tPA, but not 5 μg/mL, significantly increased MMP-9 level in cultured media while decreasing it in cellular extracts. Concurrently, tPA treatment induced a 2.3-fold increase of caveolin-1 protein levels in endothelial cells. Interestingly, knockdown of Cav-1 with siRNA inhibited tPA-induced MMP-9 mRNA up-regulation and MMP-9 increase in the conditioned media, but did not affect MMP-9 decrease in cellular extracts. These results suggest that caveolin-1 critically contributes to tPA-mediated MMP-9 up-regulation, but may not facilitate MMP-9 secretion in endothelial cells. Thrombolysis with tissue plasminogen activator (tPA) increases matrix metalloproteinase-9 (MMP-9) activity in the ischemic brain, which exacerbates ischemic blood brain barrier (BBB) injury and increases the risk of symptomatic cerebral hemorrhage. Our results suggest a novel mechanism underlying this tPA-MMP 9 axis. In response to tPA treatment, caveolin-1 protein levels increased in endothelial cells, which mediate MMP-9 mRNA up-regulation and its secretion into extracellular space. Caveolin-1 may, however, not facilitate MMP-9 secretion in endothelial cells. Our data suggest caveolin-1 as a novel therapeutic target for protecting the BBB against ischemic damage. The schematic outlines tPA-induced MMP-9 upreguation.

  5. Intrapleural low-molecular-weight urokinase or tissue plasminogen activator versus single-chain urokinase in tetracycline-induced pleural loculation in rabbits.

    PubMed

    Idell, Steven; Azghani, Ali; Chen, Shande; Koenig, Kathy; Mazar, Andrew; Kodandapani, Lalitha; Bdeir, Khalil; Cines, Douglas; Kulikovskaya, Irina; Allen, Timothy

    2007-01-01

    The authors compared the ability of a single dose of the proenzyme single-chain urokinase (scuPA), low-molecular-weight urokinase, tissue plasminogen activator (tPA), or a mutant site-inactive scuPA to resolve intrapleural loculations at 72 to 96 hours after tetracycline-induced pleural injury in rabbits. Both scuPA and tPA reversed loculations at 96 hours after injury P < or = .001, whereas low-molecular-weight urokinase and the scuPA mutant were ineffective. scuPA and tPA generated inhibitor complexes, induced fibrinolytic activity, and quenched plasminogen activator-1 activity in pleural fluids. The authors conclude that scuPA reverses loculations as effectively as tPA at clinically applied intrapleural doses, whereas low-molecular-weight urokinase was ineffective.

  6. Successful arthroscopic treatment of pigmented villonodular synovitis of the knee in a patient with congenital deficiency of plasminogen activator inhibitor-1 and recurrent haemarthrosis.

    PubMed

    Matsui, H; Takahashi, Y; Matsunaga, T; Tanaka-Horie, T; Minowa, H; Sugimoto, M; Tsukino, R; Mii, Y; Giddings, J; Yoshioka, A

    2001-01-01

    We report the arthroscopic treatment of pigmented villonodular synovitis (PVNS) in a 13-year-old Japanese boy with congenital partial deficiency of plasminogen activator inhibitor-1 (PAI-1). He was admitted to our hospital with recurrent haemarthrosis of his right knee. Characteristic abnormalities of fibrinolysis included shortened euglobulin lysis time, low PAI-1 activity and low PAI-1 antigen levels. In addition, levels of "active PAI" in the plasma, which is a measure of total PAI bound to exogenous plasminogen activator, were very low. These parameters remained low after venous occlusion. The diagnosis of PVNS was established by synovial membrane biopsy, and arthroscopic synovectomy was performed with adjuvant administration of intravenous tranexamic acid. Subsequent bleeding episodes have been well controlled by oral administration of tranexamic acid on demand.

  7. Insulin and insulin-like growth factor I exert different effects on plasminogen activator production or cell growth in the ovine thyroid cell line OVNIS.

    PubMed

    Degryse, B; Maisonobe, F; Hovsépian, S; Fayet, G

    1991-11-01

    Insulin and Insulin-like Growth Factor I (IGF-I) are evaluated for their capacity to affect cell proliferation and plasminogen activator (PA) activity production in an ovine thyroid cell line OVNIS. Insulin at physiological and supraphysiological doses induces cell proliferation and increases PA activity. IGF-I, which is also clearly mitogenic for these cells, surprisingly does not modulate PA activity. The results indicate that the growth promoting effect is mediated through the insulin and IGF-I receptors whereas PA activity is solely regulated via the insulin receptors.

  8. Oxidative modification enhances lipoprotein(a)-induced overproduction of plasminogen activator inhibitor-1 in cultured vascular endothelial cells.

    PubMed

    Ren, S; Man, R Y; Angel, A; Shen, G X

    1997-01-03

    Elevated levels of plasma lipoprotein (a) [Lp(a)] have been considered as a strong risk factor for premature cardiovascular diseases. Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of plasminogen activators (PA). Increases in PAI-1 levels with or without a reduction in PA levels have been frequently found in coronary artery disease patients. The present paper examined the effects of oxidized Lp(a) on the production of PAI-1 in cultured human umbilical vein endothelial cells (HUVEC). Lp(a) and Lp(a)-free, low density lipoprotein (LDL) were prepared using lysine-Sepharose 4B affinity chromatography. Incubations with 10(-8) M levels of native Lp(a) moderately increased the levels of biologically active PAI-1 in post-culture medium of HUVEC compared to that with equimolar concentrations of native Lp(a)-free LDL. The release of PAI-1 induced by Lp(a) was enhanced by oxidative modification with copper ion. The stimulation of oxidized Lp(a) on PAI-1 production reached plateau in EC treated with 10-20 nM oxidized Lp(a) modified by microM CuSO4. Treatment with 0.2 micrograms/ml of actinomycin D significantly reduced native and oxidized Lp(a)-induced PAI-1 overproduction in EC. Increases in the steady state levels of PAI-1 mRNA were detected in native or oxidized Lp(a)-treated EC. The effect of Lp(a)-free oxidized LDL on PAI-1 production was significantly weaker than the equimolar amount of oxidized Lp(a) but stronger than that of native LDL. Treatments with oxidized Lp(a) increased cell-associated PAI-1 to a similar extent as that in native Lp(a)-treated EC. The results of the present paper demonstrate that oxidative modification enhances Lp(a)-induced PAI-1 production in vascular endothelial cells at RNA transcription level, which suggests that oxidization potentially amplifies the anti-fibrinolytic and thrombotic effect of Lp(a).

  9. The association between the 4G/5G polymorphism in the promoter of the plasminogen activator inhibitor-1 gene and extension of postsurgical calf vein thrombosis.

    PubMed

    Ferrara, Filippo; Meli, Francesco; Raimondi, Francesco; Montalto, Salvatore; Cospite, Valentina; Novo, Giuseppina; Novo, Salvatore

    2013-04-01

    The objective of this study was to evaluate whether the presence of a plasminogen activator inhibitor type 1 (PAI-1) promoter polymorphism 4G/5G could significantly influence the proximal extension of vein thrombosis in spite of anticoagulant treatment in patients with calf vein thrombosis (CVT) following orthopaedic, urological and abdominal surgery. We studied 168 patients with CVT, who had undergone orthopaedic, urological and abdominal surgery, subdivided as follows: first, 50 patients with thrombosis progression; second, 118 patients without thrombosis progression. The 4G/5G polymorphism of the plasminogen activator inhibitor 1 was evaluated in all patients and in 70 healthy matched controls. We also studied PAI-1 activity in plasma. The presence of 4G/5G genotype was significantly increased in the group of patients with the extension of thrombotic lesions and was associated with an increase in CVT extension risk (odds ratio adjusted for sex 2.692; 95% confidence interval 1.302-4.702). Moreover, we observed a significant increase of PAI-1 plasma activity in patients with extension of thrombotic lesion vs. patients without extension (P=0.0001). Patients with 4G/5G genotype in the promoter of the plasminogen activator inhibitor - 1 gene present a higher risk of extension of thrombotic lesions.

  10. High-level expression of a novel recombinant human plasminogen activator (rhPA) in the milk of transgenic rabbits and its thrombolytic bioactivity in vitro.

    PubMed

    Song, Shaozheng; Ge, Xin; Cheng, Yaobin; Lu, Rui; Zhang, Ting; Yu, Baoli; Ji, Xueqiao; Qi, Zhengqiang; Rong, Yao; Yuan, Yuguo; Cheng, Yong

    2016-08-01

    The human tissue-type plasminogen activator (tPA) is a key kinase of fibrinolysis that plays an important role in dissolving fibrin clots to promote thrombolysis. The recombinant human plasminogen activator (rhPA) has more thrombolytic advantages than the wild type tPA. To increase the half-life and thrombolytic activity of tPA, a mutant containing only the essential K2 fibrin-binding and P activating plasminogen domains of the wild type tPA was cloned. This fragment was then inserted into goat β-casein regulatory sequences. Then, a mammary gland-specific expression vector, PCL25/rhPA, was constructed, and the transgenic rabbits were generated. In this study, 18 live transgenic founders (12♀, 6♂) were generated using pronuclear microinjection. Six transgenic rabbits were obtained, and the expression levels of rhPA in the milk had a range of 15.2-630 µg/ml. A fibrin agarose plate assay of rhPA showed that it had strong thrombolytic bioactivity in vitro, and the highest specific activity was >360 (360 times more than that of alteplase). The results indicated that the rhPA containing only the K2 and P domains is efficiently expressed with higher thrombolytic bioactivity in the milk of transgenic rabbits. Our study also demonstrated a new method for the large-scale production of clinically relevant recombinant pharmaceutical proteins in the mammary glands of transgenic rabbits.

  11. The influence of opioid peptides on matrix metalloproteinase-9 and urokinase plasminogen activator expression in three cancer cell lines.

    PubMed

    Gach, K; Wyrebska, A; Szemraj, J; Janecka, A

    2012-01-01

    Matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA) regulate proteolysis of the extracellular matrix (ECM) and as a consequence are involved in a number of physiological and pathological states, including cancer. A crucial feature of cancer progression and metastasis is the disruption of the ECM and spreading of proliferating cancer cells. Over-expression of MMPs and uPA is common for most types of cancers and correlates well with the adverse prognosis. Compounds able to modulate the activity of these proteolytic enzymes may become important agents in cancer therapy. In the present study, we examined the effect of the mu-opioid receptor selective peptide, morphiceptin, and its two synthetic analogs on mRNA and protein levels of MMP-9 and uPA in three human cancer cell lines: MCF-7, HT-29, and SH-SY5Y. Our findings indicate that in all three cell lines morphiceptin and its analogs attenuated MMP-9 expression and secretion and that this effect is not mediated by opioid receptors but is under control of the nitric oxide system. On the other hand, tested opioids up-regulated uPA levels through a mechanism that involved opioid-receptors. Different pathways by which opioid peptides exert their actionin cancer cells can explain their contradictory influence on the level of cancer markers.

  12. Fibulin-5 binds urokinase-type plasminogen activator and mediates urokinase-stimulated β1-integrin-dependent cell migration.

    PubMed

    Kapustin, Alexander; Stepanova, Victoria; Aniol, Natalia; Cines, Douglas B; Poliakov, Alexei; Yarovoi, Serge; Lebedeva, Tatiana; Wait, Robin; Ryzhakov, Grigory; Parfyonova, Yelena; Gursky, Yaroslav; Yanagisawa, Hiromi; Minashkin, Mikhail; Beabealashvilli, Robert; Vorotnikov, Alexander; Bobik, Alex; Tkachuk, Vsevolod

    2012-04-15

    uPA (urokinase-type plasminogen activator) stimulates cell migration through multiple pathways, including formation of plasmin and extracellular metalloproteinases, and binding to the uPAR (uPA receptor; also known as CD87), integrins and LRP1 (low-density lipoprotein receptor-related protein 1) which activate intracellular signalling pathways. In the present paper we report that uPA-mediated cell migration requires an interaction with fibulin-5. uPA stimulates migration of wild-type MEFs (mouse embryonic fibroblasts) (Fbln5+/+ MEFs), but has no effect on fibulin-5-deficient (Fbln5-/-) MEFs. Migration of MEFs in response to uPA requires an interaction of fibulin-5 with integrins, as MEFs expressing a mutant fibulin-5 incapable of binding integrins (Fbln(RGE/RGE) MEFs) do not migrate in response to uPA. Moreover, a blocking anti-(human β1-integrin) antibody inhibited the migration of PASMCs (pulmonary arterial smooth muscle cells) in response to uPA. Binding of uPA to fibulin-5 generates plasmin, which excises the integrin-binding N-terminal cbEGF (Ca2+-binding epidermal growth factor)-like domain, leading to loss of β1-integrin binding. We suggest that uPA promotes cell migration by binding to fibulin-5, initiating its cleavage by plasmin, which leads to its dissociation from β1-integrin and thereby unblocks the capacity of integrin to facilitate cell motility.

  13. Fibulin-5 binds urokinase-type plasminogen activator and mediates urokinase-stimulated β1-integrin-dependent cell migration

    PubMed Central

    Kapustin, Alexander; Stepanova, Victoria; Aniol, Natalia; Cines, Douglas B.; Poliakov, Alexei; Yarovoi, Serge; Lebedeva, Tatiana; Wait, Robin; Ryzhakov, Grigory; Parfyonova, Yelena; Gursky, Yaroslav; Yanagisawa, Hiromi; Minashkin, Mikhail; Beabealashvilli, Robert; Vorotnikov, Alexander; Bobik, Alex; Tkachuk, Vsevolod

    2015-01-01

    uPA (urokinase-type plasminogen activator) stimulates cell migration through multiple pathways, including formation of plasmin and extracellular metalloproteinases, and binding to the uPAR (uPA receptor; also known as CD87), integrins and LRP1 (low-density lipoprotein receptor-related protein 1) which activate intracellular signalling pathways. In the present paper we report that uPA-mediated cell migration requires an interaction with fibulin-5. uPA stimulates migration of wild-type MEFs (mouse embryonic fibroblasts) (Fbln5+/+ MEFs), but has no effect on fibulin-5-deficient (Fbln5−/−) MEFs. Migration of MEFs in response to uPA requires an interaction of fibulin-5 with integrins, as MEFs expressing a mutant fibulin-5 incapable of binding integrins (FblnRGE/RGE MEFs) do not migrate in response to uPA. Moreover, a blocking anti-(human β1-integrin) antibody inhibited the migration of PASMCs (pulmonary arterial smooth muscle cells) in response to uPA. Binding of uPA to fibulin-5 generates plasmin, which excises the integrin-binding N-terminal cbEGF (Ca2+ -binding epidermal growth factor)-like domain, leading to loss of β1-integrin binding. We suggest that uPA promotes cell migration by binding to fibulin-5, initiating its cleavage by plasmin, which leads to its dissociation from β1-integrin and thereby unblocks the capacity of integrin to facilitate cell motility. PMID:22280367

  14. A Mechanism for Assembly of Complexes of Vitronectin and Plasminogen Activator Inhibitor-1 from Sedimmentation Velocity Analysis*

    PubMed Central

    Minor, Kenneth H.; Schar, Christine R.; Blouse, Grant E.; Shore, Joseph D.; Lawrence, Daniel A.; Schuck, Peter; Peterson, Cynthia B.

    2005-01-01

    Plasminogen activator inhibitor-1 (PAI-1) and vitronectin are cofactors involved in pathological conditions such as injury, inflammation, and cancer, during which local levels of PAI-1 are increased and the active serpin forms complexes with vitronectin. These complexes become deposited into surrounding tissue matrices, where they regulate cell adhesion and peri-cellular proteolysis. The mechanism for their co-localization has not been elucidated. We hypothesize that PAI-1-vitronectin complexes form in a stepwise and concentration-dependent fashion via 1:1 and 2:1 intermediates, with the 2:1 complex serving a key role in assembly of higher order complexes. To test this hypothesis, sedimentation velocity experiments in the analytical ultracentrifuge were performed to identify different PAI-1-vitronectin complexes. Analysis of sedimentation data invoked a novel multisignal method to discern the stoichiometry of the two proteins in the higher-order complexes formed (Balbo, A., Minor, K. H., Velikovsky, C. A., Mariuzza, R. A., Peterson, C. B., and Schuck, P. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 81—86). Our results demonstrate that PAI-1 and vitronectin assemble into higher order forms via a pathway that is triggered upon saturation of the two PAI-1-binding sites of vitronectin to form the 2:1 complex. This 2:1 PAI-1-vitronectin complex, with a sedimentation coefficient of 6.5 S, is the key intermediate for the assembly of higher order complexes. PMID:15905170

  15. Risk Factors Associated with Serum Levels of the Inflammatory Biomarker Soluble Urokinase Plasminogen Activator Receptor in a General Population

    PubMed Central

    Haupt, Thomas H; Kallemose, Thomas; Ladelund, Steen; Rasmussen, Line JH; Thorball, Christian W; Andersen, Ove; Pisinger, Charlotta; Eugen-Olsen, Jesper

    2014-01-01

    The soluble urokinase plasminogen activator receptor (suPAR) is a biomarker of mortality risk in various patient populations. However, little is known about the implications of lifestyle for suPAR levels in the general population. Lifestyle, demographic, and cardiovascular disease (CVD) risk factor data were collected from 5,538 participants in the Danish population-based Inter99 study. Their suPAR levels were measured using a sandwich enzyme-linked immunosorbent assay. In the final adjusted model, smoking and morbid obesity were strongly associated with higher suPAR levels (P < 0.001). An unhealthy diet and alcohol abstinence in men were also associated with higher suPAR levels. Physical activity in leisure time had a modest impact on suPAR levels in univariate analysis, but not in the final adjusted model. In conclusion, smoking and morbid obesity were strongly associated with higher serum suPAR levels in this general population. Diet and alcohol consumption also seemed to impact suPAR levels. Lifestyle changes are likely to affect suPAR since ex-smokers had suPAR levels comparable to those of never-smokers. PMID:25574132

  16. Overexpression of SERBP1 (Plasminogen activator inhibitor 1 RNA binding protein) in human breast cancer is correlated with favourable prognosis

    PubMed Central

    2012-01-01

    Background Plasminogen activator inhibitor 1 (PAI-1) overexpression is an important prognostic and predictive biomarker in human breast cancer. SERBP1, a protein that is supposed to regulate the stability of PAI-1 mRNA, may play a role in gynaecological cancers as well, since upregulation of SERBP1 was described in ovarian cancer recently. This is the first study to present a systematic characterisation of SERBP1 expression in human breast cancer and normal breast tissue at both the mRNA and the protein level. Methods Using semiquantitative realtime PCR we analysed SERBP1 expression in different normal human tissues (n = 25), and in matched pairs of normal (n = 7) and cancerous breast tissues (n = 7). SERBP1 protein expression was analysed in two independent cohorts on tissue microarrays (TMAs), an initial evaluation set, consisting of 193 breast carcinomas and 48 normal breast tissues, and a second large validation set, consisting of 605 breast carcinomas. In addition, a collection of benign (n = 2) and malignant (n = 6) mammary cell lines as well as breast carcinoma lysates (n = 16) were investigated for SERBP1 expression by Western blot analysis. Furthermore, applying non-radioisotopic in situ hybridisation a subset of normal (n = 10) and cancerous (n = 10) breast tissue specimens from the initial TMA were analysed for SERBP1 mRNA expression. Results SERBP1 is not differentially expressed in breast carcinoma compared to normal breast tissue, both at the RNA and protein level. However, recurrence-free survival analysis showed a significant correlation (P = 0.008) between abundant SERBP1 expression in breast carcinoma and favourable prognosis. Interestingly, overall survival analysis also displayed a tendency (P = 0.09) towards favourable prognosis when SERBP1 was overexpressed in breast cancer. Conclusions The RNA-binding protein SERBP1 is abundantly expressed in human breast cancer and may represent a novel breast tumour

  17. Estriol-induced fibrinolysis due to the activation of plasminogen to plasmin by nitric oxide synthesis in platelets.

    PubMed

    Jana, Pradipta; Maiti, Smarajit; Kahn, Nighat N; Sinha, Asru K

    2015-04-01

    Estriol, an oestrogen, at 0.6 nmol/l was reported to inhibit ADP-induced platelet aggregation through nitric oxide synthesis. As nitric oxide has been reported to cause fibrinolysis due to the activation of plasminogen to plasmin, the role of estriol as a fibrinolytic agent was investigated. Also, the mechanism of estriol-induced nitric oxide synthesis in anucleated platelets was investigated. The estriol-induced lysis of platelet-rich plasma (PRP) clot was determined by photography of the clot lysis and by the assay of fibrin degradation products in the lysate and was obtained by SDS-PAGE. Nitric oxide was determined by methemoglobin method. The platelet membrane protein was isolated from the platelets by using Triton X-100 (0.05% v/v). The binding of estriol to the protein was determined by Scatchard plot by using an ELISA for estriol. Estriol at 0.6 nmol/l was found to lyse the clotted PRP due to fibrinolysis that produced fibrin degradation products in the lysate. The amino acid analysis of the platelet membrane protein, which resembles with nitric oxide synthase (NOS) activity, was activated nearly 10-fold over the control in the presence of estriol and was identified to be a human serum albumin precursor (Mr. 69 kDa) that binds to estriol with Kd1 of 6.0 × 10 mol/l and 39 ± 2 molecules of estriol bound the NOS molecule. The estriol-induced nitric oxide is capable of inducing fibrinolysis of the clotted PRP. The binding of estriol to platelet membrane NOS activated the enzyme in the absence of DNA in the platelet.

  18. Dengue Virus Nonstructural Protein 1-Induced Antibodies Cross-React with Human Plasminogen and Enhance Its Activation.

    PubMed

    Chuang, Yung-Chun; Lin, Jessica; Lin, Yee-Shin; Wang, Shuying; Yeh, Trai-Ming

    2016-02-01

    Dengue virus (DENV) infection is the most common mosquito-borne viral disease, and it can cause life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Abnormal activation of the coagulation and fibrinolysis system is one of the hallmarks of DHF/DSS. However, the mechanism underlying hemorrhage in DHF/DSS remains elusive. In previous studies, plasminogen (Plg) cross-reactive Abs, which can recognize DENV nonstructural protein (NS) 1, have been found in dengue patients. However, it is unclear whether these Abs are indeed induced by DENV NS1. Thus, we immunized mice with recombinant NS1 from both bacteria and drosophila to determine whether NS1 can induce Plg cross-reactive Abs. The results from the NS1-immunized mouse sera indicated that NS1 immunization induced Abs that could cross-react with Plg. To study the effects of these NS1-induced Plg cross-reactive Abs on fibrinolysis, we isolated several Plg cross-reactive anti-NS1 mAbs from these mice and found that some of them could enhance Plg activation. In addition, epitope mapping with a phage-displayed random peptide library revealed that one of these mAbs (2A5) could recognize NS1 C-terminal residues 305-311, which share sequence homology with Plg residues 590-597. A synthetic peptide of NS1 residues 305-311 could inhibit the binding of both 2A5 and its Fab to Plg and its enhanced activation. Thus, our results suggest that DENV NS1 can induce Plg cross-reactive Abs through molecular mimicry, which can enhance Plg activation and may contribute to the pathogenesis of DHF/DSS.

  19. The use of recombinant tissue plasminogen activator in the management of infective intracardiac thrombi in pre-term infants with thrombocytopaenia.

    PubMed

    Anderson, Ben; Urs, Prasanth; Tudehope, David; Ward, Cameron

    2009-10-01

    Bacterial endocarditis complicated by the development of intra-cardiac thrombus presents a difficult management dilemma in the pre-term infant. Here we present our experience with three infants who had this condition, all of whom were successfully managed using therapy with recombinant tissue plasminogen activator (r-TPA). Therapy in one of the infants was particularly instructive, as the condition was further complicated by severe thrombocytopaenia, making the decision to treat using r-TPA difficult.

  20. Massive Pulmonary Embolism: Treatment with Thrombus Fragmentation and Local Fibrinolysis with Recombinant Human-Tissue Plasminogen Activator

    SciTech Connect

    Stock, Klaus Wilhelm; Jacob, Augustinus Ludwig; Schnabel, Karl Jakob; Bongartz, Georg; Steinbrich, Wolfgang

    1997-09-15

    Purpose: To report the results of thrombus fragmentation in combination with local fibrinolysis using recombinant human-tissue plasminogen activator (rtPA) in patients with massive pulmonary embolism. Methods: Five patients with massive pulmonary embolism were treated with thrombus fragmentation followed by intrapulmonary injection of rtPA. Clot fragmentation was performed with a guidewire, angiographic catheter, and balloon catheter. Three patients had undergone recent surgery; one of them received a reduced dosage of rtPA. Results: All patients survived and showed clinical improvement with a resultant significant (p < 0.05) decrease in the pulmonary blood pressure (mean systolic pulmonary blood pressure before treatment, 49 mmHg; 4 hr after treatment, 28 mmHg). Angiographic follow-up in three patients revealed a decrease in thrombus material and an increase in pulmonary perfusion. Two patients developed retroperitoneal hematomas requiring transfusion. Conclusion: Clot fragmentation and local fibrinolysis with rtPA was an effective therapy for massive pulmonary embolism. Bleeding at the puncture site was a frequent complication.

  1. Plasma Plasminogen Activator Inhibitor-1 Is Associated with End-Stage Proliferative Diabetic Retinopathy in the Northern Chinese Han Population

    PubMed Central

    Zhong, Ze-Long; Chen, Song

    2012-01-01

    Objective. To identify predictors of end-stage proliferative diabetic retinopathy (PDR) in a cohort of individuals with type 2 diabetes mellitus (T2DM) from the Northern Chinese Han population. Methods. We investigated characteristics of 153 consecutive diabetic patients with end-stage PDR (62 males, 91 females), 123 consecutive PDR patients without end-stage PDR (48 males, 75 females), and 151 normal subjects (63 males, 88 females). Only one eye of each patient or healthy subject was included in this study. Univariate logistic regression models and multivariate logistic regression models were constructed to evaluate the predictors of end-stage PDR. Results. In univariate analysis, systolic blood pressure, diastolic blood pressure, duration of diabetes, family history of T2DM, and plasminogen activator inhibitor-1 (PAI-1) were significently associated with end-stage PDR. After multivariate analysis, family history of T2DM, plasma PAI-1 levels, smoking, and duration of diabetes were four positive predictors associated with end-stage PDR. Conclusions. Higher plasma levels of PAI-1 were associated with end-stage PDR in the Northern Chinese Han population with T2DM. PMID:23304115

  2. Plasminogen Activator Inhibitor-1 (PAI-1) gene 4G/5G alleles frequency distribution in the Lebanese population.

    PubMed

    Shammaa, Dina M R; Sabbagh, Amira S; Taher, Ali T; Zaatari, Ghazi S; Mahfouz, Rami A R

    2008-09-01

    Plasminogen activator inhibitor-1 (PAI-1) is an inhibitor of fibrinolysis. Increased plasma PAI-1 levels play an essential role in the pathogenesis of cardiovascular risk and other diseases associated with thrombosis. The 4G/5G polymorphism of the PAI-1 promoter region has been extensively studied in different populations. We studied 160 healthy unrelated Lebanese individuals using a reverse hybridization PCR assay to detect the 5G/5G, 4G/5G and, 4G/4G genotypes of the PAI-1 gene and the frequencies of the 4G and 5G alleles. We found that 4G/5G genotype was the most prevalent (45.6%) followed by 5G/5G (36.9%) and 4G/4G (17.5%). The frequencies of the 4G and 5G alleles were calculated to be 0.403 and 0.597, respectively. Compared to other ethnic communities, the Lebanese population was found to harbour a relatively high prevalence of the rare 4G allele. This, in turn, may predispose this population to develop cardiovascular diseases and other thrombotic clinical conditions. This study aids to enhance our understanding of the genetic features of the Lebanese population.

  3. Multifunctional roles of urokinase plasminogen activator (uPA) in cancer stemness and chemoresistance of pancreatic cancer

    PubMed Central

    Asuthkar, Swapna; Stepanova, Victoria; Lebedeva, Tatiana; Holterman, AiXuan L.; Estes, Norman; Cines, Douglas B.; Rao, Jasti S.; Gondi, Christopher S.

    2013-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is almost always lethal. One of the underlying reasons for this lethality is believed to be the presence of cancer stem cells (CSC), which impart chemoresistance and promote recurrence, but the mechanisms responsible are unclear. Recently the poor prognosis of PDAC has been correlated with increased expression of urokinase plasminogen activator (uPA). In the present study we examine the role of uPA in the generation of PDAC CSC. We observe a subset of cells identifiable as a side population (SP) when sorted by flow cytometry of MIA PaCa-2 and PANC-1 pancreatic cancer cells that possess the properties of CSC. A large fraction of these SP cells are CD44 and CD24 positive, are gemcitabine resistant, possess sphere-forming ability, and exhibit increased tumorigenicity, known characteristics of cancer stemness. Increased tumorigenicity and gemcitabine resistance decrease after suppression of uPA. We observe that uPA interacts directly with transcription factors LIM homeobox-2 (Lhx2), homeobox transcription factor A5 (HOXA5), and Hey to possibly promote cancer stemness. uPA regulates Lhx2 expression by suppressing expression of miR-124 and p53 expression by repressing its promoter by inactivating HOXA5. These results demonstrate that regulation of gene transcription by uPA contributes to cancer stemness and clinical lethality. PMID:23864708

  4. Association of Plasminogen Activator Inhibitor-1 (PAI-1) Gene Polymorphisms with Osteoporotic Vertebral Compression Fractures (OVCFs) in Postmenopausal Women

    PubMed Central

    Kim, Jung Oh; Han, Soo Hong; Lee, Yeon Ho; Ahn, Tae Keun; Lim, Jae Joon; Chung, Young Sun; Shin, Dong Eun; Lee, Woo Sik; Han, In Bo; Kim, Nam Keun

    2016-01-01

    Osteoporosis and osteoporotic fractures are strongly associated with mortality and morbidity, both in developing and developed countries. Menopause accelerates bone loss due to estrogen deficiency and age-related linear bone loss. We investigated plasminogen activator inhibitor-1 (PAI-1) gene polymorphisms in postmenopausal women with osteoporotic vertebral compression fractures (OVCFs). In this case-control study, 355 postmenopausal women were genotyped for the presence of PAI-1 gene polymorphisms −844A > G, −675 4G > 5G, 43G > A, 9785A > G, and 11053T > G. Genetic polymorphisms of PAI-1 were analyzed by the polymerization chain reaction restriction fragment length polymorphism assay, and their association with disease status and folate and homocysteine levels was determined in 158 OVCF patients and 197 control subjects. The PAI-1 −675 5G5G (adjusted odds ratio (AOR), 3.302; p = 0.017) and 43GA + AA (AOR, 2.087; p = 0.042) genotype frequencies showed significant association with the increased prevalence of OVCFs in postmenopausal women. In addition, we performed gene–environment interaction studies and demonstrated an association between PAI-1 gene polymorphisms and OVCF prevalence. Our novel finding is the identification of several PAI-1 genetic variants that increase susceptibility to OVCF. Our findings suggest that polymorphisms in PAI-1 may contribute to OVCF, and that they can be developed as biomarkers for evaluating OVCF risk. PMID:27941685

  5. Pentoxifylline Regulates Plasminogen Activator Inhibitor-1 Expression and Protein Kinase A Phosphorylation in Radiation-Induced Lung Fibrosis

    PubMed Central

    Bae, Chang-Hwan; Jin, Young-Woo; Lee, Seung-Sook

    2017-01-01

    Purpose. Radiation-induced lung fibrosis (RILF) is a serious late complication of radiotherapy. In vitro studies have demonstrated that pentoxifylline (PTX) has suppressing effects in extracellular matrix production in fibroblasts, while the antifibrotic action of PTX alone using clinical dose is yet unexplored. Materials and Methods. We used micro-computed tomography (micro-CT) and histopathological analysis to evaluate the antifibrotic effects of PTX in a rat model of RILF. Results. Micro-CT findings showed that lung density, volume loss, and mediastinal shift are significantly increased at 16 weeks after irradiation. Simultaneously, histological analysis demonstrated thickening of alveolar walls, destruction of alveolar structures, and excessive collagen deposition in the irradiated lung. PTX treatment effectively attenuated the fibrotic changes based on both micro-CT and histopathological analyses. Western analysis also revealed increased levels of plasminogen activator inhibitor- (PAI-) 1 and fibronectin (FN) and PTX treatment reduced expression of PAI-1 and FN by restoring protein kinase A (PKA) phosphorylation but not TGF-β/Smad in both irradiated lung tissues and epithelial cells. Conclusions. Our results demonstrate the antifibrotic effect of PTX on radiation-induced lung fibrosis and its effect on modulation of PKA and PAI-1 expression as possible antifibrotic mechanisms. PMID:28337441

  6. Retina Is Protected by Neuroserpin from Ischemic/Reperfusion-Induced Injury Independent of Tissue-Type Plasminogen Activator

    PubMed Central

    Gu, R. P.; Fu, L. L.; Jiang, C. H.; Xu, Y. F.; Wang, X.; Yu, J.

    2015-01-01

    The purpose of the present study was to investigate the potential neuroprotective effect of neuroserpin (NSP) on acute retinal ischemic/reperfusion-induced (IR) injury. An IR injury model was established by elevating intraocular pressure (IOP) for 60 minutes in wild type and tPA-deficient (tPA-/-) mice. Prior to IR injury, 1 μL of 20 μmol/L NSP or an equal volume of bovine serum albumin (BSA) was intravitreally administered. Retinal function was evaluated by electroretinograph (ERG) and the number of apoptotic neurons was determined via TUNEL labeling. Caspase-3, -8, -9,poly (ADP-ribose) polymerase (PARP)and their cleaved forms were subsequently analyzed. It was found that IR injury significantly damaged retinal function, inducing apoptosis in the retina, while NSP attenuated the loss of retinal function and significantly reduced the number of apoptotic neurons in both wild type and tPA-/- mice. The levels of cleaved caspase-3, cleaved PARP (the substrate of caspase-3) and caspase-9 (the modulator of the caspase-3), which had increased following IR injury, were significantly inhibited by NSP in both wild type and tPA-/- mice. NSP increased ischemic tolerance in the retina at least partially by inhibiting the intrinsic cell death signaling pathway of caspase-3. It was therefore concluded that the protective effect of neuroserpin maybe independent from its canonical interaction with a tissue-type plasminogen activator. PMID:26176694

  7. Insulin continues to induce plasminogen activator inhibitor 1 gene expression in insulin-resistant mice and adipocytes.

    PubMed Central

    Samad, F.; Pandey, M.; Bell, P. A.; Loskutoff, D. J.

    2000-01-01

    BACKGROUND: Although the association between insulin resistance and cardiovascular risk is well established, the underlying molecular mechanisms are poorly understood. The antifibrinolytic molecule plasminogen activator inhibitor 1 (PAI-1) is a cardiovascular risk factor that is consistently elevated in insulin-resistant states such as obesity and non-insulin-dependent diabetes mellitus (NIDDM). The strong positive correlation between this elevated PAI-1 and the degree of hyperinsulinemia not only implicates insulin itself in this increase, but also suggests that PAI-1 is regulated by a pathway that does not become insulin resistant. The data in this report supports this hypothesis. MATERIALS AND METHODS: We show that insulin stimulates PAI-1 gene expression in metabolically insulin-resistant ob/ob mice and in insulin-resistant 3T3-L1 adipocytes. Moreover, we provide evidence that glucose transport and PAI-1 gene expression are mediated by different insulin signaling pathways. These observations suggest that the compensatory hyperinsulinemia that is frequently associated with insulin-resistant states, directly contribute to the elevated PAI-1. CONCLUSIONS: These results provide a potential mechanism for the abnormal increases in cardiovascular risk genes in obesity, NIDDM, and polycystic ovary disease. PMID:11055587

  8. Multifunctional roles of urokinase plasminogen activator (uPA) in cancer stemness and chemoresistance of pancreatic cancer.

    PubMed

    Asuthkar, Swapna; Stepanova, Victoria; Lebedeva, Tatiana; Holterman, Aixuan L; Estes, Norman; Cines, Douglas B; Rao, Jasti S; Gondi, Christopher S

    2013-09-01

    Pancreatic ductal adenocarcinoma (PDAC) is almost always lethal. One of the underlying reasons for this lethality is believed to be the presence of cancer stem cells (CSC), which impart chemoresistance and promote recurrence, but the mechanisms responsible are unclear. Recently the poor prognosis of PDAC has been correlated with increased expression of urokinase plasminogen activator (uPA). In the present study we examine the role of uPA in the generation of PDAC CSC. We observe a subset of cells identifiable as a side population (SP) when sorted by flow cytometry of MIA PaCa-2 and PANC-1 pancreatic cancer cells that possess the properties of CSC. A large fraction of these SP cells are CD44 and CD24 positive, are gemcitabine resistant, possess sphere-forming ability, and exhibit increased tumorigenicity, known characteristics of cancer stemness. Increased tumorigenicity and gemcitabine resistance decrease after suppression of uPA. We observe that uPA interacts directly with transcription factors LIM homeobox-2 (Lhx2), homeobox transcription factor A5 (HOXA5), and Hey to possibly promote cancer stemness. uPA regulates Lhx2 expression by suppressing expression of miR-124 and p53 expression by repressing its promoter by inactivating HOXA5. These results demonstrate that regulation of gene transcription by uPA contributes to cancer stemness and clinical lethality.

  9. Urokinase type plasminogen activator mediates Interleukin-17-induced peripheral blood mesenchymal stem cell motility and transendothelial migration.

    PubMed

    Krstić, Jelena; Obradović, Hristina; Jauković, Aleksandra; Okić-Đorđević, Ivana; Trivanović, Drenka; Kukolj, Tamara; Mojsilović, Slavko; Ilić, Vesna; Santibañez, Juan F; Bugarski, Diana

    2015-02-01

    Mesenchymal stem cells (MSCs) have the potential to migrate toward damaged tissues increasing tissue regeneration. Interleukin-17 (IL-17) is a proinflammatory cytokine with pleiotropic effects associated with many inflammatory diseases. Although IL-17 can modulate MSC functions, its capacity to regulate MSC migration is not well elucidated so far. Here, we studied the role of IL-17 on peripheral blood (PB) derived MSC migration and transmigration across endothelial cells. IL-17 increased PB-MSC migration in a wound healing assay as well as cell mobilization from collagen gel. Concomitantly IL-17 induced the expression of urokinase type plasminogen activator (uPA) without affecting matrix metalloproteinase expression. The incremented uPA expression mediated the capacity of IL-17 to enhance PB-MSC migration in a ERK1,2 MAPK dependent way. Also, IL-17 induced PB-MSC migration alongside with changes in cell polarization and uPA localization in cell protrusions. Moreover, IL-17 increased PB-MSC adhesion to endothelial cells and transendothelial migration, as well as increased the capacity of PB-MSC adhesion to fibronectin, in an uPA-dependent fashion. Therefore, our data suggested that IL-17 may act as chemotropic factor for PB-MSCs by incrementing cell motility and uPA expression during inflammation development.

  10. Auranofin Suppresses Plasminogen Activator Inhibitor-2 Expression through Annexin A5 Induction in Human Prostate Cancer Cells

    PubMed Central

    Shin, Dong-Won; Kwon, Yeo-Jung; Ye, Dong-Jin; Baek, Hyoung-Seok; Lee, Joo-Eun; Chun, Young-Jin

    2017-01-01

    Auranofin has been developed as antirheumatic drugs, which is currently under clinical development for the treatment of chronic lymphocytic leukemia. Previous report showed that auranofin induced apoptosis by enhancement of annexin A5 expression in PC-3 cells. To understand the role of annexin A5 in auranofin-mediated apoptosis, we performed microarray data analysis to study annexin A5-controlled gene expression in annexin A5 knockdown PC-3 cells. Of differentially expressed genes, plasminogen activator inhibitor (PAI)-2 was increased by annexin A5 siRNA confirmed by qRT-PCR and western blot. Treatment with auranofin decreased PAI-2 and increased annexin A5 expression as well as promoting apoptosis. Furthermore, auranofin-induced apoptosis was recovered by annexin A5 siRNA but it was promoted by PAI-2 siRNA. Interestingly, knockdown of annexin A5 rescued PAI-2 expression suppressed by auranofin. Taken together, our study suggests that induction of annexin A5 by auranofin may enhance apoptosis through suppression of PAI-2 expression in PC-3 cells. PMID:27956714

  11. Cost-Effectiveness of Intraarterial Treatment as an Adjunct to Intravenous Tissue Plasminogen Activator for Acute Ischemic Stroke

    PubMed Central

    Leppert, Michelle H; Campbell, Jonathan D; Simpson, Jennifer R; Burke, James F

    2015-01-01

    Background and Purpose The objective of this study was to determine the cost-effectiveness of intraarterial treatment within the 0- to 6- hour window after intravenous (IV) tissue plasminogen activator (tPA) within 0- to 4.5-hours compared to IV tPA alone, in the US setting and from a social perspective. Methods A decision analytic model estimated the lifetime costs and outcomes associated with the additional benefit of intraarterial therapy compared to standard treatment with IV tPA alone. Model inputs were obtained from published literature, the MR CLEAN study, and claims databases in the United States. Health outcomes were measured in quality adjusted life years (QALYs). Treatment benefit was assessed by calculating the cost per QALY gained. One-way and probabilistic sensitivity analyses were performed to estimate the overall uncertainty of model results. Results The addition of intraarterial therapy compared with standard treatment alone yielded a lifetime gain of 0.7 QALY for an additional cost of $9,911, which resulted in a cost of $14,137 per QALY. Multivariable sensitivity analysis predicted cost-effectiveness (≤$50,000 per QALY) in 97.6% of simulation runs. Conclusion Intraarterial treatment after IV tPA for patients with anterior circulation strokes within the 6 hour window is likely cost effective. From a societal perspective, increased investment in access to intraarterial treatment for acute stroke may be justified. PMID:26012639

  12. The -675 4G/5G polymorphism at the Plasminogen Activator Inhibitor 1 (PAI-1) gene modulates plasma Plasminogen Activator Inhibitor 1 concentrations in response to dietary fat consumption.

    PubMed

    Pérez-Martínez, P; Adarraga-Cansino, M D; Fernández de la Puebla, R A; Blanco-Molina, A; Delgado-Lista, J; Marín, C; Ordovás, J M; López-Miranda, J; Pérez-Jiménez, F

    2008-04-01

    The objective of the study was to determine whether Plasminogen Activator Inhibitor Type 1 (PAI-1) -675 4G/5G polymorphism is associated with the response of functional plasma PAI-1 concentrations to changes in the amount and quality of dietary fat in healthy subjects. PAI-1 is the major inhibitor of fibrinolysis, and a lower level of fibrinolytic activity could be implicated in an increased risk of IHD. Fifty-nine healthy Spanish volunteers (ten 4G/4G homozygotes, twenty-eight heterozygotes 4G/5G and twenty-one 5G/5G homozygotes) consumed three diets for periods of 4 weeks each: a SFA-rich diet (38 % fat, 20 % SFA), followed by a carbohydrate-rich diet (30 % fat, 55 % carbohydrate) and a MUFA-rich diet (38 % fat, 22 % MUFA) according to a randomized crossover design. At the end of each dietary period plasma lipid and functional plasma PAI-1 concentrations were determined. Subjects carrying the 4G allele (4G/4G and 4G/5G) showed a significant decrease in PAI-1 concentrations after the MUFA diet, compared with the SFA-rich and carbohydrate-rich diets (genotype x diet interaction: P = 0.028). 5G/5G homozygotes had the lowest plasma PAI-1 concentrations compared with 4G/4G and 4G/5G subjects (genotype: P = 0.002), without any changes as a result of the amount and the quality of the dietary fat. In summary, no differences in plasma PAI-1 concentration response were found after changes in dietary fat intake in 5G/5G homozygotes, although these subjects displayed the lowest concentrations of PAI-1. On the other hand, carriers of the 4G allele are more likely to hyper-respond to the presence of MUFA in the diet because of a greater decrease in PAI-1 concentrations.

  13. Plasminogen activator inhibitor-1 is elevated, but not essential, in the development of bleomycin-induced murine scleroderma

    PubMed Central

    Matsushita, M; Yamamoto, T; Nishioka, K

    2005-01-01

    Accumulative data have demonstrated that plasminogen activator inhibitor-1 (PAI-1) plays an important role in the extracellular matrix metabolism; however, the involvement of PAI-1 in scleroderma has not been fully elucidated. In this study, we investigated the role of PAI-1 in bleomycin-induced murine scleroderma. 100 µg of bleomycin was injected subcutaneously to the back skin of C3H/HeJ mice on alternate day for 4 weeks. Histopathological findings revealed that PAI-1 was positive in macrophage-like cells and fibroblastic cells in the dermis, in parallel with the induction of dermal sclerosis. PAI-1 mRNA expression in the whole skin was up-regulated at 1 and 4 weeks. The production of active PAI-1 protein in the lesional skin was significantly increased 3 and 4 weeks after bleomycin treatment. Next, we examined whether dermal sclerosis is induced by bleomycin in PAI-1-deficient (PAI-1–/–) mice. 10 µg of bleomycin was subcutaneously injected to PAI-1–/– and wild type (WT) mice 5 days per week for 4 weeks. Histological examination revealed that dermal sclerosis was similarly induced even in PAI-1–/– as well as WT mice. Dermal thickness and collagen contents in the skin were significantly increased by bleomycin injection in both PAI-1–/– and WT mice, and the rate of increase was similar. These data suggest that PAI-1 plays an important role, possibly via TGF-β pathway activation. However, the fact that PAI-1 deficiency did not ameliorate skin sclerosis suggest that PAI-1 is not the essential factor in the development of bleomycin-induced scleroderma, and more complex biochemical effects other than PA/plasmin system are greatly suspected. PMID:15730388

  14. Expression of recombinant staphylokinase, a fibrin-specific plasminogen activator of bacterial origin, in potato (Solanum tuberosum L.) plants.

    PubMed

    Gerszberg, Aneta; Wiktorek-Smagur, Aneta; Hnatuszko-Konka, Katarzyna; Łuchniak, Piotr; Kononowicz, Andrzej K

    2012-03-01

    One of the most dynamically developing sectors of green biotechnology is molecular farming using transgenic plants as natural bioreactors for the large scale production of recombinant proteins with biopharmaceutical and therapeutic values. Such properties are characteristic of certain proteins of bacterial origin, including staphylokinase. For many years, work has been carried out on the use of this protein in thrombolytic therapy. In this study, transgenic Solanum tuberosum plants expressing a CaMV::sak-mgpf-gusA gene fusion, were obtained. AGL1 A. tumefaciens strain was used in the process of transformation. The presence of the staphylokinase gene was confirmed by PCR in 22.5% of the investigated plants. The expression of the fusion transgene was detected using the β-glucuronidase activity assay in 32 putative transgenic plants. Furthermore, on the basis of the GUS histochemical reaction, the transgene expression pattern had a strong, constitutive character in seven of the transformants. The polyacrylamide gel electrophoresis of a protein extract from the SAK/PCR-positive plants, revealed the presence of a119 kDa protein that corresponds to that of the fusion protein SAK-mGFP-GUSA. Western blot analysis, using an antibody against staphylokinase, showed the presence of the staphylokinase domain in the 119 kDa protein in six analyzed transformants. However, the enzymatic test revealed amidolytic activity characteristic of staphylokinase in the protein extract of only one plant. This is the first report on a Solanum tuberosum plant producing a recombinant staphylokinase protein, a plasminogen activator of bacterial origin.

  15. Evaluation of Fibrinolytic Inhibitors: Alpha-2-Antiplasmin and Plasminogen Activator Inhibitor 1 in Patients with Obstructive Sleep Apnoea

    PubMed Central

    Kiciński, Paweł; Przybylska-Kuć, Sylwia; Dybała, Andrzej; Myśliński, Wojciech; Pastryk, Jolanta; Tomaszewski, Tomasz; Mosiewicz, Jerzy

    2016-01-01

    Obstructive sleep apnoea (OSA) induces thrombophilia and reduces fibrinolysis. Alpha-2-antiplasmin (a-2-AP) and plasminogen activator inhibitor 1 (PAI-1) are major inhibitors of the fibrinolytic system. Increased concentrations of these factors are associated with a higher risk of cardiovascular diseases. The aim of this study was to assess plasma a-2-AP and PAI-1 in patients with OSA and evaluate correlations with the polysomnographic record and selected risk factors of cardiovascular diseases. The study group comprised 45 patients with OSA, and the control group consisted of 19 patients who did not meet the diagnostic criteria of OSA. Plasma a-2-AP and PAI-1 concentrations were assessed by enzyme-linked immunosorbent assay (ELISA). In the study group, the median value of plasma a-2-AP was higher than that of the control group (157.34 vs. 11.89 pg/ml, respectively, P<0.0001). A-2-AP concentration increased proportionally to the severity of OSA. The concentration of a-2-AP was positively correlated with the apnoea-hypopnoea index (AHI), apnoea index (AI), respiratory disturbances time (RDT), and desaturaion index (DI), and negatively correlated with mean and minimal oxygen saturation (SpO2 mean, SpO2 min, respectively). The median value of PAI-1 was higher in the study group than the control group (12.55 vs. 5.40 ng/ml, respectively, P = 0.006) and increased along with OSA severity. PAI-1 concentration was positively correlated with AHI, AI, RDT, DI, and body mass index (BMI) and negatively correlated with SpO2 mean and SpO2 min. Higher plasma concentrations of a-2-AP and PAI-1 in patients with OSA indicated that these patients had increased prothrombotic activity. OSA increases the risk of cardiovascular complications as it enhances prothrombotic activity. PMID:27861608

  16. Effects of Pharmacological Inhibition and Genetic Deficiency of Plasminogen Activator Inhibitor-1 in Radiation-Induced Intestinal Injury

    SciTech Connect

    Abderrahmani, Rym; Francois, Agnes; Buard, Valerie; Benderitter, Marc; Sabourin, Jean-Christophe; Crandall, David L.; Milliat, Fabien

    2009-07-01

    Purpose: To investigate effects of plasminogen activator inhibitor 1 (PAI-1) genetic deficiency and pharmacological PAI-1 inhibition with PAI-039 in a mouse model of radiation-induced enteropathy. Methods and Materials: Wild-type (Wt) and PAI-1{sup -/-} knockout mice received a single dose of 19 Gy to an exteriorized localized intestinal segment. Sham and irradiated Wt mice were treated orally with 1 mg/g of PAI-039. Histological modifications were quantified using a radiation injury score. Moreover, intestinal gene expression was monitored by real-time PCR. Results: At 3 days after irradiation, PAI-039 abolished the radiation-induced increase in the plasma active form of PAI-1 and limited the radiation-induced gene expression of transforming growth factor {beta}1 (TGF-{beta}1), CTGF, PAI-1, and COL1A2. Moreover, PAI-039 conferred temporary protection against early lethality. PAI-039 treatment limited the radiation-induced increase of CTGF and PAI-1 at 2 weeks after irradiation but had no effect at 6 weeks. Radiation injuries were less severe in PAI-1{sup -/-} mice than in Wt mice, and despite the beneficial effect, 3 days after irradiation, PAI-039 had no effects on microscopic radiation injuries compared to untreated Wt mice. Conclusions: A genetic deficiency of PAI-1 is associated with amelioration of late radiation enteropathy. Pharmacological inhibition of PAI-1 by PAI-039 positively impacts the early, acute phase increase in plasma PAI-1 and the associated radiation-induced gene expression of inflammatory/extracellular matrix proteins. Since PAI-039 has been shown to inhibit the active form of PAI-1, as opposed to the complete loss of PAI-1 in the knockout animals, these data suggest that a PAI-1 inhibitor could be beneficial in treating radiation-induced tissue injury in acute settings where PAI-1 is elevated.

  17. Effects of antiinflammatory agents on mouse skin tumor promotion, epidermal DNA synthesis, phorbol ester-induced cellular proliferation, and production of plasminogen activator.

    PubMed

    Viaje, A; Slaga, T J; Wigler, M; Weinstein, I B

    1977-05-01

    The antinflammatory ateroids fluocinoine acetonide, fluocinonide, and fluclorolone acetonide were found to be very effectiveinhibitory agents of mouse skin tumor promotion. These steroids also drastically inhibited epidermal DNA synthesis and epidermal cellular proliferation induced by a phorbal ester tumor promoter. In addition, these compounds were potent inhibitors, of plasminogen activator production in tumor cell cultures. The clinically used non-steroidal antiinflammatory agents oxyphenbutazone, indomethacin, and Seclazone also inhibite tumor promotion but were much less effective. Although these agents are useful against inflammatory disorders in general when given p.o., in our studies they had little effect on inflammation and epidermal cellular proliferation induced by a phorbol ester tumor promoter when given topically. The afore mentioned nonsteroidal antiinflammatory agents also had little effect on epidermal DNA synthesis. Oxyphenbutazone and indomethacin were less potent inhibitors of plasminogen activator production in tumor cells than were the antiinflammatory steroids, and Seclazone produced a negligible inhibition. There is, therefore, a general correlation in the potencies of a series of steroidal antiinflammatory agents for inhibition of tumor promotion and their ability to inhibit plasminogen activator production by tumor cell cultures and epidermal DNA synthesis.

  18. Tissue Plasminogen Activator Binding to Superparamagnetic Iron Oxide Nanoparticle—Covalent Versus Adsorptive Approach

    NASA Astrophysics Data System (ADS)

    Friedrich, Ralf P.; Zaloga, Jan; Schreiber, Eveline; Tóth, Ildikó Y.; Tombácz, Etelka; Lyer, Stefan; Alexiou, Christoph

    2016-06-01

    Functionalized superparamagnetic iron oxide nanoparticles are frequently used to develop vehicles for drug delivery, hyperthermia, and photodynamic therapy and as tools used for magnetic separation and purification of proteins or for biomolecular imaging. Depending on the application, there are various possible covalent and non-covalent approaches for the functionalization of particles, each of them shows different advantages and disadvantages for drug release and activity at the desired location.

  19. Antithrombotic activity of a monoclonal antibody inducing the substrate form of plasminogen activator inhibitor type 1 in rat models of venous and arterial thrombosis

    PubMed Central

    Berry, C N; Lunven, C; Lechaire, I; Girardot, C; O'Connor, S E

    1998-01-01

    Elevated plasminogen activator inhibitor 1 (PAI-1) is a risk factor for thrombosis, and inhibitors of the interaction between PAI-1 and tissue plasminogen activator (t-PA) have antithrombotic and pro-thrombolytic activity in animals. We describe the antithrombotic effects in the rat of a monoclonal antibody (MA33H1) which converts PAI-1 to a non-inhibitory substrate. The activity of MA33H1 against rat PAI-1 was confirmed using two-chain t-PA and a chromogenic substrate. MA33H1 was evaluated in rat venous (thromboplastin+stasis in the abdominal vena cava) and arterial (electric current applied to a carotid artery) thrombosis models. The effects on tail-transection bleeding time were studied. MA33H1 at 100 ng ml−1 inhibited both human (44.1%) and rat PAI-1 (49.7%). This effect was concentration-dependent. Its effect on human PAI-1 was not significantly inhibited by 1 μg ml−1 fibrin or a ≈amp;7 fold molar excess of vitronectin (1 nM). Inhibition of rat PAI-1 was unchanged by fibrin, but vitronectin reduced inhibition from 0.5 nM. In the venous thrombosis model, MA33H1 significantly reduced thrombus weights by 38 and 58.6% at 50 and 100 μg kg−1 min−1 i.v. respectively. This effect was inhibited by tranexamic acid. In the arterial model, MA33H1 significantly increased the delay to occlusive thrombus formation by 58 and 142% at 50 and 100 μg kg−1 min−1 i.v., and did not affect bleeding time at 300 μg kg−1 min−1 i.v. Thus, a monoclonal antibody which transforms PAI-1 to a t-PA substrate prevents thrombus formation in the rat with no effect on bleeding time at a higher dose. PMID:9776340

  20. Accelerated Dosing Frequency of a Pulmonary Formulation of Tissue Plasminogen Activator is Well-Tolerated in Mice

    PubMed Central

    Stringer, Kathleen A; Tobias, Meghan; Dunn, John S; Campos, Jackie; Van Rheen, Zachary; Mosharraf, Mitra; Nayar, Rajiv

    2009-01-01

    Summary Tissue plasminogen activator (tPA) has both fibrinolytic and anti-inflammatory activity. These properties may be useful in treating inflammatory lung diseases, such as acute respiratory distress syndrome (ARDS). We have previously demonstrated the feasibility of targeted pulmonary delivery of tPA. As part of our research to develop a clinically viable pulmonary formulation of tPA, we assessed the tolerability and incidence of haemorrhage associated with the administration of a pulmonary formulation of mouse tPA (pf-mtPA). Intratracheal doses of nebulized pf-mtPA or sterile saline were administered with increasing frequency to male and female B6C3F1 mice. After dosing, the mice entered a recovery period, after which they were killed and their lungs were lavaged and harvested. Post-mortem gross necropsy was performed and all major organs were assessed histologically for haemorrhage. The bronchoalveolar lavage fluid was assessed for markers of lung injury. Mouse tPA that was formulated to mimic a previously characterized human pf-tPA was well tolerated when given intratracheally with increasing dosing frequency. The administration of pf-mtPA did not result in any detectable haemorrhagic-related events or signs of lung injury. The results of the present longitudinal study demonstrate that a maximally feasible dose of pf-mtPA (3 mg/kg) can be given frequently over a short period of time (12 h) without haemorrhagic complications. Although these data were generated in a healthy mouse model, they provide support for the continued evaluation of pf-tPA for the treatment of pulmonary diseases, such as ARDS. PMID:18671720

  1. Impact of the 4G/5G polymorphism in the plasminogen activator inhibitor-1 gene on primary nephrotic syndrome.

    PubMed

    Luo, Yuezhong; Wang, Chao; Tu, Haitao

    2014-03-01

    The aim of the present study was to investigate whether the four guanosines (4G)/five guanosines (5G) polymorphism in the gene coding for plasminogen activator inhibitor-1 (PAI-1) affects the clinical features of primary nephrotic syndrome (PNS). A cohort of 200 biopsy-diagnosed PNS patients was studied, with 40 healthy subjects as controls. The PAI-1 gene polymorphism was detected by polymerase chain reaction and DNA sequencing. Associations between the PAI-1 4G/5G polymorphism and clinical features and pathological types of PNS were analyzed. The results indicated that the PAI-1 genotype distribution is significantly different between patients with PNS and healthy controls, with significantly higher numbers of the 4G/4G genotype and lower numbers of the 5G5G genotype detected in PNS patients compared to controls (both P<0.05). The frequency of the 4G allele was also significantly higher in PNS patients compared to healthy controls (P<0.01). Among the different pathological types of PNS, IgA nephropathy (IgAN) and membranous nephropathy (MN) were associated with significantly increased frequencies of the 4G/4G and 4G/5G genotypes, as well as of the 4G allele. The increased 4G frequency was also detected in patients with minimal change disease (MCD). Significantly increased international normalized ratio (INR) and prolonged activated partial thromboplastin time (APTT) were observed in 4G/4G compared to 5G/5G PNS subjects. The response to steroids was not significantly different among the three genotypes. In conclusion, the 4G allele of the PAI-1 gene appears to be associated with PNS, especially in MN and IgAN patients. These findings suggest that specific targeting may be required for the treatment of PNS patients with the 4G/4G genotype.

  2. The Influence of Differentially Expressed Tissue-Type Plasminogen Activator in Experimental Autoimmune Encephalomyelitis: Implications for Multiple Sclerosis

    PubMed Central

    Dahl, Lisa CM; Nasa, Zeyad; Chung, JieYu; Niego, Be’eri; Tarlac, Volga; Ho, Heidi; Galle, Adam; Petratos, Steven; Lee, Jae Young; Alderuccio, Frank; Medcalf, Robert L.

    2016-01-01

    Tissue type plasminogen activator (t-PA) has been implicated in the development of multiple sclerosis (MS) and in rodent models of experimental autoimmune encephalomyelitis (EAE). We show that levels of t-PA mRNA and activity are increased ~4 fold in the spinal cords of wild-type mice that are mice subjected to EAE. This was also accompanied with a significant increase in the levels of pro-matrix metalloproteinase 9 (pro-MMP-9) and an influx of fibrinogen. We next compared EAE severity in wild-type mice, t-PA-/- mice and T4+ transgenic mice that selectively over-express (~14-fold) mouse t-PA in neurons of the central nervous system. Our results confirm that t-PA deficient mice have an earlier onset and more severe form of EAE. T4+ mice, despite expressing higher levels of endogenous t-PA, manifested a similar rate of onset and neurological severity of EAE. Levels of proMMP-9, and extravasated fibrinogen in spinal cord extracts were increased in mice following EAE onset regardless of the absence or over-expression of t-PA wild-type. Interestingly, MMP-2 levels also increased in spinal cord extracts of T4+ mice following EAE, but not in the other genotypes. Hence, while the absence of t-PA confers a more deleterious form of EAE, neuronal over-expression of t-PA does not overtly protect against this condition with regards to symptom onset or severity of EAE. PMID:27427941

  3. Hypoxia dysregulates the production of adiponectin and plasminogen activator inhibitor-1 independent of reactive oxygen species in adipocytes

    SciTech Connect

    Chen Baoying; Lam, Karen S.L.; Wang Yu; Wu Donghai; Lam, Michael C.; Shen Jiangang; Wong Laiching; Hoo, Ruby L.C.; Zhang Jialiang; Xu Aimin . E-mail: amxu@hkucc.hku.hk

    2006-03-10

    Low plasma levels of adiponectin (hypoadiponectinemia) and elevated circulating concentrations of plasminogen activator inhibitor (PAI)-1 are causally associated with obesity-related insulin resistance and cardiovascular disease. However, the mechanism that mediates the aberrant production of these two adipokines in obesity remains poorly understood. In this study, we investigated the effects of hypoxia and reactive oxygen species (ROS) on production of adiponectin and PAI-1 in 3T3-L1 adipocytes. Quantitative PCR and immunoassays showed that ambient hypoxia markedly suppressed adiponectin mRNA expression and its protein secretion, and increased PAI-1 production in mature adipocytes. Dimethyloxallyl glycine, a stabilizer of hypoxia-inducible factor 1{alpha} (HIF-1{alpha}), mimicked the hypoxia-mediated modulations of these two adipokines. Hypoxia caused a modest elevation of ROS in adipocytes. However, ablation of intracellular ROS by antioxidants failed to alleviate hypoxia-induced aberrant production of adiponectin and PAI-1. On the other hand, the antioxidants could reverse hydrogen peroxide (H{sub 2}O{sub 2})-induced dysregulation of adiponectin and PAI-1 production. H{sub 2}O{sub 2} treatment decreased the expression levels of peroxisome proliferator-activated receptor gamma (PPAR{gamma}) and CCAAT/enhancer binding protein (C/EBP{alpha}), but had no effect on HIF-1{alpha}, whereas hypoxia stabilized HIF-1{alpha} and decreased expression of C/EBP{alpha}, but not PPAR{gamma}. Taken together, these data suggest that hypoxia and ROS decrease adiponectin production and augment PAI-1 expression in adipocytes via distinct signaling pathways. These effects may contribute to hypoadiponectinemia and elevated PAI-1 levels in obesity, type 2 diabetes, and cardiovascular diseases.

  4. Urokinase plasminogen activator released by alveolar epithelial cells modulates alveolar epithelial repair in vitro.

    PubMed

    Van Leer, Coretta; Stutz, Monika; Haeberli, André; Geiser, Thomas

    2005-12-01

    Intra-alveolar fibrin is formed following lung injury and inflammation and may contribute to the development of pulmonary fibrosis. Fibrin turnover is altered in patients with pulmonary fibrosis, resulting in intra-alveolar fibrin accumulation, mainly due to decreased fibrinolysis. Alveolar type II epithelial cells (AEC) repair the injured alveolar epithelium by migrating over the provisional fibrin matrix. We hypothesized that repairing alveolar epithelial cells modulate the underlying fibrin matrix by release of fibrinolytic activity, and that the degree of fibrinolysis modulates alveolar epithelial repair on fibrin. To test this hypothesis we studied alveolar epithelial wound repair in vitro using a modified epithelial wound repair model with human A549 alveolar epithelial cells cultured on a fibrin matrix. In presence of the inflammatory cytokine interleukin-1beta, wounds increase by 800% in 24 hours mainly due to detachment of the cells, whereas in serum-free medium wound areas decreases by 22.4 +/- 5.2% (p < 0.01). Increased levels of D-dimer, FDP and uPA in the cell supernatant of IL-1beta-stimulated A549 epithelial cells indicate activation of fibrinolysis by activation of the plasmin system. In presence of low concentrations of fibrinolysis inhibitors, including specific blocking anti-uPA antibodies, alveolar epithelial repair in vitro was improved, whereas in presence of high concentrations of fibrinolysis inhibitors, a decrease was observed mainly due to decreased spreading and migration of cells. These findings suggest the existence of a fibrinolytic optimum at which alveolar epithelial repair in vitro is most efficient. In conclusion, uPA released by AEC alters alveolar epithelial repair in vitro by modulating the underlying fibrin matrix.

  5. Plasminogen activator inhibitor 1: Mechanisms of its synergistic regulation by growth factors

    SciTech Connect

    Song, Xiaoling

    2010-01-01

    My research is on the synergistic regulation of PAI-1 by EGF and TGF-β. The mechanism of synergistic regulation of PAI-1 by EGF and TGF-β are addressed. Methods are described for effective identification of RNA accessible sites for antisense oligodexoxynucleotides (ODNs) and siRNA. In this study effective AS-ODN sequences for both Lcn2 and Bcl2 were identified by in vitro tiled microarray studies. Our results suggest that hybridization of ODN arrays to a target mRNA under physiological conditions might be used as a rapid and reliable in vitro method to accurately identify targets on mRNA molecules for effective antisense and potential siRNA activity in vivo.

  6. Statins suppress glucose-induced plasminogen activator inhibitor-1 expression by regulating RhoA and nuclear factor-κB activities in cardiac microvascular endothelial cells.

    PubMed

    Ni, Xiao-Qing; Zhu, Jian-Hua; Yao, Ning-Hua; Qian, Juan; Yang, Xiang-Jun

    2013-01-01

    The aim of this study was to investigate the possible proinflammatory signaling pathways involved in statin inhibition of glucose-induced plasminogen activator inhibitor-1 (PAI-1) expression in cardiac microvascular endothelial cells (CMECs). Primary rat CMECs were grown in the presence of 5.7 or 23 mmol/L glucose. PAI-1 mRNA and protein expression levels were measured by realtime polymerase chain reaction, Western blotting and enzyme-linked immunosorbent assay, respectively. A pull-down assay was performed to determine RhoA activity. IκBα protein expression was measured by Western blotting, nuclear factor (NF)-κB activation was detected by electrophoretic mobility shift assay and its transcription activity was determined by a dual luciferase reporter gene assay. PAI-1 mRNA and protein expression levels were both increased with high glucose concentrations, but they were significantly suppressed by simvastatin and atorvastatin treatment (P < 0.01) and the effects were reversed by mevalonate (100 μmol/L) and geranylgeranyl pyrophosphate (10 μmol/L) but not farnesyl pyrophosphate (10 μmol/L). Such effects were similar to those of a RhoA inhibitor, C3 exoenzyme (5 μg/mL), inhibitors of RhoA kinase (ROCK), Y-27632 (10 μmol/L) and hydroxyfasudil (10 μmol/L) and an NF-κB inhibitor, BAY 11-7082 (5 μmol/L). High glucose-induced RhoA and NF-κB activations in CMECs were both significantly inhibited by statins (P < 0.01). Simvastatin and atorvastatin equally suppress high glucose-induced PAI-1 expression. These effects of statins may occur partly by regulating the RhoA/ROCK-NF-κB pathway. The multifunctional roles of statins may be particularly beneficial for patients with metabolic syndrome.

  7. Substrate behavior of plasminogen activator inhibitor-1 is not associated with a lack of insertion of the reactive site loop.

    PubMed

    Gils, A; Knockaert, I; Declerck, P J

    1996-06-11

    Plasminogen activator inhibitor-1 (PAI-1) is a unique member of the serpin superfamily. In the present study, we have evaluated the effect of substitution, with a proline, at positions P5, P7, P14, P15, or P16, on the conformational flexibility and functional properties of PAI-1. These mutants (PAI-1-P5, IIe-->Pro at P5; PAI-1-P7, Ala-->Pro at P7; PAI-1-P14, Thr-->Pro at P14; PAI-1-P15, Gly-->Pro at P15; PAI-1-P16, Ser-->Pro at P16) were purified and fully characterized. WtPAI-1 had a specific activity of 68 +/- 10% (mean +/- SD, n = 6) whereas PAI-1-P5, PAI-1-P7, and PAI-1-P16 had specific activities of 34 +/- 9.3%, 42 +/- 10%, and 36 +/- 11%, respectively. PAI-1-P14 and PAI-1-P15 did not exhibit significant inhibitory activity. Conformational analysis revealed that wtPAI-1 preparations contained 12 +/- 2.0% substrate, whereas PAI-1-P5, PAI-1-P7, and PAI-1-P16 were characterized with a significantly (p < 0.001) increased substrate behavior (i.e., 43 +/- 6.1%, 42 +/- 1.5% and 22 +/- 1.7%, respectively). The inactive variants PAI-1-P14 and PAI-1-P15 behaved exclusively as substrates toward various serine proteinases. Heat denaturation studies revealed that cleavage of any noninhibitory substrate form of PAI-1 resulted in an insertion of the NH2-terminal side of the reactive site loop. Incubation with plasmin showed the presence of a unique plasmin cleavage site (Lys191-Ser192) exclusively present in all latent forms studied. We conclude that (a) the entire P5 to P16 region in PAI-1 plays an important role in the functional and conformational properties of PAI-1; (b) the substrate behavior of serpins is not associated with a lack of insertion of the reactive site loop; (c) the identification of a plasmin cleavage site in latent PAI-1 may provide new insights in the mechanisms for the inactivation of storage pools of PAI-1.

  8. Transcatheter Thrombolysis with High-Dose Bolus Tissue Plasminogen Activator in Iatrogenic Arterial Occlusion after Femoral Arterial Catheterization

    SciTech Connect

    Tsetis, Dimitrios K.; Kochiadakis, George E.; Hatzidakis, Adam A.; Skalidis, Emannuel I.; Chryssou, Evangelia G.; Tritou, Ioanna N.; Vardas, Panos E.; Gourtsoyiannis, Nicholas C.

    2002-01-15

    Purpose: To assess the efficacy of percutaneous local thrombolysis with high-dose bolus recombinant tissue plasminogen activator (rt-PA) in patients with acute limb ischemia due to arterial thrombosis after cardiac catheterization.Methods: We treated eight patients (7 men; mean age 56 years) with thrombotic occlusion of both the common femoral artery (CFA) and external iliac artery (EIA) in six patients and of the CFA only in two patients. Two 5 mg boluses of rt-PA were injected into the proximal clot through a 5 Fr end-hole catheter and subsequently two additional boluses of 5 mg rt-PA were given through a catheter with multiple side-holes. In case of a significant amount of residual thrombus, a continuous infusion of 2.5 mg/hr of rt-PA was started.Results: Successful lysis was achieved in all patients. The mean duration of lysis was 2 hr 41 min. The mean total amount of rt-PA delivered was 23.16 mg. In four patients unmasked flow-limited dissections confined to the CFA were managed by prolonged balloon dilatation, while in the remaining four patients with extension of the dissection to the external iliac artery one or two Easy Wallstents were implanted. There was prompt relief of lower limb ischemic symptoms and signs in all patients. Two groin hematomas were conservatively treated.Clinical and color Doppler flow imaging follow-up with a mean duration of 15 months, showed no reappearance of ischemic symptoms or development of restenosis in any of the patients. One patient died 6 months after thrombolysis.Conclusions: Transcatheter thrombolysis with high-dose bolus rt-PA is a safe and effective treatment inpatients with iatrogenic arterial occlusion after femoral catheterization. Underlying dissections should be treated by prolonged balloon dilatation but stent implantation is often required.

  9. Preliminary experience with air transfer of patients for rescue endovascular therapy after failure of intravenous tissue plasminogen activator.

    PubMed

    Tsujimoto, Masanori; Yoshimura, Shinichi; Enomoto, Yukiko; Yamada, Noriaki; Matsumaru, Naoki; Kumada, Keisuke; Toyoda, Izumi; Ogura, Shinji; Iwama, Toru

    2015-01-01

    The present report describes our experience with air transfer of patients with acute ischemic stroke in whom intravenous tissue plasminogen activator (IV t-PA) failed for rescue endovascular therapy (EVT). Twenty-three consecutive patients in whom IV t-PA failed were transferred to our hospital for rescue EVT between February 2011 and April 2013. The amount of time required for transfer, distance, clinical outcomes, and complications were compared between patients transferred by ground (TG group; n = 17) and by air (TA group; n = 6). Computed tomography imaging on arrival revealed hemorrhagic transformation in 1 (5.9%) patient in the TG group, whereas none of the patients in the TA group developed any type of complication. The remaining 22 patients received rescue EVT. The elapsed time from the request call to arrival at our hospital did not significantly differ between the TG and TA groups (45.8 ± 4.9 min vs. 41.6 ± 2.3 min). However, the distance from the primary hospital to our institution was significantly longer for the TA group than for the TG group (38.8 ± 10.4 km vs. 13.5 ± 1.2 km, p = 0.001). The frequency of favorable outcomes (modified Rankin Scale 0-1 at 90 days after onset) in the TG and TA groups were 25.0% and 50.0%, respectively (p = 0.267). Air transfer for patients after IV t-PA failure allowed for more rapid delivery of patients over longer distances than ground transfer.

  10. Preliminary Experience with Air Transfer of Patients for Rescue Endovascular Therapy after Failure of Intravenous Tissue Plasminogen Activator

    PubMed Central

    TSUJIMOTO, Masanori; YOSHIMURA, Shinichi; ENOMOTO, Yukiko; YAMADA, Noriaki; MATSUMARU, Naoki; KUMADA, Keisuke; TOYODA, Izumi; OGURA, Shinji; IWAMA, Toru

    2015-01-01

    The present report describes our experience with air transfer of patients with acute ischemic stroke in whom intravenous tissue plasminogen activator (IV t-PA) failed for rescue endovascular therapy (EVT). Twenty-three consecutive patients in whom IV t-PA failed were transferred to our hospital for rescue EVT between February 2011 and April 2013. The amount of time required for transfer, distance, clinical outcomes, and complications were compared between patients transferred by ground (TG group; n = 17) and by air (TA group; n = 6). Computed tomography imaging on arrival revealed hemorrhagic transformation in 1 (5.9%) patient in the TG group, whereas none of the patients in the TA group developed any type of complication. The remaining 22 patients received rescue EVT. The elapsed time from the request call to arrival at our hospital did not significantly differ between the TG and TA groups (45.8 ± 4.9 min vs. 41.6 ± 2.3 min). However, the distance from the primary hospital to our institution was significantly longer for the TA group than for the TG group (38.8 ± 10.4 km vs. 13.5 ± 1.2 km, p = 0.001). The frequency of favorable outcomes (modified Rankin Scale 0–1 at 90 days after onset) in the TG and TA groups were 25.0% and 50.0%, respectively (p = 0.267). Air transfer for patients after IV t-PA failure allowed for more rapid delivery of patients over longer distances than ground transfer. PMID:25739430

  11. Mechanisms of transport and exocytosis of dense-core granules containing tissue plasminogen activator in developing hippocampal neurons.

    PubMed

    Silverman, Michael A; Johnson, Scooter; Gurkins, Dmitri; Farmer, Meredith; Lochner, Janis E; Rosa, Patrizia; Scalettar, Bethe A

    2005-03-23

    Dense-core granules (DCGs) are organelles found in specialized secretory cells, including neuroendocrine cells and neurons. Neuronal DCGs facilitate many critical processes, including the transport and secretion of proteins involved in learning, and yet their transport and exocytosis are poorly understood. We have used wide-field and total internal reflection fluorescence microscopy, in conjunction with transport theory, to visualize the transport and exocytosis of DCGs containing a tissue plasminogen activator-green fluorescent protein hybrid in cell bodies, neurites, and growth cones of developing hippocampal neurons and to quantify the roles that diffusion, directed motion, and immobility play in these processes. Our results demonstrate that shorter-ranged transport of DCGs near sites of exocytosis in hippocampal neurons and neuroendocrine cells differs markedly. Specifically, the immobile fraction of DCGs within growth cones and near the plasma membrane of hippocampal neurons is small and relatively unaltered by actin disruption, unlike in neuroendocrine cells. Moreover, transport of DCGs in these domains of hippocampal neurons is unusually heterogeneous, being significantly rapid and directed as well as slow and diffusive. Our results also demonstrate that exocytosis is preceded by substantial movement and heterogeneous transport; this movement may facilitate delivery of DCG cargo in hippocampal neurons, given the relatively low abundance of neuronal DCGs. In addition, the extensive mobility of DCGs in hippocampal neurons argues strongly against the hypothesis that cortical actin is a major barrier to membrane-proximal DCGs in these cells. Instead, our results suggest that extended release of DCG cargo from hippocampal neurons arises from heterogeneity in DCG mobility.

  12. PET imaging of urokinase-type plasminogen activator receptor (uPAR) in prostate cancer: current status and future perspectives.

    PubMed

    Skovgaard, Dorthe; Persson, Morten; Kjaer, Andreas

    2016-01-01

    Overexpression of urokinase-type plasminogen activator receptors (uPAR) represents an important biomarker for aggressiveness in most common malignant diseases, including prostate cancer (PC). Accordingly, uPAR expression either assessed directly in malignant PC tissue or assessed directly in plasma (intact/cleaved forms)-provides independent additional clinical information to that contributed by PSA, Gleason score, and other relevant pathological and clinical parameters. In this respect, non-invasive molecular imaging by positron emission tomography (PET) offers a very attractive technology platform, which can provide the required quantitative information on the uPAR expression profile, without the need for invasive procedures and the risk of missing the target due to tumor heterogeneity. These observations support non-invasive PET imaging of uPAR in PC as a clinically relevant diagnostic and prognostic imaging method. In this review, we will focus on the recent development of uPAR PET and the relevance within prostate cancer imaging. Novel antibody and small-molecule radiotracers-targeting uPAR, including a series of uPAR-targeting PET ligands, based on the high affinity peptide ligand AE105, have been synthesized and tested in vitro and in vivo in preclinical murine xenograft models and, recently, in a first-ever clinical uPAR PET study in cancer patients, including patients with PC. In this phase I study, a high and specific uptake of the tracer (64)Cu-DOTA-AE105 was found in both primary tumors and lymph node metastases. The results are encouraging and support large-scale clinical trials to determine the utility of uPAR PET in the management of patients with PC with the goal of improving outcome.

  13. Plasminogen activator inhibitor-1 gene 4G/5G polymorphism in Turkish children with asthma and allergic rhinitis.

    PubMed

    Ozbek, Ozlem Yilmaz; Ataç, F Belgin; Ogus, Ersin; Ozbek, Namik

    2009-01-01

    Plasminogen activator inhibitor (PAI-1) has an essential role in tissue remodeling after inflammation. Recent literature revealed only one study evaluating PAI-1 4G/5G gene polymorphism in children with asthma and none in children with allergic rhinitis. We aimed to investigate distribution of PAI-1 4G/5G polymorphism in a group of Turkish children with asthma and allergic rhinitis and compare these findings with those obtained in normal peers. Patients with physician-diagnosed asthma (n = 106) and allergic rhinitis (n = 99) and 83 healthy peers were included in this study. We evaluated PAI-1 4G/5G polymorphism genotype as well as the possible association between PAI-1 4G/5G polymorphism and pulmonary function tests, serum total immunoglobulin E (IgE), total eosinophil count, and skin-prick test positivity in our study. The prevalence of the 4G allele significantly exceeded the values found in the controls both in patients with asthma (p = 0.001) and in patients with allergic rhinitis (p = 0.002). Interestingly, comparison of asthmatic patients revealed that mean baseline percent forced expiratory volume in 1 second and forced vital capacity were significantly higher in patients who bear 5G/5G genotype than in those who have 4G/4G or 4G/5G genotypes. No statistically significant relationship were found between PAI-1 polymorphism and total serum IgE levels, total eosinophil count, or selected skin test responses to aeroallergens. Our study suggests that Turkish children with asthma or allergic rhinitis have a higher prevalence of PAI-1 4G allele compared with their healthy peers.

  14. Pyruvate and cilostazol protect cultured rat cortical pericytes against tissue plasminogen activator (tPA)-induced cell death.

    PubMed

    Kim, Ha Na; Kim, Tae-Youn; Yoon, Young Hee; Koh, Jae-Young

    2015-12-02

    Since even a brief ischemia can cause permanent brain damage, rapid restoration of blood flow is critical to limiting damage. Although intravenous tPA during the acute stage is the treatment of choice for achieving reperfusion, this treatment is sometimes associated with brain hemorrhage. Agents that reduce tPA-related bleeding risk may help expand its therapeutic window. This study assessed whether zinc dyshomeostasis underlies the toxic effect of tPA on brain vascular pericytes; whether pyruvate, an inhibitor of zinc toxicity, protects pericytes against tPA-induced cell death; and whether cilostazol, which protects pericytes against tPA-induced cell death, affects zinc dyshomeostasis associated with tPA toxicity. Cultured pericytes from newborn rat brains were treated with 10-200 μg/ml tPA for 24 h, inducing cell death in a concentration-dependent manner. tPA-induced cell death was preceded by increases in intracellular free zinc levels, and was substantially attenuated by plasminogen activator inhibitor-1 (PAI-1) or TPEN. Pyruvate completely blocked direct zinc toxicity and tPA-induced pericyte cell death. Both cAMP and cilostazol, a PDE3 inhibitor that attenuates tPA-induced pericyte cell death in vitro and tPA-induced brain hemorrhage in vivo, reduced zinc- and tPA-induced pericyte cell death, suggesting that zinc dyshomeostasis may be targeted by cilostazol in tPA toxicity. These findings show that tPA-induced pericyte cell death may involve zinc dyshomeostasis, and that pyruvate and cilostazol attenuate tPA-induced cell death by reducing the toxic cascade triggered by zinc dyshomeostasis. Since pyruvate is an endogenous metabolite and cilostazol is an FDA-approved drug, in vivo testing of both as protectors against tPA-induced brain hemorrhage may be warranted. This article is part of a Special Issue entitled SI: Neuroprotection.

  15. The role of proteasome beta subunits in gastrin-mediated transcription of plasminogen activator inhibitor-2 and regenerating protein1.

    PubMed

    O'Hara, Adrian; Howarth, Alice; Varro, Andrea; Dimaline, Rod

    2013-01-01

    The hormone gastrin physiologically regulates gastric acid secretion and also contributes to maintaining gastric epithelial architecture by regulating expression of genes such as plasminogen activator inhibitor 2 (PAI-2) and regenerating protein 1 (Reg1). Here we examine the role of proteasome subunit PSMB1 in the transcriptional regulation of PAI-2 and Reg1 by gastrin, and its subcellular distribution during gastrin stimulation. We used the gastric cancer cell line AGS, permanently transfected with the CCK2 receptor (AGS-GR) to study gastrin stimulated expression of PAI-2 and Reg1 reporter constructs when PSMB1 was knocked down by siRNA. Binding of PSMB1 to the PAI-2 and Reg1 promoters was assessed by chromatin immunoprecipitation (ChIP) assay. Subcellular distribution of PSMB1 was determined by immunocytochemistry and Western Blot. Gastrin robustly increased expression of PAI-2 and Reg1 in AGS-GR cells, but when PSMB1 was knocked down the responses were dramatically reduced. In ChIP assays, following immunoprecipitation of chromatin with a PSMB1 antibody there was a substantial enrichment of DNA from the gastrin responsive regions of the PAI-2 and Reg1 promoters compared with chromatin precipitated with control IgG. In AGS-GR cells stimulated with gastrin there was a significant increase in the ratio of nuclear:cytoplasmic PSMB1 over the same timescale as recruitment of PSMB1 to the PAI-2 and Reg1 promoters seen in ChIP assays. We conclude that PSMB1 is part of the transcriptional machinery required for gastrin stimulated expression of PAI-2 and Reg1, and that its change in subcellular distribution in response to gastrin is consistent with this role.

  16. Neuroendocrinal, Neurodevelopmental, and Embryotoxic Effects of Recombinant Tissue Plasminogen Activator Treatment for Pregnant Women with Acute Ischemic Stroke

    PubMed Central

    Steinberg, Anna; Moreira, Tiago P.

    2016-01-01

    Thrombolysis with recombinant tissue plasminogen activator (rTPA) was the first evidence-based treatment approved for acute stroke. Ischemic stroke is relatively uncommon in fertile women but treatment is often delayed or not given. In randomized trials, pregnancy has been an exclusion criterion for thrombolysis. Physiologic TPA has been shown to have neuroendocrine effects namely in vasopressin secretion. Important TPA effects in brain function and development include neurite outgrowth, migration of cerebellar granular neurons and promotion of long-term potentiation, among others. Until now, no neuroendocrine side-effects have been reported in pregnant women treated with rTPA. The effects of rTPA exposure in the fetus following intravenous thrombolysis in pregnant women are still poorly understood. This depends on low case frequency, short-duration of exposure and the fact that rTPA molecule is too large to pass the placenta. rTPA has a short half-life of 4–5 min, with only 10% of its concentration remaining in circulation after 20 min, which may explain its safety at therapeutically doses. Ischemic stroke during pregnancy occurs most often in the third trimester. Complication rates of rTPA in pregnant women treated for thromboembolic conditions and ischemic stroke were found to be similar when compared to non-pregnant women (7–9% mortality). In embryos of animal models so far, no indications of a teratogenic or mutagenic potential were found. Pregnancy is still considered a relative contraindication when treating acute ischemic stroke with rTPA, however, treatment risk must be balanced against the potential of maternal disability and/or death. PMID:26941596

  17. Recombinant tissue plasminogen activator (rtPA) for the treatment of hepatic veno-occlusive disease (VOD).

    PubMed

    Kulkarni, S; Rodriguez, M; Lafuente, A; Mateos, P; Mehta, J; Singhal, S; Saso, R; Tait, D; Treleaven, J G; Powles, R L

    1999-04-01

    Seventeen patients who developed hepatic veno-occlusive disease (VOD) following hematopoietic stem cell transplantation were treated with recombinant tissue plasminogen activator (rtPA) with or without heparin. rtPA was started a median of 13 days post transplant (range 4-35). All patients received rtPA at a dose of 10 mg/day as a starting dose, and 12 patients also received heparin (1500 U bolus; then 100 U/kg/day as a continuous i.v. infusion). The median number of days of rtPA therapy was 2.5 (1-12). The median total serum bilirubin level was 116 mmol/l (range 63-194) at the beginning of treatment. Six patients showed a response to rtPA treatment (29%). It was observed that by day 2 of rtPA therapy, bilirubin levels in responders showed a downwards trend as compared to those in nonresponders. In all except one patient this response was observed after two doses of rtPA. Seven out of the 11 non-responders had a past history of liver dysfunction, compared with none of the responders. There were no differences between the two groups in terms of day of onset of liver dysfunction, manifestations of disease, maximum bilirubin and creatinine levels, and day of commencing treatment. No patient experienced severe hemorrhagic complications during therapy. Four responders survived for more than 100 days compared to none of the non-responders. Probability of survival was 33% at day 100. It is difficult to unequivocally establish the role of rtPA in the treatment of VOD. The importance of bilirubin levels on days 2 or 3 of therapy in predicting outcome should be established, as should the optimum dose of rtPA and optimum duration of therapy.

  18. Clinical experience with recombinant tissue plasminogen activator in the management of intracardiac and arterial thrombosis in children.

    PubMed

    Olgun, Hasim; Buyukavci, Mustafa; Ceviz, Naci; Sahin, Irfan Oguz; Yildirim, Zuhal Keskin; Colak, Abdurrahim; Tekgunduz, Kadir Serafettin; Caner, Ibrahim

    2014-10-01

    Thrombotic events may complicate the clinical course of many pediatric diseases. Drugs for therapeutic thrombolysis include streptokinase, urokinase and tissue plasminogen activator (t-PA). There is less experience with recombinant t-PA (rt-PA) in children. We aimed to present our experiences with rt-PA in children with intracardiac or peripheral arterial thrombus. We retrospectively reviewed the children who received rt-PA for thrombus. Twenty-two children (13 boys, 9 girls; age range: 1 day-17 years) with intracardiac (n = 5), prosthetic heart valve (n = 2) and peripheral arterial (n = 15) thrombus were evaluated. Twelve (54%) had congenital heart disease, two (9%) had rheumatic heart disease, three (14%) had leukemia and five (23%) had documented sepsis, prematurity or meconium aspiration syndrome. Ten of the 15 peripheral arterial thromboses were observed following cardiac catheterization. Three of the five intracardiac thrombi were detected in children with leukemia. All children received low-molecular-weight heparin. rt-PA (alteplase) infusion (at a dose of 0.01-0.5 mg/kg per h) was administered for different time periods (3-66 h). Ten of 11 patients with peripheral arterial occlusion and three of five patients with intracardiac thrombus showed full recovery. However, there was no response in two patients with intracardiac thrombus and in two patients with heart valve thrombus. Nose bleeding, melena and decreased serum fibrinogen concentration were observed in seven patients during the rt-PA infusion. All bleedings stopped after cessation of rt-PA infusion, and no blood transfusion was required in any patient. We conclude that rt-PA infusion seems effective and well tolerated in children for the treatment of peripheral arterial and intracardiac thrombus.

  19. Bone Marrow Urokinase Plasminogen Activator Receptor Levels are Associated with the Progress of Multiple Myeloma(△).

    PubMed

    Shou, Li-Hong; Cao, Dan; Dong, Xiao-Hui; Fang, Qiu; Xu, Bao-Lian; Fei, Ju-Ping

    2016-09-20

    Objective To determine the mRNA and protein levels of urokinase plasminogen activator receptors (uPAR) in bone marrow fluid and bone marrow tissue from multiple myeloma (MM) patients and assess association of uPAR level with prognosis of MM. Methods uPAR levels in bone marrow fluid of 22 MM patients at the stable and progressive stages and 18 iron deficiency anemia patients with normal bone marrow (control) were examined by ELISA. Furthermore, uPAR expression in bone marrow tissue was investigated by RT-PCR and Western blot, respectively. The distribution of uPAR in MM cells was examined using immunofluorescence staining. The pathological changes in different stages of MM patients were studied by HE staining. Results uPAR level in bone marrow fluid of MM patients (1.52±0.32 μg/ml) was found to be higher than that in the control group (0.98±0.15 μg/ml). Interestingly, uPAR protein (0.686±0.075 vs. 0.372±0.043, P<0.05) and mRNA (2.51±0.46 vs. 4.46±1.15, P<0.05) expression levels of MM patients at the progressive stage were significantly higher than those at the stable stage. The expression of uPAR in MM bone marrow was confirmed by immunofluorescence staining. Moreover, HE staining revealed a great increased number of nucleated cells and severe impairment of hematopoietic function in the bone marrow of patients with progressive-stage myeloma. Conclusion Our study reveals that uPAR expression is positively correlated with the development and progress of MM.

  20. Expression of urokinase-type plasminogen activator, stromelysin 1, stromelysin 3, and matrilysin genes in lung carcinomas.

    PubMed Central

    Bolon, I.; Devouassoux, M.; Robert, C.; Moro, D.; Brambilla, C.; Brambilla, E.

    1997-01-01

    We have previously shown that the extracellular-matrix-degrading enzymes, urokinase-type plasminogen activator (u-PA), stromelysin 1, stromelysin 3, and matrilysin, may play an important role in the transition from lung preneoplasia to invasive carcinoma. Using in situ hybridization and immunohistochemistry, we analyzed serial frozen sections for the expression of these enzymes in 89 lung carcinomas including 25 neuroendocrine (NE) carcinomas (10 small-cell lung carcinomas, 7 large-cell NE carcinomas, 1 atypical, and 7 typical carcinoids) and 64 non-small-cell, non-NE carcinomas (29 squamous and 7 basaloid carcinomas, 23 adenocarcinomas, and 5 large-cell carcinomas). Proteases, except matrilysin, were more often expressed in stromal than cancer cells. In non-small-cell, non-NE carcinomas, stromal co-expression of u-PA and stromelysin 3 was seen in 80 to 90% of the tumors and was highly correlated (P < 0.0001). Stromal u-PA and stromelysin 3 expression was linked to tumor size (P = 0.01 and 0.03, respectively) and lymph node involvement (P = 0.001 and 0.02, respectively). Epithelial expression of u-PA was correlated to tumor size (P = 0.04). Epithelial expression of stromelysin 3 predominated in squamous and basaloid carcinomas (P = 0.0005) and was inversely correlated to squamous differentiation (P = 0.018). Epithelial expression of matrilysin predominated in adenocarcinomas and large-cell carcinomas (P = 0.07). In NE carcinomas including small-cell lung carcinomas, stromal expression of u-PA was correlated to lymph node metastasis (P = 0.017). Epithelial expression of all enzymes were significantly less frequent in NE than in non-NE tumors. We conclude that 1) epithelial expression of matrix proteases in lung cancer is linked to cell phenotype (NE versus non-NE, squamous versus glandular) and 2) their stromal, rather than epithelial, expression influences local metastasis. Images Figure 1 PMID:9137088

  1. Serum Soluble Urokinase-Type Plasminogen Activator Receptor Levels and Idiopathic FSGS in Children: A Single-Center Report

    PubMed Central

    Price, Heather E.; Gallon, Lorenzo; Langman, Craig B.

    2013-01-01

    Summary Background and objectives FSGS is the primary cause of childhood nephrotic syndrome leading to ESRD. Permeability factors, including circulating serum soluble urokinase-type plasminogen activator receptor (suPAR), have been postulated as putative causes in adults with primary FSGS. Similar results have yet to be proven in children. Design, setting, participants, & measurements This cross-sectional single-center study assessed the association of serum suPAR in children with FSGS or other glomerular and nonglomerular kidney diseases. Results This study examined 110 samples retrieved from 99 individuals (between January 2011 and April 2012), aged 1–21 years; of these individuals, 20 had primary FSGS, 24 had non-FSGS glomerular disease, 26 had nonglomerular kidney disease, and 29 were healthy controls. suPAR levels were not significantly different in children with FSGS, non-FSGS glomerular disease, and healthy controls (P>0.05). However, suPAR levels (median [25%–75%]) were higher in children with nonglomerular kidney disease (3385 pg/ml [2695–4392]) versus FSGS (2487 pg/ml [2191–3351]; P<0.05). Female patients with nephrotic-range proteinuria (U-Pr/Cr >2) had lower suPAR levels than those without proteinuria (2380 pg/ml [2116–2571] versus 3125 pg/ml [2516–4198], respectively; P<0.001). This trend was not seen among male participants; suPAR levels in all female participants were lower than in male participants (P=0.03). Thirty-four patients studied were kidney transplant recipients; transplant status was not associated with suPAR levels in patients with FSGS or non-FSGS diagnoses, independent of proteinuria, race, or sex (P>0.05). Conclusions On the basis of these results, circulating suPAR is unlikely the leading cause for childhood idiopathic FSGS. PMID:23620441

  2. Analysis of Tissue Plasminogen Activator Eligibility by Sex in the Greater Cincinnati/Northern Kentucky Stroke Study

    PubMed Central

    Madsen, Tracy E.; Khoury, Jane C.; Alwell, Kathleen A.; Moomaw, Charles J.; Kissela, Brett M.; De Los Rios La Rosa, Felipe; Woo, Daniel; Adeoye, Opeolu; Flaherty, Matthew L.; Khatri, Pooja; Ferioli, Simona; Kleindorfer, Dawn

    2017-01-01

    Background and Purpose Sex differences in recombinant tissue-type plasminogen activator (r-tPA) administration are present in some populations. It is unknown whether this is because of eligibility differences or the modifiable exclusion criterion of severe hypertension. Our aim was to investigate sex differences in r-tPA eligibility, in individual exclusion criteria, and in the modifiable exclusion criterion, hypertension. Methods We included all ischemic stroke patients ≥18 years among residents of the Greater Cincinnati/Northern Kentucky region who presented to 16-area emergency departments in 2005. Eligibility for r-tPA and individual exclusion criteria were determined using 2013 American Heart Association (AHA) and European Cooperative Acute Stroke Study (ECASS) III guidelines. Results Of 1837 ischemic strokes, 58% were women, 24% were black. Mean age in years was 72.2 for women and 66.1 for men. Eligibility for r-tPA was similar by sex (6.8% men and 6.1% women; P=0.55), even after adjusting for age (7.0% and 5.9%; P=0.32). Similar proportions of women and men arrived beyond 3- and 4.5-hour time windows, but more women had severe hypertension. There were no sex differences in blood pressure treatment rates among those with severe hypertension (14.6% women and 20.8% men; P=0.21). More women were >80 years and had National Institutes of Health Stroke Scale (NIHSS) >25. Conclusions Within a large, biracial population, eligibility for r-tPA was similar by sex. Women were more likely to have the modifiable exclusion criterion of severe hypertension but were not more likely to be treated. Women were more likely to have 2 of the 5 ECASS III exclusion criteria. Undertreatment of hypertension in women is a potentially modifiable contributor to reported differences in r-tPA administration. PMID:25628307

  3. Upper extremity acute compartment syndrome during tissue plasminogen activator therapy for pulmonary embolism in a morbidly obese patient

    PubMed Central

    Tuna, Serkan; Duymus, Tahir Mutlu; Mutlu, Serhat; Ketenci, Ismail Emre; Ulusoy, Ayhan

    2015-01-01

    Introduction Deep vein thrombosis (DVT) and pulmonary embolism (PE) are more frequently observed in morbidly obese patients. Tissue plasminogen activator (tPA) is a thrombolytic agent which dissolves the thrombus more rapidly than conventional heparin therapy and reduces the mortality and morbidity rates associated with PE. Compartment syndrome is a well-known and documented complication of thrombolytic treatment. In awake, oriented and cooperative patients, the diagnosis of compartment syndrome is made based on clinical findings including swelling, tautness, irrational and continuous pain, altered sensation, and severe pain due to passive stretching. These clinical findings may not be able to be adequately assessed in unconscious patients. Presentation of case In this case report, we present compartment syndrome observed, for which fasciotomy was performed on the upper right extremity of a 46-year old morbidly obese, conscious female patient who was receiving tPA due to a massive pulmonary embolism. Discussion Compartment syndrome had occurred due to the damage caused by the repeated unsuccessful catheterisation attempts to the brachial artery and the accompanying tPA treatment. Thus, the bleeding that occurred in the volar compartment of the forearm and the anterior compartment of the arm led to acute compartment syndrome (ACS). After relaxation was brought about in the volar compartment of the forearm and the anterior compartment of the arm, the circulation in the limb was restored. Conclusion As soon as the diagnosis of compartment syndrome is made, an emergency fasciotomy should be performed. Close follow-up is required to avoid wound healing problems after the fasciotomy. PMID:25618841

  4. Characterization of the plasminogen activator of herpesvirus-transformed cells and examination of its correlation with the tumorigenic and metastatic ability of in vivo-derived sublines

    SciTech Connect

    Marks, G.J.

    1986-01-01

    Herpes simplex virus type 2-transformed hamster embryo fibroblasts (333-8-9 cells) produce increased amounts of plasminogen activator (PA) compared with normal hamster cells. The 333-8-9 PA activity was quantitated in comparison to a PA standard, urokinase (UK). Using a direct PA assay in which /sup 125/I-labeled plasminogen is cleaved, a linear dose-response was seen over a 1000-fold range in UK concentration when plotted on a semi-logarithmic scale. Extracellular PA activity secreted by the HSV-2-transformed cell line, 333-8-9, followed a similar dose-response slop. The optimum pH and osmolarity for detection of the 333-8-9 extracellular PA activity were pH 8.9 and approximately 150 mOsmol, respectively. Secretion of PA by the 333-8-9 cells did not vary significantly on a per cell basis over cell densities ranging from 0.1 to 8.0 x 10/sup 7/ cells/T-75 cm/sup 2/ flask. This assay was accurate, reproducible, and demonstrated that the 333-8-9 cells produced at least a 20-fold greater amount of PA activity than their normal cell counterparts. Based on the molecular weight (50-58 Kd) of the secreted 333-8-9 cell PA and lack of fibrin stimulation of the PA activity, it is concluded to be a urokinase-type PA.

  5. PACAP Interacts with PAC1 Receptors to Induce Tissue Plasminogen Activator (tPA) Expression and Activity in Schwann Cell-Like Cultures

    PubMed Central

    Castorina, Alessandro; Waschek, James A.; Marzagalli, Rubina; Cardile, Venera; Drago, Filippo

    2015-01-01

    Regeneration of peripheral nerves depends on the abilities of rejuvenating axons to migrate at the injury site through cellular debris and altered extracellular matrix, and then grow along the residual distal nerve sheath conduit and reinnervate synaptic targets. Considerable evidence suggest that glial cells participate in this process, although the mechanisms remain to be clarified. In cell culture, regenerating neurites secrete PACAP, a peptide shown to induce the expression of the protease tissue plasminogen activator (tPA) in neural cell types. In the present studies, we tested the hypothesis that PACAP can stimulate peripheral glial cells to produce tPA. More specifically, we addressed whether or not PACAP promoted the expression and activity of tPA in the Schwann cell line RT4-D6P2T, which shares biochemical and physical properties with Schwann cells. We found that PACAP dose- and time-dependently stimulated tPA expression both at the mRNA and protein level. Such effect was mimicked by maxadilan, a potent PAC1 receptor agonist, but not by the PACAP-related homolog VIP, suggesting a PAC1-mediated function. These actions appeared to be mediated at least in part by the Akt/CREB signaling cascade because wortmannin, a PI3K inhibitor, prevented peptide-driven CREB phosphorylation and tPA increase. Interestingly, treatment with BDNF mimicked PACAP actions on tPA, but acted through both the Akt and MAPK signaling pathways, while causing a robust increase in PACAP and PAC1 expression. PACAP6-38 totally blocked PACAP-driven tPA expression and in part hampered BDNF-mediated effects. We conclude that PACAP, acting through PAC1 receptors, stimulates tPA expression and activity in a Akt/CREB-dependent manner to promote proteolytic activity in Schwann-cell like cultures. PMID:25658447

  6. Synergistic and multidimensional regulation of plasminogen activator inhibitor type 1 expression by transforming growth factor type β and epidermal growth factor

    SciTech Connect

    Song, Xiaoling; Thalacker, F.W.; Nilsen-Hamilton, Marit

    2012-04-06

    The major physiological inhibitor of plasminogen activator, type I plasminogen activator inhibitor (PAI-1), controls blood clotting and tissue remodeling events that involve cell migration. Transforming growth factor type β (TGFβ) and epidermal growth factor (EGF) interact synergistically to increase PAI-1 mRNA and protein levels in human HepG2 and mink Mv1Lu cells. Other growth factors that activate tyrosine kinase receptors can substitute for EGF. EGF and TGFβ regulate PAI-1 by synergistically activating transcription, which is further amplified by a decrease in the rate of mRNA degradation, the latter being regulated only by EGF. The combined effect of transcriptional activation and mRNA stabilization results in a rapid 2-order of magnitude increase in the level of PAI-1. TGFβ also increases the sensitivity of the cells to EGF, thereby recruiting the cooperation of EGF at lower than normally effective concentrations. The contribution of EGF to the regulation of PAI-1 involves the MAPK pathway, and the synergistic interface with the TGFβ pathway is downstream of MEK1/2 and involves phosphorylation of neither ERK1/2 nor Smad2/3. Synergism requires the presence of both Smad and AP-1 recognition sites in the promoter. This work demonstrates the existence of a multidimensional cellular mechanism by which EGF and TGFβ are able to promote large and rapid changes in PAI-1 expression.

  7. Degradation of fibrin and elastin by intact human alveolar macrophages in vitro. Characterization of a plasminogen activator and its role in matrix degradation.

    PubMed Central

    Chapman, H A; Stone, O L; Vavrin, Z

    1984-01-01

    Fibrin deposition is prominent in the histopathology of a number of inflammatory lung diseases. Plasmin, activated locally in the lung, can degrade not only this fibrin but potentially structural proteins important to normal lung architecture. Because alveolar macrophages are prominent in inflammatory processes of the lung, we examined the plasminogen activator (PA) activity of human alveolar macrophages. Intact alveolar macrophages from each of 10 healthy subjects expressed PA activity. There was no difference in activity between smoking and nonsmoking individuals. The activator activity was largely cell-associated, but under certain culture conditions, macrophages released a soluble activator into the culture medium. The membrane-bound activator had an apparent molecular mass of 52-55 kD in nonreduced sodium dodecyl sulfate (SDS) gels, and monospecific antibody to urokinase neutralized the enzyme activity. Immunoprecipitation of [35S]methionine-labeled cells showed that human alveolar macrophages actually synthesize the PA in vitro. SDS-gel analysis of the immunoprecipitated material revealed the predominant species of PA to be structurally similar to reduced, active urokinase. We also examined the role of PA in the degradation of both insoluble fibrin and elastin matrices by live macrophages. Cells degraded an insoluble fibrin matrix in the presence of plasminogen whether or not the macrophages contacted the fibrin as long as proteinase inhibitors were not in the culture medium. In the presence of serum proteinase inhibitors, macrophages still degraded a fibrin matrix, but only if they were in contact with the fibrin. Live macrophages also degraded insoluble elastin only when in contact with the elastin but could do so even in the presence of serum proteinase inhibitors. In matrices containing a mixture of fibrin and elastin, cells did not degrade elastin unless plasminogen was added to the medium. These results indicate that normal alveolar macrophages

  8. In vitro and in vivo antiangiogenic activity of a novel deca-peptide derived from human tissue-type plasminogen activator kringle 2

    SciTech Connect

    Su, Li; Xu, Xun; Zhao, Hui; Gu, Qing; Zou, Haidong

    2010-06-11

    A synthetic deca-peptide corresponding to the amino acid sequence Arg{sup 54}-Trp{sup 63} of human tissue-type plasminogen activator (t-PA) kringle 2 domain, named TKII-10, is produced and tested for its ability to inhibit endothelial cell proliferation, migration, tube formation in vitro, and angiogenesis in vivo. At the same time, another peptide TKII-10S composed of the same 10 amino acids as TKII-10, but in a different sequence, is also produced and tested. The results show that TKII-10 potently inhibits VEGF-stimulated endothelial cell migration and tube formation in a dose-dependent, as well as sequence-dependent, manner in vitro while it is inactive in inhibiting endothelial cell proliferation. Furthermore, TKII-10 potently inhibits angiogenesis in chick chorioallantoic membrane and mouse cornea. The middle four amino acids DGDA in their sequence play an important role in TKII-10 angiogenesis inhibition{sub .} These results suggest that TKII-10 is a novel angiogenesis inhibitor that may serve as a prototype for antiangiogenic drug development.

  9. Soluble Urokinase-Type Plasminogen Activator Receptor Plasma Concentration May Predict Susceptibility to High Altitude Pulmonary Edema

    PubMed Central

    Zügel, Stefanie; Schoeb, Michele; Auinger, Katja; Dehnert, Christoph; Maggiorini, Marco

    2016-01-01

    Introduction. Acute exposure to high altitude induces inflammation. However, the relationship between inflammation and high altitude related illness such as high altitude pulmonary edema (HAPE) and acute mountain sickness (AMS) is poorly understood. We tested if soluble urokinase-type plasminogen activator receptor (suPAR) plasma concentration, a prognostic factor for cardiovascular disease and marker for low grade activation of leukocytes, will predict susceptibility to HAPE and AMS. Methods. 41 healthy mountaineers were examined at sea level (SL, 446 m) and 24 h after rapid ascent to 4559 m (HA). 24/41 subjects had a history of HAPE and were thus considered HAPE-susceptible (HAPE-s). Out of the latter, 10/24 HAPE-s subjects were randomly chosen to suppress the inflammatory cascade with dexamethasone 8 mg bid 24 h prior to ascent. Results. Acute hypoxic exposure led to an acute inflammatory reaction represented by an increase in suPAR (1.9 ± 0.4 at SL versus 2.3 ± 0.5 at HA, p < 0.01), CRP (0.7 ± 0.5 at SL versus 3.6 ± 4.6 at HA, p < 0.01), and IL-6 (0.8 ± 0.4 at SL versus 3.3 ± 4.9 at HA, p < 0.01) in all subjects except those receiving dexamethasone. The ascent associated decrease in PaO2 correlated with the increase in IL-6 (r = 0.46, p < 0.001), but not suPAR (r = 0.27, p = 0.08); the increase in IL-6 was not correlated with suPAR (r = 0.16, p = 0.24). Baseline suPAR plasma concentration was higher in the HAPE-s group (2.0 ± 0.4 versus 1.8 ± 0.4, p = 0.04); no difference was found for CRP and IL-6 and for subjects developing AMS. Conclusion. High altitude exposure leads to an increase in suPAR plasma concentration, with the missing correlation between suPAR and IL-6 suggesting a cytokine independent, leukocyte mediated mechanism of low grade inflammation. The correlation between IL-6 and PaO2 suggests a direct effect of hypoxia, which is not the case for suPAR. However, suPAR plasma concentration measured before hypoxic exposure may predict

  10. Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

    SciTech Connect

    Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho; Jung, Young Do

    2012-03-01

    Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H{sub 2}O{sub 2}) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H{sub 2}O{sub 2} increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells

  11. Activation of plasminogen into plasmin at the surface of endothelial microparticles: a mechanism that modulates angiogenic properties of endothelial progenitor cells in vitro

    PubMed Central

    Lacroix, Romaric; Sabatier, Florence; Mialhe, Agnes; Basire, Agnes; Pannell, Ralph; Borghi, Helene; Robert, Stephane; Lamy, Edouard; Plawinski, Laurent; Camoin-Jau, Laurence; Gurewich, Victor; Angles-Cano, Eduardo; Dignat-George, Francoise

    2007-01-01

    The regulation of plasmin generation on cell surfaces is of critical importance in the control of vascular homeostasis. Cell-derived microparticles participate in the dissemination of biological activities. However their capacity to promote plasmin generation has not been documented. In this study, we show that endothelial microparticles (EMP) from TNFα-stimulated endothelial cells, served as a surface for the generation of plasmin. The generation of plasmin involved expression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) at the surface of EMP and was further increased by their ability to bind exogenous uPA on uPAR. Plasminogen was activated at the surface of EMP in a dose-dependent, saturable and specific manner as indicated by the inhibition of plasmin formation by ε-amino-caproic acid (ε-ACA) and carboxypeptidase B. EMP-induced plasmin generation affects tube formation mediated by endothelial progenitor cells. However, low amounts of EMP increased tube formation whereas higher concentrations inhibited it. Prevention of these effects by inhibitors of either uPA or plasmin, underscore the key role of EMP-induced plasmin generation. In conclusion, we demonstrated that EMP act as vectors supporting efficient plasmin generation and dissemination, a new pathway in the regulation of endothelial proteolytic activities with potential involvement in inflammation, angiogenesis and atherosclerosis. PMID:17606760

  12. Subretinal recombinant tissue plasminogen activator and pneumatic displacement for the management of subretinal hemorrhage occurring after anti-VEGF injections for wet AMD.

    PubMed

    Tognetto, Daniele; Skiadaresi, Eirini; Cecchini, Paolo; Ravalico, Giuseppe

    2011-01-01

    We describe three cases of submacular hemorrhage that occurred two to four days after anti-VEGF intravitreal injection for occult choroidal neovascularisation in age-related macular degeneration and their management with 25 gauge pars plana vitrectomy with injection of subretinal recombinant tissue plasminogen activator (rTPA) followed by fluid-air exchange and postoperative prone position. Vitrectomy, subretinal rTPA injection and fluid-gas exchange apply as a safe and effective treatment in these cases. Functional results seem to be positive especially if surgical treatment is promptly performed.

  13. Para-aminobenzamidine linked regenerated cellulose membranes for plasminogen activator purification: effect of spacer arm length and ligand density.

    PubMed

    Fasoli, Ezio; Reyes, Yiaslin Ruiz; Guzman, Osiris Martinez; Rosado, Alexandra; Cruz, Vivian Rodriguez; Borges, Amaris; Martinez, Edmarie; Bansal, Vibha

    2013-07-01

    Despite membrane-based separations offering superior alternative to packed bed chromatographic processes, there has been a substantial lacuna in their actual application to separation processes. One of the major reasons behind this is the lack of availability of appropriately modified or end-group modifiable membranes. In this paper, an affinity membrane was developed using a commercially available serine protease inhibitor, para-aminobenzamidine (pABA). The membrane modification was optimized for protein binding capacity by varying: (i) the length of the spacer arm (SA; 5-atoms, 7-atoms, and 14-atoms) linking the ligand to membrane surface; (ii) the affinity ligand (pABA) density on membrane surface (5-25nmol/cm(2)). Resulting membranes were tested for their ability to bind plasminogen activators (PAs) from mono- and multi-component systems in batch mode. The membrane containing pABA linked through 7-atoms SA but similar ligand density as in the case of 5- or 14-atoms long SA was found to bind up to 1.6-times higher amounts of PA per nmoles of immobilized ligand from conditioned HeLa cell culture media. However, membranes with similar ligand densities but different lengths of SA, showed comparable binding capacities in mono-component system. In addition, the length of SA did not affect the selectivity of the ligand for PA. A clear inverse linear correlation was observed between ligand density and binding capacity until the point of PA binding optima was reached (11±1.0nmol/cm(2)) in mono- and multi-component systems for 7- as well as 14-atoms SA. Up to 200-fold purification was achieved in a single step separation of PA from HeLa conditioned media using these affinity membranes. The issues of ligand leaching and reuse of the membranes were also investigated. An extensive regeneration procedure allowed the preservation of approximately 95% of the PA binding capacity of the membranes even after five cycles of use.

  14. Regulatory role of microRNA-30b and plasminogen activator inhibitor-1 in the pathogenesis of cognitive impairment

    PubMed Central

    LI, XIUQIN; GAO, YONG; MENG, ZHAOYUN; ZHANG, CUI; QI, QINDE

    2016-01-01

    The present study aimed to investigate the role of plasminogen activator inhibitor-1 (PAI-1) in drug-induced early cognitive impairment and the underlying mechanism concerning microRNA (miR)-30b. A mouse model of cognitive impairment was established by intraperitoneal injection of scopolamine (2 mg/kg body weight) for 13 days. Behavioral performance was assessed using the Morris water maze (MWM) test. The mRNA expression levels of PAI-1 and miR-30b were detected using quantitative polymerase chain reaction (qPCR). The protein expression levels of PAI-1 in the hippocampus and blood were determined using western blot analysis and enzyme-linked immunosorbent assays. The MWM test demonstrated that, on days 3 and 4, the escape latency was significantly elevated in the model mice in comparison with control group (P<0.05). In addition, the length of swimming path was significantly increased (P<0.05), while the number of times of crossing the platform location was significantly reduced in the model mouse group (P<0.05) in comparison with the control group. qPCR demonstrated that the mRNA expression levels of PAI-1 in the model mice was significantly elevated in the hippocampus and blood in comparison with the control group (P<0.01). Furthermore, western blot analysis and enzyme-linked immunosorbent assay demonstrated that the protein expression levels of PAI-1 were significantly elevated in the hippocampus and blood in the model group, in comparison with the control group (P<0.05). Notably, the levels of miR-30b in the hippocampus and blood were significantly decreased in the model mice in comparison with the control group (P<0.01). To conclude, the expression levels of PAI-1 were significantly elevated in mice with scopolamine-induced cognitive impairment, which may be associated with the downregulation of miR-30b. The findings from the present study suggest that miR-30b may be involved in the regulation of PAI-1, which would contribute to the pathogenesis of cognitive

  15. Occurrence of an Inhibitor of Tissue-Type Plasminogen Activator in Seeds and in Vitro Cultures of Erythrina caffra Thunb.

    PubMed

    Meyer, H J; van Staden, J

    1991-08-01

    The level of an inhibitor of tissue-type plasminogen activator (t-PA) increased slowly during the early developmental stage of seeds of Erythrina caffra Thunb. Thereafter, the inhibitor increased exponentially until the seeds reached maturity. At maturity, the t-PA inhibitor levels in the cotyledons were 38 times higher than the levels at the onset of seed development. The t-PA inhibitor accumulated at a faster rate than the storage proteins, which reached a concentration 15 times higher than the protein concentration at the onset of seed development. During the imbibition and germination process, the t-PA inhibitor decreased gradually. The inhibitor kept on decreasing during the growth of the seedlings until the 10th day after imbibition, when it leveled off at 4.1% of that of the initial inhibitor concentration. The inhibitor remained at this level until the cotyledons were shed at day 22. The total protein in the cotyledons decreased at a slower rate than the inhibitor and reached a minimum concentration at day 20 of 3.6% of the initial protein concentration in the cotyledons. Callus cultures of root, shoot, leaf, and cotyledonary tissue was established and maintained on Murashige-Skoog medium supplemented with 3% sucrose, 10 micromolar benzyladenine, and 5 micromolar 2,4-dichlorophenoxyacetic acid. A shoot cell suspension culture was established on Murashige-Skoog medium supplemented with 3% sucrose, 1 micromolar benzyladenine, and 0.5 micromolar 2,4-dichlorophenoxyacetic acid (pH 5.7) and shaken at 60 revolutions per minute. The level of t-PA inhibitor in root, shoot, leaf, and cotyledonary callus was substantially lower than in the corresponding intact tissue. The t-PA inhibitor levels in the linear growth phase was higher than in the lag or stationary growth phases of the cell suspension culture. A hydrolysate of the cell walls of tomato and E. caffra Thunb, as well as polyamines and organic acids, did not increase the concentration of t-PA inhibitor in

  16. Collagenase and tissue plasminogen activator production in developing rat calvariae: normal progression despite fetal exposure to microgravity

    NASA Technical Reports Server (NTRS)

    Davis, B. A.; Sipe, B.; Gershan, L. A.; Fiacco, G. J.; Lorenz, T. C.; Jeffrey, J. J.; Partridge, N. C.

    1998-01-01

    Exposure to zero gravity has been shown to cause a decrease in bone formation. This implicates osteoblasts as the gravity-sensing cell in bone. Osteoblasts also are known to produce neutral proteinases, including collagenase and tissue plasminogen activator (tPA), which are thought to be important in bone development and remodeling. The present study investigated the effects of zero gravity on development of calvariae and their expression of collagenase and tPA. After in utero exposure to zero gravity for 9 days on the NASA STS-70 space shuttle mission, the calvariae of rat pups were examined by immunohistochemistry for the presence and location of these two proteinases. The ages of the pups were from gestational day 20 (G20) to postnatal (PN) day 35. Both collagenase and tPA were found to be present at all ages examined, with the greatest amount of both proteinases present in the PN14 rats. At later ages, high amounts were maintained for tPA but collagenase decreased substantially between ages PN21 to PN35. The location of collagenase was found to be associated with bone-lining cells, osteoblasts, osteocytes, and in the matrix along cement lines. In contrast, tPA was associated with endothelial cells lining the blood vessels entering bone. The presence and developmental expression of these two proteinases appeared to be unaffected by the exposure to zero gravity. The calvarial thickness of the pups was also examined; again the exposure to zero gravity showed little to no effect on the growth of the calvariae. Notably, from G20 to PN14, calvarial thickness increased dramatically, reaching a plateau after this age. It was apparent that elevated collagenase expression correlated with rapid bone growth in the period from G20 to PN14. To conclude, collagenase and tPA are present during the development of rat calvariae. Despite being produced by the same cell in vitro, i.e., the osteoblast, they are located in distinctly different places in bone in vivo. Their presence

  17. Effects of angiopoietin-1 on hemorrhagic transformation and cerebral edema after tissue plasminogen activator treatment for ischemic stroke in rats.

    PubMed

    Kawamura, Kunio; Takahashi, Tetsuya; Kanazawa, Masato; Igarashi, Hironaka; Nakada, Tsutomu; Nishizawa, Masatoyo; Shimohata, Takayoshi

    2014-01-01

    An angiogenesis factor, angiopoietin-1 (Ang1), is associated with the blood-brain barrier (BBB) disruption after focal cerebral ischemia. However, whether hemorrhagic transformation and cerebral edema after tissue plasminogen activator (tPA) treatment are related to the decrease in Ang1 expression in the BBB remains unknown. We hypothesized that administering Ang1 might attenuate hemorrhagic transformation and cerebral edema after tPA treatment by stabilizing blood vessels and inhibiting hyperpermeability. Sprague-Dawley rats subjected to thromboembolic focal cerebral ischemia were assigned to a permanent ischemia group (permanent middle cerebral artery occlusion; PMCAO) and groups treated with tPA at 1 h or 4 h after ischemia. Endogenous Ang1 expression was observed in pericytes, astrocytes, and neuronal cells. Western blot analyses revealed that Ang1 expression levels on the ischemic side of the cerebral cortex were decreased in the tPA-1h, tPA-4h, and PMCAO groups as compared to those in the control group (P = 0.014, 0.003, and 0.014, respectively). Ang1-positive vessel densities in the tPA-4h and PMCAO groups were less than that in the control group (p = 0.002 and <0.001, respectively) as well as that in the tPA-1h group (p = 0.047 and 0.005, respectively). These results suggest that Ang1-positive vessel density was maintained when tPA was administered within the therapeutic time window (1 h), while it was decreased when tPA treatment was given after the therapeutic time window (4 h). Administering Ang1 fused with cartilage oligomeric protein (COMP) to supplement this decrease has the potential to suppress hemorrhagic transformation as measured by hemoglobin content in a whole cerebral homogenate (p = 0.007) and cerebral edema due to BBB damage (p = 0.038), as compared to administering COMP protein alone. In conclusion, Ang1 might be a promising target molecule for developing vasoprotective therapies for controlling hemorrhagic transformation and cerebral edema

  18. Low-Dose Versus Standard-Dose Tissue Plasminogen Activator in Acute Ischemic Stroke in Asian Populations

    PubMed Central

    Liu, Meng-Dong; Ning, Wei-Dong; Wang, Ren-Cong; Chen, Wei; Yang, Yang; Lin, Yan; Hu, Da-Hai; Lau, Wayne-Bond; Qu, Yan

    2015-01-01

    Abstract Recent studies have investigated the most efficacious dose of intravenous tissue plasminogen activator (IV-tPA) for acute ischemic stroke (AIS) patients. There remains no definitive consensus concerning the superior efficacious IV-tPA dose (standard- vs. low-dose), prompting us to perform a meta-analysis comparing the efficacy and safety profile of standard- versus low-dose IV-tPA. We identified relevant studies pertaining to the specific aim of our meta-analysis by searching PubMed and EMBASE (January 1990–September 2015) Either a fixed- or random-effects model was employed (dependent upon data heterogeneity) to analyze the efficacy and safety outcome. Ten cohort studies involving 4389 sum patients were included in the meta-analysis. By using the random-effects model, the meta-analysis indicated no statistically significant difference in favorable functional outcome (modified Rankin scale 0–1) at 3 months (heterogeneity: χ2 = 17.45, P = 0.04, I2 = 48%; OR: 0.88 [95% CI: 0.71–1.11]; P = 0.28) and incidence of symptomatic intracranial hemorrhage (SICH) (heterogeneity: χ2 = 14.41, P = 0.11, I2 = 38%; OR: 1.19 [95% CI: 0.76 to 1.87]; P = 0.45) between the standard- and low-dose groups. The fixed-effects model demonstrated no significant difference in mortality within 3 months (heterogeneity: χ2 = 6.73, P = 0.57, I2 = 0%; OR: 0.91 [95% CI: 0.73–1.12]; P = 0.37) between the standard- and low-dose groups. Low-dose IV-tPA is comparable to standard-dose IV-tPA in both efficacy (favorable functional outcome) and safety (SICH and mortality). Confirmation of these findings through randomized trials is warranted. PMID:26717400

  19. Plasminogen activator inhibitor-1 4G/5G polymorphism and retinopathy risk in type 2 diabetes: a meta-analysis

    PubMed Central

    2013-01-01

    Background Mounting evidence has suggested that plasminogen activator inhibitor-1 (PAI-1) is a candidate for increased risk of diabetic retinopathy. Studies have reported that insertion/deletion polymorphism in the PAI-1 gene may influence the risk of this disease. To comprehensively address this issue, we performed a meta-analysis to evaluate the association of PAI-1 4G/5G polymorphism with diabetic retinopathy in type 2 diabetes. Methods Data were retrieved in a systematic manner and analyzed using Review Manager and STATA Statistical Software. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of associations. Results Nine studies with 1, 217 cases and 1, 459 controls were included. Allelic and genotypic comparisons between cases and controls were evaluated. Overall analysis suggests a marginal association of the 4G/5G polymorphism with diabetic retinopathy (for 4G versus 5G: OR 1.13, 95%CI 1.01 to 1.26; for 4G/4G versus 5G/5G: OR 1.30, 95%CI 1.04 to 1.64; for 4G/4G versus 5G/5G + 4G/5G: OR 1.26, 95%CI 1.05 to 1.52). In subgroup analysis by ethnicity, we found an association among the Caucasian population (for 4G versus 5G: OR 1.14, 95% CI 1.00 to 1.30; for 4G/4G versus 5G/5G: OR 1.33, 95%CI 1.02 to 1.74; for 4G/4G versus 5G/5G + 4G/5G: OR 1.41, 95%CI 1.13 to 1.77). When stratified by the average duration of diabetes, patients with diabetes histories longer than 10 years have an elevated susceptibility to diabetic retinopathy than those with shorter histories (for 4G/4G versus 5G/5G: OR 1.47, 95%CI 1.08 to 2.00). We also detected a higher risk in hospital-based studies (for 4G/4G versus 5G/5G+4G/5G: OR 1.27, 95%CI 1.02 to 1.57). Conclusions The present meta-analysis suggested that 4G/5G polymorphism in the PAI-1 gene potentially increased the risk of diabetic retinopathy in type 2 diabetes and showed a discrepancy in different ethnicities. A higher susceptibility in patients with longer duration of diabetes (more than 10

  20. Development of a plasminogen activator inhibitor (PAI-1) assay and comparison of plasma PAI-1 activity in hyperlipidemic/dyslipidemic dogs with either hyperadrenocorticism or diabetes mellitus, and healthy dogs.

    PubMed

    Wong, Cheryl J; Koch, Michael; Behling-Kelly, Erica L

    2017-04-01

    Thrombosis is a serious complication of many canine diseases and may be related to decreased fibrinolytic potential. Plasminogen activator inhibitor-1 (PAI-1) is the key regulator of fibrinolysis with increased levels demonstrated in states of pro-thrombosis and abnormal lipid metabolism. Our objective was to develop and validate a canine PAI-1 activity assay and test whether dogs with hyperadrenocorticism or diabetes mellitus that are hyperlipidemic/dyslipidemic have increased plasma PAI-1 activity. Functionally active PAI-1 in the plasma sample was incubated with recombinant tissue plasminogen activator (tPA), allowing the formation of a 1:1 stoichiometric inactive complex. Residual unbound tPA was then reacted with excess plasminogen in the presence of a colorimetric plasmin substrate. Plasmin production is quantified by computing the area under the curve of time (x) vs optical density (y) plot and converted to tPA IU/mL by comparison to a calibration curve of tPA standards. PAI-1 activity was determined by calculating the proportion of exogeneous tPA suppressed by PAI-1 in plasma. Assay verification included assessment of linearity, specificity, precision, sensitivity, and stability. PAI-1 activity was increased in hyperlipidemic compared to healthy dogs, but there was no significant difference between dogs with hyperadrenocorticism and diabetes mellitus. A near significant decrease in activity was detected in thawed plasma stored for 20h at 4°C. Our successfully validated assay offers a new tool for investigating fibrinolysis in dogs. Investigation of PAI-1 activity in dogs with other diseases associated with an increased risk of thrombosis would be valuable. Future studies of PAI-1 activity should consider its lability.

  1. Staurosporine induces ganglion cell differentiation in part by stimulating urokinase-type plasminogen activator expression and activation in the developing chick retina

    SciTech Connect

    Kim, Yeoun-Hee; Chang, Yongmin; Jung, Jae-Chang

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer Staurosporine mediates stimulation of RGC differentiation in vitro cultured retinal neuroblasts. Black-Right-Pointing-Pointer Staurosporine mediates uPA activation during RGC differentiation in vitro. Black-Right-Pointing-Pointer Inhibition of uPA blocks the staurosporine mediated RGC differentiation both in vitro and in ovo. Black-Right-Pointing-Pointer Thus, uPA may play a role in the staurosporine-mediated stimulation of RGC differentiation. -- Abstract: Here, we investigated whether staurosporine-mediated urokinase-type plasminogen activator (uPA) activation is involved in retinal ganglion cell (RGC) differentiation. Retinal cells were isolated from developing chick retinas at embryonic day 6 (E6). Relatively few control cells grown in serum-free medium started to form processes by 12 h. In contrast, staurosporine-treated cells had processes within 3 h, and processes were evident at 8 h. Immunofluorescence staining showed that Tuj-1-positive cells with shorter neurites could be detected in control cultures at 18 h, whereas numerous Tuj-1 positive ganglion cells with longer neuritic extensions were seen in staurosporine-treated cultures. BrdU-positive proliferating cells were more numerous in control cultures than in staurosporine-treated cultures, and the BrdU staining was not detected in post-mitotic Tuj-1 positive ganglion cells. Western blotting of cell lysates showed that staurosporine induced high levels of the active form of uPA. The staurosporine-induced uPA signal was localized predominantly in the soma, neurites and axons of Tuj-1-positive ganglion cells. Amiloride, an inhibitor of uPA, markedly reduced staurosporine-induced Tuj-1 staining, neurite length, neurite number, and uPA staining versus controls. In developing retinas in ovo, amiloride administration remarkably reduced the staurosporine-induced uPA staining and RGC differentiation. Taken together, our in vitro and in vivo data collectively indicate that

  2. Purification of c-phycocyanin from Spirulina fusiformis and its effect on the induction of urokinase-type plasminogen activator from calf pulmonary endothelial cells.

    PubMed

    Madhyastha, H K; Radha, K S; Sugiki, M; Omura, S; Maruyama, M

    2006-09-01

    c-Phycocyanin (c-pc), a blue coloured, fluorescent protein was purified from blue-green alga, Spirulina fusiformis and its effect on fibrinolytic system in vascular endothelial cells was investigated. The c-pc consisted of two subunits, alpha and beta, whose molecular masses were 16 and 17 kDa, respectively. N-terminal sequences of both subunits were well conserved compared with other blue green algal phycobiliproteins. Fibrinolytic activity in the medium conditioned by calf pulmonary arterial endothelial cells was measured by the fibrin plate method. The c-pc increased the fibrinolytic activity in dose- and time-dependent manners. Fibrin zymographic studies indicated that c-pc-induced urokinase-type plasminogen activator in the cells. These in vitro results suggest that c-pc from S. fusiformis is a potent profibrinolytic protein in the vascular endothelial system.

  3. Pneumatic Displacement with Perfluoropropane Gas and Intravitreal Tissue Plasminogen Activator for Subretinal Subfoveal Hemorrhage after Focal Laser Photocoagulation in Central Serous Chorioretinopathy

    PubMed Central

    Espinoza, Juan V.; Lasave, Andres F.; Savino-Zari, Dario; Arevalo, Fernando A.

    2014-01-01

    Objective. To report the visual and anatomic outcomes of pneumatic displacement with perfluoropropane (C3F8) gas and intravitreal tissue plasminogen activator (IVTPA) for subretinal subfoveal hemorrhage after focal laser photocoagulation in central serous chorioretinopathy (CSCR). Method. Interventional, retrospective case report of one eye (one patient). Outcome measures included visual acuity (VA), central macular thickness (CMT), and size of the lesion at two weeks of followup. Fluorescein angiography (FA) and optical coherent tomography (OCT) were used to measure anatomic outcomes. Results. A 35-year-old man with history of chronic CSCR received focal laser photocoagulation in the right eye two days before presentation. At initial examination, VA was 20/200 (ETDRS chart), CMT was 398 μ, and a subretinal subfoveal hemorrhage was seen. Tissue plasminogen activator (tPA) at a dose of 25 µg/0.1 mL was injected intravitreally before intravitreal C3F8 injection, and prone positioning was indicated postoperatively. At 24 hours, the hemorrhage had been displaced inferiorly and VA improved to 20/100. Two weeks later, VA improved to 20/80, CMT decreased to 225 μ, and the hemorrhage decreased without foveal involvement. Conclusions. The technique seems safe and effective in treating visually significant subretinal subfoveal hemorrhage. PMID:25485161

  4. Inhibitory effect of berberine on the invasion of human lung cancer cells via decreased productions of urokinase-plasminogen activator and matrix metalloproteinase-2

    SciTech Connect

    Peng, P.-L.; Hsieh, Y.-S.; Wang, C.-J.; Hsu, J.-L.; Chou, F.-P. . E-mail: fpchou@csmu.edu.tw

    2006-07-01

    Berberine, a compound isolated from medicinal herbs, has been reported with many pharmacological effects related to anti-cancer and anti-inflammation capabilities. In this study, we observed that berberine exerted a dose- and time-dependent inhibitory effect on the motility and invasion ability of a highly metastatic A549 cells under non-cytotoxic concentrations. In cancer cell migration and invasion process, matrix-degrading proteinases are required. A549 cell treated with berberine at various concentrations showed reduced ECM proteinases including matrix metalloproteinase-2 (MMP2) and urokinase-plasminogen activator (u-PA) by gelatin and casein zymography analysis. The inhibitory effect is likely to be at the transcriptional level, since the reduction in the transcripts levels was corresponding to the proteins. Moreover, berberine also exerted its action via regulating tissue inhibitor of metalloproteinase-2 (TIMP-2) and urokinase-plasminogen activator inhibitor (PAI). The upstream mediators of the effect involved c-jun, c-fos and NF-{kappa}B, as evidenced by reduced phosphorylation of the proteins. These findings suggest that berberine possesses an anti-metastatic effect in non-small lung cancer cell and may, therefore, be helpful in clinical treatment.

  5. High Expression of Urokinase-Type Plasminogen Activator Is Associated with Lymph Node Metastasis of Invasive Ductal Carcinoma of the Breast

    PubMed Central

    Kim, Eun Young; Do, Sung-Im; Hyun, Keehoon; Park, Yong Lai; Kim, Dong-Hoon; Chae, Seoung Wan; Sohn, Jin Hee

    2016-01-01

    Purpose In the present study, we evaluated the levels of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI-1) by performing immunohistochemical staining to determine whether they were reliable prognostic markers in patients with breast cancer. Methods Demographic and clinicopathological parameters of 214 patients with invasive ductal carcinoma (IDC) and 80 patients with ductal carcinoma in situ (DCIS) who were diagnosed and treated from 2006 to 2010 were analyzed. Tissue microarray was constructed and immunohistochemical staining was performed for each specimen. Results Univariate analyses showed that age at diagnosis, history of hormone replacement therapy, radiation therapy, skin and chest wall invasion, Paget disease, lymphovascular invasion, estrogen receptor positivity, and triple-negative subtype were significantly associated with patient prognosis (p<0.005). Patients with DCIS showed higher PAI-1 expression than patients with IDC (82.5% and 36.2%, respectively; p=0.012). Lymph node metastasis was more frequent in patients with high uPA levels than in patients with low uPA levels (p=0.001). Conclusion Our results suggested that PAI-1 was involved in tumor progression in the early stages of breast cancer, such as DCIS. In addition, our results suggested that high uPA levels were associated with the lymph node metastasis of IDC. PMID:27382391

  6. Plasminogen activator inhibitor type 1 gene is located at region q21. 3-q22 of chromosome 7 and genetically linked with cystic fibrosis

    SciTech Connect

    Klinger, K.W.; Winqvist, R.; Riccio, A.; Andreasen, P.A.; Sartorio, R.; Nielsen, L.S.; Stuart, N.; Stanislovitis, P.; Watkins, P.; Douglas, R.

    1987-12-01

    The regional chromosomal location of the human gene for plasminogen activator inhibitor type 1 (PAI1) was determined by three independent methods of gene mapping. PAI1 was localized first to 7cen-q32 and then to 7q21.3-q22 by Southern blot hybridization analysis of a panel of human and mouse somatic cell hybrids with a PAI1 cDNA probe and in situ hybridization, respectively. The authors frequent HindIII restriction fragment length polymorphism (RFLP) of the PAI1 gene with an information content of 0.369. In family studies using this polymorphism, genetic linkage was found between PAI1 and the loci for erythropoietin (EPO), paraoxonase (PON), the met protooncogene (MET), and cystic fibrosis (CF), all previously assigned to the middle part of the long arm of chromosome 7. The linkage with EPO was closest with an estimated genetic distance of 3 centimorgans, whereas that to CF was 20 centimorgans. A three-point genetic linkage analysis and data from previous studies showed that the most likely order of these loci is EPO, PAI1, PON, (MET, CF), with PAI1 being located centromeric to CF. The PAI1 RFLP may prove to be valuable in ordering genetic markers in the CF-linkage group and may also be valuable in genetic analysis of plasminogen activation-related diseases, such as certain thromboembolic disorders and cancer.

  7. Pneumatic displacement with perfluoropropane gas and intravitreal tissue plasminogen activator for subretinal subfoveal hemorrhage after focal laser photocoagulation in central serous chorioretinopathy.

    PubMed

    Al Rubaie, Khalid; Espinoza, Juan V; Lasave, Andres F; Savino-Zari, Dario; Arevalo, Fernando A; Arevalo, J Fernando

    2014-01-01

    Objective. To report the visual and anatomic outcomes of pneumatic displacement with perfluoropropane (C3F8) gas and intravitreal tissue plasminogen activator (IVTPA) for subretinal subfoveal hemorrhage after focal laser photocoagulation in central serous chorioretinopathy (CSCR). Method. Interventional, retrospective case report of one eye (one patient). Outcome measures included visual acuity (VA), central macular thickness (CMT), and size of the lesion at two weeks of followup. Fluorescein angiography (FA) and optical coherent tomography (OCT) were used to measure anatomic outcomes. Results. A 35-year-old man with history of chronic CSCR received focal laser photocoagulation in the right eye two days before presentation. At initial examination, VA was 20/200 (ETDRS chart), CMT was 398 μ, and a subretinal subfoveal hemorrhage was seen. Tissue plasminogen activator (tPA) at a dose of 25 µg/0.1 mL was injected intravitreally before intravitreal C3F8 injection, and prone positioning was indicated postoperatively. At 24 hours, the hemorrhage had been displaced inferiorly and VA improved to 20/100. Two weeks later, VA improved to 20/80, CMT decreased to 225 μ, and the hemorrhage decreased without foveal involvement. Conclusions. The technique seems safe and effective in treating visually significant subretinal subfoveal hemorrhage.

  8. Transgenic Over-expression of Plasminogen Activator Inhibitor-1 Results in Age-dependent and Gender-specific Increases in Bone Strength and Mineralization

    PubMed Central

    Nordstrom, S.M.; Carleton, S.M.; Carson, W.L.; Eren, M.; Phillips, C.L.; Vaughan, D.E.

    2014-01-01

    The plasminogen activation system (PAS) and its principal inhibitor, plasminogen activator inhibitor- 1 (PAI-1), are recognized modulators of matrix. In addition, the PAS has previously been implicated in the regulation of bone homeostasis. Our objective was to study the influence of active PAI-1 on geometric, biomechanical, and mineral characteristics of bone using transgenic mice that over-expresses a variant of human PAI-1 that exhibits enhanced functional stability. Femora were isolated from male and female, wildtype (WT) and transgenic (PAI-1.stab) mice at 16 and 32 weeks of age (n=10). Femora were imaged via DEXA for BMD and µCT for cortical mid-slice geometry. Torsional testing was employed for biomechanical properties. Mineral composition was analyzed via instrumental neutron activation analysis. Female femora were further analyzed for trabecular bone histomorphometry (n=11). Whole animal DEXA scans were performed on PAI-1.stab females and additional transgenic lines in which the functional domains of the PAI-1 protein were specifically disrupted. Thirty-two week female PAI-1.stab femora exhibited decreased mid-slice diameters and reduced polar moment of area compared to WT, while maintaining similar cortical bone width. Greater biomechanical strength and stiffness was demonstrated by 32 week PAI-1.stab female femora in addition to a 52% increase in BMD. PAI-1.stab trabecular bone architecture was comparable to WT. Osteoid area was decreased in PAI-1.stab mice while mineral apposition rate increased by 78% over WT. Transgenic mice expressing a reactive-site mutant form of PAI-1 showed an increase in BMD similar to PAI-1.stab, whereas transgenic mice expressing a PAI-1 with reduced affinity for vitronectin were comparable to WT. Over-expression of PAI-1 resulted in increased mineralization and biomechanical properties of mouse femora in an age-dependent and gender-specific manner. Changes in mineral preceded increases in strength/stiffness and deterred

  9. Transgenic over-expression of plasminogen activator inhibitor-1 results in age-dependent and gender-specific increases in bone strength and mineralization.

    PubMed

    Nordstrom, S M; Carleton, S M; Carson, W L; Eren, M; Phillips, C L; Vaughan, D E

    2007-12-01

    The plasminogen activation system (PAS) and its principal inhibitor, plasminogen activator inhibitor-1 (PAI-1), are recognized modulators of matrix. In addition, the PAS has previously been implicated in the regulation of bone homeostasis. Our objective was to study the influence of active PAI-1 on geometric, biomechanical, and mineral characteristics of bone using transgenic mice that over-express a variant of human PAI-1 that exhibits enhanced functional stability. Femora were isolated from male and female, wildtype (WT) and transgenic (PAI-1.stab) mice at 16 and 32 weeks of age (n=10). Femora were imaged via DEXA for BMD and muCT for cortical mid-slice geometry. Torsional testing was employed for biomechanical properties. Mineral composition was analyzed via instrumental neutron activation analysis. Female femora were further analyzed for trabecular bone histomorphometry (n=11). Whole animal DEXA scans were performed on PAI-1.stab females and additional transgenic lines in which the functional domains of the PAI-1 protein were specifically disrupted. Thirty-two week female PAI-1.stab femora exhibited decreased mid-slice diameters and reduced polar moment of area compared to WT, while maintaining similar cortical bone width. Greater biomechanical strength and stiffness were demonstrated by 32 week PAI-1.stab female femora in addition to a 52% increase in BMD. PAI-1.stab trabecular bone architecture was comparable to WT. Osteoid area was decreased in PAI-1.stab mice while mineral apposition rate increased by 78% over WT. Transgenic mice expressing a reactive-site mutant form of PAI-1 showed an increase in BMD similar to PAI-1.stab, whereas transgenic mice expressing a PAI-1 with reduced affinity for vitronectin were comparable to WT. Over-expression of PAI-1 resulted in increased mineralization and biomechanical properties of mouse femora in an age-dependent and gender-specific manner. Changes in mineral preceded increases in strength/stiffness and deterred normal

  10. Tissue plasminogen activator followed by antioxidant-loaded nanoparticle delivery promotes activation/mobilization of progenitor cells in infarcted rat brain

    PubMed Central

    Petro, Marianne; Jaffer, Hayder; Yang, Jun; Kabu, Shushi; Morris, Viola B.; Labhasetwar, Vinod

    2015-01-01

    Inherent neuronal and circulating progenitor cells play important roles in facilitating neuronal and functional recovery post stroke. However, this endogenous repair process is rather limited, primarily due to unfavorable conditions in the infarcted brain involving reactive oxygen species (ROS)-mediated oxidative stress and inflammation following ischemia/reperfusion injury. We hypothesized that during reperfusion, effective delivery of antioxidants to ischemic brain would create an environment without such oxidative stress and inflammation, thus promoting activation and mobilization of progenitor cells in the infarcted brain. We administered recombinant human tissue-type plasminogen activator (tPA) via carotid artery at 3 h post stroke in a thromboembolic rat model, followed by sequential administration of the antioxidants catalase (CAT) and superoxide dismutase (SOD), encapsulated in biodegradable nanoparticles (nano-CAT/SOD). Brains were harvested at 48 h post stroke for immunohistochemical analysis. Ipsilateral brain slices from animals that had received tPA + nano-CAT/SOD showed a widespread distribution of glial fibrillary acidic protein-positive cells (with morphology resembling radial glia-like neural precursor cells) and nestin-positive cells (indicating the presence of immature neurons); such cells were considerably fewer in untreated animals or those treated with tPA alone. Brain sections from animals receiving tPA + nano-CAT/SOD also showed much greater numbers of SOX2- and nestin-positive progenitor cells migrating from subventricular zone of the lateral ventricle and entering the rostral migratory stream than in t-PA alone treated group or untreated control. Further, animals treated with tPA + nano-CAT/SOD showed far fewer caspase-positive cells and fewer neutrophils than did other groups, as well as an inhibition of hippocampal swelling. These results suggest that the antioxidants mitigated the inflammatory response, protected neuronal cells from

  11. Effects of TGF-β1 on plasminogen activation in human dental pulp cells: Role of ALK5/Smad2, TAK1 and MEK/ERK signaling.

    PubMed

    Chang, Mei-Chi; Chang, Hsiao-Hua; Lin, Po-Shuan; Huang, Yu-An; Chan, Chiu-Po; Tsai, Yi-Ling; Lee, Shen-Yang; Jeng, Po-Yuan; Kuo, Han-Yueh; Yeung, Sin-Yuet; Jeng, Jiiang-Huei

    2016-10-09

    Transforming growth factor-β1 (TGF-β1) plays an important role in the pulpal repair and dentinogenesis. Plasminogen activation (PA) system regulates extracellular matrix turnover. In this study, we investigated the effects of TGF-β1 on PA system of dental pulp cells and its signaling pathways. Dental pulp cells were treated with different concentrations of TGF-β1. MTT assay, reverse transcription-polymerase chain reaction (RT-PCR), western blotting and enzyme-linked immunosorbant assay (ELISA) were used to detect the effect of TGF-β1 on cell viability, mRNA and protein expression of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1) as well as their secretion. The phosphorylation of Smad2 and TAK1 was analyzed by Pathscan ELISA or western blotting. Cells were pretreated with SB431542 (ALK5/Smad2/3 inhibitor), 5z-7-oxozeaenol (TAK1 inhibitor), U0126 (MEK/ERK inhibitor) for examining the related signaling. TGF-β1 slightly inhibited cell growth that was reversed by SB431542. TGF-β1 up-regulated both RNA and protein expression of PAI-1 and uPAR, whereas down-regulated uPA expression. Accordingly, TGF-β1 stimulated PAI-1 and soluble uPAR (suPAR) secretion of pulp cells, whereas uPA secretion was inhibited. TGF-β1 induced the phosphorylation of Smad2 and TAK1. In addition, SB431542, 5z-7-oxozeaenol and U0126 attenuated the TGF-β1-induced secretion of PAI-1 and suPAR. These results indicate that TGF-β1 is possibly involved in the repair/regeneration and inflammatory processes of dental pulp via regulation of PAI-1, uPA nd uPAR. These effects of TGF-β1 are related to activation of ALK5/Smad2, TAK1 and MEK/ERK signaling pathways. Clarifying the signal transduction for the effects of TGF-β1 is helpful for pulpo-dentin regeneration and tissue engineering. This article is protected by copyright. All rights reserved.

  12. Biochemical, thrombolytic and pharmacokinetic properties of rt-PA P47G, K49N, a substitution variant of human tissue-type plasminogen activator.

    PubMed

    Nelles, L; Li, X K; Vanlinthout, I; De Cock, F; Lijnen, H R; Collen, D

    1992-04-02

    rt-PA P47G, K49N, a substitution variant of recombinant human tissue-type plasminogen activator (rt-PA), in which proline at position 47 and lysine at position 49 were replaced by glycine and asparagine respectively, was previously described by Ahern et al. (J Biol Chem 1990; 265:5540-5) to have an extended in vivo half-life with unaltered in vitro fibrinolytic properties. Because this variant might possess an increased in vivo thrombolytic potency, we have constructed its cDNA, expressed it in Chinese hamster ovary cells and determined its biochemical, thrombolytic and pharmacokinetic properties relative to those of home-made rt-PA and of alteplase (Actilyse). The specific fibrinolytic activities on fibrin plates were 160,000 +/- 17,000, 210,000 +/- 88,000 and 460,000 +/- 72,000 IU/mg (mean +/- SEM) for rt-PA P47G, K49N, rt-PA and alteplase, respectively, while the catalytic efficiencies for plasminogen activation (k2/Km) in the absence of fibrin were comparable (1.1 to 1.7 x 10(-3) microM-1s-1). Fibrin enhanced the rate of plasminogen activation by rt-PA P47G, K49N 100-fold and by both wild-type molecules 390-fold. Binding of the variant rt-PA to fibrin was significantly reduced, but its affinity for lysine-Sepharose was unaltered. In an in vitro clot lysis system, consisting of a radiolabeled human plasma clot submersed in plasma, 50% clot lysis in 2 h required 0.67 +/- 0.14 micrograms/ml rt-PA P47G, K49N, 0.36 +/- 0.01 micrograms/ml rt-PA and 0.17 +/- 0.01 micrograms/ml alteplase, respectively (mean +/- SEM; n = 3 or 4). At these doses residual fibrinogen levels at 2 h were in excess of 80%.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Urokinase plasminogen activator receptor induced non-small cell lung cancer invasion and metastasis requires NHE1 transporter expression and transport activity.

    PubMed

    Provost, J J; Rastedt, D; Canine, J; Ngyuen, T; Haak, A; Kutz, C; Berthelsen, N; Slusser, A; Anderson, K; Dorsam, G; Wallert, M A

    2012-04-01

    BACKGROUND: Non-small cell lung cancers (NSLC) are aggressive cancers that are insensitive to chemotherapies and accounts for nearly 33% of all cancer deaths in the United States. Two hallmarks of cancer that allow cells to invade and metastasize are sustained proliferation and enhanced motility. In this study we investigate the relationship between urokinase plasminogen activator (uPA)/uPA receptor (uPAR) signaling and Na(+)/H(+) exchanger isoform 1 (NHE1) expression and activity. METHODS AND RESULTS: The addition of 10nM uPA increased the carcinogenic potential of three NSCLC cell lines, NCI-H358, NCI-H460, and NCI-H1299. This included an increase in the rate of cell proliferation 1.6 to 1.9 fold; an increase in the percentage of cells displaying stress fibers 3.05 to 3.17 fold; and an increase in anchorage-independent growth from 1.64 to 2.0 fold. In each of these cases the increase was blocked when the experiments were performed with NHE1 inhibited by 10 μM EIPA (ethylisopropyl amiloride). To further evaluate the role of uPA/uPAR and NHE1 in tumor progression we assessed signaling events using full-length uPA compared to the uPA amino terminal fragment (ATF). Comparing uPA and ATF signaling in H460 cells, we found that both uPA and ATF increased stress fiber formation approximately 2 fold, while uPA increased matrix metalloproteinase 9 (MMP9) activity 5.44 fold compared to 2.81 fold for ATF. To expand this signaling study, two new cell lines were generated, one with reduced NHE1 expression (H460 NHE1 K/D) and one with reduced uPAR expression (H460 uPAR K/D). Using the K/D cell lines we found that neither uPA nor ATF could stimulate stress fiber formation or MMP9 activity in cells with dramatically decreased NHE1 or uPAR expression. Finally, using in vivo tumor formation studies in athymic mice we found that when mice were injected with H460 cells 80% of mice formed tumors with an average volume of 390 mm(3). This was compared to 20% of H460 uPAR K/D injected

  14. Fluorescence Correlation Spectroscopy and Photon Counting Histogram on membrane proteins: Functional dynamics of the GPI-anchored Urokinase Plasminogen Activator Receptor

    PubMed Central

    Malengo, Gabriele; Andolfo, Annapaola; Sidenius, Nicolai; Gratton, Enrico; Zamai, Moreno; Caiolfa, Valeria R

    2009-01-01

    The oligomerization of GPI-anchored proteins is thought to regulate their association with membrane microdomains, sub-cellular sorting and activity. However, these mechanisms need to be comprehensively explored in living, unperturbed cells, without artificial clustering agents, and using fluorescent protein-tagged chimeras that are fully biologically active. We expressed in HEK293 cells a biologically active chimera of the urokinase plasminogen activator receptor (uPAR), the uPAR-mEGFP-GPI. We also produced HEK293/D2D3-mEGFP-GPI cells expressing the truncated form of the receptor, lacking biological activity. We studied the dynamics and oligomerization of the two proteins, combining FCS and PCH analyses, and using subclones with homogenously low expression levels. Overall, the mobile fractions of the two proteins, constituted by monomers and dimers, had comparable diffusion coefficients. However, only for the active receptor the diffusion coefficient decreased in monomer-enriched fractions, suggesting that uPAR monomers might be preferentially engaged in multi-protein transmembrane signaling complexes. Our approach helps in limiting the alteration of the data due to out-of-focus, and minimizing the overestimation of the molecular brightness. Joint to a careful design of the cellular model, it gives reliable estimates of diffusion coefficients and oligomerization of GPI-anchored proteins, in steady state conditions, at low expression levels, and in live, unperturbed cells. PMID:18601539

  15. Treatment of neonatal fungal infective endocarditis with recombinant tissue plasminogen: activator in a low birth weight infant case report and review of the literature.

    PubMed

    Babayigit, Aslan; Cebeci, Burcu; Buyukkale, Gokhan; Semerci, Seda Yılmaz; Bornaun, Helen; Oztarhan, Kazim; Gokce, Muge; Cetinkaya, Merih

    2015-10-01

    With advances in medical sciences, an increase in survival rates of low birth weight; increased incidence in use of catheter and antibiotics, and total parenteral nutrition are reported, therefore, the rate of fungal infections in late and very late onset neonatal sepsis have increased. Although fungal endocarditis rarely occur in newborns, it has a high morbidity and mortality. Antifungal therapy is often insufficient in cases who develop fungal endocarditis and surgical treatment is not preferred due to its difficulty and high mortality. Herein, fungal endocarditis in a preterm newborn treated with single-dose recombinant tissue plasminogen activator in addition to antifungal therapy is presented and relevant literature has been reviewed. The vegetation completely disappeared following treatment and no complication was observed.

  16. Adiponectin, C-reactive protein, fibrinogen and tissue plasminogen activator antigen levels among glucose-intolerant women with and without histories of gestational diabetes

    PubMed Central

    Kim, C.; Christophi, C. A.; Goldberg, R. B.; Perreault, L.; Dabelea, D.; Marcovina, S. M.; Pi-Sunyer, X.; Barrett-Connor, E.

    2015-01-01

    Aim To examine concentrations of biomarkers (adiponectin, C-reactive protein, fibrinogen and tissue plasminogen-activator antigen) associated with glucose homeostasis and diabetes risk by history of gestational diabetes. Methods We conducted a secondary analysis of the Diabetes Prevention Program, a randomized trial of lifestyle intervention or metformin for diabetes prevention. At baseline, participants were overweight and had impaired glucose tolerance. Biomarkers at baseline and 1 year after enrolment were compared between parous women with (n=350) and without a history of gestational diabetes (n=1466). Cox proportional hazard models evaluated whether history of gestational diabetes was associated with diabetes risk, after adjustment for baseline biomarker levels as well as for change in biomarker levels, demographic factors and anthropometrics. Results At baseline, women with histories of gestational diabetes had lower adiponectin (7.5 μg/ml vs. 8.7 μg/ml; p<0.0001) and greater log C-reactive protein (−0.90 mg/l vs. −0.78 mg/l, p=0.04) levels than women without histories of gestational diabetes, but these associations did not persist after adjustment for demographic factors. Fibrinogen and tissue plasminogen-activator antigen were similar between women with and without histories of gestational diabetes. Women with and without histories of gestational diabetes had a similar pattern of changes in biomarkers within randomization arm. Adjustment for age, race/ethnicity, baseline weight, change in weight, baseline biomarker level and change in biomarker level did not significantly alter the association between history of gestational diabetes and diabetes risk. Conclusions Among women with impaired glucose tolerance, biomarkers in women with and without histories of gestational diabetes are similar and respond similarly to lifestyle changes and metformin. Adjustment for biomarker levels did not explain the higher risk of diabetes observed in women with

  17. Recombinant Tissue Plasminogen Activator Induces Neurological Side Effects Independent on Thrombolysis in Mechanical Animal Models of Focal Cerebral Infarction: A Systematic Review and Meta-Analysis

    PubMed Central

    Wei, You-Dong; Liu, Yi-Yun; Ren, Yi-Fei; Liang, Zi-Hong; Wang, Hai-Yang; Zhao, Li-Bo; Xie, Peng

    2016-01-01

    Background and Purpose Recombinant tissue plasminogen activator (rtPA) is the only effective drug approved by US FDA to treat ischemic stroke, and it contains pleiotropic effects besides thrombolysis. We performed a meta-analysis to clarify effect of tissue plasminogen activator (tPA) on cerebral infarction besides its thrombolysis property in mechanical animal stroke. Methods Relevant studies were identified by two reviewers after searching online databases, including Pubmed, Embase, and ScienceDirect, from 1979 to 2016. We identified 6, 65, 17, 12, 16, 12 and 13 comparisons reporting effect of endogenous tPA on infarction volume and effects of rtPA on infarction volume, blood-brain barrier, brain edema, intracerebral hemorrhage, neurological function and mortality rate in all 47 included studies. Standardized mean differences for continuous measures and risk ratio for dichotomous measures were calculated to assess the effects of endogenous tPA and rtPA on cerebral infarction in animals. The quality of included studies was assessed using the Stroke Therapy Academic Industry Roundtable score. Subgroup analysis, meta-regression and sensitivity analysis were performed to explore sources of heterogeneity. Funnel plot, Trim and Fill method and Egger’s test were obtained to detect publication bias. Results We found that both endogenous tPA and rtPA had not enlarged infarction volume, or deteriorated neurological function. However, rtPA would disrupt blood-brain barrier, aggravate brain edema, induce intracerebral hemorrhage and increase mortality rate. Conclusions This meta-analysis reveals rtPA can lead to neurological side effects besides thrombolysis in mechanical animal stroke, which may account for clinical exacerbation for stroke patients that do not achieve vascular recanalization with rtPA. PMID:27387385

  18. Comparative effects of anti-platelet agents as adjuncts to tissue plasminogen activator in a dog model of occlusive coronary thrombosis.

    PubMed Central

    McAuliffe, S. J.; Moors, J. A.; Jones, H. B.

    1994-01-01

    1. This study compares a cyclo-oxygenase inhibitor (aspirin), a 5-HT2 antagonist (ZM170809) and a combined thromboxane synthase inhibitor/receptor antagonist (ZD1542) as adjuncts to tissue plasminogen activator (rt-PA). 2. Application of an anodal current (332 +/- 4.1 microA) to the stenosed left circumflex coronary artery of 20 anaesthetized dogs produced a stable platelet-rich occlusive thrombus. 3. After initial i.v. administration of recombinant human tissue type plasminogen activator (rt-PA, 3 mg bolus +2 mg kg-1 h-1 for 30 min) thrombolysis occurred in 15 out of 20 dogs. All 15 dogs reoccluded. 4. The second i.v. administration of rt-PA in the presence of either aspirin, ZM170809, ZD1542 or saline resulted in thrombolysis in all 20 dogs. 5. Both the combined thromboxane synthase inhibitor/receptor antagonist (ZD1542) and 5-HT2 antagonist (ZM170809) significantly (P < 0.05) reduced the time taken to lyse the thrombus compared with the saline group. The times were 14.4 +/- 2.7 min, 18.0 +/- 3.9 min and 36.8 +/- 6.2 min for ZD1542, ZM170809 and saline respectively. 6. Aspirin did not offer any additional benefit to using rt-PA alone. The times to thrombolysis were 36.8 +/- 8.4 min for aspirin and 36.8 +/- 6.2 min for the saline group. 7. The number of dogs in which the circumflex coronary artery reoccluded within 60 min of terminating the second infusion of rt-PA were five for saline, four for aspirin, two for ZD1542 and two for ZM170809. 8. These results indicate that both ZD1542 and ZM170809 are more effective adjuncts than aspirin in thrombolysis and may provide an improvement in current clinical practice. Images Figure 5 PMID:8032650

  19. Ochratoxin A inhibits the production of tissue factor and plasminogen activator inhibitor-2 by human blood mononuclear cells: Another potential mechanism of immune-suppression

    SciTech Connect

    Rossiello, Maria R.; Rotunno, Crescenzia; Coluccia, Addolorata; Carratu, Maria R.; Di Santo, Angelomaria; Evangelista, Virgilio; Semeraro, Nicola; Colucci, Mario

    2008-06-01

    The mycotoxin ochratoxin A (OTA), an ubiquitous contaminant of food products endowed with a wide spectrum of toxicity, affects several functions of mononuclear leukocytes. Monocytes/macrophages play a major role in fibrin accumulation associated with immune-inflammatory processes through the production of tissue factor (TF) and plasminogen activator inhibitor 2 (PAI-2). We studied the effect of OTA on TF and PAI-2 production by human blood mononuclear cells (MNC). The cells were incubated for 3 or 18 h at 37 deg. C with non toxic OTA concentrations in the absence and in the presence of lipopolysaccharide (LPS) or other inflammatory agents. TF activity was measured by a one-stage clotting test. Antigen assays were performed by specific ELISAs in cell extracts or conditioned media and specific mRNAs were assessed by RT-PCR. OTA had no direct effect on TF and PAI-2 production by MNC. However, OTA caused a dose-dependent reduction in LPS-induced TF (activity, antigen and mRNA) and PAI-2 (antigen and mRNA) production with > 85% inhibition at 1 {mu}g/ml. Similar results were obtained when monocyte-enriched preparations were used instead of MNC. TF production was also impaired by OTA (1 {mu}g/ml) when MNC were stimulated with phorbol myristate acetate (98% inhibition), IL-1{beta} (83%) or TNF-{alpha} (62%). The inhibition of TF and PAI-2 induction might represent a hitherto unrecognized mechanism whereby OTA exerts immunosuppressant activity.

  20. Controlled release of clot-dissolving tissue-type plasminogen activator from a poly(L-glutamic acid) semi-interpenetrating polymer network hydrogel.

    PubMed

    Park, Y; Liang, J; Yang, Z; Yang, V C

    2001-07-10

    With the aim of developing an effective therapeutic modality for treatment of thrombosis, a tissue-type plasminogen activator (t-PA)-loaded porous poly(L-glutamic acid) (PLGA) semi-interpenetrating polymer network (semi-IPN) hydrogel was developed as a possible local drug delivery system. Porous structure of hydrogel was essential in this system to yield a large surface area so that t-PA release could be facilitated. This semi-IPN hydrogel was prepared using the method of free-radical polymerization and crosslinking of polyethylene glycol (PEG)-methacrylate through the PLGA network. Sodium bicarbonate (NaHCO(3)) was added to function as a foaming agent under acidic conditions, rendering the semi-IPN hydrogel to be porous. While the added NaHCO(3) provided gas foam in the reaction mixture, the pH in the hydrogel increased to about 7 to 8, which stimulated the polymerization. The porous structure that was presented at both the surface and sublayer was stabilized during hydrogel formation and freeze-drying. The hydrogel thus prepared possessed a porous structure of 10-20 microm in diameter, as determined by scanning electron microscopy. Results showed that the above hydrogel preparation process did not significantly alter the specific activity of the entrapped t-PA with regard to plasminogen activation and fibrin clot lysis ability. The t-PA release from this semi-IPN hydrogel was examined by measuring the plasmin activity using the chromogenic substrate S-2251. Findings in this paper demonstrated that the porous structure of the hydrogel facilitated t-PA release when compared to the dense structure. Aside from the porous structure, other factors including the content of the crosslinker, PLGA and t-PA could all be varied to regulate t-PA release from the hydrogel. These results suggest that a porous PLGA semi-IPN hydrogel could potentially be a useful local delivery system to release active t-PA primarily at the site of a thrombus.

  1. Effects of two different fibric acid derivatives on lipoproteins, cholesteryl ester transfer, fibrinogen, plasminogen activator inhibitor and paraoxonase activity in type IIb hyperlipoproteinaemia.

    PubMed

    Durrington, P N; Mackness, M I; Bhatnagar, D; Julier, K; Prais, H; Arrol, S; Morgan, J; Wood, G N

    1998-05-01

    We have investigated the effects of two fibric acid derivatives, bezafibrate mono (400 mg daily) and gemfibrozil (600 mg b.d.), in 29 patients with type IIb hyperlipoproteinaemia. All patients received placebo and each drug for 8 weeks in randomised order in a double-blind, cross-over study designed to evaluate any different effects of the drugs on serum lipoproteins, cholesteryl ester transfer protein (CETP), cholesteryl ester transfer activity (CETA), plasma fibrinogen, plasminogen activator inhibitor-I (PAI-1) or paraoxonase. Serum cholesterol decreased (P < 0.05) with gemfibrozil, but the effect of bezafibrate on serum cholesterol did not achieve statistical significance (placebo 8.34 +/- 1.05 (mean +/- S.D.), gemfibrozil 7.70 +/- 1.23 and bezafibrate 7.8 +/- 1.37 mmol/l). Both drugs decreased the serum triglyceride concentration (both P < 0.001) (placebo 4.39 (3.13-5.75) (median (interquartile range)), bezafibrate 2.26 (1.89-3.89) and gemfibrozil 2.00 (1.30-3.30) mmol/l) and very low density lipoprotein (VLDL) cholesterol (both P < 0.001) (placebo 1.18 (0.74-2.30), bezafibrate 0.59 (0.34-0.85) and gemfibrozil 0.48 (0.34-0.68) mmol/l). Discontinuous gradient ultracentrifugation (DGU) revealed that Sf 60-400 (large VLDL) decreased by more than 50% and Sf 20-60 (small VLDL) by more than 30% with each of the drugs (both P < 0.001), neither of which affected the composition of these lipoproteins. Gemfibrozil decreased the concentration of Sf 12-20 lipoprotein (intermediate density lipoprotein; IDL) by 23% (P < 0.01), whereas the effect of bezafibrate on this lipoprotein did not achieve statistical significance. Neither drug altered the concentration of apolipoprotein B or of total Sf 0-12 lipoproteins (low density lipoprotein, (LDL)). Both, however, significantly increased the quantity of free cholesterol in Sf 0-12 lipoproteins (P < 0.05). Overall the concentration of triglycerides decreased significantly in all lipoproteins isolated by DGU (Sf 0-12, Sf 12-20, Sf

  2. Effects of a diet containing Brazilian propolis on lipopolysaccharide-induced increases in plasma plasminogen activator inhibitor-1 levels in mice

    PubMed Central

    Ohkura, Naoki; Oishi, Katsutaka; Kihara-Negishi, Fumiko; Atsumi, Gen-ichi; Tatefuji, Tomoki

    2016-01-01

    Background: Brazilian propolis has many biological activities including the ability to help prevent thrombotic diseases, but this particular effect has not been proven. Plasma levels of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, increase under inflammatory conditions such as infection, obesity and atherosclerosis and such elevated levels predispose individuals to a risk of developing thrombotic diseases. Aim: This study aimed to determine the effects of a diet containing Brazilian propolis on lipopolysaccharide (LPS)-induced increases in plasma PAI-1 levels. Materials and Methods: Mice were fed with a diet containing 0.5% (w/w) Brazilian propolis for 8 weeks. Thereafter, the mice were subcutaneously injected with saline containing 0.015 mg/kg of LPS and sacrificed 4 h later. Results: Orally administered Brazilian propolis significantly suppressed the LPS-induced increase in PAI-1 antigen and its activity in mouse plasma. Conclusion: This study indicated that Brazilian propolis contains natural products that can decrease thrombotic tendencies in mice. PMID:27757277

  3. Inhibitory effects of C-type natriuretic peptide on the differentiation of cardiac fibroblasts, and secretion of monocyte chemoattractant protein-1 and plasminogen activator inhibitor-1

    PubMed Central

    LI, ZHI-QIANG; LIU, YING-LONG; LI, GANG; LI, BIN; LIU, YANG; LI, XIAO-FENG; LIU, AI-JUN

    2015-01-01

    The present study aimed to investigate the effect of C-type natriuretic peptide (CNP) on the function of cardiac fibroblasts (CFs). Western blotting was used to investigate the expression of myofibroblast marker proteins: α-smooth muscle actin (α-SMA), extra domain-A fibronectin, collagen I and collagen III, and the activity of extracellular signal-regulated kinase 1/2 (ERK1/2). Immunofluorescence was used to examine the morphological changes; a transwell assay was used to analyze migration, and reverse transcription-quantitative polymerase chain reaction and ELISA were employed to determine the mRNA expression and protein secretion of monocyte chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1). The results demonstrated that CNP significantly reduced the protein expression of α-SMA, fibronectin, collagen I and collagen III, and suppressed the migratory ability of CFs. Additionally, the mRNA and protein expression of MCP-1 and PAI-1 was inhibited under the CNP treatment; and this effect was mediated by the inhibition of the ERK1/2 activity. In conclusion, CNP inhibited cardiac fibroblast differentiation and migration, and reduced the secretion of MCP-1 and PAI-1, which demonstrates novel mechanisms to explain the antifibrotic effect of CNP. PMID:25352084

  4. Stimulatory effect of an algal fucoidan on the release of vascular endothelial tissue-type plasminogen activator as a mechanism of fucoidan-mediated thrombolysis.

    PubMed

    Min, Soon-Ki; Han, Sung-Mi; Jang, Jae-Seok; Kim, Jong-Ki

    2016-07-01

    Identifying a pharmacological means for increasing the production of tissue-type plasminogen activator (t-PA) is always desirable to cure impaired production of this enzyme. An algal fucoidan has been shown to exhibit both novel thrombolytic and synergistic stimulatory effects in a mouse thrombosis model. The plasma levels of active t-PA were measured in mouse arterial thrombus models that were treated with various fucoidans to investigate the mechanism of thrombolysis. The mean plasma level of active t-PA after the infusion of fucoidan was 2.136 ± 0.231 ng/ml for nonthrombolytic Fucus fucoidan and 3.917 ± 0.0.529 ng/ml for thrombolytic Undaria fucoidan, which resulted in a 1.56-2.29-fold increase compared with the healthy control group (1.706 ± 0.194 ng/ml) and the untreated thrombus group (2.506 ± 0.301 ng/ml) (P < 0.01). An algal fucoidan has demonstrated to exert a thrombolytic and stimulatory effect via the induction of t-PA release in a dose-dependent manner in an arterial thrombosis model.

  5. Inhibition of endothelial nitric oxyde synthase increases capillary formation via Rac1-dependent induction of hypoxia-inducible factor-1α and plasminogen activator inhibitor-1.

    PubMed

    Petry, Andreas; BelAiba, Rachida S; Weitnauer, Michae; Görlach, Agnes

    2012-11-01

    Disruption of endothelial homeostasis results in endothelial dysfunction, characterised by a dysbalance between nitric oxide (NO) and reactive oxygen species (ROS) levels often accompanied by a prothrombotic and proproliferative state. The serine protease thrombin not only is instrumental in formation of the fibrin clot, but also exerts direct effects on the vessel wall by activating proliferative and angiogenic responses. In endothelial cells, thrombin can induce NO as well as ROS levels. However, the relative contribution of these reactive species to the angiogenic response towards thrombin is not completely clear. Since plasminogen activator inhibitor-1 (PAI-1), a direct target of the proangiogenic transcription factors hypoxia-inducible factors (HIFs), exerts prothrombotic and proangiogenic activities we investigated the role of ROS and NO in the regulation of HIF-1α, PAI-1 and capillary formation in response to thrombin. Thrombin enhanced the formation of NO as well as ROS generation involving the GTPase Rac1 in endothelial cells. Rac1-dependent ROS formation promoted induction of HIF-1α, PAI-1 and capillary formation by thrombin, while NO reduced ROS bioavailability and subsequently limited induction of HIF-1α, PAI-1 and the angiogenic response. Importantly, thrombin activation of Rac1 was diminished by NO, but enhanced by ROS. Thus, our findings show that capillary formation induced by thrombin via Rac1-dependent activation of HIF-1 and PAI-1 is limited by the concomitant release of NO which reduced ROS bioavailability. Rac1 activity is sensitive to ROS and NO, thereby playing an essential role in fine tuning the endothelial response to thrombin.

  6. A distinct basic fibroblast growth factor (FGF-2)/FGF receptor interaction distinguishes urokinase-type plasminogen activator induction from mitogenicity in endothelial cells.

    PubMed Central

    Rusnati, M; Dell'Era, P; Urbinati, C; Tanghetti, E; Massardi, M L; Nagamine, Y; Monti, E; Presta, M

    1996-01-01

    Basic fibroblast growth factor (FGF-2) induces cell proliferation and urokinase-type plasminogen activator (uPA) production in fetal bovine aortic endothelial GM 7373 cells. In the present paper we investigated the role of the interaction of FGF-2 with tyrosine-kinase (TK) FGF receptors (FGFRs) in mediating uPA up-regulation in these cells. The results show that FGF-2 antagonists suramin, protamine, heparin, the synthetic peptide FGF-2(112-155), and a soluble form of FGFR-1 do not inhibit FGF-2-mediated uPA up-regulation at concentrations that affect growth factor binding to cell surface receptors and mitogenic activity. In contrast, tyrosine phosphorylation inhibitors and overexpression of a dominant negative TK- mutant of FGFR-1 abolish the uPA-inducing activity of FGF-2, indicating that FGFR and its TK activity are essential in mediating uPA induction. Accordingly, FGF-2 induces uPA up-regulation in Chinese hamster ovary cells transfected with wild-type FGFR-1, -2, -3, or -4 but not with TK- FGFR-1 mutant. Small unilamellar phosphatidyl choline:cholesterol vesicles loaded with FGF-2 increased uPA production in GM 7373 cells in the absence of a mitogenic response. Liposome-encapsulated FGF-2 showed a limited but significant capacity, relative to free FGF-2, to interact with FGFR both at 4 degrees C and 37 degrees C and to be internalized within the cell. uPA up-regulation by liposome-encapsulated FGF-2 was quenched by neutralizing anti-FGF-2 antibodies, indicating that the activity of liposome-delivered FGF-2 is mediated by an extracellular action of the growth factor. Taken together, the data indicate that a distinct interaction of FGF-2 with FGFR, quantitatively and/or qualitatively different from the one that leads to mitogenicity, is responsible for the uPA-inducing activity of the growth factor. Images PMID:8868466

  7. The receptor for urokinase-plasminogen activator (uPAR) controls plasticity of cancer cell movement in mesenchymal and amoeboid migration style

    PubMed Central

    Taddei, Maria Letizia; Giannoni, Elisa; Laurenzana, Anna; Biagioni, Alessio; Chillà, Anastasia; Chiarugi, Paola; Fibbi, Gabriella; Rosso1, Mario Del

    2014-01-01

    The receptor for the urokinase plasminogen activator (uPAR) is up-regulated in malignant tumors. Historically the function of uPAR in cancer cell invasion is strictly related to its property to promote uPA-dependent proteolysis of extracellular matrix and to open a path to malignant cells. These features are typical of mesenchymal motility. Here we show that the full-length form of uPAR is required when prostate and melanoma cancer cells convert their migration style from the “path generating” mesenchymal to the “path finding” amoeboid one, thus conferring a plasticity to tumor cell invasiveness across three-dimensional matrices. Indeed, in response to a protease inhibitors-rich milieu, prostate and melanoma cells activated an amoeboid invasion program connoted by retraction of cell protrusions, RhoA-mediated rounding of the cell body, formation of a cortical ring of actin and a reduction of Rac-1 activation. While the mesenchymal movement was reduced upon silencing of uPAR expression, the amoeboid one was almost completely abolished, in parallel with a deregulation of small Rho-GTPases activity. In melanoma and prostate cancer cells we have shown uPAR colocalization with β1/β3 integrins and actin cytoskeleton, as well integrins-actin co-localization under both mesenchymal and amoeboid conditions. Such co-localizations were lost upon treatment of cells with a peptide that inhibits uPAR-integrin interactions. Similarly to uPAR silencing, the peptide reduced mesenchymal invasion and almost abolished the amoeboid one. These results indicate that full-length uPAR bridges the mesenchymal and amoeboid style of movement by an inward-oriented activity based on its property to promote integrin-actin interactions and the following cytoskeleton assembly. PMID:24681666

  8. Apigenin Attenuates Atherogenesis through Inducing Macrophage Apoptosis via Inhibition of AKT Ser473 Phosphorylation and Downregulation of Plasminogen Activator Inhibitor-2.

    PubMed

    Zeng, Ping; Liu, Bin; Wang, Qun; Fan, Qin; Diao, Jian-Xin; Tang, Jing; Fu, Xiu-Qiong; Sun, Xue-Gang

    2015-01-01

    Macrophage survival is believed to be a contributing factor in the development of early atherosclerotic lesions. Dysregulated apoptosis of macrophages is involved in the inflammatory process of atherogenesis. Apigenin is a flavonoid that possesses various clinically relevant properties such as anti-inflammatory, antiplatelet, and antitumor activities. Here we showed that apigenin attenuated atherogenesis in apoE (-/-) mice in an in vivo test. In vitro experiments suggested that apigenin induced apoptosis of oxidized low density lipoprotein- (OxLDL-) loaded murine peritoneal macrophages (MPMs). Proteomic analysis showed that apigenin reduced the expression of plasminogen activator inhibitor 2 (PAI-2). PAI-2 has antiapoptotic effects in OxLDL-loaded MPMs. Enhancing PAI-2 expression significantly reduced the proapoptosis effects of apigenin. Molecular docking assay with AutoDock software predicted that residue Ser473 of Akt1 is a potential binding site for apigenin. Lentiviral-mediated overexpression of Akt1 wild type weakened the proapoptosis effect of apigenin in OxLDL-loaded MPMs. Collectively, apigenin executes its anti-atherogenic effects through inducing OxLDL-loaded MPMs apoptosis. The proapoptotic effects of apigenin were at least partly attributed to downregulation of PAI-2 through suppressing phosphorylation of AKT at Ser473.

  9. Urinary-type plasminogen activator (uPA) and its receptor (uPAR) in squamous cell carcinoma of the oral cavity.

    PubMed

    Shi, Zonggao; Stack, M Sharon

    2007-10-15

    OSCC (oral squamous cell carcinoma) is the most common oral malignancy and is estimated to affect approx. 350000 new patients worldwide this year. OSCC is characterized by a high degree of morbidity and mortality, as most patients exhibit local, regional and distant metastasis at the time of diagnosis. Recent genome-wide screening efforts have identified the serine proteinase uPA (urinary-type plasminogen activator, also known as urokinase) as a strong biomarker for prediction of poor disease outcome and a key candidate for molecular classification of oral neoplasms using a 'gene signature' approach. The proteinase uPA binds a surface-anchored receptor designated uPAR (uPA receptor), focalizing proteolytic activity to the pericellular milieu. Furthermore, uPA-uPAR can interact with transmembrane proteins to modify multiple signal transduction pathways and influence a wide variety of cellular behaviours. Correlative clinical data show elevated uPA-uPAR in oral tumour tissues, with tumours exhibiting high levels of both uPA and uPAR as the most invasive. Combined in vitro, pre-clinical and clinical data support the need for further analysis of uPA-uPAR as a prognostic indicator as well as a potential therapeutic target in OSCC.

  10. Human circadian system causes a morning peak in prothrombotic plasminogen activator inhibitor-1 (PAI-1) independent of the sleep/wake cycle.

    PubMed

    Scheer, Frank A J L; Shea, Steven A

    2014-01-23

    Serious adverse cardiovascular events peak in the morning, possibly related to increased thrombosis in critical vessels. Plasminogen activator inhibitor-1 (PAI-1), which inhibits fibrinolysis, is a key circulating prothrombotic factor that rises in the morning in humans. We tested whether this morning peak in PAI-1 is caused by the internal circadian system or by behaviors that typically occur in the morning, such as altered posture and physical activity. Twelve healthy adults underwent a 2-week protocol that enabled the distinction of endogenous circadian effects from behavioral and environmental effects. The results demonstrated a robust circadian rhythm in circulating PAI-1 with a peak corresponding to ∼6:30 am. This rhythm in PAI-1 was 8-times larger than changes in PAI-1 induced by standardized behavioral stressors, including head-up tilt and 15-minute cycle exercise. If this large endogenous morning peak in PAI-1 persists in vulnerable individuals, it could help explain the morning peak in adverse cardiovascular events.

  11. Serum soluble urokinase-type plasminogen activator receptor as a biological marker of bacterial infection in adults: a systematic review and meta-analysis

    PubMed Central

    Ni, Wentao; Han, Yuliang; Zhao, Jin; Cui, Junchang; Wang, Kai; Wang, Rui; Liu, Youning

    2016-01-01

    The serum concentration of soluble urokinase-type plasminogen activator receptor (suPAR) reflects immune activation. We performed a meta-analysis to evaluate the usefulness of suPAR for the diagnosis and prognosis of bacterial infections. PubMed, Embase and Cochrane Library databases were searched for studies reporting the detection of suPAR in adult patients with bacterial infections. Seventeen studies were selected from 671 studies. The pooled sensitivity and specificity of suPAR for diagnosing infection were 0.73 and 0.79, respectively, and the area under the summary receiver operating characteristic curve (AUC) was 0.82. Subgroup analyses revealed suPAR showed similar AUC values for diagnosing sepsis and bacteremia, but the AUC for differentiating sepsis from systemic inflammatory response syndrome (SIRS) was only 0.68. Elevated suPAR levels were significantly associated with a high risk of death, with a pooled risk ratio of 3.37 (95% confidence interval, 2.60–4.38). The pooled sensitivity and specificity for predicting mortality were 0.70 and 0.72, respectivfely, with an AUC of 0.77. Serum suPAR could be a biomarker for the diagnosis and prognosis of bacterial infection, but it is relatively ineffective for differentiating sepsis from SIRS. Further investigation is required to evaluate whether using of suPAR in combination with other biomarkers can improve diagnostic efficacy. PMID:27991579

  12. Cytotoxicity of the Urokinase-Plasminogen Activator Inhibitor Carbamimidothioic Acid (4-Boronophenyl) Methyl Ester Hydrobromide (BC-11) on Triple-Negative MDA-MB231 Breast Cancer Cells.

    PubMed

    Longo, Alessandra; Librizzi, Mariangela; Chuckowree, Irina S; Baltus, Christine B; Spencer, John; Luparello, Claudio

    2015-05-28

    BC-11 is an easily synthesized simple thiouronium-substituted phenylboronic acid, which has been shown to be cytotoxic on triple negative MDA-MB231 breast cancer cells by inducing a perturbation of cell cycle when administered at a concentration equal to its ED50 at 72 h (117 μM). Exposure of cells to BC-11, either pre-absorbed with a soluble preparation of the N-terminal fragment of urokinase-plasminogen activator (uPa), or in co-treatment with two different EGFR inhibitors, indicated that: (i) BC-11 acts via binding to the N-terminus of the enzyme where uPa- and EGF receptor-recognizing sites are present, thereby abrogating the growth-sustaining effect resulting from receptor binding; and (ii) the co-presence of the EGFR inhibitor PD153035 potentiates BC-11's cytotoxicity. Exposure of cells to a higher concentration of BC-11 corresponding to its ED75 at 72 h (250 μM) caused additional impairment of mitochondrial activity, the production of reactive oxygen species and promotion of apoptosis. Therefore, BC-11 treatment appears to show potential for the development of this class of compounds in the prevention and/or therapy of "aggressive" breast carcinoma.

  13. Localization of tissue plasminogen activator in relation to morphologic changes in human saphenous veins used as coronary artery bypass autografts.

    PubMed Central

    Glas-Greenwalt, P; Dalton, B C; Astrup, T

    1975-01-01

    Employing the histochemical fibrin slide technique, we studied the localization of fibrinolytically active sites in relation to morphologic changes in saphenous veins used as coronary artery bypass autografts. Of veins from 100 patients undergoing surgery for coronary heart disease, 63 samples revealed well-demarcated intimal fibrinolytic activity. Distinct foci of lysis were present in the media and adventitia of all vein samples corresponding to the distribution of the vasa vasorum. Graft specimens obtained by autopsy from 13 patients surviving from 30 minutes to 13 days revealed considerable intimal damage with loss of fibrinolytic activity. Medial fibrinolytic activity disappeared early concomitant with disorganization of the medial muscle fibers, followed by necrosis, atrophy and fibrous transformation of the muscular structures. Adventitial activity disappeared later. One reoperation sample obtained after 8 weeks in situ showed moderate proliferative intimal fibrosis without an apparent neo-intima and without fibrinolytic activity, and one section showed evidence of valve cusp fibrosis, while most of the media had been replaced by fibrous tissue. Numerous foci of "medial" -adventitial fibrinolytic activity indicated presence of vascular structures in the venous wall. The fibrin slide technique is a convenient tool for studying the process of revascularization. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Figs. 5a-d. Fig. 6. Figs. 7a and b. Figs. 8a and b. PMID:1079445

  14. Periodicity in the levels of serum plasminogen activator inhibitor-1 is a robust prognostic factor for embryo implantation and clinical pregnancy in ongoing IVF cycles

    PubMed Central

    Mehta, Bindu N.; Nath, Nirmalendu; Chimote, Natachandra

    2014-01-01

    CONTEXT: Plasminogen activator inhibitor-1 (PAI-1) has been inversely correlated to proteolytic extracellular-matrix degradation exerted by urokinase-type (u-PA) and tissue-type plasminogen activators (t-PA). Any pathological disturbance in PAI-1 levels may lead to several pregnancy complications. AIMS: To assess the influence of periodicity in serum PAI-1 levels on embryo implantation and clinical pregnancy outcome in IVF cycles SETTINGS AND DESIGN: Prospective study of 120 IVF cycles at private infertility centre. MATERIAL AND METHODS: Endometrial response (ER) assessment by measuring Endometrial thickness (cm) and echopattern (grade). Serum PAI-1(ng/ml) measurement by ELISA method on day of hCG, day of ET and days 7 and 14 of ET. Main outcome measure was clinical pregnancy. STATISTICAL ANALYSIS: Student “t” test, ANOVA, Post-test for linear trend, Pearson Correlation. RESULTS: PAI-1 levels declined from dhCG to dET (318.8 ± 36.1 to 176.1 ± 28.4) whereas they increased steadily from dET to d7 to d14ET (176.1 ± 28.4 to 285.2 ± 30.4 to 353.5 ± 150.4; P = 0.0004) in pregnant group (n = 31). Conversely, dhCG to dET levels increased in both nonpregnant (n = 75; 173.8 ± 18.3 to 280.8 ± 26.1) and biochemical pregnancy BCP (n = 14; 172.7 ± 31.1 to 216 ± 30.1) groups. The rising pattern from dET to d7 to d14ET was not observed in non-pregnant and BCP groups. ER thickness and grade shared significant correlation with serum PAI-1 on dET (Pearson r: ER = 0.28, Grade = 0.29) and d7ET (Pearson r: ER = 0.40, Grade = 0.23). CONCLUSIONS: Periodicity in serum PAI-1 levels offers a robust prognostic factor for predicting clinical pregnancy outcome. The dhCG to dET PAI-1 transition is a decisive factor for either transferring embryos in same/ongoing cycle or cryopreserving them and postponing ET to subsequent natural cycle. PMID:25395746

  15. Enzymatic evidence of impaired reperfusion in diabetic patients after thrombolytic therapy for acute myocardial infarction: a role for plasminogen activator inhibitor?

    PubMed Central

    Gray, R P; Yudkin, J S; Patterson, D L

    1993-01-01

    OBJECTIVE--To compare the activity of plasminogen activator inhibitor (PAI-1) in diabetic and non-diabetic patients admitted with acute myocardial infarction and to determine whether PAI-1 activity influences reperfusion after thrombolytic therapy. DESIGN--Prospective study of patients admitted with acute myocardial infarction. SETTING--District general hospital. MAIN OUTCOME MEASURES--Reperfusion assessed by time to peak release of creatine kinase-MB isoenzyme. RESULTS--Baseline PAI-1 activity and antigen concentrations were significantly higher in diabetic patients (n = 45) than in non-diabetic patients (n = 110) (24.6 (6.9) v 18.6 (7.9) AU/ml (AU = arbitrary units) (p = 0.0001) and 58.8 (13.1-328.8) v 41.0 (10.9-125.4) ng/ml (p = 0.004). Time to peak release of creatine kinase-MB was calculated in 123 (80%) patients. In 98 who received thrombolytic therapy the median time to peak enzyme release was 15.5 h (7.5-24 h) in diabetic patients (n = 26) and 12 h (5-26 h) in non-diabetic patients (n = 72) (p = 0.005). In those with a time to peak release of < or = 12 h, indicating likely successful reperfusion, PAI-1 activity was 17.5 (7.8) AU/ml compared with 22.8 (7.7) AU/ml in those with a time to peak release of > 12 h (p = 0.001). In multiple regression analysis both diabetes (p = 0.0001) and PAI-1 activity at admission (p = 0.029) were independently related to successful reperfusion. In 13 patients with evidence of reinfarction in hospital PAI-1 activity on day 3 was 26.7 (6.4) AU/ml compared with 21.7 (6.3) AU/ml in those without evidence of reinfarction (p = 0.032). CONCLUSION--Both raised PAI-1 activity on admission and diabetes were associated with a reduced likelihood of enzymatic evidence of reperfusion after thrombolytic therapy. Increased PAI-1 activity on day 3 was associated with an increased risk of reinfarction. Diabetic patients had higher PAI-1 activity on admission. This may partly explain their reduced likelihood of reperfusion. PMID:8280517

  16. Very low density lipoprotein-mediated signal transduction and plasminogen activator inhibitor type 1 in cultured HepG2 cells.

    PubMed

    Banfi, C; Mussoni, L; Risé, P; Cattaneo, M G; Vicentini, L; Battaini, F; Galli, C; Tremoli, E

    1999-07-23

    In normal subjects and in patients with cardiovascular disease, plasma triglycerides are positively correlated with plasminogen activator inhibitor type 1 (PAI-1) levels. Moreover, in vitro studies indicate that VLDLs induce PAI-1 synthesis in cultured cells, ie, endothelial and HepG2 cells. However, the signaling pathways involved in the effect of VLDL on PAI-1 synthesis have not yet been investigated. We report that VLDLs induce a signaling cascade that leads to an enhanced secretion of PAI-1 by HepG2 cells. In myo-[(3)H]inositol-labeled HepG2 cells, VLDL (100 microg/mL) caused a time-dependent increase in [(3)H]inositol phosphates, the temporal sequence being tris>bis>monophosphate. VLDL brought about a time-dependent stimulation of membrane-associated protein kinase C (PKC) activity and arachidonate release. Finally, VLDL stimulated mitogen-activated protein (MAP) kinase, and this effect was reduced by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), which suggests that PKC plays a pivotal role in MAP kinase phosphorylation. VLDL-induced PAI-1 secretion was completely prevented by U73122, a specific inhibitor of phosphatidylinositol-specific phospholipase C, by H7 or by PKC downregulation, and by mepacrine (all P<0.01 versus VLDL-treated cells). 3,4,5-Trimethoxybenzoic acid 8-(diethylamino)-octyl ester, which prevents Ca2+ release from intracellular stores, inhibited VLDL-induced PAI-1 secretion by 60% (P<0.05), and the MAP kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor PD98059 completely suppressed both basal and VLDL-induced PAI-1 secretion. These data demonstrate that VLDL-induced PAI-1 biosynthesis results from a principal signaling pathway involving PKC-mediated MAP kinase activation.

  17. Overexpression of Hsp27 in a human melanoma cell line: regulation of E-cadherin, MUC18/MCAM, and plasminogen activator (PA) system

    PubMed Central

    Aldrian, Silke; Kindas-Mügge, Ingela; Trautinger, Franz; Fröhlich, Ilse; Gsur, Andrea; Herbacek, Irene; Berger, Walter; Micksche, Michael

    2003-01-01

    Hsp27 is considered a potential marker for cell differentiation in diverse tissues. Several aspects linked to the differentiation process and to the transition from high to low metastatic potential were analyzed in melanoma cells transfected with Hsp27. E-cadherin plays a central role in cell differentiation, migration, and normal development. Loss of expression or function of E-cadherin has been documented in a variety of human malignancies. We observed by fluorescence-activated cell sorter (FACS) as well as immunofluorescence (IF) analysis a pronounced expression of E-cadherin in Hsp27-transfected A375 melanoma cells compared with control melanoma cells. The expression of the adhesion molecule MUC18/MCAM correlates directly with the metastatic potential of melanoma cells. In contrast to wild-type and neotransfected melanoma cells, in Hsp27-transfected cells the expression of MUC18/MCAM could not be detected by FACS and IF analysis. The plasminogen activator (PA) system plays a central role in mediating extracellular proteolysis and also in nonproteolytic events such as cell adhesion, migration, and transmembrane signaling. Hsp27 transfectants revealed elevated messenger ribonucleic acid expression of the urokinase-type PA (uPA) and its inhibitor, PA inhibitor type 1, which might indicate a neutralization effect of the proteolytic activity of uPA. Control cells failed to express both these molecules. The influence of Hsp27 expression on uPA activity and the involvement of E-cadherin could be demonstrated by use of anti–E-cadherin–blocking antibody. Our data provide evidence for an inhibitory-regulatory role of Hsp27 in tumor progression as found in our system. PMID:14984058

  18. Genome-Wide Association Study for Circulating Tissue Plasminogen Activator (tPA) Levels and Functional Follow-up Implicates Endothelial STXBP5 and STX2

    PubMed Central

    Huang, Jie; Huffman, Jennifer E.; Yamkauchi, Munekazu; Trompet, Stella; Asselbergs, Folkert W.; Sabater-Lleal, Maria; Trégouët, David-Alexandre; Chen, Wei-Min; Smith, Nicholas L.; Kleber, Marcus E.; Shin, So-Youn; Becker, Diane M.; Tang, Weihong; Dehghan, Abbas; Johnson, Andrew D.; Truong, Vinh; Folkersen, Lasse; Yang, Qiong; Oudot-Mellakh, Tiphaine; Buckley, Brendan M.; Moore, Jason H.; Williams, Frances M.K.; Campbell, Harry; Silbernagel, Günther; Vitart, Veronique; Rudan, Igor; Tofler, Geoffrey H.; Navis, Gerjan J.; DeStefano, Anita; Wright, Alan F.; Chen, Ming-Huei; de Craen, Anton J.M.; Worrall, Bradford B.; Rudnicka, Alicja R.; Rumley, Ann; Bookman, Ebony B.; Psaty, Bruce M.; Chen, Fang; Keene, Keith L.; Franco, Oscar H.; Böhm, Bernhard O.; Uitterlinden, Andre G.; Carter, Angela M.; Jukema, J. Wouter; Sattar, Naveed; Bis, Joshua C.; Ikram, Mohammad A.; Sale, Michèle M.; McKnight, Barbara; Fornage, Myriam; Ford, Ian; Taylor, Kent; Slagboom, P. Eline; McArdle, Wendy L.; Hsu, Fang-Chi; Franco-Cereceda, Anders; Goodall, Alison H.; Yanek, Lisa R.; Furie, Karen L.; Cushman, Mary; Hofman, Albert; Witteman, Jacqueline CM.; Folsom, Aaron R.; Basu, Saonli; Matijevic, Nena; van Gilst, Wiek H.; Wilson, James F.; Westendorp, Rudi G.J.; Kathiresan, Sekar; Reilly, Muredach P.; Tracy, Russell P.; Polasek, Ozren; Winkelmann, Bernhard R.; Grant, Peter J.; Hillege, Hans L.; Cambien, Francois; Stott, David J.; Lowe, Gordon D.; Spector, Timothy D.; Meigs, James B.; Marz, Winfried; Eriksson, Per; Becker, Lewis C.; Morange, Pierre-Emmanuel; Soranzo, Nicole; Williams, Scott M.; Hayward, Caroline; van der Harst, Pim; Hamsten, Anders; Lowenstein, Charles J.; Strachan, David P.; O'Donnell, Christopher J.

    2014-01-01

    Objective Tissue plasminogen activator (tPA), a serine protease, catalyzes the conversion of plasminogen to plasmin, the major enzyme responsible for endogenous fibrinolysis. In some populations, elevated plasma levels of tPA have been associated with myocardial infarction and other cardiovascular diseases (CVD). We conducted a meta-analysis of genome-wide association studies (GWAS) to identify novel correlates of circulating levels of tPA. Approach and Results Fourteen cohort studies with tPA measures (N=26,929) contributed to the meta-analysis. Three loci were significantly associated with circulating tPA levels (P <5.0×10−8). The first locus is on 6q24.3, with the lead SNP (rs9399599, P=2.9×10−14) within STXBP5. The second locus is on 8p11.21. The lead SNP (rs3136739, P=1.3×10−9) is intronic to POLB and less than 200kb away from the tPA encoding gene PLAT. We identified a non-synonymous SNP (rs2020921) in modest LD with rs3136739 (r2 = 0.50) within exon 5 of PLAT (P=2.0×10−8). The third locus is on 12q24.33, with the lead SNP (rs7301826, P=1.0×10−9) within intron 7 of STX2. We further found evidence for association of lead SNPs in STXBP5 and STX2 with expression levels of the respective transcripts. In in vitro cell studies, silencing STXBP5 decreased release of tPA from vascular endothelial cells, while silencing of STX2 increased tPA release. Through an in-silico lookup, we found no associations of the three lead SNPs with coronary artery disease or stroke. Conclusions We identified three loci associated with circulating tPA levels, the PLAT region, STXBP5 and STX2. Our functional studies implicate a novel role for STXBP5 and STX2 in regulating tPA release. PMID:24578379

  19. Association Between Plasminogen Activator Inhibitor-1-675 4G/5G Insertion/Deletion Polymorphism and Chronic Obstructive Pulmonary Disease.

    PubMed

    Essa, Enas S; El Wahsh, Rabab A

    2016-12-01

    Molecular pathology of chronic obstructive pulmonary disease (COPD) is still being investigated to discover relationships with disease pathogenesis. Evidence of plasminogen activator inhibitor-1 (PAI-1) overexpression in the sputum and the blood of COPD patients is growing. We aimed to investigate the potential relation between PAI-1 promoter 4G/5G insertion/deletion polymorphism and COPD development. In a case-control study, we genotyped 117 COPD patients and 160 control subjects for PAI-1 promoter 4G/5G polymorphism by an allele-specific polymerase chain reaction analysis. All subjects were male smokers. In the co-dominant model, there was a significant difference in the distribution of 5G/5G, 4G/5G and 4G/4G genotypes between COPD patients and controls (p = 0.002). In the recessive model, carriers of 4G/4G genotype were significantly higher in COPD patients than controls (p = 0.01). Carriers of 4G/4G genotype were at higher risk to develop COPD than those carrying 5G/5G or 4G/5G genotypes (crude odds ratio (OR) = 2.10, 95% confidence interval (CI) = 1.19-3.73, adjusted OR = 2.5, 95% CI = 1.22-3.99). In conclusion, PAI-1 4G/5G genetic variations are associated with COPD development in males.

  20. Clinical Value of Plasma Soluble Urokinase-Type Plasminogen Activator Receptor Levels in Term Neonates with Infection or Sepsis: A Prospective Study

    PubMed Central

    Siahanidou, Tania; Margeli, Alexandra; Charoni, Stavroula; Giannaki, Maria; Vavourakis, Eustathios; Charisiadou, Athina; Papassotiriou, Ioannis

    2014-01-01

    Background. suPAR, the soluble form of the urokinase-type plasminogen activator receptor, has been identified as a biomarker of infection in adults but its properties in neonatal infection are not known. Methods. Plasma suPAR levels were determined by ELISA in 47 term neonates with infection (19 bacterial and 28 viral) and in 18 healthy neonates as controls. Thirteen out of 47 infected neonates were septic. In all infected neonates, suPAR levels were repeated at 24 hours, 48 hours, 3–5 days, and 7–10 days following admission. Results. Plasma suPAR levels were significantly increased in infected neonates upon admission, whereas they were highest in septic neonates, in comparison with controls (P < 0.001) and correlated positively with serum CRP levels (P = 0.001). At infection subsidence, suPAR concentrations decreased significantly in comparison with baseline (P < 0.001) but remained higher than in controls (P = 0.01). Receiver operating characteristic analysis resulted in significant areas under the curve for detecting either infected or septic neonates, but not for discriminating between bacterial and viral cause of infection. Conclusions. suPAR is a diagnostic biomarker of infection or sepsis in term neonates; however, it cannot discriminate bacterial from viral infections and also its utility for monitoring the response to treatment is questioned. PMID:24882949

  1. Effect of ascorbate on plasminogen activator inhibitor-1 expression and release from platelets and endothelial cells in an in-vitro model of sepsis.

    PubMed

    Swarbreck, Scott B; Secor, Dan; Ellis, Christopher G; Sharpe, Michael D; Wilson, John X; Tyml, Karel

    2015-06-01

    The microcirculation during sepsis fails due to capillary plugging involving microthrombosis. We demonstrated that intravenous injection of ascorbate reduces this plugging, but the mechanism of this beneficial effect remains unclear. We hypothesize that ascorbate inhibits the release of the antifibrinolytic plasminogen activator inhibitor-1 (PAI-1) from endothelial cells and platelets during sepsis. Microvascular endothelial cells and platelets were isolated from mice. Cells were cultured and stimulated with lipopolysaccharide (LPS), tumor necrosis factor alpha (TNFα), or thrombin (agents of sepsis), with/without ascorbate for 1-24 h. PAI-1 mRNA was determined by quantitative PCR. PAI-1 protein release into the culture medium was measured by ELISA. In platelets, PAI-1 release was measured after LPS, TNFα, or thrombin stimulation, with/without ascorbate. In endothelial cells, LPS and TNFα increased PAI-1 mRNA after 6-24 h, but no increase in PAI-1 release was observed; ascorbate did not affect these responses. In platelets, thrombin, but not LPS or TNFα, increased PAI-1 release; ascorbate inhibited this increase at low extracellular pH. In unstimulated endothelial cells and platelets, PAI-1 is released into the extracellular space. Thrombin increases this release from platelets; ascorbate inhibits it pH-dependently. The data suggest that ascorbate promotes fibrinolysis in the microvasculature under acidotic conditions in sepsis.

  2. The structure of the human tissue-type plasminogen activator gene: correlation of intron and exon structures to functional and structural domains.

    PubMed Central

    Ny, T; Elgh, F; Lund, B

    1984-01-01

    A genomic clone carrying the human tissue-type plasminogen activator (t-PA) gene was isolated from a cosmid library, and the gene structure was elucidated by restriction mapping, Southern blotting, and DNA sequencing. The cosmid contained all the coding parts of the mRNA, except for the first 58 bases in the 5' end of the mRNA, and had a total length of greater than 20 kilobases. It was separated into at least 14 exons by at least 13 introns, and the exons seemed to code for structural or functional domains. Thus, the signal peptide, the propeptide, and the domains of the heavy chain, including the regions homologous to growth factors, and to the "finger" structure of fibronectin, are all encoded by separate exons. In addition, the two kringle regions of t-PA were both coded for by two exons and were cleaved by introns at identical positions. The region coding for the light chain, comprising the serine protease part of the molecule was split by four introns, revealing a gene organization similar to other serine proteases. PMID:6089198

  3. Guidelines for the intravenous application of recombinant tissue-type plasminogen activator (alteplase), the second edition, October 2012: a guideline from the Japan Stroke Society.

    PubMed

    Minematsu, Kazuo; Toyoda, Kazunori; Hirano, Teruyuki; Kimura, Kazumi; Kondo, Rei; Mori, Etsuro; Nakagawara, Jyoji; Sakai, Nobuyuki; Shiokawa, Yoshiaki; Tanahashi, Norio; Yasaka, Masahiro; Katayama, Yasuo; Miyamoto, Susumu; Ogawa, Akira; Sasaki, Makoto; Suga, Sadao; Yamaguchi, Takenori

    2013-07-01

    In Japan, intravenous alteplase, a recombinant tissue-type plasminogen activator (rt-PA), was approved for an indication of ischemic stroke in 2005 on the basis of the results of a clinical trial with a unique dose of the drug (0.6 mg/kg). The Japan Stroke Society published the guidelines for intravenous application of rt-PA and organized training sessions for proper use all over Japan in an effort to promote the safe, widespread use of intravenous alteplase. Seven years following its approval, clinical experience with intravenous alteplase has accumulated, additional evidence of intravenous alteplase has been found in Japan and overseas, and the medical environment has substantially changed, including approvals for new drugs and medical devices. Notably, the use of alteplase in the extended therapeutic time window (within 4.5 hours of symptom onset) became covered by insurance in Japan in August 2012. To address these changing situations, we have decided to prepare the revised guidelines. In preparing the second edition, we took care to make its contents more practical by emphasizing information needed in clinical practice. While the first edition was developed with emphasis on safety in light of limited clinical experience with intravenous alteplase in Japan in 2005, this second edition is a substantial revision of the first edition mainly in terms of eligibility criteria, on the basis of accumulated evidence and the clinical experience.

  4. Coagulation alterations due to local fibrinolytic therapy with recombinant tissue-type plasminogen activator (rt-PA) in patients with peripheral arterial occlusive disease

    SciTech Connect

    Rauber, Klaus; Heidinger, Kathrin S.; Kemkes-Matthes, Bettina

    1997-05-15

    Purpose. To determine the systemic effects of local fibrinolytic therapy with low-dose recombinant tissue-type plasminogen activator (rt-PA). Methods. Ten patients received intrathrombal infusion of 20 mg rt-PA and heparin for local thrombolysis and had subsequent percutaneous transluminal angioplasty (PTA). Eight controls underwent PTA and received heparin alone. We measured t-PA, D-Dimer, and fibrinogen levels before, directly after, and 20, 40, and 60 min and 24 hr after therapy. Results. In the thrombolysis group the t-PA level peaked immediately after infusion and then declined within 1 hr. D-Dimer increased and remained elevated, whereas in the control group only t-PA levels increased, and only after 24 hr. Fibrinogen remained within the normal range in both groups. Eight of ten patients in the thrombolysis group and seven of eight with PTA had clinical improvement after the procedure. Conclusions. The increase in D-Dimer in the rt-PA group indicates a good local fibrinolytic effect. The fact that fibrinogen levels remained unchanged indicates that there is a lack of systemic fibrinogenolysis.

  5. Safety and tolerance data from the Belgian multicentre study of anisoylated plasminogen streptokinase activator complex versus heparin in acute myocardial infarction.

    PubMed

    Bossaert, L L

    1987-01-01

    In the European Multicentre Study (EMS), the safety and efficacy of a single 30U intravenous injection of anisoylated plasminogen streptokinase activator complex (APSAC) was studied in patients with acute myocardial infarction. The present study discusses the Belgian data on safety and tolerance from the EMS study. 87 patients were randomised to treatment with APSAC or heparin. The reperfusion rate was 60.5% (APSAC) versus 20.5% (heparin control), and reocclusion occurred in 21% of the reperfused APSAC patients. Drug-related adverse events consisted of bleeding problems (7 events in patients on APSAC and 1 event in a patient on heparin and moderate allergic reactions (12 events in 9 patients on APSAC and 1 event in a heparin patient). There was 1 drug-related death in the APSAC group (hypovolaemic shock due to central vein puncture during lytic state) which could have been avoided. It is concluded that thrombolytic treatment of acute myocardial infarction with APSAC is effective and safe, as long as the standard precautions for thrombolytic treatment are respected. Bleeding and allergic-type events are infrequent, usually well tolerated and easily treated.

  6. Gene Therapy to Promote Thromboresistance: Local Overexpression of Tissue Plasminogen Activator to Prevent Arterial Thrombosis in an in vivo Rabbit Model

    NASA Astrophysics Data System (ADS)

    Waugh, J. M.; Kattash, M.; Li, J.; Yuksel, E.; Kuo, M. D.; Lussier, M.; Weinfeld, A. B.; Saxena, R.; Rabinovsky, E. D.; Thung, S.; Woo, S. L. C.; Shenaq, S. M.

    1999-02-01

    Tissue-type plasminogen activator (tPA) catalyzes the rate-limiting initial step in the fibrinolytic cascade. Systemic infusion of tPA has become the standard of care for acute myocardial infarction. However, even the relatively short-duration protocols currently employed have encountered significant hemorrhagic complications, as well as complications from rebound thrombosis. Gene therapy offers a method of local high-level tPA expression over a prolonged time period to avoid both systemic hemorrhage and local rebound thrombosis. To examine the impact of local tPA overexpression, an adenoviral vector expressing tPA was created. The construct was characterized functionally in vitro, and the function of the vector was confirmed in vivo by delivery to the rabbit common femoral artery. Systemic coagulation parameters were not perturbed at any of the doses examined. The impact of local overexpression of tPA on in vivo thrombus formation was examined subsequently in a stasis/injury model of arterial thrombosis. The construct effectively prevented arterial thrombosis in treated animals, whereas viral and nonviral controls typically developed occluding thrombi. This construct thus offers a viable technique for promoting a locally thromboresistant small-caliber artery.

  7. Association of plasminogen activator inhibitor-1 and vitamin D receptor expression with the risk of keloid disease in a Chinese population.

    PubMed

    Gong, Zhen-Hua; Ji, Jian-Feng; Yang, Jun; Xiang, Tie; Zhou, Chang-Kai; Pan, Xuan-Liang; Yao, Jian

    2017-01-01

    Keloid disease (KD) is a benign fibroproliferative scarring condition of unknown etiopathogenesis. Plasminogen activator inhibitor-1 (PAI-1) and vitamin D receptor (VDR) have been shown to play important roles in the progression of tissue fibrosis; therefore, both these genes are potential susceptibility genes for KD. We aimed to determine whether the gene expression levels of PAI-1 and VDR are altered in Chinese KD patients. We measured the expression of PAI and VDR in human peripheral blood lymphocytes in 236 patients with keloid and 219 age- and sex-matched healthy controls by quantitative real-time polymerase chain reaction. We found that PAI-1 expression in peripheral blood lymphocytes was significantly higher in patients with KD than in control individuals (p < 0.0001), while VDR expression was significantly lower in KD patients than in control individuals (p < 0.0001). High levels of PAI-1 and low levels of VDR expression were significantly associated with an increased risk for KD. PAI-1 and VDR might play important roles in keloid development. Gene expression levels of PAI-1 and VDR may, therefore, be used as potential markers for the prediction of keloid development after scarring.

  8. Urokinase-type Plasminogen Activator Resulting from Endometrial Carcinogenesis Enhances Tumor Invasion and Correlates with Poor Outcome of Endometrial Carcinoma Patients

    PubMed Central

    Huang, Chia-Yen; Chang, Ming-Cheng; Huang, Wei-Yun; Huang, Ching-Ting; Tang, Yu-Chien; Huang, Hsien-Da; Kuo, Kuan-Ting; Chen, Chi-An; Cheng, Wen-Fang

    2015-01-01

    The purpose of this study was to identify the dysregulated genes involved in the tumorigenesis and progression of endometrial endometrioid adenocarcinoma (EEC), and their possible mechanisms. Endometrial specimens including normal endometrial tissues, atypical endometrial hyperplasia, and EEC were analyzed. The expression profiles were compared using GeneChip Array. The gene expression levels were determined by real-time RT-PCR in the training and testing sets to correlate the clinico-pathological parameters of EEC. Immunoblotting, in vitro cell migration and invasion assays were performed in human endometrial cancer cell lines and their transfectants. In microarray analysis, seven dysregulated genes were identified. Only the levels of urokinase-type plasminogen activator (uPA) were higher in EEC with deep myometrial invasion, positive lympho-vascular space invasion, lymph node metastasis, and advanced stages. After multivariate analysis, uPA was the only independent poor prognostic factor for disease-free survival in the EEC patients (hazard ratio: 4.65, p = 0.03). uPA may enhance the migratory and invasive capabilities of endometrial tumor cells by the phosphorylation of ERK1/2, Akt and p38 molecules. uPA is a dysregulated gene involved in the tumorigenesis, bio-pathological features and outcomes of EEC. uPA may be a potential molecule and target for the detection and treatment of EEC. PMID:26033187

  9. Is the Susceptibility Vessel Sign on 3-Tesla Magnetic Resonance T2*-Weighted Imaging a Useful Tool to Predict Recanalization in Intravenous Tissue Plasminogen Activator?

    PubMed

    Yamamoto, N; Satomi, J; Harada, M; Izumi, Y; Nagahiro, S; Kaji, R

    2016-09-01

    The aim of this study was to investigate the independent factors associated with the absence of recanalization approximately 24 h after intravenous administration of tissue-type plasminogen activator (IV TPA). The previous studies have been conducted using 1.5-Tesla (T) magnetic resonance imaging (MRI). We studied whether the characteristics of 3-T MRI findings were useful to predict outcome and recanalization after IV tPA. Patients with internal carotid artery (ICA) or middle cerebral artery (MCA) (horizontal portion, M1; Sylvian portion, M2) occlusion and treated by IV tPA were enrolled. We studied whether the presence of susceptibility vessel sign (SVS) at M1 and low clot burden score on T2*-weighted imaging (T2*-CBS) on 3-T MRI were associated with the absence of recanalization. A total of 49 patients were enrolled (27 men; mean age, 73.9 years). MR angiography obtained approximately 24 h after IV tPA revealed recanalization in 21 (42.9 %) patients. Independent factors associated with the absence of recanalization included ICA or proximal M1 occlusion (odds ratio, 69.6; 95 % confidence interval, 5.05-958.8, p = 0.002). In this study, an independent factor associated with the absence of recanalization may be proximal occlusion of the cerebral arteries rather than SVS in the MCA or low T2*-CBS on 3-T MRI.

  10. A milk protein gene promoter directs the expression of human tissue plasminogen activator cDNA to the mammary gland in transgenic mice

    SciTech Connect

    Pittius, C.W.; Hennighausen, L.; Lee, E.; Westphal, H.; Nicols, E.; Vitale, J.; Gordon, K. )

    1988-08-01

    Whey acidic protein (WAP) is a major whey protein in mouse milk. Its gene is expressed in the lactating mammary gland and is inducible by steroid and peptide hormones. A series of transgenic mice containing a hybrid gene in which human tissue plasminogen activator (tPA) cDNA is under the control of the murine WAP gene promoter had previously been generated. In this study, 21 tissues from lactating and virgin transgenic female mice containing the WAP-tPA hybrid gene were screened for the distribution of murine WAP and human tPA transcripts. Like the endogenous WAP RNA, WAP-tPA RNA was expressed predominantly in mammary gland tissue and appeared to be inducible by lactation. Whereas WAP transcripts were not detected in 22 tissues of virgin mice, low levels of WAP-tPA RNA, which were not modulated during lactation, were found in tongue, kidney, and sublingual gland. These studies demonstrate that the WAP gene promoter can target the expression of a transgene to the mammary gland and that this expression is inducible during lactation.

  11. Expression of the urokinase plasminogen activator receptor (uPAR) and its ligand (uPA) in brain tissues of human immunodeficiency virus patients with opportunistic cerebral diseases.

    PubMed

    Nebuloni, Manuela; Cinque, Paola; Sidenius, Nicolai; Ferri, Angelita; Lauri, Eleonora; Omodeo-Zorini, Elisabetta; Zerbi, Pietro; Vago, Luca

    2009-01-01

    The urokinase plasminogen activator receptor (uPAR) and its ligand (uPA) play an important role in cell migration and extracellular proteolysis. We previously described uPAR/uPA overexpression in the cerebrospinal fluid (CSF) and brain tissues of patients with human immunodeficiency virus (HIV)-related cerebral diseases. In this study, we examined uPAR/uPA expression by immunohistochemistry (IHC) in brains of HIV patients with opportunistic cerebral lesions and in HIV-positive/negative controls. uPAR was found in macrophages/microglia with the highest levels in cytomegalovirus (CMV) encephalitis, toxoplasmosis, and lymphomas; in cryptococcosis and progressive multifocal leukoencephalopathy (PML) cases, only a few positive cells were found and no positivity was observed in controls. uPA expression was demonstrated only in a few macrophages/microglia and lymphocytes in all the cases and HIV-positive controls without different pattern of distribution; no uPA immunostaining was found in cryptococcosis and HIV-negative controls. The higher expression of uPAR/uPA in most of the opportunistic cerebral lesions supports their role in these diseases, suggesting their contribution to tissue injury.

  12. Effects of tissue plasminogen activator and annexin A2 combination therapy on long-term neurological outcomes of rat focal embolic stroke

    PubMed Central

    Wang, Xiaoshu; Fan, Xiang; Yu, Zhanyang; Liao, Zhengbu; Zhao, Jianhua; Mandeville, Emiri; Guo, Shuzhen; Lo, Eng H.; Wang, Xiaoying

    2014-01-01

    Background and Purpose Tissue type plasminogen activator (tPA) in combination with recombinant annexin A2 (rA2) is known to reduce acute brain damage after focal ischemia. Here, we ask whether tPA plus rA2 combination therapy can lead to sustained long term neurological improvements as well. Methods We compared the effects of intravenous high-dose tPA alone (10mg/kg) versus a combination of low-dose tPA (5mg/kg) plus 10 mg/kg rA2 in a model of focal embolic cerebral ischemia in rats. All rats were treated at 3 hours after embolization. Brain tissue and neurological outcomes were assessed at 1 month. Surrogate biomarkers for endogenous neurovascular remodeling in peri-infarct area were analyzed by immunohistochemistry. Results Compared to high-dose tPA alone, low-dose tPA plus rA2 significantly decreased infarction and improved neurological function at 1 month post-stroke. In peri-infarct areas, tPA-plus-rA2 combination therapy also significantly augmented microvessel density, VEGF and synaptophysin expression. Conclusions Compared to conventional high-dose tPA alone, combination low-dose tPA plus rA2 therapy may provide a safe and effective way to improve long term neurological outcomes after stroke. PMID:24368559

  13. Engineering the cellular protein secretory pathway for enhancement of recombinant tissue plasminogen activator expression in Chinese hamster ovary cells: effects of CERT and XBP1s genes.

    PubMed

    Rahimpour, Azam; Vaziri, Behrouz; Moazzami, Reza; Nematollahi, Leila; Barkhordari, Farzaneh; Kokabee, Leila; Adeli, Ahmad; Mahboudi, Fereidoun

    2013-08-01

    Cell line development is the most critical and also the most time-consuming step in the production of recombinant therapeutic proteins. In this regard, a variety of vector and cell engineering strategies have been developed for generating high-producing mammalian cells; however, the cell line engineering approach seems to show various results on different recombinant protein producer cells. In order to improve the secretory capacity of a recombinant tissue plasminogen activator (t-PA)-producing Chinese hamster ovary (CHO) cell line, we developed cell line engineering approaches based on the ceramide transfer protein (CERT) and X-box binding protein 1 (XBP1) genes. For this purpose, CERT S132A, a mutant form of CERT that is resistant to phosphorylation, and XBP1s were overexpressed in a recombinant t-PA-producing CHO cell line. Overexpression of CERT S132A increased the specific productivity of t-PA-producing CHO cells up to 35%. In contrast, the heterologous expression of XBP1s did not affect the t-PA expression rate. Our results suggest that CERTS132A- based secretion engineering could be an effective strategy for enhancing recombinant t- PA production in CHO cells.

  14. Acute stroke therapy with tissue plasminogen activator (tPA) since it was approved by the U.S. Food and Drug Administration (FDA).

    PubMed

    Zivin, Justin A

    2009-07-01

    Tissue plasminogen activator (tPA) for acute ischemic stroke was approved by the U.S. Food and Drug Administration (FDA) in 1996. Since then it has been severely underutilized. At the time when most practitioners were first being exposed to the literature concerning tPA, there were many concerns about safety and the restrictions on use were quite onerous. Since then a good deal of further work has been done to loosen the restrictions and allay concerns about the risks. The true risk to benefit ratio is far better than is generally realized. Now it is mostly economic problems related to the costs of constantly supplying emergency care that is limiting access. Furthermore, in the current litigious environment, failure to treat is likely to be a more hazardous course of action than legal exposure due to poor outcomes. It must be emphasized that the drug is quite safe and highly effective, and current utilization rates are unacceptably low. Ann Neurol 2009;66:6-10.

  15. Tissue plasminogen activator contributes to morphine tolerance and induces mechanical allodynia via astrocytic IL-1β and ERK signaling in the spinal cord of mice.

    PubMed

    Berta, T; Liu, Y-C; Xu, Z-Z; Ji, R-R

    2013-09-05

    Accumulating evidence indicates that activation of spinal cord astrocytes contributes importantly to nerve injury and inflammation-induced persistent pain and chronic opioid-induced antinociceptive tolerance. Phosphorylation of extracellular signal-regulated kinase (pERK) and induction of interleukin-1 beta (IL-1β) in spinal astrocytes have been implicated in astrocytes-mediated pain. Tissue plasminogen activator (tPA) is a serine protease that has been extensively used to treat stroke. We examined the potential involvement of tPA in chronic opioid-induced antinociceptive tolerance and activation of spinal astrocytes using tPA knockout (tPA(-/-)) mice and astrocyte cultures. tPA(-/-) mice exhibited unaltered nociceptive pain and morphine-induced acute analgesia. However, the antinociceptive tolerance, induced by chronic morphine (10mg/kg/day, s.c.), is abrogated in tPA(-/-) mice. Chronic morphine induces tPA expression in glial fibrillary acidic protein (GFAP)-expressing spinal cord astrocytes. Chronic morphine also increases IL-1β expression in GFAP-expressing astrocytes, which is abolished in tPA-deficient mice. In cultured astrocytes, morphine treatment increases tPA, IL-1β, and pERK expression, and the increased IL-1β and pERK expression is abolished in tPA-deficient astrocytes. tPA is also sufficient to induce IL-1β and pERK expression in astrocyte cultures. Intrathecal injection of tPA results in up-regulation of GFAP and pERK in spinal astrocytes but not up-regulation of ionized calcium binding adapter molecule 1 in spinal microglia. Finally, intrathecal tPA elicits persistent mechanical allodynia, which is inhibited by the astroglial toxin alpha-amino adipate and the MEK (ERK kinase) inhibitor U0126. Collectively, these data suggest an important role of tPA in regulating astrocytic signaling, pain hypersensitivity, and morphine tolerance.

  16. Tissue plasminogen activator contributes to morphine tolerance and induces mechanical allodynia via astrocytic IL-1β and ERK signaling in the spinal cord of mice

    PubMed Central

    Berta, Temugin; Liu, Yen-Chin; Xu, Zhen-Zhong; Ji, Ru-Rong

    2013-01-01

    Accumulating evidence indicates that activation of spinal cord astrocytes contributes importantly to nerve injury and inflammation-induced persistent pain and chronic opioid-induced antinociceptive tolerance. Phosphorylation of extracellular signal-regulated kinase (pERK) and induction of interleukin-1 beta (IL-1β) in spinal astrocytes have been implicated in astrocytes-mediated pain. Tissue plasminogen activator (tPA) is a serine protease that has been extensively used to treat stroke. We examined the potential involvement of tPA in chronic opioid-induced antinociceptive tolerance and activation of spinal astrocytes using tPA knockout (tPA−/−) mice and astrocyte cultures. tPA−/− mice exhibited unaltered nociceptive pain and morphine-induced acute analgesia. However, the antinociceptive tolerance, induced by chronic morphine (10 mg/kg/day, s.c.), is abrogated in tPA−/− mice. Chronic morphine induces tPA expression in GFAP-expressing spinal cord astrocytes. Chronic morphine also increases IL-1β expression in GFAP-expressing astrocytes, which is abolished in tPA-deficient mice. In cultured astrocytes, morphine treatment increases tPA, IL-1β, and pERK expression, and the increased IL-1β and pERK expression is abolished in tPA-deficient astrocytes. tPA is also sufficient to induce IL-1β and pERK expression in astrocyte cultures. Intrathecal injection of tPA results in up-regulation of GFAP and pERK in spinal astrocytes but not up-regulation of IBA-1 in spinal microglia. Finally, intrathecal tPA elicits persistent mechanical allodynia, which is inhibited by the astroglial toxin alpha-amino adipate and the MEK (ERK kinase) inhibitor U0126. Collectively, these data suggest an important role of tPA in regulating astrocytic signaling, pain hypersensitivity, and morphine tolerance. PMID:23707980

  17. Dosing time-dependent effect of raloxifene on plasma plasminogen activator inhibitor-1 concentrations in post-menopausal women with osteoporosis.

    PubMed

    Ando, Hitoshi; Otoda, Toshiki; Ookami, Hitoshi; Nagai, Yukihiro; Inano, Akihiro; Takamura, Toshinari; Ushijima, Kentarou; Hosohata, Keiko; Matsushita, Eiki; Saito, Tetsuo; Kaneko, Shuichi; Fujimura, Akio

    2013-03-01

    Raloxifene, a selective oestrogen receptor modulator commonly used for the treatment of post-menopausal osteoporosis, affects the coagulation and fibrinolytic systems and consequently increases the risk of venous thromboembolism. Because both the coagulation and fibrinolytic systems exhibit circadian rhythms, the aim of the present study was to investigate the effects of dosing time of raloxifene on markers of coagulation and fibrinolysis, as well as on markers of bone metabolism. Thirty-nine post-menopausal patients with osteoporosis were randomly allocated to two groups: one received 60 mg raloxifene once daily in the morning, whereas the other received 60 mg raloxifene once daily in the evening, for 12 months. In both groups, the activity of coagulation Factors IX and XII was increased significantly after 12 months treatment compared with baseline. The activity of coagulation Factors II and V and levels of markers of bone metabolism (i.e. bone alkaline phosphatase and tartrate-resistant acid phosphatase 5b) decreased in both groups. The changes in these markers did not differ between the two groups. In contrast, the plasma concentration of plasminogen activator inhibitor (PAI)-1 increased in the group receiving the morning dose (mean change 40.9%; 95% confidence interval (CI) 9.4, 72.5), but not in the groups receiving the evening dose (mean change -0.3%; 95% CI -31.5, 30.9); these percentage changes differed significantly (P < 0.05). Because an elevated concentration of PAI-1 is known to be associated with the risk of venous thromboembolism, the findings of the present study suggest that the dosing time of raloxifene influences its safety. Further larger-scale studies are needed to determine the clinical usefulness of chronotherapy with raloxifene.

  18. The plasminogen activator system in the ovine placentome during late gestation and stage-two of parturition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The process of placental separation is not completely understood. In domestic animals, especially cattle, it is important that expulsion of the fetal membranes takes place in a timely manner in order to achieve maximal reproductive efficiency. The activity of the matrix-metalloprotease (MMP) family ...

  19. Synergistic transcriptional activation of the mouse urokinase plasminogen activator (uPA) gene and of its enhancer activator protein 1 (AP1) site by cAMP and retinoic acid.

    PubMed Central

    Mira-Y-Lopez, R; Jaramillo, S; Jing, Y

    1998-01-01

    We have investigated the mechanism whereby all-trans retinoic acid (tRA) potentiates the 8-bromo-cAMP (8-BrcAMP)-dependent transcription of the urokinase plasminogen activator (uPA) gene in SC115 mouse mammary carcinoma cells. Photoaffinity labelling experiments showed that tRA did not alter the cellular content of cAMP-dependent protein kinase regulatory subunits I and II. In agreement with this, nuclear run-on analysis in the presence of the translational inhibitor puromycin demonstrated that the effect of 8-BrcAMP and its potentiation by tRA were independent of protein synthesis. A transiently transfected 6.6 kb uPA 5'-flanking region-chloramphenicol acetyltransferase (CAT) fusion gene mimicked the response of the endogenous uPA gene. Thus 1 mM 8-BrcAMP induced a 100-200% increase in CAT content, 100 nM tRA had no effect and 100 nM tRA+1 mM 8-BrcAMP induced a 300-500% increase in cells co-transfected with tRA receptor and/or 9-cis-RA receptor. Analysis of 5'-deleted constructs showed that the tRA effect required at least two cis regions: -2657 to -2186, encompassing the 100 bp uPA enhancer, and -709 to -324, which exhibited silencing activity. Neither region contained a tRA-response element-like motif. Because tRA receptor and 9-cis-RA receptor interact with activator protein 1 (AP1), we tested whether tRA regulated the uPA enhancer AP1 site in the presence of 8-BrcAMP. We found that a dimer of this site fused to a minimal uPA-CAT fusion gene was responsive to 1 mM 8-BrcAMP (100% CAT increase), not responsive to 100 nM tRA, and synergistically responsive to 100 nM tRA+1 mM 8-BrcAMP (240% CAT increase) in cells co-transfected with Fos and Jun. Synergistic activation of the same construct and of the 6.6 kb uPA-CAT fusion gene was also obtained using tRA and 100 nM PMA. We conclude that multiple cis elements, probably including the uPA enhancer AP1 site, mediate the tRA potentiation of uPA transcription. PMID:9560322

  20. Plasminogen activator inhibitor-1 controls bone marrow-derived cells therapeutic effect through MMP9 signaling: role in physiological and pathological wound healing.

    PubMed

    Ebrahimian, Teni G; Squiban, Claire; Roque, Telma; Lugo-Martinez, Haydee; Hneino, Mohamad; Buard, Valerie; Gourmelon, Patrick; Benderitter, Marc; Milliat, Fabien; Tamarat, Radia

    2012-07-01

    We assessed the role of plasminogen activator inhibitor-1 (PAI-1) and matrix metalloproteinase 9 (MMP9) in wound healing process and in the bone marrow mononuclear cells (BMMNC)-related effects on physiological and pathological wound healing. A full thickness excision wound was created by removal of the skin on the midback of irradiated and nonirradiated animals. Angiogenesis and re-epithelialization were markedly increased in PAI-1-/- mice compared to wild-type (WT) animals. We revealed high MMP activity in tissue of PAI-1-/- animals. Of interest, the wound healing process was reduced in PAI-1-/-:MMP9-/- animals compared to PAI-1-/- mice, suggesting a key role of MMP9 in beneficial effect of PAI-1 deficiency on wound closure. To unravel the role of PAI-1 in BMMNC relative effects, mice were treated with or without local injection of BMMNC isolated from WT, PAI-1-/-, and PAI-1-/-: MMP9-/- animals for 14 days (10(6) cells, n = 6 per group). In WT nonirradiated mice, transplantation of BMMNC isolated from PAI-1-/- animals enhanced wound formation when compared with WT BMMNC. BMMNC differentiation into cells with endothelial phenotype was enhanced by PAI-1 deficiency. These effects were abrogated in PAI-1-/-:MMP9-/- and MMP9-/- BMMNC. In addition, using chimeric mice, we demonstrated that PAI-1 deficiency environment increased the BMMNC-GFP recruitment to the wound site, whereas this effect was abrogated when using PAI-1-/-:MMP9-/- BMMNC. PAI-1 deficiency, at least through MMP9 upregulation, enhanced wound healing and BMMNC therapeutic potential in irradiated and nonirradiated animals.

  1. Irradiation-Induced Regulation of Plasminogen Activator Inhibitor Type-1 and Vascular Endothelial Growth Factor in Six Human Squamous Cell Carcinoma Lines of the Head and Neck

    SciTech Connect

    Artman, Tuuli; Schilling, Daniela; Multhoff, Gabriele

    2010-02-01

    Purpose: It has been shown that plasminogen activator inhibitor type-1 (PAI-1) and vascular endothelial growth factor (VEGF) are involved in neo-angiogenesis. The aim of this study was to investigate the irradiation-induced regulation of PAI-1 and VEGF in squamous cell carcinomas of the head and neck (SCCHN) cell lines of varying radiation sensitivity. Methods and Materials: Six cell lines derived from SCCHN were investigated in vitro. The colorimetric AlamarBlue assay was used to detect metabolic activity of cell lines during irradiation as a surrogate marker for radiation sensitivity. PAI-1 and VEGF secretion levels were measured by enzyme-linked immunosorbent assay 24, 48, and 72 h after irradiation with 0, 2, 6, and 10 Gy. The direct radioprotective effect of exogenous PAI-1 was measured using the clonogenic assay. For regulation studies, transforming growth factor-beta1 (TGF-beta1), hypoxia-inducible factor-1alpha (HIF-1alpha), hypoxia-inducible factor-2alpha (HIF-2alpha), or both HIF-1alpha and HIF-2alpha were downregulated using siRNA. Results: Although baseline levels varied greatly, irradiation led to a comparable dose-dependent increase in PAI-1 and VEGF secretion in all six cell lines. Addition of exogenous stable PAI-1 to the low PAI-1-expressing cell lines, XF354 and FaDu, did not lead to a radioprotective effect. Downregulation of TGF-beta1 significantly decreased VEGF secretion in radiation-sensitive XF354 cells, and downregulation of HIF-1alpha and HIF-2alpha reduced PAI-1 and VEGF secretion in radiation-resistant SAS cells. Conclusions: Irradiation dose-dependently increased PAI-1 and VEGF secretion in all SCCHN cell lines tested regardless of their basal levels and radiation sensitivity. In addition, TGF-beta1 and HIF-1alpha could be partly responsible for VEGF and PAI-1 upregulation after irradiation.

  2. Plasminogen Activator Inhibitor-1 Deficiency Augments Visceral Mesothelial Organization, Intrapleural Coagulation, and Lung Restriction in Mice with Carbon Black/Bleomycin–Induced Pleural Injury

    PubMed Central

    Jeffers, Ann; Alvarez, Alexia; Owens, Shuzi; Koenig, Kathleen; Quaid, Brandon; Komissarov, Andrey A.; Florova, Galina; Kothari, Hema; Pendurthi, Usha; Mohan Rao, L. Vijaya; Idell, Steven

    2014-01-01

    Local derangements of fibrin turnover and plasminogen activator inhibitor (PAI)-1 have been implicated in the pathogenesis of pleural injury. However, their role in the control of pleural organization has been unclear. We found that a C57Bl/6j mouse model of carbon black/bleomycin (CBB) injury demonstrates pleural organization resulting in pleural rind formation (14 d). In transgenic mice overexpressing human PAI-1, intrapleural fibrin deposition was increased, but visceral pleural thickness, lung volumes, and compliance were comparable to wild type. CBB injury in PAI-1−/− mice significantly increased visceral pleural thickness (P < 0.001), elastance (P < 0.05), and total lung resistance (P < 0.05), while decreasing lung compliance (P < 0.01) and lung volumes (P < 0.05). Collagen, α-smooth muscle actin, and tissue factor were increased in the thickened visceral pleura of PAI-1−/− mice. Colocalization of α-smooth muscle actin and calretinin within pleural mesothelial cells was increased in CBB-injured PAI-1−/− mice. Thrombin, factor Xa, plasmin, and urokinase induced mesothelial–mesenchymal transition, tissue factor expression, and activity in primary human pleural mesothelial cells. In PAI-1−/− mice, D-dimer and thrombin–antithrombin complex concentrations were increased in pleural lavage fluids. The results demonstrate that PAI-1 regulates CBB-induced pleural injury severity via unrestricted fibrinolysis and cross-talk with coagulation proteases. Whereas overexpression of PAI-1 augments intrapleural fibrin deposition, PAI-1 deficiency promotes profibrogenic alterations of the mesothelium that exacerbate pleural organization and lung restriction. PMID:24024554

  3. Effect of Temperature Downshift on the Transcriptomic Responses of Chinese Hamster Ovary Cells Using Recombinant Human Tissue Plasminogen Activator Production Culture.

    PubMed

    Bedoya-López, Andrea; Estrada, Karel; Sanchez-Flores, Alejandro; Ramírez, Octavio T; Altamirano, Claudia; Segovia, Lorenzo; Miranda-Ríos, Juan; Trujillo-Roldán, Mauricio A; Valdez-Cruz, Norma A

    2016-01-01

    Recombinant proteins are widely used as biopharmaceuticals, but their production by mammalian cell culture is expensive. Hence, improvement of bioprocess productivity is greatly needed. A temperature downshift (TDS) from 37°C to 28-34°C is an effective strategy to expand the productive life period of cells and increase their productivity (qp). Here, TDS in Chinese hamster ovary (CHO) cell cultures, initially grown at 37°C and switched to 30°C during the exponential growth phase, resulted in a 1.6-fold increase in the qp of recombinant human tissue plasminogen activator (rh-tPA). The transcriptomic response using next-generation sequencing (NGS) was assessed to characterize the cellular behavior associated with TDS. A total of 416 (q > 0.8) and 3,472 (q > 0.9) differentially expressed transcripts, with more than a 1.6-fold change at 24 and 48 h post TDS, respectively, were observed in cultures with TDS compared to those at constant 37°C. In agreement with the extended cell survival resulting from TDS, transcripts related to cell growth arrest that controlled cell proliferation without the activation of the DNA damage response, were differentially expressed. Most upregulated genes were related to energy metabolism in mitochondria, mitochondrial biogenesis, central metabolism, and avoidance of apoptotic cell death. The gene coding for rh-tPA was not differentially expressed, but fluctuations were detected in the transcripts encoding proteins involved in the secretory machinery, particularly in glycosylation. Through NGS the dynamic processes caused by TDS were assessed in this biological system.

  4. Tissue-specific inhibition of urokinase-type plasminogen activator expression in the testes of mice by inducible lentiviral RNA interference causes male infertility.

    PubMed

    Zhao, Kai; Liu, Yan; Xiong, Zhe; Hu, Lian; Xiong, Cheng-Liang

    2017-03-16

    Urokinase-type plasminogen activator (uPA) is involved in many physiological processes, including male infertility. To explore the effects of uPA in male reproduction, we constructed an inducible uPA short hairpin RNA (shRNA) system expressed by lentiviral vectors. After proving inhibition of uPA expression in the mouse Sertoli cell line TM4 by 1μgmL-1 doxycycline (Dox), two lentivirus (pLenti4-shRNA and pLenti6/TR) were co-microinjected into mouse testes to produce TetR&shuPA mice model. Though oral gavage by 0.75mgmL-1 Dox each day for 1 week, the Plau mRNA expression, uPA protein level and uPA enzyme activity in mice testis decreased significantly in TetR&shuPA mice model. After Dox induction of 1 week, the TetR&shuPA mice mated with female mice. Our results show that the pregnancy rate was reduced by approximately 40% and the sperm motility also decreased significantly. These data indicated that downregulation of uPA could decrease the fertility of male mice, which may be caused by a reduction in sperm motility. To investigate the reversible effect and safety of the inducible uPA shRNA system, we withdraw Dox and found the mating rate and sperm motility gradually recovered after 2 weeks. The histopathology structure of the testis, epididymis, and main organs was not altered significantly. The results of the present study indicating that uPA may be regarded as a novel target for the regulation of male fertility.

  5. Effect of Temperature Downshift on the Transcriptomic Responses of Chinese Hamster Ovary Cells Using Recombinant Human Tissue Plasminogen Activator Production Culture

    PubMed Central

    Bedoya-López, Andrea; Estrada, Karel; Sanchez-Flores, Alejandro; Ramírez, Octavio T.; Altamirano, Claudia; Segovia, Lorenzo; Miranda-Ríos, Juan; Trujillo-Roldán, Mauricio A.; Valdez-Cruz, Norma A.

    2016-01-01

    Recombinant proteins are widely used as biopharmaceuticals, but their production by mammalian cell culture is expensive. Hence, improvement of bioprocess productivity is greatly needed. A temperature downshift (TDS) from 37°C to 28–34°C is an effective strategy to expand the productive life period of cells and increase their productivity (qp). Here, TDS in Chinese hamster ovary (CHO) cell cultures, initially grown at 37°C and switched to 30°C during the exponential growth phase, resulted in a 1.6-fold increase in the qp of recombinant human tissue plasminogen activator (rh-tPA). The transcriptomic response using next-generation sequencing (NGS) was assessed to characterize the cellular behavior associated with TDS. A total of 416 (q > 0.8) and 3,472 (q > 0.9) differentially expressed transcripts, with more than a 1.6-fold change at 24 and 48 h post TDS, respectively, were observed in cultures with TDS compared to those at constant 37°C. In agreement with the extended cell survival resulting from TDS, transcripts related to cell growth arrest that controlled cell proliferation without the activation of the DNA damage response, were differentially expressed. Most upregulated genes were related to energy metabolism in mitochondria, mitochondrial biogenesis, central metabolism, and avoidance of apoptotic cell death. The gene coding for rh-tPA was not differentially expressed, but fluctuations were detected in the transcripts encoding proteins involved in the secretory machinery, particularly in glycosylation. Through NGS the dynamic processes caused by TDS were assessed in this biological system. PMID:26991106

  6. TGF-β2 promotes RPE cell invasion into a collagen gel by mediating urokinase-type plasminogen activator (uPA) expression.

    PubMed

    Sugioka, Koji; Kodama, Aya; Okada, Kiyotaka; Iwata, Mihoko; Yoshida, Koji; Kusaka, Shunji; Matsumoto, Chota; Kaji, Hiroshi; Shimomura, Yoshikazu

    2013-10-01

    Transforming growth factor-beta (TGF-β) is one of the main epithelial-mesenchymal transition (EMT)-inducing factors. In general, TGF-β-induced EMT promotes cell migration and invasion. TGF-β also acts as a potent regulator of pericellular proteolysis by regulating the expression and secretion of plasminogen activators. Urokinase-type plasminogen activator (uPA) is a serine protease that binds to its cell surface receptor (uPAR) with high affinity. uPA binding to uPAR stimulates uPAR's interaction with transmembrane proteins, such as integrins, to regulate cytoskeletal reorganization and cell migration, differentiation and proliferation. However, the influence of TGF-β and the uPA/uPAR system on EMT in retinal pigment epithelial (RPE) cells is still unclear. The purpose of this study was to determine the effect of TGF-β2, which is the predominant isoform in the retina, and the uPA/uPAR system on RPE cells. In this study, we first examined the effect of TGF-β2 and/or the inhibitor of uPA (u-PA-STOP(®)) on the proliferation of a human retinal pigment epithelial cell line (ARPE-19 cells). Treatment with TGF-β2 or u-PA-STOP(®) suppressed cell proliferation. Combination treatment of TGF-β2 and u-PA-STOP(®) enhanced cell growth suppression. Furthermore, western blot analysis, fibrin zymography and real-time reverse transcription PCR showed that that TGF-β2 induced EMT in ARPE-19 cells and that the expression of uPA and uPAR expression was up-regulated during EMT. The TGF-β inhibitor SB431542 suppressed TGF-β2-stimulated uPA expression and secretion but did not suppress uPAR expression. Furthermore, we seeded ARPE-19 cells onto Transwell chambers and allowed them to invade the collagen matrix in the presence of TGF-β2 alone or with TGF-β2 and u-PA-STOP(®). TGF-β2 treatment induced ARPE-19 cell invasion into the collagen gel. Treatment with a combination of TGF-β2 and the uPA inhibitor strongly inhibited ARPE-19 cell invasion compared with treatment with

  7. CD44 stimulation by fragmented hyaluronic acid induces upregulation of urokinase-type plasminogen activator and its receptor and subsequently facilitates invasion of human chondrosarcoma cells.

    PubMed

    Kobayashi, Hiroshi; Suzuki, Mika; Kanayama, Naohiro; Nishida, Takashi; Takigawa, Masaharu; Terao, Toshihiko

    2002-12-01

    It has been established that fragmented hyaluronic acid (HA), but not native high molecular weight HA, can induce angiogenesis, cell proliferation and migration. We have studied the outside-in signal transduction pathways responsible for fragmented HA-mediated cancer cell invasion. In our study, we have studied the effects of CD44 stimulation by ligation with HA upon the expression of matrix metalloproteinases (MMPs)-2 and -9 as well as urokinase-type plasminogen activator (uPA), its receptor (uPAR) and its inhibitor (PAI-1) and the subsequent induction of invasion of human chondrosarcoma cell line HCS-2/8. Our study indicates that (i) CD44 stimulation by fragmented HA upregulates expression of uPA and uPAR mRNA and protein but does not affect MMPs secretion or PAI-1 mRNA expression; (ii) the effects of HA fragments are critically HA size dependent: high molecular weight HA is inactive, but lower molecular weight fragmented HA (Mr 3.5 kDa) is active; (iii) cells can bind avidly Mr 3.5 kDa fragmented HA through a CD44 molecule, whereas cells do not effectively bind higher Mr HA; (iv) a fragmented HA induces phosphorylation of MAP kinase proteins (MEK1/2, ERK1/2 and c-Jun) within 30 min; (v) CD44 is critical for the response (activation of MAP kinase and upregulation of uPA and uPAR expression); and (vi) cell invasion induced by CD44 stimulation with a fragmented HA is inhibited by anti-CD44 mAb, MAP kinase inhibitors, neutralizing anti-uPAR pAb, anti-catalytic anti-uPA mAb or amiloride. Therefore, our study represents the first report that CD44 stimulation induced by a fragmented HA results in activation of MAP kinase and, subsequently, enhances uPA and uPAR expression and facilitates invasion of human chondrosarcoma cells.

  8. Serum Soluble Urokinase-Type Plasminogen Activator Receptor Is Associated with Low Left Ventricular Ejection Fraction and Elevated Plasma Brain-Type Natriuretic Peptide Level

    PubMed Central

    Fujita, Shu-ichi; Tanaka, Suguru; Maeda, Daichi; Morita, Hideaki; Fujisaka, Tomohiro; Takeda, Yoshihiro; Ito, Takahide; Ishizaka, Nobukazu

    2017-01-01

    Background Recent studies have suggested that soluble urokinase plasminogen activator receptor (suPAR), a biomarker of subclinical levels of inflammation, is significantly correlated with cardiovascular events. Purpose We investigated the association between suPAR and left ventricular ejection fraction (LVEF), left ventricular mass index (LVMI), and plasma B-type natriuretic peptide (BNP) among cardiac inpatients. Methods and Results In total, 242 patients (mean age 71.3 ± 9.8 years; 70 women) admitted to the cardiology department were enrolled in the study. suPAR was significantly correlated with LVEF (R = -0.24, P<0.001), LVMI (R = 0.16, P = 0.014) and BNP (R = 0.46, P<0.001). In logistic regression analysis, the highest suPAR tertile (> 3236 pg/mL) was associated with low LVEF (< 50%) and elevated BNP (> 300 pg/mL) with an odds ratio of 3.84 (95% confidence interval [CI], 1.22–12.1) and 5.36 (95% CI, 1.32–21.8), respectively, after adjusting for age, sex, log-transformed estimated glomerular filtration rate (log(eGFR)), C-reactive protein, and diuretic use. The association between suPAR and LVMI was not statistically significant. In multivariate receiver operating characteristic analysis, addition of log(suPAR) to the combination of age, sex, log(eGFR) and CRP incrementally improved the prediction of low LVEF (area under the curve [AUC], 0.827 to 0.852, P = 0.046) and BNP ≥ 300 pg/mL (AUC, 0.869 to 0.906; P = 0.029). Conclusions suPAR was associated with low LVEF and elevated BNP, but not with left ventricular hypertrophy, independent of CRP, renal function, and diuretic use among cardiac inpatients who were not undergoing chronic hemodialysis. PMID:28135310

  9. Influence of plasminogen activator inhibitor-1 (SERPINE1) 4G/5G polymorphism on circulating SERPINE-1 antigen expression in HCC associated with viral infection.

    PubMed

    Divella, Rosa; Mazzocca, Antonio; Gadaleta, Cosimo; Simone, Giovanni; Paradiso, Angelo; Quaranta, Michele; Daniele, Antonella

    2012-01-01

    Hepatocarcinogenesis is heavily influenced by chronic hepatitis B (HBV) and C (HCV) infection. Elevated levels of plasminogen activator inhibitor-1 (SERPINE1/PAI-1) have been reported in patients with hepatocellular carcinoma (HCC) associated with viral infection. The gene encoding SERPINE1 is highly polymorphic and the frequently associated 4/5 guanosine (4G/5G) polymorphism in the gene promoter may influence its expression. Here, we investigated the distribution of genotypes and the frequency of alleles of the 4G/5G polymorphism in patients with HCC, the influence of the 4G/5G polymorphism on plasma SERPINE1 levels and its association with viral infection. A total of 75 patients with HCC were enrolled: 32 (42.6%) were HBV(+)/HCV(+), 11 (14.6%) were only HCV(+), and 32 (42.6%) were negative for both viruses. A control group of healthy donors was also enrolled (n=50). SERPINE1 plasma concentrations were determined by ELISA and the detection of the promoter 4G/5G polymorphism was performed by an allele-specific PCR analysis. We found that the frequency of both the 4G/4G genotype (p=0.02) and the 4G allele (p=0.006) were significantly higher in patients with HCC compared to the control group, and particularly higher in patients with HCC co-infected with HBV(+)/HCV(+) than in those with no viral infection. We also found that patients with the 4G/4G genotype had significantly higher plasma SERPINE1 protein levels when compared with patients with the 4G/5G or 5G/5G genotype (p<0.001). Differences in frequency of 4G allele and genetic variability of 4G/5G SERPINE1 polymorphism with a higher level of SERPINE1 protein in patients with HCC with HBV(+)/HCV(+) than those without infection, suggest the presence of two distinct pathogenic mechanisms in hepatocarcinogenesis, depending on the etiology.

  10. Impact of the -675 4G/5G polymorphism of the plasminogen activator inhibitor-1 gene on childhood IgA nephropathy.

    PubMed

    Han, Su-Ryun; Kim, Cheon-Jong; Lee, Byung-Cheol

    2012-04-01

    Plasminogen activator inhibitor-1 (PAI-1) is an important regulator of the fibrinolytic pathway and extracellular matrix (ECM) turnover. The -675 4G/5G polymorphism in the PAI-1 promoter is associated with altered PAI-1 transcription, suggesting that this polymorphism may be a candidate risk factor for diseases characterized by ECM accumulation, such as immunoglobulin A nephropathy (IgAN) and mesangial proliferative glomerulonephritis (MesPGN). We genotyped childhood patients with biopsy-confirmed IgAN (n=111) and MesPGN (n=47), and healthy control subjects (n=230) for the -675 4G/5G PAI-1 polymorphism by polymerase chain reaction-restriction fragment length polymorphism methods. The distribution of the 4G/4G (27.9%), 4G/5G (45.1%) and 5G/5G (27.0%) genotypes in IgAN patients was significantly different from the healthy controls (32.2, 54.3 and 13.5%, respectively) (p=0.0092). There was no significant difference in the genotype distributions of the 4G/5G polymorphism between MesPGN patients and the healthy controls. Regarding the impact of the polymorphism on IgAN, the 4G/4G genotype was markedly increased in patients with proteinuria (≥1,000 mg/day) and/or hypertension when compared to patients without proteinuria and hypertension (OR=5.23, 95% CI 1.34-20.38, P=0.0183). These findings indicate that the PAI-1 gene polymorphism may affect the susceptibility of childhood IgAN.

  11. Reduced carriership of 4G allele of plasminogen activator inhibitor-1 4G/5G polymorphism in very young survivors of myocardial infarction.

    PubMed

    Rallidis, Loukianos S; Gialeraki, Argyri; Merkouri, Efrosyni; Liakos, George; Dagres, Nikolaos; Sionis, Dimitrios; Travlou, Anthi; Lekakis, John; Kremastinos, Dimitrios T

    2010-05-01

    There are limited and controversial data regarding the impact of 4G/5G polymorphism of the plasminogen activator inhibitor-1 (PAI-1) gene in the pathogenesis of premature myocardial infarction (MI). We explored whether 4G/5G polymorphism of the PAI-1 gene is associated with the development of MI

  12. Admission levels of C-reactive protein and plasminogen activator inhibitor-1 in patients with acute myocardial infarction with and without cardiogenic shock or heart failure on admission.

    PubMed

    Akkus, Mehmet Necdet; Polat, Gurbuz; Yurtdas, Mustafa; Akcay, Burak; Ercetin, Neslihan; Cicek, Dilek; Doven, Oben; Sucu, Nehir

    2009-01-01

    Scarce data exist on the relationship of C-reactive protein (CRP) or plasminogen activator inhibitor-1 (PAI-1) to the occurrence of heart failure (HF) or cardiogenic shock (CS) after acute myocardial infarction (AMI) and on the relationship between these biomarkers and mortality in CS patients. Thus, we compared high-sensitivity CRP and PAI-1 antigen plasma levels on admission among 3 age- and gender-matched AMI patients groups (consisting of 60 patients with CS, 60 with HF, and 60 without HF on admission), after determining that PAI-1 levels did not vary significantly diurnally in these groups by comparing the data among subgroups which were divided according to admission time within the groups. For CS patients, we also conducted regression analyses to examine the relations of these biomarkers to mortality. CRP levels both in CS (P < 0.001) and HF (P < 0.05) patients were significantly higher compared to those without HF, PAI-1 levels in CS patients were significantly higher compared to both those with (P < 0.05) and without HF (P > 0.01), and CRP and PAI-1 were independent predictors of in-hospital (Odds ratio [OR] = 6.12, 95% confidence intervals [95%CI] = 1.47-25.54 and OR = 5.92, 95%CI = 1.31-26.77, respectively) and 1-year mortality (OR = 5.53, 95%CI = 1.21-25.17 and OR = 5.48, 95%CI = 1.09-27.52, respectively) in CS patients. In conclusion, at admission, CRP is associated with the occurrence of CS and HF and PAI-1 is associated with the occurrence of CS after AMI, and they are of prognostic value in CS complicating AMI.

  13. Soluble urokinase plasminogen activator receptor (suPAR) in acute care: a strong marker of disease presence and severity, readmission and mortality. A retrospective cohort study

    PubMed Central

    Rasmussen, Line Jee Hartmann; Ladelund, Steen; Haupt, Thomas Huneck; Ellekilde, Gertrude; Poulsen, Jørgen Hjelm; Iversen, Kasper; Eugen-Olsen, Jesper; Andersen, Ove

    2016-01-01

    Objective Soluble urokinase plasminogen activator receptor (suPAR) is an inflammatory biomarker associated with presence and progression of disease and with increased risk of mortality. We aimed to evaluate the unspecific biomarker suPAR as a prognostic marker in patients admitted to acute care. Methods This registry-based retrospective cohort study included 4343 consecutively admitted patients from the Acute Medical Unit at a large Danish university hospital. Time to readmission and death were analysed by multiple Cox regression. Results were reported as HRs for 30-day and 90-day follow-up. Results During 30-day follow-up, 782 patients (18.0%) were readmitted and 224 patients (5.2%) died. Comparing 30-day readmission and mortality between patients in the highest and lowest suPAR quartiles yielded HRs of 2.11 (95% CI 1.70 to 2.62) and 4.11 (95% CI 2.46 to 6.85), respectively, when adjusting for age, sex, Charlson score and C reactive protein. Area under the curve for receiver operating characteristics curve analysis of suPAR for 30-day mortality was 0.84 (95% CI 0.81 to 0.86). Furthermore, in the entire cohort, women had slightly higher suPAR compared with men, and suPAR was associated with age, admission time, admission to intensive care unit and Charlson score. Conclusions In this large unselected population of acute medical patients, suPAR is strongly associated with disease severity, readmission and mortality after adjusting for all other risk factors, indicating that suPAR adds information to established prognostic indicators. While patients with low suPAR levels have low risk of readmission and mortality, patients with high suPAR levels have a high risk of adverse events. PMID:27590986

  14. Remodeling of the Vessel Wall after Copper-Induced Injury Is Highly Attenuated in Mice with a Total Deficiency of Plasminogen Activator Inhibitor-1

    PubMed Central

    Ploplis, Victoria A.; Cornelissen, Ivo; Sandoval-Cooper, Mayra J.; Weeks, Lisa; Noria, Francisco A.; Castellino, Francis J.

    2001-01-01

    Clinical studies have indicated that high plasma levels of fibrinogen, or decreased fibrinolytic potential, are conducive to an increased risk of cardiovascular disease. Other investigations have shown that insoluble fibrin promotes atherosclerotic lesion formation by affecting smooth muscle cell proliferation, collagen deposition, and cholesterol accumulation. To directly assess the physiological impact of an imbalanced fibrinolytic system on both early and late stages of this disease, mice deficient for plasminogen activator inhibitor-1 (PAI-1−/−) were used in a model of vascular injury/repair, and the resulting phenotype compared to that of wild-type (WT) mice. A copper-induced arterial injury was found to generate a lesion with characteristics similar to many of the clinical features of atherosclerosis. Fibrin deposition in the injured arterial wall at early (7 days) and late (21 days) times after copper cuff placement was prevalent in WT mice, but was greatly diminished in PAI-1−/− mice. A multilayered neointima with enhanced collagen deposition was evident at day 21 in WT mice. In contrast, only diffuse fibrin was identified in the adventitial compartments of arteries from PAI-1−/− mice, with no evidence of a neointima. Neovascularization was observed in the adventitia and was more extensive in WT arteries, relative to PAI-1−/− arteries. Additionally, enhanced PAI-1 expression and fat deposition were seen only in the arterial walls of WT mice. The results of this study emphasize the involvement of the fibrinolytic system in vascular repair processes after injury and indicate that alterations in the fibrinolytic balance in the vessel wall have a profound effect on the development and progression of vascular lesion formation. PMID:11141484

  15. Regulatory role of NADPH oxidase in glycated LDL-induced upregulation of plasminogen activator inhibitor-1 and heat shock factor-1 in mouse embryo fibroblasts and diabetic mice.

    PubMed

    Zhao, Ruozhi; Le, Khuong; Moghadasian, Mohammed H; Shen, Garry X

    2013-08-01

    Cardiovascular disease is the predominant cause of death in diabetic patients. Fibroblasts are one of the major types of cells in the heart or vascular wall. Increased levels of glycated low-density lipoprotein (glyLDL) were detected in diabetic patients. Previous studies in our group demonstrated that oxidized LDL increased the amounts of NADPH oxidase (NOX), plasminogen activator inhibitor-1 (PAI-1), and heat shock factor-1 (HSF1) in fibroblasts. This study examined the expression of NOX, PAI-1, and HSF1 in glyLDL-treated wild-type or HSF1-deficient mouse embryo fibroblasts (MEFs) and in leptin receptor-knockout (db/db) diabetic mice. Treatment with physiologically relevant levels of glyLDL increased superoxide and H2O2 release and the levels of NOX4 and p22phox (an essential component of multiple NOX complexes) in wild-type or HSF1-deficient MEFs. The levels of HSF1 and PAI-1 were increased by glyLDL in wild-type MEFs, but not in HSF1-deficient MEFs. Diphenyleneiodonium (a nonspecific NOX inhibitor) or small interfering RNA for p22phox prevented glyLDL-induced increases in the levels of NOX4, HSF1, or PAI-1 in MEFs. The amounts of NOX4, HSF1, and PAI-1 were elevated in hearts of db/db diabetic mice compared to wild-type mice. The results suggest that glyLDL increased the abundance of NOX4 or p22phox via an HSF1-independent pathway, but that of PAI-1 via an HSF1-dependent manner. NOX4 plays a crucial role in glyLDL-induced expression of HSF1 and PAI-1 in mouse fibroblasts. Increased expression of NOX4, HSF1, and PAI-1 was detected in cardiovascular tissue of diabetic mice.

  16. A CASE CONTROL STUDY OF THE EFFECTIVENESS OF TISSUE PLASMINOGEN ACTIVATOR ON 6 MONTH PATIENT-REPORTED OUTCOMES AND HEALTHCARE UTILIZATION

    PubMed Central

    Lang, Catherine E.; Bland, Marghuretta D.; Cheng, Nuo; Corbetta, Maurizio; Lee, Jin-Moo

    2014-01-01

    We examined the benefit of tissue plasminogen activator (tPA), delivered as part of usual stroke management, on patient-reported outcomes and healthcare utilization. Using a case control design, patients who received tPA as part of usual stroke management were compared with patients who would have received tPA had they arrived to the hospital within the therapeutic time window. Data were collected from surveys 6 months post stroke using standardized patient-reported outcome measures and questions about healthcare utilization. Demographic and medical data were acquired from hospital records. Patients were matched on stroke severity, age, race, and gender. Matching was done with 1:2 ratio of tPA to controls. Results were compared between groups with 1-tailed tests due to directionally-specific hypothesis in favor of the tPA group. The tPA (n = 78) and control (n = 156) groups were matched across the variables, except for stroke severity, which was better in the control group; subsequent analyses controlled for this mismatch. The tPA group reported better physical function, communication, cognitive ability, depressive symptomatology, and quality of life/participation compared to the control group. Fewer people in the tPA group reported skilled nursing facility stays, emergency department visits, and re-hospitalizations after their stroke compared to controls. Reports of other post-acute services were not different between groups. While it is known that tPA reduces disability, this is the first study to demonstrate the effectiveness of tPA in improving meaningful, patient-reported outcomes. Thus, use of tPA provides a large benefit to the daily lives of people with ischemic stroke. PMID:25440368

  17. Deletion of Braun lipoprotein and plasminogen-activating protease-encoding genes attenuates Yersinia pestis in mouse models of bubonic and pneumonic plague.

    PubMed

    van Lier, Christina J; Sha, Jian; Kirtley, Michelle L; Cao, Anthony; Tiner, Bethany L; Erova, Tatiana E; Cong, Yingzi; Kozlova, Elena V; Popov, Vsevolod L; Baze, Wallace B; Chopra, Ashok K

    2014-06-01

    Currently, there is no FDA-approved vaccine against Yersinia pestis, the causative agent of bubonic and pneumonic plague. Since both humoral immunity and cell-mediated immunity are essential in providing the host with protection against plague, we developed a live-attenuated vaccine strain by deleting the Braun lipoprotein (lpp) and plasminogen-activating protease (pla) genes from Y. pestis CO92. The Δlpp Δpla double isogenic mutant was highly attenuated in evoking both bubonic and pneumonic plague in a mouse model. Further, animals immunized with the mutant by either the intranasal or the subcutaneous route were significantly protected from developing subsequent pneumonic plague. In mice, the mutant poorly disseminated to peripheral organs and the production of proinflammatory cytokines concurrently decreased. Histopathologically, reduced damage to the lungs and livers of mice infected with the Δlpp Δpla double mutant compared to the level of damage in wild-type (WT) CO92-challenged animals was observed. The Δlpp Δpla mutant-immunized mice elicited a humoral immune response to the WT bacterium, as well as to CO92-specific antigens. Moreover, T cells from mutant-immunized animals exhibited significantly higher proliferative responses, when stimulated ex vivo with heat-killed WT CO92 antigens, than mice immunized with the same sublethal dose of WT CO92. Likewise, T cells from the mutant-immunized mice produced more gamma interferon (IFN-γ) and interleukin-4. These animals had an increasing number of tumor necrosis factor alpha (TNF-α)-producing CD4(+) and CD8(+) T cells than WT CO92-infected mice. These data emphasize the role of TNF-α and IFN-γ in protecting mice against pneumonic plague. Overall, our studies provide evidence that deletion of the lpp and pla genes acts synergistically in protecting animals against pneumonic plague, and we have demonstrated an immunological basis for this protection.

  18. Effects of energy intake on type 1 plasminogen activator inhibitor levels in glomeruli of lupus-prone B/W mice.

    PubMed Central

    Troyer, D. A.; Chandrasekar, B.; Thinnes, T.; Stone, A.; Loskutoff, D. J.; Fernandes, G.

    1995-01-01

    Calorie restriction (CR) and/or reduced energy intake ameliorates the progression of autoimmune renal disease in (NZB x NZW)F1 (B/W) female mice and increases life span. Like other forms of glomerulonephritis, the lupus-like kidney disease observed in these animals is frequently accompanied by glomerular deposition of fibrin and increased accumulation of mesangial matrix. Because alterations in plasminogen activator inhibitor type 1 (PAI-1) expression or function may be involved in both fibrin deposition and accumulation of extracellular matrix, we have studied the effects of CR on the expression of PAI-1 in kidneys from female B/W mice fed either ad libitum or on a 40% CR diet. By immunohistochemistry and immunoblotting, we found that the glomerular levels of PAI-1 antigen were highest in older ad lib fed animals with more advanced glomerular disease. Increased levels of PAI-1 protein were paralleled by increased levels of PAI-1 mRNA in total RNA extracted from renal cortex and in diseased glomeruli as detected by in situ hybridization. CR diminished the accumulation of PAI-1 protein and reduced the expression of PAI-1 mRNA. Thus, glomeruli from animals fed ad lib showed much greater deposition of PAI-1 protein, increased expression of PAI-1 mRNA, and more severe histological abnormalities than animals on a CR diet. The differences between CR and ad lib animals were more pronounced in animals studied at 9 to 10 months versus those at 3 to 4 months of age. These observations indicate that the ameliorating effects of CR include diminished PAI-1 gene expression and decreased localization of PAI-1 in glomeruli. Images Figure 2 Figure 3 Figure 4 PMID:7856720

  19. Epithelial and Stromal Cell Urokinase Plasminogen Activator Receptor Expression Differentially Correlates with Survival in Rectal Cancer Stages B and C Patients

    PubMed Central

    Ahn, Seong Beom; Chan, Charles; Dent, Owen F.; Mohamedali, Abidali; Kwun, Sun Young; Clarke, Candice; Fletcher, Julie; Chapuis, Pierre H.; Nice, Edouard C.; Baker, Mark S.

    2015-01-01

    Urokinase plasminogen activator receptor (uPAR) has been proposed as a potential prognostic factor for colorectal cancer (CRC) patient survival. However, CRC uPAR expression remains controversial, especially regarding cell types where uPAR is overexpressed (e.g., epithelium (uPARE) or stroma-associated cells (uPARS)) and associated prognostic relevance. In this study, two epitope-specific anti-uPAR monoclonal antibodies (MAbs) could discriminate expression of uPARE from uPARS and were used to examine this association with survival of stages B and C rectal cancer (RC) patients. Using immunohistochemistry, MAbs #3937 and R4 were used to discriminate uPARE from uPARS respectively in the central and invasive frontal regions of 170 stage B and 179 stage C RC specimens. Kaplan-Meier and Cox regression analyses were used to determine association with survival. uPAR expression occurred in both epithelial and stromal compartments with differential expression observed in many cases, indicating uPARE and uPARS have different cellular roles. In the central and invasive frontal regions, uPARE was adversely associated with overall stage B survival (HR = 1.9; p = 0.014 and HR = 1.5; p = 0.031, respectively) reproducing results from previous studies. uPARS at the invasive front was associated with longer stage C survival (HR = 0.6; p = 0.007), reflecting studies demonstrating that macrophage peritumoural accumulation is associated with longer survival. This study demonstrates that different uPAR epitopes should be considered as being expressed on different cell types during tumour progression and at different stages in RC. Understanding how uPARE and uPARS expression affects survival is anticipated to be a useful clinical prognostic marker of stages B and C RC. PMID:25692297

  20. Two Isoforms of Yersinia pestis Plasminogen Activator Pla: Intraspecies Distribution, Intrinsic Disorder Propensity, and Contribution to Virulence

    PubMed Central

    Dentovskaya, Svetlana V.; Platonov, Mikhail E.; Svetoch, Tat’yana E.; Kopylov, Pavel Kh.; Kombarova, Tat’yana I.; Ivanov, Sergey A.; Shaikhutdinova, Rima Z.; Kolombet, Lyubov’ V.; Chauhan, Sadhana; Ablamunits, Vitaly G.; Motin, Vladimir L.; Uversky, Vladimir N.

    2016-01-01

    It has been shown previously that several endemic Y. pestis isolates with limited virulence contained the I259 isoform of the outer membrane protease Pla, while the epidemic highly virulent strains possessed only the T259 Pla isoform. Our sequence analysis of the pla gene from 118 Y. pestis subsp. microtus strains revealed that the I259 isoform was present exclusively in the endemic strains providing a convictive evidence of more ancestral origin of this isoform. Analysis of the effects of the I259T polymorphism on the intrinsic disorder propensity of Pla revealed that the I259T mutation slightly increases the intrinsic disorder propensity of the C-terminal tail of Pla and makes this protein slightly more prone for disorder-based protein-protein interactions, suggesting that the T259 Pla could be functionally more active than the I259 Pla. This assumption was proven experimentally by assessing the coagulase and fibrinolytic activities of the two Pla isoforms in human plasma, as well as in a direct fluorometric assay with the Pla peptide substrate. The virulence testing of Pla-negative or expressing the I259 and T259 Pla isoforms Y. pestis subsp. microtus and subsp. pestis strains did not reveal any significant difference in LD50 values and dose-dependent survival assays between them by using a subcutaneous route of challenge of mice and guinea pigs or intradermal challenge of mice. However, a significant decrease in time-to-death was observed in animals infected with the epidemic T259 Pla-producing strains as compared to the parent Pla-negative variants. Survival curves of the endemic I259 Pla+ strains fit between them, but significant difference in mean time to death post infection between the Pla−strains and their I259 Pla+ variants could be seen only in the isogenic set of subsp. pestis strains. These findings suggest an essential role for the outer membrane protease Pla evolution in Y. pestis bubonic infection exacerbation that is necessary for

  1. Combination of thrombin-antithrombin complex, plasminogen activator inhibitor-1, and protein C activity for early identification of severe coagulopathy in initial phase of sepsis: a prospective observational study

    PubMed Central

    2014-01-01

    Introduction Current criteria for early diagnosis of coagulopathy in sepsis are limited. We postulated that coagulopathy is already complicated with sepsis in the initial phase, and severe coagulopathy or disseminated intravascular coagulation (DIC) becomes overt after progressive consumption of platelet and coagulation factors. To determine early diagnostic markers for severe coagulopathy, we evaluated plasma biomarkers for association with subsequent development of overt DIC in patients with sepsis. Methods A single-center, prospective observational study was conducted in an adult ICU at a university hospital. Plasma samples were obtained from patients with sepsis at ICU admission. Fourteen biomarkers including global markers (platelet count, prothrombin time, activated partial thromboplastin time, fibrinogen and fibrin degradation product (FDP)); markers of thrombin generation (thrombin-antithrombin complex (TAT) and soluble fibrin); markers of anticoagulants (protein C (PC) and antithrombin); markers of fibrinolysis (plasminogen, α2-plasmin inhibitor (PI), plasmin-α2-PI complex, and plasminogen activator inhibitor (PAI)-1); and a marker of endothelial activation (soluble E-selectin) were assayed. Patients who had overt DIC at baseline were excluded, and the remaining patients were followed for development of overt DIC in 5 days, and for mortality in 28 days. Results A total of 77 patients were enrolled, and 37 developed overt DIC within the following 5 days. Most patients demonstrated hemostatic abnormalities at baseline with 98.7% TAT, 97.4% FDP and 88.3% PC. Most hemostatic biomarkers at baseline were significantly associated with subsequent development of overt DIC. Notably, TAT, PAI-1 and PC discriminated well between patients with and without developing overt DIC (area under the receiver operating characteristic curve (AUROC), 0.77 (95% confidence interval, 0.64 to 0.86); 0.87 (0.78 to 0.92); 0.85 (0.76 to 0.91), respectively), and using the three

  2. The sGC activator inhibits the proliferation and migration, promotes the apoptosis of human pulmonary arterial smooth muscle cells via the up regulation of plasminogen activator inhibitor-2

    SciTech Connect

    Zhang, Shuai; Zou, Lihui; Yang, Ting; Yang, Yuanhua; Zhai, Zhenguo; Xiao, Fei; Wang, Chen

    2015-03-15

    Background: Different types of pulmonary hypertension (PH) share the same process of pulmonary vascular remodeling, the molecular mechanism of which is not entirely clarified by far. The abnormal biological behaviors of pulmonary arterial smooth muscle cells (PASMCs) play an important role in this process. Objectives: We investigated the regulation of plasminogen activator inhibitor-2 (PAI-2) by the sGC activator, and explored the effect of PAI-2 on PASMCs proliferation, apoptosis and migration. Methods: After the transfection with PAI-2 overexpression vector and specific siRNAs or treatment with BAY 41-2272 (an activator of sGC), the mRNA and protein levels of PAI-2 in cultured human PASMCs were detected, and the proliferation, apoptosis and migration of PASMCs were investigated. Results: BAY 41-2272 up regulated the endogenous PAI-2 in PASMCs, on the mRNA and protein level. In PAI-2 overexpression group, the proliferation and migration of PASMCs were inhibited significantly, and the apoptosis of PASMCs was increased. In contrast, PAI-2 knockdown with siRNA increased PASMCs proliferation and migration, inhibited the apoptosis. Conclusions: PAI-2 overexpression inhibits the proliferation and migration and promotes the apoptosis of human PASMCs. Therefore, sGC activator might alleviate or reverse vascular remodeling in PH through the up-regulation of PAI-2. - Highlights: • sGC activator BAY41-2272 up regulated PAI-2 in PASMCs, on the mRNA and protein level. • PAI-2 overexpression inhibits the proliferation and migration of human PASMCs. • PAI-2 overexpression promotes the apoptosis of human PASMCs. • sGC activator might alleviate the vascular remodeling in pulmonary hypertension.

  3. Evaluation of Alpha 1-Antitrypsin and the Levels of mRNA Expression of Matrix Metalloproteinase 7, Urokinase Type Plasminogen Activator Receptor and COX-2 for the Diagnosis of Colorectal Cancer

    PubMed Central

    Bujanda, Luis; Sarasqueta, Cristina; Cosme, Angel; Hijona, Elizabeth; Enríquez-Navascués, José M.; Placer, Carlos; Villarreal, Eloisa; Herreros-Villanueva, Marta; Giraldez, María D.; Gironella, Meritxell; Balaguer, Francesc; Castells, Antoni

    2013-01-01

    Background Colorectal cancer (CRC) is the second most common cause of death from cancer in both men and women in the majority of developed countries. Molecular tests of blood could potentially provide this ideal screening tool. Aim Our objective was to assess the usefulness of serum markers and mRNA expression levels in the diagnosis of CRC. Methods In a prospective study, we measured mRNA expression levels of 13 markers (carbonic anhydrase, guanylyl cyclase C, plasminogen activator inhibitor, matrix metalloproteinase 7 (MMP7), urokinase-type plasminogen activator receptor (uPAR), urokinase-type plasminogen activator, survivin, tetranectin, vascular endothelial growth factor (VEGF), cytokeratin 20, thymidylate synthase, cyclooxygenase 2 (COX-2), and CD44) and three proteins in serum (alpha 1 antitrypsin, carcinoembryonic antigen (CEA) and activated C3 in 42 patients with CRC and 33 with normal colonoscopy results. Results Alpha 1-antitrypsin was the serum marker that was most useful for CRC diagnosis (1.79±0.25 in the CRC group vs 1.27±0.25 in the control group, P<0.0005). The area under the ROC curve for alpha 1-antitrypsin was 0.88 (0.79–0.96). The mRNA expression levels of five markers were statistically different between CRC cases and controls: those for which the ROC area was over 75% were MMP7 (0.81) and tetranectin (0.80), COX-2 (0.78), uPAR (0.78) and carbonic anhydrase (0.77). The markers which identified early stage CRC (Stages I and II) were alpha 1-antitrypsin, uPAR, COX-2 and MMP7. Conclusions Serum alpha 1-antitrypsin and the levels of mRNA expression of MMP7, COX-2 and uPAR have good diagnostic accuracy for CRC, even in the early stages. PMID:23300952

  4. Tissue-type Plasminogen Activator (tPA) Modulates the Postsynaptic Response of Cerebral Cortical Neurons to the Presynaptic Release of Glutamate

    PubMed Central

    Jeanneret, Valerie; Wu, Fang; Merino, Paola; Torre, Enrique; Diaz, Ariel; Cheng, Lihong; Yepes, Manuel

    2016-01-01

    Tissue-type plasminogen activator (tPA) is a serine proteinase released by the presynaptic terminal of cerebral cortical neurons following membrane depolarization (Echeverry et al., 2010). Recent studies indicate that the release of tPA triggers the synaptic vesicle cycle and promotes the exocytosis (Wu et al., 2015) and endocytic retrieval (Yepes et al., 2016) of glutamate-containing synaptic vesicles. Here we used electron microscopy, proteomics, quantitative phosphoproteomics, biochemical analyses with extracts of the postsynaptic density (PSD), and an animal model of cerebral ischemia with mice overexpressing neuronal tPA to study whether the presynaptic release of tPA also has an effect on the postsynaptic terminal. We found that tPA has a bidirectional effect on the composition of the PSD of cerebral cortical neurons that is independent of the generation of plasmin and the presynaptic release of glutamate, but depends on the baseline level of neuronal activity and the extracellular concentrations of calcium (Ca2+). Accordingly, in neurons that are either inactive or incubated with low Ca2+ concentrations tPA induces phosphorylation and accumulation in the PSD of the Ca2+/calmodulin-dependent protein kinase IIα (pCaMKIIα), followed by pCaMKIIα-mediated phosphorylation and synaptic recruitment of GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. In contrast, in neurons with previously increased baseline levels of pCaMKIIα in the PSD due to neuronal depolarization in vivo or incubation with high concentrations of either Ca2+ or glutamate in vitro, tPA induces pCaMKIIα and pGluR1 dephosphorylation and their subsequent removal from the PSD. We found that these effects of tPA are mediated by synaptic N-methyl-D-aspartate (NMDA) receptors and cyclin-dependent kinase 5 (Cdk5)-induced phosphorylation of the protein phosphatase 1 (PP1) at T320. Our data indicate that by regulating the pCaMKIIα/PP1 balance in the PSD tPA acts

  5. Activity.

    ERIC Educational Resources Information Center

    Clearing: Nature and Learning in the Pacific Northwest, 1984

    1984-01-01

    Presents three activities: (1) investigating succession in a schoolground; (2) investigating oak galls; and (3) making sun prints (photographs made without camera or darkroom). Each activity includes a list of materials needed and procedures used. (JN)

  6. The effect of dietary supplementation with limonene or myo-inositol on the induction of neoplasia and matrix metalloproteinase and plasminogen activator activities in accessory sex organs of male Lobund-Wistar rats.

    PubMed

    Wilson, Michael J; Lindgren, Bruce R; Sinha, Akhouri A

    2008-10-01

    Prostate cancer, the most prevalent non-cutaneous cancer in men, is associated with increased age. This suggests that dietary chemopreventive measures could be effective in delaying the onset or decreasing the severity of the disease. We utilized the Lobund-Wistar rat nitrosomethylurea induced, testosterone promoted (NMU-T) model of male sex accessory gland cancer to test the potential chemopreventive effects of myo-inositol and limonene on tumor incidence and associated protease activities. Tumors were found to arise in the seminal vesicles and dorsal and anterior prostate lobes. There were also some tumors that appeared to arise in both the seminal vesicles and anterior prostate, and in some cases the tissue of origin was not clear. The distribution of tumors as to site of origin in limonene or myo-inositol treated animals did not vary from that of the starch fed control animals, and the number of animals presenting with metastases did not vary significantly between treatment groups. There was a statistically significant delay in onset of tumors in myo-inositol, but not limonene fed rats, at 10 months post-induction of carcinogenesis; however, at 12 and 15 months this was not significant. The ventral prostate and seminal vesicles expressed pro-MMP-2 and plasminogen activator (PA) activities. Based on sensitivity to amiloride, the PA activities were predominately urokinase (uPA) in the ventral prostate and a mixture of tissue-type activator (tPA) and uPA in the seminal vesicles of non-treated rats. Sex accessory gland tumors, and metastases, expressed increased levels PA and pro- and active forms of MMP-2 and -9. The PA activities of the tumors were a mixture of uPA and tPA. There was no difference in the levels of these protease activities based on the tissue of tumor origin, nor in tumor vs metastasis. These studies indicate that MMP and PA activities play a role in sex accessory gland tumor biology and that dietary supplementation with myo-inositol can delay

  7. A combination of desmopressin and docetaxel inhibit cell proliferation and invasion mediated by urokinase-type plasminogen activator (uPA) in human prostate cancer cells

    SciTech Connect

    Sasaki, Hiroshi; Klotz, Laurence H.; Sugar, Linda M.; Kiss, Alexander; Venkateswaran, Vasundara

    2015-08-28

    Background: This study was designed to assess the effectiveness of a combination treatment using both desmopressin and docetaxel in prostate cancer treatment. Desmopressin is a well-known synthetic analogue of the antidiuretic hormone vasopressin. It has recently been demonstrated to inhibit tumor progression and metastasis in in vivo models. Docetaxel is widely used for the treatment of castration resistant prostate cancer (CRPC) patients. However, durable responses have been uncommon to date. In this study, we investigated the anti-tumor effect of desmopressin in combination with docetaxel in vitro and in vivo. Methods: Two prostate cancer cells (PC3, LNCaP) were treated with different concentrations of desmopressin alone, docetaxel alone, and a combination of desmopressin and docetaxel. Cell proliferation was determined by MTS assay. The anti-invasive and anti-migration potential of desmopressin and in combination with docetaxel were examined by wound healing assay, migration chamber assay, and matrigel invasion assay. Results: The combination of desmopressin and docetaxel resulted in a significant inhibition of PC3 and LNCaP cell proliferation (p < 0.01). Additionally, cell migration and invasion were also inhibited by the combination when compared to that of either treatment alone in PC3 cells (p < 0.01). The anti-tumor effect of this combination treatment was associated with down-regulation of both urokinase-type plasminogen activator (uPA) and matrix metalloproteinase (MMP-2 and MMP-9) in PC3 cells. Conclusions: We are the first to elucidate the anti-tumor and anti-metastatic potential of desmopressin in combination with docetaxel in a prostate cancer model via the uPA-MMP pathway. Our finding could potentially contribute to the therapeutic profile of desmopressin and enhance the efficacy of docetaxel based treatment for CRPC. - Highlights: • Desmopressin inhibits cell proliferation in prostate cancer cells. • The expression of cyclin A and CDK2

  8. Purification of storage granule protein-23. A novel protein identified by phage display technology and interaction with type I plasminogen activator inhibitor.

    PubMed

    Lang, I M; Chuang, T L; Barbas, C F; Schleef, R R

    1996-11-22

    Type 1 plasminogen activator inhibitor (PAI-1) is a key regulator of the fibrinolytic cascade that is stored in a rapidly releasable form within platelet alpha-granules. To identify proteins that may participate in the targeting or storage of this potent inhibitor, this report investigates the applicability of utilizing filamentous bacteriophages to display proteins expressed by cells containing a regulated secretory pathway and their enrichment based upon an interaction with PAI-1. For this purpose, RNA was extracted from AtT-20 cells (i.e. a classical model cell system for intracellular protein sorting), reverse transcribed, amplified using polymerase chain reaction primers containing internal restriction sites, and cloned into the phagemid pCOMB3H for expression as fusion constructs with the bacteriophage gene III protein. Escherichia coli was transformed with the phagemids and infected with VCSM13 helper phage, and the resulting AtT-20 cDNA-bacteriophage library was enriched by panning against solid- and solution-phase PAI-1. The enriched cDNA library was subcloned into a prokaryotic expression vector system that replaces the gene III protein with a decapeptide tag for immunologic quantitation. One novel cDNA clone (i.e. A-61), which preferentially recognized solution-phase PAI-1 and reacted positively with antibodies derived from a rabbit immunized with alpha-granules, was subcloned into the prokaryotic expression vector pTrcHis to create a construct containing an N-terminal six-histidine purification tag. This construct was expressed in E. coli, purified by nickel-chelate chromatography followed by preparative SDS-polyacrylamide gel electrophoresis, and utilized for the generation of polyclonal antibodies. Immunoblotting analysis employing antibodies against the purified A-61 construct revealed a 23-kDa protein present in the regulated secretory pathway of AtT-20 cells. The 23-kDa molecule was purified from media conditioned by AtT-20 cells by ion exchange

  9. Plasminogen activator inhibitor-1 4G/5G and the MTHFR 677C/T polymorphisms and susceptibility to polycystic ovary syndrome: a meta-analysis.

    PubMed

    Lee, Young Ho; Song, Gwan Gyu

    2014-04-01

    The aim of this study was to explore whether the plasminogen activator inhibitor-1 (PAI-1) 4G/5G and the methylenetetrahydrofolate reductase (MTHFR) 677C/T polymorphisms are associated with susceptibility to polycystic ovary syndrome (PCOS). Meta-analyses were conducted to determine the association between the PAI-1 4G/5G and MTHFR 677C/T polymorphisms and PCOS using: (1) allele contrast (2) homozygote contrast, (3) recessive, and (4) dominant models. For meta-analysis, nine studies of the PAI-1 4G/5G polymorphism with 2384 subjects (PCOS, 1615; controls, 769) and eight studies of the MTHFR 677C/T polymorphism with 1270 study subjects were included. Meta-analysis of all study subjects showed no association between PCOS and the PAI-1 4G allele (OR=0.949, 95% CI=0.671-1.343, p=0.767). Stratification by ethnicity, however, indicated a significant association between the PAI-1 4G allele and PCOS in Turkish and Asian populations (OR=0.776, 95% CI=0.602-0.999, p=0.049; OR=1.749, 95% CI=1.297-2.359, p=2.5×10(-5) respectively). In addition, meta-analysis indicated an association between PCOS and the PAI-1 4G4G+4G5G genotype in Europeans (OR=1.406, 95% CI=1.025-1.928, p=0.035). However, meta-analysis of all study subjects showed no association between PCOS and the MTHFR 677T allele (OR=0.998, 95% CI=0.762-1.307, p=0.989), including Europeans (OR=0.806, 95% CI=0.610-1.063, p=0.126). Meta-analysis showed no association between PCOS and the MTHFR 677C/T polymorphism using homozygote contrast, and recessive and dominant models. In conclusion, meta-analysis suggests the PAI-1 4G/5G polymorphism is associated with susceptibility to PCOS in European, Turkish, and Asian populations, but the MTHFR 677C/T polymorphism is not associated with susceptibility to PCOS in Europeans.

  10. Opposite and independent actions of cyclic AMP and transforming growth factor beta in the regulation of type 1 plasminogen activator inhibitor expression.

    PubMed Central

    Thalacker, F W; Nilsen-Hamilton, M

    1992-01-01

    We have investigated the mechanisms by which type 1 plasminogen activator inhibitor (PAI-1) is regulated by transforming growth factor beta (TGF-beta) and by epidermal growth factor (EGF) in CCL64 mink lung epithelial cells, BSC-1 monkey kidney epithelial cells, mouse embryo fibroblast (AKR-2B 84A) cells and normal rat kidney fibroblasts (NRK). TGF-beta increases PAI-1 expression in all four cell lines, and EGF acts synergistically with TGF-beta to increase PAI-1 expression in CCL64 cells but not in the other three cell lines. Here we show that PAI-1 expression can be regulated independently through two different signal transduction pathways. One pathway involves protein kinase C and is stimulated by the tumour promoter phorbol myristate acetate (PMA). Whereas preincubation with PMA completely eliminated PMA-induced PAI-1 synthesis and secretion in both CCL64 and BSC-1 cells, this treatment had no effect on TGF-beta- and EGF-induced PAI-1 levels. Therefore we conclude that protein kinase C does not mediate the effects of either EGF or TGF-beta on PAI-1 expression. The expression of PAI-1 was decreased by agents increasing intracellular cyclic AMP: (cAMP) cholera toxin, forskolin and dibutyryl cAMP lowered both the basal level and the TGF-beta- and PMA-induced levels of PAI-1 expression. These effects of cAMP-elevating agents and of TGF-beta on PAI-1 protein synthesis were also reflected in changes in TGF-beta-induced PAI-1 gene transcription, as measured by nuclear run-on. These results show that PAI-1 gene expression is sensitive to high levels of intracellular cAMP and that this effect occurs at the transcriptional level. Although increased intracellular cAMP concentrations decrease the absolute level of PAI-1 expression, the ability of TGF-beta and EGF to induce PAI-1 gene expression is unchanged. These results are discussed in relation to the observation that sensitivity to cAMP is a common feature of TGF-beta-regulated genes. Images Fig. 1. Fig. 2. Fig. 3. Fig

  11. A higher body temperature is associated with haemorrhagic transformation in patients with acute stroke untreated with recombinant tissue-type plasminogen activator (rtPA).

    PubMed

    Leira, Rogelio; Sobrino, Tomás; Blanco, Miguel; Campos, Francisco; Rodríguez-Yáñez, Manuel; Castellanos, Mar; Moldes, Octavio; Millán, Mónica; Dávalos, Antoni; Castillo, José

    2012-02-01

    Higher body temperature is a prognostic factor of poor outcome in acute stroke. Our aim was to study the relationship between body temperature, HT (haemorrhagic transformation) and biomarkers of BBB (blood-brain barrier) damage in patients with acute ischaemic stroke untreated with rtPA (recombinant tissue-type plasminogen activator). We studied 229 patients with ischaemic stroke <12 h from symptom onset. Body temperature was determined at admission and every 6 h during the first 3 days. HT was evaluated according to ECASS II (second European Co-operative Acute Stroke Study) criteria in a multimodal MRI (magnetic resonance imaging) at 72 h. We found that 55 patients (34.1%) showed HT. HT was associated with cardioembolic stroke (64.2% against 23.0%; P<0.0001), higher body temperature during the first 24 h (36.9°C compared with 36.5°C; P<0.0001), more severe stroke [NIHSS (National Institutes of Health Stroke Scale) score, 14 (9-20) against 10 (7-15); P=0.002], and greater DWI (diffusion-weighted imaging) lesion volume at admission (23.2 cc compared with 13.2 cc; P<0.0001). Plasma MMP-9 (matrix metalloproteinase 9) (187.3 ng/ml compared with 44.2 ng/ml; P<0.0001) and cFn (cellular fibronectin) levels (16.3 μg/ml compared with 7.1 μg/ml; P=0.001) were higher in patients with HT. Body temperature within the first 24 h was independently associated with HT {OR (odds ratio), 7.3 [95% CI (confidence interval), 2.4-22.6]; P<0.0001} after adjustment for cardioembolic stroke subtype, baseline NIHSS score and DWI lesion volume. This effect remained unchanged after controlling for MMP-9 and cFn. In conclusion, high body temperature within the first 24 h after ischaemic stroke is a risk factor for HT in patients untreated with rtPA. This effect is independent of some biological signatures of BBB damage.

  12. Role of Genetic Polymorphism of Angiotensin-Converting Enzyme, Plasminogen Activator Inhibitor-1 and Endothelial Nitric Oxide Synthase in the Prognosis of Coronary Artery Disease

    PubMed Central

    Zhang, Ai Yuan; Ji, Xiang Wu; Zhang, Ai Juan; Guan, Li Xue; Huang, Jing; Wang, Jing Xian

    2010-01-01

    Background This study was to investigate the effects of multiple genetic polymorphisms and conventional risk factors in the prognosis of coronary artery disease (CAD). Methods One hundred and fifty five patients with CAD were prospectively recruited, they were subgrouped as single vessel disease (SVD) and multiple vessel disease (MVD). All patients were detected I/D polymorphism of angiotensin-converting enzyme (ACE) gene, 4G/5G polymorphism of plasminogen activator inhibitor-1 (PAI-1) gene, and G894→T mutation of endothelial nitric oxide synthase (eNOS) gene. The patients were followed up for 10-65 months, mean 35 months. End points were major adverse cardiovascular events (MACE), including angina, myocardial infarction, and cardiac sudden death. Results During the follow-up period, MACE developed in 81 patients, 73 patients with angina, seven with myocardial infarction, and one with cardiac sudden death. CAD patients with MVD were more probable of developing MACE during follow-up. Distribution of PAI-1 gene polymorphism was significantly different between SVD and MVD patients, p < 0.001. The frequency of DD genotype of ACE and 4G/4G genotype of PAI-1 in patients with MACE were significantly higher than those in patients without MACE, p < 0.001 and p = 0.002, respectively. Incidence of diabetes mellitus was significantly higher in patients with MACE than in patients without MACE, P = 0.03. Cox regression analysis showed that diabetes mellitus (HR 2.36, 95% CI 1.33-4.46, p = 0.003), 4G/4G polymorphism of PAI-1 gene (HR 3.45, 95% CI 1.71-6.56, p = 0.009), and D/D polymorphism of ACE gene (HR 2.99, 95% CI 1.84-5.76, p = 0.005), were independent predictors of the MACE. Conclusions Our results showed that the conventional risk factors and genetic polymorphisms have significant influence on prognosis of CAD patients. CAD patients with diabetes mellitus, DD genotype of ACE, and 4G/4G genotype of PAI-1 suggested poor prognosis.

  13. Production of a urokinase plasminogen activator-IgG fusion protein (uPA-IgG) in the baculovirus expression system.

    PubMed

    Kost, T A; Ignar, D M; Clay, W C; Andrews, J; Leray, J D; Overton, L; Hoffman, C R; Kilpatrick, K E; Ellis, B; Emerson, D L

    1997-04-29

    Numerous studies have demonstrated the importance of urokinase plasminogen activator (uPA) and its receptor, uPAR, in the processes of tumor progression and metastasis. Thus, the uPA/uPAR interaction may represent an important target for inhibiting metastatic disease. The baculovirus expression system was used to produce high levels of a secreted uPA-Immunoglobulin G fusion protein (uPA-IgG) which could then be used for displacing uPA from the surface of tumor cells. The recombinant uPA-IgG fusion protein was placed under the control of either the viral polyhedrin promoter or a copy of the viral basic protein promoter. Recombinant viruses were then used to infect Sf9 and BTI-Tn-5B1-4 cells. Infection of both cell types resulted in the production of secreted uPA-IgG. The molecular mass of the secreted protein as determined by SDS-PAGE was approximately 40 kDa. The highest level of secreted uPA-IgG, 444 microg/ml, was found in the culture medium of BTI-Tn-5B1-4 cells 72 h post-infection with the basic protein promoter-uPA-IgG virus. In the case of Sf9 cells, the highest level of secreted protein was 195 microg/ml. The amount of cell-associated uPA-IgG in infected BTI-Tn-5B1-4 cells was significantly less than that of infected Sf9 cells, reflecting the superior secretory capability of the BTI-Tn-5B1-4 cells. The uPA-IgG was readily purified using a combination of zinc chelate and sephacryl S-100 column chromatography. Routinely, greater than 100 mg of greater than 95% pure protein could be obtained per liter of culture medium collected at 72 h post-infection of BTI-Tn-5B1-4 cells with the basic protein promoter virus. BIAcore analysis and competition binding assays using LOX human malignant melanoma cells expressing uPAR indicated that the purified recombinant protein possessed similar ligand binding characteristics to that of human uPA.

  14. Early treatment of acute submacular haemorrhage secondary to wet AMD using intravitreal tissue plasminogen activator, C3F8, and an anti-VEGF agent.

    PubMed

    de Silva, S R; Bindra, M S

    2016-07-01

    PurposeAcute submacular haemorrhage secondary to wet age-related macular degeneration (AMD) has a poor prognosis for which there is currently no 'gold standard' treatment. We evaluated the efficacy of early treatment using intravitreal triple therapy of tissue plasminogen activator (tPA), expansile gas, and an anti-VEGF agent.MethodsThis retrospective case series included eight patients presenting with acute submacular haemorrhage involving the fovea. All patients received treatment with 50 μg (0.05 ml) tPA, 0.3 ml 100% perfluoropropane (C3F8), and an anti-VEGF agent (0.05 mg Ranibizumab or 1.25 mg Bevacizumab in 0.05 ml) administered via intravitreal injection. An anterior chamber paracentesis post injection or vitreous tap was performed before injection to prevent retinal vascular occlusion secondary to raised intra-ocular pressure. Outcomes assessed were visual acuity, change in macular morphology, and complications.ResultsPatients presented promptly with delay between symptom onset and clinic review being 1.9±0.6 days (mean±SD). Treatment was delivered quickly with interval from presentation to treatment being 1.1±1.2 days. Symptom onset to treatment was 3.0±1.0 days. Subfoveal haemorrhage was effectively displaced in all patients. LogMAR visual acuity improved from 1.67±0.47 at presentation to 0.63±0.33 at final follow-up (P<0.0001), a mean of 7.9±4.8 months after treatment. Central retinal thickness improved from 658.1±174.2 μm at presentation to 316.6±142.4 μm at final follow-up (P=0.0028).ConclusionsEarly treatment of submacular haemorrhage using intravitreal tPA, C3F8, and anti-VEGF was effective in significantly improving visual acuity in this series of patients who presented soon after symptom onset. Treatment was well tolerated in this group of elderly and potentially frail patients.

  15. Data in support of a central role of plasminogen activator inhibitor-2 polymorphism in recurrent cardiovascular disease risk in the setting of high HDL cholesterol and C-reactive protein using Bayesian network modeling.

    PubMed

    Corsetti, James P; Salzman, Peter; Ryan, Dan; Moss, Arthur J; Zareba, Wojciech; Sparks, Charles E

    2016-09-01

    Data is presented that was utilized as the basis for Bayesian network modeling of influence pathways focusing on the central role of a polymorphism of plasminogen activator inhibitor-2 (PAI-2) on recurrent cardiovascular disease risk in patients with high levels of HDL cholesterol and C-reactive protein (CRP) as a marker of inflammation, "Influences on Plasminogen Activator Inhibitor-2 Polymorphism-Associated Recurrent Cardiovascular Disease Risk in Patients with High HDL Cholesterol and Inflammation" (Corsetti et al., 2016; [1]). The data consist of occurrence of recurrent coronary events in 166 post myocardial infarction patients along with 1. clinical data on gender, race, age, and body mass index; 2. blood level data on 17 biomarkers; and 3. genotype data on 53 presumptive CVD-related single nucleotide polymorphisms. Additionally, a flow diagram of the Bayesian modeling procedure is presented along with Bayesian network subgraphs (root nodes to outcome events) utilized as the data from which PAI-2 associated influence pathways were derived (Corsetti et al., 2016; [1]).

  16. Evaluation of serum fibrinogen, plasminogen, α2-anti-plasmin, and plasminogen activator inhibitor levels (PAI) and their correlation with presence of retinopathy in patients with type 1 DM.

    PubMed

    Polat, Sefika Burcak; Ugurlu, Nagihan; Yulek, Fatma; Simavli, Huseyin; Ersoy, Reyhan; Cakir, Bekir; Erel, Ozcan

    2014-01-01

    BACKGROUND. Diabetic retinopathy (DR) is the leading cause of blindness in the world. Retinopathy can still progress despite optimal metabolic control. The aim of the study was to determine whether different degrees of DR (proliferative or nonproliferative) were associated with abnormally modulated hemostatic parameters in patients with T1DM. METHOD. 52 T1DM patients and 40 healthy controls were enrolled in the study. Patients were subdivided into three categories. Group I was defined as those without retinopathy, group II with NPRP, and group III with PRP. We compared these subgroups with each other and the control group (Group IV) according to the serum fibrinogen, plasminogen, alpha2-anti-plasmin ( α2-anti-plasmin), and PAI. RESULTS. We detected that PAI-1, serum fibrinogen, and plasminogen levels were similar between the diabetic and control groups (P = 0.209, P = 0.224, and P = 0.244, resp.), whereas α2-anti-plasmin was higher in Groups I, II, and III compared to the control group (P < 0.01, P < 0.05, and P < 0.001, resp.). There was a positive correlation between serum α2-anti-plasmin and HbA1c levels (r = 0,268, P = 0.031). CONCLUSION. To our knowledge there is scarce data in the literature about α2-anti-plasmin levels in type 1 diabetes. A positive correlation between α2-anti-plasmin with HbA1c suggests that fibrinolytic markers may improve with disease regulation and better glycemic control.

  17. Activities.

    ERIC Educational Resources Information Center

    Bippert, Judy

    1993-01-01

    Presents activities designed to give students an opportunity to solve concrete problems involving spatial relationships and logical thinking utilizing hands-on manipulatives. Provides teacher instructions and four reproducible worksheets. (MDH)

  18. Activities.

    ERIC Educational Resources Information Center

    Kincaid, Charlene; And Others

    1993-01-01

    Presents an activity in which students collect and organize data from a real-world simulation of the scientific concept of half life. Students collect data using a marble sifter, analyze the data using a graphing calculator, and determine an appropriate mathematical model. Includes reproducible worksheets. (MDH)

  19. Transforming growth factor beta 1-responsive element: closely associated binding sites for USF and CCAAT-binding transcription factor-nuclear factor I in the type 1 plasminogen activator inhibitor gene.

    PubMed Central

    Riccio, A; Pedone, P V; Lund, L R; Olesen, T; Olsen, H S; Andreasen, P A

    1992-01-01

    Transforming growth factor beta (TGF-beta) is the name of a group of closely related polypeptides characterized by a multiplicity of effects, including regulation of extracellular proteolysis and turnover of the extracellular matrix. Its cellular mechanism of action is largely unknown. TGF-beta 1 is a strong and fast inducer of type 1 plasminogen activator inhibitor gene transcription. We have identified a TGF-beta 1-responsive element in the 5'-flanking region of the human type 1 plasminogen activator inhibitor gene and shown that it is functional both in its natural context and when fused to a heterologous nonresponsive promoter. Footprinting and gel retardation experiments showed that two different nuclear factors, present in extracts from both TGF-beta 1-treated and nontreated cells, bind to adjacent sequences contained in the responsive unit. A palindromic sequence binds a trans-acting factor(s) of the CCAAT-binding transcription factor-nuclear factor I family. A partially overlapping dyad symmetry interacts with a second protein that much evidence indicates to be USF. USF is a transactivator belonging to the basic helix-loop-helix family of transcription factors. Mutations which abolish the binding of either CCAAT-binding transcription factor-nuclear factor I or USF result in reduction of transcriptional activation upon exposure to TGF-beta 1, thus showing that both elements of the unit are necessary for the TGF-beta 1 response. We discuss the possible relationship of these findings to the complexity of the TGF-beta action. Images PMID:1549130

  20. Tumour-associated urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 in normal and neoplastic tissues of patients with squamous cell cancer of the oral cavity - clinical relevance and prognostic value.

    PubMed

    Hundsdorfer, Brigitte; Zeilhofer, Hans-Florian; Bock, Klaus Peter; Dettmar, Peer; Schmitt, Manfred; Kolk, Andreas; Pautke, Christoph; Horch, Hans-Henning

    2005-06-01

    The central role of the serine protease urokinase-type plasminogen activator (uPA) and its inhibitor, the plasminogen activator-inhibitor-1 (PAI-1), in tumour invasion and metastasis becomes more and more evident. In several studies, uPA and PAI-1 proved to be of prognostic relevance as shown for different types of cancer (e.g. breast, stomach, lung). Elevated antigen levels of uPA and/or PAI-1 predict poor outcome (relapse-free survival) for patients afflicted with cancer. For oral squamous cell carcinomas, however, the prognostic relevance of the tumour-associated proteolytic factors uPA and PAI-1 has still to be evaluated. In the present study, using tissue extracts of 79 oral cancer cases, 58 specimens of normal oral cavity mucosa and of 16 tumour positive lymph nodes taken from the same patients, uPA and PAI-1 antigen were determined by highly sensitive enzyme-linked immunosorbent assays (ELISA). A correlation was found between uPA and PAI-1 in tumour tissue, when compared with the normal mucosa of the same oral cavity. Median levels showed significant elevations in cancer tissue and in tumour positive lymph nodes versus normal oral mucosa. In patients with high levels of uPA or PAI-1, there were significantly more tumour relapses. There was no significant correlation between pathological TNM categories, grading, residual tumour category, tumour site and patient age. In summary, tumour uPA/PAI-1 content (as determined by ELISA) appears to be a strong independent prognostic factor for relapse-free survival in squamous cell cancer of the oral cavity. These observations might help to select patients with poor prognosis for additional adjuvant therapy in conjunction with complete surgical resection.

  1. Endogenous prostaglandin endoperoxides and prostacyclin modulate the thrombolytic activity of tissue plasminogen activator. Effects of simultaneous inhibition of thromboxane A2 synthase and blockade of thromboxane A2/prostaglandin H2 receptors in a canine model of coronary thrombosis.

    PubMed Central

    Golino, P; Rosolowsky, M; Yao, S K; McNatt, J; De Clerck, F; Buja, L M; Willerson, J T

    1990-01-01

    We tested the hypothesis that simultaneous inhibition of TxA2 synthase and blockade of TxA2/PHG2 receptors is more effective in enhancing thrombolysis and preventing reocclusion after discontinuat