The activation of plasminogen by Hageman factor (Factor XII) and Hageman factor fragments.

PubMed Central

Goldsmith, G H; Saito, H; Ratnoff, O S

1978-01-01

Activation of plasminogen through surface-mediated reactions is well recognized. In the presence of kaolin, purified Hageman factor (Factor XII) changed plasminogen to plasmin, as assayed upon a synthetic amide substrate and by fibrinolysis. Kinetic studies suggested an enzymatic action of Hageman factor upon its substrate, plasminogen. Hageman factor fragments, at a protein concentration equivalent to whole Hageman factor, activated plasminogen to a lesser extent. These protein preparations were not contaminated with other agents implicated in surface-mediated fibrinolysis. Diisopropyl fluorophosphate treatment of plasminogen did not inhibit its activation by Hageman factor. These studies indicate that Hageman factor has a hitherto unsuspected function, the direct activation of plasminogen. PMID:659637

Myelin basic protein stimulates plasminogen activation via tissue plasminogen activator following binding to independent l-lysine-containing domains.

PubMed

Gonzalez-Gronow, Mario; Fiedler, Jenny L; Farias Gomez, Cristian; Wang, Fang; Ray, Rupa; Ferrell, Paul D; Pizzo, Salvatore V

2017-08-26

Myelin basic protein (MBP) is a key component of myelin, the specialized lipid membrane that encases the axons of all neurons. Both plasminogen (Pg) and tissue-type plasminogen activator (t-PA) bind to MBP with high affinity. We investigated the kinetics and mechanisms involved in this process using immobilized MBP and found that Pg activation by t-PA is significantly stimulated by MBP. This mechanism involves the binding of t-PA via a lysine-dependent mechanism to the Lys 91 residue of the MBP NH 2 -terminal region Asp 82 -Pro 99 , and the binding of Pg via a lysine-dependent mechanism to the Lys 122 residue of the MBP COOH-terminal region Leu 109 -Gly 126 . In this context, MBP mimics fibrin and because MBP is a plasmin substrate, our results suggest direct participation of the Pg activation system on MBP physiology. Copyright © 2017 Elsevier Inc. All rights reserved.

Gene expression of fibrinolytic factors urokinase plasminogen activator and plasminogen activator inhibitor-1 in rabbit temporo-mandibular joint cartilage with disc displacement.

PubMed

Zhan, Jing; Gu, Zhi-yuan; Wu, Li-qun; Zhang, Yin-kai; Hu, Ji-an

2005-06-20

The urokinase plasminogen activator system is believed to play an important role in degradation of the extracellular matrix associated with cartilage and bone destruction; however its precise roles in temporomandibular disorders have not yet been clarified. The aims of this study were to investigate the gene expression of fibrinolytic factors urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) in the articular cartilage of rabbit temporomandibular joint (TMJ) with disc displacement (DD) and to probe the relationship between fibrinolytic activity and cartilage remodeling. Disc displacement of right joints was performed in 36 of 78 rabbits under investigation. The animals were sacrificed at 4 days and 1, 2, 4, 8 and 12 weeks after surgery, respectively. The right joints of these animals were harvested and processed for the examination of mRNA expression of uPA and PAI-1 in articular cartilage using in situ hybridization techniques. The expression of uPA and PAI-1 was co-expressed weakly in the chondrocytes from transitive zone to hypertrophic zone and mineralized zone, while no hybridizing signals were shown in proliferative zone and superficial zone in control rabbits. The most striking was the up-regulation of uPA and PAI-1 mRNA in 4-day rabbits postoperatively at the onset of cartilage degeneration. The strongest hybridizing signals for uPA and PAI-1 were seen in 2-week rabbits postoperatively. After 2 weeks, the expression of uPA and PAI-1 began to decrease and reached nearly normal level at 12 weeks. The expression of the uPA/PAI-1 system coincides with the pathological changes in condylar cartilage after DD. The uPA/PAI-1 system may be one of the essential mediators in articular cartilage remodeling.

Decoy Plasminogen Receptor Containing a Selective Kunitz-Inhibitory Domain

PubMed Central

2015-01-01

Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 in which P2′ residue Leu17 (bovine pancreatic trypsin inhibitor numbering) is mutated to Arg selectively inhibits the active site of plasmin with ∼5-fold improved affinity. Thrombin cleavage (24 h extended incubation at a 1:50 enzyme-to-substrate ratio) of the KD1 mutant (Leu17Arg) yielded a smaller molecule containing the intact Kunitz domain with no detectable change in the active-site inhibitory function. The N-terminal sequencing and MALDI-TOF/ESI data revealed that the starting molecule has a C-terminal valine (KD1L17R-VT), whereas the smaller molecule has a C-terminal lysine (KD1L17R-KT). Because KD1L17R-KT has C-terminal lysine, we examined whether it could serve as a decoy receptor for plasminogen/plasmin. Such a molecule might inhibit plasminogen activation as well as the active site of generated plasmin. In surface plasmon resonance experiments, tissue plasminogen activator (tPA) and Glu-plasminogen bound to KD1L17R-KT (Kd ∼ 0.2 to 0.3 μM) but not to KD1L17R-VT. Furthermore, KD1L17R-KT inhibited tPA-induced plasma clot fibrinolysis more efficiently than KD1L17R-VT. Additionally, compared to ε-aminocaproic acid KD1L17R-KT was more effective in reducing blood loss in a mouse liver-laceration injury model, where the fibrinolytic system is activated. In further experiments, the micro(μ)-plasmin–KD1L17R-KT complex inhibited urokinase-induced plasminogen activation on phorbol-12-myristate-13-acetate-stimulated U937 monocyte-like cells, whereas the μ-plasmin–KD1L17R-VT complex failed to inhibit this process. In conclusion, KD1L17R-KT inhibits the active site of plasmin as well as acts as a decoy receptor for the kringle domain(s) of plasminogen/plasmin; hence, it limits both plasmin generation and activity. With its dual function, KD1L17R-KT could serve as a preferred agent for controlling plasminogen activation in pathological processes. PMID:24383758

Decoy plasminogen receptor containing a selective Kunitz-inhibitory domain.

PubMed

Kumar, Yogesh; Vadivel, Kanagasabai; Schmidt, Amy E; Ogueli, Godwin I; Ponnuraj, Sathya M; Rannulu, Nalaka; Loo, Joseph A; Bajaj, Madhu S; Bajaj, S Paul

2014-01-28

Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 in which P2' residue Leu17 (bovine pancreatic trypsin inhibitor numbering) is mutated to Arg selectively inhibits the active site of plasmin with ∼5-fold improved affinity. Thrombin cleavage (24 h extended incubation at a 1:50 enzyme-to-substrate ratio) of the KD1 mutant (Leu17Arg) yielded a smaller molecule containing the intact Kunitz domain with no detectable change in the active-site inhibitory function. The N-terminal sequencing and MALDI-TOF/ESI data revealed that the starting molecule has a C-terminal valine (KD1L17R-VT), whereas the smaller molecule has a C-terminal lysine (KD1L17R-KT). Because KD1L17R-KT has C-terminal lysine, we examined whether it could serve as a decoy receptor for plasminogen/plasmin. Such a molecule might inhibit plasminogen activation as well as the active site of generated plasmin. In surface plasmon resonance experiments, tissue plasminogen activator (tPA) and Glu-plasminogen bound to KD1L17R-KT (Kd ∼ 0.2 to 0.3 μM) but not to KD1L17R-VT. Furthermore, KD1L17R-KT inhibited tPA-induced plasma clot fibrinolysis more efficiently than KD1L17R-VT. Additionally, compared to ε-aminocaproic acid KD1L17R-KT was more effective in reducing blood loss in a mouse liver-laceration injury model, where the fibrinolytic system is activated. In further experiments, the micro(μ)-plasmin-KD1L17R-KT complex inhibited urokinase-induced plasminogen activation on phorbol-12-myristate-13-acetate-stimulated U937 monocyte-like cells, whereas the μ-plasmin-KD1L17R-VT complex failed to inhibit this process. In conclusion, KD1L17R-KT inhibits the active site of plasmin as well as acts as a decoy receptor for the kringle domain(s) of plasminogen/plasmin; hence, it limits both plasmin generation and activity. With its dual function, KD1L17R-KT could serve as a preferred agent for controlling plasminogen activation in pathological processes.

Functional Stability of Plasminogen Activator Inhibitor-1

PubMed Central

Kuru, Pinar; Toksoy Oner, Ebru; Agirbasli, Mehmet

2014-01-01

Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of plasminogen activators, such as tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), and a major regulator of the fibrinolytic system. PAI-1 plays a pivotal role in acute thrombotic events such as deep vein thrombosis (DVT) and myocardial infarction (MI). The biological effects of PAI-1 extend far beyond thrombosis including its critical role in fibrotic disorders, atherosclerosis, renal and pulmonary fibrosis, type-2 diabetes, and cancer. The conversion of PAI-1 from the active to the latent conformation appears to be unique among serpins in that it occurs spontaneously at a relatively rapid rate. Latency transition is believed to represent a regulatory mechanism, reducing the risk of thrombosis from a prolonged antifibrinolytic action of PAI-1. Thus, relying solely on plasma concentrations of PAI-1 without assessing its function may be misleading in interpreting the role of PAI-1 in many complex diseases. Environmental conditions, interaction with other proteins, mutations, and glycosylation are the main factors that have a significant impact on the stability of the PAI-1 structure. This review provides an overview on the current knowledge on PAI-1 especially importance of PAI-1 level and stability and highlights the potential use of PAI-1 inhibitors for treating cardiovascular disease. PMID:25386620

Clot accumulation characteristics of plasminogen-bearing liposomes in a flow-system. Groningen Utrecht Institute for Drug Exploration.

PubMed

Heeremans, J L; Prevost, R; Feitsma, H; Kluft, C; Crommelin, D J

1998-01-01

In this study, the clot accumulation properties of liposome-coupled plasminogen were compared to those of free (non-liposomal) plasminogen in an in vitro, closed-loop, flow-system. After introduction of a clot into the closed system, double-radiolabelled plasminogen-liposomes were administered and the accumulation of radiolabel on the entire clot was measured. Liposomal plasminogen showed improved accumulation over free plasminogen, on both a fibrin clot and a whole blood clot. Moreover, once liposomal plasminogen was fibrin associated, it could not be washed away with buffer, in contrast to free plasminogen. Liposomal plasminogen was able to compete successfully with an excess of free plasminogen. The plateau levels for the accumulated amount of plasminogen depended on the incubated amount of plasminogen and were influenced by partial degradation of the clot. Furthermore, it was shown that a threshold liposomal plasminogen surface-density was needed for optimum clot accumulation.

Potent antitumor activity of a urokinase-activated engineered anthrax toxin

NASA Astrophysics Data System (ADS)

Liu, Shihui; Aaronson, Hannah; Mitola, David J.; Leppla, Stephen H.; Bugge, Thomas H.

2003-01-01

The acquisition of cell-surface urokinase plasminogen activator activity is a hallmark of malignancy. We generated an engineered anthrax toxin that is activated by cell-surface urokinase in vivo and displays limited toxicity to normal tissue but broad and potent tumoricidal activity. Native anthrax toxin protective antigen, when administered with a chimeric anthrax toxin lethal factor, Pseudomonas exotoxin fusion protein, was extremely toxic to mice, causing rapid and fatal organ damage. Replacing the furin activation sequence in anthrax toxin protective antigen with an artificial peptide sequence efficiently activated by urokinase greatly attenuated toxicity to mice. In addition, the mutation conferred cell-surface urokinase-dependent toxin activation in vivo, as determined by using a panel of plasminogen, plasminogen activator, plasminogen activator receptor, and plasminogen activator inhibitor-deficient mice. Surprisingly, toxin activation critically depended on both urokinase plasminogen activator receptor and plasminogen in vivo, showing that both proteins are essential cofactors for the generation of cell-surface urokinase. The engineered toxin displayed potent tumor cell cytotoxicity to a spectrum of transplanted tumors of diverse origin and could eradicate established solid tumors. This tumoricidal activity depended strictly on tumor cell-surface plasminogen activation. The data show that a simple change of protease activation specificity converts anthrax toxin from a highly lethal to a potent tumoricidal agent.

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.

Urokinase links plasminogen activation and cell adhesion by cleavage of the RGD motif in vitronectin.

PubMed

De Lorenzi, Valentina; Sarra Ferraris, Gian Maria; Madsen, Jeppe B; Lupia, Michela; Andreasen, Peter A; Sidenius, Nicolai

2016-07-01

Components of the plasminogen activation system including urokinase (uPA), its inhibitor (PAI-1) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homoeostasis. Firstly, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface plasminogen activation. Secondly, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin. We now report that uPA and plasmin induces a potent negative feedback on cell adhesion through specific cleavage of the RGD motif in vitronectin. Cleavage of vitronectin by uPA displays a remarkable receptor dependence and requires concomitant binding of both uPA and vitronectin to uPAR Moreover, we show that PAI-1 counteracts the negative feedback and behaves as a proteolysis-triggered stabilizer of uPAR-mediated cell adhesion to vitronectin. These findings identify a novel and highly specific function for the plasminogen activation system in the regulation of cell adhesion to vitronectin. The cleavage of vitronectin by uPA and plasmin results in the release of N-terminal vitronectin fragments that can be detected in vivo, underscoring the potential physiological relevance of the process. © 2016 The Authors.

X-ray crystal structure of plasmin with tranexamic acid-derived active site inhibitors.

PubMed

Law, Ruby H P; Wu, Guojie; Leung, Eleanor W W; Hidaka, Koushi; Quek, Adam J; Caradoc-Davies, Tom T; Jeevarajah, Devadharshini; Conroy, Paul J; Kirby, Nigel M; Norton, Raymond S; Tsuda, Yuko; Whisstock, James C

2017-05-09

The zymogen protease plasminogen and its active form plasmin perform key roles in blood clot dissolution, tissue remodeling, cell migration, and bacterial pathogenesis. Dysregulation of the plasminogen/plasmin system results in life-threatening hemorrhagic disorders or thrombotic vascular occlusion. Accordingly, inhibitors of this system are clinically important. Currently, tranexamic acid (TXA), a molecule that prevents plasminogen activation through blocking recruitment to target substrates, is the most widely used inhibitor for the plasminogen/plasmin system in therapeutics. However, TXA lacks efficacy on the active form of plasmin. Thus, there is a need to develop specific inhibitors that target the protease active site. Here we report the crystal structures of plasmin in complex with the novel YO ( trans -4-aminomethylcyclohexanecarbonyl-l-tyrosine- n -octylamide) class of small molecule inhibitors. We found that these inhibitors form key interactions with the S1 and S3' subsites of the catalytic cleft. Here, the TXA moiety of the YO compounds inserts into the primary (S1) specificity pocket, suggesting that TXA itself may function as a weak plasmin inhibitor, a hypothesis supported by subsequent biochemical and biophysical analyses. Mutational studies reveal that F587 of the S' subsite plays a key role in mediating the inhibitor interaction. Taken together, these data provide a foundation for the future development of small molecule inhibitors to specifically regulate plasmin function in a range of diseases and disorders.

X-ray crystal structure of plasmin with tranexamic acid–derived active site inhibitors

PubMed Central

Wu, Guojie; Leung, Eleanor W. W.; Hidaka, Koushi; Quek, Adam J.; Caradoc-Davies, Tom T.; Jeevarajah, Devadharshini; Kirby, Nigel M.; Norton, Raymond S.; Tsuda, Yuko; Whisstock, James C.

2017-01-01

The zymogen protease plasminogen and its active form plasmin perform key roles in blood clot dissolution, tissue remodeling, cell migration, and bacterial pathogenesis. Dysregulation of the plasminogen/plasmin system results in life-threatening hemorrhagic disorders or thrombotic vascular occlusion. Accordingly, inhibitors of this system are clinically important. Currently, tranexamic acid (TXA), a molecule that prevents plasminogen activation through blocking recruitment to target substrates, is the most widely used inhibitor for the plasminogen/plasmin system in therapeutics. However, TXA lacks efficacy on the active form of plasmin. Thus, there is a need to develop specific inhibitors that target the protease active site. Here we report the crystal structures of plasmin in complex with the novel YO (trans-4-aminomethylcyclohexanecarbonyl-l-tyrosine-n-octylamide) class of small molecule inhibitors. We found that these inhibitors form key interactions with the S1 and S3′ subsites of the catalytic cleft. Here, the TXA moiety of the YO compounds inserts into the primary (S1) specificity pocket, suggesting that TXA itself may function as a weak plasmin inhibitor, a hypothesis supported by subsequent biochemical and biophysical analyses. Mutational studies reveal that F587 of the S′ subsite plays a key role in mediating the inhibitor interaction. Taken together, these data provide a foundation for the future development of small molecule inhibitors to specifically regulate plasmin function in a range of diseases and disorders. PMID:29296720

Computational analysis of blood clot dissolution using a vibrating catheter tip.

PubMed

Lee, Jeong Hyun; Oh, Jin Sun; Yoon, Bye Ri; Choi, Seung Hong; Rhee, Kyehan; Jho, Jae Young; Han, Moon Hee

2012-04-01

We developed a novel concept of endovascular thrombolysis that employs a vibrating electroactive polymer actuator. In order to predict the efficacy of thrombolysis using the developed vibrating actuator, enzyme (plasminogen activator) perfusion into a clot was analyzed by solving flow fields and species transport equations considering the fluid structure interaction. In vitro thrombolysis experiments were also performed. Computational results showed that plasminogen activator perfusion into a clot was enhanced by actuator vibration at frequencies of 1 and 5 Hz. Plasminogen activator perfusion was affected by the actuator oscillation frequencies and amplitudes that were determined by electromechanical characteristics of a polymer actuator. Computed plasminogen activator perfused volumes were compared with experimentally measured dissolved clot volumes. The computed plasminogen activator perfusion volumes with threshold concentrations of 16% of the initial plasminogen activator concentration agreed well with the in vitro experimental data. This study showed the effectiveness of actuator oscillation on thrombolysis and the validity of the computational plasminogen activator perfusion model for predicting thrombolysis in complex flow fields induced by an oscillating actuator.

21 CFR 866.5715 - Plasminogen immunological test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5715 Plasminogen immunological test system. (a) Identification. A plasminogen immunological test system is a device... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Plasminogen immunological test system. 866.5715...

Leukocyte- and endothelial-derived microparticles: a circulating source for fibrinolysis

PubMed Central

Lacroix, Romaric; Plawinski, Laurent; Robert, Stéphane; Doeuvre, Loïc; Sabatier, Florence; Martinez de Lizarrondo, Sara; Mezzapesa, Anna; Anfosso, Francine; Leroyer, Aurelie S.; Poullin, Pascale; Jourde, Noémie; Njock, Makon-Sébastien; Boulanger, Chantal M.; Anglés-Cano, Eduardo; Dignat-George, Françoise

2012-01-01

Background We recently assigned a new fibrinolytic function to cell-derived microparticles in vitro. In this study we explored the relevance of this novel property of microparticles to the in vivo situation. Design and Methods Circulating microparticles were isolated from the plasma of patients with thrombotic thrombocytopenic purpura or cardiovascular disease and from healthy subjects. Microparticles were also obtained from purified human blood cell subpopulations. The plasminogen activators on microparticles were identified by flow cytometry and enzyme-linked immunosorbent assays; their capacity to generate plasmin was quantified with a chromogenic assay and their fibrinolytic activity was determined by zymography. Results Circulating microparticles isolated from patients generate a range of plasmin activity at their surface. This property was related to a variable content of urokinase-type plasminogen activator and/or tissue plasminogen activator. Using distinct microparticle subpopulations, we demonstrated that plasmin is generated on endothelial and leukocyte microparticles, but not on microparticles of platelet or erythrocyte origin. Leukocyte-derived microparticles bear urokinase-type plasminogen activator and its receptor whereas endothelial microparticles carry tissue plasminogen activator and tissue plasminogen activator/inhibitor complexes. Conclusions Endothelial and leukocyte microparticles, bearing respectively tissue plasminogen activator or urokinase-type plasminogen activator, support a part of the fibrinolytic activity in the circulation which is modulated in pathological settings. Awareness of this blood-borne fibrinolytic activity conveyed by microparticles provides a more comprehensive view of the role of microparticles in the hemostatic equilibrium. PMID:22733025

Plasminogen associates with phosphatidylserine-exposing platelets and contributes to thrombus lysis under flow

PubMed Central

Whyte, Claire S.; Swieringa, Frauke; Mastenbroek, Tom G.; Lionikiene, Ausra S.; Lancé, Marcus D.; van der Meijden, Paola E. J.; Heemskerk, Johan W. M.

2015-01-01

The interaction of plasminogen with platelets and their localization during thrombus formation and fibrinolysis under flow are not defined. Using a novel model of whole blood thrombi, formed under flow, we examine dose-dependent fibrinolysis using fluorescence microscopy. Fibrinolysis was dependent upon flow and the balance between fibrin formation and plasminogen activation, with tissue plasminogen activator-mediated lysis being more efficient than urokinase plasminogen activator-mediated lysis. Fluorescently labeled plasminogen radiates from platelet aggregates at the base of thrombi, primarily in association with fibrin. Hirudin attenuates, but does not abolish plasminogen binding, denoting the importance of fibrin. Flow cytometry revealed that stimulation of platelets with thrombin/convulxin significantly increased the plasminogen signal associated with phosphatidylserine (PS)-exposing platelets. Binding was attenuated by tirofiban and Gly-Pro-Arg-Pro amide, confirming a role for fibrin in amplifying plasminogen binding to PS-exposing platelets. Confocal microscopy revealed direct binding of plasminogen and fibrinogen to different platelet subpopulations. Binding of plasminogen and fibrinogen co-localized with PAC-1 in the center of spread platelets. In contrast, PS-exposing platelets were PAC-1 negative, and bound plasminogen and fibrinogen in a protruding “cap.” These data show that different subpopulations of platelets harbor plasminogen by diverse mechanisms and provide an essential scaffold for the accumulation of fibrinolytic proteins that mediate fibrinolysis under flow. PMID:25712989

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.

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.

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. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Successful Tissue Plasminogen Activator for a Patient with Stroke After Stanford Type A Aortic Dissection Treatment.

PubMed

Matsuzono, Kosuke; Suzuki, Masayuki; Arai, Naoto; Kim, Younhee; Ozawa, Tadashi; Mashiko, Takafumi; Shimazaki, Haruo; Koide, Reiji; Fujimoto, Shigeru

2018-07-01

Some stroke patients with the acute aortic dissection receiving thrombolysis treatment resulted in fatalities. Thus, the concurrent acute aortic dissection is the contraindication for the intravenous recombinant tissue-type plasminogen activator. However, the safety and the effectiveness of the intravenous recombinant tissue-type plasminogen activator therapy are not known in patients with stroke some days after acute aortic dissection treatment. Here, we first report a case of a man with a cardioembolism due to the nonvalvular atrial fibrillation, who received the intravenous recombinant tissue-type plasminogen activator therapy 117 days after the traumatic Stanford type A acute aortic dissection operation. Without the intravenous recombinant tissue-type plasminogen activator therapy, the prognosis was expected to be miserable. However, the outcome was good with no complication owing to the intravenous recombinant tissue-type plasminogen activator therapy. Our case suggests the effectiveness and the safety of the intravenous recombinant tissue-type plasminogen activator therapy to the ischemic stroke some days after acute aortic dissection treatment. Copyright © 2018 National Stroke Association. Published by Elsevier Inc. All rights reserved.

Tissue plasminogen activator (tPA) as a reporter gene in transient gene expression.

PubMed

Cheng, S M; Lee, S G; Kalyan, N K; McCloud, S; Levner, M; Hung, P P

1987-01-01

Using the gene coding for tissue plasminogen activator (tPA) as a reporter gene, a transient gene expression system has been established. Vectors containing the full-length cDNA of tPA with its signal sequences were introduced into mammalian recipient cells by a modified gene transfer procedure. Thirty hours after transfection, the secreted tPA was found in serum-free medium and measured by a fibrin-agarose plate assay (FAPA). In this assay, tPA converts plasminogen into plasmin which then degrades high-Mr fibrin to produce cleared zones. The sizes of these zones correspond to quantities of tPA. The combination of transient tPA expression system and the FAPA provides a quick, sensitive, quantitative and non-destructive method to examine the strength of eukaryotic regulatory elements in tissue-culture cells.

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. Copyright 2003 Wiley-Liss, Inc.

Seahorse-derived peptide suppresses invasive migration of HT1080 fibrosarcoma cells by competing with intracellular α-enolase for plasminogen binding and inhibiting uPA-mediated activation of plasminogen.

PubMed

Kim, Yong-Tae; Kim, Se-kwon; Jeon, You-Jin; Park, Sun Joo

2014-12-01

α-Enolase is a glycolytic enzyme and a surface receptor for plasminogen. α-Enolase-bound plasminogen promotes tumor cell invasion and cancer metastasis by activating plasmin and consequently degrading the extracellular matrix degradation. Therefore, α-enolase and plasminogen are novel targets for cancer therapy. We found that the amino acid sequence of a peptide purified from enzymatic hydrolysates of seahorse has striking similarities to that of α-enolase. In this study, we report that this peptide competes with cellular α-enolase for plasminogen binding and suppresses urokinase plasminogen activator (uPA)-mediated activation of plasminogen, which results in decreased invasive migration of HT1080 fibrosarcoma cells. In addition, the peptide treatment decreased the expression levels of uPA compared to that of untreated controls. These results provide new insight into the mechanism by which the seahorse-derived peptide suppresses invasive properties of human cancer cells. Our findings suggest that this peptide could emerge as a potential therapeutic agent for cancer.

Seahorse-derived peptide suppresses invasive migration of HT1080 fibrosarcoma cells by competing with intracellular α-enolase for plasminogen binding and inhibiting uPA-mediated activation of plasminogen

PubMed Central

Kim, Yong-Tae; Kim, Se-kwon; Jeon, You-Jin; Park, Sun Joo

2014-01-01

α-Enolase is a glycolytic enzyme and a surface receptor for plasminogen. α-Enolase-bound plasminogen promotes tumor cell invasion and cancer metastasis by activating plasmin and consequently degrading the extracellular matrix degradation. Therefore, α-enolase and plasminogen are novel targets for cancer therapy. We found that the amino acid sequence of a peptide purified from enzymatic hydrolysates of seahorse has striking similarities to that of α-enolase. In this study, we report that this peptide competes with cellular α-enolase for plasminogen binding and suppresses urokinase plasminogen activator (uPA)-mediated activation of plasminogen, which results in decreased invasive migration of HT1080 fibrosarcoma cells. In addition, the peptide treatment decreased the expression levels of uPA compared to that of untreated controls. These results provide new insight into the mechanism by which the seahorse-derived peptide suppresses invasive properties of human cancer cells. Our findings suggest that this peptide could emerge as a potential therapeutic agent for cancer. [BMB Reports 2014; 47(12): 691-696] PMID:24602611

Dynamic changes in plasma tissue plasminogen activator, plasminogen activator inhibitor-1 and beta-thromboglobulin content in ischemic stroke.

PubMed

Zhuang, Ping; Wo, Da; Xu, Zeng-Guang; Wei, Wei; Mao, Hui-ming

2015-07-01

The aim of this paper is to investigate the corresponding variations of plasma tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) activities, and beta-thromboglobulin (β-TG) content in patients during different stages of ischemic stroke. Ischemic stroke is a common disease among aging people and its occurrence is associated with abnormalities in the fibrinolytic system and platelet function. However, few reports focus on the dynamic changes in the plasma fibrinolytic system and β-TG content in patients with ischemic stroke. Patients were divided into three groups: acute, convalescent and chronic. Plasma t-PA and PAI-1 activities were determined by chromogenic substrate analysis and plasma β-TG content was detected by radioimmunoassay. Patients in the acute stage of ischemic stroke had significantly increased levels of t-PA activity and β-TG content, but PAI-1 activity was significantly decreased. Negative correlations were found between plasma t-PA and PAI-1 activities and between plasma t-PA activity and β-TG content in patients with acute ischemic stroke. There were significant differences in plasma t-PA and PAI-1 activities in the aged control group, as well as in the acute, convalescent and chronic groups. It can be speculated that the increased activity of t-PA in patients during the acute stage was the result of compensatory function, and that the increase in plasma β-TG level not only implies the presence of ischemic stroke but is likely a cause of ischemic stroke. During the later stages of ischemic stroke, greater attention is required in monitoring levels of PAI-1. Copyright © 2015 Elsevier Ltd. All rights reserved.

Endogenously Generated Plasmin at the Vascular Wall Injury Site Amplifies Lysine Binding Site-Dependent Plasminogen Accumulation in Microthrombi

PubMed Central

Brzoska, Tomasz; Tanaka-Murakami, Aki; Suzuki, Yuko; Sano, Hideto; Kanayama, Naohiro; Urano, Tetsumei

2015-01-01

The fibrinolytic system plays a pivotal role in the regulation of hemostasis; however, it remains unclear how and when the system is triggered to induce thrombolysis. Using intra-vital confocal fluorescence microscopy, we investigated the process of plasminogen binding to laser-induced platelet-rich microthrombi generated in the mesenteric vein of transgenic mice expressing green fluorescent protein (GFP). The accumulation of GFP-expressing platelets as well as exogenously infused Alexa Fluor 568-labeled Glu-plasminogen (Glu-plg) on the injured vessel wall was assessed by measuring the increase in the corresponding fluorescence intensities. Glu-plg accumulated in a time-dependent manner in the center of the microthrombus, where phosphatidylserine is exposed on platelet surfaces and fibrin formation takes place. The rates of binding of Glu-plg in the presence of ε-aminocaproic acid and carboxypeptidase B, as well as the rates of binding of mini-plasminogen lacking kringle domains 1-4 and lysine binding sites, were significantly lower than that of Glu-plg alone, suggesting that the binding was dependent on lysine binding sites. Furthermore, aprotinin significantly suppressed the accumulation of Glu-plg, suggesting that endogenously generated plasmin activity is a prerequisite for the accumulation. In spite of the endogenous generation of plasmin and accumulation of Glu-plg in the center of microthrombi, the microthrombi did not change in size during the 2-hour observation period. When human tissue plasminogen activator was administered intravenously, Glu-plg further accumulated and the microthrombi were lysed. Glu-plg appeared to accumulate in the center of microthrombi in the early phase of microthrombus formation, and plasmin activity and lysine binding sites were required for this accumulation. PMID:25806939

Plasminogen stimulates propagation of protease-resistant prion protein in vitro.

PubMed

Mays, Charles E; Ryou, Chongsuk

2010-12-01

To clarify the role of plasminogen as a cofactor for prion propagation, we conducted functional assays using a cell-free prion protein (PrP) conversion assay termed protein misfolding cyclic amplification (PMCA) and prion-infected cell lines. Here, we report that plasminogen stimulates propagation of the protease-resistant scrapie PrP (PrP(Sc)). Compared to control PMCA conducted without plasminogen, addition of plasminogen in PMCA using wild-type brain material significantly increased PrP conversion, with an EC(50) = ∼56 nM. PrP conversion in PMCA was substantially less efficient with plasminogen-deficient brain material than with wild-type material. The activity stimulating PrP conversion was specific for plasminogen and conserved in its kringle domains. Such activity was abrogated by modification of plasminogen structure and interference of PrP-plasminogen interaction. Kinetic analysis of PrP(Sc) generation demonstrated that the presence of plasminogen in PMCA enhanced the PrP(Sc) production rate to ∼0.97 U/μl/h and reduced turnover time to ∼1 h compared to those (∼0.4 U/μl/h and ∼2.5 h) obtained without supplementation. Furthermore, as observed in PMCA, plasminogen and kringles promoted PrP(Sc) propagation in ScN2a and Elk 21(+) cells. Our results demonstrate that plasminogen functions in stimulating conversion processes and represents the first cellular protein cofactor that enhances the hypothetical mechanism of prion propagation.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

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 notmore » 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.« less

The fibrinolytic mechanism of defibrotide: effect of defibrotide on plasmin activity.

PubMed

Echart, Cinara L; Graziadio, Barbara; Somaini, Simona; Ferro, Laura I; Richardson, Paul G; Fareed, Jawed; Iacobelli, Massimo

2009-12-01

Fibrinolytic activity has been shown to be reduced in many vascular diseases, including hepatic veno-occlusive disease after stem cell transplantation, a microangiopathy characterized by sinusoidal endothelial cell injury. Defibrotide is a polydisperse oligonucleotide with antithrombotic, profibrinolytic, anti-ischemic, and antiadhesive properties. Numerous clinical studies have shown promising activity of defibrotide in the treatment and prevention of veno-occlusive disease, with minimal toxicity. In corollary laboratory studies, defibrotide has been shown to decrease plasminogen activator inhibitor-1, increase tissue plasminogen activator levels, and increase overall plasma fibrinolytic activity in patients. Plasmin, a potent and nonspecific serine protease, plays a pivotal role in fibrinolysis by virtue of its ability to effectively degrade fibrin clots. In this study, defibrotide increases the activity of plasmin in hydrolyzing its substrate in a dose-dependent and length-dependent manner. Similar concentration-dependent effects of defibrotide were observed when plasmin was generated by tissue plasminogen activator or urokinase activation of plasminogen. In contrast, defibrotide had no direct effect on the activation of plasminogen to plasmin. Defibrotide was also able to enhance the activity of plasmin in degrading fibrin clot formed from fibrinogen, plasminogen, and thrombin. This effect was also concentration-dependent and directly correlated with the enzymatic activity of plasmin. This study therefore demonstrates that defibrotide is capable of enhancing the activity of plasmin and so contributes to its fibrinolytic activity. Taken together, these results support the effect of defibrotide in restoring the fibrinolytic vascular phenotype, in microangiopathic conditions such as veno-occlusive disease.

Effects of lipoprotein(a) on thrombolysis.

PubMed

von Hodenberg, E; Pestel, E; Kreuzer, J; Freitag, M; Bode, C

1994-01-01

Lipoprotein(a) (Lp(a)) and plasminogen share a high degree of structural homology. Therefore it has been suggested that elevated levels of Lp(a) may inhibit the profibrinolytic activity at the cell surface and increase the risk of thrombosis by competitive inhibition of plasminogen. In the present study we evaluated whether high levels of Lp(a) affect thrombolytic therapy in patients with acute myocardial infarction. Forty-one patients with acute myocardial infarction were treated with a combination of recombinant tissue-type plasminogen activator and human single-chain urokinase-type plasminogen activator. Coronary patency was assessed angiographically 90 min after initiation of treatment. Thrombolysis was successful in 30 and unsuccessful in 11 patients. Patients with high Lp(a) levels (> 25 mg/dl) (n = 9) responded equally well to thrombolytic therapy (8 of 9, patency 89%) as did patients with normal or low levels of Lp(a) (22 of 32, patency 70%, difference P > 0.1). The results demonstrate that high levels of Lp(a) do not influence thrombolysis in patients with acute myocardial infarction when low-dose pharmacologic concentrations of recombinant tissue-type plasminogen activator and human single chain urokinase-type plasminogen activator are applied in combination.

Platelets from patients with the Quebec platelet disorder contain and secrete abnormal amounts of urokinase-type plasminogen activator.

PubMed

Kahr, W H; Zheng, S; Sheth, P M; Pai, M; Cowie, A; Bouchard, M; Podor, T J; Rivard, G E; Hayward, C P

2001-07-15

The Quebec platelet disorder (QPD) is an autosomal dominant platelet disorder associated with delayed bleeding and alpha-granule protein degradation. The degradation of alpha-granule, but not plasma, fibrinogen in patients with the QPD led to the investigation of their platelets for a protease defect. Unlike normal platelets, QPD platelets contained large amounts of fibrinolytic serine proteases that had properties of plasminogen activators. Western blot analysis, zymography, and immunodepletion experiments indicated this was because QPD platelets contained large amounts of urokinase-type plasminogen activator (u-PA) within a secretory compartment. u-PA antigen was not increased in all QPD plasmas, whereas it was increased more than 100-fold in QPD platelets (P <.00009), which contained increased u-PA messenger RNA. Although QPD platelets contained 2-fold more plasminogen activator inhibitor 1 (PAI-1) (P <.0008) and 100-fold greater u-PA-PAI-1 complexes (P <.0002) than normal platelets, they contained excess u-PA activity, predominantly in the form of two chain (tcu-PA), which required additional PAI-1 for full inhibition. There was associated proteolysis of plasminogen in QPD platelets, to forms that comigrated with plasmin. When similar amounts of tcu-PA were incubated with normal platelet secretory proteins, many alpha-granule proteins were proteolyzed to forms that resembled degraded QPD platelet proteins. These data implicate u-PA in the pathogenesis of alpha-granule protein degradation in the QPD. Although patients with the QPD have normal to increased u-PA levels in their plasma, without evidence of systemic fibrinogenolysis, their increased platelet u-PA could contribute to bleeding by accelerating fibrinolysis within the hemostatic plug. QPD is the only inherited bleeding disorder in humans known to be associated with increased u-PA.

Plasmin cleaves fibrinogen and the human complement proteins C3b and C5 in the presence of Leptospira interrogans proteins: A new role of LigA and LigB in invasion and complement immune evasion.

PubMed

Castiblanco-Valencia, Mónica Marcela; Fraga, Tatiana Rodrigues; Pagotto, Ana Helena; Serrano, Solange Maria de Toledo; Abreu, Patricia Antonia Estima; Barbosa, Angela Silva; Isaac, Lourdes

2016-05-01

Plasminogen is a single-chain glycoprotein found in human plasma as the inactive precursor of plasmin. When converted to proteolytically active plasmin, plasmin(ogen) regulates both complement and coagulation cascades, thus representing an important target for pathogenic microorganisms. Leptospira interrogans binds plasminogen, which is converted to active plasmin. Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules that interact with extracellular matrix components and complement regulators, including proteins of the FH family and C4BP. In this work, we demonstrate that these multifunctional molecules also bind plasminogen through both N- and C-terminal domains. These interactions are dependent on lysine residues and are affected by ionic strength. Competition assays suggest that plasminogen does not share binding sites with C4BP or FH on Lig proteins at physiological molar ratios. Plasminogen bound to Lig proteins is converted to proteolytic active plasmin in the presence of urokinase-type plasminogen activator (uPA). Lig-bound plasmin is able to cleave the physiological substrates fibrinogen and the complement proteins C3b and C5. Taken together, our data point to a new role of LigA and LigB in leptospiral invasion and complement immune evasion. Plasmin(ogen) acquisition by these versatile proteins may contribute to Leptospira infection, favoring bacterial survival and dissemination inside the host. Copyright © 2016. Published by Elsevier GmbH.

Plasminogen replacement therapy for the treatment of children and adults with congenital plasminogen deficiency

PubMed Central

Nakar, Charles; Parker, Joseph M.; Albert, Gary R.; Moran, John E.; Thibaudeau, Karen; Thukral, Neelam; Hardesty, Brandon M.; Laurin, Pierre; Sandset, Per Morten

2018-01-01

Congenital plasminogen deficiency is caused by mutations in PLG, the gene coding for production of the zymogen plasminogen, and is an ultrarare disorder associated with abnormal accumulation or growth of fibrin-rich pseudomembranous lesions on mucous membranes. Left untreated, these lesions may impair organ function and impact quality of life. Plasminogen replacement therapy should provide an effective treatment of the manifestations of congenital plasminogen deficiency. An open-label phase 2/3 study of human Glu-plasminogen administered IV at 6.6 mg/kg every 2 to 4 days in 15 patients with congenital plasminogen deficiency is ongoing. Reported here are data on 14 patients who completed at least 12 weeks of treatment. The primary end point was an increase in trough plasminogen activity levels by at least an absolute 10% above baseline. The secondary end point was clinical success, defined as ≥50% improvement in lesion number/size or functionality impact from baseline. All patients achieved at least an absolute 10% increase in trough plasminogen activity above baseline. Clinical success was observed in all patients with clinically visible (conjunctiva and gingiva), nonvisible (nasopharynx, bronchus, colon, kidney, cervix, and vagina), and wound-healing manifestations of the disease. Therapeutic effects were rapid, as all but 2 lesions resolved or improved after 4 weeks of treatment. Human Glu-plasminogen was well tolerated in both children and adults. This study provides critical first evidence of the clinical utility of ongoing replacement therapy with human Glu-plasminogen for the treatment of children and adults with congenital plasminogen deficiency. This trial was registered at www.clinicaltrials.gov as #NCT02690714. PMID:29321155

Enzymatic properties and localization of motopsin (PRSS12), a protease whose absence causes mental retardation.

PubMed

Mitsui, Shinichi; Yamaguchi, Nozomi; Osako, Yoji; Yuri, Kazunari

2007-03-09

Motopsin (PRSS12) is a mosaic protease expressed in the central nervous system. Truncation of the human motopsin gene causes nonsyndromic mental retardation. Understanding the enzymatic properties and localization of motopsin protein in the central nervous system will help identify the molecular mechanism by which the loss of motopsin function causes mental retardation. Recombinant motopsin showed amidolytic activity against the synthetic substrate benzyloxycarbonyl-l-phenylalanyl-l-arginine 4-methyl-coumaryl-7-amide. Motopsin activated the single-chain tissue plasminogen activator precursor and exhibited gelatinolytic activity. This enzymatic activity was inhibited by typical serine protease inhibitors such as aprotinin, leupeptin, and (4-amidinophenyl) methanesulfonyl fluoride. Immunocytochemistry using anti-motopsin IgG revealed that both human and mouse motopsin proteins were distributed in discrete puncta along the dendrites and soma as well as axons in cultured hippocampal neurons. In the limbic system, including the cingulate and hippocampal pyramidal neurons and piriform cortex, high level of motopsin protein was expressed at postnatal day 10, but a very low level at 10-week-old mice. Motopsin and tissue plasminogen activator were co-expressed in the cingulate pyramidal neurons at postnatal day 10 and were distributed along dendrites of cultured pyramidal neurons. In cranial nuclei, a moderate level of motopsin protein was detected independently on the developmental stage. Our results suggest that motopsin has multiple functions, such as axon outgrowth, arranging perineuronal environment, and maintaining neuronal plasticity, partly in coordination with other proteases including tissue plasminogen activator.

Contact activation: a revision.

PubMed

Schmaier, A H

1997-07-01

In conclusion, a revised view of the contact system has been presented. This system has little to do with the initiation of hemostasis. Like lupus anticoagulants, deficiencies of contact proteins give prolonged APTTs but may be risk factors for thrombosis. BK from kininogens is a potent modulator of vascular biology inducing vasodilation, tissue plasminogen activator release, and prostacyclin liberation. Kininogens, themselves, are selective inhibitors of alpha-thrombin-induced platelet activation preventing alpha-thrombin from cleaving the cloned thrombin receptor after arginine41. Kininogens' alpha-thrombin inhibitory activity exists in intact kininogens, BK, and all of BK's breakdown products. HK also is the pivotal protein for contact protein assembly on endothelium. It is the receptor for prekallikrein which when bound to HK becomes activated to kallikrein by an endothelial cell enzyme system independent of activated forms of plasma factor XII. Prekallikrein activation on endothelial cells results in kinetically favorable single chain urokinase and plasminogen activation. Thus the "physiologic, negatively charged surface" for contact system activation is really the assembly of these proteins on cell membranes and activation by membrane-associated enzymes.

Cell Surface Translocation of Annexin A2 Facilitates Glutamate-induced Extracellular Proteolysis*

PubMed Central

Valapala, Mallika; Maji, Sayantan; Borejdo, Julian; Vishwanatha, Jamboor K.

2014-01-01

Glutamate-induced elevation in intracellular Ca2+ has been implicated in excitotoxic cell death. Neurons respond to increased glutamate levels by activating an extracellular proteolytic cascade involving the components of the plasmin-plasminogen system. AnxA2 is a Ca2+-dependent phospholipid binding protein and serves as an extracellular proteolytic center by recruiting the tissue plasminogen activator and plasminogen and mediating the localized generation of plasmin. Ratiometric Ca2+ imaging and time-lapse confocal microscopy demonstrated glutamate-induced Ca2+ influx. We showed that glutamate translocated both endogenous and AnxA2-GFP to the cell surface in a process dependent on the activity of the NMDA receptor. Glutamate-induced translocation of AnxA2 is dependent on the phosphorylation of tyrosine 23 at the N terminus, and mutation of tyrosine 23 to a non-phosphomimetic variant inhibits the translocation process. The cell surface-translocated AnxA2 forms an active plasmin-generating complex, and this activity can be neutralized by a hexapeptide directed against the N terminus. These results suggest an involvement of AnxA2 in potentiating glutamate-induced cell death processes. PMID:24742684

Role of the Yersinia pestis plasminogen activator in the incidence of distinct septicemic and bubonic forms of flea-borne plague.

PubMed

Sebbane, Florent; Jarrett, Clayton O; Gardner, Donald; Long, Daniel; Hinnebusch, B Joseph

2006-04-04

Yersinia pestis is transmitted by fleas and causes bubonic plague, characterized by severe local lymphadenitis that progresses rapidly to systemic infection and life-threatening septicemia. Here, we show that although flea-borne transmission usually leads to bubonic plague in mice, it can also lead to primary septicemic plague. However, intradermal injection of Y. pestis, commonly used to mimic transmission by fleabite, leads only to bubonic plague. A Y. pestis strain lacking the plasmid-encoded cell-surface plasminogen activator, which is avirulent by intradermal or s.c. injection, was able to cause fatal primary septicemic plague at low incidence, but not bubonic plague, when transmitted by fleas. The results clarify a long-standing uncertainty about the etiology of primary septicemic plague and support an evolutionary scenario in which plague first emerged as a flea-borne septicemic disease of limited transmissibility. Subsequent acquisition of the plasminogen activator gene by horizontal transfer enabled the bubonic form of disease and increased the potential for epidemic spread.

Plasminogen activator activity in tears of pregnant women.

PubMed

Csutak, Adrienne; Steiber, Zita; Tőzsér, József; Jakab, Attila; Berta, András; Silver, David M

2017-01-01

Plasminogen activator activity (PAA) in tears of pregnant women was investigated at various gestation times to assess the availability of plasminogen activator for aiding potential corneal wound healing processes during pregnancy. PAA was measured by a spectrophotometric method. The analysis used 91 tear samples from pregnant and non-pregnant women, supplemented with 10 additional tear PAA measurements from non-pregnant women obtained in a previous study. Tear levels of PAA in pregnant women formed a bimodal distribution. Either the tear PAA level was zero or non-zero during pregnancy. When non-zero, the tear PAA level was dissociated from gestation time and not different than non-pregnant and post-pregnant levels. The frequency of occurrence of zero level tear PAA increased with gestation: 16%, 17% and 46% had zero tear PAA in samples taken from women in the first, second and third trimester, respectively. Overall, of the tear samples taken from women during pregnancy, a total of 26% were at zero tear PAA. The remaining tear samples had non-zero tear PAA values throughout gestation equivalent to non-pregnant tear PAA values, suggesting local control of the source of PAA in tears. Given the importance of the plasminogen activator system in tears to wound healing in the cornea, and the high occurrence of zero tear PAA in our sample of pregnant women, elective corneal surgery would be contraindicated. If corneal surgery is nevertheless necessary, the tear PAA level would be worth checking and patients with low level should be closely observed during the postoperative period.

Epidermal growth factor- and hepatocyte growth factor-receptor activity in serum-free cultures of human hepatocytes.

PubMed

Runge, D M; Runge, D; Dorko, K; Pisarov, L A; Leckel, K; Kostrubsky, V E; Thomas, D; Strom, S C; Michalopoulos, G K

1999-02-01

Serum-free primary cultures of hepatocytes are a useful tool to study factors triggering hepatocyte proliferation and regeneration. We have developed a chemically defined serum-free system that allows human hepatocyte proliferation in the presence of epidermal growth factor and hepatocyte growth factor. DNA synthesis and accumulation were determined by [3H]thymidine incorporation and fluorometry, respectively. Western blot analyses and co-immunoprecipitations were used to investigate the association of proteins involved in epidermal growth factor and hepatocyte growth factor activation and signaling: epidermal growth factor receptor, hepatocyte growth factor receptor (MET), urokinase-type plasminogen activator and its receptor, and a member of the signal transducer and activator of transcription family, STAT-3. Primary human hepatocytes proliferated under serum-free conditions in a chemically defined medium for up to 12 days. Epidermal growth factor-receptor and MET were present and functional, decreasing over time. MET, urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor co-precipitated to varying degrees during the culture period. STAT-3 co-precipitated with epidermal growth factor-receptor and MET to varying degrees. Proliferation of human hepatocytes can improve by modification of a chemically defined medium originally used for rat hepatocyte cultures. In these long-term cultures of human hepatocytes, hepatocyte growth factor and epidermal growth factor can stimulate growth and differentiation by interacting with their receptors and initiating downstream signaling. This involves complex formation of the receptors with other plasma membrane components for MET (urokinase-type plasminogen activator in context of its receptor) and activation of STAT-3 for both receptors.

Plasminogen Activator Production Accompanies Loss of Anchorage Regulation in Transformation of Primary Rat Embryo Cells by Simian Virus 40

PubMed Central

Pollack, R.; Risser, R.; Conlon, S.; Rifkin, D.

1974-01-01

We have isolated several lines of rat embryo cells transformed by simian virus 40. All these lines are fully transformed with regard to saturation density and serum sensitivity, but they differ greatly in their anchorage dependence, as assayed by efficiency of plating in methyl cellulose suspension. This set of lines reveals a consistent relation of plasminogen activator production to plating efficiency in methyl cellulose. T-antigen-positive transformed lines that synthesize activator grow in methyl cellulose suspension, while T-antigen-positive transformed lines that do not synthesize activator fail to form colonies in suspension. Normal rat embryo cells produce very little plasminogen activator and do not grow in methyl cellulose. Sera that permit high levels of plasmin formation and activity support growth in semi-solid medium better than sera whose plasminogen is activated poorly and/or sera that contain inhibitors to plasmin. PMID:4373730

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

Regulation of Plasminogen Activation on Cell Surfaces and Fibrin.

PubMed

Urano, Tetsumei; Castellino, Francis J; Suzuki, Yuko

2018-05-20

The fibrinolytic system dissolves fibrin and maintains vascular patency. Recent advances in imaging analyses allowed visualization of the spatiotemporal regulatory mechanism of fibrinolysis, as well as its regulation by other plasma haemostasis cofactors. Vascular endothelial cells (VECs) retain tissue-type plasminogen activator (tPA) after secretion and maintain high plasminogen (plg) activation potential on their surfaces. As in plasma, the serpin, plasminogen activator inhibitor type 1 (PAI-1), regulates fibrinolytic potential via inhibition of the VEC surface-bound plg activator, tPA. Once fibrin is formed, plg activation by tPA is initiated and effectively amplified on the surface of fibrin, and fibrin is rapidly degraded. The specific binding of plg and tPA to lytic edges of partly degraded fibrin via newly generated C-terminal lysine residues, which amplifies fibrin digestion, is a central aspect of this pathophysiological mechanism. Thrombomodulin (TM) plays a role in the attenuation of the plg binding on fibrin and the associated fibrinolysis, which is reversed by a carboxypeptidase B inhibitor. This suggests that the plasma procarboxypeptidase B, thrombin activatable fibrinolysis inhibitor (TAFI), which is activated by thrombin bound to TM on VECs, is a critical aspect of the regulation of plg activation on VECs and subsequent fibrinolysis. Platelets also contain PAI-1, TAFI, TM and the fibrin crosslinking enzyme, Factor (F) XIIIa, and either secrete or expose these agents upon activation in order to regulate fibrinolysis. In this review, the native machinery of plg activation and fibrinolysis, as well as their spatiotemporal regulatory mechanisms, as revealed by imaging analyses, are discussed. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

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 blood clots. PMID:25635856

Reprogrammed streptokinases develop fibrin-targeting and dissolve blood clots with more potency than tissue plasminogen activator

PubMed Central

SAZONOVA, I. Y.; MCNAMEE, R. A.; HOUNG, A. K.; KING, S. M.; HEDSTROM, L.; REED, G. L.

2013-01-01

Summary Background: Given the worldwide epidemic of cardiovascular diseases, a more effective means of dissolving thrombi that cause heart attacks, could markedly reduce death, disability and healthcare costs. Plasminogen activators (PAs) such as streptokinase (SK) and tissue plasminogen activator (TPA) are currently used to dissolve fibrin thrombi. SK is cheaper and more widely available, but it appears less effective because it lacks TPA’s fibrin-targeted properties that focus plasminogen activation on the fibrin surface. Objective: We examined whether re-programming SK’s mechanism of action would create PAs with greater fibrin-targeting and potency than TPA. Methods and Results: When fibrinogen consumption was measured in human plasma, reprogrammed molecules SKΔ1 and SKΔ59 were 5-fold and > 119-fold more fibrin-dependent than SK (P < 0.0001), and 2-fold and > 50-fold more fibrin-dependent than TPA (P < 0.001). The marked fibrin-targeting of SKΔ59 was due to the fact that: (i) it did not generate plasmin in plasma, (ii) it was rapidly inhibited by α2-antiplasmin, and (iii) it only processed fibrin-bound plasminogen. To assess the fibrin-targeting and therapeutic potential of these PAs in vivo, a novel ‘humanized’ fibrinolysis model was created by reconstituting plasminogen-deficient mice with human plasminogen. When compared with TPA, SKΔ1 and SKΔ59 were 4-fold (P < 0.0001) and 2-fold (P < 0.003) more potent at dissolving blood clots in vivo, respectively, on a mass-dose basis and 2–3 logs more potent than TPA (P < 0.0001) when doses were calibrated by standard activity assays. Conclusion: These experiments suggest that reprogramming SK’s mechanism of action markedly enhances fibrin-targeting and creates, in comparison with TPA, activators with greater fibrinolytic potency. PMID:19566545

Reprogrammed streptokinases develop fibrin-targeting and dissolve blood clots with more potency than tissue plasminogen activator.

PubMed

Sazonova, I Y; McNamee, R A; Houng, A K; King, S M; Hedstrom, L; Reed, G L

2009-08-01

Given the worldwide epidemic of cardiovascular diseases, a more effective means of dissolving thrombi that cause heart attacks, could markedly reduce death, disability and healthcare costs. Plasminogen activators (PAs) such as streptokinase (SK) and tissue plasminogen activator (TPA) are currently used to dissolve fibrin thrombi. SK is cheaper and more widely available, but it appears less effective because it lacks TPA's fibrin-targeted properties that focus plasminogen activation on the fibrin surface. We examined whether re-programming SK's mechanism of action would create PAs with greater fibrin-targeting and potency than TPA. When fibrinogen consumption was measured in human plasma, reprogrammed molecules SKDelta1 and SKDelta59 were 5-fold and > 119-fold more fibrin-dependent than SK (P < 0.0001), and 2-fold and > 50-fold more fibrin-dependent than TPA (P < 0.001). The marked fibrin-targeting of SKDelta59 was due to the fact that: (i) it did not generate plasmin in plasma, (ii) it was rapidly inhibited by alpha2-antiplasmin, and (iii) it only processed fibrin-bound plasminogen. To assess the fibrin-targeting and therapeutic potential of these PAs in vivo, a novel 'humanized' fibrinolysis model was created by reconstituting plasminogen-deficient mice with human plasminogen. When compared with TPA, SKDelta1 and SKDelta59 were 4-fold (P < 0.0001) and 2-fold (P < 0.003) more potent at dissolving blood clots in vivo, respectively, on a mass-dose basis and 2-3 logs more potent than TPA (P < 0.0001) when doses were calibrated by standard activity assays. These experiments suggest that reprogramming SK's mechanism of action markedly enhances fibrin-targeting and creates, in comparison with TPA, activators with greater fibrinolytic potency.

Intracellular activation of the fibrinolytic cascade in the Quebec Platelet Disorder.

PubMed

Sheth, Prameet M; Kahr, Walter H A; Haq, M Anwar; Veljkovic, Dragoslava Kika; Rivard, Georges E; Hayward, Catherine P M

2003-08-01

The Quebec Platelet Disorder (QPD) is an unusual bleeding disorder associated with increased platelet stores of urokinase-type plasminogen activator (u-PA) and proteolysis of platelet alpha-granule proteins. The increased u-PA and proteolyzed plasminogen in QPD platelets led us to investigate possible contributions of intracellular plasmin generation to QPD alpha-granule proteolysis. ELISA indicated there were normal amounts of plasminogen and plasmin-alpha(2)-antiplasmin (PAP) complexes in QPD plasmas. Like normal platelets, QPD platelets contained only a small proportion of the blood plasminogen, however, they contained an increased amount of PAP complexes compared to normal platelets (P < 0.005). The quantities of plasminogen stored in platelets were important to induce QPD-like proteolysis of normal alpha-granule proteins by two chain u-PA (tcu-PA) in vitro. Moreover, adding supplemental plasminogen to QPD, but not to control, platelet lysates, triggered further alpha-granule protein proteolysis to forms that comigrated with plasmin degraded proteins. These data suggest the generation of increased but limiting amounts of plasmin within platelets is involved in producing the unique phenotypic changes to alpha-granule proteins in QPD platelets. The QPD is the only known bleeding disorder associated with chronic, intracellular activation of the fibrinolytic cascade.

Modulation of the malignant phenotype with the urokinase-type plasminogen activator and the type I plasminogen activator inhibitor.

PubMed

Sordat, B; Reiter, L; Cajot, J F

1990-12-02

Gene transfer techniques were utilized to evaluate the role of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) in enhancing or preventing the expression of the invasive malignant phenotype, respectively. Mouse L-cell transfectants expressing human uPA or human PAI-1 as well as mouse B16 transfectants expressing mouse uPA or human PAI-1 were generated. These transfectants were tested using a variety of experimental methods including smooth muscle cell matrix solubilization in vitro, lung colony formation in vivo and co-cultures of antagonist-expressing cells in vitro. Results from these studies provide direct evidence for an enhancing role of uPA in malignant invasion and experimental metastasis and for a modulatory role of PAI-1 in tumor cell-mediated breakdown of extracellular matrices.

Role of the Yersinia pestis plasminogen activator in the incidence of distinct septicemic and bubonic forms of flea-borne plague

PubMed Central

Sebbane, Florent; Jarrett, Clayton O.; Gardner, Donald; Long, Daniel; Hinnebusch, B. Joseph

2006-01-01

Yersinia pestis is transmitted by fleas and causes bubonic plague, characterized by severe local lymphadenitis that progresses rapidly to systemic infection and life-threatening septicemia. Here, we show that although flea-borne transmission usually leads to bubonic plague in mice, it can also lead to primary septicemic plague. However, intradermal injection of Y. pestis, commonly used to mimic transmission by fleabite, leads only to bubonic plague. A Y. pestis strain lacking the plasmid-encoded cell-surface plasminogen activator, which is avirulent by intradermal or s.c. injection, was able to cause fatal primary septicemic plague at low incidence, but not bubonic plague, when transmitted by fleas. The results clarify a long-standing uncertainty about the etiology of primary septicemic plague and support an evolutionary scenario in which plague first emerged as a flea-borne septicemic disease of limited transmissibility. Subsequent acquisition of the plasminogen activator gene by horizontal transfer enabled the bubonic form of disease and increased the potential for epidemic spread. PMID:16567636

PEGylated DX-1000: pharmacokinetics and antineoplastic activity of a specific plasmin inhibitor.

PubMed

Devy, Laetitia; Rabbani, Shafaat A; Stochl, Mark; Ruskowski, Mary; Mackie, Ian; Naa, Laurent; Toews, Mark; van Gool, Reinoud; Chen, Jie; Ley, Art; Ladner, Robert C; Dransfield, Daniel T; Henderikx, Paula

2007-11-01

Novel inhibitors of the urokinase-mediated plasminogen (plg) activation system are potentially of great clinical benefit as anticancer treatments. Using phage display, we identified DX-1000 a tissue factor pathway inhibitor-derived Kunitz domain protein which is a specific high-affinity inhibitor of plasmin (pln) (K(i) = 99 pM). When tested in vitro, DX-1000 blocks plasmin-mediated pro-matrix metalloproteinase-9 (proMMP-9) activation on cells and dose-dependently inhibits tube formation, while not significantly affecting hemostasis and coagulation. However, this low-molecular weight protein inhibitor ( approximately 7 kDa) exhibits rapid plasma clearance in mice and rabbits, limiting its potential clinical use in chronic diseases. After site-specific PEGylation, DX-1000 retains its activity and exhibits a decreased plasma clearance. This PEGylated derivative is effective in vitro, as well as potent in inhibiting tumor growth of green fluorescent protein (GFP)-labeled MDA-MB-231 cells. 4PEG-DX-1000 treatment causes a significant reduction of urokinase-type plasminogen activator (uPA) and plasminogen expressions, a reduction of tumor proliferation, and vascularization. 4PEG-DX-1000 treatment significantly decreases the level of active mitogen-activated protein kinase (MAPK) in the primary tumors and reduces metastasis incidence. Together, our results demonstrate the potential value of plasmin inhibitors as therapeutic agents for blocking breast cancer growth and metastasis.

Efficient copackaging and cotransport yields postsynaptic colocalization of neuromodulators associated with synaptic plasticity.

PubMed

Lochner, J E; Spangler, E; Chavarha, M; Jacobs, C; McAllister, K; Schuttner, L C; Scalettar, B A

2008-09-01

Recent data suggest that tissue plasminogen activator (tPA) influences long-term plasticity at hippocampal synapses by converting plasminogen into plasmin, which then generates mature brain-derived neurotrophic factor (mBDNF) from its precursor, proBDNF. Motivated by this hypothesis, we used fluorescent chimeras, expressed in hippocampal neurons, to elucidate (1) mechanisms underlying plasminogen secretion from hippocampal neurons, (2) if tPA, plasminogen, and proBDNF are copackaged and cotransported in hippocampal neurons, especially within dendritic spines, and (3) mechanisms mediating the transport of these neuromodulators to sites of release. We find that plasminogen chimeras traffic through the regulated secretory pathway of hippocampal neurons in dense-core granules (DCGs) and that tPA, plasminogen, and proBDNF chimeras are extensively copackaged in DCGs throughout hippocampal neurons. We also find that 80% of spines that contain DCGs contain chimeras of these neuromodulators in the same DCG. Finally, we demonstrate, for the first time, that neuromodulators undergo cotransport along dendrites in rapidly mobile DCGs, indicating that neuromodulators can be efficiently recruited into active spines. These results support the hypothesis that tPA mediates synaptic activation of BDNF by demonstrating that tPA, plasminogen, and proBDNF colocalize in DCGs in spines, where these neuromodulators can undergo activity-dependent release and then interact and/or mediate changes that influence synaptic efficacy. The results also raise the possibility that frequency-dependent changes in extents of neuromodulator release from DCGs influence the direction of plasticity at hippocampal synapses by altering the relative proportions of two proteins, mBDNF and proBDNF, that exert opposing effects on synaptic efficacy.

Inhibition of PAI-1 Antiproteolytic Activity Against tPA by RNA Aptamers

PubMed Central

Damare, Jared; Brandal, Stephanie

2014-01-01

Plasminogen activator inhibitor-1 (PAI-1; SERPINE1) inhibits the plasminogen activators: tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Elevated levels of PAI-1 have been correlated with an increased risk for cardiovascular disease. Pharmacologically suppressing PAI-1 might prevent, or successfully treat PAI-1 related vascular diseases. This can potentially be accomplished by using small RNA molecules (aptamers). This study's goal is to develop RNA aptamers to a region of PAI-1 that will prevent the ability of PAI-1 to interact with the plasminogen activators. The aptamers were generated through a systematic evolution of ligands via exponential enrichment approach that ensures the creation of RNA molecules that bind to our target protein, PAI-1. In vitro assays were used to determine the effect of these aptamers on PAI-1's inhibitory activity. Three aptamers that bind to PAI-1 with affinities in the nanomolar range were isolated. The aptamer clones R10-4 and R10-2 inhibited PAI-1's antiproteolytic activity against tPA and disrupted PAI-1's ability to form a stable covalent complex with tPA. Increasing aptamer concentrations correlated positively with an increase in cleaved PAI-1. To the best of our knowledge, this is the first report of RNA molecules that inhibit the antiproteolytic activity of PAI-1. PMID:24922319

Influence of natural humic acids and synthetic phenolic polymers on fibrinolysis

NASA Astrophysics Data System (ADS)

Klöcking, Hans-Peter

The influence of synthetic and natural phenolic polymers on the release of plasminogen activator was studied in an isolated, perfused, vascular preparation (pig ear). Of the tested synthetic phenolic polymers, the oxidation products of caffeic acid (KOP) and 3,4-dihydroxyphenylacetic acid (3,4-DHPOP), at a concentration of 50 µg/ml perfusate, were able to increase the plasminogen activator activity by 70%. The oxidation products of chlorogenic acid (CHOP), hydrocaffeic acid (HYKOP), pyrogallol (PYROP) and gallic acid (GALOP), at the same concentration, exerted no influence on the release of plasminogen activator. Of the naturally occurring humic acids, the influence of sodium humate was within the same order of magnitude as KOP and 3,4-DHPOP. Ammonium humate was able to increase the plasminogen activator release only at a concentration of 100 µg/ml perfusate. In rats, the t-PA activity increased after i.v. application of 10 mg/kg of KOP, Na-HS or NH4-HS.

[Clinical and etiopathogenetic role of plasminogen and metaloproteinase systems in the tumor growth. Pericellular proteolysis of extracellular matrix and tumor growth].

PubMed

Cosić, Sanda Jelisavac; Kovac, Zdenko

2011-01-01

Pericellular proteolysis is a cascade process involved in degradation of extracellular matrix. This process is included in various physiological and pathological processes. Pericellullar proteolysis has major functions like degradation of tissue stroma and weakening of intercellular connections but it also has a function in the synthesis of bioactive molecules (cytokines, growth factors and inhibitory factors). Plasminogen system is involved in fibrinolysis and starts metalloproteinase activation. Activity of proteolytic molecules is controlled by the rate of zymogenic activation, half-life of molecules, and action of inhibitory molecules. Inhibition is achieved through direct binding of inhibitor and enzyme and takes a few steps. Pericellular proteolysis is involved in tumor invasion and metastasis, inflammatory reaction, degenerative diseases and other diseases. Pathophysiological regulation of pericellular proteolysis in mentioned diseases contributes to clinical properties of diseases and has diagnostic and therapeutic importance.

[In vitro function of outer membrane protease T of Escherichia coli K1 pathogenic strain].

PubMed

Hui, Changye; Guo, Yan; Wu, Shuchi; Peng, Liang; Cao, Hong; Huang, Shenghe

2010-01-01

Plasminogen activation and antimicrobial peptide hydrolysis contribute to pathogens invasion and survival in vivo. To demonstrate the expression of outer membrane protease T in E. coli K1 pathogenic strain E44, its activity of plasminogen activator and protamine hydrolysis. After Benzamidine Sepharose Fast Flow and SOURCE 30Q chromatography, we got E44 outer membrane mixed fraction, and examined its activity of plasminogen activation with chromogenic substrate S-2251 method. An ompT deletion mutant of E44 was constructed by using the suicide vector pCVD442, termed as E44ompT. We examined 0.1 mg/mL cationic antimicrobial peptide protamine susceptibility of E44, ompT mutant strain E44ompT and E44ompT harboring pUCT, which was constructed by inserting complete ompT open reading frame into pUC13. We got about 37 kDa E44 membrane extract, which could activate plasminogen, and activation was membrane extract dose dependent. This confirmed the expression of outer membrane protease T in the outer membrane of E44. E44ompT was, more susceptible to 0.1 mg/mL protamine than E44, and E440mpT was partially complemented by pUCT. Outer membrane protease T is expressed in E. coli K1 pathogenic strain E44, and can activate plasminogen and hydrolyze protamine.

Analysis of Paracoccidioides secreted proteins reveals fructose 1,6-bisphosphate aldolase as a plasminogen-binding protein.

PubMed

Chaves, Edilânia Gomes Araújo; Weber, Simone Schneider; Báo, Sonia Nair; Pereira, Luiz Augusto; Bailão, Alexandre Melo; Borges, Clayton Luiz; Soares, Célia Maria de Almeida

2015-02-27

Despite being important thermal dimorphic fungi causing Paracoccidioidomycosis, the pathogenic mechanisms that underlie the genus Paracoccidioides remain largely unknown. Microbial pathogens express molecules that can interact with human plasminogen, a protein from blood plasma, which presents fibrinolytic activity when activated into plasmin. Additionally, plasmin exhibits the ability of degrading extracellular matrix components, favoring the pathogen spread to deeper tissues. Previous work from our group demonstrated that Paracoccidioides presents enolase, as a protein able to bind and activate plasminogen, increasing the fibrinolytic activity of the pathogen, and the potential for adhesion and invasion of the fungus to host cells. By using proteomic analysis, we aimed to identify other proteins of Paracoccidioides with the ability of binding to plasminogen. In the present study, we employed proteomic analysis of the secretome, in order to identify plasminogen-binding proteins of Paracoccidioides, Pb01. Fifteen proteins were present in the fungal secretome, presenting the ability to bind to plasminogen. Those proteins are probable targets of the fungus interaction with the host; thus, they could contribute to the invasiveness of the fungus. For validation tests, we selected the protein fructose 1,6-bisphosphate aldolase (FBA), described in other pathogens as a plasminogen-binding protein. The protein FBA at the fungus surface and the recombinant FBA (rFBA) bound human plasminogen and promoted its conversion to plasmin, potentially increasing the fibrinolytic capacity of the fungus, as demonstrated in fibrin degradation assays. The addition of rFBA or anti-rFBA antibodies was capable of reducing the interaction between macrophages and Paracoccidioides, possibly by blocking the binding sites for FBA. These data reveal the possible participation of the FBA in the processes of cell adhesion and tissue invasion/dissemination of Paracoccidioides. These data indicate that Paracoccidioides is a pathogen that has several plasminogen-binding proteins that likely play important roles in pathogen-host interaction. In this context, FBA is a protein that might be involved somehow in the processes of invasion and spread of the fungus during infection.

Ultrasound-assisted lysis using recombinant tissue plasminogen activator and the EKOS EkoSonic endovascular system for treating right atrial thrombus and massive pulmonary embolism: A case study.

PubMed

Shammas, N W; Padaria, R; Ahuja, G

2015-12-01

Right atrial thrombus in the setting of a large pulmonary embolus is rare and is associated with serious adverse events. This case report presents the role played by EKOS EkoSonic ultrasound system in successfully treating right atrial thrombus and massive pulmonary embolism. A 69-year-old female presented with a massive pulmonary embolus and a large mobile right atrial thrombus. She was treated with catheter-directed lysis using the EKOS EkoSonic ultrasound system and tissue plasminogen activator, with complete resolution of her right atrial thrombus and a marked improvement in her pulmonary embolus and hemodynamics. This case report provides a new and an effective option to treat right atrial thrombus associated with a large pulmonary embolus leading to a good outcome. © The Author(s) 2014.

The fibrinolytic system: A new target for treatment of depression with psychedelics.

PubMed

Idell, R D; Florova, G; Komissarov, A A; Shetty, S; Girard, R B S; Idell, S

2017-03-01

Current understanding of the neurobiology of depression has grown over the past few years beyond the traditional monoamine theory of depression to include chronic stress, inflammation and disrupted synaptic plasticity. Tissue plasminogen activator (tPA) is a key factor that not only promotes fibrinolysis via the activation of plasminogen, but also contributes to regulation of synaptic plasticity and neurogenesis through plasmin-mediated activation of a probrain derived neurotrophic factor (BDNF) to mature BDNF. ProBDNF activation could potentially be supressed by competition with fibrin for plasmin and tPA. High affinity binding of plasmin and tPA to fibrin could result in a decrease of proBDNF activation during brain inflammation leading to fibrosis further perpetuating depressed mood. There is a paucity of data explaining the possible role of the fibrinolytic system or aberrant extravascular fibrin deposition in depression. We propose that within the brain, an imbalance between tPA and urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) and neuroserpin favors the inhibitors, resulting in changes in neurogenesis, synaptic plasticity, and neuroinflammation that result in depressive behavior. Our hypothesis is that peripheral inflammation mediates neuroinflammation, and that cytokines such as tumor necrosis factor alpha (TNF-α) can inhibit the fibrinolytic system by up- regulating PAI-1 and potentially neuroserpin. We propose that the decrement of the activity of tPA and uPA occurs with downregulation of uPA in part involving the binding and clearance from the surface of neural cells of uPA/PAI-1 complexes by the urokinase receptor uPAR. We infer that current antidepressants and ketamine mitigate depressive symptoms by restoring the balance of the fibrinolytic system with increased activity of tPA and uPA with down-regulated intracerebral expression of their inhibitors. We lastly hypothesize that psychedelic 5-ht2a receptor agonists, such as psilocybin, can improve mood through anti- inflammatory and pro-fibrinolytic effects that include blockade of TNF-α activity leading to decreased PAI-1 activity and increased clearance. The process involves disinhibition of tPA and uPA with subsequent increased cleavage of proBDNF which promotes neurogenesis, decreased neuroinflammation, decreased fibrin deposition, normalized glial-neuronal cross-talk, and optimally functioning neuro-circuits involved in mood. We propose that psilocybin can alleviate deleterious changes in the brain caused by chronic stress leading to restoration of homeostatic brain fibrinolytic capacity leading to euthymia. Copyright © 2017 Elsevier Ltd. All rights reserved.

Role of the urokinase plasminogen activator receptor in mediating impaired efferocytosis of anti-SSA/Ro-bound apoptotic cardiocytes: Implications in the pathogenesis of congenital heart block.

PubMed

Briassouli, Paraskevi; Komissarova, Elena V; Clancy, Robert M; Buyon, Jill P

2010-08-06

Binding of maternal anti-Ro/La antibodies to cognate antigen expressed on apoptotic cardiocytes decreases clearance by healthy cardiocytes, which may contribute to the development of autoimmune associated congenital heart block and fatal cardiomyopathy. Given recent evidence implicating the urokinase plasminogen activator receptor (uPAR) as a "don't eat me" signal during efferocytosis, experiments addressed whether surface bound anti-Ro antibodies inhibit apoptotic cell removal via an effect on the expression/function of the urokinase-type plasminogen activator protease uPA/uPAR system. As assessed by flow cytometry and confocal microscopy, uPAR colocalizes and interacts with Ro60 on the surface of apoptotic human fetal cardiocytes. Blocking of uPAR enhances phagocytosis of apoptotic cardiocytes by healthy cardiocytes and reverses the anti-Ro60-dependent impaired clearance of apoptotic cardiocytes. Binding of anti-Ro60 antibodies to apoptotic cardiocytes results in increased uPAR expression, as well as enhanced uPA activity. The binding of anti-Ro60 did not alter other surface molecules involved in cell recognition (calreticulin, CD31, or CD47). These data suggest that increased uPAR expression and uPA activity induced by anti-Ro60 binding to the apoptotic fetal cardiocyte provide a molecular basis by which these antibodies inhibit efferocytosis and ultimately lead to scar of the fetal conduction system and working myocardium.

Efficient co-packaging and co-transport yields post-synaptic co-localization of neuromodulators associated with synaptic plasticity

PubMed Central

Lochner, J. E.; Spangler, E.; Chavarha, M.; Jacobs, C.; McAllister, K.; Schuttner, L. C.; Scalettar, B. A.

2009-01-01

Recent data suggest that tissue plasminogen activator (tPA) influences long-term plasticity at hippocampal synapses by converting plasminogen into plasmin, which then generates mature brain-derived neurotrophic factor (mBDNF) from its precursor, proBDNF. Motivated by this hypothesis, we used fluorescent chimeras, expressed in hippocampal neurons, to elucidate (1) mechanisms underlying plasminogen secretion from hippocampal neurons, (2) if tPA, plasminogen, and proBDNF are co-packaged and co-transported in hippocampal neurons, especially within dendritic spines, and (3) mechanisms mediating the transport of these neuromodulators to sites of release. We find that plasminogen chimeras traffic through the regulated secretory pathway of hippocampal neurons in dense-core granules (DCGs) and that tPA, plasminogen, and proBDNF chimeras are extensively co-packaged in DCGs throughout hippocampal neurons. We also find that 80% of spines that contain DCGs contain chimeras of these neuromodulators in the same DCG. Finally, we demonstrate, for the first time, that neuromodulators undergo co-transport along dendrites in rapidly mobile DCGs, indicating that neuromodulators can be efficiently recruited into active spines. These results support the hypothesis that tPA mediates synaptic activation of BDNF by demonstrating that tPA, plasminogen, and proBDNF co-localize in DCGs in spines, where these neuromodulators can undergo activity-dependent release and then interact and/or mediate changes that influence synaptic efficacy. The results also raise the possibility that frequency-dependent changes in extents of neuromodulator release from DCGs influence the direction of plasticity at hippocampal synapses by altering the relative proportions of two proteins, mBDNF and proBDNF, that exert opposing effects on synaptic efficacy. PMID:18563704

Dose-ranging study of the novel recombinant plasminogen activator BM 06.022 in healthy volunteers.

PubMed

Martin, U; von Möllendorff, E; Akpan, W; Kientsch-Engel, R; Kaufmann, B; Neugebauer, G

1991-10-01

The novel recombinant plasminogen activator BM 06.022 consists of the kringle 2 and protease domains of human tissue-type plasminogen activator and is unglycosylated because of its expression in Escherichia coli cells. Pharmacokinetics for activity and hemostatic effects of BM 06.022 were studied in 18 healthy male volunteers after an intravenous bolus injection over 2 minutes. BM 06.022 was administered successively at doses of 0.1125, 0.55, 2.2, 3.3, 4.4, and 5.5 MU to three volunteers. Plasma fibrinogen was unchanged; effects of BM 06.022 were observed on plasminogen only at higher doses, and dose-dependent effects were seen on alpha 2-antiplasmin and fibrin D-dimers. The concentration of plasminogen and alpha 2-antiplasmin was 87% +/- 3% and 79% +/- 3%, respectively, of baseline 2 hours after injection of 5.5 MU of BM 06.022. Fibrin D-dimers were highest with 1147 +/- 380 ng/ml at 5.5 MU of BM 06.022. The area under the activity concentration-time curve (AUC) increased dose-dependently and linearly. At 5.5 MU of BM 06.022, the AUC was 313 +/- 47 IU.hr.ml-1, the total plasma clearance was 306 +/- 40 ml/min, and the half-life was 14.4 +/- 1.1 minutes.

A case control study on the structural equation model of the mechanism of coagulation and fibrinolysis imbalance in chronic schistosomiasis.

PubMed

Le, Aiping; Zhang, Lunli; Liu, Wei; Li, Xiaopeng; Ren, Jianwei; Ning, An

2017-02-01

A structural equation model was used for verification with chronic schistosomiasis to investigate the coagulation-anticoagulation system imbalance and to deduce the mechanism of D-dimer (D-D) level elevation in patients with advanced schistosome hepatic disease. We detected the plasma levels of tissue-type fiber plasminogen activator (tPA), urokinase type plasminogen activator (uPA), plasmin-antiplasmin complex (PAP), plasminogen (PLG), antithrombin (AT), plasminogen activator inhibitor 1 (PAI1), D-D, factor VIII: C (FVIII:C), antithrombin-III (AT-III), PLG, protein S (PS), and protein C (PC) in the healthy people as control (69), patients with chronic schistosomiasis (150) or advanced chronic schistosomiasis (90). FVIII, PAP, D-D, tPA, and uPA plasma levels were significantly higher in the chronic group than in the control group and were also significantly higher in the advanced group. However, AT-III, PC, PS, AT, PLG, and PAI1 plasma levels in the advanced and chronic groups were significantly lower than those in the control group. With progression of disease in patients with schistosomiasis japonica, a hypercoagulable state is induced by the coagulation-anticoagulation imbalance, eventually leading to patients with high levels of D-D. Furthermore, we established a structural equation model path of a "chronic schistosomiasis disease stage-(coagulation-anticoagulation-fibrinolysis)-D-D." By using analysis of moment structures (AMOS), it was shown that the chronic schistosomiasis stage was positively related to factor VIII and had negative correlation with AT-III; a good positive correlation with PAP, tPA, and uPA; and a good negative correlation with PLG and PAI1. In addition, our results show that the path coefficient of anticoagulation-fibrinolysis system to the chronic stage of schistosomiasis or D-D levels was significantly higher than that of the coagulation system. In conclusion, the coagulation and fibrinolysis imbalance in patients with chronic schistosomiasis, especially with advanced schistosomiasis, is due to the progression of disease stages.

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

Biochemical actions of glucocorticoids on macrophages in culture. Specific inhibition of elastase, collagenase, and plasminogen activator secretion and effects on other metabolic functions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Werb, Z.

1978-01-01

The effects of glucocorticoids on biochemical functions of macrophages from man, mouse, rabbit, and guinea pig were examined. Secretion of plasminogen activator by human peripheral blood monocytes was decreased 50% with 1 nM dexamethasone. Differentiation of murine monocytic and granulocytic colonies in agar from bone marrow precursors was decreased 50% at 7 days with 20 nM dexamethasone. Secretion of elastase, collagenase, and plasminogen activator by resident and thioglycollate-elicited mouse peritoneal macrophages was decreased by dexamethasone, cortisol, and triamcinolone acetonide (1 to 1,000 nM), but not by progesterone, estradiol, and dihydrotestosterone (1,000 nM); in contast, secretion of lysozyme was not affectedmore » by glucocorticoids. The inhibition of macrophage secretion by dexamethasone was both time and dose dependent. Inhibition of macrophage secretion increased with increasing glucocorticoid concentration. Half-maximum inhibition of secretion of elastase, collagenase, and plasminogen activator was seen at dexamethasone concentrations (1 to 10 nM) similar to those that half-saturated the specific glucocorticoid receptors. At high concentrations of dexamethasone (100 to 1,000 nM) the secretion of plasminogen activator was inhibited to a greater extent (>95%) than the secretion of elastase (60 to 80%).Progesterone alone had no effect on secretion, but blocked the inhibitory effects of dexamethasone and cortisol. Secretion of collagenase, neutral proteinases, and plasminogen activator by elicited rabbit alveolar macrophages was inhibited with glucocorticoids (0.1 to 100 nM) but not with progesterone or sex steroids. Secretion of a neutral elastinolytic proteinase by guinea pig alveolar macrophages was also inhibited by dexamethasone.« less

A collaborative sequential meta-analysis of individual patient data from randomized trials of endovascular therapy and tPA vs. tPA alone for acute ischemic stroke: ThRombEctomy And tPA (TREAT) analysis: statistical analysis plan for a sequential meta-analysis performed within the VISTA-Endovascular collaboration.

PubMed

MacIsaac, Rachael L; Khatri, Pooja; Bendszus, Martin; Bracard, Serge; Broderick, Joseph; Campbell, Bruce; Ciccone, Alfonso; Dávalos, Antoni; Davis, Stephen M; Demchuk, Andrew; Diener, Hans-Christoph; Dippel, Diederik; Donnan, Geoffrey A; Fiehler, Jens; Fiorella, David; Goyal, Mayank; Hacke, Werner; Hill, Michael D; Jahan, Reza; Jauch, Edward; Jovin, Tudor; Kidwell, Chelsea S; Liebeskind, David; Majoie, Charles B; Martins, Sheila Cristina Ouriques; Mitchell, Peter; Mocco, J; Muir, Keith W; Nogueira, Raul; Saver, Jeffrey L; Schonewille, Wouter J; Siddiqui, Adnan H; Thomalla, Götz; Tomsick, Thomas A; Turk, Aquilla S; White, Philip; Zaidat, Osama; Lees, Kennedy R

2015-10-01

Endovascular treatment has been shown to restore blood flow effectively. Second-generation medical devices such as stent retrievers are now showing overwhelming efficacy in clinical trials, particularly in conjunction with intravenous recombinant tissue plasminogen activator. This statistical analysis plan utilizing a novel, sequential approach describes a prospective, individual patient data analysis of endovascular therapy in conjunction with intravenous recombinant tissue plasminogen activator agreed upon by the Thrombectomy and Tissue Plasminogen Activator Collaborative Group. This protocol will specify the primary outcome for efficacy, as 'favorable' outcome defined by the ordinal distribution of the modified Rankin Scale measured at three-months poststroke, but with modified Rankin Scales 5 and 6 collapsed into a single category. The primary analysis will aim to answer the questions: 'what is the treatment effect of endovascular therapy with intravenous recombinant tissue plasminogen activator compared to intravenous tissue plasminogen activator alone on full scale modified Rankin Scale at 3 months?' and 'to what extent do key patient characteristics influence the treatment effect of endovascular therapy?'. Key secondary outcomes include effect of endovascular therapy on death within 90 days; analyses of modified Rankin Scale using dichotomized methods; and effects of endovascular therapy on symptomatic intracranial hemorrhage. Several secondary analyses will be considered as well as expanding patient cohorts to intravenous recombinant tissue plasminogen activator-ineligible patients, should data allow. This collaborative meta-analysis of individual participant data from randomized trials of endovascular therapy vs. control in conjunction with intravenous thrombolysis will demonstrate the efficacy and generalizability of endovascular therapy with intravenous thrombolysis as a concomitant medication. © 2015 World Stroke Organization.

A novel embryo culture media supplement that improves pregnancy rates in mice.

PubMed

Highet, A R; Bianco-Miotto, T; Pringle, K G; Peura, A; Bent, S; Zhang, J; Nottle, M B; Thompson, J G; Roberts, C T

2017-03-01

The preimplantation embryo in vivo is exposed to numerous growth factors in the female reproductive tract, which are not recapitulated in embryo culture media in vitro The IGF2 and plasminogen activator systems facilitate blastocyst development. We hypothesized that the addition of IGF2 in combination with urokinase plasminogen activator (uPA) and plasminogen could improve rates of blastocyst hatching and implantation in mice. B6BcF1 and CBAB6F2 mouse embryos were divided into one of four supplemented culture media treatment groups: (1) control (media only); (2) 12.5 nM IGF2; (3) 10 µg/mL uPA and 5 µg/mL plasminogen; or (4) a combination of IGF2, uPA and plasminogen treatments. Embryo development to blastocyst stage and hatching were assessed before transfer to pseudopregnant recipient females and implantation, pregnancy rates and postnatal growth were assessed. After 90.5 h of culture, IGF2 + U + P treatment increased the percentage of B6BcF1 embryos that were hatching/hatched and percentage developing to blastocyst stage compared with controls (P < 0.02). Following B6BcF1 embryo transfer, IGF2 + U + P treatment increased implantation sites at day 8 of pregnancy compared with controls (P < 0.05). Replication in the CBAB6F2 mouse strain showed significant improvements in pregnancy rates at days 8 and 18 but not in blastocyst development. No adverse effects were seen on gestational age, litter size or birthweight, or the reproductive capacity of offspring of IGF2 + U + P treated embryos. For embryos susceptible to detrimental effects of in vitro culture, IGF2, uPA and plasminogen supplementation of culture media can improve pregnancy success, but the effect of treatment is dependent on the mouse strain. © 2017 Society for Reproduction and Fertility.

Treatment With Tissue Plasminogen Activator in the Golden Hour and the Shape of the 4.5-Hour Time-Benefit Curve in the National United States Get With The Guidelines-Stroke Population.

PubMed

Kim, Joon-Tae; Fonarow, Gregg C; Smith, Eric E; Reeves, Mathew J; Navalkele, Digvijaya D; Grotta, James C; Grau-Sepulveda, Maria V; Hernandez, Adrian F; Peterson, Eric D; Schwamm, Lee H; Saver, Jeffrey L

2017-01-10

Earlier tissue plasminogen activator treatment improves ischemic stroke outcome, but aspects of the time-benefit relationship still not well delineated are: (1) the degree of additional benefit accrued with treatment in the first 60 minutes after onset, and (2) the shape of the time-benefit curve through 4.5 hours. We analyzed patients who had acute ischemic stroke treated with intravenous tissue plasminogen activator within 4.5 hours of onset from the Get With The Guidelines-Stroke US national program. Onset-to-treatment time was analyzed as a continuous, potentially nonlinear variable and as a categorical variable comparing patients treated within 60 minutes of onset with later epochs. Among 65 384 tissue plasminogen activator-treated patients, the median onset-to-treatment time was 141 minutes (interquartile range, 110-173) and 878 patients (1.3%) were treated within the first 60 minutes. Treatment within 60 minutes, compared with treatment within 61 to 270 minutes, was associated with increased odds of discharge to home (adjusted odds ratio, 1.25; 95% confidence interval, 1.07-1.45), independent ambulation at discharge (adjusted odds ratio, 1.22; 95% confidence interval, 1.03-1.45), and freedom from disability (modified Rankin Scale 0-1) at discharge (adjusted odds ratio, 1.72; 95% confidence interval, 1.21-2.46), without increased hemorrhagic complications or in-hospital mortality. The pace of decline in benefit of tissue plasminogen activator from onset-to-treatment times of 20 through 270 minutes was mildly nonlinear for discharge to home, with more rapid benefit loss in the first 170 minutes than later, and linear for independent ambulation and in-hospital mortality. Thrombolysis started within the first 60 minutes after onset is associated with best outcomes for patients with acute ischemic stroke, and benefit declined more rapidly early after onset for the ability to be discharged home. These findings support intensive efforts to organize stroke systems of care to improve the timeliness of thrombolytic therapy in acute ischemic stroke. © 2016 American Heart Association, Inc.

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.

The Serine Protease Inhibitor Neuroserpin Is Required for Normal Synaptic Plasticity and Regulates Learning and Social Behavior

ERIC Educational Resources Information Center

Reumann, Rebecca; Vierk, Ricardo; Zhou, Lepu; Gries, Frederice; Kraus, Vanessa; Mienert, Julia; Romswinkel, Eva; Morellini, Fabio; Ferrer, Isidre; Nicolini, Chiara; Fahnestock, Margaret; Rune, Gabriele; Glatzel, Markus; Galliciotti, Giovanna

2017-01-01

The serine protease inhibitor neuroserpin regulates the activity of tissue-type plasminogen activator (tPA) in the nervous system. Neuroserpin expression is particularly prominent at late stages of neuronal development in most regions of the central nervous system (CNS), whereas it is restricted to regions related to learning and memory in the…

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

USDA-ARS?s Scientific Manuscript database

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...

Fibrin-Enhanced Canonical Wnt Signaling Directs Plasminogen Expression in Cementoblasts

PubMed Central

Rahman, Saeed Ur; Ryoo, Hyun-Mo

2017-01-01

Cementum is a mineralized layer on the tooth’s root surface and facilitates the biomechanical anchoring of fibrous connective tissues as a part of tooth-supportive complexes. Previously, we observed that OCCM30 cementoblasts cultured on fibrin matrices underwent apoptosis due to fibrin degradation through the expression of proteases. Here, we demonstrated that OCCM30 on fibrin matrices (OCCM30-fibrin) enhanced canonical Wnt signaling, which directed to plasminogen expression. The OCCM30-fibrin showed higher levels of Wnt3a expression, nuclear translocation of β-catenin, and T-cell factor (TCF) optimal motif (TOP) reporter activity than the cells on tissue culture dishes (OCCM30-TCD), indicating that the OCCM30-fibrin enhanced canonical Wnt/β-catenin signaling. Also, OCCM30-fibrin expressed biomineralization-associated markers at higher levels than OCCM30-TCD, of which levels were further increased with LiCl, a Wnt signaling activator. The OCCM30 cementoblasts simultaneously showed that high levels of plasminogen, a critical component of fibrinolysis, were expressed in the OCCM30-fibrin. Activation of canonical Wnt signaling with LiCl treatment or with forced lymphoid enhancer factor 1 (LEF1)-expression increased the expression of plasminogen. On the contrary, the inhibition of canonical Wnt signaling with siRNAs against Wnt3a or β-catenin abrogated fibrin-enhanced plasminogen expression. Furthermore, there are three conserved putative response elements for the LEF1/β-catenin complex in the plasminogen proximal promoter regions (−900 to +54). Site-directed mutations and chromatin immunoprecipitation indicated that canonical Wnt signaling directed plasminogen expression. Taken together, this study suggests that fibrin-based materials can modulate functional periodontal formations in controlling cementoblast differentiation and fibrin degradation. PMID:29120400

The Plasminogen Activation System Promotes Dendritic Spine Recovery and Improvement in Neurological Function After an Ischemic Stroke

PubMed Central

Jeanneret, Valerie; Yepes, Manuel

2016-01-01

Advances in neurocritical care and interventional neuroradiology have led to a significant decrease in acute ischemic stroke (AIS) mortality. In contrast, due to the lack of an effective therapeutic strategy to promote neuronal recovery among AIS survivors, cerebral ischemia is still a leading cause of disability in the world. Ischemic stroke has a harmful impact on synaptic structure and function, and plasticity-mediated synaptic recovery is associated with neurological improvement following an AIS. Dendritic spines (DSs) are specialized dendritic protrusions that receive most of the excitatory input in the brain. The deleterious effect of cerebral ischemia on DSs morphology and function has been associated with impaired synaptic transmission and neurological deterioration. However, these changes are reversible if cerebral blood flow is restored on time, and this recovery has been associated with neurological improvement following an AIS. Tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) are two serine proteases that besides catalyzing the conversion of plasminogen into plasmin in the intravascular and pericellular environment, respectively, are also are efficient inductors of synaptic plasticity. Accordingly, recent evidence indicates that both, tPA and uPA, protect DSs from the metabolic stress associated with the ischemic injury, and promote their morphological and functional recovery during the recovery phase from an AIS. Here we will review data indicating that plasticity-induced changes in DSs and the associated post-synaptic density play a pivotal role in the recovery process from AIS, making special emphasis on the role of tPA and uPA in this process. PMID:26846991

Penicillin binding protein 3 of Staphylococcus aureus NCTC 8325-4 binds and activates human plasminogen.

PubMed

Kylväjä, Riikka; Ojalehto, Tuomas; Kainulainen, Veera; Virkola, Ritva; Westerlund-Wikström, Benita

2016-08-04

Staphylococcus aureus is a versatile pathogen expressing a number of virulence-associated adhesive molecules. In a previous study, we generated in a secretion-competent Escherichia coli strain a library of random FLAG-tag positive (FTP) polypeptides of S. aureus. To identify adhesive proteins and gain additional knowledge on putative virulence factors of S. aureus, we here screened the FTP library against human serum proteins. Staphylococcus aureus NCTC 8325-4, origin of the FTP library, adhered to immobilized plasminogen in vitro. In an enzyme-linked immunoassay a C-terminal part of penicillin binding protein 3 (PBP3), included in the FTP library, bound to immobilized plasminogen. We expressed and purified full-length PBP3 and its C-terminal fragments as recombinant proteins. In a time-resolved fluorometry-based assay the PBP3 polypeptides bound to immobilized plasminogen. The polypeptides enhanced formation of plasmin from plasminogen as analyzed by cleavage of a chromogenic plasmin substrate. The present findings, although preliminary, demonstrate reliably that S. aureus NCTC 8325-4 adheres to immobilized plasminogen in vitro and that the adhesion may be mediated by a C-terminal fragment of the PBP3 protein. The full length PBP3 and the penicillin binding C-terminal domain of PBP3 expressed as recombinant proteins bound plasminogen and activated plasminogen to plasmin. These phenomena were inhibited by the lysine analogue ε-aminocaproic acid suggesting that the binding is mediated by lysine residues. A detailed molecular description of surface molecules enhancing the virulence of S. aureus will aid in understanding of its pathogenicity and help in design of antibacterial drugs in the future.

Effects of TGF-β1 on plasminogen activation in human dental pulp cells: Role of ALK5/Smad2, TAK1 and MEK/ERK signalling.

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

2018-04-01

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 signalling pathways. Dental pulp cells were treated with different concentrations of TGF-β1. MTT assay, reverse transcription-polymerase chain reaction, 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 analysed by Pathscan ELISA or Western blotting. Cells were pretreated with SB431542 (ALK5/Smad2/3 inhibitor), 5z-7-oxozeaenol (TAK1 inhibitor) and U0126 (MEK/ERK inhibitor) for examining the related signalling. TGF-β1 slightly inhibited cell growth that was reversed by SB431542. TGF-β1 upregulated both RNA and protein expression of PAI-1 and uPAR, whereas it downregulated 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 and uPAR. These effects of TGF-β1 are related to activation of ALK5/Smad2, TAK1 and MEK/ERK signalling pathways. Clarifying the signal transduction for the effects of TGF-β1 is helpful for pulpo-dentin regeneration and tissue engineering. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Fibrinolytic and procoagulant activities of Yersinia pestis and Salmonella enterica.

PubMed

Korhonen, T K

2015-06-01

Pla of the plague bacterium Yersinia pestis and PgtE of the enteropathogen Salmonella enterica are surface-exposed, transmembrane β-barrel proteases of the omptin family that exhibit a complex array of interactions with the hemostatic systems in vitro, and both proteases are established virulence factors. Pla favors fibrinolysis by direct activation of plasminogen, inactivation of the serpins plasminogen activator inhibitor-1 and α2-antiplasmin, inactivation of the thrombin-activable fibrinolysis inhibitor, and activation of single-chain urokinase. PgtE is structurally very similar but exhibits partially different functions and differ in expression control. PgtE proteolysis targets control aspects of fibrinolysis, and mimicry of matrix metalloproteinases enhances cell migration that should favor the intracellular spread of the bacterium. Enzymatic activity of both proteases is strongly influenced by the environment-induced variations in lipopolysaccharide that binds to the β-barrel. Both proteases cleave the tissue factor pathway inhibitor and thus also express procoagulant activity. © 2015 International Society on Thrombosis and Haemostasis.

Plasminogen activator inhibitor links obesity and thrombotic cerebrovascular diseases: The roles of PAI-1 and obesity on stroke.

PubMed

Chen, Rui; Yan, Jinchuan; Liu, Peijing; Wang, Zhongqun; Wang, Cuiping

2017-06-01

One of the global socioeconomic phenomena occurred during the last decades is the increased prevalence of obesity, with direct consequence on the risk of developing thrombotic disorders. As the physiological inhibitor of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1) is well known for its role in fibrinolysis. More and more evidences have shown that PAI-1 involves in physiopathologic mechanisms of many diseases and metabolic disorder. Increased serum level of PAI-1 has been observed in obesity and it also contributes to the development of adipose tissue and then has effects on obesity. Meantime, obesity affects also the PAI-1 levels. These evidences indicate the complicated interaction between PAI-1 and obesity. Many clinic studies have confirmed that obesity relates to the stroke outcome although there are many contradictory results. Simultaneously, correlation is found between plasma PAI-1 and thrombotic cerebrovascular diseases. This article reviews contemporary knowledge regarding the complex interplay of obesity, PAI-1 and stroke.

Structural Basis of Interaction between Urokinase-Type Plasminogen Activator and Its Receptor

PubMed Central

Barinka, Cyril; Parry, Graham; Callahan, Jennifer; Shaw, David E.; Kuo, Alice; Bdeir, Khalil; Cines, Douglas B.; Mazar, Andrew; Lubkowski, Jacek

2009-01-01

Summary Recent studies indicate that binding of 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 as to the exact mode of uPAR-uPA interactions and the presumed conformational changes that accompanying 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 Å. We also report the 1.9 Å crystal structure of the 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. PMID:16979660

Highly potent fibrinolytic serine protease from Streptomyces.

PubMed

Uesugi, Yoshiko; Usuki, Hirokazu; Iwabuchi, Masaki; Hatanaka, Tadashi

2011-01-05

We introduce a highly potent fibrinolytic serine protease from Streptomyces omiyaensis (SOT), which belongs to the trypsin family. The fibrinolytic activity of SOT was examined using in vitro assays and was compared with those of known fibrinolytic enzymes such as plasmin, tissue-type plasminogen activator (t-PA), urokinase, and nattokinase. Compared to other enzymes, SOT showed remarkably higher hydrolytic activity toward mimic peptides of fibrin and plasminogen. The fibrinolytic activity of SOT is about 18-fold higher than that of plasmin, and is comparable to that of t-PA by fibrin plate assays. Furthermore, SOT had some plasminogen activator-like activity. Results show that SOT and nattokinase have very different fibrinolytic and fibrinogenolytic modes, engendering significant synergetic effects of SOT and nattokinase on fibrinolysis. These results suggest that SOT presents important possibilities for application in the therapy of thrombosis. Copyright © 2010 Elsevier Inc. All rights reserved.

Lingual Haematoma due to Tenecteplase in a Patient with Acute Myocardial Infarction

PubMed Central

Bal, Muhlis; Salturk, Ziya; Ateş, Ahmet Hakan; Yağcı, Serkan; Coşkun Bal, Gökçen

2013-01-01

The use of intravenous thrombolytic agents has revolutionised the treatment of acute myocardial infarction. However, the improvement in mortality rate achieved with these drugs is tempered by the risk of serious bleeding complications, including intracranial haemorrhage. Tenecteplase is a genetically engineered mutant tissue plasminogen activator. Haemorrhagic complications of tissue plasminogen activator (tPA) are well known. Compared to other tPAs, tenecteplase use leads to lower rates of bleeding complications. Here, we report a case of unusual site of spontaneous bleeding, intralingual haematoma during tenecteplase therapy following acute myocardial infarction, which caused significant upper airway obstruction and required tracheotomy to maintain the patient's airway. Clinical dilemmas related to securing the airway or reversing the effects of tissue plasminogen activator are discussed. PMID:23862086

Subunits of the Pyruvate Dehydrogenase Cluster of Mycoplasma pneumoniae Are Surface-Displayed Proteins that Bind and Activate Human Plasminogen

PubMed Central

Gründel, Anne; Friedrich, Kathleen; Pfeiffer, Melanie; Jacobs, Enno; Dumke, Roger

2015-01-01

The dual role of glycolytic enzymes in cytosol-located metabolic processes and in cell surface-mediated functions with an influence on virulence is described for various micro-organisms. Cell wall-less bacteria of the class Mollicutes including the common human pathogen Mycoplasma pneumoniae possess a reduced genome limiting the repertoire of virulence factors and metabolic pathways. After the initial contact of bacteria with cells of the respiratory epithelium via a specialized complex of adhesins and release of cell-damaging factors, surface-displayed glycolytic enzymes may facilitate the further interaction between host and microbe. In this study, we described detection of the four subunits of pyruvate dehydrogenase complex (PDHA-D) among the cytosolic and membrane-associated proteins of M. pneumoniae. Subunits of PDH were cloned, expressed and purified to produce specific polyclonal guinea pig antisera. Using colony blotting, fractionation of total proteins and immunofluorescence experiments, the surface localization of PDHA-C was demonstrated. All recombinant PDH subunits are able to bind to HeLa cells and human plasminogen. These interactions can be specifically blocked by the corresponding polyclonal antisera. In addition, an influence of ionic interactions on PDHC-binding to plasminogen as well as of lysine residues on the association of PDHA-D with plasminogen was confirmed. The PDHB subunit was shown to activate plasminogen and the PDHB-plasminogen complex induces degradation of human fibrinogen. Hence, our data indicate that the surface-associated PDH subunits might play a role in the pathogenesis of M. pneumoniae infections by interaction with human plasminogen. PMID:25978044

Cpa, the outer membrane protease of Cronobacter sakazakii, activates plasminogen and mediates resistance to serum bactericidal activity.

PubMed

Franco, A A; Kothary, M H; Gopinath, G; Jarvis, K G; Grim, C J; Hu, L; Datta, A R; McCardell, B A; Tall, B D

2011-04-01

Cronobacter spp. are emerging neonatal pathogens in humans, associated with outbreaks of meningitis and sepsis. To cause disease, they must survive in blood and invade the central nervous system by penetrating the blood-brain barrier. C. sakazakii BAA-894 possesses an ~131-kb plasmid (pESA3) that encodes an outer membrane protease (Cpa) that has significant identity to proteins that belong to the Pla subfamily of omptins. Members of this subfamily of proteins degrade a number of serum proteins, including circulating complement, providing protection from the complement-dependent serum killing. Moreover, proteins of the Pla subfamily can cause uncontrolled plasmin activity by converting plasminogen to plasmin and inactivating the plasmin inhibitor α2-antiplasmin (α2-AP). These reactions enhance the spread and invasion of bacteria in the host. In this study, we found that an isogenic cpa mutant showed reduced resistance to serum in comparison to its parent C. sakazakii BAA-894 strain. Overexpression of Cpa in C. sakazakii or Escherichia coli DH5α showed that Cpa proteolytically cleaved complement components C3, C3a, and C4b. Furthermore, a strain of C. sakazakii overexpressing Cpa caused a rapid activation of plasminogen and inactivation of α2-AP. These results strongly suggest that Cpa may be an important virulence factor involved in serum resistance, as well as in the spread and invasion of C. sakazakii.

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.

Uninvolved Skin from Psoriatic Patients Develops Signs of Involved Psoriatic Skin after Being Grafted onto Nude Mice

NASA Astrophysics Data System (ADS)

Fraki, Jorma E.; Briggaman, Robert A.; Lazarus, Gerald S.

1982-02-01

Clinically involved psoriatic epidermis maintains its histological appearance, increased labeling index, and increased level of plasminogen activator after being grafted onto athymic nude mice. Uninvolved psoriatic epidermis develops increases in plasminogen activator activity after being grafted onto athymic nude mice; this is accompanied by an increased labeling index. Thus, psoriatic skin can develop markers of psoriasis independent of the host.

Effects of Korean Red Ginseng extract on tissue plasminogen activator and plasminogen activator inhibitor-1 expression in cultured rat primary astrocytes

PubMed Central

Ko, Hyun Myung; Joo, So Hyun; Kim, Pitna; Park, Jin Hee; Kim, Hee Jin; Bahn, Geon Ho; Kim, Hahn Young; Lee, Jongmin; Han, Seol-Heui; Shin, Chan Young; Park, Seung Hwa

2013-01-01

Korean Red Ginseng (KRG) is an oriental herbal preparation obtained from Panax ginseng Meyer (Araliaceae). To expand our understanding of the action of KRG on central nervous system (CNS) function, we examined the effects of KRG on tissue plasminogen activator (tPA)/plasminogen activator inhibitor-1 (PAI-1) expression in rat primary astrocytes. KRG extract was treated in cultured rat primary astrocytes and neuron in a concentration range of 0.1 to 1.0 mg/mL and the expression of functional tPA/PAI-1 was examined by casein zymography, Western blot and reverse transcription-polymerase chain reaction. KRG extracts increased PAI-1 expression in rat primary astrocytes in a concentration dependent manner (0.1 to 1.0 mg/mL) without affecting the expression of tPA itself. Treatment of 1.0 mg/mL KRG increased PAI-1 protein expression in rat primary astrocytes to 319.3±65.9% as compared with control. The increased PAI-1 expression mediated the overall decrease in tPA activity in rat primary astrocytes. Due to the lack of PAI-1 expression in neuron, KRG did not affect tPA activity in neuron. KRG treatment induced a concentration dependent activation of PI3K, p38, ERK1/2, and JNK in rat primary astrocytes and treatment of PI3K or MAPK inhibitors such as LY294002, U0126, SB203580, and SP600125 (10 μM each), significantly inhibited 1.0 mg/mL KRG-induced expression of PAI- 1 and down-regulation of tPA activity in rat primary astrocytes. Furthermore, compound K but not other ginsenosides such as Rb1 and Rg1 induced PAI-1 expression. KRG-induced up-regulation of PAI-1 in astrocytes may play important role in the regulation of overall tPA activity in brain, which might underlie some of the beneficial effects of KRG on CNS such as neuroprotection in ischemia and brain damaging condition as well as prevention or recovery from addiction. PMID:24235858

Differential Regulation of PAI-1 in Hantavirus Cardiopulmonary Syndrome and Hemorrhagic Fever With Renal Syndrome.

PubMed

Bellomo, Carla; Korva, Miša; Papa, Anna; Mäkelä, Satu; Mustonen, Jukka; Avšič-Županc, Tatjana; Vaheri, Antti; Martinez, Valeria P; Strandin, Tomas

2018-02-01

We analyzed the levels of circulating tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI)-1 in acute hantavirus cardiopulmonary syndrome (HCPS) and hemorrhagic fever with renal syndrome (HFRS). The levels of tPA commonly increased in both diseases, whereas PAI-1 correlated with disease severity in HCPS but not in HFRS.

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

USDA-ARS?s Scientific Manuscript database

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...

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

USDA-ARS?s Scientific Manuscript database

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...

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eren, Elif; Murphy, Megan; Goguen, Jon

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 changesmore » 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.« less

Dose dependency of outcomes of intrapleural fibrinolytic therapy in new rabbit empyema models

PubMed Central

Florova, Galina; Azghani, Ali O.; Buchanan, Ann; Boren, Jake; Allen, Timothy; Rahman, Najib M.; Koenig, Kathleen; Chamiso, Mignote; Karandashova, Sophia; Henry, James; Idell, Steven

2016-01-01

The incidence of empyema (EMP) is increasing worldwide; EMP generally occurs with pleural loculation and impaired drainage is often treated with intrapleural fibrinolytic therapy (IPFT) or surgery. A number of IPFT options are used clinically with empiric dosing and variable outcomes in adults. To evaluate mechanisms governing intrapleural fibrinolysis and disease outcomes, models of Pasteurella multocida and Streptococcus pneumoniae were generated in rabbits and the animals were treated with either human tissue (tPA) plasminogen activator or prourokinase (scuPA). Rabbit EMP was characterized by the development of pleural adhesions detectable by chest ultrasonography and fibrinous coating of the pleura. Similar to human EMP, rabbits with EMP accumulated sizable, 20- to 40-ml fibrinopurulent pleural effusions associated with extensive intrapleural organization, significantly increased pleural thickness, suppression of fibrinolytic and plasminogen-activating activities, and accumulation of high levels of plasminogen activator inhibitor 1, plasminogen, and extracellular DNA. IPFT with tPA (0.145 mg/kg) or scuPA (0.5 mg/kg) was ineffective in rabbit EMP (n = 9 and 3 for P. multocida and S. pneumoniae, respectively); 2 mg/kg tPA or scuPA IPFT (n = 5) effectively cleared S. pneumoniae-induced EMP collections in 24 h with no bleeding observed. Although intrapleural fibrinolytic activity for up to 40 min after IPFT was similar for effective and ineffective doses of fibrinolysin, it was lower for tPA than for scuPA treatments. These results demonstrate similarities between rabbit and human EMP, the importance of pleural fluid PAI-1 activity, and levels of plasminogen in the regulation of intrapleural fibrinolysis and illustrate the dose dependency of IPFT outcomes in EMP. PMID:27343192

Extracellular Collagen Promotes Interleukin-1β-Induced Urokinase-Type Plasminogen Activator Production by Human Corneal Fibroblasts.

PubMed

Sugioka, Koji; Kodama-Takahashi, Aya; Yoshida, Koji; Aomatsu, Keiichi; Okada, Kiyotaka; Nishida, Teruo; Shimomura, Yoshikazu

2017-03-01

Keratocytes maintain homeostasis of the corneal stroma through synthesis, secretion, and degradation of collagen fibrils of the extracellular matrix. Given that these cells are essentially embedded in a collagen matrix, keratocyte-collagen interactions may play a key role in regulation of the expression or activation of enzymes responsible for matrix degradation including urokinase-type plasminogen activator (uPA), plasmin, and matrix metalloproteinases (MMPs). We examined the effect of extracellular collagen on the production of uPA by corneal fibroblasts (activated keratocytes) stimulated with the proinflammatory cytokine interleukin-1β (IL-1β). Human corneal fibroblasts were cultured either on plastic or in a three-dimensional gel of type I collagen. Plasminogen activators were detected by fibrin zymography, whereas the IL-1 receptor (IL-1R) and MMPs were detected by immunoblot analysis. Collagen degradation by corneal fibroblasts was assessed by measurement of hydroxyproline in acid hydrolysates of culture supernatants. Collagen and IL-1β synergistically increased the synthesis and secretion of uPA in corneal fibroblasts. Collagen also upregulated IL-1R expression in the cells in a concentration-dependent manner. The conversion of extracellular plasminogen to plasmin, as well as the plasminogen-dependent activation of MMP-1 and MMP-3 and degradation of collagen apparent in three-dimensional cultures of corneal fibroblasts exposed to IL-1β, were all abolished by a selective uPA inhibitor. Collagen and IL-1β cooperate to upregulate uPA production by corneal fibroblasts. Furthermore, IL-1β-induced collagen degradation by these cells appears to be strictly dependent on uPA expression and mediated by a uPA-plasmin-MMP pathway.

Dose dependency of outcomes of intrapleural fibrinolytic therapy in new rabbit empyema models.

PubMed

Komissarov, Andrey A; Florova, Galina; Azghani, Ali O; Buchanan, Ann; Boren, Jake; Allen, Timothy; Rahman, Najib M; Koenig, Kathleen; Chamiso, Mignote; Karandashova, Sophia; Henry, James; Idell, Steven

2016-08-01

The incidence of empyema (EMP) is increasing worldwide; EMP generally occurs with pleural loculation and impaired drainage is often treated with intrapleural fibrinolytic therapy (IPFT) or surgery. A number of IPFT options are used clinically with empiric dosing and variable outcomes in adults. To evaluate mechanisms governing intrapleural fibrinolysis and disease outcomes, models of Pasteurella multocida and Streptococcus pneumoniae were generated in rabbits and the animals were treated with either human tissue (tPA) plasminogen activator or prourokinase (scuPA). Rabbit EMP was characterized by the development of pleural adhesions detectable by chest ultrasonography and fibrinous coating of the pleura. Similar to human EMP, rabbits with EMP accumulated sizable, 20- to 40-ml fibrinopurulent pleural effusions associated with extensive intrapleural organization, significantly increased pleural thickness, suppression of fibrinolytic and plasminogen-activating activities, and accumulation of high levels of plasminogen activator inhibitor 1, plasminogen, and extracellular DNA. IPFT with tPA (0.145 mg/kg) or scuPA (0.5 mg/kg) was ineffective in rabbit EMP (n = 9 and 3 for P. multocida and S. pneumoniae, respectively); 2 mg/kg tPA or scuPA IPFT (n = 5) effectively cleared S. pneumoniae-induced EMP collections in 24 h with no bleeding observed. Although intrapleural fibrinolytic activity for up to 40 min after IPFT was similar for effective and ineffective doses of fibrinolysin, it was lower for tPA than for scuPA treatments. These results demonstrate similarities between rabbit and human EMP, the importance of pleural fluid PAI-1 activity, and levels of plasminogen in the regulation of intrapleural fibrinolysis and illustrate the dose dependency of IPFT outcomes in EMP. Copyright © 2016 the American Physiological Society.

Intrapleural Adenoviral Delivery of Human Plasminogen Activator Inhibitor–1 Exacerbates Tetracycline-Induced Pleural Injury in Rabbits

PubMed Central

Karandashova, Sophia; Florova, Galina; Azghani, Ali O.; Komissarov, Andrey A.; Koenig, Kathy; Tucker, Torry A.; Allen, Timothy C.; Stewart, Kris; Tvinnereim, Amy

2013-01-01

Elevated concentrations of plasminogen activator inhibitor–1 (PAI-1) are associated with pleural injury, but its effects on pleural organization remain unclear. A method of adenovirus-mediated delivery of genes of interest (expressed under a cytomegalovirus promoter) to rabbit pleura was developed and used with lacZ and human (h) PAI-1. Histology, β-galactosidase staining, Western blotting, enzymatic and immunohistochemical analyses of pleural fluids (PFs), lavages, and pleural mesothelial cells were used to evaluate the efficiency and effects of transduction. Transduction was selective and limited to the pleural mesothelial monolayer. The intrapleural expression of both genes was transient, with their peak expression at 4 to 5 days. On Day 5, hPAI-1 (40–80 and 200–400 nM of active and total hPAI-1 in lavages, respectively) caused no overt pleural injury, effusions, or fibrosis. The adenovirus-mediated delivery of hPAI-1 with subsequent tetracycline-induced pleural injury resulted in a significant exacerbation of the pleural fibrosis observed on Day 5 (P = 0.029 and P = 0.021 versus vehicle and adenoviral control samples, respectively). Intrapleural fibrinolytic therapy (IPFT) with plasminogen activators was effective in both animals overexpressing hPAI-1 and control animals with tetracycline injury alone. An increase in intrapleural active PAI-1 (from 10–15 nM in control animals to 20–40 nM in hPAI-1–overexpressing animals) resulted in the increased formation of PAI-1/plasminogen activator complexes in vivo. The decrease in intrapleural plasminogen-activating activity observed at 10 to 40 minutes after IPFT correlates linearly with the initial concentration of active PAI-1. Therefore, active PAI-1 in PFs affects the outcome of IPFT, and may be both a biomarker of pleural injury and a molecular target for its treatment. PMID:23002099

Network of Surface-Displayed Glycolytic Enzymes in Mycoplasma pneumoniae and Their Interactions with Human Plasminogen

PubMed Central

Gründel, Anne; Pfeiffer, Melanie; Jacobs, Enno

2015-01-01

In different bacteria, primarily cytosolic and metabolic proteins are characterized as surface localized and interacting with different host factors. These moonlighting proteins include glycolytic enzymes, and it has been hypothesized that they influence the virulence of pathogenic species. The presence of surface-displayed glycolytic enzymes and their interaction with human plasminogen as an important host factor were investigated in the genome-reduced and cell wall-less microorganism Mycoplasma pneumoniae, a common agent of respiratory tract infections of humans. After successful expression of 19 glycolytic enzymes and production of polyclonal antisera, the localization of proteins in the mycoplasma cell was characterized using fractionation of total proteins, colony blot, mild proteolysis and immunofluorescence of M. pneumoniae cells. Eight glycolytic enzymes, pyruvate dehydrogenases A to C (PdhA-C), glyceraldehyde-3-phosphate dehydrogenase (GapA), lactate dehydrogenase (Ldh), phosphoglycerate mutase (Pgm), pyruvate kinase (Pyk), and transketolase (Tkt), were confirmed as surface expressed and all are able to interact with plasminogen. Plasminogen bound to recombinant proteins PdhB, GapA, and Pyk was converted to plasmin in the presence of urokinase plasminogen activator and plasmin-specific substrate d-valyl-leucyl-lysine-p-nitroanilide dihydrochloride. Furthermore, human fibrinogen was degraded by the complex of plasminogen and recombinant protein PdhB or Pgm. In addition, surface-displayed proteins (except PdhC) bind to human lung epithelial cells, and the interaction was reduced significantly by preincubation of cells with antiplasminogen. Our results suggest that plasminogen binding and activation by different surface-localized glycolytic enzymes of M. pneumoniae may play a role in successful and long-term colonization of the human respiratory tract. PMID:26667841

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.

Activation of cardiac renin-angiotensin system and plasminogen activator inhibitor-1 gene expressions in oral contraceptive-induced cardiometabolic disorder.

PubMed

Olatunji, Lawrence A; Usman, Taofeek O; Seok, Young-Mi; Kim, In-Kyeom

2017-02-01

Clinical studies have shown that combined oral contraceptive (COC) use is associated with cardiometabolic disturbances. Elevated renin-angiotensin system (RAS) and plasminogen activator inhibitor-1 (PAI-1) have also been implicated in the development of cardiometabolic events. To determine the effect of COC treatment on cardiac RAS and PAI-1 gene expressions, and whether the effect is circulating aldosterone or corticosterone dependent. Female rats were treated (p.o.) with olive oil (vehicle) or COC (1.0 µg ethinylestradiol and 10.0 µg norgestrel) daily for six weeks. COC treatment led to increases in blood pressure, HOMA-IR, Ace1 mRNA, Atr1 mRNA, Pai1 mRNA, cardiac PAI-1, plasma PAI-1, C-reactive protein, uric acid, insulin and corticosterone. COC treatment also led to dyslipidemia, decreased glucose tolerance and plasma 17β-estradiol. These results demonstrates that hypertension and insulin resistance induced by COC is associated with increased cardiac RAS and PAI-1 gene expression, which is likely to be through corticosterone-dependent but not aldosterone-dependent mechanism.

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

USDA-ARS?s Scientific Manuscript database

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...

Plasminogen fragments K 1-3 and K 5 bind to different sites in fibrin fragment DD.

PubMed

Grinenko, T V; Kapustianenko, L G; Yatsenko, T A; Yusova, O I; Rybachuk, V N

2016-01-01

Specific plasminogen-binding sites of fibrin molecule are located in Аα148-160 regions of C-terminal domains. Plasminogen interaction with these sites initiates the activation process of proenzyme and subsequent fibrin lysis. In this study we investigated the binding of plasminogen fragments K 1-3 and K 5 with fibrin fragment DD and their effect on Glu-plasminogen interaction with DD. It was shown that the level of Glu-plasminogen binding to fibrin fragment DD is decreased by 50-60% in the presence of K 1-3 and K 5. Fragments K 1-3 and K 5 have high affinity to fibrin fragment DD (Kd is 0.02 for K 1-3 and 0.054 μМ for K 5). K 5 interaction is independent and K 1-3 is partly dependent on C-terminal lysine residues. K 1-3 interacts with complex of fragment DD-immobilized K 5 as well as K 5 with complex of fragment DD-immobilized K 1-3. The plasminogen fragments do not displace each other from binding sites located in fibrin fragment DD, but can compete for the interaction. The results indicate that fibrin fragment DD contains different binding sites for plasminogen kringle fragments K 1-3 and K 5, which can be located close to each other. The role of amino acid residues of fibrin molecule Аα148-160 region in interaction with fragments K 1-3 and K 5 is discussed.

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.

Tissue plasminogen activator (tPA) and matrix metalloproteinases in the pathogenesis of stroke: therapeutic strategies.

PubMed

Adibhatla, Rao Muralikrishna; Hatcher, James F

2008-06-01

Today there exists only one FDA-approved treatment for ischemic stroke; i.e., the serine protease tissue-type plasminogen activator (tPA). In the aftermath of the failed stroke clinical trials with the nitrone spin trap/radical scavenger, NXY-059, a number of articles raised the question: are we doing the right thing? Is the animal research truly translational in identifying new agents for stroke treatment? This review summarizes the current state of affairs with plasminogen activators in thrombolytic therapy. In addition to therapeutic value, potential side effects of tPA also exist that aggravate stroke injury and offset the benefits provided by reperfusion of the occluded artery. Thus, combinational options (ultrasound alone or with microspheres/nanobubbles, mechanical dissociation of clot, activated protein C (APC), plasminogen activator inhibitor-1 (PAI-1), neuroserpin and CDP-choline) that could offset tPA toxic side effects and improve efficacy are also discussed here. Desmoteplase, a plasminogen activator derived from the saliva of Desmodus rotundus vampire bat, antagonizes vascular tPA-induced neurotoxicity by competitively binding to low-density lipoprotein related-receptors (LPR) at the blood-brain barrier (BBB) interface, minimizing the tPA uptake into brain parenchyma. tPA can also activate matrix metalloproteinases (MMPs), a family of endopeptidases comprised of 24 mammalian enzymes that primarily catalyze the turnover and degradation of the extracellular matrix (ECM). MMPs have been implicated in BBB breakdown and neuronal injury in the early times after stroke, but also contribute to vascular remodeling, angiogenesis, neurogenesis and axonal regeneration during the later repair phase after stroke. tPA, directly or by activation of MMP-9, could have beneficial effects on recovery after stroke by promoting neurovascular repair through vascular endothelial growth factor (VEGF). However, any treatment regimen directed at MMPs must consider their pleiotropic nature and the likelihood of either beneficial or detrimental effects that might depend on the timing of the treatment in relation to the stage of brain injury.

Co-ordinated spatial propagation of blood plasma clotting and fibrinolytic fronts

PubMed Central

Zhalyalov, Ansar S.; Panteleev, Mikhail A.; Gracheva, Marina A.; Ataullakhanov, Fazoil I.

2017-01-01

Fibrinolysis is a cascade of proteolytic reactions occurring in blood and soft tissues, which functions to disintegrate fibrin clots when they are no more needed. In order to elucidate its regulation in space and time, fibrinolysis was investigated using an in vitro reaction-diffusion experimental model of blood clot formation and dissolution. Clotting was activated by a surface with immobilized tissue factor in a thin layer of recalcified blood plasma supplemented with tissue plasminogen activator (TPA), urokinase plasminogen activator or streptokinase. Formation and dissolution of fibrin clot was monitored by videomicroscopy. Computer systems biology model of clot formation and lysis was developed for data analysis and experimental planning. Fibrin clot front propagated in space from tissue factor, followed by a front of clot dissolution propagating from the same source. Velocity of lysis front propagation linearly depended on the velocity clotting front propagation (correlation r2 = 0.91). Computer model revealed that fibrin formation was indeed the rate-limiting step in the fibrinolysis front propagation. The phenomenon of two fronts which switched the state of blood plasma from liquid to solid and then back to liquid did not depend on the fibrinolysis activator. Interestingly, TPA at high concentrations began to increase lysis onset time and to decrease lysis propagation velocity, presumably due to plasminogen depletion. Spatially non-uniform lysis occurred simultaneously with clot formation and detached the clot from the procoagulant surface. These patterns of spatial fibrinolysis provide insights into its regulation and might explain clinical phenomena associated with thrombolytic therapy. PMID:28686711

The plasminogen activator system modulates sympathetic nerve function.

PubMed

Schaefer, Ulrich; Machida, Takuji; Vorlova, Sandra; Strickland, Sidney; Levi, Roberto

2006-09-04

Sympathetic neurons synthesize and release tissue plasminogen activator (t-PA). We investigated whether t-PA modulates sympathetic activity. t-PA inhibition markedly reduced contraction of the guinea pig vas deferens to electrical field stimulation (EFS) and norepinephrine (NE) exocytosis from cardiac synaptosomes. Recombinant t-PA (rt-PA) induced exocytotic and carrier-mediated NE release from cardiac synaptosomes and cultured neuroblastoma cells; this was a plasmin-independent effect but was potentiated by a fibrinogen cleavage product. Notably, hearts from t-PA-null mice released much less NE upon EFS than their wild-type (WT) controls (i.e., a 76.5% decrease; P<0.01), whereas hearts from plasminogen activator inhibitor-1 (PAI-1)-null mice released much more NE (i.e., a 275% increase; P<0.05). Furthermore, vasa deferentia from t-PA-null mice were hyporesponsive to EFS (P<0.0001) but were normalized by the addition of rt-PA. In contrast, vasa from PAI-1-null mice were much more responsive (P<0.05). Coronary NE overflow from hearts subjected to ischemia/reperfusion was much smaller in t-PA-null than in WT control mice (P<0.01). Furthermore, reperfusion arrhythmias were significantly reduced (P<0.05) in t-PA-null hearts. Thus, t-PA enhances NE release from sympathetic nerves and contributes to cardiac arrhythmias in ischemia/reperfusion. Because the risk of arrhythmias and sudden cardiac death is increased in hyperadrenergic conditions, targeting the NE-releasing effect of t-PA may have valuable therapeutic potential.

Further analyses of human kidney cell populations separated on the Space Shuttle

NASA Technical Reports Server (NTRS)

Stewart, Robin M.; Todd, Paul; Cole, Kenneth D.; Morrison, Dennis R.

1992-01-01

Cultured human embryonic kidney cells were separated into electrophoretic subpopulations in laboratory experiments and in two separation experiments on the STS-8 (Challenger) Space Shuttle flight using the mid-deck Continuous Flow Electrophoretic Separator (CFES). Populations of cells from each fraction were cultured for the lifetime of the cells, and supernatant medium was withdrawn and replaced at 4-day intervals. Withdrawn medium was frozen at -120 C for subsequent analysis. Enzyme assays, antibodies and gel electrophoresis were used as analytical tools for the detection and quantization of plasminogen activators in these samples. These assays of frozen-culture supernatant fluids confirmed the electrophoretic separation of plasminogen-activator-producing cells from nonproducing cells, the isolation of cells capable of sustained production, and the separation of cells that produce different plasminogen activators from one other.

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

USDA-ARS?s Scientific Manuscript database

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...

Clustering of haemostatic variables and the effect of high cashew and walnut diets on these variables in metabolic syndrome patients.

PubMed

Pieters, Marlien; Oosthuizen, Welma; Jerling, Johann C; Loots, Du Toit; Mukuddem-Petersen, Janine; Hanekom, Susanna M

2005-09-01

We investigated the effect of a high walnut and cashew diet on haemostatic variables in people with the metabolic syndrome. Factor analysis was used to determine how the haemostatic variables cluster with other components of the metabolic syndrome and multiple regression to determine possible predictors. This randomized, control, parallel, controlled-feeding trial included 68 subjects who complied with the Third National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol criteria. After a 3-week run-in following the control diet, subjects were divided into three groups receiving either walnuts or cashews (20 energy%) or a control diet for 8 weeks. The nut intervention had no significant effect on von Willebrand factor antigen, fibrinogen, factor VII coagulant activity, plasminogen activator inhibitor 1 activity, tissue plasminogen activator activity or thrombin activatable fibrinolysis inhibitor. Statistically, fibrinogen clustered with the body-mass-correlates and acute phase response factors, and factor VII coagulant activity clustered with high-density lipoprotein cholesterol (HDL-C). Tissue plasminogen activator activity, plasminogen activator inhibitor 1 activity and von Willebrand factor antigen clustered into a separate endothelial function factor. HDL-C and markers of obesity were the strongest predictors of the haemostatic variables. We conclude that high walnut and cashew diets did not influence haemostatic factors in this group of metabolic syndrome subjects. The HDL-C increase and weight loss may be the main focus of dietary intervention for the metabolic syndrome. Furthermore, diet composition may have only limited effects if weight loss is not achieved.

The evolution of recombinant thrombolytics: Current status and future directions

PubMed Central

Adivitiya; Khasa, Yogender Pal

2017-01-01

ABSTRACT Cardiovascular disorders are on the rise worldwide due to alcohol abuse, obesity, hypertension, raised blood lipids, diabetes and age-related risks. The use of classical antiplatelet and anticoagulant therapies combined with surgical intervention helped to clear blood clots during the inceptive years. However, the discovery of streptokinase and urokinase ushered the way of using these enzymes as thrombolytic agents to degrade the fibrin network with an issue of systemic hemorrhage. The development of second generation plasminogen activators like anistreplase and tissue plasminogen activator partially controlled this problem. The third generation molecules, majorly t-PA variants, showed desirable properties of improved stability, safety and efficacy with enhanced fibrin specificity. Plasmin variants are produced as direct fibrinolytic agents as a futuristic approach with targeted delivery of these drugs using liposome technlogy. The novel molecules from microbial, plant and animal origin present the future of direct thrombolytics due to their safety and ease of administration. PMID:27696935

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.

Proteases induce secretion of collagenase and plasminogen activator by fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Werb, Z.; Aggeler, J.

1978-04-01

We have observed that treatment of rabbit synovial fibroblasts with proteolytic enzymes can induce secretion of collagenase (EC 3.4.24.7) and plasminogen activator (EC 3.4.21.-). Cells treated for 2 to 24 hr with plasmin, trypsin, chymotrypsin, pancreatic elastase, papain, bromelain, thermolysin, or ..cap alpha..-protease but not with thrombin or neuraminidase secreted detectable amounts of collagenase within 16 to 48 hr. Treatment of fibroblasts with trypsin also induced secretion of plasminogen activator. Proteases initiated secretion of collagenase (up to 20 units per 10/sup 6/ cells per 24 hr) only when treatment produced decreased cell adhesion. Collagenase production did not depend on continuedmore » presence of proteolytic activity or on subsequent cell adhesion, spreading, or proliferation. Routine subculturing with crude trypsin also induced collagenase secretion by cells. Secretion of collagenase was prevented and normal spreading was obtained if the trypsinized cells were placed into medium containing fetal calf serum. Soybean trypsin inhibitor, ..cap alpha../sub 1/-antitrypsin, bovine serum albumin, collagen, and fibronectin did not inhibit collagenase production. Although proteases that induced collagenase secretion also removed surface glycoprotein, the kinetics of induction of cell protease secretion were different from those for removal of fibronectin. Physiological inducers of secretion of collagenase and plasminogen activator by cells have not been identified. These results suggest that extracellular proteases in conjunction with plasma proteins may govern protease secretion by cells.« less

Anti-Urokinase Receptor Antisense Oligonucleotide (uPAR-aODN) to Prevent and Cure Long-Term Space Exploration-Related Retinal Pathological Angiogenesis

NASA Astrophysics Data System (ADS)

Lazzarano, Stefano; Lulli, Matteo; Fibbi, Gabriella; Margheri, Francesca; Papucci, Laura; Serrati, Simona; Witort, Ewa; Chilla, Anastasia; Lapucci, Andrea; Donnini, Martino; Quaglierini, Paolo; Romiti, Alice; Specogna, Rebecca; Del Rosso, Mario; Capaccioli, Sergio

2008-06-01

Angiogenesis underlies a variety of physiological processes and its possible deregulation during long term space exploration needs to be investigated. Angiogenesis is a multistep process of new blood capillary formation, where degradation of the extracellular matrix (ECM) by proteolytic enzymes, including uPA (urokinase plasminogen activator) and opening the way to migration of endothelial cells (EC), is critical. Plasminogen activation system regulates angiogenesis by both uPA-driven ECM degradation and uPA receptor (uPAR). Microgravity and low dose irradiations promote tissue neoangiogeenesis and neovascularization is often common occurence in ophthalmologic pathologies. We have designed and patented the uPAR antisense oligonucleotide (aODN) and evaluated its antiangiogenetic activity by EC cellular migration and capillary morphogenesis assays. The uPAR aODN treatment caused a 75% inhibition of human microvascular EC migration and a complete inhibition of capillary morphogenesis, suggesting its therapeutic application to prevent neoangiogenesis-related ophthalmologic pathologies during space exploration.

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.

Fibrin-specific and effective clot lysis requires both plasminogen activators and for them to be in a sequential rather than simultaneous combination.

PubMed

Pannell, R; Li, S; Gurewich, V

2017-08-01

Thrombolysis with tissue plasminogen activator (tPA) has been a disappointment and has now been replaced by an endovascular procedure whenever possible. Nevertheless, thrombolysis remains the only means by which circulation in a thrombosed artery can be restored rapidly. In contrast to tPA monotherapy, endogenous fibrinolysis uses both tPA and urokinase plasminogen activator (uPA), whose native form is a proenzyme, prouPA. This combination is remarkably effective as evidenced by the fibrin degradation product, D-dimer, which is invariably present in plasma. The two activators have complementary mechanisms of plasminogen activation and are synergistic in combination. Since tPA initiates fibrinolysis when released from the vessel wall and prouPA is in the blood, they induce fibrinolysis sequentially. It was postulated that this may be more effective and fibrin-specific. The hypothesis was tested in a model of clot lysis in plasma in which a clot was first exposed to tPA for 5 min, washed and incubated with prouPA. Lysis was compared with that of clots incubated with both activators simultaneously. The sequential combination was almost twice as effective and caused less fibrinogenolysis than the simultaneous combination (p < 0.0001) despite having significantly less tPA, as a result of the wash. A mechanism is described by which this phenomenon can be explained. The findings are believed to have significant therapeutic implications.

A novel clot lysis assay for recombinant plasminogen activator.

PubMed

Jamialahmadi, Oveis; Fazeli, Ahmad; Hashemi-Najafabadi, Sameereh; Fazeli, Mohammad Reza

2015-03-01

Recombinant plasminogen activator (r-PA, reteplase) is an engineered variant of alteplase. When expressed in E. coli, it appears as inclusion bodies that require refolding to recover its biological activity. An important step following refolding is to determine the activity of refolded protein. Current methods for enzymatic activity of thrombolytic drugs are costly and complex. Here a straightforward and low-cost clot lysis assay was developed. It quantitatively measures the activity of the commercial reteplase and is also capable of screening refolding conditions. As evidence for adequate accuracy and sensitivity of the current assay, r-PA activity measurements are shown to be comparable to those obtained from chromogenic substrate assay.

Protease-activated receptor 2 (PAR2) is upregulated by Acanthamoeba plasminogen activator (aPA) and induces proinflammatory cytokine in human corneal epithelial cells.

PubMed

Tripathi, Trivendra; Abdi, Mahshid; Alizadeh, Hassan

2014-05-29

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. 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. 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. Acanthamoeba plasminogen activator and PAR2 agonists stimulated IL-8 mRNA expression and protein production, which is significantly diminished by PAR2 antagonist (P < 0.05). Protease-activated receptor 1 antagonist did not alter aPA-stimulated IL-8 mRNA expression and protein production in HCE cells. Flow cytometry and immunocytochemistry showed that aPA and SLIGRL-NH2 (PAR2 agonist) upregulated PAR2 surface protein as compared to that in unstimulated HCE cells. Thrombin, but not aPA, stimulated PAR1 surface protein in HCE cells. Acanthamoeba plasminogen activator specifically induces expression and production of IL-8 in HCE cells via PAR2 pathway, and PAR2 antagonists may be used as a therapeutic target in AK. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

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.

Increased expression of urokinase plasminogen activator in Quebec platelet disorder is linked to megakaryocyte differentiation

PubMed Central

Veljkovic, D. Kika; Rivard, Georges E.; Diamandis, Maria; Blavignac, Jessica; Cramer-Bordé, Elisabeth M.

2009-01-01

Quebec platelet disorder (QPD) is an inherited bleeding disorder associated with increased urokinase plasminogen activator (uPA) in platelets but not in plasma, intraplatelet plasmin generation, and α-granule protein degradation. These abnormalities led us to investigate uPA expression by QPD CD34+ progenitors, cultured megakaryocytes, and platelets, and whether uPA was stored in QPD α-granules. Although QPD CD34+ progenitors expressed normal amounts of uPA, their differentiation into megakaryocytes abnormally increased expression of the uPA gene but not the flanking genes for vinculin or calcium/calmodulin-dependent protein kinase IIγ on chromosome 10. The increased uPA production by cultured QPD megakaryocytes mirrored their production of α-granule proteins, which was normal. uPA was localized to QPD α-granules and it showed extensive colocalization with α-granule proteins in both cultured QPD megakaryocytes and platelets, and with plasminogen in QPD platelets. In QPD megakaryocytes, cultured without or with plasma as a source of plasminogen, α-granule proteins were stored undegraded and this was associated with much less uPA-plasminogen colocalization than in QPD platelets. Our studies indicate that the overexpression of uPA in QPD emerges with megakaryocyte differentiation, without altering the expression of flanking genes, and that uPA is costored with α-granule proteins prior to their proteolysis in QPD. PMID:19029443

Increased expression of urokinase plasminogen activator in Quebec platelet disorder is linked to megakaryocyte differentiation.

PubMed

Veljkovic, D Kika; Rivard, Georges E; Diamandis, Maria; Blavignac, Jessica; Cramer-Bordé, Elisabeth M; Hayward, Catherine P M

2009-02-12

Quebec platelet disorder (QPD) is an inherited bleeding disorder associated with increased urokinase plasminogen activator (uPA) in platelets but not in plasma, intraplatelet plasmin generation, and alpha-granule protein degradation. These abnormalities led us to investigate uPA expression by QPD CD34(+) progenitors, cultured megakaryocytes, and platelets, and whether uPA was stored in QPD alpha-granules. Although QPD CD34(+) progenitors expressed normal amounts of uPA, their differentiation into megakaryocytes abnormally increased expression of the uPA gene but not the flanking genes for vinculin or calcium/calmodulin-dependent protein kinase IIgamma on chromosome 10. The increased uPA production by cultured QPD megakaryocytes mirrored their production of alpha-granule proteins, which was normal. uPA was localized to QPD alpha-granules and it showed extensive colocalization with alpha-granule proteins in both cultured QPD megakaryocytes and platelets, and with plasminogen in QPD platelets. In QPD megakaryocytes, cultured without or with plasma as a source of plasminogen, alpha-granule proteins were stored undegraded and this was associated with much less uPA-plasminogen colocalization than in QPD platelets. Our studies indicate that the overexpression of uPA in QPD emerges with megakaryocyte differentiation, without altering the expression of flanking genes, and that uPA is costored with alpha-granule proteins prior to their proteolysis in QPD.

Proteolysis of plasminogen activator inhibitor-1 by Yersinia pestis remodulates the host environment to promote virulence.

PubMed

Eddy, J L; Schroeder, J A; Zimbler, D L; Caulfield, A J; Lathem, W W

2016-09-01

Essentials Effect of plasminogen activator inhibitor (PAI)-1 on plague and its Y. pestis cleavage is unknown. An intranasal mouse model of infection was used to determine the role of PAI-1 in pneumonic plague. PAI-1 is cleaved and inactivated by the Pla protease of Y. pestis in the lung airspace. PAI-1 impacts both bacterial outgrowth and the immune response to respiratory Y. pestis infection. Click to hear Dr Bock discuss pathogen activators of plasminogen. Background The hemostatic regulator plasminogen activator inhibitor-1 (PAI-1) inactivates endogenous plasminogen activators and aids in the immune response to bacterial infection. Yersinia pestis, the causative agent of plague, produces the Pla protease, a virulence factor that is required during plague. However, the specific hemostatic proteins cleaved by Pla in vivo that contribute to pathogenesis have not yet been fully elucidated. Objectives To determine whether PAI-1 is cleaved by the Pla protease during pneumonic plague, and to define the impact of PAI-1 on Y. pestis respiratory infection in the presence or absence of Pla. Methods An intranasal mouse model of pneumonic plague was used to assess the levels of total and active PAI-1 in the lung airspace, and the impact of PAI-1 deficiency on bacterial pathogenesis, the host immune response and plasmin generation following infection with wild-type or ∆pla Y. pestis. Results We found that Y. pestis cleaves and inactivates PAI-1 in the lungs in a Pla-dependent manner. The loss of PAI-1 enhances Y. pestis outgrowth in the absence of Pla, and is associated with increased conversion of plasminogen to plasmin. Furthermore, we found that PAI-1 regulates immune cell recruitment, cytokine production and tissue permeability during pneumonic plague. Conclusions Our data demonstrate that PAI-1 is an in vivo target of the Pla protease in the lungs, and that PAI-1 is a key regulator of the pulmonary innate immune response. We conclude that the inactivation of PAI-1 by Y. pestis alters the host environment to promote virulence during pneumonic plague. © 2016 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.

Bioconjugation of recombinant tissue plasminogen activator to magnetic nanocarriers for targeted thrombolysis

PubMed Central

Yang, Hung-Wei; Hua, Mu-Yi; Lin, Kun-Ju; Wey, Shiaw-Pyng; Tsai, Rung-Ywan; Wu, Siao-Yun; Lu, Yi-Ching; Liu, Hao-Li; Wu, Tony; Ma, Yunn-Hwa

2012-01-01

Low-toxicity magnetic nanocarriers (MNCs) composed of a shell of poly [aniline-co-N-(1-one-butyric acid) aniline] over a Fe3O4 magnetic nanoparticle core were developed to carry recombinant tissue plasminogen activator (rtPA) in MNC-rtPA for targeted thrombolysis. With an average diameter of 14.8 nm, the MNCs exerted superparamagnetic properties. Up to 276 μg of active rtPA was immobilized per mg of MNCs, and the stability of the immobilized rtPA was greatly improved during storage at 4°C and 25°C. In vitro thrombolysis testing with a tubing system demonstrated that magnet-guided MNC-rtPA showed significantly improved thrombolysis compared with free rtPA and reduced the clot lysis time from 39.2 ± 3.2 minutes to 10.8 ± 4.2 minutes. In addition, magnet-guided MNC-rtPA at 20% of the regular rtPA dose restored blood flow within 15–25 minutes of treatment in a rat embolism model without triggering hematological toxicity. In conclusion, this improved system is based on magnetic targeting accelerated thrombolysis and is potentially amenable to therapeutic applications in thromboembolic diseases. PMID:23055728

Cpa, the Outer Membrane Protease of Cronobacter sakazakii, Activates Plasminogen and Mediates Resistance to Serum Bactericidal Activity▿

PubMed Central

Franco, A. A.; Kothary, M. H.; Gopinath, G.; Jarvis, K. G.; Grim, C. J.; Hu, L.; Datta, A. R.; McCardell, B. A.; Tall, B. D.

2011-01-01

Cronobacter spp. are emerging neonatal pathogens in humans, associated with outbreaks of meningitis and sepsis. To cause disease, they must survive in blood and invade the central nervous system by penetrating the blood-brain barrier. C. sakazakii BAA-894 possesses an ∼131-kb plasmid (pESA3) that encodes an outer membrane protease (Cpa) that has significant identity to proteins that belong to the Pla subfamily of omptins. Members of this subfamily of proteins degrade a number of serum proteins, including circulating complement, providing protection from the complement-dependent serum killing. Moreover, proteins of the Pla subfamily can cause uncontrolled plasmin activity by converting plasminogen to plasmin and inactivating the plasmin inhibitor α2-antiplasmin (α2-AP). These reactions enhance the spread and invasion of bacteria in the host. In this study, we found that an isogenic cpa mutant showed reduced resistance to serum in comparison to its parent C. sakazakii BAA-894 strain. Overexpression of Cpa in C. sakazakii or Escherichia coli DH5α showed that Cpa proteolytically cleaved complement components C3, C3a, and C4b. Furthermore, a strain of C. sakazakii overexpressing Cpa caused a rapid activation of plasminogen and inactivation of α2-AP. These results strongly suggest that Cpa may be an important virulence factor involved in serum resistance, as well as in the spread and invasion of C. sakazakii. PMID:21245266

Inhibitory Monoclonal Antibodies against Mouse Proteases Raised in Gene-Deficient Mice Block Proteolytic Functions in vivo

PubMed Central

Lund, Ida K.; Rasch, Morten G.; Ingvarsen, Signe; Pass, Jesper; Madsen, Daniel H.; Engelholm, Lars H.; Behrendt, Niels; Høyer-Hansen, Gunilla

2012-01-01

Identification of targets for cancer therapy requires the understanding of the in vivo roles of proteins, which can be derived from studies using gene-targeted mice. An alternative strategy is the administration of inhibitory monoclonal antibodies (mAbs), causing acute disruption of the target protein function(s). This approach has the advantage of being a model for therapeutic targeting. mAbs for use in mouse models can be obtained through immunization of gene-deficient mice with the autologous protein. Such mAbs react with both species-specific epitopes and epitopes conserved between species. mAbs against proteins involved in extracellular proteolysis, including plasminogen activators urokinase plasminogen activator (uPA), tissue-type plasminogen activator (tPA), their inhibitor PAI-1, the uPA receptor (uPAR), two matrix metalloproteinases (MMP9 and MMP14), as well as the collagen internalization receptor uPARAP, have been developed. The inhibitory mAbs against uPA and uPAR block plasminogen activation and thereby hepatic fibrinolysis in vivo. Wound healing, another plasmin-dependent process, is delayed by an inhibitory mAb against uPA in the adult mouse. Thromboembolism can be inhibited by anti-PAI-1 mAbs in vivo. In conclusion, function-blocking mAbs are well-suited for targeted therapy in mouse models of different diseases, including cancer. PMID:22754528

Plasminogen-induced aggregation of PANC-1 cells requires conversion to plasmin and is inhibited by endogenous plasminogen activator inhibitor-1.

PubMed

Deshet, Naamit; Lupu-Meiri, Monica; Espinoza, Ingrid; Fili, Oded; Shapira, Yuval; Lupu, Ruth; Gershengorn, Marvin C; Oron, Yoram

2008-09-01

PANC-1 cells express proteinase-activated receptors (PARs)-1, -2, and respond to their activation by transient elevation of cytosolic [Ca(2+)] and accelerated aggregation (Wei et al., 2006, J Cell Physiol 206:322-328). We studied the effect of plasminogen (PGN), an inactive precursor of the PAR-1-activating protease, plasmin (PN) on aggregation of pancreatic adenocarcinoma (PDAC) cells. A single dose of PGN time- and dose-dependently promoted PANC-1 cells aggregation in serum-free medium, while PN did not. PANC-1 cells express urokinase plasminogen activator (uPA), which continuously converted PGN to PN. This activity and PGN-induced aggregation were inhibited by the uPA inhibitor amiloride. PGN-induced aggregation was also inhibited by alpha-antiplasmin and by the PN inhibitor epsilon-aminocaproic acid (EACA). Direct assay of uPA activity revealed very low rate, markedly enhanced in the presence of PGN. Moreover, in PGN activator inhibitor 1-deficient PANC-1 cells, uPA activity and PGN-induced aggregation were markedly potentiated. Two additional human PDAC cell lines, MiaPaCa and Colo347, were assayed for PGN-induced aggregation. Both cell lines responded by aggregation and exhibited PGN-enhanced uPA activity. We hypothesized that the continuous conversion of PGN to PN by endogenous uPA is limited by PN's degradation and negatively controlled by endogenously produced PAI-1. Indeed, we found that PANC-1 cells inactivate PN with t1/2 of approximately 7 h, while the continuous addition of PN promoted aggregation. Our data suggest that PANC-1 cells possess intrinsic, PAI-1-sensitive mechanism for promotion of aggregation and differentiation by prolonged exposure to PGN and, possibly, additional precursors of PARs agonists.

Variable Resistance to Plasminogen Activator Initiated Fibrinolysis for Intermediate-Risk Pulmonary Embolism.

PubMed

Stubblefield, William B; Alves, Nathan J; Rondina, Matthew T; Kline, Jeffrey A

2016-01-01

We examine the clinical significance and biomarkers of tissue plasminogen activator (tPA)-catalyzed clot lysis time (CLT) in patients with intermediate-risk pulmonary embolism (PE). Platelet-poor, citrated plasma was obtained from patients with PE. Healthy age- and sex-matched patients served as disease-negative controls. Fibrinogen, α2-antiplasmin, plasminogen, thrombin activatable fibrinolysis inhibitor (TAFI), plasminogen activator Inhibitor 1 (PAI-1), thrombin time and D-dimer were quantified. Clotting was induced using CaCl2, tissue factor, and phospholipid. Lysis was induced using 60 ng/mL tPA. Time to 50% clot lysis (CLT) was assessed by both thromboelastography (TEG) and turbidimetry (A405). Compared with disease-negative controls, patients with PE exhibited significantly longer mean CLT on TEG (+2,580 seconds, 95% CI 1,380 to 3,720 sec). Patients with PE and a short CLT who were treated with tenecteplase had increased risk of bleeding, whereas those with long CLT had significantly worse exercise tolerance and psychometric testing for quality of life at 3 months. A multivariate stepwise removal regression model selected PAI-1 and TAFI as predictive biomarkers of CLT. The CLT from TEG predicted increased risk of bleeding and clinical failure with tenecteplase treatment for intermediate-risk PE. Plasmatic PAI-1 and TAFI were independent predictors of CLT.

Interaction of Trypanosoma evansi with the plasminogen-plasmin system.

PubMed

Acosta, Héctor; Rondón-Mercado, Rocío; Avilán, Luisana; Concepción, Juan Luis

2016-08-15

Trypanosoma evansi is a widely-distributed haemoflagellated parasite of veterinary importance that infects a variety of mammals including horses, mules, camels, buffalos, cattle and deer. It is the causal agent of a trypanosomiasis known as Surra which produces epidemics of great economic importance in Africa, Asia and South America. The main pathology includes an enlarged spleen with hypertrophy of lymphoid follicles, congested lungs, neuronal degeneration and meningoencephalitis, where migration of the parasites from the blood to the tissues is essential. Most cells, including pathogenic cells, use diverse strategies for tissue invasion, such as the expression of surface receptors to bind plasminogen or plasmin. In this work, we show that T. evansi is able to bind plasminogen and plasmin on its surface. The analysis of this binding revealed a high affinity dissociation constant (Kd of 0.080±0.009μM) and 1×10(5) plasminogen binding sites per cell. Also a second population of receptors with a Kd of 0.255±0.070μM and 3.2×10(4) plasminogen binding sites per cell was determined. Several proteins with molecular masses between ∼18 and ∼70kDa are responsible for this binding. This parasite-plasminogen interaction may be important in the establishment of the infection in the vertebrate host, where the physiological concentration of available plasminogen is around 2μM. Copyright © 2016 Elsevier B.V. All rights reserved.

Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

PubMed Central

Mazar, Andrew P.; Koenig, Kathy; Kurdowska, Anna K.; Idell, Steven

2009-01-01

The proenzyme single-chain urokinase plasminogen activator (scuPA) more effectively resolved intrapleural loculations in rabbits with tetracycline (TCN)-induced loculation than a range of clinical doses of two-chain uPA (Abbokinase) and demonstrated a trend toward greater efficacy than single-chain tPA (Activase) (Idell S et al., Exp Lung Res 33: 419, 2007.). scuPA more slowly generates durable intrapleural fibrinolytic activity than Abbokinase or Activase, but the interactions of these agents with inhibitors in pleural fluids (PFs) have been poorly understood. PFs from rabbits with TCN-induced pleural injury treated with intrapleural scuPA, its inactive Ser195Ala mutant, Abbokinase, Activase, or vehicle, were analyzed to define the mechanism by which scuPA induces durable fibrinolysis. uPA activity was elevated in PFs of animals treated with scuPA, correlated with the ability to clear pleural loculations, and resisted (70–80%) inhibition by PAI-1. α-macroglobulin (αM) but not urokinase receptor complexes immunoprecipitated from PFs of scuPA-treated rabbits retained uPA activity that resists PAI-1 and activates plasminogen. Conversely, little plasminogen activating or enzymatic activity resistant to PAI-1 was detectable in PFs of rabbits treated with Abbokinase or Activase. Consistent with these findings, PAI-1 interacts with scuPA much slower than with Activase or Abbokinase in vitro. An equilibrium between active and inactive scuPA (kon = 4.3 h−1) limits the rate of its inactivation by PAI-1, favoring formation of complexes with αM. These observations define a newly recognized mechanism that promotes durable intrapleural fibrinolysis via formation of αM/uPA complexes. These complexes promote uPA-mediated plasminogen activation in scuPA-treated rabbits with TCN-induced pleural injury. PMID:19666776

Enhanced functional stability of plasminogen activator inhibitor-1 in patients with livedoid vasculopathy.

PubMed

Agirbasli, Mehmet; Eren, Mesut; Eren, Fatih; Murphy, Sheila B; Serdar, Zehra A; Seckin, Dilek; Zara, Tuba; Cem Mat, M; Demirkesen, Cuyan; Vaughan, Douglas E

2011-07-01

Livedoid vasculopathy (LV) is a chronic, recurrent, painful cutaneous disease with distinctive clinical features and an uncertain etiology. The skin lesions are recognizable by focal purpura, depigmentation and shallow ulcers. Thrombophilic conditions occur frequently in patients with LV. While no definitive treatment exists for LV, smoking cessation, antiplatelet therapy, immunosuppressive treatment, and anabolic steroids are often included in the therapeutic ladder. Recently, a possible link between LV and impaired fibrinolysis was established as cutaneous LV lesions responded to tissue plasminogen activator (t-PA) infusion suggesting that inhibition of the fibrinolysis through plasminogen activator inhibitor-1 (PAI-1) activity may determine the disease course in patients with LV. In this study, we investigated PAI-1 antigen (Ag) and activity levels in 20 patients with biopsy proven LV (mean age 26 ± 11, M/F = 7/13, median disease duration 3.5 years). All patients received antiplatelet treatment with aspirin and/or dipyrimadole and 14 patients received anabolic steroids or immunosuppressive treatment. Fasting PAI-1 Ag and activity levels were measured at 9 AM in all patients. Both Ag (34 (26) ng/ml) (median (interquartile range)) and specific activity (17 (23) IU/fmole) levels of PAI-1 were moderately elevated in LV patients compared to the controls, however, PAI-1 kinetic studies demonstrated markedly enhanced stability of PAI-1 activity in plasma from patients with LV. Specific activity at 16 h was significantly higher than expected specific activity levels (7 (11) vs. 0.07 (0.09) IU/fmole, P < 0.01). While the exact mechanism of increased stability of PAI-1 activity is not known, it may be due to post-translational modifications or increased binding affinity for a stabilizing cofactor. In conclusion, enhanced stability of PAI-1 may contribute to the pathophysiology of LV, and systemic or local treatment with PAI-1 inhibitors may offer a potential treatment alternative in patients with LV.

Tissue plasminogen activator mediates amyloid-induced neurotoxicity via Erk1/2 activation

PubMed Central

Medina, Manel G; Ledesma, Maria Dolores; Domínguez, Jorge E; Medina, Miguel; Zafra, Delia; Alameda, Francesc; Dotti, Carlos G; Navarro, Pilar

2005-01-01

Tissue plasminogen activator (tPA) is the main activator of plasminogen into plasmin in the brain where it may have beneficial roles but also neurotoxic effects that could be plasmin dependent or not. Little is known about the substrates and pathways that mediate plasmin-independent tPA neurotoxicity. Here we show in primary hippocampal neurons that tPA promotes a catalytic-independent activation of the extracellular regulated kinase (Erk)1/2 signal transduction pathway through the N-methyl-D-aspartate receptor, G-proteins and protein kinase C. This results in GSK3 activation in a process that requires de novo synthesis of proteins, and leads to tau aberrant phosphorylation, microtubule destabilization and apoptosis. Similar effects are produced by amyloid aggregates in a tPA-dependent manner, as demonstrated by pharmacological treatments and in wt and tPA−/− mice neurons. Consistently, in Alzheimer's disease (AD) patients' brains, high levels of tPA colocalize with amyloid-rich areas, activated Erk1/2 and phosphorylated tau. This is the first demonstration of an intracellular pathway by which tPA triggers kinase activation, tau phosphorylation and neurotoxicity, suggesting a key role for this molecule in AD pathology. PMID:15861134

Solitaire™ with the Intention for Thrombectomy as Primary Endovascular Treatment for Acute Ischemic Stroke (SWIFT PRIME) trial: protocol for a randomized, controlled, multicenter study comparing the Solitaire revascularization device with IV tPA with IV tPA alone in acute ischemic stroke.

PubMed

Saver, Jeffrey L; Goyal, Mayank; Bonafe, Alain; Diener, Hans-Christoph; Levy, Elad I; Pereira, Vitor M; Albers, Gregory W; Cognard, Christophe; Cohen, David J; Hacke, Werner; Jansen, Olav; Jovin, Tudor G; Mattle, Heinrich P; Nogueira, Raul G; Siddiqui, Adnan H; Yavagal, Dileep R; Devlin, Thomas G; Lopes, Demetrius K; Reddy, Vivek; du Mesnil de Rochemont, Richard; Jahan, Reza

2015-04-01

Early reperfusion in patients experiencing acute ischemic stroke is critical, especially for patients with large vessel occlusion who have poor prognosis without revascularization. Solitaire™ stent retriever devices have been shown to immediately restore vascular perfusion safely, rapidly, and effectively in acute ischemic stroke patients with large vessel occlusions. The aim of the study was to demonstrate that, among patients with large vessel, anterior circulation occlusion who have received intravenous tissue plasminogen activator, treatment with Solitaire revascularization devices reduces degree of disability 3 months post stroke. The study is a global multicenter, two-arm, prospective, randomized, open, blinded end-point trial comparing functional outcomes in acute ischemic stroke patients who are treated with either intravenous tissue plasminogen activator alone or intravenous tissue plasminogen activator in combination with the Solitaire device. Up to 833 patients will be enrolled. Patients who have received intravenous tissue plasminogen activator are randomized to either continue with intravenous tissue plasminogen activator alone or additionally proceed to neurothrombectomy using the Solitaire device within six-hours of symptom onset. The primary end-point is 90-day global disability, assessed with the modified Rankin Scale (mRS). Secondary outcomes include mortality at 90 days, functional independence (mRS ≤ 2) at 90 days, change in National Institutes of Health Stroke Scale at 27 h, reperfusion at 27 h, and thrombolysis in cerebral infarction 2b/3 flow at the end of the procedure. Statistical analysis will be conducted using simultaneous success criteria on the overall distribution of modified Rankin Scale (Rankin shift) and proportions of subjects achieving functional independence (mRS 0-2). © 2015 The Authors. International Journal of Stroke published by John Wiley & Sons Ltd on behalf of World Stroke Organization.

Antiplatelet, anticoagulant, and profibrinolytic activities of withaferin A.

PubMed

Ku, Sae-Kwang; Bae, Jong-Sup

2014-03-01

Withaferin A (WFA), an active compound from Withania somnifera, is widely researched for its anti-inflammatory, cardioactive and central nervous system effects. However, antiplatelet, anticoagulant, and profibrinolytic properties of WFA have not been studied. In this study, the anticoagulant activities of WFA were measured by monitoring activated partial thromboplastin-time (aPTT), prothrombin time (PT), fibrin polymerization, platelet aggregation, thrombus formation, and the activities of cell-based thrombin and activated factor X (FXa). The effects of WFA on the expressions of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were also tested in tumor necrosis factor-α (TNF-α) activated human umbilical vein endothelial cells (HUVECs). Our data showed that WFA inhibited thrombin-catalyzed fibrin polymerization and platelet aggregation, FeCl3-induced thrombus formation, prolonged aPTT and PT significantly and inhibited the activities and production of thrombin and FXa. WFA prolonged in vivo and ex vivo bleeding time and inhibited TNF-α induced PAI-1 production. Furthermore, PAI-1/t-PA ratio was significantly decreased by WFA. Collectively, these results indicate that WFA possesses antithrombotic activities and suggest that the current study could provide bases for the development of new anticoagulant agents. Copyright © 2014 Elsevier Inc. All rights reserved.

21 CFR 866.5715 - Plasminogen immunological test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Section 866.5715 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... substance from which plasmin, a blood-clotting factor, is formed) in serum, other body fluids, and tissues. Measurement of plasminogen levels may aid in the diagnosis of fibrinolytic (blood-clotting) disorders. (b...

21 CFR 866.5715 - Plasminogen immunological test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... Section 866.5715 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... substance from which plasmin, a blood-clotting factor, is formed) in serum, other body fluids, and tissues. Measurement of plasminogen levels may aid in the diagnosis of fibrinolytic (blood-clotting) disorders. (b...

21 CFR 866.5715 - Plasminogen immunological test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... Section 866.5715 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... substance from which plasmin, a blood-clotting factor, is formed) in serum, other body fluids, and tissues. Measurement of plasminogen levels may aid in the diagnosis of fibrinolytic (blood-clotting) disorders. (b...

21 CFR 866.5715 - Plasminogen immunological test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... Section 866.5715 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... substance from which plasmin, a blood-clotting factor, is formed) in serum, other body fluids, and tissues. Measurement of plasminogen levels may aid in the diagnosis of fibrinolytic (blood-clotting) disorders. (b...

Persons with Quebec platelet disorder have a tandem duplication of PLAU, the urokinase plasminogen activator gene.

PubMed

Paterson, Andrew D; Rommens, Johanna M; Bharaj, Bhupinder; Blavignac, Jessica; Wong, Isidro; Diamandis, Maria; Waye, John S; Rivard, Georges E; Hayward, Catherine P M

2010-02-11

Quebec platelet disorder (QPD) is an autosomal dominant bleeding disorder linked to a region on chromosome 10 that includes PLAU, the urokinase plasminogen activator gene. QPD increases urokinase plasminogen activator mRNA levels, particularly during megakaryocyte differentiation, without altering expression of flanking genes. Because PLAU sequence changes were excluded as the cause of this bleeding disorder, we investigated whether the QPD mutation involved PLAU copy number variation. All 38 subjects with QPD had a direct tandem duplication of a 78-kb genomic segment that includes PLAU. This mutation was specific to QPD as it was not present in any unaffected family members (n = 114), unrelated French Canadians (n = 221), or other persons tested (n = 90). This new information on the genetic mutation will facilitate diagnostic testing for QPD and studies of its pathogenesis and prevalence. QPD is the first bleeding disorder to be associated with a gene duplication event and a PLAU mutation.

Impaired glymphatic perfusion after strokes revealed by contrast-enhanced MRI: a new target for fibrinolysis?

PubMed

Gaberel, Thomas; Gakuba, Clement; Goulay, Romain; Martinez De Lizarrondo, Sara; Hanouz, Jean-Luc; Emery, Evelyne; Touze, Emmanuel; Vivien, Denis; Gauberti, Maxime

2014-10-01

The aim of the present study was to investigate the impact of different stroke subtypes on the glymphatic system using MRI. We first improved and characterized an in vivo protocol to measure the perfusion of the glymphatic system using MRI after minimally invasive injection of a gadolinium chelate within the cisterna magna. Then, the integrity of the glymphatic system was evaluated in 4 stroke models in mice including subarachnoid hemorrhage (SAH), intracerebral hemorrhage, carotid ligature, and embolic ischemic stroke. We were able to reliably evaluate the glymphatic system function using MRI. Moreover, we provided evidence that the glymphatic system was severely impaired after SAH and in the acute phase of ischemic stroke, but was not altered after carotid ligature or in case of intracerebral hemorrhage. Notably, this alteration in glymphatic perfusion reduced brain clearance rate of low-molecular-weight compounds. Interestingly, glymphatic perfusion after SAH can be improved by intracerebroventricular injection of tissue-type plasminogen activator. Moreover, spontaneous arterial recanalization was associated with restoration of the glymphatic function after embolic ischemic stroke. SAH and acute ischemic stroke significantly impair the glymphatic system perfusion. In these contexts, injection of tissue-type plasminogen activator either intracerebroventricularly to clear perivascular spaces (for SAH) or intravenously to restore arterial patency (for ischemic stroke) may improve glymphatic function. © 2014 American Heart Association, Inc.

Antihypertensive treatment prolongs tissue plasminogen activator door-to-treatment time: secondary analysis of the INSTINCT trial.

PubMed

Skolarus, Lesli E; Scott, Phillip A; Burke, James F; Adelman, Eric E; Frederiksen, Shirley M; Kade, Allison M; Kalbfleisch, Jack D; Ford, Andria L; Meurer, William J

2012-12-01

Identifying modifiable tissue plasminogen activator treatment delays may improve stroke outcomes. We hypothesized that prethrombolytic antihypertensive treatment (AHT) may prolong door-to-treatment time (DTT). We performed an analysis of consecutive tissue plasminogen activator-treated patients at 24 randomly selected community hospitals in the Increasing Stroke Treatment through Interventional Behavior Change Tactics (INSTINCT) trial between 2007 and 2010. DTT among stroke patients who received prethrombolytic AHT were compared with those who did not receive prethrombolytic AHT. We then calculated a propensity score for the probability of receiving prethrombolytic AHT using logistic regression with demographics, stroke risk factors, home medications, stroke severity (National Institutes of Health Stroke Scale), onset-to-door time, admission glucose, pretreatment blood pressure, emergency medical service transport, and location at time of stroke as independent variables. A paired t test was performed to compare the DTT between the propensity-matched groups. Of 534 tissue plasminogen activator-treated stroke patients analyzed, 95 received prethrombolytic AHT. In the unmatched cohort, patients who received prethrombolytic AHT had a longer DTT (mean increase, 9 minutes; 95% confidence interval, 2-16 minutes) than patients who did not. After propensity matching, patients who received prethrombolytic AHT had a longer DTT (mean increase, 10.4 minutes; 95% confidence interval, 1.9-18.8) than patients who did not receive prethrombolytic AHT. Prethrombolytic AHT is associated with modest delays in DTT. This represents a potential target for quality-improvement initiatives. Further research evaluating optimum prethrombolytic hypertension management is warranted.

Physiological regulation of MMPs and tPA/PAI in the arterial wall of rats by noradrenergic tone and angiotensin II.

PubMed

Dab, Houcine; Hachani, Rafik; Dhaouadi, Nedra; Hodroj, Wassim; Sakly, Mohsen; Randon, Jacques; Bricca, Giampiero; Kacem, Kamel

2012-03-01

The interactions between the sympathetic nervous system (SNS) and angiotensin II (ANG II), and their direct effects in vitro on the enzymes involved in vascular extracellular matrix (ECM) degradation, were examined. Rats were treated with guanethidine, losartan or the combined treatments. mRNA, protein and activity of matrix metalloproteinase (MMP)-2 and MMP-9 and mRNA of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) were quantified in abdominal aorta (AA) and femoral artery (FA). Norepinephrine (NE) or ANG II with adrenergic (β, α1 and α2) or losartan antagonists was tested for MMP mRNA response in cultured vascular smooth muscle cells (VSMCs). Combined treatment enhances the inhibition of MMP-2 mRNA and protein level induced by simple treatment in AA. However MMP-9 in AA and MMP mRNA in FA were reduced in the same order by treatments. MMP activities were not affected by treatments. The t-PA/PAI-1 ratio, which reflects the fibrinolytic balance, remained higher after treatments. In cultured VSMCs, NE induced stimulation of MMP mRNA via α2 and β adrenergic receptors and MMP-2 activity via β adrenergic receptors, while ANG II-induced stimulation was abrogated by losartan. Overall, there is a synergic inhibition of both systems on the level of MMP-2 in AA.

The Plasminogen Activation System Modulates Differently Adipogenesis and Myogenesis of Embryonic Stem Cells

PubMed Central

Hadadeh, Ola; Barruet, Emilie; Peiretti, Franck; Verdier, Monique; Bernot, Denis; Hadjal, Yasmine; Yazidi, Claire El; Robaglia-Schlupp, Andrée; De Paula, Andre Maues; Nègre, Didier; Iacovino, Michelina; Kyba, Michael; Alessi, Marie-Christine; Binétruy, Bernard

2012-01-01

Regulation of the extracellular matrix (ECM) plays an important functional role either in physiological or pathological conditions. The plasminogen activation (PA) system, comprising the uPA and tPA proteases and their inhibitor PAI-1, is one of the main suppliers of extracellular proteolytic activity contributing to tissue remodeling. Although its function in development is well documented, its precise role in mouse embryonic stem cell (ESC) differentiation in vitro is unknown. We found that the PA system components are expressed at very low levels in undifferentiated ESCs and that upon differentiation uPA activity is detected mainly transiently, whereas tPA activity and PAI-1 protein are maximum in well differentiated cells. Adipocyte formation by ESCs is inhibited by amiloride treatment, a specific uPA inhibitor. Likewise, ESCs expressing ectopic PAI-1 under the control of an inducible expression system display reduced adipogenic capacities after induction of the gene. Furthermore, the adipogenic differentiation capacities of PAI-1−/− induced pluripotent stem cells (iPSCs) are augmented as compared to wt iPSCs. Our results demonstrate that the control of ESC adipogenesis by the PA system correspond to different successive steps from undifferentiated to well differentiated ESCs. Similarly, skeletal myogenesis is decreased by uPA inhibition or PAI-1 overexpression during the terminal step of differentiation. However, interfering with uPA during days 0 to 3 of the differentiation process augments ESC myotube formation. Neither neurogenesis, cardiomyogenesis, endothelial cell nor smooth muscle formation are affected by amiloride or PAI-1 induction. Our results show that the PA system is capable to specifically modulate adipogenesis and skeletal myogenesis of ESCs by successive different molecular mechanisms. PMID:23145071

Spin filter perfusion system for high density cell culture: production of recombinant urinary type plasminogen activator in CHO cells.

PubMed

Avgerinos, G C; Drapeau, D; Socolow, J S; Mao, J I; Hsiao, K; Broeze, R J

1990-01-01

We have used a 20 liter stirred tank fermentor, equipped with a 127 mesh ethylene-tetrafluoroethylene rotating screen for cell recycle, for the continuous production of recombinant single chain urokinase-type plasminogen activator (rscu-PA) from Chinese hamster ovary (CHO) cells. Viable cell densities between 60 and 74 million per ml were maintained at medium perfusion rates of 3.0 to 4.0 fermentor volumes per day. Cells were retained by the 120 micron nominal opening filter through the formation of "clumped" cell aggregates of 200 to 600 microns in size, which did not foul the filter. In 31 days of culture, a total of 51 grams of rscu-PA were produced in 1,000 liters of medium. The rscu-PA produced over the course of this continuous culture was purified and characterized both in vitro and in vivo and shown to be comparable to natural scu-PA produced from the transformed human kidney cell line, TCL-598.

Pelvic adhesion and gonadotropin-releasing hormone analogue: effects of triptorelin acetate depot on coagulation and fibrinolytic activities.

PubMed

Di Nardo, Maria Antonietta; Annunziata, Maria Laura; Ammirabile, Massimiliano; Di Minno, Matteo Nicola Dario; Ruocco, Anna Lilia; De Falco, Marianna; Di Lieto, Andrea

2012-06-01

The study investigated the impact of gonadotropin-releasing hormone analogue (GnRH-a) on coagulation and fibrinolytic activities and its effectiveness in the prevention of pelvic adhesion after myomectomy. Thirty-two infertile women underwent myomectomy followed by adhesion evaluation surgery with a second-look laparoscopy. Before myomectomy, 15 women were treated with triptorelin acetate for 3 months and 17 received no treatment. Plasminogen activator inhibitor (PAI), thrombin activatable fibrinolysis inhibitor (TAFI), protein C (PC), plasminogen, α2-antiplasmin were determined by enzyme-linked immunosorbent assays and the activity of coagulation factors V and VIII by coagulometric methods. Patients treated with GnRH-a showed significant decrease in PAI, TAFI, factors V, and VIII (P < .05) and increased PC (P < .05), but no significant change in plasminogen and α2-antiplasmin levels compared with control group. The incidence, extent, and severity of adhesions were significantly lower in GnRH-a-treated patients compared with control group (P < .05), suggesting a possible critical role of the GnRH-a therapy in preventing postoperative adhesion development.

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

Effect of pH and glucose on cultured human peritoneal mesothelial cells.

PubMed

Shao, J C; Yorioka, N; Nishida, Y; Yamakido, M

1999-08-01

We investigated the effects of various pH and glucose concentrations on the growth of human peritoneal mesothelial cells and on coagulation and fibrinolytic factors. Cells were cultured at various pH values in Ham's F-12 medium containing 1.0% foetal calf serum and supplemented with D-glucose or D-mannitol at various concentrations. After 4-48 h, cell proliferation and 3H-thymidine incorporation were determined. Coagulation and fibrinolytic factors were measured after 48 h. Glucose caused concentration-dependent inhibition of cell growth at all pH values, but the deleterious effect of low pH on cell proliferation was faster and stronger than that of high glucose. At a similar osmolality, mannitol caused less inhibition of cell proliferation than glucose. There was a glucose concentration-dependent increase of thrombin-antithrombin III complex production at all pH values. At pH 5.2, tissue-type plasminogen activator production was far lower than at higher pH values, and production of the plasminogen activator inhibitor showed a glucose concentration-dependent increase. At pH 6.5 or 7.3, however, the plasminogen activator inhibitor production decreased and tissue-type plasminogen activator production increased in a glucose concentration-dependent manner. Low pH and/or high glucose culture medium had an inhibitory effect on peritoneal mesothelial cells, with the effect of high glucose being partially related to hyperosmolality. These cells may modulate peritoneal coagulant and fibrinolytic activity, with the balance between coagulation and fibrinolysis being disturbed by low pH and/or high glucose.

Variable Resistance to Plasminogen Activator Initiated Fibrinolysis for Intermediate-Risk Pulmonary Embolism

PubMed Central

Stubblefield, William B.; Alves, Nathan J.; Rondina, Matthew T.; Kline, Jeffrey A.

2016-01-01

Background We examine the clinical significance and biomarkers of tissue plasminogen activator (tPA)-catalyzed clot lysis time (CLT) in patients with intermediate-risk pulmonary embolism (PE). Methods Platelet-poor, citrated plasma was obtained from patients with PE. Healthy age- and sex-matched patients served as disease-negative controls. Fibrinogen, α2-antiplasmin, plasminogen, thrombin activatable fibrinolysis inhibitor (TAFI), plasminogen activator Inhibitor 1 (PAI-1), thrombin time and D-dimer were quantified. Clotting was induced using CaCl2, tissue factor, and phospholipid. Lysis was induced using 60 ng/mL tPA. Time to 50% clot lysis (CLT) was assessed by both thromboelastography (TEG) and turbidimetry (A405). Results Compared with disease-negative controls, patients with PE exhibited significantly longer mean CLT on TEG (+2,580 seconds, 95% CI 1,380 to 3,720 sec). Patients with PE and a short CLT who were treated with tenecteplase had increased risk of bleeding, whereas those with long CLT had significantly worse exercise tolerance and psychometric testing for quality of life at 3 months. A multivariate stepwise removal regression model selected PAI-1 and TAFI as predictive biomarkers of CLT. Conclusion The CLT from TEG predicted increased risk of bleeding and clinical failure with tenecteplase treatment for intermediate-risk PE. Plasmatic PAI-1 and TAFI were independent predictors of CLT. PMID:26866684

Hydronephrosis is associated with elevated plasmin in urine in pediatric patients and rats and changes in NCC and γ-ENaC abundance in rat kidney.

PubMed

Zachar, Rikke; Al-Mashhadi, Ammar; Dimke, Henrik; Svenningsen, Per; Jensen, Boye L; Carlström, Mattias

2018-05-16

Obstruction of urine flow at the level of the pelvo-ureteric junction (UPJO) and subsequent development of hydronephrosis is one of the most common congenital renal malformations. UPJO is associated with development of salt-sensitive hypertension, which is set by the obstructed kidney, and with a stimulated renin-angiotensin-aldosterone system (RAAS) in rodent models. This study aimed at investigating the hypothesis that i) in pediatric patients with UPJO the RAAS is activated prior to surgical relief of the obstruction; ii) in rats with UPJO the RAAS activation is reflected by increased abundance of renal aldosterone-stimulated Na+ transporters; and iii) the injured UPJO kidney allows aberrant filtration of plasminogen leading to proteolytic activation of the epithelial sodium channel gamma subunit (γ-ENaC). Hydronephrosis due to UPJO in pediatric patients and rats was associated with increased urinary plasminogen/creatinine ratio. In pediatric patients, plasma renin, angiotensin II, urine and plasma aldosterone and urine soluble pro-renin receptor did not differ significantly before and after surgery, or compared with controls. Increased plasmin/plasminogen ratio was seen in UPJO rats. Intact γ-ENaC abundance was not changed in UPJO kidney while low-molecular cleavage product abundance increased. The Na-Cl cotransporter (NCC) displayed significantly lower abundance in the UPJO kidney compared to the non-obstructed contralateral kidney. The Na-K-ATPase alpha-subunit was unaltered. Treatment with an angiotensin-converting enzyme inhibitor (8 days, captopril) significantly lowered blood pressure in UPJO rats. It is concluded that the RAAS contributes to hypertension following partial obstruction of urine flow at the pelvo-ureteric junction with potential contribution from proteolytic activation of ENaC.

A study of the possible association of plasminogen activator inhibitor type 1 4G/5G insertion/deletion polymorphism with susceptibility to schizophrenia and in its subtypes.

PubMed

Yenilmez, C; Ozdemir Koroglu, Z; Kurt, H; Yanas, M; Colak, E; Degirmenci, I; Gunes, H V

2017-02-01

Inhibition of the fibrinolytic system may occur at the level of plasminogen activation, mainly by PAI-1. Mental and physical stress caused to alterations of platelet function, and also decreased to fibrinolytic activity. Furthermore, stress-induced thrombosis regulation was proposed to be by PAI-1 in schizophrenia patients. In this study, the distribution of genotypes and frequency of alleles of the plasminogen activator inhibitor type 1 (PAI-1) gene 4G/5G polymorphism in different Turkish clinical schizophrenia subtypes was investigated for its role in schizophrenia development. The clinical schizophrenia subtypes include paranoid, catatonic, disorganized, undifferentiated and residual, as diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders-Fourth Edition IV (DSM-IV). Samples of genomic DNA (250 total, including 150 schizophrenia patients and 100 healthy subjects) were analysed. PAI-1 4G/5G genotyping was performed by polymerase chain reaction-allele-specific amplification. PCR products were separated by 2% agarose gel electrophoresis and then visualized. The genotype distributions (P = 0·136) and allele frequencies (P = 0·721 for 4G, P = 0. 097 for 5G) were not significantly different between patients with schizophrenia and control subjects for the 4G/5G polymorphism. Similar results were also found for the genotype distributions (P = 0·640) and allele frequencies (P = 0·763 for 4G, P = 0·448 for 5G) in the clinical schizophrenia subtypes compared to the each other. We conclude that PAI-1 4G/5G polymorphism was not significantly associated with schizophrenia or its subtypes in the Turkish population. However, we recognize that with our sample sizes, we cannot exclude weak associations. © 2016 John Wiley & Sons Ltd.

Numerical Simulation of Rheological, Chemical and Hydromechanical Processes of Thrombolysis

NASA Astrophysics Data System (ADS)

Khramchenkov, E.; Khramchenkov, M.

2015-04-01

Mathematical model of clot lysis in blood vessels is developed on the basis of equations of convection-diffusion. Fibrin of the clot is considered stationary solid phase, and plasminogen, plasmin and plasminogen-activators - as dissolved fluid phases. As a result of numerical solution of the model predictions of lysis process are gained. Important influence of clot swelling on the process of lysis is revealed.

Chronic dietary fat intake modifies the postprandial response of hemostatic markers to a single fatty test meal.

PubMed

Delgado-Lista, Javier; Lopez-Miranda, Jose; Cortés, Begoña; Perez-Martinez, Pablo; Lozano, Aquiles; Gomez-Luna, Rafael; Gomez, Purificacion; Gomez, Maria Jose; Criado, Juan; Fuentes, Francisco; Perez-Jimenez, Francisco

2008-02-01

Hemostasis is the result of a complex equilibrium between coagulation and fibrinolysis, and the influence of different dietary models on this equilibrium is not entirely known. The objective was to compare the effects of the chronic intake of different dietary models on postprandial hemostasis. In a randomized crossover design, 20 healthy men consumed for 28 d each diets rich in monounsaturated fatty acids (MUFAs), saturated fatty acids (SFAs), and carbohydrates plus n-3 fatty acids (CHO/N3). Fasting and postprandial hemostatic factors (factor VII coagulant activity, plasminogen activator inhibitor-1, tissue-type plasminogen activator, d-dimer, and thromboxane B(2)) were measured; meal tests for the postprandial measures were based on butter, virgin olive oil, and walnuts for the SFA, MUFA, and CHO/N3 diets, respectively. There were no differences in the fasting variables after the dietary periods. After the 3 fatty meals were consumed, we observed an increase in thromboxane B(2) and d-dimer and a reduction in tissue plasminogen activator, irrespective of the dietary model. The MUFA or CHO/N3 meals lowered postprandial concentrations of factor VII coagulant activity, although the reduction was greater after the MUFA-enriched meal. The concentration of plasminogen activator inhibitor-1 was greater after the SFA meal than after the other 2 meals. The administration of a fatty meal induces a postprandial procoagulant tendency, irrespective of the type of fat consumed. However, the use of a dietary model rich in SFA creates a more procoagulant environment than does a model that includes MUFA or CHO/N3 as the source of fatty acids.

Clotting and fibrinolytic changes after firefighting activities.

PubMed

Smith, Denise L; Horn, Gavin P; Petruzzello, Steven J; Fahey, George; Woods, Jeffrey; Fernhall, Bo

2014-03-01

Approximately 45%-50% of all duty-related deaths among firefighters are due to sudden cardiovascular events, and a disproportionate number of these fatalities occur after strenuous fire suppression activities. The objective of this study is to evaluate the effect of strenuous firefighting activities on platelets, coagulation, and fibrinolytic activity and to document the extent to which these variables recovered 2 h after completion of the firefighting activity. Firefighters performed 18 min of simulated firefighting activities in a training structure that contained live fires. After firefighting activities, firefighters were provided with fluid and allowed to cool down and then recovered for 2 h in an adjacent room. Blood samples were obtained prefirefighting, postfirefighting, and 2 h postfirefighting. Platelet number, platelet activity, and coagulatory potential increased immediately postfirefighting and many variables (platelet function, partial thromboplastin time, and factor VIII) reflected a procoagulatory state even after 2 h of recovery. Fibrinolysis, as reflected by tissue plasminogen activator, also was enhanced immediately postfirefighting but returned to baseline values by 2 h postfirefighting. In contrast, inhibition of fibrinolysis, as evidenced by a reduction in plasminogen activator inhibitor-1, was depressed at 2 h postfirefighting. Firefighting resulted in elevated coagulatory and fibrinolytic activity. However, 2 h postfirefighting, tissue plasminogen activator returned to baseline and coagulatory potential remained elevated. The procoagulatory state that exists after firefighting may provide a mechanistic link to the reports of sudden cardiac events after strenuous fire suppression activities.

Subdural Instillation of a Thrombolytic Agent for Treatment of Recurrent Subdural Hematoma.

PubMed

Frenkel, Mark B; Sarwal, Aarti; Wren, Mary Petrulis; Newey, Christopher R; Couture, Daniel E

This study aims to report the case of a patient with recurrent subdural hemorrhage (SDH) who was administered tissue plasminogen activator through a subdural drain to enhance drainage and prevent recurrence. An 85-year-old man was treated for subacute over chronic SDH that kept on reaccumulating despite serial twist drill drainage, burr hole drainage, and craniotomy. No coagulopathy was identified with adequate blood pressure control. Treatment with tissue plasminogen activator resulted in successful drainage of the SDH, and the patient had no further recurrence at 9-month follow-up.

Interaction of Leptospira Elongation Factor Tu with Plasminogen and Complement Factor H: A Metabolic Leptospiral Protein with Moonlighting Activities

PubMed Central

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. PMID:24312361

Stimulation of cell-surface urokinase-type plasminogen activator activity and cell migration in vascular endothelial cells by a novel hexapeptide analogue of neurotensin.

PubMed

Ushiro, S; Mizoguchi, K; Yoshida, S; Jimi, S; Fujiwara, T; Yoshida, M; Wei, E T; Kitabgi, P; Amagaya, S; Ono, M; Kuwano, M

1997-12-01

To investigate if neurotensin (NT) could induce activation of urokinase-type plasminogen activator (uPA) in vascular endothelial cells, we utilized the acetyl-NT (8-13) analogue, TJN-950, in which the C-terminal leucine is reduced to leucinol. TJN-950 inhibited the binding of 125I-NT to membranes of newborn rat brains and of COS-7 cells transfected with rat NT receptor cDNA, but at 10(4) higher doses than NT (8-13). However, TJN-950 was as effective as NT in inducing the fibrinolytic activity in bovine vascular aortic and human umbilical vein endothelial cells, and enhanced the migration of vascular endothelial cells. Moreover, administration of TJN-950 induced neovascularization in the rat cornea in vivo. TJN-950 had no effect on expression of uPA, plasminogen activator inhibitor-1 or uPA receptor mRNA. The binding of 125I-TJN-950 to cell membranes was blocked by unlabeled uPA and TJN-950, but not the amino-terminal or 12-32 fragment of uPA. TJN-950 may enhance uPA activity in vascular endothelial cells by interacting with the uPA receptor, resulting in induction of angiogenesis.

Tranexamic acid in treatment of melasma: A comprehensive review of clinical studies.

PubMed

Taraz, Mohammad; Niknam, Somayeh; Ehsani, Amir Houshang

2017-05-01

Melasma is a human melanogenesis dysfunction that results in localized, chronic acquired hyperpigmentation of the skin. It has a significant impact on appearance, causing psychosocial and emotional distress, and reducing the quality of life of the affected patients. Tranexamic acid (TA) is a plasmin inhibitor used to prevent abnormal fibrinolysis to reduce blood loss and exerts its effect by reversibly blocking lysine binding sites on plasminogen molecules, thus inhibiting plasminogen activator (PA) from converting plasminogen to plasmin. As plasminogen also exists in human epidermal basal cells and cultured human keratinocyte are known to produce PA, there is basic rationale that TA will affect keratinocyte function and interaction. A thorough literature review indicates that while TA is used through various route of administration including oral, topical, and intradermal injection and as adjutant therapy with laser to treat melasma, its efficacy is not established adequately. Further studies are needed to clarify the role of TA in treatment of melasma. © 2017 Wiley Periodicals, Inc.

Effects of Heparin and ε-Aminocaproic Acid in Dogs on Plasmin- 125I Generation in Response to Urokinase Injections and Venous Injury

PubMed Central

Takeda, Y.; Parkhill, T. R.; Nakabayashi, M.

1972-01-01

The isotopic method described previously for quantification of plasmin- 125I by disc gel electrophoresis was modified by inclusion of euglobulin precipitation to expand its applicability to plasmas containing low radioactivity of plasmin- 125I and plasminogen- 125I. It was found that the euglobulin precipitation method precipitates 72.4±2.1 (sd)% of both plasmin- 125I and plasminogen- 125I. Using this method and plasminogen- 125I as a tracer, studies were first made of the effects of heparin and ε-aminocaproic acid in dogs on plasmin- 125I generation in responese to a single injection of urokinase and to venous injury; second, of the effects of venous occlusion and thrombosis on plasmin- 125I generation; and third, in vitro studies of plasminogen- 125I affinity to fibrin and its activation in blood clots. The venous injury was produced by the damage of venous endothelium by an injection of 90% phenol and the thrombosis by a thrombin injection into an occluded vein. Heparin and ε-aminocaproic acid under the present experimental conditions inhibited about 78 and 100%, respectively of plasmin- 125I generation by the urokinase injection. Similar inhibitory effects of heparin and ε-aminocaproic acid were observed on plasmin- 125I generation in response to venous injury. The venous occlusion caused a small degree of plasmin- 125I generation, but thrombin thrombosis did not seem to stimulate the generation of plasmin- 125I. The in vitro studies showed that plasminogen- 125I does not have a specific affinity to fibrin and is incorporated into blood clots in approximately equal concentrations as those in serum during clotting processes, and that blood clots per se do not stimulate plasmin- 125I generation. These results suggest that injured veins release considerable amounts of vascular plasminogen activators into circulation and that these play an important role in thrombus dissolution in vivo. PMID:4262519

Prothrombotic state and impaired fibrinolysis in bullous pemphigoid, the most frequent autoimmune blistering disease

PubMed Central

Marzano, A V; Tedeschi, A; Polloni, I; Crosti, C; Cugno, M

2013-01-01

Bullous pemphigoid (BP) is a potentially life-threatening autoimmune blistering disease that is burdened with an increased risk of cardiovascular events. In BP, there is an interplay between inflammation and coagulation both locally, which contributes to skin damage, and systemically, which leads to a prothrombotic state. Fibrinolysis is an important defence mechanism against thrombosis, but has only been studied locally in BP and no systemic data are available. The aim of this observational study was to evaluate systemic fibrinolysis and coagulation activation in patients with BP. We measured parameters of fibrinolysis and coagulation by immunoenzymatic methods in plasma from 20 patients with BP in an active phase and during remission after corticosteroid treatment. The controls were 20 age- and sex-matched healthy subjects. Plasma levels of plasminogen activator inhibitor type 1 (PAI-1) antigen, PAI-1 activity and tissue plasminogen activator (t-PA) antigen were significantly higher in the BP patients with active disease than in healthy controls (P = 0·0001 for all), as were the plasma levels of the fibrin fragment d-dimer and prothrombin fragment F1+2 (P = 0·0001 for both). During remission after treatment, levels of PAI-1 antigen and PAI-1 activity decreased significantly (P = 0·008 and P = 0·006, respectively), and there was also a significant decrease in plasma levels of d-dimer (P = 0·0001) and F1+2 (P = 0·0001). Fibrinolysis is inhibited in patients with active BP, due mainly to an increase in plasma levels of PAI-1. Corticosteroids not only induce the regression of BP lesions, but also reduce the inhibition of fibrinolysis, which may contribute to decreasing thrombotic risk. PMID:23199326

The Association of Plasminogen Activator Inhibitor Type 1 (PAI-1) Level and PAI-1 4G/5G Gene Polymorphism with the Formation and the Grade of Endometrial Cancer.

PubMed

Yıldırım, Malik Ejder; Karakuş, Savas; Kurtulgan, Hande Küçük; Kılıçgün, Hasan; Erşan, Serpil; Bakır, Sevtap

2017-08-01

Plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor (Serpine 1), and it inhibits both tissue plasminogen activator and urokinase plasminogen activator which are important in fibrinolysis. We aimed to find whether there is a possible association between PAI-1 level, PAI-1 4G/5G polymorphism, and endometrial cancer. PAI-1 levels in peripheral blood were determined in 82 patients with endometrial carcinoma and 76 female healthy controls using an enzyme-linked immunoassay (ELISA). Then, the genomic DNA was extracted and screened by reverse hybridization procedure (Strip assay) to detect PAI 1 4G/5G polymorphism. The levels of PAI-1 in the patients were higher statistically in comparison to controls (P < 0.001). The distribution of PAI-1 4G/5G polymorphism was quite different between patients and controls (P = 0.008), and 4G allelic frequency was significantly higher in the patients of endometrial cancer than in controls (P = 0.026). We found significant difference between Grade 1 and Grade 2+3 patients in terms of the PAI-1 levels (P = 0.047). There was no association between PAI-1 4G/5G polymorphism and the grades of endometrial cancer (P = 0.993). Our data suggest that the level of PAI-1 and PAI-1 4G/5G gene polymorphism are effective in the formation of endometrial cancer. PAI-1 levels are also associated with the grades of endometrial cancer.

Alveolar fibrin formation caused by enhanced procoagulant and depressed fibrinolytic capacities in severe pneumonia. Comparison with the acute respiratory distress syndrome.

PubMed

Günther, A; Mosavi, P; Heinemann, S; Ruppert, C; Muth, H; Markart, P; Grimminger, F; Walmrath, D; Temmesfeld-Wollbrück, B; Seeger, W

2000-02-01

Changes in the alveolar hemostatic balance in severe pneumonia were compared with those in the acute respiratory distress syndrome (ARDS). Analysis was performed in bronchoalveolar lavage fluids (BALF) of patients with ARDS triggered by nonpulmonary underlying events in the absence of lung infection (ARDS; n = 25), pneumonia demanding mechanical ventilation (PNEU-vent; n = 114), spontaneously breathing patients with pneumonia (PNEU-spon; n = 40), and ARDS in combination with lung infection (ARDS+PNEU; n = 43); comparison with healthy control subjects (n = 35) was performed. In all groups of patients, BALF total procoagulant activity was increased by nearly two orders of magnitude, being largely attributable to the tissue factor pathway of coagulation. Concomitantly, markedly reduced overall fibrinolytic capacity (fibrin plate assay) was noted in the lavage fluids of all patients. BALF levels of urokinase-type plasminogen activator were significantly reduced throughout, whereas the lavage concentrations of tissue-type plasminogen activator did not differ from those in control subjects. In addition, markedly enhanced levels of plasminogen activator- inhibitor I and alpha(2)-antiplasmin were noted in ARDS, ARDS+PNEU, and PNEU-vent, but not in PNEU-spon. In all groups of patients, the changes in the lavage enzymatic activities were paralleled by manifold increased BALF concentrations of fibrinopeptide A and D-dimer, reflecting in vivo coagulation processes. Within the overall number of patients with pneumonia, changes in the alveolar hemostatic balance were more prominent in alveolar and interstitial pneumonia than in bronchopneumonia. Acute inflammatory lung injury, whether triggered by nonpulmonary systemic events or primary lung infection, is thus consistently characterized by both enhanced procoagulant and depressed fibrinolytic activities in the alveolar lining layer, with the appearance of fibrin formation in this compartment. Profile and extent of changes in severe pneumonia demanding respirator therapy are virtually identical to those in ARDS, whereas somewhat less prominent alterations of the alveolar hemostatic balance are noted in spontaneously breathing patients with pneumonia.

Molecular adaptation of a plant-bacterium outer membrane protease towards plague virulence factor Pla

PubMed Central

2011-01-01

Background Omptins are a family of outer membrane proteases that have spread by horizontal gene transfer in Gram-negative bacteria that infect vertebrates or plants. Despite structural similarity, the molecular functions of omptins differ in a manner that reflects the life style of their host bacteria. To simulate the molecular adaptation of omptins, we applied site-specific mutagenesis to make Epo of the plant pathogenic Erwinia pyrifoliae exhibit virulence-associated functions of its close homolog, the plasminogen activator Pla of Yersinia pestis. We addressed three virulence-associated functions exhibited by Pla, i.e., proteolytic activation of plasminogen, proteolytic degradation of serine protease inhibitors, and invasion into human cells. Results Pla and Epo expressed in Escherichia coli are both functional endopeptidases and cleave human serine protease inhibitors, but Epo failed to activate plasminogen and to mediate invasion into a human endothelial-like cell line. Swapping of ten amino acid residues at two surface loops of Pla and Epo introduced plasminogen activation capacity in Epo and inactivated the function in Pla. We also compared the structure of Pla and the modeled structure of Epo to analyze the structural variations that could rationalize the different proteolytic activities. Epo-expressing bacteria managed to invade human cells only after all extramembranous residues that differ between Pla and Epo and the first transmembrane β-strand had been changed. Conclusions We describe molecular adaptation of a protease from an environmental setting towards a virulence factor detrimental for humans. Our results stress the evolvability of bacterial β-barrel surface structures and the environment as a source of progenitor virulence molecules of human pathogens. PMID:21310089

Identification and characterization of Taenia solium enolase as a plasminogen-binding protein.

PubMed

Ayón-Núñez, Dolores A; Fragoso, Gladis; Espitia, Clara; García-Varela, Martín; Soberón, Xavier; Rosas, Gabriela; Laclette, Juan P; Bobes, Raúl J

2018-06-01

The larval stage of Taenia solium (cysticerci) is the causal agent of human and swine cysticercosis. When ingested by the host, T. solium eggs are activated and hatch in the intestine, releasing oncospheres that migrate to various tissues and evolve into cysticerci. Plasminogen (Plg) receptor proteins have been reported to play a role in migration processes for several pathogens. This work is aimed to identify Plg-binding proteins in T. solium cysticerci and determine whether T. solium recombinant enolase (rTsEnoA) is capable of specifically binding and activating human Plg. To identify Plg-binding proteins, a 2D-SDS-PAGE ligand blotting was performed, and recognized spots were identified by MS/MS. Seven proteins from T. solium cysticerci were found capable of binding Plg: fascicilin-1, fasciclin-2, enolase, MAPK, annexin, actin, and cytosolic malate dehydrogenase. To determine whether rTsEnoA binds human Plg, a ligand blotting was performed and the results were confirmed by ELISA both in the presence and absence of εACA, a competitive Plg inhibitor. Finally, rTsEnoA-bound Plg was activated to plasmin in the presence of tPA. To better understand the evolution of enolase isoforms in T. solium, a phylogenetic inference analysis including 75 enolase amino acid sequences was conducted. The origin of flatworm enolase isoforms, except for Eno4, is independent of their vertebrate counterparts. Therefore, herein we propose to designate tapeworm protein isoforms as A, B, C, and 4. In conclusion, recombinant enolase showed a strong plasminogen binding and activating activity in vitro. T. solium enolase could play a role in parasite invasion along with other plasminogen-binding proteins. Copyright © 2018 Elsevier B.V. All rights reserved.

THE ENHANCEMENT OF CHLOROFORM-INDUCED PLASMA PROTEOLYTIC ACTIVITY BY EPSILON AMINOCAPROIC ACID

PubMed Central

Donaldson, Virginia H.; Ratnoff, Oscar D.

1962-01-01

The proteolytic activity in chloroform-treated plasma euglobulins has been attributed to plasmin. Plasmin can digest both casein and fibrin. Epsilon aminocaproic acid, which inhibits the activation of plasminogen, the precursor of plasmin, by streptokinase, urokinase, and tissue activators enhanced the development of casein hydrolytic activity in a mixture of chloroform and plasma euglobulins. Fibrinolytic activity was also enhanced, but this was evident only if the epsilon aminocaproic acid was removed from the chloroform-treated euglobulins prior to assay. The reasons for the paradoxical enhancement of chloroform-induced casein hydrolysis by euglobulins containing epsilon aminocaproic acid are unclear. However, studies of optimal pH, heat stability, and the effect of ionic strength on the activation of the precursor of this proteolytic enzyme do not differentiate it from plasminogen. PMID:13887179

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

Interaction of fucoidan with proteases and inhibitors of coagulation and fibrinolysis.

PubMed

Minix, R; Doctor, V M

1997-09-01

The interactions of fucoidan with glutamic plasminogen (Glu-Plg), two-chain tissue plasminogen activator (t-PA), LMwt-urokinase, thrombin, and antithrombin III (AT-III) were investigated using fucoidan-sepharose affinity chromatography. The results showed 1) a high degree of affinity between fucoidan-sepharose and Glu-Plg; Lmwt-urokinase and thrombin while t-Pa and AT-III did not bind with fucoidan-sepharose. 2) The double reciprocal plot for the LMwt-urokinase activation of Glu-Plg showed that plasminogen activator inhibitor (PAI-1) inhibited this reaction in a noncompetitive manner and that the presence of fucoidan decreased Km for this interaction by 50% and increased Kcat by 30-fold, 3) The double reciprocal plot for the t-PA activation of Glu-Plg showed that PAI-1 inhibited this reaction in a competitive manner and that fucoidan in conjunction with 6-aminohexanoic acid (6-AH) increased Kcat for this interaction by 5-fold without affecting Km. 4) Fucoidan enhanced the interaction of thrombin with both AT-III and heparin cofactor II (HC-II) and it was more effective than unfractionated heparin of LMwt-heparin in enhancing the interaction of HC-II with thrombin.

Endotoxin-induced intravascular coagulation in rabbits: effect of tissue plasminogen activator vs urokinase of PAI generation, fibrin deposits and mortality.

PubMed

Paloma, M J; Páramo, J A; Rocha, E

1995-12-01

We have evaluated the effect of plasminogen activators (t-PA and urokinase) on an experimental model of disseminated intravascular coagulation (DIC) in rabbits by injection of 20 micrograms/kg/h of E. coli lipopolysaccharide during 6 h t-PA (0.2 mg/kg and 0.7 mg/kg), urokinase (3000 U/kg/h) and saline (control) were given simultaneously with endotoxin. Results indicated that urokinase and low dose of t-PA significantly reduced the increase of plasminogen activator inhibitor (PAI) activity observed 2 h after endotoxin (p < 0.001). High t-PA dose also diminished the PAI levels at 6 h (p < 0.001). A significant reduction of fibrin deposits in kidneys was observed din both t-PA treated groups as compared with findings in the group of rabbits infused with saline solution (p < 0.005), whereas urokinase had no significant effect on the extent of fibrin deposition. Finally, the mortality rate in the control group (70%) was reduced to 50% in rabbits receiving high doses of t-PA. In conclusion, treatment with t-PA resulted in reduced PAI generation, fibrin deposits and mortality in endotoxin-treated rabbits.

Dysfunction of annexin A2 contributes to hyperglycaemia-induced loss of human endothelial cell surface fibrinolytic activity.

PubMed

Dai, Haibin; Yu, Zhanyang; Fan, Xiang; Liu, Ning; Yan, Min; Chen, Zhong; Lo, Eng H; Hajjar, Katherine A; Wang, Xiaoying

2013-06-01

Hyperglycaemia impairs fibrinolytic activity on the surface of endothelial cells, but the underlying mechanisms are not fully understood. In this study, we tested the hypothesis that hyperglycaemia causes dysfunction of the endothelial membrane protein annexin A2, thereby leading to an overall reduction of fibrinolytic activity. Hyperglycaemia for 7 days significantly reduced cell surface fibrinolytic activity in human brain microvascular endothelial cells (HBMEC). Hyperglycaemia also decreased tissue type plasminogen activator (t-PA), plasminogen, and annexin A2 mRNA and protein expression, while increasing plasminogen activator inhibitor-1 (PAI-1). No changes in p11 mRNA or protein expression were detected. Hyperglycaemia significantly increased AGE-modified forms of total cellular and membrane annexin A2. The hyperglycemia-associated reduction in fibrinolytic activity was fully restored upon incubation with recombinant annexin A2 (rA2), but not AGE-modified annexin A2 or exogenous t-PA. Hyperglycaemia decreased t-PA, upregulated PAI-1 and induced AGE-related disruption of annexin A2 function, all of which contributed to the overall reduction in endothelial cell surface fibrinolytic activity. Further investigations to elucidate the underlying molecular mechanisms and pathophysiological implications of A2 derivatisation might ultimately lead to a better understanding of mechanisms of impaired vascular fibrinolysis, and to development of new interventional strategies for the thrombotic vascular complications in diabetes.

Dysfunction of annexin A2 contributes to hyperglycaemia-induced loss of human endothelial cell surface fibrinolytic activity

PubMed Central

Dai, Haibin; Yu, Zhanyang; Fan, Xiang; Liu, Ning; Yan, Min; Chen, Zhong; Lo, Eng H.; Hajjar, Katherine A.; Wang, Xiaoying

2014-01-01

Summary Hyperglycaemia impairs fibrinolytic activity on the surface of endothelial cells, but the underlying mechanisms are not fully understood. In this study, we tested the hypothesis that hyperglycaemia causes dysfunction of the endothelial membrane protein annexin A2, thereby leading to an overall reduction of fibrinolytic activity. Hyperglycaemia for 7 days significantly reduced cell surface fibrinolytic activity in human brain microvascular endothelial cells (HBMEC). Hyperglycaemia also decreased tissue type plasminogen activator (t-PA), plasminogen, and annexin A2 mRNA and protein expression, while increasing plasminogen activator inhibitor-1 (PAI-1). No changes in p11 mRNA or protein expression were detected. Hyperglycaemia significantly increased AGE-modified forms of total cellular and membrane annexin A2. The hyperglycemia-associated reduction in fibrinolytic activity was fully restored upon incubation with recombinant annexin A2 (rA2), but not AGE-modified annexin A2 or exogenous t-PA. Hyperglycaemia decreased t-PA, upregulated PAI-1 and induced AGE-related disruption of annexin A2 function, all of which contributed to the overall reduction in endothelial cell surface fibrinolytic activity. Further investigations to elucidate the underlying molecular mechanisms and pathophysiological implications of A2 derivatisation might ultimately lead to a better understanding of mechanisms of impaired vascular fibrinolysis, and to development of new interventional strategies for the thrombotic vascular complications in diabetes. PMID:23572070

The Effect of Levonorgestrel on Fibrinolytic Factors in Human Endometrial Endothelial Cells.

PubMed

Pakrashi, Tarita; Taylor, Joelle E; Nelson, Ashley; Archer, David F; Jacot, Terry

2016-11-01

The levonorgestrel-releasing intrauterine system is considered a highly effective treatment of heavy menstrual bleeding (HMB). While LNG has established effects on the stromal and glandular compartments of the endometrial tissue, its effect on the endometrial endothelial cells has not been investigated. We examined whether LNG regulates fibrinolytic factors, tissue plasminogen activator (tPA), and urokinase plasminogen activator (uPA) secreted by human endometrial endothelial cells (HEECs) and determined the steroid receptor through which LNG exerts its effect on the endothelium. The HEECs were treated with LNG or progesterone and levels of tPA and plasminogen activator inhibitor 1 (PAI-1) measured. The HEECs were specifically examined for the presence of androgen receptors through Western blot. Levonorgestrel ± flutamide were added to HEECs and the levels of tPA and uPA were examined. An enzyme-linked immunosorbent assay performed on culture media confirmed a statistically significant decrease in tPA levels in cells treated with LNG (77.80% ± 8.0% of control; n = 5, P < .05 vs control) but not progesterone. The androgen receptor (110 kDa) was detected in HEEC lysates. The decrease in tPA was blocked by the addition of flutamide (101.3% ± 16% of control), a classic nonsteroidal androgen receptor blocker. There was no change in uPA or PAI-1 levels in cells treated with LNG. Levonorgestrel decreases tPA levels through the androgen receptor in HEECs. Thus, LNG inhibits tPA secretion by the endometrial endothelial cell. This response suggests reduction in HMB with LNG-IUS could reflect an LNG-mediated promotion of hemostasis. © The Author(s) 2016.

Sickle Mice Are Sensitive to Hypoxia/Ischemia-Induced Stroke but Respond to Tissue-Type Plasminogen Activator Treatment.

PubMed

Sun, Yu-Yo; Lee, Jolly; Huang, Henry; Wagner, Mary B; Joiner, Clinton H; Archer, David R; Kuan, Chia-Yi

2017-12-01

The effects of lytic stroke therapy in patients with sickle cell anemia are unknown, although a recent study suggested that coexistent sickle cell anemia does not increase the risk of cerebral hemorrhage. This finding calls for systemic analysis of the effects of thrombolytic stroke therapy, first in humanized sickle mice, and then in patients. There is also a need for additional predictive markers of sickle cell anemia-associated vasculopathy. We used Doppler ultrasound to examine the carotid artery of Townes sickle mice tested their responses to repetitive mild hypoxia-ischemia- and transient hypoxia-ischemia-induced stroke at 3 or 6 months of age, respectively. We also examined the effects of tPA (tissue-type plasminogen activator) treatment in transient hypoxia-ischemia-injured sickle mice. Three-month-old sickle cell (SS) mice showed elevated resistive index in the carotid artery and higher sensitivity to repetitive mild hypoxia-ischemia-induced cerebral infarct. Six-month-old SS mice showed greater resistive index and increased flow velocity without obstructive vasculopathy in the carotid artery. Instead, the cerebral vascular wall in SS mice showed ectopic expression of PAI-1 (plasminogen activator inhibitor-1) and P-selectin, suggesting a proadhesive and prothrombotic propensity. Indeed, SS mice showed enhanced leukocyte and platelet adherence to the cerebral vascular wall, broader fibrin deposition, and higher mortality after transient hypoxia-ischemia. Yet, post-transient hypoxia-ischemia treatment with tPA reduced thrombosis and mortality in SS mice. Sickle mice are sensitive to hypoxia/ischemia-induced cerebral infarct but benefit from thrombolytic treatment. An increased resistive index in carotid arteries may be an early marker of sickle cell vasculopathy. © 2017 American Heart Association, Inc.

Bolus dose response characteristics of single chain urokinase plasminogen activator and tissue plasminogen activator in a dog model of arterial thrombosis.

PubMed

Badylak, S F; Voytik, S; Klabunde, R E; Henkin, J; Leski, M

1988-11-15

Tissue plasminogen activator (t-PA) and single chain urokinase-plasminogen activator (scu-PA) are relatively "fibrin-specific" thrombolytic drugs with short plasma half lives of 6-8 minutes. Most treatment regimens with these agents utilize a bolus injection followed by continuous drug infusion, usually combined with anticoagulant therapy. The purpose of this study was to establish the dose-response characteristics for scu-PA and t-PA, when given as a single intravenous bolus injection, in a dog model of arterial thrombosis. Eight groups of 6 dogs each were given one of the following doses of scu-PA (mg/kg): 0.20, 0.50, 1.00, 2.00; or t-PA: 0.05, 0.10, 0.20; or an equivalent amount of saline (control group). All doses were given as a single bolus injection 60 minutes after formation of a totally occlusive femoral artery thrombus. Thrombolysis was measured by monitoring the continuous decrement of 125I activity from a radiolabelled thrombus. Ninety minutes after drug injection, all scu-PA treated dogs showed greater thrombolysis (30%, 45%, 56%, and 67%, respectively) than the control group (15%, p less than 0.01). The 0.10 and 0.20 mg/kg t-PA treated dogs showed greater thrombolysis (35% and 49%, respectively) than the control group (15%, p less than 0.01). Both scu-PA and t-PA caused a partial and dose-dependent decrease in alpha 2-antiplasmin activity but scu-PA caused a greater depletion (72% vs. 18%, respectively, p less than 0.05) at 60 minutes after the highest dose of drug administration. Both drugs showed a longer than expected thrombolytic effect based upon the known half lives. Neither drug caused significant changes in the prothrombin time, activated partial thromboplastin time, thrombin time, hematocrit, platelet count, or fibrin degradation product concentration. Single bolus injections of scu-PA and t-PA produce safe and effective thrombolysis in this dog model of arterial thrombosis.

Tissue Plasminogen Activator Induction in Purkinje Neurons After Cerebellar Motor Learning

NASA Astrophysics Data System (ADS)

Seeds, Nicholas W.; Williams, Brian L.; Bickford, Paula C.

1995-12-01

The cerebellar cortex is implicated in the learning of complex motor skills. This learning may require synaptic remodeling of Purkinje cell inputs. An extracellular serine protease, tissue plasminogen activator (tPA), is involved in remodeling various nonneural tissues and is associated with developing and regenerating neurons. In situ hybridization showed that expression of tPA messenger RNA was increased in the Purkinje neurons of rats within an hour of their being trained for a complex motor task. Antibody to tPA also showed the induction of tPA protein associated with cerebellar Purkinje cells. Thus, the induction of tPA during motor learning may play a role in activity-dependent synaptic plasticity.

Proteolytic inactivation of tissue factor pathway inhibitor by bacterial omptins

PubMed Central

Yun, Thomas H.; Cott, Jessica E.; Tapping, Richard I.; Slauch, James M.

2009-01-01

The immune response to infection includes activation of the blood clotting system, leading to extravascular fibrin deposition to limit the spread of invasive microorganisms. Some bacteria have evolved mechanisms to counteract this host response. Pla, a member of the omptin family of Gram-negative bacterial proteases, promotes the invasiveness of the plague bacterium, Yersinia pestis, by activating plasminogen to plasmin to digest fibrin. We now show that the endogenous anticoagulant tissue factor pathway inhibitor (TFPI) is also highly sensitive to proteolysis by Pla and its orthologs OmpT in Escherichia coli and PgtE in Salmonella enterica serovar Typhimurium. Using gene deletions, we demonstrate that bacterial inactivation of TFPI requires omptin expression. TFPI inactivation is mediated by proteolysis since Western blot analysis showed that TFPI cleavage correlated with loss of anticoagulant function in clotting assays. Rates of TFPI inactivation were much higher than rates of plasminogen activation, indicating that TFPI is a better substrate for omptins. We hypothesize that TFPI has evolved sensitivity to proteolytic inactivation by bacterial omptins to potentiate procoagulant responses to bacterial infection. This may contribute to the hemostatic imbalance in disseminated intravascular coagulation and other coagulopathies accompanying severe sepsis. PMID:18988866

[Cell-derived microparticles unveil their fibrinolytic and proteolytic function].

PubMed

Doeuvre, Loïc; Angles-Cano, Eduardo

2009-01-01

Cell-derived microparticles (MP) are membrane microvesicles, 0.1-1 microm in size, shed by cells following activation or during apoptosis in a variety of pathological conditions. MPs released by blood cells or by vascular endothelial cells display molecular signatures that allow their identification and functional characterization. In addition, they provide tissue factor (TF) and a procoagulant phospholipid surface. Therefore, at present, the most strongly established applied research on MPs is their procoagulant activity as a determinant of thrombotic risk in various clinical conditions. Previous studies have indicated that MPs derived from malignant cells express matrix metalloproteinases, urokinase and its receptor (uPA/uPAR) that, in the presence of plasminogen, may act in concert to degrade extracellular matrix proteins. Recently, it was shown that MPs from TNFa-stimulated endothelial cells served as a surface for interaction with plasminogen and its conversion into plasmin by the uPA/uPAR system expressed at their surface. This capacity of MPs to promote plasmin generation confers them a new profibrinolytic and proteolytic function that may be of relevance in fibrinolysis, cell migration, angiogenesis, dissemination of malignant cells, cell detachment and apoptosis.

Quebec platelet disorder is linked to the urokinase plasminogen activator gene (PLAU) and increases expression of the linked allele in megakaryocytes

PubMed Central

Diamandis, Maria; Paterson, Andrew D.; Rommens, Johanna M.; Veljkovic, D. Kika; Blavignac, Jessica; Bulman, Dennis E.; Waye, John S.; Derome, Francine; Rivard, Georges E.

2009-01-01

Quebec platelet disorder (QPD) is an autosomal dominant disorder with high penetrance that is associated with increased risks for bleeding. The hallmark of QPD is a gain-of-function defect in fibrinolysis due to increased platelet content of urokinase plasminogen activator (uPA) without systemic fibrinolysis. We hypothesized that increased expression of uPA by differentiating QPD megakaryocytes is linked to PLAU. Genetic marker analyses indicated that QPD was significantly linked to a 2-Mb region on chromosome 10q containing PLAU with a maximum multipoint logarithm of the odds (LOD) score of +11 between markers D10S1432 and D10S1136. Analysis of PLAU by sequencing and Southern blotting excluded mutations within PLAU and its known regulatory elements as the cause of QPD. Analyses of uPA mRNA indicated that QPD distinctly increased transcript levels of the linked PLAU allele with megakaryocyte differentiation. These findings implicate a mutation in an uncharacterized cis element near PLAU as the cause of QPD. PMID:18988861

Quebec platelet disorder is linked to the urokinase plasminogen activator gene (PLAU) and increases expression of the linked allele in megakaryocytes.

PubMed

Diamandis, Maria; Paterson, Andrew D; Rommens, Johanna M; Veljkovic, D Kika; Blavignac, Jessica; Bulman, Dennis E; Waye, John S; Derome, Francine; Rivard, Georges E; Hayward, Catherine P M

2009-02-12

Quebec platelet disorder (QPD) is an autosomal dominant disorder with high penetrance that is associated with increased risks for bleeding. The hallmark of QPD is a gain-of-function defect in fibrinolysis due to increased platelet content of urokinase plasminogen activator (uPA) without systemic fibrinolysis. We hypothesized that increased expression of uPA by differentiating QPD megakaryocytes is linked to PLAU. Genetic marker analyses indicated that QPD was significantly linked to a 2-Mb region on chromosome 10q containing PLAU with a maximum multipoint logarithm of the odds (LOD) score of +11 between markers D10S1432 and D10S1136. Analysis of PLAU by sequencing and Southern blotting excluded mutations within PLAU and its known regulatory elements as the cause of QPD. Analyses of uPA mRNA indicated that QPD distinctly increased transcript levels of the linked PLAU allele with megakaryocyte differentiation. These findings implicate a mutation in an uncharacterized cis element near PLAU as the cause of QPD.

Activated fibrinolytic enzymes in the synovial fluid during acute arthritis induced by urate crystal injection in dogs.

PubMed

Morimoto, N; Sumi, H; Tsushima, H; Etou, Y; Yoshida, E; Mihara, H

1991-10-01

To identify the relationship of the severity of inflammation and fibrinolytic activity in arthritis, the fibrinolytic activity of synovial fluid was studied in acute experimental arthritis induced by injecting monosodium urate crystals into dogs' knee joints. The maximum activity in the synovial fluid was observed 6 h after crystal injection. It was inferred that the fibrinolytic activity was mainly due to plasminogen activator based on fibrin plate assays, substrate specificity, inhibitor effects and zymography. On the other hand, the activity of lysosomal enzymes (beta-glucuronidase and cathepsin G) reached a peak in the synovia after 12 h. Histological examination of the synovial membrane after 12 h also showed greater inflammation than at 6 h. The peak in fibrinolytic activity preceded the peak of lysosomal enzymes and histological changes. These results suggest that an increase in fibrinolytic activity by plasminogen activator may contribute to the development of an acute inflammatory response.

12/15-Lipoxygenase Inhibition or Knockout Reduces Warfarin-Associated Hemorrhagic Transformation After Experimental Stroke.

PubMed

Liu, Yu; Zheng, Yi; Karatas, Hulya; Wang, Xiaoying; Foerch, Christian; Lo, Eng H; van Leyen, Klaus

2017-02-01

For stroke prevention, patients with atrial fibrillation typically receive oral anticoagulation. The commonly used anticoagulant warfarin increases the risk of hemorrhagic transformation (HT) when a stroke occurs; tissue-type plasminogen activator treatment is therefore restricted in these patients. This study was designed to test the hypothesis that 12/15-lipoxygenase (12/15-LOX) inhibition would reduce HT in warfarin-treated mice subjected to experimental stroke. Warfarin was dosed orally in drinking water, and international normalized ratio values were determined using a Coaguchek device. C57BL6J mice or 12/15-LOX knockout mice were subjected to transient middle cerebral artery occlusion with 3 hours severe ischemia (model A) or 2 hours ischemia and tissue-type plasminogen activator infusion (model B), with or without the 12/15-LOX inhibitor ML351. Hemoglobin was determined in brain homogenates, and hemorrhage areas on the brain surface and in brain sections were measured. 12/15-LOX expression was detected by immunohistochemistry. Warfarin treatment resulted in reproducible increased international normalized ratio values and significant HT in both models. 12/15-LOX knockout mice suffered less HT after severe ischemia, and ML351 reduced HT in wild-type mice. When normalized to infarct size, ML351 still independently reduced hemorrhage. HT after tissue-type plasminogen activator was similarly reduced by ML351. In addition to its benefits in infarct size reduction, 12/15-LOX inhibition also may independently reduce HT in warfarin-treated mice. ML351 should be further evaluated as stroke treatment in anticoagulated patients suffering a stroke, either alone or in conjunction with tissue-type plasminogen activator. © 2017 American Heart Association, Inc.

Twelve-Month Clinical and Quality-of-Life Outcomes in the Interventional Management of Stroke III Trial.

PubMed

Palesch, Yuko Y; Yeatts, Sharon D; Tomsick, Thomas A; Foster, Lydia D; Demchuk, Andrew M; Khatri, Pooja; Hill, Michael D; Jauch, Edward C; Jovin, Tudor G; Yan, Bernard; von Kummer, Rüdiger; Molina, Carlos A; Goyal, Mayank; Schonewille, Wouter J; Mazighi, Mikael; Engelter, Stefan T; Anderson, Craig; Spilker, Judith; Carrozzella, Janice; Ryckborst, Karla J; Janis, L Scott; Simpson, Annie; Simpson, Kit N; Broderick, Joseph P

2015-05-01

Randomized trials have indicated a benefit for endovascular therapy in appropriately selected stroke patients at 3 months, but data regarding outcomes at 12 months are currently lacking. We compared functional and quality-of-life outcomes at 12 months overall and by stroke severity in stroke patients treated with intravenous tissue-type plasminogen activator followed by endovascular treatment as compared with intravenous tissue-type plasminogen activator alone in the Interventional Management of Stroke III Trial. The key outcome measures were a modified Rankin Scale score ≤2 (functional independence) and the Euro-QoL EQ-5D, a health-related quality-of-life measure. 656 subjects with moderate-to-severe stroke (National Institutes of Health Stroke Scale ≥8) were enrolled at 58 centers in the United States (41 sites), Canada (7), Australia (4), and Europe (6). There was an interaction between treatment group and stroke severity in the repeated measures analysis of modified Rankin Scale ≤2 outcome (P=0.039). In the 204 participants with severe stroke (National Institutes of Health Stroke Scale ≥20), a greater proportion of the endovascular group had a modified Rankin Scale ≤2 (32.5%) at 12 months as compared with the intravenous tissue-type plasminogen activator group (18.6%, P=0.037); no difference was seen for the 452 participants with moderately severe strokes (55.6% versus 57.7%). In participants with severe stroke, the endovascular group had 35.2 (95% confidence interval: 2.1, 73.3) more quality-adjusted-days over 12 months as compared with intravenous tissue-type plasminogen activator alone. Endovascular therapy improves functional outcome and health-related quality-of-life at 12 months after severe ischemic stroke. URL: http://www.clinicaltrials.gov. Unique identifier: NCT00359424. © 2015 American Heart Association, Inc.

Epistatic Effects of Polymorphisms in Genes from the Renin-Angiotensin, Bradykinin, and Fibrinolytic Systems on Plasma t-PA and PAI-1 Levels

PubMed Central

Asselbergs, Folkert W.; Williams, Scott M.; Hebert, Patricia R.; Coffey, Christopher S.; Hillege, Hans L.; Navis, Gerjan; Vaughan, Douglas E.; van Gilst, Wiek H.; Moore, Jason H.

2007-01-01

Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) directly influence thrombus formation and degradation and thereby risk for arterial thrombosis. Activation of the renin-angiotensin system has been linked to the production of PAI-1 expression via the angiotensin II type 1 receptor (AT1R). In addition, bradykinin can induce the release of t-PA through a B2 receptor mechanism. In the present study, we aimed to investigate the epistatic effects of polymorphisms in genes from the renin-angiotensin, bradykinin and fibrinolytic systems on plasma t-PA and PAI-1 levels in a large population-based sample (n=2,527). We demonstrated a strong significant interaction within genetic variations of the bradykinin B2 gene (p=0.002) and between ACE and bradykinin B2 (p=0.003) polymorphisms on t-PA levels in females. In males, polymorphisms in the bradykinin B2 and AT1R gene showed the most strong effect on t-PA levels (p=0.006). In both females as well as males, the bradykinin B2 gene interacted with AT1R gene on plasma PAI-1 levels (p=0.026 and p=0.039, respectively). In addition, the current study found a borderline significant interaction between PAI 4G5G and ACE I/D on plasma t-PA and PAI-1 levels. These results support the idea that the interplay between the renin-angiotensin, bradykinin, and fibrinolytic systems might play an important role in t-PA and PAI-1 biology. PMID:17207964

Lytic and mechanical stability of clots composed of fibrin and blood vessel wall components.

PubMed

Rottenberger, Z; Komorowicz, E; Szabó, L; Bóta, A; Varga, Z; Machovich, R; Longstaff, C; Kolev, K

2013-03-01

Proteases expressed in atherosclerotic plaque lesions generate collagen fragments, release glycosaminoglycans (chondroitin sulfate [CS] and dermatan sulfate [DS]) and expose extracellular matrix (ECM) proteins (e.g. decorin) at sites of fibrin formation. Here we address the effect of these vessel wall components on the lysis of fibrin by the tissue plasminogen activator (tPA)/plasminogen system and on the mechanical stability of clots. MMP-8-digested collagen fragments, isolated CS, DS, glycosylated decorin and its core protein were used to prepare mixed matrices with fibrin (additives present at a 50-fold lower mass concentration than fibrinogen). Scanning electron microscopy (SEM) showed that the presence of ECM components resulted in a coarse fibrin structure, most pronounced for glycosylated decorin causing an increase in the median fiber diameter from 85 to 187 nm. Rheological measurements indicated that these structural alterations were coupled to decreased shear resistance (1.8-fold lower shear stress needed for gel/fluid transition of the clots containing glycosylated decorin) and rigidity (reduction of the storage modulus from 54.3 to 33.2 Pa). The lytic susceptibility of the modified fibrin structures was increased. The time to 50% lysis by plasmin was reduced approximately 2-fold for all investigated ECM components (apart from the core protein of decorin which produced a moderate reduction of the lysis time by 25%), whereas fibrin-dependent plasminogen activation by tPA was inhibited by up to 30%. ECM components compromise the chemical and mechanical stability of fibrin as a result of changes in its ultrastructure. © 2012 International Society on Thrombosis and Haemostasis.

Postoperative bleeding in cardiac surgery: the role of tranexamic acid in patients homozygous for the 5G polymorphism of the plasminogen activator inhibitor-1 gene.

PubMed

Iribarren, Jose L; Jimenez, Juan J; Hernández, Domingo; Brouard, Maitane; Riverol, Debora; Lorente, Leonardo; de La Llana, Ramiro; Nassar, Ibrahim; Perez, Rosalia; Martinez, Rafael; Mora, Maria L

2008-04-01

Plasminogen activator inhibitor 1 (PAI-1) attenuates the conversion of plasminogen to plasmin. Polymorphisms of the PAI-1 gene are associated with varying PAI-1 levels and risk of prothrombotic events in nonsurgical patients. The purpose of this study, a secondary analysis of a clinical trial, was to investigate whether PAI-1 genotype affects the efficacy of tranexamic acid (TA) in reducing postoperative chest tube blood loss of patients undergoing cardiopulmonary bypass. Fifty patients were classified according to PAI-1 genotype (4G/4G, 4G/5G, or 5G/5G). Twenty-four received 2 g TA before and after cardiopulmonary bypass, whereas 26 received placebo. The authors recorded data related to coagulation, fibrinolysis, and bleeding before surgery, at admission to the intensive care unit (0 h), and 4 and 24 h later. In patients not receiving TA, those with the 5G/5G genotype had significantly higher chest tube blood loss and transfusion requirements compared with patients with the other genotypes at all time points. Patients with the 5G/5G genotype receiving TA showed significantly lower blood loss compared with the placebo group. There were no significant differences in blood loss or transfusion requirements between patients with the 4G/4G genotype when TA was used. Plasminogen activator inhibitor-1 5G/5G homozygotes who did not receive TA showed significantly greater postoperative bleeding than patients with other PAI-1 genotypes. 5G/5G homozygotes who received TA showed the greatest blood-sparing benefit.

Quality aspects of fibrinolytic agents based on biochemical characterization.

PubMed

Werner, R G; Bassarab, S; Hoffmann, H; Schlüter, M

1991-11-01

The purity, composition and in vitro fibrinolytic activity of four commercially available fibrinolytic agents, alteplase (recombinant tissue plasminogen activator, rt-PA, Actilyse; CAS 105857-23-6), streptokinase, urokinase and anistreplase (ansioyl-plasminogen-streptokinase activator-complex, APSAC), have been compared in this investigation. The fibrinolytic activity was measured in an in vitro thrombolytic assay. In this assay a human blood thrombus is dissolved in an environment of human plasma. This assay is representative for the in vivo situation, where plasminogen activation is also a limiting step in thrombolysis. In the in vitro thrombolytic assay alteplase is about 10 times more effective in clot lysis than either streptokinase or urokinase and more than 300 times more active than anistreplase. In addition, the ratio of active ingredient to total protein content in the preparations was analysed by RP-HPLC, SDS-PAGE, GPC-HPLC and amino acid analysis. The portion of active ingredient per total protein was 99.9% for alteplase, 55% for anistreplase, 20% for urokinase and 1% for streptokinase. This demonstrates that alteplase is the only fibrinolytic agent tested which is essentially free of protein additives of human origine and potential contaminants associated therewith. The superior purity of alteplase compared to the other fibrinolytics was confirmed by SDS-PAGE, RP-HPLC, and HPLC-GPC. Significant levels of aggregates were detected in streptokinase and urokinase preparations, whereas alteplase and anistreplase were essentially free of aggregates. These data demonstrate that there are significant differences in composition, purity and in vitro activity between different fibrinolytic agents.

Molecular Disruption of Breast Tumor Angiogenesis

DTIC Science & Technology

2004-07-01

human breast cancer . During the period of this grant, we carried out studies to confirm that targeted ablation of PAI- 1 gene expression resulted in a...microvessel endothelial cells. Breast cancer -derived factors (TGF-P, EGF)were found to be important contributors of continued PAI-I expression and long...various culture model systems implicated both urokinase plasminogen activator (uPA) and its fast-acting type-i inhibitor (PAl-l) as necessary to achieve

Gene polymorphisms of fibrinolytic enzymes in coal workers' pneumoconiosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, L.C.; Tseng, J.C.; Hua, C.C.

2006-03-15

The authors assessed the gene polymorphisms of missense C/T polymorphism in exon 6 of the urokinase-plasminogen activator (PLAU) gene (PLAU P141L), A/u-repeat in intron 8 of the tissue-type plasminogen activator (PLAT) gene (PLAT TPA25 Alu insertion), and 4G/5G in the promoter region of the serine proteinase inhibitor, clade E (SERPINE) or plasminogen activator inhibitor type 1 gene (SERPINE1 -675 4G/5G) in 153 healthy volunteers and 154 retired coal miners with coal miners' pneumoconiosis (CWP). The CWP subjects included 94 individuals with simple pneumoconiosis and 60 individuals with progressive massive fibrosis presenting with worse pulmonary function. The distributions of genotypes ofmore » these three genes did not differ between the control and CWP subjects or between subjects with simple pneumoconiosis and those with progressive massive fibrosis. However, by assessing duration of work and its interaction with genotypes by means of logistic regression, the authors found the missense C/T polymorphism in exon 6 of the PLAU gene to be an effect modifier of the association between work duration and the development of progressive massive fibrosis.« less

[Tumor-associated prognostic factors of the plasminogen activator family: determination and clinical value of u-PA, t-PA, PAI-1, and PAI-2].

PubMed

Mengele, K; Harbeck, N; Reuning, U; Magdolen, V; Schmitt, M

2005-08-01

Proteolytic factors belonging t the plasminogen activator family (plasmin, u-PA, t-PA, u-PAR, PAI-1, and PAI-2), which usually are involved in blood clotting and degradation of blood clots, are also present in healthy and diseased tissue of the kidney, lung, liver, gastro-intestinal tract, breast, prostate, ovary, and brain. These factors are engaged in brain development, angiogenesis and vascular invasion, wound healing as well as in placenta development and embryogenesis. Plasminogen activators u-PA and t-PA, their inhibitors PAI-1 and PAI-2, and the u-PA-receptor (u-PAR, CD87) are often elevated in solid malignant tumour tissues compared to their normal counterparts. In breast cancer patients, an elevated tumour tissue extract antigen content of u-PA, PAI-1, and u-PAR is associated with increased tumour aggressiveness and poor prognosis; in contrary, an elevated content of t-PA and PAI-2 indicates a favourable prognosis. For clinical relevant determination of these proteolytic factors in tumour tissue extracts, only enzymo-immunometric tests (ELISA) are recommended. Enzymometric and enzymographic tests are actually conducted only in an experimental, preclinical context.

Platelet activation independent of pulmonary inflammation contributes to diesel exhaust particulate-induced promotion of arterial thrombosis.

PubMed

Tabor, Caroline M; Shaw, Catherine A; Robertson, Sarah; Miller, Mark R; Duffin, Rodger; Donaldson, Ken; Newby, David E; Hadoke, Patrick W F

2016-02-09

Accelerated thrombus formation induced by exposure to combustion-derived air pollution has been linked to alterations in endogenous fibrinolysis and platelet activation in response to pulmonary and systemic inflammation. We hypothesised that mechanisms independent of inflammation contribute to accelerated thrombus formation following exposure to diesel exhaust particles (DEP). Thrombosis in rats was assessed 2, 6 and 24 h after administration of DEP, carbon black (CB; control carbon nanoparticle), DQ12 quartz microparticles (to induce pulmonary inflammation) or saline (vehicle) by either intra-tracheal instillation (0.5 mg, except Quartz; 0.125 mg) or intravenous injection (0.5 mg/kg). Thrombogenicity was assessed by carotid artery occlusion, fibrinolytic variables and platelet-monocyte aggregates. Measures of inflammation were determined in plasma and bronchoalveolar lavage fluid. Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI)-1 were measured following direct in vitro exposure of human umbilical vein endothelial cells (HUVECs) to DEP (10-150 μg/mL). Instillation of DEP reduced the time to thrombotic occlusion in vivo, coinciding with the peak of DEP-induced pulmonary inflammation (6 h). CB and DQ12 produced greater inflammation than DEP but did not alter time to thrombotic occlusion. Intravenous DEP produced an earlier (2 h) acceleration of thrombosis (as did CB) without pulmonary or systemic inflammation. DEP inhibited t-PA and PAI-1 release from HUVECs, and reduced the t-PA/PAI-1 ratio in vivo; similar effects in vivo were seen with CB and DQ12. DEP, but not CB or DQ12, increased platelet-monocyte aggregates. DEP accelerates arterial thrombus formation through increased platelet activation. This effect is dissociated from pulmonary and systemic inflammation and from impaired fibrinolytic function.

Influence of racial differences on outcomes after thrombolytic therapy in acute ischemic stroke.

PubMed

Mishra, Nishant K; Mandava, Pitchaiah; Chen, Christopher; Grotta, James; Lees, Kennedy R; Kent, Thomas A

2014-07-01

The National Institutes of Neurological Disorders and Stroke and the European Co-operative Acute Stroke III trials enrolled a largely Caucasian population, but the results are often extrapolated onto non-Caucasians. A limited number of nonrandomized studies have proposed that non-Caucasian patients show differential response to tissue plasminogen activator. We examined if non-Caucasian patients of mixed national origin within the Virtual International Stroke Trials Archives neuroprotection trials responded differently to tissue plasminogen activator compared with Caucasians. We matched patients within each race-subtype for age, baseline National Institutes of Health Stroke Scales, and diabetes status, and excluded outliers. We tested for an interaction of race ethnicity with tissue plasminogen activator on predicting outcomes at α = 0·05. We compared 90-day ordinal outcome (modified Rankin Scale; primary analysis) and dichotomized outcomes (modified Rankin Scale 0-1; modified Rankin Scale 0-2; survival) within individual race ethnicity. One thousand nine hundred forty-six thrombolysed patients (125 Blacks, 39 Asians, and 1821 Caucasians) were matched with 1946 non-thrombolysed patients in each race ethnicity group. Postmatching, there were no imbalances in baseline National Institutes of Health Stroke Scales and age between the groups (P > 0·05). The interaction of tissue plasminogen activator with race ethnicity was nonsignificant in ordinal (P = 0·4) and in dichotomized outcome models (P > 0·05). Ordinal odds for improved outcomes were 1·5 for all patients (P < 0·05). Ordinal odds for Caucasians were 1·5 (P < 0·05); for Blacks, 2·1 (P < 0·05); and for Asians, 1·2 (P > 0·05; 1·6 after 1:2 matching with nonthrombolysed, because of small numbers). Dichotomized functional outcomes improved after thrombolysis overall, in Caucasians, in Blacks (modified Rankin Scale 0-2 only), and in Asians (after 1:2 matching; P > 0·05). Odds for survival were consistent across all groups. These results do not suggest a differential response to tissue plasminogen activator based on race ethnicity. Among Asians, data were particularly sparse, and results should be interpreted with caution. © 2013 The Authors. International Journal of Stroke © 2013 World Stroke Organization.

Close relationship of tissue plasminogen activator-plasminogen activator inhibitor-1 complex with multiple organ dysfunction syndrome investigated by means of the artificial pancreas

PubMed Central

Hoshino, Masami; Haraguchi, Yoshikura; Hirasawa, Hiroyuki; Sakai, Motohiro; Saegusa, Hiroshi; Hayashi, Kazushiro; Horita, Naoki; Ohsawa, Hiroyuki

2001-01-01

Background: Glucose tolerance (GT) has not been taken into consideration in investigations concerning relationships between coagulopathy and multiple organ dysfunction syndrome (MODS), and endothelial cell activation/endothelial cell injury (ECA/ECI) in septic patients, although coagulopathy is known to be influenced by blood glucose level. We investigated those relationships under strict blood glucose control and evaluation of GT with the glucose clamp method by means of the artificial pancreas in nine septic patients with glucose intolerance. The relationships between GT and blood stress related hormone levels (SRH) were also investigated. Methods: The amount of metabolized glucose (M value), as the parameter of GT, was measured by the euglycemic hyperinsulinemic glucose clamp method, in which the blood glucose level was clamped at 80 mg/dl under a continuous insulin infusion rate of 1.12 mU/kg per min, using the artificial pancreas, STG-22. Multiple organ failure (MOF) score was calculated using the MOF criteria of Japanese Association for Critical Care Medicine. Regarding coagulopathy, the following parameters were used: disseminated intravascular coagulation (DIC) score (calculated from the DIC criteria of the Ministry of Health and Welfare of Japan) and the parameters used for calculating DIC score, protein-C, protein-S, plasminogen, antithrombin III (AT-III), plasminogen activator inhibitor-1 (PAI-1), and tissue plasminogen activator-PAI-1 (tPA-PAI-1) complex. Thrombomodulin (TM) was measured as the indicator of ECI. Results: There were no significant correlations between M value and SRH, parameters indicating coagulopathy and the MOF score. The MOF score and blood TM levels were positively correlated with DIC score, thrombin-AT-III complex and tPA-PAI-1 complex, and negatively correlated with blood platelet count. Conclusions: GT was not significantly related to SRH, coagulopathy and MODS under strict blood glucose control. Hypercoagulability was closely related to MODS and ECI. Among the parameters indicating coagulopathy, tPA-PAI-1 complex, which is considered to originate from ECA, seemed to be a sensitive parameter of MODS and ECI, and might be a predictive marker of MODS. The treatment for reducing hypercoagulability and ECA/ECI were thought to be justified as one of the therapies for acutely ill septic patients. PMID:11299067

Antibiotic modulation of the plasminogen binding ability of viridans group streptococci.

PubMed

Teles, Cristina; Smith, Andrew; Lang, Sue

2012-01-01

The ability of viridans group streptococci to bind human plasminogen and its subsequent activation into plasmin may contribute to the pathogenesis of infective endocarditis (IE) by leading to a decreased stability of the streptococcal vegetation and facilitating dehiscence of emboli. At levels greater than or equal to their MICs, penicillin, vancomycin, and linezolid are efficacious in the treatment of streptococcal endocarditis. However, at sub-MICs, antibiotics can modulate the expression of bacterial genes, including virulence-associated genes, which can have counterproductive effects on the treatment of endocarditis. The effects of 1/8× and 1/4× MICs of penicillin, vancomycin, and linezolid on the plasminogen binding ability of IE isolates Streptococcus mitis 881/956, Streptococcus oralis 12601, and Streptococcus sanguinis 12403 were assessed phenotypically and the expression of plasminogen receptors α-enolase and glyceraldehyde 3-phosphate dehydrogenase of S. oralis 12601 when exposed to 1/4× MIC of penicillin, was analyzed through quantitative reverse transcription (qRT)-PCR. The plasminogen binding ability of S. mitis 881/956 and S. sanguinis 12403 remained unaffected by exposure to sub-MICs of all of the antibiotics tested, while that of S. oralis 12601 was significantly enhanced by all of the antibiotics tested at sub-MICs. qRT-PCR analysis of S. oralis 12601 demonstrated an upregulation of the eno and gapdh genes, indicating an overexpression of plasminogen receptors. These findings suggest that for some endocarditis isolates, the effect of antibiotic sub-MICs, in addition to a reduced antibacterial effect, may influence the clinical response to nonsurgical therapy. It remains difficult to accurately predict isolate responses to sub-MIC antimicrobials since there appears to be interspecies variation.

Lytic resistance of fibrin containing red blood cells

PubMed Central

Wohner, Nikolett; Sótonyi, Péter; Machovich, Raymund; Szabó, László; Tenekedjiev, Kiril; Silva, Marta M.C.G.; Longstaff, Colin; Kolev, Krasimir

2012-01-01

Objective Arterial thrombi contain variable amounts of red blood cell (RBC), which interact with fibrinogen through an eptifibatide-sensitive receptor and modify the structure of fibrin. Here we evaluate the modulator role of RBCs in the lytic susceptibility of fibrin. Methods and Results If fibrin is formed at increasing RBC counts, scanning electron microscopy evidenced a decrease in fiber diameter from 150 nm to 96 nm at 40 %(v/v) RBC, an effect susceptible to eptifibatide inhibition (restoring 140 nm diameter). RBC prolonged the lysis time in a homogeneous-phase fibrinolytic assay with tissue plasminogen activator (tPA) by up to 22.7±1.6 %, but not in the presence of eptifibatide. Confocal laser microscopy using green fluorescent protein (GFP)-labeled tPA and orange fluorescent fibrin showed that 20-40 %(v/v) RBC significantly slowed down the dissolution of the clots. tPA-GFP did not accumulate on the surface of fibrin containing RBC at any cell count above 10 %. The presence of RBC in the clot suppressed the tPA-induced plasminogen activation resulting in a 45 % less plasmin generated after 30 min activation at 40 %(v/v) RBC. Conclusion RBCs confer lytic resistance to fibrin resulting from modified fibrin structure and impaired plasminogen activation through a mechanism that involves eptifibatide-sensitive fibrinogen-RBC interactions. PMID:21737785

Arrhenius temperature dependence of in vitro tissue plasminogen activator thrombolysis.

PubMed

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

2007-06-07

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

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.

Cloning, expression and activity analysis of a novel fibrinolytic serine protease from Arenicola cristata

NASA Astrophysics Data System (ADS)

Zhao, Chunling; Ju, Jiyu

2015-06-01

The full-length cDNA of a protease gene from a marine annelid Arenicola cristata was amplified through rapid amplification of cDNA ends technique and sequenced. The size of the cDNA was 936 bp in length, including an open reading frame encoding a polypeptide of 270 amino acid residues. The deduced amino acid sequnce consisted of pro- and mature sequences. The protease belonged to the serine protease family because it contained the highly conserved sequence GDSGGP. This protease was novel as it showed a low amino acid sequence similarity (< 40%) to other serine proteases. The gene encoding the active form of A. cristata serine protease was cloned and expressed in E. coli. Purified recombinant protease in a supernatant could dissolve an artificial fibrin plate with plasminogen-rich fibrin, whereas the plasminogen-free fibrin showed no clear zone caused by hydrolysis. This result suggested that the recombinant protease showed an indirect fibrinolytic activity of dissolving fibrin, and was probably a plasminogen activator. A rat model with venous thrombosis was established to demonstrate that the recombinant protease could also hydrolyze blood clot in vivo. Therefore, this recombinant protease may be used as a thrombolytic agent for thrombosis treatment. To our knowledge, this study is the first of reporting the fibrinolytic serine protease gene in A. cristata.

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

PubMed

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

2011-05-01

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.

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.

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.

Plasminogen activation independent of uPA and tPA maintains wound healing in gene-deficient mice

PubMed Central

Lund, Leif R; Green, Kirsty A; Stoop, Allart A; Ploug, Michael; Almholt, Kasper; Lilla, Jennifer; Nielsen, Boye S; Christensen, Ib J; Craik, Charles S; Werb, Zena; Danø, Keld; Rømer, John

2006-01-01

Simultaneous ablation of the two known activators of plasminogen (Plg), urokinase-type (uPA) and the tissue-type (tPA), results in a substantial delay in skin wound healing. However, wound closure and epidermal re-epithelialization are significantly less impaired in uPA;tPA double-deficient mice than in Plg-deficient mice. Skin wounds in uPA;tPA-deficient mice treated with the broad-spectrum matrix metalloproteinase (MMP) inhibitor galardin (N-[(2R)-2-(hydroxamido-carbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide) eventually heal, whereas skin wounds in galardin-treated Plg-deficient mice do not heal. Furthermore, plasmin is biochemically detectable in wound extracts from uPA;tPA double-deficient mice. In vivo administration of a plasma kallikrein (pKal)-selective form of the serine protease inhibitor ecotin exacerbates the healing impairment of uPA;tPA double-deficient wounds to a degree indistinguishable from that observed in Plg-deficient mice, and completely blocks the activity of pKal, but not uPA and tPA in wound extracts. These findings demonstrate that an additional plasminogen activator provides sufficient plasmin activity to sustain the healing process albeit at decreased speed in the absence of uPA, tPA and galardin-sensitive MMPs and suggest that pKal plays a role in plasmin generation. PMID:16763560

Binding of high molecular weight kininogen to human endothelial cells is mediated via a site within domains 2 and 3 of the urokinase receptor.

PubMed Central

Colman, R W; Pixley, R A; Najamunnisa, S; Yan, W; Wang, J; Mazar, A; McCrae, K R

1997-01-01

The urokinase receptor (uPAR) binds urokinase-type plasminogen activator (u-PA) through specific interactions with uPAR domain 1, and vitronectin through interactions with a site within uPAR domains 2 and 3. These interactions promote the expression of cell surface plasminogen activator activity and cellular adhesion to vitronectin, respectively. High molecular weight kininogen (HK) also stimulates the expression of cell surface plasminogen activator activity through its ability to serve as an acquired receptor for prekallikrein, which, after its activation, may directly activate prourokinase. Here, we report that binding of the cleaved form of HK (HKa) to human umbilical vein endothelial cells (HUVEC) is mediated through zinc-dependent interactions with uPAR. These occur through a site within uPAR domains 2 and 3, since the binding of 125I-HKa to HUVEC is inhibited by vitronectin, anti-uPAR domain 2 and 3 antibodies and soluble, recombinant uPAR (suPAR), but not by antibody 7E3, which recognizes the beta chain of the endothelial cell vitronectin receptor (integrin alphavbeta3), or fibrinogen, another alphavbeta3 ligand. We also demonstrate the formation of a zinc-dependent complex between suPAR and HKa. Interactions of HKa with endothelial cell uPAR may underlie its ability to promote kallikrein-dependent cell surface plasmin generation, and also explain, in part, its antiadhesive properties. PMID:9294114

Association of late-onset Alzheimer disease with a genotype of PLAU, the gene encoding urokinase-type plasminogen activator on chromosome 10q22.2.

PubMed

Finckh, U; van Hadeln, K; Müller-Thomsen, T; Alberici, A; Binetti, G; Hock, C; Nitsch, R M; Stoppe, G; Reiss, J; Gal, A

2003-08-01

Urokinase-type plasminogen activator (uPA) converts plasminogen to plasmin. Plasmin is involved in processing of amyloid precursor protein and degrades secreted and aggregated amyloid-beta, a hallmark of Alzheimer disease (AD). PLAU, the gene encoding uPA, maps to chromosome 10q22.2 between two regions showing linkage to late-onset AD (LOAD). We genotyped a frequent C/T single nucleotide polymorphism in codon 141 of PLAU (P141L) in 347 patients with LOAD and 291 control subjects. LOAD was associated with homozygous C/C PLAU genotype in the whole sample (chi2=15.7, P=0.00039, df 2), as well as in all sub-samples stratified by gender or APOE epsilon4 carrier status (chi2> or = 6.84, P< or =0.033, df 2). Odds ratio for LOAD due to homozygosity C/C was 1.89 (95% confidence interval 1.37-2.61). PLAU is a promising new candidate gene for LOAD, with allele C (P141) being a recessive risk allele or allele T (L141) conferring protection.

Changes in the haemostatic system after thermoneutral and hyperthermic water immersion.

PubMed

Boldt, Leif-Hendrik; Fraszl, Waltraud; Röcker, Lothar; Schefold, Jörg Christian; Steinach, Mathias; Noack, Thilo; Gunga, Hanns-Christian

2008-03-01

Warm water bathing is a popular recreational activity and is frequently used in rehabilitation medicine. Although well tolerated in most cases, there are reports indicating an increased risk of thrombotic events after hot tub bathing. The effects of a 45 min thermoneutral bath followed by a 50 min bath with increasing water temperature (maximum 41 degrees C) until reaching a body core temperature of 39 degrees C on factors of blood coagulation and fibrinolysis were studied in eight healthy male volunteers. Blood was obtained after a 45-min resting period as control and after the thermoneutral and hyperthermic bath as well as after another 45 min recovery period at the end of the study. Hyperthermic immersion (HI) lead to a shortening of activated partial thromboplastin time (aPTT) (P < 0.05). Fibrinogen concentration decreased immediately after HI (P < 0.05) but increased during recovery (P < 0.05). Plasminogen activator inhibitor (PAI) activity decreased during HI (P < 0.05), D-dimer concentration was not found to change. Thrombocyte count increased (P < 0.05) during HI. The increases in tissue-type plasminogen activator concentration as well as leucocyte count during HI were due to haemoconcentration. Prothrombin time, PAI-activity and granulocyte count decreased during thermoneutral immersion (P < 0.05). Warm water bathing leads to haemoconcentration and minimal activation of coagulation. The PAI-1 activity is decreased. A marked risk for thrombotic or bleeding complications during warm water bathing in healthy males could not be ascertained.

Late night activity regarding stroke codes: LuNAR strokes.

PubMed

Tafreshi, Gilda; Raman, Rema; Ernstrom, Karin; Rapp, Karen; Meyer, Brett C

2012-08-01

There is diurnal variation for cardiac arrest and sudden cardiac death. Stroke may show a similar pattern. We assessed whether strokes presenting during a particular time of day or night are more likely of vascular etiology. To compare emergency department stroke codes arriving between 22:00 and 8:00 hours (LuNAR strokes) vs. others (n-LuNAR strokes). The purpose was to determine if late night strokes are more likely to be true strokes or warrant acute tissue plasminogen activator evaluations. We reviewed prospectively collected cases in the University of California, San Diego Stroke Team database gathered over a four-year period. Stroke codes at six emergency departments were classified based on arrival time. Those arriving between 22:00 and 8:00 hours were classified as LuNAR stroke codes, the remainder were classified as 'n-LuNAR'. Patients were further classified as intracerebral hemorrhage, acute ischemic stroke not receiving tissue plasminogen activator, acute ischemic stroke receiving tissue plasminogen activator, transient ischemic attack, and nonstroke. Categorical outcomes were compared using Fisher's Exact test. Continuous outcomes were compared using Wilcoxon's Rank-sum test. A total of 1607 patients were included in our study, of which, 299 (19%) were LuNAR code strokes. The overall median NIHSS was five, higher in the LuNAR group (n-LuNAR 5, LuNAR 7; P=0·022). There was no overall differences in patient diagnoses between LuNAR and n-LuNAR strokes (P=0·169) or diagnosis of acute ischemic stroke receiving tissue plasminogen activator (n-LuNAR 191 (14·6%), LuNAR 42 (14·0%); P=0·86). Mean arrival to computed tomography scan time was longer during LuNAR hours (n-LuNAR 54·9±76·3 min, LuNAR 62·5±87·7 min; P=0·027). There was no significant difference in 90-day mortality (n-LuNAR 15·0%, LuNAR 13·2%; P=0·45). Our stroke center experience showed no difference in diagnosis of acute ischemic stroke between day and night stroke codes. This similarity was further supported in similar rates of tissue plasminogen activator administration. Late night strokes may warrant a more rapid stroke specialist evaluation due to the longer time elapsed from symptom onset and the longer time to computed tomography scan. © 2011 The Authors. International Journal of Stroke © 2011 World Stroke Organization.

Surface-Expressed Enolase Contributes to the Pathogenesis of Clinical Isolate SSU of Aeromonas hydrophila▿

PubMed Central

Sha, Jian; Erova, Tatiana E.; Alyea, Rebecca A.; Wang, Shaofei; Olano, Juan P.; Pancholi, Vijay; Chopra, Ashok K.

2009-01-01

In this study, we demonstrated that the surface-expressed enolase from diarrheal isolate SSU of Aeromonas hydrophila bound to human plasminogen and facilitated the latter's tissue-type plasminogen activator-mediated activation to plasmin. The bacterial surface-bound plasmin was more resistant to the action of its specific physiological inhibitor, the antiprotease α2-antiplasmin. We found that immunization of mice with purified recombinant enolase significantly protected the animals against a lethal challenge dose of wild-type (WT) A. hydrophila. Minimal histological changes were noted in organs from mice immunized with enolase and then challenged with WT bacteria compared to severe pathological changes found in the infected and nonimmunized group of animals. This correlated with the smaller bacterial load of WT bacteria in the livers and spleens of enolase-immunized mice than that found in the nonimmunized controls. We also showed that the enolase gene could potentially be important for the viability of A. hydrophila SSU as we could delete the chromosomal copy of the enolase gene only when another copy of the targeted gene was supplied in trans. By site-directed mutagenesis, we altered five lysine residues located at positions 343, 394, 420, 427, and 430 of enolase in A. hydrophila SSU; the mutated forms of enolase were hyperexpressed in Escherichia coli, and the proteins were purified. Our results indicated that lysine residues at positions 420 and 427 of enolase were crucial in plasminogen-binding activity. We also identified a stretch of amino acid residues (252FYDAEKKEY260) in the A. hydrophila SSU enolase involved in plasminogen binding. To our knowledge, this is the first report of the direct involvement of surface-expressed enolase in the pathogenesis of A. hydrophila SSU infections and of any gram-negative bacteria in general. PMID:19270100

Binding of human plasminogen by the lipoprotein LipL46 of Leptospira interrogans.

PubMed

Santos, Jadson V; Pereira, Priscila R M; Fernandes, Luis G V; Siqueira, Gabriela Hase; de Souza, Gisele O; Souza Filho, Antônio; Vasconcellos, Silvio A; Heinemann, Marcos B; Chapola, Erica G B; Nascimento, Ana L T O

2018-02-01

Leptospirosis is a widespread zoonosis caused by pathogenic Leptospira. Bacteria disseminate via the bloodstream and colonize the renal tubules of reservoir hosts. Leptospiral surface-exposed proteins are important targets, because due to their location they can elicit immune response and mediate adhesion and invasion processes. LipL46 has been previously reported to be located at the leptospiral outer membrane and recognized by antibodies present in serum of infected hamsters. In this study, we have confirmed the cellular location of this protein by immunofluorescence and FACS. We have cloned and expressed the recombinant protein LipL46 in its soluble form. LipL46 was recognized by confirmed leptospirosis human serum, suggesting its expression during infection. Binding screening of LipL46 with extracellular matrix (ECM) and plasma components showed that this protein interacts with plasminogen. The binding is dose-dependent on protein concentration, but saturation was not reached with the range of protein concentration used. Kringle domains of plasminogen and lysine residues of the recombinant protein are involved in the binding because the lysine analog, amino caproic acid (ACA) almost totally inhibited the reaction. The interaction of LipL46 with plasminogen generates plasmin in the presence of plasminogen activator uPA. Because plasmin generated at the leptospiral surface can degrade ECM molecules and decrease opsonophagocytosis, we tentatively infer that Lip46 has a role in helping the invasion process of pathogenic Leptospira. Copyright © 2017. Published by Elsevier Ltd.

Clinical evaluation of multiple inflammation biomarkers for diagnosis and prognosis for patients with systemic inflammatory response syndrome.

PubMed

Reichsoellner, M; Raggam, R B; Wagner, J; Krause, R; Hoenigl, M

2014-11-01

A multiplexed biomarker bundle consisting of nine different inflammation markers was evaluated regarding their diagnostic and prognostic performances in 159 adult systemic inflammatory response syndrome (SIRS) patients enrolled at the emergency department. Fibronectin, interleukin-8 (IL-8), biotin, and neutrophil gelatinase-associated lipocalin (NGAL) were the most robust markers but were not superior to the already established markers IL-6, C-reactive protein (CRP), procalcitonin (PCT), and soluble urokinase plasminogen activator receptor (suPAR). Copyright © 2014, American Society for Microbiology. All Rights Reserved.

A call for formal telemedicine training during stroke fellowship

PubMed Central

Jia, Judy; Gildersleeve, Kasey; Ankrom, Christy; Cai, Chunyan; Rahbar, Mohammad; Savitz, Sean I.; Wu, Tzu-Ching

2016-01-01

During the 20 years since US Food and Drug Administration approval of IV tissue plasminogen activator for acute ischemic stroke, vascular neurology consultation via telemedicine has contributed to an increased frequency of IV tissue plasminogen activator administration and broadened geographic access to the drug. Nevertheless, a growing demand for acute stroke coverage persists, with the greatest disparity found in rural communities underserved by neurologists. To provide efficient and consistent acute care, formal training in telemedicine during neurovascular fellowship is warranted. Herein, we describe our experiences incorporating telestroke into the vascular neurology fellowship curriculum and propose recommendations on integrating formal telemedicine training into the Accreditation Council for Graduate Medical Education vascular neurology fellowship. PMID:27016522

A call for formal telemedicine training during stroke fellowship.

PubMed

Jagolino, Amanda L; Jia, Judy; Gildersleeve, Kasey; Ankrom, Christy; Cai, Chunyan; Rahbar, Mohammad; Savitz, Sean I; Wu, Tzu-Ching

2016-05-10

During the 20 years since US Food and Drug Administration approval of IV tissue plasminogen activator for acute ischemic stroke, vascular neurology consultation via telemedicine has contributed to an increased frequency of IV tissue plasminogen activator administration and broadened geographic access to the drug. Nevertheless, a growing demand for acute stroke coverage persists, with the greatest disparity found in rural communities underserved by neurologists. To provide efficient and consistent acute care, formal training in telemedicine during neurovascular fellowship is warranted. Herein, we describe our experiences incorporating telestroke into the vascular neurology fellowship curriculum and propose recommendations on integrating formal telemedicine training into the Accreditation Council for Graduate Medical Education vascular neurology fellowship. © 2016 American Academy of Neurology.

The Kringle-2 domain of tissue plasminogen activator significantly reduces mortality and brain infarction in middle cerebral artery occlusion rats.

PubMed

Zhang, Haitao; Bi, Feng; Xiao, Chunlan; Liu, Jianxia; Wang, Zhixia; Liu, Jian-Ning; Zhang, Jing

2010-08-01

Tissue plasminogen activator (TPA) showed brain-protective activity within the first 15 min after cerebral ischemia in rats. To understand its molecular mechanism, TPA derivates were intracerebroventricularly administered at 15 min before, and 15, 90, 120 min after middle cerebral artery occlusion (MCAO) in rats. The reduction in mortality and cerebral infarction at 24 h was seen only with TPA administered at 15 min after MCAO. The down-regulation of endogenous TPA by the intracerebroventricular injection of TPA was found to be responsible for the protective effect on the integrity of blood-brain barrier after MCAO, as well as for the reduction in mortality and cerebral infarction. Moreover, for the first time we have found that the Kringle-2 domain is essential for the brain-protective activity of TPA.

Plasminogen activator inhibitor-1 4G/5G polymorphism, factor V Leiden, prothrombin mutations and the risk of VTE recurrence.

PubMed

Sundquist, Kristina; Wang, Xiao; Svensson, Peter J; Sundquist, Jan; Hedelius, Anna; Larsson Lönn, Sara; Zöller, Bengt; Memon, Ashfaque A

2015-11-25

Plasminogen-activator inhibitor (PAI)-1 is an important inhibitor of the plasminogen/plasmin system. PAI-1 levels are influenced by the 4G/5G polymorphism in the PAI-1 promoter. We investigated the relationship between the PAI-1 polymorphism and VTE recurrence, and its possible modification by factor V Leiden (FVL) and prothrombin (PTM) mutations. Patients (n=1,069) from the Malmö Thrombophilia Study were followed from discontinuation of anticoagulant treatment until diagnosis of VTE recurrence or the end of the study (maximum follow-up 9.8 years). One hundred twenty-seven patients (11.9 %) had VTE recurrence. PAI-1 was genotyped by TaqMan PCR. Cox regression analysis adjusted for age, sex and acquired risk factors of VTE showed no evidence of an association between PAI-1 genotype and risk of VTE recurrence in the study population as a whole. However, by including an interaction term in the analysis we showed that FVL but not PTM modified the effect of PAI-1 genotype: patients with the 4G allele plus FVL had a higher risk of VTE recurrence [hazard ratio (HR) =2.3, 95 % confidence interval (CI) =1.5-3.3] compared to patients with the 4G allele but no FVL (reference group) or FVL irrespective of PAI-1 genotype (HR=1.8, 95 % CI=1.3-2.5). Compared to reference group, 5G allele irrespective of FVL was associated with lower risk of VTE recurrence only when compared with 4G allele together with FVL. In conclusion, FVL has a modifying effect on PAI-1 polymorphism in relation to risk of VTE recurrence. The role of PAI-1 polymorphism as a risk factor of recurrent VTE may be FVL dependent.

Coagulation Management in Jersey Calves: An ex vivo Study.

PubMed

Gröning, Sabine; Maas, Judith; van Geul, Svenja; Rossaint, Rolf; Steinseifer, Ulrich; Grottke, Oliver

2017-01-01

Jersey calves are frequently used as an experimental animal model for in vivo testing of cardiac assist devices or orthopedic implants. In this ex vivo study, we analyzed the coagulation system of the Jersey calves and the potential of human-based coagulation management to circumvent perioperative bleeding complications during surgery. Experimental Procedure: Blood from 7 Jersey calves was subjected to standard laboratory tests and thromboelastometry analysis. An ex vivo model of dilutional coagulopathy was used to study the effects of fibrinogen or prothrombin complex concentrate supplementation. Fibrinolysis was induced with tissue plasminogen activator to identify potential therapeutic strategies involving tranexamic acid or aprotinin. Furthermore, anticoagulation strategies were evaluated by incubating the blood samples with dabigatran or rivaroxaban. Baseline values for thromboelastometry and standard laboratory parameters, including prothrombin time, activated partial thromboplastin time, fibrinogen, antithrombin III, and D-dimers, were established. Fifty percent diluted blood showed a statistically significant impairment of hemostasis. The parameters significantly improved after the administration of fibrinogen or prothrombin complex concentrate. Tranexamic acid and aprotinin ameliorated tissue plasminogen activator-induced fibrinolysis. Both dabigatran and rivaroxaban significantly prolonged the coagulation parameters. In this ex vivo study, coagulation factors, factor concentrate, antifibrinolytic reagents, and anticoagulants regularly used in the clinic positively impacted coagulation parameters in Jersey calf blood. © 2017 S. Karger AG, Basel.

Interaction Effects of Temperature and Ozone on Lung Function and Markers of Systemic Inflammation, Coagulation, and Fibrinolysis: A Crossover Study of Healthy Young Volunteers

PubMed Central

Kahle, Juliette J.; Neas, Lucas M.; Devlin, Robert B.; Case, Martin W.; Schmitt, Michael T.; Madden, Michael C.

2014-01-01

Background: Trends in climate suggest that extreme weather events such as heat waves will become more common. High levels of the gaseous pollutant ozone are associated with elevated temperatures. Ozone has been associated with respiratory diseases as well as cardiovascular morbidity and mortality and can reduce lung function and alter systemic markers of fibrinolysis. The interaction between ozone and temperature is unclear. Methods: Sixteen healthy volunteers were exposed in a randomized crossover study to 0.3 ppm ozone and clean air for 2 hr at moderate (22°C) temperature and again at an elevated temperature (32.5°C). In each case lung function was performed and blood taken before and immediately after exposure and the next morning. Results: Ozone exposure at 22°C resulted in a decrease in markers of fibrinolysis the next day. There was a 51.8% net decrease in PAI-1 (plasminogen activator inhibitor-1), a 12.1% net decrease in plasminogen, and a 17.8% net increase in D-dimer. These significantly differed from the response at 32.5°C, where there was a 44.9% (p = 0.002) and a 27.9% (p = 0.001) increase in PAI-1 and plasminogen, respectively, and a 12.5% (p = 0.042) decrease in D-dimer. In contrast, decrements in lung function following ozone exposure were comparable at both moderate and elevated temperatures (forced expiratory volume in 1 sec, –12.4% vs. –7.5%, p > 0.05). No changes in systemic markers of inflammation were observed for either temperature. Conclusion: Ozone-induced systemic but not respiratory effects varied according to temperature. Our study suggests that at moderate temperature ozone may activate the fibrinolytic pathway, while at elevated temperature ozone may impair it. These findings provide a biological basis for the interaction between temperature and ozone on mortality observed in some epidemiologic studies. Citation: Kahle JJ, Neas LM, Devlin RB, Case MW, Schmitt MT, Madden MC, Diaz-Sanchez D. 2015. Interaction effects of temperature and ozone on lung function and markers of systemic inflammation, coagulation, and fibrinolysis: a crossover study of healthy young volunteers. Environ Health Perspect 123:310–316; http://dx.doi.org/10.1289/ehp.1307986 PMID:25514459

Delayed activation of PPAR-β/δ improves long-term survival in mouse sepsis: effects on organ inflammation and coagulation.

PubMed

Busch, Daniel; Kapoor, Amar; Rademann, Pia; Hildebrand, Frank; Bahrami, Soheyl; Thiemermann, Christoph; Osuchowski, Marcin F

2018-05-01

Activation of peroxisome proliferator-activated receptor (PPAR)-β/δ reduces tissue injury in murine endotoxemia. We hypothesized that the PPAR-β/δ-agonist GW0742 improves long-term outcome after sepsis caused by cecal ligation and puncture (CLP). Fifty-one CD-1 female mice underwent CLP and received either vehicle (control), GW0742 (0.03 mg/kg/injection; five post-CLP i.v. injections), GSK0660 (PPAR-β/δ-antagonist) or both and were monitored for 28 d. Another 20 CLP mice treated with GW0742 and vehicle were sacrificed 24 h post-CLP to assess coagulopathy. Compared to vehicle, survival of CLP-mice treated with GW0742 was higher by 35% at d 7 and by 50% at d 28. CLP mice treated with GW0742 had 60% higher IFN-γ but circulating monocyte chemoattractant protein-1 and chemokine ligand were lower at 48 h post-CLP. Compared to vehicle, CLP mice treated with GW0742 exhibited a 50% reduction in the circulating plasminogen activator inhibitor-1 associated with an increase in platelet number at 24 h post-CLP (but no changes occurred in anti-thrombin-III, plasminogen, fibrinogen and clotting-times). CLP mice treated with GW0742 exhibited a similar increase in most of the biochemical markers of organ injury/dysfunction (lactate dehydrogenase, alanine aminotransferase, creatine kinase, creatinine, blood urea nitrogen, and triglycerides) measured. Treatment with GW0742 consistently improved long-term survival in septic CD-1 mice by partially modulating the post-CLP systemic cytokine response and coagulation systems.

A label-free photoelectrochemical biosensor for urokinase-type plasminogen activator detection based on a g-C3N4/CdS nanocomposite.

PubMed

Liu, Xing-Pei; Chen, Jing-Shuai; Mao, Chang-Jie; Niu, He-Lin; Song, Ji-Ming; Jin, Bao-Kang

2018-09-26

Herein, we established a novel ultrasensitive photoelectrochemical biosensor for detecting urokinase-type plasminogen activator (u-PA), based on a g-C 3 N 4 /CdS nanocomposite. The prepared nanocomposite was characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, ultraviolet-visible absorption spectroscopy, and Fourier transform infrared spectroscopy, thus indicating that the nanocomposite was prepared successfully. In the typical process, the prepared nanocomposite was deposited on the surface of a bare FTO electrode. After being air-dried, the g-C 3 N 4 /CdS nanocomposite modified electrode was successively incubated with antibody against urokinase-type plasminogen activator and the blocking agent BSA to produce a photoelectrochemical biosensor for u-PA. In the presence of target u-PA antigen, the photocurrent response of the prepared biosensor electrode decreased significantly. The proposed novel photoelectrochemical biosensor exhibited good sensitivity, specificity, and reproducibility for u-PA detection, and a low detection limit of 33 fg mL -1 , ranging from 1 μg mL -1 -0.1 pg mL -1 . The proposed strategy should provide a promising method for detection of other biomarkers. Copyright © 2018 Elsevier B.V. All rights reserved.

Inhibitory effect of berberine on the invasion of human lung cancer cells via decreased productions of urokinase-plasminogen activator and matrix metalloproteinase-2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peng, P.-L.; Hsieh, Y.-S.; Wang, C.-J.

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 reductionmore » 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.« less

Submacular hemorrhage in neovascular age-related macular degeneration: A synthesis of the literature.

PubMed

Stanescu-Segall, Dinu; Balta, Florian; Jackson, Timothy L

2016-01-01

Large submacular hemorrhage, an uncommon manifestation of neovascular age-related macular degeneration, may also occur with idiopathic polypoidal choroidal vasculopathy. Submacular hemorrhage damages photoreceptors owing to iron toxicity, fibrin meshwork contraction, and reduced nutrient flux, with subsequent macular scarring. Clinical and experimental studies support prompt treatment, as tissue damage can occur within 24 hours. Without treatment the natural history is poor, with a mean final visual acuity (VA) of 20/1600. Reported treatments include retinal pigment epithelial patch, macular translocation, pneumatic displacement, intravitreal or subretinal tissue plasminogen activator, intravitreal anti-vascular endothelial growth factor (VEGF) drugs, and combinations thereof. In the absence of comparative studies, we combined eligible studies to assess the VA change before and after each treatment option. The greatest improvement occurred after combined pars plana vitrectomy, subretinal tissue plasminogen activator, intravitreal gas, and anti-vascular endothelial growth factor treatment, with VA improving from 20/1000 to 20/400. The best final VA occurred using combined intravitreal tissue plasminogen activator, gas, and anti-vascular endothelial growth factor therapy, with VA improving from 20/200 to 20/100. Both treatments had an acceptable safety profile, but most studies were small, and larger randomized controlled trials are needed to determine both safety and efficacy. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

Pharmacokinetic and hemostatic properties of the recombinant plasminogen activator bm 06.022 in healthy volunteers.

PubMed

Martin, U; von Möllendorff, E; Akpan, W; Kientsch-Engel, R; Kaufmann, B; Neugebauer, G

1991-11-01

In a randomized, single-blind, placebo-controlled, cross-over Phase-I study pharmacokinetic and hemostatic properties of BM 06.022 were investigated in seven healthy, male human volunteers. The novel recombinant plasminogen activator BM 06.022 consists of the kringle 2 domain and the protease domain of human t-PA and is unglycosylated due to its expression in Escherichia coli cells. Vehicle or 6 MU (= 10.4 mg) BM 06.022 was administered as a single i.v. bolus injection of 10 ml over 2 min. BM 06.022 was well tolerated. Fibrinogen levels and clotting times remained unchanged at baseline levels after 6 MU BM 06.022; plasminogen and alpha 2-antiplasmin (collected on chloromethylketone) decreased maximally to 83 +/- 1% and 64 +/- 3%, respectively, of baseline. D-dimers and fibrinogen degradation products increased to 1,006 +/- 234 ng/ml and 555 +/- 155 ng/ml, respectively, after BM 06.022. Half-life of BM 06.022-activity was 11.2 +/- 0.4 min and of antigen was 13.9 +/- 0.7 min, followed by a terminal half-life only for antigen of 173 +/- 33 min. Plasma clearance of BM 06.022 was 371 +/- 13 ml/min for activity and 183 +/- 15 ml/min for antigen. Thus, BM 06.022 is not fibrinogenolytic at 6 MU and is a fibrinolytic agent with a longer half-life than t-PA.

Successful silencing of plasminogen activator inhibitor-1 in human vascular endothelial cells using small interfering RNA.

PubMed

Hecke, Anneke; Brooks, Hilary; Meryet-Figuière, Matthieu; Minne, Stephanie; Konstantinides, Stavros; Hasenfuss, Gerd; Lebleu, Bernard; Schäfer, Katrin

2006-05-01

Clinical as well as experimental evidence suggests that vascular overexpression of plasminogen activator inhibitor (PAI)-1, the primary physiological inhibitor of both urokinase and tissue-type plasminogen activator, may be involved in the pathophysiology of atherosclerosis and cardiovascular disease. We investigated the feasibility, efficacy and functional effects of PAI-1 gene silencing in human vascular endothelial cells using small interfering RNA. Double-stranded 21 bp-RNA molecules targeted at sequences within the human PAI-1 gene were constructed. Successful siRNA transfection of HUVEC was confirmed using fluorescence microscopy and flow cytometry. One of five candidate siRNA sequences reduced PAI-1 mRNA and protein in a concentration- and time-dependent manner. Suppression of PAI-1 mRNA was detected up to 72 hours after transfection. Moreover, siRNA treatment reduced the activity of PAI-1 released from HUVEC, and prevented the oxLDL- or LPS-induced upregulation of PAI-1 secretion. Importantly, siRNA treatment did not affect the expression of other endothelial-cell markers. Moreover, downregulation of PAI-1 significantly enhanced the ability of endothelial cells to adhere to vitronectin, and this effect could be reversed upon addition of recombinant PAI-1. SiRNA-mediated reduction of PAI-1 expression may be a promising strategy for dissecting the effects of PAI-1 on vascular homeostasis.

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

Use of Biomarkers to Guide Decisions on Adjuvant Systemic Therapy for Women With Early-Stage Invasive Breast Cancer: American Society of Clinical Oncology Clinical Practice Guideline.

PubMed

Harris, Lyndsay N; Ismaila, Nofisat; McShane, Lisa M; Andre, Fabrice; Collyar, Deborah E; Gonzalez-Angulo, Ana M; Hammond, Elizabeth H; Kuderer, Nicole M; Liu, Minetta C; Mennel, Robert G; Van Poznak, Catherine; Bast, Robert C; Hayes, Daniel F

2016-04-01

To provide recommendations on appropriate use of breast tumor biomarker assay results to guide decisions on adjuvant systemic therapy for women with early-stage invasive breast cancer. A literature search and prospectively defined study selection sought systematic reviews, meta-analyses, randomized controlled trials, prospective-retrospective studies, and prospective comparative observational studies published from 2006 through 2014. Outcomes of interest included overall survival and disease-free or recurrence-free survival. Expert panel members used informal consensus to develop evidence-based guideline recommendations. The literature search identified 50 relevant studies. One randomized clinical trial and 18 prospective-retrospective studies were found to have evaluated the clinical utility, as defined by the guideline, of specific biomarkers for guiding decisions on the need for adjuvant systemic therapy. No studies that met guideline criteria for clinical utility were found to guide choice of specific treatments or regimens. In addition to estrogen and progesterone receptors and human epidermal growth factor receptor 2, the panel found sufficient evidence of clinical utility for the biomarker assays Oncotype DX, EndoPredict, PAM50, Breast Cancer Index, and urokinase plasminogen activator and plasminogen activator inhibitor type 1 in specific subgroups of breast cancer. No biomarker except for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 was found to guide choices of specific treatment regimens. Treatment decisions should also consider disease stage, comorbidities, and patient preferences. © 2016 by American Society of Clinical Oncology.

Use of Biomarkers to Guide Decisions on Adjuvant Systemic Therapy for Women With Early-Stage Invasive Breast Cancer: American Society of Clinical Oncology Clinical Practice Guideline

PubMed Central

Harris, Lyndsay N.; McShane, Lisa M.; Andre, Fabrice; Collyar, Deborah E.; Gonzalez-Angulo, Ana M.; Hammond, Elizabeth H.; Kuderer, Nicole M.; Liu, Minetta C.; Mennel, Robert G.; Van Poznak, Catherine; Bast, Robert C.; Hayes, Daniel F.

2016-01-01

Purpose To provide recommendations on appropriate use of breast tumor biomarker assay results to guide decisions on adjuvant systemic therapy for women with early-stage invasive breast cancer. Methods A literature search and prospectively defined study selection sought systematic reviews, meta-analyses, randomized controlled trials, prospective-retrospective studies, and prospective comparative observational studies published from 2006 through 2014. Outcomes of interest included overall survival and disease-free or recurrence-free survival. Expert panel members used informal consensus to develop evidence-based guideline recommendations. Results The literature search identified 50 relevant studies. One randomized clinical trial and 18 prospective-retrospective studies were found to have evaluated the clinical utility, as defined by the guideline, of specific biomarkers for guiding decisions on the need for adjuvant systemic therapy. No studies that met guideline criteria for clinical utility were found to guide choice of specific treatments or regimens. Recommendations In addition to estrogen and progesterone receptors and human epidermal growth factor receptor 2, the panel found sufficient evidence of clinical utility for the biomarker assays Oncotype DX, EndoPredict, PAM50, Breast Cancer Index, and urokinase plasminogen activator and plasminogen activator inhibitor type 1 in specific subgroups of breast cancer. No biomarker except for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 was found to guide choices of specific treatment regimens. Treatment decisions should also consider disease stage, comorbidities, and patient preferences. PMID:26858339

Analysis of a two-domain binding site for the urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex in low-density-lipoprotein-receptor-related protein.

PubMed

Andersen, O M; Petersen, H H; Jacobsen, C; Moestrup, S K; Etzerodt, M; Andreasen, P A; Thøgersen, H C

2001-07-01

The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each approximately 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca(2+) cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. alpha(2)-macroglobulin (alpha(2)M)-proteinase complexes, lipoprotein-containing particles and serine proteinase-inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA-PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3-CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA-PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp(958,CR5), Asp(999,CR6), Trp(953,CR5) and Trp(994,CR6)), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP) - the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA-PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA-PAI-1 as well as for the binding of RAP.

Strokes with minor symptoms: an exploratory analysis of the National Institute of Neurological Disorders and Stroke recombinant tissue plasminogen activator trials.

PubMed

Khatri, Pooja; Kleindorfer, Dawn O; Yeatts, Sharon D; Saver, Jeffrey L; Levine, Steven R; Lyden, Patrick D; Moomaw, Charles J; Palesch, Yuko Y; Jauch, Edward C; Broderick, Joseph P

2010-11-01

The pivotal National Institute of Neurological Disorders and Stroke recombinant tissue plasminogen activator trials excluded patients with ischemic stroke with specific minor presentations or rapidly improving symptoms. The recombinant tissue plasminogen activator product label notes that its use for minor neurological deficit or rapidly improving stroke symptoms has not been evaluated. As a result, patients with low National Institutes of Health Stroke Scale scores are not commonly treated in clinical practice. We sought to further characterize the patients with minor stroke who were included in the National Institute of Neurological Disorders and Stroke trials. Minor strokes were defined as National Institutes of Health Stroke Scale score ≤ 5 at baseline for this retrospective analysis, because this subgroup is most commonly excluded from treatment in clinical practice and trials. Clinical stroke syndromes were defined based on prespecified National Institutes of Health Stroke Scale item score clusters. Clinical outcomes were reviewed generally and within these cluster subgroups. Only 58 cases had National Institutes of Health Stroke Scale scores of 0 to 5 in the National Institute of Neurological Disorders and Stroke trials (42 recombinant tissue plasminogen activator and 16 placebo), and 2971 patients were excluded from the trials due to "rapidly improving" or "minor symptoms" as the primary reason. No patients were enrolled with isolated motor symptoms, isolated facial droop, isolated ataxia, dysarthria, isolated sensory symptoms, or with only symptoms/signs not captured by the National Institutes of Health Stroke Scale score (ie, National Institutes of Health Stroke Scale=0). There were ≤ 3 patients with each of the other isolated deficits enrolled in the trial. The National Institute of Neurological Disorders and Stroke trials excluded a substantial number of strokes with minor presentations, those that were included were small in number, and conclusions about outcomes based on specific syndromes cannot be drawn. Further prospective, systematic study of this subgroup is needed.

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

PubMed

Akazawa, Munetoshi; Nishida, Makoto

2017-05-01

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. © 2017 Nordic Federation of Societies of Obstetrics and Gynecology.

Estimated cost savings of increased use of intravenous tissue plasminogen activator for acute ischemic stroke in Canada.

PubMed

Yip, Todd R; Demaerschalk, Bart M

2007-06-01

Intravenous tissue plasminogen activator (tPA) is an economically worthwhile but underused treatment option for acute ischemic stroke. We sought to identify the extent of tPA use in Canadian medical centers and the potential savings associated with increased use nationally and by province. We determined the nationwide annual incidence of ischemic stroke from the Canadian Institute of Health Information. The proportion of all ischemic stroke patients who received tPA was derived from published data. Economic analyses that report the expected annual cost savings of tPA were consulted. The analysis was conducted from the perspective of a universal health care system during 1 year. We estimated cost-savings with incrementally (eg, 2%, 4%, 6%, 8%, 10%, 15%, and 20%) increased use of tPA for acute ischemic stroke nationally and provincially. The current average national tPA utilization is 1.4%. For every increase of 2 percentage points in utilization, $757,204 (Canadian) could possibly be saved annually (95% CI maximum loss of $3,823,992 to a maximum savings of $2,201,252). With a 20% rate, >$7.5 million (Canadian) could be saved nationwide the first year. We estimate that even small increases in the proportion of all Canadian ischemic stroke patients receiving tPA could result in substantial realized savings for Canada's health care system.

IMD-4690, a novel specific inhibitor for plasminogen activator inhibitor-1, reduces allergic airway remodeling in a mouse model of chronic asthma via regulating angiogenesis and remodeling-related mediators.

PubMed

Tezuka, Toshifumi; Ogawa, Hirohisa; Azuma, Masahiko; Goto, Hisatsugu; Uehara, Hisanori; Aono, Yoshinori; Hanibuchi, Masaki; Yamaguchi, Yoichi; Fujikawa, Tomoyuki; Itai, Akiko; Nishioka, Yasuhiko

2015-01-01

Plasminogen activator inhibitor (PAI)-1 is the principal inhibitor of plasminogen activators, and is responsible for the degradation of fibrin and extracellular matrix. IMD-4690 is a newly synthesized inhibitor for PAI-1, whereas the effect on allergic airway inflammation and remodeling is still unclear. We examined the in vivo effects by using a chronic allergen exposure model of bronchial asthma in mice. The model was generated by an immune challenge for 8 weeks with house dust mite antigen, Dermatophagoides pteronyssinus (Dp). IMD-4690 was intraperitoneally administered during the challenge. Lung histopathology, hyperresponsiveness and the concentrations of mediators in lung homogenates were analyzed. The amount of active PAI-1 in the lungs was increased in mice treated with Dp. Administration with IMD-4690 reduced an active/total PAI-1 ratio. IMD-4690 also reduced the number of bronchial eosinophils in accordance with the decreased expressions of Th2 cytokines in the lung homogenates. Airway remodeling was inhibited by reducing subepithelial collagen deposition, smooth muscle hypertrophy, and angiogenesis. The effects of IMD-4690 were partly mediated by the regulation of TGF-β, HGF and matrix metalloproteinase. These results suggest that PAI-1 plays crucial roles in airway inflammation and remodeling, and IMD-4690, a specific PAI-1 inhibitor, may have therapeutic potential for patients with refractory asthma due to airway remodeling.

The urokinase plasminogen activator system components are regulated by vascular endothelial growth factor D in bovine oviduct.

PubMed

García, Daniela C; Russo-Maenza, Agostina; Miceli, Dora C; Valdecantos, Pablo A; Roldán-Olarte, Mariela

2018-06-08

SummaryThe mammalian oviduct plays a pivotal role in the success of early reproductive events. The urokinase plasminogen activator system (uPAS) is present in the bovine oviduct and is involved in extracellular matrix remodelling through plasmin generation. This system can be regulated by several members of the vascular endothelial growth factors (VEGF) and their receptors. In this study, the VEGF-D effect on the regulation of uPAS was evaluated. First, RT-polymerase chain reaction (PCR) analyses were used to evidence the expression of VEGF-D and its receptors in oviductal epithelial cells (BOEC). VEGF-D, VEGFR2 and VEGFR3 transcripts were found in ex vivo and in vitro BOEC, while only VEGFR2 mRNA was present after in vitro conditions. VEGF-D showed a regulatory effect on uPAS gene expression in a dose-dependent manner, inducing an increase in the expression of both uPA and its receptor (uPAR) at 24 h post-induction and decreases in the expression of its inhibitor (PAI-1). In addition, the regulation of cell migration induced by VEGF-D and uPA in BOEC monolayer cultures was analyzed. The wound areas of monolayer cultures incubated with VEGF-D 10 ng/ml or uPA 10 nM were modified and significant differences were found at 24 h for both stimulations. These results indicated that uPAS and VEGF-D systems can modify the arrangement of the bovine oviductal epithelium and contribute to the correct maintenance of the oviductal microenvironment.

Serine proteases in rodent hippocampus.

PubMed

Davies, B J; Pickard, B S; Steel, M; Morris, R G; Lathe, R

1998-09-04

Brain serine proteases are implicated in developmental processes, synaptic plasticity, and in disorders including Alzheimer's disease. The spectrum of the major enzymes expressed in brain has not been established previously. We now present a systematic study of the serine proteases expressed in adult rat and mouse hippocampus. Using a combination of techniques including polymerase chain reaction amplification and Northern blotting we show that tissue-type plasminogen activator (t-PA) is the major species represented. Unexpectedly, the next most abundant species were RNK-Met-1, a lymphocyte protease not reported previously in brain, and two new family members, BSP1 (brain serine protease 1) and BSP2. We report full-length sequences of the two new proteases; homologies indicate that these are of tryptic specificity. Although BSP2 is expressed in several brain regions, BSP1 expression is strikingly restricted to hippocampus. Other enzymes represented, but at lower levels, included elastase IV, proteinase 3, complement C2, chymotrypsin B, chymotrypsin-like protein, and Hageman factor. Although thrombin and urokinase-type plasminogen activator were not detected in the primary screen, low level expression was confirmed using specific polymerase chain reaction primers. In contrast, and despite robust expression of t-PA, the usual t-PA substrate plasminogen was not expressed at detectable levels.

PAI-1 (Plasminogen Activator Inhibitor-1) Expression Renders Alternatively Activated Human Macrophages Proteolytically Quiescent

PubMed Central

Hohensinner, Philipp J.; Baumgartner, Johanna; Kral-Pointner, Julia B.; Uhrin, Pavel; Ebenbauer, Benjamin; Thaler, Barbara; Doberer, Konstantin; Stojkovic, Stefan; Demyanets, Svitlana; Fischer, Michael B.; Huber, Kurt; Schabbauer, Gernot; Speidl, Walter S.

2017-01-01

Objective— Macrophages are versatile immune cells capable of polarizing into functional subsets depending on environmental stimulation. In atherosclerotic lesions, proinflammatory polarized macrophages are associated with symptomatic plaques, whereas Th2 (T-helper cell type 2) cytokine–polarized macrophages are inversely related with disease progression. To establish a functional cause for these observations, we analyzed extracellular matrix degradation phenotypes in polarized macrophages. Approach and Results— We provide evidence that proinflammatory polarized macrophages rely on membrane-bound proteases including MMP-14 (matrix metalloproteinase-14) and the serine protease uPA (urokinase plasminogen activator) together with its receptor uPAR for extracellular matrix degradation. In contrast, Th2 cytokine alternatively primed macrophages do not show different proteolytic activity in comparison to unpolarized macrophages and lack increased localization of MMP-14 and uPA receptor to the cell membrane. Nonetheless, they express the highest amount of the serine protease uPA. However, uPA activity is blocked by similarly increased expression of its inhibitor PAI-1 (plasminogen activator inhibitor 1). When inhibiting PAI-1 or when analyzing macrophages deficient in PAI-1, Th2 cytokine–polarized macrophages display the same matrix degradation capability as proinflammatory-primed macrophages. Within atherosclerotic lesions, macrophages positive for the alternative activation marker CD206 express high levels of PAI-1. In addition, to test changed tissue remodeling capacities of alternatively activated macrophages, we used a bleomycin lung injury model in mice reconstituted with PAI-1−/− bone marrow. These results supported an enhanced remodeling phenotype displayed by increased fibrosis and elevated MMP activity in the lung after PAI-1 loss. Conclusions— We were able to demonstrate matrix degradation dependent on membrane-bound proteases in proinflammatory stimulated macrophages and a forced proteolytical quiescence in alternatively polarized macrophages by the expression of PAI-1. PMID:28818858

Residual vein thrombosis and onset of post-thrombotic syndrome: influence of the 4G/5G polymorphism of plasminogen activator inhibitor-1 gene.

PubMed

Incalcaterra, Egle; Meli, Francesco; Muratori, Ida; Corrado, Egle; Amato, Corrado; Canino, Baldassare; Ferrara, Filippo

2014-03-01

Plasminogen activator inhibitor-1 (PAI-1) is the most important inhibitor of plasminogen activator. The functional 4G/5G polymorphism of the gene coding for PAI-1 may affect PAI-1 plasmatic activity, influencing the imbalance between coagulation and fibrinolysis cascades. In this prospective cohort analytic study, we investigated the role of this single nucleotide polymorphism in the persistence of thrombotic lesion and the occurrence of post-thrombotic syndrome. In a group of 168 patients with post-surgical deep vein thrombosis of the legs, we analyzed the 4G/5G polymorphism in the promoter of PAI-1 gene and plasmatic PAI-1 activity. Enrolled patients were divided in two groups: patients with 4G/5G polymorphism and increased PAI-1 activity (n=85) and patients without 4G/5G polymorphism and normal PAI-1 activity (n=83). All patients were treated according to current protocols and re-examined after 3, 12 and 36 months in order to evaluate the persistence of thrombotic lesion and the occurrence of post-thrombotic syndrome. We found a significantly increased PAI activity in carrier of the 4G allele, who experienced much more frequently a persistence of thrombosis after 3, 12 and 36 months and/or the development of post-thrombosis syndrome, in spite of the anticoagulant treatment. These data not only confirm the role played by PAI-1 activity and by the 4G/5G SNP of the PAI-1 gene, but also suggest that current therapeutic protocols, recommending the administration of low weight molecular heparin and oral anticoagulant for the treatment of deep vein thrombosis, could be non sufficient for patients genetically predisposed to a less efficient clot lysis. Copyright © 2013. Published by Elsevier Ltd.

The story of an exceptional serine protease, tissue-type plasminogen activator (tPA).

PubMed

Hébert, M; Lesept, F; Vivien, D; Macrez, R

2016-03-01

The only acute treatment of ischemic stroke approved by the health authorities is tissue recombinant plasminogen activator (tPA)-induced thrombolysis. Under physiological conditions, tPA, belonging to the serine protease family, is secreted by endothelial and brain cells (neurons, astrocytes, microglia, oligodendrocytes). Although revascularisation induced by tPA is beneficial during a stroke, research over the past 20 years shows that tPA can also be deleterious for the brain parenchyma. Thus, in this review of the literature, after a brief history on the discovery of tPA, we reviewed current knowledge of mechanisms by which tPA can influence brain function in physiological and pathological conditions. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Ultrasound-enhanced thrombolysis using Definity as a cavitation nucleation agent.

PubMed

Datta, Saurabh; Coussios, Constantin-C; Ammi, Azzdine Y; Mast, T Douglas; de Courten-Myers, Gabrielle M; Holland, Christy K

2008-09-01

Ultrasound has been shown previously to act synergistically with a thrombolytic agent, such as recombinant tissue plasminogen activator (rt-PA) to accelerate thrombolysis. In this in vitro study, a commercial contrast agent, Definity, was used to promote and sustain the nucleation of cavitation during pulsed ultrasound exposure at 120 kHz. Ultraharmonic signals, broadband emissions and harmonics of the fundamental were measured acoustically by using a focused hydrophone as a passive cavitation detector and used to quantify the level of cavitation activity. Human whole blood clots suspended in human plasma were exposed to a combination of rt-PA, Definity and ultrasound at a range of ultrasound peak-to-peak pressure amplitudes, which were selected to expose clots to various degrees of cavitation activity. Thrombolytic efficacy was determined by measuring clot mass loss before and after the treatment and correlated with the degree of cavitation activity. The penetration depth of rt-PA and plasminogen was also evaluated in the presence of cavitating microbubbles using a dual-antibody fluorescence imaging technique. The largest mass loss (26.2%) was observed for clots treated with 120-kHz ultrasound (0.32-MPa peak-to-peak pressure amplitude), rt-PA and stable cavitation nucleated by Definity. A significant correlation was observed between mass loss and ultraharmonic signals (r = 0.85, p < 0.0001, n = 24). The largest mean penetration depth of rt-PA (222 microm) and plasminogen (241 microm) was observed in the presence of stable cavitation activity. Stable cavitation activity plays an important role in enhancement of thrombolysis and can be monitored to evaluate the efficacy of thrombolytic treatment.

Enhanced venous thrombus resolution in plasminogen activator inhibitor type-2 deficient mice.

PubMed

Siefert, S A; Chabasse, C; Mukhopadhyay, S; Hoofnagle, M H; Strickland, D K; Sarkar, R; Antalis, T M

2014-10-01

The resolution of deep vein thrombosis requires an inflammatory response and mobilization of proteases, such as urokinase-type plasminogen activator (uPA) and matrix metalloproteinases (MMPs), to degrade the thrombus and remodel the injured vein wall. Plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor (serpin) with unique immunosuppressive and cell survival properties that was originally identified as an inhibitor of uPA. To investigate the role of PAI-2 in venous thrombus formation and resolution. Venous thrombus resolution was compared in wild-type C57BL/6, PAI-2(-/-) , and PAI-1(-/-) mice using the stasis model of deep vein thrombosis. Formed thrombi were harvested, thrombus weights were recorded, and tissue was analyzed for uPA and MMP activities, PAI-1 expression, and the nature of inflammatory cell infiltration. We found that the absence of PAI-2 enhanced venous thrombus resolution, while thrombus formation was unaffected. Enhanced venous thrombus resolution in PAI-2(-/-) mice was associated with increased uPA activity and reduced levels of PAI-1, with no significant effect on MMP-2 and -9 activities. PAI-1 deficiency resulted in an increase in thrombus resolution similar to PAI-2 deficiency, but additionally reduced venous thrombus formation and altered MMP activity. PAI-2-deficient thrombi had increased levels of the neutrophil chemoattractant CXCL2, which was associated with early enhanced neutrophil recruitment. These data identify PAI-2 as a novel regulator of venous thrombus resolution, which modulates several pathways involving both inflammatory and uPA activity mechanisms, distinct from PAI-1. Further examination of these pathways may lead to potential therapeutic prospects in accelerating thrombus resolution. © 2014 International Society on Thrombosis and Haemostasis.

The Role of Osteopontin in the Malignancy of Human Breast Carcinoma

DTIC Science & Technology

1998-06-01

1997): The urokinase-type plasminogen activator system in cancer metastasis: a review. Int. J. Cancer 72:1-22. 59. Yebra M, Parry GCN, Str6mblad S...involved in the motility response) (Andreasen et al., 1997; Yebra et al., 1996). The finding that human breast epithelial cells upregulated for OPN...247-255. Xuan JW, Hota C, Shigeyama Y, D’Errico JA, Somerman MJ, and Chambers AF. (1995). J. Cell Biochem., 57, 680-690. Yebra M, Parry GCN

76 FR 31616 - Government-Owned Inventions; Availability for Licensing

Federal Register 2010, 2011, 2012, 2013, 2014

2011-06-01

... DD. Ultrasound-facilitated thrombolysis using tissue-plasminogen activator-loaded echogenic liposomes... al. Pulsed-high intensity focused ultrasound enhances thrombolysis in an in vitro model. Radiology...

Thrombin stimulates increased plasminogen activator inhibitor-1 release from liver compared to lung endothelium.

PubMed

Huebner, Benjamin R; Moore, Ernest E; Moore, Hunter B; Gonzalez, Eduardo; Kelher, Marguerite R; Sauaia, Angela; Banerjee, Anirban; Silliman, Christopher C

2018-05-01

Plasminogen activator inhibitor-1 (PAI-1) is a major regulator of the fibrinolytic system, covalently binding to tissue plasminogen activator and blocking its activity. Fibrinolysis shutdown is evident in the majority of severely injured patients in the first 24 h and is thought to be due to PAI-1. The source of this PAI-1 is thought to be predominantly endothelial cells, but there are known organ-specific differences, with higher levels thought to be in the liver. Thrombin generation is also elevated in injured patients and is a potent stimulus for PAI-1 release in human umbilical endothelial cells. We hypothesize that thrombin induces liver endothelial cells to release increased amounts of PAI-1, versus pulmonary endothelium, consisting of both stored PAI-1 and a larger contribution from de novo PAI-1 synthesis. Human liver sinusoidal endothelial cells (LSECs) and human microvascular lung endothelial cells (HMVECs) were stimulated in vitro ± thrombin (1 and 5 IU/mL) for 15-240 min, the supernatants were collected, and PAI-1 was measured by enzyme-linked immunosorbent assays. To elucidate the PAI-1 contribution from storage versus de novo synthesis, cycloheximide (10 μg/mL) was added before thrombin in separate experiments. While both LSECs and HMVECs rapidly stimulated PAI-1 release, LSECs released more PAI-1 than HMVECs in response to high-dose thrombin, whereas low-dose thrombin did not provoke immediate release. LSECs continued to release PAI-1 over the ensuing 240 min, whereas HMVECs did not. Cycloheximide did not inhibit early PAI-1 release from LSECs but did at the later time points (30-240 min). Thrombin elicits increased amounts of PAI-1 release from liver endothelium compared with lung, with a small presynthesized stored contribution and a later, larger increase in PAI-1 release via de novo synthesis. This study suggests that the liver may be an important therapeutic target for inhibition of the hypercoagulable surgical patient and the associated complications that result. Copyright © 2017 Elsevier Inc. All rights reserved.

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

Inhibition of plasminogen activator inhibitor-1 binding to endocytosis receptors of the low-density-lipoprotein receptor family by a peptide isolated from a phage display library

PubMed Central

Jensen, Jan K.; Malmendal, Anders; Schiøtt, Birgit; Skeldal, Sune; Pedersen, Katrine E.; Celik, Leyla; Nielsen, Niels Chr.; Andreasen, Peter A.; Wind, Troels

2006-01-01

The functions of the serpin PAI-1 (plasminogen activator inhibitor-1) are based on molecular interactions with its target proteases uPA and tPA (urokinase-type and tissue-type plasminogen activator respectively), with vitronectin and with endocytosis receptors of the low-density-lipoprotein family. Understanding the significance of these interactions would be facilitated by the ability to block them individually. Using phage display, we have identified the disulfide-constrained peptide motif CFGWC with affinity for natural human PAI-1. The three-dimensional structure of a peptide containing this motif (DVPCFGWCQDA) was determined by liquid-state NMR spectroscopy. A binding site in the so-called flexible joint region of PAI-1 was suggested by molecular modelling and validated through binding studies with various competitors and site-directed mutagenesis of PAI-1. The peptide with an N-terminal biotin inhibited the binding of the uPA–PAI-1 complex to the endocytosis receptors low-density-lipoprotein-receptor-related protein 1A (LRP-1A) and very-low-density-lipoprotein receptor (VLDLR) in vitro and inhibited endocytosis of the uPA–PAI-1 complex in U937 cells. We conclude that the isolated peptide represents a novel approach to pharmacological interference with the functions of PAI-1 based on inhibition of one specific molecular interaction. PMID:16813566

Reduced Plasminogen Binding and Delayed Activation Render γ′-Fibrin More Resistant to Lysis than γA-Fibrin*

PubMed Central

Kim, Paul Y.; Vu, Trang T.; Leslie, Beverly A.; Stafford, Alan R.; Fredenburgh, James C.; Weitz, Jeffrey I.

2014-01-01

Fibrin (Fn) clots formed from γ′-fibrinogen (γ′-Fg), a variant with an elongated γ-chain, are resistant to lysis when compared with clots formed from the predominant γA-Fg, a finding previously attributed to differences in clot structure due to delayed thrombin-mediated fibrinopeptide (FP) B release or impaired cross-linking by factor XIIIa. We investigated whether slower lysis of γ′-Fn reflects delayed plasminogen (Pg) binding and/or activation by tissue plasminogen activator (tPA), reduced plasmin-mediated proteolysis of γ′-Fn, and/or altered cross-linking. Clots formed from γ′-Fg lysed more slowly than those formed from γA-Fg when lysis was initiated with tPA/Pg when FPA and FPB were both released, but not when lysis was initiated with plasmin, or when only FPA was released. Pg bound to γ′-Fn with an association rate constant 22% lower than that to γA-Fn, and the lag time for initiation of Pg activation by tPA was longer with γ′-Fn than with γA-Fn. Once initiated, however, Pg activation kinetics were similar. Factor XIIIa had similar effects on clots formed from both Fg isoforms. Therefore, slower lysis of γ′-Fn clots reflects delayed FPB release, which results in delayed binding and activation of Pg. When clots were formed from Fg mixtures containing more than 20% γ′-Fg, the upper limit of the normal level, the delay in lysis was magnified. These data suggest that circulating levels of γ′-Fg modulate the susceptibility of clots to lysis by slowing Pg activation by tPA and provide another example of the intimate connections between coagulation and fibrinolysis. PMID:25128532

Tissue factor levels and the fibrinolytic system in thin and thick intraluminal thrombus and underlying walls of abdominal aortic aneurysms.

PubMed

Siennicka, Aldona; Zuchowski, Marta; Kaczmarczyk, Mariusz; Cnotliwy, Miłosław; Clark, Jeremy Simon; Jastrzębska, Maria

2018-03-20

The hemostatic system cooperates with proteolytic degradation in processes allowing abdominal aortic aneurysm (AAA) formation. In previous studies, it has been suggested that aneurysm rupture depends on intraluminal thrombus (ILT) thickness, which varies across each individual aneurysm. We hypothesized that hemostatic components differentially accumulate in AAA tissue in relation to ILT thickness. Thick (A1) and thin (B1) segments of ILTs and aneurysm wall sections A (adjacent to A1) and B (adjacent to B1) from one aneurysm sac were taken from 35 patients undergoing elective repair. Factor levels were measured using enzyme-linked immunosorbent assay of protein extract. Tissue factor (TF) activities were significantly higher in thinner segments of AAA (B1 vs A1, P = .003; B vs A, P < .001; B vs A1, P < .001; B vs B1, P = .001). Significantly higher tissue plasminogen activator was found in thick thrombus-covered wall segments (A) than in B, A1, and B1 (P = .015, P < .001, and P < .001, respectively). Plasminogen concentrations were highest in ILT. Concentrations of α 2 -antiplasmin in thin ILT adjacent walls (B) were higher compared with wall (A) adjacent to thick ILT (P = .021) and thick ILT (A1; P < .001). Significant correlations between levels of different factors were mostly found in thick ILT (A1). However, no correlations were found at B sites, except for a correlation between plasmin and TF activities (r = 0.55; P = .004). These results suggest that higher TF activities are present in thinner AAA regions. These parameters and local fibrinolysis may be part of the processes leading to destruction of the aneurysm wall. Copyright © 2018 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.

Plasminogen Activator of the Blood Vessels in Tumours and in Carrageenin-induced Granulomas

PubMed Central

Pick, C. R.; Cater, D. B.

1971-01-01

Fibrinolytic activity in tumours was studied by the fibrin slide technique. The tumour cells were inactive and fibrinolysis was seen only in areas with young blood vessels. In carrageenin-induced granulomas at 6 days the fibrinolytic activity was small and confined to mature veins, but from 7-14 days activity was high in zones containing young vessels supplying the terminal capillary buds; these latter showed no activity. In old fibrosed granulomas there was no fibrinolytic activity. The vascular permeability changes of inflammation (detected by the colloidal carbon technique) showed no correlation with fibrinolytic activity, and systemic injection of inflammatory agents had no effect on the fibrinolytic activity of the vessels. These findings are discussed in relationship to tumour vascularization. ImagesFigs. 5-8Figs. 1-4 PMID:5547651

Games network and application to PAs system.

PubMed

Chettaoui, C; Delaplace, F; Manceny, M; Malo, M

2007-02-01

In this article, we present a game theory based framework, named games network, for modeling biological interactions. After introducing the theory, we more precisely describe the methodology to model biological interactions. Then we apply it to the plasminogen activator system (PAs) which is a signal transduction pathway involved in cancer cell migration. The games network theory extends game theory by including the locality of interactions. Each game in a games network represents local interactions between biological agents. The PAs system is implicated in cytoskeleton modifications via regulation of actin and microtubules, which in turn favors cell migration. The games network model has enabled us a better understanding of the regulation involved in the PAs system.

Interactions between iodinated contrast media and tissue plasminogen activator: In vitro comparison study.

PubMed

Vörös, Eszter; Deres, László; Halmosi, Róbert; Várady, Edit; Tóth, Kálmán; Battyáni, István

2017-01-01

Iodinated contrast media (Xenetix®, Ultravist®, Omnipaque®, Visipaque® and Iomeron®) used for computed tomography (CT) may decrease fibrinolysis by recombinant tissue plasminogen activator (rt-PA). We hypothesized that receiving iodinated contrast media before rt-PA may impair thrombolysis as measured by a new model system. Whole blood from Wistar Kyoto rats (n = 10) was obtained and allowed to form blood clots. Thrombolysis was performed by placing individually the prepared clots into 15 mL tubes and adding 5 mL saline buffer, 100μg rt-PA and a different contrast media; adjusting the quantity of iodine to either 30 mg or 60 mg. The thrombolytic efficacy was quantified by measuring the optical density (OD415) of the supernatant at different time points, namely at 0, 30, 60, and 90 min. There was a significant decrease in clot lysis efficiency observed in presence of iodine containing contrast media comparing to positive control group. Moreover, when the quantity of iodine was increased from 30 mg to 60 mg; the dissolution rate downturned with additional ∼50%. In conclusion, our study suggests that high dose of iodine potentially could negatively affect the efficiency of the thrombolytic therapy performed by rt-PA.

Node-Negative Breast Cancer: Which Patients Should Be Treated?

PubMed Central

Schmidt, Marcus

2008-01-01

Summary Adjuvant systemic therapy has led to markedly improved outcome in early-stage breast cancer. However, the absolute gains from chemotherapy might be modest in node-negative patients. Adjuvant chemotherapy is the only option for triple-negative breast cancer patients and should be used with trastuzumab in HER2-positive patients. Considering the large group of patients with some degree of endocrine responsiveness, adding chemotherapy according to risk is an option. At present, we guide our therapeutic decisions using clinicopathologic risk classifications like the St. Gallen risk category or Adjuvant! online. A downside of these risk estimations is a low specificity and consequently the risk for overtreatment of a considerable number of patients. To spare patients unnecessary toxicities we need more reliable prognostic factors or tumor markers. From the plethora of tumor markers, only urokinase-type plasminogen activator (uPA)/plasminogen activator inhibitor 1 (PAI-1) and certain multiparameter gene expression assays are recommended by the American Society of Clinical Oncology. These tumor markers are presently investigated in clinical trials in node-negative breast cancer (NNBC-3, MINDACT, TAILORx). These studies will hopefully allow us to quantify the risk of progression in the individual patient and to tailor treatment accordingly. This should lead to a more personalized treatment recommendation. PMID:21076603

Emergency physician and stroke specialist beliefs and expectations regarding telestroke.

PubMed

Moskowitz, Ari; Chan, Yu-Feng Yvonne; Bruns, John; Levine, Steven R

2010-04-01

Telestroke has been effective in the management of acute ischemic stroke. This study characterizes and compares stroke specialist (SS) and emergency physician (EP) perceptions of telestroke and identifies barriers preventing increased implementation. A survey was developed and distributed nationwide to 382 SSs through an online survey system and in paper form to 226 EPs attending the 2008 American College of Emergency Physicians national conference. Stroke specialists perceived themselves to be more knowledgeable about telemedicine and telestroke (P<0.001 and P=0.010, respectively). A large majority of physicians in both specialties either strongly agreed or agreed that telestroke will reduce geographical differences in stroke management and that it is superior to telephone consultation. EPs perceived patient preference (P<0.001), recombinant tissue plasminogen activator side effects (P<0.001), level of technology (P=0.005), and recombinant tissue plasminogen activator not the standard of care (P<0.001) to be more significant obstacles to increased implementation of telestroke than SSs. However, SSs found increased personal work to be a greater barrier than EPs (P<0.001). SSs and EPs report positive beliefs regarding telestroke; however, perceived obstacles exist to implementation. Differences between barriers perceived by EPs and SSs need to be addressed to enhance acute ischemic stroke treatment.

Plasminogen activator inhibitor-1 4G/5G gene polymorphism and primary open-angle glaucoma

PubMed Central

Weger, Martin; Faschinger, Christoph; Schmut, Otto; Renner, Wilfried

2008-01-01

Purpose Alterations of the plasmin system have been suggested to participate in the multifactorial pathogenesis of primary open-angle glaucoma (POAG). The main physiological inhibitor of the plasmin system is plasminogen activator inhibitor-1 (PAI-1), which leads to decreased degradation of extracellular material. Interestingly, elevated PAI-1 levels in the aqueous humor of patients with POAG have been reported. A common polymorphism within the promoter region (PAI-1 4G/5G) has previously been shown to reduce the gene transcription rate of PAI-1. The purpose of the present study was to investigate a hypothesized association between PAI-1 4G/5G and the presence of POAG in a Caucasian population. Methods The present case-control study comprised 212 unrelated patients with POAG and 212 healthy control subjects, matched for age and sex. Genotyping of PAI-1 4G/5G polymorphisms was done using polymerase chain reaction. Results Allelic frequencies and genotype distributions of PAI-1 4G/5G did not significantly differ between patients with POAG and control subjects (PAI-1 4G/5G: 29.7% versus 29.7%). Presence of the PAI-1 4G-allele was associated with a nonsignificant odds ratio of 0.98 (95% confidence interval: 0.74–1.30) for POAG. Conclusions Our data suggest that PAI-1 4G/5G itself is unlikely to be a major risk factor among Caucasian patients with POAG. PMID:18615155

SERPINE1: A Molecular Switch in the Proliferation-Migration Dichotomy in Wound-“Activated” Keratinocytes

PubMed Central

Simone, Tessa M.; Higgins, Craig E.; Czekay, Ralf-Peter; Law, Brian K.; Higgins, Stephen P.; Archambeault, Jaclyn; Kutz, Stacie M.; Higgins, Paul J.

2014-01-01

Significance: A highly interactive serine protease/plasmin/matrix metalloproteinase axis regulates stromal remodeling in the wound microenvironment. Current findings highlight the importance of stringent controls on protease expression and their topographic activities in cell proliferation, migration, and tissue homeostasis. Targeting elements in this cascading network may lead to novel therapeutic approaches for fibrotic diseases and chronic wounds. Recent Advances: Matrix-active proteases and their inhibitors orchestrate wound site tissue remodeling, cell migration, and proliferation. Indeed, the serine proteases urokinase plasminogen activator and tissue-type plasminogen activator (uPA/tPA) and their major phsyiological inhibitor, plasminogen activator inhibitor-1 (PAI-1; serine protease inhibitor clade E member 1 [SERPINE1]), are upregulated in several cell types during injury repair. Coordinate expression of proteolytic enzymes and their inhibitors in the wound bed provides a mechanism for fine control of focal proteolysis to facilitate matrix restructuring and cell motility in complex environments. Critical Issues: Cosmetic and tissue functional consequences of wound repair anomalies affect the quality of life of millions of patients in the United States alone. The development of novel therapeutics to manage individuals most affected by healing anomalies will likely derive from the identification of critical, translationally accessible, control elements in the wound site microenvironment. Future Directions: Activation of the PAI-1 gene early after wounding, its prominence in the repair transcriptome and varied functions suggest a key role in the global cutaneous injury response program. Targeting PAI-1 gene expression and/or PAI-1 function with molecular genetic constructs, neutralizing antibodies or small molecule inhibitors may provide a novel, therapeutically relevant approach, to manage the pathophysiology of wound healing disorders associated with deficient or excessive PAI-1 levels. PMID:24669362

Influence of decreased fibrinolytic activity and plasminogen activator inhibitor-1 4G/5G polymorphism on the risk of venous thrombosis.

PubMed

Vuckovic, Biljana A; Djeric, Mirjana J; Tomic, Branko V; Djordjevic, Valentina J; Bajkin, Branislav V; Mitic, Gorana P

2018-01-01

: Objective of our study is to determine whether decreased fibrinolytic activity or plasminogen activator inhibitor (PAI)-1 4G/5G polymorphism influence the risk of venous thrombosis.Our case-control study included 100 patients with venous thrombosis, and 100 random controls. When patients were compared with random controls, unconditional logistic regression was used to calculate odds ratios (ORs) with 95% confidence intervals (CIs).Decreased fibrinolytic activity yielded a 2.7-fold increase in risk for venous thrombosis than physiological fibrinolytic activity (OR 2.70; 95% CI 1.22-5.98), when comparing patients with random controls. Adjustment for several putative confounders did not change the estimate (OR 3.02; 95% CI 1.26-7.22). Analysis of venous thrombotic risk influenced by PAI-1 genotype, showed no influence of PAI-1 4G/5G gene variant in comparison with 5G/5G genotype (OR 0.57 95% CI; 0.27-1.20).Decreased fibrinolytic activity increased, whereas PAI-1 4G/5G polymorphism did not influence venous thrombosis risk in this study.

Aβ delays fibrin clot lysis by altering fibrin structure and attenuating plasminogen binding to fibrin

PubMed Central

Zamolodchikov, Daria

2012-01-01

Alzheimer disease is characterized by the presence of increased levels of the β-amyloid peptide (Aβ) in the brain parenchyma and cerebral blood vessels. This accumulated Aβ can bind to fibrin(ogen) and render fibrin clots more resistant to degradation. Here, we demonstrate that Aβ42 specifically binds to fibrin and induces a tighter fibrin network characterized by thinner fibers and increased resistance to lysis. However, Aβ42-induced structural changes cannot be the sole mechanism of delayed lysis because Aβ overlaid on normal preformed clots also binds to fibrin and delays lysis without altering clot structure. In this regard, we show that Aβ interferes with the binding of plasminogen to fibrin, which could impair plasmin generation and fibrin degradation. Indeed, plasmin generation by tissue plasminogen activator (tPA), but not streptokinase, is slowed in fibrin clots containing Aβ42, and clot lysis by plasmin, but not trypsin, is delayed. Notably, plasmin and tPA activities, as well as tPA-dependent generation of plasmin in solution, are not decreased in the presence of Aβ42. Our results indicate the existence of 2 mechanisms of Aβ42 involvement in delayed fibrinolysis: (1) through the induction of a tighter fibrin network composed of thinner fibers, and (2) through inhibition of plasmin(ogen)–fibrin binding. PMID:22238323

Selection of High-Affinity Peptidic Serine Protease Inhibitors with Increased Binding Entropy from a Back-Flip Library of Peptide-Protease Fusions.

PubMed

Sørensen, Hans Peter; Xu, Peng; Jiang, Longguang; Kromann-Hansen, Tobias; Jensen, Knud J; Huang, Mingdong; Andreasen, Peter A

2015-09-25

We have developed a new concept for designing peptidic protein modulators, by recombinantly fusing the peptidic modulator, with randomized residues, directly to the target protein via a linker and screening for internal modulation of the activity of the protein. We tested the feasibility of the concept by fusing a 10-residue-long, disulfide-bond-constrained inhibitory peptide, randomized in selected positions, to the catalytic domain of the serine protease murine urokinase-type plasminogen activator. High-affinity inhibitory peptide variants were identified as those that conferred to the fusion protease the lowest activity for substrate hydrolysis. The usefulness of the strategy was demonstrated by the selection of peptidic inhibitors of murine urokinase-type plasminogen activator with a low nanomolar affinity. The high affinity could not have been predicted by rational considerations, as the high affinity was associated with a loss of polar interactions and an increased binding entropy. Copyright © 2015 Elsevier Ltd. All rights reserved.

Galectin-8 induces partial epithelial–mesenchymal transition with invasive tumorigenic capabilities involving a FAK/EGFR/proteasome pathway in Madin–Darby canine kidney cells

PubMed Central

Oyanadel, Claudia; Holmes, Christopher; Pardo, Evelyn; Retamal, Claudio; Shaughnessy, Ronan; Smith, Patricio; Cortés, Priscilla; Bravo-Zehnder, Marcela; Metz, Claudia; Feuerhake, Teo; Romero, Diego; Roa, Juan Carlos; Montecinos, Viviana; Soza, Andrea; González, Alfonso

2018-01-01

Epithelial cells can acquire invasive and tumorigenic capabilities through epithelial–mesenchymal-transition (EMT). The glycan-binding protein galectin-8 (Gal-8) activates selective β1-integrins involved in EMT and is overexpressed by certain carcinomas. Here we show that Gal-8 overexpression or exogenous addition promotes proliferation, migration, and invasion in nontumoral Madin–Darby canine kidney (MDCK) cells, involving focal-adhesion kinase (FAK)-mediated transactivation of the epidermal growth factor receptor (EGFR), likely triggered by α5β1integrin binding. Under subconfluent conditions, Gal-8–overexpressing MDCK cells (MDCK-Gal-8H) display hallmarks of EMT, including decreased E-cadherin and up-regulated expression of vimentin, fibronectin, and Snail, as well as increased β-catenin activity. Changes related to migration/invasion included higher expression of α5β1 integrin, extracellular matrix-degrading MMP13 and urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) protease systems. Gal-8–stimulated FAK/EGFR pathway leads to proteasome overactivity characteristic of cancer cells. Yet MDCK-Gal-8H cells still develop apical/basolateral polarity reverting EMT markers and proteasome activity under confluence. This is due to the opposite segregation of Gal-8 secretion (apical) and β1-integrins distribution (basolateral). Strikingly, MDCK-Gal-8H cells acquired tumorigenic potential, as reflected in anchorage-independent growth in soft agar and tumor generation in immunodeficient NSG mice. Therefore, Gal-8 can promote oncogenic-like transformation of epithelial cells through partial and reversible EMT, accompanied by higher proliferation, migration/invasion, and tumorigenic properties. PMID:29298841

A study of proteases and protease-inhibitor complexes in biological fluids

PubMed Central

Granelli-Piperno, A; Reich, E

1978-01-01

We have (a) screened a variety of cell lines and body fluids for plasminogen activators and (b) studied the activity of proteases bound to α2- macroglobulin after exposing the complexes to partial degradation and/or denaturing procedures to unmask proteolytic activity. The respective results show (a) that the plasminogen activators in urine and cell culture media are generally of lower molecular weight than those in plasma; and (b) that proteases bound to α2-macroglobulin recover the ability to attack macromolecular substrates after exposure to sodium dodecyl sulfate while retaining the electrophoretic mobility of the protease inhibitor complex. This indicates that the protease and inhibitor are probably linked by covalent bonds. In contrast, other complexes formed between proteases and inhibitors of lower molecular weight (such as soybean or Kunitz inhibitors) are fully dissociated by sodium dodecyl sulfate (SDS). The experiments described were based on a new procedure for detecting proteolytic enzyme activity in SDS-polyacrylamide gels. The method relies on solutions of nonionic detergents for extracting SDS, after which the electrophoretic gel is applied to an indicator gel consisting of a fibrin- agar mixture. The method is sensitive, permitting the detection of proteinases in less than 1 μl of fresh plasma, and it is effective for resolving small differences in molecular weight. The procedure can be quantitated and, with minor modifications appropriate to each particular system, it has been applied to a broad spectrum of serine enzymes and proenzymes, including some that function in the pathways of fibrinolysis, coagulation and kinin-generation. Other potential applications appear likely. PMID:78958

Effect of obesity on acute hemostatic responses to live-fire training drills.

PubMed

Smith, Denise L; Horn, Gavin P; Petruzzello, Steven J; Freund, Gregory G; Woods, Jeffrey A; Cook, Marc D; Goldstein, Eric; Fernhall, Bo

2014-12-01

The objective of this study was to evaluate the impact of obesity and firefighting activities on coagulation and fibrinolytic activity in relatively young, apparently healthy firefighters. Firefighters performed simulated firefighting activities for 18 minutes in a live-fire training structure. Blood samples were obtained at baseline, before firefighting, and within a few minutes of completing the activity. Nearly all markers of coagulation and fibrinolytic activity increased immediately after firefighting with an overall shift toward a procoagulatory profile. Obese firefighters exhibited lower levels of tissue plasminogen activator activity (0.98 vs 0.63 IU/ml) and higher levels of plasminogen activator inhibitor-1 activity (2.2 vs 4.5 ng/ml) at baseline compared with normal-weight firefighters, suggesting that fibrinolytic activity was lower in obese firefighters. There were few interactions between body mass index and firefighting activity, thus our findings suggest that obese firefighters did not exhibit a greater procoagulatory response to live firefighting compared with normal-weight firefighters. Acute live firefighting produced increases in both fibrinolytic and coagulatory responses; although obesity was associated with a reduced fibrinolytic profile at baseline, the changes produced by acute firefighting were similar in obese and nonobese firefighters. Copyright © 2014 Elsevier Inc. All rights reserved.

Inhibitory effect of amiloride on the urokinase plasminogen activators in prostatic cancer.

PubMed

Ray, P; Bhatti, R; Gadarowski, J; Bell, N; Nasruddin, S

1998-01-01

The diuretic drug amiloride (AMLD), which competitively inhibits the catalytic activity of urokinase plasminogen activators (UPA), was used to study its effects on the proteolytic enzymes implicated in the invasiveness and metastases in a prostatic tumor model carrying two different sublines of adenocarcinoma of the prostate. Our data showed that UPA activity was significantly higher, both in the cytosol and pellet of R3327-AT3, a fast-growing highly metastatic and androgen-insensitive tumor, as compared to the G3327-G subline, a slow-growing nonmetastatic tumor of the prostate. The UPA activity in AT3 tumor dropped when the rats were treated with AMLD for 3 weeks. The UPA activity in the sera and tumor effusions from rats with AT3 tumor was significantly higher as compared to those with G subline tumor. The number of pulmonary metastatic foci was the same in untreated rats as compared to those treated with AMLD. The lymph node inspection after 3 weeks revealed no secondary tumor in the AMLD-treated group. The role of UPA in the metastases of prostate cancer is discussed.

Plasmin-dependent proteolysis of Tissue Factor Pathway Inhibitor in a mouse model of endotoxemia

PubMed Central

Lupu, Cristina; Herlea, Oana; Tang, Haiwang; Lijnen, Roger H.; Lupu, Florea

2012-01-01

Summary Background Development of a procoagulant state in sepsis, due to aberrant expression of tissue factor (TF) and sharp decrease of its major inhibitor tissue factor pathway inhibitor (TFPI), could lead to microthrombotic organ failure. The mechanism for the decline of TFPI activity in the lung could involve plasmin-mediated cleavage of the inhibitor. Objective To investigate the effect of plasmin generation on lung-associated TFPI activity, in normal conditions and during infusion of endotoxin (LPS) in mice. Methods Plasmin generation and TFPI activity were assayed in the lungs of mice deficient of tissue-type plasminogen activator (t-PA) or plasminogen (Plg), at 2-hrs after LPS or saline injection. Results The sharp loss of lung-associated TFPI activity at 2-hrs post LPS paralleled the abrupt increase of plasmin generation. TFPI activity was significantly retained in both t-PA-/- and Plg-/- mice, which are unable to generate plasmin. Conclusion The increased plasmin generation during the early stages of sepsis could cleave/inactivate TFPI and thus lead to thrombotic complications. PMID:23106863

Urokinase plasminogen activator mRNA is induced by IL-1alpha and TNF-alpha in in vitro acantholysis.

PubMed

Feliciani, Claudio; Toto, Paola; Wang, Binghe; Sauder, Daniel N; Amerio, Pierluigi; Tulli, Antonio

2003-08-01

The role of urokinase type plasminogen activator (uPA) has been well documented in the pathogenesis of pemphigus vulgaris (PV). Activation of plasminogen into active serine protease plasmin initiates extracellular proteolysis leading to acantholysis but the mechanisms underlying this process are not clearly understood. We have previously shown that keratinocyte derived cytokines IL-1alpha and TNF-alpha are involved in PV-induced acantholysis. In the present study we sought to examine whether keratinocyte-derived IL-1alpha and TNF-alpha are correlated with uPA induction in keratinocytes during acantholysis. Normal human keratinocytes were incubated with diluted PV serum. mRNAs for IL-1alpha, TNF-alpha and uPA were examined with RT-PCR at various time points and acantholysis was measured. IL-1alpha, TNF-alpha and uPA mRNAs were all induced in keratinocytes following PV serum stimulation; IL-1alpha/TNF-alpha mRNAs' expression was earlier than the expression of uPA mRNA. To further examine the role of IL-1alpha, TNF-alpha and uPA in acantholysis, we performed antibody blocking studies. Anti-IL-1alpha, anti-TNF-alpha and anti-uPA antibodies suppressed acantholysis by 76%, 80% and 90%, respectively. In addition, anti-IL-1alpha and anti-TNF-alpha antibodies inhibited uPA mRNA induction, whereas anti-uPA antibodies did not alter IL-1alpha/TNF-alpha mRNAs' expression. Our results confirm the role of uPA in acantholysis and suggest an involvement of IL-1alpha/TNF-alpha in uPA induction.

Plasmin-dependent modulation of the blood–brain barrier: a major consideration during tPA-induced thrombolysis?

PubMed Central

Niego, Be'eri; Medcalf, Robert L

2014-01-01

Plasmin, the principal downstream product of tissue-type plasminogen activator (tPA), is known for its potent fibrin-degrading capacity but is also recognized for many non-fibrinolytic activities. Curiously, plasmin has not been conclusively linked to blood–brain barrier (BBB) disruption during recombinant tPA (rtPA)-induced thrombolysis in ischemic stroke. This is surprising given the substantial involvement of tPA in the modulation of BBB permeability and the co-existence of tPA and plasminogen in both blood and brain throughout the ischemic event. Here, we review the work that argues a role for plasmin together with endogenous tPA or rtPA in BBB alteration, presenting the overall controversy around the topic yet creating a rational case for an involvement of plasmin in this process. PMID:24896566

Plasminogen activator inhibitor type 1 gene is located at region q21. 3-q22 of chromosome 7 and genetically linked with cystic fibrosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Klinger, K.W.; Winqvist, R.; Riccio, A.

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 metmore » 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.« less

Plasminogen activator inhibitor-1 is an independent prognostic factor of ovarian cancer and IMD-4482, a novel plasminogen activator inhibitor-1 inhibitor, inhibits ovarian cancer peritoneal dissemination.

PubMed

Nakatsuka, Erika; Sawada, Kenjiro; Nakamura, Koji; Yoshimura, Akihito; Kinose, Yasuto; Kodama, Michiko; Hashimoto, Kae; Mabuchi, Seiji; Makino, Hiroshi; Morii, Eiichi; Yamaguchi, Yoichi; Yanase, Takeshi; Itai, Akiko; Morishige, Ken-Ichirou; Kimura, Tadashi

2017-10-27

In the present study, the therapeutic potential of targeting plasminogen activator inhibitor-1 (PAI-1) in ovarian cancer was tested. Tissues samples from 154 cases of ovarian carcinoma were immunostained with anti-PAI-1 antibody, and the prognostic value was analyzed. Among the samples, 67% (104/154) showed strong PAI-1 expression; this was significantly associated with poor prognosis (progression-free survival: 20 vs. 31 months, P = 0.0033). In particular, among patients with stage II-IV serous adenocarcinoma, PAI-1 expression was an independent prognostic factor. The effect of a novel PAI-1 inhibitor, IMD-4482, on ovarian cancer cell lines was assessed and its therapeutic potential was examined using a xenograft mouse model of ovarian cancer. IMD-4482 inhibited in vitro cell adhesion to vitronectin in PAI-1-positive ovarian cancer cells, followed by the inhibition of extracellular signal-regulated kinase and focal adhesion kinase phosphorylation through dissociation of the PAI-urokinase receptor complex from integrin αVβ3. IMD-4482 caused G0/G1 cell arrest and inhibited the proliferation of PAI-1-positive ovarian cancer cells. In the xenograft model, IMD-4482 significantly inhibited peritoneal dissemination with the reduction of PAI-1 expression and the inhibition of focal adhesion kinase phosphorylation. Collectively, the functional inhibition of PAI-1 significantly inhibited ovarian cancer progression, and targeting PAI-1 may be a potential therapeutic strategy in ovarian cancer.

Plasminogen activator inhibitor-1 is an independent prognostic factor of ovarian cancer and IMD-4482, a novel plasminogen activator inhibitor-1 inhibitor, inhibits ovarian cancer peritoneal dissemination

PubMed Central

Nakatsuka, Erika; Sawada, Kenjiro; Nakamura, Koji; Yoshimura, Akihito; Kinose, Yasuto; Kodama, Michiko; Hashimoto, Kae; Mabuchi, Seiji; Makino, Hiroshi; Morii, Eiichi; Yamaguchi, Yoichi; Yanase, Takeshi; Itai, Akiko; Morishige, Ken-ichirou; Kimura, Tadashi

2017-01-01

In the present study, the therapeutic potential of targeting plasminogen activator inhibitor-1 (PAI-1) in ovarian cancer was tested. Tissues samples from 154 cases of ovarian carcinoma were immunostained with anti-PAI-1 antibody, and the prognostic value was analyzed. Among the samples, 67% (104/154) showed strong PAI-1 expression; this was significantly associated with poor prognosis (progression-free survival: 20 vs. 31 months, P = 0.0033). In particular, among patients with stage II-IV serous adenocarcinoma, PAI-1 expression was an independent prognostic factor. The effect of a novel PAI-1 inhibitor, IMD-4482, on ovarian cancer cell lines was assessed and its therapeutic potential was examined using a xenograft mouse model of ovarian cancer. IMD-4482 inhibited in vitro cell adhesion to vitronectin in PAI-1-positive ovarian cancer cells, followed by the inhibition of extracellular signal-regulated kinase and focal adhesion kinase phosphorylation through dissociation of the PAI-urokinase receptor complex from integrin αVβ3. IMD-4482 caused G0/G1 cell arrest and inhibited the proliferation of PAI-1-positive ovarian cancer cells. In the xenograft model, IMD-4482 significantly inhibited peritoneal dissemination with the reduction of PAI-1 expression and the inhibition of focal adhesion kinase phosphorylation. Collectively, the functional inhibition of PAI-1 significantly inhibited ovarian cancer progression, and targeting PAI-1 may be a potential therapeutic strategy in ovarian cancer. PMID:29163796

Comparison between the clot-protecting activity of a mutant plasminogen activator inhibitor-1 with a very long half-life and 6-aminocaproic acid.

PubMed

Kindell, Daniel Glenn; Keck, Rick Wayne; Jankun, Jerzy

2015-06-01

Plasminogen activator inhibitor (PAI)-1 is a serpin glycoprotein that can stabilize blood clots by inhibiting fibrinolysis. However, wild-type PAI-1 has the disadvantage of a short half-life of ∼2 h. A very long half-life (VLHL) PAI-1 mutant was developed previously with an active-form half-life of >700 h, making it a possible candidate for use in hemorrhagic therapy. Current treatments for mitigating hemorrhage, other than inducers of blood clotting, are limited to lysine analog antifibrinolytics, including 6-aminocaproic acid and tranexamic acid. VLHL PAI-1 has been previously demonstrated to limit bleeding; however, the efficacy of this protein compared with lysine analog antifibrinolytics has not been investigated. The aim of the current study was to compare the clot stabilizing properties of the novel antifibrinolytic VLHL PAI-1 with those of 6-aminocaproic acid in reference plasma. Using thromboelastographic analysis, VLHL PAI-1 exhibited an IC 50 (half maximal inhibitory concentration) of 8.8×10 -8 mol/l, while 6-aminocaproic acid showed an IC 50 of 1.6×10 -4 mol/l. However, at doses of >9.0×10 -7 mol/l, VLHL PAI-1 exhibited a delay in the onset of clot formation, which may be attributed to thrombin inhibition by excess PAI-1. The inhibition of tissue plasminogen activator by VLHL PAI-1 demonstrated improved efficacy over 6-aminocaproic acid in mitigating hemorrhage. In addition, patients with a PAI-1 deficiency, which causes blood clots to lyse rapidly resulting in profuse bleeding, may benefit from the application of VLHL PAI-1 as an antihemorrhagic therapy.

Comparison between the clot-protecting activity of a mutant plasminogen activator inhibitor-1 with a very long half-life and 6-aminocaproic acid

PubMed Central

KINDELL, DANIEL GLENN; KECK, RICK WAYNE; JANKUN, JERZY

2015-01-01

Plasminogen activator inhibitor (PAI)-1 is a serpin glycoprotein that can stabilize blood clots by inhibiting fibrinolysis. However, wild-type PAI-1 has the disadvantage of a short half-life of ∼2 h. A very long half-life (VLHL) PAI-1 mutant was developed previously with an active-form half-life of >700 h, making it a possible candidate for use in hemorrhagic therapy. Current treatments for mitigating hemorrhage, other than inducers of blood clotting, are limited to lysine analog antifibrinolytics, including 6-aminocaproic acid and tranexamic acid. VLHL PAI-1 has been previously demonstrated to limit bleeding; however, the efficacy of this protein compared with lysine analog antifibrinolytics has not been investigated. The aim of the current study was to compare the clot stabilizing properties of the novel antifibrinolytic VLHL PAI-1 with those of 6-aminocaproic acid in reference plasma. Using thromboelastographic analysis, VLHL PAI-1 exhibited an IC50 (half maximal inhibitory concentration) of 8.8×10−8 mol/l, while 6-aminocaproic acid showed an IC50 of 1.6×10−4 mol/l. However, at doses of >9.0×10−7 mol/l, VLHL PAI-1 exhibited a delay in the onset of clot formation, which may be attributed to thrombin inhibition by excess PAI-1. The inhibition of tissue plasminogen activator by VLHL PAI-1 demonstrated improved efficacy over 6-aminocaproic acid in mitigating hemorrhage. In addition, patients with a PAI-1 deficiency, which causes blood clots to lyse rapidly resulting in profuse bleeding, may benefit from the application of VLHL PAI-1 as an antihemorrhagic therapy. PMID:26136983

Asphyxia by Drowning Induces Massive Bleeding Due To Hyperfibrinolytic Disseminated Intravascular Coagulation.

PubMed

Schwameis, Michael; Schober, Andreas; Schörgenhofer, Christian; Sperr, Wolfgang Reinhard; Schöchl, Herbert; Janata-Schwatczek, Karin; Kürkciyan, Erol Istepan; Sterz, Fritz; Jilma, Bernd

2015-11-01

To date, no study has systematically investigated the impact of drowning-induced asphyxia on hemostasis. Our objective was to test the hypothesis that asphyxia induces bleeding by hyperfibrinolytic disseminated intravascular coagulation. Observational study. A 2,100-bed tertiary care facility in Vienna, Austria, Europe. All cases of drowning-induced asphyxia (n=49) were compared with other patients with cardiopulmonary resuscitation (n=116) and to patients with acute promyelocytic leukemia (n=83). Six drowning victims were investigated prospectively. To study the mechanism, a forearm-ischemia model was used in 20 volunteers to investigate whether hypoxia releases tissue plasminogen activator. None. Eighty percent of patients with drowning-induced asphyxia developed overt disseminated intravascular coagulation within 24 hours. When compared with nondrowning cardiac arrest patients, drowning patients had a 13 times higher prevalence of overt disseminated intravascular coagulation at admission (55% vs 4%; p<0.001). Despite comparable disseminated intravascular coagulation scores, acute promyelocytic leukemia patients had higher fibrinogen but lower d-dimer levels and platelet counts than drowning patients (p<0.001). Drowning victims had a three-fold longer activated partial thromboplastin time (124 s; p<0.001) than both nondrowning cardiac arrest and acute promyelocytic leukemia patients. Hyperfibrinolysis was reflected by up to 1,000-fold increased d-dimer levels, greater than 5-fold elevated plasmin antiplasmin levels, and a complete absence of thrombelastometric clotting patterns, which was reversed by antifibrinolytics and heparinase. Thirty minutes of forearm-ischemia increased tissue plasminogen activator 31-fold (p<0.001). The vast majority of drowning patients develops overt hyperfibrinolytic disseminated intravascular coagulation, partly caused by hypoxia induced tissue plasminogen activator release. Antifibrinolytics and heparinase partially reverse the abnormal clotting patterns. Severe activated partial thromboplastin time prolongation may be a marker of combined hyperfibrinolytic afibrinogenemia and autoheparinization in drowning-related asphyxia.

A Small Molecule Inhibitor of Plasminogen Activator Inhibitor-1 Reduces Brain Amyloid-β Load and Improves Memory in an Animal Model of Alzheimer's Disease.

PubMed

Akhter, Hasina; Huang, Wen-Tan; van Groen, Thomas; Kuo, Hui-Chien; Miyata, Toshio; Liu, Rui-Ming

2018-01-01

Alzheimer's disease (AD) is a major cause of dementia in the elderly with no effective treatment. Accumulation of amyloid-β peptide (Aβ) in the brain is a pathological hallmark of AD and is believed to be a central disease-causing and disease-promoting event. In a previous study, we showed that deletion of plasminogen activator inhibitor 1 (PAI-1), a primary inhibitor of tissue type and urokinase type plasminogen activators (tPA and uPA), significantly reduced brain Aβ load in APP/PS1 mice, an animal model of familial AD. In this study, we further show that oral administration of TM5275, a small molecule inhibitor of PAI-1, for a period of 6 weeks, inhibits the activity of PAI-1 and increases the activities of tPA and uPA as well as plasmin, which is associated with a reduction of Aβ load in the hippocampus and cortex and improvement of learning/memory function in APP/PS1 mice. Protein abundance of low density lipoprotein related protein-1 (LRP-1), a multi ligand endocytotic receptor involved in transporting Aβ out of the brain, as well as plasma Aβ42 are increased, whereas the expression and processing of full-length amyloid-β protein precursor is not affected by TM5275 treatment in APP/PS1 mice. In vitro studies further show that PAI-1 increases, whereas TM5275 reduces, Aβ40 level in the culture medium of SHSY5Y-APP neuroblastoma cells. Collectively, our data suggest that TM5275 improves memory function of APP/PS1 mice, probably by reducing brain Aβ accumulation through increasing plasmin-mediated degradation and LRP-1-mediated efflux of Aβ in the brain.

Characterization of a Novel Class of Polyphenolic Inhibitors of Plasminogen Activator Inhibitor-1*

PubMed Central

Cale, Jacqueline M.; Li, Shih-Hon; Warnock, Mark; Su, Enming J.; North, Paul R.; Sanders, Karen L.; Puscau, Maria M.; Emal, Cory D.; Lawrence, Daniel A.

2010-01-01

Plasminogen activator inhibitor type 1, (PAI-1) the primary inhibitor of the tissue-type (tPA) and urokinase-type (uPA) plasminogen activators, has been implicated in a wide range of pathological processes, making it an attractive target for pharmacologic inhibition. Currently available small-molecule inhibitors of PAI-1 bind with relatively low affinity and do not inactivate PAI-1 in the presence of its cofactor, vitronectin. To search for novel PAI-1 inhibitors with improved potencies and new mechanisms of action, we screened a library selected to provide a range of biological activities and structural diversity. Five potential PAI-1 inhibitors were identified, and all were polyphenolic compounds including two related, naturally occurring plant polyphenols that were structurally similar to compounds previously shown to provide cardiovascular benefit in vivo. Unique second generation compounds were synthesized and characterized, and several showed IC50 values for PAI-1 between 10 and 200 nm. This represents an enhanced potency of 10–1000-fold over previously reported PAI-1 inactivators. Inhibition of PAI-1 by these compounds was reversible, and their primary mechanism of action was to block the initial association of PAI-1 with a protease. Consistent with this mechanism and in contrast to previously described PAI-1 inactivators, these compounds inactivate PAI-1 in the presence of vitronectin. Two of the compounds showed efficacy in ex vivo plasma and one blocked PAI-1 activity in vivo in mice. These data describe a novel family of high affinity PAI-1-inactivating compounds with improved characteristics and in vivo efficacy, and suggest that the known cardiovascular benefits of dietary polyphenols may derive in part from their inactivation of PAI-1. PMID:20061381

Plasminogen activator inhibitor 1 is elevated in the children of men with premature myocardial infarction.

PubMed

Rallidis, L S; Megalou, A A; Papageorgakis, N H; Trikas, A G; Chatzidimitriou, G I; Tsitouris, G K

1996-09-01

To assess whether plasminogen activator inhibitor 1 (PAI-1) activity is elevated in the progeny of young coronary men, 193 young subjects were recruited and divided into two groups. Group A consisted of 104 children whose fathers had suffered a myocardial infarction before the age of 55 ("cases"). Eighty-nine young subjects matched for age, sex, body mass index (BMI) and smoking habits without familial history of coronary artery disease (CAD) served as controls (group B). Children with a family history of diabetes mellitus or hypertension were excluded from both groups. We measured PAI-1 activity, tissue-type plasminogen activator (t-PA) antigen, a2-antiplasmin, fibrinogen, lipids and apolipoproteins in both groups. PAI-1 activity levels were also determined in the men who suffered a premature myocardial infarction 4 months after their discharge. PAI-1 activity levels were higher in cases compared to controls (3.13 +/- 1.9 vs 2.17 +/- 1.9 U/ml, p = 0.0014). t-PA antigen and a2-antiplasmin did not differ significantly between the two groups, while fibrinogen, total cholesterol, low-density lipoprotein cholesterol, apolipoprotein B and lipoprotein(a) were significantly higher in group A. PAI-1 was positively correlated with triglycerides (r = 0.22, p = 0.024), apolipoprotein B (r = 0.21, p = 0.039) and fibrinogen (r = 0.22, p = 0.029) in cases and with BMI in both cases (r = 0.37, p = 0.0003) and controls (r = 0.23, p = 0.044). In stepwise multiple regression analysis, only apolipoprotein B (p = 0.008) and BMI (p = 0.0014) were significant determinants of PAI-1 activity in cases. There was also a positive correlation between PAI-1 activity levels of the affected fathers and their children (r = 0.30, p = 0.01). The present data support the hypothesis that elevated PAI-1 levels in the offspring of men with premature myocardial infarction impair their fibrinolytic capacity contributing to their familial predisposition to CAD.

Prognostic value of tissue-type plasminogen activator (tPA) and its complex with the type-1 inhibitor (PAI-1) in breast cancer

PubMed Central

Witte, J H de; Sweep, C G J; Klijn, J G M; Grebenschikov, N; Peters, H A; Look, M P; Tienoven, ThH van; Heuvel, J J T M; Vries, J Bolt-De; Benraad, ThJ; Foekens, J A

1999-01-01

The prognostic value of tissue-type plasminogen activator (tPA) measured in samples derived from 865 patients with primary breast cancer using a recently developed enzyme-linked immunosorbent assay (ELISA) was evaluated. Since the assay could easily be adapted to the assessment of the complex of tPA with its type-1 inhibitor (PAI-1), it was investigated whether the tPA:PAI-1 complex also provides prognostic information. To this end, cytosolic extracts and corresponding detergent extracts of 100 000 g pellets obtained after ultracentrifugation when preparing the cytosolic fractions for routine steroid hormone receptor determination were assayed. Statistically significant correlations were found between the cytosolic levels and those determined in the pellet extracts (Spearman correlation coefficient rs = 0.75, P < 0.001 for tPA and r = 0.50, P < 0.001 for tPA:PAI-1 complex). In both Cox univariate and multivariate analysis elevated levels of (total) tPA determined in the pellet extracts, but not in cytosols, were associated with prolonged relapse-free (RFS) and overall survival (OS). In contrast, high levels of the tPA:PAI-1 complex measured in cytosols, but not in the pellet extracts, were associated with a poor RFS and OS. The prognostic information provided by the cytosolic tPA:PAI-1 complex was comparable to that provided by cytosolic (total) PAI-1. Furthermore, the estimated levels of free, uncomplexed tPA and PAI-1, in cytosols and in pellet extracts, were related to patient prognosis in a similar way as the (total) levels of tPA and PAI-1 respectively. Determination of specific forms of components of the plasminogen activation system, i.e. tPA:PAI-1 complex and free, uncomplexed tPA and/or PAI-1, may be considered a useful adjunct to the analyses of the separate components (tPA and/or PAI-1) and provide valuable additional prognostic information with respect to survival of breast cancer patients. © 1999 Cancer Research Campaign PMID:10390010

Enhancement of the thrombolytic efficacy of prourokinase by lys-plasminogen in a dog model of arterial thrombosis.

PubMed

Badylak, S F; Voytik, S L; Henkin, J; Burke, S E; Sasahara, A A; Simmons, A

1991-05-01

Current findings suggest that the efficacy of thrombolytic therapy may be limited by the availability of active forms of plasminogen at the thrombus site. The purpose of this study was to determine if the systemic administration of 0.5 mg kg-1 glu-plasminogen (glu-plg) or 0.5 mg kg-1 lys-plasminogen (lys-plg) could safely increase the efficacy of a single intravenous bolus injection of 50,000 U kg-1 prourokinase (proUK) in a dog model of arterial thrombosis. Thrombolysis was measured by monitoring the continuous decrement of 125I-gamma emissions from a radiolabeled thrombus. Reflow was evaluated by direct visual examination. Forty dogs (mean wt 10.3 +/- 2 kg) were randomly sorted into 4 groups of 10 each. The dogs in each group were given either saline plus saline, saline plus proUK, glu-plg plus proUK, or lys-plg plus proUK 60 minutes after formation of an occlusive arterial thrombus. Ninety minutes after drug administration the dogs receiving saline plus proUK, glu-plg plus proUK, and the lys-plg plus proUK showed greater thrombolysis (41%, 43%, and 66%, respectively) than the control (saline plus saline) group (15%, P less than 0.01). The lys-plg plus proUK treatment caused greater lysis than the saline plus proUK or the glu-plg plus proUK treatment (P less than 0.05). All of the dogs (10/10) receiving lys-plg plus proUK had patent vessels at the end of the 90 minute monitoring period, whereas only 4/10 and 5/10 vessels were patent in the saline plus proUK and glu-plg plus proUK groups, respectively. None of the dogs in the saline plus saline group had patent vessels. No significant changes were observed in the various coagulation parameters tested for any of the 4 treatment groups. The results show that lys-plg can safely increase the thrombolytic efficacy of proUK.

Systemic hyperfibrinolysis after trauma: a pilot study of targeted proteomic analysis of superposed mechanisms in patient plasma.

PubMed

Banerjee, Anirban; Silliman, Christopher C; Moore, Ernest E; Dzieciatkowska, Monika; Kelher, Marguerite; Sauaia, Angela; Jones, Kenneth; Chapman, Michael P; Gonzalez, Eduardo; Moore, Hunter B; D'Alessandro, Angelo; Peltz, Erik; Huebner, Benjamin E; Einerson, Peter; Chandler, James; Ghasabayan, Arsen; Hansen, Kirk

2018-06-01

Viscoelastic measurements of hemostasis indicate that 20% of seriously injured patients exhibit systemic hyperfibrinolysis, with increased early mortality. These patients have normal clot formation with rapid clot lysis. Targeted proteomics was applied to quantify plasma proteins from hyperfibrinolytic (HF) patients to elucidate potential pathophysiology. Blood samples were collected in the field or at emergency department arrival and thrombelastography (TEG) was used to characterize in vitro clot formation under native and tissue plasminogen activator (tPA)-stimulated conditions. Ten samples were taken from injured patients exhibiting normal lysis time at 30 min (Ly30), "eufibrinolytic" (EF), 10 from HF patients, defined as tPA-stimulated TEG Ly30 >50%, and 10 from healthy controls. Trauma patient samples were analyzed by targeted proteomics and ELISA assays for specific coagulation proteins. HF patients exhibited increased plasminogen activation. Thirty-three proteins from the HF patients were significantly decreased compared with healthy controls and EF patients; 17 were coagulation proteins with anti-protease consumption (p < 0.005). The other 16 decreased proteins indicate activation of the alternate complement pathway, depletion of carrier proteins, and four glycoproteins. CXC7 was elevated in all injured patients versus healthy controls (p < 0.005), and 35 proteins were unchanged across all groups (p > 0.1 and fold change of concentrations of 0.75-1.3). HF patients had significant decreases in specific proteins and support mechanisms known in trauma-induced hyperfibrinolysis and also unexpected decreases in coagulation factors, factors II, X, and XIII, without changes in clot formation (SP, R times, or angle). Decreased clot stability in HF patients was corroborated with tPA-stimulated TEGs. Prognostic, level III.

Genetics Home Reference: complete plasminogen activator inhibitor 1 deficiency

MedlinePlus

... Affected females may have excessive bleeding associated with menstruation (menorrhagia) and abnormal bleeding in pregnancy and childbirth. ... frequently in females because of its effects on menstruation, pregnancy, and childbirth. Related Information What information about ...

Fibrin Accumulation Secondary to Loss of Plasmin-Mediated Fibrinolysis Drives Inflammatory Osteoporosis in Mice

PubMed Central

Cole, Heather A.; Ohba, Tetsuro; Nyman, Jeffry S.; Hirotaka, Haro; Cates, Justin M. M.; Flick, Matthew J.; Degen, Jay L.; Schoenecker, Jonathan G.

2015-01-01

Objective Osteoporosis is a skeletal disorder characterized by low bone mass and increased bone fragility associated with aging, menopause, smoking, obesity, or diabetes. Persistent inflammation has been identified as an instigating factor in progressive bone loss. In addition to the role of fibrin in coagulation, inordinate fibrin deposition within a tissue matrix results in increased local inflammation. Given that fibrin accumulation is a hallmark of osteoporosis-related co-morbidities, we undertook this study to test the hypothesis that persistent fibrin deposition causes inflammatory osteoporosis. Methods Multiple imaging modalities, bone integrity metrics, and histologic analyses were employed to evaluate skeletal derangements in relation to fibrin deposition, circulating fibrinogen levels, and systemic markers of inflammation in mice that were plasminogen deficient and in plasminogen-deficient mice that were concomitantly either fibrinogen deficient or carrying a mutant form of fibrinogen lacking the αMβ2 binding motif. Results Mice generated with a genetic deficit in the key fibrinolytic protease, plasmin, uniformly developed severe osteoporosis. Furthermore, the development of osteoporosis was fibrin(ogen) dependent, and the derangements in the bone remodeling unit were mechanistically tied to fibrin(ogen)-mediated activation of osteoclasts via activation of the leukocyte integrin receptor αMβ2 on monocytes and secondary stimulation of osteoblasts by RANKL. Notably, the genetic elimination of fibrin(ogen) or the expression of a mutant form of fibrinogen retaining clotting function but lacking the αMβ2 binding motif prevented the degenerative skeletal phenotypes, resulting in normal local and systemic cytokine levels. Conclusion Taken together, these data reveal for the first time that fibrin promotes inflammation-driven systemic osteoporosis, which suggests a novel association between hemostasis, inflammation, and bone biology. PMID:24664548

Fibrin accumulation secondary to loss of plasmin-mediated fibrinolysis drives inflammatory osteoporosis in mice.

PubMed

Cole, Heather A; Ohba, Tetsuro; Nyman, Jeffry S; Hirotaka, Haro; Cates, Justin M M; Flick, Matthew J; Degen, Jay L; Schoenecker, Jonathan G

2014-08-01

Osteoporosis is a skeletal disorder characterized by low bone mass and increased bone fragility associated with aging, menopause, smoking, obesity, or diabetes. Persistent inflammation has been identified as an instigating factor in progressive bone loss. In addition to the role of fibrin in coagulation, inordinate fibrin deposition within a tissue matrix results in increased local inflammation. Given that fibrin accumulation is a hallmark of osteoporosis-related comorbidities, we undertook this study to test the hypothesis that persistent fibrin deposition causes inflammatory osteoporosis. Multiple imaging modalities, bone integrity metrics, and histologic analyses were employed to evaluate skeletal derangements in relation to fibrin deposition, circulating fibrinogen levels, and systemic markers of inflammation in mice that were plasminogen deficient and in plasminogen-deficient mice that were concomitantly either fibrinogen deficient or carrying a mutant form of fibrinogen lacking the αM β2 binding motif. Mice generated with a genetic deficit in the key fibrinolytic protease, plasmin, uniformly developed severe osteoporosis. Furthermore, the development of osteoporosis was fibrin(ogen) dependent, and the derangements in the bone remodeling unit were mechanistically tied to fibrin(ogen)-mediated activation of osteoclasts via activation of the leukocyte integrin receptor αM β2 on monocytes and secondary stimulation of osteoblasts by RANKL. Notably, the genetic elimination of fibrin(ogen) or the expression of a mutant form of fibrinogen retaining clotting function but lacking the αM β2 binding motif prevented the degenerative skeletal phenotypes, resulting in normal local and systemic cytokine levels. Taken together, these data reveal for the first time that fibrin promotes inflammation-driven systemic osteoporosis, which suggests a novel association between hemostasis, inflammation, and bone biology. Copyright © 2014 by the American College of Rheumatology.

Effects of gestational hypertension and pre-eclampsia in mRNA expression of fibrinolysis genes in primary cultured human umbilical vein endothelial cells.

PubMed

Poblete-Naredo, Irais; Rodríguez-Yáñez, Yury; Corona-Núñez, Rogelio O; González-Monroy, Stuart; Salinas, Juan E; Albores, Arnulfo

2018-05-17

Hypertension disorders (HD) and pre-eclampsia (PRE) are leading causes of maternal deaths worldwide. PRE is associated with vascular endothelial dysfunction and with deregulation of the fibrinolysis pathway genes. Fibrinolysis is the fibrin clot hydrolysis process catalyzed by plasmin, a proteolytic enzyme formed from plasminogen. Plasminogen is cleaved by tissue-type (tPA) and urokinase-type (uPA) activators and inhibited by the plasminogen activator inhibitors type-1 (PAI-1) and type-2 (PAI-2). The whole process maintains blood hemostasis. This study aims to assess PAI-1, PAI-2, tPA and uPA mRNA expression in primary cultured human umbilical vein endothelial cells (HUVEC) isolated and cultured from healthy, HD and PRE women. Results show that PAI-1 and PAI-2 mRNA decreased in HD-HUVEC, whereas PAI-1 and uPA decreased in PRE-HUVEC cultures compared to control ones. Notably, the expression ratio between pro- and anti-fibrinolytic actors remained unchanged among the studied groups. It seems that newborn's hemostasis is maintained balanced probably by a compensatory mechanism that involves changes in the fibrinolysis gene expression profile. The real impact of these changes in mRNA expression is unknown, however, it is suggested that these changes could be associated with an increased predisposition to vascular disease development in the progeny. Copyright © 2018. Published by Elsevier Ltd.

Defibrotide blunts the prothrombotic effect of thalidomide on endothelial cells.

PubMed

Echart, C L; Somaini, S; Distaso, M; Palumbo, A; Richardson, P G; Fareed, J; Iacobelli, M

2012-01-01

Patients with multiple myeloma (MM) are at relatively high risk of developing thromboembolic events such deep venous thrombosis (DVT) where thalidomide therapy has been identified to increase this risk. Defibrotide (DF), a polydisperse oligonucleotide, showed previously to counteract the alterations in endothelial cells (ECs) induced by lipopolysaccharide. It prompts us to investigate the impact of thalidomide on ECs and whether DF modulates changes in fibrinolysis induced by thalidomide. In this in vitro study, MM by itself alters the profibrinolytic potential of ECs decreasing the tissue plasminogen activator (t-PA) and increasing the plasminogen activator inhibitor 1 (PAI-1) levels which is potentiated by thalidomide. Defibrotide was able to counteract these effects. Additionally, DF upregulated the t-PA and downregulated PAI-1 gene expression modulated by thalidomide. Defibrotide also protects ECs from thalidomide-mediated cell death without interfering with its antitumor effects. These findings support DF clinical use for the prevention of DVT induced by immunomodulatory drugs.

Plasminogen activator: analysis of enzyme induction by ultraviolet irradiation mapping

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miskin, R.; Reich, E.; Dixon, K.

1981-10-01

Ultraviolet irradiation mapping techniques have previously been used to study the organization of eucaryotic gene classes and transcription units. We used the same method to probe some regulatory phenomena observed in the induction of plasminogen activator (PA) biosynthesis: PA synthesis in chicken embryo fibroblasts is induced by tumor-promoting phorbol esters and by retinoic acid; furthermore, PA induction by phorbol esters is synergistic with transformation, being 10- to 20-fold greater in virus-transformed cells than in normal cells. We found that the ultraviolet irradiation inactivation cross sections for PA induction by phorbol esters and by retinoate differed significantly, suggesting that these agentsmore » induce PA biosynthesis by different mechanisms. On the other hand, the ultraviolet irradiation sensitivity of phorbol ester induction in normal chicken embryo fibroblasts was the same as in transformed cells, indicating that the synergism of transformation and phorbol esters is probably not due to different pathways of PA induction.« less

Clinical utility of level-of-evidence-1 disease forecast cancer biomarkers uPA and its inhibitor PAI-1.

PubMed

Schmitt, Manfred; Mengele, Karin; Napieralski, Rudolf; Magdolen, Viktor; Reuning, Ute; Gkazepis, Apostolos; Sweep, Fred; Brünner, Nils; Foekens, John; Harbeck, Nadia

2010-11-01

The prognostic and/or predictive value of the cancer biomarkers, urokinase-type plasminogen activator (uPA) and its inhibitor (plasminogen activator inhibitor [PAI]-1), determined by ELISA in tumor-tissue extracts, was demonstrated for several cancer types in numerous clinically relevant retrospective or prospective studies, including a multicenter breast cancer therapy trial (Chemo-N0). Consequently, for the first time ever for any cancer biomarker for breast cancer, uPA and PAI-1 have reached the highest level of evidence, level-of-evidence-1. At present, two other breast cancer therapy trials, NNBC-3 and Plan B, also incorporating uPA and PAI-1 as treatment-assignment tools are in effect. Furthermore, small synthetic molecules targeting uPA are currently in Phase II clinical trials in patients afflicted with advanced cancer of the ovary, breast or pancreas.

Differentiating the Influences of Aging and Adiposity on Brain Weights, Levels of Serum and Brain Cytokines, Gastrointestinal Hormones, and Amyloid Precursor Protein.

PubMed

Banks, William A; Abrass, Christine K; Hansen, Kim M

2016-01-01

Aging and obesity exert important effects on disease. Differentiating these effects is difficult, however, because weight gain often accompanies aging. Here, we used a nested design of aged, calorically restricted, and refed rats to measure changes in brain and blood levels of cytokines and gastrointestinal hormones, brain amyloid precursor protein levels, and brain and body weights. By comparing groups and using path analysis, we found divergent influences of chronological aging versus body weight, our main findings being (i) changes in whole brain weight and serum macrophage colony-stimulating factor levels correlated better with body weight than with chronological aging, (ii) a decrease in brain cytokines and brain plasminogen activator inhibitor levels correlated better with chronological aging than with body weight, (iii) serum erythropoietin levels were influenced by both body weight and aging, (iv) serum plasminogen activator inhibitor, serum cytokines, and brain tumor necrosis factor were not influenced by aging or body weight, and (v) brain amyloid precursor protein more closely related to body weight and serum levels of gastrointestinal hormones than to brain weight, chronological aging, or cytokines. These findings show that although aging and body weight interact, their influences are distinct not only among various cytokines and hormones but also between the central nervous system and the peripheral tissue compartments. Published by Oxford University Press on behalf of the Gerontological Society of America 2014.

Utilization of intravenous tissue plasminogen activator for ischemic stroke: are there sex differences?

PubMed

Allen, Norrina B; Myers, Daniela; Watanabe, Emi; Dostal, Jackie; Sama, Danny; Goldstein, Larry B; Lichtman, Judith H

2009-01-01

We evaluated whether there were sex-related differences in the administration of intravenous tissue plasminogen activator (IV-tPA) to patients with acute ischemic stroke admitted to US academic medical centers. Medical records were abstracted for consecutive ischemic stroke patients admitted to 32 academic medical centers from January through June, 2004, as part of the University HealthSystem Consortium Ischemic Stroke Benchmarking Project. Multivariate logistic models were used to test for sex-related differences in the receipt of IV-tPA with adjustment for demographic and clinical factors. The study included 1,234 patients (49% women; mean age 66.6 years; 56% white). IV-tPA was given to 7% (6.5% of women versus 7.5% of men, p = 0.49). Women and men were equally likely to receive IV-tPA in risk-adjusted analyses (OR 1.02, 95% CI 0.64-1.64). Approximately 77% of women and men who did not receive IV-tPA did not meet the 3-hour treatment window or their time of onset was unknown. Women admitted to academic hospitals receive IV-tPA as often as men; however, a substantial percentage of both women and men are not arriving within the 3-hour time window required for diagnostic assessment and administration of intravenous thrombolytic therapy. Additional efforts are needed to improve the rapid identification, evaluation and treatment of stroke patients.

Receptor-Targeted Nanoparticles for In Vivo Imaging of Breast Cancer

PubMed Central

Yang, Lily; Peng, Xiang-Hong; Wang, Y. Andrew; Wang, Xiaoxia; Cao, Zehong; Ni, Chunchun; Karna, Prasanthi; Zhang, Xinjian; Wood, William C.; Gao, Xiaohu; Nie, Shuming; Mao, Hui

2009-01-01

Purpose Cell surface receptor-targeted magnetic iron oxide (IO) nanoparticles provide molecular magnetic resonance imaging (MRI) contrast agents for improving specificity of the detection of human cancer. Experimental design The present study reports the development of a novel targeted IO nanoparticle using a recombinant peptide containing the amino-terminal fragment (ATF) of urokinase plasminogen activator conjugated to IO nanoparticles (ATF-IO). This nanoparticle targets urokinase plasminogen activator receptor (uPAR), which is overexpressed in breast cancer tissues. Results ATF-IO nanoparticles are able to specifically bind to and be internalized by uPAR-expressing tumor cells. Systemic delivery of ATF-IO nanoparticles into mice bearing subcutaneous and intraperitoneal mammary tumors leads to the accumulation of the particles in tumors, generating a strong MRI contrast detectable by a clinical MRI scanner at a field strength of 3 Tesla. Target specificity of ATF-IO nanoparticles demonstrated by in vivo MRI is further confirmed by near infrared (NIR) fluorescence imaging of the mammary tumors using NIR dye-labeled ATF peptides conjugated to IO nanoparticles. Furthermore, mice administered ATF-IO nanoparticles exhibit lower uptake of the particles in the liver and spleen compared to those receiving non-targeted IO nanoparticles. Conclusions Our results suggest that uPAR-targeted ATF-IO nanoparticles have potential as molecularly-targeted, dual modality imaging agents for in vivo imaging of breast cancer. PMID:19584158

Association Between Arsenic Exposure From Drinking Water and Plasma Levels of Cardiovascular Markers

PubMed Central

Wu, Fen; Jasmine, Farzana; Kibriya, Muhammad G.; Liu, Mengling; Wójcik, Oktawia; Parvez, Faruque; Rahaman, Ronald; Roy, Shantanu; Paul-Brutus, Rachelle; Segers, Stephanie; Slavkovich, Vesna; Islam, Tariqul; Levy, Diane; Mey, Jacob L.; van Geen, Alexander; Graziano, Joseph H.; Ahsan, Habibul; Chen, Yu

2012-01-01

The authors conducted a cross-sectional study to assess the relation between arsenic exposure from drinking water and plasma levels of markers of systemic inflammation and endothelial dysfunction (matrix metalloproteinase-9, myeloperoxidase, plasminogen activator inhibitor-1, soluble E-selectin, soluble intercellular adhesion molecule-1 (ICAM-1), and soluble vascular adhesion molecule-1 (VCAM-1)) using baseline data from 668 participants (age, >30 years) in the Health Effects of Arsenic Longitudinal Study in Bangladesh (2007–2008). Both well water arsenic and urinary arsenic were positively associated with plasma levels of soluble VCAM-1. For every 1-unit increase in log-transformed well water arsenic (ln μg/L) and urinary arsenic (ln μg/g creatinine), plasma soluble VCAM-1 was 1.02 (95% confidence interval: 1.01, 1.03) and 1.04 (95% confidence interval: 1.01, 1.07) times greater, respectively. There was a significant interaction between arsenic exposure and higher body mass index, such that the increased levels of plasminogen activator inhibitor-1 and soluble VCAM-1 associated with arsenic exposure were stronger among people with higher body mass index. The findings indicate an effect of chronic arsenic exposure from drinking water on vascular inflammation and endothelial dysfunction that could be modified by body mass index and also suggest a potential mechanism underlying the association between arsenic exposure and cardiovascular disease. PMID:22534204

Association between arsenic exposure from drinking water and plasma levels of cardiovascular markers.

PubMed

Wu, Fen; Jasmine, Farzana; Kibriya, Muhammad G; Liu, Mengling; Wójcik, Oktawia; Parvez, Faruque; Rahaman, Ronald; Roy, Shantanu; Paul-Brutus, Rachelle; Segers, Stephanie; Slavkovich, Vesna; Islam, Tariqul; Levy, Diane; Mey, Jacob L; van Geen, Alexander; Graziano, Joseph H; Ahsan, Habibul; Chen, Yu

2012-06-15

The authors conducted a cross-sectional study to assess the relation between arsenic exposure from drinking water and plasma levels of markers of systemic inflammation and endothelial dysfunction (matrix metalloproteinase-9, myeloperoxidase, plasminogen activator inhibitor-1, soluble E-selectin, soluble intercellular adhesion molecule-1 (ICAM-1), and soluble vascular adhesion molecule-1 (VCAM-1)) using baseline data from 668 participants (age, >30 years) in the Health Effects of Arsenic Longitudinal Study in Bangladesh (2007-2008). Both well water arsenic and urinary arsenic were positively associated with plasma levels of soluble VCAM-1. For every 1-unit increase in log-transformed well water arsenic (ln μg/L) and urinary arsenic (ln μg/g creatinine), plasma soluble VCAM-1 was 1.02 (95% confidence interval: 1.01, 1.03) and 1.04 (95% confidence interval: 1.01, 1.07) times greater, respectively. There was a significant interaction between arsenic exposure and higher body mass index, such that the increased levels of plasminogen activator inhibitor-1 and soluble VCAM-1 associated with arsenic exposure were stronger among people with higher body mass index. The findings indicate an effect of chronic arsenic exposure from drinking water on vascular inflammation and endothelial dysfunction that could be modified by body mass index and also suggest a potential mechanism underlying the association between arsenic exposure and cardiovascular disease.

Quality of milk and of Canestrato Pugliese cheese from ewes exposed to different ventilation regimens.

PubMed

Albenzio, Marzia; Marino, Rosaria; Caroprese, Mariangela; Santillo, Antonella; Annicchiarico, Giovanni; Sevi, Agostino

2004-11-01

Effects of ventilation regimen on the quality of ewes' milk and on proteolysis in Canestrato Pugliese cheese during ripening were studied. Cheeses were manufactured from the bulk milk of Comisana ewes subjected to three different ventilation regimens, which were designated low (LOV, 23 m3/h per ewe), moderate (MOV, 47 m3/h per ewe) and programmed ventilation regimen (PROV, 73 m3/h per ewe; fan set to maintain 70% relative humidity). Bulk milk was analysed for chemical and microbial composition, renneting parameters and plasmin-plasminogen activities. At 1, 15, 30 and 45 d of ripening, the cheeses were analysed for gross chemical composition, nitrogen fractions, and plasmin and plasminogen activities. The pH 4.6-insoluble nitrogen fractions were analysed by urea-PAGE. Free amino acid content was determined at the end of ripening. Lower concentrations of bulk milk somatic cell count (BMSCC) and of mesophilic bacteria were found in the MOV group than in the LOV and the PROV groups. A lower plasminogen (PG) to plasmin (PL) ratio (PG/PL) was observed in the MOV and PROV than in the LOV cheeses. Irrespective of treatment, PL activity in cheeses was higher at 15d of ripening, while a sudden decrease of PL and PG activities was observed at 30 d, which was associated with a marked increase in non-protein nitrogen. The peptide profile characterized in the urea-PAGE showed a greater intensity of alpha- and beta-CN hydrolysis in the MOV than in the PROV and LOV cheeses. The results provide evidence that a proper ventilation regimen is critical for optimizing the hygienic quality of milk and the proteolysis of Canestrato Pugliese cheese during ripening.

Identification and functional characterization of alpha-enolase from Taenia pisiformis metacestode.

PubMed

Zhang, Shaohua; Guo, Aijiang; Zhu, Xueliang; You, Yanan; Hou, Junling; Wang, Qiuxia; Luo, Xuenong; Cai, Xuepeng

2015-04-01

Enolase belongs to glycolytic enzymes with moonlighting functions. The role of enolase in Taenia species is still poorly understood. In this study, the full length of cDNA encoding for Taenia pisiformis alpha-enolase (Tpeno) was cloned from larval parasites and soluble recombinant Tpeno protein (rTpeno) was produced. Western blot indicated that both rTpeno and the native protein in excretion-secretion antigens from the larvae were recognized by anti-rTpeno monoclonal antibodies (MAbs). The primary structure of Tpeno showed the presence of a highly conserved catalytic site for substrate binding and an enolase signature motif. rTpeno enzymatic activities of catalyzing the reversible dehydration of 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate (PEP) and vice versa were shown to be 30.71 ± 2.15 U/mg (2-PGA to PEP) and 11.29 ± 2.38 U/mg (PEP to 2-PGA), respectively. Far-Western blotting showed that rTpeno could bind to plasminogen, however its binding ability was inhibited by ϵ-aminocaproic acid (ϵACA) in a competitive ELISA test. Plasminogen activation assay showed that plasminogen bound to rTpeno could be converted into active plasmin using host-derived activators. Immunohistochemistry and immunofluorescence indicated that Tpeno was distributed in the bladder wall of the metacestode and the periphery of calcareous corpuscles. In addition, a vaccine trial showed that the enzyme could produce a 36.4% protection rate in vaccinated rabbits against experimental challenges from T. pisiformis eggs. These results suggest that Tpeno with multiple functions may play significant roles in the migration, growth, development and adaptation of T. pisiformis for survival in the host environment. Copyright © 2015 Elsevier B.V. All rights reserved.

Podocyte injury: the role of proteinuria, urinary plasminogen, and oxidative stress

PubMed Central

Tian, Runxia; Wong, Jenny S.; He, John C.; Campbell, Kirk N.

2016-01-01

Podocytes are the key target for injury in proteinuric glomerular diseases that result in podocyte loss, progressive focal segmental glomerular sclerosis (FSGS), and renal failure. Current evidence suggests that the initiation of podocyte injury and associated proteinuria can be separated from factors that drive and maintain these pathogenic processes leading to FSGS. In nephrotic urine aberrant glomerular filtration of plasminogen (Plg) is activated to the biologically active serine protease plasmin by urokinase-type plasminogen activator (uPA). In vivo inhibition of uPA mitigates Plg activation and development of FSGS in several proteinuric models of renal disease including 5/6 nephrectomy. Here, we show that Plg is markedly increased in the urine in two murine models of proteinuric kidney disease associated with podocyte injury: Tg26 HIV-associated nephropathy and the Cd2ap−/− model of FSGS. We show that human podocytes express uPA and three Plg receptors: uPAR, tPA, and Plg-RKT. We demonstrate that Plg treatment of podocytes specifically upregulates NADPH oxidase isoforms NOX2/NOX4 and increases production of mitochondrial-dependent superoxide anion (O2−) that promotes endothelin-1 synthesis. Plg via O2− also promotes expression of the B scavenger receptor CD36 and subsequent increased intracellular cholesterol uptake resulting in podocyte apoptosis. Taken together, our findings suggest that following disruption of the glomerular filtration barrier at the onset of proteinuric disease, podocytes are exposed to Plg resulting in further injury mediated by oxidative stress. We suggest that chronic exposure to Plg could serve as a “second hit” in glomerular disease and that Plg is potentially an attractive target for therapeutic intervention. PMID:27335373

Serine proteases, inhibitors and receptors in renal fibrosis

PubMed Central

Eddy, Allison A.

2011-01-01

Summary Chronic kidney disease (CKD) is estimated to affect one in eight adults. Their kidney function progressively deteriorates as inflammatory and fibrotic processes damage nephrons. New therapies to prevent renal functional decline must build on basic research studies that identify critical cellular and molecular mediators. Plasminogen activator inhibitor-1 (PAI-1), a potent fibrosis-promoting glycoprotein, is one promising candidate. Absent from normal kidneys, PAI-1 is frequently expressed in injured kidneys. Studies in genetically engineered mice have demonstrated its potency as a pro-fibrotic molecule. Somewhat surprising, its ability to inhibit serine protease activity does not appear to be its primary pro-fibrotic effect in CKD. Both tissue-type plasminogen activator and plasminogen deficiency significantly reduced renal fibrosis severity after ureteral obstruction, while genetic urokinase (uPA) deficiency had no effect. PAI-1 expression is associated with enhanced recruitment of key cellular effectors of renal fibrosis – interstitial macrophages and myofibroblasts. The ability of PAI-1 to promote cell migration involves interactions with the low-density lipoprotein receptor-associate protein-1 and also complex interactions with uPA bound to its receptor (uPAR) and several leukocyte and matrix integrins that associate with uPAR as co-receptors. uPAR is expressed by several cell types in damaged kidneys, and studies in uPAR-deficient mice have shown that its serves a protective role. uPAR mediates additional anti-fibrotic effects - it interacts with specific co-receptors to degrade PAI-1 and extracellular collagens, and soluble uPAR has leukocyte chemoattractant properties. Molecular pathways activated by serine proteases and their inhibitor, PAI-1, are promising targets for future anti-fibrotic therapeutic agents. PMID:19350108

Multipotent mesenchymal stromal cells decrease transforming growth factor β1 expression in microglia/macrophages and down-regulate plasminogen activator inhibitor 1 expression in astrocytes after stroke.

PubMed

Xin, Hongqi; Chopp, Michael; Shen, Li Hong; Zhang, Rui Lan; Zhang, Li; Zhang, Zheng Gang; Li, Yi

2013-05-10

Multipotent mesenchymal stromal cells (MSCs) decrease the expression of transforming growth factor β1 (TGFβ1) in astrocytes and subsequently decrease astrocytic plasminogen activator inhibitor 1 (PAI-1) level in an autocrine manner. Since activated microglia/macrophages are also a source of TGFβ1 after stroke, we therefore tested whether MSCs regulate TGFβ1 expression in microglia/macrophages and subsequently alters PAI-1 expression after ischemia. TGFβ1 and its downstream effector phosphorylated SMAD 2/3 (p-SMAD 2/3) were measured in mice subjected to middle cerebral artery occlusion (MCAo). MSC treatment significantly decreased TGFβ1 protein expression in both astrocytes and microglia/macrophages in the ischemic boundary zone (IBZ) at day 14 after stroke. However, the p-SMAD 2/3 was only detected in astrocytes and decreased after MSC treatment. In vitro, RT-PCR results showed that the TGFβ1 mRNA level was increased in both astrocytes and microglia/macrophages in an astrocyte-microglia/macrophage co-culture system after oxygen-glucose deprived (OGD) treatment. MSCs treatment significantly decreased the above TGFβ1 mRNA level under OGD conditions, respectively. OGD increased the PAI-1 mRNA in astrocytes in the astrocyte-microglia/macrophage co-culture system, and MSC administration significantly decreased this level. PAI-1 mRNA was very low in microglia/macrophages compared with that in astrocytes under different conditions. Western blot results also verified that MSC administration significantly decreased p-SMAD 2/3 and PAI-1 level in astrocytes in astrocyte-microglia/macrophage co-culture system under OGD conditions. Our in vivo and in vitro data, in concert, suggest that MSCs decrease TGFβ1 expression in microglia/macrophages in the IBZ which contribute to the down-regulation of PAI-1 level in astrocytes. Published by Elsevier Ireland Ltd.

Ashitaba (Angelica Keiskei) Exudate Prevents Increases in Plasminogen Activator Inhibitor-1 Induced by Obesity in Tsumura Suzuki Obese Diabetic Mice.

PubMed

Ohta, Mitsuhiro; Fujinami, Aya; Oishi, Katsutaka; Kobayashi, Norihiro; Ohnishi, Katsunori; Ohkura, Naoki

2018-04-30

Angelica keiskei koidzumi (ashitaba) is consumed as a traditional folk medicine and health food in Japan. Ashitaba extract contains abundant flavonoids containing chalcones. Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of tissue plasminogen activator. Excessive amounts of PAI-1 in plasma disrupt the fibrinolytic balance and promote a prothrombotic state with which thrombosis and cardiovascular diseases are associated. In the present study, we investigated the effects of ashitaba yellow exudate (AE) on enhanced PAI-1 levels in Tsumura Suzuki obese diabetic (TSOD) mice. AE significantly decreased food efficiency and plasma PAI-1 in TSOD mice but did not affect lean control Tsumura Suzuki nonobese (TSNO) mice. AE also decreased some parameters in the plasma, such as glucose, insulin, tumor necrosis factor alpha (TNF-α) and gains in body weight, subcutaneous, mesenteric fat weight in TSOD mice but had little effect on these parameters in TSNO mice. Levels of adipose PAI-1 were significantly higher in TSOD than in TSNO mice. Major sources of plasma PAI-1 are thought to be adipose tissue and liver. AE significantly suppressed PAI-1 protein levels in the livers of both TSOD and TSNO mice. These results suggest that AE decreased plasma PAI-1 levels by suppressing both the adipose tissue retention of PAI-1 protein and liver PAI-1 production in TSOD mice. Supplementing the diet with AE might help to prevent thrombotic diseases or alleviate the risk of thrombotic diseases as well as to suppress metabolic state in obese individuals.

The effective concentration of epsilon-aminocaproic Acid for inhibition of fibrinolysis in neonatal plasma in vitro.

PubMed

Yurka, Heather G; Wissler, Richard N; Zanghi, Christine N; Liu, Xiang; Tu, Xin; Eaton, Michael P

2010-07-01

Pediatric patients, particularly neonates, are at high risk for bleeding complications after cardiovascular surgery because of their immature hemostatic system, small size, and the complex operations they require. Activation of intravascular fibrinolysis is one of the principle effects of cardiopulmonary bypass that causes poor postoperative hemostasis. This complication has long been recognized and treated with antifibrinolytic medications, including the lysine analog epsilon aminocaproic acid (EACA). The therapeutic plasma concentration of EACA has been scientifically determined for the adult population, but the current recommended dosage for neonates has been empirically derived from adult studies. Therefore, we investigated the appropriate concentration of EACA for neonates undergoing bypass. We conducted an in vitro study using neonatal plasma derived from the placenta/cord units from 20 term, elective cesarean deliveries. Graded concentrations of EACA were added to aliquots of the plasma pool before activating fibrinolysis with tissue-type plasminogen activator. Standard kaolin-activated thromboelastograms were then run with the primary outcome variable being estimated percent lysis. These procedures were repeated on samples of commercially available pooled adult normal plasma for comparison. We found that neonatal plasma required significantly lower concentrations of EACA to completely prevent fibrinolysis than did adult plasma (44.2 microg/mL and 47.8 microg/mL for neonatal plasma and 94.4 and 131.4 microg/mL in adult plasma for 400 and 1000 U/mL of plasminogen activator, respectively, P < 0.001). Our data establish the minimal effective concentration of EACA necessary to completely prevent fibrinolysis in neonatal blood in vitro. This concentration is significantly less than that targeted by current dosing schemes, indicating that neonates are possibly being exposed to greater levels of EACA than is clinically necessary.

Binding of anti-SSA antibodies to apoptotic fetal cardiocytes stimulates urokinase plasminogen activator (uPA)/uPA receptor-dependent activation of TGF-β and potentiates fibrosis.

PubMed

Briassouli, Paraskevi; Rifkin, Daniel; Clancy, Robert M; Buyon, Jill P

2011-11-15

In congenital heart block (CHB), binding of maternal anti-SSA/Ro Abs to fetal apoptotic cardiocytes impairs their removal by healthy cardiocytes and increases urokinase plasminogen activator (uPA)/uPA receptor (uPAR)-dependent plasmin activation. Because the uPA/uPAR system plays a role in TGF-β activation, we evaluated whether anti-Ro binding to apoptotic cardiocytes enhances plasmin-mediated activation of TGF-β, thereby promoting a profibrosing phenotype. Supernatants from cocultures of healthy cardiocytes and apoptotic cardiocytes bound by IgG from a mother whose child had CHB (apoptotic-CHB-IgG [apo-CHB-IgG]) exhibited significantly increased levels of active TGF-β compared with supernatants from cocultures of healthy cardiocytes and apoptotic cardiocytes preincubated with IgG from a healthy donor. Treatment of the culture medium with anti-TGF-β Ab or TGF-β inhibitor (SB431542) abrogated the luciferase response, thereby confirming TGF-β dependency. Increased uPA levels and activity were present in supernatants generated from cocultures of healthy cardiocytes and apo-CHB-IgG cardiocytes compared with healthy cardiocytes and apoptotic cardiocytes preincubated with IgG from a healthy donor, respectively. Treatment of apo-CHB-IgG cardiocytes with anti-uPAR or anti-uPA Abs or plasmin inhibitor aprotinin prior to coculturing with healthy cardiocytes attenuated TGF-β activation. Supernatants derived from cocultures of healthy cardiocytes and apo-CHB-IgG cardiocytes promoted Smad2 phosphorylation and fibroblast transdifferentiation, as evidenced by increased smooth muscle actin and collagen expression, which decreased when fibroblasts were treated with supernatants from cocultures pretreated with uPAR Abs. These data suggested that binding of anti-Ro Abs to apoptotic cardiocytes triggers TGF-β activation, by virtue of increasing uPAR-dependent uPA activity, thus initiating and amplifying a cascade of events that promotes myofibroblast transdifferentiation and scar.

Increased Levels of Metalloproteinase 10 and Hemostatic Markers in Patients With Noncomplicated Primary Varicose Veins.

PubMed

Dzieciuchowicz, Lukasz; Espinosa, Gaudencio; Páramo, José A

2015-10-01

The purpose of the study was to analyze a systemic activation of hemostasis and concentration of matrix metalloproteinase 10 (MMP-10) in patients with primary varicose veins (PVVs). A study group consisted of 41 patients with noncomplicated PVVs. A control group consisted of 30 age- and sex-matched healthy individuals without varicose veins. The concentration of d-dimers (DD), prothrombin fragments 1 and 2 (F1+2), antigen of von Willebrand factor (vWF), and activity of plasminogen activator inhibitor (PAI-1) in plasma and concentration of MMP-10 in serum were analyzed. In patients with PVVs, higher concentrations of DD (P < .001), F1+2 (P < .001), vWF (P = .027), MMP-10 (P = .006), and higher activity of PAI-1 (P < .001) were observed. However, no correlation between the concentrations of MMP-10 and prothrombotic markers was found. Noncomplicated PVVs are associated with systemic, prothrombotic activation of hemostasis and increased concentration of MMP-10, suggesting a prothrombotic and proinflammatory state. © The Author(s) 2014.

Helodermatine, a kallikrein-like, hypotensive enzyme from the venom of Heloderma horridum horridum (Mexican beaded lizard)

PubMed Central

1986-01-01

We have purified and characterized the major N-benzoyl-L-arginine ethyl ester hydrolase from the venom of Heloderma horridum horridum. The enzyme belongs to the serine proteinase family, and its activity vs. peptide amide substrates and human high-molecular-weight kininogen suggests a similarity to the family of kallikreins. This interpretation is corroborated by its reactivity with the natural inhibitors soybean trypsin inhibitor and Kunitz-type bovine pancreatic trypsin inhibitor (aprotinin). Injection of the enzyme (2-16 micrograms/kg) into anesthetized rabbits leads to a rapid dose-dependent transient decrease of the arterial blood pressure. Like glandular kallikrein it specifically converts single-chain tissue type plasminogen activator into its double chain form. In contrast to other kallikrein-like enzymes from snake venoms it shows no thrombin-like or plasminogen activator activity. The enzyme is a single-chain glycoprotein (Mr 63,000). The N-terminal sequence revealed significant homology to pig pancreatic kallikrein and to kallikrein like enzymes from Crotalus atrox and Crotalus adamanteus venom. This enzyme, which we name Helodermatine, is the first purified from Sauria with kallikrein-like properties. PMID:3537191

Biochemical Assays of Cultured Cells

NASA Technical Reports Server (NTRS)

Barlow, G. H.

1985-01-01

Subpopulations of human embryonic kidney cells isolated from continuous flow electrophoresis experiments performed at McDonnell Douglas and on STS-8 have been analyzed. These analyses have included plasminogen activator assays involving indirect methodology on fibrin plated and direct methodology using chromogenic substrates. Immunological studies were performed and the conditioned media for erythropoietin activity and human granulocyte colony stimulating (HGCSF) activity was analyzed.

Activity of Nanobins Targeted to the Urokinase Plasminogen Activator System

NASA Astrophysics Data System (ADS)

Hankins, Patrick Leon

While innovations in nanotechnology have resulted in numerous medical advancements for the treatment of cancer, there remains an urgent unmet need for safe and efficient molecular platforms that facilitate the delivery of potent therapeutics to solid tumors. Nanoscale formulations help to overcome the poor bioavailability and systemic organ toxicity associated with many small molecule drugs. Of these nanoparticle drug delivery systems, the greatest clinical successes to date have employed simple nanoscale lipid bilayer assemblies which encase large payloads of chemotherapeutic. While the nanobin platform we have developed has seen initial success through the passive accumulation into tumors, actively targeting nanobins to tumor specific antigens has the potential to increase the therapeutic index of these nanoparticle drugs. We have identified the urokinase plasminogen activator (uPA) and its cell surface bound receptor (uPAR) as ideal targets for drug delivery due to their selective overexpression in metastatic cancers and their important role in tumor progression. From a panel of monoclonal antibodies targeted to uPA and uPAR, we have selected ATN291 and ATN658 as lead candidates for nanobin targeting based on their tumor cell binding and ability to be internalized by cells. A novel method of conjugating antibodies to liposomes was developed for our nanobin platform that preserves the high binding affinity and specificity of these antibodies. We evaluated these uPA- and uPAR-targeted nanobins in several xenograft tumor models and found that they were well-tolerated over a wide range of doses and demonstrated significantly increased antitumor efficacy over untargeted nanobins in multiple tumor types. Preliminary studies suggest that uPA-targeted nanobins are readily internalized by tumor cells, and we believe this is the mechanism for their increased antitumor effect. A method for radiolabeling nanobins with gallium-67 was developed, and preliminary SPECT-CT imaging studies showed the preferential accumulation of these nanobins in an orthotopic model of breast cancer. Due to their biocompatibility, robustness, and extensive history in the clinic, liposomes are an ideal drug delivery vehicle for the development of targeted therapies. The data presented in this thesis demonstrates the potential for active targeting to increase the therapeutic index of nanoscale drug delivery systems by increasing antitumor effect while simultaneously preventing drug uptake in peripheral tissue. In particular, targeting nanoparticles to the uPA system is a promising strategy for the treatment of many advanced, metastatic cancers.

Reduced endothelial activation after exercise is associated with improved HbA1c in patients with type 2 diabetes and coronary artery disease.

PubMed

Byrkjeland, Rune; Njerve, Ida U; Arnesen, Harald; Seljeflot, Ingebjørg; Solheim, Svein

2017-03-01

We have previously reported insignificant changes in HbA 1c after exercise in patients with both type 2 diabetes and coronary artery disease. In this study, we investigated the effect of exercise on endothelial function and possible associations between changes in endothelial function and HbA 1c . Patients with type 2 diabetes and coronary artery disease ( n = 137) were randomised to 12 months exercise or standard follow-up. Endothelial function was assessed by circulating biomarkers (E-selectin, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, von Willebrand factor, tissue plasminogen activator antigen, asymmetric dimethylarginine and L-arginine/asymmetric dimethylarginine ratio). Differences between the randomised groups were analysed by analysis of covariance and correlations by Spearman's rho or Pearson's correlation. No effect of exercise on endothelial function was demonstrated. The changes in HbA 1c in the exercise group correlated with changes in E-selectin ( r = 0.56, p < 0.001), intercellular adhesion molecule-1 ( r = 0.27, p = 0.052), vascular cell adhesion molecule-1 ( r = 0.32, p = 0.022) and tissue plasminogen activator antigen ( r = 0.35, p =  0.011). HbA 1c decreased significantly more in patients with versus without a concomitant reduction in E-selectin ( p =  0.002), intercellular adhesion molecule-1 ( p =  0.011), vascular cell adhesion molecule-1 ( p =  0.028) and tissue plasminogen activator antigen ( p =  0.009). Exercise did not affect biomarkers of endothelial function in patients with both type 2 diabetes and coronary artery disease. However, changes in biomarkers of endothelial activation correlated with changes in HbA 1c , and reduced endothelial activation was associated with improved HbA 1c after exercise.

Novel actions of tissue-type plasminogen activator in chronic kidney disease: a paradigm shift

PubMed Central

Hu, Kebin; Mars, Wendy M.; Liu, Youhua

2009-01-01

Tissue-type plasminogen activator (tPA) is traditionally viewed as a simple serine protease whose main function is to convert plasminogen into biologically active plasmin. As a protease, tPA plays a crucial role in regulating blood fibrinolysis, in maintaining the homeostasis of extracellular matrix (ECM) and in modulating the post-translational activation of growth factors. However, emerging evidence indicates that tPA may also function as a cytokine that transmits its signal across the cell membrane, initiates a diverse array of intracellular signaling, and dictates gene expression in the nuclei. Structurally, tPA is a kringle-containing protein that shares significant similarity to other classic cytokines such as hepatocyte growth factor (HGF) and macrophage-stimulating protein (MSP). Although there is no dedicated receptor, tPA binds to the cell membrane low density lipoprotein (LDL) receptor-related protein-1 (LRP-1), triggers LRP-1 tyrosine phosphorylation, and activates various intracellular signaling. As a cytokine, tPA plays a pivotal role in the pathogenesis of renal interstitial fibrosis through diverse mechanisms. It induces matrix matelloproteinase-9 (MMP-9) gene expression in renal interstitial fibroblasts, which causes the destruction of the tubular basement membrane (TBM), thereby facilitating tubular epithelial to mesenchymal transition (EMT). tPA also potentiates myofibroblast activation from quiescent interstitial fibroblasts through LRP-1-mediated recruitment of β1 integrin signaling. Furthermore, tPA acts as a survival factor that protects renal interstitial fibroblasts/myofibroblasts from apoptosis, thereby resulting in an expansion of myofibroblast populations in diseased kidney. Together, a growing body of evidence has implicated tPA as a fibrogenic cytokine that promotes the progression of kidney diseases. These new findings have radically changed our conception of tPA in renal fibrogenesis and represent a paradigm shift towards uncovering its cytokine function. A better understanding of renal tPA biology will ultimately translate into more rational therapeutic remedies for patients with chronic kidney fibrosis. PMID:18508579

Extracellular proteases as targets for drug development

PubMed Central

Cudic, Mare

2015-01-01

Proteases constitute one of the primary targets in drug discovery. In the present review, we focus on extracellular proteases (ECPs) because of their differential expression in many pathophysiological processes, including cancer, cardiovascular conditions, and inflammatory, pulmonary, and periodontal diseases. Many new ECP inhibitors are currently under clinical investigation and a significant increase in new therapies based on protease inhibition can be expected in the coming years. In addition to directly blocking the activity of a targeted protease, one can take advantage of differential expression in disease states to selectively deliver therapeutic or imaging agents. Recent studies in targeted drug development for the metalloproteases (matrix metalloproteinases, adamalysins, pappalysins, neprilysin, angiotensin-converting enzyme, metallocarboxypeptidases, and glutamate carboxypeptidase II), serine proteases (elastase, coagulation factors, tissue/urokinase plasminogen activator system, kallikreins, tryptase, dipeptidyl peptidase IV), cysteine proteases (cathepsin B), and renin system are discussed herein. PMID:19689354

Fibrinogen catabolism within the procoagulant VX-2 tumor of rabbit lung in vivo: Effluxing fibrin(ogen) fragments contain antiangiogenic activity.

PubMed

Hatton, Mark W C; Southward, Suzanne M R; Legault, Kimberly J; Ross, Bonnie L; Clarke, Bryan J; Bajzar, Laszlo; Blajchman, Morris A; Singh, Gurmit; Richardson, Mary

2004-04-01

Many types of solid tumors are known to be procoagulant environments. This is partly because a hyperpermeable vascular system within the tumor allows plasma hemostatic factors to accumulate in relatively high concentrations in the stroma, and many solid-tumor cells express tissue factor or a procoagulant factor. These circumstances appear to exist in the VX-2 lung tumor of the New Zealand White (NZW) rabbit, and they sustain a measurable turnover of stromal deposits of fibrin(ogen). We have measured the turnover of fibrinogen within tumors of the VX-2 tumor-burdened rabbit and analysed the catabolic products of fibrin(ogen) and the status of fibrinolysis in tumor-derived interpleural effusate. Using intravenously injected (125)I-labeled rabbit fibrinogen as a marker, we found that fibrinogen (approximate blood concentration 1740 microg/mL) passed from blood to VX-2 tumor stroma, saturating the tumor at a concentration of approximately 348 microg fibrinogen/g in approximately 12 hours. We measured fibrin(ogen) fragments, at a concentration of approximately 292 microg/mL, in interpleural effusates that we recovered from 13% of the VX-2-burdened rabbits. Unreduced fibrin(ogen) fragments consisted of 4 major components with a relative molecular mass of approximately 250,000 (assumed to be fragment X; approximately 9% of total fragments from densitometry of immunoblots), 200,000 (d-dimer; 41%), 110,000 (fragment D; 49%), and 50,000 to 55,000 (fragment E; 1%-2%) kD. Total fibrin(ogen) fragments immunopurified from effusates exhibited an antiangiogenic effect when subjected to a chick embryo chorioallantoic membrane procedure. Interpleural effusates were devoid of plasmin activity or active plasminogen activator inhibitor-1 but contained plasmin complexes and active urokinase-like plasminogen activator (uPA), alpha(2)-antiplasmin, and thrombin-activatable fibrinolysis inhibitor. We speculate that VX-2 cells release uPA to activate fibrinolysis within the tumor stroma. Catabolic products of hemostasis (eg, fibrinolytic fragments, angiostatin) flux from the stroma into the interpleural space, thereby providing a net antiangiogenic property to the effusate and ultimately to the lymphatic and circulatory systems.

The Mast Cell, Contact, and Coagulation System Connection in Anaphylaxis

PubMed Central

Guilarte, Mar; Sala-Cunill, Anna; Luengo, Olga; Labrador-Horrillo, Moisés; Cardona, Victoria

2017-01-01

Anaphylaxis is the most severe form of allergic reaction, resulting from the effect of mediators and chemotactic substances released by activated cells. Mast cells and basophils are considered key players in IgE-mediated human anaphylaxis. Beyond IgE-mediated activation of mast cells/basophils, further mechanisms are involved in the occurrence of anaphylaxis. New insights into the potential relevance of pathways other than mast cell and basophil degranulation have been unraveled, such as the activation of the contact and the coagulation systems. Mast cell heparin released upon activation provides negatively charged surfaces for factor XII (FXII) binding and auto-activation. Activated FXII, the initiating serine protease in both the contact and the intrinsic coagulation system, activates factor XI and prekallikrein, respectively. FXII-mediated bradykinin (BK) formation has been proven in the human plasma of anaphylactic patients as well as in experimental models of anaphylaxis. Moreover, the severity of anaphylaxis is correlated with the increase in plasma heparin, BK formation and the intensity of contact system activation. FXII also activates plasminogen in the fibrinolysis system. Mast cell tryptase has been shown to participate in fibrinolysis through plasmin activation and by facilitating the degradation of fibrinogen. Some usual clinical manifestations in anaphylaxis, such as angioedema or hypotension, or other less common, such as metrorrhagia, may be explained by the direct effect of the activation of the coagulation and contact system driven by mast cell mediators. PMID:28798744

Microglia activation due to obesity programs metabolic failure leading to type two diabetes.

PubMed

Maldonado-Ruiz, R; Montalvo-Martínez, L; Fuentes-Mera, L; Camacho, A

2017-03-20

Obesity is an energy metabolism disorder that increases susceptibility to the development of metabolic diseases. Recently, it has been described that obese subjects have a phenotype of chronic inflammation in organs that are metabolically relevant for glucose homeostasis and energy. Altered expression of immune system molecules such as interleukins IL-1, IL-6, IL-18, tumor necrosis factor alpha (TNF-α), serum amyloid A (SAA), and plasminogen activator inhibitor-1 (PAI-1), among others, has been associated with the development of chronic inflammation in obesity. Chronic inflammation modulates the development of metabolic-related comorbidities like metabolic syndrome (insulin resistance, glucose tolerance, hypertension and hyperlipidemia). Recent evidence suggests that microglia activation in the central nervous system (CNS) is a priority in the deregulation of energy homeostasis and promotes increased glucose levels. This review will cover the most significant advances that explore the molecular signals during microglia activation and inflammatory stage in the brain in the context of obesity, and its influence on the development of metabolic syndrome and type two diabetes.

Fibrin Formation, Structure and Properties

PubMed Central

Weisel, John W.; Litvinov, Rustem I.

2017-01-01

Fibrinogen and fibrin are essential for hemostasis and are major factors in thrombosis, wound healing, and several other biological functions and pathological conditions. The X-ray crystallographic structure of major parts of fibrin(ogen), together with computational reconstructions of missing portions and numerous biochemical and biophysical studies, have provided a wealth of data to interpret molecular mechanisms of fibrin formation, its organization, and properties. On cleavage of fibrinopeptides by thrombin, fibrinogen is converted to fibrin monomers, which interact via knobs exposed by fibrinopeptide removal in the central region, with holes always exposed at the ends of the molecules. The resulting half-staggered, double-stranded oligomers lengthen into protofibrils, which aggregate laterally to make fibers, which then branch to yield a three-dimensional network. Much is now known about the structural origins of clot mechanical properties, including changes in fiber orientation, stretching and buckling, and forced unfolding of molecular domains. Studies of congenital fibrinogen variants and post-translational modifications have increased our understanding of the structure and functions of fibrin(ogen). The fibrinolytic system, with the zymogen plasminogen binding to fibrin together with tissue-type plasminogen activator to promote activation to the active proteolytic enzyme, plasmin, results in digestion of fibrin at specific lysine residues. In spite of a great increase in our knowledge of all these interconnected processes, much about the molecular mechanisms of the biological functions of fibrin(ogen) remains unknown, including some basic aspects of clotting, fibrinolysis, and molecular origins of fibrin mechanical properties. Even less is known concerning more complex (patho)physiological implications of fibrinogen and fibrin. PMID:28101869

Mechanisms regulating plasminogen activators in transformed retinal ganglion cells

PubMed Central

Rock, Nathan; Chintala, Shravan K.

2008-01-01

Irreversible loss of retinal ganglion cells (RGCs) is a major clinical issue in glaucoma, but the mechanisms that lead to RGC death are currently unclear. We have previously reported that elevated levels of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) cause the death of RGCs in vivo and transformed retinal ganglion cells (RGC-5) in vitro. Yet, it is unclear how secreted proteases such as tPA and uPA directly cause RGCs' death. In this study, by employing RGC-5 cells, we report that tPA and uPA elicit their direct effect through the low-density lipoprotein-related receptor-1 (LRP-1). We also show that blockade of protease-LRP-1 interaction leads to a compete reduction in autocrine synthesis of tPA and uPA, and prevents protease-mediated death of RGC-5 cells. RGC-5 cells were cultured in serum-free medium and treated with 2.0 uM Staurosporine to induce their differentiation. Neurite outgrowth was observed by a phase contrast microscope and quantified by NeuroJ imaging software. Proteolytic activities of tPA and uPA were determined by zymography assays. Cell viability was determined by MTT assays. Compared to untreated RGC-5 cells, cells treated with Staurosporine differentiated, synthesized and secreted elevated levels of tPA and uPA, and underwent cell death. In contrast, when RGC-5 cells were treated with Staurosporine along with the receptor associated protein (RAP), proteolytic activities of both tPA and uPA were significantly reduced. Under these conditions, a significant number of RGC-5 cells survived and showed increased neurite outgrowth. These results indicate that LRP-1 regulates autocrine synthesis of tPA and uPA in RGC-5 cells and suggest that the use of RAP to antagonize the effect of proteases may be a way to prevent RGC death in glaucoma. PMID:18243176

16(R)-hydroxyeicosatetraenoic acid, a novel cytochrome P450 product of arachidonic acid, suppresses activation of human polymorphonuclear leukocyte and reduces intracranial pressure in a rabbit model of thromboembolic stroke.

PubMed

Bednar, M M; Gross, C E; Russell, S R; Fuller, S P; Ahern, T P; Howard, D B; Falck, J R; Reddy, K M; Balazy, M

2000-12-01

Activated polymorphonuclear leukocytes (PMNs) have been suggested to contribute to the development of increased intracranial pressure (ICP). We recently demonstrated that human PMNs produce a novel cytochrome P450-derived arachidonic acid metabolite, 1 6(R)-hydroxyeicosatetraenoic acid [16(R)-HETE], that modulates their function. It was thus of interest to examine this novel mediator in an acute stroke model. 16-HETE was assessed initially in a variety of human PMN and platelet in vitro assays and subsequently in an established rabbit model of thromboembolic stroke. A total of 50 rabbits completed a randomized, blinded, four-arm study, receiving 16(R)-HETE, tissue plasminogen activator, both, or neither. Experiments were completed 7 hours after autologous clot embolization. The primary end point for efficacy was the suppression of increased ICP. In in vitro assays, 16(R)-HETE selectively inhibited human PMN adhesion and aggregation and leukotriene B4 synthesis. In the thromboembolic stroke model, animals that received 16(R)-HETE demonstrated significant suppression of increased ICP (7.7 +/- 1.2 to 13.1 +/- 2.7 mm Hg, baseline versus final 7-h time point, mean +/- standard error), compared with either the vehicle-treated group (7.7 +/- 0.9 to 15.8 +/- 2.6 mm Hg) or the tissue plasminogen activator-treated group (7.6 +/- 0.6 to 13.7 +/- 2.1 mm Hg). The group that received the combination of 16(R)-HETE plus tissue plasminogen activator demonstrated no significant change in ICP for the duration of the protocol (8.6 +/- 0.6 to 11.1 +/- 1.2 mm Hg). 16(R)-HETE suppresses the development of increased ICP in a rabbit model of thromboembolic stroke and may serve as a novel therapeutic strategy in ischemic and inflammatory pathophysiological states.

Administration of antioxidant vitamins does not alter plasma fibrinolytic activity in subjects with central obesity.

PubMed

Rifici, V A; Schneider, S H; Chen, Y; Khachadurian, A K

1997-09-01

In vitro studies suggest that oxidized low density lipoprotein inhibits fibrinolysis by stimulating the production of plasminogen activator inhibitor -1 (PAI). We assessed the effects of dietary antioxidant vitamins for four weeks on three indices of copper mediated oxidation of very low and low density lipoproteins (VLDL+LDL) and plasma fibrinolytic activities in 15 male subjects with central obesity, a condition associated with increased PAI activity. Vitamin administration resulted in a decrease in production of thiobarbituric acid reactive substances from 29.3 +/- 3.9 to 13.6 +/- 3.5 nmoles/mg VLDL + LDL protein (mean +/- SE, p <0.003), an increase in the lag phase of conjugated diene formation from 94.8 +/- 5.5 to 225.0 +/- 31.9 min (p <0.001) and an increase in reactivity of lysine residues from 73.6% +/- 4.8% to 86.8% +/- 3.6% (p <0.034) demonstrating a reduction in the susceptibility of the lipoproteins to oxidation. However, antioxidant vitamins had no effect on plasma PAI activity, PAI antigen, tissue-type plasminogen activator activity and antigen, fibrinogen and fibrin degradation products. These results do not support the hypothesis that lipoprotein oxidation is a significant cause of impaired fibrinolysis in men with central obesity.

Circadian variability of fibrinolytic markers and endothelial function in patients with obstructive sleep apnea.

PubMed

Bagai, Kanika; Muldowney, James A S; Song, Yanna; Wang, Lily; Bagai, Jayant; Artibee, Kay J; Vaughan, Douglas E; Malow, Beth A

2014-02-01

Obstructive sleep apnea (OSA) is strongly associated with cardiovascular disease, including stroke and acute coronary syndromes. Plasminogen activator inhibitor-1 (PAI-1), the principal inhibitor of tissue-type plasminogen activator (t-PA), has a pronounced circadian rhythm and is elevated in both OSA and cardiovascular disease and may be an important link between the two conditions. Endothelial dysfunction is one of the underlying pathophysiological mechanisms of cardiovascular disease, and may be altered in OSA. Our primary aim was to compare circadian variability of PAI-1 and t-PA in patients with OSA and normal controls by determining the amplitude (peak level) and mesor (rhythm adjusted mean) of PAI-1 and t-PA in serial blood samples over a 24-h period. The secondary aim was to measure markers of endothelial function (brachial and radial artery flow) in patients with OSA compared with normal controls. Cross-sectional cohort study. Subjects age 18 y or older, with a body mass index of 25-45 kg/m(2), with or without evidence of untreated OSA. Plasma samples were collected every 2 h, in OSA patients and matched controls, over a 24-h period. PAI-1 and t-PA antigen and activity were measured. The presence or absence of OSA (apnea-hypopnea index of 5 or greater) was confirmed by overnight polysomnography. Endothelial function was measured via brachial artery flow mediated vasodilatation and computerized arterial pulse waveform analysis. The rhythm-adjusted mean levels of PAI-1 antigen levels in the OSA group (21.8 ng/mL, 95% confidence level [CI], 18 to 25.7) were significantly higher as compared to the non-OSA group (16 ng/mL, 95% CI, 12.2 to 19.8; P = 0.03). The rhythm-adjusted mean levels of PAI-1 activity levels in the OSA group (23.9 IU/mL, 95% CI, 21.4 to 26.5) were also significantly higher than in the non-OSA group (17.2 IU/ mL, 95% CI, 14.6 to 19.9; P < 0.001).There were strong correlations between amplitude of PAI-1 activity and severity of OSA as measured by AHI (P = 0.02), and minimum oxygen levels during sleep (P = 0.04). Endothelial function parameters did not differ significantly between the two groups. The presence of obstructive sleep apnea adversely affects circadian fibrinolytic balance with higher mean plasminogen activator inhibitor-1 activity and antigen, and significantly lower mean tissue-type plasminogen activator activity compared with controls. This perturbation may be an important mechanism for increased cardiovascular events in patients with obstructive sleep apnea. Intermittent hypoxia and changes in circadian clock gene activity in obstructive sleep apnea may be responsible for these findings and warrant further study. Favorable changes in fibrinolytic balance may underlie the reduction in cardiovascular events observed with the treatment of obstructive sleep apnea.

Outcomes after tissue plasminogen activator administration under the drip and ship paradigm may differ according to the regional stroke care system.

PubMed

Cha, Jae-Kwan; Nah, Hyun-Wook; Kang, Myung-Jin; Kim, Dae-Hyun; Park, Hyun-Seok; Kim, Sang-Beom; Jeong, Eun Hwan; Huh, Jae-Taeck

2014-01-01

The drip and ship paradigm for stroke patients enhances the rate of using intravenous tissue plasminogen activator (IVT) in community hospitals. The safety and outcomes of patients treated with IVT for acute ischemic stroke (AIS) under the drip and ship paradigm were compared with patients directly treated at a comprehensive stroke center in the Busan metropolitan area of Korea. This was a retrospective study of patients with AIS treated with IVT between January 2009 and January 2012. Information on patients' baseline characteristics, neuroimaging, symptomatic intracerebral hemorrhage (sICH), and outcome 90 days after using IVT was obtained from our stroke registry. We surveyed stroke neurologists regarding their pattern of post-thrombolysis care. During the observation periods, we selected 317 patients using IVT. Among these, 239 patients received IVT at our stroke center, and 78 were treated at 21 community hospitals under the drip and ship paradigm. Initial neurologic deficits and the size of ischemic lesions on magnetic resonance imaging were much more severe in patients treated with IVT under the drip and ship paradigm compared with patients treated at our comprehensive stroke center. The prevalence of a poor outcome (modified Rankin Scale score 3-6) 90 days after IVT was much higher in patients treated with the drip and ship paradigm than in those treated at our comprehensive stroke center. Regarding the occurrence of sICH, there was no significant difference between the 2 groups. The clinical characteristics and outcomes after using IVT under the drip and ship paradigm may differ greatly among stroke care systems. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

Plasminogen Activator Inhibitor-1 Antagonist TM5484 Attenuates Demyelination and Axonal Degeneration in a Mice Model of Multiple Sclerosis.

PubMed

Pelisch, Nicolas; Dan, Takashi; Ichimura, Atsuhiko; Sekiguchi, Hiroki; Vaughan, Douglas E; van Ypersele de Strihou, Charles; Miyata, Toshio

2015-01-01

Multiple sclerosis (MS) is characterized by inflammatory demyelination and deposition of fibrinogen in the central nervous system (CNS). Elevated levels of a critical inhibitor of the mammalian fibrinolitic system, plasminogen activator inhibitor 1 (PAI-1) have been demonstrated in human and animal models of MS. In experimental studies that resemble neuroinflammatory disease, PAI-1 deficient mice display preserved neurological structure and function compared to wild type mice, suggesting a link between the fibrinolytic pathway and MS. We previously identified a series of PAI-1 inhibitors on the basis of the 3-dimensional structure of PAI-1 and on virtual screening. These compounds have been reported to provide a number of in vitro and in vivo benefits but none was tested in CNS disease models because of their limited capacity to penetrate the blood-brain barrier (BBB). The existing candidates were therefore optimized to obtain CNS-penetrant compounds. We performed an in vitro screening using a model of BBB and were able to identify a novel, low molecular PAI-1 inhibitor, TM5484, with the highest penetration ratio among all other candidates. Next, we tested the effects on inflammation and demyelination in an experimental allergic encephalomyelitis mice model. Results were compared to either fingolimod or 6α-methylprednisolone. Oral administration of TM5484 from the onset of signs, ameliorates paralysis, attenuated demyelination, and axonal degeneration in the spinal cord of mice. Furthermore, it modulated the expression of brain-derived neurotrophic factor, which plays a protective role in neurons against various pathological insults, and choline acetyltransferase, a marker of neuronal density. Taken together, these results demonstrate the potential benefits of a novel PAI-1 inhibitor, TM5484, in the treatment of MS.

Effect of plasminogen activator inhibitor-1 on adipogenesis in vivo.

PubMed

Scroyen, Ilse; Jacobs, Frank; Cosemans, Leen; De Geest, Bart; Lijnen, H Roger

2009-02-01

To study the functional role of plasminogen activator inhibitor-1 (PAI-1) in obesity, the effect of its overexpression on de novo adipogenesis was evaluated in murine models in vivo. Therefore, 3T3-F442A preadipocytes expressing murine PAI-1 (mPAI-1) or control cells were injected in the back of male NUDE mice, which were fed a high-fat diet (HFD) for four weeks. De novo fat pads that formed from the PAI-1 expressing cells were larger (21 +/- 2.4 mg vs. 14 +/- 1.4 mg; p = 0.017) and showed a higher adipocyte density (373 +/- 28 mm(-2) vs. 301 +/- 12 mm(-2); p = 0.03) as compared to those formed from control cells. In a second model, male NUDE mice were injected in the tail vein with an adenoviral construct expressing mPAI-1 or with the empty vector, and three days later with 3T3-F442A cells. After four weeks of HFD, total body weight and de novo fat pad weight were comparable for both groups. Mild adipocyte hypotrophy was observed in the de novo fat pads of the PAI-1 overexpressing mice (1180 +/- 33 microm(2) vs. 1285 +/- 32 microm(2); p = 0.024), whereas the blood vessel size was significantly smaller than in controls (30 +/- 1.8 microm(2) vs. 63 +/- 3.6 microm(2); p < 0.0001). Thus, the effect of local or systemic PAI-1 (over)expression on adipocyte or blood vessel size and density of de novo formed fat pads appears to be different, and concentration-dependent. Whereas local expression resulted in larger fat pads, systemic overexpression had no effect on de novo adipogenesis, although angiogenesis appeared to be impaired.

Effect of helicopter transport on neurological outcomes in a mouse model of embolic stroke with reperfusion: AIR-MICE pilot study.

PubMed

Leira, Enrique C; Zaheer, Asgar; Schnell, Thomas; Torner, James C; Olalde, Heena M; Pieper, Andrew A; Ortega-Gutierrez, Santiago; Nagaraja, Nandakumar; Marks, Nancy L; Adams, Harold P

2015-10-01

Patients often suffer a stroke at a significant distance from a center capable of delivering endovascular therapy, thus requiring rapid transport by helicopter emergency medical services while receiving a recombinant tissue plasminogen activator infusion that was initiated locally. But little is known about how a helicopter flight may impact the safety and efficacy of recombinant tissue plasminogen activator-induced reperfusion and patient outcomes. To establish a new animal method to address with fidelity the safety and overall effect of helicopter emergency medical services during thrombolysis. Prospective randomized open blinded end-point study of an actual helicopter flight exposure. Adult C57BL/6 male mice were treated with a 10 mg/kg recombinant tissue plasminogen activator infusion two-hours after an embolic middle cerebral artery occlusion. Mice were randomized in pairs to simultaneously receive the infusion during a local helicopter flight or in a ground hangar. Eighteen mice (nine pairs) were analyzed. The paired t-test analysis showed nonsignificant smaller infarction volumes in the helicopter-assigned animals (mean pair difference 33 mm(3) , P = 0·33). The amount of hemorrhagic transformation between the helicopter and ground groups was 4·08 vs. 4·56 μl, respectively (paired t-test, P = 0·45). This study shows that helicopter emergency medical services do not have an inherent adverse effect on outcome in a mouse model of ischemic stroke with reperfusion. These results endorse the safety of the practice of using helicopter emergency medical services in stroke patients. The observed potential synergistic effect of helicopter-induced factors, such as vibration and changes in altitude, with reperfusion merits further exploration in animal experimental models and in stroke patients. © 2015 World Stroke Organization.

Thrombectomy in patients ineligible for iv tPA (THRILL).

PubMed

Bendszus, Martin; Thomalla, Götz; Knauth, Michael; Hacke, Werner; Bonekamp, Susanne; Fiehler, Jens

2015-08-01

A relevant proportion of patients with acute ischemic stroke are ineligible for intravenous thrombolysis with recombinant tissue plasminogen activator. Mechanical thrombectomy offers a treatment alternative for these patients; however, only few data are available on its safety and efficacy. The aim of this study was to compare safety and efficacy of stent retrievers as device class with best medical care alone in acute stroke patients with large intracranial vessel occlusion in the anterior circulation who are not eligible for intravenous thrombolysis with recombinant tissue plasminogen activator up to eight-hours of symptom onset. 'Thrombectomy in patients ineligible for iv tPA' is a prospective, open-label, blinded end-point, binational (Germany and Austria), two-arm, randomized, controlled, post-market study. Primary end-point is the modified Rankin Score shift analysis 90 days (±14) after stroke. Secondary end-points are excellent neurological outcomes (modified Rankin Score ≤ 1), good neurological outcomes (modified Rankin Score ≤ 2 or National Institutes of Health Stroke Scale improvement ≥ 10), difference between predicted infarct volume and actual core infarct volume (computed tomography or magnetic resonance imaging) at 30 (±6) h post-ictus, successful recanalization (thrombolysis in cerebral infarction score 2b or 3), functional health status 90 (±14) days after stroke (European Quality of Life-5 Dimensions) as well as common safety end-points (adverse event, serious adverse event, symptomatic intracranial haemorrhage at 30 (±6) h, death, or dependency). Whether mechanical thrombectomy in patients with acute ischemic stroke who are not eligible for intravenous thrombolysis with recombinant tissue plasminogen activator improves clinical outcomes is unclear. 'Thrombectomy in patients ineligible for iv tPA' may change clinical practice by providing evidence of an effective and safe treatment for such patients. © 2015 World Stroke Organization.

The Use of Tissue Plasminogen Activator in the Treatment of Wallenberg Syndrome Caused by Vertebral Artery Dissection.

PubMed

Salerno, Alexis; Cotter, Bradford V; Winters, Michael E

2017-05-01

Acute cerebrovascular accident (CVA) is a devastating cause of patient morbidity and mortality. Up to 10% of acute CVAs in young patients are caused by dissection of the vertebral or carotid artery. Wallenberg syndrome results from a CVA in the vertebral or posterior inferior artery of the cerebellum and manifests as various degrees of cerebellar dysfunction. The administration of a thrombolytic medication has been recommended in the treatment of patients with stroke caused by cervical artery dissection. Surprisingly, there is scant literature on the use of this medication in the treatment of this condition. We describe a 42-year-old man with the sudden onset of headache, left-sided neck pain, vomiting, nystagmus, and ataxia 1 h after completing a weightlifting routine. Computed tomography angiography revealed a grade IV left vertebral artery injury with a dissection flap extending distally and resulting in complete occlusion. Subsequent magnetic resonance imaging and angiography demonstrated acute left cerebellar and lateral medullary infarcts, consistent with Wallenberg syndrome. The patient was treated with tissue plasminogen activator, which failed to resolve his symptoms. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Emergency physicians frequently manage patients with acute CVAs. For select patients, the administration of tissue plasminogen activator can improve outcomes. However, the risk of major hemorrhage with this medication is significant. Cervical artery dissection is an important cause of acute stroke in young patients and is often missed on initial presentation. It is imperative for the emergency physician to consider acute cervical artery dissection as a cause of stroke and to be knowledgeable regarding the efficacy of thrombolytic medications for this condition. Copyright © 2017 Elsevier Inc. All rights reserved.

Adolescents with Classical Polycystic Ovary Syndrome Have Alterations in the Surrogate Markers of Cardiovascular Disease but Not in the Endothelial Function. The Possible Benefits of Metformin.

PubMed

Fruzzetti, Franca; Ghiadoni, Lorenzo; Virdis, Agostino; De Negri, Ferdinando; Perini, Daria; Bucci, Fiorella; Giannarelli, Chiara; Gadducci, Angiolo; Taddei, Stefano

2016-10-01

To study whether adolescents with the classical form of polycystic ovary syndrome (PCOS) have alterations in metabolic and vascular structure and function. The effect of metformin was evaluated. Controlled study. University outpatient clinic. Eighteen nonobese adolescents with PCOS were enrolled. Seventeen healthy age-matched adolescents were recruited as control subjects. The metabolic profile and the endothelial structure and function were evaluated. Hormonal and lipid profile, blood pressure (BP) measurement, fasting glucose and insulin levels, C-reactive protein (CRP), homocysteine, tissue-type plasminogen activator, plasminogen activator inhibitor-1 (PAI-1), and plasmin-antiplasmin complexes (PAP) were measured. Flow mediated dilation (FMD), central pulse wave velocity (PWV), radial artery pulse wave, and common carotid intima-media thickness (IMT) were also assessed. Girls with PCOS were also studied 6 months after treatment with metformin (850 mg twice per day). Adolescents with PCOS were insulin resistant and/or hyperinsulinemic and they had higher BP values and levels of CRP and PAI-1 than the control subjects. The levels of tissue-type plasminogen activator and PAP were similar in both groups. FMD, PWV, and IMT were also similar. Metformin significantly (P < .05) reduced insulin, BP, CRP, and PAI-1 levels. The PAP levels significantly (P < .05) increased. Radial artery pulse wave was significantly reduced after metformin treatment. No modifications in FMD, PWV, and IMT were observed. Adolescents with classical PCOS have alterations in some surrogate markers of cardiovascular risk and they are ameliorated by metformin. No deterioration of vascular structure and function has been detected, probably because of the short duration of exposure to the disease. Copyright © 2016 North American Society for Pediatric and Adolescent Gynecology. Published by Elsevier Inc. All rights reserved.

Ethnic Comparison of Clinical Characteristics and Ischemic Stroke Subtypes Among Young Adult Patients With Stroke in Hawaii.

PubMed

Nakagawa, Kazuma; Ito, Cherisse S; King, Sage L

2017-01-01

Native Hawaiians and other Pacific Islanders (NHOPI) with ischemic stroke have younger age of stroke onset compared with whites. However, ethnic differences in stroke subtypes in this population have been inadequately studied. Consecutive young adult patients (aged ≤55 years) who were hospitalized for ischemic stroke between 2006 and 2012 at a tertiary center in Honolulu were studied. Clinical characteristics and stroke subtypes based on pathophysiological TOAST classification (Trial of Org 10172) of NHOPI and Asians were compared with whites. A total of 427 consecutive young adult (mean age, 46.7±7.8 years) patients (NHOPI 45%, Asians 38%, and whites 17%) were studied. NHOPI had a higher prevalence of hypertension, diabetes mellitus, prosthetic valve, higher body mass index, hemoglobin A1c, and lower high-density lipoprotein than whites (all P<0.05). Stroke subtype distribution was not different between the ethnic groups. Specifically, the prevalence of small-vessel disease was similar between NHOPI (26.6%), whites (28.4%), and Asians (24.8%). In the univariate analyses, the use of intravenous tissue-type plasminogen activator was lower among NHOPI (4.7%; P=0.01) and Asians (3.1%; P=0.002) than among whites (12.5%). In the multivariable model, NHOPI (odds ratio, 0.35; 95% confidence interval, 0.12-0.98) and Asians (odds ratio, 0.23; 95% confidence interval, 0.07-0.74) were less likely to be treated with intravenous tissue-type plasminogen activator than whites. NHOPI have greater cardiovascular risk factors than whites, but there were no differences in stroke subtypes between the ethnic groups. Furthermore, NHOPI and Asians may be less likely to be treated with intravenous tissue-type plasminogen activator than whites. © 2016 American Heart Association, Inc.

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

Dong, Mei-Xue; Hu, Qing-Chuan; Shen, Peng; Pan, Jun-Xi; Wei, You-Dong; Liu, Yi-Yun; Ren, Yi-Fei; Liang, Zi-Hong; Wang, Hai-Yang; Zhao, Li-Bo; Xie, Peng

2016-01-01

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. 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. 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. 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.

Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor.

PubMed

Antalis, T M; Clark, M A; Barnes, T; Lehrbach, P R; Devine, P L; Schevzov, G; Goss, N H; Stephens, R W; Tolstoshev, P

1988-02-01

Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding the remainder of the mPAI-2 mRNA was obtained by primer extension of U937 poly(A)+ RNA using a probe complementary to the mPAI-2 coding region. The coding sequence for mPAI-2 was placed under the control of the lambda PL promoter, and the protein expressed in Escherichia coli formed a complex with urokinase that could be detected immunologically. By nucleotide sequence analysis, mPAI-2 cDNA encodes a protein containing 415 amino acids with a predicted unglycosylated Mr of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 (3 amino acid differences) and shows extensive homology with members of the serine protease inhibitor (serpin) superfamily. mPAI-2 was found to be more homologous to ovalbumin (37%) than the endothelial plasminogen activator inhibitor, PAI-1 (26%). Like ovalbumin, mPAI-2 appears to have no typical amino-terminal signal sequence. The 3' untranslated region of the mPAI-2 cDNA contains a putative regulatory sequence that has been associated with the inflammatory mediators.

Enzyme immunoassay for measurement of murine plasminogen activator inhibitor-1, employing a specific antibody produced by the DNA vaccine method.

PubMed

Yamada, Takayuki; Takagi, Akira; Takeshita, Kyosuke; Yamamoto, Koji; Ito, Masafumi; Matsushita, Tadashi; Murate, Takashi; Saito, Hidehiko; Kojima, Tetsuhito

2003-01-01

We developed a sensitive immunoassay to determine the concentration of mouse plasminogen activator inhibitor-1. The assay was a non-competitive sandwich enzyme-linked immunosorbent assay (ELISA) based on the production of a specific polyclonal antibody against mouse plasminogen activator inhibitor type-1 (PAI-1) used both as a trapping and detecting antibody. This antibody was raised in a rabbit by direct introduction of the expression vector plasmid DNA encoding mouse PAI-1, instead of conventional immunization with the purified protein. The standard curve was constructed with a recombinant glutathione S-transferase (GST)-mouse PAI-1 fusion protein (GST-mPAI-1) and dose-response of the assay was linear for GST-mPAI-1 between 6.25 and 100 pM. In order to assess the consistency of the assay, we measured PAI-1 antigen in normal mouse pooled plasma several times. We found that the intra-assay and inter-assay coefficients of variation (CV) were 4.8% and 9.2%, respectively, indicating that the ELISA would be sufficiently repeatable and reproducible. In this assay, lipopolysaccharide (LPS)-injected mice showed substantially higher levels (22-fold) of plasma PAI-1 antigen than did control mice (12.5+/-2.4 vs. 0.58+/-0.16 nM), similar to results reported elsewhere. Taken together, the DNA vaccine method is extremely useful for preparing specific antibodies against mouse PAI-1, which can be utilized to establish the ELISA and analyze the profile of PAI-1 distributions in mice under various conditions. This approach might also be useful for immunological investigation of other coagulation factors and related proteins.

Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor.

PubMed Central

Antalis, T M; Clark, M A; Barnes, T; Lehrbach, P R; Devine, P L; Schevzov, G; Goss, N H; Stephens, R W; Tolstoshev, P

1988-01-01

Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding the remainder of the mPAI-2 mRNA was obtained by primer extension of U937 poly(A)+ RNA using a probe complementary to the mPAI-2 coding region. The coding sequence for mPAI-2 was placed under the control of the lambda PL promoter, and the protein expressed in Escherichia coli formed a complex with urokinase that could be detected immunologically. By nucleotide sequence analysis, mPAI-2 cDNA encodes a protein containing 415 amino acids with a predicted unglycosylated Mr of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 (3 amino acid differences) and shows extensive homology with members of the serine protease inhibitor (serpin) superfamily. mPAI-2 was found to be more homologous to ovalbumin (37%) than the endothelial plasminogen activator inhibitor, PAI-1 (26%). Like ovalbumin, mPAI-2 appears to have no typical amino-terminal signal sequence. The 3' untranslated region of the mPAI-2 cDNA contains a putative regulatory sequence that has been associated with the inflammatory mediators. Images PMID:3257578

Improving door-to-needle times: a single center validation of the target stroke hypothesis.

PubMed

Ruff, Ilana M; Ali, Syed F; Goldstein, Joshua N; Lev, Michael; Copen, William A; McIntyre, Joyce; Rost, Natalia S; Schwamm, Lee H

2014-02-01

National guidelines recommend imaging within 25 minutes of emergency department arrival and intravenous tissue-type plasminogen activator within 60 minutes of emergency department arrival for patients with acute stroke. In 2007, we implemented a new institutional acute stroke care model to include 10 best practices and evaluated the effect of this intervention on improving door-to-computed tomography (CT) and door-to-needle (DTN) times at our hospital. We compared patients who presented directly to our hospital with acute ischemic stroke in the preintervention (2003-2006) and postintervention (2008-2011) periods. We did not include 2007, the year that the new protocol was established. Predictors of DTN ≤60 minutes before and after the intervention were assessed using χ(2) for categorical variables, and t test and Wilcoxon signed-rank test for continuous variables. Among 2595 patients with acute stroke, 284 (11%) received intravenous tissue-type plasminogen activator. For patients arriving within an intravenous tissue-type plasminogen activator window, door-to-CT <25 improved from 26.7% pre intervention to 52.3% post intervention (P<0.001). Similarly, the percentage of patients with DTN <60 doubled from 32.4% to 70.3% (P<0.001). Patients with DTN ≤60 did not differ significantly with respect to demographics, comorbidities, or National Institutes of Health Stroke Scale score in comparison with those treated after 60 minutes. Door-to-CT and DTN times improved dramatically after applying 10 best practices, all of which were later incorporated into the Target Stroke Guidelines created by the American Heart Association. The only factor that significantly affected DTN60 was the intervention itself, indicating that these best practices can result in improved DTN times.

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

Low-dose spironolactone ameliorates insulin resistance and suppresses elevated plasminogen activator inhibitor-1 during gestational testosterone exposure.

PubMed

Olatunji, Lawrence A; Usman, Taofeek O; Akinade, Aminat I; Adeyanju, Oluwaseun A; Kim, InKyeom; Soladoye, Ayodele O

2017-12-01

Elevated gestational circulating testosterone has been associated with pathological pregnancies that increase the risk of development of cardiometabolic disorder in later life. We hypothesised that gestational testosterone exposure, in late pregnancy, causes glucose deregulation and atherogenic dyslipidaemia that would be accompanied by high plasminogen activator inhibitor-1 (PAI-1). The study also hypothesise that low-dose spironolactone treatment would ameliorate these effects. Pregnant Wistar rats received vehicle, testosterone (0.5 mg/kg; sc), spironolactone (0.5 mg/kg, po) or testosterone and spironolactone daily between gestational days 15 and 19. Gestational testosterone exposure led to increased HOMA-IR, circulating insulin, testosterone, 1-h post-load glucose, atherogenic dyslipidaemia, PLR, PAI-1 and MDA. However, all these effects, except that of circulating testosterone, were ameliorated by spironolactone. These results demonstrate that low-dose spironolactone ameliorates glucose deregulation and atherogenic dyslipidaemia during elevated gestational testosterone exposure, at least in part, by suppressing elevated PAI-1.

Plasminogen Activators and Ischemic Stroke: Conditions for Acute Delivery

PubMed Central

del Zoppo, Gregory J

2013-01-01

Appropriate acute treatment with plasminogen activators (PAs) can significantly increase the probability of minimal or no disability in selected ischemic stroke patients. There is a great deal of evidence showing that intravenous recombinant tissue PAs (rt-PA) infusion accomplishes this goal, recanalization with other PAs has also been demonstrated in the development of this treatment. Recanalization of symptomatic, documented carotid or vertebrobasilar arterial territory occlusions have also been achieved by local intra-arterial PA delivery, although only a single prospective double-blinded randomized placebo-controlled study has been reported. The increase in intracerebral hemorrhage with these agents by either delivery approach underscores the need for careful patient selection, dose-appropriate safety and efficacy, proper clinical trial design, and an understanding of the evolution of cerebral tissue injury due to focal ischemia. Principles underlying the evolution of focal ischemia have been expanded by experience with acute PA intervention. Several questions remain open that concern the manner in which PAs can be applied acutely in ischemic stroke and how injury development can be limited. PMID:23539414

Plasma plasminogen activator inhibitor-1 levels and nonalcoholic fatty liver in individuals with features of metabolic syndrome.

PubMed

de Larrañaga, Gabriela; Wingeyer, Silvia Perés; Graffigna, Mabel; Belli, Susana; Bendezú, Karla; Alvarez, Silvia; Levalle, Oscar; Fainboim, Hugo

2008-07-01

Fatty liver represents the liver component of metabolic syndrome and may be involved in plasminogen activator inhibitor-1 (PAI-1) synthesis. We studied plasma PAI-1 levels and relationships with risk factors for metabolic syndrome, including fatty liver, in 170 patients. Liver ultrasound scan was performed on all patients, and a liver biopsy was performed on those patients with chronically elevated transaminase levels. Plasma PAI-1 levels correlated significantly (P < .05) with body mass index, degree of steatosis, insulin resistance, insulin level, waist circumference, triglycerides, and high-density lipoprotein (HDL) -cholesterol. However, only body mass index (beta = .455) and HDL-cholesterol (beta = .293) remained predictors of PAI-1 levels. Liver biopsy revealed a significant correlation (P < .05) between insulin resistance (r = 0.381) or insulin level (r = 0.519) and liver fibrosis. In patients presenting features of metabolic syndrome, plasma PAI-1 levels were mainly conditioned by the whole-body fat content.

Association between plasminogen activator inhibitor-1 4G/5G gene polymorphism and immunoglobulin A nephropathy susceptibility.

PubMed

Zhou, Tian-Biao; Jiang, Zong-Pei

2015-02-01

The association between plasminogen activator inhibitor-1 (PAI-1) 4 G/5 G gene polymorphism and immunoglobulin A nephropathy (IgAN) risk is still controversial. A meta-analysis was performed to evaluate the association between PAI-1 4 G/5 G gene polymorphism and IgAN susceptibility. A predefined literature search and selection of eligible relevant studies were performed to collect data from electronic database. Four articles were identified for the analysis of association between PAI-1 4 G/5 G gene polymorphism and IgAN risk. 4 G allele was not associated with IgAN susceptibility in overall populations and in Asians. Furthermore, 4 G/4 G and 5 G/5 G genotype were not associated with IgAN for overall populations, Asians. In conclusion, PAI-1 4 G/5 G gene polymorphism was not associated with IgAN risk in overall populations and in Asians. However, more studies should be performed in the future.

4G/5G polymorphism of plasminogen activator inhibitor-1 gene is associated with polycystic ovary syndrome in Chinese patients: a meta-analysis.

PubMed

Wang, Li-Hong; Wang, Li-Mei; Zhou, Na

2015-09-01

To date, case-control studies on the association between a single-nucleotide polymorphism (SNP) in the plasminogen activator inhibitor-1 (PAI-1) gene and polycystic ovary syndrome (PCOS) have provided controversial results. The electronic databases PubMed, Embase, Web of Science, and CNKI (China National Knowledge Infrastructure) were searched for studies to include in the present meta-analysis. The fixed effects and random effects models showed that the 4G allele was associated with a risk of PCOS compared with the 5G allele in Chinese patients (OR = 2.05; 95 % CI = 1.56-2.69), but not in Caucasian patients (OR = 1.05; 95 % CI = 0.81-1.37). The contrast of homozygotes and the recessive and dominant models produced the same pattern of results as the allele contrast. Our pooled data suggest evidence for a major role of PAI-1 gene 4G/5G polymorphism in the pathogenesis of PCOS among Chinese patients.

Hyperbaric oxygen increases tissue-plasminogen activator-induced thrombolysis in vitro, and reduces ischemic brain damage and edema in rats subjected to thromboembolic brain ischemia.

PubMed

Chazalviel, Laurent; Haelewyn, Benoit; Degoulet, Mickael; Blatteau, Jean-Eric; Vallée, Nicolas; Risso, Jean-Jacques; Besnard, Stéphane; Abraini, Jacques H

2016-01-01

Recent data have shown that normobaric oxygen (NBO) increases the catalytic and thrombolytic efficiency of recombinant tissue plasminogen activator (rtPA) in vitro , and is as efficient as rtPA at restoring cerebral blood flow in rats subjected to thromboembolic brain ischemia. Therefore, in the present study, we studied the effects of hyperbaric oxygen (HBO) (i) on rtPA-induced thrombolysis in vitro and (ii) in rats subjected to thromboembolic middle cerebral artery occlusion-induced brain ischemia. HBO increases rtPA-induced thrombolysis in vitro to a greater extent than NBO; in addition, HBO treatment of 5-minute duration, but not of 25-minute duration, reduces brain damage and edema in vivo . In line with the facilitating effect of NBO on cerebral blood flow, our findings suggest that 5-minute HBO could have provided neuroprotection by promoting thrombolysis. The lack of effect of HBO exposure of longer duration is discussed.

Slow fusion pore expansion creates a unique reaction chamber for co-packaged cargo

PubMed Central

Bittner, Mary A.; Lawrence, Daniel A.

2017-01-01

A lumenal secretory granule protein, tissue plasminogen activator (tPA), greatly slows fusion pore dilation and thereby slows its own discharge. We investigated another outcome of the long-lived narrow fusion pore: the creation of a nanoscale chemical reaction chamber for granule contents in which the pH is suddenly neutralized upon fusion. Bovine adrenal chromaffin cells endogenously express both tPA and its primary protein inhibitor, plasminogen activator inhibitor 1 (PAI). We found by immunocytochemistry that tPA and PAI are co-packaged in the same secretory granule. It is known that PAI irreversibly and covalently inactivates tPA at neutral pH. We demonstrate with zymography that the acidic granule lumen protects tPA from inactivation by PAI. Immunocytochemistry, total internal reflection fluorescence (TIRF) microscopy, and polarized TIRF microscopy demonstrated that co-packaged PAI and tPA remain together in granules for many seconds in the nanoscale reaction chamber, more than enough time to inhibit tPA and create a new secreted protein species. PMID:28882880

Rosuvastatin reduced deep vein thrombosis in ApoE gene deleted mice with hyperlipidemia through non-lipid lowering effects

PubMed Central

Patterson, K.A.; Zhang, X.; Wrobleski, S.K.; Hawley, A.E.; Lawrence, D. A.; Wakefield, T.W.; Myers, D.D.; Diaz, J.A.

2013-01-01

Introduction Statins, particularly rosuvastatin, have recently become relevant in the setting of venous thrombosis. The objective of this study was to study the non-lipid lowering effects of rosuvastatin in venous thrombosis in mice with hyperlipidemia. Materials and Methods An inferior vena cava ligation model of venous thrombosis in mice was utilized. Saline or 5mg/kg of rosuvastatin was administered by gavage 48hs previous thrombosis. Blood, the inferior vena cava, thrombus, and liver were harvested 3, 6 hours, and 2 days post-thrombosis. Thrombus weight, inflammatory markers, and plasminogen activator inhibitor-1 expression and plasma levels were measured and neutrophil migration to the IVC was assessed. Results Rosuvastatin significantly decreased thrombus weight, plasminogen activator inhibitor-1 expression and plasma levels, expression of molecules related to the interleukin-6 pathway, and neutrophil migration into the vein wall. Conclusions This work supports the beneficial effects of rosuvastatin on venous thrombosis in mice with hyperlipidemia due to its non-lipid lowering effects. PMID:23276528

Sepsis-Induced Coagulation in the Baboon Lung Is Associated with Decreased Tissue Factor Pathway Inhibitor

PubMed Central

Tang, Haiwang; Ivanciu, Lacramioara; Popescu, Narcis; Peer, Glenn; Hack, Erik; Lupu, Cristina; Taylor, Fletcher B.; Lupu, Florea

2007-01-01

Increased tissue factor (TF)-dependent procoagulant activity in sepsis may be partly due to decreased expression or function of tissue factor pathway inhibitor (TFPI). To test this hypothesis, baboons were infused with live Escherichia coli and sacrificed after 2, 8, or 24 hours. Confocal and electron microscopy revealed increased leukocyte infiltration and fibrin deposition in the intravascular and interstitial compartments. Large amounts of TF were detected by immunostaining in leukocytes and platelet-rich microthrombi. TF induction was documented by quantitative reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and coagulation assays. Lung-associated TFPI antigen and mRNA decreased during sepsis, and TFPI activity diminished abruptly at 2 hours. Blocking antibodies against TFPI increased fibrin deposition in septic baboon lungs, suggesting that TF-dependent coagulation might be aggravated by reduced endothelial TFPI. Decreased TFPI activity coincided with the release of tissue plasminogen activator and the peak of plasmin generation, suggesting that TFPI could undergo proteolytic inactivation by plasmin. Enhanced plasmin produced in septic baboons by infusion of blocking antibodies against plasminogen activator inhibitor-1 led to decreased lung-associated TFPI and unforeseen massive fibrin deposition. We conclude that activation of TF-driven coagulation not adequately countered by TFPI may underlie the widespread thrombotic complications of sepsis. PMID:17640967

Examination of the signal transduction pathways leading to upregulation of tissue type plasminogen activator by Porphyromonas endodontalis in human pulp cells.

PubMed

Huang, F-M; Chen, Y-J; Chou, M-Y; Chang, Y-C

2005-12-01

To investigate the tissue type plasminogen activator (t-PA) activity in human pulp cells stimulated with Porphyromonas endodontalis (P. endodontalis) in the absence or presence of p38 inhibitor SB203580, mitogen-activated protein kinase kinase (MEK) inhibitor U0126 and phosphatidylinositaol 3-kinase (PI3K) inhibitor LY294002. The supernatants of P. endodontalis were used to evaluate t-PA activity in human pulp cells using casein zymography and enzyme-linked immunosorbent assay (ELISA). Furthermore, to search for possible signal transduction pathways, SB203580, U0126 and LY294002 were added to test how they modulated the t-PA activity. The main casein secreted by human pulp cells migrated at 70 kDa and represented t-PA. Secretion of t-PA was found to be stimulated with P. endodontalis during 2-day cultured period (P < 0.05). From the results of casein zymography and ELISA, SB203580 and U0126 significantly reduced the P. endodontalis stimulated t-PA production respectively (P < 0.05). However, LY294002 lacked the ability to change the P. endodontalis stimulated t-PA production (P > 0.05). Porphyromonas endodontalis enhances t-PA production in human pulp cells, and the signal transduction pathways p38 and MEK are involved in the inhibition of t-PA.

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

Up-regulation of milk secretion with modified microclimate through manipulating plasminogen-plasmin system in Murrah buffaloes during hot dry season

NASA Astrophysics Data System (ADS)

Haque, N.; Singh, M.; Hossain, S. A.

2016-12-01

The present study was aimed at determining changes in milk yield and composition along with the plasminogen-plasmin system of milk, plasma hormones, and metabolites of buffaloes during hot dry season (air temperature range 39.7 to 44.8 °C) under two different management systems. Buffaloes were divided in two groups of six animals each: control and treatment, where treatment group animals accessed benefit of mist and fan cooling from 9:30 a.m. to 5:00 p.m., while control group animals were devoid of it. Duration of experiment was 6 weeks. Under mist and fan cooling system, buffaloes experienced better comfort by alleviating environmental stress as their physiological responses such as rectal temperature, respiration rate, pulse rate, and forehead and middorsal temperatures were significantly ( P < 0.05) reduced compared to control, which subsequently resulted higher milk yield by 4.44 % ( P < 0.001). Analysis of milk samples revealed higher concentration of plasminogen (7.99 vs 6.27 μg/ml; P < 0.01) and β-casein (1.09 vs 0.92 g/dl; P < 0.001) and lower plasmin level (0.178 vs 0.194 μg/ml; P < 0.05) in buffaloes under the treatment group compared to that under the control. Plasma glucose level was higher ( P < 0.001) by 21.08 %, whereas cortisol, norepinephrine, and NEFA levels were lower ( P < 0.001) by 19.19, 15.38, and 11.41 %, respectively, in treatment animals. However, exposure of buffaloes to cooling system did not alter composition and calcium content of milk, GH, and epinephrine level in plasma. Hence, it may be concluded that provision of cooling system during summer was effective to minimize environmental stress and improve milk production by manipulation of the PG-PL system in buffaloes.

Up-regulation of milk secretion with modified microclimate through manipulating plasminogen-plasmin system in Murrah buffaloes during hot dry season.

PubMed

Haque, N; Singh, M; Hossain, S A

2016-12-01

The present study was aimed at determining changes in milk yield and composition along with the plasminogen-plasmin system of milk, plasma hormones, and metabolites of buffaloes during hot dry season (air temperature range 39.7 to 44.8 °C) under two different management systems. Buffaloes were divided in two groups of six animals each: control and treatment, where treatment group animals accessed benefit of mist and fan cooling from 9:30 a.m. to 5:00 p.m., while control group animals were devoid of it. Duration of experiment was 6 weeks. Under mist and fan cooling system, buffaloes experienced better comfort by alleviating environmental stress as their physiological responses such as rectal temperature, respiration rate, pulse rate, and forehead and middorsal temperatures were significantly (P < 0.05) reduced compared to control, which subsequently resulted higher milk yield by 4.44 % (P < 0.001). Analysis of milk samples revealed higher concentration of plasminogen (7.99 vs 6.27 μg/ml; P < 0.01) and β-casein (1.09 vs 0.92 g/dl; P < 0.001) and lower plasmin level (0.178 vs 0.194 μg/ml; P < 0.05) in buffaloes under the treatment group compared to that under the control. Plasma glucose level was higher (P < 0.001) by 21.08 %, whereas cortisol, norepinephrine, and NEFA levels were lower (P < 0.001) by 19.19, 15.38, and 11.41 %, respectively, in treatment animals. However, exposure of buffaloes to cooling system did not alter composition and calcium content of milk, GH, and epinephrine level in plasma. Hence, it may be concluded that provision of cooling system during summer was effective to minimize environmental stress and improve milk production by manipulation of the PG-PL system in buffaloes.

Non-Lethal Endotoxin Injection: A Rat Model of Hypercoagulability.

PubMed

Brooks, Marjory B; Turk, James R; Guerrero, Abraham; Narayanan, Padma K; Nolan, John P; Besteman, Elizabeth G; Wilson, Dennis W; Thomas, Roberta A; Fishman, Cindy E; Thompson, Karol L; Ellinger-Ziegelbauer, Heidrun; Pierson, Jennifer B; Paulman, April; Chiang, Alan Y; Schultze, Albert E

2017-01-01

Systemic inflammation co-activates coagulation, which unchecked culminates in a lethal syndrome of multi-organ microvascular thrombosis known as disseminated intravascular coagulation (DIC). We studied an endotoxin-induced inflammatory state in rats to identify biomarkers of hemostatic imbalance favoring hypercoagulability. Intraperitoneal injection of LPS at 15 mg/kg body weight resulted in peripheral leukopenia and widespread neutrophilic sequestration characteristic of an acute systemic inflammatory response. Early indicators of hemostatic pathway activation developed within 4 hours, including increased circulating concentrations of procoagulant extracellular vesicles (EVs), EVs expressing endothelial cell and platelet membrane markers, and high concentration of soluble intercellular adhesion molecule-1 (sICAM-1), plasminogen activator inhibitor-1 (PAI-1), and D-dimers. Inflammation persisted throughout the 48-hour observation period; however, increases were found in a subset of serum microRNA (miRNA) that coincided with gradual resolution of hemostatic protein abnormalities and reduction in EV counts. Dose-adjusted LPS treatment in rats provides a time-course model to develop biomarker profiles reflecting procoagulant imbalance and rebalance under inflammatory conditions.

Small molecule antagonists of the urokinase (uPA): urokinase receptor (uPAR) interaction with high reported potencies show only weak effects in cell-based competition assays employing the native uPAR ligand.

PubMed

De Souza, Melissa; Matthews, Hayden; Lee, Jodi A; Ranson, Marie; Kelso, Michael J

2011-04-15

Binding of the urokinase-type plasminogen activator (uPA) to its cell-surface-bound receptor uPAR and upregulation of the plasminogen activation system (PAS) correlates with increased metastasis and poor prognosis in several tumour types. Disruptors of the uPA:uPAR interaction represent promising anti-tumour/metastasis agents and several approaches have been explored for this purpose, including the use of small molecule antagonists. Two highly potent non-peptidic antagonists 1 and 2 (IC(50)1=0.8 nM, IC(50)2=33 nM) from the patent literature were reportedly identified using competition assays employing radiolabelled uPAR-binding uPA fragments and appeared as useful pharmacological tools for studying the PAS. Before proceeding to such studies, confirmation was sought that 1 and 2 retained their potencies in physiologically relevant cell-based competition assays employing uPAR's native binding partner high molecular weight uPA (HMW-uPA). This study describes a new solution phase synthesis of 1, a mixed solid/solution phase synthesis of 2 and reports the activities of 1 and 2 in semi-quantitative competition flow cytometry assays and quantitative cell-based uPA activity assays that employed HMW-uPA as the competing ligand. The flow cytometry experiments revealed that high concentrations of 2 (10-100 μM) are required to compete with HMW-uPA for uPAR binding and that 1 shows no antagonist effects at 100 μM. The cell-based enzyme activity assays similarly revealed that 1 and 2 are poor inhibitors of cell surface-bound HMW-uPA activity (IC(50) >100 μM for 1 and 2). The report highlights the dangers of identifying false-positive lead uPAR antagonists from competition assays employing labelled competing ligands other than the native HMW-uPA. Copyright © 2011 Elsevier Ltd. All rights reserved.

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.

Plasminogen Activator Inhibitor-2 Polymorphism Associates with Recurrent Coronary Event Risk in Patients with High HDL and C-Reactive Protein Levels

PubMed Central

Corsetti, James P.; Salzman, Peter; Ryan, Dan; Moss, Arthur J.; Zareba, Wojciech; Sparks, Charles E.

2013-01-01

The objective of this work was to investigate whether fibrinolysis plays a role in establishing recurrent coronary event risk in a previously identified group of postinfarction patients. This group of patients was defined as having concurrently high levels of high-density lipoprotein cholesterol (HDL-C) and C-reactive protein (CRP) and was previously demonstrated to be at high-risk for recurrent coronary events. Potential risk associations of a genetic polymorphism of plasminogen activator inhibitor-2 (PAI-2) were probed as well as potential modulatory effects on such risk of a polymorphism of low-density lipoprotein receptor related protein (LRP-1), a scavenger receptor known to be involved in fibrinolysis in the context of cellular internalization of plasminogen activator/plansminogen activator inhibitor complexes. To this end, Cox multivariable modeling was performed as a function of genetic polymorphisms of PAI-2 (SERPINB, rs6095) and LRP-1 (LRP1, rs1800156) as well as a set of clinical parameters, blood biomarkers, and genetic polymorphisms previously demonstrated to be significantly and independently associated with risk in the study population including cholesteryl ester transfer protein (CETP, rs708272), p22phox (CYBA, rs4673), and thrombospondin-4 (THBS4, rs1866389). Risk association was demonstrated for the reference allele of the PAI-2 polymorphism (hazard ratio 0.41 per allele, 95% CI 0.20-0.84, p=0.014) along with continued significant risk associations for the p22phox and thrombospondin-4 polymorphisms. Additionally, further analysis revealed interaction of the LRP-1 and PAI-2 polymorphisms in generating differential risk that was illustrated using Kaplan-Meier survival analysis. We conclude from the study that fibrinolysis likely plays a role in establishing recurrent coronary risk in postinfarction patients with concurrently high levels of HDL-C and CRP as manifested by differential effects on risk by polymorphisms of several genes linked to key actions involved in the fibrinolytic process. PMID:23874812

Flow cytometry of human embryonic kidney cells: A light scattering approach

NASA Technical Reports Server (NTRS)

Kunze, M. E.; Goolsby, C. L.; Todd, P. W.; Morrison, D. R.; Lewis, M. L.

1985-01-01

The mammalian kidney contains cells that transport water, convert vitamin D to active forms, synthesize hormones such a renin and erythropoietin, and produce enzymes such as urokinase, a plasminogen activator. Several of these functions are maintained by human embryonic kidney cells (HEK) cultivated in vitro. Biochemical study of these functions in their individual cell types in vitro requires purified populations of cells. Light-scattering activated cell sorting (LACS) was explored as a means of achieving such purifications. It was found that HEK cells at the first 1 to 5 passages in culture were heterogeneous with respect to 2-parameter light scattering intensity distribution, in which combined measurements included forward angle scattering (2.5 to 19 deg), 90 deg scattering, and time-of-flight size measurements. Size was measured at a resolution of 0.15 microns/channel in 256 channels using pulse-height independent pulse-width measurements. Two-parameter distributions combining these measurements were obtained for HEK cell subpopulations that had been purified by microgravity electrophoresis and subsequently propagated in culture. These distributions contained at least 3 subpopulations in all purified fractions, and results of experiments with prepurified cultured HEK cells indicated that subpopulations of living cells that were high in plasminogen-activator activity also contained the highest per cent of cells with high 90 deg light scatter intensity.

Accelerated Tumor Cell Death by Angiogenic Modifiers

DTIC Science & Technology

2003-08-01

complex process Two huge molecules called plasminogen-related growth is regulated by a delicate balance of angiogenesis in- factors ( PRGFs ...Increased activator(s) and/or decreased inhibi- cancer progression. PRGF -1 is also called hepatocyte tor(s) alter the balance and lead to the growth of...new growth factor/scatter factor (HGF/SF). PRGF -2 is also blood vessels (Hanahan, 1997). Several growth factors, known as macrophage-stimulating

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. © 2015 International Society for Neurochemistry.

Emergency medicine in the Veterans Health Administration-results from a nationwide survey.

PubMed

Ward, Michael J; Collins, Sean P; Pines, Jesse M; Dill, Curt; Tyndall, Gary; Kessler, Chad S

2015-07-01

We describe emergency physician staffing, capabilities, and academic practices in US Veterans Health Administration (VHA) emergency departments (EDs). As part of an ongoing process improvement effort for the VHA emergency care system, VHA-wide surveys are conducted among ED medical directors every 3 years. Web-based surveys of VHA ED directors were conducted in 2013 on clinical operations and academic program development. We describe the results from the 2013 survey. When available, we compare responses with the previously administered survey from 2010. A total of 118 of 118 ED directors filled out the survey in 2013 (100% response rate). Respondents reported that 45.5% of VHA emergency physicians are board certified in emergency medicine, and 95% spend most their time in direct patient care. Clinical care is also provided by part-time (<0.5 full-time employee equivalent) emergency physicians in 59.3% of EDs. More than half of EDs (57%) provide on-site tissue plasminogen activator for acute ischemic stroke patients, and only 39% can administer tissue plasminogen activator 24 hours per day, 7 days per week. Less than half (48.3%) of EDs have emergency Obstetrics and Gynecology consultation availability. Most VHA EDs (78.8%) have a university affiliation, but only 21.5% participated in the respective academic emergency medicine program. Veterans Health Administration emergency physicians have primarily clinical responsibilities, and less than half have formal emergency medicine board certification. Despite most VHA EDs having university affiliations, traditional academic activities (eg, teaching and research) are performed in only 1 in 3 VHA EDs. Less than half of VHA EDs have availability of consulting services, including advanced stroke care and women's health. Published by Elsevier Inc.

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

Endometrial haemostasis and menstruation.

PubMed

Davies, Joanna; Kadir, Rezan A

2012-12-01

Under normal physiological circumstances menstruation is a highly regulated, complex process that is under strict hormonal control. During normal menstruation, progesterone withdrawal initiates menstruation. The cessation of menstrual bleeding is achieved by endometrial haemostasis via platelet aggregation, fibrin deposition and thrombus formation. Local endocrine, immunological and haemostatic factors interact at a molecular level to control endometrial haemostasis. Tissue factor and thrombin play a key role locally in the cessation of menstrual bleeding through instigation of the coagulation factors. On the other hand, fibrinolysis prevents clot organisation within the uterine cavity while plasminogen activator inhibitors (PAI) and thrombin-activatable fibrinolysis inhibitors control plasminogen activators and plasmin activity. Abnormalities of uterine bleeding can result from imbalance of the haemostatic factors. The most common abnormality of uterine bleeding is heavy menstrual bleeding (HMB). Modern research has shown that an undiagnosed bleeding disorder, in particular von Willebrand disease (VWD) and platelet function disorders, can be an underlying cause of HMB. This has led to a change in the approach to the management of HMB. While full haemostatic assessment is not required for all women presenting with HMB, menstrual score and bleeding score can help to discriminate women who are more likely to have a bleeding disorder and benefit from laboratory haemostatic evaluation. Haemostatic agents (tranexamic acid and DDAVP) enhance systemic and endometrial haemostasis and are effective in reducing menstrual blood loss in women with or without bleeding disorders. Further research is required to enhance our understanding of the complex interactions of haemostatic factors in general, and specifically within the endometrium. This will lead to the development of more targeted interventions for the management of abnormal uterine bleeding in the future.

Extravascular plasminogen activator and inhibitor activities detected at the site of a chronic mycobacterial-induced inflammation.

PubMed Central

O'Rourke, J.; Wang, W. P.; Donnelly, L.; Wang, E.; Kreutzer, D. L.

1987-01-01

Levels of extravascular tissue plasminogen activator activity (PA) and those of inhibitors of PA and of urokinase (UK) present within the anterior chamber of normal and inflamed feline eyes were assessed with the use of a direct PA assay of microsamples of aqueous humor. Purposes of the study were, first, to confirm prior indirect evidence that this extravascular space normally contains higher levels of uninhibited PA, but lower levels of inhibitor activity, than does plasma and, second, to determine patterns of change in these activities under in vivo conditions imposed by a chronic mycobacterial-induced uveitis (CMIU) disease model. The PA assay utilized a 125I-plasminogen substrate whose cleavage by PA contained in samples was both visualized during gel electrophoreis, and quantified by gamma counting. The results provided the first direct evidence that the higher fibrinolytic activity previously observed in normal aqueous in comparison with plasma is in fact associated with higher levels of available (uninhibited) PA (P less than 0.01) The data also indicated that normal aqueous contains a much higher level of PA inhibitor activity than previously suspected--roughly 40 times more than available PA levels. These normal values for PA and inhibitors occupied a relatively narrow, threefold range, in contrast to the wide scattering of individual values that appeared during 18-20 weeks of the chronic inflammation disease model. Despite this, however, the general pattern of observation for all individual eyes during CMIU was a significant increase in levels of both PA and inhibitors. The net effect of CMIU was thus to cause the 1:40 ratio noted above to be tilted more strongly in favor of inhibitor activity, ie, up to 1:80. Increases in local vasopermeability in this disease model were believed contributory to this change. However, local generations of PA and APA in vivo by inflammatory cells, especially monocyte-macrophages, must also be considered. Assays for UK inhibitor showed levels of activity and directions of change that closely followed those of PA inhibitor, which suggests the possibility that they may be identical. It is surmised that the above patterns, along with results of our prior studies, indicate an apparent need for a multistep, strict inhibitory control of plasmin generation and proteolysis in vivo within normal extravascular spaces such as the anterior chamber.(ABSTRACT TRUNCATED AT 400 WORDS) Images Figure 2 PMID:3493701

[Can dexpanthenol prevent peritoneal adhesion formation? An experimental study].

PubMed

Akdeniz, Yusuf; Tarhan, Omer Ridvan; Barut, Ibrahim

2007-04-01

Peritoneum has an intrinsic fibrinolytic activity that breaks the peritoneal adhesions. Ischemic peritoneal injuries interfere with this fibrinolytic activity. Local application of dexpanthenol, the alcohol form of pantothenic acid (vitamin B5) accelerates wound healing by increasing mitosis. We hypothesized that dexpanthenol would decrease peritoneal adhesions. In rats, antimesenteric border of cecum was abraded with gauze. No medication was given to the control group (n=15). Dexpanthenol was administered intraperitoneally (IP) (n=15, 25 mg/kg, before abdominal closure) or intravenously (IV) (n=15, 25 mg/kg, for 9 days after operation) in the experiment groups. On postoperative day 10, adhesions were graded; activities and concentrations of tissue plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI-1), tPA/PAI-1 complex and hydroxyproline contents were determined in peritoneum. Adhesion formation was decreased in IP dexpanthenol group compared with control group (p=0.034). tPA concentration and activity and tPA/PAI-1 complex levels were increased in the treated groups compared to controls. PAI-1 levels were similar among the three groups. Peritoneal hydroxyproline levels were lower in animals receiving IV dexpanthenol compared with control animals and in addition, they remained unchanged in IP dexpanthenol treated group (p=0.009, p=0.84, respectively). Our results suggest that dexpanthenol administration through IP may reduce peritoneal adhesion formation probably by altering peritoneal fibrinolytic activity.

[Construction and expression of a fusion protein made of tissue-type plasminogen activator and hirudin in Pichia pastoris].

PubMed

Yu, Ai-Ping; Shi, Bing-Xing; Dong, Chun-Na; Jiang, Zhong-Hua; Wu, Zu-Ze

2005-07-01

To combine the fibrinolytic with anticoagulant activities for therapy of thrombotic deseases, a fusion protein made of tissue-type plasminogen activator (t-PA) and hirudin was constructed and expressed in chia pastoris. To improve thrombolytic properties of t-PA and reduce bleeding side effect of hirudin, FXa-recognition sequence was introduced between t-PA and hirudin molecules.The anticoagulant activity of hirudin can be target-released through cleavage of FXa at thrombus site. t-PA gene and hirudin gene with FXa-recognition sequence at its 5'-terminal were obtained by RT-PCR and PCR respectively. The fusion protein gene was cloned into plasmid pIC9K and electroporated into the genome of Pichia pastoris GS115. The expression of fusion protein was induced by methanol in shaking flask and secreted into the culture medium. Two forms of the fusion protein, single-chain and double-chain linked by a disulfide bond (due to the cleveage of t-PA at Arg275-Ile276), were obtained. The intact fusion protein retained the fibrinolytic activity but lacked any anticoagulant activity. After cleavage by FXa, the fusion protein liberated intact free hirudin to exert its anticoagulant activity. So, the fusion protein is a bifunctional molecule having good prospect to develop into a new targeted therapeutic agent with reduced bleeding side effect for thrombotic diseases.

Plasminogen activator inhibitor-2 in patients with monocytic leukemia.

PubMed

Scherrer, A; Kruithof, E K; Grob, J P

1991-06-01

Plasma and tumor cells from 103 patients with leukemia or lymphoma at initial presentation were investigated for the presence of plasminogen activator inhibitor-2 (PAI-2) antigen, a potent inhibitor of urokinase. PAI-2 was detected in plasma and leukemic cells of the 21 patients with leukemia having a monocytic component [acute myelomonocytic (M4), acute monoblastic (M5), and chronic myelomonocytic leukemias], and in the three patients with acute undifferentiated myeloblastic leukemia (M0). In contrast, this serine protease inhibitor was undetectable in 79 patients with other subtypes of acute myeloid leukemia or other hematological malignancies. Serial serum PAI-2 determinations in 16 patients with acute leukemia at presentation, during therapy, remission, and relapse revealed that in the five patients with M4-M5, elevated PAI-2 levels rapidly normalized under therapy and during remission, but increased again in the patients with a relapse associated with an M4-M5 phenotype. Thus, PAI-2 seems to be a marker highly specific for the active stages of monocytic leukemia, i.e. presentation and relapse. The presence of PAI-2 in the plasma and cells of patients with M0 may give a clue to a monocytic origin of these cells.

Monomer–dimer dynamics and distribution of GPI-anchored uPAR are determined by cell surface protein assemblies

PubMed Central

Caiolfa, Valeria R.; Zamai, Moreno; Malengo, Gabriele; Andolfo, Annapaola; Madsen, Chris D.; Sutin, Jason; Digman, Michelle A.; Gratton, Enrico; Blasi, Francesco; Sidenius, Nicolai

2007-01-01

To search for functional links between glycosylphosphatidylinositol (GPI) protein monomer–oligomer exchange and membrane dynamics and confinement, we studied urokinase plasminogen activator (uPA) receptor (uPAR), a GPI receptor involved in the regulation of cell adhesion, migration, and proliferation. Using a functionally active fluorescent protein–uPAR in live cells, we analyzed the effect that extracellular matrix proteins and uPAR ligands have on uPAR dynamics and dimerization at the cell membrane. Vitronectin directs the recruitment of dimers and slows down the diffusion of the receptors at the basal membrane. The commitment to uPA–plasminogen activator inhibitor type 1–mediated endocytosis and recycling modifies uPAR diffusion and induces an exchange between uPAR monomers and dimers. This exchange is fully reversible. The data demonstrate that cell surface protein assemblies are important in regulating the dynamics and localization of uPAR at the cell membrane and the exchange of monomers and dimers. These results also provide a strong rationale for dynamic studies of GPI-anchored molecules in live cells at steady state and in the absence of cross-linker/clustering agents. PMID:18056417

Regulated Proteolytic Processing of Reelin through Interplay of Tissue Plasminogen Activator (tPA), ADAMTS-4, ADAMTS-5, and Their Modulators

PubMed Central

Krstic, Dimitrije; Rodriguez, Myriam; Knuesel, Irene

2012-01-01

The extracellular signaling protein Reelin, indispensable for proper neuronal migration and cortical layering during development, is also expressed in the adult brain where it modulates synaptic functions. It has been shown that proteolytic processing of Reelin decreases its signaling activity and promotes Reelin aggregation in vitro, and that proteolytic processing is affected in various neurological disorders, including Alzheimer's disease (AD). However, neither the pathophysiological significance of dysregulated Reelin cleavage, nor the involved proteases and their modulators are known. Here we identified the serine protease tissue plasminogen activator (tPA) and two matrix metalloproteinases, ADAMTS-4 and ADAMTS-5, as Reelin cleaving enzymes. Moreover, we assessed the influence of several endogenous protease inhibitors, including tissue inhibitors of metalloproteinases (TIMPs), α-2-Macroglobulin, and multiple serpins, as well as matrix metalloproteinase 9 (MMP-9) on Reelin cleavage, and described their complex interplay in the regulation of this process. Finally, we could demonstrate that in the murine hippocampus, the expression levels and localization of Reelin proteases largely overlap with that of Reelin. While this pattern remained stable during normal aging, changes in their protein levels coincided with accelerated Reelin aggregation in a mouse model of AD. PMID:23082219

Microglia activation due to obesity programs metabolic failure leading to type two diabetes

PubMed Central

Maldonado-Ruiz, R; Montalvo-Martínez, L; Fuentes-Mera, L; Camacho, A

2017-01-01

Obesity is an energy metabolism disorder that increases susceptibility to the development of metabolic diseases. Recently, it has been described that obese subjects have a phenotype of chronic inflammation in organs that are metabolically relevant for glucose homeostasis and energy. Altered expression of immune system molecules such as interleukins IL-1, IL-6, IL-18, tumor necrosis factor alpha (TNF-α), serum amyloid A (SAA), and plasminogen activator inhibitor-1 (PAI-1), among others, has been associated with the development of chronic inflammation in obesity. Chronic inflammation modulates the development of metabolic-related comorbidities like metabolic syndrome (insulin resistance, glucose tolerance, hypertension and hyperlipidemia). Recent evidence suggests that microglia activation in the central nervous system (CNS) is a priority in the deregulation of energy homeostasis and promotes increased glucose levels. This review will cover the most significant advances that explore the molecular signals during microglia activation and inflammatory stage in the brain in the context of obesity, and its influence on the development of metabolic syndrome and type two diabetes. PMID:28319103

Mosaic serine proteases in the mammalian central nervous system.

PubMed

Mitsui, Shinichi; Watanabe, Yoshihisa; Yamaguchi, Tatsuyuki; Yamaguchi, Nozomi

2008-01-01

We review the structure and function of three kinds of mosaic serine proteases expressed in the mammalian central nervous system (CNS). Mosaic serine proteases have several domains in the proenzyme fragment, which modulate proteolytic function, and a protease domain at the C-terminus. Spinesin/TMPRSS5 is a transmembrane serine protease whose presynaptic distribution on motor neurons in the spinal cord suggests that it is significant for neuronal plasticity. Cell type-specific alternative splicing gives this protease diverse functions by modulating its intracellular localization. Motopsin/PRSS12 is a mosaic protease, and loss of its function causes mental retardation. Recent reports indicate the significance of this protease for cognitive function. We mention the fibrinolytic protease, tissue plasminogen activator (tPA), which has physiological and pathological functions in the CNS.

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. Copyright 2009 S. Karger AG, Basel.

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

PubMed

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

2017-04-01

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. © 2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Lsa30, a novel adhesin of Leptospira interrogans binds human plasminogen and the complement regulator C4bp.

PubMed

Souza, Natalie M; Vieira, Monica L; Alves, Ivy J; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

2012-09-01

Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Surface proteins have the potential to promote several activities, including adhesion. This work aimed to study the leptospiral coding sequence (CDS) LIC11087, genome annotated as hypothetical outer membrane protein. The LIC11087 gene was cloned and expressed in Escherichia coli BL21 (DE3) strain by using the expression vector pAE. The recombinant protein tagged with N-terminal 6XHis was purified by metal-charged chromatography and characterized by circular dichroism (CD) spectroscopy. The recombinant protein has the ability to mediate attachment to the extracellular matrix (ECM) components, laminin and plasma fibronectin, and was named Lsa30 (Leptospiral surface adhesin of 30 kDa). Lsa30 binds to laminin and to plasma fibronectin in a dose-dependent and saturable manner, with dissociation equilibrium constants (K(D)) of 292 ± 24 nm and 157 ± 35 nm, respectively. Moreover, the Lsa30 is a plasminogen (PLG) receptor, capable of generating plasmin, in the presence of activator. This protein may interfere with the complement cascade by interacting with C4bp regulator. The Lsa30 is probably a new surface protein of Leptospira as revealed by immunofluorescence assays with living organisms and the reactivity with antibodies present in serum samples of experimentally infected hamsters. Thus, Lsa30 is a novel versatile protein that may play a role in mediating adhesion and may help pathogenic Leptospira to overcome tissue barriers and to escape the immune system. Copyright © 2012 Elsevier Ltd. All rights reserved.

Diagnostic value of decoy receptor 3 combined with procalcitonin and soluble urokinase-type plasminogen activator receptor for sepsis.

PubMed

Zhao, Jing-Jing; Lou, Xiao-Li; Chen, Hong-Wei; Zhu, Feng-Ting; Hou, Yan-Qiang

2018-01-01

The levels of decoy receptor 3 (DcR3), soluble urokinase type plasminogen activator receptor (suPAR) and procalcitonin (PCT) are significantly increased in sepsis. We investigated the diagnostic value of DcR3 combined with suPAR and PCT in sepsis. Patients with sepsis, non-infectious systemic inflammatory response comprehensive syndrome (SIRS) and healthy controls were recruited according to the diagnostic standard. We measured DcR3, suPAR, PCT, interleukin-6 (IL-6) and C-reactive protein (CRP), and the diagnostic value was evaluated by receiver operating characteristics (ROC) curves. In our analysis, serum DcR3, suPAR and PCT levels of the sepsis group were significantly higher than those of the SIRS and control groups. However, IL-6, CRP and WBC showed no significant difference between the SIRS group and the sepsis group. The serum DcR3 level was positively correlated with the serum suPAR level ( r  = 0.37, p  = 0.0022) and PCT level ( r  = 0.37, p  = 0.0021). Using DcR3, suPAR and PCT to distinguish SIRS from sepsis, the area under the curve (AUC) values were 0.892, 0.778 and 0.692. When DcR3, suPAR and PCT combined were used for diagnosis of sepsis, the AUC was 0.933, at a cut-off point of 0.342. This combination improved the sensitivity and specificity of diagnosis of sepsis, suggesting that use of the combination of three indexes enhanced the efficiency of sepsis diagnosis.

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

Thrombus resolution and hemodynamic recovery using ultrasound-accelerated thrombolysis in acute pulmonary embolism.

PubMed

Kennedy, Robert J; Kenney, Hai H; Dunfee, Brian L

2013-06-01

To evaluate retrospectively the safety profile and clinical success of ultrasound-accelerated thrombolysis for acute pulmonary embolism (PE) with a standard lytic infusion protocol. A retrospective study was performed at a single center treating patients with acute PE between October 2009 and April 2012. On diagnosis of submassive or massive PE by pulmonary computed tomography angiography or ventilation/perfusion scan, all patients received anticoagulation and treatment using the EkoSonic endovascular system (EKOS Corporation, Bothell, Washington). The ultrasound-accelerated thrombolytic infusion catheters were placed into the affected pulmonary arteries to facilitate administration of recombinant tissue plasminogen activator at 0.5-1.0mg/h/catheter. Treatment of 60 patients (35 men, 25 women; age 61 y±16; 53 bilateral PE; 48 submassive PE) resulted in complete thrombus clearance (≥90%) in 57% and near-complete (50%-90%) clearance in 41% of patients after infusion of 35.1 mg±11.1 of recombinant tissue plasminogen activator over 19.6 hours±6.0. Measurements before and after treatment showed a decrease in pulmonary artery pressure (47 mm Hg±15 to 38 mm Hg±12 [systolic], P<.001) and Miller score (25±3 to 17±6, P<.001). There were 57 patients who survived to discharge. All three patients who died in the hospital presented with massive PE. On 90-day follow-up, 56 patients (93%) were alive. The current study demonstrates effectiveness and safety of ultrasound-accelerated thrombolysis in patients with acute PE with a large thrombus burden. Copyright © 2013 SIR. Published by Elsevier Inc. All rights reserved.

Neural stem cells rescue nervous purkinje neurons by restoring molecular homeostasis of tissue plasminogen activator and downstream targets.

PubMed

Li, Jianxue; Imitola, Jaime; Snyder, Evan Y; Sidman, Richard L

2006-07-26

Neural stem cells (NSCs) offer special therapeutic prospects because they can be isolated from the CNS, expanded ex vivo, and re-implanted into diseased CNS where they not only migrate and differentiate according to cues from host tissue but also appear to be capable of affecting host cells. In nervous (nr) mutant mice Purkinje neuron (PN) mitochondria become abnormal by the second postnatal week, and a majority of PNs die in the fourth to fifth weeks. We previously identified in nr cerebellum a 10-fold increase in tissue plasminogen activator (tPA) as a key component of the mechanism causing nr PN death. Here we report that undifferentiated wild-type murine NSCs, when transplanted into the newborn nr cerebellar cortex, do not replace host PNs but contact imperiled PNs and support their mitochondrial function, dendritic growth, and synaptogenesis, subsequently leading to the rescue of host PNs and restoration of motor coordination. This protection of nr PNs also is verified by an in vitro organotypic slice model in which nr cerebellar slices are cocultured with NSCs. Most importantly, the integrated NSCs in young nr cerebellum rectify excessive tPA mRNA and protein to close to normal levels and protect the mitochondrial voltage-dependent anion channel and neurotrophins, downstream targets of the tPA/plasmin proteolytic system. This report demonstrates for the first time that NSCs can rescue imperiled host neurons by rectifying their gene expression, elevating somatic stem cell therapeutic potential beyond solely cell replacement strategy.

Plasminogen binding inhibitors demonstrate unwanted activities on GABAA and glycine receptors in human iPSC derived neurons.

PubMed

Kristensson, Lisbeth; Lundin, Anders; Gustafsson, David; Fryklund, Jan; Fex, Tomas; Louise, Delsing; Ryberg, Erik

2018-05-11

Plasminogen binding inhibitors (PBIs) reduce the risk of bleeding in hemorrhagic conditions. However, generic PBIs are also associated with an increased risk of seizures, an adverse effect linked to unwanted activities towards inhibitory neuronal receptors. Development of novel PBIs serve to remove compounds with such properties, but progress is limited by a lack of higher throughput methods with human translatability. Herein we apply human induced pluripotent stem cell (hiPSC) derived neurons in combination with dynamic mass redistribution (DMR) technology to demonstrate robust and reproducible modulation of both GABA A and glycine receptors. These cells respond to GABA (EC 50 0.33 ± 0.18 μM), glycine (EC 50 11.0 ± 3.7 μM) and additional ligands in line with previous reports from patch clamp technologies. Additionally, we identify and characterize a competitive antagonistic behavior of the prototype inhibitor and drug tranexamic acid (TXA). Finally, we demonstrate proof of concept for effective counter-screening of lead series compounds towards unwanted GABA A receptor activities. No activity was observed for a previously identified PBI candidate drug, AZD6564, whereas a discontinued analog, AZ13267257, could be characterized as a potent GABA A receptor agonist. Copyright © 2018. Published by Elsevier B.V.

The Role of Plasminogen Activator Inhibitor-1 and Angiotensin-Converting Enzyme Gene Polymorphisms in Bronchopulmonary Dysplasia

PubMed Central

Atac, Fatma Belgin; Ozkiraz, Servet; Dilmen, Ugur; Gulcan, Hande; Tarcan, Aylin; Ozbek, Namik

2010-01-01

Background: Bronchopulmonary dysplasia (BPD) is a multifactorial disease of preterm infants that is characterized by airway injury, inflammation, and parencymal remodeling. Activation of the coagulation cascade leads to intraalveolar fibrin deposition in many inflammatory pulmonary disorders. Increased fibrin formation or decreased fibrinolysis may cause extravascular fibrin deposition. Extravascular fibrin deposits in septae and alveoli due to the altered fibrin turnover are the pathological hallmarks of BPD, which strongly indicate the importance of the imbalance in the competing activities of coagulation and fibrinolysis. Objective: We investigated the predictive value of variations in plasminogen activator inhibitor-1 (PAI-1) and angiotensin-converting enzyme (ACE) genes as molecular determinants for BPD in neonates. Methods: The study group comprised 98 preterm infants with BPD and a control group including 94 preterm infants without BPD. Restriction fragment size analyses were performed by visualizing digested polymerase chain reaction products for ACE and PAI-1 genotypes. Results: No significant associations were found between ACE, PAI-1 gene polymorphisms, and BPD phenotype in our population. Conclusions: The two gene polymorphisms (PAI-1 and ACE) had no role in the development of BPD in our study. Further studies with other genes are required for the identification of molecular predisposing factors for BPD that may help in the development of new treatments. PMID:20818980

THE ROLE OF MICROVASCULAR THROMBOSIS IN PARTICULATE MATTER (PM) AND PM COMPONENT-INDUCED CARDIOVASCULAR EFFECTS: OXIDATIVE STRESS AS A MEDIATOR OF THROMBOSIS

EPA Science Inventory

Particulate matter (PM) exposure has been associated with increased plasma fibrinogen. We have found that Spontaneously hypertensive rats respond to PM by increasing fibrinogen and plasminogen activator inhibitor -1 at PM concentration that would cause minimal changes in healthy ...

[Managment of subretinal heamorrhages within the macular area using intravitreal injections of recombined tissue plasminogen activator, sulphur hexafluoride and ranihizumab--preliminary report].

PubMed

Miniewicz, Joanna; Kubicka-Trząska, Agnieszka; Karska-Basta, Izabella; Romanowska-Dixon, Boźena

2015-01-01

Submacular hemorrhages cause serious vision impairment. Patient observation, waiting for the spontaneous blood reabsorption and resolution of the haemorrhage leads to the severe damage to retinal tissue as a result of scar formation. The paper presents 7 cases of patients with submacular haemorrhages treated with intravitreal injections of recombined tissue plasminogen activator (rtPA) and sulphur hexafluoride (SFG). In 4 cases, the haemorrhage was secondary to AMD, in two cases to trauma, and it was idiopathic in one case. All patients were treated with intravitreal injections of rtPA and SF6 for thrombolysis and pneumatic displacement of haemorrhage outside macular structures. Ranibizumab was additionally administered to patients with age-related macular degeneration. Such treatment improved visual acuity in all patients, reducing the central retinal thickness as shown in follow-up optical coherence tomography. The presented treatment of submacular hemorrhages with intravitreal injections of rtPA and SF6 provided good results, but in order to develop a standard management algorithm for this disease, the analysis of larger patient sample is required.

Isolation and purification of recombinant human plasminogen Kringle 5 by liquid chromatography and ammonium sulfate salting-out.

PubMed

Bian, Liujiao; Ji, Xu; Hu, Wei

2014-07-01

In this work, a novel method was established to isolate and purify Human plasminogen Kringle 5 (HPK5) as a histidine-tagged fusion protein expressed in Escherichia coli BL21 (DE3). This method consisted of sample extraction using a Ni-chelated Sepharose Fast-Flow affinity column, ammonium sulfate salting-out and Sephadex G-75 size-exclusion column in turn. The purity analysis by SDS-PAGE, high-performance size-exclusion and reversed-phase chromatographies showed that the obtained recombinant fusion HPK5 was homogeneous and its purity was higher than 96%; the activity analysis by chorioallantoic membrane model of chicken embryos revealed that the purified recombinant HPK5 exhibited an obvious anti-angiogenic activity under the effective range of 5.0-25.0 µg/mL. Through this procedure, about 19 mg purified recombinant fusion HPK5 can be obtained from 1 L of original fermentation solution. Approximate 32% of the total recombinant fusion HPK5 can be captured and the total yield was approximately 11%. Copyright © 2013 John Wiley & Sons, Ltd.

The high-level expression of human tissue plasminogen activator in the milk of transgenic mice with hybrid gene locus strategy.

PubMed

Zhou, Yanrong; Lin, Yanli; Wu, Xiaojie; Xiong, Fuyin; Lv, Yuemeng; Zheng, Tao; Huang, Peitang; Chen, Hongxing

2012-02-01

Transgene expression for the mammary gland bioreactor aimed at producing recombinant proteins requires optimized expression vector construction. Previously we presented a hybrid gene locus strategy, which was originally tested with human lactoferrin (hLF) as target transgene, and an extremely high-level expression of rhLF ever been achieved as to 29.8 g/l in mice milk. Here to demonstrate the broad application of this strategy, another 38.4 kb mWAP-htPA hybrid gene locus was constructed, in which the 3-kb genomic coding sequence in the 24-kb mouse whey acidic protein (mWAP) gene locus was substituted by the 17.4-kb genomic coding sequence of human tissue plasminogen activator (htPA), exactly from the start codon to the end codon. Corresponding five transgenic mice lines were generated and the highest expression level of rhtPA in the milk attained as to 3.3 g/l. Our strategy will provide a universal way for the large-scale production of pharmaceutical proteins in the mammary gland of transgenic animals.

Biomarkers of Inflammation, Thrombogenesis, and Collagen Turnover in Patients With Atrial Fibrillation.

PubMed

Jabati, Sallu; Fareed, Jawed; Liles, Jeffrey; Otto, Abigail; Hoppensteadt, Debra; Bontekoe, Jack; Phan, Trung; Walborn, Amanda; Syed, Mushabbar

2018-07-01

The purpose of this study was to determine whether there are any differences in the levels of inflammatory, thrombotic, and collagen turnover biomarkers between individuals with atrial fibrillation (AF) and healthy volunteers. Circulating plasma levels of plasminogen activator inhibitor 1 (PAI-1), CD40-ligand (CD40-L), nucleosomes (which are indicators of cell death), C-reactive protein (CRP), procollagen III N-terminal propeptide (PIIINP), procollagen III C-terminal propeptide (PIIICP), procollagen I N-terminal propeptide, tissue plasminogen activator, and von Willebrand factor were analyzed as potential biomarkers of AF. Baseline plasma was collected from patients with AF prior to ablation surgery at Loyola University Medical Center. Individuals with AF had statistically significantly increased levels of PAI-1, CD40-L, and nucleosomes, when compared to the normal population ( P < .0001). Additionally, there was a statistically significant increase in the CRP ( P = .01), PIIINP ( P = .04), and PIIICP ( P = .0008) when compared to normal individuals. From this study, it is concluded that the prothrombotic, inflammatory, and collagen turnover biomarkers PAI-1, CD40-L, nucleosomes, CRP, PIIICP, and PIIINP are elevated in AF.

Inhibitory Effects of North American Wild Rice on Monocyte Adhesion and Inflammatory Modulators in Low-Density Lipoprotein Receptor-Knockout Mice.

PubMed

Moghadasian, Mohammed H; Zhao, Ruozhi; Ghazawwi, Nora; Le, Khuong; Apea-Bah, Franklin B; Beta, Trust; Shen, Garry X

2017-10-18

The present study examined the effects of wild rice on monocyte adhesion, inflammatory and fibrinolytic mediators in low-density lipoprotein receptor-knockout (LDLr-KO) mice. Male LDLr-KO mice received a cholesterol (0.06%, w/w)-supplemented diet with or without white or wild rice (60%, w/w) for 20 weeks. White rice significantly increased monocyte adhesion and abundances of monocyte chemoattractant protein-1, tissue necrosis factor-α, intracellular cell adhesion molecule-1, plasminogen activator inhibitor-1, urokinase plasminogen activator (uPA), and uPA receptor in aortae and hearts of LDLr-KO mice compared to the control diet. Wild rice inhibited monocyte adhesion to the aorta, atherosclerosis, and abundances of the inflammatory and fibrinolytic regulators in the cardiovascular tissue of LDLr-KO mice compared to white rice. White or wild rice did not significantly alter the levels of cholesterol, triglycerides, or antioxidant enzymes in plasma. The anti-atherosclerotic effect of wild rice may result from its inhibition on monocyte adhesion and inflammatory modulators in LDLr-KO mice.

Telemedicine Can Replace the Neurologist on a Mobile Stroke Unit.

PubMed

Wu, Tzu-Ching; Parker, Stephanie A; Jagolino, Amanda; Yamal, Jose-Miguel; Bowry, Ritvij; Thomas, Abraham; Yu, Amy; Grotta, James C

2017-02-01

The BEST-MSU study (Benefits of Stroke Treatment Delivered Using a Mobile Stroke Unit) is a comparative effectiveness trial in patients randomized to mobile stroke unit or standard management. A substudy tested interrater agreement for tissue-type plasminogen activator eligibility between a telemedicine vascular neurologist and onboard vascular neurologist. On scene, both the telemedicine vascular neurologist and onboard vascular neurologist independently evaluated the patient, documenting their tissue-type plasminogen activator treatment decision, National Institutes of Health Stroke Scale score, and computed tomographic interpretation. Agreement was determined using Cohen κ statistic. Telemedicine-related technical failures that impeded remote assessment were recorded. Simultaneous and independent telemedicine vascular neurologist and onboard vascular neurologist assessment was attempted in 174 patients. In 4 patients (2%), the telemedicine vascular neurologist could not make a decision because of technical problems. The telemedicine vascular neurologist agreed with the onboard vascular neurologist on 88% of evaluations (κ=0.73). Remote telemedicine vascular neurologist assessment is reliable and accurate, supporting either telemedicine vascular neurologist or onboard vascular neurologist assessment on our mobile stroke unit. URL: http://www.clinicaltrials.gov. Unique identifier: NCT02190500. © 2017 American Heart Association, Inc.

Plasminogen activator inhibitor-1 4G/5G polymorphism is associated with type 2 diabetes risk

PubMed Central

Zhao, Luqian; Huang, Ping

2013-01-01

A number of studies were performed to assess the association between plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphism and susceptibility to type 2 diabetes (T2DM). However, the results were inconsistent and inconclusive. In the present study, the possible association was investigated by a meta-analysis. Eligible articles were identified for the period up to June 2013. Pooled odds ratios (OR) with 95% confidence intervals (CI) were appropriately derived from random-effects models or fixed-effects models. Fourteen case-control studies with a total of 2487 cases and 3538 controls were eligible. In recessive model, PAI-1 4G/5G polymorphism was associated with T2DM risk (OR = 1.23; 95% CI 1.07-1.41; P = 0.004). In the subgroup analysis by ethnicity, a significant association was found among Asians (OR = 1.27; 95% CI 1.08-1.51; P = 0.005). This meta-analysis suggested that PAI-1 4G/5G polymorphism may be associated with T2DM development. PMID:24040470

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.

Rationale for the selective administration of tranexamic acid to inhibit fibrinolysis in the severely injured patient.

PubMed

Moore, Ernest E; Moore, Hunter B; Gonzalez, Eduardo; Sauaia, Angela; Banerjee, Anirban; Silliman, Christopher C

2016-04-01

Postinjury fibrinolysis can manifest as three distinguishable phenotypes: 1) hyperfibrinolysis, 2) physiologic, and 3) hypofibrinolysis (shutdown). Hyperfibrinolysis is associated with uncontrolled bleeding due to clot dissolution; whereas, fibrinolysis shutdown is associated with organ dysfunction due to microvascular occlusion. The incidence of fibrinolysis phenotypes at hospital arrival in severely injured patients is: 1) hyperfibrinolysis 18%, physiologic 18%, and shutdown 64%. The mechanisms responsible for dysregulated fibrinolysis following injury remain uncertain. Animal work suggests hypoperfusion promotes fibrinolysis, while tissue injury inhibits fibrinolysis. Clinical experience is consistent with these observations. The predominant mediator of postinjury hyperfibrinolysis appears to be tissue plasminogen activator (tPA) released from ischemic endothelium. The effects of tPA are accentuated by impaired hepatic clearance. Fibrinolysis shutdown, on the other hand, may occur from inhibition of circulating tPA, enhanced clot strength impairing the binding of tPA and plasminogen to fibrin, or the inhibition of plasmin. Plasminogen activator inhibitor -1 (PAI-1) binding of circulating tPA appears to be a major mechanism for postinjury shutdown. The sources of PAI-1 include endothelium, platelets, and organ parenchyma. The laboratory identification of fibrinolysis phenotype, at this moment, is best determined with viscoelastic hemostatic assays (TEG, ROTEM). While D-dimer and plasmin antiplasmin (PAP) levels corroborate fibrinolysis, they do not provide real-time assessment of the circulating blood capacity. Our clinical studies indicate that fibrinolysis is a very dynamic process and our experimental work suggests plasma first resuscitation reverses hyperfibrinolysis. Collectively, we believe recent clinical and experimental work suggest antifibrinolytic therapy should be employed selectively in the acutely injured patient, and optimally guided by TEG or ROTEM. © 2016 AABB.

Adiponectin, C-reactive protein, fibrinogen and tissue plasminogen activator antigen levels among glucose-intolerant women with and without histories of gestational diabetes.

PubMed

Kim, C; Christophi, C A; Goldberg, R B; Perreault, L; Dabelea, D; Marcovina, S M; Pi-Sunyer, X; Barrett-Connor, E

2016-01-01

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 (GDM). 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 histories of GDM (n = 1466). Cox proportional hazard models evaluated whether history of GDM was associated with diabetes risk, after adjustment for baseline biomarker levels as well as for change in biomarker levels, demographic factors and anthropometrics. At baseline, women with histories of GDM 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 GDM, 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 GDM. Women with and without histories of GDM 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 GDM, and diabetes risk. Among women with impaired glucose tolerance, biomarkers in women with and without histories of GDM 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 histories of GDM. © 2015 The Authors. Diabetic Medicine © 2015 Diabetes UK.

Differential Effects of Nebivolol and Metoprolol on Insulin Sensitivity and Plasminogen Activator Inhibitor in the Metabolic Syndrome

PubMed Central

Ayers, Katie; Byrne, Loretta M.; DeMatteo, Anthony; Brown, Nancy J.

2012-01-01

Early generation β-blockers lower blood pressure and reduce cardiovascular morality in coronary artery disease and congestive heart failure, but worsen glucose homeostasis and fibrinolytic balance. Nebivolol is a third-generation β-blocker which increases the bioavailability of nitric oxide. We compared the effect of nebivolol (5mg/d) and the β1-selective antagonist metoprolol (100mg/d) on glucose homeostasis and markers of fibrinolysis in 46 subjects with metabolic syndrome. Subjects underwent a frequently sampled intravenous glucose tolerance test after 3-week washout and placebo treatment, and following randomized treatment with study drug. After 12-week treatment, nebivolol and metoprolol equivalently decreased systolic blood pressure, diastolic blood pressure, and heart rate. Neither drug affected beta cell function, disposition index, or acute insulin response to glucose. Metoprolol significantly decreased the insulin sensitivity index. In contrast, nebivolol did not affect insulin sensitivity, and the decrease in sensitivity was significantly greater following metoprolol than nebivolol (-1.5±2.5 × 10-4 × min-1 per mU/L versus 0.04±2.19 × 10-4 × min-1 per mU/L after nebivolol, P=0.03). Circulating plasminogen activator inhibitor also increased following treatment with metoprolol (from 9.8±6.8 to 12.3±7.8 ng/mL), but not nebivolol (from 10.8±7.8 to 10.5±6.2 ng/mL, P=0.05 versus metoprolol). Metoprolol, but not nebivolol, increased F2-isoprostane concentrations. In summary, treatment with metoprolol decreased insulin sensitivity and increased oxidative stress and the antifibrinolytic plasminogen activator inhibitor-1in patients with metabolic syndrome, whereas nebivolol lacked detrimental metabolic effects. Large clinical trials are needed to compare effects of nebivolol and the β1 receptor antagonist metoprolol on clinical outcomes in patients with hypertension and the metabolic syndrome. PMID:22353614

Safety of tPA in stroke mimics and neuroimaging-negative cerebral ischemia(Podcast)(e–Pub ahead of print)(CME)

PubMed Central

Chernyshev, O.Y.; Martin-Schild, S.; Albright, K.C.; Barreto, A.; Misra, V.; Acosta, I.; Grotta, J.C.; Savitz, S.I.

2010-01-01

Background: Patients with acute neurologic symptoms may have other causes simulating ischemic stroke, called stroke mimics (SM), but they may also have averted strokes that do not appear as infarcts on neuroimaging, which we call neuroimaging-negative cerebral ischemia (NNCI). We determined the safety and outcome of IV thrombolysis within 3 hours of symptom onset in patients with SM and NNCI. Methods: Patients treated with IV tissue plasminogen activator (tPA) within 3 hours of symptom onset were identified from our stroke registry from June 2004 to October 2008. We collected admission NIH Stroke Scale (NIHSS) score, modified Rankin score (mRS), length of stay (LOS), symptomatic intracerebral hemorrhage (sICH), and discharge diagnosis. Results: Among 512 treated patients, 21% were found not to have an infarct on follow-up imaging. In the SM group (14%), average age was 55 years, median admission NIHSS was 7, median discharge NIHSS was 0, median LOS was 3 days, and there were no instances of sICH. The most common etiologies were seizure, complicated migraine, and conversion disorder. In the NNCI group (7%), average age was 61 years, median admission NIHSS was 7, median discharge NIHSS was 0, median LOS was 3 days, and there were no instances of sICH. Nearly all SM (87%) and NNCI (91%) patients were functionally independent on discharge (mRS 0–1). Conclusions: Our data support the safety of administering IV tissue plasminogen activator to patients with suspected acute cerebral ischemia within 3 hours of symptom onset, even when the diagnosis ultimately is found not to be stroke or imaging does not show an infarct. GLOSSARY AIS = acute ischemic stroke; CI = confidence interval; DWI = diffusion-weighted imaging; ED = emergency department; LOS = length of stay; mRS = modified Rankin score; NIHSS = NIH Stroke Scale; NNCI = neuroimaging-negative cerebral ischemia; OR = odds ratio; sICH = symptomatic intracerebral hemorrhage; SM = stroke mimics; tPA = tissue plasminogen activator. PMID:20335564

Triglycerides as an early pathophysiological marker of endothelial dysfunction in nondiabetic women with a previous history of gestational diabetes.

PubMed

Sokup, Alina; Góralczyk, Barbara; Góralczyk, Krzysztof; Rość, Danuta

2012-02-01

To investigate whether baseline triglyceride levels are associated with early glucose dysregulation and/or cardiovascular risk in women with a previous history of gestational diabetes. Prospective postpregnancy cohort study. Polish university hospitals. Participants included 125 women with previous gestational diabetes and 40 women with normal glucose regulation during pregnancy. All women were studied 2-24 months (mean 12 ± 10 months) after the index pregnancy. Women with previous gestational diabetes were divided into tertiles in accordance with baseline triglyceride levels. We assessed glucose regulation (oral glucose tolerance test), insulin resistance (homeostasis model assessment), markers of endothelial dysfunction (soluble: intercellular adhesion molecule-1, vascular cell adhesion molecule-1, E-selectin, tissue plasminogen activator antigen, von Willebrand factor antigen), fibrinolysis (plasminogen activator inhibitor antigen), inflammation (high-sensitivity C-reactive protein) and lipid levels. Women with previous gestational diabetes (78% normal glucose regulation, 22% impaired glucose tolerance) had a high cardiometabolic risk profile compared with control women (100% normal glucose regulation). Baseline triglycerides >0.83 mmol/l were associated with a higher prevalence of impaired glucose tolerance, higher high-sensitivity C-reactive protein and triglyceride/high-density lipoprotein-cholesterol ratio. Triglycerides >1.22 mmol/l were associated with higher body fat indexes, higher insulin resistance, higher levels of endothelial dysfunction biomarkers, higher plasminogen activator inhibitor antigen and dyslipidemia. Only E-selectin was independently associated with triglyceride levels. Baseline triglyceride levels are a cardiovascular risk marker as well as a pathophysiological parameter independently associated with endothelial dysfunction in nondiabetic women with previous gestational diabetes at 2-24 months after an index pregnancy. Normalization of triglycerides should be included in preventive therapy after a pregnancy complicated by gestational diabetes. © 2012 The Authors Acta Obstetricia et Gynecologica Scandinavica© 2012 Nordic Federation of Societies of Obstetrics and Gynecology.

Urokinase-Type Plasminogen Activator Receptor Is Internalized by Different Mechanisms in Polarized and Nonpolarized Madin–Darby Canine Kidney Epithelial Cells

PubMed Central

Vilhardt, Frederik; Nielsen, Morten; Sandvig, Kirsten; van Deurs, Bo

1999-01-01

Accumulated data indicate that endocytosis of the glycosylphosphatidyl-inositol-anchored protein urokinase plasminogen activator receptor (uPAR) depends on binding of the ligand uPA:plasminogen activator inhibitor-1 (PAI-1) and subsequent interaction with internalization receptors of the low-density lipoprotein receptor family, which are internalized through clathrin-coated pits. This interaction is inhibited by receptor-associated protein (RAP). We show that uPAR with bound uPA:PAI-1 is capable of entering cells in a clathrin-independent process. First, HeLaK44A cells expressing mutant dynamin efficiently internalized uPA:PAI-1 under conditions in which transferrin endocytosis was blocked. Second, in polarized Madin–Darby canine kidney (MDCK) cells, which expressed human uPAR apically, the low basal rate of uPAR ligand endocytosis, which could not be inhibited by RAP, was increased by forskolin or phorbol ester (phorbol 12-myristate 13-acetate), which selectively up-regulate clathrin-independent endocytosis from the apical domain of epithelial cells. Third, in subconfluent nonpolarized MDCK cells, endocytosis of uPA:PAI-1 was only decreased marginally by RAP. At the ultrastructural level uPAR was largely excluded from clathrin-coated pits in these cells and localized in invaginated caveolae only in the presence of cross-linking antibodies. Interestingly, a larger fraction of uPAR in nonpolarized relative to polarized MDCK cells was insoluble in Triton X-100 at 0°C, and by surface labeling with biotin we also show that internalized uPAR was mainly detergent insoluble, suggesting a correlation between association with detergent-resistant membrane microdomains and higher degree of clathrin-independent endocytosis. Furthermore, by cryoimmunogold labeling we show that 5–10% of internalized uPAR in nonpolarized, but not polarized, MDCK cells is targeted to lysosomes by a mechanism that is regulated by ligand occupancy. PMID:9880335

Magnetic Resonance Imaging-DRAGON score: 3-month outcome prediction after intravenous thrombolysis for anterior circulation stroke.

PubMed

Turc, Guillaume; Apoil, Marion; Naggara, Olivier; Calvet, David; Lamy, Catherine; Tataru, Alina M; Méder, Jean-François; Mas, Jean-Louis; Baron, Jean-Claude; Oppenheim, Catherine; Touzé, Emmanuel

2013-05-01

The DRAGON score, which includes clinical and computed tomographic scan parameters, showed a high specificity to predict 3-month outcome in patients with acute ischemic stroke treated by intravenous tissue plasminogen activator. We adapted the score for patients undergoing MRI as the first-line diagnostic tool. We reviewed patients with consecutive anterior circulation ischemic stroke treated ≤ 4.5 hour by intravenous tissue plasminogen activator between 2003 and 2012 in our center, where MRI is systematically implemented as first-line diagnostic work-up. We derived the MRI-DRAGON score keeping all clinical parameters of computed tomography-DRAGON (age, initial National Institutes of Health Stroke Scale and glucose level, prestroke handicap, onset to treatment time), and considering the following radiological variables: proximal middle cerebral artery occlusion on MR angiography instead of hyperdense middle cerebral artery sign, and diffusion-weighted imaging Alberta Stroke Program Early Computed Tomography Score (DWI ASPECTS) ≤ 5 instead of early infarct signs on computed tomography. Poor 3-month outcome was defined as modified Rankin scale >2. We calculated c-statistics as a measure of predictive ability and performed an internal cross-validation. Two hundred twenty-eight patients were included. Poor outcome was observed in 98 (43%) patients and was significantly associated with all parameters of the MRI-DRAGON score in multivariate analysis, except for onset to treatment time (nonsignificant trend). The c-statistic was 0.83 (95% confidence interval, 0.78-0.88) for poor outcome prediction. All patients with a MRI-DRAGON score ≤ 2 (n=22) had a good outcome, whereas all patients with a score ≥ 8 (n=11) had a poor outcome. The MRI-DRAGON score is a simple tool to predict 3-month outcome in acute stroke patients screened by MRI then treated by intravenous tissue plasminogen activator and may help for therapeutic decision.

Number needed to treat to benefit and to harm for intravenous tissue plasminogen activator therapy in the 3- to 4.5-hour window: joint outcome table analysis of the ECASS 3 trial.

PubMed

Saver, Jeffrey L; Gornbein, Jeffrey; Grotta, James; Liebeskind, David; Lutsep, Helmi; Schwamm, Lee; Scott, Phillip; Starkman, Sidney

2009-07-01

Measures of a therapy's effect size are important guides to clinicians, patients, and policy-makers on treatment decisions in clinical practice. The ECASS 3 trial demonstrated a statistically significant benefit of intravenous tissue plasminogen activator for acute cerebral ischemia in the 3- to 4.5-hour window, but an effect size estimate incorporating benefit and harm across all levels of poststroke disability has not previously been derived. Joint outcome table specification was used to derive number needed to treat to benefit (NNTB) and number needed to treat to harm (NNTH) values summarizing treatment impact over the entire outcome range on the modified Rankin scale of global disability, including both expert-dependent and expert-independent (algorithmic and repeated random sampling) array generation. For the full 7-category modified Rankin scale, algorithmic analysis demonstrated that the NNTB for 1 additional patient to have a better outcome by >or=1 grades than with placebo must lie between 4.0 and 13.0. In bootstrap simulations, the mean NNTB was 7.1. Expert joint outcome table analyses indicated that the NNTB for improved final outcome was 6.1 (95% CI, 5.6-6.7) and the NNTH 37.5 (95% CI, 34.6-40.5). Benefit per 100 patients treated was 16.3 and harm per 100 was 2.7. The likelihood of help to harm ratio was 6.0. Treatment with tissue plasminogen activator in the 3- to 4.5-hour window confers benefit on approximately half as many patients as treatment <3 hours, with no increase in the conferral of harm. Approximately 1 in 6 patients has a better and 1 in 35 has a worse outcome as a result of therapy.

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

PubMed

Serce, Nuran Bektas; Boesl, Andreas; Klaman, Irina; von Serényi, Sonja; Noetzel, Erik; Press, Michael F; Dimmler, Arno; Hartmann, Arndt; Sehouli, Jalid; Knuechel, Ruth; Beckmann, Matthias W; Fasching, Peter A; Dahl, Edgar

2012-12-13

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. 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. 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. The RNA-binding protein SERBP1 is abundantly expressed in human breast cancer and may represent a novel breast tumour marker with prognostic significance. Its potential involvement in the plasminogen activator protease cascade warrants further investigation.

Gastric Expression of Plasminogen Activator Inhibitor (PAI)-1 Is Associated with Hyperphagia and Obesity in Mice

PubMed Central

Kenny, Susan; Gamble, Joanne; Lyons, Suzanne; Vlatković, Nikolina; Dimaline, Rod; Varro, Andrea

2013-01-01

The adipokine plasminogen activator inhibitor (PAI)-1 is increased in plasma of obese individuals and exhibits increased expression in the stomachs of individuals infected with Helicobacter. To investigate the relevance of gastric PAI-1, we used 1.1 kb of the H+/K+β subunit promoter to overexpress PAI-1 specifically in mouse gastric parietal cells (PAI-1-H/Kβ mice). We studied the physiological, biochemical, and behavioral characteristics of these and mice null for PAI-1 or a putative receptor, urokinase plasminogen activator receptor (uPAR). PAI-1-H/Kβ mice had increased plasma concentrations of PAI-1 and increased body mass, adiposity, and hyperphagia compared with wild-type mice. In the latter, food intake was inhibited by cholecystokinin (CCK)8s, but PAI-1-H/Kβ mice were insensitive to the satiating effects of CCK8s. PAI-1-H/Kβ mice also had significantly reduced expression of c-fos in the nucleus tractus solitarius in response to CCK8s and refeeding compared with wild-type mice. Exogenous PAI-1 reversed the effects of CCK8s on food intake and c-fos levels in the nucleus tractus solitarius of wild-type mice, but not uPAR-null mice. Infection of C57BL/6 mice with Helicobacter felis increased gastric abundance of PAI-1 and reduced the satiating effects of CCK8s, whereas the response to CCK8s was maintained in infected PAI-1–null mice. In cultured vagal afferent neurons, PAI-1 inhibited stimulation of neuropeptide Y type 2 receptor (Y2R) expression by CCK8s. Thus, gastric expression of PAI-1 is associated with hyperphagia, moderate obesity, and resistance to the satiating effects of CCK indicating a new role in suppressing signals from the upper gut that inhibit food intake. PMID:23254194

Gastric expression of plasminogen activator inhibitor (PAI)-1 is associated with hyperphagia and obesity in mice.

PubMed

Kenny, Susan; Gamble, Joanne; Lyons, Suzanne; Vlatkovic, Nikolina; Dimaline, Rod; Varro, Andrea; Dockray, Graham J

2013-02-01

The adipokine plasminogen activator inhibitor (PAI)-1 is increased in plasma of obese individuals and exhibits increased expression in the stomachs of individuals infected with Helicobacter. To investigate the relevance of gastric PAI-1, we used 1.1 kb of the H(+)/K(+)β subunit promoter to overexpress PAI-1 specifically in mouse gastric parietal cells (PAI-1-H/Kβ mice). We studied the physiological, biochemical, and behavioral characteristics of these and mice null for PAI-1 or a putative receptor, urokinase plasminogen activator receptor (uPAR). PAI-1-H/Kβ mice had increased plasma concentrations of PAI-1 and increased body mass, adiposity, and hyperphagia compared with wild-type mice. In the latter, food intake was inhibited by cholecystokinin (CCK)8s, but PAI-1-H/Kβ mice were insensitive to the satiating effects of CCK8s. PAI-1-H/Kβ mice also had significantly reduced expression of c-fos in the nucleus tractus solitarius in response to CCK8s and refeeding compared with wild-type mice. Exogenous PAI-1 reversed the effects of CCK8s on food intake and c-fos levels in the nucleus tractus solitarius of wild-type mice, but not uPAR-null mice. Infection of C57BL/6 mice with Helicobacter felis increased gastric abundance of PAI-1 and reduced the satiating effects of CCK8s, whereas the response to CCK8s was maintained in infected PAI-1-null mice. In cultured vagal afferent neurons, PAI-1 inhibited stimulation of neuropeptide Y type 2 receptor (Y2R) expression by CCK8s. Thus, gastric expression of PAI-1 is associated with hyperphagia, moderate obesity, and resistance to the satiating effects of CCK indicating a new role in suppressing signals from the upper gut that inhibit food intake.

Endovascular thrombectomy for acute ischemic stroke in failed intravenous tissue plasminogen activator versus non-intravenous tissue plasminogen activator patients: revascularization and outcomes stratified by the site of arterial occlusions.

PubMed

Shi, Zhong-Song; Loh, Yince; Walker, Gary; Duckwiler, Gary R

2010-06-01

Intracranial mechanical thrombectomy is a therapeutic option for acute ischemic stroke patients failing intravenous tissue plasminogen activator (IV tPA). We compared patients treated by mechanical embolus removal in cerebral ischemia (MERCI) thrombectomy after failed IV tPA with those treated with thrombectomy alone. We pooled MERCI and Multi MERCI study patients, grouped them either as failed IV tPA or non-IV tPA, and assessed revascularization rates, procedural complications, symptomatic hemorrhage rates, clinical outcomes, and mortality. We also evaluated outcomes stratified by the occlusion site and final revascularization. Among 305 patients, 48 failed, and 257 were ineligible for IV tPA. Nonresponders to IV tPA trended toward a higher revascularization rate (73% versus 63%) and less mortality (27.7% versus 40.1%) and had similar rates of symptomatic hemorrhage and procedural complications. Favorable 90-day outcomes were similar in failed and non-IV tPA patients (38% versus 31%), with no difference according to occlusion site. Among patients failing IV tPA, good outcomes tended to occur more frequently in revascularized patients (47.1% versus 15.4%), although this relationship was attributable solely to middle cerebral artery and not internal carotid artery occlusions, with no difference in mortality. Among IV tPA-ineligible patients, revascularization correlated with good outcome (47.4% versus 4.4%) and less mortality (28.5% versus 59.6%). The risks of hemorrhage and procedure-related complications after mechanical thrombectomy do not differ with respect to previous IV tPA administration. Thrombectomy after IV tPA achieves similar rates of good outcomes, a tendency toward lower mortality, and similar revascularization rates when stratified by clot location. Good outcomes correlate with successful revascularization except with internal carotid artery occlusions in tPA-nonresponders.

Years of disability-adjusted life gained as a result of thrombolytic therapy for acute ischemic stroke.

PubMed

Hong, Keun-Sik; Saver, Jeffrey L

2010-03-01

Disability-adjusted life year (DALY) metric reflects years of healthy life lost because of living with disability and years of life lost because of premature mortality. Widely used in epidemiological analyses, DALY has not been applied to acute stroke trials. From previous studies, we derived, for each modified Rankin Scale level, disability weights, disability-linked mortality hazard ratios, and age-specific life expectancies. We then analyzed patient level data from the 2 publicly available National Institute of Neurological Disorders and Stroke (NINDS) recombinant tissue plasminogen activator trials. For each subject, we abstracted age, treatment assignment, and 3-month modified Rankin Scale outcome and calculated the DALYs lost resulting from the qualifying stroke. The disability-linked hazard ratios for premature annual mortality for a modified Rankin Scale score of 0 to 5 were 1.53, 1.52, 2.17, 3.18, 4.55, and 6.55, respectively. In the NINDS recombinant tissue plasminogen activator trials, DALYs (mean+/-SE) lost as a result of the qualifying stroke were substantially less with recombinant tissue plasminogen activator than with placebo (4.64+/-0.17 versus 5.91+/-0.21; P<0.0001), a finding that remained robust after adjustment for baseline prognostic factors. When DALYs gained were apportioned to the 29% of patients experiencing any benefit from lytic therapy, each patient gained an average of 4.4 DALYs. DALY analysis showed greater power than dichotomized modified Rankin Scale analysis in discriminating treatment effects overall and in patients >or=70 years of age. For patients who benefit from treatment, <3-hour thrombolytic therapy adds the equivalent of 4.4 years of healthy life, free of disability. The DALY metric provides a continuous scale that increases statistical power, is intuitively understandable, and is applicable to a wide range of conditions and treatments.

The metabolic syndrome is associated with a higher resistance to intravenous thrombolysis for acute ischemic stroke in women than in men.

PubMed

Arenillas, Juan F; Sandoval, Patricio; Pérez de la Ossa, Natalia; Millán, Mónica; Guerrero, Cristina; Escudero, Domingo; Dorado, Laura; López-Cancio, Elena; Castillo, José; Dávalos, Antoni

2009-02-01

The metabolic syndrome (MetS) might confer a higher resistance to intravenous thrombolysis in acute middle cerebral artery (MCA) ischemic stroke. MetS increases the risk of stroke in women to a greater extent than in men. We aimed to investigate whether there might be sex differences in the impact of MetS on the response to intravenous thrombolysis for acute MCA ischemic stroke. We prospectively studied consecutive ischemic stroke patients, treated with intravenous tissue-type plasminogen activator according to SITS-MOST criteria, with an MCA occlusion on prebolus transcranial Doppler examination. Resistance to thrombolysis was defined as the absence of complete MCA recanalization 24 hours after tissue-type plasminogen activator infusion by transcranial Doppler criteria. MetS was diagnosed according to the criteria established by the American Heart Association/National Heart, Lung, and Blood Institute 2005 statement. A total of 125 patients (75 men, 50 women; mean age, 67.6+/-11 years) were included. MetS was diagnosed in 76 (61%) patients. Resistance to clot lysis at 24 hours was observed in 53 (42%) patients. Two multivariate-adjusted, logistic-regression models identified that MetS was associated with a higher resistance to tissue-type plasminogen activator, independently of other significant baseline variables (odds ratio=9.8; 95% CI, 3.5 to 27.8; P=0.0001) and of the individual components of the MetS. The MetS was associated with a significantly higher odds of resistance to thrombolysis in women (odds ratio=17.5; 95% CI, 1.9 to 163.1) than in men (odds ratio=5.1; 95% CI, 1.6 to 15.6; P for interaction=0.0004). The effect of MetS on the resistance to intravenous thrombolysis for acute MCA ischemic stroke appears to be more pronounced in women than in men.

Engineering streptokinase for generation of active site-labeled plasminogen analogs*

PubMed Central

Laha, Malabika; Panizzi, Peter; Nahrendorf, Matthias; Bock, Paul E.

2011-01-01

We previously demonstrated that streptokinase (SK) can be used to generate active site-labeled fluorescent analogs of plasminogen (Pg) by virtue of its non-proteolytic activation of the zymogen. The method is versatile and allows for stoichiometric and active site-specific incorporation of any one of many molecular probes. The limitation of the labeling approach is that it is both time-consuming and low yield. Here we demonstrate an improved method for the preparation of labeled Pg analogs by the use of an engineered SK mutant fusion protein with both COOH- and NH2-terminal His6-tags. The NH2-terminal tag is followed by a tobacco etch virus proteinase cleavage site to ensure that the SK Ile1 residue, essential for conformational activation of Pg, is preserved. The SK COOH-terminal Lys414 residue and residues Arg253-Leu260 in the SK β-domain were deleted to prevent cleavage by plasmin (Pm), and to disable Pg substrate binding to the SK·Pg*/Pm catalytic complexes, respectively. Near-elimination of Pm generation with the SKΔ(R253-L260)ΔK414-His6 mutant increased the yield of labeled Pg 2.6-fold and reduced the time required >2-fold. The versatility of the labeling method was extended to the application of Pg labeled with a near-infrared probe to quantitate Pg receptors on immune cells by flow cytometry. PMID:21570944

Plasma markers of inflammation and hemostatic and endothelial activity in naturally overweight and obese dogs.

PubMed

Barić Rafaj, R; Kuleš, J; Marinculić, A; Tvarijonaviciute, A; Ceron, J; Mihaljević, Ž; Tumpa, A; Mrljak, V

2017-01-06

Obesity is one of the most prevalent health problems in the canine population. While haemostatic parameters and markers of endothelial function have been evaluated in various disease conditions in dogs, there are no studies of these markers in canine obesity. This study was designed to evaluate the effect of naturally gained weight excess and obesity on inflammatory, hemostatic and endothelial biomarkers in dogs. A total of 37 overweight and obese dogs were compared with 28 normal weight dogs. Overweight and obese dogs had significantly elevated concentrations of serum interleukin-6 (IL-6) and C-reactive protein (hsCRP). Number of platelets, activity of factor X and factor VII were significantly higher, while activated partial thromboplastine time (aPTT) and soluble plasminogen activator receptor (suPAR) were significantly decreased. Statistical analysis of high mobility group box - 1 protein (HMGB-1), soluble intercellular adhesive molecule -1 (sICAM-1) and plasminogen activator inhibitor type 1 (PAI-1) concentrations did not show significant differences between the total overweight and obese group and the normal weight group of dogs. Analytical changes in the dogs in our study reflects that weight excess in dogs can be associated with a chronic low degree of inflammation and a hypercoagulable state, where primary and secondary hemostasis are both affected. However obesity is not associated with impairment of endothelial function in dogs.

Activity-dependent release of tissue plasminogen activator from the dendritic spines of hippocampal neurons revealed by live-cell imaging.

PubMed

Lochner, Janis E; Honigman, Leah S; Grant, Wilmon F; Gessford, Sarah K; Hansen, Alexis B; Silverman, Michael A; Scalettar, Bethe A

2006-05-01

Tissue plasminogen activator (tPA) has been implicated in a variety of important cellular functions, including learning-related synaptic plasticity and potentiating N-methyl-D-aspartate (NMDA) receptor-dependent signaling. These findings suggest that tPA may localize to, and undergo activity-dependent secretion from, synapses; however, conclusive data supporting these hypotheses have remained elusive. To elucidate these issues, we studied the distribution, dynamics, and depolarization-induced secretion of tPA in hippocampal neurons, using fluorescent chimeras of tPA. We found that tPA resides in dense-core granules (DCGs) that traffic to postsynaptic dendritic spines and that can remain in spines for extended periods. We also found that depolarization induced by high potassium levels elicits a slow, partial exocytotic release of tPA from DCGs in spines that is dependent on extracellular Ca(+2) concentrations. This slow, partial release demonstrates that exocytosis occurs via a mechanism, such as fuse-pinch-linger, that allows partial release and reuse of DCG cargo and suggests a mechanism that hippocampal neurons may rely upon to avoid depleting tPA at active synapses. Our results also demonstrate release of tPA at a site that facilitates interaction with NMDA-type glutamate receptors, and they provide direct confirmation of fundamental hypotheses about tPA localization and release that bear on its neuromodulatory functions, for example, in learning and memory.

Interhospital Transfer Before Thrombectomy Is Associated With Delayed Treatment and Worse Outcome in the STRATIS Registry (Systematic Evaluation of Patients Treated With Neurothrombectomy Devices for Acute Ischemic Stroke)

PubMed Central

Saver, Jeffrey L.; Zaidat, Osama O.; Jahan, Reza; Aziz-Sultan, Mohammad Ali; Klucznik, Richard P.; Haussen, Diogo C.; Hellinger, Frank R.; Yavagal, Dileep R.; Yao, Tom L.; Liebeskind, David S.; Jadhav, Ashutosh P.; Gupta, Rishi; Hassan, Ameer E.; Martin, Coleman O.; Bozorgchami, Hormozd; Kaushal, Ritesh; Nogueira, Raul G.; Gandhi, Ravi H.; Peterson, Eric C.; Dashti, Shervin R.; Given, Curtis A.; Mehta, Brijesh P.; Deshmukh, Vivek; Starkman, Sidney; Linfante, Italo; McPherson, Scott H.; Kvamme, Peter; Grobelny, Thomas J.; Hussain, Muhammad S.; Thacker, Ike; Vora, Nirav; Chen, Peng Roc; Monteith, Stephen J.; Ecker, Robert D.; Schirmer, Clemens M.; Sauvageau, Eric; Abou-Chebl, Alex; Derdeyn, Colin P.; Maidan, Lucian; Badruddin, Aamir; Siddiqui, Adnan H.; Dumont, Travis M.; Alhajeri, Abdulnasser; Taqi, M. Asif; Asi, Khaled; Carpenter, Jeffrey; Boulos, Alan; Jindal, Gaurav; Puri, Ajit S.; Chitale, Rohan; Deshaies, Eric M.; Robinson, David H.; Kallmes, David F.; Baxter, Blaise W.; Jumaa, Mouhammad A.; Sunenshine, Peter; Majjhoo, Aniel; English, Joey D.; Suzuki, Shuichi; Fessler, Richard D.; Delgado Almandoz, Josser E.; Martin, Jerry C.; Mueller-Kronast, Nils H.

2017-01-01

Background: Endovascular treatment with mechanical thrombectomy (MT) is beneficial for patients with acute stroke suffering a large-vessel occlusion, although treatment efficacy is highly time-dependent. We hypothesized that interhospital transfer to endovascular-capable centers would result in treatment delays and worse clinical outcomes compared with direct presentation. Methods: STRATIS (Systematic Evaluation of Patients Treated With Neurothrombectomy Devices for Acute Ischemic Stroke) was a prospective, multicenter, observational, single-arm study of real-world MT for acute stroke because of anterior-circulation large-vessel occlusion performed at 55 sites over 2 years, including 1000 patients with severe stroke and treated within 8 hours. Patients underwent MT with or without intravenous tissue plasminogen activator and were admitted to endovascular-capable centers via either interhospital transfer or direct presentation. The primary clinical outcome was functional independence (modified Rankin Score 0–2) at 90 days. We assessed (1) real-world time metrics of stroke care delivery, (2) outcome differences between direct and transfer patients undergoing MT, and (3) the potential impact of local hospital bypass. Results: A total of 984 patients were analyzed. Median onset-to-revascularization time was 202.0 minutes for direct versus 311.5 minutes for transfer patients (P<0.001). Clinical outcomes were better in the direct group, with 60.0% (299/498) achieving functional independence compared with 52.2% (213/408) in the transfer group (odds ratio, 1.38; 95% confidence interval, 1.06–1.79; P=0.02). Likewise, excellent outcome (modified Rankin Score 0–1) was achieved in 47.4% (236/498) of direct patients versus 38.0% (155/408) of transfer patients (odds ratio, 1.47; 95% confidence interval, 1.13–1.92; P=0.005). Mortality did not differ between the 2 groups (15.1% for direct, 13.7% for transfer; P=0.55). Intravenous tissue plasminogen activator did not impact outcomes. Hypothetical bypass modeling for all transferred patients suggested that intravenous tissue plasminogen activator would be delayed by 12 minutes, but MT would be performed 91 minutes sooner if patients were routed directly to endovascular-capable centers. If bypass is limited to a 20-mile radius from onset, then intravenous tissue plasminogen activator would be delayed by 7 minutes and MT performed 94 minutes earlier. Conclusions: In this large, real-world study, interhospital transfer was associated with significant treatment delays and lower chance of good outcome. Strategies to facilitate more rapid identification of large-vessel occlusion and direct routing to endovascular-capable centers for patients with severe stroke may improve outcomes. Clinical Trial Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT02239640. PMID:28943516

Interhospital Transfer Before Thrombectomy Is Associated With Delayed Treatment and Worse Outcome in the STRATIS Registry (Systematic Evaluation of Patients Treated With Neurothrombectomy Devices for Acute Ischemic Stroke).

PubMed

Froehler, Michael T; Saver, Jeffrey L; Zaidat, Osama O; Jahan, Reza; Aziz-Sultan, Mohammad Ali; Klucznik, Richard P; Haussen, Diogo C; Hellinger, Frank R; Yavagal, Dileep R; Yao, Tom L; Liebeskind, David S; Jadhav, Ashutosh P; Gupta, Rishi; Hassan, Ameer E; Martin, Coleman O; Bozorgchami, Hormozd; Kaushal, Ritesh; Nogueira, Raul G; Gandhi, Ravi H; Peterson, Eric C; Dashti, Shervin R; Given, Curtis A; Mehta, Brijesh P; Deshmukh, Vivek; Starkman, Sidney; Linfante, Italo; McPherson, Scott H; Kvamme, Peter; Grobelny, Thomas J; Hussain, Muhammad S; Thacker, Ike; Vora, Nirav; Chen, Peng Roc; Monteith, Stephen J; Ecker, Robert D; Schirmer, Clemens M; Sauvageau, Eric; Abou-Chebl, Alex; Derdeyn, Colin P; Maidan, Lucian; Badruddin, Aamir; Siddiqui, Adnan H; Dumont, Travis M; Alhajeri, Abdulnasser; Taqi, M Asif; Asi, Khaled; Carpenter, Jeffrey; Boulos, Alan; Jindal, Gaurav; Puri, Ajit S; Chitale, Rohan; Deshaies, Eric M; Robinson, David H; Kallmes, David F; Baxter, Blaise W; Jumaa, Mouhammad A; Sunenshine, Peter; Majjhoo, Aniel; English, Joey D; Suzuki, Shuichi; Fessler, Richard D; Delgado Almandoz, Josser E; Martin, Jerry C; Mueller-Kronast, Nils H

2017-12-12

Endovascular treatment with mechanical thrombectomy (MT) is beneficial for patients with acute stroke suffering a large-vessel occlusion, although treatment efficacy is highly time-dependent. We hypothesized that interhospital transfer to endovascular-capable centers would result in treatment delays and worse clinical outcomes compared with direct presentation. STRATIS (Systematic Evaluation of Patients Treated With Neurothrombectomy Devices for Acute Ischemic Stroke) was a prospective, multicenter, observational, single-arm study of real-world MT for acute stroke because of anterior-circulation large-vessel occlusion performed at 55 sites over 2 years, including 1000 patients with severe stroke and treated within 8 hours. Patients underwent MT with or without intravenous tissue plasminogen activator and were admitted to endovascular-capable centers via either interhospital transfer or direct presentation. The primary clinical outcome was functional independence (modified Rankin Score 0-2) at 90 days. We assessed (1) real-world time metrics of stroke care delivery, (2) outcome differences between direct and transfer patients undergoing MT, and (3) the potential impact of local hospital bypass. A total of 984 patients were analyzed. Median onset-to-revascularization time was 202.0 minutes for direct versus 311.5 minutes for transfer patients ( P <0.001). Clinical outcomes were better in the direct group, with 60.0% (299/498) achieving functional independence compared with 52.2% (213/408) in the transfer group (odds ratio, 1.38; 95% confidence interval, 1.06-1.79; P =0.02). Likewise, excellent outcome (modified Rankin Score 0-1) was achieved in 47.4% (236/498) of direct patients versus 38.0% (155/408) of transfer patients (odds ratio, 1.47; 95% confidence interval, 1.13-1.92; P =0.005). Mortality did not differ between the 2 groups (15.1% for direct, 13.7% for transfer; P =0.55). Intravenous tissue plasminogen activator did not impact outcomes. Hypothetical bypass modeling for all transferred patients suggested that intravenous tissue plasminogen activator would be delayed by 12 minutes, but MT would be performed 91 minutes sooner if patients were routed directly to endovascular-capable centers. If bypass is limited to a 20-mile radius from onset, then intravenous tissue plasminogen activator would be delayed by 7 minutes and MT performed 94 minutes earlier. In this large, real-world study, interhospital transfer was associated with significant treatment delays and lower chance of good outcome. Strategies to facilitate more rapid identification of large-vessel occlusion and direct routing to endovascular-capable centers for patients with severe stroke may improve outcomes. URL: https://www.clinicaltrials.gov. Unique identifier: NCT02239640. © 2017 The Authors.

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.

A prospective multicenter cohort study of the association between global tissue hypoxia and coagulation abnormalities during early sepsis resuscitation.

PubMed

Trzeciak, Stephen; Jones, Alan E; Shapiro, Nathan I; Pusateri, Anthony E; Arnold, Ryan C; Rizzuto, Michael; Arora, Tanisha; Parrillo, Joseph E; Dellinger, R Phillip

2010-04-01

Coagulation activation is an integral part of sepsis pathogenesis. Experimental data suggest that endothelial exposure to hypoxia activates coagulation. We aimed to test the hypothesis that the quantity of exposure to global tissue hypoxia is associated with the degree of coagulation activation during early sepsis resuscitation. Prospective, multicenter cohort study. Emergency department and intensive care unit of three academic hospitals. Inclusion criteria were age older than 17, acute infection with two or more signs of systemic inflammation, hypotension despite fluid challenge (or lactate >4 mM), and continuous central venous oxygen saturation (Scvo2) monitoring for quantitative resuscitation. Exclusion criteria were anticoagulant or blood product administration. We recorded central venous oxygen saturation continuously for 0 to 6 hrs of resuscitation and calculated the area under the curve for central venous oxygen saturation <70%. We defined hypoxia exposure as exceeding the median area under the curve for the entire cohort. At 0, 6, and 24 hrs, we measured conventional coagulation biomarkers plus thrombin-antithrombin complex, plasmin-antiplasmin complex, tissue plasminogen activator, plasminogen activator inhibitor-1, protein C, antithrombin, and endothelial markers (E-selectin, intracellular adhesion molecule-1, thrombomodulin). We compared changes during 0 to 6 hrs and 0 to 24 hrs in biomarkers between hypoxia exposure and nonexposure groups. We enrolled 40 patients (60% requiring vasopressors; 30% mortality). We found that exposure to hypoxia alone was not associated with a significant degree of coagulation activation. However, in secondary analyses we found that exposure to arterial hypotension induced E-selectin and thrombin-antithrombin complex, whereas concomitant exposure to both hypotension and hypoxia was associated with amplification of E-selectin and thrombomodulin, and a reduction in protein C. In this sample of patients undergoing quantitative resuscitation for sepsis, we found that exposure to global tissue hypoxia (as quantified by low central venous oxygen saturation) was not associated with major coagulation activation. Further investigation to elucidate the clinical factors that trigger or intensify the procoagulant response to sepsis is warranted.

Plasminogen Activator Inhibitor: A Regulator of Ancrod-Induced Fibrin Deposition in Rabbits

DTIC Science & Technology

1987-03-01

Acad Sci Invest 51:2841. 1972 82:8710. 1985 15. Alving BM, Evait BI., Bell WR: Stimulation of librinogen 4. Colucci M, Paramo JA. Collen D: Generation in... Paramo IA. Stassen .1M. Collen ): Influence of 1986 the fast-acting inhibitor on in vivo thrombolysis induced b, tissue- 7. Wiman B. Ljungberg B

The cardioprotective effect of wine on human blood chemistry.

PubMed

van Velden, David P; Mansvelt, Erna P G; Fourie, Elba; Rossouw, Marietjie; Marais, A David

2002-05-01

We investigated the in vivo effects of regular consumption of red and white wine on the serum lipid profile, plasma plasminogen activator-1, homocysteine levels, and total antioxidant status. This study confirmed that moderate consumption of wine, red more than white, exerts cardioprotective effects through beneficial changes in lipid profiles and plasma total antioxidant status.

Hepatitis virus infection affects DNA methylation in mice with humanized livers.

PubMed

Okamoto, Yasuyuki; Shinjo, Keiko; Shimizu, Yasuhiro; Sano, Tsuyoshi; Yamao, Kenji; Gao, Wentao; Fujii, Makiko; Osada, Hirotaka; Sekido, Yoshitaka; Murakami, Shuko; Tanaka, Yasuhito; Joh, Takashi; Sato, Shinya; Takahashi, Satoru; Wakita, Takaji; Zhu, Jingde; Issa, Jean-Pierre J; Kondo, Yutaka

2014-02-01

Cells of tumors associated with chronic inflammation frequently have altered patterns of DNA methylation, including hepatocellular carcinomas. Chronic hepatitis has also been associated with aberrant DNA methylation, but little is known about their relationship. Pyrosequencing was used to determine the methylation status of cultured Huh7.5.1 hepatoma cells after hepatitis C virus (HCV) infection. We also studied mice with severe combined immunodeficiency carrying the urokinase-type plasminogen activator transgene controlled by an albumin promoter (urokinase-type plasminogen activator/severe combined immunodeficient mice), in which up to 85% of hepatocytes were replaced by human hepatocytes (chimeric mice). Mice were given intravenous injections of hepatitis B virus (HBV) or HCV, liver tissues were collected, and DNA methylation profiles were determined at different time points after infection. We also compared methylation patterns between paired samples of hepatocellular carcinomas and adjacent nontumor liver tissues from patients. No reproducible changes in DNA methylation were observed after infection of Huh7.5.1 cells with HCV. Livers from HBV- and HCV-infected mice had genome-wide, time-dependent changes in DNA methylation, compared with uninfected urokinase-type plasminogen activator/severe combined immunodeficient mice. There were changes in 160 ± 63 genes in HBV-infected and 237 ± 110 genes in HCV-infected mice. Methylation of 149 common genes increased in HBV- and HCV-infected mice; methylation of some of these genes also increased in hepatocellular carcinoma samples from patients compared with nontumor tissues. Expression of Ifng, which is expressed by natural killer cells, increased significantly in chimeric livers, in concordance with induction of DNA methylation, after infection with HBV or HCV. Induction of Ifng was reduced after administration of an inhibitor of natural killer cell function (anti-asialo GM1). In chimeric mice with humanized livers, infection with HBV and HCV appears to activate a natural kill cell-dependent innate immune response. This contributes to the induction and accumulation of aberrant DNA methylation in human hepatocytes. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Compaction of fibrin clots reveals the antifibrinolytic effect of factor XIII.

PubMed

Rijken, D C; Abdul, S; Malfliet, J J M C; Leebeek, F W G; Uitte de Willige, S

2016-07-01

Essentials Factor XIIIa inhibits fibrinolysis by forming fibrin-fibrin and fibrin-inhibitor cross-links. Conflicting studies about magnitude and mechanisms of inhibition have been reported. Factor XIIIa most strongly inhibits lysis of mechanically compacted or retracted plasma clots. Cross-links of α2-antiplasmin to fibrin prevent the inhibitor from being expelled from the clot. Background Although insights into the underlying mechanisms of the effect of factor XIII on fibrinolysis have improved considerably in the last few decades, in particular with the discovery that activated FXIII (FXIIIa) cross-links α2 -antiplasmin to fibrin, the topic remains a matter of debate. Objective To elucidate the mechanisms of the antifibrinolytic effect of FXIII. Methods and Results Platelet-poor plasma clot lysis, induced by the addition of tissue-type plasminogen activator, was measured in the presence or absence of a specific FXIIIa inhibitor. Both in a turbidity assay and in a fluorescence assay, the FXIIIa inhibitor had only a small inhibitory effect: 1.6-fold less tissue-type plasminogen activator was required for 50% clot lysis in the presence of the FXIIIa inhibitor. However, when the plasma clot was compacted by centrifugation, the FXIIIa inhibitor had a strong inhibitory effect, with 7.7-fold less tissue-type plasminogen activator being required for 50% clot lysis in the presence of the FXIIIa inhibitor. In both experiments, the effects of the FXIIIa inhibitor were entirely dependent on the cross-linking of α2 -antiplasmin to fibrin. The FXIIIa inhibitor reduced the amount of α2 -antiplasmin present in the compacted clots from approximately 30% to < 4%. The results were confirmed with experiments in which compaction was achieved by platelet-mediated clot retraction. Conclusions Compaction or retraction of fibrin clots reveals the strong antifibrinolytic effect of FXIII. This is explained by the cross-linking of α2 -antiplasmin to fibrin by FXIIIa, which prevents the plasmin inhibitor from being fully expelled from the clot during compaction/retraction. © 2016 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.

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.

N-Arachidonoyl Dopamine Modulates Acute Systemic Inflammation via Nonhematopoietic TRPV1

PubMed Central

Lawton, Samira K.; Xu, Fengyun; Tran, Alphonso; Wong, Erika; Schumacher, Mark; Wilhelmsen, Kevin

2017-01-01

N-Arachidonoyl dopamine (NADA) is an endogenous lipid that potently activates the transient receptor potential vanilloid 1 (TRPV1), which mediates pain and thermosensation. NADA is also an agonist of cannabinoid receptors 1 and 2. We have reported that NADA reduces the activation of cultured human endothelial cells by LPS and TNF-α. Thus far, in vivo studies using NADA have focused on its neurologic and behavioral roles. In this article, we show that NADA potently decreases in vivo systemic inflammatory responses and levels of the coagulation intermediary plasminogen activator inhibitor 1 in three mouse models of inflammation: LPS, bacterial lipopeptide, and polymicrobial intra-abdominal sepsis. We also found that the administration of NADA increases survival in endotoxemic mice. Additionally, NADA reduces blood levels of the neuropeptide calcitonin gene-related peptide but increases the neuropeptide substance P in LPS-treated mice. We demonstrate that the anti-inflammatory effects of NADA are mediated by TRPV1 expressed by nonhematopoietic cells and provide data suggesting that neuronal TRPV1 may mediate NADA’s anti-inflammatory effects. These results indicate that NADA has novel TRPV1-dependent anti-inflammatory properties and suggest that the endovanilloid system might be targeted therapeutically in acute inflammation. PMID:28701511

Deficiency in Serine Protease Inhibitor Neuroserpin Exacerbates Ischemic Brain Injury by Increased Postischemic Inflammation

PubMed Central

Ludewig, Peter; Bernreuther, Christian; Krasemann, Susanne; Arunachalam, Priyadharshini; Gerloff, Christian; Glatzel, Markus; Magnus, Tim

2013-01-01

The only approved pharmacological treatment for ischemic stroke is intravenous administration of plasminogen activator (tPA) to re-canalize the occluded cerebral vessel. Not only reperfusion but also tPA itself can induce an inflammatory response. Microglia are the innate immune cells of the central nervous system and the first immune cells to become activated in stroke. Neuroserpin, an endogenous inhibitor of tPA, is up-regulated following cerebral ischemia. To examine neuroserpin-dependent mechanisms of neuroprotection in stroke, we studied neuroserpin deficient (Ns−/−) mice in an animal model of temporal focal ischemic stroke. Infarct size and neurological outcome were worse in neuroserpin deficient mice even though the fibrinolytic activity in the ischemic brain was increased. The increased infarct size was paralleled by a selective increase in proinflammatory microglia activation in Ns−/− mice. Our results show excessive microglial activation in Ns−/− mice mediated by an increased activity of tPA. This activation results in a worse outcome further underscoring the potential detrimental proinflammatory effects of tPA. PMID:23658802

α-Enolase Causes Proinflammatory Activation of Pulmonary Microvascular Endothelial Cells and Primes Neutrophils Through Plasmin Activation of Protease-Activated Receptor 2.

PubMed

Bock, Ashley; Tucker, Nicole; Kelher, Marguerite R; Khan, Samina Y; Gonzalez, Eduardo; Wohlauer, Max; Hansen, Kirk; Dzieciatkowska, Monika; Sauaia, Angels; Banerjee, Anirban; Moore, Ernest E; Silliman, Christopher C

2015-08-01

Proinflammatory activation of vascular endothelium leading to increased surface expression of adhesion molecules and neutrophil (PMN) sequestration and subsequent activation is paramount in the development of acute lung injury and organ injury in injured patients. We hypothesize that α-enolase, which accumulates in injured patients, primes PMNs and causes proinflammatory activation of endothelial cells leading to PMN-mediated cytotoxicity. Proteomic analyses of field plasma samples from injured versus healthy patients were used for protein identification. Human pulmonary microvascular endothelial cells (HMVECs) were incubated with α-enolase or thrombin, and intercellular adhesion molecule-1 surface expression was measured by flow cytometry. A two-event in vitro model of PMN cytotoxicity HMVECs activated with α-enolase, thrombin, or buffer was used as targets for lysophosphatidylcholine-primed or buffer-treated PMNs. The PMN priming activity of α-enolase was completed, and lysates from both PMNs and HMVECs were immunoblotted for protease-activated receptor 1 (PAR-1) and PAR-2 and coprecipitation of α-enolase with PAR-2 and plasminogen/plasmin. α-Enolase increased 10.8-fold in injured patients (P < 0.05). Thrombin and α-enolase significantly increased intercellular adhesion molecule-1 surface expression on HMVECs, which was inhibited by antiproteases, induced PMN adherence, and served as the first event in the two-event model of PMN cytotoxicity. α-Enolase coprecipitated with PAR-2 and plasminogen/plasmin on HMVECs and PMNs and induced PMN priming, which was inhibited by tranexamic acid, and enzymatic activity was not required. α-Enolase increases after injury and may activate pulmonary endothelial cells and prime PMNs through plasmin activity and PAR-2 activation. Such proinflammatory endothelial activation may predispose to PMN-mediated organ injury.

Can the activation of plasminogen/plasmin system of the host by metabolic products of Dirofilaria immitis participate in heartworm disease endarteritis?

PubMed

González-Miguel, Javier; Morchón, Rodrigo; Carretón, Elena; Montoya-Alonso, José Alberto; Simón, Fernando

2015-04-01

Proliferative endarteritis is one of the key pathological mechanisms of cardiopulmonary dirofilariosis, a cosmopolitan parasitosis caused by Dirofilaria immitis affecting dogs and cats around the world. It has been shown that the excretory/secretory antigens from D. immitis adult worms (DiES) bind plasminogen (PLG) and activate fibrinolysis, which can lead to a survival mechanism for the parasite in its intravascular environment. However, overproduction of plasmin (final product of the route) has been related to pathological processes similar to those described in proliferative endarteritis. The aim of this study is to relate the appearance of this pathological condition with the activation of the PLG/plasmin system of the host by DiES. Cell proliferation through the crystal violet technique, cell migration by wound healing assay and degradation of the extracellular matrix by measuring collagen degradation and levels of matrix metalloproteinases were studied in an "in vitro" model using canine vascular endothelial and smooth muscle cells. These cells were treated with a mixture of DiES + PLG. Untreated cells, cells only stimulated with DiES or with PLG, or with a mixture of DiES + PLG + εACA (an inhibitor of the PLG-plasmin conversion) were employed as controls. In addition, the effect of DiES on the expression of the fibrinolytic activators tPA and uPA, the inhibitor PAI-1 and the PLG receptor Annexin A2 was analyzed in both types of cultures by western blot. Plasmin generated by DiES + PLG binding produced a significant increase in the cell proliferation and migration of the endothelial and smooth muscle cells, as well as an increase in the destruction of the extracellular matrix based on a further degradation of Type I Collagen and an increased level of matrix metalloproteinase-2. DiES also induce an increase in the expression of tPA and uPA in endothelial cells in culture, as well as a decrease in the expression of PAI-1 in both types of cells. Our study reports an interrelationship between plasmin caused by fibrinolysis activation by metabolic products of D. immitis and the appearance of pathological events similar to those described in the emergence of proliferative endarteritis in the cardiopulmonary dirofilariosis.

A continuous-exchange cell-free protein synthesis system based on extracts from cultured insect cells.

PubMed

Stech, Marlitt; Quast, Robert B; Sachse, Rita; Schulze, Corina; Wüstenhagen, Doreen A; Kubick, Stefan

2014-01-01

In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds.

A Continuous-Exchange Cell-Free Protein Synthesis System Based on Extracts from Cultured Insect Cells

PubMed Central

Stech, Marlitt; Quast, Robert B.; Sachse, Rita; Schulze, Corina; Wüstenhagen, Doreen A.; Kubick, Stefan

2014-01-01

In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds. PMID:24804975

Regulatory elements involved in constitutive and phorbol ester-inducible expression of the plasminogen activator inhibitor type 2 gene promoter.

PubMed Central

Cousin, E; Medcalf, R L; Bergonzelli, G E; Kruithof, E K

1991-01-01

Gene transcription rates and mRNA levels of plasminogen activator inhibitor type 2 (PAI-2) are markedly induced by the tumor promoting agent phorbol 12-myristate 13-acetate (PMA) in human HT1080 fibrosarcoma cells. To identify promoter elements required for basal-, and phorbol ester-inducible expression, deletion mutants of the PAI-1 promoter fused to the chloramphenicol acetyl transferase (CAT) reporter gene, were transiently expressed in HT1080 cells. Constitutive CAT activity was expressed from constructs containing more than 215 bp of promoter sequence, whereas deletion to position -91 bp abolished CAT gene expression. Treatment of transfected cells with PMA resulted in a three- to ten-fold increase in CAT expression from all constructs except from the construct shortened to position -91. DNAse1 protection analysis of the promoter region between -215 and the transcription initiation site revealed numerous protected regions, including two AP1-like binding sites (AP1a and AP1b) and one CRE-like element. Site-directed mutagenesis of the AP1a site or of the CRE-like site resulted in the loss of basal CAT activity and abolished the PMA effect, whereas mutagenesis of AP1b only partially inhibited basal and PMA-mediated expression. Our results suggest that the PAI-2 promoter contains at least two elements required for basal gene transcription and PMA-mediated induction. Images PMID:1650454

Multi-Carotenoids at Physiological Levels Inhibit VEGF-Induced Tube Formation of Endothelial Cells and the Possible Mechanisms of Action Both In Vitro and Ex Vivo.

PubMed

Huang, Chien-Hao; Huang, Chin-Shiu; Hu, Miao-Lin; Chuang, Cheng-Hung

2018-01-01

Carotenoids have been shown to exhibit antiangiogenic activities. Several studies have indicated that carotenoids used in combination were more effective on antioxidation and anticancer actions than carotenoids used singly. However, it is unclear whether multi-carotenoids have antiangiogenic effects. We investigated the effects of multi-carotenoids at physiological plasma levels of Taiwanese (abbreviated as MCT, with a total of 1.4 μM) and Americans (abbreviated as MCA, with a total of 1.8 μM), and of post-supplemental plasma levels (abbreviated as HMC with a total of 3.55 μM) on vascular endothelial growth factor (VEGF)-induced tube formation in human umbilical vein endothelial cells (HUVECs) and rat aortic rings. MCT, MCA, and HMC inhibited VEGF-induced migration, invasion, and tube formation of HUVECs as well as new vessels formation in rat aortic rings. MCT, MCA, and HMC inhibited activities o\\f matrix metalloproteinase (MMP)-2, urokinase plasminogen activator, and phosphorylation of VEGF receptor 2 induced by VEGF. Moreover, MCT, MCA, and HMC significantly upregulated protein expression of tissue inhibitors of MMP-2 and plasminogen activator inhibitor-1. These results demonstrate the antiangiogenic effect of multi-carotenoids both in vitro and ex vivo with possible mechanistic actions involving attenuation of VEGF receptor 2 phosphorylation and extracellular matrix degradation.

Xenon is an inhibitor of tissue-plasminogen activator: adverse and beneficial effects in a rat model of thromboembolic stroke

PubMed Central

David, Hélène N; Haelewyn, Benoît; Risso, Jean-Jacques; Colloc'h, Nathalie; Abraini, Jacques H

2010-01-01

Preclinical evidence in rodents has proven that xenon may be a very promising neuroprotective agent for treating acute ischemic stroke. This has led to the general thinking that clinical trials with xenon could be initiated in acute stroke patients in a next future. However, an unappreciated physicochemical property of xenon has been that this gas also binds to the active site of a series of serine proteases. Because the active site of serine proteases is structurally conserved, we have hypothesized and investigated whether xenon may alter the catalytic efficiency of tissue-type plasminogen activator (tPA), a serine protease that is the only approved therapy for acute ischemic stroke today. Here, using molecular modeling and in vitro and in vivo studies, we show (1) xenon is a tPA inhibitor; (2) intraischemic xenon dose dependently inhibits tPA-induced thrombolysis and subsequent reduction of ischemic brain damage; (3) postischemic xenon virtually suppresses ischemic brain damage and tPA-induced brain hemorrhages and disruption of the blood–brain barrier. Taken together, these data indicate (1) xenon should not be administered before or together with tPA therapy; (2) xenon could be a golden standard for treating acute ischemic stroke if given after tPA-induced reperfusion, with both unique neuroprotective and antiproteolytic (anti-hemorrhaging) properties. PMID:20087367

The FAST-ED App: A Smartphone Platform for the Field Triage of Patients With Stroke.

PubMed

Nogueira, Raul G; Silva, Gisele S; Lima, Fabricio O; Yeh, Yu-Chih; Fleming, Carol; Branco, Daniel; Yancey, Arthur H; Ratcliff, Jonathan J; Wages, Robert Keith; Doss, Earnest; Bouslama, Mehdi; Grossberg, Jonathan A; Haussen, Diogo C; Sakano, Teppei; Frankel, Michael R

2017-05-01

The Emergency Medical Services field triage to stroke centers has gained considerable complexity with the recent demonstration of clinical benefit of endovascular treatment for acute ischemic stroke. We sought to describe a new smartphone freeware application designed to assist Emergency Medical Services professionals with the field assessment and destination triage of patients with acute ischemic stroke. Review of the application's platform and its development as well as the different variables, assessments, algorithms, and assumptions involved. The FAST-ED (Field Assessment Stroke Triage for Emergency Destination) application is based on a built-in automated decision-making algorithm that relies on (1) a brief series of questions assessing patient's age, anticoagulant usage, time last known normal, motor weakness, gaze deviation, aphasia, and hemineglect; (2) a database of all regional stroke centers according to their capability to provide endovascular treatment; and (3) Global Positioning System technology with real-time traffic information to compute the patient's eligibility for intravenous tissue-type plasminogen activator or endovascular treatment as well as the distances/transportation times to the different neighboring stroke centers in order to assist Emergency Medical Services professionals with the decision about the most suitable destination for any given patient with acute ischemic stroke. The FAST-ED smartphone application has great potential to improve the triage of patients with acute ischemic stroke, as it seems capable to optimize resources, reduce hospital arrivals times, and maximize the use of both intravenous tissue-type plasminogen activator and endovascular treatment ultimately leading to better clinical outcomes. Future field studies are needed to properly evaluate the impact of this tool in stroke outcomes and resource utilization. © 2017 American Heart Association, Inc.

Differences in Acute Ischemic Stroke Quality of Care and Outcomes by Primary Stroke Center Certification Organization.

PubMed

Man, Shumei; Cox, Margueritte; Patel, Puja; Smith, Eric E; Reeves, Mathew J; Saver, Jeffrey L; Bhatt, Deepak L; Xian, Ying; Schwamm, Lee H; Fonarow, Gregg C

2017-02-01

Primary stroke center (PSC) certification was established to identify hospitals providing evidence-based care for stroke patients. The numbers of PSCs certified by Joint Commission (JC), Healthcare Facilities Accreditation Program, Det Norske Veritas, and State-based agencies have significantly increased in the past decade. This study aimed to evaluate whether PSCs certified by different organizations have similar quality of care and in-hospital outcomes. The study population consisted of acute ischemic stroke patients who were admitted to PSCs participating in Get With The Guidelines-Stroke between January 1, 2010, and December 31, 2012. Measures of care quality and outcomes were compared among the 4 different PSC certifications. A total of 477 297 acute ischemic stroke admissions were identified from 977 certified PSCs (73.8% JC, 3.7% Det Norske Veritas, 1.2% Healthcare Facilities Accreditation Program, and 21.3% State-based). Composite care quality was generally similar among the 4 groups of hospitals, although State-based PSCs underperformed JC PSCs in a few key measures, including intravenous tissue-type plasminogen activator use. The rates of tissue-type plasminogen activator use were higher in JC and Det Norske Veritas (9.0% and 9.8%) and lower in State and Healthcare Facilities Accreditation Program certified hospitals (7.1% and 5.9%) (P<0.0001). Door-to-needle times were significantly longer in Healthcare Facilities Accreditation Program hospitals. State PSCs had higher in-hospital risk-adjusted mortality (odds ratio 1.23, 95% confidence intervals 1.07-1.41) compared with JC PSCs. Among Get With The Guidelines-Stroke hospitals with PSC certification, acute ischemic stroke quality of care and outcomes may differ according to which organization provided certification. These findings may have important implications for further improving systems of care. © 2016 American Heart Association, Inc.

Analysis of Plasminogen Genetic Variants in Multiple Sclerosis Patients

PubMed Central

Sadovnick, A. Dessa; Traboulsee, Anthony L.; Bernales, Cecily Q.; Ross, Jay P.; Forwell, Amanda L.; Yee, Irene M.; Guillot-Noel, Lena; Fontaine, Bertrand; Cournu-Rebeix, Isabelle; Alcina, Antonio; Fedetz, Maria; Izquierdo, Guillermo; Matesanz, Fuencisla; Hilven, Kelly; Dubois, Bénédicte; Goris, An; Astobiza, Ianire; Alloza, Iraide; Antigüedad, Alfredo; Vandenbroeck, Koen; Akkad, Denis A.; Aktas, Orhan; Blaschke, Paul; Buttmann, Mathias; Chan, Andrew; Epplen, Joerg T.; Gerdes, Lisa-Ann; Kroner, Antje; Kubisch, Christian; Kümpfel, Tania; Lohse, Peter; Rieckmann, Peter; Zettl, Uwe K.; Zipp, Frauke; Bertram, Lars; Lill, Christina M; Fernandez, Oscar; Urbaneja, Patricia; Leyva, Laura; Alvarez-Cermeño, Jose Carlos; Arroyo, Rafael; Garagorri, Aroa M.; García-Martínez, Angel; Villar, Luisa M.; Urcelay, Elena; Malhotra, Sunny; Montalban, Xavier; Comabella, Manuel; Berger, Thomas; Fazekas, Franz; Reindl, Markus; Schmied, Mascha C.; Zimprich, Alexander; Vilariño-Güell, Carles

2016-01-01

Multiple sclerosis (MS) is a prevalent neurological disease of complex etiology. Here, we describe the characterization of a multi-incident MS family that nominated a rare missense variant (p.G420D) in plasminogen (PLG) as a putative genetic risk factor for MS. Genotyping of PLG p.G420D (rs139071351) in 2160 MS patients, and 886 controls from Canada, identified 10 additional probands, two sporadic patients and one control with the variant. Segregation in families harboring the rs139071351 variant, identified p.G420D in 26 out of 30 family members diagnosed with MS, 14 unaffected parents, and 12 out of 30 family members not diagnosed with disease. Despite considerably reduced penetrance, linkage analysis supports cosegregation of PLG p.G420D and disease. Genotyping of PLG p.G420D in 14446 patients, and 8797 controls from Canada, France, Spain, Germany, Belgium, and Austria failed to identify significant association with disease (P = 0.117), despite an overall higher prevalence in patients (OR = 1.32; 95% CI = 0.93–1.87). To assess whether additional rare variants have an effect on MS risk, we sequenced PLG in 293 probands, and genotyped all rare variants in cases and controls. This analysis identified nine rare missense variants, and although three of them were exclusively observed in MS patients, segregation does not support pathogenicity. PLG is a plausible biological candidate for MS owing to its involvement in immune system response, blood-brain barrier permeability, and myelin degradation. Moreover, components of its activation cascade have been shown to present increased activity or expression in MS patients compared to controls; further studies are needed to clarify whether PLG is involved in MS susceptibility. PMID:27194806

Gender-specific association of the plasminogen activator inhibitor-1 4G/5G polymorphism with central arterial blood pressure.

PubMed

Björck, Hanna M; Eriksson, Per; Alehagen, Urban; De Basso, Rachel; Ljungberg, Liza U; Persson, Karin; Dahlström, Ulf; Länne, Toste

2011-07-01

The functional plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphism has previously been associated with hypertension. In recent years, central blood pressure, rather than brachial has been argued a better measure of cardiovascular damage and clinical outcome. The aim of this study was to investigate the possible influence of the 4G/5G polymorphism on central arterial blood pressure in a cohort of elderly individuals. We studied 410 individuals, 216 men and 194 women, aged 70-88. Central pressures and pulse waveforms were calculated from the radial artery pressure waveform by the use of the SphygmoCor system and a generalized transfer function. Brachial pressure was recorded using oscillometric technique (Dinamap, Critikon, Tampa, FL). PAI-1 antigen was determined in plasma. The results showed that central pressures were higher in women carrying the PAI-1 4G/4G genotype compared to female carriers of the 5G/5G genotype, (P = 0.025, P = 0.002, and P = 0.002 for central systolic-, diastolic-, and mean arterial pressure, respectively). The association remained after adjustment for potentially confounding factors related to hypertension. No association of the PAI-1 genotype with blood pressure was found in men. Multiple regression analysis revealed an association between PAI-1 genotype and plasma PAI-1 levels (P = 0.048). Our findings show a gender-specific association of the PAI-1 4G/5G polymorphism with central arterial blood pressure. The genotype effect was independent of other risk factors related to hypertension, suggesting that impaired fibrinolytic potential may play an important role in the development of central hypertension in women.

A high-protein diet during hospitalization is associated with an accelerated decrease in soluble urokinase plasminogen activator receptor levels in acutely ill elderly medical patients with SIRS.

PubMed

Tavenier, Juliette; Haupt, Thomas H; Andersen, Aino L; Buhl, Sussi F; Langkilde, Anne; Andersen, Jens R; Jensen, Jens-Erik B; Pedersen, Mette M; Petersen, Janne; Andersen, Ove

2017-05-01

Acute illness and hospitalization in elderly individuals are often accompanied by the systemic inflammatory response syndrome (SIRS) and malnutrition, both associated with wasting and mortality. Nutritional support and resistance training were shown to increase muscle anabolism and reduce inflammation in healthy elderly. We hypothesized that nutritional support and resistance training would accelerate the resolution of inflammation in hospitalized elderly patients with SIRS. Acutely admitted patients aged >65 years with SIRS were randomized to an intervention consisting of a high-protein diet (1.7 g/kg per day) during hospitalization, and daily protein supplement (18.8 g) and 3 weekly resistance training sessions for 12 weeks after discharge (Intervention, n=14), or to standard-care (Control, n=15). Plasma levels of the inflammatory biomarkers soluble urokinase plasminogen activator receptor (suPAR), interleukin-6, C-reactive protein (CRP), and albumin were measured at admission, discharge, and 4 and 13 weeks after discharge. The Intervention group had an earlier decrease in suPAR levels than the Control group: -15.4% vs. +14.5%, P=.007 during hospitalization, and -2.4% vs. -28.6%, P=.007 between discharge and 4 weeks. There were no significant effects of the intervention on the other biomarkers. All biomarkers improved significantly between admission and 13 weeks, although with different kinetics (suPAR: -22%, interleukin-6: -86%, CRP: -89%, albumin: +11%). Nutritional support during hospitalization was associated with an accelerated decrease in suPAR levels, whereas the combined nutrition and resistance training intervention after discharge did not appear to affect the inflammatory state. Our results indicate that improved nutritional care during hospitalization may accelerate recovery in acutely ill elderly medical patients. Copyright © 2017 Elsevier Inc. All rights reserved.

PAI-1 promoter 4G/5G polymorphism (rs1799768) contributes to tumor susceptibility: Evidence from meta-analysis.

PubMed

Xu, Xin; Xie, Yanqi; Lin, Yiwei; Xu, Xianglai; Zhu, Yi; Mao, Yeqing; Hu, Zhenghui; Wu, Jian; Chen, Hong; Zheng, Xiangyi; Qin, Jie; Xie, Liping

2012-12-01

Plasminogen activator inhibitor-1 (PAI-1), belonging to the urokinase plasminogen activation (uPA) system, is involved in cancer development and progression. The PAI-1 promoter 4G/5G polymorphism was shown to contribute to genetic susceptibility to cancer, although the results were inconsistent. To assess this relationship more precisely, a meta-analysis was performed. The electronic databases PubMed, Scopus, Web of Science and Chinese National Knowledge Infrastructure (CNKI) were searched; data were extracted and analyzed independently by two reviewers. Ultimately, 21 eligible case-control studies with a total of 8,415 cancer cases and 9,208 controls were included. The overall odds ratio (OR) with its 95% confidence interval (CI) showed a statistically significant association between the PAI-1 promoter 4G/5G polymorphism and cancer risk (4G/4G vs. 5G/5G: OR=1.25, 95% CI=1.07-1.47, P(heterogeneity)=0.001; 4G/4G vs. 4G/5G+5G/5G: OR=1.10, 95% CI=1.03-1.17, P(heterogeneity)=0.194; 4G/4G+4G/5G vs. 5G/5G: OR=1.17, 95% CI=1.01-1.35, P(heterogeneity)=0.041). In further subgroup analyses, the increased risk of cancer was observed in a subgroup of Caucasians with regards to endometrial cancer. Our meta-analysis suggests that the PAI-1 4G/5G polymorphism most likely contributes to susceptibility to cancer, particularly in Caucasians. Furthermore, the 4G allele may be associated with an increased risk of endometrial cancer.

PAI-1 promoter 4G/5G polymorphism (rs1799768) contributes to tumor susceptibility: Evidence from meta-analysis

PubMed Central

XU, XIN; XIE, YANQI; LIN, YIWEI; XU, XIANGLAI; ZHU, YI; MAO, YEQING; HU, ZHENGHUI; WU, JIAN; CHEN, HONG; ZHENG, XIANGYI; QIN, JIE; XIE, LIPING

2012-01-01

Plasminogen activator inhibitor-1 (PAI-1), belonging to the urokinase plasminogen activation (uPA) system, is involved in cancer development and progression. The PAI-1 promoter 4G/5G polymorphism was shown to contribute to genetic susceptibility to cancer, although the results were inconsistent. To assess this relationship more precisely, a meta-analysis was performed. The electronic databases PubMed, Scopus, Web of Science and Chinese National Knowledge Infrastructure (CNKI) were searched; data were extracted and analyzed independently by two reviewers. Ultimately, 21 eligible case-control studies with a total of 8,415 cancer cases and 9,208 controls were included. The overall odds ratio (OR) with its 95% confidence interval (CI) showed a statistically significant association between the PAI-1 promoter 4G/5G polymorphism and cancer risk (4G/4G vs. 5G/5G: OR=1.25, 95% CI=1.07–1.47, Pheterogeneity=0.001; 4G/4G vs. 4G/5G+5G/5G: OR=1.10, 95% CI=1.03–1.17, Pheterogeneity=0.194; 4G/4G+4G/5G vs. 5G/5G: OR=1.17, 95% CI=1.01–1.35, Pheterogeneity=0.041). In further subgroup analyses, the increased risk of cancer was observed in a subgroup of Caucasians with regards to endometrial cancer. Our meta-analysis suggests that the PAI-1 4G/5G polymorphism most likely contributes to susceptibility to cancer, particularly in Caucasians. Furthermore, the 4G allele may be associated with an increased risk of endometrial cancer. PMID:23226787

Uric acid therapy improves the outcomes of stroke patients treated with intravenous tissue plasminogen activator and mechanical thrombectomy.

PubMed

Chamorro, Ángel; Amaro, Sergio; Castellanos, Mar; Gomis, Meritxell; Urra, Xabier; Blasco, Jordi; Arenillas, Juan F; Román, Luis S; Muñoz, Roberto; Macho, Juan; Cánovas, David; Marti-Fabregas, Joan; Leira, Enrique C; Planas, Anna M

2017-06-01

Background Numerous neuroprotective drugs have failed to show benefit in the treatment of acute ischemic stroke, making the search for new treatments imperative. Uric acid is an endogenous antioxidant making it a drug candidate to improve stroke outcomes. Aim To report the effects of uric acid therapy in stroke patients receiving intravenous thrombolysis and mechanical thrombectomy. Methods Forty-five patients with proximal vessel occlusions enrolled in the URICO-ICTUS trial received intravenous recombinant tissue plasminogen activator within 4.5 h after stroke onset and randomized to intravenous 1000 mg uric acid or placebo (NCT00860366). These patients also received mechanical thrombectomy because a brain computed tomogaphy angiography confirmed the lack of proximal recanalization at the end of systemic thrombolysis. The primary outcome was good functional outcome at 90 days (modified Rankin Score 0-2). Safety outcomes included mortality, symptomatic intracerebral bleeding, and gout attacks. Results The rate of successful revascularization was >80% in the uric acid and the placebo groups but good functional outcome was observed in 16 out of 24 (67%) patients treated with uric acid and 10 out of 21 (48%) treated with placebo (adjusted Odds Ratio, 6.12 (95% CI 1.08-34.56)). Mortality was observed in two out of 24 (8.3%) patients treated with uric acid and one out of 21 (4.8%) treated with placebo (adjusted Odds Ratio, 3.74 (95% CI 0.06-226.29)). Symptomatic cerebral bleeding and gout attacks were similar in both groups. Conclusions Uric acid therapy was safe and improved stroke outcomes in stroke patients receiving intravenous thrombolysis followed by thrombectomy. Validation of this simple strategy in a larger trial is urgent.

Freeze-dried plasma enhances clot formation and inhibits fibrinolysis in the presence of tissue plasminogen activator similar to pooled liquid plasma.

PubMed

Huebner, Benjamin R; Moore, Ernest E; Moore, Hunter B; Sauaia, Angela; Stettler, Gregory; Dzieciatkowska, Monika; Hansen, Kirk; Banerjee, Anirban; Silliman, Christopher C

2017-08-01

Systemic hyperfibrinolysis is an integral part of trauma-induced coagulopathy associated with uncontrolled bleeding. Recent data suggest that plasma-first resuscitation attenuates hyperfibrinolysis; however, the availability, transport, storage, and administration of plasma in austere environments remain challenging and have limited its use. Freeze-dried plasma (FDP) is a potential alternative due to ease of storage, longer shelf life, and efficient reconstitution. FDP potentially enhances clot formation and resists breakdown better than normal saline (NS) and albumin and similar to liquid plasma. Healthy volunteers underwent citrated blood draw followed by 50% dilution with NS, albumin, pooled plasma (PP), or pooled freeze-dried plasma (pFDP). Citrated native and tissue plasminogen activator (t-PA)-challenge (75 ng/mL) thrombelastography were done. Proteins in PP, pFDP, and albumin were analyzed by mass spectroscopy. pFDP and PP had superior clot-formation rates (angle) and clot strength (maximum amplitude) compared with NS and albumin in t-PA-challenge thrombelastographies (angle: pFDP, 67.9 degrees; PP, 67.8 degrees; NS, 40.6 degrees; albumin, 35.8 degrees; maximum amplitude: pFDP, 62.4 mm; PP, 63.5 mm; NS, 44.8 mm; albumin, 41.1 mm). NS and albumin dilution increased susceptibility to t-PA-induced hyperfibrinolysis compared with pFDP and PP (NS, 62.4%; albumin, 62.6%; PP, 8.5%; pFDP, 6.7%). pFDP was similar to PP in the attenuation of t-PA-induced fibrinolysis. Most proteins (97%) were conserved during the freeze-dry process, with higher levels in 12% of pFDP proteins compared with PP. pFDP enhances clot formation and attenuates hyperfibrinolysis better than NS and albumin and is a potential alternative to plasma resuscitation in the treatment of hemorrhagic shock. © 2017 AABB.

Anti-invasion effects of 6-shogaol and 6-gingerol, two active components in ginger, on human hepatocarcinoma cells.

PubMed

Weng, Chia-Jui; Wu, Cheng-Feng; Huang, Hsiao-Wen; Ho, Chi-Tang; Yen, Gow-Chin

2010-11-01

Hepatocellular carcinoma is the most common type of liver cancer and is highly metastatic. Metastasis is considered to be the major cause of death in cancer patients. Ginger is a natural dietary rhizome with anti-oxidative, anti-inflammatory, and anti-carcinogenic activities. The aims of this study were to evaluate the anti-invasion activity of 6-shogaol and 6-gingerol, two compounds found in ginger, on hepatoma cells. The migratory and invasive abilities of phorbol 12-myristate 13-acetate (PMA)-treated HepG2 and PMA-untreated Hep3B cells were both reduced in a dose-dependent manner by treatment with 6-shogaol and 6-gingerol. Upon incubation of PMA-treated HepG2 cells and PMA-untreated Hep3B cells with 6-shogaol and 6-gingerol, matrix metalloproteinase (MMP)-9 activity decreased, whereas the expression of tissue inhibitor metalloproteinase protein (TIMP)-1 increased in both cell types. Additionally, urokinase-type plasminogen activator activity was dose-dependently decreased in Hep3B cells after incubation with 6-shogaol for 24 h. Analysis with semi-quantitative reverse transcription-PCR showed that the regulation of MMP-9 by 6-shogaol and 6-gingerol and the regulation of TIMP-1 by 6-shogaol in Hep3B cells may on the transcriptional level. These results suggest that 6-shogaol and 6-gingerol might both exert anti-invasive activity against hepatoma cells through regulation of MMP-9 and TIMP-1 and that 6-shogaol could further regulate urokinase-type plasminogen activity.

Molecular mechanism inhibiting human hepatocarcinoma cell invasion by 6-shogaol and 6-gingerol.

PubMed

Weng, Chia-Jui; Chou, Chai-Ping; Ho, Chi-Tang; Yen, Gow-Chin

2012-08-01

We previously demonstrated that 6-shogaol and 6-gingerol, two active compounds in ginger (Zingiber officinale), possess antiinvasive activity against highly metastatic hepatoma cells. The aims of this study were to evaluate the inhibitory effect and molecular mechanism underlying the transcription and translation of matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA) in Hep3B cells as well as the antiangiogenic activity of 6-gingerol and 6-shogaol. By gelatin zymography and luciferase reporter gene assays, we found that 6-gingerol and 6-shogaol regulate MMP-2/-9 transcription. Moreover, 6-gingerol directly decreased expression of uPA, but the 6-shogaol-mediated decrease in uPA was accompanied by up-regulation of plasminogen activator inhibitor (PAI)-1. 6-Gingerol and 6-shogaol concentrations of ≥ 10 μM and ≥ 2.5 μM, respectively, significantly inhibited the phosphorylation of mitogen-activated protein kinase (MAPK) and PI3K/Akt signaling, the activation of NF-κB, and the translocation of NF-κB and STAT3. Incubation of 6-gingerol or 6-shogaol with human umbilical vein endothelial cells or rat aortas significantly attenuated tube formation. 6-Shogaol and 6-gingerol effectively inhibit invasion and metastasis of hepatocellular carcinoma through diverse molecular mechanisms, including inhibition of the MAPK and PI3k/Akt pathways and NF-κB and STAT3 activities to suppress expression of MMP-2/-9 and uPA and block angiogenesis. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Spontaneous hemothorax following thrombolytic and anticoagulant therapy for massive pulmonary embolism.

PubMed

Wang, Chung-Cheng; Ng, Chip-Jin; Seak, Chen-Ken; Seak, Chen-June

2013-06-01

Massive spontaneous hemothorax following combined thrombolytic and anticoagulant therapy for pulmonary embolism(PE) is a rare event that is little documented in the literature. Here, we describe a rare case of spontaneous hemothorax in a 23-year-old woman with underlying systemic lupus erythematosus following combined administration of tissue plasminogen activator and low-molecular-weight heparin for massive PE. This report of our successful treatment of this case by video-assisted thoracoscopic thoracotomy demonstrates that although the occurrence is rare, massive hemothorax following anticoagulant and/or thrombolytic therapy for PE should be suspected if patients experience chest pain, dyspnea, or signs of anemia, and follow-up physical examination and hemogram should be performed to facilitate diagnosis of this life-threatening complication.

The biology of the peroxisome proliferator-activated receptor system in the female reproductive tract.

PubMed

Vélez, Leandro Martín; Abruzzese, Giselle Adriana; Motta, Alicia Beatriz

2013-01-01

Fuel sensors such as glucose, insulin or leptin, are known to be directly involved in the regulation of fertility at each level of the hypothalamic-pituitary-gonadal axis. The discovery of the peroxisome proliferator-activated receptor (PPAR) family of transcription factors has revealed the link between lipid/glucose availability and long-term metabolic adaptation. By binding to specific regions of DNA in heterodimers with the retinoid X receptors (RXRs), the members of the PPAR family (α, β/δ, γ) are able to regulate the gene expressions of several key regulators of energy homeostasis including several glucose regulators (glucose transporters, insulin receptor, substrate insulin receptor, etc), and also metabolic and endocrine pathways like lipogenesis, steroidogenesis, ovulation, oocyte maturation, maintenance of the corpus luteum, nitric oxide system, several proteases and plasminogen activator among others. All the three PPAR isoforms are expressed in different tissues of the female reproductive tract and regulate gametogenesis, ovulation, corpus luteum regression and the implantation process among others. The present review discusses the mechanisms involved in PPAR activation focusing on endogenous and synthetic ligands of PPAR not only in physiological but also in pathological conditions (such as polycystic ovary syndrome, pathologies of implantation process, chronic anovulation, etc).

N2 non-thermal atmospheric pressure plasma promotes wound healing in vitro and in vivo: Potential modulation of adhesion molecules and matrix metalloproteinase-9.

PubMed

Kang, Sung Un; Choi, Jae Won; Chang, Jae Won; Kim, Kang Il; Kim, Yeon Soo; Park, Ju Kyeong; Kim, Yang Eun; Lee, Yun Sang; Yang, Sang Sik; Kim, Chul-Ho

2017-02-01

Advances in physics and biology have made it possible to apply non-thermal atmospheric pressure plasma (NTP) in the biomedical field. Although accumulating evidence suggests that NTP has various medicinal effects, such as facilitating skin wound healing on exposed tissue while minimizing undesirable tissue damage, the underlying molecular mechanisms are not fully understood. In this study, NTP generated from N 2 optimized wound healing in the scratch wound healing assay. In addition, matrix metalloproteinase (MMP)-9 expression and enzyme activity increased and the urokinase-type plasminogen activator (uPA) system was activated after NTP treatment. We also showed that NTP treatment increased Slug and TCF8/ZEB1 expression and decreased that of E-cadherin, suggesting induction of the epithelial-to-mesenchymal transition (EMT). The effect of N 2 NTP was verified on rat wound model. Taken together, these results suggest that N 2 NTP promotes wound healing by inducing the EMT and activating the MMP-9/uPA system. These findings show the therapeutic potential of NTP for skin wound healing. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Full-length soluble urokinase plasminogen activator receptor down-modulates nephrin expression in podocytes

PubMed Central

Alfano, Massimo; Cinque, Paola; Giusti, Guido; Proietti, Silvia; Nebuloni, Manuela; Danese, Silvio; D’Alessio, Silvia; Genua, Marco; Portale, Federica; Lo Porto, Manuela; Singhal, Pravin C.; Rastaldi, Maria Pia; Saleem, Moin A.; Mavilio, Domenico; Mikulak, Joanna

2015-01-01

Increased plasma level of soluble urokinase-type plasminogen activator receptor (suPAR) was associated recently with focal segmental glomerulosclerosis (FSGS). In addition, different clinical studies observed increased concentration of suPAR in various glomerular diseases and in other human pathologies with nephrotic syndromes such as HIV and Hantavirus infection, diabetes and cardiovascular disorders. Here, we show that suPAR induces nephrin down-modulation in human podocytes. This phenomenon is mediated only by full-length suPAR, is time-and dose-dependent and is associated with the suppression of Wilms’ tumor 1 (WT-1) transcription factor expression. Moreover, an antagonist of αvβ3 integrin RGDfv blocked suPAR-induced suppression of nephrin. These in vitro data were confirmed in an in vivo uPAR knock out Plaur−/− mice model by demonstrating that the infusion of suPAR inhibits expression of nephrin and WT-1 in podocytes and induces proteinuria. This study unveiled that interaction of full-length suPAR with αvβ3 integrin expressed on podocytes results in down-modulation of nephrin that may affect kidney functionality in different human pathologies characterized by increased concentration of suPAR. PMID:26380915

Pericyte protection by edaravone after tissue plasminogen activator treatment in rat cerebral ischemia

PubMed Central

Deguchi, Kentaro; Liu, Ning; Liu, Wentao; Omote, Yoshio; Kono, Syoichiro; Yunoki, Taijun; Deguchi, Shoko; Yamashita, Toru; Ikeda, Yoshio; Abe, Koji

2014-01-01

Pericytes play a pivotal role in contraction, mediating inflammation and regulation of blood flow in the brain. In this study, changes of pericytes in the neurovascular unit (NVU) were examined in relation to the effects of exogenous tissue plasminogen activator (tPA) and a free radical scavenger, edaravone. Immunohistochemistry and Western blot analyses showed that the overlap between platelet-derived growth factor receptor β-positive pericytes and N-acetylglucosamine oligomers (NAGO)-positive endothelial cells increased significantly at 4 days after 90 min of transient middle cerebral artery occlusion (tMCAO). The number of pericytes and the overlap with NAGO decreased with tPA but recovered with edaravone 4 days after tMCAO with proliferation. Thus, tPA treatment damaged pericytes, resulting in the detachment from astrocytes and a decrease in glial cell line-derived neurotrophic factor secretion. However, treatment with edaravone greatly improved tPA-induced damage to pericytes. The present study demonstrates that exogenous tPA strongly damages pericytes and destroys the integrity of the NVU, but edaravone treatment can greatly ameliorate such damage after acute cerebral ischemia in rats. © 2014 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc. PMID:24938625

The urea decomposition product cyanate promotes endothelial dysfunction

PubMed Central

El-Gamal, Dalia; Rao, Shailaja Prabhakar; Holzer, Michael; Hallström, Seth; Haybaeck, Johannes; Gauster, Martin; Wadsack, Christian; Kozina, Andrijana; Frank, Saša; Schicho, Rudolf; Schuligoi, Rufina; Heinemann, Akos; Marsche, Gunther

2014-01-01

The dramatic cardiovascular mortality of chronic kidney disease patients is attributable in a significant proportion to endothelial dysfunction. Cyanate, a reactive species in equilibrium with urea, is formed in excess in chronic kidney disease. Cyanate is thought to have a causal role in promoting cardiovascular disease, but the underlying mechanisms remain unclear. Immunohistochemical analysis performed in the present study revealed that carbamylated epitopes associate mainly with endothelial cells in human atherosclerotic lesions. Cyanate treatment of human coronary artery endothelial cells reduced expression of endothelial nitric oxide synthase and increased tissue factor and plasminogen activator inhibitor-1 expression. In mice, administration of cyanate - promoting protein carbamylation at levels observed in uremic patients - attenuated arterial vasorelaxation of aortic rings in response to acetylcholine, without affecting sodium nitroprusside-induced relaxation. Total endothelial nitric oxide synthase and nitric oxide production were significantly reduced in aortic tissue of cyanate-treated mice. This coincided with a marked increase of tissue factor and plasminogen activator inhibitor-1 protein levels in aortas of cyanate-treated mice. These data provide evidence that cyanate compromises endothelial functionality in vitro and in vivo and may contribute to the dramatic cardiovascular risk of patients suffering from chronic kidney disease. PMID:24940796

Evaluation of chest tube administration of tissue plasminogen activator to treat retained hemothorax.

PubMed

Stiles, P J; Drake, Rachel M; Helmer, Stephen D; Bjordahl, Paul M; Haan, James M

2014-06-01

When retained hemothorax occurs, video-assisted thoracoscopy or thoracotomy is performed, but recently, tissue plasminogen activator (tPA) has been used. This study evaluated intrapleural tPA use for retained traumatic hemothoraces. A retrospective review was conducted of trauma patients treated with intrapleural tPA for retained hemothorax. Data included demographics, past medical and surgical histories, injury details, treatment details, and outcomes. Seven patients (median age = 47 years, male = 6, blunt trauma = 6) met study criteria. All patients received a chest tube. Six patients later received computed tomography-guided drains for tPA infusion. Number of tPA treatments per patient varied from 1 to 5. Median total tPA dosage was 24 mg. Median time from injury to chest tube placement was 11 days and from chest tube placement to first tPA treatment was 4 days. No patients required a video-assisted thoracoscopy; however, 1 patient required thoracotomy. There were no deaths or bleeding complications attributed to intrapleural tPA. Although future studies are needed to identify optimum treatment guidelines, intrapleural tPA appears to be a safe and efficacious treatment option. Copyright © 2014 Elsevier Inc. All rights reserved.

Meta-analysis of the association between plasminogen activator inhibitor-1 4G/5G polymorphism and recurrent pregnancy loss.

PubMed

Li, Xuejiao; Liu, Yukun; Zhang, Rui; Tan, Jianping; Chen, Libin; Liu, Yinglin

2015-04-11

The association between plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphism and recurrent pregnancy loss (RPL) risk is still contradictory. We thus performed a meta-analysis. Relevant studies were searched for in PubMed, Web of Science, Embase, and Cochrane Library. An odds ratio (OR) with a 95% confidence interval (CI) was used to assess the association between PAI-1 4G/5G polymorphism and RPL risk. A total of 22 studies with 4306 cases and 3076 controls were included in this meta-analysis. We found that PAI-1 4G/5G polymorphism was significantly associated with an increased RPL risk (OR=1.89; 95% CI 1.34-2.67; P=0.0003). In the subgroup analysis by race, PAI-1 4G/5G polymorphism was significantly associated with an increased RPL risk in Caucasians (OR=2.23; 95% CI 1.44-3.46; P=0.0003). However, no significant association was observed in Asians (OR=1.47; 95% CI 0.84-2.59; P=0.18). In conclusion, this meta-analysis suggests that PAI-1 4G/5G polymorphism might be associated with RPL development in Caucasians.

Association between plasminogen activator inhibitor-1 -675 4G/5G polymorphism and sepsis: a meta-analysis.

PubMed

Li, Li; Nie, Wei; Zhou, Hongfeng; Yuan, Weifeng; Li, Weifeng; Huang, Wenjie

2013-01-01

Several studies have evaluated the association between plasminogen activator inhibitor-1 (PAI-1) -675 4G/5G polymorphism and sepsis in different populations. However, the available results are conflicting. A search of Pubmed and EMBASE databases was performed to identify relevant studies for inclusion in the meta-analysis. Odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were determined using a random-effects model. Twelve case-control studies and three cohort studies were included. Overall, a significant association between 4G/5G polymorphism and sepsis risk was observed for 4G/4G vs. 4G/5G +5G/5G (OR = 1.30, 95% CI 1.08-1.56, P = 0.006). In addition, there was a significant association between PAI-1 4G/5G polymorphism and sepsis-related mortality (OR = 1.72, 95% CI 1.27-2.33, P = 0.0005). In subgroup analyses, increased sepsis risk and mortality risk were found in Caucasians and in patients with sepsis. This meta-analysis suggested that the PAI-1 -675 4G/5G polymorphism was a risk factor for sepsis and sepsis mortality.

Co-morbid conditions in use of recombinant tissue plasminogen activator (rt-PA) for the treatment of acute ischaemic stroke.

PubMed

Nathaniel, Thomas I; Cochran, Thomas; Chaves, Jose; Fulmer, Eric; Sosa, Crystal; Yi, Sara; Fredwall, Megan; Sternberg, Shannon; Blackhurst, Dawn; Nelson, Alfred; Leacock, Rodney

2016-01-01

The objective of this study was to characterize the comorbidities in a population of patients with an acute ischaemic stroke, comparing patients that received recombinant tissue plasminogen activator (rt-PA) to those that did not receive rt-PA. In a retrospective sample of 663 patients admitted for acute ischaemic stroke, this study analysed the effects of co-morbid conditions in the use of rt-PA. It determined non-cerebrovascular risk factors (comorbidities) that differentiate patients who received rt-PA from those who did not receive rt-PA. Patients with a history of carotid stenosis, CHF and previous strokes are significantly (p < 0.05) associated with high risk of not receiving rt-PA. A significant number of patients with a history of hypertension and smoking received rt-PA (p < 0.05). The findings indicate that certain risk factors including carotid stenosis, CHF and previous stroke history impact the treatment of patients with acute ischaemic stroke, specifically the decision to administer rt-PA. Treatment with rt-PA is dependent on stroke severity and onset to treatment time, but the findings suggest that rt-PA use may also depend on patient comorbidities.

Increased alveolar plasminogen activator in early asbestosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cantin, A.; Allard, C.; Begin, R.

1989-03-01

Alveolar macrophage-derived plasminogen activator (PA) activity is decreased in some chronic interstitial lung diseases such as idiopathic pulmonary fibrosis and sarcoidosis but increased in experimental models of acute alveolitis. Although asbestos fibers can stimulate alveolar macrophages (AM) to release PA in vitro, the effect of chronic asbestos exposure of the lower respiratory tract on lung PA activity remains unknown. The present study was designed to evaluate PA activity of alveolar macrophages and bronchoalveolar lavage (BAL) fluid in asbestos-exposed sheep and asbestos workers. Forty-three sheep were exposed to either 100 mg UICC chrysotile B asbestos in 100 ml phosphate-buffered saline (PBS)more » or to 100 ml PBS by tracheal infusion every 2 wk for 18 months. At Month 18, chest roentgenograms were analyzed and alveolar macrophage and extracellular fluid PA activity were measured in samples obtained by BAL. Alveolar macrophage PA activity was increased in the asbestos-exposed sheep compared to control sheep (87.2 +/- 17.3 versus 41.1 +/- 7.2 U/10(5) AM-24 h, p less than 0.05) as was the BAL fluid PA activity (674.9 +/- 168.4 versus 81.3 +/- 19.7 U/mg alb-24 h, p less than 0.01). Among the asbestos-exposed sheep, 10 had normal chest roentgenograms (Group SA) and 15 had irregular interstitial opacities (Group SB). Strikingly, whereas Group SA did not differ from the control group in BAL cellularity or PA activity, Group SB had marked increases in alveolar macrophages (p less than 0.005), AM PA activity (p less than 0.02), and BAL PA activity (p less than 0.001) compared to the control group.« less

Expression profiles of glyceraldehyde-3-phosphate dehydrogenase from Clonorchis sinensis: a glycolytic enzyme with plasminogen binding capacity.

PubMed

Hu, Yue; Zhang, Erhong; Huang, Lisi; Li, Wenfang; Liang, Pei; Wang, Xiaoyun; Xu, Jin; Huang, Yan; Yu, Xinbing

2014-12-01

Globally, 15-20 million people are infected with Clonorchis sinensis (C. sinensis) which results in clonorchiasis. In China, clonorchiasis is considered to be one of the fastest-growing food-borne parasitic diseases. That more key molecules of C. sinensis are characterized will be helpful to understand biology and pathogenesis of the carcinogenic liver fluke. Glyceraldehyde-3-phosphate dehydrogenases (GAPDHs) from many species have functions other than their catalytic role in glycolysis. In the present study, we analyzed the sequence and structure of GAPDH from C. sinensis (CsGAPDH) by using bioinformatics tools and obtained its recombinant protein by prokaryotic expression system, to learn its expression profiles and molecular property. CsGAPDH could bind to human intrahepatic biliary epithelial cell in vivo and in vitro by the method of immunofluorescence assays. CsGAPDH also disturbed in lumen of biliary tract near to the parasite in the liver of infected rat. Western blotting analysis together with immunofluorescence assay indicated that CsGAPDH was a component of excretory/secretory proteins (CsESPs) and a surface-localized protein of C. sinensis. Quantitative real-time PCR (Q-PCR) and Western blotting demonstrated that CsGAPDHs are expressed at the life stages of adult worm, metacercaria, and egg, but the expression levels were different from each other. Recombinant CsGAPDH (rCsGAPDH) was confirmed to have the capacity to catalyze the conversion of glyceraldehyde 3-phosphate to D-glycerate 1,3-bisphosphate which was inhibited by AMP in a dose-dependent manner. In addition, rCsGAPDH was able to interact with human plasminogen in a dose-dependent manner by ELISA. The interaction could be inhibited by lysine. The plasminogen binding capacity of rCsGAPDH along with the distribution of CsGAPDH in vivo and in the liver of C. sinensis-infected rat hinted that surface-localized CsGAPDH might play an important role in host invasion of the worm besides its glycolytic activity. Our work will be a cornerstone for getting more messages about CsGAPDH and its role in biology and parasitism of C. sinensis.

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

2017-12-01

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. © 2017 Blackwell Verlag GmbH.

Downregulation of Extracellular Matrix Metalloproteinase Inducer by scFv-M6-1B9 Intrabody Suppresses Cervical Cancer Invasion Through Inhibition of Urokinase-Type Plasminogen Activator.

PubMed

Panich, Tipattaraporn; Tragoolpua, Khajornsak; Pata, Supansa; Tayapiwatana, Chatchai; Intasai, Nutjeera

2017-02-01

Overexpression of extracellular matrix metalloproteinase inducer (EMMPRIN) accelerates tumor invasion and metastasis via activation of matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA) expression. The authors were interested in whether the scFv-M6-1B9 intrabody against EMMPRIN that retains EMMPRIN in endoplasmic reticulum could be a potential tool to suppress cervical cancer invasion through inhibition of uPA. The chimeric adenoviral vector Ad5/F35-scFv-M6-1B9 was transferred into human cervical carcinoma HeLa cells to produce the scFv-M6-1B9 intrabody against EMMPRIN. Cell surface expression of EMMPRIN, the membrane-bound uPA, the enzymatic activity of secreted uPA, and the invasion ability were analyzed. The scFv-M6-1B9 intrabody successfully diminished the cell surface expression of EMMPRIN and the membrane-bound uPA on HeLa cells. uPA activity from tissue culture media of EMMPRIN-downregulated HeLa cells was decreased. The invasion ability of HeLa cells harboring scFv-M6-1B9 intrabody was also suppressed. These results suggested that the scFv-M6-1B9 intrabody might represent a potential approach for invasive cervical cancer treatment. The application of scFv-M6-1B9 intrabody in animal experiments and preclinical studies would be investigated further.

Abrogating fibrinolysis does not improve bleeding or rFVIIa/rFVIII treatment in a non-mucosal venous injury model in haemophilic rodents.

PubMed

Stagaard, Rikke; Flick, Matthew J; Bojko, Barbara; Goryński, Krzysztof; Goryńska, Paulina Z; Ley, Carsten D; Olsen, Lisbeth H; Knudsen, Tom

2018-05-14

Fibrinolysis may exacerbate bleeding in haemophilia A (HA). Accordingly, antifibrinolytics have been used to help maintain haemostatic control. While antifibrinolytic drugs have been proven effective in the treatment of mucosal bleeds in the oral cavity, their efficacy in non-mucosal tissues remain an open question of significant clinical interest. To determine whether inhibiting fibrinolysis improves the outcome in non-mucosal haemophilic tail vein transection (TVT) bleeding models, and to determine whether a standard ex vivo clotting/fibrinolysis assay can be used as a predictive surrogate for in vivo efficacy. A highly sensitive tail vein transection model was employed in haemophilic rodents with a suppressed fibrinolytic system to examine the effect of inhibiting fibrinolysis on bleeding in a non-mucosal tissue. In mice induced- and congenital haemophilia models were combined with fibrinolytic attenuation achieved either genetically or pharmacologically (tranexamic acid; TXA) induced. In haemophilic rats, tail bleeding was followed by whole blood rotational thromboelastometry (ROTEM) evaluation of the same animals to gauge the predictive value of such assays. The beneficial effect of systemic TXA therapy observed ex vivo could not be confirmed in vivo in haemophilic rats. Furthermore, neither intravenously administered TXA nor congenital knockout of the fibrinolytic genes plasminogen or tissue-type plasminogen activator markedly improved the TVT bleeding phenotype or response to factor therapy in haemophilic mice. The finding here suggest that inhibition of fibrinolysis is not effective in limiting the TVT bleeding phenotype of haemophilia A rodents in non-mucosal tissues. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Alterations in coagulatory and fibrinolytic systems following an ultra-marathon.

PubMed

Kupchak, Brian R; Volk, Brittanie M; Kunces, Laura J; Kraemer, William J; Hoffman, Martin D; Phinney, Stephen D; Volek, Jeff S

2013-11-01

The aim of this study was to examine coagulatory and fibrinolytic responses to the Western States Endurance Run (WSER, June 23 to 24, 2012). The WSER is a 161-km (100 mile) trail foot race through the Sierra Nevada Mountains that involves 6,030 m of climb and 7,001 m of descent. We examined 12 men and 4 women [mean (95 % CI), age 44.6 years (38.7-50.6)] who completed the race (24.64 h; range 16.89-29.46). Blood samples were collected the morning before the race, immediately post-race, and 1 (D1) and 2 (D2) days post-race (corresponding to 51-54 h and 75-78 h from the start of the race, respectively). Hypercoagulable state was characterized by prothrombin fragment 1+2 (PTF 1+2) and thrombin-antithrombin complex (TAT). Fibrinolytic state was assessed by plasminogen activator inhibitor antigen (PAI-1 Ag), tissue plasminogen activator antigen (tPA Ag), and D-Dimer. Muscle damage was assessed by serum creatine kinase (CK) and myoglobin concentrations. Significant (P ≤ 0.05) increases were observed immediately post-race for thrombin generation markers, PTF 1+2 (3.9-fold) and TAT (2.4-fold); markers of fibrinolysis, tPA Ag (4.0-fold), PAI-1 Ag (4.5-fold), and D-Dimer (2.2-fold); and muscle damage markers, CK (154-fold) and myoglobin (114-fold). Most markers continued to be elevated at D1, as seen by PTF 1+2, TAT (1.5- and 1.3-fold increase at D1), and D-Dimer (2.5- and 2.1-fold increase at D1 and D2, respectively). Additionally, PTF 1+2:tPA and TAT:tPA ratios, which assessed balance between coagulation and fibrinolysis, were slightly, but significantly increased at D1 (69 and 36 %) and D2 (19 and 31 %). CK and myoglobin also remained elevated at D1 (54- and 7-fold) and D2 (25- and 2-fold) time points. The WSER produced extensive muscle damage and activated the coagulation and fibrinolytic systems. Since we observed a slight imbalance response between the two systems, a limited potential for thrombotic episodes is apparent in these highly trained athletes.

[The influence of lactate Ringer solution versus hydroxyethyl starch on coagulation and fibrinolytic system in patients with septic shock].

PubMed

Lv, Jie; Zhao, Hui-ying; Liu, Fang; An, You-zhong

2012-01-01

To investigate the influence of lactate Ringer solution (RL) versus hydroxyethyl starch 130/0.4 (HES130/0.4) solution on coagulation and fibrinolytic system in the patients with septic shock. Forty-two consecutive patients with septic shock diagnosed between September 2009 and June 2011 were randomized to two study groups: RL resuscitation group (RL group) with 20 patients, and HES130/0.4 resuscitation group (HES group) with 22 patients. In all of them peripheral blood was collected at four points of time: before resuscitation, 6, 12, 24 hours after resuscitation, and then prothrombin time (PT), activated partial thromboplastin time (APTT) and levels of plasma tissue plasminogen activator (t-PA), and plasminogen activator inhibitor (PAI) were determined. Meanwhile, the patients' outcome and the length of intensive care unit stay (ICU-LOS) were recorded. ICU-LOS (days) in HES group was significantly shorter than the RL group (12.5 ± 8.8 vs. 17.1 ± 16.6, P < 0.01). Meanwhile, the volume of fluid (L: 2.77 ± 0.59) as well as vasoactive drugs [μg×kg(-1)×min(-1): 0.56 ± 0.15] used in the HES group were significantly lower than RL group (3.46 ± 0.73, 0.81 ± 0.41, both P < 0.01). In RL group, 12 patients died and 8 patients survived, while in HES group, 7 patients died and 15 patients survived, showing no difference between two groups. PT, APTT and the levels of t-PA showed no significant differences between two groups at different time points, but the levels of plasma PAI (μg/L) of the HES group decreased gradually, and was significantly lower than that before resuscitation and RL group at 24 hours after resuscitation (41.76 ± 25.95 vs. 89.11 ± 14.27, 55.08 ± 35.43, both P < 0.05). Both RL and HES130/0.4 fluid resuscitation did not affect the outcome of the patients with septic shock, but the resuscitation efficiency of HES130/0.4 is much better than RL. Both type of fluids did not show the effect on coagulability of the septic patients, but colloid fluid resuscitation may protect the vascular endothelial cell, reduce the inhibition of fibrinolytic system, and alleviate hypercoagulability state of patients in early stage.

Proteomic analysis to unravel the effect of heat stress on gene expression and milk synthesis in bovine mammary epithelial cells.

PubMed

Li, Lian; Wang, Yiru; Li, Chengmin; Wang, Genlin

2017-12-01

Heat stress can play a negative effect on milk yield and composition of dairy cattle, leading to immeasurable economic loss. The basic components of the mammary gland are the alveoli; these alveolar mammary epithelial cells reflect the milk producing ability of dairy cows. In this study, we exposed bovine mammary epithelial cells to heat stress and compared them to a control group using isobaric tags for relative and absolute quantitation combined with liquid chromatography coupled with tandem mass spectrometry. Compared with a control group, 104 differentially elevated proteins (>1.3-fold) and 167 decreased proteins (<0.77-fold) were identified in the heat treatment group. Gene Ontology analysis identified a majority of the differentially expressed proteins are associated in cell-substrate junction assembly, catabolic processes and metabolic processes. Some of these significantly regulated proteins were related to the synthesis and secretion of milk, such as milk protein and fat. This finding was further supported by the results obtained from the reduced β-casein expression through the system of plasminogen activator - plasminogen - plasmin and decreased fatty acid synthase could partly explain why milk fat synthesis ability of dairy cows decreased under heat stress. Our results highlight the effects of heat stress on synthesis of milk protein and fat, thus providing additional clues for further studies of heat stress on dairy milk production. © 2017 Japanese Society of Animal Science.

The interactions between hemostasis and resistance training: a review

PubMed Central

Nascimento, Dahan da Cunha; Neto, Frederico Ribeiro; de Santana, Frederico Santos; da Silva, Renato André Sousa; dos Santos-Neto, Leopoldo; Balsamo, Sandor

2012-01-01

Physical inactivity is considered a risk factor for cardiovascular disease and is strongly associated with changes in arterial structure. Regular physical activity and exercise contributes to the prevention of coronary artery disease. Therefore, cardiovascular and resistance training improve hemostatic parameters and promote a less thrombotic blood profile. This review highlights the studies, mechanisms, and outcomes relating to the effectiveness of resistance training on the process of hemostasis. The Pubmed, Scopus, Medline, Scielo, Lilacs, Ibecs, and Cochrane databases were used to locate the original articles. Seventeen studies were found during the research process. Of these, ten articles were excluded. Those protocols using a high volume of training for young adults showed a greater fibrinolytic response, and training protocols with intensities above 80% of 1 maximum repetition showed an increased platelet activity. In subjects with coronary artery disease, just one session of resistance training resulted in improvement in the fibrinolytic system (tissue plasminogen activator) without raising potential thrombotic markers. PMID:22419885

Neutrophil interaction with the hemostatic system contributes to liver injury in rats cotreated with lipopolysaccharide and ranitidine.

PubMed

Deng, Xiaomin; Luyendyk, James P; Zou, Wei; Lu, Jingtao; Malle, Ernst; Ganey, Patricia E; Roth, Robert A

2007-08-01

Cotreatment of rats with nontoxic doses of ranitidine (RAN) and lipopolysaccharide (LPS) causes liver injury, and this drug-inflammation interaction might be a model for idiosyncratic adverse drug responses in humans. Both polymorphonuclear neutrophils (PMNs) and the hemostatic system have been shown to be important in the injury. We tested the hypothesis that PMNs cause liver injury by interacting with the hemostatic system and producing subsequent hypoxia. In rats cotreated with LPS/RAN, PMN depletion by anti-PMN serum reduced fibrin deposition and hypoxia in the liver. PMN depletion also reduced the plasma concentration of active plasminogen activator inhibitor-1 (PAI-1), a major down-regulator of the fibrinolytic system. This suggests that PMNs promote fibrin deposition by increasing PAI-1 concentration. PMNs were activated in the livers of LPS/RAN-cotreated rats as evidenced by increased staining for hypochlorous acid-modified proteins generated by the myeloperoxidase-hydrogen peroxide-chloride system of activated phagocytes. Antiserum against the PMN adhesion molecule CD18 protected against LPS/RAN-induced liver injury. Because CD18 is important for PMN transmigration and activation, these results suggest that PMN activation is required for the liver injury. Furthermore, anti-CD18 serum reduced biomarkers of hemostasis and hypoxia, suggesting the necessity for PMN activation in the interaction between PMNs and the hemostatic system/hypoxia. Liver injury, liver fibrin, and plasma PAI-1 concentration were also reduced by eglin C, an inhibitor of proteases released by activated PMNs. In summary, PMNs are activated in LPS/RAN-cotreated rats and participate in the liver injury in part by contributing to hemostasis and hypoxia.

Effects of astaxanthin on blood coagulation, fibrinolysis and platelet aggregation in hyperlipidemic rats.

PubMed

Deng, Zu-Yue; Shan, Wei-Guang; Wang, Shen-Feng; Hu, Meng-Mei; Chen, Yan

2017-12-01

Astaxanthin (ASTX) is a xanthophyll carotenoid that reduces hemostasis in hyperlipidemic organisms. Its antihemostatic mechanisms remain unclear. The effects of ASTX on coagulation, the fibrinolytic system and platelet aggregation were investigated in hyperlipidemic rats. Different doses of ASTX (5, 10 and 30 mg/kg/day, p.o.) were administered for four weeks to high-fat diet-induced hyperlipidemic rats. Serum lipid and lipoprotein levels were measured with an automatic biochemical analyzer. The prothrombin time (PT), activated partial thromboplastin time (APTT) and maximum platelet aggregation rate (MAR) were determined by a coagulation analyzer. The activities of the tissue-type plasminogen activator (t-PA), type-1 plasminogen activator inhibitor (PAI-1) and endothelial nitric oxide synthase (eNOS), as well as the levels of thromboxane B(2) [TXB(2)], 6-keto prostaglandin F(1α) [6-keto-PGF(1α)] and platelet granule membrane protein (GMP-140), were measured with enzyme-linked immunosorbent assay kits. Gene and protein expression levels were analyzed by reverse transcriptase polymerase chain reaction and Western blot, respectively. ASTX (30 mg/kg) treatment in hyperlipidemic rats reduced serum TG (0.58 ± 0.14 versus 1.12 ± 0.24 mmol/L), serum TC (1.77 ± 0.22 versus 2.24 ± 0.21 mmol/L), serum LDL-C (1.13 ± 0.32 versus 2.04 ± 0.48 mmol/L), serum MDA (69%), plasma MAR (55%), serum TXB2/6-keto-PGF1α (34%) and serum GMP-140 levels (25%), plasma PAI-1 activity (48%) and downregulated the mRNA (33%) and protein (23%) expression of aorta eNOS, the mRNA (79%) and protein (72%) expression levels of aorta PAI-1. However, ASTX (30 mg/kg/d) treatment increased serum SOD activity (2.1 fold), serum GPx activity (1.8 fold), plasma PT (1.3 fold), plasma APTT (1.7 fold), serum NO (1.4-fold), serum 6-keto-PGF1α (1.3 fold). ASTX reduced blood coagulation and platelet aggregation and promoted fibrinolytic activity in hyperlipidemic rats. These activities were closely correlated with ASTX, maintaining the balance of t-PA/PAI-1, NO/ROS and TXA2/PGI2 in vivo.

In vitro regulation of pericellular proteolysis in prostatic tumor cells treated with bombesin.

PubMed

Festuccia, C; Guerra, F; D'Ascenzo, S; Giunciuglio, D; Albini, A; Bologna, M

1998-01-30

Bombesin is a potent inducer of signal trasduction pathways involved in the proliferation and invasion of androgen-insensitive prostatic tumor cells. This study examines the bombesin-mediated modulation of pericellular proteolysis, monitoring cell capability to migrate and invade basement membranes, using a chemo-invasion assay and analyzing protease production. The results suggest that bombesin could modulate the invasive potential of prostatic cell lines regulating secretion and cell-surface uptake of uPA and MMP-9 activation. In fact, in PC3 and DU145 cells but not in LNCaP cells, urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) are induced by bombesin treatment. Bombesin also stimulates cell proliferation and this effect can be inhibited blocking uPA by antibodies and/or uPA inhibitor p-aminobenzamidine. Moreover, HMW-uPA induces cell proliferation in LNCaP cells, which do not produce uPA in the basal conditions, while PC3 and DU145 cell growth is supported by autocrine production of uPA. The increment of uPA activity on the external plasma membrane causes an increased pericellular plasmin activation. This effect is inhibited by antibodies against uPA and by p-aminobenzamidine. Similarly to EGF, bombesin stimulates secretion and activation of MMP-9 and TIMP-1 production. MMP-9 activation can be also obtained by HMW-uPA treatment, suggesting that plasma-membrane-bound uPA can start a proteolytic cascade involving MMP-9. Therefore, in in vitro assays, bombesin is able to modulate pericellular proteolysis and cell proliferation, differently distributing and activating proteolytic activities. This effect can be related to the "non-random" degradation of the extracellular matrix in which membrane uPA-uPAreceptor complexes could start bombesin-induced directional protein degradation during metastatic spread.

The effect of recombinant tissue plasminogen activator on MMP-2 and MMP-9 activities in vitro.

PubMed

Golab, Piotr; Kielbus, Michal; Bielewicz, Joanna; Kurzepa, Jacek

2015-01-01

One of the most significant side effects during recombinant tissue plasminogen activator (rtPA) for acute stroke treatment is intracranial bleeding. Gelatinases [matrix metalloproteinase (MMP)-2 and MMP-9] are one of the agents involved in the blood-brain barrier destruction resulting in secondary bleeding into the ischemic area during stroke. Previous papers revealed that patients with high baseline MMP-9 serum level have higher risk of intracranial bleeding after thrombolytic therapy. Our objective was to evaluate rtPA influence on serum MMP-2 and MMP-9 activities in vitro. Nine sera obtained from healthy donors were applied for experiment. The commercially available rtPA (Actylise) were diluted with included solvent and additionally with phosphate-buffered saline (PBS) to get concentrations: 2, 4, 8, and 16 μg/ml. Next, 100 μl of serum was mixed with equal proportion with different concentrations of rtPA to obtain final rtPA concentrations: 1, 2, 4, and 8 μg/ml. The sera together with rtPA were incubated for 1 or 2 hours at 37 °C. The activity of gelatinases was estimated with zymography. The activities of MMP-9 (92 kDa) and MMP-2 (72 kDa) were increased by incubation with rtPA in a dose-dependent manner. Simultaneously, the activity of band at 200 kDa (MMP-9/MMP-9 homodimer) was decreased. The activity of gelatinases incubated for 2 hours was elevated in comparison with 1-hour incubation; however, the increase was observed even for sample without rtPA. In conclusion, this study showed that rtPA can increase the biological activity of MMP-2 and MMP-9 on posttranslational level.

Monoclonal Antibody Testing for Cancer Metastasis

NASA Technical Reports Server (NTRS)

1993-01-01

Malignant cells are characterized by the ability to invade surrounding normal tissues. Tumor invasion is abetted by proteolytic enzymes that have been correlated with recurrent disease and metastasis. These enzymes are involved in a cascade of proteolytic interactions with other enzymes and inhibitors which allow cancer cells to dissolve surrounding extracellular matrix, thereby enabling the cells to rapidly invade adjacent tissues and migrate to metastatic sites distant from the primary tumor. Among these proteases are the plasminogen activators (PA), collagenase IV, faminase, and in some cases cathepsin D, which together mediate key steps in the invasion process of metastasis. Cells which have the selective advantage for invasion and metastasis are those capable of regulating their proteolytic activity and proliferation. Cells in the process of invasion would be probably down-regulated for proliferation, but subsequent to attachment and adhesion at a distant site, would then be in a proliferative mode, up-regulating DNA replication. Urokinase (uPA) can be present in the tissues in several molecular forms. The inactive proenzyme is a single chain protein (scuPA) that is cleaved at Lys. 158 to form the double chain, high molecular weight active form (HMW-uPA) of 54 kD. A low molecular weight form (LMW-uPA) can also be produced by cleavage of the HMW-U PA at Lys. 135 - Lys. 136 giving a 35 kD active enzyme. Recently, it has been shown that the HMW active form of urokinase, bound to the tumor cell membrane, is responsible for the local lysis of the extracellular matrix, hence the tissue invasion mechanism for metastasis (Andreasen et al, 19861. Receptor- (membrane) bound uPA is twice as efficient (catalytically) as free fluid-phase uPA. Tho unbound uPA and the LMW form is not responsible for most of the local dissolution of extracellular matrix in the immediate vicinity of the metastatic tumor cell. High levels of urokinase (greater than 3.49 ng/mg of total protein) extracted from breast tumor tissues have recently been shown, together with plasminogen activator inhibitor 1 (PAI-1), to be a good prognostic indicator for high risk of recurrence and shorter patient survival times. In this project, we have attempted to develop immunocytochemical methodologies for the clinical assessment of the expression of urokinase plasminogen activator, which has been implicated to be important for initial steps in tumor invasion, and to relate it to cell proliferation and DNA replication at the single-cell level.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu Shuqing; Sun Mingzhong; 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 indicatemore » 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.« less

Recombinant interferon-gamma secreted by Chinese hamster ovary-320 cells cultivated in suspension in protein-free media is protected against extracellular proteolysis by the expression of natural protease inhibitors and by the addition of plant protein hydrolysates to the culture medium.

PubMed

Mols, J; Peeters-Joris, C; Wattiez, R; Agathos, S N; Schneider, Y-J

2005-01-01

Biosafety requirements increasingly restrict the cultivation of mammalian cells producing therapeutic glycoproteins to conditions that are devoid of any compound of animal origin. On cultivation in serum-free media, the proteases inhibitors, usually found in serum, cannot protect secreted recombinant proteins against unwanted endogenous proteolysis. Chinese hamster ovary (CHO) cells, secreting recombinant human interferon-gamma (CHO-320 cell line) and cultivated in suspension in an original protein-free medium, expressed at least two members of the matrix metalloproteinases (MMP), either at the cell surface (proMMP-14 and MMP-14) or secreted (proMMP-9). In addition, tissue- and urinary-type plasminogen activators were also secreted in such culture conditions. At the cell surface, dipeptidyl peptidase IV and tripeptidyl peptidase II (TPPII) activities were also detected, and their activities decreased during time course of batch cultures. The proteolytic activities of these proteins were counterbalanced by (1) their expression as zymogens (proMMP-9, proMMP-14), (2) the expression of their natural inhibitors, tissue inhibitors of metalloproteinases-1 and -2 and plasminogen activator inhibitor-1 (PAI-1), or (3) the addition of plant protein hydrolysates to the culture medium, acting as a nonspecific source of TPPII inhibitors. This study points out that, even in protein-free media, recombinant proteins secreted by CHO cells are actively protected against physiological and unwanted extracellular proteolysis either by endogenous or by exogenous inhibitors.

Determinants of hypofibrinolysis in patients with digestive tract cancer.

PubMed

Gronostaj, Katarzyna; Richter, Piotr; Nowak, Wojciech; Undas, Anetta

2016-01-01

Recently, we demonstrated that digestive tract cancer (DTC) is associated with reduced fibrin clot permeability and impaired fibrinolysis. We investigated determinants of fibrinolysis in DTC patients. In 44 consecutive patients with DTC and 47 controls matched for age, sex, and cardiovascular risk, we evaluated fibrinolysis proteins, platelet activation markers, thrombin formation, together with plasma clot lysis time assays in the absence (CLT) and presence of carboxypeptidase potato inhibitor (CLT CPI) that blocks thrombin activatable fibrinolysis inhibitor (TAFI). In the DTC group CLT (by 22.3%) and CLT CPI (by 27.4%) were longer compared with controls. The DTC patients had higher plasma fibrinolysis inhibitors, plasminogen activator inhibitor 1 (PAI-1) (by 18.2%), TAFI activity (by 17.3%), and antigen (by 11.2%). The patients had markedly increased platelet markers - soluble CD40 ligand (by 338%) and P-selectin (by 97%), together with von Willebrand factor (vWF) antigen (by 61%). Thrombin-antithrombin complexes (TAT) (by 48.7%) and soluble thrombomodulin (sTM) (by 17.2%) were also increased in the DTC group (all p < 0.05). Patients with high-grade tumours (n = 26) compared with remainders (n = 18) had longer CLT, higher tissue-type plasminogen activator antigen, both TAFI antigen and activity levels, vWF, and sTM. Multiple regression analysis after adjustment for potential confounders showed that independent predictors of CLT in DTC patients were TAT, TAFI activity, and vWF. The only independent predictor of CLT CPI was TAT. Hypofibrinolysis in DTC patients is largely driven by enhanced thrombin generation, TAFI, and endothelial injury.

Specificity determinants in the interaction of apolipoprotein(a) kringles with tetranectin and LDL.

PubMed

Caterer, Nigel R; Graversen, Jonas H; Jacobsen, Christian; Moestrup, Søren K; Sigurskjold, Bent W; Etzerodt, Michael; Thøgersen, Hans C

2002-11-01

Lipoprotein(a) is composed of low density lipoprotein and apolipoprotein(a). Apolipoprotein(a) has evolved from plasminogen and contains 10 different plasminogen kringle 4 homologous domains [KIV(1-110)]. Previous studies indicated that lipoprotein(a) non-covalently binds the N-terminal region of lipoprotein B100 and the plasminogen kringle 4 binding plasma protein tetranectin. In this study recombinant KIV(2), KIV(7) and KIV(10) derived from apolipoprotein(a) were produced in E. coli and the binding to tetranectin and low density lipoprotein was examined. Only KIV(10) bound to tetranectin and binding was similar to that of plasminogen kringle 4 to tetranectin. Only KIV(7) bound to LDL. In order to identify the residues responsible for the difference in specificity between KIV(7) and KIV(10), a number of surface-exposed residues located around the lysine binding clefts were exchanged. Ligand binding analysis of these derivatives showed that Y62, and to a minor extent W32 and E56, of KIV(7) are important for LDL binding to KIV(7), whereas R32 and D56 of KIV(10) are required for tetranectin binding of KIV(10).

Comparative Proteomics of Tandem Mass Spectrometry Analyses for Bacterial Strains Identification and Differentiation

DTIC Science & Technology

2012-02-01

risk, bio -terrorism utility, Homeland Security, agricultural monitoring, quality of foodstuffs, environmental monitoring, and biological warfare agents...CAL19717 Putative surface antigen CAL21872 Putative sigma 54 modulation protein NP_395233 Plasminogen activator protease precursor CAL19882 OMP...S. (2005). Chemical and biological weapons : current concepts for future defenses. Johns Hopkins APL Tech. Digest, 26, 321-333. Dworzanski, J.P

Journey During Acute Ischemic Stroke: A Physician’s Experience

PubMed Central

Hoong, Low Chen; Sharma, Vijay K.

2010-01-01

Acute ischemic stroke is a potentially devastating condition. What follows is a true narration of the experience of a doctor-patient during his treatment for acute ischemic stroke and how the experience changed him. Described is the temporal sequence of events, starting from home to infusion of tissue plasminogen activator, which, when coupled with a multimodal therapeutic approach, resulted in an excellent clinical recovery. PMID:20458112

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

PubMed

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

2016-08-01

Essentials C57BL/6J-tissue plasminogen activator (tPA)-deficient mice are widely used to study tPA function. Congenic C57BL/6J-tPA-deficient mice harbor large 129-derived chromosomal segments. The 129-derived chromosomal segments contain gene mutations that may confound data interpretation. Passenger mutation-free isogenic tPA-deficient mice were generated for study of tPA function. 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 single nucleotide polymorphism (SNP) analysis, bioinformatics and genome editing were 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 into 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. © 2016 International Society on Thrombosis and Haemostasis.

4G/5G Plasminogen Activator Inhibitor-1 Polymorphisms and Haplotypes Are Associated with Pneumonia

PubMed Central

Yende, Sachin; Angus, Derek C.; Ding, Jingzhong; Newman, Anne B.; Kellum, John A.; Li, Rongling; Ferrell, Robert E.; Zmuda, Joseph; Kritchevsky, Stephen B.; Harris, Tamara B.; Garcia, Melissa; Yaffe, Kristine; Wunderink, Richard G.

2007-01-01

Rationale: Plasminogen activator inhibitor (PAI)-1 inhibits urokinase and tissue plasminogen activator, required for host response to infection. Whether variation within the PAI-1 gene is associated with increased susceptibility to infection is unknown. Objectives: To ascertain the role of the 4G/5G polymorphism and other genetic variants within the PAI-1 gene. We hypothesized that variants associated with increased PAI-1 expression would be associated with an increased occurrence of community-acquired pneumonia (CAP). Methods: Longitudinal analysis (>12 yr) of the Health, Aging, and Body Composition cohort, aged 65–74 years at start of analysis. Measurements and Main Results: We genotyped the 4G/5G PAI-1 polymorphism and six additional single nucleotide polymorphisms. Of the 3,075 subjects, 272 (8.8%) had at least one hospitalization for CAP. Among whites, variants at the PAI4G,5G, PAI2846, and PAI7343 sites had higher risk of CAP (P = 0.018, 0.021, and 0.021, respectively). At these sites, variants associated with higher PAI-1 expression were associated with increased CAP susceptibility. Compared with the 5G/5G genotypes at PAI4G,5G site, the 4G/4G and 4G/5G genotypes were associated with a 1.98-fold increased risk of CAP (95% confidence interval, 1.2–3.2; P = 0.006). In whole blood stimulation assay, subjects with a 4G allele had 3.3- and 1.9-fold increased PAI-1 expression (P = 0.043 and 0.034, respectively). In haplotype analysis, the 4G/G/C/A haplotype at the PAI4G,5G, PAI2846, PAI4588, and PAI7343 single nucleotide polymorphisms was associated with higher CAP susceptibility, whereas the 5G/G/C/A haplotype was associated with lower CAP susceptibility. No associations were seen among blacks. Conclusions: Genotypes associated with increased expression of PAI-1 were associated with increased susceptibility to CAP in elderly whites. PMID:17761618

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 marker with prognostic significance. Its potential involvement in the plasminogen activator protease cascade warrants further investigation. PMID:23236990

Cadmium exposure is associated with soluble urokinase plasminogen activator receptor, a circulating marker of inflammation and future cardiovascular disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fagerberg, Björn, E-mail: bjorn.fagerberg@wlab.gu.

Background: Diet and smoking are the main sources of cadmium exposure in the general population. Cadmium increases the risk of cardiovascular diseases, and experimental studies show that it induces inflammation. Blood cadmium levels are associated with macrophages in human atherosclerotic plaques. Soluble urokinase-type plasminogen activator receptor (suPAR) is an emerging biomarker for cardiovascular events related to inflammation and atherosclerotic plaques. The aim was to examine whether blood cadmium levels are associated with circulating suPAR and other markers of inflammation. Methods: A population sample of 4648 Swedish middle-aged women and men was examined cross-sectionally in 1991–1994. Plasma suPAR was assessed bymore » ELISA, leukocytes were measured by standard methods, and blood cadmium was analysed by inductively coupled plasma mass spectrometry. Prevalent cardiovascular disease, ultrasound-assessed carotid plaque occurrence, and several possible confounding factors were recorded. Results: After full adjustment for risk factors and confounding variables, a 3-fold increase in blood cadmium was associated with an 10.9% increase in suPAR concentration (p<0.001). In never-smokers, a 3-fold increase in blood cadmium was associated with a 3.7% increase in suPAR concentration (p<0.01) after full adjustment. Blood cadmium was not associated with C-reactive protein, white blood cell count and Lp-PLA2 but with neutrophil/lymphocyte ratio in one of two statistical models. Conclusions: Exposure to cadmium was associated with increased plasma suPAR in the general population, independently of smoking and cardiovascular disease. These results imply that cadmium is a possible cause for raised levels of this inflammatory marker. - Highlights: • Cadmium is a toxic proinflammatory, proatherosclerotic metal. • Soluble urokinase-type plasminogen activator receptor (suPAR) in plasma is a promising proinflammatory marker of atherosclerosis. • Blood cadmium and plasma suPAR were measured in a cohort of 4648 Swedish men and women. • Blood cadmium was positively associated with plasma suPAR, also in never smokers.« less

Markers of endothelial dysfunction and severity of hypoxaemia in the Eisenmenger syndrome.

PubMed

de P S Soares, Rosangela; Maeda, Nair Y; Bydlowski, Sérgio P; Lopes, Antonio Augusto

2005-10-01

Endothelial dysfunction has been reported in hypoxaemic patients with the Eisenmenger syndrome, but a direct correlation between levels of endothelial markers and the severity of hypoxaemia has not been explored. With this in mind, we compared the levels in the plasma of tissue-type plasminogen activator, thrombomodulin, and von Willebrand factor in 25 patients with the Eisenmenger syndrome. They had a median age of 31 years, and were divided into 2 groups according to their recent clinical history. Thus, 18 patients were stable, being in functional class II or III, seen as outpatients, and having peripheral saturations of oxygen of 89 plus or minus 5 percent. In contrast, 7 patients were unstable, showing episodes of symptoms placing them in functional class IV, requiring care in hospital, and manifesting saturations of oxygen of 77 plus or minus 5 percent. We were able to follow 12 patients, 8 who were stable and 4 unstable, for 24 months. At baseline, levels of von Willebrand factor were higher in the unstable patients when compared to those who were stable, at 142 plus or minus 29 and 110 plus or minus 25 units per decilitre, respectively (p equal to 0.013). This correlated positively with oxygen desaturation (p less than 0.020). The structural abnormalities also correlated positively with the magnitude of hypoxaemia (p less than 0.020). Levels remained higher in the unstable patients throughout the period of follow-up (p equal to 0.006). Tissue-type plasminogen activator was also increased, at 14.3 plus or minus 8.4 versus 6.5 plus or minus 2.7 nanograms per millilitre in controls (p less than 0.001), whereas thrombomodulin was decreased, with values of 14.4 versus 34.6 nanograms per millilitre in controls (p for median values of less than 0.001). There was no correlation with saturations of oxygen. We conclude that measurement of von Willebrand factor, as compared with tissue-type plasminogen activator and thrombomodulin, will prove a better marker of endothelial response to hypoxaemia in patients with the Eisenmenger syndrome.

Glucose deprivation reversibly down-regulates tissue plasminogen activator via proteasomal degradation in rat primary astrocytes.

PubMed

Cho, Kyu Suk; Joo, So Hyun; Choi, Chang Soon; Kim, Ki Chan; Ko, Hyun Myung; Park, Jin Hee; Kim, Pitna; Hur, Jun; Lee, Sung Hoon; Bahn, Geon Ho; Ryu, Jong Hoon; Lee, Jongmin; Han, Seol-Heui; Kwon, Kyoung Ja; Shin, Chan Young

2013-05-20

Tissue plasminogen activator (tPA) is an essential neuromodulator whose involvement in multiple functions such as synaptic plasticity, cytokine-like immune function and regulation of cell survival mandates rapid and tight tPA regulation in the brain. We investigated the possibility that a transient metabolic challenge induced by glucose deprivation may affect tPA activity in rat primary astrocytes, the main cell type responsible for metabolic regulation in the CNS. Rat primary astrocytes were incubated in serum-free DMEM without glucose. Casein zymography was used to determine tPA activity, and tPA mRNA was measured by RT-PCR. The signaling pathways regulating tPA activity were identified by Western blotting. Glucose deprivation rapidly down-regulated the activity of tPA without affecting its mRNA level in rat primary astrocytes; this effect was mimicked by translational inhibitors. The down-regulation of tPA was accompanied by increased tPA degradation, which may be modulated by a proteasome-dependent degradation pathway. Glucose deprivation induced activation of PI3K-Akt-GSK3β, p38 and AMPK, and inhibition of these pathways using LY294002, SB203580 and compound C significantly inhibited glucose deprivation-induced tPA down-regulation, demonstrating the essential role of these pathways in tPA regulation in glucose-deprived astrocytes. Rapid and reversible regulation of tPA activity in rat primary astrocytes during metabolic crisis may minimize energy-requiring neurologic processes in stressed situations. This effect may thereby increase the opportunity to invest cellular resources in cell survival and may allow rapid re-establishment of normal cellular function after the crisis. Copyright © 2013 Elsevier Inc. All rights reserved.

A neurokinin 1 receptor antagonist decreases postoperative peritoneal adhesion formation and increases peritoneal fibrinolytic activity.

PubMed

Reed, Karen L; Fruin, A Brent; Gower, Adam C; Stucchi, Arthur F; Leeman, Susan E; Becker, James M

2004-06-15

Fibrous adhesions remain a major sequela of abdominal surgery. The proinflammatory peptide substance P (SP), known to participate in inflammatory events, may play a key role in adhesion formation. This hypothesis was tested by using an antagonist, CJ-12,255 (Pfizer), that blocks the binding of SP to the neurokinin 1 receptor (NK-1R). Adhesion formation was surgically induced in the peritoneum of rats receiving daily doses of the NK-1R antagonist (NK-1RA; 5.0 or 10.0 mg/kg per day) or saline. On postoperative day 7, both the low and high doses of NK-1RA significantly (P < 0.05) reduced adhesion formation by 45% and 53%, respectively, compared with controls. Subsequently, the effect of NK-1RA administration on peritoneal fibrinolytic activity was investigated to determine a potential mechanism for SP action in the peritoneum. Samples were collected from nonoperated controls and from animals 24 h postsurgery that were administered either NK-1RA or saline. Fibrinolytic activity in peritoneal fluid was assayed by zymography, and expression of tissue plasminogen activator (tPA) and plasminogen activator inhibitor 1, both regulators of fibrinolytic activity, was assessed in peritoneal tissue and fluid by RT-PCR and bioassay, respectively. NK-1RA administration led to a marked (P < 0.05) increase in tPA mRNA levels in peritoneal tissue compared with nonoperated and saline-administered animals. Likewise, NK-1RA administration significantly (P < 0.05) increased tPA in the peritoneal fluid. These data suggest that activation of the NK-1R promotes peritoneal adhesion formation by limiting fibrinolytic activity in the postoperative peritoneum, thus enabling fibrinous adhesions to persist.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Shuai; Beijing Key Laboratory of Respiratory and Pulmonary Circulation Disorders, 8 Gongti South Rd, Beijing; Zou, Lihui

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 humanmore » 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.« less

Discovery of novel urokinase plasminogen activator (uPA) inhibitors using ligand-based modeling and virtual screening followed by in vitro analysis.

PubMed

Al-Sha'er, Mahmoud A; Khanfar, Mohammad A; Taha, Mutasem O

2014-01-01

Urokinase plasminogen activator (uPA)-a serine protease-is thought to play a central role in tumor metastasis and angiogenesis and, therefore, inhibition of this enzyme could be beneficial in treating cancer. Toward this end, we explored the pharmacophoric space of 202 uPA inhibitors using seven diverse sets of inhibitors to identify high-quality pharmacophores. Subsequently, we employed genetic algorithm-based quantitative structure-activity relationship (QSAR) analysis as a competition arena to select the best possible combination of pharmacophoric models and physicochemical descriptors that can explain bioactivity variation within the training inhibitors (r (2) 162 = 0.74, F-statistic = 64.30, r (2) LOO = 0.71, r (2) PRESS against 40 test inhibitors = 0.79). Three orthogonal pharmacophores emerged in the QSAR equation suggesting the existence of at least three binding modes accessible to ligands within the uPA binding pocket. This conclusion was supported by receiver operating characteristic (ROC) curve analyses of the QSAR-selected pharmacophores. Moreover, the three pharmacophores were comparable with binding interactions seen in crystallographic structures of bound ligands within the uPA binding pocket. We employed the resulting pharmacophoric models and associated QSAR equation to screen the national cancer institute (NCI) list of compounds. The captured hits were tested in vitro. Overall, our modeling workflow identified new low micromolar anti-uPA hits.

The Soluble Plasminogen Activator Receptor as a Biomarker on Monitoring the Therapy Progress of Pulmonary TB-AFB(+) Patients

PubMed Central

Mardining Raras, Tri Yudani; Noor Chozin, Iin

2010-01-01

The role of soluble soluble urokinase-type plasminogen activator receptor (suPAR) as a biological marker for TB treatment efficacy on active pulmonary TB-AFB(+) patients was investigated. Twenty pulmonary TB-AFB(+) patients participated in a cohort study for six months. The plasma suPAR level was measured using ELISA method before treatment, two months, four months and six months after treatment. At the same time clinical parameters were also measured. Results indicated that all patients (n = 20) showed highest plasma suPAR levels before treatment (median 12.775 ng/mL) and significantly decreased ( P = .0001<.05, R 2 = .890) after 2 months (median 8.019 ng/mL) and 4 months (median 5.771 ng/mL) of treatment, respectively. However, only slightly declined after 6 months therapy (median 5.009 ng/mL), near control group level (median 4.772 ng/mL). Interestingly, the significant reduced of suPAR level was parallel to treatment efficacy and correlated with other clinical and laboratory parameters, that is, decreasing of patients' complaints, increasing of BMI (r = −0.281), thoracic imaging improvement, sputum conversion, decreasing of ESR (r = 0.577) and monocytes count (r = 0.536) with exception the width of lesion in thoracic imaging. In conclusion, the suPAR level in could reflect the progress of TB therapy. PMID:22567258

Plasminogen activator inhibitor-1 as regulator of tumor-initiating cell properties in head and neck cancers.

PubMed

Lee, Yueh-Chun; Yu, Cheng-Chia; Lan, Chih; Lee, Che-Hsin; Lee, Hsueh-Te; Kuo, Yu-Liang; Wang, Po-Hui; Chang, Wen-Wei

2016-04-01

The existence of tumor-initiating cells (TICs) has been described in head and neck cancers. Plasminogen activator inhibitor-1 (PAI-1) has been demonstrated to act as a prognostic factor in head and neck cancers. Tiplaxtinin (PAI-039), a specific inhibitor of PAI-1, and PAI-1-specific siRNA were used to examine the role of PAI-1 in the self-renewal property of head and neck cancer-TICs by tumorsphere formation. Western blot, real-time polymerase chain reaction, and luciferase-based reporter assay were used to study the effect of PAI-039 in the sex-determining region Y-box 2 (Sox2) expression. PAI-039 suppressed the self-renewal capability of head and neck cancer-TICs derived from head and neck cancer cell lines through the inhibition of Sox2 expression. PAI-039 decreased the activity of the core promoter and the enhancer of the Sox2 gene in head and neck cancer-TICs. Knockdown of PAI-1 expression also inhibited self-renewal and radioresistance properties of head and neck cancer-TICs. The inhibition of PAI-1 by PAI-039 or siRNA could suppress head and neck cancer-TICs within head and neck cancer cell lines through the downregulation of Sox2. © 2015 Wiley Periodicals, Inc. Head Neck 38: E895-E904, 2016. © 2015 Wiley Periodicals, Inc.

Leukemia inhibitory factor promote trophoblast invasion via urokinase-type plasminogen activator receptor in preeclampsia.

PubMed

Zheng, Qin; Dai, Kuixing; Cui, Xinyuan; Yu, Ming; Yang, Xuesong; Yan, Bin; Liu, Shuai; Yan, Qiu

2016-05-01

Preeclampsia is a pregnancy-related syndrome which can cause perinatal mortality and morbidity. Inadequate invasion by trophoblast cells may lead to poor perfusion of the placenta, even result in preeclampsia. Understanding the molecular mechanisms underlying placentation facilitates the better intervention of preeclampsia. Urokinase-type plasminogen activator receptor (uPAR) is involved in the physiological and pathological processes. Leukemia inhibitory factor (LIF) is an important regulator in the establishment of pregnancy. However, the expression of uPAR in preeclamptic patients and its relationship with LIF remains unclear. In the current study, we found that the level of uPAR was relatively lower in the placentas from preeclamptic patients as compared with normal pregnant women. LIF promoted trophoblast cell outgrowth by upregulating uPAR in an explants culture, and LIF also enhanced migration and invasion potential through uPAR in trophoblast JAR and JEG-3 cell lines, and with increased gelatinolytic activities of matrix metalloproteinase 2 (MMP-2). The effect of LIF and uPAR on trophoblast migration and invasion was mediated by PI3K/AKT signaling pathway. Our data indicates the roles of LIF in promoting trophoblast migration and invasion through uPAR and suggest that abnormal expression of uPAR might be associated with the etiology of preeclampsia. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Tissue-type plasminogen activator-induced fibrinolysis is enhanced in patients with breast, lung, pancreas and colon cancer.

PubMed

Nielsen, Vance G; Matika, Ryan W; Ley, Michele L B; Waer, Amy L; Gharagozloo, Farid; Kim, Samuel; Nfonsam, Valentine N; Ong, Evan S; Jie, Tun; Warneke, James A; Steinbrenner, Evangelina B

2014-04-01

Although cancer-mediated changes in hemostatic proteins unquestionably promote hypercoagulation, the effects of neoplasia on fibrinolysis in the circulation are less well defined. The goals of the present investigation were to determine if plasma obtained from patients with breast, lung, pancreas and colon cancer was less or more susceptible to lysis by tissue-type plasminogen activator (tPA) compared to plasma obtained from normal individuals. Archived plasma obtained from patients with breast (n = 18), colon/pancreas (n = 27) or lung (n = 19) was compared to normal individual plasma (n = 30) using a thrombelastographic assay that assessed fibrinolytic vulnerability to exogenously added tPA. Plasma samples were activated with tissue factor/celite, had tPA added, and had data collected until clot lysis occurred. Additional, similar samples had potato carboxypeptidase inhibitor added to assess the role played by thrombin-activatable fibrinolysis inhibitor in cancer-modulated fibrinolysis. Rather than inflicting a hypofibrinolytic state, the three groups of cancers demonstrated increased vulnerability to tPA (e.g. decreased time to lysis, increased speed of lysis, decreased clot lysis time). However, hypercoagulation manifested as increased speed of clot formation and strength compensated for enhanced fibrinolytic vulnerability, resulting in a clot residence time that was not different from normal individual thrombi. In sum, enhanced hypercoagulability associated with cancer was in part diminished by enhanced fibrinolytic vulnerability to tPA.

Relationship between post-SARS osteonecrosis and PAI-1 4G/5G gene polymorphisms.

PubMed

Sun, Wei; Li, Zirong; Shi, Zhengcai; Wang, Bailiang; Gao, Fuqiang; Yang, Yurun; Guo, Wanshou

2014-05-01

To explore the correlation between post-severe acute respiratory symptom (SARS) patients with osteonecrosis, investigate the etiology of post-SARS osteonecrosis and select the sensitive molecular symbols for early diagnosis and distinguish the high-risk population. The studied subjects were divided into two groups. Sixty-two post-SARS patients with osteonecrosis were one group, and 52 age- and sex-matched healthy people were as normal controlled group. Empty stomach blood samples from cubital veins were collected from both groups. Plasminogen activator inhibitor (PAI) by means of enzyme-linked immunosorbent assay and PAI-1 4G/5G polymorphism was detected by polymerase chain reaction and solid phase oligonucleotide assay. The blood agents of post-SARS patients changed obviously with 15.64 ± 13.85 U/ml while the control group 7.96 ± 4.27 U/ml; 4G/4G genotype for the PAI-1 polymorphism detected in post-SARS group was more than that of the control group, but had no statistical significance. The plasma PAI activity was related to homozygote 4G/4G genotype. This reveals that homozygote 4G/4G genotype may be a susceptible gene mark to Chinese osteonecrosis patients. Plasminogen activator inhibitor-1 is sensitive blood symbol for screening high-risk susceptible population; 4G/4G PAI-1 genotype may be an etiological factor in osteonecrosis.

VEGF correlates with inflammation and fibrosis in tuberculous pleural effusion.

PubMed

Bien, Mauo-Ying; Wu, Ming-Ping; Chen, Wei-Lin; Chung, Chi-Li

2015-01-01

To investigate the relationship among angiogenic cytokines, inflammatory markers, and fibrinolytic activity in tuberculous pleural effusion (TBPE) and their clinical importance. Forty-two patients diagnosed with TBPE were studied. Based on chest ultrasonography, there were 26 loculated and 16 nonloculated TBPE patients. The effusion size radiological scores and effusion vascular endothelial growth factor (VEGF), interleukin- (IL-) 8, plasminogen activator inhibitor type-1 (PAI-1), and tissue type plasminogen activator (tPA) were measured. Treatment outcome and pleural fibrosis, defined as radiological residual pleural thickening (RPT), were assessed at 6-month follow-up. The effusion size and effusion lactate dehydrogenase (LDH), VEGF, IL-8, PAI-1, and PAI-1/tPA ratio were significantly higher, while effusion glucose, pH value, and tPA were significantly lower, in loculated than in nonloculated TBPE. VEGF and IL-8 correlated positively with LDH and PAI-1/tPA ratio and negatively with tPA in both loculated and nonloculated TBPE. Patients with higher VEGF or greater effusion size were prone to develop RPT (n=14; VEGF, odds ratio 1.28, P=0.01; effusion size, odds ratio 1.01, P=0.02), and VEGF was an independent predictor of RPT in TBPE (receiver operating characteristic curve AUC=0.985, P<0.001). Effusion VEGF correlates with pleural inflammation and fibrosis and may be targeted for adjunct therapy for TBPE.

VEGF Correlates with Inflammation and Fibrosis in Tuberculous Pleural Effusion

PubMed Central

Bien, Mauo-Ying; Wu, Ming-Ping; Chen, Wei-Lin; Chung, Chi-Li

2015-01-01

Objective. To investigate the relationship among angiogenic cytokines, inflammatory markers, and fibrinolytic activity in tuberculous pleural effusion (TBPE) and their clinical importance. Methods. Forty-two patients diagnosed with TBPE were studied. Based on chest ultrasonography, there were 26 loculated and 16 nonloculated TBPE patients. The effusion size radiological scores and effusion vascular endothelial growth factor (VEGF), interleukin- (IL-) 8, plasminogen activator inhibitor type-1 (PAI-1), and tissue type plasminogen activator (tPA) were measured. Treatment outcome and pleural fibrosis, defined as radiological residual pleural thickening (RPT), were assessed at 6-month follow-up. Results. The effusion size and effusion lactate dehydrogenase (LDH), VEGF, IL-8, PAI-1, and PAI-1/tPA ratio were significantly higher, while effusion glucose, pH value, and tPA were significantly lower, in loculated than in nonloculated TBPE. VEGF and IL-8 correlated positively with LDH and PAI-1/tPA ratio and negatively with tPA in both loculated and nonloculated TBPE. Patients with higher VEGF or greater effusion size were prone to develop RPT (n = 14; VEGF, odds ratio 1.28, P = 0.01; effusion size, odds ratio 1.01, P = 0.02), and VEGF was an independent predictor of RPT in TBPE (receiver operating characteristic curve AUC = 0.985, P < 0.001). Conclusions. Effusion VEGF correlates with pleural inflammation and fibrosis and may be targeted for adjunct therapy for TBPE. PMID:25884029

Temporary dietary iron restriction affects the process of thrombus resolution in a rat model of deep vein thrombosis.

PubMed

Oboshi, Makiko; Naito, Yoshiro; Sawada, Hisashi; Hirotani, Shinichi; Iwasaku, Toshihiro; Okuhara, Yoshitaka; Morisawa, Daisuke; Eguchi, Akiyo; Nishimura, Koichi; Fujii, Kenichi; Mano, Toshiaki; Ishihara, Masaharu; Masuyama, Tohru

2015-01-01

Deep vein thrombosis (DVT) is a major cause of pulmonary thromboembolism and sudden death. Thus, it is important to consider the pathophysiology of DVT. Recently, iron has been reported to be associated with thrombotic diseases. Hence, in this study, we investigate the effects of dietary iron restriction on the process of thrombus resolution in a rat model of DVT. We induced DVT in 8-week-old male Sprague-Dawley rats by performing ligations of their inferior venae cavae. The rats were then given either a normal diet (DVT group) or an iron-restricted diet (DVT+IR group). Thrombosed inferior venae cavae were harvested at 5 days after ligation. The iron-restricted diet reduced venous thrombus size compared to the normal diet. Intrathrombotic collagen content was diminished in the DVT+IR group compared to the DVT group. In addition, intrathrombotic gene expression and the activity of matrix metalloproteinase-9 were increased in the DVT+IR group compared to the DVT group. Furthermore, the DVT+IR group had greater intrathrombotic neovascularization as well as higher gene expression levels of urokinase-type plasminogen activator and tissue-type plasminogen activator than the DVT group. The iron-restricted diet decreased intrathrombotic superoxide production compared to the normal diet. These results suggest that dietary iron restriction affects the process of thrombus resolution in DVT.

Temporary Dietary Iron Restriction Affects the Process of Thrombus Resolution in a Rat Model of Deep Vein Thrombosis

PubMed Central

Oboshi, Makiko; Naito, Yoshiro; Sawada, Hisashi; Hirotani, Shinichi; Iwasaku, Toshihiro; Okuhara, Yoshitaka; Morisawa, Daisuke; Eguchi, Akiyo; Nishimura, Koichi; Fujii, Kenichi; Mano, Toshiaki; Ishihara, Masaharu; Masuyama, Tohru

2015-01-01

Background Deep vein thrombosis (DVT) is a major cause of pulmonary thromboembolism and sudden death. Thus, it is important to consider the pathophysiology of DVT. Recently, iron has been reported to be associated with thrombotic diseases. Hence, in this study, we investigate the effects of dietary iron restriction on the process of thrombus resolution in a rat model of DVT. Methods We induced DVT in 8-week-old male Sprague-Dawley rats by performing ligations of their inferior venae cavae. The rats were then given either a normal diet (DVT group) or an iron-restricted diet (DVT+IR group). Thrombosed inferior venae cavae were harvested at 5 days after ligation. Results The iron-restricted diet reduced venous thrombus size compared to the normal diet. Intrathrombotic collagen content was diminished in the DVT+IR group compared to the DVT group. In addition, intrathrombotic gene expression and the activity of matrix metalloproteinase-9 were increased in the DVT+IR group compared to the DVT group. Furthermore, the DVT+IR group had greater intrathrombotic neovascularization as well as higher gene expression levels of urokinase-type plasminogen activator and tissue-type plasminogen activator than the DVT group. The iron-restricted diet decreased intrathrombotic superoxide production compared to the normal diet. Conclusions These results suggest that dietary iron restriction affects the process of thrombus resolution in DVT. PMID:25962140

Neuroserpin Differentiates Between Forms of Tissue Type Plasminogen Activator via pH Dependent Deacylation

PubMed Central

Carlson, Karen-Sue B.; Nguyen, Lan; Schwartz, Kat; Lawrence, Daniel A.; Schwartz, Bradford S.

2016-01-01

Tissue-type plasminogen activator (t-PA), initially characterized for its critical role in fibrinolysis, also has key functions in both physiologic and pathologic processes in the CNS. Neuroserpin (NSP) is a t-PA specific serine protease inhibitor (serpin) found almost exclusively in the CNS that regulates t-PA’s proteolytic activity and protects against t-PA mediated seizure propagation and blood–brain barrier disruption. This report demonstrates that NSP inhibition of t-PA varies profoundly as a function of pH within the biologically relevant pH range for the CNS, and reflects the stability, rather than the formation of NSP: t-PA acyl-enzyme complexes. Moreover, NSP differentiates between the zymogen-like single chain form (single chain t-PA, sct-PA) and the mature protease form (two chain t-PA, tct-PA) of t-PA, demonstrating different pH profiles for protease inhibition, different pH ranges over which catalytic deacylation occurs, and different pH dependent profiles of deacylation rates for each form of t-PA. NSP’s pH dependent inhibition of t-PA is not accounted for by differential acylation, and is specific for the NSP-t-PA serpin-protease pair. These results demonstrate a novel mechanism for the differential regulation of the two forms of t-PA in the CNS, and suggest a potential specific regulatory role for CNS pH in controlling t-PA proteolytic activity. PMID:27378851

Hemostasis in Hypothyroidism and Autoimmune Thyroid Disorders.

PubMed

Ordookhani, Arash; Burman, Kenneth D

2017-04-01

There are contradictory results on the effect of hypothyroidism on the changes in hemostasis. Inadequate population-based studies limited their clinical implications, mainly on the risk of venous thromboembolism (VTE). This paper reviews the studies on laboratory and population-based findings regarding hemostatic changes and risk of VTE in hypothyroidism and autoimmune thyroid disorders. A comprehensive literature search was conducted employing MEDLINE database. The following words were used for the search: Hypothyroidism; thyroiditis, autoimmune; blood coagulation factors; blood coagulation tests; hemostasis, blood coagulation disorders; thyroid hormones; myxedema; venous thromboembolism; fibrinolysis, receptors thyroid hormone. The papers that were related to hypothyroidism and autoimmune thyroid disorder and hemostasis are used in this review. Overt hypothyroidism is more associated with a hypocoagulable state. Decreased platelet count, aggregation and agglutination, von Willebrand factor antigen and activity, several coagulation factors such as factor VIII, IX, XI, VII, and plasminogen activator-1 are detected in overt hypothyrodism. Increased fibrinogen has been detected in subclinical hypothyroidism and autoimmune thyroid disease rendering a tendency towards a hypercoagulability state. Increased factor VII and its activity, and plasminogen activator inhibitor-1 are among several findings contributing to a prothrombotic state in subclinical hypothyroidism. Overt hypothyroidism is associated with a hypocoagulable state and subclinical hypothyroidism and autoimmune thyroid disorders may induce a prothrombotic state. However, there are contradictory findings for the abovementioned thyroid disorders. Prospective studies on the risk of VTE in various levels of hypofunctioning of the thyroid and autoimmune thyroid disorders are warranted.

Hemostasis in Hypothyroidism and Autoimmune Thyroid Disorders

PubMed Central

Ordookhani, Arash; Burman, Kenneth D.

2017-01-01

Context There are contradictory results on the effect of hypothyroidism on the changes in hemostasis. Inadequate population-based studies limited their clinical implications, mainly on the risk of venous thromboembolism (VTE). This paper reviews the studies on laboratory and population-based findings regarding hemostatic changes and risk of VTE in hypothyroidism and autoimmune thyroid disorders. Evidence Acquisition A comprehensive literature search was conducted employing MEDLINE database. The following words were used for the search: Hypothyroidism; thyroiditis, autoimmune; blood coagulation factors; blood coagulation tests; hemostasis, blood coagulation disorders; thyroid hormones; myxedema; venous thromboembolism; fibrinolysis, receptors thyroid hormone. The papers that were related to hypothyroidism and autoimmune thyroid disorder and hemostasis are used in this review. Results Overt hypothyroidism is more associated with a hypocoagulable state. Decreased platelet count, aggregation and agglutination, von Willebrand factor antigen and activity, several coagulation factors such as factor VIII, IX, XI, VII, and plasminogen activator-1 are detected in overt hypothyrodism. Increased fibrinogen has been detected in subclinical hypothyroidism and autoimmune thyroid disease rendering a tendency towards a hypercoagulability state. Increased factor VII and its activity, and plasminogen activator inhibitor-1 are among several findings contributing to a prothrombotic state in subclinical hypothyroidism. Conclusions Overt hypothyroidism is associated with a hypocoagulable state and subclinical hypothyroidism and autoimmune thyroid disorders may induce a prothrombotic state. However, there are contradictory findings for the abovementioned thyroid disorders. Prospective studies on the risk of VTE in various levels of hypofunctioning of the thyroid and autoimmune thyroid disorders are warranted. PMID:29026409

Substance P increases sympathetic activity during combined angiotensin-converting enzyme and dipeptidyl peptidase-4 inhibition.

PubMed

Devin, Jessica K; Pretorius, Mias; Nian, Hui; Yu, Chang; Billings, Frederic T; Brown, Nancy J

2014-05-01

Dipeptidyl peptidase-4 inhibitors prevent the degradation of incretin hormones and reduce postprandial hyperglycemia in patients with type 2 diabetes mellitus. Dipeptidyl peptidase-4 degrades other peptides with a penultimate proline or alanine, including bradykinin and substance P, which are also substrates of angiotensin-converting enzyme (ACE). During ACE inhibition, substance P is inactivated primarily by dipeptidyl peptidase-4, whereas bradykinin is first inactivated by aminopeptidase P. This study tested the hypothesis that dipeptidyl peptidase-4 inhibition potentiates vasodilator and fibrinolytic responses to substance P when ACE is inhibited. Twelve healthy subjects participated in this randomized, double-blinded, placebo-controlled crossover study. On each study day, subjects received sitagliptin 200 mg by mouth or placebo. Substance P and bradykinin were infused via brachial artery before and during intra-arterial enalaprilat. Sitagliptin and enalaprilat each reduced forearm vascular resistance and increased forearm blood flow without affecting mean arterial pressure, but there was no interactive effect of the inhibitors. Enalaprilat increased bradykinin-stimulated vasodilation and tissue plasminogen activator release; sitagliptin did not affect these responses to bradykinin. The vasodilator response to substance P was unaffected by sitagliptin and enalaprilat; however, substance P increased heart rate and vascular release of norepinephrine during combined ACE and dipeptidyl peptidase-4 inhibition. In women, sitagliptin diminished tissue plasminogen activator release in response to substance P both alone and during enalaprilat. Substance P increases sympathetic activity during combined ACE and dipeptidyl peptidase-4 inhibition. - URL: http://www.clinicaltrials.gov. Unique identifier: NCT01413542.

Substance P increases Sympathetic Activity during Combined Angiotensin Converting Enzyme and Dipeptidyl Peptidase-4 Inhibition

PubMed Central

Devin, Jessica K.; Pretorius, Mias; Nian, Hui; Yu, Chang; Billings, Frederic T.; Brown, Nancy J.

2014-01-01

Dipeptidyl peptidase-4 inhibitors prevent the degradation of incretin hormones and reduce post-prandial hyperglycemia in patients with type 2 diabetes mellitus. Dipeptidyl peptidase-4 degrades other peptides with a penultimate proline or alanine, including bradykinin and substance P, which are also substrates of angiotensin-converting enzyme. During angiotensin-converting enzyme inhibition, substance P is inactivated primarily by dipeptidyl peptidase-4, while bradykinin is first inactivated by aminopeptidase P. This study tested the hypothesis that dipeptidyl peptidase-4 inhibition potentiates vasodilator and fibrinolytic responses to substance P when angiotensin-converting enzyme is inhibited. Twelve healthy subjects participated in this randomized, double-blinded, placebo-controlled crossover study. On each study day, subjects received sitagliptin 200 mg p.o. or placebo. Substance P and bradykinin were infused via brachial artery before and during intra-arterial enalaprilat. Sitagliptin and enalaprilat each reduced forearm vascular resistance and increased forearm blood flow without affecting mean arterial pressure, but there was no interactive effect of the inhibitors. Enalaprilat increased bradykinin-stimulated vasodilation and tissue plasminogen activator release; sitagliptin did not affect these responses to bradykinin. The vasodilator response to substance P was unaffected by sitagliptin and enalaprilat, however, substance P increased heart rate and vascular release of norepinephrine during combined angiotensin-converting enzyme and dipeptidyl peptidase-4 inhibition. In women, sitagliptin diminished tissue plasminogen activator release in response to substance P both alone and during enalaprilat. Substance P increases sympathetic activity during combined angiotensin-converting enzyme and dipeptidyl peptidase-4 inhibition. PMID:24516103

Vibrio parahaemolyticus enolase is an adhesion-related factor that binds plasminogen and functions as a protective antigen.

PubMed

Jiang, Wei; Han, Xiangan; Wang, Quan; Li, Xintong; Yi, Li; Liu, Yongjie; Ding, Chan

2014-06-01

Vibrio parahaemolyticus, an emerging food and waterborne pathogen, is a leading cause of seafood poisoning worldwide. Surface proteins can directly participate in microbial virulence by facilitating pathogen dissemination via interactions with host factors. Screening and identification of protective antigens is important for developing therapies against V. parahaemolyticus infections. Here, we systematically characterized a novel immunogenic enolase of V. parahaemolyticus. The enolase gene of V. parahaemolyticus ATCC33847 was cloned, sequenced, and expressed in Escherichia coli BL21. Enzymatic assays revealed that the purified recombinant V. parahaemolyticus enolase protein catalyzes the dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate. Western blot analysis showed that V. parahaemolyticus enolase was detectable in the extracellular, outer membrane (OM) and cytoplasmic protein fractions using antibodies against the recombinant enolase. Surface expression of enolase was further confirmed by immunogold staining and mass spectrometry (liquid chromatography-tandem mass spectrometry) analysis of OM protein profiles. Notably, V. parahaemolyticus enolase was identified as a human plasminogen-binding protein with the enzyme-linked immunosorbent assay. The values obtained for adherence and inhibition suggest a role of surface-exposed enolase in epithelial adherence of V. parahaemolyticus. We further showed that enolase confers efficient immunity against challenge with a lethal dose of V. parahaemolyticus in a mouse model. To our knowledge, this is the first study to demonstrate the plasminogen-binding activity of enolase that is an adhesion-related factor of V. parahaemolyticus. Our findings collectively imply that enolase plays important roles in pathogenicity, supporting its utility as a novel vaccine candidate against V. parahaemolyticus infection.

Alteration of Antithrombin III and D-dimer Levels in Clinically Localized Prostate Cancer

PubMed Central

Ko, Dong Woo; Park, Juhyun; Kim, In Sung; Doo, Seung Hwan; Yoon, Cheol Yong; Park, Hongzoo; Lee, Won Ki; Kim, Dae Sung; Jeong, Seong Jin; Byun, Seok-Soo; Lee, Sang Eun

2010-01-01

Purpose We performed a comparative analysis of the plasma levels of antithrombin (AT) III, plasminogen, fibrinogen, and D-dimer among patients with and without clinically localized prostate cancer to investigate the clinical significance of the coagulation profile in prostate cancer. Materials and Methods A prospective study was performed in which plasma levels of AT III, plasminogen, fibrinogen, and D-dimer were assessed in patients before they underwent prostate biopsy. According to the results of the biopsy, the patients were categorized into the cancer group or the control group. Levels of the four coagulation factors were then compared between the cancer and control groups. Also, levels of the four coagulation factors were correlated with tumor stage and grade in the cancer group. Results The cancer group had significantly lower levels of AT III activity and higher plasma D-dimer levels than did the control group (p=0.007 and p=0.018, respectively). Within the cancer group, no significant differences were observed in the levels of AT III, plasminogen, fibrinogen, or D-dimer between those with a pathological Gleason score of ≥7 and otherwise. Regarding pathologic stage of prostate cancer, the subjects with organ-confined disease and those with extraprostatic extension of a tumor demonstrated no significant differences in the preoperative levels of the four coagulation factors analyzed. Conclusions Our results suggest that plasma levels of AT III and D-dimer are altered in patients with prostate cancer. Further study is needed to elucidate the underlying mechanism and clinical significances of such a phenomenon among patients with clinically localized prostate cancer. PMID:20414406

Free radical activity and hemostatic factors in NIDDM patients with and without microalbuminuria.

PubMed

Collier, A; Rumley, A; Rumley, A G; Paterson, J R; Leach, J P; Lowe, G D; Small, M

1992-08-01

In non-insulin-dependent diabetes mellitus (NIDDM) patients, microalbuminuria predicts early mortality, predominantly from cardiovascular disease. Increased free radical activity and abnormalities in hemostasis have been implicated in the development of vascular disease. Therefore, we measured markers of free radical activity (nonperoxide-conjugated diene isomer of linoleic acid [PL-9,11-LA'] and lipid peroxides expressed as malondialdehyde [MDA]) along with the hemostatic variables: fibrinogen, von Willebrand factor (vWf), plasminogen activator inhibitor (PAI-1), tissue plasminogen activator (t-PA), and plasmin activity (B beta 15-42) in 24 NIDDM patients (12 patients with microalbuminuria and 12 without microalbuminuria) and in 12 age-matched control subjects. There were no differences in linoleic acid (PL-9,12-LA) concentrations between the three groups. PL-9,11-LA' was elevated in the microalbuminuric patients compared with control subjects (P less than 0.05), but there was no difference between the two diabetic groups. MDA was elevated in the microalbuminuric diabetic patients compared with those patients without microalbuminuria (P less than 0.05) and control subjects (P less than 0.001). MDA was also increased in the patients without microalbuminuria compared with control subjects (P less than 0.01). Except for B beta 15-42, all the hemostatic variables were increased (P less than 0.05) in the diabetic patients compared with control subjects. The microalbuminuric diabetic patients had further increases in vWf (P less than 0.03) and t-PA (P less than 0.03) compared with patients with microalbuminuria. Our study suggests that there is an increase in free radical activity and abnormalities in hemostatic variables favoring a hypercoagulable state in NIDDM, especially in those with microalbuminuria.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced Venous Thrombus Resolution in Plasminogen Activator Inhibitor Type-2 Deficient Mice

PubMed Central

Siefert, Suzanne A; Chabasse, Christine; Mukhopadhyay, Subhradip; Hoofnagle, Mark H; Strickland, Dudley K; Sarkar, Rajabrata; Antalis, Toni M

2014-01-01

Background The resolution of deep vein thrombosis (DVT) requires an inflammatory response and mobilization of proteases, such as urokinase-type plasminogen activator (uPA) and matrix metalloproteinases (MMPs), to degrade the thrombus and remodel the injured vein wall. PAI-2 is a serine protease inhibitor (serpin) with unique immunosuppressive and cell survival properties that was originally identified as an inhibitor of uPA. Objective To investigate the role of PAI-2 in venous thrombus formation and resolution. Methods Venous thrombus resolution was compared in wild type C57BL/6, PAI-2 -/- and PAI-1 -/- mice using the stasis model of DVT. Formed thrombi were harvested, thrombus weights were recorded, and tissue was analyzed for uPA, and MMP activities, PAI-1 expression, and the nature of inflammatory cell infiltration. Results We found that absence of PAI-2 enhanced venous thrombus resolution, while thrombus formation was unaffected. Enhanced venous thrombus resolution in PAI-2 -/- mice was associated with increased uPA activity and reduced levels of PAI-1, with no significant effect on MMP-2 and -9 activities. PAI-1 deficiency resulted in an increase in thrombus resolution similar to PAI-2 deficiency, but additionally reduced venous thrombus formation and altered MMP activity. PAI-2 deficient thrombi had increased levels of the neutrophil chemoattractant, CXCL2, which was associated with early enhanced neutrophil recruitment. Conclusions These data identify PAI-2 as a novel regulator of venous thrombus resolution, which modulates several pathways involving both inflammatory and uPA activity mechanisms, distinct from PAI-1. Further examination of these pathways may lead to potential therapeutic prospects in accelerating thrombus resolution. PMID:25041188

Mechanisms of Nattokinase in protection of cerebral ischemia.

PubMed

Ji, Hongrui; Yu, Liang; Liu, Keyu; Yu, Zhigang; Zhang, Qian; Zou, Fengjuan; Liu, Bo

2014-12-15

In vivo, the level of cyclic Adenosine Monophosphate (cAMP) and the pathway of the Janus Kinase1/Signal Transducers and Activators of Transcription1 (JAK1/STAT1) were studied. In vitro, the Ca(2+) mobilization in human platelet stimulated by thrombin was observed. In addition, vasomotion of vascular smooth muscle was measured by adding KCl or norepinephrine(NE) under the Ca(2+) contained bath solutions. The effect induced by NE in the presence of N-nitro-L-arginine methyl ester (L-NAME) or indometacin (Indo) was also detected. At last, the levels of tissue plasminogen activator (t-PA) and Plasminogen activator inhibitor-1 (PAI-1) in cultured supernatans in Human umbilical vein endothelial cells (Huvecs) were measured by means of ELISA kit. Results showed that Nattokinase (NK) significantly increased the cAMP level, activated the signal passage of JAK1/STAT1 in injured part and inhibited remarkably the rise of platelet intracellular Ca(2+) ([Ca(2+)]i) in human platelet. Furthermore, NK relaxed rat thoracic aortic artery in the dose-dependent manner and in the endothelium dependent manner and its effect could be attenuated by L-NAME. Also, the secretion of t-PA and PAI-1 were reduced stimulated by Adr on Huvecs. These data indicated that the neuroprotective effect of NK was associated with its antiplatelet activity by elevating cAMP level and attenuating the calcium release from calcium stores; with its anti-apoptotic effect through the activation of JAK1/STAT1 pathway; with its relaxing vascular smooth muscle by promoting synthesis and release of NO, reducing ROC calcium ion influx and with its protection on endothelial cells through increasing fibrinolytic activity and facilitating spontaneous thrombolysis. Copyright © 2014 Elsevier B.V. All rights reserved.

Immunohistochemical analysis of the gingiva with periodontitis of type I plasminogen deficiency compared to gingiva with gingivitis and periodontitis and healthy gingiva.

PubMed

Kurtulus Waschulewski, Idil; Gökbuget, Aslan Y; Christiansen, Nina M; Ziegler, Maike; Schuster, Volker; Wahl, Gerhard; Götz, Werner

2016-12-01

Type I plasminogen deficiency (Plgdef) is an uncommon chronic inflammation of mucous membranes. Gingival enlargements usually proceed with progressive periodontal destruction and tooth-loss. Plasmin(ogen)-independent enzymatic mechanisms for fibrin clearance have already been discussed in the literature. Our primary objective was to verify, immunohistochemically, the occurrence of different enzymatic factors involved in tissue breakdown of inflamed compared to healthy gingiva. Secondly, we tried to find out, if these patients have a similar microbiological profile to the patients with known gingivitis and periodontitis. Immunohistochemical analysis of enzymes elastase, plasminogen (plg), cathepsin G, matrix-metalloproteinase (MMP)-3 and MMP-7 and of glycoprotein fibrinogen were performed with gingival tissues from 3 healthy controls, 8 patients with Plgdef and 3 patients with gingivitis and periodontitis. Furthermore, plaque from 5 patients with plasminogen deficiency were also obtained to determine the microbiological profile. Significantly high numbers of elastase positive leukocytes were detected in all samples. Staining for MMP-3 and MMP-7 was seen in samples with gingivitis and periodontitis with a stronger staining in samples with periodontitis by Plgdef. Fibrinogen was detectable in all samples. Staining for plg was stronger in samples with periodontitis than in other samples. Staining for cathepsin G was weak in gingivitis and periodontitis. Subgingival microbial flora showed elevated colony forming units of Prevotella intermedia/nigrescens, Fusobacterium spp., Eikenella corrodens, Porphyromonas gingivalis and viridans streptococci. Strong staining of elastase, MMP-3 and MMP-7 and weak staining of plg in Plgdef samples supports the plasmin(ogen) - independent fibrin clearance. Similar subgingival microbiological flora was observed in periodontitis with Plgdef as in other periodontal diseases. Further investigations should determine the exact pathomechanism and focus on effective treatment methods of this entity. Copyright © 2016. Published by Elsevier Ltd.

Role of altered coagulation-fibrinolytic system in the pathophysiology of diabetic retinopathy.

PubMed

Behl, Tapan; Velpandian, Thirumurthy; Kotwani, Anita

2017-05-01

The implications of altered coagulation-fibrinolytic system in the pathophysiology of several vascular disorders, such as stroke and myocardial infarction, have been well researched upon and established. However, its role in the progression of diabetic retinopathy has not been explored much. Since a decade, it is known that hyperglycemia is associated with a hypercoagulated state and the various impairments it causes are well acknowledged as independent risk factors for the development of cardiovascular diseases. But recent studies suggest that the hypercoagulative state and diminished fibrinolytic responses might also alter retinal homeostasis and induce several deleterious molecular changes in retinal cells which aggravate the already existing hyperglycemia-induced pathological conditions and thereby lead to the progression of diabetic retinopathy. The major mediators of coagulation-fibrinolytic system whose concentration or activity get altered during hyperglycemia include fibrinogen, antithrombin-III (AT-III), plasminogen activator inhibitor-1 (PAI-1) and von Willebrand factor (vWF). Inhibiting the pathways by which these altered mediators get involved in the pathophysiology of diabetic retinopathy can serve as potential targets for the development of an adjuvant novel alternative therapy for diabetic retinopathy. Copyright © 2017 Elsevier Inc. All rights reserved.

Weight loss improves biomarkers endothelial function and systemic inflammation in obese postmenopausal Saudi women.

PubMed

Abd El-Kader, Shehab Mahmoud; Saiem Al-Dahr, Mohammed H

2016-06-01

Although postmenopausal associated disorders are important public health problems worldwide, to date limited studies evaluated the endothelial function and systemic inflammation response to weight loss in obese postmenopausal women. This study was done to evaluate the endothelial function and systemic inflammation response to weight loss in obese postmenopausal Saudi women. Eighty postmenopausal obese Saudi women (mean age 52.64±6.13 year) participated in two groups: Group (A) received aerobic exercise on treadmill and diet whereas, group (B) received no intervention. Markers of inflammation and endothelial function were measured before and after 3 months at the end of the study. The values of body mass index(BMI), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP), inter-cellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1) and plasminogen activator inhibitor-1 activity (PAI-1:Ac) were significantly decreased in group (A), while changes were not significant in group (B). Also, there were significant differences between mean levels of the investigated parameters in group (A) and group (B) after treatment. Weight loss ameliorates inflammatory cytokines and markers of endothelial function in obese postmenopausal Saudi women.

The Pathogenesis of Traumatic Coagulopathy

DTIC Science & Technology

2015-01-01

reduction in death due to haemorrhage in trauma patients given tranexamic acid (TXA), which inhibits activation of plasminogen to plasmin [36, 37]. Other...in combination with red cells and tranexamic acid , with extremely limited use of colloid or crystalloid infusions [76–82], a practice known as...blocked by tranexamic acid . Journal of Trauma and Acute Care Surgery 2013; 74: 482–8. 15. Martini WZ, Pusateri AE, Uscilowicz JM, Delgado AV, Holcomb

Endovascular therapy for acute ischemic stroke.

PubMed

Broderick, Joseph P

2009-03-01

To review advances in endovascular therapy for acute ischemic stroke. Data from primate studies, randomized studies of intravenous recombinant tissue-type plasminogen activator, and nonrandomized and randomized studies of endovascular therapy were reviewed. Clinical trial data demonstrate the superiority of endovascular treatment with thrombolytic medication or mechanical methods to reopen arteries compared with control patients from the PROACT II Trial treated with heparin alone. However, these same clinical trials, as well as preclinical primate models, indicate that recanalization, whether by endovascular approaches or standard-dose recombinant tissue-type plasminogen activator, is unlikely to improve clinical outcome after a certain time point. Although the threshold beyond which reperfusion has no or little benefit has yet to be conclusively defined, accumulated data to this point indicate an overall threshold of approximately 6 to 7 hours. In addition, although the risk of symptomatic intracerebral hemorrhage is similar in trials of intravenous lytics and endovascular approaches, endovascular approaches have distinctive risk profiles that can impact outcome. The treatment of acute ischemic stroke is evolving with new tools to reopen arteries and salvage the ischemic brain. Ongoing randomized trials of these new approaches are prerequisite next steps to demonstrate whether reperfusion translates into clinical effectiveness. Physiologic time to reperfusion will remain critical no matter which tools prove most effective and safest.

Berberine suppresses in vitro migration and invasion of human SCC-4 tongue squamous cancer cells through the inhibitions of FAK, IKK, NF-kappaB, u-PA and MMP-2 and -9.

PubMed

Ho, Yung-Tsuan; Yang, Jai-Sing; Li, Tsai-Chung; Lin, Jen-Jyh; Lin, Jaung-Geng; Lai, Kuang-Chi; Ma, Chia-Yu; Wood, W Gibson; Chung, Jing-Gung

2009-07-08

There is increasing evidence that urokinase-type plasminogen activator (u-PA) and matrix metalloproteinases (MMPs) play an important role in cancer metastasis and angiogenesis. Inhibition of u-PA and MMPs could suppress migration and invasion of cancer cells. Berberine, one of the main constituents of the plant Rhizoma coptidis, is a type of isoquinoline alkaloid, reported to have anti-cancer effects in different human cancer cell lines. There is however, no available information on effects of berberine on migration and invasion of human tongue cancer cells. Here, we report that berberine inhibited migration and invasion of human SCC-4 tongue squamous carcinoma cells. This action was mediated by the p-JNK, p-ERK, p-p38, IkappaK and NF-kappaB signaling pathways resulting in inhibition of MMP-2 and -9 in human SCC-4 tongue squamous carcinoma cells. Our Western blowing analysis also showed that berberine inhibited the levels of urokinase-plasminogen activator (u-PA). These results suggest that berberine down-regulates u-PA, MMP-2 and -9 expressions in SCC-4 cells through the FAK, IKK and NF-kappaB mediated pathways and a novel function of berberine is to inhibit the invasive capacity of malignant cells.

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

PubMed

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.

Plasminogen activator inhibitor-1 -675 4G/5G polymorphism and polycystic ovary syndrome risk: a meta analysis.

PubMed

Liu, Ying; Sun, Mei-Guo; Jiang, Rong; Ding, Rui; Che, Zhen; Chen, Yan-Yan; Yao, Ci-Jiang; Zhu, Xiao-Xia; Cao, Ji-Yu

2014-03-01

Several studies have reported that excessive amounts of plasminogen activator inhibitor-1(PAI-1) might increase the incidence of polycystic ovary syndrome(PCOS), but so far the published results were inconsistent. The aim of this study was to further investigate the association between PAI-1 gene polymorphism and the susceptibility to PCOS by performing a meta-analysis. A comprehensive literature search for relevant studies was conducted on google scholar, PubMed, the Chinese National Knowledge Infrastructure (CNKI) and the Chinese Biomedical Literature Database (CBM). This meta-analysis was performed using the STATA 11.0 software and the pooled odds ratio (OR) with 95% confidence interval (CI) was calculated. Ten case-control studies were included in this meta-analysis with a total of 2,079 cases and 1,556 controls. The results showed that PAI-1 -675 4G/5G polymorphism may increase the risk of PCOS, especially among Asian populations. However, there was no statistically significant association between the polymorphism and PCOS risk in Caucasians. Our meta-analysis suggests that PAI-1 -675 4G/5G polymorphism may contribute to increasing susceptibility to PCOS in Asians. Detection of the PAI-1 gene polymorphism might be a promising biomarker for the susceptibility of PCOS.

The effects of a low-dose monophasic preparation of levonorgestrel and ethinyl estradiol on coagulation and other hemostatic factors.

PubMed

Archer, D F; Mammen, E F; Grubb, G S

1999-11-01

This study was undertaken to evaluate the effects on hemostatic factors of a low-dose preparation of levonorgestrel and ethinyl estradiol in a 12-cycle study. Thirty healthy women began taking 100 microg levonorgestrel and 20 microg ethinyl estradiol on the first day of the menstrual cycle, continued to take the preparation for the next 21 days, and then took placebo for 7 days. Mean changes in prothrombin time, partial thromboplastin time, and levels of factors VII and X, antithrombin, plasminogen, fibrinogen, protein S, thrombin-antithrombin complexes, and D-dimer were analyzed at baseline and at cycles 3, 6, and 12 with paired Student t tests. Factor X, plasminogen antigen and activity, and D-dimer levels were significantly increased (P

Comparison of the effects of Korean mindfulness-based stress reduction, walking, and patient education in diabetes mellitus.

PubMed

Jung, Hee Young; Lee, Haejung; Park, Jina

2015-12-01

The purpose of this study was to compare the effects of Korean mindfulness-based stress reduction (K-MBSR), walking, and patient education regarding diabetes mellitus (DM) on stress response, glycemic control, and vascular inflammation in patients with diabetes mellitus. A cluster randomized trial including 56 adults with diabetes mellitus (K-MBSR group = 21, walking group = 18, patient education group = 17) was conducted between 13 July and 14 September 2012. The questionnaire included the Diabetes Distress Scale and Perceived Stress Response Inventory. Fasting blood samples were used to measure levels of cortisol, blood glucose, plasminogen activator inhibitor-1 (PAI-1), and tissue plasminogen activator (t-PA). There were no statistically significant differences between the effects of K-MBSR, walking, and patient education on stress, glycemic control, or vascular inflammation. However, in the K-MBSR and walking groups, significant reductions in the levels of serum cortisol and PAI-1 were observed. A significant reduction in psychological responses to stress was observed in the walking and patient education groups. Longitudinal studies could provide better insight into the impact of K-MBSR, walking, and patient education on health outcomes in adults with diabetes mellitus. © 2015 Wiley Publishing Asia Pty Ltd.

Meta-Analysis of the Association between Plasminogen Activator Inhibitor-1 4G/5G Polymorphism and Recurrent Pregnancy Loss

PubMed Central

Li, Xuejiao; Liu, Yukun; Zhang, Rui; Tan, Jianping; Chen, Libin; Liu, Yinglin

2015-01-01

Background The association between plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphism and recurrent pregnancy loss (RPL) risk is still contradictory. We thus performed a meta-analysis. Material/Methods Relevant studies were searched for in PubMed, Web of Science, Embase, and Cochrane Library. An odds ratio (OR) with a 95% confidence interval (CI) was used to assess the association between PAI-1 4G/5G polymorphism and RPL risk. Results A total of 22 studies with 4306 cases and 3076 controls were included in this meta-analysis. We found that PAI-1 4G/5G polymorphism was significantly associated with an increased RPL risk (OR=1.89; 95% CI 1.34–2.67; P=0.0003). In the subgroup analysis by race, PAI-1 4G/5G polymorphism was significantly associated with an increased RPL risk in Caucasians (OR=2.23; 95% CI 1.44–3.46; P=0.0003). However, no significant association was observed in Asians (OR=1.47; 95% CI 0.84–2.59; P=0.18). Conclusions In conclusion, this meta-analysis suggests that PAI-1 4G/5G polymorphism might be associated with RPL development in Caucasians. PMID:25862335

Association between Plasminogen Activator Inhibitor-1 -675 4G/5G Polymorphism and Sepsis: A Meta-Analysis

PubMed Central

Yuan, Weifeng; Li, Weifeng; Huang, Wenjie

2013-01-01

Background Several studies have evaluated the association between plasminogen activator inhibitor-1 (PAI-1) -675 4G/5G polymorphism and sepsis in different populations. However, the available results are conflicting. Methods A search of Pubmed and EMBASE databases was performed to identify relevant studies for inclusion in the meta-analysis. Odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were determined using a random-effects model. Results Twelve case-control studies and three cohort studies were included. Overall, a significant association between 4G/5G polymorphism and sepsis risk was observed for 4G/4G vs. 4G/5G +5G/5G (OR = 1.30, 95% CI 1.08–1.56, P = 0.006). In addition, there was a significant association between PAI-1 4G/5G polymorphism and sepsis-related mortality (OR = 1.72, 95% CI 1.27–2.33, P = 0.0005). In subgroup analyses, increased sepsis risk and mortality risk were found in Caucasians and in patients with sepsis. Conclusions This meta-analysis suggested that the PAI-1 -675 4G/5G polymorphism was a risk factor for sepsis and sepsis mortality. PMID:23382992

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.

Mitogenic signaling of urokinase receptor-deficient kidney fibroblasts: actions of an alternative urokinase receptor and LDL receptor-related protein.

PubMed

Zhang, Guoqiang; Cai, Xiaohe; López-Guisa, Jesús M; Collins, Sarah J; Eddy, Allison A

2004-08-01

The urokinase receptor (uPAR) attenuates myofibroblast recruitment and fibrosis in the kidney. This study examined the role of uPAR and its co-receptor LDL receptor-related protein (LRP) in the regulation of kidney fibroblast proliferation and extracellular signal-regulated kinase (ERK) signaling. Compared with uPAR+/+ cells, uPAR-/- kidney fibroblasts were hyperproliferative. UPAR-/- fibroblast proliferation was 60% inhibited by an ERK kinase inhibitor. LRP protein was reduced and extracellular accumulation of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) proteins were greater in uPAR-/- cultures. Addition of functional uPA protein or LRP antisense RNA significantly increased ERK signaling and cell mitosis in both genotypes. Enhanced uPAR-/- fibroblast proliferation was reversed by a recombinant nonfunctional uPA peptide. The density of cell-bound fluor-uPA was similar between uPAR-/- and uPAR+/+ fibroblasts (78 +/- 6 versus 92 +/- 16 units). These data suggest that uPAR-deficient kidney fibroblasts express lower levels of its scavenger co-receptor LRP, resulting in greater extracellular accumulation of uPA and PAI-1. Enhanced proliferation of uPAR-/- fibroblasts seems to be mediated by uPA-dependent ERK signaling via an alternative urokinase receptor.

Topical tissue plasminogen activator appears ineffective for the clearance of intraocular fibrin.

PubMed

Zwaan, J; Latimer, W B

1998-06-01

To determine the efficacy of topical tissue plasminogen activator (tPA) for the resolution of postoperative or inflammatory intraocular fibrinous exudates. Each treatment consisted of drops of 1 mg/ml tPA given 9 times 5 minutes apart. Records were reviewed and the results at 24 and 48 hours were recorded. Sixty-two patients had a total of 94 treatments. Fibrin exudates following intraocular surgery in 34 patients were treated 44 times. In 6 patients there was a positive result. Fibrin associated with intraocular infection was treated in 9 patients. None showed clear improvement. Nineteen patients had a total of 34 treatments for poorly controlled intraocular pressure (IOP) after glaucoma surgery. Five patients showed adequate control of the IOP, 12 did not change, and 2 had a questionable improvement. Eleven patients had adequate IOP control after additional treatment. Seven required suture lysis, 2 ab interno bleb revision, and 2 YAG capsulotomy or iridotomy to reduce the IOP to an acceptable level. Within the limits of this retrospective study and taking into account that fibrin may resolve spontaneously, it appears that topical tPA drops are not effective for the liquefaction of intraocular fibrin after surgery or in association with intraocular inflammation. They did not improve IOP control after glaucoma surgery.

Unusual cause of aborted sudden cardiac death in a teen athlete: homozygosity for the 4G allele of the plasminogen activase inhibitor type 1 gene.

PubMed

Phillips, Susie B; Batlivala, Sarosh; Knudson, Jarrod D

2015-10-01

Common aetiologies of sudden cardiac death in children include coronary anomalies, channelopathies, and cardiomyopathies. Less frequently, hypercoagulable states cause sudden arrest. We report an unusual case of aborted sudden cardiac death in a teenager, ultimately found to have homozygosity for the 4G allele of the plasminogen activase inhibitor type 1 gene.

Insulin-like growth factor binding proteins initiate cell death and extracellular matrix remodeling in the mammary gland.

PubMed

Flint, D J; Boutinaud, M; Tonner, E; Wilde, C J; Hurley, W; Accorsi, P A; Kolb, A F; Whitelaw, C B A; Beattie, J; Allan, G J

2005-08-01

We have demonstrated that insulin-like growth factor binding protein-5 (IGFBP-5) production by mammary epithelial cells increases dramatically during forced involution of the mammary gland in rats, mice and pigs. We proposed that growth hormone (GH) increases the survival factor IGF-I, whilst prolactin (PRL) enhances the effects of GH by decreasing the concentration of IGFBP-5, which would otherwise inhibit the actions of IGFs. To demonstrate a causal relationship between IGFBP-5 and cell death, we created transgenic mice expressing IGFBP-5, specifically, in the mammary gland. DNA content in the mammary glands of transgenic mice was decreased as early as day 10 of pregnancy. Mammary cell number and milk synthesis were both decreased by approximately 50% during the first 10 days of lactation. The concentrations of the pro-apoptotic molecule caspase-3 was increased in transgenic animals whilst the concentrations of two pro-survival molecules Bcl-2 and Bcl-x were both decreased. In order to examine whether IGFBP-5 acts by inhibiting the survival effect of IGF-I, we examined IGF receptor- and Akt-phoshorylation and showed that both were inhibited. These studies also indicated that the effects of IGFBP-5 could be mediated in part by IGF-independent effects involving potential interactions with components of the extracellular matrix involved in tissue remodeling, such as components of the plasminogen system, and the matrix metallo-proteinases (MMPs). Mammary development was normalised in transgenic mice by R3-IGF-I, an analogue of IGF-I which binds weakly to IGFBPs, although milk production was only partially restored. In contrast, treatment with prolactin was able to inhibit early involutionary processes in normal mice but was unable to prevent this in mice over-expressing IGFBP-5, although it was able to inhibit activation of MMPs. Thus, IGFBP-5 can simultaneously inhibit IGF action and activate the plasminogen system thereby coordinating cell death and tissue remodeling processes. The ability to separate these properties, using mutant IGFBPs, is currently under investigation.