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Sample records for activated partial thrombin

  1. Influence of fibrinogen degradation products on thrombin time, activated partial thromboplastin time and prothrombin time of canine plasma.

    PubMed

    Mischke, R; Wolling, H

    2000-01-01

    To investigate how thrombin time, activated partial thromboplastin time (APTT) and prothrombin time are influenced by fibrinogen degradation products (FDP), different concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8 and 1.0 mg/ml) of the purified FDP X, Y, D and E were added to the plasma of healthy dogs. If fragment Y was added to the plasma a considerable inhibitory effect could be demonstrated for all three test systems. A significant prolongation (p < 0.05) was found for concentrations of > or =0.1 mg/ml (thrombin time, APTT) and > or =0.2 mg/ml (prothrombin time). With FDP Y concentrations from >0.185 mg/ml (prothrombin time) to >0.24 mg/ml (APTT) coagulation time was prolonged beyond the respective reference range. As regards the other fragments, a comparable inhibitory effect could only be shown for fragment X added to the thrombin time test system. This effect can most probably be explained by the competition of the FDP X and fibrinogen for the fibrinogen binding sites of thrombin, rather than by a fibrin polymerization disorder. The results demonstrate that for plasma with normal fibrinogen concentration the group tests are only prolonged beyond the reference range at FDP concentrations very rarely found in spontaneous hyperfibrinolysis. PMID:11014962

  2. Thrombin generation, ProC®Global, prothrombin time and activated partial thromboplastin time in thawed plasma stored for seven days and after methylene blue/light pathogen inactivation

    PubMed Central

    Thiele, Thomas; Hron, Gregor; Kellner, Sarah; Wasner, Christina; Westphal, Antje; Warkentin, Theodore E.; Greinacher, Andreas; Selleng, Kathleen

    2016-01-01

    Background Methylene blue pathogen inactivation and storage of thawed plasma both lead to changes in the activity of several clotting factors. We investigated how this translates into a global loss of thrombin generation potential and alterations in the protein C pathway. Materials and methods Fifty apheresis plasma samples were thawed and each divided into three subunits. One subunit was stored for 7 days at 4 °C, one was stored for 7 days at 22 °C and one was stored at 4 °C after methylene blue/light treatment. Thrombin generation parameters, ProC®Global-NR, prothrombin time and activated partial thromboplastin time were assessed on days 0 and 7. Results The velocity of thrombin generation increased significantly after methylene blue treatment (increased thrombin generation rate; time to peak decreased) and decreased after storage (decreased thrombin generation rate and peak thrombin; increased lag time and time to peak). The endogenous thrombin generation potential remained stable after methylene blue treatment and storage at 4 °C. Methylene blue treatment and 7 days of storage at 4 °C activated the protein C pathway, whereas storage at room temperature and storage after methylene blue treatment decreased the functional capacity of the protein C pathway. Prothrombin time and activated partial thromboplastin time showed only modest alterations. Discussion The global clotting capacity of thawed plasma is maintained at 4 °C for 7 days and directly after methylene blue treatment of thawed plasma. Thrombin generation and ProC®Global are useful tools for investigating the impact of pathogen inactivation and storage on the clotting capacity of therapeutic plasma preparations. PMID:26192785

  3. Inhibition of thrombin activity with DNA-aptamers.

    PubMed

    Dobrovolsky, A B; Titaeva, E V; Khaspekova, S G; Spiridonova, V A; Kopylov, A M; Mazurov, A V

    2009-07-01

    The effects of two DNA aptamers (oligonucleotides) 15TBA and 31TBA (15- and 31-mer thrombin-binding aptamers, respectively) on thrombin activity were studied. Both aptamers added to human plasma dose-dependently increased thrombin time (fibrin formation upon exposure to exogenous thrombin), prothrombin time (clotting activation by the extrinsic pathway), and activated partial thromboplastin time (clotting activation by the intrinsic pathway). At the same time, these aptamers did not modify amidolytic activity of thrombin evaluated by cleavage of synthetic chromogenic substrate. Aptamers also inhibited thrombin-induced human platelet aggregation. The inhibitory effects of 31TBA manifested at lower concentrations than those of 15TBA in all tests. These data indicate that the studied antithrombin DNA aptamers effectively suppress its two key reactions, fibrin formation and stimulation of platelet aggregation, without modifying active center of the thrombin molecule. PMID:19902090

  4. Thrombin

    PubMed Central

    Di Cera, Enrico

    2008-01-01

    Thrombin is a Na+-activated, allosteric serine protease that plays opposing functional roles in blood coagulation. Binding of Na+ is the major driving force behind the procoagulant, prothrombotic and signaling functions of the enzyme, but is dispensable for cleavage of the anticoagulant protein C. The anticoagulant function of thrombin is under the allosteric control of the cofactor thrombomodulin. Much has been learned on the mechanism of Na+ binding and recognition of natural substrates by thrombin. Recent structural advances have shed light on the remarkable molecular plasticity of this enzyme and the molecular underpinnings of thrombin allostery mediated by binding to exosite I and the Na+ site. This review summarized our current understanding of the molecular basis of thrombin function and allosteric regulation. The basic information emerging from recent structural, mutagenesis and kinetic investigation of this important enzyme is that thrombin exists in three forms, E*, E and E:Na+, that interconvert under the influence of ligand binding to distinct domains. The transition between the Na+-free slow from E and the Na+-bound fast form E:Na+ involves the structure of the enzyme as a whole, and so does the interconversion between the two Na+-free forms E* and E. E* is most likely an inactive form of thrombin, unable to interact with Na+ and substrate. The complexity of thrombin function and regulation has gained this enzyme pre-eminence as the prototypic allosteric serine protease. Thrombin is now looked upon as a model system for the quantitative analysis of biologically important enzymes. PMID:18329094

  5. Novel thrombin inhibitors incorporating non-basic partially saturated heterobicyclic P1-arginine mimetics.

    PubMed

    Peterlin-Masic, Lucija; Mlinsek, Gregor; Solmajer, Tomaz; Trampus-Bakija, Alenka; Stegnar, Mojca; Kikelj, Danijel

    2003-03-10

    The design, synthesis and biological activity of non-covalent thrombin inhibitors incorporating 4,5,6,7-tetrahydroindazole, 2-methyl-4,5,6,7-tetrahydroindazole, 4,5,6,7-tetrahydroisoindole, 5,6,7,8-tetrahydroquinazoline and 5,6,7,8-tetrahydroquinazolin-2-amine as novel, partially saturated, heterobicyclic P(1)-arginine side-chain mimetics is described. The binding mode of the most potent candidate in the series co-crystallized with human alpha-thrombin, which exhibited an in vitro K(i) of 140nM and more that 478-fold selectivity against trypsin, is discussed. PMID:12617892

  6. Allosteric Partial Inhibition of Monomeric Proteases. Sulfated Coumarins Induce Regulation, not just Inhibition, of Thrombin

    PubMed Central

    Verespy III, Stephen; Mehta, Akul Y.; Afosah, Daniel; Al-Horani, Rami A.; Desai, Umesh R.

    2016-01-01

    Allosteric partial inhibition of soluble, monomeric proteases can offer major regulatory advantages, but remains a concept on paper to date; although it has been routinely documented for receptors and oligomeric proteins. Thrombin, a key protease of the coagulation cascade, displays significant conformational plasticity, which presents an attractive opportunity to discover small molecule probes that induce sub-maximal allosteric inhibition. We synthesized a focused library of some 36 sulfated coumarins to discover two agents that display sub-maximal efficacy (~50%), high potency (<500 nM) and high selectivity for thrombin (>150-fold). Michaelis-Menten, competitive inhibition, and site-directed mutagenesis studies identified exosite 2 as the site of binding for the most potent sulfated coumarin. Stern-Volmer quenching of active site-labeled fluorophore suggested that the allosteric regulators induce intermediate structural changes in the active site as compared to those that display ~80–100% efficacy. Antithrombin inactivation of thrombin was impaired in the presence of the sulfated coumarins suggesting that allosteric partial inhibition arises from catalytic dysfunction of the active site. Overall, sulfated coumarins represent first-in-class, sub-maximal inhibitors of thrombin. The probes establish the concept of allosteric partial inhibition of soluble, monomeric proteins. This concept may lead to a new class of anticoagulants that are completely devoid of bleeding. PMID:27053426

  7. Thrombin stimulates insulin secretion via protease-activated receptor-3

    PubMed Central

    Hänzelmann, Sonja; Wang, Jinling; Güney, Emre; Tang, Yunzhao; Zhang, Enming; Axelsson, Annika S; Nenonen, Hannah; Salehi, Albert S; Wollheim, Claes B; Zetterberg, Eva; Berntorp, Erik; Costa, Ivan G; Castelo, Robert; Rosengren, Anders H

    2015-01-01

    The disease mechanisms underlying type 2 diabetes (T2D) remain poorly defined. Here we aimed to explore the pathophysiology of T2D by analyzing gene co-expression networks in human islets. Using partial correlation networks we identified a group of co-expressed genes (‘module’) including F2RL2 that was associated with glycated hemoglobin. F2Rl2 is a G-protein-coupled receptor (GPCR) that encodes protease-activated receptor-3 (PAR3). PAR3 is cleaved by thrombin, which exposes a 6-amino acid sequence that acts as a ‘tethered ligand’ to regulate cellular signaling. We have characterized the effect of PAR3 activation on insulin secretion by static insulin secretion measurements, capacitance measurements, studies of diabetic animal models and patient samples. We demonstrate that thrombin stimulates insulin secretion, an effect that was prevented by an antibody that blocks the thrombin cleavage site of PAR3. Treatment with a peptide corresponding to the PAR3 tethered ligand stimulated islet insulin secretion and single β-cell exocytosis by a mechanism that involves activation of phospholipase C and Ca2+ release from intracellular stores. Moreover, we observed that the expression of tissue factor, which regulates thrombin generation, was increased in human islets from T2D donors and associated with enhanced β-cell exocytosis. Finally, we demonstrate that thrombin generation potential in patients with T2D was associated with increased fasting insulin and insulinogenic index. The findings provide a previously unrecognized link between hypercoagulability and hyperinsulinemia and suggest that reducing thrombin activity or blocking PAR3 cleavage could potentially counteract the exaggerated insulin secretion that drives insulin resistance and β-cell exhaustion in T2D. PMID:26742564

  8. Thrombin time and anti-IIa dabigatran's activity: hypothesis of thrombin time's predictive value.

    PubMed

    Le Guyader, Maïlys; Kaabar, Mohammed; Lemaire, Pierre; Pineau Vincent, Fabienne

    2015-01-01

    Dabigatran etexilate (Pradaxa®) is a new oral anticoagulant, competitive inhibitor, selective, fast, direct and reversible of thrombin. Dabigatran has an impact on a large panel of used coagulation tests. There is no relationship between thrombin time's lengthening and anti-IIa activity. This study defines thrombin time's predictive value, when its time is normal. The result of negative value is 97,6%. 255 patients were studied between January 2013 and July 2014. Thrombin time and anti-IIa activity were dosed for each patient. This study can be an assistant for therapeutic decision for laboratories without specialized test. PMID:26489812

  9. Multiple active forms of thrombin. IV. Relative activities of meizothrombins

    SciTech Connect

    Doyle, M.F.; Mann, K.G. )

    1990-06-25

    The prothrombin activation intermediates meizothrombin and meizothrombin(desF1) (meizothrombin that has been autoproteolyzed to remove fragment 1) have been obtained in a relatively pure, active form with minimal autolysis, making them suitable for enzymatic characterization. When compared at equimolar concentrations, alpha-thrombin, fragment 1.2+ alpha-thrombin, meizothrombin(desF1), and meizothrombin have approximately 100, 100, 10, and 1% activity, respectively, toward the macromolecular substrates factor V, fibrinogen, and platelets. The difference in activity of these four enzymes cannot be attributed to alterations in the catalytic triad, as all four enzymes have nearly identical catalytic efficiency toward the chromogenic substrate S2238. Further, the ability of meizothrombin and meizothrombin(desF1) to activate protein C was 75% of the activity exhibited by alpha-thrombin or fragment 1.2+ alpha-thrombin. All four enzymes bind to thrombomodulin, as judged by the enhanced rate of protein C activation upon preincubation of the enzymes with thrombomodulin. The extent of rate enhancement varied, with meizothrombin/thrombomodulin exhibiting only 50% of the alpha-thrombin/thrombomodulin rate. This difference in rate is not due to a decreased affinity of the meizothrombin for thrombomodulin since the apparent dissociation constants for the alpha-thrombin-thrombomodulin complex and the meizothrombin-thrombomodulin complex are virtually identical. The difference in the observed rate is due in part to the higher Km for protein C exhibited by the meizothrombin-thrombomodulin complex. Incubation of the thrombomodulin-enzyme complex with phospholipid vesicles caused an increase in the protein C activation rates. The kinetic constants for protein C activation in the presence of phospholipid are virtually identical for these enzyme-thrombomodulin complexes.

  10. Thrombin induces endothelial arginase through AP-1 activation.

    PubMed

    Zhu, Weifei; Chandrasekharan, Unni M; Bandyopadhyay, Smarajit; Morris, Sidney M; DiCorleto, Paul E; Kashyap, Vikram S

    2010-04-01

    Arterial thrombosis is a common disease leading to severe ischemia beyond the obstructing thrombus. Additionally, endothelial dysfunction at the site of thrombosis can be rescued by l-arginine supplementation or arginase blockade in several animal models. Exposure of rat aortic endothelial cells (RAECs) to thrombin upregulates arginase I mRNA and protein levels. In this study, we further investigated the molecular mechanism of thrombin-induced arginase changes in endothelial cells. Thrombin strikingly increased arginase I promoter and enzyme activity in primary cultured RAECs. Using different deletion and point mutations of the promoter, we demonstrated that the activating protein-1 (AP-1) consensus site located at -3,157 bp in the arginase I promoter was a thrombin-responsive element. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay further confirmed that upon thrombin stimulation, c-Jun and activating transcription factor-2 (ATF-2) bound to the AP-1 site, which initiated the transactivation. Moreover, loss-of-function studies using small interfering RNA confirmed that recruitment of these two transcription factors to the AP-1 site was required for thrombin-induced arginase upregulation. In the course of defining the signaling pathway leading to the activation of AP-1 by thrombin, we found thrombin-induced phosphorylation of stress-activated protein kinase/c-Jun-NH(2)-terminal kinase (SAPK/JNK or JNK1/2/3) and p38 mitogen-activated protein kinase, which were followed by the phosphorylation of both c-Jun and ATF-2. These findings reveal the basis for thrombin induction of endothelial arginase I and indicate that arginase inhibition may be an attractive therapeutic alternative in the setting of arterial thrombosis and its associated endothelial dysfunction. PMID:20032511

  11. Thrombomodulin Binding Selects the Catalytically Active Form of Thrombin.

    PubMed

    Handley, Lindsey D; Treuheit, Nicholas A; Venkatesh, Varun J; Komives, Elizabeth A

    2015-11-01

    Human α-thrombin is a serine protease with dual functions. Thrombin acts as a procoagulant, cleaving fibrinogen to make the fibrin clot, but when bound to thrombomodulin (TM), it acts as an anticoagulant, cleaving protein C. A minimal TM fragment consisting of the fourth, fifth, and most of the sixth EGF-like domain (TM456m) that has been prepared has much improved solubility, thrombin binding capacity, and anticoagulant activity versus those of previous TM456 constructs. In this work, we compare backbone amide exchange of human α-thrombin in three states: apo, D-Phe-Pro-Arg-chloromethylketone (PPACK)-bound, and TM456m-bound. Beyond causing a decreased level of amide exchange at their binding sites, TM and PPACK both cause a decreased level of amide exchange in other regions including the γ-loop and the adjacent N-terminus of the heavy chain. The decreased level of amide exchange in the N-terminus of the heavy chain is consistent with the historic model of activation of serine proteases, which involves insertion of this region into the β-barrel promoting the correct conformation of the catalytic residues. Contrary to crystal structures of thrombin, hydrogen-deuterium exchange mass spectrometry results suggest that the conformation of apo-thrombin does not yet have the N-terminus of the heavy chain properly inserted for optimal catalytic activity, and that binding of TM allosterically promotes the catalytically active conformation. PMID:26468766

  12. Activation of human factor V by factor Xa and thrombin

    SciTech Connect

    Monkovic, D.D.; Tracy, P.B. )

    1990-02-06

    The activation of human factor V by factor Xa and thrombin was studied by functional assessment of cofactor activity and sodium dodecyl sulfate-polycarylamide gel electrophoresis followed by either autoradiography of {sup 125}I-labeled factor V activation products or Western blot analyses of unlabeled factor V activation products. Cofactor activity was measured by the ability of the factor V/Va peptides to support the activation of prothrombin. The factor Xa catalyzed cleavage of factor V was observed to be time, phospholipid, and calcium ion dependent, yielding a cofactor with activity equal to that of thrombin-activated factor V (factor Va). The cleavage pattern differed markedly from the one observed in the bovine system. The factor Xa activated factor V subunits expressing cofactor activity were isolated and found to consist of peptides of M{sub r} 220,000 and 105,000. Although thrombin cleaved the M{sub r} 220,000 peptide to yield peptides previously shown to be products of thrombin activation, cofactor activity did not increase. N-Terminal sequence analysis confirmed that both factor Xa and thrombin cleave factor V at the same bond to generate the M{sub r} 220,000 peptide. The factor Xa dependent functional assessment of {sup 125}I-labeled factor V coupled with densitometric analyses of the cleavage products indicated that the cofactor activity of factor Xa activated factor V closely paralleled the appearance of the M{sub r} 220,000 peptide. The data indicate that factor Xa is as efficient an enzyme toward factor V as thrombin.

  13. The thrombin receptor extracellular domain contains sites crucial for peptide ligand-induced activation.

    PubMed Central

    Bahou, W F; Coller, B S; Potter, C L; Norton, K J; Kutok, J L; Goligorsky, M S

    1993-01-01

    A thrombin receptor (TR) demonstrating a unique activation mechanism has recently been isolated from a megakaryocytic (Dami) cell line. To further study determinants of peptide ligand-mediated activation phenomenon, we have isolated, cloned, and stably expressed the identical receptor from a human umbilical vein endothelial cell (HUVEC) library. Chinese hamster ovary (CHO) cells expressing a functional TR (CHO-TR), platelets, and HUVECs were then used to specifically characterize alpha-thrombin- and peptide ligand-induced activation responses using two different antibodies: anti-TR34-52 directed against a 20-amino acid peptide spanning the thrombin cleavage site, and anti-TR1-160 generated against the NH2-terminal 160 amino acids of the TR expressed as a chimeric protein in Escherichia coli. Activation-dependent responses to both alpha-thrombin (10 nM) and peptide ligand (20 microM) were studied using fura 2-loaded cells and microspectrofluorimetry. Whereas preincubation of CHO-TR with anti-TR34-52 abolished only alpha-thrombin-induced [Ca2+]i transients, preincubation with anti-TR1-160 abrogated both alpha-thrombin- and peptide ligand-induced responses. This latter inhibitory effect was dose dependent and similar for both agonists, with an EC50 of approximately 90 micrograms/ml. Anti-TR1-160 similarly abolished peptide ligand-induced [Ca2+]i transients in platelets and HUVECs, whereas qualitatively different responses characterized by delayed but sustained elevations in [Ca2+]i transients were evident using alpha-thrombin. Platelet aggregation to low concentrations of both ligands was nearly abolished by anti-TR1-160, although some shape change remained; anti-TR34-52 only inhibited alpha-thrombin-induced aggregation. These data establish that a critical recognition sequence for peptide ligand-mediated receptor activation is contained on the NH2-terminal portion of the receptor, upstream from the first transmembrane domain. Furthermore, alpha-thrombin

  14. Thrombin A-Chain: Activation Remnant or Allosteric Effector?

    PubMed Central

    Carter, Isis S. R.; Vanden Hoek, Amanda L.; Pryzdial, Edward L. G.; MacGillivray, Ross T. A.

    2010-01-01

    Although prothrombin is one of the most widely studied enzymes in biology, the role of the thrombin A-chain has been neglected in comparison to the other domains. This paper summarizes the current data on the prothrombin catalytic domain A-chain region and the subsequent thrombin A-chain. Attention is given to biochemical characterization of naturally occurring prothrombin A-chain mutations and alanine scanning mutants in this region. While originally considered to be simply an activation remnant with little physiologic function, the thrombin A-chain is now thought to play a role as an allosteric effector in enzymatic reactions and may also be a structural scaffold to stabilize the protease domain. PMID:22084659

  15. Thrombin stimulates fibroblast procollagen production via proteolytic activation of protease-activated receptor 1.

    PubMed Central

    Chambers, R C; Dabbagh, K; McAnulty, R J; Gray, A J; Blanc-Brude, O P; Laurent, G J

    1998-01-01

    Thrombin is a multifunctional serine protease that has a crucial role in blood coagulation. It is also a potent mesenchymal cell mitogen and chemoattractant and might therefore have an important role in the recruitment and local proliferation of mesenchymal cells at sites of tissue injury. We hypothesized that thrombin might also affect the deposition of connective tissue proteins at these sites by directly stimulating fibroblast procollagen production. To address this hypothesis, the effect of thrombin on procollagen production and gene expression by human foetal lung fibroblasts was assessed over 48 h. Thrombin stimulated procollagen production at concentrations of 1 nM and above, with maximal increases of between 60% and 117% at 10 nM thrombin. These effects of thrombin were, at least in part, due to increased steady-state levels of alpha1(I) procollagen mRNA. They could furthermore be reproduced with thrombin receptor-activating peptides for the protease-activated receptor 1 (PAR-1) and were completely abolished when thrombin was rendered proteolytically inactive with the specific inhibitors d-Phe-Pro-ArgCH2Cl and hirudin, indicating that thrombin is mediating these effects via the proteolytic activation of PAR-1. These results suggest that thrombin might influence the deposition of connective tissue proteins during normal wound healing and the development of tissue fibrosis by stimulating fibroblast procollagen production. PMID:9639571

  16. Thrombin-receptor agonist peptides, in contrast to thrombin itself, are not full agonists for activation and signal transduction in human platelets in the absence of platelet-derived secondary mediators.

    PubMed Central

    Lau, L F; Pumiglia, K; Côté, Y P; Feinstein, M B

    1994-01-01

    are partial agonists for the thrombin receptor and produce incomplete receptor desensitization in keeping with their lower intrinsic activity; (2) thrombin's effects in platelets, even in TRP-desensitized platelets, are entirely mediated through the recently cloned G-protein linked receptor, and (3) thrombin's ability to produce sustained signals, compared with TRPs, may require the continued progressive proteolytic activation of naive thrombin receptors. Images Figure 3 PMID:7526841

  17. Metabolic Plasticity in Resting and Thrombin Activated Platelets

    PubMed Central

    Ravi, Saranya; Chacko, Balu; Sawada, Hirotaka; Kramer, Philip A.; Johnson, Michelle S.; Benavides, Gloria A.; O’Donnell, Valerie; Marques, Marisa B.; Darley-Usmar, Victor M.

    2015-01-01

    Platelet thrombus formation includes several integrated processes involving aggregation, secretion of granules, release of arachidonic acid and clot retraction, but it is not clear which metabolic fuels are required to support these events. We hypothesized that there is flexibility in the fuels that can be utilized to serve the energetic and metabolic needs for resting and thrombin-dependent platelet aggregation. Using platelets from healthy human donors, we found that there was a rapid thrombin-dependent increase in oxidative phosphorylation which required both glutamine and fatty acids but not glucose. Inhibition of fatty acid oxidation or glutamine utilization could be compensated for by increased glycolytic flux. No evidence for significant mitochondrial dysfunction was found, and ATP/ADP ratios were maintained following the addition of thrombin, indicating the presence of functional and active mitochondrial oxidative phosphorylation during the early stages of aggregation. Interestingly, inhibition of fatty acid oxidation and glutaminolysis alone or in combination is not sufficient to prevent platelet aggregation, due to compensation from glycolysis, whereas inhibitors of glycolysis inhibited aggregation approximately 50%. The combined effects of inhibitors of glycolysis and oxidative phosphorylation were synergistic in the inhibition of platelet aggregation. In summary, both glycolysis and oxidative phosphorylation contribute to platelet metabolism in the resting and activated state, with fatty acid oxidation and to a smaller extent glutaminolysis contributing to the increased energy demand. PMID:25875958

  18. A novel histochemical method for the visualization of thrombin activity in the nervous system.

    PubMed

    Bushi, D; Gera, O; Kostenich, G; Shavit-Stein, E; Weiss, R; Chapman, J; Tanne, D

    2016-04-21

    Although thrombin has an important role in both central and peripheral nerve diseases, characterization of the anatomical distribution of its proteolytic activity has been limited by available methods. This study presents the development, challenges, validation and implementation of a novel histochemical method for visualization of thrombin activity in the nervous system. The method is based on the cleavage of the substrate, Boc-Asp(OBzl)-Pro-Arg-4MβNA by thrombin to liberate free 4-methoxy-2-naphthylamine (4MβNA). In the presence of 5-nitrosalicylaldehyde, free 4MβNA is captured, yielding an insoluble yellow fluorescent precipitate which marks the site of thrombin activity. The sensitivity of the method was determined in vitro using known concentrations of thrombin while the specificity was verified using a highly specific thrombin inhibitor. Using this method we determined the spatial distribution of thrombin activity in mouse brain following transient middle cerebral artery occlusion (tMCAo) and in mouse sciatic nerve following crush injury. Fluorescence microscopy revealed well-defined thrombin activity localized to the right ischemic hemisphere in cortical areas and in the striatum compared to negligible thrombin activity contralaterally. The histochemical localization of thrombin activity following tMCAo was in good correlation with the infarct areas per triphenyltetrazolium chloride staining and to thrombin activity measured biochemically in tissue punches (85 ± 35 and 20 ± 3 mU/ml, in the cortical and striatum areas respectively, compared to 7 ± 2 and 13 ± 2 mU/ml, in the corresponding contralateral areas; mean ± SEM; p<0.05). In addition, 24 h following crush injury, focal areas of highly elevated thrombin activity were detected in teased sciatic fibers. This observation was supported by the biochemical assay and western blot technique. The histochemical method developed in this study can serve as an important tool for studying the role of thrombin

  19. Real Colorimetric Thrombin Aptasensor by Masking Surfaces of Catalytically Active Gold Nanoparticles.

    PubMed

    Chen, Zhengbo; Tan, Lulu; Hu, Liangyu; Zhang, Yimeng; Wang, Shaoxiong; Lv, Fanyi

    2016-01-13

    We presented a simple, cost-effective, and ultrasensitive colorimetric approach for visually detecting thrombin by the catalytic amplification of gold nanoparticles (AuNPs) and aptamer-thrombin recognition. Thrombin can be quantified in the presence of catalytic AuNP surface by using color-change time of 4-nitrophenol. Without thrombin, yellow 4-nitrophenol can freely access the surface of AuNP and becomes colorless 4-aminophenol. With the addition of thrombin, aptamer-thrombin with large size interaction masks the partial surfaces of AuNPs, and increases the reduction time of 4-nitrophenol to 4-aminophenol. The maximum number of bound thrombin fully mask the catalytic AuNP surface, and thus 4-nitrophenol cannot approach to AuNP surface, the color of the solution remains yellow. The limit of detection (LOD) of 0.1 nM can be achieved with naked eyes. Of note, the method was further applied for the detection of thrombin in human serum samples, showing the results in agreement with those values obtained in an immobilization buffer by the colorimetric method. PMID:26558607

  20. Mechanism of the Anticoagulant Activity of Thrombin Mutant W215A/E217A

    SciTech Connect

    Gandhi, Prafull S.; Page, Michael J.; Chen, Zhiwei; Bush-Pelc, Leslie; Di Cera, Enrico

    2009-09-15

    The thrombin mutant W215A/E217A (WE) is a potent anticoagulant both in vitro and in vivo. Previous x-ray structural studies have shown that WE assumes a partially collapsed conformation that is similar to the inactive E* form, which explains its drastically reduced activity toward substrate. Whether this collapsed conformation is genuine, rather than the result of crystal packing or the mutation introduced in the critical 215-217 {beta}-strand, and whether binding of thrombomodulin to exosite I can allosterically shift the E* form to the active E form to restore activity toward protein C are issues of considerable mechanistic importance to improve the design of an anticoagulant thrombin mutant for therapeutic applications. Here we present four crystal structures of WE in the human and murine forms that confirm the collapsed conformation reported previously under different experimental conditions and crystal packing. We also present structures of human and murine WE bound to exosite I with a fragment of the platelet receptor PAR1, which is unable to shift WE to the E form. These structural findings, along with kinetic and calorimetry data, indicate that WE is strongly stabilized in the E* form and explain why binding of ligands to exosite I has only a modest effect on the E*-E equilibrium for this mutant. The E* {yields} E transition requires the combined binding of thrombomodulin and protein C and restores activity of the mutant WE in the anticoagulant pathway.

  1. Thrombin-induced conversion of fibrinogen to fibrin results in rapid platelet trapping which is not dependent on platelet activation or GPIb

    PubMed Central

    Jarvis, Gavin E; Atkinson, Ben T; Frampton, Jon; Watson, Steve P

    2003-01-01

    Activation of human platelets by thrombin is mediated by the proteolytic cleavage of two G-protein coupled protease-activated receptors, PAR-1 and PAR-4. However, thrombin also binds specifically to the platelet surface glycoprotein GPIb. It has been claimed that thrombin can induce aggregation of platelets via a novel GPIb-mediated pathway, which is independent of PAR activation and fibrinogen binding to αIIbβ3 integrin, but dependent upon polymerizing fibrin and the generation of intracellular signals. In the presence of both fibrinogen and the αIIbβ3 receptor antagonist lotrafiban, thrombin induced a biphasic platelet aggregation response. The initial primary response was small but consistent and associated with the release of platelet granules. The delayed secondary response was more substantial and was abolished by the fibrin polymerization blocking peptide GPRP. Cleavage of the extracellular portion of GPIb by mocarhagin partially inhibited thrombin-induced αIIbβ3-dependent aggregation and release, but had no effect on the secondary fibrin-dependent response. Fixing of the platelets abolished αIIbβ3-dependent aggregation and release of adenine nucleotides, whereas the fibrin-dependent response remained, indicating that platelet activation and intracellular signalling are not necessary for this secondary ‘aggregation'. In conclusion, the secondary fibrin-dependent ‘aggregation' response observed in the presence of fibrinogen and lotrafiban is a platelet trapping phenomenon dependent primarily on the conversion of soluble fibrinogen to polymerizing fibrin by thrombin. PMID:12598411

  2. Signaling-mediated cooperativity between glycoprotein Ib-IX and protease-activated receptors in thrombin-induced platelet activation.

    PubMed

    Estevez, Brian; Kim, Kyungho; Delaney, M Keegan; Stojanovic-Terpo, Aleksandra; Shen, Bo; Ruan, Changgeng; Cho, Jaehyung; Ruggeri, Zaverio M; Du, Xiaoping

    2016-02-01

    Thrombin-induced cellular response in platelets not only requires protease-activated receptors (PARs), but also involves another thrombin receptor, the glycoprotein Ib-IX complex (GPIb-IX). It remains controversial how thrombin binding to GPIb-IX stimulates platelet responses. It was proposed that GPIb-IX serves as a dock that facilitates thrombin cleavage of protease-activated receptors, but there are also reports suggesting that thrombin binding to GPIb-IX induces platelet activation independent of PARs. Here we show that GPIb is neither a passive thrombin dock nor a PAR-independent signaling receptor. We demonstrate a novel signaling-mediated cooperativity between PARs and GPIb-IX. Low-dose thrombin-induced PAR-dependent cell responses require the cooperativity of GPIb-IX signaling, and conversely, thrombin-induced GPIb-IX signaling requires cooperativity of PARs. This mutually dependent cooperativity requires a GPIb-IX-specific 14-3-3-Rac1-LIMK1 signaling pathway, and activation of this pathway also requires PAR signaling. The cooperativity between GPIb-IX signaling and PAR signaling thus drives platelet activation at low concentrations of thrombin, which are important for in vivo thrombosis. PMID:26585954

  3. A peptide with a cysteine terminus: probe for label-free fluorescent detection of thrombin activity.

    PubMed

    Feng, Jingjing; Zhuo, Caixia; Ma, Xuejuan; Li, Shuangqin; Zhang, Yaodong

    2016-07-21

    Thrombin has been implicated in atherosclerotic disease development. However, thrombin activity detection is currently limited because of the lack of convenient fluorescent probes. We developed a label-free fluorescent method to assay thrombin activity on the basis of a designed peptide probe with a thrombin-cleavable peptide sequence and a cysteine terminus. The peptide probe can be conjugated to DNA-templated silver nanoclusters (DNA-AgNCs) through Ag-S bonding; as a result, the fluorescence of DNA-AgNCs was enhanced. As the DNA-AgNCs-peptide conjugate was adsorbed to graphene oxide (GO), the enhanced fluorescence of DNA-AgNCs was quenched. Once the peptide probe was cleaved by thrombin, the resulting release of the DNA-AgNCs from the surface of GO restored the enhanced fluorescence. Thrombin can be determined with a linear range of 0.0-50.0 nM with a detection limit of 1 nM. The thrombin-sensitive probe with a cysteine terminus may be developed into probes to detect other proteases. PMID:27187619

  4. Fibrinogen blocks the autoactivation and thrombin-mediated activation of factor XI on dextran sulfate.

    PubMed Central

    Scott, C F; Colman, R W

    1992-01-01

    The intrinsic pathway of blood coagulation is activated when factor XIa, one of the three contact-system enzymes, is generated and then activates factor IX. Factor XI has been shown to be efficiently activated in vitro by surface-bound factor XIIa after factor XI is transported to the surface by its cofactor, high molecular weight kininogen (HK). However, individuals lacking any of the three contact-system proteins--namely, factor XII, prekallikrein, and HK--do not suffer from bleeding abnormalities. This mystery has led several investigators to search for an "alternate" activation pathway for factor XI. Recently, factor XI has been reported to be autoactivated on the soluble "surface" dextran sulfate, and thrombin was shown to accelerate the autoactivation. However, it was also reported that HK, the cofactor for factor XIIa-mediated activation of factor XI, actually diminishes the thrombin-catalyzed activation rate of factor XI. Nonetheless, it was suggested that thrombin was a more efficient activator than factor XIIa. In this report we investigated the effect of fibrinogen, the major coagulation protein in plasma, on the activation rate of factor XI. Fibrinogen, the preferred substrate for thrombin in plasma, virtually prevented autoactivation of factor XI as well as the thrombin-mediated activation of factor XI, while having no effect on factor XIIa-catalyzed activation. HK dramatically curtailed the autoactivation of factor XI in addition to the thrombin-mediated activation. These data indicate that factor XI would not be autoactivated in a plasma environment, and thrombin would, therefore, be unlikely to potentiate the activation. We believe that the "missing pathway" for factor XI activation remains an enigma that warrants further investigation. PMID:1454798

  5. A direct thrombin inhibitor suppresses protein C activation and factor Va degradation in human plasma: Possible mechanisms of paradoxical enhancement of thrombin generation.

    PubMed

    Kamisato, Chikako; Furugohri, Taketoshi; Morishima, Yoshiyuki

    2016-05-01

    We have demonstrated that antithrombin (AT)-independent thrombin inhibitors paradoxically increase thrombin generation (TG) in human plasma in a thrombomodulin (TM)- and protein C (PC)-dependent manner. We determined the effects of AT-independent thrombin inhibitors on the negative-feedback system, activation of PC and production and degradation of factor Va (FVa), as possible mechanisms underlying the paradoxical enhancement of TG. TG in human plasma containing 10nM TM was assayed by means of the calibrated automated thrombography. As an index of PC activation, plasma concentration of activated PC-PC inhibitor complex (aPC-PCI) was measured. The amounts of FVa heavy chain and its degradation product (FVa(307-506)) were examined by western blotting. AT-independent thrombin inhibitors, melagatran and dabigatran (both at 25-600nM) and 3-30μg/ml active site-blocked thrombin (IIai), increased peak levels of TG. Melagatran, dabigatran and IIai significantly decreased plasma concentration of aPC-PCI complex at 25nM or more, 75nM or more, and 10 and 30μg/ml, respectively. Melagatran (300nM) significantly increased FVa and decreased FVa(307-506). In contrast, a direct factor Xa inhibitor edoxaban preferentially inhibited thrombin generation (≥25nM), and higher concentrations were required to inhibit PC activation (≥150nM) and FVa degradation (300nM). The present study suggests that the inhibitions of protein C activation and subsequent degradation of FVa and increase in FVa by antithrombin-independent thrombin inhibitors may contribute to the paradoxical TG enhancement, and edoxaban may inhibit PC activation and FVa degradation as a result of TG suppression. PMID:26974491

  6. Membrane lipid peroxidation in neurodegeneration: Role of thrombin and proteinase-activated receptor-1.

    PubMed

    Citron, Bruce A; Ameenuddin, Syed; Uchida, K; Suo, William Z; SantaCruz, Karen; Festoff, Barry W

    2016-07-15

    Thrombin and membrane lipid peroxidation (MLP) have been implicated in various central nervous system (CNS) disorders from CNS trauma to stroke, Alzheimer's (AD) and Parkinson's (PD) diseases. Because thrombin also induces MLP in platelets and its involvement in neurodegenerative diseases we hypothesized that its deleterious effects might, in part, involve formation of MLP in neuronal cells. We previously showed that thrombin induced caspase-3 mediated apoptosis in motor neurons, via a proteinase-activated receptor (PAR1). We have now investigated thrombin's influence on the oxidative state of neurons leading to induction of MLP-protein adducts. Translational relevance of thrombin-induced MLP is supported by increased levels of 4-hydroxynonenal-protein adducts (HNEPA) in AD and PD brains. We now report for the first time that thrombin dose-dependently induces formation of HNEPA in NSC34 mouse motor neuron cells using anti-HNE and anti-acrolein monoclonal antibodies. The most prominent immunoreactive band, in SDS-PAGE, was at ∼54kDa. Membrane fractions displayed higher amounts of the protein-adduct than cytosolic fractions. Thrombin induced MLP was mediated, at least in part, through PAR1 since a PAR1 active peptide, PAR1AP, also elevated HNEPA levels. Of interest, glutamate and Fe2SO4 also increased the ∼54kDa HNEPA band in these cells but to a lesser extent. Taken together our results implicate the involvement of thrombin and MLP in neuronal cell loss observed in various CNS degenerative and traumatic pathologies. PMID:27138068

  7. Interaction of hirudin with thrombin: Identification of a minimal binding domain of hirudin that inhibits clotting activity

    SciTech Connect

    Mao, S.J.T.; Yates, M.T.; Owen, T.J.; Krstenansky, J.L. )

    1988-10-18

    Hirudin, isolated from the European leech Hirudo medicinalis, is a potent inhibitor of thrombin, forming an almost irreversible thrombin-hirudin complex. Previously, the authors have shown that the carboxyl terminus of hirudin (residues 45-65) inhibits clotting activity and without binding to the catalytic site of thrombin. In the present study, a series of peptides corresponding to this carboxyl-terminal region of hirudin have been synthesized, and their anticoagulant activity and binding properties to thrombin were examined. Binding was assessed by their ability to displace {sup 125}I-hirudin 45-65 from Sepharose-immobilized thrombin and by isolation of peptide-thrombin complexes. They show that the carboxyl-terminal 10 amino acid residues 56-65 (Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln) are minimally required for binding to thrombin and inhibition of clotting. Phe-56 was critical for maintaining anticoagulant activity as demonstrated by the loss of activity when Phe-56 was substituted with D-Phe, Glu, or Leu. In addition, they found that the binding of the carboxyl-terminal peptide of hirudin with thrombin was associated with a significant conformational change of thrombin as judged by circular dichroism. This conformational change might be responsible for the loss of clotting activity of thrombin.

  8. Thrombin stimulates albumin transcytosis in lung microvascular endothelial cells via activation of acid sphingomyelinase.

    PubMed

    Kuebler, Wolfgang M; Wittenberg, Claudia; Lee, Warren L; Reppien, Eike; Goldenberg, Neil M; Lindner, Karsten; Gao, Yizhuo; Winoto-Morbach, Supandi; Drab, Marek; Mühlfeld, Christian; Dombrowsky, Heike; Ochs, Matthias; Schütze, Stefan; Uhlig, Stefan

    2016-04-15

    Transcellular albumin transport occurs via caveolae that are abundant in lung microvascular endothelial cells. Stimulation of albumin transcytosis by proinflammatory mediators may contribute to alveolar protein leak in lung injury, yet the regulation of albumin transport and its underlying molecular mechanisms are so far incompletely understood. Here we tested the hypothesis that thrombin may stimulate transcellular albumin transport across lung microvascular endothelial cells in an acid-sphingomyelinase dependent manner. Thrombin increased the transport of fluorescently labeled albumin across confluent human lung microvascular endothelial cell (HMVEC-L) monolayers to an extent that markedly exceeds the rate of passive diffusion. Thrombin activated acid sphingomyelinase (ASM) and increased ceramide production in HMVEC-L, but not in bovine pulmonary artery cells, which showed little albumin transport in response to thrombin. Thrombin increased total caveolin-1 (cav-1) content in both whole cell lysates and lipid rafts from HMVEC-L, and this effect was blocked by inhibition of ASM or de novo protein biosynthesis. Thrombin-induced uptake of albumin into lung microvascular endothelial cells was confirmed in isolated-perfused lungs by real-time fluorescence imaging and electron microscopy of gold-labeled albumin. Inhibition of ASM attenuated thrombin-induced albumin transport both in confluent HMVEC-L and in intact lungs, whereas HMVEC-L treatment with exogenous ASM increased albumin transport and enriched lipid rafts in cav-1. Our findings indicate that thrombin stimulates transcellular albumin transport in an acid sphingomyelinase-dependent manner by inducing de novo synthesis of cav-1 and its recruitment to membrane lipid rafts. PMID:26851257

  9. Comparison of laser-activated tissue solders and thrombin-activated cryoprecipitate for wound closure

    NASA Astrophysics Data System (ADS)

    Kayton, Mark L.; Libutti, Steven K.; Bessler, Marc; Allendorf, John D. F.; Eiref, Simon D.; Marx, Gerard; Mou, Xiaode; Morales, Alfredo M.; Treat, Michael R.; Nowygrod, Roman

    1994-09-01

    To determine the relative strengths of various biologic adhesives at several timepoints, we compared thrombin-activated SD (solvent-detergent treated) cryoprecipitate with laser- activated SD cryoprecipitate and a laser-activated, albumin-based glue. Male Sprague-Dawley rats (n equals 79) received four, 3-cm, dorsal skin incisions which were closed with either laser- activated cryoprecipitate, laser-activated albumin solder, thrombin-activated cryoprecipitate, or standard skin staples. The cryoprecipitate was derived from pooled human plasma and was treated with a solvent-detergent process, rendering it free of envelope-coated viruses (i.e., HBV, HIV). An 808-nm diode laser was used to activate each solder with an average duration of exposure of 75 seconds per incision. Animals were sacrificed for evaluation of wound tensile strength and histology at 0 hours, 2 hours, 4 hours, and 4 days. At all timepoints tested, laser-activated solders were significantly stronger than thrombin-activated cryoprecipitate (p < 0.03) and control wounds (p < 0.003). There was no significant difference in tensile strength between the two types of laser-activated solder at any timepoint.

  10. Purification and variability in thrombin-like activity of Bothrops atrox venom from different geographic regions.

    PubMed

    Cavinato, R A; Remold, H; Kipnis, T L

    1998-02-01

    Bothrops atrox snake venoms from two different Amazon regions, i.e., Manaus, AM (3 degrees 0.6'40"S; 60 degrees 0.1'6.0"W) and Tucurui, PA (3 degrees 0.42'30"S; 49 degrees 0.41'45"W), were analyzed with respect to the thrombin-like activity component by elution profile on gel-filtration and reverse phase HPLC chromatography, electrophoretic mobility on SDS-PAGE, and enzymatic activity on fibrinogen. Despite some individual discrepancies among venom specimens, the thrombin-like activity present in the Manaus pool was eluted earlier compared with the Tucurui pool but its enzymatic specific activity on thrombin was lower (s.a. = 6.0) than that observed in the Tucurui pool (s.a. = 134.0). However, the electrophoretic mobilities of the pools were similar, with most protein bands being concentrated around three main regions, i.e., protein bands with an apparent mr of 100 kDa, of 38-37 kDa and 30 kDa. However, no significant differences were observed in amidolytic activity on the synthetic substrate Tos-Gly-Pro-Arg-pNa, and there was no correlation between thrombin-like and amidolytic activities. A 32 kDa protein endowed with thrombin-like activity and specific activity of 2444 recognized and neutralized by horse anti-B. atrox antivenom, was purified by the successive use of gel filtration, electrofocusing and reverse phase HPLC. PMID:9620574

  11. Leptospira interrogans reduces fibrin clot formation by modulating human thrombin activity via exosite I.

    PubMed

    Fernandes, Luis G; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

    2015-06-01

    Pathogenic bacteria of the genus Leptospira are the etiological agents of leptospirosis, a disease that affects humans and animals worldwide. Although there are an increasing number of studies on the biology of Leptospira, the mechanisms of pathogenesis are not yet understood. We report in this work that Leptospira interrogans FIOCRUZ L1-130 virulent, M20 culture attenuated and the saprophyte L. biflexa Patoc 1 strains do not bind prothrombin. Leptospiral binding to thrombin was detected with the virulent, followed by culture-attenuated M20, and practically none was observed with the saprophyte strain. The interaction of Leptospira with thrombin mostly occurs via exosite I, with a minor participation of catalytic site, as determined by employing the thrombin inhibitors hirugen, hirudin and argatroban. Leptospira interrogans binding to thrombin inhibits its catalytic activity reducing fibrin clot formation in thrombin-catalyzed reaction of fibrinogen. This inhibition was more efficient with the virulent FIOCRUZ L1-130 than with the M20 culture attenuated, while none was seen with the saprophyte strain, suggesting that this binding might be important for bacterial virulence. This is the first study reporting the binding of pathogenic Leptospira to thrombin promoting a decrease in fibrin clotting that could lead to hemorrhage, helping bacteria dissemination. PMID:25834144

  12. Calcium inhibition of the activation of protein C by thrombin. Role of the P3 and P3' residues.

    PubMed

    Rezaie, A R; Esmon, C T

    1994-07-15

    Protein C, a precursor to a natural plasma anticoagulant, and the platelet thrombin receptor, involved in cell activation, both require proteolytic cleavage to be activated. In humans, the sequences adjacent to the scissile bond of protein C, DPR/LID and the thrombin receptor DPR/SFL are similar. Previous studies with Asp-->Gly mutants indicated that both the P3 and the P3' Asp residues make either peptides or protein C a poor substrate for free thrombin, but thrombin interaction with thrombomodulin overcomes these inhibitory interactions. Similar mechanisms are probably operative in the thrombin receptor. In rodents, the P3 Asp residue of the human thrombin receptor is replaced by Asn and in protein C, the P3' residue is Asn. To determine the functional significance of these Asp-->Asn substitutions, the Asp in the P3 or P3' position of human protein C was changed to Asn. The resultant mutants were still resistant to activation by thrombin, and still required Ca2+ for activation by thrombin-thrombomodulin complex. We conclude that, unlike activation of the Asp-->Gly mutants by thrombin, activation of the P3 and P3' Asp-->Asn mutants is still potently inhibited by physiological Ca2+. Furthermore, even though the charge has been deleted, thrombomodulin acceleration is retained. PMID:8055928

  13. Differential effects on glial activation by a direct versus an indirect thrombin inhibitor.

    PubMed

    Marangoni, M Natalia; Braun, David; Situ, Annie; Moyano, Ana L; Kalinin, Sergey; Polak, Paul; Givogri, Maria I; Feinstein, Douglas L

    2016-08-15

    Thrombin is a potent regulator of brain function in health and disease, modulating glial activation and brain inflammation. Thrombin inhibitors, several of which are in clinical use as anti-coagulants, can reduce thrombin-dependent neuroinflammation in pathological conditions. However, their effects in a healthy CNS are largely unknown. In adult healthy mice, we compared the effects of treatment by the direct thrombin inhibitor dabigatran etexilate (DE), to those of warfarin, which acts by preventing vitamin K recycling essential for coagulation. After 4weeks, warfarin increased both astrocyte GFAP and microglia Iba-1 staining throughout the CNS; whereas DE reduced expression of both markers. Warfarin, but not DE, reduced sulfatide levels; and warfarin showed longer lasting changes in cerebellar gene expression. DE also reduced glial activation in a mouse model of Alzheimer's disease, although no changes in amyloid plaque burden were observed. These results suggest that treatment with direct thrombin inhibitors may be preferable to those agents which reduce vitamin K levels and have the potential to increase glial activation. PMID:27397090

  14. Antibodies against thrombin in dengue patients contain both anti-thrombotic and pro-fibrinolytic activities.

    PubMed

    Chuang, Yung-Chun; Lin, Yee-Shin; Liu, Hsiao-Sheng; Wang, Jen-Reng; Yeh, Trai-Ming

    2013-08-01

    Dengue virus (DENV) infection may result in severe life-threatening Dengue haemorrhagic fever (DHF). The mechanisms causing haemorrhage in those with DHF are unclear. In this study, we demonstrated that antibodies against human thrombin were increased in the sera of Dengue patients but not in that of patients infected with other viruses. To further characterise the properties of these antibodies, affinity-purified anti-thrombin antibodies (ATAs) were collected from Dengue patient sera by thrombin and protein A/L affinity columns. Most of the ATAs belonged to the IgG class and recognized DENV nonstructural protein 1 (NS1). In addition, we found that dengue patient ATAs also cross-reacted with human plasminogen (Plg). Functional studies in vitro indicated that Dengue patient ATAs could inhibit thrombin activity and enhance Plg activation. Taken together, these results suggest that DENV NS1-induced thrombin and Plg cross-reactive antibodies may contribute to the development of haemorrhage in patients with DHF by interfering with coagulation and fibrinolysis. PMID:23740201

  15. A balance between TFPI and thrombin-mediated platelet activation is required for murine embryonic development

    PubMed Central

    Ellery, Paul E. R.; Maroney, Susan A.; Cooley, Brian C.; Luyendyk, James P.; Zogg, Mark; Weiler, Hartmut

    2015-01-01

    Tissue factor pathway inhibitor (TFPI) is a critical anticoagulant protein present in endothelium and platelets. Mice lacking TFPI (Tfpi−/−) die in utero from disseminated intravascular coagulation. They are rescued by concomitant tissue factor (TF) deficiency, demonstrating that TFPI modulates TF function in vivo. Recent studies have found TFPI inhibits prothrombinase activity during the initiation of coagulation and limits platelet accumulation during thrombus formation, implicating TFPI in modulating platelet procoagulant activity. To examine whether altered platelet function would compensate for the lack of TFPI and rescue TFPI-null embryonic lethality, Tfpi+/− mice lacking the platelet thrombin receptor, protease activated receptor 4 (PAR4; Par4−/−), or its coreceptor, PAR3, were mated. PAR3 deficiency did not rescue Tfpi−/− embryos, but >40% of expected Tfpi−/−:Par4−/− offspring survived to adulthood. Adult Tfpi−/−:Par4−/− mice did not exhibit overt thrombosis. However, they had focal sterile inflammation with fibrin(ogen) deposition in the liver and elevated plasma thrombin-antithrombin complexes, indicating activation of coagulation at baseline. Tfpi−/−:Par4−/− mice have platelet and fibrin accumulation similar to Par4−/− mice following venous electrolytic injury but were more susceptible than Par4−/− mice to TF-induced pulmonary embolism. In addition, ∼30% of the Tfpi−/−:Par4−/− mice were born with short tails. Tfpi−/−:Par4−/− mice are the first adult mice described that lack TFPI with unaltered TF. They demonstrate that TFPI physiologically modulates thrombin-dependent platelet activation in a manner that is required for successful embryonic development and identify a role for TFPI in dampening intravascular procoagulant stimuli that lead to thrombin generation, even in the absence of thrombin-mediated platelet activation. PMID:25954015

  16. Granzyme A released upon stimulation of cytotoxic T lymphocytes activates the thrombin receptor on neuronal cells and astrocytes.

    PubMed Central

    Suidan, H S; Bouvier, J; Schaerer, E; Stone, S R; Monard, D; Tschopp, J

    1994-01-01

    Granzymes are a family of serine proteases that are harbored in cytoplasmic granules of activated T lymphocytes and are released upon target cell interaction. Immediate and complete neurite retraction was induced in a mouse neuronal cell line when total extracts of granule proteins were added. This activity was isolated and identified as granzyme A. This protease not only induced neurite retraction at nanomolar concentrations but also reversed the stellation of astrocytes. Both effects were critically dependent on the esterolytic activity of granzyme A. As neurite retraction is known to be induced by thrombin, possible cleavage and activation of the thrombin receptor were investigated. A synthetic peptide spanning the N-terminal thrombin receptor activation sequence was cleaved by granzyme A at the authentic thrombin cleavage site Leu-Asp-Pro-Arg-Ser. Antibodies to the thrombin receptor inhibited both thrombin and granzyme A-mediated neurite retraction. Thus, T-cell-released granzyme A induces cellular responses by activation of the thrombin receptor. As brain-infiltrating CD4+ lymphocytes are the effector cells in experimental allergic encephalomyelitis, granzyme A released in the brain may contribute to the etiology of autoimmune disorders in the nervous system. Images PMID:8058766

  17. Thrombin activation and liver inflammation in advanced hepatitis C virus infection

    PubMed Central

    González-Reimers, Emilio; Quintero-Platt, Geraldine; Martín-González, Candelaria; Pérez-Hernández, Onán; Romero-Acevedo, Lucía; Santolaria-Fernández, Francisco

    2016-01-01

    Hepatitis C virus (HCV) infection is associated with increased thrombotic risk. Several mechanisms are involved including direct endothelial damage by the HCV virus, with activation of tissue factor, altered fibrinolysis and increased platelet aggregation and activation. In advanced stages, chronic HCV infection may evolve to liver cirrhosis, a condition in which alterations in the portal microcirculation may also ultimately lead to thrombin activation, platelet aggregation, and clot formation. Therefore in advanced HCV liver disease there is an increased prevalence of thrombotic phenomena in portal vein radicles. Increased thrombin formation may activate hepatic stellate cells and promote liver fibrosis. In addition, ischemic changes derived from vascular occlusion by microthrombi favor the so called parenchymal extinction, a process that promotes collapse of hepatocytes and the formation of gross fibrous tracts. These reasons may explain why advanced HCV infection may evolve more rapidly to end-stage liver disease than other forms of cirrhosis. PMID:27182154

  18. Thrombin activation and liver inflammation in advanced hepatitis C virus infection.

    PubMed

    González-Reimers, Emilio; Quintero-Platt, Geraldine; Martín-González, Candelaria; Pérez-Hernández, Onán; Romero-Acevedo, Lucía; Santolaria-Fernández, Francisco

    2016-05-14

    Hepatitis C virus (HCV) infection is associated with increased thrombotic risk. Several mechanisms are involved including direct endothelial damage by the HCV virus, with activation of tissue factor, altered fibrinolysis and increased platelet aggregation and activation. In advanced stages, chronic HCV infection may evolve to liver cirrhosis, a condition in which alterations in the portal microcirculation may also ultimately lead to thrombin activation, platelet aggregation, and clot formation. Therefore in advanced HCV liver disease there is an increased prevalence of thrombotic phenomena in portal vein radicles. Increased thrombin formation may activate hepatic stellate cells and promote liver fibrosis. In addition, ischemic changes derived from vascular occlusion by microthrombi favor the so called parenchymal extinction, a process that promotes collapse of hepatocytes and the formation of gross fibrous tracts. These reasons may explain why advanced HCV infection may evolve more rapidly to end-stage liver disease than other forms of cirrhosis. PMID:27182154

  19. Mutant N143P Reveals How Na[superscript +] Activates Thrombin

    SciTech Connect

    Niu, Weiling; Chen, Zhiwei; Bush-Pelc, Leslie A.; Bah, Alaji; Gandhi, Prafull S.; Di Cera, Enrico

    2010-01-12

    The molecular mechanism of thrombin activation by Na{sup +} remains elusive. Its kinetic formulation requires extension of the classical Botts-Morales theory for the action of a modifier on an enzyme to correctly account for the contribution of the E*, E, and E:Na{sup +} forms. The extended scheme establishes that analysis of k{sub cat} unequivocally identifies allosteric transduction of Na{sup +} binding into enhanced catalytic activity. The thrombin mutant N143P features no Na{sup +}-dependent enhancement of k{sub cat} yet binds Na{sup +} with an affinity comparable to that of wild type. Crystal structures of the mutant in the presence and absence of Na{sup +} confirm that Pro{sup 143} abrogates the important H-bond between the backbone N atom of residue 143 and the carbonyl O atom of Glu{sup 192}, which in turn controls the orientation of the Glu{sup 192}-Gly{sup 193} peptide bond and the correct architecture of the oxyanion hole. We conclude that Na{sup +} activates thrombin by securing the correct orientation of the Glu{sup 192}-Gly{sup 193} peptide bond, which is likely flipped in the absence of cation. Absolute conservation of the 143-192 H-bond in trypsin-like proteases and the importance of the oxyanion hole in protease function suggest that this mechanism of Na{sup +} activation is present in all Na{sup +}-activated trypsin-like proteases.

  20. Identification of small peptide analogues having agonist and antagonist activity at the platelet thrombin receptor.

    PubMed

    Ruda, E M; Petty, A; Scrutton, M C; Tuffin, D P; Manley, P W

    1988-06-15

    phosphatidylethanolamine. SC40476 causes no detectable hydrolysis of glycoprotein V as detected by release of the proteolytic product (glycoprotein VFR). The results indicate that SC40476 and SC42619 interact selectively with the platelet thrombin receptor. Both peptide analogues act as effective antagonists for this receptor but also possess weak agonist activity which may also result from interaction with the thrombin receptor. The molecular basis for this latter activity has not been defined. SC42619 non-selectively inhibits Ca2+ influx induced by several agonists but this effect does not appear to contribute to the observed inhibition of the aggregatory and secretory responses. PMID:2839193

  1. Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper.

    PubMed

    Pérez, A V; Rucavado, A; Sanz, L; Calvete, J J; Gutiérrez, J M

    2008-01-01

    A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13% of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 microg) and fibrinogen (minimum coagulant dose = 4.2 microg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 microg). In addition, when injected intravenously in mice at doses of 5 and 10 microg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the ;gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms. PMID:17994164

  2. Evidence for the presence of a protease-activated receptor distinct from the thrombin receptor in human keratinocytes.

    PubMed Central

    Santulli, R J; Derian, C K; Darrow, A L; Tomko, K A; Eckardt, A J; Seiberg, M; Scarborough, R M; Andrade-Gordon, P

    1995-01-01

    Thrombin receptor activation was explored in human epidermal keratinocytes and human dermal fibroblasts, cells that are actively involved in skin tissue repair. The effects of thrombin, trypsin, and the receptor agonist peptides SFLLRN and TFRIFD were assessed in inositolphospholipid hydrolysis and calcium mobilization studies. Thrombin and SFLLRN stimulated fibroblasts in both assays to a similar extent, whereas TFRIFD was less potent. Trypsin demonstrated weak efficacy in these assays in comparison with thrombin. Results in fibroblasts were consistent with human platelet thrombin receptor activation. Keratinocytes, however, exhibited a distinct profile, with trypsin being a far better activator of inositolphospholipid hydrolysis and calcium mobilization than thrombin. Furthermore, SFLLRN was more efficacious than thrombin, whereas no response was observed with TFRIFD. Since our data indicated that keratinocytes possess a trypsin-sensitive receptor, we addressed the possibility that these cells express the human homologue of the newly described murine protease-activated receptor, PAR-2 [Nystedt, S., Emilsson, K., Wahlestedt, C. & Sundelin, J. (1994) Proc. Natl. Acad. Sci. USA 91, 9208-9212]. PAR-2 is activated by nanomolar concentrations of trypsin and possesses the tethered ligand sequence SLIGRL. SLIGRL was found to be equipotent with SFLLRN in activating keratinocyte inositolphospholipid hydrolysis and calcium mobilization. Desensitization studies indicated that SFLLRN, SLIGRL, and trypsin activate a common receptor, PAR-2. Northern blot analyses detected a transcript of PAR-2 in total RNA from keratinocytes but not fibroblasts. Levels of thrombin receptor message were equivalent in the two cell types. Our results indicate that human keratinocytes possess PAR-2, suggesting a potential role for this receptor in tissue repair and/or skin-related disorders. Images Fig. 6 PMID:7568091

  3. Biological activities of the peptides obtained by digestion of troponin C and calmodulin with thrombin.

    PubMed Central

    Wall, C M; Grand, R J; Perry, S V

    1981-01-01

    1. Troponin C and calmodulin were not digested by thrombin at a significant rate in the presence of Ca2+. 2. In the presence of EGTA, troponin C was digested by thrombin to yield three peptides, TH1 (residues 1--120), TH3 (residues 1--100) and TH2 (residues 121--159). 3. In the presence of EGTA calmodulin was digested by thrombin giving two peptides, TM1 (residues 1--106) and TM2 (residues 107--148). 4. The electrophoretic mobilities of peptides TH1 and TM1 were increased at pH 8.6 by Ca2+ both in the presence and absence of urea. The mobilities of peptides TH2 and TM2 were unaltered under these conditions. 5. Peptides TH1, TH2 and tM1 formed complexes with troponin I on polyacrylamide gels at pH 8.6 in the presence of Ca2+. 6. The phosphorylation of troponin I by cyclic AMP-dependent protein kinase was significantly inhibited by peptides TH1 and TH3 and to a lesser extent by peptide TM1. 7. The calmodulin peptide TM1 activated myosin light-chain kinase when present in large molar excess. Peptide TM2 did not activate the enzyme. Images Fig. 1. Fig. 4. PMID:6895466

  4. Differential proteolytic activation of factor VIII-von Willebrand factor complex by thrombin

    SciTech Connect

    Hill-Eubanks, D.C.; Parker, C.G.; Lollar, P. )

    1989-09-01

    Blood coagulation factor VIII (fVIII) is a plasma protein that is decreased or absent in hemophilia A. It is isolated as a mixture of heterodimers that contain a variably sized heavy chain and a common light chain. Thrombin catalyzes the activation of fVIII in a reaction that is associated with cleavages in both types of chain. The authors isolated a serine protease from Bothrops jararacussu snake venom that catalyzes thrombin-like heavy-chain cleavage but not light-chain cleavage in porcine fVIII as judged by NaDodSO{sub 4}/PAGE and N-terminal sequence analysis. Using a plasma-free assay of the ability of activated {sup 125}I-fVIII to function as a cofactor in the activation of factor X by factor IXa, they found that fVIII is activated by the venom enzyme. The venom enzyme-activated fVIII was isolated in stable form by cation-exchange HPLC. von Willebrand factor inhibited venom enzyme-activated fVIII but not thrombin-activated fVIII. These results suggest that the binding of fVIII to von Willebrand factor depends on the presence of an intact light chain and that activated fVIII must dissociate from von Willebrand factor to exert its cofactor effect. Thus, proteolytic activation of fVIII-von Willebrand factor complex appears to be differentially regulated by light-chain cleavage to dissociate the complex and heavy-chain cleavage to activate the cofactor function.

  5. Rapid Detection of Thrombin and Other Protease Activity Directly in Whole Blood

    NASA Astrophysics Data System (ADS)

    Yu, Johnson Chung Sing

    Thrombin is a serine protease that plays a key role in the clotting cascade to promote hemostasis following injury to the endothelium. From a clinical diagnostic perspective, in-vivo thrombin activity is linked to various blood clotting disorders, as well as cardiovascular disease (DVT, arteriosclerosis, etc). Thus, the ability to rapidly measure protease activity directly in whole blood will provide important new diagnostics, and clinical researchers with a powerful tool to further elucidate the relationship between circulating protease levels and disease. The ultimate goal is to design novel point of care (POC) diagnostic devices that are capable of monitoring protease activities directly in whole blood and biological sample. A charge-changing substrate specific to the thrombin enzyme was engineered and its functionality was confirmed by a series of experiments. This led to the preliminary design, construction, and testing of two device platforms deemed fully functional for the electrophoretic separation and focusing of charged peptide fragments. The concept of using the existing charge-changing substrate platform for bacterial protease detection was also investigated. Certain strains of E coli are associated with severe symptoms such as abdominal cramps, bloody diarrhea, and vomiting. The OmpT protease is expressed on the outer membrane of E coli and plays a role in the cleavage of antimicrobial peptides, the degradation of recombinant heterologous proteins, and the activation of plasminogen in the host. Thus, a synthetic peptide substrate specific to the OmpT protease was designed and modeled for the purpose of detecting E coli in biological sample.

  6. Phosphoproteomic Analysis of Platelets Activated by Pro-Thrombotic Oxidized Phospholipids and Thrombin

    PubMed Central

    Zimman, Alejandro; Titz, Bjoern; Komisopoulou, Evangelia; Biswas, Sudipta; Graeber, Thomas G.; Podrez, Eugene A.

    2014-01-01

    Specific oxidized phospholipids (oxPCCD36) promote platelet hyper-reactivity and thrombosis in hyperlipidemia via the scavenger receptor CD36, however the signaling pathway(s) induced in platelets by oxPCCD36 are not well defined. We have employed mass spectrometry-based tyrosine, serine, and threonine phosphoproteomics for the unbiased analysis of platelet signaling pathways induced by oxPCCD36 as well as by the strong physiological agonist thrombin. oxPCCD36 and thrombin induced differential phosphorylation of 115 proteins (162 phosphorylation sites) and 181 proteins (334 phosphorylation sites) respectively. Most of the phosphoproteome changes induced by either agonist have never been reported in platelets; thus they provide candidates in the study of platelet signaling. Bioinformatic analyses of protein phosphorylation dependent responses were used to categorize preferential motifs for (de)phosphorylation, predict pathways and kinase activity, and construct a phosphoproteome network regulating integrin activation. A putative signaling pathway involving Src-family kinases, SYK, and PLCγ2 was identified in platelets activated by oxPCCD36. Subsequent ex vivo studies in human platelets demonstrated that this pathway is downstream of the scavenger receptor CD36 and is critical for platelet activation by oxPCCD36. Our results provide multiple insights into the mechanism of platelet activation and specifically in platelet regulation by oxPCCD36. PMID:24400094

  7. A new peptide (Ruviprase) purified from the venom of Daboia russelii russelii shows potent anticoagulant activity via non-enzymatic inhibition of thrombin and factor Xa.

    PubMed

    Thakur, Rupamoni; Kumar, Ashok; Bose, Biplab; Panda, Dulal; Saikia, Debashree; Chattopadhyay, Pronobesh; Mukherjee, Ashis K

    2014-10-01

    Compounds showing dual inhibition of thrombin and factor Xa (FXa) are the subject of great interest owing to their broader specificity for effective anticoagulation therapy against cardiovascular disorders. This is the first report on the functional characterization and assessment of therapeutic potential of a 4423.6 Da inhibitory peptide (Ruviprase) purified from Daboia russelii russelii venom. The secondary structure of Ruviprase is composed of α-helices (61.9%) and random coils (38.1%). The partial N-terminal sequence (E(1)-V(2)-X(3)-W(4)-W(5)-W(6)-A(7)-Q(8)-L(9)-S(10)) of Ruviprase demonstrated significant similarity (80.0%) with an internal sequence of apoptosis-stimulating protein reported from the venom of Ophiophagus hannah and Python bivittatus; albeit Ruviprase did not show sequence similarity with existing thrombin/FXa inhibitors, suggesting its uniqueness. Ruviprase demonstrated a potent in vitro anticoagulant property and inhibited both thrombin and FXa following slow binding kinetics. Ruviprase inhibited thrombin by binding to its active site via an uncompetitive mechanism with a Ki value and dissociation constant (KD) of 0.42 μM and 0.46 μM, respectively. Conversely, Ruviprase demonstrated mixed inhibition (Ki = 0.16 μM) of FXa towards its physiological substrate prothrombin. Furthermore, the biological properties of Ruviprase could not be neutralized by commercial polyvalent or monovalent antivenom. Ruviprase at a dose of 2.0 mg/kg was non-toxic and showed potent in vivo anticoagulant activity after 6 h of intraperitoneal treatment in mice. Because of the potent anticoagulant property as well as non-toxic nature of Ruviprase, the possible application of the peptide as an antithrombotic agent for combating thrombosis-associated ailments appears promising. PMID:25038567

  8. Cysteine proteases from the Asclepiadaceae plants latex exhibited thrombin and plasmin like activities.

    PubMed

    Shivaprasad, H V; Riyaz, M; Venkatesh Kumar, R; Dharmappa, K K; Tarannum, Shaista; Siddesha, J M; Rajesh, R; Vishwanath, B S

    2009-10-01

    In the present study we evaluated the presence of cysteine protease from the latex of four plants Asclepias curassavica L., Calotropis gigantea R.Br., Pergularia extensa R.Br. and Cynanchum puciflorum R.Br. belongs to the family Asclepiadaceae. Cysteine proteases from these plants latex exhibited both thrombin and plasmin like activities. Latex enzyme fraction in a concentration dependent manner induced the formation of clot in citrated blood plasma. Direct incubation of fibrinogen with latex enzyme fraction resulted in the formation of fibrin clot similar to thrombin enzyme. However prolonged incubation resulted in degradation of the formed fibrin clot suggesting plasmin like activity. Latex enzyme fraction preferentially hydrolyzed Aalpha and Bbeta chains of fibrinogen to form fibrin clot. Latex enzyme fraction also hydrolyzed the subunits of fully cross linked fibrin efficiently, the order of hydrolysis was alpha-polymer > alpha-chains > beta-chain and gamma-gamma dimer. Cysteine proteases from all the four Asclepiadaceae plants latex exhibited similar action on fibrinogen and fibrin. This study scientifically validate the use of plant latex in stop bleeding and wound healing by traditional healers all over the world. PMID:18979066

  9. [Modifications induced on thrombin and plasmin activity by voluntary interruption of pregnancy].

    PubMed

    Manoni, F; Gessoni, G; Antico, A; Finesso, P; Rossito, G; Sartori, R

    1993-05-01

    Pregnancy is characterized by plasmatic variations of coagulative factors' concentration and by different haemostatic-fibrinolytic balance. At present it is possible, with EIA methods, to measure fibrinogen (FgDP) and fibrin (FbDP) degradation products with precision and accuracy, as direct indexes of fibrinolysis and the thrombin-antithrombin III complex (TAT) as indirect index of thrombophilia. We have considered the course of those indexes in 61 pregnant women within the tenth week of gestation, before and after voluntary pregnancy interruption (VPI) resulted without complications. The results don't show any peculiar variation of the examined parameters between the pregnant women before VPI and a control group. Comparing the basal data with those obtained three hours after VPI, all indexes are increased, particularly FbDP. After 24 hours the concentration of FgDP, FbDP and TDP decreased in comparison with the three hours control drawing, nevertheless staying higher than the values obtained in the basal drawing. The evolution of FDP and of TAT, in our study, points out that, in the first weeks of pregnancy, the haemostatic-fibrinolytic balance does not differ significantly from the physiological balance. Three hours after VPI fibrinolytic mechanisms prevail as regards the fibrinogenolytic ones. TAT increases after 3 hours and returns to the rules after 24 hours, proposing itself as an indirect index of thrombinic activation and as a direct index of antithrombinic activity. PMID:8351063

  10. Thrombin-activated human platelets acutely generate oxidized docosahexaenoic-acid-containing phospholipids via 12-lipoxygenase.

    PubMed

    Morgan, Lloyd T; Thomas, Christopher P; Kühn, Hartmut; O'Donnell, Valerie B

    2010-10-01

    Arachidonate-containing oxidized phospholipids are acutely generated by 12-LOX (12-lipoxygenase) in agonist-activated platelets. In the present study, formation of structurally related lipids by oxidation of DHA (docosahexaenoic acid)-containing phospholipids is demonstrated using lipidomic approaches. Precursor scanning reverse-phase LC (liquid chromatography)-MS/MS (tandem MS) identified a new family of lipids that comprise phospholipid-esterified HDOHE (hydroxydocosahexaenoic acid). Two diacyl and two plasmalogen PEs (phosphatidylethanolamines) containing predominantly the 14-HDOHE positional isomer (18:0p/14-HDOHE-PE, 18:0a/14-HDOHE-PE, 16:0a/14-HDOHE-PE and 16:0p/14-HDOHE-PE) were structurally characterized using MS/MS and by comparison with biogenic standards. An involvement of 12-LOX was indicated as purified recombinant human 12-LOX also generated the 14-HDOHE isomer from DHA. Pharmacological studies using inhibitors and recombinant platelet 12-LOX indicate that they form via esterification of newly formed non-esterified HDOHE. HDOHE-PEs formed at significant rates (2-4 ng/4×10(7) cells) within 2-180 min of thrombin stimulation, and their formation was blocked by calcium chelation. In summary, a new family of oxidized phospholipid was identified in thrombin-activated human platelets. PMID:20653566

  11. Thrombin-activatable fibrinolysis inhibitor.

    PubMed

    Marx, Pauline F

    2004-09-01

    The coagulation system is a potent mechanism that prevents blood loss after vascular injury. It consists of a number of linked enzymatic reactions resulting in thrombin generation. Thrombin converts soluble fibrinogen into a fibrin clot. The clot is subsequently removed by the fibrinolytic system upon wound healing. Thrombin-activatable fibrinolysis inhibitor (TAFI), which is identical to the previously identified proteins procarboxypeptidase B, R, and U, forms a link between blood coagulation and fibrinolysis. TAFI circulates as an inactive proenzyme in the bloodstream, and becomes activated during blood clotting. The active form, TAFIa, inhibits fibrinolysis by cleaving off C-terminal lysine residues from partially degraded fibrin that stimulates the tissue-type plasminogen activator-mediated conversion of plasminogen to plasmin. Consequently, removal of these lysines leads to less plasmin formation and subsequently to protection of the fibrin clot from break down. Moreover, TAFI may also play a role in other processes such as, inflammation and tissue repair. In this review, recent developments in TAFI research are discussed. PMID:15379716

  12. Activity of thrombin-activatable fibrinolysis inhibitor in the plasma of patients with abdominal aortic aneurysm.

    PubMed

    Dubis, Joanna; Zuk, Natalia; Grendziak, Ryszard; Zapotoczny, Norbert; Pfanhauser, Monika; Witkiewicz, Wojciech

    2014-04-01

    Patients with abdominal aortic aneurysm (AAA) experience impaired balance between fibrinolysis and coagulation, manifested by increased prothrombotic tendency and intensified inflammatory processes. The aim of this study was to evaluate the TAFI activity level (thrombin activatable fibrinolysis inhibitor) in the plasma of AAA patients. Plasma levels of PAI-1 (plasminogen activator inhibitor type 1), urokinase-type plasminogen activator and uPAR (urokinase-type plasminogen activator receptor) were measured as markers of fibrinolytic activity. The study showed that the activity of the thrombin-activatable fibrinolysis inhibitor in the plasma of AAA patients was significantly lower than in the plasma of the control individuals (64.6 ± 10.1 vs. 54.2 ± 10.9%, P < 0.0001). TAFI activity positively correlated with the white blood cell count (r = 0.486, P < 0.005). The uPAR concentration in the AAA patients was statistically significantly higher than in the control group and positively correlated with TAFI activity (r = 0.409, P = 0.02). The levels of PAI-1 and D-dimers (fibrin fragments) were significantly higher in patients with AAA than in the control group (44.3 ± 17.5 vs. 21.7 ± 8.7 ng/ml and 1869.6 ± 1490.1 vs. 181.5 ± 188.6 ng/ml, respectively). Lowered activity of the fibrinolysis inhibitor TAFI may heighten the blood fibrinolytic potential in AAA patients and contribute to the development of comorbidities. Therefore, TAFI participation in AAA pathogenesis cannot be excluded. PMID:24378973

  13. Thrombin Time

    MedlinePlus

    ... monitor unfractionated heparin therapy and to detect heparin contamination in a blood sample. While it is still ... thrombin time may sometimes be ordered when heparin contamination of a sample is suspected or when a ...

  14. Anticoagulant Activity of a Unique Sulfated Pyranosic (1→3)-β-l-Arabinan through Direct Interaction with Thrombin*

    PubMed Central

    Fernández, Paula V.; Quintana, Irene; Cerezo, Alberto S.; Caramelo, Julio J.; Pol-Fachin, Laercio; Verli, Hugo; Estevez, José M.; Ciancia, Marina

    2013-01-01

    A highly sulfated 3-linked β-arabinan (Ab1) with arabinose in the pyranose form was obtained from green seaweed Codium vermilara (Bryopsidales). It comprised major amounts of units sulfated on C-2 and C-4 and constitutes the first polysaccharide of this type isolated in the pure form and fully characterized. Ab1 showed anticoagulant activity by global coagulation tests. Less sulfated arabinans obtained from the same seaweed have less or no activity. Ab1 exerts its activity through direct and indirect (antithrombin- and heparin cofactor II-mediated) inhibition of thrombin. Direct thrombin inhibition was studied in detail. By native PAGE, it was possible to detect formation of a complex between Ab1 and human thrombin (HT). Ab1 binding to HT was measured by fluorescence spectroscopy. CD spectra of the Ab1 complex suggested that ligand binding induced a small conformational change on HT. Ab1-thrombin interactions were studied by molecular dynamic simulations using the persulfated octasaccharide as model compound. Most carbohydrate-protein contacts would occur by interaction of sulfate groups with basic amino acid residues on the surface of the enzyme, more than 60% of them being performed by the exosite 2-composing residues. In these interactions, the sulfate groups on C-2 were shown to interact more intensely with the thrombin structure. In contrast, the disulfated oligosaccharide does not promote major conformational modifications at the catalytic site when complexed to exosite 1. These results show that this novel pyranosic sulfated arabinan Ab1 exerts its anticoagulant activity by a mechanism different from those found previously for other sulfated polysaccharides and glycosaminoglycans. PMID:23161548

  15. Plasma Thrombin Generation and Sensitivity to Activated Protein C Among Patients With Myeloma and Monoclonal Gammopathy of Undetermined Significance.

    PubMed

    Crowley, Maeve P; Kevane, Barry; O'Shea, Susan I; Quinn, Shane; Egan, Karl; Gilligan, Oonagh M; Ní Áinle, Fionnuala

    2016-09-01

    The etiology of the prothrombotic state in myeloma has yet to be definitively characterized. Similarly, while recent evidence suggests that patients with monoclonal gammopathy of undetermined significance (MGUS) may also be at increased risk of thrombosis, the magnitude and the etiology of this risk have also yet to be defined. The present study aims to characterize patterns of plasma thrombin generation and sensitivity to the anticoagulant activity of activated protein C (APC) at the time of initial diagnosis of myeloma and in response to therapy in comparison to that observed among patients with MGUS and matched, healthy volunteers. Patients presenting with newly diagnosed/newly relapsed myeloma (n = 8), MGUS (n = 8), and matched healthy volunteers (n = 8) were recruited. Plasma thrombin generation was determined by calibrated automated thrombography. Peak thrombin generation was significantly higher in patients with myeloma (383.4 ± 33.4 nmol/L) and MGUS (353.4 ± 16.5 nmol/L) compared to healthy volunteers (276.7 ± 20.8 nmol/L; P < .05). In the presence of APC, endogenous thrombin potential was significantly lower in control plasma (228.6 ± 44.5 nmol/L × min) than in either myeloma (866.2 ± 241.3 nmol/L × min, P = .01) or MGUS plasma (627 ± 91.5 nmol/L × min, P = .003). Within the myeloma cohort, peak thrombin generation was significantly higher at diagnosis (353.2 ± 15.9 nmol/L) than following completion of the third cycle of therapy (282.1 ± 15.2 nmol/L; P < .005). Moreover, sensitivity to APC increased progressively with each cycle of chemotherapy. Further study of the etiology and evolving patterns of hypercoagulability among patients with these conditions is warranted and may have future implications for thromboprophylaxis strategies. PMID:26759370

  16. GDP beta S enhances the activation of phospholipase C caused by thrombin in human platelets: evidence for involvement of an inhibitory GTP-binding protein

    SciTech Connect

    Oberdisse, E.; Lapetina, E.G.

    1987-05-14

    Guanosine 5'-O-thiotriphosphate (GTP gamma S) and thrombin stimulate the activity of phospholipase C in platelets that have been permeabilized with saponin and whose inositol phospholipids have been prelabeled with (/sup 3/H)inositol. Ca/sup 2 +/ has opposite effects on the formation of (/sup 3/H)inositol phosphates induced by thrombin or GTP gamma S. While the action of GTP gamma S on the formation of (/sup 3/H)inositol phosphates is inhibited by Ca/sup 2 +/, action of thrombin is stimulated by Ca/sup 2 +/. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which inhibits the function of GTP-binding proteins, also inhibits the effect of GTP gamma S on phospholipase C stimulation but, surprisingly, increases the effect of thrombin. Ca/sup 2 +/ increases the inhibitory effect of GDP beta S on GTP gamma S activation of phospholipase C, but Ca/sup 2 +/ further enhances the stimulatory effect of GDP beta S on the thrombin activation of phospholipase C. This indicates that two mechanisms are responsible for the activation of phospholipase C in platelets. A GTP-binding protein is responsible for regulation of phospholipase C induced by GTP gamma S, while the effect of thrombin on the stimulation of phospholipase C is independent of GTP-binding proteins. However, the effect of thrombin may be modulated by the action of an inhibitory GTP-binding protein.

  17. The Crystal Structure of Thrombin-activable Fibrinolysis Inhibitor (TAFI) Provides the Structural Basis for Its Intrinsic Activity and the Short Half-life of TAFIa*♦

    PubMed Central

    Anand, Kanchan; Pallares, Irantzu; Valnickova, Zuzana; Christensen, Trine; Vendrell, Josep; Wendt, K. Ulrich; Schreuder, Herman A.; Enghild, Jan J.; Avilés, Francesc X.

    2008-01-01

    Mature thrombin-activable fibrinolysis inhibitor (TAFIa) is a highly unstable metallocarboxypeptidase that stabilizes blood clots by clipping C-terminal lysine residues from partially degraded fibrin. In accordance with its in vitro antifibrinolytic activity, animal studies have reported that inhibition of mature TAFI aids in the prevention of thrombosis. The level of TAFI activity is stringently regulated through (i) controlled proteolytic truncation of the zymogen (TAFI), generating the mature enzyme, TAFIa, and (ii) the short half-life of TAFIa. TAFI itself exhibits an intrinsic enzymatic activity, which is likely required to provide a baseline level of antifibrinolytic activity. The novel crystal structure presented here reveals that the active site of TAFI is accessible, providing the structural explanation for the its intrinsic activity. It also supports the notion that an “instability region” exists, in agreement with site-directed mutagenesis studies. Sulfate ions, bound to this region, point toward a potential heparin-binding site and could explain how heparin stabilizes TAFIa. PMID:18669641

  18. Study of the Differential Activity of Thrombin Inhibitors Using Docking, QSAR, Molecular Dynamics, and MM-GBSA

    PubMed Central

    Mena-Ulecia, Karel; Tiznado, William; Caballero, Julio

    2015-01-01

    Non-peptidic thrombin inhibitors (TIs; 177 compounds) with diverse groups at motifs P1 (such as oxyguanidine, amidinohydrazone, amidine, amidinopiperidine), P2 (such as cyanofluorophenylacetamide, 2-(2-chloro-6-fluorophenyl)acetamide), and P3 (such as phenylethyl, arylsulfonate groups) were studied using molecular modeling to analyze their interactions with S1, S2, and S3 subsites of the thrombin binding site. Firstly, a protocol combining docking and three dimensional quantitative structure–activity relationship was performed. We described the orientations and preferred active conformations of the studied inhibitors, and derived a predictive CoMSIA model including steric, donor hydrogen bond, and acceptor hydrogen bond fields. Secondly, the dynamic behaviors of some selected TIs (compounds 26, 133, 147, 149, 162, and 177 in this manuscript) that contain different molecular features and different activities were analyzed by creating the solvated models and using molecular dynamics (MD) simulations. We used the conformational structures derived from MD to accomplish binding free energetic calculations using MM-GBSA. With this analysis, we theorized about the effect of van der Waals contacts, electrostatic interactions and solvation in the potency of TIs. In general, the contents reported in this article help to understand the physical and chemical characteristics of thrombin-inhibitor complexes. PMID:26599107

  19. Study of the Differential Activity of Thrombin Inhibitors Using Docking, QSAR, Molecular Dynamics, and MM-GBSA.

    PubMed

    Mena-Ulecia, Karel; Tiznado, William; Caballero, Julio

    2015-01-01

    Non-peptidic thrombin inhibitors (TIs; 177 compounds) with diverse groups at motifs P1 (such as oxyguanidine, amidinohydrazone, amidine, amidinopiperidine), P2 (such as cyanofluorophenylacetamide, 2-(2-chloro-6-fluorophenyl)acetamide), and P3 (such as phenylethyl, arylsulfonate groups) were studied using molecular modeling to analyze their interactions with S1, S2, and S3 subsites of the thrombin binding site. Firstly, a protocol combining docking and three dimensional quantitative structure-activity relationship was performed. We described the orientations and preferred active conformations of the studied inhibitors, and derived a predictive CoMSIA model including steric, donor hydrogen bond, and acceptor hydrogen bond fields. Secondly, the dynamic behaviors of some selected TIs (compounds 26, 133, 147, 149, 162, and 177 in this manuscript) that contain different molecular features and different activities were analyzed by creating the solvated models and using molecular dynamics (MD) simulations. We used the conformational structures derived from MD to accomplish binding free energetic calculations using MM-GBSA. With this analysis, we theorized about the effect of van der Waals contacts, electrostatic interactions and solvation in the potency of TIs. In general, the contents reported in this article help to understand the physical and chemical characteristics of thrombin-inhibitor complexes. PMID:26599107

  20. Thrombin produces phosphorylation of cytosolic phospholipase A2 by a mitogen-activated protein kinase kinase-independent mechanism in the human astrocytoma cell line 1321N1.

    PubMed Central

    Hernández, M; Bayón, Y; Sánchez Crespo, M; Nieto, M L

    1997-01-01

    The release of [3H]arachidonic acid was studied in the 1321N1 astrocytoma cell line upon stimulation with thrombin. The effect of thrombin was antagonized by hirudin only when both compounds were added simultaneously, which suggests activation of thrombin receptor. Evidence that the cytosolic phospholipase A2 (cPLA2) takes part in thrombin-induced arachidonate release was provided by the finding that thrombin induced retardation of the mobility of cPLA2 in SDS/polyacrylamide gels, which is a feature of the activation of cPLA2 by mitogen-activated protein (MAP) kinases. Thrombin induced activation of two members of the MAP kinase family whose consensus primary sequence appears in cPLA2, namely p42-MAP kinase and c-Jun kinase. However, the activation of c-Jun kinase preceded the phosphorylation of cPLA2 more clearly than the activation of p42-MAK kinase did. Both cPLA2 and c-Jun kinase activation were not affected by PD-98059, a specific inhibitor of MAP kinase kinases, which indeed completely blocked p42-MAP kinase shift. Heat shock, a well-known activator of c-Jun kinase, also phosphorylated cPLA2 but not p42-MAP kinase. These data indicate the existence in astrocytoma cells of a signalling pathway triggered by thrombin receptor stimulation that activates a kinase cascade acting on the Pro-Leu-Ser-Pro consensus primary sequence, activates cPLA2, and associates the release of arachidonate with nuclear signalling pathways. PMID:9359863

  1. Thrombin-initiated platelet activation in vivo is vWF independent during thrombus formation in a laser injury model

    PubMed Central

    Dubois, Christophe; Panicot-Dubois, Laurence; Gainor, Justin F.; Furie, Barbara C.; Furie, Bruce

    2007-01-01

    Adhesion of platelets to an injured vessel wall and platelet activation are critical events in the formation of a thrombus. Of the agonists involved in platelet activation, thrombin, collagen, and vWF are known to induce in vitro calcium mobilization in platelets. Using a calcium-sensitive fluorochrome and digital multichannel intravital microscopy to image unstimulated and stimulated platelets, calcium mobilization was monitored as a reporter of platelet activation (as distinct from platelet accumulation) during thrombus formation in live mice. In the absence of vWF, platelet activation was normal, but platelet adherence and aggregation were attenuated during thrombus formation following laser-induced injury in the cremaster muscle microcirculation. In WT mice treated with lepirudin, platelet activation was blocked, and platelet adherence and aggregation were inhibited. The kinetics of platelet activation and platelet accumulation were similar in FcRγ–/– mice lacking glycoprotein VI (GPVI), GPVI-depleted mice, and WT mice. Our results indicate that the tissue factor–mediated pathway of thrombin generation, but not the collagen-induced GPVI-mediated pathway, is the major pathway leading to platelet activation after laser-induced injury under the conditions employed. In the tissue factor–mediated pathway, vWF plays a role in platelet accumulation during thrombus formation but is not required for platelet activation in vivo. PMID:17380206

  2. PAR-1 activation by thrombin is protective in human pulmonary artery endothelial cells if endothelial protein C receptor is occupied by its natural ligand

    PubMed Central

    Bae, Jong-Sup; Rezaie, Alireza R.

    2008-01-01

    Summary We recently demonstrated that the occupancy of endothelial protein C receptor (EPCR) by its natural ligand activated protein C (APC)/protein C switches the protease activated receptor 1 (PAR-1)-dependent signaling specificity of thrombin from a disruptive to a protective effect in cultured human umbilical vein endothelial cells. Given the phenotypic differences between endothelial cells in venular and arterial beds, in this study we evaluated the signaling function of thrombin in human pulmonary artery endothelial cells (HPAECs) before and after treating them with PC-S195A which lacks catalytic activity but exhibits a normal affinity for EPCR. As expected, both thrombin and thrombin receptor agonist peptide (TRAP) enhanced the permeability barrier of HPAECs, however, both PAR-1 agonists exhibited a potent barrier protective effect when the cells were treated with PC-S195A prior to stimulation by the agonists. Interestingly, similar to APC, thrombin exhibited a potent cytoprotective activity in the LPS-induced permeability and TNF-α-induced apoptosis and adhesion assays in the PC-S195A treated HPAECs. Treatment of HPAECs with the cholesterol depleting molecule methyl-β-cyclodextrin eliminated the protective effect of both APC and thrombin. These results suggest that the occupancy of EPCR by its natural ligand recruits PAR-1 to a protective signaling pathway within lipid rafts of HPAECs. Based on these results we conclude that the activation of PAR-1 by thrombin would initiate a protective response in intact arterial vascular cells expressing EPCR. These findings may have important ramifications for understanding the mechanism of the participation of the vascular PAR-1 in pathophysiology of the inflammatory disorders. PMID:18612544

  3. Thrombostatin FM compounds: direct thrombin inhibitors – mechanism of action in vitro and in vivo

    PubMed Central

    Nieman, M. T.; Burke, F.; Warnock, M.; Zhou, Y.; Sweigart, J.; Chen, A.; Ricketts, D.; Lucchesi, B. R.; Chen, Z.; Di Cera, E.; Hilfinger, J.; Kim, J. S.; Mosberg, H. I.; Schmaier, A. H.

    2009-01-01

    Summary Background Novel pentapeptides called Thrombostatin FM compounds consisting mostly of D-isomers and unusual amino acids were prepared based upon the stable angiotensin converting enzyme breakdown product of bradykinin – RPPGF. Methods and Results These peptides are direct thrombin inhibitors prolonging the thrombin clotting time, activated partial thromboplastin time, and prothrombin time at ≥0.78, 1.6, and 1.6 µm, respectively. They competitively inhibit α-thrombin-induced cleavage of a chromogenic substrate at 4.4–8.2 µm. They do not significantly inhibit plasma kallikrein, factor (F) XIIa, FXIa, FIXa, FVIIa-TF, FXa, plasmin or cathepsin G. One form, FM19 [rOicPaF(p-Me)], blocks α-thrombin-induced calcium flux in fibroblasts with an IC50 of 6.9 ± 1.2 µm. FM19 achieved 100% inhibition of threshold α- or γ-thrombin-induced platelet aggregation at 8.4 ± 4.7 µm and 16 ± 4 µm, respectively. The crystal structure of thrombin in complex with FM19 shows that the N-terminal D-Arg retrobinds into the S1 pocket, its second residue Oic interacts with His-57, Tyr-60a and Trp-60d, and its C-terminal p-methyl Phe engages thrombin_s aryl binding site composed of Ile-174, Trp-215, and Leu-99. When administered intraperitoneal, intraduodenal, or orally to mice, FM19 prolongs thrombin clotting times and delays carotid artery thrombosis. Conclusion FM19, a low affinity reversible direct thrombin inhibitor, might be useful as an add-on agent to address an unmet need in platelet inhibition in acute coronary syndromes in diabetics and others who with all current antiplatelet therapy still have reactive platelets. PMID:18315550

  4. Test of hirudin activity by tracking the binding of hirudin to thrombin in the presence of BS3 cross-linking.

    PubMed

    Liu, Yanfang; Yang, Jian; Wang, Jiangmin; Huang, Qingmei; Yang, Xiaohong; Zhang, Jianhua

    2015-10-01

    Hirudin has a great potential in inhibiting thrombin, and its antithrombin activity has direct bearing on its clinical application. Using bovine alpha-thrombin and recombinant hirudin of Poecilobdella javanica purified from Phichia pastoris as materials, this study introduced a novel method to testing antithrombin activity of hirudin visually and dynamically by tracking the binding of hirudin to thrombin. After incubating the mixture of thrombin and hirudin at 37 °C for 5 min, the binding of hirudin to thrombin was cross-linked by bis[sulfosuccinimidyl] suberate for 30 min and visualized by SDS-polyacrylamide gel electrophoresis. With the aid of image analysis on the basis of INRA-Noésis E1D analysis software, antithrombin activity of hirudin was calculated through intensity variations of protein bands of either thrombin-hirudin compound, unbound thrombin, or unbound hirudin. In this regard, activity of the given hirudin was tested to be 5625 ATU/mg based on a single reaction, and 5675.3 ATU/mg based on a series of reactions in a stepwise manner, close to the result of 6000 ATU/mg concluded by titration method. The superiorities of the method include good accuracy (the minimum testable concentration of hirudin is 1.5 μg/ml) and little sample consumption (sample consumption of hirudin is generally 1-11.5 μl using the apparatus of Mini Protean 3 Cell). Easy operation, low input, and equipment requirement also grant it as an effective way. PMID:26332983

  5. Prospective evaluation of hemostatic system activation and thrombin potential in healthy pregnant women with and without factor V Leiden.

    PubMed

    Eichinger, S; Weltermann, A; Philipp, K; Hafner, E; Kaider, A; Kittl, E M; Brenner, B; Mannhalter, C; Lechner, K; Kyrle, P A

    1999-10-01

    Normal pregnancy is associated with alterations of the hemostatic system towards a hypercoagulable state and an increased risk of venous thromboembolism. The risk of venous thrombosis is higher in pregnant women with factor V Leiden (FVL) than in those with wildtype factor V. Routine laboratory assays are not useful to detect hypercoagulable conditions. A prospective and systematic evaluation of hemostatic system activation in women with and without FVL during an uncomplicated pregnancy employing more sensitive markers of hypercoagulability, such as prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), D-Dimer, or the endogenous thrombin potential (ETP), an indicator of the plasma's potential to generate thrombin, has not been performed. We prospectively followed 113 pregnant women with (n = 11) and without (n = 102) FVL and measured F1+2. TAT, D-Dimer and the ETP at the 12th, 22nd and 34th gestational week as well as 3 months after delivery (baseline) in each subject. None of the women developed clinical signs of venous thromboembolism during pregnancy or postpartum. Pregnant women with and without FVL exhibited substantial activation of the coagulation and fibrinolytic system as indicated by a gradual increase of F1+2, TAT and D-Dimer throughout uncomplicated pregnancy up to levels similar to those found in acute thromboembolic events (p < 0.0001 by analysis of variance for each parameters). Levels of F1+2 and TAT were comparable between women with and without FVL, but levels of D-Dimer were significantly higher in women with FVL than in those without the mutation (p = 0.0005). The ETP remained unchanged in both women with and without FVL at all timepoints. Our data demonstrate a substantial coagulation and fibrinolytic system activation in healthy women with and without FVL during uncomplicated pregnancy. An elevated F1+2, TAT or D-Dimer level during pregnancy is not necessarily indicative for an acute thromboembolic event. The normal ETP in both

  6. The 29-kDa proteins phosphorylated ion thrombin-activated human platelets are forms of the estrogen receptor-related 27-kDa heat shock protein

    SciTech Connect

    Mendelsohn, M.E.; Yan Zhu; O'Neill, S. )

    1991-12-15

    Thrombin plays a critical role in platelet activation, hemostasis, and thrombosis. Cellular activation by thrombin leads to the phosphorylation of multiple proteins, most of which are unidentified. The authors have characterized several 29-kDa proteins that are rapidly phosphorylated following exposure of intact human platelets to thrombin. A murine monoclonal antibody raised to an unidentified estrogen receptor-related 29-kDa protein selectively recognized these proteins as well as a more basic, unphosphorylated 27-kDa protein. Cellular activation by thrombin led to a marked shift in the proportion of protein from the 27-kDa unphosphorylated form to the 29-kDa phosphoprotein species. Using this antibody, they isolated and sequenced a human cDNA clone encoding a protein that was identical to the mammalian 27-kDa heat shock protein (HSP27), a protein of uncertain function that is known to be phosphorylated to several forms and to be transcriptionally induced by estrogen. The 29-kDa proteins were confirmed to be phosphorylated forms of HSP27 by immunoprecipitation studies. Thus, the estrogen receptor-related protein is HSP27, and the three major 20-kDa proteins phosphorylated in thrombin-activated platelets are forms of HSP27. These data suggest a role for HSP27 in the signal transduction events of platelet activation.

  7. Recombinant human activated protein C resets thrombin generation in patients with severe sepsis – a case control study

    PubMed Central

    de Pont, Anne-Cornélie JM; Bakhtiari, Kamran; Hutten, Barbara A; de Jonge, Evert; Vroom, Margreeth B; Meijers, Joost CM; Büller, Harry R; Levi, Marcel

    2005-01-01

    Introduction Recombinant human activated protein C (rhAPC) is the first drug for which a reduction of mortality in severe sepsis has been demonstrated. However, the mechanism by which this reduction in mortality is achieved is still not clearly defined. The aim of the present study was to evaluate the dynamics of the anticoagulant, anti-inflammatory and pro-fibrinolytic action of rhAPC in patients with severe sepsis, by comparing rhAPC-treated patients with case controls. Methods In this prospectively designed multicenter case control study, 12 patients who were participating in the ENHANCE study, an open-label study of rhAPC in severe sepsis, were treated intravenously with rhAPC at a constant rate of 24 μg/kg/h for a total of 96 h. Twelve controls with severe sepsis matching the inclusion criteria received standard therapy. The treatment was started within 48 h after the onset of organ failure. Blood samples were taken before the start of the infusion and at 4, 8, 24, 48, 96 and 168 h, for determination of parameters of coagulation and inflammation. Results Sepsis-induced thrombin generation as measured by thrombin-antithrombin complexes and prothrombin fragment F1+2, was reset by rhAPC within the first 8 h of infusion. The administration of rhAPC did not influence parameters of fibrinolysis and inflammation. There was no difference in outcome or occurrence of serious adverse events between the treatment group and the control group. Conclusion Sepsis-induced thrombin generation in severely septic patients is reset by rhAPC within the first 8 h of infusion without influencing parameters of fibrinolysis and inflammation. PMID:16277710

  8. Thrombin-Mediated Platelet Activation of Lysed Whole Blood and Platelet-Rich Plasma: A Comparison Between Platelet Activation Markers and Ultrastructural Alterations.

    PubMed

    Augustine, Tanya N; van der Spuy, Wendy J; Kaberry, Lindsay L; Shayi, Millicent

    2016-06-01

    Platelet ultrastructural alterations representing spurious activation have been identified in pathological conditions. A limitation of platelet studies is that sample preparation may lead to artifactual activation processes which may confound results, impacting the use of scanning electron microscopy as a supplemental diagnostic tool. We used scanning electron microscopy and flow cytometry to analyze platelet activation in platelet-rich plasma (PRP) and whole blood (WB) samples. PRP generated using a single high g force centrifugation, and WB samples treated with a red blood cell lysis buffer, were exposed to increasing concentrations of the agonist thrombin. Platelets in lysed WB samples responded to thrombin by elevating the activation marker CD62p definitively, with corresponding ultrastructural changes indicating activation. Conversely, CD62p expression in PRP preparations remained static. Ultrastructural analysis revealed fully activated platelets even under low concentration thrombin stimulation, with considerable fibrin deposition. It is proposed that the method for PRP production induced premature platelet activation, preventable by using an inhibitor of platelet aggregation and fibrin polymerization. Nevertheless, our results show a definitive correspondence between flow cytometry and scanning electron microscopy in platelet activation studies, highlighting the potential of the latter technique as a supplemental diagnostic tool. PMID:27329313

  9. Interaction of the 268-282 region of glycoprotein Ibalpha with the heparin-binding site of thrombin inhibits the enzyme activation of factor VIII.

    PubMed Central

    De Cristofaro, R; De Filippis, V

    2003-01-01

    Activation of factor VIII (FVIII) by thrombin plays a fundamental role in the amplification of the coagulation cascade and takes place through specific proteolytic cleavages at Arg(372), Arg(740) and Arg(1689). Full FVIII activation requires cleavage at Arg(372), a process involving the alpha-thrombin exosite-II; referred to as heparin-binding site (HBS). The present study was aimed at investigating the effect of glycoprotein Ibalpha (GpIbalpha; 1-282 fragment) binding to thrombin HBS on FVIII activation. Similar experiments were also performed using a synthetic peptide modelled on the 268-282 sequence of GpIbalpha, and sulphated successfully at all tyrosine residues present along its sequence, at positions 276, 278 and 279. Both GpIbalpha 1-282 and the sulphated GpIb 268-282 peptides induced a progressive decrease (up to 70%) in activated FVIII generation, assessed by coagulation and FXa-generation assays. Furthermore, SDS/PAGE and Western-blot experiments showed that the specific appearance of the 44 kDa A2 domain on cleavage of the FVIII Arg(372)-Ser(373) peptide bond was delayed significantly in the presence of either GpIbalpha 1-282 or GpIb 268-282 peptide. Moreover, the effect of the latter on thrombin-mediated hydrolysis of a peptide having the sequence 341-376 of FVIII was investigated using reverse-phase HPLC. The k (cat)/ K (m) values of the FVIII 341-376 peptide hydrolysis by thrombin decreased linearly as a function of the GpIbalpha 268-282 peptide concentration, according to a competitive inhibition effect. Taken together, these experiments suggest that the sulphated 268-282 region of GpIbalpha binds to thrombin HBS, and is responsible for the inhibition of the Arg(372)-Ser(373) bond cleavage and activation of FVIII. PMID:12689334

  10. Facile fabrication of an aptasensor for thrombin based on graphitic carbon nitride/TiO2 with high visible-light photoelectrochemical activity.

    PubMed

    Fan, Dawei; Guo, Cuijuan; Ma, Hongmin; Zhao, Di; Li, Yina; Wu, Dan; Wei, Qin

    2016-01-15

    A novel aptasensor for thrombin with high visible-light activity was facilely fabricated based on graphitic carbon nitride/TiO2 (g-C3N4/TiO2) photoelectrochemical (PEC) composite. Crystallization of TiO2 nanoparticles (NPs) and their strong interaction with g-C3N4 sheet were confirmed by high-resolution transmission electron microscope (HR-TEM), both of which contributed to the high photocurrent intensity under visible-light irradiation. Carboxyl functionalized thrombin aptamers were first successfully bound to the g-C3N4/TiO2 modified electrode as proven by photoelectrochemical test and electrochemical impedance spectroscopy (EIS) analysis. Ascorbic acid was utilized as the electron donor for scavenging photo-generated holes and inhibiting light driven electron-hole pair recombination. The specific recognition between thrombin aptamer and thrombin led to the linear decrease of photocurrent with the increase of logarithm of thrombin concentration in the range of 5.0×10(-13)molL(-1) to 5.0×10(-9)molL(-1) with a detection limit of 1.2×10(-13)molL(-1). This proposed low-cost, convenient and sensitive aptasensor showed promising applications in biosensor and photoelectrochemical analysis. PMID:26301999

  11. Thrombostatin FM compounds: direct thrombin inhibitors - mechanism of action in vitro and in vivo

    SciTech Connect

    Nieman, M T; Burke, F; Warnock, M; Zhou, Y; Sweigart, J; Chen, A; Ricketts, D; Lucchesi, B R; Chen, Z; Cera, E Di; Hilfinger, J; Kim, J S; Mosberg, H I; Schmaier, A H

    2008-04-29

    Novel pentapeptides called Thrombostatin FM compounds consisting mostly of D-isomers and unusual amino acids were prepared based upon the stable angiotensin converting enzyme breakdown product of bradykinin - RPPGF. These peptides are direct thrombin inhibitors prolonging the thrombin clotting time, activated partial thromboplastin time, and prothrombin time at ≥0.78, 1.6, and 1.6 μm, respectively. They competitively inhibit α-thrombin-induced cleavage of a chromogenic substrate at 4.4--8.2 μm. They do not significantly inhibit plasma kallikrein, factor (F) XIIa, FXIa, FIXa, FVIIa-TF, FXa, plasmin or cathepsin G. One form, FM19 [rOicPaF(p-Me)], blocks α-thrombin-induced calcium flux in fibroblasts with an IC50 of 6.9 ± 1.2 μm. FM19 achieved 100% inhibition of threshold α- or γ-thrombin-induced platelet aggregation at 8.4 ± 4.7 μm and 16 ± 4 μm, respectively. The crystal structure of thrombin in complex with FM19 shows that the N-terminal D-Arg retrobinds into the S1 pocket, its second residue Oic interacts with His-57, Tyr-60a and Trp-60d, and its C-terminal p-methyl Phe engages thrombin's aryl binding site composed of Ile-174, Trp-215, and Leu-99. When administered intraperitoneal, intraduodenal, or orally to mice, FM19 prolongs thrombin clotting times and delays carotid artery thrombosis. FM19, a low affinity reversible direct thrombin inhibitor, might be useful as an add-on agent to address an unmet need in platelet inhibition in acute coronary syndromes in diabetics and others who with all current antiplatelet therapy still have reactive platelets.

  12. Zinc modulates thrombin adsorption to fibrin

    SciTech Connect

    Hopmeier, P.; Halbmayer, M.; Fischer, M.; Marx, G. )

    1990-05-01

    Human thrombin with high affinity to Sepharose insolubilized fibrin monomers (high-affinity thrombin) was used to investigate the effect of Zn(II) on the thrombin adsorption to fibrin. Results showed that at Zn(II) concentrations exceeding 100 mumols/l, thrombin binding to fibrin was decreased concomitant with the Zn(II) concentration and time; at lower Zn(II) concentrations, thrombin adsorption was enhanced. Experimental results were identical by using 125I-labelled high-affinity alpha-thrombin or by measuring the thrombin activity either by chromogenic substrate or by a clotting time method. In contrast, Ca(II) alone (final conc. 3 mmol/l) or in combination with Zn(II) was not effective. However, at higher Ca(II) concentrations (7.5-15 mmol/l), thrombin adsorption was apparently decreased. Control experiments revealed that Zn(II) had no impact on the clottability of fibrinogen, and that the results of the experiments with Ca(II) were not altered by possible cross-linking of fibrin. We conclude that unlike Ca(II), Zn(II) is highly effective in modulating thrombin adsorption to fibrin.

  13. Hemin/G-quadruplexes as DNAzymes for the fluorescent detection of DNA, aptamer-thrombin complexes, and probing the activity of glucose oxidase.

    PubMed

    Golub, Eyal; Freeman, Ronit; Niazov, Angelica; Willner, Itamar

    2011-11-01

    Hemin/G-quadruplex catalyzes the H(2)O(2)-mediated oxidation of Amplex Red to the fluorescent product resorufin. This process is implemented to develop hairpin nucleic acid structures for the detection of DNA, to probe the catalytic activity of glucose oxidase, to use the thrombin-aptamer complex as a catalytic readout structure, and to quantitatively analyze telomere chain composition. PMID:21881641

  14. Isolation, Co-Crystallization and Structure-Based Characterization of Anabaenopeptins as Highly Potent Inhibitors of Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa)

    PubMed Central

    Schreuder, Herman; Liesum, Alexander; Lönze, Petra; Stump, Heike; Hoffmann, Holger; Schiell, Matthias; Kurz, Michael; Toti, Luigi; Bauer, Armin; Kallus, Christopher; Klemke-Jahn, Christine; Czech, Jörg; Kramer, Dan; Enke, Heike; Niedermeyer, Timo H. J.; Morrison, Vincent; Kumar, Vasant; Brönstrup, Mark

    2016-01-01

    Mature thrombin activatable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase that stabilizes fibrin clots by removing C-terminal arginines and lysines from partially degraded fibrin. Inhibition of TAFIa stimulates the degradation of fibrin clots and may help to prevent thrombosis. Applying a lead finding approach based on literature-mining, we discovered that anabaenopeptins, cyclic peptides produced by cyanobacteria, were potent inhibitors of TAFIa with IC50 values as low as 1.5 nM. We describe the isolation and structure elucidation of 20 anabaenopeptins, including 13 novel congeners, as well as their pronounced structure-activity relationships (SAR) with respect to inhibition of TAFIa. Crystal structures of the anabaenopeptins B, C and F bound to the surrogate protease carboxypeptidase B revealed the binding modes of these large (~850 Da) compounds in detail and explained the observed SAR, i.e. the strong dependence of the potency on a basic (Arg, Lys) exocyclic residue that addressed the S1’ binding pocket, and a broad tolerance towards substitutions in the pentacyclic ring that acted as a plug of the active site. PMID:27604544

  15. Evaluation of a multifunctional staphylokinase variant with thrombin inhibition and antiplatelet aggregation activities produced from salt-inducible E. coli GJ1158.

    PubMed

    Kumar, Anmol; Pulicherla, Krishna Kanth; Mayuren, Candasamy; Kotra, Seetharam; Rao, Krothapalli Rajasurya Sambasiva

    2013-10-01

    Reocclusion is one of the major root causes for secondary complications that arise during thrombolytic therapy. A multifunctional staphylokinase variant SRH (staphylokinase (SAK) linked with tripeptide RGD and didecapeptide Hirulog) with antiplatelet and antithrombin activities in addition to clot specific thrombolytic function, was developed to address the reocclusion problem. We preferred to use Escherichia coli GJ1158 as the host in this study for economic production of SRH by osmotic (0.3 mol/L sodium chloride) induction, to overcome the problems associated with the yeast expression system. The therapeutic potential of SRH was evaluated in the murine model of vascular thrombosis. The SAK protein (1 mg/kg body mass) and SRH protein (1 mg/kg and 2 mg/kg) were administered intravenously to the different treatment groups. The results have shown a dose-dependent antithrombotic effect in carrageenan-induced mouse tail thrombosis. The thrombin time, activated partial thromboplastin time, and prothrombin time were significantly prolonged (p < 0.05) in the SRH-infused groups. Moreover, SRH inhibited platelet aggregation in a dose-dependent manner (p < 0.05), while the bleeding time was significantly (p < 0.05) prolonged. All of these results inferred that the osmotically produced multifunctional fusion protein SRH (SAK-RGD-Hirulog) is a promising thrombolytic agent, and one which sustained its multifunctionality in the animal models. PMID:24144055

  16. Isolation, Co-Crystallization and Structure-Based Characterization of Anabaenopeptins as Highly Potent Inhibitors of Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa).

    PubMed

    Schreuder, Herman; Liesum, Alexander; Lönze, Petra; Stump, Heike; Hoffmann, Holger; Schiell, Matthias; Kurz, Michael; Toti, Luigi; Bauer, Armin; Kallus, Christopher; Klemke-Jahn, Christine; Czech, Jörg; Kramer, Dan; Enke, Heike; Niedermeyer, Timo H J; Morrison, Vincent; Kumar, Vasant; Brönstrup, Mark

    2016-01-01

    Mature thrombin activatable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase that stabilizes fibrin clots by removing C-terminal arginines and lysines from partially degraded fibrin. Inhibition of TAFIa stimulates the degradation of fibrin clots and may help to prevent thrombosis. Applying a lead finding approach based on literature-mining, we discovered that anabaenopeptins, cyclic peptides produced by cyanobacteria, were potent inhibitors of TAFIa with IC50 values as low as 1.5 nM. We describe the isolation and structure elucidation of 20 anabaenopeptins, including 13 novel congeners, as well as their pronounced structure-activity relationships (SAR) with respect to inhibition of TAFIa. Crystal structures of the anabaenopeptins B, C and F bound to the surrogate protease carboxypeptidase B revealed the binding modes of these large (~850 Da) compounds in detail and explained the observed SAR, i.e. the strong dependence of the potency on a basic (Arg, Lys) exocyclic residue that addressed the S1' binding pocket, and a broad tolerance towards substitutions in the pentacyclic ring that acted as a plug of the active site. PMID:27604544

  17. Dabigatran and Argatroban Diametrically Modulate Thrombin Exosite Function

    PubMed Central

    Yeh, Calvin H.; Stafford, Alan R.; Leslie, Beverly A.; Fredenburgh, James C.; Weitz, Jeffrey I.

    2016-01-01

    Thrombin is a highly plastic molecule whose activity and specificity are regulated by exosites 1 and 2, positively-charged domains that flank the active site. Exosite binding by substrates and cofactors regulates thrombin activity by localizing thrombin, guiding substrates, and by inducing allosteric changes at the active site. Although inter-exosite and exosite-to-active-site allostery have been demonstrated, the impact of active site ligation on exosite function has not been examined. To address this gap, we used surface plasmon resonance to determine the effects of dabigatran and argatroban, active site-directed inhibitors, on thrombin binding to immobilized γA/γA-fibrin or glycoprotein Ibα peptide via exosite 1 and 2, respectively, and thrombin binding to γA/γ′-fibrin or factor Va, which is mediated by both exosites. Whereas dabigatran attenuated binding, argatroban increased thrombin binding to γA/γA- and γA/γ′-fibrin and to factor Va. The results with immobilized fibrin were confirmed by examining the binding of radiolabeled thrombin to fibrin clots. Thus, dabigatran modestly accelerated the dissociation of thrombin from γA/γA-fibrin clots, whereas argatroban attenuated dissociation. Dabigatran had no effect on thrombin binding to glycoprotein Ibα peptide, whereas argatroban promoted binding. These findings not only highlight functional effects of thrombin allostery, but also suggest that individual active site-directed thrombin inhibitors uniquely modulate exosite function, thereby identifying potential novel mechanisms of action. PMID:27305147

  18. Structural stability of human alpha-thrombin studied by disulfide reduction and scrambling.

    PubMed

    Rajesh Singh, R; Chang, Jui Yoa

    2003-09-23

    Human alpha-thrombin is a very important plasma serine protease, which is involved in physiologically vital processes like hemostasis, thrombosis, and activation of platelets. Knowledge regarding the structural stability of alpha-thrombin is essential for understanding its biological regulation. Here, we investigated the structural and conformational stability of alpha-thrombin using the techniques of disulfide reduction and disulfide scrambling. alpha-Thrombin is composed of a light A-chain (36 residues) and a heavy B-chain (259 residues) linked covalently by an inter-chain disulfide bond (Cys(1)-Cys(122)). The B-chain is stabilized by three intra-chain disulfide bonds (Cys(42)-Cys(58), Cys(168)-Cys(182), and Cys(191)-Cys(220)) (Chymotrypsinogen nomenclature). Upon reduction with dithiothreitol (DTT), alpha-thrombin unfolded in a 'sequential' manner with sequential reduction of Cys(168)-Cys(182) within the B-chain followed by the inter-chain disulfide, generating two distinct partially reduced intermediates, I-1 and I-2, respectively. Conformational stability of alpha-thrombin was investigated by the technique of disulfide scrambling. alpha-Thrombin denatures by scrambling its native disulfide bonds in the presence of denaturant [urea, guanidine hydrochloride (GdmCl) or guanidine thiocyanate (GdmSCN)] and a thiol initiator. During the process, cleavage of the inter-chain disulfide bond and release of the A-chain from B-chain was the foremost event. The three disulfides in the B-chain subsequently scrambled to form three major isomers (designated as X-Ba, X-Bb, and X-Bc). Complete denaturation of alpha-thrombin was observed at low concentrations of denaturants (0.5 M GdmSCN, 1.5 M GdmCl, or 3 M urea) indicating low conformational stability of the protease. PMID:14499592

  19. Thrombin Induces Tumor Cell Cycle Activation and Spontaneous Growth by Down-regulation of p27Kip1, in Association with the Up-regulation of Skp2 and MiR-222

    PubMed Central

    Hu, Liang; Ibrahim, Sherif; Liu, Cynthia; Skaar, Jeffrey; Pagano, Michele; Karpatkin, Simon

    2009-01-01

    The effect of thrombin on tumor cell cycle activation and spontaneous growth was examined in synchronized serum-starved tumor cell lines and a model of spontaneous prostate cancer development in TRAMP mice. BrdUrd incorporation and propidium iodide staining of prostate LNCaP cells arrested in G0 and treated with thrombin or serum revealed a 48- and 29-fold increase in S phase cells, respectively, at 8 hours. Similar results were obtained with TRAMP cells and a glioblastoma cell line, T98G. Cell cycle kinases and inhibitors in synchronized tumor cells revealed high levels of p27Kip1 and low levels of Skp2 and cyclins D1 and A. Addition of thrombin, TFLLRN, or serum down-regulated p27Kip1 with concomitant induction of Skp2, Cyclin D1, and Cyclin A with similar kinetics. LNCaP p27Kip1-transfected cells or Skp2 knockdown cells were refractory to thrombin-induced cell cycle activation. MicroRNA 222, an inhibitor of p27Kip1, was robustly up-regulated by thrombin. The in vitro observations were tested in vivo with transgenic TRAMP mice. Repetitive thrombin injection enhanced prostate tumor volume 6- to 8-fold (P < 0.04). Repetitive hirudin, a specific potent antithrombin, decreased tumor volume 13- to 24-fold (P < 0.04). Thus, thrombin stimulates tumor cell growth in vivo by down-regulation of p27Kip1. PMID:19351827

  20. Enhancement by heparin of thrombin-induced antithrombin III proteolysis: its relation to the molecular weight and anticoagulant activity of heparin

    SciTech Connect

    Marciniak, E.; Gora-Maslak, G.

    1982-11-01

    Previous findings indicated that binding of heparin to antithrombin III (AT III) facilitates thrombin-induced proteolysis of the inhibitor. Researchers now studied this property of heparin in regard to its molecular weight and anticoagulant activity. Commercial heparin was resolved on Sephadex G-200 into six fractions of decreasing molecular weight. From each fraction high affinity (HA) heparin was isolated by chromatography on AT III-Sepharose and examined in reaction of alpha-thrombin with a molar excess of /sup 125/I AT III. Proteolysis of the inhibitor was assessed by SDS polyacrylamide gel electrophoresis. In the presence of the HA heparin from 18% to 38% of AT III participating in reaction appeared in the form of inactive 50,000-dalton fragment, as opposed to 7% of AT III fragmented in the absence of heparin. Although the ability to potentiate proteolysis was at its peak in the medium-molecular-size heparin fraction, the amount of degraded inhibitor relative to anticoagulant activity increased with decreasing molecular weight of the polysaccharide. These findings are consistent with the possibility that the ability of bound heparin to facilitate the cleavage of AT III by thrombin is generally less contingent upon secondary characteristics of the polysaccharide than the anticoagulant activity.

  1. Thrombin-activatable fibrinolysis inhibitor activity and global fibrinolytic capacity in Type 1 diabetes: evidence for normal fibrinolytic state.

    PubMed

    Harmanci, Ayla; Kandemir, Nurgun; Dagdelen, Selcuk; Gonc, Nazli; Buyukasik, Yahya; Alikasifoglu, Ayfer; Kirazli, Serafettin; Ozon, Alev; Gurlek, Alper

    2006-01-01

    Hypofibrinolysis is a state that is commonly observed in type 2 diabetic patients, a finding also possibly related to obesity and insulin resistance. There is little information, however, regarding the status of fibrinolytic system in Type 1 diabetes, in particular as reflected by thrombin-activatable fibrinolysis inhibitor (TAFI) activity and global fibrinolytic capacity (GFC). To provide information in this respect, 30 Type 1 diabetic patients (median age=16) and 28 healthy controls (median age=14) were enrolled in this study. The median duration of diabetes was 7 years, and median HbA(1c) was 8.85% (range: 5.5-11.9%) in the diabetic group. None of the patients had macrovascular complications. Microvascular complications were present in a total of eight patients (nephropathy: n=5; retinopathy: n=3). A comparison of the TAFI activity between the patient (median 84.9, range: 71.5-103.3%) and the control groups (median=83.3, range: 63.7-97.4%) yielded no statistically significant difference (P=.950). Similarly, GFC was comparable between the two groups (median=8.22, range: 0.72-22.38 microg/ml, and median=13.32, range: 3.0-23.22 microg/ml, respectively, in the diabetic and control groups, P=.086). TAFI activity did not significantly correlate with age, albumin excretion, fasting plasma glucose, HbA(1c), D-dimer, and fibrinogen by Spearman rank correlation test. There was as a significant inverse correlation between GFC and TAFI activity (r=-.414, P=.006). Contrary to the previous observations in Type 2 diabetes, our data suggest that fibrinolytic activity is not adversely affected by Type 1 diabetes, and it has no relationship with the degree of metabolic control. PMID:16389166

  2. Structure of the thrombin complex with triabin, a lipocalin-like exosite-binding inhibitor derived from a triatomine bug.

    PubMed

    Fuentes-Prior, P; Noeske-Jungblut, C; Donner, P; Schleuning, W D; Huber, R; Bode, W

    1997-10-28

    Triabin, a 142-residue protein from the saliva of the blood-sucking triatomine bug Triatoma pallidipennis, is a potent and selective thrombin inhibitor. Its stoichiometric complex with bovine alpha-thrombin was crystallized, and its crystal structure was solved by Patterson search methods and refined at 2.6-A resolution to an R value of 0.184. The analysis revealed that triabin is a compact one-domain molecule essentially consisting of an eight-stranded beta-barrel. The eight strands A to H are arranged in the order A-C-B-D-E-F-G-H, with the first four strands exhibiting a hitherto unobserved up-up-down-down topology. Except for the B-C inversion, the triabin fold exhibits the regular up-and-down topology of lipocalins. In contrast to the typical ligand-binding lipocalins, however, the triabin barrel encloses a hydrophobic core intersected by a unique salt-bridge cluster. Triabin interacts with thrombin exclusively via its fibrinogen-recognition exosite. Surprisingly, most of the interface interactions are hydrophobic. A prominent exception represents thrombin's Arg-77A side chain, which extends into a hydrophobic triabin pocket forming partially buried salt bridges with Glu-128 and Asp-135 of the inhibitor. The fully accessible active site of thrombin in this complex is in agreement with its retained hydrolytic activity toward small chromogenic substrates. Impairment of thrombin's fibrinogen converting activity or of its thrombomodulin-mediated protein C activation capacity upon triabin binding is explained by usage of overlapping interaction sites of fibrinogen, thrombomodulin, and triabin on thrombin. These data demonstrate that triabin inhibits thrombin via a novel and unique mechanism that might be of interest in the context of potential therapeutic applications. PMID:9342325

  3. Thrombin regulates the function of human blood dendritic cells

    SciTech Connect

    Yanagita, Manabu; Kobayashi, Ryohei; Kashiwagi, Yoichiro; Shimabukuro, Yoshio; Murakami, Shinya E-mail: ipshinya@dent.osaka-u.ac.jp

    2007-12-14

    Thrombin is the key enzyme in the coagulation cascade and activates endothelial cells, neutrophils and monocytes via protease-activated receptors (PARs). At the inflammatory site, immune cells have an opportunity to encounter thrombin. However little is known about the effect of thrombin for dendritic cells (DC), which are efficient antigen-presenting cells and play important roles in initiating and regulating immune responses. The present study revealed that thrombin has the ability to stimulate blood DC. Plasmacytoid DC (PDC) and myeloid DC (MDC) isolated from PBMC expressed PAR-1 and released MCP-1, IL-10, and IL-12 after thrombin stimulation. Unlike blood DC, monocyte-derived DC (MoDC), differentiated in vitro did not express PAR-1 and were unresponsive to thrombin. Effects of thrombin on blood DC were significantly diminished by the addition of anti-PAR-1 Ab or hirudin, serine protease inhibitor. Moreover, thrombin induced HLA-DR and CD86 expression on DC and the thrombin-treated DC induced allogenic T cell proliferation. These findings indicate that thrombin plays a role in the regulation of blood DC functions.

  4. Thrombin-like activity in snake venoms from Peruvian Bothrops and Lachesis genera.

    PubMed

    Orejuela, P; Zavaleta, A; Salas, M; Marsh, N

    1991-01-01

    Venoms from Lachesis muta muta, Bothrops pictus, B. barnetti, B. atrox and B. hyoprorus coagulate in vitro canine fibrinogen, and both bovine fibrinogen and bovine plasma. B. barnetti and L. muta muta venoms have greater activity on canine fibrinogen and B. atrox and B. hyoprorus venoms, a greater activity on bovine fibrinogen. Gel filtration showed one peak of coagulant activity in all venoms except B. atrox venom which possessed two peaks. The apparent mol. wt of these enzymes ranged from 45,000 to 69,000. PMID:1796478

  5. Inhibition of thrombin-mediated cellular effects by triabin, a highly potent anion-binding exosite thrombin inhibitor.

    PubMed

    Glusa, E; Bretschneider, E; Daum, J; Noeske-Jungblut, C

    1997-06-01

    Triabin, a 17 kDa protein from the saliva of the assassin bug Triatoma pallidipennis is a potent thrombin inhibitor interfering with the anion-binding exosite of the enzyme. The recombinant protein, produced by the baculovirus/insect cell system, was used to study the inhibitory effect on thrombin-mediated cellular responses. The thrombin (1 nM)-stimulated aggregation of washed human platelets and the rise in cytoplasmic calcium in platelets were inhibited by triabin at nanomolar concentrations. In contrast, the rise in calcium induced by the thrombin receptor-activating peptide (10 microM) was not suppressed by triabin. In isolated porcine pulmonary arteries, preconstricted with PGF 2 alpha thrombin (2 nM) elicited an endothelium-dependent relaxation which was inhibited by triabin in the same concentration range as found for the inhibition of platelet aggregation. Higher concentrations of triabin were required to diminish the contractile response of endotheliumdenuded pulmonary vessels to thrombin (10 nM). In cultured bovine coronary smooth muscle cells, the mitogenic activity of thrombin (3 nM), measured by [3H]thymidine incorporation, was also suppressed by triabin. In all these assays, the inhibitory effect of triabin was dependent on the thrombin concentration used. These studies suggest that the new anion-binding exosite thrombin inhibitor triabin is one of the most potent inhibitors of thrombin-mediated cellular effects. PMID:9241757

  6. Human alpha-thrombin inhibition by the active site titrant N alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester: a comparative kinetic and X-ray crystallographic study.

    PubMed

    Nardini, M; Pesce, A; Rizzi, M; Casale, E; Ferraccioli, R; Balliano, G; Milla, P; Ascenzi, P; Bolognesi, M

    1996-05-24

    Kinetics for the hydrolysis of the chromogenic active site titrant N alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp) catalyzed by bovine beta-trypsin, bovine alpha-thrombin, human alpha-thrombin, human Lys77-plasmin, human urinary kallikrein, the M(r) 33,000 and M(r) 54,000 species of human urokinase, as well as by porcine pancreatic beta-kallikrein-A and B have been obtained between pH 6.0 and 8.0, at 21.0 degrees C. Moreover, the three dimensional structure of the human alpha-thrombin-(hirugen).Dmc-azaLys acyl.enzyme complex has been analyzed and refined by X-ray crystallography at 2.0 A resolution (R-factor = 0.168). As observed for bovine beta-trypsin, the acylating inhibitor molecule is covalently bound to the Ser195 catalytic residue, filling the human alpha-thrombin S1 primary specificity subsite with its lysyl side-group. However, the carbonyl group of the scissile human alpha-thrombin.Dmc-azaLys acyl bond does not occupy properly the oxyanion binding hole. At variance from the bovine beta-trypsin.Dmc-azaLys acyl.enzyme structure, a second, not covalently bound, inhibitor molecule, partly shielded by the 60-insertion loop of human alpha-thrombin, is contacting the enzyme "aryl-binding site". PMID:8637015

  7. SLOW THROMBIN IS ZYMOGEN-LIKE

    PubMed Central

    Huntington, James A.

    2009-01-01

    Summary Blood coagulation is the result of a cascade of zymogen activation events, however, its initiation is allosteric. Factor VIIa circulates in a zymogen-like state and is allosterically activated by binding to tissue factor. Thrombin, the final protease generated in the blood coagulation cascade, has also been shown to exist in a low activity state in the absence of cofactors, and the structural features of this ‘slow’ form has been studied for many years. In this manuscript I will review the general features that render zymogens inactive and how proteolytic cleavage results in activation, but I will also show how this distinction is blurred by zymogens that have activity (protease-like zymogens) and proteases with low activity (zymogen-like proteases). This will then be applied in the analysis of slow thrombin to reveal how allosteric activation of thrombin simply reflects the conversion from a zymogen-like enzyme to an active serine protease. PMID:19630791

  8. The ligand occupancy of endothelial protein C receptor switches the protease-activated receptor 1-dependent signaling specificity of thrombin from a permeability-enhancing to a barrier-protective response in endothelial cells

    PubMed Central

    Bae, Jong-Sup; Yang, Likui; Manithody, Chandrashekhara

    2007-01-01

    Recent studies have indicated that activated protein C (APC) may exert its cytoprotective and anti-inflammatory activities through the endothelial protein C receptor (EPCR)-dependent cleavage of protease-activated receptor 1 (PAR-1) on vascular endothelial cells. Noting that (1) the activation of protein C on endothelial cells requires thrombin, (2) relative to APC, thrombin cleaves PAR-1 with approximately 3 to 4 orders of magnitude higher catalytic efficiency, and (3) PAR-1 is a target for the proinflammatory activity of thrombin, it is not understood how APC can elicit a protective signaling response through the cleavage of PAR-1 when thrombin is present. In this study, we demonstrate that EPCR is associated with caveolin-1 in lipid rafts of endothelial cells and that its occupancy by the γ-carboxyglutamic acid (Gla) domain of protein C/APC leads to its dissociation from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway through coupling of PAR-1 to the pertussis toxin–sensitive Gi-protein. Thus, when EPCR is bound by protein C, the PAR-1 cleavage-dependent protective signaling responses in endothelial cells can be mediated by either thrombin or APC. These results provide a new paradigm for understanding how PAR-1 and EPCR participate in protective signaling events in endothelial cells. PMID:17823308

  9. Motor neuron cell death in wobbler mutant mice follows overexpression of the G-protein-coupled, protease-activated receptor for thrombin.

    PubMed Central

    Festoff, B. W.; D'Andrea, M. R.; Citron, B. A.; Salcedo, R. M.; Smirnova, I. V.; Andrade-Gordon, P.

    2000-01-01

    BACKGROUND: Mechanisms underlying neurodegeneration are actively sought for new therapeutic strategies. Transgenic, knockout and genetic mouse models greatly aid our understanding of the mechanisms for neuronal cell death. A naturally occurring, autosomal recessive mutant, known as wobbler, and mice transgenic for familial amyotrophic lateral sclerosis (FALS) superoxide dismutase (SOD)1 mutations are available, but the molecular mechanisms remain equally unknown. Both phenotypes are detectable after birth. Wobbler is detectable in the third week of life, when homozygotes (wr/wr) exhibit prominent gliosis and significant motor neuron loss in the cervical, but not in lumbar, spinal cord segments. To address molecular mechanisms, we evaluated "death signals" associated with the multifunctional serine protease, thrombin, which leads to apoptotic motor neuronal cell death in culture by cleavage of a G-protein coupled, protease-activated receptor 1 (PAR-1). MATERIALS AND METHODS: Thrombin activities were determined with chromogenic substrate assays, Western immunoblots and immunohistochemistry were performed with anti-PAR-1 to observe localizations of the receptor and anti-GFAP staining was used to monitor astrocytosis. PAR-1 mRNA levels and locations were determined by reverse transcription polymerase chain reaction (qRT-PCR) and in situ hybridizations. Cell death was monitored with in situ DNA fragmentation assays. RESULTS: In preliminary studies we found a 5-fold increase in PAR-1 mRNA in cervical spinal cords from wr/wr, compared with wild-type (wt) littermates. Our current studies suggested that reactive astrocytosis and motor neuron cell death were causally linked with alterations in thrombin signaling. PAR-1 protein expression was increased, as demonstrated by immunocytochemistry and confirmed with in situ hybridization, in phenotypic wr/wr motor neurons, compared with wt, but not in astrocytes. This increase was much greater in cervical, compared with lumbar

  10. Role of thrombin signalling in platelets in haemostasis and thrombosis

    NASA Astrophysics Data System (ADS)

    Sambrano, Gilberto R.; Weiss, Ethan J.; Zheng, Yao-Wu; Huang, Wei; Coughlin, Shaun R.

    2001-09-01

    Platelets are critical in haemostasis and in arterial thrombosis, which causes heart attacks and other events triggered by abnormal clotting. The coagulation protease thrombin is a potent activator of platelets ex vivo. However, because thrombin also mediates fibrin deposition and because multiple agonists can trigger platelet activation, the relative importance of platelet activation by thrombin in haemostasis and thrombosis is unknown. Thrombin triggers cellular responses at least in part through protease-activated receptors (PARs). Mouse platelets express PAR3 and PAR4 (ref. 9). Here we show that platelets from PAR4-deficient mice failed to change shape, mobilize calcium, secrete ATP or aggregate in response to thrombin. This result demonstrates that PAR signalling is necessary for mouse platelet activation by thrombin and supports the model that mouse PAR3 (mPAR3) does not by itself mediate transmembrane signalling but instead acts as a cofactor for thrombin cleavage and activation of mPAR4 (ref. 10). Importantly, PAR4-deficient mice had markedly prolonged bleeding times and were protected in a model of arteriolar thrombosis. Thus platelet activation by thrombin is necessary for normal haemostasis and may be an important target in the treatment of thrombosis.

  11. The refined 1.9-A X-ray crystal structure of D-Phe-Pro-Arg chloromethylketone-inhibited human alpha-thrombin: structure analysis, overall structure, electrostatic properties, detailed active-site geometry, and structure-function relationships.

    PubMed Central

    Bode, W.; Turk, D.; Karshikov, A.

    1992-01-01

    Thrombin is a multifunctional serine proteinase that plays a key role in coagulation while exhibiting several other key cellular bioregulatory functions. The X-ray crystal structure of human alpha-thrombin was determined in its complex with the specific thrombin inhibitor D-Phe-Pro-Arg chloromethylketone (PPACK) using Patterson search methods and a search model derived from trypsinlike proteinases of known spatial structure (Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S.R., & Hofsteenge, J., 1989, EMBO J. 8, 3467-3475). The crystallographic refinement of the PPACK-thrombin model has now been completed at an R value of 0.156 (8 to 1.92 A); in particular, the amino- and the carboxy-termini of the thrombin A-chain are now defined and all side-chain atoms localized; only proline 37 was found to be in a cis-peptidyl conformation. The thrombin B-chain exhibits the characteristic polypeptide fold of trypsinlike serine proteinases; 195 residues occupy topologically equivalent positions with residues in bovine trypsin and 190 with those in bovine chymotrypsin with a root-mean-square (r.m.s.) deviation of 0.8 A for their alpha-carbon atoms. Most of the inserted residues constitute novel surface loops. A chymotrypsinogen numbering is suggested for thrombin based on the topological equivalences. The thrombin A-chain is arranged in a boomeranglike shape against the B-chain globule opposite to the active site; it resembles somewhat the propeptide of chymotrypsin(ogen) and is similarly not involved in substrate and inhibitor binding. Thrombin possesses an exceptionally large proportion of charged residues. The negatively and positively charged residues are not distributed uniformly over the whole molecule, but are clustered to form a sandwichlike electrostatic potential; in particular, two extended patches of mainly positively charged residues occur close to the carboxy-terminal B-chain helix (forming the presumed heparin-binding site) and on the surface of loop segment 70

  12. Bidirectional functions of thrombin on fibrinolysis: Evidence of thrombin-dependent enhancement of fibrinolysis provided by spontaneous plasma clot lysis.

    PubMed

    Tomczyk, Martyna; Suzuki, Yuko; Sano, Hideto; Brzoska, Tomasz; Tanaka, Hiroki; Urano, Tetsumei

    2016-07-01

    Besides procoagulant activity, thrombin exhibits anticoagulant and profibrinolytic activities. We demonstrated that the euglobulin clot lysis time (ECLT) was shortened by endogenously generated thrombin as a result of the inactivation of plasminogen activator inhibitor type 1 (PAI-1). In contrast, thrombin suppressed fibrinolytic activity through the activation of thrombin activatable fibrinolysis inhibitor (TAFI). Here, using three different clot lysis assays of the ECLT, the tissue plasminogen activator supplemented plasma clot lysis time (tPA-PCLT) and the spontaneous plasma clot lysis time (s-PCLT), we analyzed how the coagulation process modifies fibrinolysis. The ECLT was shortened by exogenously supplemented thrombin in a dose-dependent manner in the absence of calcium ion (Ca(++)), whereas this shortening was not observed in the presence of Ca(++) where endogenous prothrombin was effectively activated to thrombin. This shortening was also not observed for the tPA-PCLT, in which tPA is supplemented in excess and PAI-1 activity is mostly lost. On the contrary, thrombin dose-dependently prolonged the tPA-PCLT, which was mostly abolished by inhibitors of carboxypeptidase and activated FXIII, suggesting that the prolongation is TAFI- and Factor XIII-dependent. The s-PCLT was shortened when thrombin generation was boosted by supplementing tissue factor and phosphatidylserine together with Ca(++), which was more apparent in the presence of inhibitors of activated FXIII and activated TAFI. Thus, thrombin appeared to express its enhancing effect on fibrinolysis even in plasma, in addition to its inhibiting effect. These bidirectional functions of thrombin on fibrinolysis seem to take place on demand under different environments to maintain adequate vascular blood flow. PMID:27179129

  13. Thrombin or Ca(++)-ionophore-mediated fall in endothelial ATP levels independent of poly(ADP-Ribose) polymerase activity and NAD levels--comparison with the effects of hydrogen peroxide.

    PubMed

    Halldórsson, Haraldur; Thors, Brynhildur; Thorgeirsson, Gudmundur

    2015-01-01

    To test the hypothesis that a fall in cellular ATP following stimulation of endothelial cells with thrombin is secondary to a decrease in NAD levels caused by poly(ADP-Ribose)polymerase (PARP), we measured the levels of NAD and ATP in endothelial cells after treatment with thrombin, the Ca(++)-ionophore A23187, or hydrogen peroxide (H2O2), and compared the effects of inhibitors of PARP, NAD synthesis, and ADP-ribose breakdown on these responses. Neither thrombin nor A23187 caused a reduction in endothelial NAD levels and A23187 affected ATP levels independently of NAD levels or PARP activity. H2O2 induced lowering of NAD caused modest lowering of ATP but marked additional ATP-lowering, independent of PARP and NAD, was also demonstrated. We conclude that in endothelial cells ATP levels are largely independent of NAD and PARP, which do not play a role in thrombin or Ca(++)-ionophore-mediated lowering of ATP. H2O2 caused ATP lowering through a similar mechanism as thrombin and A23187 but, additionally, caused a further ATP lowering through its intense stimulation of PARP and marked lowering of NAD. PMID:25774718

  14. Thrombin Cleavage of Osteopontin Modulates Its Activities in Human Cells In Vitro and Mouse Experimental Autoimmune Encephalomyelitis In Vivo

    PubMed Central

    Boggio, Elena; Gigliotti, Casimiro Luca; Soluri, Maria Felicia; Clemente, Nausicaa; Toth, Erika; Raineri, Davide; Ferrara, Benedetta; Chiocchetti, Annalisa

    2016-01-01

    Osteopontin is a proinflammatory cytokine and plays a pathogenetic role in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), by recruiting autoreactive T cells into the central nervous system. Osteopontin functions are modulated by thrombin cleavage generating N- and C-terminal fragment, whose individual roles are only partly known. Published data are difficult to compare since they have been obtained with heterogeneous approaches. Interestingly, thrombin cleavage of osteopontin unmasks a cryptic domain of interaction with α4β1 integrin that is the main adhesion molecule involved in lymphocyte transmigration to the brain and is the target for natalizumab, the most potent drug preventing relapses. We produced recombinant osteopontin and its N- and C-terminal fragments in an eukaryotic system in order to allow their posttranslational modifications. We investigated, in vitro, their effect on human cells and in vivo in EAE. We found that the osteopontin cleavage plays a key role in the function of this cytokine and that the two fragments exert distinct effects both in vitro and in vivo. These findings suggest that drugs targeting each fragment may be used to fine-tune the pathological effects of osteopontin in several diseases. PMID:27478856

  15. Thrombin Cleavage of Osteopontin Modulates Its Activities in Human Cells In Vitro and Mouse Experimental Autoimmune Encephalomyelitis In Vivo.

    PubMed

    Boggio, Elena; Dianzani, Chiara; Gigliotti, Casimiro Luca; Soluri, Maria Felicia; Clemente, Nausicaa; Cappellano, Giuseppe; Toth, Erika; Raineri, Davide; Ferrara, Benedetta; Comi, Cristoforo; Dianzani, Umberto; Chiocchetti, Annalisa

    2016-01-01

    Osteopontin is a proinflammatory cytokine and plays a pathogenetic role in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), by recruiting autoreactive T cells into the central nervous system. Osteopontin functions are modulated by thrombin cleavage generating N- and C-terminal fragment, whose individual roles are only partly known. Published data are difficult to compare since they have been obtained with heterogeneous approaches. Interestingly, thrombin cleavage of osteopontin unmasks a cryptic domain of interaction with α 4 β 1 integrin that is the main adhesion molecule involved in lymphocyte transmigration to the brain and is the target for natalizumab, the most potent drug preventing relapses. We produced recombinant osteopontin and its N- and C-terminal fragments in an eukaryotic system in order to allow their posttranslational modifications. We investigated, in vitro, their effect on human cells and in vivo in EAE. We found that the osteopontin cleavage plays a key role in the function of this cytokine and that the two fragments exert distinct effects both in vitro and in vivo. These findings suggest that drugs targeting each fragment may be used to fine-tune the pathological effects of osteopontin in several diseases. PMID:27478856

  16. Crystal structure of two new bifunctional nonsubstrate type thrombin inhibitors complexed with human alpha-thrombin.

    PubMed Central

    Féthière, J.; Tsuda, Y.; Coulombe, R.; Konishi, Y.; Cygler, M.

    1996-01-01

    The crystal structures of two new thrombin inhibitors, P498 and P500, complexed with human alpha-thrombin have been determined at 2.0 A resolution and refined to crystallographic R-factors of 0.170 and 0.169, respectively. These compounds, with picomolar binding constants, belong to a family of potent bifunctional inhibitors that bind thrombin at two remote sites: the active site and the fibrinogen recognition exosite (FRE). The inhibitors incorporate a nonsubstrate type active site binding fragment: Dansyl-Arg-(D)Pipecolic acid (Dns-Arg-(D)Pip), reminiscent of the active-site directed inhibitors MD-805 and MQPA, rendering them resistant to thrombin-induced hydrolysis. The FRE binding fragment of these inhibitors corresponds to the hirudin55-65 sequence. They differ in the chemical nature of the nonpeptidyl linker bridging these two functional activities. In both cases, the active site binding fragment is well defined in the electron density. The DnsH1, ArgH2, and (D)PipH3 groups occupy the S3, S1, and S2 subsites of thrombin, respectively, in a way similar to that observed in the thrombin-MQPA complexes. Binding in the active site of thrombin is characterized by numerous van der Waals contacts and ring-ring system interactions. Unlike in the substrate-like inhibitors, ArgH2 enters the S1 specificity pocket from the P2 position and adopts a bent conformation to make an hydrogen bond to the carboxylate of Asp189. In this noncanonical position, its carbonyl points away from the oxyanion hole, which is now occupied by well-ordered solvent molecules. The linkers fit in the groove extending from the active site to the FRE. The C-terminal fragments of both inhibitors bind in the same way as analogous FRE binding elements in previously described complexes. PMID:8762149

  17. Partial pressure measurements with an active spectrometer

    SciTech Connect

    Brooks, N.H.; Jensen, T.H.; Colchin, R.J.; Maingi, R.; Wade, M.R.; Finkenthal, D.F.; Naumenko, N.; Tugarinov, S.

    1998-07-01

    Partial pressure neutral ga measurements have been made using a commercial Penning gauge in conjunction with an active spectrometer. In prior work utilizing bandpass filters and conventional spectrometers, trace concentrations of the hydrogen isotopes H, D, T and of the noble gases He, Ne and Ar were determined from characteristic spectral lines in the light emitted by the neutral species of these elements. For all the elements mentioned, the sensitivity was limited by spectral contamination from a pervasive background of molecular hydrogen radiation. The active spectrometer overcomes this limitations by means of a digital lock-in method and correlation with reference spectra. Preliminary measurements of an admixture containing a trace amount of neon in deuterium show better than a factor of 20 improvement in sensitivity over conventional techniques. This can be further improved by correlating the relative intensities of multiple lines to sets of reference spectra.

  18. Inhibition of thrombin generation in plasma by fibrin formation (Antithrombin I).

    PubMed

    de Bosch, N B; Mosesson, M W; Ruiz-Sáez, A; Echenagucia, M; Rodriguez-Lemoin, A

    2002-08-01

    The adsorption of thrombin to fibrin during clotting defines "Antithrombin I" activity. We confirmed that thrombin generation in afibrinogenemic or in Reptilase defibrinated normal plasma was higher than in normal plasma. Repletion of these fibrinogen-deficient plasmas with fibrinogen 1 (gamma A/gamma A), whose fibrin has two "low affinity" non-substrate thrombin binding sites, resulted in moderately reduced thrombin generation by 29-37%. Repletion with fibrinogen 2 (gamma'/gamma A), which in addition to low affinity thrombin-binding sites in fibrin, has a "high affinity" non-substrate thrombin binding site in the carboxy-terminal region of its gamma' chain, was even more effective and reduced thrombin generation by 57-67%. Adding peptides that compete for thrombin binding to fibrin [S-Hir53-64 (hirugen) or gamma'414-427] caused a transient delay in the onset of otherwise robust thrombin generation, indicating that fibrin formation is necessary for full expression of Antithrombin I activity. Considered together, 1) the increased thrombin generation in afibrinogenemic or fibrinogen-depleted normal plasma that is mitigated by fibrinogen replacement; 2) evidence that prothrombin activation is increased in afibrinogenemia and normalized by fibrinogen replacement; 3) the severe thrombophilia that is associated with defective thrombin-binding in dysfibrinogenemias Naples I and New York I, and 4) the association of afibrinogenemia or hypofibrinogenemia with venous or arterial thromboembolism, indicate that Antithrombin I (fibrin) modulates thromboembolic potential by inhibiting thrombin generation in blood. PMID:12195697

  19. Anticoagulant activity of original synthetic peptide derivatives.

    PubMed

    Drozd, N N; Tolstenkov, A S; Makarov, V A; Miphtakhova, N T; Voyushina, T L; Sergeev, M E

    2008-01-01

    Original synthetic peptide derivatives exhibit anticoagulant activity in vitro and in vivo. They delayed fibrin clot formation from human blood plasma in tests for the intrinsic coagulation pathway (activated partial thromboplastin time) and final stage of plasma coagulation (thrombin time) and inhibited amidolytic activity of thrombin. We determined the minimum effective dose of the most active compound providing a 2-fold lengthening of blood clotting time (activated partial thromboplastin time test and thrombin time test), which persisted for 2-3 h. PMID:19024001

  20. [THE TEST OF GENERATION OF THROMBIN IN DYNAMICS IN PATIENTS AFTER TRANSCUTANEOUS CORONARY INTERVENTION].

    PubMed

    Napalkova, O S; Emanuel, V L; Karpenko, M A; Berezovskaia, G A; Iakovlev, A N; Yudina, V A; Vasilieva, E Yu; Lapin, S V; Tishkov, A V; Hisheva, N A

    2015-04-01

    The article presents results of observation of generation of thrombin in patients with ischemic heart disease in different terms after transcutaneous coronary intervention. The sampling included 37 patients with stable ischemic heart disease. The control group included 30 healthy individuals. To study system of hemostasis of this category of patients the test of generation of thrombin and its modification with added thrombomodulin were applied for evaluating anti-coagulant activity of system of protein C. The comparison of indicators of test of generation of thrombin in patients with ischemic heart disease before operation and in individuals of control group revealed no reliable differences (p > 0.05). The observation of patients with stable ischemic heart disease in various time-frame after mechanical re-vascularization of myocardium established significant increasing of generation of thrombin and decreasing of anticoagulant activity of system of protein C at 1-3 day after operation (p < 0.05). The positive correlation was established between endogenous thrombin potential and annexin 5, an early marker of dysfunction of endothelium in mentioned time-frame after operation (p = 0.0008; r = 0.57). The significant increasing of content of anti-inflammatory markers of C-reactive protein and fibrinogen was observed at 1-3 day after transcutaneous coronary intervention (p < 0.05). In that way, the study data give evidence to hyper-coagulation in patients with stable ischemic heart disease in early time-frame after operation despite normal values of activated partial thromboplastin time, prothrombin time, D-dimer and applied standard disaggregant therapy. PMID:26189289

  1. Thrombin Receptor-Activating Protein (TRAP)-Activated Akt Is Involved in the Release of Phosphorylated-HSP27 (HSPB1) from Platelets in DM Patients

    PubMed Central

    Tokuda, Haruhiko; Kuroyanagi, Gen; Tsujimoto, Masanori; Matsushima-Nishiwaki, Rie; Akamatsu, Shigeru; Enomoto, Yukiko; Iida, Hiroki; Otsuka, Takanobu; Ogura, Shinji; Iwama, Toru; Kojima, Kumi; Kozawa, Osamu

    2016-01-01

    It is generally known that heat shock protein 27 (HSP27) is phosphorylated through p38 mitogen-activated protein (MAP) kinase. We have previously reported that HSP27 is released from human platelets associated with collagen-induced phosphorylation. In the present study, we conducted an investigation into the effect of thrombin receptor-activating protein (TRAP) on the release of HSP27 in platelets in type 2 diabetes mellitus (DM) patients. The phosphorylated-HSP27 levels induced by TRAP were directly proportional to the aggregation of platelets. The levels of phosphorylated-HSP27 (Ser-78) were correlated with the levels of phosphorylated-p38 MAP kinase and phosphorylated-Akt in the platelets stimulated by 10 µM TRAP but not with those of phosphorylated-p44/p42 MAP kinase. The levels of HSP27 released from the TRAP (10 µM)-stimulated platelets were correlated with the levels of phosphorylated-HSP27 in the platelets. The released platelet-derived growth factor-AB (PDGF-AB) levels were in parallel with the HSP27 levels released from the platelets stimulated by 10 µM TRAP. Although the area under the curve (AUC) of small aggregates (9–25 µm) induced by 10 µM TRAP showed no significant correlation with the released HSP27 levels, AUC of medium aggregates (25–50 µm), large aggregates (50–70 µm) and light transmittance were significantly correlated with the released HSP27 levels. TRAP-induced phosphorylation of HSP27 was truly suppressed by deguelin, an inhibitor of Akt, in the platelets from a healthy subject. These results strongly suggest that TRAP-induced activation of Akt in addition to p38 MAP kinase positively regulates the release of phosphorylated-HSP27 from human platelets, which is closely related to the platelet hyper-aggregation in type 2 DM patients. PMID:27187380

  2. Thrombin Receptor-Activating Protein (TRAP)-Activated Akt Is Involved in the Release of Phosphorylated-HSP27 (HSPB1) from Platelets in DM Patients.

    PubMed

    Tokuda, Haruhiko; Kuroyanagi, Gen; Tsujimoto, Masanori; Matsushima-Nishiwaki, Rie; Akamatsu, Shigeru; Enomoto, Yukiko; Iida, Hiroki; Otsuka, Takanobu; Ogura, Shinji; Iwama, Toru; Kojima, Kumi; Kozawa, Osamu

    2016-01-01

    It is generally known that heat shock protein 27 (HSP27) is phosphorylated through p38 mitogen-activated protein (MAP) kinase. We have previously reported that HSP27 is released from human platelets associated with collagen-induced phosphorylation. In the present study, we conducted an investigation into the effect of thrombin receptor-activating protein (TRAP) on the release of HSP27 in platelets in type 2 diabetes mellitus (DM) patients. The phosphorylated-HSP27 levels induced by TRAP were directly proportional to the aggregation of platelets. The levels of phosphorylated-HSP27 (Ser-78) were correlated with the levels of phosphorylated-p38 MAP kinase and phosphorylated-Akt in the platelets stimulated by 10 µM TRAP but not with those of phosphorylated-p44/p42 MAP kinase. The levels of HSP27 released from the TRAP (10 µM)-stimulated platelets were correlated with the levels of phosphorylated-HSP27 in the platelets. The released platelet-derived growth factor-AB (PDGF-AB) levels were in parallel with the HSP27 levels released from the platelets stimulated by 10 µM TRAP. Although the area under the curve (AUC) of small aggregates (9-25 µm) induced by 10 µM TRAP showed no significant correlation with the released HSP27 levels, AUC of medium aggregates (25-50 µm), large aggregates (50-70 µm) and light transmittance were significantly correlated with the released HSP27 levels. TRAP-induced phosphorylation of HSP27 was truly suppressed by deguelin, an inhibitor of Akt, in the platelets from a healthy subject. These results strongly suggest that TRAP-induced activation of Akt in addition to p38 MAP kinase positively regulates the release of phosphorylated-HSP27 from human platelets, which is closely related to the platelet hyper-aggregation in type 2 DM patients. PMID:27187380

  3. Role of clot-associated (-derived) thrombin in cell proliferation induced by fibrin clots in vitro

    PubMed Central

    Gandossi, E; Lunven, C; Berry, C N

    2000-01-01

    Thrombin is a potent mitogenic agent. Clot-associated thrombin retains its amidolytic and pro-aggregant activity. We therefore studied the ability of fibrin clots to induce proliferation in CCL39 cells (Chinese hamster lung fibroblasts), in the absence and presence of the thrombin inhibitors PPACK, recombinant hirudin (rHV2 Lys47) and heparin:antithrombin III. Fibrin clots incubated for 48 h with CCL39 cells led to significant cell proliferation, which was dependent on the concentration of thrombin used to prepare the clots. Thus, clots prepared with 91 nmol l−1 thrombin produced a similar proliferation (231±21%) to that obtained with 50 nmol l−1 thrombin in solution (213±29%). Rabbit plasma clots led to a 499±41% increase in cell number under identical conditions. Fibrin clot-induced cell proliferation was inhibited by all three thrombin inhibitors with no difference in IC50 values compared to those obtained against thrombin in solution, suggesting that cell proliferation be due to thrombin leaching from the clots. We found a time-dependent increase in thrombin release from the clots attaining a plateau at 24 h (∼61% of the total thrombin used in clot formation). Clots separated from the cells using porous cell culture chamber inserts led to similar proliferation to that of clots in contact with the cells. Thus fibrin-clot induced CCL39 proliferation is due to thrombin released from the clots. PMID:10696104

  4. Thrombin immobilized to extracellular matrix is a potent mitogen for vascular smooth muscle cells: nonenzymatic mode of action.

    PubMed Central

    Bar-Shavit, R; Benezra, M; Eldor, A; Hy-Am, E; Fenton, J W; Wilner, G D; Vlodavsky, I

    1990-01-01

    Esterolytically inactive diisopropyl fluorophosphate-conjugated thrombin (DIP-alpha-thrombin) stimulated 3H-thymidine incorporation and proliferation of growth-arrested vascular smooth muscle cells (SMCs), similar to native alpha-thrombin. Half-maximal mitogenic response of SMCs was obtained at 1 nM thrombin and was specifically blocked by the leech-derived, high-affinity thrombin inhibitor, hirudin. Native thrombin and a variety of thrombin species that were chemically modified to alter thrombin procoagulant or esterolytic functions were found to induce 3H-thymidine incorporation to a similar extent. Exposure of SMCs to DIP-alpha-thrombin caused a rapid and transient expression of the c-fos protooncogene, determined by Northern blot analysis. These results indicate that thrombin is a potent mitogen for SMCs through a distinct non-enzymatic domain. Binding of 125I-alpha-thrombin to SMC cultures revealed an apparent dissociation constant of 6 nM and an estimated 5.4 x 10(5) binding sites per cell. This binding was inhibited to the same extent by native thrombin and by its nonenzymatic form, DIP-alpha-thrombin. Moreover, the chemotactic fragment of thrombin (CB67-129), which failed to elicit a mitogenic response, competed for 125I-alpha-thrombin binding to SMCs. Cross-linking analysis of 125I-alpha-thrombin to SMCs revealed a specific cell-surface binding site 55 kDa in size. Finally, thrombin immobilized to a naturally produced extracellular matrix retained potent mitogenic activity toward SMCs. These observations lend support to the possibility that in vivo, subendothelial basement membranes sequester thrombin (as well as other bioactive molecules), which may stimulate localized and persistent growth of arterial SMCs. Thrombin may thus be involved directly in progression of atherosclerotic plaque formation. Images PMID:1963793

  5. Expression screening of bacterial libraries of recombinant alpha-1 proteinase inhibitor variants for candidates with thrombin inhibitory capacity.

    PubMed

    Bhakta, Varsha; Gierczak, Richard F; Sheffield, William P

    2013-12-01

    Exhaustive mutagenesis studies of the reactive centre loop (RCL), a key structural component of proteins belonging to the serpin superfamily of protease inhibitors, are complicated by the size of the RCL, serpin conformational complexity, and, for most serpins, the lack of a serpin-dependent phenotype of expressing cells. Here, we describe a thrombin capture assay that distinguished thrombin-inhibitory recombinant human alpha-1 proteinase inhibitor (API M358R) from non-inhibitory API variants in Escherichia coli lysates prepared from either single clones or pools. Binding of API proteins in the lysates to thrombin immobilized on microtiter plate wells was quantified via colour generated by a peroxidase-coupled anti-API antibody. Bacterial expression plasmids encoding inhibitory API M358R were mixed 1:99 with plasmids encoding non-inhibitory API T345R/M358R and the resulting library screened in pools of 10. All above-background signals arising from pools or subsequently re-probed single clones were linked to the presence of plasmids encoding API M358R. Screening of a portion of another expression library encoding hypervariable API with all possibilities at codons 352-358 also yielded only novel, thrombin-inhibitory variants. Probing a smaller library expressing all possible codons at Ala347 yielded the wild type, 6 different functional variants, one partially active variant, and two variants with no thrombin-inhibitory activity. API antigen levels varied considerably less among Ala347 variants than activity levels, and comparison of rate constants of inhibition of purified API variants to their corresponding thrombin capture assay lysate values was used to establish the sensitivity and specificity of the assay. The results indicate that the approach is sufficiently robust to correctly identify functional versus non-functional candidates in API expression libraries, and could be of value in systematically probing structure/function relationships not only in the API

  6. [Human thrombin: enzymatic properties, stability and standardization of preparation].

    PubMed

    Kolodzeĭskaia, M V; Chernyshenko, T M

    2002-01-01

    The work deals with estimation of thrombin preparation having such features as: sedimentation activity 3000-3200 NIH un. per 1 mg of protein and 97% of active centres. The enzyme isolated has been estimated according to the amidolytic activity on synthetic substrates S-2160 and BAPNA being equal 5200 and 185 milli un/mg of protein, respectively. According to the electrophoresis in PAAG in the presence of Ds-Na the preparation is homogenous, its molecular mass is 36000. The fibrinogen sedimentation time dependence on the isolated thrombin concentration has been estimated as well as the comparative analysis with the thrombin of the firm "Sigma" with the previously calibrated activity using the international standartion (coded P4) has been conducted. The absence of proportionality between the substrate sedimentation time and the preparation concentration has been determined. It has been revealed, that if the experimental findings are presented in the units 1/t against the thrombin units NIH the right lines are received within the limits used. The defreezing and secondary freezing of the preparation preserved under -20 degrees C have been showed as rendering an essential effect on thrombin activity. In order of the enzyme stabilizing at preserving the thrombin isolated has been concentrated applying the amycon membranes (MWCo: 30,000). While applying the thrombin water-saline solution in the conditions selected the preparation has showed itself practically stable during a year without utilizing any admixtures. The essential effect on thrombin has been found from the side of 1% glycin, 0.5% PEG, 1% saccharose and so on. The thrombin isolated high functional homogeneity, its stability permit to recommend the preparation as an operative standard. PMID:12916152

  7. The interaction of thrombin with platelet protease nexin

    SciTech Connect

    Knupp, C.L. )

    1989-10-01

    Thrombin interacts with a platelet protein which is immunologically related to fibroblast protease nexin and has been termed platelet protease nexin I (PNI). Conflicting hypotheses about the relationship of the thrombin-PNI complex formation to platelet activation have been proposed. The studies presented here demonstrate that the platelet-associated and supernatant complexes with added 125I-thrombin are formed only under conditions which produce platelet activation in normal and chymotrypsin-modified platelets. The platelet-associated complex is formed prior to the appearance of complexes in supernatants. Appearance of the supernatant complex coincides with the appearance of thrombospondin in the reaction supernatants. Excess native thrombin, dansylarginine N-(3-ethyl-1,5-pentanediyl) amide or hirudin can prevent radiolabeled platelet-associated complex formation if added before 125I-thrombin. DAPA or hirudin can prevent or dissociate complex formation if added up to one minute after thrombin but not at later time points. The surface associated complex is accessible to trypsin although a portion remains with the cytoskeletal proteins when thrombin-activated platelets are solubilized with Triton X 100. The surface-associated complex formation parallels many aspects of the specific measurable thrombin binding, yet it does not appear to involve other identified surface glycoprotein thrombin receptors or substrates. Although the time course of appearance of the complexes in supernatants is consistent with other data which suggest that PNI may be released from platelet granules during platelet activation, other explanations for the appearance of PNI on the platelet surface and in supernatants during platelet activation are possible.

  8. Interaction of thrombin des-ETW with antithrombin III, the Kunitz inhibitors, thrombomodulin and protein C. Structural link between the autolysis loop and the Tyr-Pro-Pro-Trp insertion of thrombin.

    PubMed

    Le Bonniec, B F; Guinto, E R; Esmon, C T

    1992-09-25

    X-ray diffraction studies of human thrombin revealed that compared with trypsin, two insertions (B and C) potentially limit access to the active site groove. When amino acids Glu146, Thr147, and Trp148, adjacent to the C-insertion (autolysis loop), are deleted the resulting thrombin (des-ETW) has dramatically altered interaction with serine protease inhibitors. Whereas des-ETW resists antithrombin III inactivation with a rate constant (Kon) approximately 350-fold slower than for thrombin, des-ETW is remarkably sensitive to the Kunitz inhibitors, with inhibition constants (Ki) decreased from 2.6 microM to 34 nM for the soybean trypsin inhibitor and from 52 microM to 1.8 microM for the bovine pancreatic trypsin inhibitor. The affinity for hirudin (Ki = 5.6 pM) is weakened at least 30-fold compared with recombinant thrombin. The mutation affects the charge stabilizing system and the primary binding pocket of thrombin as depicted by a decrease in Kon for diisopropylfluorophosphate (9.5-fold) and for N alpha-p-tosyl-L-lysine-chloromethyl ketone (51-fold) and a 39-fold increase in the Ki for benzamidine. With peptidyl p-nitroanilide substrates, the des-ETW deletion results in changes in the Michaelis (Km) and/or catalytic (kcat) constants, worsened as much as 85-fold (Km) or 100-fold (kcat). The specific clotting activity of des-ETW is less than 5% that of thrombin and the kcat/Km for protein C activation in the absence of cofactor less than 2%. Thrombomodulin binds to des-ETW with a dissociation constant of approximately 2.5 nM and partially restores its ability to activate protein C since, in the presence of the cofactor, kcat/Km rises to 6.5% that of thrombin. This study suggests that the ETW motif of thrombin prevents (directly or indirectly) its interaction with the two Kunitz inhibitors and is not essential for the thrombomodulin-mediated enhancement of protein C activation. PMID:1326550

  9. Effect of thrombin on maturing human megakaryocytes.

    PubMed Central

    Cramer, E. M.; Massé, J. M.; Caen, J. P.; Garcia, I.; Breton-Gorius, J.; Debili, N.; Vainchenker, W.

    1993-01-01

    Thrombin causes platelet activation and secretion. In some nucleated cells, it is mitogenic. In this study, we have investigated how human megakaryocytes (MKs) respond to this agonist and whether the response depends on the maturation stage. MKs were cultured from bone marrow precursors in liquid culture in the presence of normal plasma. To determine whether thrombin can activate MKs, 14-day MK cultures were incubated with thrombin for 5 minutes, and cells were studied by electron microscopy, either by standard techniques or after embedding in glycol-methacrylate for immunoelectron microscopy. Ultrastructural examination of thrombin-treated MKs revealed dramatic morphological changes reminiscent of those found in platelets, including shape change and organelle centralization that involved immature as well as mature cells. MKs were also able to secrete alpha-granule proteins in the dilated cisternae of the demarcation membrane system, as shown by immunogold staining for thrombospondin and glycoprotein Ib. These changes were rapid (less than 5 minutes) but despite them, MKs remained viable for more than 24 hours. To determine whether thrombin has a mitogenic activity, it was added to the culture of MKs from day 3 to day 10 of culture at concentrations varying from 0.1 to 10 U/ml. Cells were subsequently studied by a double staining technique using flow cytometry to determine MK number and ploidy. No changes were observed in these two parameters, showing that thrombin is not mitogenic for MKs at the concentrations used. In conclusion, this study confirms for human MKs previous observations made about guinea pig MKs (Fedorko et al, Lab Invest 1977, 36:32). In addition, it demonstrates that immature MKs are able to respond to thrombin and that more mature cells can secrete alpha-granule proteins into the demarcation membrane system, which is in continuity with the extracellular space. This phenomenon may have implications for pathological states such as myelofibrosis

  10. Site-specific replacement of the thymine methyl group by fluorine in thrombin binding aptamer significantly improves structural stability and anticoagulant activity

    PubMed Central

    Virgilio, Antonella; Petraccone, Luigi; Vellecco, Valentina; Bucci, Mariarosaria; Varra, Michela; Irace, Carlo; Santamaria, Rita; Pepe, Antonietta; Mayol, Luciano; Esposito, Veronica; Galeone, Aldo

    2015-01-01

    Here we report investigations, based on circular dichroism, nuclear magnetic resonance spectroscopy, molecular modelling, differential scanning calorimetry and prothrombin time assay, on analogues of the thrombin binding aptamer (TBA) in which individual thymidines were replaced by 5-fluoro-2′-deoxyuridine residues. The whole of the data clearly indicate that all derivatives are able to fold in a G-quadruplex structure very similar to the ‘chair-like’ conformation typical of the TBA. However, only ODNs TBA-F4 and TBA-F13 have shown a remarkable improvement both in the melting temperature (ΔTm ≈ +10) and in the anticoagulant activity in comparison with the original TBA. These findings are unusual, particularly considering previously reported studies in which modifications of T4 and T13 residues in TBA sequence have clearly proven to be always detrimental for the structural stability and biological activity of the aptamer. Our results strongly suggest the possibility to enhance TBA properties through tiny straightforward modifications. PMID:26582916

  11. The Catalytic Subunit of Protein Phosphatase 1 Gamma Regulates Thrombin-Induced Murine Platelet αIIbβ3 Function

    PubMed Central

    Gushiken, Francisca C.; Hyojeong, Han; Pradhan, Subhashree; Langlois, Kimberly W.; Alrehani, Nawaf; Cruz, Miguel A.; Rumbaut, Rolando E.; Vijayan, K. Vinod

    2009-01-01

    Background Hemostasis and thrombosis are regulated by agonist-induced activation of platelet integrin αIIbβ3. Integrin activation, in turn is mediated by cellular signaling via protein kinases and protein phosphatases. Although the catalytic subunit of protein phosphatase 1 (PP1c) interacts with αIIbβ3, the role of PP1c in platelet reactivity is unclear. Methodology/Principal Findings Using γ isoform of PP1c deficient mice (PP1cγ−/−), we show that the platelets have moderately decreased soluble fibrinogen binding and aggregation to low concentrations of thrombin or protease-activated receptor 4 (PAR4)-activating peptide but not to adenosine diphosphate (ADP), collagen or collagen-related peptide (CRP). Thrombin-stimulated PP1cγ−/− platelets showed decreased αIIbβ3 activation despite comparable levels of αIIbβ3, PAR3, PAR4 expression and normal granule secretion. Functions regulated by outside-in integrin αIIbβ3 signaling like adhesion to immobilized fibrinogen and clot retraction were not altered in PP1cγ−/− platelets. Thrombus formation induced by a light/dye injury in the cremaster muscle venules was significantly delayed in PP1cγ−/− mice. Phosphorylation of glycogen synthase kinase (GSK3)β-serine 9 that promotes platelet function, was reduced in thrombin-stimulated PP1cγ−/− platelets by an AKT independent mechanism. Inhibition of GSK3β partially abolished the difference in fibrinogen binding between thrombin-stimulated wild type and PP1cγ−/− platelets. Conclusions/Significance These studies illustrate a role for PP1cγ in maintaining GSK3β-serine9 phosphorylation downstream of thrombin signaling and promoting thrombus formation via fibrinogen binding and platelet aggregation. PMID:20016849

  12. JAK2-STAT3 signaling pathway mediates thrombin-induced proinflammatory actions of microglia in vitro.

    PubMed

    Huang, Chengfang; Ma, Rong; Sun, Shenggang; Wei, Guirong; Fang, Yuan; Liu, Rengang; Li, Gang

    2008-11-15

    The present study shows that JAK2-STAT3 inflammatory signaling mediates thrombin-stimulated microglia activation. In rat primary microglia, thrombin rapidly activated JAK2 and induced phosphorylation of STAT3. In addition, thrombin increased transcription of the inflammation-associated genes tumor necrosis factor (TNF)-alpha, inducible nitric oxide synthase (iNOS), production of TNF-alpha, NO and induced neurodegeneration of dopaminergic neurons in mesencephalic cultures. AG490, a JAK inhibitor, markedly reduced activation of JAK2 and STAT3 in thrombin-treated microglia. AG490 also inhibited thrombin-induced transcription and expression of TNF-alpha, iNOS and/or NO release, moreover rescued dopaminergic neurons. These results suggest that JAK2-STAT3 signaling pathway plays a critical role in mediating thrombin-induced activation of microglia and degeneration of dopaminergic neurons. PMID:18710787

  13. Stanniocalcin-1 inhibits thrombin-induced signaling and protects from bleomycin-induced lung injury

    PubMed Central

    Huang, Luping; Zhang, Lin; Ju, Huiming; Li, Qingtian; Pan, Jenny Szu-Chin; Al-Lawati, Zahraa; Sheikh-Hamad, David

    2015-01-01

    Thrombin-induced and proteinase-activated receptor 1 (PAR1)-mediated signaling increases ROS production, activates ERK, and promotes inflammation and fibroblast proliferation in bleomycin-induced lung injury. Stanniocalcin-1 (STC1) activates anti-oxidant pathways, inhibits inflammation and provides cytoprotection; hence, we hypothesized that STC1 will inhibit thrombin/PAR1 signaling and protect from bleomycin-induced pneumonitis. We determined thrombin level and activity, thrombin-induced PAR-1-mediated signaling, superoxide generation and lung pathology after intra-tracheal administration of bleomycin to WT and STC1 Tg mice. Lungs of bleomycin-treated WT mice display: severe pneumonitis; increased generation of superoxide; vascular leak; increased thrombin protein abundance and activity; activation of ERK; greater cytokine/chemokine release and infiltration with T-cells and macrophages. Lungs of STC1 Tg mice displayed none of the above changes. Mechanistic analysis in cultured pulmonary epithelial cells (A549) suggests that STC1 inhibits thrombin-induced and PAR1-mediated ERK activation through suppression of superoxide. In conclusion, STC1 blunts bleomycin-induced rise in thrombin protein and activity, diminishes thrombin-induced signaling through PAR1 to ERK, and inhibits bleomycin-induced pneumonitis. Moreover, our study identifies a new set of cytokines/chemokines, which play a role in the pathogenesis of bleomycin-induced lung injury. These findings broaden the array of potential therapeutic targets for the treatment of lung diseases characterized by thrombin activation, oxidant stress and inflammation. PMID:26640170

  14. Antithrombotic effects of bromophenol, an alga-derived thrombin inhibitor

    NASA Astrophysics Data System (ADS)

    Shi, Dayong; Li, Xiaohong; Li, Jing; Guo, Shuju; Su, Hua; Fan, Xiao

    2010-01-01

    Thrombin, the ultimate proteinase of the coagulation cascade, is an attractive target for the treatment of a variety of cardiovascular diseases. A bromophenol derivative named (+)-3-(2,3-dibromo-4, 5-dihydroxy-phenyl)-4-bromo-5,6-dihydroxy-1,3-dihydroiso-benzofuran 1, isolated from the brown alga Leathesia nana exhibited significant thrombin inhibitory activity. In this study, we investigated the inhibition of human thrombin in vitro with this bromophenol derivative, and its antithrombotic efficacy in vivo using the arteriovenous shunt model and the ferric chloride-induced arterial thrombosis model in rats. The results show that the bromophenol derivative is a potential inhibitor of thrombin (IC50=1.03 nmol/L). In antithrombotic experiments in vivo, the bromophenol derivative also shows good effect comparing with the control group. These data indicate that the bromophenol derivative is a potential drug for prophylaxis and the treatment of thrombotic diseases.

  15. Development of a Novel Fluorescence Assay Based on the Use of the Thrombin-Binding Aptamer for the Detection of O-Alkylguanine-DNA Alkyltransferase Activity.

    PubMed

    Tintoré, Maria; Aviñó, Anna; Ruiz, Federico M; Eritja, Ramón; Fàbrega, Carme

    2010-01-01

    Human O(6)-alkylguanine-DNA alkyltransferase (hAGT) is a DNA repair protein that reverses the effects of alkylating agents by removing DNA adducts from the O(6) position of guanine. Here, we developed a real-time fluorescence hAGT activity assay that is based on the detection of conformational changes of the thrombin-binding aptamer (TBA). The quadruplex structure of TBA is disrupted when a central guanine is replaced by an O(6)-methyl-guanine. The sequence also contains a fluorophore (fluorescein) and a quencher (dabsyl) attached to the opposite ends. In the unfolded structure, the fluorophore and the quencher are separated. When hAGT removes the methyl group from the central guanine of TBA, it folds back immediately into its quadruplex structure. Consequently, the fluorophore and the quencher come into close proximity, thereby resulting in decreased fluorescence intensity. Here, we developed a new method to quantify the hAGT without using radioactivity. This new fluorescence resonance energy transfer assay has been designed to detect the conformational change of TBA that is induced by the removal of the O(6)-methyl group. PMID:20936180

  16. Role of Krev Interaction Trapped-1 in Prostacyclin-Induced Protection against Lung Vascular Permeability Induced by Excessive Mechanical Forces and Thrombin Receptor Activating Peptide 6.

    PubMed

    Meliton, Angelo; Meng, Fanyong; Tian, Yufeng; Shah, Alok A; Birukova, Anna A; Birukov, Konstantin G

    2015-12-01

    Mechanisms of vascular endothelial cell (EC) barrier regulation during acute lung injury (ALI) or other pathologies associated with increased vascular leakiness are an active area of research. Adaptor protein krev interaction trapped-1 (KRIT1) participates in angiogenesis, lumen formation, and stabilization of EC adherens junctions (AJs) in mature vasculature. We tested a role of KRIT1 in the regulation of Rho-GTPase signaling induced by mechanical stimulation and barrier dysfunction relevant to ventilator-induced lung injury and investigated KRIT1 involvement in EC barrier protection by prostacyclin (PC). PC stimulated Ras-related protein 1 (Rap1)-dependent association of KRIT1 with vascular endothelial cadherin at AJs, with KRIT1-dependent cortical cytoskeletal remodeling leading to EC barrier enhancement. KRIT1 knockdown exacerbated Rho-GTPase activation and EC barrier disruption induced by pathologic 18% cyclic stretch and thrombin receptor activating peptide (TRAP) 6 and attenuated the protective effects of PC. In the two-hit model of ALI caused by high tidal volume (HTV) mechanical ventilation and TRAP6 injection, KRIT1 functional deficiency in KRIT1(+/-) mice increased basal lung vascular leak and augmented vascular leak and lung injury caused by exposure to HTV and TRAP6. Down-regulation of KRIT1 also diminished the protective effects of PC against TRAP6/HTV-induced lung injury. These results demonstrate a KRIT1-dependent mechanism of vascular EC barrier control in basal conditions and in the two-hit model of ALI caused by excessive mechanical forces and TRAP6 via negative regulation of Rho activity and enhancement of cell junctions. We also conclude that the stimulation of the Rap1-KRIT1 signaling module is a major mechanism of vascular endothelial barrier protection by PC in the injured lung. PMID:25923142

  17. Thrombin regulation of synaptic transmission and plasticity: implications for health and disease

    PubMed Central

    Ben Shimon, Marina; Lenz, Maximilian; Ikenberg, Benno; Becker, Denise; Shavit Stein, Efrat; Chapman, Joab; Tanne, David; Pick, Chaim G.; Blatt, Ilan; Neufeld, Miri; Vlachos, Andreas; Maggio, Nicola

    2015-01-01

    Thrombin, a serine protease involved in the blood coagulation cascade has been shown to affect neural function following blood-brain barrier breakdown. However, several lines of evidence exist that thrombin is also expressed in the brain under physiological conditions, suggesting an involvement of thrombin in the regulation of normal brain functions. Here, we review ours’ as well as others’ recent work on the role of thrombin in synaptic transmission and plasticity through direct or indirect activation of Protease-Activated Receptor-1 (PAR1). These studies propose a novel role of thrombin in synaptic plasticity, both in physiology as well as in neurological diseases associated with increased brain thrombin/PAR1 levels. PMID:25954157

  18. Sucrose Octasulfate Selectively Accelerates Thrombin Inactivation by Heparin Cofactor II*

    PubMed Central

    Sarilla, Suryakala; Habib, Sally Y.; Kravtsov, Dmitri V.; Matafonov, Anton; Gailani, David; Verhamme, Ingrid M.

    2010-01-01

    Inactivation of thrombin (T) by the serpins heparin cofactor II (HCII) and antithrombin (AT) is accelerated by a heparin template between the serpin and thrombin exosite II. Unlike AT, HCII also uses an allosteric interaction of its NH2-terminal segment with exosite I. Sucrose octasulfate (SOS) accelerated thrombin inactivation by HCII but not AT by 2000-fold. SOS bound to two sites on thrombin, with dissociation constants (KD) of 10 ± 4 μm and 400 ± 300 μm that were not kinetically resolvable, as evidenced by single hyperbolic SOS concentration dependences of the inactivation rate (kobs). SOS bound HCII with KD 1.45 ± 0.30 mm, and this binding was tightened in the T·SOS·HCII complex, characterized by Kcomplex of ∼0.20 μm. Inactivation data were incompatible with a model solely depending on HCII·SOS but fit an equilibrium linkage model employing T·SOS binding in the pathway to higher order complex formation. Hirudin-(54–65)(SO3−) caused a hyperbolic decrease of the inactivation rates, suggesting partial competitive binding of hirudin-(54–65)(SO3−) and HCII to exosite I. Meizothrombin(des-fragment 1), binding SOS with KD = 1600 ± 300 μm, and thrombin were inactivated at comparable rates, and an exosite II aptamer had no effect on the inactivation, suggesting limited exosite II involvement. SOS accelerated inactivation of meizothrombin 1000-fold, reflecting the contribution of direct exosite I interaction with HCII. Thrombin generation in plasma was suppressed by SOS, both in HCII-dependent and -independent processes. The ex vivo HCII-dependent process may utilize the proposed model and suggests a potential for oversulfated disaccharides in controlling HCII-regulated thrombin generation. PMID:20053992

  19. Antiplatelet therapy: thrombin receptor antagonists

    PubMed Central

    Tello-Montoliu, Antonio; Tomasello, Salvatore D; Ueno, Masafumi; Angiolillo, Dominick J

    2011-01-01

    Activated platelets stimulate thrombus formation in response to rupture of an atherosclerotic plaque or endothelial cell erosion, promoting atherothrombotic disease. Multiple pathways contribute to platelet activation. Aspirin, an irreversible inhibitor of thromboxane A2 synthesis, in combination with clopidogrel, an inhibitor of P2Y12 adenosine diphosphate platelet receptors, represent the current standard-of-care of antiplatelet therapy for patients with acute coronary syndrome and for those undergoing percutaneous coronary intervention. Although these agents have demonstrated significant clinical benefit, the increased risk of bleeding and the recurrence of thrombotic events represent substantial limitations. Thrombin is one of the most important platelet activators. The inhibition of protease-activated receptor 1 showed a good safety profile in preclinical studies. In fact, phase II studies with vorapaxar (SCH530348) and atopaxar (E5555) showed no increase of bleeding events in addition to the current standard-of-care of antiplatelet therapy. Although the results of phase III trials for both drugs are awaited, this family is a promising new addition to the current clinical practice for patients with atherothrombotic disease, not only as an alternative, but also as additional therapy. PMID:21906120

  20. Thrombin Generation in Zebrafish Blood

    PubMed Central

    Hemker, Coenraad; Lindhout, Theo; Kelchtermans, Hilde; de Laat, Bas

    2016-01-01

    To better understand hypercoagulability as an underlying cause for thrombosis, the leading cause of death in the Western world, new assays to study ex vivo coagulation are essential. The zebrafish is generally accepted as a good model for human hemostasis and thrombosis, as the hemostatic system proved to be similar to that in man. Their small size however, has been a hurdle for more widespread use in hemostasis related research. In this study we developed a method that enables the measurement of thrombin generation in a single drop of non-anticoagulated zebrafish blood. Pre-treatment of the fish with inhibitors of FXa and thrombin, resulted in a dose dependent diminishing of thrombin generation, demonstrating the validity of the assay. In order to establish the relationship between whole blood thrombin generation and fibrin formation, we visualized the resulting fibrin network by scanning electron microscopy. Taken together, in this study we developed a fast and reliable method to measure thrombin generation in whole blood collected from a single zebrafish. Given the similarities between coagulation pathways of zebrafish and mammals, zebrafish may be an ideal animal model to determine the effect of novel therapeutics on thrombin generation. Additionally, because of the ease with which gene functions can be silenced, zebrafish may serve as a model organism for mechanistical research in thrombosis and hemostasis. PMID:26872266

  1. Human platelets stimulated by thrombin produce platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) when the degrading enzyme acetyl hydrolase is blocked.

    PubMed Central

    Touqui, L; Hatmi, M; Vargaftig, B B

    1985-01-01

    It has been shown [Touqui, Jacquemin & Vargaftig (1983) Thromb. Haemostasis 50, 163; Touqui, Jacquemin & Vargaftig (1983) Biochem. Biophys. Res. Commun. 110, 890-893; Alam, Smith & Melvin (1983) Lipids 18, 534-538; Pieroni & Hanahan (1983) Arch. Biochem. Biophys. 224, 485-493] that rabbit platelets inactivate exogenous PAF (platelet-activating factor, PAF-acether) by a deacetylation-reacylation mechanism. The deacetylation step is catalysed by an acetyl hydrolase sensitive to the serine-hydrolase inhibitor PMSF (phenylmethanesulphonyl fluoride) [Touqui, Jacquemin, Dumarey & Vargaftig (1985) Biochim. Biophys. Acta 833, 111-118]. We report here that human platelets can produce PAF on thrombin stimulation. This production is marginal and transient, reaching a maximum at 10 min and decreasing thereafter. In contrast, 10-12 times more PAF is produced when platelets are treated with PMSF and stimulated with thrombin. Under these conditions, the maximum formation is observed at 30 min and no decline occurs for up to 60 min after stimulation. In addition, these platelets (treated with PMSF and stimulated with thrombin) incorporate exogenous labelled acetate in the 2-position of PAF, probably by an acetyltransferase-dependent mechanism. Production of PAF by human platelets during physiological stimulation can be demonstrated when PAF degradation is suppressed by the acetyl-hydrolase inhibitor PMSF. PMID:4052028

  2. Isolation and characterization of a new serine protease with thrombin-like activity (TLBm) from the venom of the snake Bothrops marajoensis.

    PubMed

    Vilca-Quispe, Augusto; Ponce-Soto, Luis Alberto; Winck, Flavia Vischi; Marangoni, Sergio

    2010-04-01

    The thrombin-like serine protease TLBm from Bothrops marajoensis was isolated in one chromatographic step in reverse phase HPLC. Its molecular mass was 33239.95 Da, as based on the determined primary structure and confirmed experimentally by MALDI-TOF mass spectrometry (33332.5 Da) and it contains 12 half-cysteine residues. This TLBm exhibited high specificity for BArhoNA, Michaelis-Menten behavior with K(m) 2.3x10(-1)M and the V(max) 0.52x10(-1) nmoles rho-NA/lt/min for this substrate. TLBm also showed ability to coagulate bovine fibrinogen and was inhibited by soybean trypsin inhibitor, EDTA and S(Dm) from the serum of the species Didelphis marsupialis. The primary structure of TLBm showed the presence of His(45), Asp(103) and Ser(228) residues in the corresponding positions of the catalytic triad established in the serine proteases and Ser(228) are inhibited by phenylmethylsulfonyl fluoride (PMSF). Amino acid analysis showed a high content of Asp, Glu, Gly, Ser, Ala and Pro as well as 12 half-cysteine residues and calculated pI of 6.47; TLBm presented 285 amino acid residues. In this work, we investigated the ability of TLBm to degrade fibrinogen and we observed that it is able to cause alpha- and beta-chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with PMSF, a specific inhibitor of serine protease. Also, TLBm induced platelet aggregation in washed and platelet-rich plasma, and in both cases, PMSF inhibited its activity. PMID:19931298

  3. PDZ-RhoGEF and LARG Are Essential for Embryonic Development and Provide a Link between Thrombin and LPA Receptors and Rho Activation*

    PubMed Central

    Mikelis, Constantinos M.; Palmby, Todd R.; Simaan, May; Li, Wenling; Szabo, Roman; Lyons, Ruth; Martin, Daniel; Yagi, Hiroshi; Fukuhara, Shigetomo; Chikumi, Hiroki; Galisteo, Rebeca; Mukouyama, Yoh-suke; Bugge, Thomas H.; Gutkind, J. Silvio

    2013-01-01

    G protein-coupled receptors (GPCRs) linked to both members of the Gα12 family of heterotrimeric G proteins α subunits, Gα12 and Gα13, regulate the activation of Rho GTPases, thereby contributing to many key biological processes. Multiple Rho GEFs have been proposed to link Gα12/13 GPCRs to Rho activation, including PDZ-RhoGEF (PRG), leukemia-associated Rho GEF (LARG), p115-RhoGEF (p115), lymphoid blast crisis (Lbc), and Dbl. PRG, LARG, and p115 share the presence of a regulator of G protein signaling homology (RGS) domain. There is limited information on the biological roles of this RGS-containing family of RhoGEFs in vivo. p115-deficient mice are viable with some defects in the immune system and gastrointestinal motor dysfunctions, whereas in an initial study we showed that mice deficient for Larg are viable and resistant to salt-induced hypertension. Here, we generated knock-out mice for Prg and observed that these mice do not display any overt phenotype. However, deficiency in Prg and Larg leads to complex developmental defects and early embryonic lethality. Signaling from Gα11/q-linked GPCRs to Rho was not impaired in mouse embryonic fibroblasts defective in all three RGS-containing RhoGEFs. However, a combined lack of Prg, Larg, and p115 expression abolished signaling through Gα12/13 to Rho and thrombin-induced cell proliferation, directional migration, and nuclear signaling through JNK and p38. These findings provide evidence of an essential role for the RGS-containing RhoGEF family in signaling to Rho by Gα12/13-coupled GPCRs, which may likely play a critical role during embryonic development. PMID:23467409

  4. Hirudin as a molecular probe for thrombin in vitro and during systemic coagulation in the pig.

    PubMed Central

    Zoldhelyi, P; Chesebro, J H; Owen, W G

    1993-01-01

    The amount of thrombin active in vivo in the intravascular space (blood and endothelial surface), both basally and in experimental intravascular coagulation, is measured by way of the accessibility of thrombin to intravascular hirudin. Blood samples from pigs given intravenous 125I-labeled hirudin contain 125I-labeled hirudin-thrombin complex in concentrations indicative of a basal thrombin concentration in vivo of 0.5 nmol/liter. Intravenous infusion of Salmonella endotoxin elicits an increase in the circulating concentration of hirudin-thrombin complex that begins within 15 min and is 20-30 times basal after 4 hr. Induction of mild intravascular coagulation is evidenced by a modest reduction in plasma fibrinogen concentrations. It is concluded that there is a basal pool of hirudin-accessible thrombin in the intravascular space that, were it free in the plasma phase, would be sufficient in principle to sustain intravascular coagulation. Images Fig. 5 PMID:8446595

  5. Nanocomplexation of thrombin with cationic amylose derivative for improved stability and hemostatic efficacy.

    PubMed

    Zhuang, Baoxiong; Li, Zhihua; Pang, Jiadong; Li, Wenbin; Huang, Pinbo; Wang, Jie; Zhou, Yu; Lin, Qing; Zhou, Quanbo; Ye, Xiao; Ye, Huilin; Liu, Yimin; Zhang, Li-Ming; Chen, Rufu

    2015-01-01

    As a topical hemostatic agent, thrombin has wide application for many surgical treatments. However, native thrombin always suffers from its physical and chemical instabilities. In this work, a nanocomplexation strategy was developed for modifying the stability and hemostatic efficacy of thrombin, in which a water-soluble cationic amylose derivative containing poly(l-lysine) dendrons was prepared by a click reaction and then used to complex thrombin in an aqueous system. For resultant thrombin nanocomplexes, their morphology and particle size distribution were investigated. Their stabilities were studied in terms of activity retention percentages under different storage time, pH values, and illumination time. In addition, their ability to achieve in vitro fibrinogen and blood coagulation were evaluated. Via a rat hepatic hemorrhage model and a rat iliac artery hemorrhage model, these thrombin nanocomplexes were confirmed to have good tissue biocompatibility and in vivo hemostatic effectiveness. PMID:25673989

  6. Nanocomplexation of thrombin with cationic amylose derivative for improved stability and hemostatic efficacy

    PubMed Central

    Zhuang, Baoxiong; Li, Zhihua; Pang, Jiadong; Li, Wenbin; Huang, Pinbo; Wang, Jie; Zhou, Yu; Lin, Qing; Zhou, Quanbo; Ye, Xiao; Ye, Huilin; Liu, Yimin; Zhang, Li-Ming; Chen, Rufu

    2015-01-01

    As a topical hemostatic agent, thrombin has wide application for many surgical treatments. However, native thrombin always suffers from its physical and chemical instabilities. In this work, a nanocomplexation strategy was developed for modifying the stability and hemostatic efficacy of thrombin, in which a water-soluble cationic amylose derivative containing poly(l-lysine) dendrons was prepared by a click reaction and then used to complex thrombin in an aqueous system. For resultant thrombin nanocomplexes, their morphology and particle size distribution were investigated. Their stabilities were studied in terms of activity retention percentages under different storage time, pH values, and illumination time. In addition, their ability to achieve in vitro fibrinogen and blood coagulation were evaluated. Via a rat hepatic hemorrhage model and a rat iliac artery hemorrhage model, these thrombin nanocomplexes were confirmed to have good tissue biocompatibility and in vivo hemostatic effectiveness. PMID:25673989

  7. Synthesis and biochemical evaluation of triazole/tetrazole-containing sulfonamides against thrombin and related serine proteases

    PubMed Central

    Siles, Rogelio; Kawasaki, Yuko; Ross, Patrick; Freire, Ernesto

    2011-01-01

    A small library of 25 triazole/tetrazole-based sulfonamides have been synthesized and further evaluated for their inhibitory activity against thrombin, trypsin, tryptase and chymase. In general, the triazole-based sulfonamides inhibited thrombin more efficiently than the tetrazole counterparts. Particularly, compound 26 showed strong thrombin inhibition (Ki =880 nM) and significant selectivity against other human related serine proteases like trypsin (Ki =729 µM). Thrombin binding affinity of the same compound was determined by ITC and demonstrated that the binding of this new triazole-based scaffold is enthalpically driven, making it a good candidate for further development. PMID:21807511

  8. A comparative evaluation of assays for markers of activated coagulation and/or fibrinolysis: thrombin-antithrombin complex, D-dimer and fibrinogen/fibrin fragment E antigen.

    PubMed

    Boisclair, M D; Lane, D A; Wilde, J T; Ireland, H; Preston, F E; Ofosu, F A

    1990-04-01

    Measurements were made of levels of D-dimer in plasma and serum, thrombin-antithrombin complex (TAT) in plasma and fibrinogen/fibrin fragment E antigen (FgE) in serum in a normal healthy control group and in patients with a range of disorders associated with hypercoagulability. Levels were determined in 31 normal healthy controls, 30 patients with disseminated intravascular coagulation (DIC), 21 patients with deep venous thrombosis (DVT), 27 patients with myocardial infarction (MI), 26 patients with acute leukaemia and 56 patients with liver disease. Considering all subjects, significant correlations were established between the results of all assays. Notably high correlations (r greater than 0.9) were established between plasma and serum levels of D-dimer, between plasma levels of D-dimer and serum levels of FgE, and between serum levels of D-dimer and FgE. All assays showed very high discrimination (sensitivity) between the normal control group and patients with DIC (97-100%), but there were marked differences between the assays in sensitivity for DVT and MI. In general, the FgE assay was more sensitive than the D-dimer assay, whilst both the FgE and D-dimer assays were more sensitive than the TAT assay. The same trends were apparent in the capability of the assays to discriminate between the normal control group and patients with acute leukaemia and liver disease: disorders with an unknown prevalence of activation of coagulation/fibrinolysis. Our results indicated that measurements of fibrinogen/fibrin degradation products (FDPs) in serum were almost unaffected by artefacts. The data further suggested that the broad-spectrum FgE assay was better than the more specific D-dimer assay in detecting clinical hypercoagulability. Our study showed that, in the clinical conditions examined, FDPs were more effective markers of hypercoagulability than TAT. PMID:2189490

  9. Monitoring Low Molecular Weight Heparins at Therapeutic Levels: Dose-Responses of, and Correlations and Differences between aPTT, Anti-Factor Xa and Thrombin Generation Assays

    PubMed Central

    Thomas, Owain; Lybeck, Emanuel; Strandberg, Karin; Tynngård, Nahreen; Schött, Ulf

    2015-01-01

    Background Low molecular weight heparins (LMWH’s) are used to prevent and treat thrombosis. Tests for monitoring LMWH’s include anti-factor Xa (anti-FXa), activated partial thromboplastin time (aPTT) and thrombin generation. Anti-FXa is the current gold standard despite LMWH’s varying affinities for FXa and thrombin. Aim To examine the effects of two different LMWH’s on the results of 4 different aPTT-tests, anti-FXa activity and thrombin generation and to assess the tests’ concordance. Method Enoxaparin and tinzaparin were added ex-vivo in concentrations of 0.0, 0.5, 1.0 and 1.5 anti-FXa international units (IU)/mL, to blood from 10 volunteers. aPTT was measured using two whole blood methods (Free oscillation rheometry (FOR) and Hemochron Jr (HCJ)) and an optical plasma method using two different reagents (ActinFSL and PTT-Automat). Anti-FXa activity was quantified using a chromogenic assay. Thrombin generation (Endogenous Thrombin Potential, ETP) was measured on a Ceveron Alpha instrument using the TGA RB and more tissue-factor rich TGA RC reagents. Results Methods’ mean aPTT at 1.0 IU/mL LMWH varied between 54s (SD 11) and 69s (SD 14) for enoxaparin and between 101s (SD 21) and 140s (SD 28) for tinzaparin. ActinFSL gave significantly shorter aPTT results. aPTT and anti-FXa generally correlated well. ETP as measured with the TGA RC reagent but not the TGA RB reagent showed an inverse exponential relationship to the concentration of LMWH. The HCJ-aPTT results had the weakest correlation to anti-FXa and thrombin generation (Rs0.62–0.87), whereas the other aPTT methods had similar correlation coefficients (Rs0.80–0.92). Conclusions aPTT displays a linear dose-respone to LMWH. There is variation between aPTT assays. Tinzaparin increases aPTT and decreases thrombin generation more than enoxaparin at any given level of anti-FXa activity, casting doubt on anti-FXa’s present gold standard status. Thrombin generation with tissue factor-rich activator is

  10. Crystal Structure of Thrombin Bound to the Uncleaved Extracellular Fragment of PAR1

    SciTech Connect

    Gandhi, Prafull S.; Chen, Zhiwei; Di Cera, Enrico

    2010-05-11

    Abundant structural information exists on how thrombin recognizes ligands at the active site or at exosites separate from the active site region, but remarkably little is known about how thrombin recognizes substrates that bridge both the active site and exosite I. The case of the protease-activated receptor PAR1 is particularly relevant in view of the plethora of biological effects associated with its activation by thrombin. Here, we present the 1.8 {angstrom} resolution structure of thrombin S195A in complex with a 30-residue long uncleaved extracellular fragment of PAR1 that documents for the first time a productive binding mode bridging the active site and exosite I. The structure reveals two unexpected features of the thrombin-PAR1 interaction. The acidic P3 residue of PAR1, Asp{sup 39}, does not hinder binding to the active site and actually makes favorable interactions with Gly{sup 219} of thrombin. The tethered ligand domain shows a considerable degree of disorder even when bound to thrombin. The results fill a significant gap in our understanding of the molecular mechanisms of recognition by thrombin in ways that are relevant to other physiological substrates.

  11. Intracellular Ascorbate Prevents Endothelial Barrier Permeabilization by Thrombin.

    PubMed

    Parker, William H; Qu, Zhi-chao; May, James M

    2015-08-28

    Intracellular ascorbate (vitamin C) has previously been shown to tighten the endothelial barrier and maintain barrier integrity during acute inflammation in vitro. However, the downstream effectors of ascorbate in the regulation of endothelial permeability remain unclear. In this study, we evaluated ascorbate as a mediator of thrombin-induced barrier permeabilization in human umbilical vein endothelial cells and their immortalized hybridoma line, EA.hy926. We found that the vitamin fully prevented increased permeability to the polysaccharide inulin by thrombin in a dose-dependent manner, and it took effect both before and after subjection to thrombin. Thrombin exposure consumed intracellular ascorbate but not the endogenous antioxidant GSH. Likewise, the antioxidants dithiothreitol and tempol did not reverse permeabilization. We identified a novel role for ascorbate in preserving cAMP during thrombin stimulation, resulting in two downstream effects. First, ascorbate maintained the cortical actin cytoskeleton in a Rap1- and Rac1-dependent manner, thus preserving stable adherens junctions between adjacent cells. Second, ascorbate prevented actin polymerization and formation of stress fibers by reducing the activation of RhoA and phosphorylation of myosin light chain. Although ascorbate and thrombin both required calcium for their respective effects, ascorbate did not prevent thrombin permeabilization by obstructing calcium influx. However, preservation of cAMP by ascorbate was found to depend on both the production of nitric oxide by endothelial nitric-oxide synthase, which ascorbate is known to activate, and the subsequent generation cGMP by guanylate cyclase. Together, these data implicate ascorbate in the prevention of inflammatory endothelial barrier permeabilization and explain the underlying signaling mechanism. PMID:26152729

  12. An aptamer assay using rolling circle amplification coupled with thrombin catalysis for protein detection.

    PubMed

    Guo, Limin; Hao, Lihua; Zhao, Qiang

    2016-07-01

    We describe a sensitive aptamer-based sandwich assay for protein detection on microplate by using rolling circle amplification (RCA) coupled with thrombin catalysis. This assay takes advantage of RCA generating long DNA oligonucleotides with repeat thrombin-binding aptamer sequence, specific aptamer affinity binding to achieve multiple thrombin labeling, and enzyme activity of thrombin for signal generation. Protein target is specifically captured by antibody-coated microplate. Then, an oligonucleotide containing an aptamer for protein and a primer sequence is added to form a typical sandwich structure. Following a template encoded with complementary sequence of aptamer for thrombin, RCA reaction extends the primer sequence into a long oligonucleotide. Many thrombin molecules bind with the RCA product. Thrombin catalyzes the conversion of its chromogenic or fluorogenic peptide substrates into detectable products for final quantification of protein targets. We applied this strategy to the detection of a model protein target, platelet-derived growth factor-BB (PDGF-BB). Due to double signal amplifications from RCA and thrombin catalysis, this assay enabled the detection of PDGF-BB as low as 3.1 pM when a fluorogenic peptide substrate was used. This assay provides a new way for signal generation in RCA-involved assay through direct thrombin labeling, circumventing time-consuming preparation of enzyme-conjugate and affinity probes. This method has promise for a variety of analytical applications. PMID:27108282

  13. Structure and expression of human thrombomodulin, a thrombin receptor on endothelium acting as a cofactor for protein C activation.

    PubMed Central

    Suzuki, K; Kusumoto, H; Deyashiki, Y; Nishioka, J; Maruyama, I; Zushi, M; Kawahara, S; Honda, G; Yamamoto, S; Horiguchi, S

    1987-01-01

    We have deduced the entire 575-amino acid sequence of the human thrombomodulin precursor from cDNA clones. The precursor starts with an 18-residue signal peptide domain, followed by the NH2-terminal domain, a domain with six epidermal growth factor-like structures, an O-glycosylation site-rich domain, a 24-residue transmembrane domain and a cytoplasmic domain. Simian COS cells transfected with the expression vector pSV2 containing thrombomodulin cDNA synthesized immunoreactive and functionally active thrombomodulin. Images Fig. 1. Fig. 7. PMID:2820710

  14. Association of thrombospondin-1 with the actin cytoskeleton of human thrombin-activated platelets through an alphaIIbbeta3- or CD36-independent mechanism.

    PubMed Central

    Saumet, Anne; Jesus, Nando de; Legrand, Chantal; Dubernard, Véronique

    2002-01-01

    Thrombospondin-1 (TSP-1) is an adhesive glycoprotein which, when secreted from alpha-granules of activated platelets, can bind to the cell surface and participate in platelet aggregate formation. In this study, we show that thrombin activation leads to the rapid and specific association of a large amount of secreted alpha-granular TSP-1 with the actin cytoskeleton. This cytoskeletal association of TSP-1 was correlated with platelet secretion, but not aggregation, and was inhibited by cytochalasin D, an inhibitor of actin polymerization. Association of TSP-1 with the actin cytoskeleton was mediated by membrane receptors, as shown by using MAII, a TSP-1-specific monoclonal antibody that inhibited both TSP-1 surface binding to activated platelets and cytoskeletal association. TSP-1 and its potential membrane receptors, e.g. alphaIIbbeta3 integrin, CD36 and CD47, concomitantly associated with the actin cytoskeleton. However, studies on platelets from a patient with type I Glanzmann's thrombasthenia lacking alphaIIbbeta3 and another with barely detectable CD36 showed normal TSP-1 surface expression and association with the actin cytoskeleton. Likewise, no involvement of CD47 in TSP-1 association with the actin cytoskeleton could be inferred from experiments with control platelets using the function-blocking anti-CD47 antibody B6H12. Finally, assembly of signalling complexes, as observed through translocation of tyrosine-phosphorylated proteins and kinases to the actin cytoskeleton, was found to occur in concert with cytoskeletal association of TSP-1, in control platelets as well as in thrombasthenic and CD36-deficient platelets. Our results imply a role for the actin cytoskeleton in the membrane-surface expression process of TSP-1 molecules and suggest a possible coupling of TSP-1 receptors to signalling events occurring independently of alphaIIbbeta3 or CD36. These results provide new insights into the link between surface-bound TSP-1 and the contractile actin

  15. [Thrombin--a regulator of reparative processes in wound healing].

    PubMed

    Strukova, S M; Dugina, T N; Chistov, I V; Markvicheva, E A; Kuptsova, S V; Kolokol'chikova, E G; Rumsh, L D; Zubov, V P; Gluza, E

    1998-04-01

    Thrombin, binding to receptors of the protease activated receptor (PAR) family, is involved in wound healing by inducing the reparation processes and regulating the activity of mast cells, which secrete mediators of inflammation. Using thrombin receptor agonist peptide (TRAP-6) for the activation of rat mast cells, effect of several receptors, including PAR-1, on mast cells was demonstrated. It was shown that TRAP increases the concentration of Ca2+ in the cytoplasm of mast cells and regulates cell degranulation, while releasing nitrogen oxide. Thrombin encapsulated in poly(N-vinyl caprolactam)-calcium alginate (PVCL-Ca-Alg) hydrogel films promotes wound healing in rats as demonstrated by the acceleration of fibroblast proliferation and neovascularization. PMID:9612571

  16. Evaluation of Potential Thrombin Inhibitors from the White Mangrove (Laguncularia racemosa (L.) C.F. Gaertn.).

    PubMed

    Rodrigues, Caroline Fabri Bittencourt; Gaeta, Henrique Hessel; Belchor, Mariana Novo; Ferreira, Marcelo José Pena; Pinho, Marcus Vinícius Terashima; Toyama, Daniela de Oliveira; Toyama, Marcos Hikari

    2015-07-01

    The aim of this work was to verify the effects of methanol (MeOH) and hydroalcoholic (HA) extracts and their respective partition phases obtained from white mangrove (Laguncularia racemosa (L.) C.F. Gaertn.) leaves on human thrombin activity. Among the extracts and phases tested, only the ethyl acetate and butanolic partitions significantly inhibited human thrombin activity and the coagulation of plasma in the presence of this enzyme. Chromatographic analyses of the thrombin samples incubated with these phases revealed that different compounds were able to interact with thrombin. The butanolic phase of the MeOH extract had the most potent inhibitory effects, reducing enzymatic activity and thrombin-induced plasma coagulation. Two glycosylated flavonoids in this partition were identified as the most potent inhibitors of human thrombin activity, namely quercetin-3-O-arabinoside (QAra) and quercetin-3-O-rhamnoside (Qn). Chromatographic analyses of thrombin samples incubated with these flavonoids demonstrated the chemical modification of this enzyme, suggesting that the MeOH extract contained other compounds that both induced structural changes in thrombin and diminished its activity. In this article, we show that despite the near absence of the medical use of mangrove compounds, this plant contains natural compounds with potential therapeutic applications. PMID:26197325

  17. Inhibition of prostaglandin E2 receptor EP3 mitigates thrombin-induced brain injury.

    PubMed

    Han, Xiaoning; Lan, Xi; Li, Qiang; Gao, Yufeng; Zhu, Wei; Cheng, Tian; Maruyama, Takayuki; Wang, Jian

    2016-06-01

    Prostaglandin E2 EP3 receptor is the only prostaglandin E2 receptor that couples to multiple G-proteins, but its role in thrombin-induced brain injury is unclear. In the present study, we exposed mouse hippocampal slice cultures to thrombin in vitro and injected mice with intrastriatal thrombin in vivo to investigate the role of EP3 receptor in thrombin-induced brain injury and explore its underlying cellular and molecular mechanisms. In vitro, EP3 receptor inhibition reduced thrombin-induced hippocampal CA1 cell death. In vivo, EP3 receptor was expressed in astrocytes and microglia in the perilesional region. EP3 receptor inhibition reduced lesion volume, neurologic deficit, cell death, matrix metalloproteinase-9 activity, neutrophil infiltration, and the number of CD68(+) microglia, but increased the number of Ym-1(+) M2 microglia. RhoA-Rho kinase levels were increased after thrombin injection and were decreased by EP3 receptor inhibition. In mice that received an intrastriatal injection of autologous arterial blood, inhibition of thrombin activity with hirudin decreased RhoA expression compared with that in vehicle-treated mice. However, EP3 receptor activation reversed this effect of hirudin. These findings show that prostaglandin E2 EP3 receptor contributes to thrombin-induced brain damage via Rho-Rho kinase-mediated cytotoxicity and proinflammatory responses. PMID:26661165

  18. Thrombin induces endothelial cell growth via both a proteolytic and a non-proteolytic pathway.

    PubMed Central

    Herbert, J M; Dupuy, E; Laplace, M C; Zini, J M; Bar Shavit, R; Tobelem, G

    1994-01-01

    Binding of 125I-thrombin to human umbilical vein endothelial cells (HUVECs) was specifically displaced by the synthetic tetradecapeptide SFLLRNPNDKYEPF, named thrombin receptor agonist peptide (TRAP), which has recently been described as a peptide mimicking the new N-terminus created by cleavage of the thrombin receptor, and F-14, a tetradecapeptide representing residues 365-378 of the human alpha-thrombin B chain. Binding of 125I-TRAP to HUVECs was time-dependent, reversible and saturable, showing high affinity (KD = 1.5 +/- 0.4 microM) and high binding capacity (Bmax. = 7.1 +/- 0.6 x 10(6) sites/cell) (n = 3). Unlabelled thrombin and TRAP competitively and selectively inhibited the specific binding of 125I-TRAP with IC50 values of 5.8 +/- 0.7 nM and 2.8 +/- 0.4 microM respectively, whereas F-14 remained ineffective at displacing 125I-TRAP from its binding sites, suggesting the presence of at least two different types of thrombin-binding sites on HUVECs. TRAP was a potent mitogen for HUVECs in culture. Both TRAP and alpha-thrombin stimulated the proliferation of HUVECs with half-maximum mitogenic responses between 1 and 10 nM. F-14 also promoted HUVEC growth. The mitogenic effects of F-14 and TRAP were additive. N alpha-(2-Naphthylsulphonylglycyl)-DL-p-amidinophenylalanylpiper idine (NAPAP) and hirudin (two specific inhibitors of the enzyme activity of thrombin) specifically inhibited thrombin-induced HUVEC growth (IC50 values 400 +/- 60 and 52 +/- 8 nM respectively) but remained without effect on the mitogenic effect of TRAP or F-14. This demonstrated that the mitogenic effect of alpha-thrombin for HUVECs was intimately linked to its esterolytic activity but also showed that thrombin can stimulate HUVEC growth via another non-enzymic pathway. This hypothesis was further reinforced by the fact that F-14-induced proliferation of HUVECs remained unaltered by two antibodies directed against TRAP or the cleavage site on the extracellular portion of the thrombin

  19. Fibrinogen Substrate Recognition by Staphylocoagulase·(Pro)thrombin Complexes*

    PubMed Central

    Panizzi, Peter; Friedrich, Rainer; Fuentes-Prior, Pablo; Richter, Klaus; Bock, Paul E.; Bode, Wolfram

    2008-01-01

    Thrombin generation and fibrinogen (Fbg) clotting are the ultimate proteolytic reactions in the blood coagulation pathway. Staphylocoagulase (SC), a protein secreted by the human pathogen Staphylococcus aureus, activates prothrombin (ProT) without proteolysis. The SC·(pro)thrombin complex recognizes Fbg as a specific substrate, converting it directly into fibrin. The crystal structure of a fully active SC fragment containing residues 1–325 (SC-(1–325)) bound to human prethrombin 2 showed previously that SC inserts its Ile1-Val2 N terminus into the Ile16 pocket of prethrombin 2, inducing a functional active site in the cognate zymogen conformationally. Exosite I of α-thrombin, the Fbg recognition site, and proexosite I on ProT are blocked by domain 2 of SC-(1–325). In the present studies, active site-labeled fluorescent ProT analogs were used to quantitate Fbg binding to the SC-(1–325)·ProT complex. Fbg binding and cleavage are mediated by expression of a new Fbg-binding exosite on the SC-(1–325)·ProT complex, resulting in formation of an (SC-(1–325)·ProT)2·Fbg pentameric complex with a dissociation constant of 8–34 nM. In both crystal structures, the SC-(1–325)·(pre)thrombin complexes form dimers, with both pro-teinases/zymogens facing each other over a large U-shaped cleft, through which the Fbg substrate could thread. On this basis, a molecular model of the pentameric (SC-(1–325)·thrombin)2·Fbg encounter complex was generated, which explains the coagulant properties and efficient Fbg conversion. The results provide new insight into the mechanism that mediates high affinity Fbg binding and cleavage as a substrate of SC·(pro)thrombin complexes, a process that is central to the molecular pathology of S. aureus endocarditis. PMID:16230339

  20. Screening of direct thrombin inhibitors from Radix Salviae Miltiorrhizae by a peak fractionation approach.

    PubMed

    Lu, Jun; Song, Hui-Peng; Li, Ping; Zhou, Ping; Dong, Xin; Chen, Jun

    2015-05-10

    Thrombin plays a significant role in thromboembolic disease. In this work, a peak fractionation approach combined with an activity assay method was used to screen direct thrombin inhibitors from Radix Salviae Miltiorrhizae (RSM), a famous herbal remedy for the treatment of cardiovascular diseases in China. A total of 91 fractions were collected from the RSM extract, and 19 fractions out of them showed thrombin inhibitory effects with dose-effect relationship. Among them, three compounds were unambiguously identified as 15, 16-dihydrotanshinone I, cryptotanshinone and tanshinone IIA with IC50 values of 29.39, 81.11 and 66.60μM, respectively. The three compounds were reported with direct thrombin inhibition activities for the first time and their ligand-thrombin interactions were explored by a molecular docking research. These results may contribute to explain the medical benefit of RSM for the prevention and treatment of cardiovascular diseases. PMID:25819728

  1. A peptide (P2) derived from the variable heavy chain of an anti-P-selectin monoclonal antibody (LYP20) inhibits leucocyte adhesion to thrombin-activated platelets and endothelial cells.

    PubMed

    Murphy, Joseph F; McGregor, John L

    2003-02-01

    P-selectin, a member of the selectin family of adhesion molecules, is present in endothelial Weibel-Palade bodies and platelet alpha-granules, and is rapidly expressed on their surface upon activation, resulting in leucocyte adhesion. LYP20 is a functional monoclonal antibody previously generated in our laboratory that binds with high affinity and specificity directed against P-selectin. This binding is largely imparted by the specific sequence of amino acids present on the hypervariable portions of the IgG chains. We now show that a peptide derived from the heavy chain of mAb LYP20 dose dependently inhibits the adhesion of poly morphonuclear cells to resting and thrombin-activated endothelial cells (EC) and platelets. The scrambled form of this peptide, identical in amino acid composition to the authentic peptide but with altered sequence, was not inhibitory at corresponding concentrations. Binding studies revealed that this peptide also dose dependently bound to both resting and thrombin-activated EC and platelets. Our results may prove useful for the development of new therapeutic inhibitors to modulate leucocyte interactions in inflammatory disorders. PMID:12588346

  2. Thrombin-Responsive Gated Silica Mesoporous Nanoparticles As Coagulation Regulators.

    PubMed

    Bhat, Ravishankar; Ribes, Àngela; Mas, Núria; Aznar, Elena; Sancenón, Félix; Marcos, M Dolores; Murguía, Jose R; Venkataraman, Abbaraju; Martínez-Máñez, Ramón

    2016-02-01

    The possibility of achieving sophisticated actions in complex biological environments using gated nanoparticles is an exciting prospect with much potential. We herein describe new gated mesoporous silica nanoparticles (MSN) loaded with an anticoagulant drug and capped with a peptide containing a thrombin-specific cleavage site. When the coagulation cascade was triggered, active thrombin degraded the capping peptidic sequence and induced the release of anticoagulant drugs to delay the clotting process. The thrombin-dependent response was assessed and a significant increase in coagulation time in plasma from 2.6 min to 5 min was found. This work broadens the application of gated silica nanoparticles and demonstrates their ability to act as controllers in a complex scenario such as hemostasis. PMID:26794474

  3. The Importance of Thrombin in Cerebral Injury and Disease.

    PubMed

    Krenzlin, Harald; Lorenz, Viola; Danckwardt, Sven; Kempski, Oliver; Alessandri, Beat

    2016-01-01

    There is increasing evidence that prothrombin and its active derivative thrombin are expressed locally in the central nervous system. So far, little is known about the physiological and pathophysiological functions exerted by thrombin in the human brain. Extra-hepatic prothrombin expression has been identified in neuronal cells and astrocytes via mRNA measurement. The actual amount of brain derived prothrombin is expected to be 1% or less compared to that in the liver. The role in brain injury depends upon its concentration, as higher amounts cause neuroinflammation and apoptosis, while lower concentrations might even be cytoprotective. Its involvement in numerous diseases like Alzheimer's, multiple sclerosis, cerebral ischemia and haemorrhage is becoming increasingly clear. This review focuses on elucidation of the cerebral thrombin expression, local generation and its role in injury and disease of the central nervous system. PMID:26761005

  4. The Importance of Thrombin in Cerebral Injury and Disease

    PubMed Central

    Krenzlin, Harald; Lorenz, Viola; Danckwardt, Sven; Kempski, Oliver; Alessandri, Beat

    2016-01-01

    There is increasing evidence that prothrombin and its active derivative thrombin are expressed locally in the central nervous system. So far, little is known about the physiological and pathophysiological functions exerted by thrombin in the human brain. Extra-hepatic prothrombin expression has been identified in neuronal cells and astrocytes via mRNA measurement. The actual amount of brain derived prothrombin is expected to be 1% or less compared to that in the liver. The role in brain injury depends upon its concentration, as higher amounts cause neuroinflammation and apoptosis, while lower concentrations might even be cytoprotective. Its involvement in numerous diseases like Alzheimer’s, multiple sclerosis, cerebral ischemia and haemorrhage is becoming increasingly clear. This review focuses on elucidation of the cerebral thrombin expression, local generation and its role in injury and disease of the central nervous system. PMID:26761005

  5. Detection and partial characterization of an inhibitor of plasminogen activator in human platelets.

    PubMed Central

    Erickson, L A; Ginsberg, M H; Loskutoff, D J

    1984-01-01

    In this study, we demonstrate the presence of a previously undescribed fibrinolytic inhibitor in human serum. It has an apparent molecular weight of 50,000 and is not detected in serum derived from platelet-poor plasma, suggesting that it originates from platelets. This conclusion is supported by a number of observations. For example, extracts of washed, gel-filtered human platelets contain an inhibitor of similar activity and size, and physiological concentrations of thrombin induce its release from the platelets. Moreover, the kinetics and dose dependency of this release are similar to those observed for the release of platelet factor 4, and the release of both molecules is blocked by pretreating the platelets with prostaglandin E1 and theophylline. Mixing experiments, which were devised to investigate the specificity of the inhibitor, showed that the fibrinolytic activity initiated by both urokinase and tissue-type plasminogen activator was blocked by platelet releasate in a dose-dependent manner. In both cases, the amount of inhibition increased when the releasates were preincubated with the purified activators, indicating a direct interaction between the activators and an inhibitor(s). The inhibitory activity was removed by preincubating the releasates with antiserum prepared against an antiactivator purified from cultured bovine aortic endothelial cells. These results indicate that platelets contain an inhibitor which is released by thrombin, inhibits both urokinase and tissue-type plasminogen activator, and is immunologically similar to an inhibitor produced by endothelial cells. This molecule may represent a new class of inhibitors, the antiactivators, which function together with alpha 2-antiplasmin to regulate the fibrinolytic system of the blood. Its release from platelets by thrombin may protect the growing thrombus against premature dissolution initiated by plasminogen activators released by the endothelium. Images PMID:6434594

  6. Thrombin-linked aptamer assay for detection of platelet derived growth factor BB on magnetic beads in a sandwich format.

    PubMed

    Guo, Limin; Zhao, Qiang

    2016-09-01

    Here we describe a thrombin-linked aptamer assay (TLAA) for protein by using thrombin as an enzyme label, harnessing enzyme activity of thrombin and aptamer affinity binding. TLAA converts detection of specific target proteins to the detection of thrombin by using a DNA sequence that consists of two aptamers with the first aptamer binding to the specific target protein and the second aptamer binding to thrombin. Through the affinity binding, the thrombin enzyme is labeled on the protein target, and thrombin catalyzes the hydrolysis of small peptide substrate into product, generating signals for quantification. As a proof of principle, we show a sandwich TLAA for platelet derived growth factor BB (PDGF-BB) by using anti-PDGF-BB antibody coated on magnetic beads and an oligonucleotide containing the aptamer for PDGF-BB and the aptamer for thrombin. The binding of PDGF-BB to both the antibody and the aptamer results in labeling the complex with thrombin. We achieved detection of PDGF-BB at 16 pM. This TLAA contributes a new application of thrombin and its aptamer in bioanalysis, and shows potentials in assay developments. PMID:27343590

  7. Translational success stories: development of direct thrombin inhibitors.

    PubMed

    Coppens, Michiel; Eikelboom, John W; Gustafsson, David; Weitz, Jeffrey I; Hirsh, Jack

    2012-09-14

    Anticoagulants are the cornerstone of therapy for conditions associated with arterial and venous thrombosis. Direct thrombin inhibitors (DTIs) are anticoagulants that bind to thrombin and block its enzymatic activity. The bivalent parenteral DTIs hirudin and bivalirudin were based on the observation that the salivary extracts of medicinal leeches prevented blood from clotting. Key events that facilitated the subsequent development of small molecule active site inhibitors, such as argatroban, were the observation that fibrinopeptide A had antithrombotic properties and determination of the crystal structure of thrombin. Hirudin and argatroban have found their niche for the treatment of patients with heparin-induced thrombocytopenia, whereas bivalirudin is approved as an alternative to heparin for patients undergoing percutaneous coronary intervention. The development of orally active direct thrombin inhibitors was challenging because of the need to convert water-soluble, poorly absorbable, active site inhibitors into fat-soluble prodrugs that were then transformed back to the active drug after intestinal absorption. Dabigatran etexilate was the first new oral anticoagulant to be approved for long-term anticoagulant treatment in 6 decades. This Review highlights the development of DTIs as a translational success story; an example in which the combination of scientific ingenuity, structure-based design, and rigorous clinical trials has created a new class of anticoagulants that has improved patient care. PMID:22982873

  8. Nanostructured bioluminescent sensor for rapidly detecting thrombin.

    PubMed

    Chen, Longyan; Bao, Yige; Denstedt, John; Zhang, Jin

    2016-03-15

    Thrombin plays a key role in thrombosis and hemostasis. The abnormal level of thrombin in body fluids may lead to different diseases, such as rheumatoid arthritis, glomerulonephritis, etc. Detection of thrombin level in blood and/or urine is one of important methods for medical diagnosis. Here, a bioluminescent sensor is developed for non-invasively and rapidly detecting thrombin in urine. The sensor is assembled through conjugating gold nanoparticles (Au NPs) and a recombinant protein containing Renilla luciferase (pRluc) by a peptide, which is thrombin specific substrate. The luciferase-catalyzed bioluminescence can be quenched by peptide-conjugating Au NPs. In the presence of thrombin, the short peptide conjugating luciferase and Au NPs is digested and cut off, which results in the recovery of bioluminescence due to the release of luciferase from Au NPs. The bioluminescence intensity at 470 nm is observed, and increases with increasing concentration of thrombin. The bioluminescence intensity of this designed sensor is significantly recovered when the thrombin digestion time lasts for 10 min. In addition, a similar linear relationship between luminescence intensity and the concentration of thrombin is found in the range of 8 nM to 8 μM in both buffer and human urine spiked samples. The limit of detection is as low as 80 pM. It is anticipated that our nanosensor could be a promising tool for clinical diagnosis of thrombin in human urine. PMID:26397418

  9. N-ethoxycarbonyl-D-phenylalanyl-L-prolyl-alpha-azalysine p-nitrophenyl ester: a novel, high selective and optimal chromogenic active site titrant for human and bovine alpha-, beta- and gamma-thrombin.

    PubMed

    Balliano, G; Milla, P; Giordano, C; Gallina, C; Coletta, M; Menegatti, E; Rizzi, M; Bolognesi, M; Ascenzi, P

    1996-08-14

    The serine proteinase catalyzed hydrolysis of N-ethoxycarbonyl-D-phenylalanyl-L-prolyl-alpha-azalysine p- nitrophenyl ester (Eoc-D-Phe-Pro-azaLys-ONp) was investigated at pH 6.2 and 21.0 degrees C. The results are consistent with the minimum three-step catalytic mechanism. The acylation step is rate limiting for human (Lys 77 species) and porcine plasmin, and for bovine beta-trypsin, the deacylation rate being limiting, on the other hand, for human and bovine alpha-, beta- and gamma-thrombin. Moreover the M(r) 33,000 species of human urokinase and the neuraminidase-treated porcine pancreatic beta-kallikrein-B do not catalyze the hydrolysis of the tripeptide. According to the specificity properties of the serine proteinases considered. Eoc-D-Phe- Pro-azaLys-ONp shows the characteristics of a novel, high selective and optimal chromogenic active site titrant for human and bovine alpha-, beta- and gamma-thrombin. PMID:8753800

  10. Endothelial Angiogenesis and Barrier Function in Response to Thrombin Require Ca2+ Influx through the Na+/Ca2+ Exchanger.

    PubMed

    Andrikopoulos, Petros; Kieswich, Julius; Harwood, Steven M; Baba, Akemichi; Matsuda, Toshio; Barbeau, Olivier; Jones, Keith; Eccles, Suzanne A; Yaqoob, Muhammad M

    2015-07-24

    Thrombin acts on the endothelium by activating protease-activated receptors (PARs). The endothelial thrombin-PAR system becomes deregulated during pathological conditions resulting in loss of barrier function and a pro-inflammatory and pro-angiogenic endothelial phenotype. We reported recently that the ion transporter Na(+)/Ca(2+) exchanger (NCX) operating in the Ca(2+)-influx (reverse) mode promoted ERK1/2 activation and angiogenesis in vascular endothelial growth factor-stimulated primary human vascular endothelial cells. Here, we investigated whether Ca(2+) influx through NCX was involved in ERK1/2 activation, angiogenesis, and endothelial barrier dysfunction in response to thrombin. Reverse-mode NCX inhibitors and RNAi-mediated NCX1 knockdown attenuated ERK1/2 phosphorylation in response to thrombin or an agonist of PAR-1, the main endothelial thrombin receptor. Conversely, promoting reverse-mode NCX by suppressing Na(+)-K(+)-ATPase activity enhanced ERK1/2 activation. Reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced primary human vascular endothelial cell angiogenesis, quantified as proliferation and tubular differentiation. Reverse-mode NCX inhibitors or NCX1 knockdown preserved barrier integrity upon thrombin stimulation in vitro. Moreover, the reverse-mode NCX inhibitor SEA0400 suppressed Evans' blue albumin extravasation to the lung and kidneys and attenuated edema formation and ERK1/2 activation in the lungs of mice challenged with a peptide activator of PAR-1. Mechanistically, thrombin-induced ERK1/2 activation required NADPH oxidase 2-mediated reactive oxygen species (ROS) production, and reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced ROS production. We propose that reverse-mode NCX is a novel mechanism contributing to thrombin-induced angiogenesis and hyperpermeability by mediating ERK1/2 activation in a ROS-dependent manner. Targeting reverse-mode NCX could be beneficial in pathological conditions

  11. Planar Hall magnetoresistive aptasensor for thrombin detection.

    PubMed

    Sinha, B; Ramulu, T S; Kim, K W; Venu, R; Lee, J J; Kim, C G

    2014-09-15

    The use of aptamer-based assays is an emerging and attractive approach in disease research and clinical diagnostics. A sensitive aptamer-based sandwich-type sensor is presented to detect human thrombin using a planar Hall magnetoresistive (PHR) sensor in cooperation with superparamagnetic labels. A PHR sensor has the great advantages of a high signal-to-noise ratio, a small offset voltage and linear response in the low-field region, allowing it to act as a high-resolution biosensor. In the system presented here, the sensor has an active area of 50 µm × 50 µm with a 10-nm gold layer deposited onto the sensor surface prior to the binding of thiolated DNA primary aptamer. A polydimethylsiloxane well of 600-µm radius and 1-mm height was prepared around the sensor surface to maintain the same specific area and volume for each sensor. The sensor response was traced in real time upon the addition of streptavidin-functionalized magnetic labels on the sensor. A linear response to the thrombin concentration in the range of 86 pM-8.6 µM and a lower detection limit down to 86 pM was achieved by the proposed present method with a sample volume consumption of 2 µl. The proposed aptasensor has a strong potential for application in clinical diagnosis. PMID:24727201

  12. Catabolism of hirudin and thrombin-hirudin complexes in the rat.

    PubMed Central

    Bichler, J; Baynes, J W; Thorpe, S R

    1993-01-01

    The metabolic fate of the anticoagulant protein, hirudin, and its complex with thrombin are presently unknown. Therefore we have labelled hirudin and human thrombin-hirudin complex with the residualizing label dilactitol-125I-tyramine (*I-DLT) in order to identify their tissue sites of catabolism in the rat. The rapid plasma clearance of hirudin after intravenous injection was unaffected by *I-DLT labelling, and by 2 h 6% or less of the injected dose remained in the blood. The majority (80.3 +/- 4.0%, n = 2) of *I-DLT-hirudin radioactivity recovered in tissues was found in kidney, and kidney was also at least 150 times more active in taking up hirudin, on a weight basis, than any other tissue examined (liver, spleen, skin, muscle, intestine, fat, lung). *I-DLT-hirudin which bound to thrombin was isolated by chromatography on concanavalin A-Sepharose; hirudin itself does not bind to concanavalin A. Radioactivity from thrombin-*I-DLT-hirudin was precipitable by anti-thrombin antibody and *I-DLT-thrombin-hirudin was precipitable by anti-hirudin antibody. By 1 h after injection of labelled thrombin-hirudin complexes, the recoveries of radioactivity from hirudin and thrombin in liver were comparable (38.6 +/- 3.0 and 36.4 +/- 4.1%, n = 3), whereas more radioactivity was recovered in kidney from hirudin than from thrombin (27.6 +/- 8.7 compared with 13.6 +/- 4.5%) and less was recovered in lung (0.4 +/- 0.2 compared with 17.7 +/- 2.9%). We conclude that hirudin is catabolized predominantly in kidney, whereas the thrombin-hirudin complex is catabolized by both liver and kidney. PMID:8280076

  13. Thrombin selectively induces transcription of genes in human monocytes involved in inflammation and wound healing.

    PubMed

    López, Mercedes L; Bruges, Gustavo; Crespo, Gustavo; Salazar, Victor; Deglesne, Pierre-Antoine; Schneider, Heike; Cabrera-Fuentes, Hector; Schmitz, M Lienhard; Preissner, Klaus T

    2014-11-01

    Thrombin is essential for blood coagulation but functions also as a mediator of cellular signalling. Gene expression microarray experiments in human monocytes revealed thrombin-induced upregulation of a limited subset of genes, which are almost exclusively involved in inflammation and wound healing. Among these, the expression of F3 gene encoding for tissue factor (TF) was enhanced indicating that this physiological initiator of coagulation cascade may create a feed-forward loop to enhance blood coagulation. Activation of protease-activated receptor type 1 (PAR1) was shown to play a main role in promoting TF expression. Moreover, thrombin induced phosphorylation of ERK1/2, an event that is required for expression of thrombin-regulated genes. Thrombin also increased the expression of TF at the protein level in monocytes as evidenced by Western blot and immunostaining. Furthermore, FXa generation induced by thrombin-stimulated monocytes was abolished by a TF blocking antibody and therefore it is entirely attributable to the expression of tissue factor. This cellular activity of thrombin provides a new molecular link between coagulation, inflammation and wound healing. PMID:25057055

  14. Tissue factor and thrombin mediate myocardial ischemia-reperfusion injury.

    PubMed

    Chong, Albert J; Pohlman, Timothy H; Hampton, Craig R; Shimamoto, Akira; Mackman, Nigel; Verrier, Edward D

    2003-02-01

    Reperfusion of the ischemic heart is necessary to prevent irreversible injury of the myocardium, which leads to permanent organ dysfunction. However, reperfusion in itself leads to myocardial ischemia/reperfusion (I/R) injury, which is characterized by an acute inflammatory response mediated by activated inflammatory cells, chemokines, cytokines, and adhesion molecules. The molecular mechanisms of myocardial I/R injury are not completely known. Tissue factor (TF) and thrombin, two potent procoagulant and proinflammatory mediators, are recognized to play significant roles in myocardial I/R injury. To investigate the role of TF and thrombin in myocardial I/R injury, we used rabbit and murine in situ coronary artery ligation models. Increased TF mRNA, antigen, and activity were found in ischemic cardiomyocytes. Administration of an inhibitory antirabbit TF monoclonal antibody before or during the onset of ischemia resulted in a significant reduction in infarct size. Functional inhibition of thrombin with hirudin also reduced the infarct size. However, defibrinogenating rabbits with ancrod had no effect on infarct size, suggesting a requirement of thrombin generation but not fibrin deposition in myocardial I/R injury. PMID:12607707

  15. Immobilized thrombin receptor agonist peptide accelerates wound healing in mice.

    PubMed

    Strukova, S M; Dugina, T N; Chistov, I V; Lange, M; Markvicheva, E A; Kuptsova, S; Zubov, V P; Glusa, E

    2001-10-01

    To accelerate the healing processes in wound repair, attempts have been repeatedly made to use growth factors including thrombin and its peptide fragments. Unfortunately, the employment of thrombin is limited because of its high liability and pro-inflammatory actions at high concentrations. Some cellular effects of thrombin in wound healing are mediated by the activation of protease activated receptor-1 (PAR-1). The thrombin receptor agonist peptide (TRAP:SFLLRN) activates this receptor and mimics the effects of thrombin, but TRAP is a relatively weak agonist. We speculated that the encapsulated peptide may be more effective for PAR-1 activation than nonimmobilized peptide and developed a novel method for TRAP encapsulation in hydrogel films based on natural and synthetic polymers. The effects of an encapsulated TRAP in composite poly(N-vinyl caprolactam)-calcium alginate (PVCL) hydrogel films were investigated in a mouse model of wound healing. On day 7 the wound sizes decreased by about 60% under TRAP-chitosan-containing PVCL films, as compared with control films without TRAP. In the case of TRAP-polylysine-containing films no significant decrease in wound sizes was found. The fibroblast/macrophage ratio increased under TRAP-containing films on day 3 and on day 7. The number of proliferating fibroblasts increased to 150% under TRAP-chitosan films on day 7 as compared with control films. The number of [3H]-thymidine labeled endothelial and epithelial cells in granulation tissues was also enhanced. Thus, the immobilized TRAP to PVCL-chitosan hydrogel films were found to promote wound healing following the stimulation of fibroblast and epithelial cell proliferation and neovascularization. Furthermore, TRAP was shown to inhibit the secretion of the inflammatory mediator PAF from stimulated rat peritoneal mast cells due to augmentation of NO release from the mast cells. The encapsulated TRAP is suggested to accelerate wound healing due to the anti-inflammatory effects

  16. [Interaction of human alpha-thrombin with organic ligands of ionic nature].

    PubMed

    Kolodzeĭskaia, M V; Chumachenko, Iu V; Volkov, G L

    2003-01-01

    Investigations results of human thrombin interaction with organic ligands of ion nature containing nonpolar groups are presented. It is shown that electrostatic interaction is the basic one under enzyme binding, while hydrophobic binding is only additional function in the reaction enzyme-ligand, this fact is confirmed by the absence of interaction between thrombin and rivanol which has a positive charge side by side with cumbrous hydrophobic group. New data are presented about the ligand specificity of binding sites of thrombin active centre. The importance of relative arrangement of hydrophobic ligand groups for interaction with enzyme is shown. It is supposed that thrombin binding with organic ligands occurs owing anionic site of beta-domain of active thrombin centre with the major aminoacids arginine and lysine (Lys 68, Arg 78, Arg 77, Arg 66 etc.). It is shown that the compounds containing negative group SO3 and have some cunbours hydrophobic groups interact more intensively with the enzyme. Thus, rosseline--with symmetrical hydrophobic nucleus (four benzene rings)--is the most efficient ligand for the binding with thrombin. The obtained investigation results evidence for bacteriostatical and stabilizing effect of low-molecular asobenzene ligands on rather labile thrombin molecules. PMID:15143519

  17. Investigation of the selectivity of thrombin-binding aptamers for thrombin titration in murine plasma.

    PubMed

    Trapaidze, Ana; Hérault, Jean-Pascal; Herbert, Jean-Marc; Bancaud, Aurélien; Gué, Anne-Marie

    2016-04-15

    Detection of thrombin in plasma raises timely challenges to enable therapeutic management of thrombosis in patients under vital threat. Thrombin binding aptamers represent promising candidates as sensing elements for the development of real-time thrombin biosensors; however implementation of such biosensor requires the clear understanding of thrombin-aptamer interaction properties in real-like environment. In this study, we used Surface Plasmon Resonance technique to answer the questions of specificity and sensitivity of thrombin detection by the thrombin-binding aptamers HD1, NU172 and HD22. We systematically characterized their properties in the presence of thrombin, as well as interfering molecular species such as the thrombin precursor prothrombin, thrombin in complex with some of its natural inhibitors, nonspecific serum proteins, and diluted plasma. Kinetic experiments show the multiple binding modes of HD1 and NU172, which both interact with multiple sites of thrombin with low nanomolar affinities and show little specificity of interaction for prothrombin vs. thrombin. HD22, on the other hand, binds specifically to thrombin exosite II and has no affinity to prothrombin at all. While thrombin in complex with some of its inhibitors could not be recognized by any aptamer, the binding of HD1 and NU172 properties is compromised by thrombin inhibitors alone, as well as with serum albumin. Finally, the complex nature of plasma was overwhelming for HD1, but we define conditions for the thrombin detection at 10nM range in 100-fold diluted plasma by HD22. Consequently HD22 showed key advantage over HD1 and NU172, and appears as the only alternative to design an aptasensor. PMID:26594887

  18. Thrombin-inhibiting nanoparticles rapidly constitute versatile and detectable anticlotting surfaces

    NASA Astrophysics Data System (ADS)

    Wheatley Myerson, Jacob; He, Li; Allen, John Stacy; Williams, Todd; Lanza, Gregory; Tollefsen, Douglas; Caruthers, Shelton; Wickline, Samuel

    2014-09-01

    Restoring an antithrombotic surface to suppress ongoing thrombosis is an appealing strategy for treatment of acute cardiovascular disorders such as erosion of atherosclerotic plaque. An antithrombotic surface would present an alternative to systemic anticoagulation with attendant risks of bleeding. We have designed thrombin-targeted nanoparticles (NPs) that bind to sites of active clotting to extinguish local thrombin activity and inhibit platelet deposition while exhibiting only transient systemic anticoagulant effects. Perfluorocarbon nanoparticles (PFC NP) were functionalized with thrombin inhibitors (either D-phenylalanyl-L-prolyl-L-arginyl-chloromethyl ketone or bivalirudin) by covalent attachment of more than 15 000 inhibitors to each PFC NP. Fibrinopeptide A (FPA) ELISA demonstrated that thrombin-inhibiting NPs prevented cleavage of fibrinogen by both free and clot-bound thrombin. Magnetic resonance imaging (MRI) confirmed that a layer of thrombin-inhibiting NPs prevented growth of clots in vitro. Thrombin-inhibiting NPs were administered in vivo to C57BL6 mice subjected to laser injury of the carotid artery. NPs significantly delayed thrombotic occlusion of the artery, whereas an equivalent bolus of free inhibitor was ineffective. For thrombin-inhibiting NPs, only a short-lived (˜10 min) systemic effect on bleeding time was observed, despite prolonged clot inhibition. Imaging and quantification of in vivo antithrombotic NP layers was demonstrated by MRI of the PFC NP. 19F MRI confirmed colocalization of particles with arterial thrombi, and quantitative 19F spectroscopy demonstrated specific binding and retention of thrombin-inhibiting NPs in injured arteries. The ability to rapidly form and image a new antithrombotic surface in acute vascular syndromes while minimizing risks of bleeding would permit a safer method of passivating active lesions than current systemic anticoagulant regimes.

  19. Formation of thrombin-antithrombin III complex using polyamide and hemophan dialyzers.

    PubMed

    Schultze, G; Hollmann, S; Sinah, P

    1992-06-01

    The recently developed ELISA for the thrombin-antithrombin III complex (TAT) is a sensitive, specific, and simplified means of detecting intravascular coagulation. For further evaluation of the thrombogenicity of a polyamide (P) and a Hemophan (H) hollow-fibre dialyzer a cross-over study was done in ten stable patients on maintenance hemodialysis. At the same doses of heparin (mean bolus of 30 U/kg bw and maintenance doses of 86 U/kg bw), thrombin time and partial thromboplastin time were significantly lower using H. At the end of dialysis TAT was significantly higher in H (mean +/- SEM before HD 3.57 +/- .56, at 240 min 14.9 +/- 6.5 ng/ml, p less than 0.05, Wilcoxon-test) than in P (before HD 4.36 +/- .98, at 240 min 8.95 +/- 3.0 ng/ml, p less than 0.05 H 240 vs. P 240, Wilcoxon-test). Visible clotting was more pronounced in the H filter. Among other favourable features of blood compatibility the polyamide/polyvinylpyrrolidone copolymer with a hydrophilic/hydrophobic microdomain structure has less thrombogenicity. The modified cellulosic membrane H has advantages in complement activation and leukocyte depression, but thrombogenicity seems less favourable since the incorporated diethyl-amino-ethyl groups with their positive charge bind and inactivate negatively charged heparin. PMID:1639530

  20. The modification of the thrombin generation test for the clinical assessment of dabigatran etexilate efficiency.

    PubMed

    Gribkova, Irina V; Lipets, Elena N; Rekhtina, Irina G; Bernakevich, Alex I; Ayusheev, Dorzho B; Ovsepyan, Ruzanna A; Ataullakhanov, Fazoil I; Sinauridze, Elena I

    2016-01-01

    A new oral anticoagulant, dabigatran etexilate (DE, a prodrug of direct thrombin inhibitor (DTI) dabigatran), has been used clinically to prevent thrombosis. The assessment of dabigatran efficiency is necessary in some clinical cases, such as renal insufficiency, risk of bleeding, and drug interactions. However, a specific thrombin generation test (TGT) that is one of the most informative and sensitive to anticoagulant therapy (calibrated automated thrombinography (САТ)) shows a paradoxical increase of test parameters, such as endogenous thrombin potential (ETP) and peak thrombin, in patients receiving DE. The paradoxical behaviour of ETP and peak thrombin in these patients in the presence of DTIs is mostly caused by a decrease in the activity of thrombin in the α2-macroglobulin-thrombin complex that is used as a calibrator in CAT. For a correct estimation of the TGT parameters in patient's plasma containing DTIs we proposed to use our previously described alternative calibration method that is based on the measurement of the fluorescence signal of a well-known concentration of the reaction product (7-amino-4-methylcoumarin). In this study, the validity of such approach was demonstrated in an ex vivo study in patients with knee replacement and two special patients with multiple myeloma, who received DE for thrombosis prophylaxis. PMID:27377013

  1. Heparin coating of tantalum coronary stents reduces surface thrombin generation but not factor IXa generation.

    PubMed

    Blezer, R; Cahalan, L; Cahalan, P T; Lindhout, T

    1998-07-01

    In the present study we used an in-vitro technique to examine initiation and propagation of blood coagulation at the surface of tantalum coronary stents exposed to flowing platelet-rich and platelet-free plasma. The time course of factor IXa production at the surface of the stent was not influenced by platelets. In spite of a significant factor IXa production, no thrombin activity was detected when the tantalum stent was exposed to platelet-free plasma; only when the stent was exposed to platelet-rich plasma was extensive thrombin production observed. These findings indicate that tantalum triggers blood coagulation, but that (adherent) platelets are essential for thrombin generation. Heparin-coated tantalum stents exposed to flowing platelet-rich plasma showed that factor IXa generation was slightly reduced compared with the bare stent. However, the heparin coating drastically delayed the onset of thrombin generation and largely reduced the steady-state production of thrombin. We found a clear relationship between the antithrombin binding capacity and the antithrombogenic potential of the heparin-coated stents. The mode of action of immobilized heparin is thought to abrogate thrombin generation by inhibiting thrombin-dependent positive feedback reactions at the surface of the coronary stent. PMID:9712292

  2. Thrombin-unique coagulation system protein with multifaceted impacts on cancer and metastasis.

    PubMed

    Wojtukiewicz, Marek Z; Hempel, Dominika; Sierko, Ewa; Tucker, Stephanie C; Honn, Kenneth V

    2016-06-01

    The association between blood coagulation and cancer development is well recognized. Thrombin, the pleiotropic enzyme best known for its contribution to fibrin formation and platelet aggregation during vascular hemostasis, may also trigger cellular events through protease-activated receptors, PAR-1 and PAR-4, leading to cancer progression. Our pioneering findings provided evidence that thrombin contributes to cancer metastasis by increasing adhesive potential of malignant cells. However, there is evidence that thrombin regulates every step of cancer dissemination: (1) cancer cell invasion, detachment from primary tumor, migration; (2) entering the blood vessel; (3) surviving in vasculature; (4) extravasation; (5) implantation in host organs. Recent studies have provided new molecular data about thrombin generation in cancer patients and the mechanisms by which thrombin contributes to transendothelial migration, platelet/tumor cell interactions, angiogenesis, and other processes. Though a great deal is known regarding the role of thrombin in cancer dissemination, there are new data for multiple thrombin-mediated events that justify devoting focus to this topic with a comprehensive approach. PMID:27189210

  3. The modification of the thrombin generation test for the clinical assessment of dabigatran etexilate efficiency

    PubMed Central

    Gribkova, Irina V.; Lipets, Elena N.; Rekhtina, Irina G.; Bernakevich, Alex I.; Ayusheev, Dorzho B.; Ovsepyan, Ruzanna A.; Ataullakhanov, Fazoil I.; Sinauridze, Elena I.

    2016-01-01

    A new oral anticoagulant, dabigatran etexilate (DE, a prodrug of direct thrombin inhibitor (DTI) dabigatran), has been used clinically to prevent thrombosis. The assessment of dabigatran efficiency is necessary in some clinical cases, such as renal insufficiency, risk of bleeding, and drug interactions. However, a specific thrombin generation test (TGT) that is one of the most informative and sensitive to anticoagulant therapy (calibrated automated thrombinography (САТ)) shows a paradoxical increase of test parameters, such as endogenous thrombin potential (ETP) and peak thrombin, in patients receiving DE. The paradoxical behaviour of ETP and peak thrombin in these patients in the presence of DTIs is mostly caused by a decrease in the activity of thrombin in the α2-macroglobulin-thrombin complex that is used as a calibrator in CAT. For a correct estimation of the TGT parameters in patient’s plasma containing DTIs we proposed to use our previously described alternative calibration method that is based on the measurement of the fluorescence signal of a well-known concentration of the reaction product (7-amino-4-methylcoumarin). In this study, the validity of such approach was demonstrated in an ex vivo study in patients with knee replacement and two special patients with multiple myeloma, who received DE for thrombosis prophylaxis. PMID:27377013

  4. PAR1-dependent and independent increases in COX-2 and PGE2 in human colonic myofibroblasts stimulated by thrombin.

    PubMed

    Seymour, Michelle L; Zaidi, Nosheen F; Hollenberg, Morley D; MacNaughton, Wallace K

    2003-05-01

    Subepithelial myofibroblast-derived prostaglandin E(2) (PGE(2)) regulates epithelial chloride secretion in the intestine. Thrombin is elevated in inflammatory conditions of the bowel. Therefore, we sought to determine a role for thrombin in regulating PGE(2) synthesis by colonic myofibroblasts. Incubation of cultured CCD-18Co colonic myofibroblasts with thrombin, the proteinase-activated receptor 1 (PAR(1))-activating peptide (Cit-NH(2)), and peptides corresponding to 2 noncatalytic regions of thrombin (TP367 and TP508) for 18 h increased both cyclooxygenase (COX)-2 expression (immunocytochemistry) and PGE(2) synthesis (enzyme immunoassay). Inhibition of thrombin by D-Phe-Pro-Arg-chloromethylketone (PPACK) did not significantly reduce PGE(2) synthesis, which remained elevated compared with control. We also investigated the basic fibroblast growth factor (bFGF) dependence of thrombin-induced PGE(2) elevations. Recombinant human bFGF concentration dependently increased PGE(2) synthesis, and a bFGF neutralizing antibody inhibited PGE(2) synthesis induced by TP367 and TP508 (approximately 40%) and by thrombin (approximately 20%) (but not Cit-NH(2)). Thrombin, therefore, upregulates COX-2-derived PGE(2) synthesis by both catalytic cleavage of PAR(1) and bFGF-dependent noncatalytic activity. This presents a novel mechanism by which intestinal myofibroblasts might regulate epithelial chloride secretion. PMID:12505789

  5. Voltage is a partial activator of rat thermosensitive TRP channels

    PubMed Central

    Matta, José A; Ahern, Gerard P

    2007-01-01

    TRPV1 and TRPM8 are sensory nerve ion channels activated by heating and cooling, respectively. A variety of physical and chemical stimuli activate these receptors in a synergistic manner but the underlying mechanisms are unclear. Both channels are voltage sensitive, and temperature and ligands modulate this voltage dependence. Thus, a voltage-sensing mechanism has become an attractive model to explain the generalized gating of these and other thermo-sensitive TRP channels. We show here using whole-cell and single channel measurements that voltage produces only a partial activation of TRPV1 and TRPM8. At room temperature (20–25°C) membrane depolarization evokes responses that saturate at ∼50–60% of the maximum open probability. Furthermore, high concentrations of capsaicin (10 μm), resiniferatoxin (5 μm) and menthol (6 mm) reveal voltage-independent gating. Similarly, other modes of TRPV1 regulation including heat, protein kinase C-dependent phosphorylation, and protons enhance both the efficacy and sensitivity of voltage activation. In contrast, the TRPV1 antagonist capsazepine produces the opposite effects. These data can be explained by an allosteric model in which voltage, temperature, agonists and inverse agonists are independently coupled, either positively or negatively, to channel gating. Thus, voltage acts separately but in concert with other stimuli to regulate channel activation, and, therefore, a voltage-sensitive mechanism is unlikely to represent a final, gating mechanism for these channels. PMID:17932142

  6. Targeting thrombin long-term after an acute coronary syndrome: Opportunities and challenges.

    PubMed

    De Caterina, Raffaele; Goto, Shinya

    2016-06-01

    Patients after an acute coronary syndrome (ACS) are at increased risk of recurrent thrombotic events, justifying the search for additional antithrombotic treatments. The pathophysiology of ACS involves arterial thrombus formation, in turn occurring because of a combination of platelet activation and fibrin formation, with thrombin playing a key role in both. Antiplatelet therapy, targeting the thromboxane pathway and the ADP P2Y12 receptor has been widely accepted for secondary prevention after an ACS. Now, data from recent clinical trials in such patients also encourage the pursuit of inhibiting thrombin formation or thrombin-mediated platelet activation in addition to antiplatelet therapy. This "triple pathway inhibition", including inhibition of thrombin activity or thrombin receptor(s), is currently an option in pure ACS, but already a must in the setting of ACS accompanied by atrial fibrillation (AF), where anticoagulants have been shown to be much more effective than antiplatelet agents in preventing stroke. We here discuss the challenges of managing combined thrombin activity or receptor inhibition and antiplatelet therapy in all such patients. Translating this into practice still requires further studies and patient tailoring to fully exploit its potential. PMID:26994821

  7. Thrombin Ca(2+)-dependently stimulates protein tyrosine phosphorylation in BC3H1 muscle cells.

    PubMed Central

    Offermanns, S; Bombien, E; Schultz, G

    1993-01-01

    The proteinase thrombin, known to act via heptahelical G-protein-coupled receptors, is a mitogenic agent for different cell types, including the mouse muscle cell line BC3H1. In this study, the effect of thrombin on tyrosine phosphorylation was examined using anti-phosphotyrosine antibodies. Thrombin was found to induce phosphorylation of 65-70 and 110-120 kDa proteins in BC3H1 cells. The effect of thrombin was concentration-dependent, being half-maximal and maximal at concentrations of 0.03 and 1 unit/ml respectively. The thrombin-induced increase in phosphorylation was rapid (< or = 10 s) and transient, with a peak response after about 1-2 min. The effect of thrombin could be mimicked by the thrombin receptor agonist peptide SFLLRN-NH2. Preincubation of cells with pertussis toxin (PT) had no effect on thrombin-induced tyrosine phosphorylation. Epidermal growth factor, platelet-derived growth factor and insulin stimulated tyrosine phosphorylation of different proteins, among which were 65-70 and 110-120 kDa proteins. The phorbol ester 12-myristate 13-acetate (PMA) as well as the Ca2+ ionophore A23187 both stimulated tyrosine phosphorylation of proteins identical to those phosphorylated by thrombin, suggesting that activation of protein kinase C (PKC) and elevation of the cytosolic Ca2+ concentration alone are sufficient to induce tyrosine phosphorylation. However, calphostin C and other PKC inhibitors, which completely inhibited tyrosine phosphorylation induced by PMA, had no influence on the effect of thrombin, whereas loading of cells with the intracellular Ca2+ chelator bis-(O-aminophenoxy)ethane-NNN'N'-tetra-acetic acid totally blocked thrombin-stimulated tyrosine phosphorylation. Thus tyrosine phosphorylation stimulated by thrombin is an early PT-insensitive cellular response which is either directly mediated by elevation of cytosolic Ca2+ concentration or by a presently unknown mechanism that requires an elevated cytosolic Ca2+ concentration. Images Figure 1

  8. Identification and Mechanistic Analysis of a Novel Tick-Derived Inhibitor of Thrombin

    PubMed Central

    Jablonka, Willy; Kotsyfakis, Michalis; Mizurini, Daniella M.; Monteiro, Robson Q.; Lukszo, Jan; Drake, Steven K.; Ribeiro, José M. C.; Andersen, John F.

    2015-01-01

    A group of peptides from the salivary gland of the tick Hyalomma marginatum rufipes, a vector of Crimean Congo hemorrhagic fever show weak similarity to the madanins, a group of thrombin-inhibitory peptides from a second tick species, Haemaphysalis longicornis. We have evaluated the anti-serine protease activity of one of these H. marginatum peptides that has been given the name hyalomin-1. Hyalomin-1 was found to be a selective inhibitor of thrombin, blocking coagulation of plasma and inhibiting S2238 hydrolysis in a competitive manner with an inhibition constant (Ki) of 12 nM at an ionic strength of 150 mM. It also blocks the thrombin-mediated activation of coagulation factor XI, thrombin-mediated platelet aggregation, and the activation of coagulation factor V by thrombin. Hyalomin-1 is cleaved at a canonical thrombin cleavage site but the cleaved products do not inhibit coagulation. However, the C-terminal cleavage product showed non-competitive inhibition of S2238 hydrolysis. A peptide combining the N-terminal parts of the molecule with the cleavage region did not interact strongly with thrombin, but a 24-residue fragment containing the cleavage region and the C-terminal fragment inhibited the enzyme in a competitive manner and also inhibited coagulation of plasma. These results suggest that the peptide acts by binding to the active site as well as exosite I or the autolysis loop of thrombin. Injection of 2.5 mg/kg of hyalomin-1 increased arterial occlusion time in a mouse model of thrombosis, suggesting this peptide could be a candidate for clinical use as an antithrombotic. PMID:26244557

  9. Inhibition of thrombin and factor Xa by Fucus evanescens fucoidan and its modified analogs.

    PubMed

    Lapikova, E S; Drozd, N N; Tolstenkov, A S; Makarov, V A; Zvyagintseva, T N; Shevchenko, N M; Bakunina, I U; Besednova, N N; Kuznetsova, T A

    2008-09-01

    Specimens of fucoidan extracted from Fucus evanescens were purified from protein and polyphenols, deacetylated and depolymerized by fucoidanase for evaluation of their biological activity. Deacetylation did not modify the capacity of fucoidan to inhibit thrombin and factor Xa, while purification from protein and polyphenols reduced this capacity. Depolymerization of fucoidan increased its capacity to inhibit thrombin mainly through heparin cofactor II. All the studied specimens formed complexes with protamine sulfate. PMID:19240852

  10. Substitution of valine for glycine-558 in the congenital dysthrombin thrombin Quick II alters primary substrate specificity

    SciTech Connect

    Henriksen, R.A.; Mann, K.G. )

    1989-03-07

    Thrombin Quick II is one of two dysfunctional forms of thrombin derived from the previously described congenital dysprothrombin prothrombin Quick. Thrombin Quick II does not clot fibrinogen, hydrolyze p-nitroanilide substrates of thrombin, or bind N{sup 2}-(5-(dimethylamino)naphthalene-1-sulfonyl)arginine N,N-(3-ethyl-1,5-pentanediyl)amide, a high-affinity competitive inhibitor of thrombin. To determine the structural alteration in thrombin Quick II, the reduced, carboxymethylated protein was hydrolyzed by a lysyl endopeptidase. A peptide not present in a parallel thrombin hydrolysate was identified by reverse-phase chromatography. This Gly residue, which is highly conserved in the chymotrypsin family of serine proteases, forms part of the substrate binding pocket for bulky aromatic and basic side chains in chymotrypsin and trypsin, respectively. However, in porcine elastase 1, the corresponding residue is threonine. Consistent with the identified structural alteration, thrombin Quick II incorporates ({sup 3}H)diisopropyl fluorophosphate stoichiometrically and hydrolyzes the elastase substrate succinyl-Ala-Ala-Pro-Leu-p-nitroanilide with a relative k{sub cat}/K{sub M} of 0.14 when compared to thrombin. This results from a 3-fold increase in K{sub M} and a 2.5-fold decrease in k{sub cat} for thrombin Quick II when compared to thrombin acting on the same substrate. These results and those of other investigators studying mutant trypsins support the conclusion that the catalytic activity of serine proteases is very sensitive to structural alterations in the primary substrate binding pocket.

  11. Equilibrium binding of thrombin to recombinant human thrombomodulin: Effect of hirudin, fibrinogen, factor Va, and peptide analogues

    SciTech Connect

    Tsiang, Manuel; Lentz, S.R.; Dittman, W.A.; Wen, D.; Scarpati, E.M.; Sadler, J.E. )

    1990-11-01

    Thrombomodulin is an endothelial cell surface receptor for thrombin that acts as a physiological anticoagulant. The properties of recombinant human thrombomodulin were studied in COS-7, CHO, CV-1, and K562 cell lines. Thrombomudlin was expressed on the cell surface as shown by the acquisition of thrombin-dependent protein C activation. Like native thrombomodulin, recombinant thrombomodulin contained N-linked oligosaccharides, had M{sub r} {approximately} 100 000, and was inhibited or immunoprecipitated by anti-thrombomodulin antibodies. Binding studies demonstrated that nonrecombinant thrombomodulin expressed by A549 carcinoma cells and recombinant thrombomodulin expressed by CV-1 and K562 cells had similar K{sub d}'s for thrombin of 1.3 nM, 3.3 nM, and 4.7 nM, respectively. The K{sub d} for DIP-thrombin binding to recombinant thrombomodulin on CV-1(18A) cells was identical with that of thrombin. Increasing concentrations of hirudin or fibrinogen progressively inhibited the binding of {sup 125}I-DIP-thrombin, while factor Va did not inhibit binding. Three synthetic peptides were tested for ability to inhibit DIP-thrombin, while factor Va did not inhibit binding. Three synthetic peptides were tested for ability to inhibit DIP-thrombin binding. Both the hirudin peptide Hir{sup 53-64} and the thrombomodulin fifth-EGF-domain peptide Tm{sup 426-444} displaced DIP-thrombin from thrombomodulin, but the factor V peptide FacV{sup 30-43} which is similar in composition and charge to Hir{sup 53-64} showed no binding inhibition. The data exclude the significant formation of a ternary complex consisting of thrombin, thrombomodulin, and hirudin. These studies are consistent with a model in which thrombomodulin, hirudin, and fibrinogen compete for binding to DIP-thrombin at the same site.

  12. Baicalin protects against thrombin induced cell injury in SH-SY5Y cells

    PubMed Central

    Ju, Xiao-Ning; Mu, Wei-Na; Liu, Yuan-Tao; Wang, Mei-Hong; Kong, Feng; Sun, Chao; Zhou, Qing-Bo

    2015-01-01

    Baicalin, an extract from the dried root of Scutellaria baicalensis Georgi, was shown to be neuroprotective. However, the precise mechanisms are incompletely known. In this study, we determined the effect of baicalin on thrombin induced cell injury in SH-SY5Y cells, and explored the possible mechanisms. SH-SY5Y cells was treated with thrombin alone or pre-treated with baicalin (5, 10, 20 μM) for 2 h followed by thrombin treatment. Cells without thrombin and baicalin treatment were used as controls. Cell viability was detected by MTT assay. Cell apoptosis was analyzed by flow cytometry. Real-time PCR was performed to determine the mRNA expression of protease-activated receptor-1 (PAR-1). Western blotting was conducted to determine the protein expression of PAR-1, Caspase-3 and NF-κB. Baicalin reduced cell death following thrombin treatment in a dose-dependent manner, with concomitant inhibition of NF-κB activation and suppression of PAR-1 expression. In addition, baicalin reduced Caspase-3 expression. The above findings indicated that baicalin prevents against cell injury after thrombin stimulation possibly through inhibition of PAR-1 expression and NF-κB activation. PMID:26823714

  13. Ezrin/radixin/moesin proteins differentially regulate endothelial hyperpermeability after thrombin.

    PubMed

    Adyshev, Djanybek M; Dudek, Steven M; Moldobaeva, Nurgul; Kim, Kyung-mi; Ma, Shwu-Fan; Kasa, Anita; Garcia, Joe G N; Verin, Alexander D

    2013-08-01

    Endothelial cell (EC) barrier disruption induced by inflammatory agonists such as thrombin leads to potentially lethal physiological dysfunction such as alveolar flooding, hypoxemia, and pulmonary edema. Thrombin stimulates paracellular gap and F-actin stress fiber formation, triggers actomyosin contraction, and alters EC permeability through multiple mechanisms that include protein kinase C (PKC) activation. We previously have shown that the ezrin, radixin, and moesin (ERM) actin-binding proteins differentially participate in sphingosine-1 phosphate-induced EC barrier enhancement. Phosphorylation of a conserved threonine residue in the COOH-terminus of ERM proteins causes conformational changes in ERM to unmask binding sites and is considered a hallmark of ERM activation. In the present study we test the hypothesis that ERM proteins are phosphorylated on this critical threonine residue by thrombin-induced signaling events and explore the role of the ERM family in modulating thrombin-induced cytoskeletal rearrangement and EC barrier function. Thrombin promotes ERM phosphorylation at this threonine residue (ezrin Thr567, radixin Thr564, moesin Thr558) in a PKC-dependent fashion and induces translocation of phosphorylated ERM to the EC periphery. Thrombin-induced ERM threonine phosphorylation is likely synergistically mediated by protease-activated receptors PAR1 and PAR2. Using the siRNA approach, depletion of either moesin alone or of all three ERM proteins significantly attenuates thrombin-induced increase in EC barrier permeability (transendothelial electrical resistance), cytoskeletal rearrangements, paracellular gap formation, and accumulation of phospho-myosin light chain. In contrast, radixin depletion exerts opposing effects on these indexes. These data suggest that ERM proteins play important differential roles in the thrombin-induced modulation of EC permeability, with moesin promoting barrier dysfunction and radixin opposing it. PMID:23729486

  14. The direct thrombin inhibitor hirudin.

    PubMed

    Greinacher, Andreas; Warkentin, Theodore E

    2008-05-01

    This review discusses the pharmacology and clinical applications of hirudin, a bivalent direct thrombin inhibitor (DTI). Besides the current major indication for hirudin--anticoagulation of patients with heparin-induced thrombocytopenia (HIT)--the experience with hirudin in other indications, especially acute coronary syndromes, are briefly presented. Hirudins have been formally studied prior to their regulatory approval; however, important information on their side effects and relevant preventative measures only became available later. Therefore, current recommendations and dosing schedules for hirudin differ considerably from the information given in the package inserts. Drawbacks of hirudin and important precautions for avoiding potential adverse effects are discussed in detail in the third part of this review. PMID:18449411

  15. Magnesium as a regulator of thrombin formation in bovine ovarian follicular fluid.

    PubMed

    Yamada, M; Hirakushi, K; Inoue, K; Horiuchi, T; Sakai, J; Okada, T; Sugie, I

    1998-07-01

    In the ovarian follicular fluid (FF) of Holstein cows, calcium (Ca) and magnesium (Mg) levels and their roles on thrombin generation were examined and compared with the blood samples. Total Ca levels in FF increased while the total Mg levels decreased with follicular development from preantral to preovulatory stage of follicles. These changes resulted in Ca values being significantly (p < 0.05) higher in FF from the most developed follicles and the Mg values being significantly higher (p < 0.05) in the least developed follicles. To determine whether the high level of Mg might function to regulate thrombin generation in FF as occurs in plasma, the influence of Mg supplementation of FF from various types of follicles was examined. In FF from small size follicles, Mg accelerated the prothrombin time, an estimation of the overall rate of thrombin production, although a similar effects was not observed in FF from medium and large size follicles. The addition of Mg to FF from all sizes of follicles resulted an inhibition in factor X activation. Since activation of factor X is a precursor step for thrombin formation it is concluded that Mg can function as a slow accelerator of thrombin generation in FF from follicles at the antral stage of development. It is likely to have a more important role in regulating the rate of thrombin generation as the follicle develops. PMID:9713812

  16. Comparison of several activated partial thromboplastin time methods.

    PubMed

    Morin, R J; Willoughby, D

    1975-08-01

    Activated partial thromboplastin times (APTT's) performed with a semi-automated electrical-conductivity type of clot timer on plasmas from patients with hepatic disease and intravascular coagulation, and on warfarin or heparin therapy, were significantly lower than when done on the same plasmas with either a manual optical method or an automated optical-endpoint instrument. Results of APTT's done on normal plasmas by the three methods were not significantly different. Substitution of different activator-phospholipid reagents resulted in some variability in results, but these differences were less than those between the different done with both the electrical clot timer and the automated optical instrument on prepared plasmas containing 5.0 or 1.0% of factor II, V, VIII, IX, OR X revealed shorter times with the electrical clot timer only in the case of factor II- and factor V-deficient plasmas. APTT's done on normal plasmas to which 0.1 or 0.3 units per ml. of heparin had been added vitro also were shorter with the electrical clot itmer than the automatic optical instrument. Prothrombin times done on normal and abnormal control plasmas and on a series of plasmas from patients on warfarin therapy showed no significant difference between the two methods. PMID:239589

  17. Follow-up of thrombin generation after prostate cancer surgery: global test for increased hypercoagulability.

    PubMed

    Benyo, Matyas; Flasko, Tibor; Molnar, Zsuzsanna; Kerenyi, Adrienne; Batta, Zoltan; Jozsa, Tamas; Harsfalvi, Jolan

    2012-01-01

    Recent studies provided evidence that evaluation of thrombin generation identifies patients at thrombotic risk. Thrombin generation has a central role in hemorrhage control and vascular occlusion and its measurement provides new metrics of these processes providing sufficient evaluation of an individual's hemostatic competence and response to anticoagulant therapy. The objective of the study is to assess a new measure of hypercoagulability that predisposes to venous thromboembolism in the postoperative period after radical prostatectomy. Pre- (day-1) and postoperative (hour 1, day 6, month 1 and 10) blood samples of 24 patients were tested for plasma thrombin generation (peak thrombin), routine hematology and hemostasis. Patients received low molecular weight heparin for thromboprophylaxis. Peak thrombin levels were higher in patients compared to controls at baseline (p<0.001), and elevated further in the early postoperative period (p<0.001). Longer general anesthesia and high body mass index were associated with increased thrombin generation after surgery (p = 0.024 and p = 0.040). D dimer and fibrinogen levels were higher after radical prostatectomy (p = 0.001 and p<0.001). Conventional clotting tests remained within the reference range. Our study contributed to the cognition of the hypercoagulable state in cancer patients undergoing pelvic surgery and revealed the course of thrombin generation after radical prostatectomy. Whilst it is unsurprising that thrombin generation increases after tissue trauma, further evaluation of this condition during the postoperative period would lead urologists to an international and well-supported consensus regarding thromboprophylaxis in order to provide better clinical outcome. Considering the routine evaluation of procoagulant activity and extending prophylactic anticoagulant therapy accordingly may potentially prevent late thrombotic events. PMID:23236465

  18. Gold nanoparticles doped conducting polymer nanorod electrodes: ferrocene catalyzed aptamer-based thrombin immunosensor.

    PubMed

    Rahman, Md Aminur; Son, Jung Ik; Won, Mi-Sook; Shim, Yoon-Bo

    2009-08-15

    Au nanoparticles-doped conducting polymer nanorods electrodes (AuNPs/CPNEs) were prepared by coating Au nanorods (AuNRs) with a conducting polymer layer. The AuNRs were prepared through an electroless deposition method using the polycarbonate membrane (pore diameter, 50 nm, pore density, 6 x 10(8) pores/cm(2)) as a template. The AuNPs/CPNEs combining catalytic activity of ferrocene to ascorbic acid were used for the fabrication of an ultrasensitive aptamer sensor for thrombin detection. The AuNPs/3D-CPNEs were characterized employing cyclic voltammetry (CV), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and atomic force microscopy (AFM). Sandwiched immunoassay for alpha-human thrombin with NH(2)-functionalized-thrombin binding aptamer (Apt) immobilized on AuNPs/3D-CPNEs was studied through the electrocatalytic oxidation of ascorbic acid by the ferrocene moiety that was bound with an antithrombin antibody and attached with the Apt/3D-CPNEs probe through target binding. Various experimental parameters affecting thrombin detection were optimized, and the performance of the thrombin aptamer sensor was examined. The Apt/AuNPs/3D-CPNEs based thrombin sensor exhibited a wide dynamic range of 5-2000 ng L(-1) and a low detection limit of 5 ng L(-1) (0.14 pM). The selectivity and the stability of the proposed thrombin aptamer sensor were excellent, and it was tested in a real human serum sample for the detection of spiked concentrations of thrombin. PMID:20337374

  19. Energetics of thrombin-fibrinogen interaction.

    PubMed

    Hopfner, K P; Di Cera, E

    1992-11-24

    The kinetic mechanism of thrombin-fibrinogen interaction has been elucidated by steady-state measurements of synthetic substrate hydrolysis by human alpha-thrombin in the presence of human fibrinogen used as a competitive inhibitor and sucrose used as a viscogenic agent. Sucrose greatly affects the FKm for thrombin-fibrinogen interaction, without altering the intrinsic properties of the system. Under conditions of pH 7.5 and 0.1 M NaCl, fibrinogen behaves like a sticky substrate for thrombin, with acylation being comparable to dissociation in the temperature range 20-37 degrees C. In the same temperature range, deacylation is much faster than acylation. The van't Hoff enthalpy of binding for thrombin-fibrinogen interaction is -24 +/- 3 kcal/mol and the entropy is -55 +/- 11 cal mol-1 deg-1. A chemical compensation effect is present in the binding of fibrinogen and synthetic amide substrates to thrombin, with the delta H and delta G values being linked through a linear relationship. PMID:1445891

  20. PROTON BRIDGING IN THE INTERACTIONS OF THROMBIN WITH SMALL INHIBITORS†

    PubMed Central

    Kovach, Ildiko M.; Kelley, Paul; Eddy, Carol; Jordan, Frank; Baykal, Ahmet

    2009-01-01

    Thrombin is the pivotal serine protease enzyme in the blood cascade system. Phe-Pro-Arg-chloromethylketone (PPACK), phosphate and phosphonate ester inhibitors form a covalent bond with the active-site Ser of thrombin. PPACK, a mechanism-based inhibitor, and the phosphate/phosphonate esters form adducts that mimic intermediates formed in reactions catalyzed by thrombin. Therefore, the dependence of the inhibition of human α-thrombin on the concentration of these inhibitors, pH, and temperature was investigated. The second-order rate constant, ki/Ki, and the inhibition constant, Ki, for inhibition of human α-thrombin by PPACK are (1.1 ± 0.2) × 107 M−1 s−1 and (2.4 ± 1.3) × 10−8 M at pH 7.00 in 0.05 M phosphate buffer, 0.15 M NaCl, and 25.0 ± 0.1˚C, and in good agreement with previous reports. The activation parameters at pH 7.00, 0.05M phosphate buffer 0.15 M NaCl, are ΔH‡ = 10.6 ± 0.7 kcal/mol and ΔS‡ = 9 ± 2 cal/mol deg. The pH dependence of the second-order rate constants of inhibition is bell shaped. Values of pKa1 and pKa2 are 7.3 ± 0.2 and 8.8 ± 0.3, respectively, at 25.0 ± 0.1 °C. A phosphate and a phosphonate ester inhibitor gave higher values, 7.8 and 8.0, for pKa1 and 9.3 and 8.6 for pKa2. They inhibit thrombin over six orders of magnitude less efficiently than PPACK does. The deuterium solvent isotope effect for the second-order rate constant at pH 7.0 and 8.3 at 25.0 ± 0.1°C is unity within experimental error in all three cases, indicating the absence of proton transfer in the rate-determining step for the association of thrombin with the inhibitors. But in a 600 MHz 1H NMR spectrum of the inhibition adduct at pH 6.7 and 30 °C, a peak at 18.10 ppm with respect to TSP appears with PPACK, which is absent in the 1H NMR spectrum of a solution of the enzyme between pH 5.3–8.5. The peak at low field is an indication of the presence an SSHB at the active site in the adduct. The deuterium isotope effect on this hydrogen bridge is 2

  1. Inhibitory effect of apixaban compared with rivaroxaban and dabigatran on thrombin generation assay.

    PubMed

    Wong, Pancras C; White, Andrew; Luettgen, Joseph

    2013-02-01

    The effect of the oral direct activated factor X (factor Xa) inhibitor apixaban on tissue factor-induced thrombin generation in human plasma was investigated in vitro using the calibrated automated thrombogram (CAT) method and compared with the oral direct factor Xa inhibitor rivaroxaban and the direct thrombin inhibitor dabigatran. Pooled citrated, anticoagulated, platelet-poor human plasma was spiked with apixaban, rivaroxaban, or dabigatran at concentrations of 0.01 to 10 μM. The inhibitory potencies of the compounds were quantified by 5 CAT parameters: the control thrombin lag time (LT) and time to thrombin peak (TTP) for the doubling of inhibitor concentration (IC2x); and the control endogenous thrombin potential (ETP), thrombin peak, and maximum rate of thrombin generation (Vmax) for the inhibitor concentration, which inhibited 50% (IC50). The inhibitors modified CAT concentration dependently. Their inhibitory potencies, expressed as IC2x LT, IC2x TTP, IC50 ETP, IC50 peak thrombin, and IC50 Vmax, were as follows: 0.10 ± 0.01, 0.19 ± 0.02, 0.65 ± 0.11, 0.089 ± 0.019, and 0.049 ± 0.007 μM for apixaban; 0.049 ± 0.007, 0.070 ± 0.009, 0.43 ± 0.07, 0.048 ± 0.008, and 0.022 ± 0.005 μM for rivaroxaban; and 0.063 ± 0.019, 0.18 ± 0.06, 0.50 ± 0.08, 0.55 ± 0.06, and 0.57 ± 0.27 μM for dabigatran. In summary, apixaban, rivaroxaban, and dabigatran have similar potencies in the prolongation of LT and TTP. The CAT parameters that are related to the rate of thrombin generation during the propagation phase (ie, peak thrombin and Vmax) are more sensitive to activities of apixaban and rivaroxaban than dabigatran. The ETP is the least sensitive parameter for measuring the activities of these inhibitors. Recombinant activated factor VII at 5 and 50 μg/mL reversed the anticoagulant effects of apixaban more at 0.2 μM than at 2 μM. Our study suggests that the CAT method is a sensitive assay to monitor the pharmacodynamic and pharmacokinetic properties of

  2. Thrombin-induced contraction in alveolar epithelial cells probed by traction microscopy.

    PubMed

    Gavara, Núria; Sunyer, Raimon; Roca-Cusachs, Pere; Farré, Ramon; Rotger, Mar; Navajas, Daniel

    2006-08-01

    Contractile tension of alveolar epithelial cells plays a major role in the force balance that regulates the structural integrity of the alveolar barrier. The aim of this work was to study thrombin-induced contractile forces of alveolar epithelial cells. A549 alveolar epithelial cells were challenged with thrombin, and time course of contractile forces was measured by traction microscopy. The cells exhibited basal contraction with total force magnitude 55.0 +/- 12.0 nN (mean +/- SE, n = 12). Traction forces were exerted predominantly at the cell periphery and pointed to the cell center. Thrombin (1 U/ml) induced a fast and sustained 2.5-fold increase in traction forces, which maintained peripheral and centripetal distribution. Actin fluorescent staining revealed F-actin polymerization and enhancement of peripheral actin rim. Disruption of actin cytoskeleton with cytochalasin D (5 microM, 30 min) and inhibition of myosin light chain kinase with ML-7 (10 microM, 30 min) and Rho kinase with Y-27632 (10 microM, 30 min) markedly depressed basal contractile tone and abolished thrombin-induced cell contraction. Therefore, the contractile response of alveolar epithelial cells to the inflammatory agonist thrombin was mediated by actin cytoskeleton remodeling and actomyosin activation through myosin light chain kinase and Rho kinase signaling pathways. Thrombin-induced contractile tension might further impair alveolar epithelial barrier integrity in the injured lung. PMID:16675616

  3. [Sodium ions as the effector of catalytic action of alpha-thrombin].

    PubMed

    Kolodzeĭskaia, M V; Volkov, G L

    2007-01-01

    A process of thrombin interaction with synthetic and natural substrates in the presence of Na+ ions has been analyzed in the survey. Molecular bases of this interaction have been presented, interrelation between the structure and function of thrombin has been noted; the nature of the unique site of its active centre which determines high thrombin affinity for the substrates and increase of its catalytic activity defined by the term of "specificity to univalent cations" have been considered in detail. Na+ ions play the role of allosteric effector in realization of two informational states of thrombin which penform, respectively, two fundamental and competing functions in the process of hemostasis. The molecular basis of the process of Na+ binding with thrombin is rather simple and depends only on the single site which importance for the enzyme function is marked by numerous investigations of a number of authors, and it is shown that Na(+)-binding site is distributed in the other zone of thrombin molecule as compared to exosites I and II, which do not take part in Na(+)-binding and allosteric transduction. Considerable attention was given to conformational conversions of a thrombin molecule caused by Na+ ions binding. It was shown that the transition slow <--> fast of the enzyme forms leads to formation of the ion pair Arg-187: Asp-222, optimal orientation of Asp-189 and Ser-195 for binding of substrates and considerable shift of the lateral chain Glu-192 determined by the disturbance of the lattice of water molecules which connects Na(+)-binding site with aminoacid Ser-195 of the active centre of the enzyme. New data have been presented which indicate that the changes in the lattice of water molecules and allosteric nucleus of Na(+)-binding site of the enzyme are the basic link of raising the affinity between the thrombin and substrate and mechanism of the enzyme activation by Na(+)-ions. The survey touches some problems of creation of allosteric inhibitors of

  4. Allogeneic Platelet Releasate Preparations Derived via a Novel Rapid Thrombin Activation Process Promote Rapid Growth and Increased BMP-2 and BMP-4 Expression in Human Adipose-Derived Stem Cells

    PubMed Central

    McLaughlin, Michael; Gagnet, Paul; Cunningham, Elizabeth; Yeager, Randi; D'Amico, Michael; Guski, Katie; Scarpone, Michael; Kuebler, Daniel

    2016-01-01

    The administration of human adipose-derived stem cells (ASCs) represents a promising regenerative therapy for the treatment of orthopedic injuries. While ASCs can be easily isolated from liposuction-derived adipose tissue, most clinical applications will likely require in vitro culture expansion of these cells using nonxenogeneic components. In this study, platelet releasate was generated using a novel rapid thrombin activation method (tPR). ASCs grown in media supplemented with tPR proliferated much faster than ASCs grown in media supplemented with 10% fetal bovine serum. The cells also retained the ability to differentiate along chondrogenic, adipogenic, and osteogenic lineages. The tPR cultured ASCs displayed elevated expression of BMP-4 (5.7 ± 0.97-fold increase) and BMP-2 (4.7 ± 1.3-fold increase) and decreased expression of PDGF-B (4.0 ± 1.4-fold decrease) and FGF-2 (33 ± 9.0-fold decrease). No significant changes in expression were seen with TGF-β and VEGF. This pattern of gene expression was consistent across different allogeneic tPR samples and different ASC lines. The use of allogeneic rapidly activated tPR to culture ASCs is associated with both an increased cell yield and a defined gene expression profile making it an attractive option for cell expansion prior to cell-based therapy for orthopedic applications. PMID:26823671

  5. Low-Level Resistance of Staphylococcus aureus to Thrombin-Induced Platelet Microbicidal Protein 1 In Vitro Associated with qacA Gene Carriage Is Independent of Multidrug Efflux Pump Activity

    PubMed Central

    Bayer, A. S.; Kupferwasser, L. I.; Brown, M. H.; Skurray, R. A.; Grkovic, S.; Jones, T.; Mukhopadhay, K.; Yeaman, M. R.

    2006-01-01

    Thrombin-induced platelet microbial protein 1 (tPMP-1), a cationic antimicrobial polypeptide released from thrombin-stimulated rabbit platelets, targets the Staphylococcus aureus cytoplasmic membrane to initiate its microbicidal effects. In vitro resistance to tPMP-1 correlates with survival advantages in vivo. In S. aureus, the plasmid-carried qacA gene encodes a multidrug transporter, conferring resistance to organic cations (e.g., ethidium [Et]) via proton motive force (PMF)-energized export. We previously showed that qacA also confers a tPMP-1-resistant (tPMP-1r) phenotype in vitro. The current study evaluated whether (i) transporters encoded by the qacB and qacC multidrug resistance genes also confer tPMP-1r and (ii) tPMP-1r mediated by qacA is dependent on efflux pump activity. In contrast to tPMP-1r qacA-bearing strains, the parental strain and its isogenic qacB- and qacC-containing strains were tPMP-1 susceptible (tPMP-1s). Efflux pump inhibition by cyanide m-chlorophenylhydrazone abrogated Etr, but not tPMP-1r, in the qacA-bearing strain. In synergy assays, exposure of the qacA-bearing strain to tPMP-1 did not affect the susceptibility of Et (ruling out Et-tPMP-1 cotransport). The following cytoplasmic membrane parameters did not differ significantly between the qacA-bearing and parental strains: contents of the major phospholipids; asymmetric distributions of the positively charged species, lysyl-phosphotidylglycerol; fatty acid composition; and relative surface charge. Of note, the qacA-bearing strain exhibited greater membrane fluidity than that of the parental, qacB-, or qacC-bearing strain. In conclusion, among these families of efflux pumps, only the multidrug transporter encoded by qacA conferred a tPMP-1r phenotype. These data suggest that qacA-encoded tPMP-1r results from the impact of a specific transporter upon membrane structure or function unrelated to PMF-dependent peptide efflux. PMID:16801425

  6. Adenosine regulates the proinflammatory signaling function of thrombin in endothelial cells

    PubMed Central

    Hassanian, Seyed Mahdi; Dinarvand, Peyman; Rezaie, Alireza R.

    2014-01-01

    The plasma level of the regulatory metabolite adenosine increases during the activation of coagulation and inflammation. Here we investigated the effect of adenosine on modulation of thrombin-mediated proinflammatory responses in HUVECs. We found that adenosine inhibits the barrier-disruptive effect of thrombin in HUVECs by a concentration-dependent manner. Analysis of cell surface expression of adenosine receptors revealed that A2A and A2B are expressed at the highest level among the four receptor subtypes (A2B>A2A>A1>A3) on HUVECs. The barrier-protective effect of adenosine in response to thrombin was recapitulated by the A2A specific agonist, CGS 21680, and abrogated both by the siRNA knockdown of the A2A receptor and by the A2A-specific antagonists, ZM-241385 and SCH-58261. The thrombin-induced RhoA activation and its membrane translocation were both inhibited by adenosine in a cAMP-dependent manner, providing a molecular mechanism through which adenosine exerts a barrier-protective function. Adenosine also inhibited thrombin-mediated activation of NF-κB and decreased adhesion of monocytic THP-1 cells to stimulated HUVECs via down-regulation of expression of cell surface adhesion molecules, VCAM-1, ICAM-1 and E-selectin. Moreover, adenosine inhibited thrombin-induced elevated expression of proinflammatory cytokines, IL-6 and HMGB-1; and chemokines, MCP-1, CXCL-1 and CXCL-3. Taken together, these results suggest that adenosine may inhibit thrombin-mediated proinflammatory signaling responses, thereby protecting the endothelium from injury during activation of coagulation and inflammation. PMID:24477600

  7. Thrombin Inhibition with Dabigatran Protects against High-Fat Diet–Induced Fatty Liver Disease in Mice

    PubMed Central

    Kopec, Anna K.; Joshi, Nikita; Towery, Keara L.; Kassel, Karen M.; Sullivan, Bradley P.; Flick, Matthew J.

    2014-01-01

    Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of obesity and metabolic syndrome. Robust coagulation cascade activation is common in obese patients with NAFLD. We identified a critical temporal relationship between thrombin generation and the manifestation of hepatic steatosis, inflammation, and injury in C57BL/6J mice fed a high-fat diet (HFD) for 1, 2, and 3 months. Mice fed a HFD exhibited dramatic increases in hepatocellular injury and inflammation over time. Hepatic fibrin deposition preceded an increase in serum alanine aminotransferase, and the most dramatic changes in liver histopathology occurred in conjunction with a detectable increase in plasma thrombin-antithrombin levels at 3 months. To directly determine whether thrombin activity promotes NAFLD pathogenesis, mice were fed a HFD and simultaneously treated with the direct thrombin inhibitor dabigatran etexilate for 3 months. Notably, dabigatran treatment significantly reduced hepatic fibrin deposition, hepatic inflammation, hepatocellular injury, and steatosis in mice fed a HFD. Of interest, dabigatran treatment also significantly attenuated HFD-induced body weight gain. Gene expression analysis suggested that thrombin potentially drives NAFLD pathogenesis by altering the expression of genes associated with lipid metabolism and bile acid synthesis. Collectively, the results suggest that thrombin activity is central to HFD-induced body weight gain, liver injury, and inflammation and provide the proof-of-principle evidence that pharmacological thrombin inhibition could be effective in limiting NAFLD and associated pathologies. PMID:25138021

  8. Thrombin inhibition by the highly selective 'reversible suicide substrate' N-ethoxycarbonyl-D-phenylalanyl-L-prolyl-alpha-azalysine p-nitrophenyl ester.

    PubMed

    Ascenzi, Paolo; Gallina, Carlo; Bolognesi, Martino

    2005-07-01

    Thrombin is the last enzyme in the blood coagulation cascade. All pharmacological aspects support the use of thrombin inhibitors as antithrombotic agents. Here, we review the unusual inhibition behavior of the highly selective 'reversible suicide substrate' N-ethoxycarbonyl-D-phenylalanyl-L-prolyl-alpha-azalysine p-nitrophenyl ester (Eoc-D-Phe-Pro-azaLys-ONp) targeted to the active center of human alpha-thrombin. Eoc-D-Phe-Pro-azaLys-ONp is an acylating agent, but its hydrolysis product 1(N-ethoxycarbonyl-D-phenylalanyl-L-prolyl)-2(4-aminobutyl) hydrazine behaves as a highly selective human alpha-thrombin competitive inhibitor. PMID:16029155

  9. Protein C inhibits endocytosis of thrombin-thrombomodulin complexes in A549 lung cancer cells and human umbilical vein endothelial cells

    SciTech Connect

    Maruyama, I.; Majerus, P.W.

    1987-05-01

    We investigated the effect of protein C on the endocytosis of thrombin-thrombomodulin complexes. We previously showed that exposure of umbilical vein endothelial cells to thrombin stimulated the internalization and degradation of thrombin. A similar internalization was stimulated by a monoclonal antithrombomodulin antibody. We have repeated these studies in the presence of protein C and found that endocytosis of /sup 125/I-thrombin-thrombomodulin complexes, but not /sup 125/I-antithrombomodulin-thrombomodulin complexes, is inhibited. Activated protein C did not inhibit endocytosis of thrombin-thrombomodulin complexes. Protein C inhibited both internalization and degradation of /sup 125/I-thrombin and diisopropylphosphoryl (DIP) /sup 125/I-thrombin in human lung cancer cells (A549). These effects were observed at protein C concentrations found in human plasma. Protein S had no effect on the inhibition of endocytosis of thrombin-thrombomodulin complexes by protein C. We propose that protein C may regulate the rate of endocytosis of thrombin-thrombomodulin complexes in vivo and thereby control the capacity for endothelium to activate protein C.

  10. Antithrombotic effects of synthetic peptides targeting various functional domains of thrombin.

    PubMed Central

    Kelly, A B; Maraganore, J M; Bourdon, P; Hanson, S R; Harker, L A

    1992-01-01

    To determine in vivo functional roles for thrombin's structural domains, we have compared the relative antithrombotic and antihemostatic effects of (i) catalytic-site antithrombin peptide, D-Phe-Pro-Arg; (ii) exosite antithrombin peptide, the C-terminal tyrosine-sulfated dodecapeptide of hirudin; and (iii) bifunctional antithrombin peptide, a 20-mer peptide combining catalytic-site antithrombin peptide and exosite antithrombin peptide with a polyglycyl linker. All three peptides inhibited thrombin-mediated platelet aggregation and fibrin formation in vitro. In vivo thrombus formation was measured in real time as 111In-labeled platelet deposition and 125I-labeled fibrin accumulation on thrombogenic segments incorporated into chronic exteriorized arteriovenous access shunts in baboons. Under low flow conditions, the continuous infusion of peptides reduced thrombus formation onto collagen-coated tubing by half at doses (ID50) and corresponding concentrations (IC50) of 800 nmol per kg per min and 400 nmol/ml for catalytic-site antithrombin peptide, greater than 1250 nmol per kg per min and greater than 1500 mumol/ml for exosite antithrombin peptide, and 50 nmol per kg per min and 25 nmol/ml for bifunctional antithrombin peptide. Under arterial flow conditions, systemically administered bifunctional antithrombin peptide decreased thrombus formation in a dose-dependent manner for segments of collagen-coated tubing or prosthetic vascular graft ID50 and IC50 values of 120 nmol per kg per min and 15 nmol/ml; this dose also produced intermediate inhibition of hemostatic function [bleeding time, 21 +/- 3 min vs. 4.5 +/- 0.5 min (baseline values); P less than 0.001; activated partial thromboplastin time, 285 +/- 13 sec vs. 31 +/- 3 sec (baseline), P less than 0.001]. In contrast, thrombus formation onto segments of endarterectomized aorta was potently decreased by bifunctional antithrombin peptide with an ID50 value of 2.4 nmol per kg per min and an IC50 value of 0.75 nmol

  11. Positive cooperativity between the thrombin and bradykinin B2 receptors enhances arachidonic acid release

    PubMed Central

    Hecquet, Claudie; Biyashev, Dauren; Tan, Fulong; Erdös, Ervin G.

    2006-01-01

    Bradykinin (BK) or kallikreins activate B2 receptors (R) which couple Gαi and Gαq proteins to release arachidonic acid (AA) and elevate [Ca2+]i. Thrombin cleaves the protease-activated-receptor-1 (PAR1) that couples Gαi, Gαq and Gα12/13 proteins. In CHO cells stably transfected with human B2R, thrombin liberated little AA, but it significantly potentiated AA release by B2R agonists. We explored mechanisms of cooperativity between constitutively expressed PAR1 and B2R. We also examined human endothelial cells expressing both Rs constitutively. The PAR1 agonist hexapeptide (TRAP) was as effective as thrombin. Inhibitors of components of Gαi, Gαq and Gα12/13 signaling pathways, and a PKCα inhibitor, Gö6976 blocked potentiation while phorbol, an activator, enhanced it. Several inhibitors, including a RhoA kinase inhibitor, a [Ca2+]i antagonist, and an inositol-(1,3,4)-trisphosphate R antagonist, reduced mobilization of [Ca2+]i by thrombin and blocked potentiation of AA release by B2R agonists. Because either a non-selective inhibitor (isotetrandrine) of phospholipase A2 (PLA2) or a Ca2+-dependent PLA2 inhibitor abolished potentiation of AA release by thrombin, while a Ca2+-independent PLA2 inhibitor did not, we concluded that the mechanism involves Ca2+-dependent PLA2 activation. Both thrombin and TRAP modified activation and phosphorylation of the B2R induced by BK. In lower concentrations they enhanced it, while higher concentrations inhibited phosphorylation and diminished B2R activation. Protection of the N-terminal Ser1-Phe2 bond of TRAP by an aminopeptidase inhibitor made this peptide much more active than the unprotected agonist. Thus, PAR1 activation enhances AA release by B2R agonists through signal transduction pathway. PMID:16183725

  12. Lysosomotropic agents selectively potentiate thrombin-induced acid hydrolase secretion from platelets.

    PubMed Central

    Van Oost, B A; Smith, J B; Holmsen, H; Vladutiu, G D

    1985-01-01

    Thrombin induces partial secretion (up to 60%) of beta-N-acetyl-D-hexosaminidase (EC 3.2.1.52) from untreated platelets. Preincubation of platelets with 10 mM NH4Cl for up to 2 hr resulted in a time-dependent and marked stimulation of thrombin-induced secretion of both this enzyme and other acid glycosidases from platelets. The enhancement of the thrombin-induced secretion was not due to cell lysis, and NH4Cl alone did not cause leakage of lysosomal enzymes into the medium. The effect could be reversed by reincubating the platelets in NH4Cl-free medium. Stimulation of thrombin-induced secretion also was produced by a series of aliphatic primary amines from methylamine to butylamine, and by micromolar concentrations of chloroquine. The effect of weak bases on platelets appeared to be quite specific for enhancing lysosomal enzyme secretion. Thrombin-induced secretion of adenine nucleotides from dense granules and of beta-thromboglobulin from alpha granules was slightly enhanced by NH4Cl but was slightly inhibited by methylamine. The only direct effect of the weak bases on platelets was the displacement of serotonin from dense granules. Accumulation of weak bases in acidic pools in the platelets (e.g., lysosomes) might, therefore, be responsible for the enhanced secretion of lysosomal enzymes. By using controlled digitonin-induced platelet lysis, it was found that preincubation of platelets with NH4Cl lowered the digitonin concentration required for enzyme solubilization. We suggest that loading of lysosomes with weak bases dissociates already bound enzyme inside the lysosomes, resulting in a more effective discharge upon stimulation by thrombin. PMID:3157989

  13. In vitro study of the role of thrombin in platelet rich plasma (PRP) preparation: utility for gel formation and impact in growth factors release

    PubMed Central

    Huber, Stephany Cares; Cunha Júnior, José Luiz Rosenberis; Montalvão, Silmara; da Silva, Letícia Queiroz; Paffaro, Aline Urban; da Silva, Francesca Aparecida Ramos; Rodrigues, Bruno Lima; Lana, José Fabio Santos Duarte; Annichino-Bizzacchi, Joyce Maria

    2016-01-01

    Introduction: The use of PRP has been studied for different fields, with promising results in regenerative medicine. Until now, there is no study in the literature evaluating thrombin levels in serum, used as autologous thrombin preparation. Therefore, in the present study we evaluated the role played by different thrombin concentrations in PRP and the impact in the release of growth factors. Also, different activators for PRP gel formation were evaluated. Methods: Thrombin levels were measured in different autologous preparations: serum, L-PRP (PRP rich in leukocytes) and T-PRP (thrombin produced through PRP added calcium gluconate). L-PRP was prepared according to the literature, with platelets and leukocytes being quantified. The effect of autologous thrombin associated or not with calcium in PRP gel was determined by measuring the time of gel formation. The relationship between thrombin concentration and release of growth factors was determined by growth factors (PDGF-AA, VEGF and EGF) multiplex analysis. Results: A similar concentration of thrombin was observed in serum, L-PRP and T-PRP (8.13 nM, 8.63 nM and 7.56 nM, respectively) with a high variation between individuals (CV%: 35.07, 43 and 58.42, respectively). T-PRP and serum with calcium chloride showed similar results in time to promote gel formation. The increase of thrombin concentrations (2.66, 8 and 24 nM) did not promote an increase in growth factor release. Conclusions: The technique of using serum as a thrombin source proved to be the most efficient and reproducible for promoting PRP gel formation, with some advantages when compared to other activation methods, as this technique is easier and quicker with no need of consuming part of PRP. Noteworthy, PRP activation using different thrombin concentrations did not promote a higher release of growth factors, appearing not to be necessary when PRP is used as a suspension. PMID:27397996

  14. Shc adaptor proteins are key transducers of mitogenic signaling mediated by the G protein-coupled thrombin receptor.

    PubMed Central

    Chen, Y; Grall, D; Salcini, A E; Pelicci, P G; Pouysségur, J; Van Obberghen-Schilling, E

    1996-01-01

    The serine protease thrombin activates G protein signaling systems that lead to Ras activation and, in certain cells, proliferation. Whereas the steps leading to Ras activation by G protein-coupled receptors are not well defined, the mechanisms of Ras activation by receptor tyrosine kinases have recently been elucidated biochemically and genetically. The present study was undertaken to determine whether common signaling components are used by these two distinct classes of receptors. Here we report that the adaptor protein Shc, is phosphorylated on tyrosine residues following stimulation of the thrombin receptor in growth-responsive CCL39 fibroblasts. Shc phosphorylation by thrombin or the thrombin receptor agonist peptide is maximal by 15 min and persists for > or = 2 h. Following thrombin stimulation, phosphorylated Shc is recruited to Grb2 complexes. One or more pertussis toxin-insensitive proteins appear to mediate this effect, since (i) pertussis toxin pre-treatment of cells does not blunt the action of thrombin and (ii) Shc phosphorylation on tyrosine can be stimulated by the muscarinic m1 receptor. Shc phosphorylation does not appear to involve protein kinase C, since the addition of 4-beta-phorbol-12,13-dibutyrate has no effect. Rather, thrombin-induced Shc phosphorylation is enhanced in cells depleted of phorbol ester-sensitive protein kinase C isoforms. Expression of mutant Shc proteins defective in Grb2 binding displays a dominant-negative effect on thrombin-stimulated p44 MAP kinase activation, gene induction and cell growth. From these data, we conclude that Shc represents a crucial point of convergence between signaling pathways activated by receptor tyrosine kinases and G protein-coupled receptors. Images PMID:8605873

  15. Multiple Signaling Pathways Contribute to the Thrombin-induced Secretory Phenotype in Vascular Smooth Muscle Cells

    PubMed Central

    Jeong, Ji Young; Son, Younghae; Kim, Bo-Young; Eo, Seong-Kug; Rhim, Byung-Yong

    2015-01-01

    We attempted to investigate molecular mechanisms underlying phenotypic change of vascular smooth muscle cells (VSMCs) by determining signaling molecules involved in chemokine production. Treatment of human aortic smooth muscle cells (HAoSMCs) with thrombin resulted not only in elevated transcription of the (C-C motif) ligand 11 (CCL11) gene but also in enhanced secretion of CCL11 protein. Co-treatment of HAoSMCs with GF109230X, an inhibitor of protein kinase C, or GW5074, an inhibitor of Raf-1 kinase, caused inhibition of ERK1/2 phosphorylation and significantly attenuated expression of CCL11 at transcriptional and protein levels induced by thrombin. Both Akt phosphorylation and CCL11 expression induced by thrombin were attenuated in the presence of pertussis toxin (PTX), an inhibitor of Gi protein-coupled receptor, or LY294002, a PI3K inhibitor. In addition, thrombin-induced production of CCL11 was significantly attenuated by pharmacological inhibition of Akt or MEK which phosphorylates ERK1/2. These results indicate that thrombin is likely to promote expression of CCL11 via PKC/Raf-1/ERK1/2 and PTX-sensitive protease-activated receptors/PI3K/Akt pathways in HAoSMCs. We propose that multiple signaling pathways are involved in change of VSMCs to a secretory phenotype. PMID:26557022

  16. Multiple Signaling Pathways Contribute to the Thrombin-induced Secretory Phenotype in Vascular Smooth Muscle Cells.

    PubMed

    Jeong, Ji Young; Son, Younghae; Kim, Bo-Young; Eo, Seong-Kug; Rhim, Byung-Yong; Kim, Koanhoi

    2015-11-01

    We attempted to investigate molecular mechanisms underlying phenotypic change of vascular smooth muscle cells (VSMCs) by determining signaling molecules involved in chemokine production. Treatment of human aortic smooth muscle cells (HAoSMCs) with thrombin resulted not only in elevated transcription of the (C-C motif) ligand 11 (CCL11) gene but also in enhanced secretion of CCL11 protein. Co-treatment of HAoSMCs with GF109230X, an inhibitor of protein kinase C, or GW5074, an inhibitor of Raf-1 kinase, caused inhibition of ERK1/2 phosphorylation and significantly attenuated expression of CCL11 at transcriptional and protein levels induced by thrombin. Both Akt phosphorylation and CCL11 expression induced by thrombin were attenuated in the presence of pertussis toxin (PTX), an inhibitor of Gi protein-coupled receptor, or LY294002, a PI3K inhibitor. In addition, thrombin-induced production of CCL11 was significantly attenuated by pharmacological inhibition of Akt or MEK which phosphorylates ERK1/2. These results indicate that thrombin is likely to promote expression of CCL11 via PKC/Raf-1/ERK1/2 and PTX-sensitive protease-activated receptors/PI3K/Akt pathways in HAoSMCs. We propose that multiple signaling pathways are involved in change of VSMCs to a secretory phenotype. PMID:26557022

  17. Random Forests Are Able to Identify Differences in Clotting Dynamics from Kinetic Models of Thrombin Generation

    PubMed Central

    2016-01-01

    Current methods for distinguishing acute coronary syndromes such as heart attack from stable coronary artery disease, based on the kinetics of thrombin formation, have been limited to evaluating sensitivity of well-established chemical species (e.g., thrombin) using simple quantifiers of their concentration profiles (e.g., maximum level of thrombin concentration, area under the thrombin concentration versus time curve). In order to get an improved classifier, we use a 34-protein factor clotting cascade model and convert the simulation data into a high-dimensional representation (about 19000 features) using a piecewise cubic polynomial fit. Then, we systematically find plausible assays to effectively gauge changes in acute coronary syndrome/coronary artery disease populations by introducing a statistical learning technique called Random Forests. We find that differences associated with acute coronary syndromes emerge in combinations of a handful of features. For instance, concentrations of 3 chemical species, namely, active alpha-thrombin, tissue factor-factor VIIa-factor Xa ternary complex, and intrinsic tenase complex with factor X, at specific time windows, could be used to classify acute coronary syndromes to an accuracy of about 87.2%. Such a combination could be used to efficiently assay the coagulation system. PMID:27171403

  18. Random Forests Are Able to Identify Differences in Clotting Dynamics from Kinetic Models of Thrombin Generation.

    PubMed

    Arumugam, Jayavel; Bukkapatnam, Satish T S; Narayanan, Krishna R; Srinivasa, Arun R

    2016-01-01

    Current methods for distinguishing acute coronary syndromes such as heart attack from stable coronary artery disease, based on the kinetics of thrombin formation, have been limited to evaluating sensitivity of well-established chemical species (e.g., thrombin) using simple quantifiers of their concentration profiles (e.g., maximum level of thrombin concentration, area under the thrombin concentration versus time curve). In order to get an improved classifier, we use a 34-protein factor clotting cascade model and convert the simulation data into a high-dimensional representation (about 19000 features) using a piecewise cubic polynomial fit. Then, we systematically find plausible assays to effectively gauge changes in acute coronary syndrome/coronary artery disease populations by introducing a statistical learning technique called Random Forests. We find that differences associated with acute coronary syndromes emerge in combinations of a handful of features. For instance, concentrations of 3 chemical species, namely, active alpha-thrombin, tissue factor-factor VIIa-factor Xa ternary complex, and intrinsic tenase complex with factor X, at specific time windows, could be used to classify acute coronary syndromes to an accuracy of about 87.2%. Such a combination could be used to efficiently assay the coagulation system. PMID:27171403

  19. TMJ function after partial condylectomy in active mandibular condylar hyperplasia

    PubMed Central

    Olate, Sergio; Martinez, Felipe; Uribe, Francisca; Pozzer, Leandro; Cavalieri-Pereira, Lucas; de Moraes, Marcio

    2014-01-01

    Condylar hyperplasia is a frequent pathology that causes severe facial asymmetries. The partial condylectomy generally halts the disease. The aim of this research was to examine post-condylectomy TMJ function; 14 patients were included in this study, 6 male and 8 female. The average age was 21 years old. In all, the partial condylectomy was performed with preauricular or endaural access and the osteotomy were performed with drills, saw or an ultrasonic system. The patients were assessed with 3 or more time after surgery and were considering maximum mouth opening, right and left lateralities, presence of pain, noises, alterations in the facial nerve (VII) and esthetic alteration from the scar. The analysis was performed with the visual analog scale (VAS) and with a 7 cm metallic rule. Data analysis was descriptive plus chi-square test considering p value < 0.05 for statistical differences. With an average of 11 month after surgery, the results showed that the open mouth (over 35 mm) and lateralities (average 9 mm for the both right and left side) were normal and without statistical differences between the right or left side. Noise was observed in 3 patients and pain was observed in two patients with level 2 and 1 (VAS score). Scar was not related to problem with patient and the temporal branch of facial nerve was observed with limitations but without problem for patients. It can be concluded that the condylectomy is a safe and effective procedure with low morbidity for patients. PMID:24753777

  20. Dabigatran, a direct thrombin inhibitor, blocks differentiation of normal fibroblasts to a myofibroblast phenotype and demonstrates anti-fibrotic effects on scleroderma lung fibroblasts

    PubMed Central

    Bogatkevich, Galina S.; Ludwicka-Bradley, Anna; Silver, Richard M.

    2010-01-01

    Myofibroblasts are the principal mesenchymal cells responsible for tissue remodeling, collagen deposition, and the restrictive nature of lung parenchyma associated with pulmonary fibrosis. We previously reported that thrombin activates protease-activated receptor (PAR)-1 thereby inducing normal lung fibroblasts to differentiate to a myofibroblast phenotype resembling scleroderma lung myofibroblasts. Here we demonstrate that the thrombin inhibitor dabigatran inhibits in a dose-dependant manner thrombin's induction of myofibroblasts. Dabigatran inhibits thrombin-induced cell proliferation, α-smooth muscle actin (α-SMA) expression and organization, and the production of collagen and connective tissue growth factor (CTGF). Moreover, when treated with dabigatran scleroderma lung myofibroblasts produce less CTGF, α-SMA, and collagen type I. We conclude that dabigatran restrains important profibrotic events in lung fibroblasts and that this oral direct thrombin inhibitor warrants study as a potential anti-fibrotic drug for the treatment of fibrosing lung diseases, e.g. scleroderma lung disease and idiopathic pulmonary fibrosis. PMID:19877031

  1. Design, synthesis and structural exploration of novel fluorinated dabigatran derivatives as direct thrombin inhibitors.

    PubMed

    Li, Mei-Lin; Ren, Yu-Jie; Dong, Ming-Hui; Ren, Wei-Xin

    2015-01-01

    Twenty-one fluorinated dabigatran derivatives were designed based on the bioisosteric principle. All derivatives were synthesised and evaluated for their thrombin inhibitory activity in vitro. Among these compounds, 14h, 14m, 14s and 14t were potent and the activity was in the range of reference drug, dabigatran. Three structural changes were introduced in these 21 compounds to elucidate the structure-activity relationship of the drugs. In addition, prodrugs of compounds 14h and 14s were developed to investigate their anticoagulant activities in vivo. In these experiments, compound 16 showed a fairly strong inhibitory effect on thrombin-induced platelet aggregation, and demonstrated potent activity for inhibiting arteriovenous thrombosis with an inhibition rate of (73 ± 6) %, which was comparable to that of dabigatran etexilate (76 ± 2) %. Moreover, molecular docking studies were performed to understand the binding interactions of active compounds 14h, 14s and 14t with thrombin protein (PDB ID:1KTS). Contour maps obtained from the 3D-QSAR model are meaningful in designing more active molecules to act as direct inhibitors of thrombin. PMID:25874337

  2. Cloning, purification and biochemical characterization of dipetarudin, a new chimeric thrombin inhibitor.

    PubMed

    López, M; Mende, K; Steinmetzer, T; Nowak, G

    2003-03-25

    The development of thrombin inhibitors could provide invaluable progress for antithrombotic therapy. In this paper, we report the cloning, purification and biochemical characterization of dipetarudin, a chimeric thrombin inhibitor composed of the N-terminal head structure of dipetalogastin II, the strongest inhibitor from the assassin bug Dipetalogaster maximus, and the exosite 1 blocking segment of hirudin, connected through a five glycine linker. The cloning of dipetarudin was performed by a simple method which had not been used previously to clone chimeras. Biochemical characterization of dipetarudin revealed that it is a slow, tight-binding inhibitor with a molecular mass (M(r)=7560) and a thrombin inhibitory activity (K(i)=446 fM) comparable to r-hirudin. PMID:12651003

  3. The polymerization and thrombin-binding properties of des-(B beta 1-42)-fibrin.

    PubMed

    Siebenlist, K R; DiOrio, J P; Budzynski, A Z; Mosesson, M W

    1990-10-25

    Multiple factors affect the thrombin-catalyzed conversion of fibrinogen to fibrin, including: fibrinopeptide (FPA and FPB) release leading to exposure of two types of polymerization domains ("A" and "B," respectively) in the central portion of the molecule, and exposure of a noncatalytic "secondary" thrombin-binding site in fibrin. Fibrinogen containing the FPA sequence but lacking the B beta 1-42 sequence ("des-(B beta 1-42)-fibrinogen"), was compared to native fibrinogen (containing both FPA and FPB) to investigate the role played by B beta 1-42 in the polymerization of alpha-fibrin (i.e. fibrin lacking FPA), to compare reptilase and thrombin cleavage of FPA from fibrinogen, and to explore the location and function of the secondary thrombin-binding site. Electron microscopy of evolving polymer structures (mu, 0.14; pH 7.4) plus turbidity measurements, showed that early thin fibril formation as well as subsequent lateral fibril associations were impaired in des-(B beta 1-42)-alpha-fibrin, thus indicating that the B beta 1-42 sequence contributes to the A polymerization site. Reptilase-activated des-(B beta 1-42)-alpha-fibrin polymerized even more slowly than thrombin-activated des-(B beta 1-42)-alpha-fibrin, differences that disappeared when repolymerization of preformed fibrin monomers was carried out. Since existing data indicate that thrombin releases FPA in a concerted manner, resulting in relatively rapid evolution of fully functional divalent alpha-fibrin monomers, it can be inferred that delayed fibrin assembly of reptilase fibrin is due to slower formation of divalent alpha-fibrin monomers. Thrombin-activated des-(B beta 1-42)-alpha-fibrin polymerized more rapidly at low ionic strength (mu, 0.04) than did native alpha,beta-fibrin, a reversal of their behavior at physiological ionic strength (mu, 0.14). Concomitant measurement of FPA release revealed modest slowing of release at low ionic strength from des-(B beta 1-42)-fibrinogen (t1/2, 36.5 versus 21

  4. Thrombin drives tumorigenesis in colitis-associated colon cancer

    PubMed Central

    Rosenfeldt, Leah; Kombrinck, Keith; Flick, Matthew J.; Steinbrecher, Kris A.; Harmel-Laws, Eleana; Mullins, Eric S.; Shaw, Maureen; Witte, David P.; Revenko, Alexey; Monia, Brett; Palumbo, Joseph S.

    2014-01-01

    The established association between inflammatory bowel disease and colorectal cancer underscores the importance of inflammation in colon cancer development. Based on evidence that hemostatic proteases are powerful modifiers of both inflammatory pathologies and tumor biology, gene-targeted mice carrying low levels of prothrombin were used to directly test the hypothesis that prothrombin contributes to tumor development in colitis-associated colon cancer (CAC). Remarkably, imposing a modest 50% reduction in circulating prothrombin in fII+/− mice, a level that carries no significant bleeding risk, dramatically decreased adenoma formation following an azoxymethane/dextran sodium sulfate challenge. Similar results were obtained with pharmacological inhibition of prothrombin expression or inhibition of thrombin proteolytic activity. Detailed longitudinal analyses showed that the role of thrombin in tumor development in CAC was temporally associated with the antecedent inflammatory colitis. However, direct studies of the antecedent colitis showed that mice carrying half-normal prothrombin levels were comparable to control mice in mucosal damage, inflammatory cell infiltration and associated local cytokine levels. These results suggest that thrombin supports early events coupled to inflammation-mediated tumorigenesis in CAC that are distinct from overall inflammation-induced tissue damage and inflammatory cell trafficking. That prothrombin is linked to early events in CAC was strongly inferred by the observation that prothrombin deficiency dramatically reduced the formation of very early, pre-cancerous aberrant crypt foci. Given the importance of inflammation in the development of colon cancer, these studies suggest that therapeutic interventions at the level of hemostatic factors may be an effective means to prevent and/or impede colitis-associated colon cancer progression. PMID:24710407

  5. The antithrombotic effect of potent bifunctional thrombin inhibitors based on hirudin sequence, P551 and P532, on He-Ne laser-induced thrombosis in rat mesenteric microvessels.

    PubMed

    Yamashita, T; Tsuda, Y; Konishi, Y; Okada, Y; Matsuoka, A; Giddings, J C; Yamamoto, J

    1998-06-01

    The antithrombotic effect of potent synthetic bifunctional non-substrate type thrombin inhibitors based on hirudin sequences, P551 and P532, on Helium-Neon laser-induced thrombosis was investigated in rat mesenteric microvessels and compared with other types of thrombin inhibitors. P551 and P532, when given intravenously, inhibited platelet-rich thrombus formation in both arterioles and venules in a dose-dependent manner. The inhibitory effect was maximal immediately after intravenous administration and persisted for 20-30 minutes in both arterioles and venules. The minimal effective doses of P551 and P532 were 1.0 mg/ kg (intravenously) in both. However, the time course of the antithrombotic effect was not in keeping with the inhibitory effect measured by an activated partial thromboplastin time and was similar to other types of inhibitors in spite of different half-lives. The current findings show that P551 and P532 had significant inhibitory effects on platelet-rich thrombus formation and suggest that these bifunctional thrombin inhibitors could be potent antithrombotic agents. PMID:9694241

  6. Rigidification of the autolysis loop enhances Na[superscript +] binding to thrombin

    SciTech Connect

    Pozzi, Nicola; Chen, Raymond; Chen, Zhiwei; Bah, Alaji; Di Cera, Enrico

    2011-09-20

    Binding of Na{sup +} to thrombin ensures high activity toward physiological substrates and optimizes the procoagulant and prothrombotic roles of the enzyme in vivo. Under physiological conditions of pH and temperature, the binding affinity of Na{sup +} is weak due to large heat capacity and enthalpy changes associated with binding, and the K{sub d} = 80 mM ensures only 64% saturation of the site at the concentration of Na{sup +} in the blood (140 mM). Residues controlling Na{sup +} binding and activation have been identified. Yet, attempts to improve the interaction of Na{sup +} with thrombin and possibly increase catalytic activity under physiological conditions have so far been unsuccessful. Here we report how replacement of the flexible autolysis loop of human thrombin with the homologous rigid domain of the murine enzyme results in a drastic (up to 10-fold) increase in Na{sup +} affinity and a significant improvement in the catalytic activity of the enzyme. Rigidification of the autolysis loop abolishes the heat capacity change associated with Na{sup +} binding observed in the wild-type and also increases the stability of thrombin. These findings have general relevance to protein engineering studies of clotting proteases and trypsin-like enzymes.

  7. Exploiting the antithrombotic effect of the (pro)thrombin inhibitor bothrojaracin.

    PubMed

    Assafim, Mariane; Frattani, Flávia S; Ferreira, Marcos S; Silva, Dione M; Monteiro, Robson Q; Zingali, Russolina B

    2016-09-01

    Bothrojaracin is a 27 kDa C-type lectin-like protein from Bothrops jararaca snake venom. It behaves as a potent thrombin inhibitor upon high-affinity binding to thrombin exosites. Bothrojaracin also forms a stable complex with prothrombin that can be detected in human plasma. Formation of the zymogen-inhibitor complex severely decreases prothrombin activation and contributes to the anticoagulant activity of bothrojaracin. In the present study, we employed two rodent models to evaluate the antithrombotic effect of bothrojaracin in vivo: stasis-induced thrombosis and thrombin-induced pulmonary thromboembolism. It was observed that bothrojaracin interacts with rat prothrombin in plasma. Ex-vivo assays showed stable complex formation even after 24 h of a single bothrojaracin dose. As a result, bothrojaracin showed significant antithrombotic activity in a rat venous thrombosis model elicited by thromboplastin combined with stasis. The antithrombotic activity of bothrojaracin (1 mg/kg) persisted for up to 24 h and it was associated with moderate bleeding as assessed by a tail transection method. Formation of bothrojaracin-prothrombin complex has been also observed following intravenous administration of the inhibitor into mice. As a result, bothrojaracin effectively protected mice from thrombin-induced fatal thromboembolism. We conclude that bothrojaracin is a potent antithrombotic agent in vivo and may serve as a prototype for the development of new zymogen-directed drugs that could result in prolonged half-life and possible decreased hemorrhagic risk. PMID:27179421

  8. Regulation of Thrombin-Induced Lung Endothelial Cell Barrier Disruption by Protein Kinase C Delta

    PubMed Central

    Xie, Lishi; Chiang, Eddie T.; Kelly, Gabriel T.; Kanteti, Prasad; Singleton, Patrick A.; Camp, Sara M.; Zhou, Tingting; Dudek, Steven M.; Natarajan, Viswanathan; Wang, Ting; Black, Steven M.; Garcia, Joe G. N.; Jacobson, Jeffrey R.

    2016-01-01

    Protein Kinase C (PKC) plays a significant role in thrombin-induced loss of endothelial cell (EC) barrier integrity; however, the existence of more than 10 isozymes of PKC and tissue–specific isoform expression has limited our understanding of this important second messenger in vascular homeostasis. In this study, we show that PKCδ isoform promotes thrombin-induced loss of human pulmonary artery EC barrier integrity, findings substantiated by PKCδ inhibitory studies (rottlerin), dominant negative PKCδ construct and PKCδ silencing (siRNA). In addition, we identified PKCδ as a signaling mediator upstream of both thrombin-induced MLC phosphorylation and Rho GTPase activation affecting stress fiber formation, cell contraction and loss of EC barrier integrity. Our inhibitor-based studies indicate that thrombin-induced PKCδ activation exerts a positive feedback on Rho GTPase activation and contributes to Rac1 GTPase inhibition. Moreover, PKD (or PKCμ) and CPI-17, two known PKCδ targets, were found to be activated by PKCδ in EC and served as modulators of cytoskeleton rearrangement. These studies clarify the role of PKCδ in EC cytoskeleton regulation, and highlight PKCδ as a therapeutic target in inflammatory lung disorders, characterized by the loss of barrier integrity, such as acute lung injury and sepsis. PMID:27442243

  9. A major phospholipase A2 from Daboia russelii russelii venom shows potent anticoagulant action via thrombin inhibition and binding with plasma phospholipids.

    PubMed

    Mukherjee, Ashis K

    2014-04-01

    This is the first report on antithrombin effects of a phospholipase A2 (RVAPLA2) purified from venom of Daboia russelii russelii. The N-terminal sequence as well as in-gel tryptic digested peptides of RVAPLA2 showed significant homology with PLA2s from Russell's viper venom. RVAPLA2 demonstrated highest specific activity in hydrolyzing phosphatidylcholine (1.8 × 10(6) U/mg) with Km and Vmax values of 0.61 mM and 132.3 μmol/min, respectively. RVAPLA2 exerted dose-dependent catalytic and strong anticoagulant activities; however, studies indicated dissociation of its catalytic and anticoagulant sites. The anticoagulant action of RVAPLA2 was partially contributed by catalytic hydrolysis of plasma phospholipids. RVAPLA2 showed strong anticoagulant effect via thrombin inhibition with a Ki value of 380 nM as well as by binding to pro-coagulant phospholipids of plasma. In ex-vivo conditions, RVAPLA2 (1.0 μM) was non-hemolytic and non-cytotoxic to mammalian cells. It did not inhibit the collagen-induced aggregation of platelets. RVAPLA2 at a dose of 5 mg/kg was not lethal to mice after 48 h of injection. It demonstrated in vivo anticoagulant activity possibly due to targeting thrombin and binding with plasma phospholipids. PMID:24333043

  10. Targeting the GPIbα Binding Site of Thrombin To Simultaneously Induce Dual Anticoagulant and Antiplatelet Effects

    PubMed Central

    2015-01-01

    Exosite 2 of human thrombin contributes to two opposing pathways, the anticoagulant pathway and the platelet aggregation pathway. We reasoned that an exosite 2 directed allosteric thrombin inhibitor should simultaneously induce anticoagulant and antiplatelet effects. To assess this, we synthesized SbO4L based on the sulfated tyrosine-containing sequence of GPIbα. SbO4L was synthesized in three simple steps in high yield and found to be a highly selective, direct inhibitor of thrombin. Michelis–Menten kinetic studies indicated a noncompetitive mechanism of inhibition. Competitive inhibition studies suggested ideal competition with heparin and glycoprotein Ibα, as predicted. Studies with site-directed mutants of thrombin indicated that SbO4L binds to Arg233, Lys235, and Lys236 of exosite 2. SbO4L prevented thrombin-mediated platelet activation and aggregation as expected on the basis of competition with GPIbα. SbO4L presents a novel paradigm of simultaneous dual anticoagulant and antiplatelet effects achieved through the GPIbα binding site of thrombin. PMID:24635452

  11. Acceleration of thrombin-antithrombin complex formation in rat hindquarters via heparinlike molecules bound to the endothelium.

    PubMed Central

    Marcum, J A; McKenney, J B; Rosenberg, R D

    1984-01-01

    We have examined the role of heparinlike molecules in the regulation of coagulation by perfusing rat hindquarters with purified human thrombin and with its plasma inhibitor, antithrombin. Our data indicate that contact of the hemostatic components with the endothelium enhances the rate of thrombin-antithrombin complex formation by as much as 19-fold over the uncatalyzed rate of enzyme-inhibitor interaction. Heparinlike molecules are responsible for the antithrombin accelerating activity. The amount of thrombin-antithrombin complex generated within the hindlimb preparation after pretreatment of the vasculature with purified Flavobacterium heparinase or with addition of platelet Factor IV to the hemostatic components, was equal to the uncatalyzed levels. These heparinlike molecules appear to be tightly bound to the luminal surface of the endothelium, since they could not be detected within the physiologic buffer that was perfused through the animal. The above mucopolysaccharides function in a manner similar to commercial heparin, since modification of antithrombin at a site critical for heparin-dependent acceleration of the protease inhibitor resulted in a level of interaction product identical to the uncatalyzed amount. Finally, addition of diisofluorophosphate-thrombin to the enzyme perfusion stream reduced the amount of thrombin-antithrombin complex formed in the animal by 30-40%, which suggested that thrombin bound to the endothelium as well as enzyme free in solution are accessible to antithrombin that has interacted with heparinlike molecules present on the endothelium. PMID:6746897

  12. pHAST (pH-Driven Aptamer Switch for Thrombin) Catch-and-Release of Target Protein.

    PubMed

    McConnell, E M; Bolzon, R; Mezin, P; Frahm, G; Johnston, M; DeRosa, M C

    2016-06-15

    A pH-driven DNA nanomachine based on the human α-thrombin binding aptamer was designed for the specific catch-and-release of human α-thrombin at neutral and acidic pH, respectively. In neutral conditions, the thrombin aptamer component of the nanomachine is exposed and exists in the G-quadruplex conformation required to bind to the target protein. At slightly acidic pH, the polyadenine tail of the nanomachine becomes partially protonated and A+(anti)•G(syn) mispairing results in a conformational change, causing the target protein to be released. Förster resonance energy transfer (FRET) was used to monitor conformational switching over multiple pH cycles. Electrophoretic mobility shift assay (EMSA) and fluorescence anisotropy were used to show pH dependent protein binding and release by the nanomachine. This approach could be applied generally to existing G-rich aptamers to develop novel biosensors, theranostics, and nanoswitches. PMID:27115292

  13. Transposon disruption of the complex I NADH oxidoreductase gene (snoD) in Staphylococcus aureus is associated with reduced susceptibility to the microbicidal activity of thrombin-induced platelet microbicidal protein 1.

    PubMed

    Bayer, Arnold S; McNamara, Peter; Yeaman, Michael R; Lucindo, Natalie; Jones, Tiffanny; Cheung, Ambrose L; Sahl, Hans-Georg; Proctor, Richard A

    2006-01-01

    The cationic molecule thrombin-induced platelet microbicidal protein 1 (tPMP-1) exerts potent activity against Staphylococcus aureus. We previously reported that a Tn551 S. aureus transposon mutant, ISP479R, and two bacteriophage back-transductants, TxA and TxB, exhibit reduced in vitro susceptibility to tPMP-1 (tPMP-1(r)) compared to the parental strain, ISP479C (V. Dhawan, M. R. Yeaman, A. L. Cheung, E. Kim, P. M. Sullam, and A. S. Bayer, Infect. Immun. 65:3293-3299, 1997). In the current study, the genetic basis for tPMP-1(r) in these mutants was identified. GenBank homology searches using sequence corresponding to chromosomal DNA flanking Tn551 mutant strains showed that the fourth gene in the staphylococcal mnh operon (mnhABCDEFG) was insertionally inactivated. This operon was previously reported to encode a Na(+)/H(+) antiporter involved in pH tolerance and halotolerance. However, the capacity of ISP479R to grow at pH extremes and in high NaCl concentrations (1 to 3 M), coupled with its loss of transmembrane potential (DeltaPsi) during postexponential growth, suggested that the mnh gene products are not functioning as a secondary (i.e., passive) Na(+)/H(+) antiporter. Moreover, we identified protein homologies between mnhD and the nuo genes of Escherichia coli that encode components of a complex I NADH:ubiquinone oxidoreductase. Consistent with these data, exposures of tPMP-1-susceptible (tPMP-1(s)) parental strains (both clinical and laboratory derived) with either CCCP (a proton ionophore which collapses the proton motive force) or pieracidin A (a specific complex I enzyme inhibitor) significantly reduced tPMP-induced killing to levels seen in the tPMP-1(r) mutants. To reflect the energization of the gene products encoded by the mnh operon, we have renamed the locus sno (S. aureus nuo orthologue). These novel findings indicate that disruption of a complex I enzyme locus can confer reduced in vitro susceptibility to tPMP-1 in S. aureus. PMID:16352837

  14. Transposon Disruption of the Complex I NADH Oxidoreductase Gene (snoD) in Staphylococcus aureus Is Associated with Reduced Susceptibility to the Microbicidal Activity of Thrombin-Induced Platelet Microbicidal Protein 1

    PubMed Central

    Bayer, Arnold S.; McNamara, Peter; Yeaman, Michael R.; Lucindo, Natalie; Jones, Tiffanny; Cheung, Ambrose L.; Sahl, Hans-Georg; Proctor, Richard A.

    2006-01-01

    The cationic molecule thrombin-induced platelet microbicidal protein 1 (tPMP-1) exerts potent activity against Staphylococcus aureus. We previously reported that a Tn551 S. aureus transposon mutant, ISP479R, and two bacteriophage back-transductants, TxA and TxB, exhibit reduced in vitro susceptibility to tPMP-1 (tPMP-1r) compared to the parental strain, ISP479C (V. Dhawan, M. R. Yeaman, A. L. Cheung, E. Kim, P. M. Sullam, and A. S. Bayer, Infect. Immun. 65:3293-3299, 1997). In the current study, the genetic basis for tPMP-1r in these mutants was identified. GenBank homology searches using sequence corresponding to chromosomal DNA flanking Tn551 mutant strains showed that the fourth gene in the staphylococcal mnh operon (mnhABCDEFG) was insertionally inactivated. This operon was previously reported to encode a Na+/H+ antiporter involved in pH tolerance and halotolerance. However, the capacity of ISP479R to grow at pH extremes and in high NaCl concentrations (1 to 3 M), coupled with its loss of transmembrane potential (ΔΨ) during postexponential growth, suggested that the mnh gene products are not functioning as a secondary (i.e., passive) Na+/H+ antiporter. Moreover, we identified protein homologies between mnhD and the nuo genes of Escherichia coli that encode components of a complex I NADH:ubiquinone oxidoreductase. Consistent with these data, exposures of tPMP-1-susceptible (tPMP-1s) parental strains (both clinical and laboratory derived) with either CCCP (a proton ionophore which collapses the proton motive force) or pieracidin A (a specific complex I enzyme inhibitor) significantly reduced tPMP-induced killing to levels seen in the tPMP-1r mutants. To reflect the energization of the gene products encoded by the mnh operon, we have renamed the locus sno (S. aureus nuo orthologue). These novel findings indicate that disruption of a complex I enzyme locus can confer reduced in vitro susceptibility to tPMP-1 in S. aureus. PMID:16352837

  15. Targeting the thrombin receptor modulates inflammation and astrogliosis to improve recovery after spinal cord injury.

    PubMed

    Radulovic, Maja; Yoon, Hyesook; Wu, Jianmin; Mustafa, Karim; Scarisbrick, Isobel A

    2016-09-01

    The deregulation of serine protease activity is a common feature of neurological injury, but little is known regarding their mechanisms of action or whether they can be targeted to facilitate repair. In this study we demonstrate that the thrombin receptor (Protease Activated Receptor 1, (PAR1)) serves as a critical translator of the spinal cord injury (SCI) proteolytic microenvironment into a cascade of pro-inflammatory events that contribute to astrogliosis and functional decline. PAR1 knockout mice displayed improved locomotor recovery after SCI and reduced signatures of inflammation and astrogliosis, including expression of glial fibrillary acidic protein (GFAP), vimentin, and STAT3 signaling. SCI-associated elevations in pro-inflammatory cytokines such as IL-1β and IL-6 were also reduced in PAR1-/- mice and co-ordinate improvements in tissue sparing and preservation of NeuN-positive ventral horn neurons, and PKCγ corticospinal axons, were observed. PAR1 and its agonist's thrombin and neurosin were expressed by perilesional astrocytes and each agonist increased the production of IL-6 and STAT3 signaling in primary astrocyte cultures in a PAR1-dependent manner. In turn, IL-6-stimulated astrocytes increased expression of PAR1, thrombin, and neurosin, pointing to a model in which PAR1 activation contributes to increased astrogliosis by feedforward- and feedback-signaling dynamics. Collectively, these findings identify the thrombin receptor as a key mediator of inflammation and astrogliosis in the aftermath of SCI that can be targeted to reduce neurodegeneration and improve neurobehavioral recovery. PMID:27145117

  16. Flow and delta-P dictate where thrombin, fibrin, and von Willebrand Factor will be found.

    PubMed

    Diamond, Scott L

    2016-05-01

    Hemostasis occurs in two different topological scenarios: complete severing of a vessel or disruption of the vessel wall. Either to meet the daily rigors of active life or during an acute trauma, hemostasis involves the regulated and self-limiting production of thrombin to stop bleeding. In contrast, arterial and venous thrombosis typically involves the unregulated, intraluminal growth of a clot, in the absence of bleeding. For either hemostasis or thrombosis, the presence of flow and pressure gradients (delta-P, ΔP) dictates when and where thrombin and fibrin are located and in what quantity. For hemostatic clots, fibrin formation helped limit clot growth. We found that γ'-fibrinogen had a role in limiting clot growth via anti-thrombin activity at venous, but not arterial conditions. For hemophilic blood, severe factor deficiency (<1% healthy) led to a defect in both platelet and fibrin deposition under flow. However, moderate deficiency, which is associated with a less severe bleeding phenotype, had normalized platelet function but still lacked fibrin production. We conclude signaling levels of thrombin can be generated during moderate hemophilia to sufficiently activate platelets to achieve primary hemostasis, even if fibrin formation remains defective. Finally, as a clot grows, shear stresses can become sufficiently extreme in diseased arteries to drive von Willebrand Factor self-association into massive fibers, potentially the final burst of clot growth towards full thrombotic occlusion. PMID:27207416

  17. Effect of Electronic Polarization to Human α-Thrombin

    NASA Astrophysics Data System (ADS)

    Duan, Li-Li; Li, Zong-Chao; He, Xiang; Zhang, Qing-Gang

    2014-04-01

    The polarized protein-specific charges (PPC) of human α-thrombin (thrombin) and its inhibitor (L86) are made possible by employing the recently developed molecular fractionation with conjugate caps approach incorporated the Poisson—Boltzmann model. Molecular dynamics (MD) simulations of thrombin have been carried out to investigate the dynamics and stability of the thrombin-inhibitor using PPC and AMBER charges respectively. Detailed analysis and comparison of MD results show that the PPC can correctly describe the polarized state of the thrombin and L86. Especially, the root-mean-square deviation of backbone atoms and the hydrogen bonds using PPC are more stable than the AMBER charge. The present results indicate that protein polarization plays critical roles in maintaining the compact structure of thrombin.

  18. Hypersensitivity to thrombin of platelets from hypercholesterolemic rats

    SciTech Connect

    Winocour, P.D.; Rand, M.L.; Kinlough-Rathbone, R.L.; Mustard, J.F.

    1986-03-01

    Hypersensitivity of platelets to thrombin has been associated with hypercholesterolemia. The authors have examined the mechanisms involved in this hypersensitivity. Rats were given diets rich in milk fat and containing added cholesterol and taurocholate to produce hypercholesterolemia (HC) (262 +/- 25 mg%) or added sitosterol as a normocholesterolemic control (NC) (89 +/- 6 mg%). Washed platelets were prelabelled with /sup 14/C-serotonin. In the presence of acetylsalicyclic acid (ASA) (to inhibit thromboxane A/sub 2/ (TXA/sub 2/) formation) and creatine phosphate/creatine phosphokinase (CP/CPK) (to remove released ADP), HC platelets aggregated more (26 +/- 1%) and released more /sup 14/C (9.1 +/- 2.0%) than NC platelets (aggregation: 0%, p < 0.001; /sup 14/C release: 1.5 +/- 0.5%, p < 0.002) in response to thrombin (0.075 U/ml). Thus, a pathway independent of released ADP or TXA/sub 2/ formation is involved in the hypersensitivity of HC platelets to thrombin. Total binding of /sup 125/I-thrombin to HC platelets was less than that to NC platelets but HC platelets were smaller and had less protein than NC platelets; the thrombin binding per mg platelet protein was the same for HC and NC platelets, indicating that hypersensitivity to thrombin of HC platelets does not result from increased thrombin binding. Thus, hypersensitivity of HC platelets to thrombin is not due to TXA/sub 2/ formation, the action of released ADP or increased thrombin binding.

  19. A non-aggregation colorimetric assay for thrombin based on catalytic properties of silver nanoparticles.

    PubMed

    Li, Jie; Li, Wei; Qiang, Weibing; Wang, Xi; Li, Hui; Xu, Danke

    2014-01-01

    In this paper, we developed a simple and rapid colorimetric assay for protein detection based on the reduction of dye molecules catalyzed by silver nanoparticles (AgNPs). Aptamer-modified magnetic particles and aptamer-functionalized AgNPs were employed as capture and detection probes, respectively. Introduction of thrombin as target protein could form a sandwich-type complex involving catalytically active AgNPs, whose catalytic activity was monitored on the catalytic reduction of rhodamine B (RhB) by sodium borohydride (NaBH4). The amount of immobilized AgNPs on the complex increased along with the increase of the thrombin concentration, thus the detection of thrombin was achieved via recording the decrease in absorbance corresponding to RhB. This method has adopted several advantages from the key factors involved, i.e., the sandwich binding of affinity aptamers contributed to the increased specificity; magnetic particles could result in rapid capture and separation processes; the conjugation of AgNPs would lead to a clear visual detection. It allows for the detection limit of thrombin down to picomolar level by the naked eye, with remarkable selectivity over other proteins. Moreover, it is possible to apply this method to the other targets with two binding sites as well. PMID:24356228

  20. Comparative pharmacodynamics and pharmacokinetics of oral direct thrombin and factor xa inhibitors in development.

    PubMed

    Eriksson, Bengt I; Quinlan, Daniel J; Weitz, Jeffrey I

    2009-01-01

    For the past five decades, there has been little progress in the development of oral anticoagulants, with the choices being limited to the vitamin K antagonists (VKAs). The situation is changing with the development of orally active small molecules that directly target thrombin or activated factor X (FXa). The two agents in the most advanced stages of development are dabigatran etexilate and rivaroxaban, which inhibit thrombin and FXa, respectively. Both are approved in the EU and Canada for venous thromboprophylaxis in patients undergoing elective hip- or knee-replacement surgery. Other agents in the early stages of development include several FXa inhibitors (apixaban, DU 176b, LY 517717, YM 150, betrixaban, eribaxaban [PD 0348292] and TAK 442) and one thrombin inhibitor (AZD 0837). With a predictable anticoagulant response and low potential for drug-drug interactions, these new agents can be given in fixed doses without coagulation monitoring. This renders them more convenient than VKAs. While the anticoagulant effect of the new thrombin and FXa inhibitors is similar, differences in the pharmacokinetic and pharmacodynamic parameters may influence their use in clinical practice. Here, we compare the pharmacokinetic and pharmacodynamic features of these new oral agents. PMID:19071881

  1. Different duplex/quadruplex junctions determine the properties of anti-thrombin aptamers with mixed folding.

    PubMed

    Russo Krauss, Irene; Spiridonova, Vera; Pica, Andrea; Napolitano, Valeria; Sica, Filomena

    2016-01-29

    Mixed duplex/quadruplex oligonucleotides have attracted great interest as therapeutic targets as well as effective biomedical aptamers. In the case of thrombin-binding aptamer (TBA), the addition of a duplex motif to the G-quadruplex module improves the aptamer resistance to biodegradation and the affinity for thrombin. In particular, the mixed oligonucleotide RE31 is significantly more effective than TBA in anticoagulation experiments and shows a slower disappearance rate in human plasma and blood. In the crystal structure of the complex with thrombin, RE31 adopts an elongated structure in which the duplex and quadruplex regions are perfectly stacked on top of each other, firmly connected by a well-structured junction. The lock-and-key shape complementarity between the TT loops of the G-quadruplex and the protein exosite I gives rise to the basic interaction that stabilizes the complex. However, our data suggest that the duplex motif may have an active role in determining the greater anti-thrombin activity in biological fluids with respect to TBA. This work gives new information on mixed oligonucleotides and highlights the importance of structural data on duplex/quadruplex junctions, which appear to be varied, unpredictable, and fundamental in determining the aptamer functional properties. PMID:26673709

  2. Different duplex/quadruplex junctions determine the properties of anti-thrombin aptamers with mixed folding

    PubMed Central

    Russo Krauss, Irene; Spiridonova, Vera; Pica, Andrea; Napolitano, Valeria; Sica, Filomena

    2016-01-01

    Mixed duplex/quadruplex oligonucleotides have attracted great interest as therapeutic targets as well as effective biomedical aptamers. In the case of thrombin-binding aptamer (TBA), the addition of a duplex motif to the G-quadruplex module improves the aptamer resistance to biodegradation and the affinity for thrombin. In particular, the mixed oligonucleotide RE31 is significantly more effective than TBA in anticoagulation experiments and shows a slower disappearance rate in human plasma and blood. In the crystal structure of the complex with thrombin, RE31 adopts an elongated structure in which the duplex and quadruplex regions are perfectly stacked on top of each other, firmly connected by a well-structured junction. The lock-and-key shape complementarity between the TT loops of the G-quadruplex and the protein exosite I gives rise to the basic interaction that stabilizes the complex. However, our data suggest that the duplex motif may have an active role in determining the greater anti-thrombin activity in biological fluids with respect to TBA. This work gives new information on mixed oligonucleotides and highlights the importance of structural data on duplex/quadruplex junctions, which appear to be varied, unpredictable, and fundamental in determining the aptamer functional properties. PMID:26673709

  3. Immunomodulatory activity and partial characterisation of polysaccharides from Momordica charantia.

    PubMed

    Deng, Yuan-Yuan; Yi, Yang; Zhang, Li-Fang; Zhang, Rui-Fen; Zhang, Yan; Wei, Zhen-Cheng; Tang, Xiao-Jun; Zhang, Ming-Wei

    2014-01-01

    Momordica charantia Linn. is used as an edible and medicinal vegetable in sub-tropical areas. Until now, studies on its composition and related activities have been confined to compounds of low molecular mass, and no data have been reported concerning the plant's polysaccharides. In this work, a crude polysaccharide of M. charantia (MCP) fruit was isolated by hot water extraction and then purified using DEAE-52 cellulose anion-exchange chromatography to produce two main fractions MCP1 and MCP2. The immunomodulatory effects and physicochemical characteristics of these fractions were investigated in vitro and in vivo. The results showed that intragastric administration of 150 or 300 mg·kg-·d⁻¹ of MCP significantly increased the carbolic particle clearance index, serum haemolysin production, spleen index, thymus index and NK cell cytotoxicity to normal control levels in cyclophosphamide (Cy)-induced immunosuppressed mice. Both MCP1 and MCP2 effectively stimulated normal and concanavalin A-induced splenic lymphocyte proliferation in vitro at various doses. The average molecular weights of MCP1 and MCP2, which were measured using high-performance gel permeation chromatography, were 8.55×10⁴ Da and 4.41×10⁵ Da, respectively. Both fractions exhibited characteristic polysaccharide bands in their Fourier transform infrared spectrum. MCP1 is mainly composed of glucose and galactose, and MCP2 is mainly composed of glucose, mannose and galactose. The results indicate that MCP and its fractions have good potential as immunotherapeutic adjuvants. PMID:25178064

  4. A blood meal-induced Ixodes scapularis tick saliva serpin inhibits trypsin and thrombin, and interferes with platelet aggregation and blood clotting.

    PubMed

    Ibelli, Adriana M G; Kim, Tae K; Hill, Creston C; Lewis, Lauren A; Bakshi, Mariam; Miller, Stephanie; Porter, Lindsay; Mulenga, Albert

    2014-05-01

    Ixodes scapularis is a medically important tick species that transmits causative agents of important human tick-borne diseases including borreliosis, anaplasmosis and babesiosis. An understanding of how this tick feeds is needed prior to the development of novel methods to protect the human population against tick-borne disease infections. This study characterizes a blood meal-induced I. scapularis (Ixsc) tick saliva serine protease inhibitor (serpin (S)), in-house referred to as IxscS-1E1. The hypothesis that ticks use serpins to evade the host's defense response to tick feeding is based on the assumption that tick serpins inhibit functions of protease mediators of the host's anti-tick defense response. Thus, it is significant that consistent with hallmark characteristics of inhibitory serpins, Pichia pastoris-expressed recombinant IxscS-1E1 (rIxscS-1E1) can trap thrombin and trypsin in SDS- and heat-stable complexes, and reduce the activity of the two proteases in a dose-responsive manner. Additionally, rIxscS-1E1 also inhibited, but did not apparently form detectable complexes with, cathepsin G and factor Xa. Our data also show that rIxscS-1E1 may not inhibit chymotrypsin, kallikrein, chymase, plasmin, elastase and papain even at a much higher rIxscS-1E1 concentration. Native IxscS-1E1 potentially plays a role(s) in facilitating I. scapularis tick evasion of the host's hemostatic defense as revealed by the ability of rIxscS-1E1 to inhibit adenosine diphosphate- and thrombin-activated platelet aggregation, and delay activated partial prothrombin time and thrombin time plasma clotting in a dose-responsive manner. We conclude that native IxscS-1E1 is part of the tick saliva protein complex that mediates its anti-hemostatic, and potentially inflammatory, functions by inhibiting the actions of thrombin, trypsin and other yet unknown trypsin-like proteases at the tick-host interface. PMID:24583183

  5. Selectively active markers for solving of the partial occlusion problem in matchmoving and chromakeying workflow

    NASA Astrophysics Data System (ADS)

    Mazurek, Przemysław

    2013-09-01

    Matchmoving (Match Moving) is the process used for the estimation of camera movements for further integration of acquired video image with computer graphics. The estimation of movements is possible using pattern recognition, 2D and 3D tracking algorithms. The main problem for the workflow is the partial occlusion of markers by the actor, because manual rotoscoping is necessary for fixing of the chroma-keyed footage. In the paper, the partial occlusion problem is solved using the invented, selectively active electronic markers. The sensor network with multiple infrared links detects occlusion state (no-occlusion, partial, full) and switch LED's based markers.

  6. Two heads are better than one: crystal structure of the insect derived double domain Kazal inhibitor rhodniin in complex with thrombin.

    PubMed Central

    van de Locht, A; Lamba, D; Bauer, M; Huber, R; Friedrich, T; Kröger, B; Höffken, W; Bode, W

    1995-01-01

    Rhodniin is a highly specific inhibitor of thrombin isolated from the assassin bug Rhodnius prolixus. The 2.6 Angstrum crystal structure of the non-covalent complex between recombinant rhodniin and bovine alpha-thrombin reveals that the two Kazal-type domains of rhodniin bind to different sites of thrombin. The amino-terminal domain binds in a substrate-like manner to the narrow active-site cleft of thrombin; the imidazole group of the P1 His residue extends into the S1 pocket to form favourable hydrogen/ionic bonds with Asp189 at its bottom, and additionally with Glu192 at its entrance. The carboxy-terminal domain, whose distorted reactive-site loop cannot adopt the canonical conformation, docks to the fibrinogen recognition exosite via extensive electrostatic interactions. The rather acidic polypeptide linking the two domains is displaced from the thrombin surface, with none of its residues involved in direct salt bridges with thrombin. The tight (Ki = 2 x 10(-13) M) binding of rhodniin to thrombin is the result of the sum of steric and charge complementarity of the amino-terminal domain towards the active-site cleft, and of the electrostatic interactions between the carboxy-terminal domain and the exosite. Images PMID:7489704

  7. Two heads are better than one: crystal structure of the insect derived double domain Kazal inhibitor rhodniin in complex with thrombin.

    PubMed

    van de Locht, A; Lamba, D; Bauer, M; Huber, R; Friedrich, T; Kröger, B; Höffken, W; Bode, W

    1995-11-01

    Rhodniin is a highly specific inhibitor of thrombin isolated from the assassin bug Rhodnius prolixus. The 2.6 Angstrum crystal structure of the non-covalent complex between recombinant rhodniin and bovine alpha-thrombin reveals that the two Kazal-type domains of rhodniin bind to different sites of thrombin. The amino-terminal domain binds in a substrate-like manner to the narrow active-site cleft of thrombin; the imidazole group of the P1 His residue extends into the S1 pocket to form favourable hydrogen/ionic bonds with Asp189 at its bottom, and additionally with Glu192 at its entrance. The carboxy-terminal domain, whose distorted reactive-site loop cannot adopt the canonical conformation, docks to the fibrinogen recognition exosite via extensive electrostatic interactions. The rather acidic polypeptide linking the two domains is displaced from the thrombin surface, with none of its residues involved in direct salt bridges with thrombin. The tight (Ki = 2 x 10(-13) M) binding of rhodniin to thrombin is the result of the sum of steric and charge complementarity of the amino-terminal domain towards the active-site cleft, and of the electrostatic interactions between the carboxy-terminal domain and the exosite. PMID:7489704

  8. Loop Electrostatics Asymmetry Modulates the Preexisting Conformational Equilibrium in Thrombin.

    PubMed

    Pozzi, Nicola; Zerbetto, Mirco; Acquasaliente, Laura; Tescari, Simone; Frezzato, Diego; Polimeno, Antonino; Gohara, David W; Di Cera, Enrico; De Filippis, Vincenzo

    2016-07-19

    Thrombin exists as an ensemble of active (E) and inactive (E*) conformations that differ in their accessibility to the active site. Here we show that redistribution of the E*-E equilibrium can be achieved by perturbing the electrostatic properties of the enzyme. Removal of the negative charge of the catalytic Asp102 or Asp189 in the primary specificity site destabilizes the E form and causes a shift in the 215-217 segment that compromises substrate entrance. Solution studies and existing structures of D102N document stabilization of the E* form. A new high-resolution structure of D189A also reveals the mutant in the collapsed E* form. These findings establish a new paradigm for the control of the E*-E equilibrium in the trypsin fold. PMID:27347732

  9. Neuronal Activity in the Subthalamic Cerebrovasodilator Area under Partial-Gravity Conditions in Rats

    PubMed Central

    Zeredo, Jorge L.; Toda, Kazuo; Kumei, Yasuhiro

    2014-01-01

    The reduced-gravity environment in space is known to cause an upward shift in body fluids and thus require cardiovascular adaptations in astronauts. In this study, we recorded in rats the neuronal activity in the subthalamic cerebrovasodilator area (SVA), a key area that controls cerebral blood flow (CBF), in response to partial gravity. “Partial gravity” is the term that defines the reduced-gravity levels between 1 g (the unit gravity acceleration on Earth) and 0 g (complete weightlessness in space). Neuronal activity was recorded telemetrically through chronically implanted microelectrodes in freely moving rats. Graded levels of partial gravity from 0.4 g to 0.01 g were generated by customized parabolic-flight maneuvers. Electrophysiological signals in each partial-gravity phase were compared to those of the preceding 1 g level-flight. As a result, SVA neuronal activity was significantly inhibited by the partial-gravity levels of 0.15 g and lower, but not by 0.2 g and higher. Gravity levels between 0.2–0.15 g could represent a critical threshold for the inhibition of neurons in the rat SVA. The lunar gravity (0.16 g) might thus trigger neurogenic mechanisms of CBF control. This is the first study to examine brain electrophysiology with partial gravity as an experimental parameter. PMID:25370031

  10. Neuronal Activity in the Subthalamic Cerebrovasodilator Area under Partial-Gravity Conditions in Rats.

    PubMed

    Zeredo, Zeredo L; Toda, Kazuo; Kumei, Yasuhiro

    2014-01-01

    The reduced-gravity environment in space is known to cause an upward shift in body fluids and thus require cardiovascular adaptations in astronauts. In this study, we recorded in rats the neuronal activity in the subthalamic cerebrovasodilator area (SVA), a key area that controls cerebral blood flow (CBF), in response to partial gravity. "Partial gravity" is the term that defines the reduced-gravity levels between 1 g (the unit gravity acceleration on Earth) and 0 g (complete weightlessness in space). Neuronal activity was recorded telemetrically through chronically implanted microelectrodes in freely moving rats. Graded levels of partial gravity from 0.4 g to 0.01 g were generated by customized parabolic-flight maneuvers. Electrophysiological signals in each partial-gravity phase were compared to those of the preceding 1 g level-flight. As a result, SVA neuronal activity was significantly inhibited by the partial-gravity levels of 0.15 g and lower, but not by 0.2 g and higher. Gravity levels between 0.2-0.15 g could represent a critical threshold for the inhibition of neurons in the rat SVA. The lunar gravity (0.16 g) might thus trigger neurogenic mechanisms of CBF control. This is the first study to examine brain electrophysiology with partial gravity as an experimental parameter. PMID:25370031

  11. Protein kinase C is differentially regulated by thrombin, insulin, and epidermal growth factor in human mammary tumor cells

    SciTech Connect

    Gomez, M.L.; Tellez-Inon, M.T. ); Medrano, E.E.; Cafferatta, E.G.A. )

    1988-03-01

    The exposure of serum-deprived mammary tumor cells MCF-7 and T-47D to insulin, thrombin, and epidermal growth factor (EGF) resulted in dramatic modifications in the activity and in the translocation capacity of protein kinase C from cytosol to membrane fractions. Insulin induces a 600% activation of the enzyme after 5 h of exposure to the hormone in MCF-7 cells; thrombin either activates (200% in MCF-7) or down-regulates (in T-47D), and EGF exerts only a moderate effect. Thus, the growth factors studied modulate differentially the protein kinase C activity in human mammary tumor cells. The physiological significance of the results obtained are discussed in terms of the growth response elicited by insulin, thrombin, and EGF.

  12. Mutant B-Raf(V600E) Promotes Melanoma Paracellular Transmigration by Inducing Thrombin-mediated Endothelial Junction Breakdown.

    PubMed

    Zhang, Pu; Feng, Shan; Liu, Gentao; Wang, Heyong; Zhu, Huifeng; Ren, Qiao; Bai, Huiyuan; Fu, Changliang; Dong, Cheng

    2016-01-29

    Tumor invasiveness depends on the ability of tumor cells to breach endothelial barriers. In this study, we investigated the mechanism by which the adhesion of melanoma cells to endothelium regulates adherens junction integrity and modulates tumor transendothelial migration (TEM) by initiating thrombin generation. We found that the B-Raf(V600E) mutation in metastatic melanoma cells up-regulated tissue factor (TF) expression on cell membranes and promoted thrombin production. Co-culture of endothelial monolayers with metastatic melanoma cells mediated the opening of inter-endothelial spaces near melanoma cell contact sites in the presence of platelet-free plasma (PFP). By using small interfering RNA (siRNA), we demonstrated that B-Raf(V600E) and TF silencing attenuated the focal disassembly of adherens junction induced by tumor contact. Vascular endothelial-cadherin (VE-cadherin) disassembly was dependent on phosphorylation of p120-catenin on Ser-879 and VE-cadherin on Tyr-658, Tyr-685, and Tyr-731, which can be prevented by treatment with the thrombin inhibitor, hirudin, or by silencing the thrombin receptor, protease-activated receptor-1, in endothelial cells. We also provided strong evidence that tumor-derived thrombin enhanced melanoma TEM by inducing ubiquitination-coupled VE-cadherin internalization, focal adhesion formation, and actin assembly in endothelium. Confocal microscopic analysis of tumor TEM revealed that junctions transiently opened and resealed as tumor cells accomplished TEM. In addition, in the presence of PFP, tumor cells preferentially transmigrated via paracellular routes. PFP supported melanoma transmigration under shear conditions via a B-Raf(V600E)-thrombin-dependent mechanism. We concluded that the activation of thrombin generation by cancer cells in plasma is an important process regulating melanoma extravasation by disrupting endothelial junction integrity. PMID:26504080

  13. Improved thrombin binding aptamer by incorporation of a single unlocked nucleic acid monomer

    PubMed Central

    Pasternak, Anna; Hernandez, Frank J.; Rasmussen, Lars M.; Vester, Birte; Wengel, Jesper

    2011-01-01

    A 15-mer DNA aptamer (named TBA) adopts a G-quadruplex structure that strongly inhibits fibrin-clot formation by binding to thrombin. We have performed thermodynamic analysis, binding affinity and biological activity studies of TBA variants modified by unlocked nucleic acid (UNA) monomers. UNA-U placed in position U3, U7 or U12 increases the thermodynamic stability of TBA by 0.15–0.50 kcal/mol. In contrast, modification of any position within the two G-quartet structural elements is unfavorable for quadruplex formation. The intramolecular folding of the quadruplexes is confirmed by Tm versus ln c analysis. Moreover, circular dichroism and thermal difference spectra of the modified TBAs displaying high thermodynamic stability show bands that are characteristic for antiparallel quadruplex formation. Surface plasmon resonance studies of the binding of the UNA-modified TBAs to thrombin show that a UNA monomer is allowed in many positions of the aptamer without significantly changing the thrombin-binding properties. The biological effect of a selection of the modified aptamers was tested by a thrombin time assay and showed that most of the UNA-modified TBAs possess anticoagulant properties, and that the construct with a UNA-U monomer in position 7 is a highly potent inhibitor of fibrin-clot formation. PMID:20870750

  14. Bound Na(+) is a Negative Effecter for Thrombin-Substrate Stereospecific Complex Formation.

    PubMed

    Kurisaki, Ikuo; Takayanagi, Masayoshi; Nagaoka, Masataka

    2016-05-26

    Thrombin has been studied as a paradigmatic protein of Na(+)-activated allosteric enzymes. Earlier structural studies suggest that Na(+)-binding promotes the thrombin-substrate association reaction. However, it is still elusive because (1) the structural change, driven by Na(+)-binding, is as small as the thermal fluctuation, and (2) the bound Na(+) is close to Asp189 in the primary substrate binding pocket (S1-pocket), possibly preventing substrate access via repulsive interaction. It still remains a matter of debate whether Na(+)-binding actually promotes the reaction. To solve this problem, we examined the effect of Na(+) on the reaction by employing molecular dynamics (MD) simulations. By executing independent 210 MD simulations of apo and holo systems, we obtained 80 and 26 trajectories undergoing substrate access to S1-pocket, respectively. Interestingly, Na(+)-binding results in a 3-fold reduction of the substrate access. Furthermore, we examined works for the substrate access and release, and found that Na(+)-binding is disadvantageous for the presence of the substrate in the S1-pocket. These observations provide the insight that the bound Na(+) is essentially a negative effecter in thrombin-substrate stereospecific complex formation. The insight rationalizes an enigmatic feature of thrombin, relatively low Na(+)-binding affinity. This is essential to reduce the disadvantage of Na(+)-binding in the substrate-binding. PMID:27164318

  15. Structural and Functional Characterization of Hirudin P6 Derived Novel Bivalent Thrombin Inhibitors - Studying the Effect of Linker Length and Glycosylation on Their Function.

    PubMed

    Shabareesh, Prv; Kaur, Kanwal J

    2016-07-01

    HirudinP6 is a glycosylated and sulfated high affinity thrombin inhibitory protein isolated from Hirudineria manillensis. In this study, designing of novel bivalent thrombin inhibitory peptides based on this hirudin isoform is described. The structural and functional impact of varying linker length and glycosylation on their inhibitory potencies and binding kinetics were assessed. The bivalent peptides were obtained by tethering an active site blocking fPRP motif with the carboxy terminal 22 residue segment of hirudin P6 (HP642-63 ) by varying number of glycine residues in the linker region. Among them, analog BiG1 -HP6 inhibited thrombin with a Ki of 5.12 nm which was comparable to that of glycosylated (disaccharide bearing) and non-sulfated full length hirudin P6 protein (Ki = 6.38 nm). Binding kinetics studies revealed increasing linker length can decrease the association rates of peptide─thrombin interactions. Similarly, glycosylation was found to negatively modulate the inhibitory potencies of these peptides by decreasing their rates of association with thrombin. Molecular docking studies revealed that increasing linker length can compromise the electrostatic interactions with the prime subsite residues of thrombin and provided structural explanation for the observed effect of linker length on association rates. These findings thus enhance our understanding of thrombin─(glyco)peptide interactions and provide key insights into the designing of efficient thrombin inhibitors and allosteric modulators of therapeutic potential. PMID:26850929

  16. 21 CFR 864.7875 - Thrombin time test.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Thrombin time test. 864.7875 Section 864.7875 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7875 Thrombin time test....

  17. 21 CFR 864.7875 - Thrombin time test.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Thrombin time test. 864.7875 Section 864.7875 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7875 Thrombin time test....

  18. 21 CFR 864.7875 - Thrombin time test.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Thrombin time test. 864.7875 Section 864.7875 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7875 Thrombin time test....

  19. 21 CFR 864.7875 - Thrombin time test.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Thrombin time test. 864.7875 Section 864.7875 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7875 Thrombin time test....

  20. 21 CFR 864.7875 - Thrombin time test.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Thrombin time test. 864.7875 Section 864.7875 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7875 Thrombin time test....

  1. Acidosis, magnesium and acetylsalicylic acid: Effects on thrombin

    NASA Astrophysics Data System (ADS)

    Borisevich, Nikolaj; Loznikova, Svetlana; Sukhodola, Aleksandr; Halets, Inessa; Bryszewska, Maria; Shcharbin, Dzmitry

    2013-03-01

    Thrombin, an enzyme from the hydrolase family, is the main component of the blood coagulation system. In ischemic stroke it acts as a serine protease that converts soluble fibrinogen into insoluble strands of fibrin forming blood clots in the brain. It has been found to phosphoresce at room temperature in the millisecond and microsecond ranges. The phosphorescence of thrombin was studied under physiological conditions, in acidosis (decrease of pH from 8.0 to 5.0) and on the addition of salts (magnesium sulfate and sodium chloride) and of acetylsalicylic acid, and its connection with thrombin function is discussed. Acidosis significantly increased the internal dynamics of thrombin. We propose that lactate-acidosis plays a protective role in stroke, preventing the formation of clots. The addition of NaCl and MgSO4 in different concentrations increased the internal dynamics of thrombin. Also, the addition of MgSO4 decreased thrombin-induced platelet aggregation. However, magnesium sulfate and acetylsalicylic acid in the therapeutic concentrations used for treatment of ischemic stroke had no effect on thrombin internal dynamics. The data obtained will help to elucidate the conformational stability of thrombin under conditions modulating lactate-acidosis and in the presence of magnesium sulfate.

  2. Bare magnetic nanoparticles as fluorescence quenchers for detection of thrombin.

    PubMed

    Yu, Jiemiao; Yang, Liangrong; Liang, Xiangfeng; Dong, Tingting; Liu, Huizhou

    2015-06-21

    Rapid and sensitive detection of thrombin has very important significance in clinical diagnosis. In this work, bare magnetic iron oxide nanoparticles (magnetic nanoparticles) without any modification were used as fluorescence quenchers. In the absence of thrombin, a fluorescent dye (CY3) labeled thrombin aptamer (named CY3-aptamer) was adsorbed on the surface of magnetic nanoparticles through interaction between a phosphate backbone of the CY3-aptamer and hydroxyl groups on the bare magnetic nanoparticles in binding solution, leading to fluorescence quenching. Once thrombin was introduced, the CY3-aptamer formed a G-quartet structure and combined with thrombin, which resulted in the CY3-aptamer being separated from the magnetic nanoparticles and restoration of fluorescence. This proposed assay took advantage of binding affinity between the CY3-aptamer and thrombin for specificity, and bare magnetic nanoparticles for fluorescence quenching. The fluorescence signal had a good linear relationship with thrombin concentration in the range of 1-60 nM, and the limit of detection for thrombin was estimated as low as 0.5 nM. Furthermore, this method could be applied for other target detection using the corresponding fluorescence labeled aptamer. PMID:25894923

  3. Thrombin Regulates Soluble fms-Like Tyrosine Kinase-1 (sFlt-1) Expression in First Trimester Decidua

    PubMed Central

    Lockwood, Charles J.; Toti, Paolo; Arcuri, Felice; Norwitz, Errol; Funai, Edmund F.; Huang, Se-Te J.; Buchwalder, Lynn F.; Krikun, Graciela; Schatz, Frederick

    2007-01-01

    The primary placental defect in preeclampsia is shallow trophoblast invasion of the decidua leading to incomplete vascular transformation and inadequate uteroplacental perfusion. Soluble fms-like tyrosine kinase-1 (sFlt-1) seems to interfere with these events by inhibiting local angiogenesis and/or by impeding trophoblast invasion. Preeclampsia is also associated with maternal thrombophilias and decidual hemorrhage, which form thrombin from decidual cell-expressed tissue factor. Although sFlt-1 is highly expressed by trophoblasts, sFlt-1 expression has not been studied in decidual cells, which are the predominant cell type encountered by invading trophoblasts. Here, we demonstrate that isolated decidual cells express sFlt-1 mRNA, suggesting that they can synthesize sFlt-1. Moreover, in first trimester decidual cells, thrombin enhanced sFlt-1 mRNA levels, as measured by quantitative reverse transcriptase-polymerase chain reaction, and levels of secreted sFlt-1 protein, as measured by enzyme-linked immunosorbent assay. The thrombin antagonist hirudin blocked this effect, demonstrating that active thrombin is required. Emphasizing the specificity of the thrombin response, neither interleukin-1β nor tumor necrosis factor-α affected sFlt-1 expression in the decidual cells. In contrast to first trimester decidual cells, thrombin did not affect sFlt-1 levels in cultured term decidual cells. In early pregnancy, thrombin may act as an autocrine/paracrine enhancer of sFlt-1 expression by decidual cells to promote pre-eclampsia by interfering with local vascular transformation. PMID:17392178

  4. Mechanistic Modeling of the Effects of Acidosis on Thrombin Generation

    PubMed Central

    Mitrophanov, Alexander Y.; Rosendaal, Frits R.

    2015-01-01

    BACKGROUND: Acidosis, a frequent complication of trauma and complex surgery, results from tissue hypoperfusion and IV resuscitation with acidic fluids. While acidosis is known to inhibit the function of distinct enzymatic reactions, its cumulative effect on the blood coagulation system is not fully understood. Here, we use computational modeling to test the hypothesis that acidosis delays and reduces the amount of thrombin generation in human blood plasma. Moreover, we investigate the sensitivity of different thrombin generation parameters to acidosis, both at the individual and population level. METHODS: We used a kinetic model to simulate and analyze the generation of thrombin and thrombin–antithrombin complexes (TAT), which were the end points of this study. Large groups of temporal thrombin and TAT trajectories were simulated and used to calculate quantitative parameters, such as clotting time (CT), thrombin peak time, maximum slope of the thrombin curve, thrombin peak height, area under the thrombin trajectory (AUC), and prothrombin time. The resulting samples of parameter values at different pH levels were compared to assess the acidosis-induced effects. To investigate intersubject variability, we parameterized the computational model using the data on clotting factor composition for 472 subjects from the Leiden Thrombophilia Study. To compare acidosis-induced relative parameter changes in individual (“virtual”) subjects, we estimated the probabilities of relative change patterns by counting the pattern occurrences in our virtual subjects. Distribution overlaps for thrombin generation parameters at distinct pH levels were quantified using the Bhattacharyya coefficient. RESULTS: Acidosis in the range of pH 6.9 to 7.3 progressively increased CT, thrombin peak time, AUC, and prothrombin time, while decreasing maximum slope of the thrombin curve and thrombin peak height (P < 10–5). Acidosis delayed the onset and decreased the amount of TAT generation (P

  5. TEMPERATURE-HYDROSTATIC PRESSURE STUDIES ON PARTIALLY PURIFIED INORGANIC PYROPHOSPHATASE ACTIVITY.

    PubMed

    MORITA, R Y; MATHEMEIER, P F

    1964-12-01

    Morita, Richard Y. (Oregon State University, Corvallis), and Paul F. Mathemeier. Temperature-hydrostatic pressure studies on partially purified inorganic pyrophosphatase activity. J. Bacteriol. 88:1667-1671. 1964.-Partially purified inorganic pyrophosphatase from Bacillus stearothermophilus demonstrated increased activity at 90 C with hydrostatic pressures of 100 to 1,100 atm. At 90 C, optimal activity was at 700 atm. No enzyme activity could be demonstrated at 100 C at 1 atm; however, at 105 C moderate hydrostatic pressures favored the enzyme reaction, resulting in increased activity. The increased enzyme activity is explained on the basis of pressure counteracting the molecular volume increase which results from elevated temperature. Thermal denaturation studies at 90 C show inorganic pyrophosphatase to be more stable in the presence of cofactor than substrate, and indicate an enyzme-cofactor intermediate in the hydrolysis. PMID:14240955

  6. Human activities recognition by head movement using partial recurrent neural network

    NASA Astrophysics Data System (ADS)

    Tan, Henry C. C.; Jia, Kui; De Silva, Liyanage C.

    2003-06-01

    Traditionally, human activities recognition has been achieved mainly by the statistical pattern recognition methods or the Hidden Markov Model (HMM). In this paper, we propose a novel use of the connectionist approach for the recognition of ten simple human activities: walking, sitting down, getting up, squatting down and standing up, in both lateral and frontal views, in an office environment. By means of tracking the head movement of the subjects over consecutive frames from a database of different color image sequences, and incorporating the Elman model of the partial recurrent neural network (RNN) that learns the sequential patterns of relative change of the head location in the images, the proposed system is able to robustly classify all the ten activities performed by unseen subjects from both sexes, of different race and physique, with a recognition rate as high as 92.5%. This demonstrates the potential of employing partial RNN to recognize complex activities in the increasingly popular human-activities-based applications.

  7. Acute ischemic stroke treated with intravenous tissue plasminogen activator in a patient taking dabigatran with radiographic evidence of recanalization.

    PubMed

    Sangha, Navdeep; El Khoury, Ramy; Misra, Vivek; Lopez, George

    2012-11-01

    Dabigatran etexelate is a new oral direct thrombin inhibitor that has been approved by the US Food and Drug Administration to prevent stroke in patients with nonvalvular atrial fibrillation. A 51-year-old man with a history of atrial fibrillation who was taking dabigatran presented with an acute ischemic stroke. The patient had a normal international normalized ratio, activated partial thromboplastin time, and an elevated thrombin time of 26.4 seconds. Recanalization of the middle cerebral artery with intravenous tissue plasminogen activator was apparent on digital subtraction angiography, and there was no evidence of intracerebral hemorrhage on the repeat computed tomographic scan. This is the first report of a patient who was taking dabigatran etexilate and who had an ischemic stroke caused by a middle cerebral artery occlusion, with an elevated thrombin time and radiographic recanalization with intravenous tissue plasminogen activator without evidence of hemorrhagic transformation. PMID:22683118

  8. Elevated Cytokines, Thrombin and PAI-1 in Severe HCPS Patients Due to Sin Nombre Virus

    PubMed Central

    Bondu, Virginie; Schrader, Ron; Gawinowicz, Mary Ann; McGuire, Paul; Lawrence, Daniel A.; Hjelle, Brian; Buranda, Tione

    2015-01-01

    Sin Nombre Hantavirus (SNV, Bunyaviridae Hantavirus) is a Category A pathogen that causes Hantavirus Cardiopulmonary Syndrome (HCPS) with case fatality ratios generally ranging from 30% to 50%. HCPS is characterized by vascular leakage due to dysregulation of the endothelial barrier function. The loss of vascular integrity results in non-cardiogenic pulmonary edema, shock, multi-organ failure and death. Using Electric Cell-substrate Impedance Sensing (ECIS) measurements, we found that plasma samples drawn from University of New Mexico Hospital patients with serologically-confirmed HCPS, induce loss of cell-cell adhesion in confluent epithelial and endothelial cell monolayers grown in ECIS cultureware. We show that the loss of cell-cell adhesion is sensitive to both thrombin and plasmin inhibitors in mild cases, and to thrombin only inhibition in severe cases, suggesting an increasing prothrombotic state with disease severity. A proteomic profile (2D gel electrophoresis and mass spectrometry) of HCPS plasma samples in our cohort revealed robust antifibrinolytic activity among terminal case patients. The prothrombotic activity is highlighted by acute ≥30 to >100 fold increases in active plasminogen activator inhibitor (PAI-1) which, preceded death of the subjects within 48 h. Taken together, this suggests that PAI-1 might be a response to the severe pathology as it is expected to reduce plasmin activity and possibly thrombin activity in the terminal patients. PMID:25674766

  9. ISOLATION AND PARTIAL CHARACTERIZATION OF AN ACID PHOSPHATASE ACTIVITY FROM SPIRODELA OLIGORHIZA

    EPA Science Inventory

    An acid phosphatase activity from the aquatic plant Spirodela oligorhiza (duckweed) was isolated and partially characterized. S. oligorhiza was grown in a hydroponic growth medium, harvested, and ground up in liquid nitrogen. The ground plant material was added to a biological ...

  10. Optimization of the production of triabin, a novel thrombin inhibitor, in High Five™ insect cells infected with a recombinant baculovirus.

    PubMed

    Vallazza, M; Petri, T

    1999-03-01

    The isolation of a new type of thrombin inhibitor, called triabin, from the saliva of the hematophagous bug Triatoma pallidipennis, has recently been described. In the in vitro platelet aggregation inhibition assay triabin has a similar potency as the thrombin inhibitor hirudin now in phase III clinical trials. However, in another in vitro assay using a low molecular weight substrate for thrombin, triabin does not inhibit thrombin completely even at 6 fold higher molar doses in comparison with hirudin. This means that triabin has a novel mode of action towards thrombin making triabin into an interesting candidate as a therapeutic agent. Recently it has been shown that a recombinant baculovirus can be efficiently used for the triabin production in insect cells and that the yields in adherent cultures of High Five™ cells (approx. 20 mg l-1) were about 7 fold higher than in adherent cultures of Sf9 cells (approx. 3 mg l- 1). To optimize the triabin yield from the baculovirus/insect cell expression system, experiments were performed with suspension adapted cultures of High Five™ cells to investigate the effects of the state of the host cell, of the multiplicity of infection, of the cell density at the time of infection and of supplementation of the medium with nutrients and oxygen. Triabin yields of up to 200 mg l-1, as determined by an activity assay, could finally be obtained here. PMID:22359057

  11. Partial purification of a Bacillus licheniformis levansucrase producing levan with antitumor activity.

    PubMed

    Dahech, Imen; Belghith, Karima Srih; Belghith, Hafedh; Mejdoub, Hafedh

    2012-10-01

    The extracellular fructosyltransferase (FTase) of a novel strain of Bacillus licheniformis capable of producing fructooligosaccharides (FOS) and a polysaccharide type levan was obtained and partially purified. The purification was achieved by ammonium sulfate precipitation, DEAE cellulose and gel filtration chromatographies. The enzyme was partially purified as determined by SDS-PAGE, and the specific activity reached was 67.5, representing a purification factor of 177 and yield of 40%. Levan was isolated from the cultures of B. licheniformis. The levan was composed mainly of fructose residues when analyzed by TLC after acid hydrolysis and NMR analysis. In a previous study, the levan produced exhibited a hypoglycemiant activity. The present paper deals with the study of the antitumor and anti-cytotoxic effect of levan produced by B. licheniformis strain. In the in vitro antitumor activity test of levan against some tumor cell lines, relatively the significantly high activity was observed against the HepG(2). PMID:22579870

  12. Label-free impedimetric biosensor for thrombin using the thrombin-binding aptamer as receptor

    NASA Astrophysics Data System (ADS)

    Frense, D.; Kang, S.; Schieke, K.; Reich, P.; Barthel, A.; Pliquett, U.; Nacke, T.; Brian, C.; Beckmann, D.

    2013-04-01

    This study presents the further establishment of impedimetric biosensors with aptamers as receptors. Aptamers are short single-stranded oligonucleotides which bind analytes with a specific region of their 3D structure. Electrical impedance spectroscopy is a sensitive method for analyzing changes on the electrode surface, e.g. caused by receptor-ligand-interactions. Fast and inexpensive prototyping of electrodes on the basis of commercially available compact discs having a 24 carat gold reflective layer was investigated. Electrode structures (CDtrodes [1]) in the range from few millimetres down to 100 microns were realized. The well-studied thrombin-binding aptamer (TBA) was used as receptor for characterizing these micro- and macro-electrodes. The impedance signal showed a linear correlation for concentrations of thrombin between 1.0 nM to 100 nM. This range corresponds well with most of the references and may be useful for the point-of-care testing (POCT).

  13. APTAMER-BASED SERRS SENSOR FOR THROMBIN DETECTION

    SciTech Connect

    Cho, H; Baker, B R; Wachsmann-Hogiu, S; Pagba, C V; Laurence, T A; Lane, S M; Lee, L P; Tok, J B

    2008-07-02

    We describe an aptamer-based Surface Enhanced Resonance Raman Scattering (SERRS) sensor with high sensitivity, specificity, and stability for the detection of a coagulation protein, human a-thrombin. The sensor achieves high sensitivity and a limit of detection of 100 pM by monitoring the SERRS signal change upon the single step of thrombin binding to immobilized thrombin binding aptamer. The selectivity of the sensor is demonstrated by the specific discrimination of thrombin from other protein analytes. The specific recognition and binding of thrombin by the thrombin binding aptamer is essential to the mechanism of the aptamer-based sensor, as shown through measurements using negative control oligonucleotides. In addition, the sensor can detect 1 nM thrombin in the presence of complex biofluids, such as 10% fetal calf serum, demonstrating that the immobilized, 5{prime}-capped, 3{prime}-capped aptamer is sufficiently robust for clinical diagnostic applications. Furthermore, the proposed sensor may be implemented for multiplexed detection using different aptamer-Raman probe complexes.

  14. Aptamer-based SERRS sensor for thrombin detection.

    PubMed

    Cho, Hansang; Baker, Brian R; Wachsmann-Hogiu, Sebastian; Pagba, Cynthia V; Laurence, Ted A; Lane, Stephen M; Lee, Luke P; Tok, Jeffrey B H

    2008-12-01

    We describe an aptamer-based surface enhanced resonance Raman scattering (SERRS) sensor with high sensitivity, specificity, and stability for the detection of a coagulation protein, human alpha-thrombin. The sensor achieves high sensitivity and a limit of detection of 100 pM by monitoring the SERRS signal change upon the single-step of thrombin binding to immobilized thrombin binding aptamer. The selectivity of the sensor is demonstrated by the specific discrimination of thrombin from other protein analytes. The specific recognition and binding of thrombin by the thrombin binding aptamer is essential to the mechanism of the aptamer-based sensor, as shown through measurements using negative control oligonucleotides. In addition, the sensor can detect 1 nM thrombin in the presence of complex biofluids, such as 10% fetal calf serum, demonstrating that the immobilized, 5'-capped, 3'-capped aptamer is sufficiently robust for clinical diagnostic applications. Furthermore, the proposed sensor may be implemented for multiplexed detection using different aptamer-Raman probe complexes. PMID:19367849

  15. The inhibition of thrombin-dependent positive-feedback reactions is critical to the expression of the anticoagulant effect of heparin.

    PubMed Central

    Ofosu, F A; Sie, P; Modi, G J; Fernandez, F; Buchanan, M R; Blajchman, M A; Boneu, B; Hirsh, J

    1987-01-01

    Heparin catalyses the inhibition of two key enzymes of blood coagulation, namely Factor Xa and thrombin, by enhancing the antiproteinase activities of plasma antithrombin III and heparin cofactor II. In addition, heparin can directly inhibit the activation of Factor X and prothrombin. The contributions of each of these effects to the anticoagulant activity of heparin have not been delineated. We therefore performed experiments to assess how each of these effects of heparin contributes to its anticoagulant activity by comparing the effects of heparin, pentosan polysulphate and D-Phe-Pro-Arg-CH2Cl on the intrinsic pathway of coagulation. Unlike heparin, pentosan polysulphate catalyses only the inhibition of thrombin by plasma. D-Phe-Pro-Arg-CH2Cl is rapid enough an inhibitor of thrombin so that when added to plasma no complexes of thrombin with its inhibitors are formed, whether or not the plasma also contains heparin. Heparin (0.66 microgram/ml) and pentosan polysulphate (6.6 micrograms/ml) completely inhibited the intrinsic-pathway activation of 125I-prothrombin to 125I-prothrombin fragment 1 + 2 and 125I-thrombin. On the addition of thrombin, a good Factor V activator, to the plasma before each sulphated polysaccharide, the inhibition of prothrombin activation was demonstrable only in the presence of higher concentrations of the sulphated polysaccharide. D-Phe-Pro-Arg-CH2Cl also completely inhibited the intrinsic-pathway activation of prothrombin in normal plasma. The inhibitory effect of D-Phe-Pro-Arg-CH2Cl was reversed if thrombin was added to the plasma before D-Phe-Pro-Arg-CH2Cl. The inhibition of the activation of prothrombin by the three agents was also abolished with longer times with re-added Ca2+. Reversal of the inhibitory effects of heparin and pentosan polysulphate was associated with the accelerated formation of 125I-thrombin-antithrombin III and 125I-thrombin-heparin cofactor complexes respectively. These results suggest that the anticoagulant

  16. Effect of heparin on TAFI-dependent inhibition of fibrinolysis: relative importance of TAFIa generated by clot-bound and fluid phase thrombin.

    PubMed

    Colucci, Mario; Pentimone, Anna; Binetti, Bianca M; Cramarossa, Marialisa; Piro, Donatella; Semeraro, Nicola

    2002-08-01

    Heparin has been proposed to enhance thrombolysis by inhibiting thrombin-dependent generation of activated TAFI (thrombin activatable fibrinolysis inhibitor), a carboxypeptidase that inhibits fibrinolysis. We evaluated the effect of heparin in an in vitro thrombolysis model consisting of a radiolabelled blood clot submerged in defibrinated plasma. Fibrinolysis was induced by adding t-PA (250 ng/ml) and calcium to the plasma bath. Control experiments indicated that thrombin generation induced by recalcification caused significant TAFI activation and inhibited clot lysis. Heparin (up to 1 U/ml), added to the plasma bath, failed to enhance clot lysis. Thrombin generation in the fluid phase was totally inhibited by heparin at concentrations > 0.5 U/ml. In contrast, thrombin generation on the clot surface was not inhibited by heparin (1 U/ml). TAFIa generation did occur in heparin-containing samples (1 U/ml) and amounted to about 10% of TAFIa formed in control samples. This low amount of TAFIa did exert antifibrinolytic activity as indicated by the observation that the addition of a specific TAFIa inhibitor (PTI) along with heparin enhanced clot lysis. Hirudin (10 micrograms/ml), at variance with heparin, inhibited clot-bound thrombin and enhanced clot lysis. These data show that heparin is unable to stimulate fibrinolysis through a TAFI-dependent mechanism, most likely because of its inefficiency in inhibiting thrombin generation on the clot surface. Moreover, they suggest that clot-bound thrombin plays a major role in TAFI-mediated inhibition of fibrinolysis through "localized" TAFIa generation. PMID:12195701

  17. Use of dressing with human fibrin and thrombin during resection of a right atrial angiosarcoma

    PubMed Central

    Bochenek, Maciej; Kapelak, Bogusław; Bartuś, Krzysztof; Urbańczyk, Małgorzata; Sadowski, Jerzy

    2015-01-01

    Primary malignant cardiac tumors are rare and are usually detected at an advanced stage of disease. Their location and infiltration often hinder surgical resection. Tissue sarcomas, especially angiosarcomas, are composed of irregular and delicate vascular tissue. The resection of such tumors from the heart is associated with a high risk of life-threatening bleeding that cannot be stopped with traditional surgical methods. We present a case report of the application of a dressing containing human fibrin and thrombin in order to prevent bleeding during the partial resection of advanced cardiac angiosarcoma in a 40-year-old patient. PMID:26336498

  18. Comparison of the ‘Chemical’ and ‘Structural’ Approaches to the Optimization of the Thrombin-Binding Aptamer

    PubMed Central

    Tatarinova, Olga; Tsvetkov, Vladimir; Basmanov, Dmitry; Barinov, Nikolay; Smirnov, Igor; Timofeev, Edward; Kaluzhny, Dmitry; Chuvilin, Andrey; Klinov, Dmitry; Varizhuk, Anna; Pozmogova, Galina

    2014-01-01

    Noncanonically structured DNA aptamers to thrombin were examined. Two different approaches were used to improve stability, binding affinity and biological activity of a known thrombin-binding aptamer. These approaches are chemical modification and the addition of a duplex module to the aptamer core structure. Several chemically modified aptamers and the duplex-bearing ones were all studied under the same conditions by a set of widely known and some relatively new methods. A number of the thrombin-binding aptamer analogs have demonstrated improved characteristics. Most importantly, the study allowed us to compare directly the two approaches to aptamer optimization and to analyze their relative advantages and disadvantages as well as their potential in drug design and fundamental studies. PMID:24586736

  19. The involvement of a novel mechanism distinct from the thrombin receptor in the vasocontraction induced by trypsin.

    PubMed

    Komuro, T; Miwa, S; Minowa, T; Okamoto, Y; Enoki, T; Ninomiya, H; Zhang, X F; Uemura, Y; Kikuchi, H; Masaki, T

    1997-03-01

    1. The vasocontracting effect of a serine protease trypsin and its mechanisms were investigated by monitoring the isometric tension in endothelium-denuded rings of rabbit thoracic aortae and its effects on intracellular free Ca2+ concentrations ([Ca2+]i) in dispersed rabbit vascular smooth muscle cells with a Ca2+ indicator fura-2. The actions of trypsin were compared with those of thrombin. 2. Both thrombin and trypsin reversibly contracted aortic rings without endothelium in a concentration-dependent manner. The vasocontraction induced by trypsin was well correlated with the protease activity of trypsin actually added to the tissue baths containing the aortic rings and was completely blocked by soybean trypsin inhibitor and phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor. 3. The trypsin-induced contractions of the aortic rings were not the result of irreversible damage to vascular smooth muscle cells, since the contractile responses induced by noradrenaline or 30 mM KCl were unaffected by pretreatment with trypsin. 4. The contractions induced by either thrombin or trypsin were reduced to about 30% of control responses after removal of extracellular Ca2+, indicating that most of the contraction is dependent on extracellular Ca2+. By contrast, the contractions induced by either of the proteases were reduced by an antagonist of L-type voltage-operated Ca2+ channels, nifedipine, to about 70% of control responses, indicating that both nifedipine-sensitive and -resistant Ca2+ channels are involved in these contractions. 5. In the aortic rings precontracted by a maximally effective concentration of thrombin, the second application of thrombin virtually failed to induce contractions but trypsin could still induce contractions amounting to 10% of control values by it's protease activity. 6. After the first application of a maximal concentration of thrombin, the second application of thrombin could not induce an increase in [Ca2+]i, but an application of

  20. Pseudomonas aeruginosa elastase cleaves a C-terminal peptide from human thrombin that inhibits host inflammatory responses

    PubMed Central

    van der Plas, Mariena J. A.; Bhongir, Ravi K. V.; Kjellström, Sven; Siller, Helena; Kasetty, Gopinath; Mörgelin, Matthias; Schmidtchen, Artur

    2016-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen known for its immune evasive abilities amongst others by degradation of a large variety of host proteins. Here we show that digestion of thrombin by P. aeruginosa elastase leads to the release of the C-terminal thrombin-derived peptide FYT21, which inhibits pro-inflammatory responses to several pathogen-associated molecular patterns in vitro and in vivo by preventing toll-like receptor dimerization and subsequent activation of down-stream signalling pathways. Thus, P. aeruginosa ‘hijacks' an endogenous anti-inflammatory peptide-based mechanism, thereby enabling modulation and circumvention of host responses. PMID:27181065

  1. Pseudomonas aeruginosa elastase cleaves a C-terminal peptide from human thrombin that inhibits host inflammatory responses.

    PubMed

    van der Plas, Mariena J A; Bhongir, Ravi K V; Kjellström, Sven; Siller, Helena; Kasetty, Gopinath; Mörgelin, Matthias; Schmidtchen, Artur

    2016-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen known for its immune evasive abilities amongst others by degradation of a large variety of host proteins. Here we show that digestion of thrombin by P. aeruginosa elastase leads to the release of the C-terminal thrombin-derived peptide FYT21, which inhibits pro-inflammatory responses to several pathogen-associated molecular patterns in vitro and in vivo by preventing toll-like receptor dimerization and subsequent activation of down-stream signalling pathways. Thus, P. aeruginosa 'hijacks' an endogenous anti-inflammatory peptide-based mechanism, thereby enabling modulation and circumvention of host responses. PMID:27181065

  2. A label-free electrochemiluminescence aptasensor for thrombin detection based on host-guest recognition between tris(bipyridine)ruthenium(II)-β-cyclodextrin and aptamer.

    PubMed

    Chen, Qiong; Chen, Hong; Zhao, Yingying; Zhang, Fan; Yang, Fan; Tang, Jie; He, Pingang

    2014-04-15

    An ultrasensitive label-free electrochemiluminescence (ECL) aptasensor for the detection of thrombin was developed based on the specific recognition between tris(bipyridine)ruthenium(II)-β-cyclodextrin (tris(bpyRu)-β-CD) and the anti-thrombin aptamer (aptamer). The NH2-aptamer was first immobilized on the activated glassy carbon electrode (GCE) by coupling interaction. By use of the specific recognition between tris(bpyRu)-β-CD and aptamer, tris(bpyRu)-β-CD was then attached on the surface of GCE. Resulting from the outstanding photoactive properties of tris(bpyRu)-β-CD, the fabricated GCE performed strong ECL signal with the coreactant of 2-(dibutylamino)ethanol (DBAE). However, in the presence of thrombin, aptamer-thrombin bioaffinity complexes were formed, which restricted the recognition activities between aptamer and tris(bpyRu)-β-CD. Thus, fewer tris(bpyRu)-β-CD could be attached on the surface of GCE and led to an obvious decrease of ECL signal. Fortunately, the difference of ECL intensity before and after combination with thrombin was logarithmically linear with the concentration of thrombin in a wide range of 10 nM-1 pM. Meantime, a detection limit of 0.1 pM without any other signal labeling or amplifying procedures indicated that the biosensor performed excellent sensitivity, operability and simplicity. PMID:24321886

  3. Evolution of thrombin and other hemostatic proteases by survey of protochordate, hemichordate, and echinoderm genomes.

    PubMed

    Ponczek, Michal B; Bijak, Michal Z; Nowak, Pawel Z

    2012-06-01

    Protochordate genomes enable a prevalence of hemostasis evolution. Broad searches were performed for homologs of human serine proteases of hemostasis on the genomes of Branchiostoma floridae, Saccoglossus kowalevskii, and Strongylocentrotus purpuratus. Sequences were analyzed by multiple bioinformatic tools. The survey revealed numerous homologous components. Amphioxus was rich in some serine proteases not accompanied by gamma-carboxyglutamic or kringle domains similar more to thrombin than to other coagulation factors. The serine proteases found in amphioxus exhibited the attributes similar to those of thrombin by phylogeny relationships, sequence conservation, gene synteny, spatial structure, and ligand docking. A few plasminogen- and plasminogen activators-like proteases with kringles were also present. Those serine proteases demonstrated the greatest proximity rather to plasminogen or plasminogen activators than to thrombin. Searching for homologs of serine protease hemostatic factors in acorn worm and sea urchin revealed several components similar to those found in amphioxus. Hypothetically, the common ancestor of chordates had three separate serine proteases that evolved independently into immunoglobulin-like and kringle proteases in lancelets, and prothrombin, plasminogen activators, and plasminogen in vertebrates. Ancestral proteases evolved in vertebrates into hemostasis factors after merging the proper N-terminal domains and duplications. PMID:22752046

  4. Bifunctional combined aptamer for simultaneous separation and detection of thrombin.

    PubMed

    Bing, Tao; Liu, Xiangjun; Cheng, Xiaohong; Cao, Zehui; Shangguan, Dihua

    2010-02-15

    Here we report on the construction and evaluation of a bifunctional combined aptamer (BCA) that consists of a DNA streptavidin-binding aptamer (SBA), a DNA thrombin-binding aptamer (TBA) and a fluorophore. The BCA adopts a new conformation that is very different from simply linking the conformations of the two individual aptamers together, so that it does not bind to streptavidin in the absence of thrombin. Binding of this novel DNA aptamer to streptavidin is triggered by the thrombin binding and depends on the concentration of thrombin. Meanwhile, fluorescence from the streptavidin captured BCA reflects the quantity of the target molecule in the sample. This aptamer combination strategy based on the SBA holds good potential for applications in simultaneous detection and separation of targets of aptamers or certain DNA and RNA targets. PMID:19959350

  5. [Expression of snake venom thrombin-like enzyme calobin in Pichia pastoris].

    PubMed

    Yuan, Shengling; Wang, Peng; Tao, Haoxia; Zhan, Dewen; Wang, Yanchun; Wang, Lingchun; Liu, Chunjie; Zhang, Zhaoshan

    2009-04-01

    Thrombin-like enzymes (TLEs) are studied widely because of their therapeutic potential in myocardial infarction and thrombotic diseases. We synthesized the DNA fragment encoding thrombin-like enzyme calobin from Agkistrodon caliginosus (Korean Viper) venom by fusion PCR and expressed it in Pichia pastoris. After induction by 0.5% methanol for 48 h, the expression level of recombinant calobin reached 3.5 g/L in medium. The recombinant calobin was purified by Q-Sepharose Fast Flow ion-exchange chromatography and Sephacryl-S-100 gel filtration chromatography. Purified sample had a molecular weight of 32 kD shown in SDS-PAGE. It hydrolyzed fibrinogen and formed a light white hydrolysis circle in fibrinogen plate. SDS-PAGE analysis showed that recombinant calobin cleaved Aalpha-chain of fibrinogen specifically, and produced an appropriately 40 kD new band. However, we failed to find its fibrin-clot formation activity. PMID:19637626

  6. An electrochemical aptasensor based on TiO2/MWCNT and a novel synthesized Schiff base nanocomposite for the ultrasensitive detection of thrombin.

    PubMed

    Heydari-Bafrooei, Esmaeil; Amini, Maryam; Ardakani, Mehdi Hatefi

    2016-11-15

    A sensitive aptasensor based on a robust nanocomposite of titanium dioxide nanoparticles, multiwalled carbon nanotubes (MWCNT), chitosan and a novel synthesized Schiff base (SB) (TiO2/MWCNT/CHIT/SB) on the surface of a glassy carbon electrode (GCE) was developed for thrombin detection. The resultant nanocomposite can provide a large surface area, excellent electrocatalytic activity, and high stability, which would improve immobilization sites for biological molecules, allow remarkable amplification of the electrochemical signal and contribute to improved sensitivity. Thrombin aptamers were simply immobilized onto the TiO2-MWCNT/CHIT-SB nanocomposite matrix through simple π - π stacking and electrostatic interactions between CHIT/SB and aptamer strands. The electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to analyze the surface characterization of unmodified GCE and TiO2-MWCNT/CHIT-SB modified GCE, and also the interaction between aptamer and thrombin. In the presence of thrombin, the aptamer on the adsorbent layer captures the target on the electrode interface, which makes a barrier for electrons and inhibits electron transfer, thereby resulting in decreased DPV and increased impedance signals of the TiO2-MWCNT/CHIT-SB modified GCE. Furthermore, the proposed aptasensor has a very low LOD of 1.0fmolL(-1) thrombin within the detection range of 0.00005-10nmolL(-1). The aptasensor also presents high specificity and reproducibility for thrombin, which is unaffected by the coexistence of other proteins. Clinical application was performed with analysis of the thrombin levels in blood and CSF samples obtained from patients with MS, Parkinson, Epilepsy and Polyneuropathy using both the aptasensor and commercial ELISA kit. The results revealed the proposed system to be a promising candidate for clinical analysis of thrombin. PMID:27295570

  7. Pharmacology, pharmacokinetics, and pharmacodynamics of dabigatran etexilate, an oral direct thrombin inhibitor.

    PubMed

    Stangier, Joachim; Clemens, Andreas

    2009-01-01

    Dabigatran etexilate is a novel, oral reversible direct thrombin inhibitor that is rapidly absorbed and converted to its active form, dabigatran. Dabigatran has been shown to be a potent, competitive, and reversible inhibitor of thrombin, inhibiting both thrombin activity and generation. Studies in healthy volunteers and in patients undergoing orthopedic surgery indicate that dabigatran has a predictable pharmacokinetic profile, allowing for a fixed-dose regimen without the need for coagulation monitoring. In healthy volunteers, peak plasma concentrations of dabigatran are reached approximately 2 hours after oral administration. The elimination half-life is 12 to 14 hours, with clearance predominantly occurring via renal excretion of unchanged drug. Dabigatran is not metabolized by cytochrome P450 isoenzymes, has no interactions with food, and also has a low potential for drug-drug interactions. The pharmacokinetic profile of dabigatran is consistent across a broad range of different patient populations and is unaffected by gender, body weight, ethnic origin, obesity, and mild-to-moderate hepatic impairment. Small differences in dabigatran pharmacokinetics associated with age are attributable to variation in renal function. Dabigatran etexilate produces a predictable pharmacodynamic effect and requires no coagulation monitoring. It has been approved in the European Union (EU) and Canada for prophylaxis of thromboembolism in patients undergoing total knee or hip arthroplasty. Ongoing clinical trials are investigating its use in the treatment of venous thromboembolism, prevention of stroke in patients with nonvalvular atrial fibrillation, and treatment of thromboembolic complications, following acute coronary syndromes. PMID:19696042

  8. The Importance of Exosite Interactions for Substrate Cleavage by Human Thrombin

    PubMed Central

    Chahal, Gurdeep; Thorpe, Michael; Hellman, Lars

    2015-01-01

    Thrombin is a serine protease of the chymotrypsin family that acts both as a procoagulant and as an anticoagulant by cleaving either factor VIII, factor V and fibrinogen or protein C, respectively. Numerous previous studies have shown that electropositive regions at a distance from the active site, so called exosites, are of major importance for the cleavage by human thrombin. Upstream of all the known major cleavage sites for thrombin in factor VIII, factor V and fibrinogen are clusters of negatively charged amino acids. To study the importance of these sites for the interaction with the exosites and thereby the cleavage by thrombin, we have developed a new type of recombinant substrate. We have compared the cleavage rate of the minimal cleavage site, involving only 8-9 amino acids (typically the P4-P4’ positions) surrounding the cleavage site, with the substrates also containing the negatively charged regions upstream of the cleavage sites. The results showed that addition of these regions enhanced the cleavage rate by more than fifty fold. However, the enhancement was highly dependent on the sequence of the actual cleavage site. A minimal site that showed poor activity by itself could be cleaved as efficiently as an optimal cleavage site when presented together with these negatively charged regions. Whereas sites conforming closely to the optimal site were only minimally enhanced by the addition of these regions. The possibility to mimic this interaction for the sites in factor V and factor VIII by recombinant substrates, which do not have the same folding as the full size target, indicates that the enhancement was primarily dependent on a relatively simple electrostatic interaction. However, the situation was very different for fibrinogen and protein C where other factors than only charge is of major importance. PMID:26110612

  9. Modifying the substrate specificity of Carcinoscorpius rotundicauda serine protease inhibitor domain 1 to target thrombin.

    PubMed

    Giri, Pankaj Kumar; Tang, Xuhua; Thangamani, Saravanan; Shenoy, Rajesh T; Ding, Jeak Ling; Swaminathan, Kunchithapadam; Sivaraman, J

    2010-01-01

    Protease inhibitors play a decisive role in maintaining homeostasis and eliciting antimicrobial activities. Invertebrates like the horseshoe crab have developed unique modalities with serine protease inhibitors to detect and respond to microbial and host proteases. Two isoforms of an immunomodulatory two-domain Kazal-like serine protease inhibitor, CrSPI-1 and CrSPI-2, have been recently identified in the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. Full length and domain 2 of CrSPI-1 display powerful inhibitory activities against subtilisin. However, the structure and function of CrSPI-1 domain-1 (D1) remain unknown. Here, we report the crystal structure of CrSPI-1-D1 refined up to 2.0 Å resolution. Despite the close structural homology of CrSPI-1-D1 to rhodniin-D1 (a known thrombin inhibitor), the CrSPI-1-D1 does not inhibit thrombin. This prompted us to modify the selectivity of CrSPI-1-D1 specifically towards thrombin. We illustrate the use of structural information of CrSPI-1-D1 to modify this domain into a potent thrombin inhibitor with IC(50) of 26.3 nM. In addition, these studies demonstrate that, besides the rigid conformation of the reactive site loop of the inhibitor, the sequence is the most important determinant of the specificity of the inhibitor. This study will lead to the significant application to modify a multi-domain inhibitor protein to target several proteases. PMID:21188150

  10. Modifying the Substrate Specificity of Carcinoscorpius rotundicauda Serine Protease Inhibitor Domain 1 to Target Thrombin

    PubMed Central

    Giri, Pankaj Kumar; Tang, Xuhua; Thangamani, Saravanan; Shenoy, Rajesh T.; Ding, Jeak Ling; Swaminathan, Kunchithapadam; Sivaraman, J.

    2010-01-01

    Protease inhibitors play a decisive role in maintaining homeostasis and eliciting antimicrobial activities. Invertebrates like the horseshoe crab have developed unique modalities with serine protease inhibitors to detect and respond to microbial and host proteases. Two isoforms of an immunomodulatory two-domain Kazal-like serine protease inhibitor, CrSPI-1 and CrSPI-2, have been recently identified in the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. Full length and domain 2 of CrSPI-1 display powerful inhibitory activities against subtilisin. However, the structure and function of CrSPI-1 domain-1 (D1) remain unknown. Here, we report the crystal structure of CrSPI-1-D1 refined up to 2.0 Å resolution. Despite the close structural homology of CrSPI-1-D1 to rhodniin-D1 (a known thrombin inhibitor), the CrSPI-1-D1 does not inhibit thrombin. This prompted us to modify the selectivity of CrSPI-1-D1 specifically towards thrombin. We illustrate the use of structural information of CrSPI-1-D1 to modify this domain into a potent thrombin inhibitor with IC50 of 26.3 nM. In addition, these studies demonstrate that, besides the rigid conformation of the reactive site loop of the inhibitor, the sequence is the most important determinant of the specificity of the inhibitor. This study will lead to the significant application to modify a multi-domain inhibitor protein to target several proteases. PMID:21188150

  11. Changes in the nuclear protein kinase activities in the regenerating liver of partially irradiated rat

    SciTech Connect

    Asami, K.; Kobayashi, H.; Fujiwara, A.; Yasumasu, I. )

    1989-09-01

    X rays (4.8 Gy) inhibit both DNA synthesis and phosphorylation of histone H1 in the regenerating liver of the rat. To determine the cause of the inhibition of histone H1 phosphorylation, changes in the nuclear protein kinase activities during the prereplicative phase of regeneration were measured. The cAMP-dependent protein kinase activity was low during regeneration, and the changes in the activity were not statistically significant. The cAMP-independent protein kinase activity increased at 15 h, decreased at 18 h, and increased again at 24 h after partial hepatectomy. X irradiation prior to partial hepatectomy did not inhibit the increase at 15 h, but it did inhibit the increase at 24 h. The activity was not inhibited by isoquinolinesulfonamide inhibitors such as H-7, and it was activated by a commercial preparation of an inhibitor protein of the cAMP-dependent kinase. It was also inhibited by quercetin. The possibility that the radiation-sensitive nuclear protein kinase is a nuclear cAMP-independent protein kinase specific for histone H1 is considered.

  12. Sensitive and selective detection of thrombin by using a cyclic peptide as affinity ligand.

    PubMed

    Zhao, Qiang; Gao, Jie

    2015-01-15

    Here we describe a sensitive assay for thrombin by using a high binding-affinity cyclic peptide against thrombin as affinity ligand. The cyclic peptide is immobilized on the magnetic beads or microplates to selectively capture thrombin. The enriched thrombin then catalyzes the cleavage of a substrate of thrombin to a detectable product. The detection of thrombin is finally achieved by measuring the generated product. This assay enables the detection of thrombin at tens fM in 100 µL of sample solution when fluorogenic substrate was applied, while detection limits reached pM level when chromogenic substrate was used. Thrombin in plasma sample can be detected with this assay. This cyclic peptide affinity ligand shows potentials for thrombin analysis in other detection formats. PMID:25048449

  13. Partially Oxidized Sub-10 nm MnO Nanocrystals with High Activity for Water Oxidation Catalysis

    PubMed Central

    Jin, Kyoungsuk; Chu, Arim; Park, Jimin; Jeong, Donghyuk; Jerng, Sung Eun; Sim, Uk; Jeong, Hui-Yun; Lee, Chan Woo; Park, Yong-Sun; Yang, Ki Dong; Kumar Pradhan, Gajendra; Kim, Donghun; Sung, Nark-Eon; Hee Kim, Sun; Nam, Ki Tae

    2015-01-01

    The oxygen evolution reaction (OER) is considered a major bottleneck in the overall water electrolysis process. In this work, highly active manganese oxide nano-catalysts were synthesized via hot injection. Facile surface treatment generated Mn(III) species on monodisperse 10 nm MnO nanocrystals (NCs). Size dependency of MnO NCs on OER activity was also investigated. Surprisingly, the partially oxidized MnO NCs only required 530 mV @ 5 mA cm−2 under near neutral conditions. PMID:25998696

  14. Partially Oxidized Sub-10 nm MnO Nanocrystals with High Activity for Water Oxidation Catalysis

    NASA Astrophysics Data System (ADS)

    Jin, Kyoungsuk; Chu, Arim; Park, Jimin; Jeong, Donghyuk; Jerng, Sung Eun; Sim, Uk; Jeong, Hui-Yun; Lee, Chan Woo; Park, Yong-Sun; Yang, Ki Dong; Kumar Pradhan, Gajendra; Kim, Donghun; Sung, Nark-Eon; Hee Kim, Sun; Nam, Ki Tae

    2015-05-01

    The oxygen evolution reaction (OER) is considered a major bottleneck in the overall water electrolysis process. In this work, highly active manganese oxide nano-catalysts were synthesized via hot injection. Facile surface treatment generated Mn(III) species on monodisperse 10 nm MnO nanocrystals (NCs). Size dependency of MnO NCs on OER activity was also investigated. Surprisingly, the partially oxidized MnO NCs only required 530 mV @ 5 mA cm-2 under near neutral conditions.

  15. Comparison of the structures of the cyclotheonamide A complexes of human alpha-thrombin and bovine beta-trypsin.

    PubMed Central

    Ganesh, V.; Lee, A. Y.; Clardy, J.; Tulinsky, A.

    1996-01-01

    Thrombin, a trypsin-like serine protease present in blood, plays a central role in the regulation of thrombosis and hemostasis. A cyclic pentapeptide, cyclotheonamide A (CtA), isolated from sponges of the genus Theonella, inhibits thrombin, trypsin, and certain other serine proteases. Enzyme inhibition data for CtA indicate that it is a moderate inhibitor of alpha-thrombin (K(i) = 1.0 nM), but substantially more potent toward trypsin (K(i) = 0.2 nM). The comparative study of the crystal structures of the CtA complexes of alpha-thrombin and beta-trypsin reported here focuses on structure-function relationships in general and the enhanced specificity of trypsin, in particular. The crystal structures of the CtA complexes of thrombin and trypsin were solved and refined at 1.7 and 2.0 A resolution, respectively. The structures show that CtA occupies the active site with the Pro-Arg motif positioned in the S2 and S1 binding sites. The alpha-keto group of CtA is involved in a tetrahedral intermediate hemiketal structure with Ser 195 OG of the catalytic triad and is positioned within bonding distance from, and orthogonal to, the re-face of the carbonyl of the arginine of CtA. As in other productive binding modes of serine proteases, the Ser 214-Gly 216 segment runs in a twisted antiparallel beta-strand manner with respect to the diaminopropionic acid (Dpr)-Arg segment of CtA. The Tyr 60A-Thr 60I insertion loop of thrombin makes a weak aromatic stacking interaction with the v-Tyr of CtA through Trp 60D. The Glu 39 Tyr and Leu 41 Phe substitutions in trypsin produce an enhanced aromatic interaction with D-Phe of CtA, which also leads to different orientations of the side chains of D-Phe and the v-Tyr. The comparison of the CtA complexes of thrombin and trypsin shows that the gross structural features of both in the active site region are the same, whereas the differences observed are mainly due to minor insertions and substitutions. In trypsin, the substitution of Ile 174

  16. Development of gel-forming lyophilized formulation with recombinant human thrombin.

    PubMed

    Murányi, Andrej; Bartoš, Peter; Tichý, Eduard; Lazová, Jana; Pšenková, Jana; Žabka, Marián

    2015-01-01

    The objective of this work was development and evaluation of gel-forming lyophilized formulation with recombinant human thrombin for topical administration. The influence of pH, ionic strength and buffer type on protein stability was evaluated as part of the pre-formulation screening studies. Results indicated an optimal pH from 6.0 to 7.0 and increased stability with increasing content of sodium chloride. The tested buffer types had no significant effect on thrombin stability. For further development, thermosensitive Pluronic® F-127 was employed as a bulking and gelling agent. Physical and mechanical characterization and viscosity measurement confirmed the gel-forming properties of the formulation at the application temperature of 32 °C. Several techniques (addition of well-soluble polyols, different freezing protocols and reconstitution under vacuum) were tested to decrease the reconstitution time. The obtained results revealed that a vacuum in the vial headspace is crucial for acceptable reconstitution. The freeze drying process has no negative impact on recombinant thrombin stability, and this was confirmed by reverse-phase-HPLC, activity assay and optical density measurements. PMID:25347143

  17. Thrombin generation assay: a new tool to predict and optimize clinical outcome in cardiovascular patients?

    PubMed

    Campo, Gianluca; Pavasini, Rita; Pollina, Alberto; Fileti, Luca; Marchesini, Jlenia; Tebaldi, Matteo; Ferrari, Roberto

    2012-12-01

    Antithrombotic therapy (including antiplatelet and anticoagulant drugs) is the cornerstone of the current medical treatment of patients with acute coronary syndromes (ACS). This therapy and particularly the new antiplatelet and anticoagulant drugs have significantly reduced the ischemic risk, but have increased bleeding complications. Recently, several studies have emphasized the negative prognostic impact on long-term mortality of these bleeding adverse events. Thus, new assays to estimate the bleeding risk and the efficacy of these antithrombotic drugs are clearly in demand. Regarding the anticoagulant drugs, new promising data have emerged about the thrombin generation assay (TGA). TGA measures the ability of plasma to generate thrombin. TGA may be used to check coagulation function, to value risk of thrombosis and to compare the efficacy of different anticoagulants employed in clinical management of patients with ACS. The TGA result is a curve which describes the variation of thrombin's amount during the activation of the coagulation cascade. All available anticoagulant drugs influence the principal parameters generated by TGA and so it is possible to evaluate the effects of the medical treatment. In this review we provide a brief description of the assay and we summarize the principals of previous studies by analyzing the relationship between anticoagulant drugs and TGA. Moreover, a brief summary of its ability to predict ischemic and bleeding risks has been provided. PMID:22688556

  18. Molecular modeling studies, synthesis and biological evaluation of dabigatran analogues as thrombin inhibitors.

    PubMed

    Dong, Ming-Hui; Chen, Hai-Feng; Ren, Yu-Jie; Shao, Fang-Ming

    2016-01-15

    In this work, 48 thrombin inhibitors based on the structural scaffold of dabigatran were analyzed using a combination of molecular modeling techniques. We generated three-dimensional quantitative structure-activity relationship (3D-QSAR) models based on three alignments for both comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) to highlight the structural requirements for thrombin protein inhibition. In addition to the 3D-QSAR study, Topomer CoMFA model also was established with a higher leave-one-out cross-validation q(2) and a non-cross-validation r(2), which suggest that the three models have good predictive ability. The results indicated that the steric, hydrophobic and electrostatic fields play key roles in QSAR model. Furthermore, we employed molecular docking and re-docking simulation explored the binding relationship of the ligand and the receptor protein in detail. Molecular docking simulations identified several key interactions that were also indicated through 3D-QSAR analysis. On the basis of the obtained results, two compounds were designed and predicted by three models, the biological evaluation in vitro (IC50) demonstrated that these molecular models were effective for the development of novel potent thrombin inhibitors. PMID:26690913

  19. Core-shell nanostructures for ultrasensitive detection of α-thrombin

    NASA Astrophysics Data System (ADS)

    Chen, Xia; Liu, Hongli; Zhou, Xiaodong; Hu, Jiming

    2010-12-01

    We have synthesized a stable, sensitive and specific surface-enhanced Raman tag, and demonstrated its application in human α-thrombin detection. The tag consists of aptamer-modified core-shell nanoparticles with hydrophobic Au@Ag as core and silica as shell encapsulating Raman active molecules. By taking advantage of the Raman signal enhancement effect by metallic nanostructures, high stability and robustness of glass-coated core-shell nanostructures and the recognition capabilities of aptamers, we designed a sandwich detection for protein identification with high selectivity and sensitivity. In this way, we realized the ultrasensitive detection of α-thrombin. GDNs (glass-coated, dye-tagged nanoparticles), which were conjugated with oligonucleotides or antibodies, were extremely soluble in water, and had mechanical and chemical stability, easily controllable-size distribution and friendly biocompatibility. Specifically, the glass coating renders the particles amenable to use in many solvents without altering the Raman spectral response and makes agglomeration a nonfactor. All these merits open the door of the real applications in diagnostics or medical investigations in complex biofluids, such as human plasma and serum. Using the aptamer-modified GDNs as Raman tags, we successfully performed the detection of α-thrombin in human plasma. Furthermore, the overall method have been proved effective and selective, and may be implemented for multiplex target analysis simultaneously.

  20. INFLUENCE OF SUBSTRATE-COFACTOR RATIOS ON PARTIALLY PURIFIED INORGANIC PYROPHOSPHATASE ACTIVITY AT ELEVATED TEMPERATURES.

    PubMed

    MATHEMEIER, P F; MORITA, R Y

    1964-12-01

    Mathemeier, Paul F. (Oregon State University, Corvallis), and Richard Y. Morita. Influence of substrate-cofactor ratios on partially purified inorganic pyrophosphatase activity at elevated temperatures. J. Bacteriol. 88:1661-1666. 1964.-Inorganic pyrophosphatase of Bacillus stearothermophilus was studied for optimal substrate-cofactor ratios at 60 to 100 C. Mg(++) was the primary cofactor, and Co(++) resulted in 50% enzyme activity at 60 C. The pH optima differed for the Mg(++) activated and Co(++) activated pyrophosphatase. At 80 C and above, Co(++) replaced Mg(++) as the optimal cofactor in the enzyme reaction. The optimal ratio of pyrophosphate to Mg(++) varied from 2 to 0.25, dependent on enzyme concentration. The optimal pyrophosphate-cobalt ratio was constant at 1.0. The enzyme catalyzed appreciable pyrophosphate hydrolysis at 95 C. PMID:14240954

  1. Direct characterization of factor VIII in plasma: detection of a mutation altering a thrombin cleavage site (arginine-372----histidine).

    PubMed Central

    Arai, M; Inaba, H; Higuchi, M; Antonarakis, S E; Kazazian, H H; Fujimaki, M; Hoyer, L W

    1989-01-01

    An immunoadsorbent method has been developed for the direct analysis of normal and variant plasma factor VIII. Using this method, the molecular defect responsible for mild hemophilia A has been identified for a patient whose plasma factor VIII activity is 0.05 unit/ml, even though the factor VIII antigen content is 3.25 units/ml. Although the variant factor VIII has an apparently normal molecular mass and chain composition, the 92-kDa heavy chain accumulates when the variant protein is incubated with thrombin and the 44-kDa heavy chain fragment cannot be detected. In contrast, thrombin cleavage of the 80-kDa light chain to the 72-kDa fragment is normal. As these data indicate a loss of factor VIII cleavage by thrombin at arginine-372, the genetic defect was determined by polymerase-chain-reaction amplification of exon 8 of the factor VIII gene and direct sequencing of the amplified product. A single-base substitution (guanine----adenine) was identified that produces an arginine to histidine substitution at amino acid residue 372. These data identify the molecular basis of an abnormal factor VIII, "factor VIII-Kumamoto," that lacks procoagulant function because of impaired thrombin activation. Images PMID:2498882

  2. Nitrogenase Activity Is Affected by Reduced Partial Pressures of N2 and NO3- 1.

    PubMed Central

    Blumenthal, J. M.; Russelle, M. P.; Vance, C. P.

    1997-01-01

    Optimal use of legumes in cropping systems requires a thorough understanding of the interaction between inorganic N nutrition and symbiotic N2 fixation. Our objective was to test the hypothesis that increased NO3- uptake by alfalfa (Medicago sativa L.) would compensate for lower N2 fixation caused by low partial pressure of N2. Root systems of hydroponically grown alfalfa at 2 mg L-1 NO3--N were exposed to (a) 80% N2, (b) 7% N2, (c) 2% N2, or (d) 0% N2. Exposure to reduced partial pressures of N2 reduced total nitrogenase activity (TNA, measured as H2 production in 20% O2 and 80% Ar) by 40% within less than 30 min, followed by a recovery period over the next 30 min to initial activity. Five hours after treatments began, the TNA of plants exposed to 7 and 2% N2 was substantially higher than pretreatment activities, whereas the TNA of plants exposed either to 0 or 80% N2 did not differ from pretreatment values. The decline in TNA due to NO3- exposure over 4 d was not affected by reduced partial pressure of N2. During the 1st h the proportion of electrons used for the reduction of N2 fell from 0.52 to 0.23 for plants exposed to 7% N2, and to 0.09 for plants exposed to 2% N2, and remained unchanged for the rest of the experiment. Although the hypothesis that alfalfa compensated with increased NO3- uptake for lower N2 fixation was not validated by our results, we unexpectedly demonstrated that the decline in TNA upon exposure to NO3- was independent of the N2-fixing efficiency (i.e. the amount of N2 reduced by nitrogenase) of the symbiosis. PMID:12223779

  3. Rational design and characterization of D-Phe-Pro-D-Arg-derived direct thrombin inhibitors.

    PubMed

    Figueiredo, Ana C; Clement, Cristina C; Zakia, Sheuli; Gingold, Julian; Philipp, Manfred; Pereira, Pedro J B

    2012-01-01

    The tremendous social and economic impact of thrombotic disorders, together with the considerable risks associated to the currently available therapies, prompt for the development of more efficient and safer anticoagulants. Novel peptide-based thrombin inhibitors were identified using in silico structure-based design and further validated in vitro. The best candidate compounds contained both L- and D-amino acids, with the general sequence D-Phe(P3)-Pro(P2)-D-Arg(P1)-P1'-CONH₂. The P1' position was scanned with L- and D-isomers of natural or unnatural amino acids, covering the major chemical classes. The most potent non-covalent and proteolysis-resistant inhibitors contain small hydrophobic or polar amino acids (Gly, Ala, Ser, Cys, Thr) at the P1' position. The lead tetrapeptide, D-Phe-Pro-D-Arg-D-Thr-CONH₂, competitively inhibits α-thrombin's cleavage of the S2238 chromogenic substrate with a K(i) of 0.92 µM. In order to understand the molecular details of their inhibitory action, the three-dimensional structure of three peptides (with P1' L-isoleucine (fPrI), L-cysteine (fPrC) or D-threonine (fPrt)) in complex with human α-thrombin were determined by X-ray crystallography. All the inhibitors bind in a substrate-like orientation to the active site of the enzyme. The contacts established between the D-Arg residue in position P1 and thrombin are similar to those observed for the L-isomer in other substrates and inhibitors. However, fPrC and fPrt disrupt the active site His57-Ser195 hydrogen bond, while the combination of a P1 D-Arg and a bulkier P1' residue in fPrI induce an unfavorable geometry for the nucleophilic attack of the scissile bond by the catalytic serine. The experimental models explain the observed relative potency of the inhibitors, as well as their stability to proteolysis. Moreover, the newly identified direct thrombin inhibitors provide a novel pharmacophore platform for developing antithrombotic agents by exploring the conformational

  4. Partially Hydrogenated Graphene Materials Exhibit High Electrocatalytic Activities Related to Unintentional Doping with Metallic Impurities.

    PubMed

    Jankovský, Ondřej; Libánská, Alena; Bouša, Daniel; Sedmidubský, David; Matějková, Stanislava; Sofer, Zdeněk

    2016-06-13

    Partially hydrogenated graphene materials, synthesized by the chemical reduction/hydrogenation of two different graphene oxides using zinc powder in acidic environment or aluminum powder in alkaline environment, exhibit high electrocatalytic activities, as well as electrochemical sensing properties. The starting graphene oxides and the resultant hydrogenated graphenes were characterized in detail. Their electrocatalytic activity was examined in the oxygen reduction reaction, whereas sensing properties towards explosives were tested by using picric acid as a redox probe. Findings indicate that the high electrocatalytic performance originates not only from the hydrogenation of graphene, but also from unintentional contamination of graphene with manganese and other metals during synthesis. A careful evaluation of the obtained data and a detailed chemical analysis are necessary to identify the origin of this anomalous electrocatalytic activity. PMID:27167069

  5. A 2D-DIGE-based proteomic analysis reveals differences in the platelet releasate composition when comparing thrombin and collagen stimulations

    PubMed Central

    Vélez, Paula; Izquierdo, Irene; Rosa, Isaac; García, Ángel

    2015-01-01

    Upon stimulation, platelets release a high number of proteins (the releasate). There are clear indications that these proteins are involved in the pathogenesis of several diseases, such as atherosclerosis. In the present study we compared the platelet releasate following platelet activation with two major endogenous agonists: thrombin and collagen. Proteome analysis was based on 2D-DIGE and LC-MS/MS. Firstly, we showed the primary role of thrombin and collagen receptors in platelet secretion by these agonists; moreover, we demonstrated that GPVI is the primary responsible for collagen-induced platelet activation/aggregation. Proteomic analysis allowed the detection of 122 protein spots differentially regulated between both conditions. After excluding fibrinogen spots, down-regulated in the releasate of thrombin-activated platelets, 84 differences remained. From those, we successfully identified 42, corresponding to 37 open-reading frames. Many of the differences identified correspond to post-translational modifications, primarily, proteolysis induced by thrombin. Among others, we show vitamin K-dependent protein S, an anticoagulant plasma protein, is up-regulated in thrombin samples. Our results could have pathological implications given that platelets might be playing a differential role in various diseases and biological processes through the secretion of different subsets of granule proteins and microvesicles following a predominant activation of certain receptors. PMID:25645904

  6. A Novel Photoelectrochemical Biosensor for Tyrosinase and Thrombin Detection

    PubMed Central

    Chen, Jiexia; Liu, Yifan; Zhao, Guang-Chao

    2016-01-01

    A novel photoelectrochemical biosensor for step-by-step assay of tyrosinase and thrombin was fabricated based on the specific interactions between the designed peptide and the target enzymes. A peptide chain with a special sequence which contains a positively charged lysine-labeled terminal, tyrosine at the other end and a cleavage site recognized by thrombin between them was designed. The designed peptide can be fixed on surface of the CdTe quantum dots (QDs)-modified indium-tin oxide (ITO) electrode through electrostatic attraction to construct the photoelectrochemical biosensor. The tyrosinase target can catalyze the oxidization of tyrosine by oxygen into ortho-benzoquinone residues, which results in a decrease in the sensor photocurrent. Subsequently, the cleavage site could be recognized and cut off by another thrombin target, restoring the sensor photocurrent. The decrease or increase of photocurrent in the sensor enables us to assay tyrosinase and thrombin. Thus, the detection of tyrosinase and thrombin can be achieved in the linear range from 2.6 to 32 μg/mL and from 4.5 to 100 μg/mL with detection limits of 1.5 μg/mL and 1.9 μg/mL, respectively. Most importantly, this strategy shall allow us to detect different classes of enzymes simultaneously by designing various enzyme-specific peptide substrates. PMID:26805846

  7. A Novel Photoelectrochemical Biosensor for Tyrosinase and Thrombin Detection.

    PubMed

    Chen, Jiexia; Liu, Yifan; Zhao, Guang-Chao

    2016-01-01

    A novel photoelectrochemical biosensor for step-by-step assay of tyrosinase and thrombin was fabricated based on the specific interactions between the designed peptide and the target enzymes. A peptide chain with a special sequence which contains a positively charged lysine-labeled terminal, tyrosine at the other end and a cleavage site recognized by thrombin between them was designed. The designed peptide can be fixed on surface of the CdTe quantum dots (QDs)-modified indium-tin oxide (ITO) electrode through electrostatic attraction to construct the photoelectrochemical biosensor. The tyrosinase target can catalyze the oxidization of tyrosine by oxygen into ortho-benzoquinone residues, which results in a decrease in the sensor photocurrent. Subsequently, the cleavage site could be recognized and cut off by another thrombin target, restoring the sensor photocurrent. The decrease or increase of photocurrent in the sensor enables us to assay tyrosinase and thrombin. Thus, the detection of tyrosinase and thrombin can be achieved in the linear range from 2.6 to 32 μg/mL and from 4.5 to 100 μg/mL with detection limits of 1.5 μg/mL and 1.9 μg/mL, respectively. Most importantly, this strategy shall allow us to detect different classes of enzymes simultaneously by designing various enzyme-specific peptide substrates. PMID:26805846

  8. Medium Chain Fatty Acids Are Selective Peroxisome Proliferator Activated Receptor (PPAR) γ Activators and Pan-PPAR Partial Agonists

    PubMed Central

    Ayers, Steven D.; Lin, Jean Z.; Cvoro, Aleksandra; Silveira, Rodrigo L.; Martínez, Leandro; Souza, Paulo C. T.; Saidemberg, Daniel; Deng, Tuo; Amato, Angela Angelica; Togashi, Marie; Hsueh, Willa A.; Phillips, Kevin; Palma, Mário Sérgio; Neves, Francisco A. R.; Skaf, Munir S.; Webb, Paul; Polikarpov, Igor

    2012-01-01

    Thiazolidinediones (TZDs) act through peroxisome proliferator activated receptor (PPAR) γ to increase insulin sensitivity in type 2 diabetes (T2DM), but deleterious effects of these ligands mean that selective modulators with improved clinical profiles are needed. We obtained a crystal structure of PPARγ ligand binding domain (LBD) and found that the ligand binding pocket (LBP) is occupied by bacterial medium chain fatty acids (MCFAs). We verified that MCFAs (C8–C10) bind the PPARγ LBD in vitro and showed that they are low-potency partial agonists that display assay-specific actions relative to TZDs; they act as very weak partial agonists in transfections with PPARγ LBD, stronger partial agonists with full length PPARγ and exhibit full blockade of PPARγ phosphorylation by cyclin-dependent kinase 5 (cdk5), linked to reversal of adipose tissue insulin resistance. MCFAs that bind PPARγ also antagonize TZD-dependent adipogenesis in vitro. X-ray structure B-factor analysis and molecular dynamics (MD) simulations suggest that MCFAs weakly stabilize C-terminal activation helix (H) 12 relative to TZDs and this effect is highly dependent on chain length. By contrast, MCFAs preferentially stabilize the H2-H3/β-sheet region and the helix (H) 11-H12 loop relative to TZDs and we propose that MCFA assay-specific actions are linked to their unique binding mode and suggest that it may be possible to identify selective PPARγ modulators with useful clinical profiles among natural products. PMID:22649490

  9. Mechanism of poly(acrylic acid) acceleration of antithrombin inhibition of thrombin: implications for the design of novel heparin mimics.

    PubMed

    Monien, Bernhard H; Cheang, Kai I; Desai, Umesh R

    2005-08-11

    The bridging mechanism of antithrombin inhibition of thrombin is a dominant mechanism contributing a massive approximately 2500-fold acceleration in the reaction rate and is also a key reason for the clinical usage of heparin. Our recent study of the antithrombin-activating properties of a carboxylic acid-based polymer, poly(acrylic acid) (PAA), demonstrated a surprisingly high acceleration in thrombin inhibition (Monien, B. H.; Desai, U. R. J. Med. Chem. 2005, 48, 1269). To better understand this interesting phenomenon, we have studied the mechanism of PAA-dependent acceleration in antithrombin inhibition of thrombin. Competitive binding studies with low-affinity heparin and a heparin tetrasaccharide suggest that PAA binds antithrombin in both the pentasaccharide- and the extended heparin-binding sites, and these results are corroborated by molecular modeling. The salt-dependence of the K(D) of the PAA-antithrombin interaction shows the formation of five ionic interactions. In contrast, the contribution of nonionic forces is miniscule, resulting in an interaction that is significantly weaker than that observed for heparins. A bell-shaped profile of the observed rate constant for antithrombin inhibition of thrombin as a function of PAA concentration was observed, suggesting that inhibition proceeds through the "bridging" mechanism. The knowledge gained in this mechanistic study highlights important rules for the rational design of orally available heparin mimics. PMID:16078853

  10. Scanning electrochemical microscopy for study of aptamer-thrombin interfacial interactions on gold disk microelectrodes.

    PubMed

    Bai, Huei-Yu; del Campo, F Javier; Tsai, Yu-Chen

    2014-03-01

    A feasibility for the determination of thrombin on gold disk microelectrodes (GDMs) using scanning electrochemical microscopy (SECM) is reported. The assembly process step-by-step of thrombin aptasensor on GDMs is monitored by SECM. SECM analysis reveals the immobilization of thrombin aptamers on GDMs. The interaction between thrombin aptamers and thrombin on GDMs is imaged by SECM with feedback mode using ferrocenemethanol as an electrochemical mediator. The formation of thrombin/thrombin aptamer complex on GDMs results in a decrease in the tip peak current on spatial SECM images. This method is able to linearly and selectively detect thrombin over a linear range from 10(-12) to 10(-5)M with a detection limit of 6.07 fM. PMID:24407695

  11. trans-Resveratrol inhibits calcium influx in thrombin-stimulated human platelets

    PubMed Central

    Dobrydneva, Yuliya; Williams, Roy L; Blackmore, Peter F

    1999-01-01

    The phytoestrogenic compound trans-resveratrol (trans-3,5,4′-trihydroxystilbene) is found in appreciable quantities in grape skins and wine. It has been shown that both products rich in trans-resveratrol and pure trans-resveratrol inhibit platelet aggregation both in vivo and in vitro. However the mechanism of this action still remains unknown. An essential component of the aggregation process in platelets is an increase in intracellular free Ca2+ ([Ca2+]i). Ca2+ must enter the cell from the external media through specific and tightly regulated Ca2+ channels in the plasma membrane. The objective of this study was to characterize what effect trans-resveratrol had on the Ca2+ channels in thrombin stimulated platelets. In this study we showed that trans-resveratrol immediately inhibited Ca2+ influx in thrombin-stimulated platelets with an IC50 of 0.5 μM. trans-Resveratrol at 0.1, 1.0 and 10.0 μM produced 20±6, 37±6 and 57±4% inhibition respectively of the effect of thrombin (0.01 u  ml−1) to increase [Ca2+]i. trans-Resveratrol also inhibited spontaneous Ba2+ entry into Fura-2 loaded platelets, with 0.1, 1.0 and 10.0 μM trans-resveratrol producing 10±5, 30±5 and 50±7% inhibition respectively. This indicated that trans-resveratrol directly inhibited Ca2+ channel activity in the platelets in the absence of agonist stimulation. trans-Resveratrol also inhibited thapsigargin-mediated Ca2+ influx into platelets. This suggests that the store-operated Ca2+ channels are one of the possible targets of trans-resveratrol. These channels rely on the emptying of the internal Ca2+ stores to initiate influx of Ca2+ into the cell. The phytoestrogens genistein, daidzein, apigenin and genistein-glucoside (genistin) produced inhibitory effects against thrombin similar to those seen with trans-resveratrol. We conclude that trans-resveratrol is an inhibitor of store-operated Ca2+ channels in human platelets. This accounts for the ability of trans-resveratrol to

  12. Antitcoagulant and antiplatelet activities of scolymoside

    PubMed Central

    Yoon, Eun-Kyung; Ku, Sae-Kwang; Lee, Wonhwa; Kwak, Soyoung; Kang, Hyejin; Jung, Byeongjin; Bae, Jong-Sup

    2015-01-01

    Cyclopia subternata is a medicinal plant commonly used in traditional medicine to relieve pain. Here, the anticoagulant effects of scolymoside, an active compound in C. subternata, were examined by monitoring activated partial thromboplastin time (aPTT), prothrombin time (PT), and the activities of thrombin and activated factor X (FXa). The effects of scolymoside on plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) expression were evaluated in tumor necrosis factor (TNF)-α-activated human endothelial cells. Treatment with scolymoside resulted in prolonged aPTT and PT and the inhibition of thrombin and FXa activities and production. In addition, scolymoside inhibited thrombin-catalyzed fibrin polymerization and platelet aggregation. Scolymoside also elicited anticoagulant effects in mice, including a significant reduction in the PAI-1 to t-PA ratio. Collectively, these findings indicate that scolymoside possesses anticoagulant activities and could be developed as a novel anticoagulant. [BMB Reports 2015; 48(10): 577-582] PMID:25887749

  13. Development and Optimization of a Thrombin Sandwich Aptamer Microarray

    PubMed Central

    Meneghello, Anna; Sosic, Alice; Antognoli, Agnese; Cretaio, Erica; Gatto, Barbara

    2012-01-01

    A sandwich microarray employing two distinct aptamers for human thrombin has been optimized for the detection of subnanomolar concentrations of the protein. The aptamer microarray demonstrates high specificity for thrombin, proving that a two-site binding assay with the TBA1 aptamer as capture layer and the TBA2 aptamer as detection layer can ensure great specificity at times and conditions compatible with standard routine analysis of biological samples. Aptamer microarray sensitivity was evaluated directly by fluorescent analysis employing Cy5-labeled TBA2 and indirectly by the use of TBA2-biotin followed by detection with fluorescent streptavidin. Sub-nanomolar LODs were reached in all cases and in the presence of serum, demonstrating that the optimized aptamer microarray can identify thrombin by a low-cost, sensitive and specific method.

  14. Deformation of partially pumped active mirrors for high average-power diode-pumped solid-state lasers.

    PubMed

    Albach, Daniel; LeTouzé, Geoffroy; Chanteloup, Jean-Christophe

    2011-04-25

    We discuss the deformation of a partially pumped active mirror amplifier as a free standing disk, as implemented in several laser systems. We rely on the Lucia laser project to experimentally evaluate the analytical and numerical deformation models. PMID:21643092

  15. Participation of the hypophyseal-adrenal cortex system in thrombin clearance during immobilization stress

    NASA Technical Reports Server (NTRS)

    Kudryashov, B. A.; Uljanov, A. M.; Shapiro, F. B.; Bazazyan, G. G.

    1981-01-01

    Thrombin marked with I-131 resulted in a considerable increase of the thrombined clearance rate in healthy male rats during stress caused by an immobilization lasting 30 minutes, and in an increase of thrombin clearance occurred by a combination of immobilization and administration of adrenocorticotropin (ACTH). Contrary to ACTH, the thrombin clearance is not stimulated in healthy animals by hydrocortisone. The results of the examination are presented.

  16. Anticoagulation with the oral direct thrombin inhibitor dabigatran does not enlarge hematoma volume in experimental intracerebral hemorrhage

    PubMed Central

    Lauer, Arne; Cianchetti, Flor A.; Van Cott, Elizabeth M.; Schlunk, Frieder; Schulz, Elena; Pfeilschifter, Waltraud; Steinmetz, Helmuth; Schaffer, Chris B.; Lo, Eng H.; Foerch, Christian

    2013-01-01

    Background The direct thrombin inhibitor dabigatran etexilate (DE) may constitute a future replacement of vitamin K antagonists for long-term anticoagulation. Whereas warfarin pre-treatment is associated with greater hematoma expansion following intracerebral hemorrhage (ICH), it remains unclear what effect direct thrombin inhibitors would have. Using different experimental models of ICH, this study compared hematoma volume between DE treated mice, warfarin treated mice and controls. Methods and Results CD-1 mice were fed with DE or warfarin. Sham-treated mice served as controls. At the time point of ICH induction, DE mice revealed an increased activated partial thromboplastin time as compared to controls (46.1±5.0 vs. 18.0±1.5sec; p=0.022), whereas warfarin pre-treatment resulted in a prothrombin time prolongation (51.4±17.9 vs. 10.4±0.3sec; p<0.001). Twenty-four hours after collagenase-induced ICH formation, hematoma volume was 3.8±2.9μL in controls, 4.8±2.7μL in DE mice, and 14.5±11.8μL in warfarin mice (n=16; Welch's ANOVA between group differences p=0.007, post-hoc analysis with Dunnett's method: DE vs. controls, p=0.899; warfarin vs. controls, p<0.001; DE vs. warfarin, p=0.001). In addition, a model of laser-induced cerebral microhemorrhage was applied, and the distances which red blood cells and blood plasma were pushed into the brain were quantified. Warfarin mice showed enlarged red blood cell- and blood plasma diameters as compared to controls, but no difference was found between DE mice and controls. Conclusions In contrast to warfarin, pretreatment with DE did not increase hematoma volume in two different experimental models of ICH. In terms of safety, this observation may represent a potential advantage of anticoagulation with DE over warfarin. PMID:21911784

  17. Vitamin K deficiency amplifies anticoagulation response to ximelagatran: possible implications for direct thrombin inhibitors and their clinical safety.

    PubMed

    Kamali, Farhad; Wood, Peter; Ward, Alan

    2009-02-01

    Dietary vitamin K is known to influence the anticoagulation response to warfarin. It is possible that dietary vitamin K availability also influences the pharmacological activity of other oral anticoagulants, which target the vitamin-K dependent clotting proteins in the coagulation cascade. This study examined whether vitamin K insufficiency affected anticoagulation response to the direct thrombin inhibitor, ximelagatran. Anticoagulation response to ximelagatran and warfarin in rats on a normal diet was compared to those on a vitamin K deficient diet. Ximelagatran and warfarin increased prothrombin time (PT) by 1.4- and 1.3-fold, activated partial thromboplastin time (APTT) by 1.8- and 1.4-fold and ecarin clotting time (ECT) by 6.8- and 1.2-fold, respectively, in rats on normal diet. Vitamin K deficient diet alone caused modest increases in PT, APTT and ECT. The anticoagulant activity of both ximelagatran and warfarin was significantly greater in rats on vitamin K deficient diet (6.1- and 12.3-fold for PT, 2.6- and 5.1-fold for APTT and 2.9- and 1.6-fold for ECT, respectively) compared to those on normal diet. Factor II activity was reduced by both ximelagatran (58%) and warfarin (44%) in rats on normal diet. However, factor II activity was virtually abolished (<0.1%) by both drugs in rats on vitamin K deficient diet. The results suggest that oral anticoagulant drugs, whose primary site of action is not within the vitamin K cycle, may also exhibit variability in clinical response due to dietary variation as the established coumarin drugs such as warfarin. PMID:18716776

  18. Elucidation of Nonadditive Effects in Protein-Ligand Binding Energies: Thrombin as a Case Study.

    PubMed

    Calabrò, Gaetano; Woods, Christopher J; Powlesland, Francis; Mey, Antonia S J S; Mulholland, Adrian J; Michel, Julien

    2016-06-23

    Accurate predictions of free energies of binding of ligands to proteins are challenging partly because of the nonadditivity of protein-ligand interactions; i.e., the free energy of binding is the sum of numerous enthalpic and entropic contributions that cannot be separated into functional group contributions. In principle, molecular simulations methodologies that compute free energies of binding do capture nonadditivity of protein-ligand interactions, but efficient protocols are necessary to compute well-converged free energies of binding that clearly resolve nonadditive effects. To this end, an efficient GPU-accelerated implementation of alchemical free energy calculations has been developed and applied to two congeneric series of ligands of the enzyme thrombin. The results show that accurate binding affinities are computed across the two congeneric series and positive coupling between nonpolar R(1) substituents and a X = NH3(+) substituent is reproduced, albeit with a weaker trend than experimentally observed. By contrast, a docking methodology completely fails to capture nonadditive effects. Further analysis shows that the nonadditive effects are partly due to variations in the strength of a hydrogen-bond between the X = NH3(+) ligands family and thrombin residue Gly216. However, other partially compensating interactions occur across the entire binding site, and no single interaction dictates the magnitude of the nonadditive effects for all the analyzed protein-ligand complexes. PMID:27248478

  19. Factor XIa and Thrombin Generation Are Elevated in Patients with Acute Coronary Syndrome and Predict Recurrent Cardiovascular Events

    PubMed Central

    Loeffen, Rinske; van Oerle, René; Leers, Mathie P. G.; Kragten, Johannes A.; Crijns, Harry; Spronk, Henri M. H.; ten Cate, Hugo

    2016-01-01

    Objective In acute coronary syndrome (ACS) cardiac cell damage is preceded by thrombosis. Therefore, plasma coagulation markers may have additional diagnostic relevance in ACS. By using novel coagulation assays this study aims to gain more insight into the relationship between the coagulation system and ACS. Methods We measured plasma thrombin generation, factor XIa and D-dimer levels in plasma from ACS (n = 104) and non-ACS patients (n = 42). Follow-up measurements (n = 73) were performed at 1 and 6 months. Associations between coagulation markers and recurrent cardiovascular events were calculated by logistic regression analysis. Results Thrombin generation was significantly enhanced in ACS compared to non-ACS patients: peak height 148±53 vs. 122±42 nM. There was a significantly diminished ETP reduction (32 vs. 41%) and increased intrinsic coagulation activation (25 vs. 7%) in ACS compared to non-ACS patients. Furthermore, compared to non-ACS patients factor XIa and D-dimer levels were significantly elevated in ACS patients: 1.9±1.1 vs. 1.4±0.7 pM and 495(310–885) vs. 380(235–540) μg/L. Within the ACS spectrum, ST-elevated myocardial infarction patients had the highest prothrombotic profile. During the acute event, thrombin generation was significantly increased compared to 1 and 6 months afterwards: peak height 145±52 vs. 100±44 vs. 98±33 nM. Both peak height and factor XIa levels on admission predicted recurrent cardiovascular events (OR: 4.9 [95%CI 1.2–20.9] and 4.5 [1.1–18.9]). Conclusion ACS patients had an enhanced prothrombotic profile, demonstrated by an increased thrombin generation potential, factor XIa and D-dimer levels. This study is the first to demonstrate the positive association between factor XIa, thrombin generation and recurrent cardiovascular events. PMID:27419389

  20. Thrombin-cleaved Fragments of Osteopontin Are Overexpressed in Malignant Glial Tumors and Provide a Molecular Niche with Survival Advantage*

    PubMed Central

    Yamaguchi, Yasuto; Shao, Zhifei; Sharif, Shadi; Du, Xiao-Yan; Myles, Timothy; Merchant, Milton; Harsh, Griffith; Glantz, Michael; Recht, Lawrence; Morser, John; Leung, Lawrence L. K.

    2013-01-01

    Osteopontin (OPN), which is highly expressed in malignant glioblastoma (GBM), possesses inflammatory activity modulated by proteolytic cleavage by thrombin and plasma carboxypeptidase B2 (CPB2) at a highly conserved cleavage site. Full-length OPN (OPN-FL) was elevated in cerebrospinal fluid (CSF) samples from all cancer patients compared with noncancer patients. However, thrombin-cleaved OPN (OPN-R) and thrombin/CPB2-double-cleaved OPN (OPN-L) levels were markedly increased in GBM and non-GBM gliomas compared with systemic cancer and noncancer patients. Cleaved OPN constituted ∼23 and ∼31% of the total OPN in the GBM and non-GBM CSF samples, respectively. OPN-R was also elevated in GBM tissues. Thrombin-antithrombin levels were highly correlated with cleaved OPN, but not OPN-FL, suggesting that the cleaved OPN fragments resulted from increased thrombin and CPB2 in this extracellular compartment. Levels of VEGF and CCL4 were increased in CSF of GBM and correlated with the levels of cleaved OPN. GBM cell lines were more adherent to OPN-R and OPN-L than OPN-FL. Adhesion to OPN altered gene expression, in particular genes involved with cellular processes, cell cycle regulation, death, and inflammation. OPN and its cleaved forms promoted motility of U-87 MG cells and conferred resistance to apoptosis. Although functional mutation of the RGD motif in OPN largely abolished these functions, OPNRAA-R regained significant cell binding and signaling function, suggesting that the SVVYGLR motif in OPN-R may substitute for the RGD motif if the latter becomes inaccessible. OPN cleavage contributes to GBM development by allowing more cells to bind in niches where they acquire anti-apoptotic properties. PMID:23204518

  1. Thrombin enhances the adhesion and migration of human colon adenocarcinoma cells via increased beta 3-integrin expression on the tumour cell surface and their inhibition by the snake venom peptide, rhodostomin.

    PubMed Central

    Chiang, H. S.; Yang, R. S.; Huang, T. F.

    1996-01-01

    The interactions between tumour cells and the microvasculature, including the adhesion of tumour cells to endothelium and extracellular matrix (ECM) as well as their migratory ability, are prerequisites for metastasis to occur. In this study we showed that thrombin is capable of enhancing in vitro tumour cell metastatic potential in terms of adhesive properties and migratory response. Following exposure to subclotting concentrations of thrombin, SW-480 human colon adenocarcinoma cells exhibited increased adhesion to both the endothelium and ECM component (i.e. fibronectin). Likewise, the pretreatment of thrombin enhanced the migratory ability of SW-480 cells. The enhanced adhesion was significantly inhibited by complexing of thrombin with its inhibitor hirudin, or by serine proteinase inhibition with 3,4-DCI, but was unaffected by pretreatment of tumour cells with actinomycin D or cycloheximide. The effect of thrombin resulted in an upregulated cell-surface expression of beta 3 integrins, a group of receptors mediating interactions between tumour cells and endothelial cells, and between tumour cells and ECM. Antibodies against beta 3 integrins effectively blocked both the enhanced adhesion and migration. This thrombin-mediated up-regulation of beta 3 integrins involved the activation of protein kinase C (PKC) as thrombin-enhanced adhesion was diminished by PKC inhibition. Rhodostomin, an Arg-Gly-Asp-containing antiplatelet snake venom peptide that antagonises the binding of ECM toward beta 3 integrins on SW-480 cells, was about 600 and 500 times, more potent that RGDS in inhibiting thrombin-enhanced adhesion and migration respectively. Our data suggest that PKC inhibitors as well as rhodostomin may serve as inhibitory agents in the prevention of thrombin-enhanced metastasis. PMID:8611404

  2. Cerebral blood flow during paroxysmal EEG activation induced by sleep in patients with complex partial seizures

    SciTech Connect

    Gozukirmizi, E.; Meyer, J.S.; Okabe, T.; Amano, T.; Mortel, K.; Karacan, I.

    1982-01-01

    Cerebral blood flow (CBF) measurements were combined with sleep polysomnography in nine patients with complex partial seizures. Two methods were used: the 133Xe method for measuring regional (rCBF) and the stable xenon CT method for local (LCBF). Compared to nonepileptic subjects, who show diffuse CBF decreases during stages I-II, non-REM sleep onset, patients with complex partial seizures show statistically significant increases in CBF which are maximal in regions where the EEG focus is localized and are predominantly seen in one temporal region but are also propagated to other cerebral areas. Both CBF methods gave comparable results, but greater statistical significance was achieved by stable xenon CT methodology. CBF increases are more diffuse than predicted by EEG paroxysmal activity recorded from scalp electrodes. An advantage of the 133Xe inhalation method was achievement of reliable data despite movement of the head. This was attributed to the use of a helmet which maintained the probes approximated to the scalp. Disadvantages were poor resolution (7 cm3) and two-dimensional information. The advantage of stable xenon CT method is excellent resolution (80 mm3) in three dimensions, but a disadvantage is that movement of the head in patients with seizure disorders may limit satisfactory measurements.

  3. Allosteric activation of the Par-6 PDZ via a partial unfolding transition

    PubMed Central

    Whitney, Dustin S.; Peterson, Francis C.; Kovrigin, Evgenii L.; Volkman, Brian F.

    2013-01-01

    Proteins exist in a delicate balance between the native and unfolded states, where thermodynamic stability may be sacrificed to attain the flexibility required for efficient catalysis, binding or allosteric control. Par-6 regulates the Par polarity complex by transmitting a GTPase signal through the CRIB-PDZ module that alters PDZ lig-and binding. Allosteric activation of the PDZ is achieved by local rearrangement of the L164 and K165 side chains to stabilize the interdomain CRIB-PDZ interface and reposition a conserved element of the ligand binding pocket. However, microsecond to millisecond dynamics measurements revealed that L164/K165 exchange requires a larger rearrangement than expected. The margin of thermodynamic stability for the PDZ domain is modest (~3 kcal/mol) and further reduced by transient interactions with the disordered CRIB domain. Measurements of local structural stability revealed that tertiary contacts within the PDZ are disrupted by a partial unfolding transition that enables interconversion of the L/K switch. The unexpected participation of partial PDZ unfolding in the allosteric mechanism of Par-6 suggests that native-state unfolding may be essential for the function of other marginally stable proteins. PMID:23705660

  4. Cerebral blood flow during paroxysmal EEG activation induced by sleep in patients with complex partial seizures.

    PubMed

    Gozukirmizi, E; Meyer, J S; Okabe, T; Amano, T; Mortel, K; Karacan, I

    1982-01-01

    Cerebral blood flow (CBF) measurements were combined with sleep polysomnography in nine patients with complex partial seizures. Two methods were used: the 133Xe method for measuring regional (rCBF) and the stable xenon CT method for local (LCBF). Compared to nonepileptic subjects, who show diffuse CBF decreases during stages I-II, non-REM sleep onset, patients with complex partial seizures show statistically significant increases in CBF which are maximal in regions where the EEG focus is localized and are predominantly seen in one temporal region but are also propagated to other cerebral areas. Both CBF methods gave comparable results, but greater statistical significance was achieved by stable xenon CT methodology. CBF increases are more diffuse than predicted by EEG paroxysmal activity recorded from scalp electrodes. An advantage of the 133Xe inhalation method was achievement of reliable data despite movement of the head. This was attributed to the use of a helmet which maintained the probes approximated to the scalp. Disadvantages were poor resolution (7 cm3) and two-dimensional information. The advantage of stable xenon CT method is excellent resolution (80 mm3) in three dimensions, but a disadvantage is that movement of the head in patients with seizure disorders may limit satisfactory measurements. PMID:7163722

  5. The structure-function relationship of thrombin-like enzymes from the green pit viper (Trimeresurus albolabris).

    PubMed

    Pradniwat, Paweena; Rojnuckarin, Ponlapat

    2015-06-15

    Pit viper venoms can decrease fibrinogen levels in snakebite patients. Studies have shown that the hypofibrinogenemia is a consequence of snake venom thrombin-like enzymes (TLEs), the serine proteases that have the potential to be both diagnostic and therapeutic agents. Exosites of thrombin are the molecular regions that determine the substrate specificities, but its presence and significance in TLEs are unclear. Therefore, the putative exosites of recombinant TLEs derived from Green pit viper (Trimeresurus albolabris), GPV-TL1 and GPV-TL2, were mutated in a Pichia pastoris system. In a previous report, GPV-TL1 showed a strong fibrinogenolytic activity on the Aα and Bβ chains of fibrinogen, as well as a plasma clotting activity. Compared with GPV-TL1, the GPV-TL1m mutated in the putative exosite (TRN to RRR at residues 60-62) showed a weaker fibrinogenolytic activity with a similar clotting activity of 207.1 thrombin units/mg. GPV-TL2 contained two-residue differences from GPV-TL1 in the putative exosite (N73M and V74Y). GPV-TL2 selectively cleaved only the Aα chain of fibrinogen without detectable clotting activity. The mutated GPV-TL2 (GPV-TL2m) showed a weaker fibrinogenolytic activity compared with that of the wild type. These results support the important roles of the putative exosite in snake venom TLE activities. This information is helpful for future protein engineering. PMID:25912946

  6. Two Acidic, Anticoagulant PLA2 Isoenzymes Purified from the Venom of Monocled Cobra Naja kaouthia Exhibit Different Potency to Inhibit Thrombin and Factor Xa via Phospholipids Independent, Non-Enzymatic Mechanism

    PubMed Central

    Mukherjee, Ashis K.; Kalita, Bhargab; Thakur, Rupamoni

    2014-01-01

    Background The monocled cobra (Naja kaouthia) is responsible for snakebite fatality in Indian subcontinent and in south-western China. Phospholipase A2 (PLA2; EC 3.1.1.4) is one of the toxic components of snake venom. The present study explores the mechanism and rationale(s) for the differences in anticoagulant potency of two acidic PLA2 isoenzymes, Nk-PLA2α (13463.91 Da) and Nk-PLA2β (13282.38 Da) purified from the venom of N. kaouthia. Principal Findings By LC-MS/MS analysis, these PLA2s showed highest similarity (23.5% sequence coverage) with PLA2 III isolated from monocled cobra venom. The catalytic activity of Nk-PLA2β exceeds that of Nk-PLA2α. Heparin differentially regulated the catalytic and anticoagulant activities of these Nk-PLA2 isoenzymes. The anticoagulant potency of Nk-PLA2α was comparable to commercial anticoagulants warfarin, and heparin/antithrombin-III albeit Nk-PLA2β demonstrated highest anticoagulant activity. The anticoagulant action of these PLA2s was partially contributed by a small but specific hydrolysis of plasma phospholipids. The strong anticoagulant effect of Nk-PLA2α and Nk-PLA2β was achieved via preferential, non-enzymatic inhibition of FXa (Ki = 43 nM) and thrombin (Ki = 8.3 nM), respectively. Kinetics study suggests that the Nk-PLA2 isoenzymes inhibit their “pharmacological target(s)” by uncompetitive mechanism without the requirement of phospholipids/Ca2+. The anticoagulant potency of Nk-PLA2β which is higher than that of Nk-PLA2α is corroborated by its superior catalytic activity, its higher capacity for binding to phosphatidylcholine, and its greater strength of thrombin inhibition. These PLA2 isoenzymes thus have evolved to affect haemostasis by different mechanisms. The Nk-PLA2β partially inhibited the thrombin-induced aggregation of mammalian platelets suggesting its therapeutic application in the prevention of unwanted clot formation. Conclusion/Significance In order to develop peptide-based superior

  7. Phage display of the serpin alpha-1 proteinase inhibitor randomized at consecutive residues in the reactive centre loop and biopanned with or without thrombin.

    PubMed

    Scott, Benjamin M; Matochko, Wadim L; Gierczak, Richard F; Bhakta, Varsha; Derda, Ratmir; Sheffield, William P

    2014-01-01

    In spite of the power of phage display technology to identify variant proteins with novel properties in large libraries, it has only been previously applied to one member of the serpin superfamily. Here we describe phage display of human alpha-1 proteinase inhibitor (API) in a T7 bacteriophage system. API M358R fused to the C-terminus of T7 capsid protein 10B was directly shown to form denaturation-resistant complexes with thrombin by electrophoresis and immunoblotting following exposure of intact phages to thrombin. We therefore developed a biopanning protocol in which thrombin-reactive phages were selected using biotinylated anti-thrombin antibodies and streptavidin-coated magnetic beads. A library consisting of displayed API randomized at residues 357 and 358 (P2-P1) yielded predominantly Pro-Arg at these positions after five rounds of thrombin selection; in contrast the same degree of mock selection yielded only non-functional variants. A more diverse library of API M358R randomized at residues 352-356 (P7-P3) was also probed, yielding numerous variants fitting a loose consensus of DLTVS as judged by sequencing of the inserts of plaque-purified phages. The thrombin-selected sequences were transferred en masse into bacterial expression plasmids, and lysates from individual colonies were screening for API-thrombin complexing. The most active candidates from this sixth round of screening contained DITMA and AAFVS at P7-P3 and inhibited thrombin 2.1-fold more rapidly than API M358R with no change in reaction stoichiometry. Deep sequencing using the Ion Torrent platform confirmed that over 800 sequences were significantly enriched in the thrombin-panned versus naïve phage display library, including some detected using the combined phage display/bacterial lysate screening approach. Our results show that API joins Plasminogen Activator Inhibitor-1 (PAI-1) as a serpin amenable to phage display and suggest the utility of this approach for the selection of "designer

  8. Responsiveness of G protein-coupled odorant receptors is partially attributed to the activation mechanism

    PubMed Central

    Yu, Yiqun; de March, Claire A.; Ni, Mengjue J.; Adipietro, Kaylin A.; Golebiowski, Jérôme; Matsunami, Hiroaki; Ma, Minghong

    2015-01-01

    Mammals detect and discriminate numerous odors via a large family of G protein-coupled odorant receptors (ORs). However, little is known about the molecular and structural basis underlying OR response properties. Using site-directed mutagenesis and computational modeling, we studied ORs sharing high sequence homology but with different response properties. When tested in heterologous cells by diverse odorants, MOR256-3 responded broadly to many odorants, whereas MOR256-8 responded weakly to a few odorants. Out of 36 mutant MOR256-3 ORs, the majority altered the responses to different odorants in a similar manner and the overall response of an OR was positively correlated with its basal activity, an indication of ligand-independent receptor activation. Strikingly, a single mutation in MOR256-8 was sufficient to confer both high basal activity and broad responsiveness to this receptor. These results suggest that broad responsiveness of an OR is at least partially attributed to its activation likelihood. PMID:26627247

  9. Antithrombotic actions of the thrombin inhibitor, argatroban, in a canine model of coronary cyclic flow: comparison with heparin.

    PubMed Central

    Duval, N.; Lunven, C.; O'Brien, D. P.; Grosset, A.; O'Connor, S. E.; Berry, C. N.

    1996-01-01

    1. The antithrombotic action of argatroban, a synthetic thrombin inhibitor, was studied in a canine model of coronary cyclic flow having some of the characteristics of acute unstable angina. Heparin was studied as a reference anticoagulant. 2. Localized endothelial damage was induced in the circumflex coronary artery of anaesthetized open-chest foxhounds and a critical stenosis was applied by use of a Lexan constrictor placed around the artery at the site of endothelial damage. An electro-magnetic flow probe was placed distal to the lesion, and cyclic flow variations (CFVs) were observed, as thrombi formed at the site of the arterial lesion and were dislodged. Test compounds were administered by i.v. infusion commencing 1 h after the appearance of CFVs, and maintained for 1 h. On termination of the treatments, coronary flow was observed for a further 60 min. A series of blood samples were taken at predetermined times throughout each experiment in order to determine the coagulation parameters, thrombin time (TT) activated partial thromboplastin time (aPTT) and for the determination of fibrinopeptide A (FpA) levels before, during and post-treatment. 3. Argatroban and heparin showed antithrombotic effects in this model. Argatroban dose-dependently increased the minimum coronary flow at the nadir of the CFVs from 5.4 +/- 1.7 to 9.1 +/- 2.1 ml min-1 (30 micrograms kg-1 min-1, P = 0.041) and from 2.9 +/- 0.9 to 16.3 +/- 4.5 ml min-1 (100 micrograms kg-1 min-1, P = 0.023, n = 8 dogs at each dose level). Heparin (5 and 15 iu kg-1 min-1) also increased minimum flow, but the increase was not statistically significant at the 5% level, although the P value in animals treated with 15 iu kg-1 min-1 (P = 0.0521, n = 6 dogs) fell just outside this limit. Although neither compound significantly decreased the overall CFV frequency, argatroban (100 micrograms kg-1 min-1) significantly (P < 0.01) decreased the number of large amplitude CFVs (minimum coronary flow < 10 ml min-1) by 63

  10. Label-free electrochemical aptasensor for sensitive thrombin detection using layer-by-layer self-assembled multilayers with toluidine blue-graphene composites and gold nanoparticles.

    PubMed

    Xie, Shunbi; Yuan, Ruo; Chai, Yaqin; Bai, Lijuan; Yuan, Yali; Wang, Yan

    2012-08-30

    In the present study, toluidine blue-graphene (Tb-Gra) nanocomposites were prepared to design a Lable-free electrochemical aptasensor for highly sensitive detection of thrombin based on layer-by-layer (LBL) technology. The nanocomposites with excellent redox electrochemical activities were first immobilized on the gold nanoparticles (nano-Au) modified glassy carbon electrodes (GCE). Then, the LBL structure was performed by electrostatic adsorption between the positively charged Tb-Gra and negatively charged nano-Au, which formed {Tb-Gra/nano-Au}(n) multilayer films for electroactive species enrichment and biomolecule immobilization. Subsequently, the thiolated thrombin binding aptamer (TBA) was assembled on the nano-Au surface through Au-S bond. In the presence of target thrombin (TB), the TBA on the multilayer could catch the thrombin onto the electrode surface, which resulted in a barrier for electro-transfer, leading to the decrease of the electrochemical signal of Tb-Gra nanocomposites. Under the optimal conditions, a wide detection range from 0.001 nM to 80 nM and a low detection limit of 0.33 pM (defined as S/N=3) for thrombin were obtained. In addition, the sensor exhibited excellent selectivity against other proteins. PMID:22939121

  11. Anticoagulant activities of piperlonguminine in vitro and in vivo.

    PubMed

    Lee, Wonhwa; Yoo, Hayoung; Ku, Sae-Kwang; Kim, Jeong Ah; Bae, Jong-Sup

    2013-10-01

    Piperlonguminine (PL), an important component of Piper longum fruits, is known to exhibit anti-hyperlipidemic, anti-platelet and anti-melanogenic activities. Here, the anticoagulant activities of PL were examined by monitoring activated-partial-thromboplastin-time (aPTT), prothrombin-time (PT), and the activities of thrombin and activated factor X (FXa). The effects of PL 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 HUVECs. The results showed that PL prolonged aPTT and PT significantly and inhibited the activities of thrombin and FXa. PL inhibited the generation of thrombin and FXa in HUVECs. In accordance with these anticoagulant activities, PL prolonged in vivo bleeding time and inhibited TNF-α induced PAI-1 production. Furthermore, PAI-1/t-PA ratio was significan- tly decreased by PL. Collectively, our results suggest that PL possesses antithrombotic activities and that the current study could provide bases for the development of new anticoagulant agents. PMID:24148768

  12. Interictal regional cerebral blood flow during non specific activation test in partial epilepsy.

    PubMed Central

    Valmier, J; Touchon, J; Baldy-Moulinier, M

    1989-01-01

    In order to investigate, during activation testing, the interictal cortical cerebral blood flows (rCBF) of epileptic patients suffering from complex partial seizures, 40 epileptic patients (divided into "lesional", this is, with abnormal CT findings, and "non lesional", that is, with normal CT findings) were submitted to rCBF measurements with the 133 xenon intravenous technique, at rest and during intermittent light stimulation (ILS). The findings compared with normal volunteers seem to demonstrate that, during ILS, (1) in non lesional patients, the suspected epileptic focus shows a significant rCBF increase (2) in lesional patients, the significant rCBF increases were not in the region of the suspected epileptic focus but in adjacent or in contralateral ones. It was concluded that activation interictal rCBF measurements are more useful than resting ones for the determination of epileptic foci when CT findings are normal and that the nature of the epileptic focus influences markedly the interhemispheric activation pattern. PMID:2926422

  13. Mex3c mutation reduces adiposity partially through increasing physical activity.

    PubMed

    Han, Changjie; Jiao, Yan; Zhao, Qingguo; Lu, Baisong

    2014-06-01

    MEX3C is an RNA-binding protein with unknown physiological function. We have recently reported that a Mex3c mutation in mice causes growth retardation and reduced adiposity, but how adiposity is reduced remains unclear. Herein, we show that homozygous Mex3c gene trap mice have increased physical activity. The Mex3c mutation consistently conferred full protection from diet-induced obesity, hyperglycemia, insulin resistance, hyperlipidemia, and hepatic steatosis. In ob/ob mice with leptin deficiency, the Mex3c mutation also increased physical activity and improved glucose and lipid profiles. Expressing cre in the neurons of Mex3c gene trap mice, an attempt to partially restoring neuronal Mex3c expression, significantly increased white adipose tissue deposition, but had no effects on body length. Our data suggest that one way in which Mex3c regulates adiposity is through controlling physical activity, and that neuronal Mex3c expression could play an important role in this process. PMID:24741071

  14. Isotherm-Based Thermodynamic Models for Solute Activities of Organic Acids with Consideration of Partial Dissociation.

    PubMed

    Nandy, Lucy; Ohm, Peter B; Dutcher, Cari S

    2016-06-23

    Organic acids make up a significant fraction of the organic mass in atmospheric aerosol particles. The calculation of gas-liquid-solid equilibrium partitioning of the organic acid is therefore critical for accurate determination of atmospheric aerosol physicochemical properties and processes such as new particle formation and activation to cloud condensation nuclei. Previously, an adsorption isotherm-based statistical thermodynamic model was developed for capturing solute concentration-activity relationships for multicomponent aqueous solutions over the entire concentration range (Dutcher et al. J. Phys. Chem. C/A 2011, 2012, 2013), with model parameters for energies of adsorption successfully related to dipole-dipole electrostatic forces in solute-solvent and solvent-solvent interactions for both electrolytes and organics (Ohm et al. J. Phys. Chem. A 2015). However, careful attention is needed for weakly dissociating semivolatile organic acids. Dicarboxylic acids, such as malonic acid and glutaric acid are treated here as a mixture of nondissociated organic solute (HA) and dissociated solute (H(+) + A(-)). It was found that the apparent dissociation was greater than that predicted by known dissociation constants alone, emphasizing the effect of dissociation on osmotic and activity coefficient predictions. To avoid additional parametrization from the mixture approach, an expression was used to relate the Debye-Hückel hard-core collision diameter to the adjustable solute-solvent intermolecular distance. An improved reference state treatment for electrolyte-organic aqueous mixtures, such as that observed here with partial dissociation, has also been proposed. This work results in predictive correlations for estimation of organic acid and water activities for which there is little or no activity data. PMID:27222917

  15. Effects of thrombin on the integrity of monolayers of cultured human endothelial cells

    SciTech Connect

    Galdal, K.S.; Evensen, S.A.; Brosstad, F.

    1982-09-01

    /sup 51/Cr-prelabelled endothelial cells (EC) in confluent monolayers were incubated in RPMI 1640 + foetal calf serum 20% (v/v) to which purified thrombin was added. Thrombin (greater than or equal to 0.1 NIH U/ml) significantly accelerated /sup 51/Cr-release and caused extensive but reversible cell contraction. Thrombin-exposed EC reacted to a new dose of thrombin with no appreciable shape change, but /sup 51/Cr-efflux was again accelerated. EC exposed to thrombin pretreated with N-bromosuccinimide (modifying the macromolecular site) or phenylmethylsulfonyl fluoride (blocking the serine site) retained normal morphology and did not leak excess amounts of /sup 51/Cr. Antithrombin III also inhibited the effect of thrombin. Pretreatment of EC with either indomethacin, aspirin, sulfinpyrazone, pronase or neuraminidase did not influence the effect of subsequent thrombin exposure.

  16. The phosphorylation status of extracellular-regulated kinase 1/2 in astrocytes and neurons from rat hippocampus determines the thrombin-induced calcium release and ROS generation.

    PubMed

    Zündorf, Gregor; Reiser, Georg

    2011-12-01

    Challenge of protease-activated receptors induces cytosolic Ca(2+) concentration ([Ca(2+) ](c)) increase, mitogen-activated protein kinase activation and reactive oxygen species (ROS) formation with a bandwidth of responses in individual cells. We detected in this study in situ the thrombin-induced [Ca(2+) ](c) rise and ROS formation in dissociated hippocampal astrocytes and neurons in a mixed culture. In identified cells, single cell responses were correlated with extracellular-regulated kinase (ERK)1/2 phosphorylation level. On average, in astrocytes, thrombin induced a transient [Ca(2+) ](c) rise with concentration-dependent increase in amplitude and extrusion rate and high ERK1/2 phosphorylation level. Correlation analysis of [Ca(2+) ](c) response characteristics of single astrocytes reveals that astrocytes with nuclear phosphoERK1/2 localization have a smaller Ca(2+) amplitude and extrusion rate compared with cells with a cytosolic phosphoERK1/2 localization. In naive neurons, without thrombin challenge, variable ERK1/2 phosphorylation patterns are observed. ROS were detected by hydroethidine. Only in neurons with increased ERK1/2 phosphorylation level, we see sustained intracellular rise in fluorescence of the dye lasting over several minutes. ROS formation was abolished by pre-incubation with the NADPH oxidase inhibitor apocynin. Additionally, thrombin induced an immediate, transient hydroethidine fluorescence increase. This was interpreted as NADPH oxidase-mediated O(2) (•-) -release into the extracellular milieu, because it was decreased by pre-incubation with apocynin, and could be eluted by superfusion. In conclusion, the phosphorylation status of ERK1/2 determines the thrombin-dependent [Ca(2+) ](c) increase and ROS formation and, thus, influences the capacity of thrombin to regulate neuroprotection or neurodegeneration. PMID:21988180

  17. Benzyloxycarbonyl-D-Phe-Pro-methoxypropylboroglycine: a novel inhibitor of thrombin with high selectivity containing a neutral side chain at the P1 position.

    PubMed Central

    Claeson, G; Philipp, M; Agner, E; Scully, M F; Metternich, R; Kakkar, V V; DeSoyza, T; Niu, L H

    1993-01-01

    Thrombin, the blood-clotting enzyme, is a serine proteinase with trypsin-like specificity and is able to cleave Arg-Xaa peptide bonds but only in a very limited number of substrates (and sites therein). For the prevention and treatment of thrombosis the control of thrombin activity is a key target, and a variety of synthetic inhibitors have been introduced recently, all of which have a positive charge at the P1 site. We report the synthesis of the first example of a new class of inhibitor containing a neutral side chain at the P1 site, the peptide benzyloxycarbonyl-D-Phe-Pro- methoxypropylboroglycine. The peptide is a potent inhibitor of thrombin [Ki (limiting) = 7 nM] and is highly selective for its target enzyme in respect of other serine proteinases. This may be expected to confer considerable advantage in terms of specificity of action and reduced toxicity over conventional, positively charged, inhibitors. PMID:8452516

  18. Identification of BRCA1 missense substitutions that confer partial functional activity: potential moderate risk variants?

    PubMed Central

    Lovelock, Paul K; Spurdle, Amanda B; Mok, Myth TS; Farrugia, Daniel J; Lakhani, Sunil R; Healey, Sue; Arnold, Stephen; Buchanan, Daniel; Investigators, kConFab; Couch, Fergus J; Henderson, Beric R; Goldgar, David E; Tavtigian, Sean V; Chenevix-Trench, Georgia; Brown, Melissa A

    2007-01-01

    Introduction Many of the DNA sequence variants identified in the breast cancer susceptibility gene BRCA1 remain unclassified in terms of their potential pathogenicity. Both multifactorial likelihood analysis and functional approaches have been proposed as a means to elucidate likely clinical significance of such variants, but analysis of the comparative value of these methods for classifying all sequence variants has been limited. Methods We have compared the results from multifactorial likelihood analysis with those from several functional analyses for the four BRCA1 sequence variants A1708E, G1738R, R1699Q, and A1708V. Results Our results show that multifactorial likelihood analysis, which incorporates sequence conservation, co-inheritance, segregation, and tumour immunohistochemical analysis, may improve classification of variants. For A1708E, previously shown to be functionally compromised, analysis of oestrogen receptor, cytokeratin 5/6, and cytokeratin 14 tumour expression data significantly strengthened the prediction of pathogenicity, giving a posterior probability of pathogenicity of 99%. For G1738R, shown to be functionally defective in this study, immunohistochemistry analysis confirmed previous findings of inconsistent 'BRCA1-like' phenotypes for the two tumours studied, and the posterior probability for this variant was 96%. The posterior probabilities of R1699Q and A1708V were 54% and 69%, respectively, only moderately suggestive of increased risk. Interestingly, results from functional analyses suggest that both of these variants have only partial functional activity. R1699Q was defective in foci formation in response to DNA damage and displayed intermediate transcriptional transactivation activity but showed no evidence for centrosome amplification. In contrast, A1708V displayed an intermediate transcriptional transactivation activity and a normal foci formation response in response to DNA damage but induced centrosome amplification. Conclusion

  19. Mnemonic strategy training partially restores hippocampal activity in patients with mild cognitive impairment

    PubMed Central

    Hampstead, Benjamin M.; Stringer, Anthony Y.; Stilla, Randall F.; Giddens, Michelle; Sathian, K.

    2012-01-01

    Learning and memory deficits typify patients with mild cognitive impairment (MCI) and are generally attributed to medial temporal lobe dysfunction. Although the hippocampus is perhaps the most commonly studied neuroanatomical structure in these patients, there have been few attempts to identify rehabilitative interventions that facilitate its functioning. Here, we present results from a randomized, controlled, single-blind study in which patients with MCI and healthy elderly controls (HEC) were randomized to either 3 sessions of mnemonic strategy training (MS) or a matched-exposure control group (XP). All participants underwent pre- and post-training fMRI scanning as they encoded and retrieved object-location associations. For the current report, fMRI analyses were restricted to the hippocampus, as defined anatomically. Before training, MCI patients showed reduced hippocampal activity during both encoding and retrieval, relative to HEC. Following training, the MCI MS group demonstrated increased activity during both encoding and retrieval. There were significant differences between the MCI MS and MCI XP groups during retrieval, especially within the right hippocampus. Thus, MS facilitated hippocampal functioning in a partially restorative manner. We conclude that cognitive rehabilitation techniques may help mitigate hippocampal dysfunction in MCI patients. PMID:22368035

  20. Rational design of orally-active, pyrrolidine-based progesterone receptor partial agonists

    SciTech Connect

    Thompson, Scott K.; Washburn, David G.; Frazee, James S.; Madauss, Kevin P.; Hoang, Tram H.; Lapinski, Leahann; Grygielko, Eugene T.; Glace, Lindsay E.; Trizna, Walter; Williams, Shawn P.; Duraiswami, Chaya; Bray, Jeffrey D.; Laping, Nicholas J.

    2010-09-03

    Using the X-ray crystal structure of an amide-based progesterone receptor (PR) partial agonist bound to the PR ligand binding domain, a novel PR partial agonist class containing a pyrrolidine ring was designed. Members of this class of N-alkylpyrrolidines demonstrate potent and highly selective partial agonism of the progesterone receptor, and one of these analogs was shown to be efficacious upon oral dosing in the OVX rat model of estrogen opposition.

  1. Urinary Thrombin: A Novel Marker of Glomerular Inflammation for the Diagnosis of Crescentic Glomerulonephritis (Prospective Observational Study)

    PubMed Central

    Kitamoto, Yasunori; Arizono, Kenji; Fukui, Hiroyoshi; Tomita, Kimio; Kitamura, Hiroshi; Taguma, Yoshio; Imamura, Takahisa

    2015-01-01

    Background Crescentic glomerulonephritis (CresGN), an uncommon rapidly progressive disease, is characterized by severe glomerular inflammation with fibrin deposition. The lack of specific CresGN biomarkers delays diagnosis and threatens life. Because fibrin deposits in CresGN glomeruli indicate thrombin generation, we hypothesized that thrombin is excreted in urine and is a specific CresGN biomarker. Methods We measured urinary thrombin activity in 200 untreated patients (17 with CresGN, 183 with primary glomerulonephritis) and controls (8 patients with healed CresGN, 11 with nephrosclerosis, and 10 with tubulointerstitial nephritis, and 66 healthy volunteers). CresGN types included 15 pauci-immune and 2 immune complex. We assessed the diagnostic accuracy of thrombinuria in 169 patients with hematuria and proteinuria. Renal biopsy tissues were immunostained for tissue factor and fibrin. We analyzed the relationship of thrombinuria to plasma thrombin-antithrombin complex, hematuria, proteinuria, glomerular filtration rate, glomerular fibrin deposition, antineutrophil cytoplasmic antibodies (ANCAs), and C-reactive protein (CRP). We studied changes in thrombin activities after glucocorticoid treatment in 12 patients with thrombinuria. Results The highest thrombinuria occurrence was in CresGN (70.6%), followed by membranoproliferative glomerulonephritis (41.7%), IgA nephropathy (9.2%), and acute glomerulonephritis (0%). More than 75% of patients with nonproliferative glomerulonephritis manifested no thrombinuria. No controls had thrombinuria. Thrombinuria showed high CresGN specificity (90.1%) and moderate sensitivity (70.6%) and was detected in 4 of 7 patients with ANCA-negative CresGN. In CresGN, thrombinuria was associated with fibrin deposition in glomerular extracapillary tissue, where monocytes/macrophages expressed tissue factor. Thrombinuria in CresGN was unrelated to plasma thrombin-antithrombin complex, hematuria, proteinuria, glomerular filtration rate, and

  2. Comparison of mammalian and bacterial expression library screening to detect recombinant alpha-1 proteinase inhibitor variants with enhanced thrombin inhibitory capacity.

    PubMed

    Gierczak, Richard F; Bhakta, Varsha; Xie, Michael; Sheffield, William P

    2015-08-20

    Serpins are a widely distributed family of serine proteases. A key determinant of their specificity is the reactive centre loop (RCL), a surface motif of ∼20 amino acids in length. Expression libraries of variant serpins could be rapidly probed with proteases to develop novel inhibitors if optimal systems were available. The serpin variant alpha-1 proteinase inhibitor M358R (API M358R) inhibits the coagulation protease thrombin, but at sub-maximal rates compared to other serpins. Here we compared two approaches to isolate functional API variants from serpin expression libraries, using the same small library of API randomized at residue 358 (M358X): flow cytometry of transfected HEK 293 cells expressing membrane-displayed API; and a thrombin capture assay (TCA) performed on pools of bacterial lysates expressing soluble API. No enrichment for specific P1 residues was observed when the RCL codons of the 1% of sorted transfected 293 cells with the highest fluorescent thrombin-binding signals were subcloned and sequenced. In contrast, screening of 16 pools of bacterial API-expressing transformants led to the facile identification of API M358R and M358K as functional variants. Kinetic characterization showed that API M358R inhibited thrombin 17-fold more rapidly than API M358K. Reducing the incubation time with immobilized thrombin improved the sensitivity of TCA to detect supra-active API M358R variants and was used to screen a hypervariable library of API variants expressing 16 different amino acids at residues 352-357. The most active variant isolated, with TLSATP substituted for FLEAI, inhibited thrombin 2.9-fold more rapidly than API M358R. Our results indicate that flow cytometric approaches used in protein engineering of antibodies are not appropriate for serpins, and highlight the utility of the optimized TCA for serpin protein engineering. PMID:26043905

  3. Association of aortic valve sclerosis with thrombin generation in hypertensive patients.

    PubMed

    Iida, M; Yamamoto, M; Yamazaki, M; Sawaguchi, M; Honjo, H; Kodama, I; Kamiya, K

    2008-11-01

    Aortic valve sclerosis (AVS) may predispose to a prothrombotic state, as AVS is predictor of cardiovascular events in hypertensive populations. Thrombin exerts non-thrombotic effects such as vessel tone regulation, progression of atherosclerosis and stimulation of atrial natriuretic peptide (ANP) secretion. We hypothesized that hypertensive patients with AVS may have a persistently activated thrombin generation. We studied 234 asymptomatic never-treated hypertensive patients (73 of them with AVS). Prothrombin F1+2 (F1+2), as a marker of thrombin generation and fibrin D-dimer, as a marker of thrombus formation, ANP and brain natriuretic peptide (BNP) were measured. Presence of AVS, aortic jet velocity and left ventricular diameter at diastole were determined by echocardiography. Glomerular filtration rate was estimated using the Modification of Diet in Renal Disease formula. F1+2 (median and interquartile range (IQR) = 1.05, 0.87-1.38 nM vs. 0.93, 0.72-1.16) and ANP (22, 14-37 pg ml(-1) vs. 17, 11-25) levels were greater, and glomerular filtration rate values (65+/-9 ml min(-1)/1.73 m2 vs. 68+/-11) were lower in hypertensive patients with AVS than in those without AVS. F1+2 (odds ratio, 95% CI = 2.94, 1.07-8.6) was independently associated with AVS after being adjusted for age, gender and the variables of cardiorenal functions measured. After 6 months of treatment using valsartan, F1+2 levels remained elevated in hypertensive patients with AVS (1.14, 0.83-1.42 nM vs. 1.07, 0.84-1.5, n=19), but decreased in those without AVS (1.01, 0.85-1.31 vs. 0.8, 0.84-1.78, n=27). Thrombin generation was associated with AVS in untreated hypertensive patients, and this association was persistent after blood-pressure-lowering treatment using valsartan. PMID:18633427

  4. Computational study of some benzamidine-based inhibitors of thrombin-like snake venom proteinases

    NASA Astrophysics Data System (ADS)

    Henriques, Elsa S.; Nascimento, Marco A. C.; Ramos, Maria João

    Pit viper venoms contain a number of serine proteinases that, despite their observed coagulant thrombin-like action in vitro, exhibit a paradoxical benign defibrinogenating (anticoagulant) action in vivo, with clinical applications in preventing thrombi and improved blood circulation. Considering that several benzamidine-based inhibitors, some highly selective to thrombin, also inhibit the enzymatic activity of such venombins, the modeling of their enzyme-inhibitor interactions could provide valuable information on the topological factors that determine the divergences in activity. The first step, and the object of the present study, was to derive the necessary set of parameters, consistent with the CHARMM force field, and to perform molecular dynamics (MD) simulations on a few selected representatives of the inhibitors in question under physiological conditions. Bonding and van der Waals parameters were derived by analogy to similar ones in the existing force field. Net atomic charges were obtained with a restrained fitting to the molecular electrostatic potential generated at B3LYP/6-31G(d) level. The parameters were refined to reproduce the available experimental geometries and crystal data, and the MD simulations of the free inhibitors in aqueous solution at 298 K provided an insightful description of their available conformational space.

  5. Estimation of active pharmaceutical ingredients content using locally weighted partial least squares and statistical wavelength selection.

    PubMed

    Kim, Sanghong; Kano, Manabu; Nakagawa, Hiroshi; Hasebe, Shinji

    2011-12-15

    Development of quality estimation models using near infrared spectroscopy (NIRS) and multivariate analysis has been accelerated as a process analytical technology (PAT) tool in the pharmaceutical industry. Although linear regression methods such as partial least squares (PLS) are widely used, they cannot always achieve high estimation accuracy because physical and chemical properties of a measuring object have a complex effect on NIR spectra. In this research, locally weighted PLS (LW-PLS) which utilizes a newly defined similarity between samples is proposed to estimate active pharmaceutical ingredient (API) content in granules for tableting. In addition, a statistical wavelength selection method which quantifies the effect of API content and other factors on NIR spectra is proposed. LW-PLS and the proposed wavelength selection method were applied to real process data provided by Daiichi Sankyo Co., Ltd., and the estimation accuracy was improved by 38.6% in root mean square error of prediction (RMSEP) compared to the conventional PLS using wavelengths selected on the basis of variable importance on the projection (VIP). The results clearly show that the proposed calibration modeling technique is useful for API content estimation and is superior to the conventional one. PMID:22001843

  6. Exogenous Magnesium Chloride Reduces the Activated Partial Thromboplastin Times of Lupus Anticoagulant-Positive Patients

    PubMed Central

    Tokutake, Takayoshi; Baba, Hisami; Shimada, Yuji; Takeda, Wataru; Sato, Keijiro; Hiroshima, Yuki; Kirihara, Takehiko; Shimizu, Ikuo; Nakazawa, Hideyuki; Kobayashi, Hikaru; Ishida, Fumihiro

    2016-01-01

    The activated partial thromboplastin time (APTT) assay is a basic hemostatic assay based on the time it takes for clots to form in plasma samples after the addition of calcium chloride. It is used to screen for various coagulation disorders. Several previous reports have suggested that magnesium (Mg) might contribute to coagulation reactions by binding to specific coagulation proteins. We investigated the effects of Mg on the APTT. In healthy controls, the APTT was significantly prolonged in proportion to the increase in the concentration of magnesium chloride in the range from 2.1 to 16.7 mmol/L. Among eight samples from patients with various disorders that exhibited prolonged APTT, two samples demonstrated shorter APTT when Mg was added, both of which were from patients that were positive for lupus anticoagulant. When we examined 206 clinical APTT samples, we found that Mg shortened the APTT of two samples. These two samples were also from lupus anticoagulant-positive patients (p-value: <0.003). Our findings regarding the unique effects of exogenous Mg on the APTT of lupus anticoagulant-positive patients might shed light on the role of Mg in APTT assays and lead to the development of a novel screening method for lupus anticoagulant. PMID:27355205

  7. Brain Endothelial Cells Synthesize Neurotoxic Thrombin in Alzheimer’s Disease

    PubMed Central

    Yin, Xiangling; Wright, Jill; Wall, Trevor; Grammas, Paula

    2010-01-01

    Alzheimer’s disease (AD) is characterized by neuronal death; thus, identifying neurotoxic proteins and their source is central to understanding and treating AD. The multifunctional protease thrombin is neurotoxic and found in AD senile plaques. The objective of this study was to determine whether brain endothelial cells can synthesize thrombin and thus be a source of this neurotoxin in AD brains. Microvessels were isolated from AD patient brains and from age-matched controls. Reverse transcription-PCR demonstrated that thrombin message was highly expressed in microvessels from AD brains but was not detectable in control vessels. Similarly, Western blot analysis of microvessels showed that the thrombin protein was highly expressed in AD- but not control-derived microvessels. In addition, high levels of thrombin were detected in cerebrospinal fluid obtained from AD but not control patients, and sections from AD brains showed reactivity to thrombin antibody in blood vessel walls but not in vessels from controls. Finally, we examined the ability of brain endothelial cells in culture to synthesize thrombin and showed that oxidative stress or cell signaling perturbations led to increased expression of thrombin mRNA in these cells. The results demonstrate, for the first time, that brain endothelial cells can synthesize thrombin, and suggest that novel therapeutics targeting vascular stabilization that prevent or decrease release of thrombin could prove useful in treating this neurodegenerative disease. PMID:20150433

  8. Identification of PPARgamma Partial Agonists of Natural Origin (II): In Silico Prediction in Natural Extracts with Known Antidiabetic Activity

    PubMed Central

    Guasch, Laura; Sala, Esther; Mulero, Miquel; Valls, Cristina; Salvadó, Maria Josepa; Pujadas, Gerard; Garcia-Vallvé, Santiago

    2013-01-01

    Background Natural extracts have played an important role in the prevention and treatment of diseases and are important sources for drug discovery. However, to be effectively used in these processes, natural extracts must be characterized through the identification of their active compounds and their modes of action. Methodology/Principal Findings From an initial set of 29,779 natural products that are annotated with their natural source and using a previously developed virtual screening procedure (carefully validated experimentally), we have predicted as potential peroxisome proliferators-activated receptor gamma (PPARγ) partial agonists 12 molecules from 11 extracts known to have antidiabetic activity. Six of these molecules are similar to molecules with described antidiabetic activity but whose mechanism of action is unknown. Therefore, it is plausible that these 12 molecules could be the bioactive molecules responsible, at least in part, for the antidiabetic activity of the extracts containing them. In addition, we have also identified as potential PPARγ partial agonists 10 molecules from 16 plants with undescribed antidiabetic activity but that are related (i.e., they are from the same genus) to plants with known antidiabetic properties. None of the 22 molecules that we predict as PPARγ partial agonists show chemical similarity with a group of 211 known PPARγ partial agonists obtained from the literature. Conclusions/Significance Our results provide a new hypothesis about the active molecules of natural extracts with antidiabetic properties and their mode of action. We also suggest plants with undescribed antidiabetic activity that may contain PPARγ partial agonists. These plants represent a new source of potential antidiabetic extracts. Consequently, our work opens the door to the discovery of new antidiabetic extracts and molecules that can be of use, for instance, in the design of new antidiabetic drugs or functional foods focused towards the

  9. Streptococcal SpeB Cleaved PAR-1 Suppresses ERK Phosphorylation and Blunts Thrombin-Induced Platelet Aggregation

    PubMed Central

    Ender, Miriam; Andreoni, Federica; Zinkernagel, Annelies Sophie; Schuepbach, Reto Andreas

    2013-01-01

    Background The family of 4 related protease-activated receptors (PAR-1, 2, 3 & 4) expressed by mammalian cells allow to sense for and react to extracellular proteolytic activity. Since major human bacterial pathogens secret a wide array of protease(-s) we investigated whether they interfere with human PAR function. Methodology/Principal Findings Supernatants from cultures of major human bacterial pathogens were assayed for the presence of protease(-s) capable to cleave overexpressed human PAR-1, 2, 3 and 4 reporter constructs. Group A streptococcus (GAS) was found to secret a PAR-1-cleaving protease. Experiments involving genetical and pharmacological gain and loss of function identified streptococcal pyrogenic exotoxin B SpeB as the protease responsible. On the host’s side analysis of overexpressed PAR-1 carrying alanine substitutions and deletions showed the amino acid residue leucine44 on PAR-1’s extracellular N-terminus to be the only cleavage site. Complementary studies on endogenously expressed PAR-1 using PAR-1 blocking antibodies further supported our conclusion. Through PAR-1 cleavage SpeB efficiently blunted thrombin-induced induction of the ERK-pathway in endothelial cells and prevented platelets aggregation in response to thrombin. Conclusions/Significance Our results identify a novel function of the streptococcal virulence factor SpeB. By cleaving human PAR-1 at the N-terminal amino acid residue leucine44 SpeB rendered endothelial cells unresponsive to thrombin and prevented human platelets from thrombin-induced aggregation. These results suggest that by blunting PAR-1 signaling, SpeB modulates various innate host responses directed against invasive GAS potentially helping the invasive bacteria to escape. This may allow to tailor additional treatments in the future since upon invasion of the blood stream endothelial cells as well as platelets and mononuclear cells respond to PAR-1 agonists aiming to prevent further bacterial dissemination. PMID

  10. Reversible thrombin detection by aptamer functionalized STING sensors

    PubMed Central

    Actis, Paolo; Rogers, Adam; Nivala, Jeff; Vilozny, Boaz; Seger, R. Adam; Jejelowo, Olufisayo; Pourmand, Nader

    2011-01-01

    Signal Transduction by Ion NanoGating (STING) is a label-free technology based on functionalized quartz nanopipettes. The nanopipette pore can be decorated with a variety of recognition elements and the molecular interaction is transduced via a simple electrochemical system. A STING sensor can be easily and reproducibly fabricated and tailored at the bench starting from inexpensive quartz capillaries. The analytical application of this new biosensing platform, however, was limited due to the difficult correlation between the measured ionic current and the analyte concentration in solution. Here we show that STING sensors functionalized with aptamers allow the quantitative detection of thrombin. The binding of thrombin generates a signal that can be directly correlated to its concentration in the bulk solution. PMID:21636261

  11. Antithrombotic and antiplatelet activities of small-molecule alkaloids from Scolopendra subspinipes mutilans

    PubMed Central

    Lee, Wonhwa; Lee, JungIn; Kulkarni, Roshan; Kim, Mi-Ae; Hwang, Jae Sam; Na, MinKyun; Bae, Jong-Sup

    2016-01-01

    The aim of this study was to discover small-molecule anticoagulants from Scolopendra subspinipes mutilans (SSM). A new acylated polyamine (1) and a new sulfated quinoline alkaloid (2) were isolated from SSM. Treatment with the new alkaloids 1, 2, and indole acetic acid 4 prolonged the activated partial thromboplastin time and prothrombin time and inhibited the activity and production of thrombin and activated factor X. Furthermore, compounds 1, 2, and 4 inhibited thrombin-catalyzed fibrin polymerization and platelet aggregation. In accordance with these potential in vitro antiplatelet activities, compounds 1, 2, and 4 showed enhanced antithrombotic effects in an in vivo pulmonary embolism and arterial thrombosis model. Compounds 1, 2, and 4 also elicited anticoagulant effects in mice. Collectively, this study may serve as the groundwork for commercializing SSM or compounds 1, 2, and 4 as functional food components for the prevention and treatment of pathogenic conditions and serve as new scaffolds for the development of anticoagulants. PMID:26905699

  12. An electrochemical aptasensor electrocatalyst for detection of thrombin.

    PubMed

    Tian, Rong; Chen, Xiaojun; Li, Qingwen; Yao, Cheng

    2016-05-01

    This work reports a novel signal amplification strategy based on three-dimensional ordered macroporous C60-poly(3,4-ethylenedioxythiophene)-1-butyl-3-methylimidazolium hexafluorophosphate (3DOM C60-PEDOT-[BMIm][BF6]) for ultrasensitive detection of thrombin by cascade catalysis of Au-PEDOT@SiO2 microspheres and alcohol dehydrogenase (ADH). Au-PEDOT@SiO2 microspheres were constructed not only as nanocarriers to anchor the large amounts of secondary thrombin aptamers but also as nanocatalysts to catalyze the oxidation of ethanol efficiently. Significantly, the electrochemical signal was greatly enhanced based on cascade catalysis: First, ADH catalyzed the oxidation of ethanol to acetaldehyde with the concomitant generation of NADH in the presence of β-nicotinamide adenine dinucleotide hydrate (NAD(+)). Then, gold nanoparticles (AuNPs) as nanocatalysts could effectively catalyze NADH to produce NAD(+) with the help of PEDOT as redox probe. Under the optimal conditions, the proposed aptasensor exhibits a linear range of 2 × 10(-13) to 2 × 10(-8) M with a low detection limit of 2 × 10(-14) M for thrombin detection and shows high sensitivity and good specificity. PMID:26869084

  13. Porcine pituitary peptides with opiate-like activity: partial purification and effects in the rat after intraventricular injection.

    PubMed

    Teschemacher, H; Bläsig, J; Kromer, W

    1976-09-01

    A peptide material with opiate-like activity in the guinea-pig ileum was extracted from porcine pituitaries using a hot glacial acetic acid extraction method and was partially purified by gel filtration. When injected intraventricularly in rats, these purified peptides induced strong analgesia, catelepsy, respiratory depression and other opiate-like effects, which lasted for several hours. PMID:1034219

  14. Effect of partial agonist activity in beta blockers in severe angina pectoris: a double blind comparison of pindolol and atenolol.

    PubMed

    Quyyumi, A A; Wright, C; Mockus, L; Fox, K M

    1984-10-13

    The use of beta adrenoceptor blockade in the treatment of rest angina is controversial, and the effects on severe angina of partial agonist activity in beta blockers are unknown. Eight patients with effort angina and seven with effort and nocturnal angina and severe coronary artery disease were studied initially when they were not taking any antianginal drugs. Pindolol 5 mg thrice daily (with partial agonist activity) and atenolol 100 mg daily (without partial agonist activity) were given for five days each in a double blind randomised manner. Diaries of angina were kept and treadmill exercise testing and ambulatory ST monitoring performed during the last 48 hours of each period of treatment. Daytime and nocturnal resting heart rates and the frequency of angina were significantly reduced by atenolol compared with pindolol (p less than 0.01). The duration of exercise was significantly increased and the frequency, duration, and magnitude of daytime and nocturnal episodes of ST segment depression on ambulatory monitoring were reduced by atenolol. Reduction in resting heart rate is important in the treatment of both effort and nocturnal angina. Partial agonist activity in beta adrenoceptor antagonists may be deleterious in patients with severe angina pectoris. PMID:6148991

  15. Differential pathway coupling efficiency of the activated insulin receptor drives signaling selectivity by xmeta, an allosteric partial agonist antibody

    Technology Transfer Automated Retrieval System (TEKTRAN)

    XMetA, an anti-insulin receptor (IR) monoclonal antibody, is an allosteric partial agonist of the IR. We have previously reported that XMetA activates the “metabolic-biased” Akt kinase signaling pathway while having little or no effect on the “mitogenic” MAPK signaling pathwayof ERK 1/2. To inves...

  16. In-depth study of tripeptide-based alpha-ketoheterocycles as inhibitors of thrombin. Effective utilization of the S1' subsite and its implications to structure-based drug design.

    PubMed

    Costanzo, Michael J; Almond, Harold R; Hecker, Leonard R; Schott, Mary R; Yabut, Stephen C; Zhang, Han-Cheng; Andrade-Gordon, Patricia; Corcoran, Thomas W; Giardino, Edward C; Kauffman, Jack A; Lewis, Joan M; de Garavilla, Lawrence; Haertlein, Barbara J; Maryanoff, Bruce E

    2005-03-24

    Thrombin inhibitors are potentially useful in medicine for their anticoagulant and antithrombotic effects. We synthesized and evaluated diverse heterocycle-activated ketones based on the d-Phe-Pro-Arg, and related thrombin active-site recognition motifs, as candidate inhibitors. The peptide-based alpha-ketoheterocycles were typically prepared by either an imidate or a Weinreb amide route (Schemes 1 and 2), the latter of which proved to be more general. Test compounds were generally assayed for inhibition of human alpha-thrombin and bovine trypsin. From a structure-based design standpoint, the heterocycle allows one to explore and adjust interactions within the S1' subsite of thrombin. The preferred alpha-ketoheterocycle is a pi-rich 2-substituted azole with at least two heteroatoms proximal to the carbon bearing the keto group, and a preferred thrombin inhibitor is 2-ketobenzothiazole 3, with a potent K(i) value of 0.2 nM and ca. 15-fold selectivity over trypsin. 2-Ketobenzothiazole 13 exhibited exceedingly potent thrombin inhibition (K(i) = 0.000 65 nM; slow tight binding). Several alpha-ketoheterocycles had thrombin K(i) values in the range 0.1-400 nM. The "Arg" unit in the alpha-ketoheterocycles can be sensitive to stereomutation under mildy basic conditions. For example, 2-ketothiazoles 4 and 59 readily epimerize at pH 7.4, although they are fairly stable stereochemically at pH 3-4; thus, suitable conditions had to be selected for the enzymatic assays. Lead d-Phe-Pro-Arg 2-benzothiazoles 3, 4, and 68 displayed good selectivity for thrombin over other key coagulation enzymes (e.g., factor Xa, plasmin, protein Ca, uPA, tPA, and streptokinase); however, their selectivity for thrombin over trypsin was modest (<25-fold). Compounds 3, 4, and 68 exhibited potent in vitro antithrombotic activity as measured by inhibition of gel-filtered platelet aggregation induced by alpha-thrombin (IC(50) = 30-40 nM). They also proved to be potent anticoagulant/antithrombotic agents

  17. A sensitive HIV-1 envelope induced fusion assay identifies fusion enhancement of thrombin

    SciTech Connect

    Cheng, De-Chun; Zhong, Guo-Cai; Su, Ju-Xiang; Liu, Yan-Hong; Li, Yan; Wang, Jia-Ye; Hattori, Toshio; Ling, Hong; Zhang, Feng-Min

    2010-01-22

    To evaluate the interaction between HIV-1 envelope glycoprotein (Env) and target cell receptors, various cell-cell-fusion assays have been developed. In the present study, we established a novel fusion system. In this system, the expression of the sensitive reporter gene, firefly luciferase (FL) gene, in the target cells was used to evaluate cell fusion event. Simultaneously, constitutively expressed Renilla luciferase (RL) gene was used to monitor effector cell number and viability. FL gave a wider dynamic range than other known reporters and the introduction of RL made the assay accurate and reproducible. This system is especially beneficial for investigation of potential entry-influencing agents, for its power of ruling out the false inhibition or enhancement caused by the artificial cell-number variation. As a case study, we applied this fusion system to observe the effect of a serine protease, thrombin, on HIV Env-mediated cell-cell fusion and have found the fusion enhancement activity of thrombin over two R5-tropic HIV strains.

  18. The role of structural information in the discovery of direct thrombin and factor Xa inhibitors.

    PubMed

    Nar, Herbert

    2012-05-01

    The quest for novel medications to treat thromboembolic disorders such as venous thrombosis, pulmonary embolism and stroke received a boost when the 3D structures of two major players in the blood coagulation cascade were determined in 1989 and 1993. Structure-guided design of inhibitors of thrombin (factor IIa, fIIa) and factor Xa (fXa) eventually led to the discovery of potent, selective, efficacious, orally active and safe compounds that proved successful in clinical studies. In 2008, the direct thrombin inhibitor dabigatran etexilate developed by Boehringer Ingelheim became the first novel antithrombotic molecular entity to enter the market in 50 years. Additional compounds targeting factor Xa were subsequently granted marketing authorization or are in late-stage clinical studies. In this review, I use selected case studies to describe the discovery of novel fIIa and fXa inhibitors, with a particular emphasis on the pre-eminent role that structural information played in this process. PMID:22503439

  19. Aggregate size and architecture determine microbial activity balance for one-stage partial nitritation and anammox.

    PubMed

    Vlaeminck, Siegfried E; Terada, Akihiko; Smets, Barth F; De Clippeleir, Haydée; Schaubroeck, Thomas; Bolca, Selin; Demeestere, Lien; Mast, Jan; Boon, Nico; Carballa, Marta; Verstraete, Willy

    2010-02-01

    Aerobic ammonium-oxidizing bacteria (AerAOB) and anoxic ammonium-oxidizing bacteria (AnAOB) cooperate in partial nitritation/anammox systems to remove ammonium from wastewater. In this process, large granular microbial aggregates enhance the performance, but little is known about granulation so far. In this study, three suspended-growth oxygen-limited autotrophic nitrification-denitrification (OLAND) reactors with different inoculation and operation (mixing and aeration) conditions, designated reactors A, B, and C, were used. The test objectives were (i) to quantify the AerAOB and AnAOB abundance and the activity balance for the different aggregate sizes and (ii) to relate aggregate morphology, size distribution, and architecture putatively to the inoculation and operation of the three reactors. A nitrite accumulation rate ratio (NARR) was defined as the net aerobic nitrite production rate divided by the anoxic nitrite consumption rate. The smallest reactor A, B, and C aggregates were nitrite sources (NARR, >1.7). Large reactor A and C aggregates were granules capable of autonomous nitrogen removal (NARR, 0.6 to 1.1) with internal AnAOB zones surrounded by an AerAOB rim. Around 50% of the autotrophic space in these granules consisted of AerAOB- and AnAOB-specific extracellular polymeric substances. Large reactor B aggregates were thin film-like nitrite sinks (NARR, <0.5) in which AnAOB were not shielded by an AerAOB layer. Voids and channels occupied 13 to 17% of the anoxic zone of AnAOB-rich aggregates (reactors B and C). The hypothesized granulation pathways include granule replication by division and budding and are driven by growth and/or decay based on species-specific physiology and by hydrodynamic shear and mixing. PMID:19948857

  20. Human alpha-thrombin inhibition by the highly selective compounds N-ethoxycarbonyl-D-Phe-Pro-alpha-azaLys p-nitrophenyl ester and N-carbobenzoxy-Pro-alpha-azaLys p-nitrophenyl ester: a kinetic, thermodynamic and X-ray crystallographic study.

    PubMed

    De Simone, G; Balliano, G; Milla, P; Gallina, C; Giordano, C; Tarricone, C; Rizzi, M; Bolognesi, M; Ascenzi, P

    1997-06-20

    Kinetics, thermodynamics and structural aspects of human alpha-thrombin (thrombin) inhibition by newly synthesized low molecular weight derivatives of alpha-azalysine have been investigated. The thrombin catalyzed hydrolysis of N-ethoxycarbonyl-D-Phe-Pro-alpha-azaLys p-nitrophenyl ester (Eoc-D-Phe-Pro-azaLys-ONp) and N-carbobenzoxy-Pro-alpha-azaLys p-nitrophenyl ester (Cbz-Pro-azaLys-ONp) was investigated at pH 6.2 and 21.0 degrees C, and analyzed in parallel with that of N-alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp). Decarboxylation following the enzymatic hydrolysis of these p-nitrophenyl esters gave the corresponding 1-peptidyl-2(4-aminobutyl) hydrazines (peptidyl-Abh) showing properties of thrombin competitive inhibitors. Therefore, thermodynamics for the reversible binding of D-Phe-Pro-Abh, Cbz-Pro-Abh and Dmc-Abh to thrombin was examined. These results are consistent with the minimum four-step catalytic mechanism for product inhibition of serine proteinases. Eoc-D-Phe-Pro-azaLys-ONp and Eoc-D-Phe-Pro-Abh display a sub-micromolar affinity for thrombin together with a high selectivity versus homologous plasmatic and pancreatic serine proteinases acting on cationic substrates. The three-dimensional structures of the reversible non-covalent thrombin:Eoc-D-Phe-Pro-Abh and thrombin:Cbz-Pro-Abh complexes have been determined by X-ray crystallography at 2.0 A resolution (R-factor = 0.169 and 0.179, respectively), and analyzed in parallel with that of the thrombin:Dmc-azaLys acyl-enzyme adduct. Both Eoc-D-Phe-Pro-Abh and Cbz-Pro-Abh competitive inhibitors are accommodated in the thrombin active center, spanning the region between the aryl binding site and the S1 primary specificity subsite. PMID:9217260

  1. Inhibition of collagen, and thrombin-induced platelet aggregation by Lansberg's hognose pit viper (Porthidium lansbergii hutmanni) venom.

    PubMed

    López-Johnston, Juan C; de Bosch, Norma; Scannone, Héctor; Rodríguez-Acosta, Alexis

    2007-12-01

    The Porthidium genus is represented by the P. lansbergii rozei and P. lansbergii hutmanni (Plh) subspecies in Venezuela. The venom components of these have been little studied, probably due to the low incidence of reported accidents, although acute and serious local effects such as invasive edema and disseminated ecchymosis are present during human envenonation. The aim of this work was to characterize the in vitro effects of crude P. l. hutmanni venom, and its fractions, on platelet aggregation triggered by two physiologic agonists: thrombin and collagen. The effects of thrombin and collagen were observed on a platelet-rich plasma (PRP) solution (3 x 10(5) platelets/microL) using serial dilutions of P. l. hutmanni venom (0.625-40 microg). The crude venom was fractionated by anionic exchange chromatography and two peaks obtained. Crude venom and both fractions were highly inhibitory on platelet aggregation mediated by the two agonists. The anti-aggregating dose (AD(50)) for both agonists was determined. PRP collagen-triggered aggregation was most inhibited by the crude venom (AD(50) = 0.67 microg) when compared with PRP thrombin-triggered aggregation (AD(50) = 4.92 microg). Collagen-induced aggregation was more intensely inhibited by venom than thrombin-induced aggregation. In conclusion, to specify the inhibition mechanisms involved for each of the active components in the venom from these subspecies, we must characterize and purify the inhibitors of aggregation from P. l. hutmanni venom, with the purpose of suggesting new pharmacological substances to be incorporated into the therapeutic arsenal to treat hemostatic pathologies related to high levels of platelet aggregation. PMID:17486300

  2. An electrochemical aptasensor for thrombin detection based on direct electrochemistry of glucose oxidase using a functionalized graphene hybrid for amplification.

    PubMed

    Bai, Lijuan; Yan, Bin; Chai, Yaqin; Yuan, Ruo; Yuan, Yali; Xie, Shunbi; Jiang, Liping; He, Ying

    2013-11-01

    In this work, we reported a new label-free electrochemical aptasensor for highly sensitive detection of thrombin using direct electron transfer of glucose oxidase (GOD) as a redox probe and a gold nanoparticle-polyaniline-graphene (Au-PANI-Gra) hybrid for amplification. The Au-PANI-Gra hybrid with large surface area provided a biocompatible sensing platform for the immobilization of GOD. GOD was encapsulated into the three-dimensional netlike (3-mercaptopropyl)trimethoxysilane (MPTS) to form the MPTS-GOD biocomposite, which not only retained the native functions and properties, but also exhibited tunable porosity, high thermal stability, and chemical inertness. With abundant thiol tail groups on MPTS, MPTS-GOD was able to chemisorb onto the surface of the Au-PANI-Gra modified electrode through the strong affinity of the Au-S bond. The electrochemical signal originated from GOD, avoiding the addition or labeling of other redox mediators. After immobilizing the thiolated thrombin binding aptamer through gold nanoparticles (AuNPs), GOD as a blocking reagent was employed to block the remaining active sites of the AuNPs and avoid the nonspecific adsorption. The proposed method avoided the labeling process of redox probes and increased the amount of electroactive GOD. The concentration of thrombin was monitored based on the decrease of current response through cyclic voltammetry (CV) in 0.1 M PBS (pH 7.4). With the excellent direct electron transfer of double layer GOD membranes, the resulting aptasensor exhibited high sensitivity for detection of thrombin with a wide linear range from 1.0 × 10(-12) to 3.0 × 10(-8) M. The proposed aptasensor also showed good stability, satisfactory reproducibility and high specificity, which provided a promising strategy for electrochemical aptamer-based detection of other biomolecules. PMID:24003439

  3. Simultaneous measurement of thrombin generation and fibrin formation in whole blood under flow conditions.

    PubMed

    Kelchtermans, Hilde; Pelkmans, Leonie; Bouwhuis, Anne; Schurgers, Evelien; Lindhout, Theo; Huskens, Dana; Miszta, Adam; Hemker, H Coenraad; Lancé, Marcus D; de Laat, Bas

    2016-07-01

    Assays based on the formation of thrombin and fibrin are frequently used, and results are considered exchangeable in research/clinical settings. However, thrombin generation and fibrin formation do not always go hand in hand and flow profoundly influences thrombus formation. We describe the technical/clinical evaluation of an assay to simultaneously measure thrombin generation and fibrin formation under conditions of flow. Introduction of a fluorometer into a 'cone and base principle'-based rheometer allowed the measurement of thrombin generation (using a thrombin-sensitive substrate) and fibrin formation (changes in viscosity), while applying a linear shear flow. Increasing shear rates inversely related with thrombin generation and fibrin formation. Increasing fibrinogen concentrations in defibrinated plasma resulted in increased thrombin generation and fibrin formation. In pre-operative samples of 70 patients undergoing cardiothoracic surgery, fibrin formation and thrombin generation parameters correlated with fibrinogen content, rotational thromboelastometry (ROTEM) and whole blood Calibrated Automated Thrombinography (CAT) parameters, respectively. Upon dividing patients into two groups based on the median clot strength, a significant difference in perioperative/total blood loss was established. In conclusion, we clinically evaluated a method capable of simultaneously measuring thrombin generation and fibrin formation in plasma/whole blood under continuous flow, rendering our method one step closer to physiology. Importantly, our test proved to be indicative for the amount of blood loss during/after cardiothoracic surgery. PMID:27074907

  4. Binding modes of thrombin binding aptamers investigated by simulations and experiments

    NASA Astrophysics Data System (ADS)

    Trapaidze, A.; Bancaud, A.; Brut, M.

    2015-01-01

    Thrombin binding aptamers HD1 and HD22 are the most studied aptamers, both for therapeutic and sensing purposes. Yet, there is still no commercialized aptamer-based sensor device for thrombin detection, suggesting that the binding modes of these aptamers remain to be precisely described. Here, we investigate thrombin-aptamer interactions with molecular dynamics simulations, and show that the different solved structures of HD1-thrombin complex are energetically similar and consequently possibly co-existing. Conversely, HD22 folding is much more stable, and its binding energy with thrombin is significantly larger than that of HD1 complexes. These results are confronted to experiments, which consist in monitoring aggregation of aptamer-functionalized gold nanoparticles triggered by thrombin. HD1 alone, but not HD22, can trigger aggregation, meaning that this aptamer has multiple sites of interactions with thrombin. Furthermore, pre-incubation of HD22 with thrombin impedes HD1 aggregation, suggesting that HD1 and HD22 have competing affinities for the same binding site. Altogether, this study shows that the characterization of aptamer-thrombin interactions by structural and kinetic experiments joined to simulations is necessary for the development of biosensors.

  5. Thrombin Maybe Plays an Important Role in MK Differentiation into Platelets

    PubMed Central

    Yang, Xiao-Lei; Ge, Meng-Kai; Mao, De-Kui; Lv, Ying-Tao; Sun, Shu-Yan; Yu, Ai-Ping

    2016-01-01

    Objectives. After development and differentiation, megakaryocytes (MKs) can produce platelets. As is well known, thrombopoietin (TPO) can induce MKs to differentiate. The effect of thrombin on MKs differentiation is not clear. In this study, we used a human megakaryoblastic leukemia cell line (Meg-01) to assess the effect of thrombin on MKs differentiation. Methods. In order to interrogate the role of thrombin in Meg-01 cells differentiation, the changes of morphology, cellular function, and expression of diverse factors were analyzed. Results. The results show that thrombin suppresses Meg-01 cells proliferation and induces apoptosis and cell cycle arrest. Thrombin upregulates the expression of CD41b, which is one of the most important MK markers. Globin transcription factor 1 (GATA-1), an important transcriptional regulator, controls MK development and maturation. The expression of GATA-1 is also upregulated by thrombin in Meg-01 cells. The expression of B-cell lymphoma 2 (Bcl-2), an apoptosis-inhibitory protein, is downregulated by thrombin. Phosphorylated protein kinase B (p-AKT) and phosphorylated extracellular signal-regulated kinase (p-ERK) were upregulated by thrombin in Meg-01 cells. All the results are consistent with Meg-01 cells treated with TPO. Discussion and Conclusion. In conclusion, all these data indicate that thrombin maybe plays an important role in MK differentiation into platelets. However, whether the platelet-like particles are certainly platelets remains unknown. PMID:27064425

  6. PAR-1 and thrombin: the ties that bind the microenvironment to melanoma metastasis.

    PubMed

    Zigler, Maya; Kamiya, Takafumi; Brantley, Emily C; Villares, Gabriel J; Bar-Eli, Menashe

    2011-11-01

    Progression of melanoma is dependent on cross-talk between tumor cells and the adjacent microenvironment. The thrombin receptor, protease-activated receptor-1 (PAR-1), plays a key role in exerting this function during melanoma progression. PAR-1 and its activating factors, which are expressed on tumor cells and the surrounding stroma, induce not only coagulation but also cell signaling, which promotes the metastatic phenotype. Several adhesion molecules, cytokines, growth factors, and proteases have recently been identified as downstream targets of PAR-1 and have been shown to modulate interactions between tumor cells and the microenvironment in the process of melanoma growth and metastasis. Inhibiting such interactions by targeting PAR-1 could potentially be a useful therapeutic modality for melanoma patients. PMID:22009534

  7. PAR-1 and Thrombin: The Ties that Bind the Microenvironment to Melanoma Metastasis*

    PubMed Central

    Zigler, Maya; Kamiya, Takafumi; Brantley, Emily C.; Villares, Gabriel J.; Bar-Eli, Menashe

    2011-01-01

    Progression of melanoma is dependent on cross-talk between tumor cells and the adjacent microenvironment. The thrombin receptor, Protease Activated Receptor-1 (PAR-1) plays a key role in exerting this function during melanoma progression. PAR-1 and its activating factors, which are expressed on tumor cells and the surrounding stroma, induce not only coagulation, but also cell signaling which promotes the metastatic phenotype. Several adhesion molecules, cytokines, growth factors and proteases have recently been identified as downstream targets of PAR-1 and have been shown to modulate interactions between tumor cells and the microenvironment in the process of melanoma growth and metastasis. Inhibition of these interactions by targeting PAR-1 could be utilized as a potential therapeutic modality for melanoma patients. PMID:22009534

  8. The Emerging Role of the Thrombin Receptor (PAR-1) in Melanoma Metastasis - a Possible Therapeutic Target

    PubMed Central

    Villares, Gabriel J.; Zigler, Maya; Bar-Eli, Menashe

    2011-01-01

    Melanoma remains as the deadliest form of skin cancer with limited and inefficient treatment options available for patients with metastatic disease. Within the last decade, the thrombin receptor, Protease Activated Receptor-1, has been described as an essential gene involved in the progression of human melanoma. PAR-1 is known to activate adhesive, invasive and angiogenic factors to promote melanoma metastasis. It is overexpressed not only in metastatic melanoma cell lines but is also highly expressed in metastatic lesions as compared to primary nevi and normal skin. Recently, PAR-1 has been described to regulate the gap junction protein Connexin 43 and the tumor suppressor gene Maspin to promote the metastatic melanoma phenotype. Herein, we review the role of PAR-1 in the progression of melanoma as well as utilizing PAR-1-regulated genes as potential therapeutic targets for melanoma treatment. PMID:21378407

  9. Charged particle detectors with active detector surface for partial energy deposition of the charged particles and related methods

    DOEpatents

    Gerts, David W; Bean, Robert S; Metcalf, Richard R

    2013-02-19

    A radiation detector is disclosed. The radiation detector comprises an active detector surface configured to generate charge carriers in response to charged particles associated with incident radiation. The active detector surface is further configured with a sufficient thickness for a partial energy deposition of the charged particles to occur and permit the charged particles to pass through the active detector surface. The radiation detector further comprises a plurality of voltage leads coupled to the active detector surface. The plurality of voltage leads is configured to couple to a voltage source to generate a voltage drop across the active detector surface and to separate the charge carriers into a plurality of electrons and holes for detection. The active detector surface may comprise one or more graphene layers. Timing data between active detector surfaces may be used to determine energy of the incident radiation. Other apparatuses and methods are disclosed herein.

  10. Proximity Binding and Metal Ion-Dependent DNAzyme Cyclic Amplification-Integrated Aptasensor for Label-Free and Sensitive Electrochemical Detection of Thrombin.

    PubMed

    Yang, Jianmei; Dou, Baoting; Yuan, Ruo; Xiang, Yun

    2016-08-16

    Thrombin plays important roles for the diagnosis of neurodegenerative and cardiovascular diseases. By integrating proximity binding-induced strand displacement and metal ion-dependent DNAzyme recycling amplification, we demonstrate here the development of a simple and sensitive strategy for the detection of thrombin in human serums. The binding of the two distinct aptamers to the thrombin targets increases the local concentration of the aptamers and facilitates the release of the enzymatic sequences through proximity binding-induced strand displacement. The liberated enzymatic sequences further hybridize with the G-quadruplex containing and hairpin-structured substrate sequences on the sensor electrode to form the metal-ion dependent DNAzymes. Subsequently, the metal ions catalyze the cleavage of the substrate sequences to unlock the G-quadruplex forming sequences and to release the enzymatic sequences to trigger another cleavage cycle. Such metal ion-dependent DNAzyme recycling amplification leads to the formation of many active G-quadruplex forming sequences, which associate with hemin to form G-quadruplex/hemin complexes on the electrode surface. Direct electron transfer of hemin to the electrode during the potential scan can thus generate significantly amplified current for sensitive detection of thrombin at the low picomolar level. The work demonstrated here can thus offer new opportunities for the development of convenient signal amplification strategies for detecting various protein targets. PMID:27436431

  11. Synthesis and structure-activity relationships of novel indazolyl glucocorticoid receptor partial agonists.

    PubMed

    Gilmore, John L; Sheppeck, James E; Wang, Jim; Dhar, T G Murali; Cavallaro, Cullen; Doweyko, Arthur M; Mckay, Lorraine; Cunningham, Mark D; Habte, Sium F; Nadler, Steven G; Dodd, John H; Somerville, John E; Barrish, Joel C

    2013-10-01

    SAR was used to further develop an indazole class of non-steroidal glucocorticoid receptor agonists aided by a GR LBD (ligand-binding domain)-agonist co-crystal structure described in the accompanying paper. Progress towards discovering a dissociated GR agonist guided by human in vitro assays biased the optimization of this compound series towards partial agonists that possessed excellent selectivity against other nuclear hormone receptors. PMID:23916594

  12. Changes in neuronal activity and gene expression in guinea-pig auditory brainstem after unilateral partial hearing loss.

    PubMed

    Dong, S; Mulders, W H A M; Rodger, J; Robertson, D

    2009-03-31

    Spontaneous neural hyperactivity in the central auditory pathway is often associated with deafness, the most common form of which is partial hearing loss. We quantified both peripheral hearing loss and spontaneous activity in single neurons of the contralateral inferior colliculus in a guinea-pig model 1 week after a unilateral partial deafness induced by cochlear mechanical lesion. We also measured mRNA levels of candidate genes in the same animals using quantitative real-time PCR. Spontaneous hyperactivity was most marked in the frequency region of the peripheral hearing loss. Expression of glutamate decarboxylase 1 (GAD1), GABA-A receptor subunit alpha-1 (GABRA1), and potassium channel subfamily K member 15 (KCNK15) was decreased ipsilaterally in the cochlear nucleus and bilaterally in the inferior colliculus. A member of RAB family of small GTPase (RAB3A) was decreased in both ipsilateral cochlear nucleus and contralateral inferior colliculus. RAB3 GTPase activating protein subunit 1 (RAB3GAP1) and glycine receptor subunit alpha-1 (GLRA1) were reduced ipsilaterally in the cochlear nucleus only. These results suggest that a decrease in inhibitory neurotransmission and an increase in membrane excitability may contribute to elevated neuronal spontaneous activity in the auditory brainstem following unilateral partial hearing loss. PMID:19356697

  13. Coronary artery rupture during balloon angioplasty, rescued with localized thrombin injection and coil embolization.

    PubMed

    Fischell, Tim A; Carter, Andrew J; Ashraf, Kamal; Birdsall, Joseph; Smoker, Sandy

    2006-08-01

    Distal intracoronary thrombin injection has been used successfully to seal very small, guidewire related, coronary artery perforations during percutaneous coronary intervention. This case report expands upon this therapeutic approach, by describing the use of high dose distal thrombin injection for the successful (non-surgical) management of balloon-induced coronary artery rupture, with an intrapericardial leak. PMID:16819769

  14. A plasmonic aptasensor for ultrasensitive detection of thrombin via arrested rolling circle amplification.

    PubMed

    Wang, Sai; Bi, Sai; Wang, Zonghua; Xia, Jianfei; Zhang, Feifei; Yang, Min; Gui, Rijun; Li, Yanhui; Xia, Yanzhi

    2015-05-01

    A sensitive signal generation mechanism for gold nanoparticle growth by reducing gold ions with hydrogen peroxide is applied in a plasmonic aptasensor, achieving naked-eye detection of thrombin at the single-molecule level based on the specific interaction of aptamer-thrombin via an arrested rolling circle amplification to yield horseradish peroxidase (HRP)-mimicking DNAzymes as biocatalysts. PMID:25864665

  15. Thrombin and fibrinogen γ' impact clot structure by marked effects on intrafibrillar structure and protofibril packing.

    PubMed

    Domingues, Marco M; Macrae, Fraser L; Duval, Cédric; McPherson, Helen R; Bridge, Katherine I; Ajjan, Ramzi A; Ridger, Victoria C; Connell, Simon D; Philippou, Helen; Ariëns, Robert A S

    2016-01-28

    Previous studies have shown effects of thrombin and fibrinogen γ' on clot structure. However, structural information was obtained using electron microscopy, which requires sample dehydration. Our aim was to investigate the role of thrombin and fibrinogen γ' in modulating fibrin structure under fully hydrated conditions. Fibrin fibers were studied using turbidimetry, atomic force microscopy, electron microscopy, and magnetic tweezers in purified and plasma solutions. Increased thrombin induced a pronounced decrease in average protofibril content per fiber, with a relatively minor decrease in fiber size, leading to the formation of less compact fiber structures. Atomic force microscopy under fully hydrated conditions confirmed that fiber diameter was only marginally decreased. Decreased protofibril content of the fibers produced by high thrombin resulted in weakened clot architecture as analyzed by magnetic tweezers in purified systems and by thromboelastometry in plasma and whole blood. Fibers produced with fibrinogen γ' showed reduced protofibril packing over a range of thrombin concentrations. High-magnification electron microscopy demonstrated reduced protofibril packing in γ' fibers and unraveling of fibers into separate protofibrils. Decreased protofibril packing was confirmed in plasma for high thrombin concentrations and fibrinogen-deficient plasma reconstituted with γ' fibrinogen. These findings demonstrate that, in fully hydrated conditions, thrombin and fibrinogen γ' have dramatic effects on protofibril content and that protein density within fibers correlates with strength of the fibrin network. We conclude that regulation of protofibril content of fibers is an important mechanism by which thrombin and fibrinogen γ' modulate fibrin clot structure and strength. PMID:26608329

  16. Peptide mimetics of the thrombin-bound structure of fibrinopeptide A.

    PubMed Central

    Nakanishi, H; Chrusciel, R A; Shen, R; Bertenshaw, S; Johnson, M E; Rydel, T J; Tulinsky, A; Kahn, M

    1992-01-01

    Recent work has suggested that the thrombin-bound conformation of fibrinopeptide A exhibits a strand-turn-strand motif, with a beta-turn centered at residues Glu-11 and Gly-12. Our molecular modeling analysis indicates that the published fibrinopeptide conformation cannot bind reasonably to thrombin but that reorientation of two residues by alignment with bovine pancreatic trypsin inhibitor provides a good fit within the deep thrombin cleft and satisfies all of the experimental nuclear Overhauser effect data. Based on this analysis, we have successfully designed and synthesized hybrid peptide mimetic substrates and inhibitors that mimic the proposed beta-turn structure. The results indicate that the turn conformation is an important aspect of thrombin specificity and that our turn mimetic design successfully mimics the thrombin-bound conformation of fibrinopeptide. Images PMID:1542664

  17. Partial Fractionation of Venoms from Two Iranian Vipers, Echis carinatus and Cerastes persicus Fieldi and Evaluation of Their Antiplatelet Activity.

    PubMed

    Mehdizadeh Kashani, Toktam; Vatanpour, Hossein; Zolfagharian, Hossein; Hooshdar Tehrani, Hasan; Heydari, Mohammad Hossein; Kobarfard, Farzad

    2012-01-01

    Platelet aggregation inhibitory effect and anticoagulant properties of fractions separated from the venoms of Cerastes persicus fieldi and Echis carinatus were investigated. The partial fractionation was performed on a Sephadex G-100 column. Two fractions separated from Cerastes persicus fieldi showed anti platelet aggregation activity on ADP (200 μM)-induced platelet aggregation (ca 80% inhibition). Attempts to measure the antiplatelet aggregation activity of crude Echis carinatus venom and its fractions were not successful due to the protein coagulation of the plasma samples after the addition of venom. Anticoagulant activities of venoms were also evaluated. Total venom of Echis carinatus showed anti coagulant activity in PT test, while its fractions showed procoagulant activity. PMID:24250552

  18. Partial Fractionation of Venoms from Two Iranian Vipers, Echis carinatus and Cerastes persicus Fieldi and Evaluation of Their Antiplatelet Activity

    PubMed Central

    Mehdizadeh Kashani, Toktam; Vatanpour, Hossein; Zolfagharian, Hossein; Hooshdar Tehrani, Hasan; Heydari, Mohammad Hossein; Kobarfard, Farzad

    2012-01-01

    Platelet aggregation inhibitory effect and anticoagulant properties of fractions separated from the venoms of Cerastes persicus fieldi and Echis carinatus were investigated. The partial fractionation was performed on a Sephadex G-100 column. Two fractions separated from Cerastes persicus fieldi showed anti platelet aggregation activity on ADP (200 μM)-induced platelet aggregation (ca 80% inhibition). Attempts to measure the antiplatelet aggregation activity of crude Echis carinatus venom and its fractions were not successful due to the protein coagulation of the plasma samples after the addition of venom. Anticoagulant activities of venoms were also evaluated. Total venom of Echis carinatus showed anti coagulant activity in PT test, while its fractions showed procoagulant activity. PMID:24250552

  19. Fluorimetric determination of the active form of tetracycline, chloretetracycline and oxytetracycline in partially decomposed solutions.

    PubMed

    Regosz, A

    1977-11-01

    The content of tetracycline (1), chlortetracycline (2) and oxytetracycline (3) has been determined by use of the fluorimetric method in partially decomposed acqueous solutions of different pH values. The procedure consisted in the extraction of fluorescent calcium and 5.5-diethyl-barbituric acid complexes of 1 and 3 (with 2 calcium complex only) into an organic solvent. In the method, only complexes with undecomposed 1--3 show a strong fluorescence. Products of decomposition of the antibiotics did not affect significantly analytical results. Comparative investigations have been carried out with 1--3 using t.l.c. and turbidimetry. PMID:24855

  20. Polyacetylenes from Notopterygium incisum–New Selective Partial Agonists of Peroxisome Proliferator-Activated Receptor-Gamma

    PubMed Central

    Liu, Xin; Noha, Stefan M.; Malainer, Clemens; Kramer, Matthias P.; Cocic, Amina; Kunert, Olaf; Schinkovitz, Andreas; Heiss, Elke H.; Schuster, Daniela

    2013-01-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator of glucose and lipid metabolism and therefore an important pharmacological target to combat metabolic diseases. Since the currently used full PPARγ agonists display serious side effects, identification of novel ligands, particularly partial agonists, is highly relevant. Searching for new active compounds, we investigated extracts of the underground parts of Notopterygium incisum, a medicinal plant used in traditional Chinese medicine, and observed significant PPARγ activation using a PPARγ-driven luciferase reporter model. Activity-guided fractionation of the dichloromethane extract led to the isolation of six polyacetylenes, which displayed properties of selective partial PPARγ agonists in the luciferase reporter model. Since PPARγ activation by this class of compounds has so far not been reported, we have chosen the prototypical polyacetylene falcarindiol for further investigation. The effect of falcarindiol (10 µM) in the luciferase reporter model was blocked upon co-treatment with the PPARγ antagonist T0070907 (1 µM). Falcarindiol bound to the purified human PPARγ receptor with a Ki of 3.07 µM. In silico docking studies suggested a binding mode within the ligand binding site, where hydrogen bonds to Cys285 and Glu295 are predicted to be formed in addition to extensive hydrophobic interactions. Furthermore, falcarindiol further induced 3T3-L1 preadipocyte differentiation and enhanced the insulin-induced glucose uptake in differentiated 3T3-L1 adipocytes confirming effectiveness in cell models with endogenous PPARγ expression. In conclusion, we identified falcarindiol-type polyacetylenes as a novel class of natural partial PPARγ agonists, having potential to be further explored as pharmaceutical leads or dietary supplements. PMID:23630612

  1. Phthalide Derivatives with Anticoagulation Activities from Angelica sinensis.

    PubMed

    Zhang, Lai-Bin; Lv, Jie-Li; Liu, Jun-Wei

    2016-07-22

    Two new phthalide derivatives, angesinenolides A and B (1 and 2), were isolated from the roots of Angelica sinensis. Their structures were elucidated using HRMS, NMR, and X-ray crystallographic data. Compound 1 is the first example of a phthalide trimer presumably formed through two [2+2] cycloaddition reactions. Compound 2 is a unique dimeric phthalide with a peroxy bridge between C-3a and C-6. Both phthalides were evaluated for in vitro anticoagulation activities. Compound 1 reduced the level of fibrinogen (FIB). Compound 2 significantly extended thrombin time and activated partial thromboplastin time, as well as markedly reduced the content of FIB. PMID:27400088

  2. Aptamer and PNIPAAm co-conjugated nanoparticles regulate activity of enzyme with different temperature.

    PubMed

    Yu, Jiemiao; Yang, Liangrong; Liang, Xiangfeng; Dong, Tingting; Qu, Hongnan; Rong, Meng; Liu, Huizhou

    2016-10-01

    In this paper, we described a temperature responsive nano-system that can regulate activity of enzyme with different temperature. Temperature responsive polymer poly(N-isopropylacrylamide) (PNIPAAm), with low critical solution temperature of 32°C, was synthesized with thiol modification. PNIPAAm and thrombin aptamer were co-functionalized on the surface of gold nanoparticles for effective regulation of thrombin activity with different temperature. On the one hand, we studied the thermal responsive properties of this inhibitor via UV-visible spectroscopy. On the other hand, we investigated the regulation of thrombin activity by this platform with different temperature. The PNIPAAm chains could extend and shrink with different temperature, which suggested that PNIPAAm on the surface of gold nanoparticles could regulate interaction between thrombin and aptamer according to temperature changing. At 25°C, PNIPAAm was hydrophilic extended state, which blocked the interaction between thrombin and aptamer on the surface of gold nanoparticles, therefore thrombin activity had no change. On the contrary, at 37°C, PNIPAAm transformed from hydrophilic extended state to hydrophobic shrank state, allowing the aptamer to capture thrombin, inhibiting the activity of thrombin. More interestingly, this regulation was reverse to normal condition, where 37°C was always the optimum reaction temperature for most of human enzymes. This system we prepared was opposite, which was capable of inhibiting the thrombin activity at 37°C. Furthermore, this was the first report of regulation of thrombin activity using this temperature responsive platform. PMID:27474278

  3. The Acute Coagulopathy of Trauma is due to Impaired Initial Thrombin Generation but not Clot Formation or Clot Strength

    PubMed Central

    Harr, Jeffrey N.; Moore, Ernest E.; Wohlauer, Max V.; Droz, Nathan; Fragoso, Miguel; Banerjee, Anirban; Silliman, Christopher C.

    2011-01-01

    The Acute Coagulopathy of Trauma (ACOT) has been described as a very early hypocoagulable state, but the mechanism remains controversial. One proposed mechanism is tissue hypoperfusion leading to protein C activation, with subsequent inhibition of Factors V and VIII. Variability in trauma has impeded the use of clinical data towards the elucidation of the mechanisms of ACOT, but thrombelastography (TEG) may provide insight by assessing hemostatic function from initial thrombin activation to fibrinolysis. We hypothesized that, in a controlled animal model of trauma/hemorrhagic shock, clotting factor dysfunction is the predominant mechanism in early ACOT. Methods Rats anesthetized by inhaled isoflurane (n=6) underwent laparotomy, and hemorrhage was induced to maintain a MAP of 35 mmHg for 30 minutes. Rats were then resuscitated with twice their shed blood volume in normal saline. TEG was performed at baseline, shock, and post-resuscitation periods. No heparin was given. Statistical analysis was performed by ANOVA with post-hoc Fisher’s test. Results Coagulation factor function was significantly impaired in the early stages of trauma/hemorrhagic shock. TEG R and SP-values were significantly increased from baseline to shock (p<0.001) and from shock to post-resuscitation periods (p<0.05). Delta (R-SP), a measure of thrombin generation, showed a significant increase (p<0.05) from baseline to shock. No significant changes were found in K, Angle, MA, and LY30 values. Conclusion Clotting factor derangement leading to impaired thrombin generation is the principle etiology of ACOT in this model and not the dynamics of clot formation, fibrin cross-linking, clot strength/platelet function, or fibrinolysis. PMID:21550061

  4. A convenient sandwich assay of thrombin in biological media using nanoparticle-enhanced fluorescence polarization.

    PubMed

    Yue, Qiaoli; Shen, Tongfei; Wang, Lei; Xu, Shuling; Li, Haibo; Xue, Qingwang; Zhang, Yuanfu; Gu, Xiaohong; Zhang, Shuqiu; Liu, Jifeng

    2014-06-15

    A new aptamer biosensor was presented for the detection of thrombin in this work, which was based on fluorescence polarization (FP) using silica nanoparticles as enhancement probe. The silica nanoparticles covered by streptavidin were tagged with a thrombin aptamer (5'-biotin-GGTTGGTGTGGTTGG-3'), which was bound to the surface of silica nanoparticle through the specific interaction between streptavidin and biotin. In the presence of thrombin, it induced the aptamer to form quadruplex structure. When the other thrombin aptamer labeled with fluorescein (5'-FAM-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3') was added to the above system, a sandwich structure can form at the surface of silica nanoparticles. The fluorescence polarization was therefore enhanced and quantification between fluorescence polarization signal and concentration of thrombin was built. The sensor provided a linear range from 0.6 to 100 nM for thrombin with a detection limit of 0.20 nM (3.29 SB/m, according to the recent recommendation of IUPAC) in a homogeneous media. The same linear range was obtained in spiked human serum samples with a slightly higher detection limit (0.26 nM), demonstrating high anti-interference of the sensor in a complex biological sample matrix. And the sensor can be used to monitor spiked concentration of thrombin level in real human plasma with satisfactory results obtained. PMID:24508546

  5. Solubilization, partial purification, and reconstitution of glutamate- and N-methyl-D-aspartate-activated cation channels from brain synaptic membranes

    SciTech Connect

    Ly, A.M.; Michaelis, E.K. )

    1991-04-30

    L-Glutamate-activated cation channel proteins from rat brain synaptic membranes were solubilized, partially purified, and reconstituted into liposomes. Optimal conditions for solubilization and reconstitution included treatment of the membranes with nonionic detergents in the presence of neutral phospholipids plus glycerol. Quench-flow procedures were developed to characterize the rapid kinetics of ion flux induced by receptor agonists. ({sup 14}C)Methylamine, a cation that permeates through the open channel of both vertebrate and invertebrate glutamate receptors, was used to measure the activity of glutamate receptor-ion channel complexes in reconstituted liposomes. L-Glutamate caused an increase in the rate of ({sup 14}C)methylamine influx into liposomes reconstituted with either solubilized membrane proteins or partially purified glutamate-binding proteins. Of the major glutamate receptor agonists, only N-methyl-D-aspartate activated cation fluxes in liposomes reconstituted with glutamate-binding proteins. In liposomes reconstituted with glutamate-binding proteins, N-methyl-D-aspartate- or glutamate-induced influx of NA{sup +} led to a transient increase in the influx of the lipid-permeable anion probe S{sup 14}CN{sup {minus}}. These results indicate the functional reconstitution of N-methyl-D-aspartate-sensitive glutamate receptors and the role of the {approximately}69-kDa protein in the function of these ion channels.

  6. TORC1 activity is partially reduced under nitrogen starvation conditions in sake yeast Kyokai no. 7, Saccharomyces cerevisiae.

    PubMed

    Nakazawa, Nobushige; Sato, Aya; Hosaka, Masahiro

    2016-03-01

    Industrial yeasts are generally unable to sporulate but treatment with the immunosuppressive drug rapamycin restores this ability in a sake yeast strain Kyokai no. 7 (K7), Saccharomyces cerevisiae. This finding suggests that TORC1 is active under sporulation conditions. Here, using a reporter gene assay, Northern and Western blots, we tried to gain insight into how TORC1 function under nitrogen starvation conditions in K7 cells. Similarly to a laboratory strain, RPS26A transcription was repressed and Npr1 was dephosphorylated in K7 cells, indicative of the expected loss of TORC1 function under nitrogen starvation. The expression of nitrogen catabolite repression-sensitive genes, however, was not induced, the level of Cln3 remained constant, and autophagy was more slowly induced than in a laboratory strain, all suggestive of active TORC1. We conclude that TORC1 activity is partially reduced under nitrogen starvation conditions in K7 cells. PMID:26272416

  7. Uveal Melanoma Cell Growth Is Inhibited by Aminoimidazole Carboxamide Ribonucleotide (AICAR) Partially Through Activation of AMP-Dependent Kinase

    PubMed Central

    Al-Moujahed, Ahmad; Nicolaou, Fotini; Brodowska, Katarzyna; Papakostas, Thanos D.; Marmalidou, Anna; Ksander, Bruce R.; Miller, Joan W.; Gragoudas, Evangelos; Vavvas, Demetrios G.

    2014-01-01

    Purpose. To evaluate the effects and mechanism of aminoimidazole carboxamide ribonucleotide (AICAR), an AMP-dependent kinase (AMPK) activator, on the growth of uveal melanoma cell lines. Methods. Four different cell lines were treated with AICAR (1–4 mM). Cell growth was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. Cell cycle analysis was conducted by flow cytometry; additionally, expression of cell-cycle control proteins, cell growth transcription factors, and downstream effectors of AMPK were determined by RT-PCR and Western blot. Results. Aminoimidazole carboxamide ribonucleotide inhibited cell growth, induced S-phase arrest, and led to AMPK activation. Aminoimidazole carboxamide ribonucleotide treatment was associated with inhibition of eukaryotic translation initiation factor 4E-BP1 phosphorylation, a marker of mammalian target of rapamycin (mTOR) pathway activity. Aminoimidazole carboxamide ribonucleotide treatment was also associated with downregulation of cyclins A and D, but had minimal effects on the phosphorylation of ribosomal protein S6 or levels of the macroautophagy marker LC3B. The effects of AICAR were abolished by treatment with dipyridamole, an adenosine transporter inhibitor that blocks the entry of AICAR into cells. Treatment with adenosine kinase inhibitor 5-iodotubericidin, which inhibits the conversion of AICAR to its 5′-phosphorylated ribotide 5-aminoimidazole-4-carboxamide-1-D-ribofuranosyl-5′-monophosphate (ZMP; the direct activator of AMPK), reversed most of the growth-inhibitory effects, indicating that some of AICAR's antiproliferative effects are mediated at least partially through AMPK activation. Conclusions. Aminoimidazole carboxamide ribonucleotide inhibited uveal melanoma cell proliferation partially through activation of the AMPK pathway and downregulation of cyclins A1 and D1. PMID:24781943

  8. Salivary gland extracts of partially fed Dermacentor reticulatus ticks decrease natural killer cell activity in vitro.

    PubMed Central

    Kubes, M; Fuchsberger, N; Labuda, M; Zuffová, E; Nuttall, P A

    1994-01-01

    The salivary glands and saliva of ticks (Arachnida, Acari, Ixodida) play a vital role in blood feeding, including manipulation of the host's immune response to tick infestation. Furthermore, a diverse number of tick-borne pathogens are transmitted to vertebrate hosts via tick saliva. A factor synthesized in the salivary glands of feeding ticks potentiates the transmission of certain tick-borne viruses. We show that salivary gland extracts (SGE) derived from Dermacentor reticulatus female ticks fed for 6 days on laboratory mice (SGED6) induced a decrease in the natural killer (NK) activity of effector cells obtained from 16 healthy blood donors. The decreased activity ranged from 14 to 69% of NK activity observed with the respective untreated effector cells. Such a decrease was not observed after treatment of effector cells with SGE from unfed ticks. Ten-fold dilution of SGED6 significantly reduced the capacity to decrease NK activity and a further 10-fold dilution almost eliminated the effect. After addition of IFN-alpha 2, the SGED6-induced decrease in NK activity was restored to activity levels approaching those of untreated cells. The apparent reversibility of the inhibition indicates that the effect of SGED6 on NK activity was not due to cytotoxicity. The results demonstrate the presence of a factor(s) in the salivary gland products of feeding D. reticulatus female ticks that influences human NK activity in vitro. These data suggest a possible mechanism by which tick SGE potentiates the transmission of some tick-borne viruses through suppression of NK activity. PMID:8045588

  9. [Polymer coatings with immobilized thrombin and peptides: preparation and use for wound healing].

    PubMed

    Markvicheva, E A; Kuptsova, S V; Rumsh, L D; Dugina, T N; Lange, M A; Chistov, I V; Strukova, S M; Zubov, V P

    2002-01-01

    Polymer dressings with encapsulated thrombin or synthetic peptides which can mimic thrombin action are employed for wound healing. Paper describes the method for preparation of these hydrogel composites of PVCL-CaAlg [poly(N-vinyl caprolactam-calcium alginate). The effect of encapsulated thrombin/peptides on tissue repair process have beet investigatat in vivo experiments using a mouse model of wound healing. The developed dressings accelerated wound healing: thascan be used as a basis for creation of novel formulations with controlled drug release for wound therapy. PMID:12698556

  10. Sulfated polysaccharides from Loligo vulgaris skin: potential biological activities and partial purification.

    PubMed

    Abdelmalek, Baha Eddine; Sila, Assaâd; Krichen, Fatma; Karoud, Wafa; Martinez-Alvarez, Oscar; Ellouz-Chaabouni, Semia; Ayadi, Mohamed Ali; Bougatef, Ali

    2015-01-01

    The characteristics, biological properties, and purification of sulfated polysaccharides extracted from squid (Loligo vulgaris) skin were investigated. Their chemical and physical characteristics were determined using X-ray diffraction and infrared spectroscopic analysis. Sulfated polysaccharides from squid skin (SPSS) contained 85.06% sugar, 2.54% protein, 1.87% ash, 8.07% sulfate, and 1.72% uronic acid. The antioxidant properties of SPSS were investigated based on DPPH radical-scavenging capacity (IC50 = 19.42 mg mL(-1)), hydrogen peroxide-scavenging activity (IC50 = 0.91 mg mL(-1)), and β-carotene bleaching inhibition (IC50 = 2.79 mg mL(-1)) assays. ACE-inhibitory activity of SPSS was also investigated (IC50 = 0.14 mg mL(-1)). Further antimicrobial activity assays indicated that SPSS exhibited marked inhibitory activity against the bacterial and fungal strains tested. Those polysaccharides did not display hemolytic activity towards bovine erythrocytes. Fractionation by DEAE-cellulose column chromatography showed three major absorbance peaks. Results of this study suggest that sulfated polysaccharides from squid skin are attractive sources of polysaccharides and promising candidates for future application as dietary ingredients. PMID:25301697

  11. Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca²⁺ signalling in human platelets.

    PubMed

    Lever, Robert A; Hussain, Azhar; Sun, Benjamin B; Sage, Stewart O; Harper, Alan G S

    2015-12-01

    Rises in cytosolic Ca(2+) concentration ([Ca(2+)]cyt) are central in platelet activation, yet many aspects of the underlying mechanisms are poorly understood. Most studies examine how experimental manipulations affect agonist-evoked rises in [Ca(2+)]cyt, but these only monitor the net effect of manipulations on the processes controlling [Ca(2+)]cyt (Ca(2+) buffering, sequestration, release, entry and removal), and cannot resolve the source of the Ca(2+) or the transporters or channels affected. To investigate the effects of protein kinase C (PKC) on platelet Ca(2+) signalling, we here monitor Ca(2+) flux around the platelet by measuring net Ca(2+) fluxes to or from the extracellular space and the intracellular Ca(2+) stores, which act as the major sources and sinks for Ca(2+) influx into and efflux from the cytosol, as well as monitoring the cytosolic Na(+) concentration ([Na(+)]cyt), which influences platelet Ca(2+) fluxes via Na(+)/Ca(2+) exchange. The intracellular store Ca(2+) concentration ([Ca(2+)]st) was monitored using Fluo-5N, the extracellular Ca(2+) concentration ([Ca(2+)]ext) was monitored using Fluo-4 whilst [Ca(2+)]cyt and [Na(+)]cyt were monitored using Fura-2 and SFBI, respectively. PKC inhibition using Ro-31-8220 or bisindolylmaleimide I potentiated ADP- and thrombin-evoked rises in [Ca(2+)]cyt in the absence of extracellular Ca(2+). PKC inhibition potentiated ADP-evoked but reduced thrombin-evoked intracellular Ca(2+) release and Ca(2+) removal into the extracellular medium. SERCA inhibition using thapsigargin and 2,5-di(tert-butyl) l,4-benzohydroquinone abolished the effect of PKC inhibitors on ADP-evoked changes in [Ca(2+)]cyt but only reduced the effect on thrombin-evoked responses. Thrombin evokes substantial rises in [Na(+)]cyt which would be expected to reduce Ca(2+) removal via the Na(+)/Ca(2+) exchanger (NCX). Thrombin-evoked rises in [Na(+)]cyt were potentiated by PKC inhibition, an effect which was not due to altered changes in non

  12. Effect of short term external perturbations on bacterial ecology and activities in a partial nitritation and anammox reactor.

    PubMed

    Wu, Sha; Bhattacharjee, Ananda S; Weissbrodt, David G; Morgenroth, Eberhard; Goel, Ramesh

    2016-11-01

    This research investigated the short term effects of temperature changes (lasting 2-4weeks each) from 35±2°C to 21±2°C and 13±2°C and sulfide toxicity on partial nitrification-anammox (PN/A) system. Temperatures below 20°C and sulfide content as low as 5mgSL(-1) affected both aerobic and anaerobic catabolic activities of ammonia oxidation and the expression of related functional gene markers. The activity of AOB was inversely correlated with ammonium monooxygenase (amoA) gene expression. In contrast, the activity of AMX bacteria was positively correlated with the expression of their hydrazine synthase (hzsA) gene. Although the overall activities of AMX bacteria decreased at lower temperatures, the AMX bacteria were still active at the low temperatures. The inverse correlation between amoA gene expressions and the corresponding AOB activities was surprising. 16S rDNA based high throughput amplicon sequencing revealed the dominance of Chloroflexi, Planctomycetes and Proteobacteria phyla the distribution of which changed with temperature changes. PMID:27522119

  13. Evaluation of cytotoxic and anti-tumor activity of partially purified serine protease isolate from the Indian earthworm Pheretima posthuma

    PubMed Central

    Verma, Mahendra Kumar; Xavier, Francies; Verma, Yogendra Kumar; Sobha, Kota

    2013-01-01

    Objective To isolate, partially purify and evaluate the cytotoxic and antitumor activity of a serine protease from the chosen Indian earthworm Pheretima posthuma. Methods Whole animal extract was prepared and purified its protein constituents by size and charge based chromatographic separation techniques using Sephadex G-50 and DEAE-Cellulose resin respectively. Average molecular weight of the protein isolate was determined and analyzed for its cytotoxic property against Vero cells in different dilutions (1: 20 and 1: 40) and anti-tumor activity by MTT assay (a colorimetric assay) using breast cancer cell line MCF-7, with tamoxifen as standard. Results One of the protein constituents after purification was characterized as serine protease by Caseinolytic plate diffusion assay. Average molecular weight of this purified isolate was determined, by SDS-PAGE analysis with standard protein ladder, as of 15 kDa. The performed tests suggested that the 15kDa fraction has potent cytotoxic activity and satisfactory antitumor activity as well in vitro. Conclusions Exact molecular mechanism of the cytotoxic and antitumor activities is yet to be explored and currently we are working on ultra-purification and biophysical characterization of this fraction. Further investigation into the mechanism(s) of cytotoxic and antitumor activities at molecular level would be useful in treatment of various classes of cancer and viral infections in future.

  14. Limitation in tidal volume expansion partially determines the intensity of physical activity in COPD.

    PubMed

    Kortianou, Eleni A; Aliverti, Andrea; Louvaris, Zafeiris; Vasilopoulou, Maroula; Nasis, Ioannis; Asimakos, Andreas; Zakynthinos, Spyros; Vogiatzis, Ioannis

    2015-01-01

    In patients with chronic obstructive pulmonary disease (COPD), reduced levels of daily physical activity are associated with the degree of impairment in lung, peripheral muscle, and central hemodynamic function. There is, however, limited evidence as to whether limitations in tidal volume expansion also, importantly, determine daily physical activity levels in COPD. Eighteen consecutive patients with COPD [9 active (forced expiratory volume in 1 s, FEV1: 1.59 ± 0.64 l) with an average daily movement intensity >1.88 m/s(2) and 9 less active patients (FEV1: 1.16 ± 0.41 l) with an average intensity <1.88 m/s(2)] underwent a 4-min treadmill test at a constant speed corresponding to each individual patient's average movement intensity, captured by a triaxial accelerometer during a preceding 7-day period. When chest wall volumes, captured by optoelectronic plethysmography, were expressed relative to comparable levels of minute ventilation (ranging between 14.5 ± 4.3 to 33.5 ± 4.4 l/min), active patients differed from the less active ones in terms of the lower increase in end-expiratory chest wall volume (by 0.15 ± 0.17 vs. 0.45 ± 0.21 l), the greater expansion in tidal volume (by 1.76 ± 0.58 vs. 1.36 ± 0.24 l), and the larger inspiratory reserve chest wall volume (IRVcw: by 0.81 ± 0.25 vs. 0.39 ± 0.27 l). IRVcw (r(2) = 0.420), expiratory flow (r(2) change = 0.174), and Borg dyspnea score (r(2) change = 0.123) emerged as the best contributors, accounting for 71.7% of the explained variance in daily movement intensity. Patients with COPD exhibiting greater ability to expand tidal volume and to maintain adequate inspiratory reserve volume tend to be more physically active. Thus interventions aiming at mitigating restrictions on operational chest wall volumes are expected to enhance daily physical activity levels in COPD. PMID:25398190

  15. Sustained Oxidative Stress Inhibits NF-kB Activation Partially via Inactivating the Proteasome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    NF-kB is a family of important transcription factors involved in many cellular functions, such as cell survival, proliferation and stress responses. Many studies indicate that NF-kB is a stress sensitive transcription factor and its activation is regulated by reactive oxygen species. In previous s...

  16. Antifatigue Activity of Liquid Cultured Tricholoma matsutake Mycelium Partially via Regulation of Antioxidant Pathway in Mouse

    PubMed Central

    Li, Quan; Wang, Yanzhen; Cai, Guangsheng; Kong, Fange; Wang, Xiaohan; Liu, Yang; Yang, Chuanbin; Wang, Di; Teng, Lirong

    2015-01-01

    Tricholoma matsutake has been popular as food and biopharmaceutical materials in Asian countries for its various pharmacological activities. The present study aims to analyze the antifatigue effects on enhancing exercise performance of Tricholoma matsutake fruit body (ABM) and liquid cultured mycelia (TM) in mouse model. Two-week Tricholoma matsutake treatment significantly enhances the exercise performance in weight-loaded swimming, rotating rod, and forced running test. In TM- and ABM-treated mice, some factors were observed at 60 min after swimming compared with nontreated mice, such as the increased levels of adenosine triphosphate (ATP), antioxidative enzymes, and glycogen and the reduced levels of malondialdehyde and reactive oxygen species in muscle, liver, and/or serum. Further data obtained from western blot show that CM and ABM have strongly enhanced the activation of 5′-AMP-activated protein kinase (AMPK), and the expressions of peroxisome proliferator have activated receptor γ coactivator-1α (PGC-1α) and phosphofructokinase-1 (PFK-1) in liver. Our data suggest that both Tricholoma matsutake fruit body and liquid cultured mycelia possess antifatigue effects related to AMPK-linked antioxidative pathway. The information uncovered in our study may serve as a valuable resource for further identification and provide experimental evidence for clinical trials of Tricholoma matsutake as an effective agent against fatigue related diseases. PMID:26697489

  17. 75 FR 70208 - Certain Activated Carbon From the People's Republic of China: Final Results and Partial...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-17

    ... Second Antidumping Duty Administrative Review, and Preliminary Rescission in Part, 75 FR 26927 (May 13... review was requested. See Initiation of Antidumping and Countervailing Duty Administrative Reviews, 74 FR... Administrative Review, 74 FR 31690 (July 2, 2009). \\3\\ See Certain Activated Carbon From the People's Republic...

  18. Effect of Fagonia arabica on thrombin induced release of t-PA and complex of PAI-1 tPA in cultured HUVE cells.

    PubMed

    Aloni, Prutha D; Nayak, Amit R; Chaurasia, Sweta R; Deopujari, Jayant Y; Chourasia, Chhaya; Purohit, Hemant J; Taori, Girdhar M; Daginawala, Hatim F; Kashyap, Rajpal S

    2016-07-01

    Fagonia arabica (FA) possesses a thrombolytic property which has been earlier reported in our laboratory. Current study was undertaken to investigate the effect of aqueous extract of FA on thrombin-induced tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) release from cultured human umbilical vein endothelial cell line (HUVE) for studying its clot lytic activity. For this, establishment of cell line model has been done by isolating the cells from human umbilical cord. Cell toxicity was evaluated using XTT assay. Estimation of t-PA and PAI-1 t-PA complex were done using ELISA technique. Thrombin treatment induces the t-PA and PAI-1 release from HUVE cell line, and FA treatment was found to antagonize the thrombin induced t-PA and PAI-1 release. Our preliminary results suggest that FA may be used as an alternative to thrombolytic drug. However, study demands further experiments using animal model of thrombosis to establish the role of FA as a novel thrombolytic drug. PMID:27419084

  19. An Investigation of the Characteristics of the Enzyme Thrombin, Suitable for Classwork

    ERIC Educational Resources Information Center

    Blofield, B. Ann

    1972-01-01

    Shows how a simple investigation of the enzyme, thrombin, can provide a series of experiments giving information on enzyme characteristics. The results also provide a basis for discussion of the coagulation mechanism and related phenomena. (Author/AL)

  20. Human Thrombin Detection Through a Sandwich Aptamer Microarray: Interaction Analysis in Solution and in Solid Phase

    PubMed Central

    Sosic, Alice; Meneghello, Anna; Cretaio, Erica; Gatto, Barbara

    2011-01-01

    We have developed an aptamer-based microarray for human thrombin detection exploiting two non-overlapping DNA thrombin aptamers recognizing different exosites of the target protein. The 15-mer aptamer (TBA1) binds the fibrinogen-binding site, whereas the 29-mer aptamer (TBA2) binds the heparin binding domain. Extensive analysis on the complex formation between human thrombin and modified aptamers was performed by Electrophoresis Mobility Shift Assay (EMSA), in order to verify in solution whether the chemical modifications introduced would affect aptamers/protein recognition. The validated system was then applied to the aptamer microarray, using the solid phase system devised by the solution studies. Finally, the best procedure for Sandwich Aptamer Microarray (SAM) and the specificity of the sandwich formation for the developed aptasensor for human thrombin were optimized. PMID:22163703

  1. Partial in vitro analysis of toxic and antigenic activities of eleven Peruvian pitviper snake venoms.

    PubMed

    Guerra-Duarte, C; Lopes-Peixoto, J; Fonseca-de-Souza, B R; Stransky, S; Oliveira, D; Schneider, F S; Lopes-de-Souza, L; Bonilla, C; Silva, W; Tintaya, B; Yarleque, A; Chávez-Olórtegui, C

    2015-12-15

    This work used eleven Peruvian snake venoms (Bothrops andianus, Bothrops atrox, Bothrops barnetti, Bothrops castelnaudi, Bothriopsis chloromelas, Bothrocophias microphthalmus, Bothrops neuwiedi, Bothriopsis oligolepis, Bothriopsis peruviana, Bothrops pictus and Bothriopsis taeniata) to perform in vitro experimentation and determine its main characteristics. Hyaluronidase (HYAL), phospholipase A2 (PLA2), snake venom metalloproteinase (SVMP), snake venom serine protease (SVSP) and L-amino acid oxidase (LAAO) activities; toxicity by cell viability assays using MGSO3, VERO and HeLa cell lineages; and crossed immunoreactivity with Peruvian (PAV) and Brazilian (BAV) antibothropic polyvalent antivenoms, through ELISA and Western Blotting assays, were determined. Results show that the activities tested in this study were not similar amongst the venoms and each species present their own peculiarities, highlighting the diversity within Bothrops complex. All venoms were capable of reducing cell viability of all tested lineages. It was also demonstrated the crossed recognition of all tested venoms by both antivenoms. PMID:26365916

  2. Activity-dependent changes in partial VAMP complexes during neurotransmitter release.

    PubMed

    Hua, S Y; Charlton, M P

    1999-12-01

    The temporal sequence of SNARE protein interactions that cause exocytosis is unknown. Blockade of synaptic neurotransmitter release through cleavage of VAMP/synaptobrevin by tetanus toxin light chain (TeNT-LC) was accelerated by nerve stimulation. Botulinum/B neurotoxin light chain (BoNT/B-LC), which cleaves VAMP at the same site as TeNT-LC, did not require stimulation. Because TeNT-LC requires the N-terminal coil domain of VAMP for binding but BoNT/B-LC requires the C-terminal coil domain, it seems that, before nerve activity, the N-terminal domain is shielded in a protein complex, but the C-terminal domain is exposed. This N-terminal complex lasts until nerve activity occurs and may serve to cock synaptic vesicles for immediate exocytosis upon Ca2+ entry. PMID:10570484

  3. Reconstitution of glucotoxic HIT-T15 cells with somatostatin transcription factor-1 partially restores insulin promoter activity.

    PubMed

    Harmon, J S; Tanaka, Y; Olson, L K; Robertson, R P

    1998-06-01

    activity after STF-1 transfection. We conclude that loss of RIPE-3b1 activity precedes loss of STF-1 activity in glucotoxic HIT-T15 cells and that defective promoter activity can be partially restored by STF-1 transfection and predict that eventual cloning of the RIPE-3b1 gene will allow cotransfection studies with both factors that will allow full reconstitution of insulin promoter activity. PMID:9604866

  4. Linking mutagenic activity to micropollutant concentrations in wastewater samples by partial least square regression and subsequent identification of variables.

    PubMed

    Hug, Christine; Sievers, Moritz; Ottermanns, Richard; Hollert, Henner; Brack, Werner; Krauss, Martin

    2015-11-01

    We deployed multivariate regression to identify compounds co-varying with the mutagenic activity of complex environmental samples. Wastewater treatment plant (WWTP) effluents with a large share of industrial input of different sampling dates were evaluated for mutagenic activity by the Ames Fluctuation Test and chemically characterized by a screening for suspected pro-mutagens and non-targeted software-based peak detection in full scan data. Areas of automatically detected peaks were used as predictor matrix for partial least squares projections to latent structures (PLS) in combination with measured mutagenic activity. Detected peaks were successively reduced by the exclusion of all peaks with lowest variable importance until the best model (high R(2) and Q(2)) was reached. Peaks in the best model co-varying with the observed mutagenicity showed increased chlorine, bromine, sulfur, and nitrogen abundance compared to original peak set indicating a preferential selection of anthropogenic compounds. The PLS regression revealed four tentatively identified compounds, newly identified 4-(dimethylamino)-pyridine, and three known micropollutants present in domestic wastewater as co-varying with the mutagenic activity. Co-variance between compounds stemming from industrial wastewater and mutagenic activity supported the application of "virtual" EDA as a statistical tool to separate toxicologically relevant from less relevant compounds. PMID:26070082

  5. Purification, crystallization and preliminary X-ray diffraction analysis of saxthrombin, a thrombin-like enzyme from Gloydius saxatilis venom

    SciTech Connect

    Wei, Wenqing; Zhao, Wei; Wang, Xiaoping; Teng, Maikun Niu, Liwen

    2007-08-01

    The thrombin-like enzyme saxthrombin has been purified from G. saxatilis snake venom. Crystallization conditions were found and a data set was obtained to 1.43 Å. The snake-venom thrombin-like enzymes (SVTLEs) are a class of serine proteinases that show fibrinogen-clotting and esterolytic activities. Most TLEs convert fibrinogen to fibrin by releasing either fibrinopeptide A or fibrinopeptide B and cannot activate factor XIII. The enzymes hydrolyze fibrinogen to produce non-cross-linked fibrins, which are susceptible to the lytic action of plasmin. Because of these physiological properties, TLEs have important medical applications in myocardial infarction, ischaemic stroke and thrombotic diseases. Here, a three-step chromatography procedure was used to purify saxthrombin (AAP20638) from Gloydius saxatilis venom to homogeneity. Its molecular weight is about 30 kDa as estimated by SDS–PAGE. A saxthrombin crystal was obtained using the hanging-drop vapour-diffusion method and diffracted to a resolution limit of 1.43 Å. The crystal belongs to space group C2, with unit-cell parameters a = 97.23, b = 52.21, c = 50.10 Å, β = 96.72°, and the Matthews coefficient (V{sub M}) was calculated to be 2.13 Å{sup 3} Da{sup −1} with one molecule in the asymmetric unit.

  6. Percutaneous Repair of Radial Artery Pseudoaneurysm in a Hemodialysis Patient Using Sonographically Guided Thrombin Injection

    SciTech Connect

    Corso, Rocco Rampoldi, Antonio; Vercelli, Ruggero; Leni, Davide; Vanzulli, Angelo

    2006-02-15

    We report a case of a radial artery pseudoaneurysm complicating an incorrect puncture of a Brescia-Cimino hemodialysis fistula that was treated with percutaneous ultrasound-guided thrombin injection. The pseudoaneurysm recurred after the initial successful thrombin injection. With a second injection we obtained permanent pseudoaneurysm occlusion. Our case illustrates that this procedure is an effective treatment in this type of arteriovenous fistula complication. We compare this case with the only similar one we could find in the literature.

  7. Study of a mechanism responsible for potential antidepressant activity of EMD 386088, a 5-HT6 partial agonist in rats.

    PubMed

    Jastrzębska-Więsek, Magdalena; Siwek, Agata; Partyka, Anna; Antkiewicz-Michaluk, Lucyna; Michaluk, Jerzy; Romańska, Irena; Kołaczkowski, Marcin; Wesołowska, Anna

    2016-08-01

    It was shown that 5-HT6 receptor agonists can exert pharmacological activity due to various modifications in monoamines' level and metabolism activity in rats' brain structures. This finding was correlated with antidepressant- or anxiolytic-like properties of these compounds. The study was designed to establish a possible mechanism of the antidepressant-like activity of the partial 5-HT6 receptor agonist EMD386088 (5-chloro-2-methyl-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole hydrochloride) in rats. The concentrations of monoamines (dopamine (DA), noradrenaline (NA), and serotonin (5-HT)) and the rate of their metabolism were measured ex vivo in the brain structures (hippocampus, nucleus accumbens, striatum) using high-performance liquid chromatography (HPLC). The rats were killed after the forced swim test (FST); the collected tissue samples were used to ex vivo experiments. The potency of EMD386088 to blockade dopamine transporter (DAT) was tested in a functional in vitro study. FST was used to assess the involvement of D1- and D2-like receptor subfamilies in antidepressant-like properties of EMD386088. Neurochemical data from ex vivo experiments showed that antiimmobility activity of EMD386088 may be connected with the activation of dopaminergic system, while neither noradrenergic nor serotonergic ones are involved in its effect. EMD386088 also possesses a significant affinity for DAT which may be a mechanism in the abovementioned effect. Behavioral data seem to confirm the importance of dopaminergic system activation in antidepressant-like activity of EMD386088, since this effect, observed in the FST, was abolished by the preferential D1- and D2-like receptor subfamily antagonists SCH23390 and sulpiride, respectively. Dopaminergic system is involved in antidepressant-like activity of EMD386088. PMID:27106213

  8. Partial purification and characterization of an antimicrobial activity from the wood extract of mangrove plant Ceriops decandra

    PubMed Central

    Simlai, Aritra; Mukherjee, Kalishankar; Mandal, Anurup; Bhattacharya, Kashinath; Samanta, Amalesh; Roy, Amit

    2016-01-01

    The development of resistance towards the antibiotics in use today has been a source of growing concern in the modern healthcare system around the world. To counter this major threat, there is an urgent need for discovery of new antimicrobials. Many plants, like mangroves, possess highly diversified list of natural phytochemicals which are known to have wide range of bioactivities. These phytochemicals can be good sources for the discovery of new drugs. In this study, we report the partial phytochemical characterization and antimicrobial activities of a semi-purified fraction isolated from the wood tissue of Ceriops decandra, a mangrove plant. This fraction named CD-3PM was chromatographically separated from C. decandra wood extract and was subjected to different spectral analyses to determine its partial chemical nature. The structural investigation indicates the presence of two diterpenoids, i) 3β, 13β-Dihydroxy-8-abietaen-7-one and ii) 3β-Hydroxy-8,13-abietadien-7-one in the CD-3PM fraction. The antimicrobial potential of this fraction was evaluated by microdilution-MTT assay against several organisms. Among the nine microorganisms found to be sensitive to the CD-3PM fraction, six organisms are reported to be pathogenic in nature. The CD-3PM fraction with broad spectrum antimicrobial efficacy revealed the presence of two diterpenoids and possesses potential applications in drug discovery process and food processing industries. PMID:27065777

  9. Partial purification and characterization of an antimicrobial activity from the wood extract of mangrove plant Ceriops decandra.

    PubMed

    Simlai, Aritra; Mukherjee, Kalishankar; Mandal, Anurup; Bhattacharya, Kashinath; Samanta, Amalesh; Roy, Amit

    2016-01-01

    The development of resistance towards the antibiotics in use today has been a source of growing concern in the modern healthcare system around the world. To counter this major threat, there is an urgent need for discovery of new antimicrobials. Many plants, like mangroves, possess highly diversified list of natural phytochemicals which are known to have wide range of bioactivities. These phytochemicals can be good sources for the discovery of new drugs. In this study, we report the partial phytochemical characterization and antimicrobial activities of a semi-purified fraction isolated from the wood tissue of Ceriops decandra, a mangrove plant. This fraction named CD-3PM was chromatographically separated from C. decandra wood extract and was subjected to different spectral analyses to determine its partial chemical nature. The structural investigation indicates the presence of two diterpenoids, i) 3β, 13β-Dihydroxy-8-abietaen-7-one and ii) 3β-Hydroxy-8,13-abietadien-7-one in the CD-3PM fraction. The antimicrobial potential of this fraction was evaluated by microdilution-MTT assay against several organisms. Among the nine microorganisms found to be sensitive to the CD-3PM fraction, six organisms are reported to be pathogenic in nature. The CD-3PM fraction with broad spectrum antimicrobial efficacy revealed the presence of two diterpenoids and possesses potential applications in drug discovery process and food processing industries. PMID:27065777

  10. Semi-active control of a sandwich beam partially filled with magnetorheological elastomer

    NASA Astrophysics Data System (ADS)

    Dyniewicz, Bartłomiej; Bajkowski, Jacek M.; Bajer, Czesław I.

    2015-08-01

    The paper deals with the semi-active control of vibrations of structural elements. Elastomer composites with ferromagnetic particles that act as magnetorheological fluids are used. The damping coefficient and the shear modulus of the elastomer increases when it is exposed to an electro-magnetic field. The control of this process in time allows us to reduce vibrations more effectively than if the elastomer is permanently exposed to a magnetic field. First the analytical solution for the vibrations of a sandwich beam filled with an elastomer is given. Then the control problem is defined and applied to the analytical formula. The numerical solution of the minimization problem results in a periodic, perfectly rectangular control function if free vibrations are considered. Such a temporarily acting magnetic field is more efficient than a constantly acting one. The surplus reaches 20-50% or more, depending on the filling ratio of the elastomer. The resulting control was verified experimentally in the vibrations of a cantilever sandwich beam. The proposed semi-active control can be directly applied to engineering vibrating structural elements, for example helicopter rotors, aircraft wings, pads under machines, and vehicles.

  11. Characterization of DicB by partially masking its potent inhibitory activity of cell division.

    PubMed

    Yang, Shaoyuan; Pei, Hairun; Zhang, Xiaoying; Wei, Qiang; Zhu, Jia; Zheng, Jimin; Jia, Zongchao

    2016-07-01

    DicB, a protein encoded by the Kim (Qin) prophage in Escherichia coli, inhibits cell division through interaction with MinC. Thus far, characterization of DicB has been severely hampered owing to its potent activity which ceases cell division and leads to cell death. In this work, through fusing maltose-binding protein to the N-terminus of DicB (MBP-DicB), we successfully expressed and purified recombinant DicB that enabled in vitro analysis for the first time. More importantly, taking advantage of the reduced inhibitory activity of MBP-DicB, we were able to study its effects on cell growth and morphology. Inhibition of cell growth by MBP-DicB was systematically evaluated using various DicB constructs, and their corresponding effects on cell morphology were also investigated. Our results revealed that the N-terminal segment of DicB plays an essential functional role, in contrast to its C-terminal tail. The N-terminus of DicB is of critical importance as even the first amino acid (following the initial Met) could not be removed, although it could be mutated. This study provides the first glimpse of the molecular determinants underlying DicB's function. PMID:27466443

  12. Characterization of DicB by partially masking its potent inhibitory activity of cell division

    PubMed Central

    Yang, Shaoyuan; Pei, Hairun; Zhang, Xiaoying; Wei, Qiang; Zhu, Jia; Zheng, Jimin; Jia, Zongchao

    2016-01-01

    DicB, a protein encoded by the Kim (Qin) prophage in Escherichia coli, inhibits cell division through interaction with MinC. Thus far, characterization of DicB has been severely hampered owing to its potent activity which ceases cell division and leads to cell death. In this work, through fusing maltose-binding protein to the N-terminus of DicB (MBP–DicB), we successfully expressed and purified recombinant DicB that enabled in vitro analysis for the first time. More importantly, taking advantage of the reduced inhibitory activity of MBP–DicB, we were able to study its effects on cell growth and morphology. Inhibition of cell growth by MBP–DicB was systematically evaluated using various DicB constructs, and their corresponding effects on cell morphology were also investigated. Our results revealed that the N-terminal segment of DicB plays an essential functional role, in contrast to its C-terminal tail. The N-terminus of DicB is of critical importance as even the first amino acid (following the initial Met) could not be removed, although it could be mutated. This study provides the first glimpse of the molecular determinants underlying DicB's function. PMID:27466443

  13. Thrombin use in surgery: an evidence-based review of its clinical use

    PubMed Central

    Ham, Sung W; Lew, Wesley K; Weaver, Fred A

    2010-01-01

    When surgical ligation of bleeding fails, or is not possible, surgeons rely on a number of hemostatic aids, including thrombin. This review discusses the history, pharmacology and clinical application of thrombin as a surgical hemostat. The initial thrombin was bovine in origin, but its use has been complicated by the formation of antibodies that cross-react with human coagulation factors. This has been associated with life-threatening bleeding and in some circumstances anaphylaxis and death. Human thrombin, isolated from pooled plasma of donors, was developed in an effort to minimize these risks, but its downsides are its limited availability and the potential for transmitting blood-borne pathogens. Recently a recombinant thrombin has been developed, and approved for use by the FDA. It has the advantage of being minimally antigenic and devoid of the risk of viral transmission. Thrombin is often used in conjunction with other hemostatic aids, including absorbable agents such as Gelfoam, and with fibrinogen in fibrin glues. The last part of this review will discuss these agents in detail, and review their clinical applications. PMID:22282693

  14. Sensitive and antifouling impedimetric aptasensor for the determination of thrombin in undiluted serum sample.

    PubMed

    Qi, Honglan; Shangguan, Li; Li, Congcong; Li, Xiaoxia; Gao, Qiang; Zhang, Chengxiao

    2013-01-15

    A highly sensitive and attractive antifouling impedimetric aptasensor for the determination of thrombin in undiluted serum sample was developed. The aptasensor was fabricated by co-assembling thiol-modified anti-thrombin binding aptamer, dithiothreitol and mercaptohexanol on the surface of gold electrode. The performance of aptasensor was characterized by atomic force microscopy, contact angle and electrochemical impedance spectroscopy. In the measurement of thrombin, the change in interfacial electron transfer resistance of aptasensor was monitored using a redox couple of Fe(CN)(6)(3-/4-). The increase in the electron transfer resistance was linearly proportional to the concentration of thrombin in the range from 1.0 to 20ng/mL and a detection limit of 0.3ng/mL thrombin was achieved. The fabricated aptasensor displayed attractive antifouling properties and allowed direct quantification of extrinsic thrombin down to 0.08ng/mL in undiluted serum sample. This work provides a promising strategy for clinical application with impressive sensitivity and antifouling characteristics. PMID:22884002

  15. An exonuclease-assisted amplification electrochemical aptasensor of thrombin coupling "signal on/off" strategy.

    PubMed

    Bao, Ting; Wen, Wei; Zhang, Xiuhua; Wang, Shengfu

    2015-02-20

    In this work, a dual-signaling electrochemical aptasensor based on exonuclease-catalyzed target recycling was developed for thrombin detection. The proposed aptasensor coupled "signal-on" and "signal-off" strategies. As to the construction of the aptasensor, ferrocene (Fc) labeled thrombin binding aptamer (TBA) could perfectly hybridize with the methylene blue (MB) modified thiolated capture DNA to form double-stranded structure, hence emerged two different electrochemical signals. In the presence of thrombin, TBA could form a G-quadruplex structure with thrombin, leading to the dissociation of TBA from the duplex DNA and capture DNA formed hairpin structure. Exonuclease could selectively digest single-stranded TBA in G-quadruplex structure and released thrombin to realize target recycling. As a consequence, the electrochemical signal of MB enhanced significantly, which realized "signal on" strategy, meanwhile, the deoxidization peak current of Fc decreased distinctly, which realized "signal off" strategy. The employment of exonuclease and superposition of two signals significantly improved the sensitivity of the aptasensor. In this way, an aptasensor with high sensitivity, good stability and selectivity for quantitative detection of thrombin was constructed, which exhibited a good linear range from 5 pM to 50 nM with a detection limit of 0.9 pM (defined as S/N=3). In addition, this design strategy could be applied to the detection of other proteins and small molecules. PMID:25682249

  16. Constitutive Activation of the Nlrc4 Inflammasome Prevents Hepatic Fibrosis and Promotes Hepatic Regeneration after Partial Hepatectomy

    PubMed Central

    DeSantis, David A.; Ko, Chih-Wei; Wang, Lan; Lee, Peter; Croniger, Colleen M.

    2015-01-01

    TThe molecular mechanisms responsible for the development of hepatic fibrosis are not fully understood. The Nlrc4 inflammasome detects cytosolic presence of bacterial components, activating inflammatory cytokines to facilitate clearance of pathogens and infected cells. We hypothesized that low-grade constitutive activation of the Nlrc4 inflammasome may lead to induced hepatocyte proliferation and prevent the development of hepatic fibrosis. The gene of Nlrc4 contains two single nucleotide polymorphisms (SNPs), one located within the Nlrc4 promoter and one contained within exon 5. These SNPs regulate Nlrc4 gene transcription and activation as measured through gene reporter assays and IL-1β secretion. The 17C-6 mice have increased IL-1β in plasma after chronic carbon tetrachloride (CCl4) administration compared to B6 mice. After two-thirds partial hepatectomy (2/3PH) 17C-6 mice have earlier restoration of liver mass with greater cyclin D1 protein and BrdU incorporation compared to B6 mice at several time points. These data reveal mild constitutive activation of the Nlrc4 inflammasome as the results of two SNPs, which leads to the stimulation of hepatocyte proliferation. The increased liver regeneration induces rapid liver mass recovery after hepatectomy and may prevent the development of hepatotoxin-induced liver fibrosis. PMID:26635450

  17. Vipegitide: a folded peptidomimetic partial antagonist of α2β1 integrin with antiplatelet aggregation activity

    PubMed Central

    Momic, Tatjana; Katzhendler, Jehoshua; Shai, Ela; Noy, Efrat; Senderowitz, Hanoch; Eble, Johannes A; Marcinkiewicz, Cezary; Varon, David; Lazarovici, Philip

    2015-01-01

    Linear peptides containing the sequence WKTSRTSHY were used as lead compounds to synthesize a novel peptidomimetic antagonist of α2β1 integrin, with platelet aggregation-inhibiting activity, named Vipegitide. Vipegitide is a 13-amino acid, folded peptidomimetic molecule, containing two α-aminoisobutyric acid residues at positions 6 and 8 and not stable in human serum. Substitution of glycine and tryptophan residues at positions 1 and 2, respectively, with a unit of two polyethylene glycol (PEG) molecules yielded peptidomimetic Vipegitide-PEG2, stable in human serum for over 3 hours. Vipegitide and Vipegitide-PEG2 showed high potency (7×10−10 M and 1.5×10−10 M, respectively) and intermediate efficacy (40% and 35%, respectively) as well as selectivity toward α2 integrin in inhibition of adhesion of α1/α2 integrin overexpressing cells toward respective collagens. Interaction of both peptidomimetics with extracellular active domain of α2 integrin was confirmed in cell-free binding assay with recombinant α2 A-domain. Integrin α2β1 receptor is found on the platelet membrane and triggers collagen-induced platelet aggregation. Vipegitide and Vipegitide-PEG2 inhibited α2β1 integrin-mediated adhesion of human and murine platelets under the flow condition, by 50%. They efficiently blocked adenosine diphosphate- and collagen I-induced platelet aggregation in platelet rich plasma and whole human blood. Higher potency of Vipegitide than Vipegitide-PEG2 is consistent with results of computer modeling of the molecules in water. These peptidomimetic molecules were acutely tolerated in mice upon intravenous bolus injection of 50 mg/kg. These results underline the potency of Vipegitide and Vipegitide-PEG2 molecules as platelet aggregation-inhibiting drug lead compounds in antithrombotic therapy. PMID:25609915

  18. Partial Activation of SA- and JA-Defensive Pathways in Strawberry upon Colletotrichum acutatum Interaction.

    PubMed

    Amil-Ruiz, Francisco; Garrido-Gala, José; Gadea, José; Blanco-Portales, Rosario; Muñoz-Mérida, Antonio; Trelles, Oswaldo; de Los Santos, Berta; Arroyo, Francisco T; Aguado-Puig, Ana; Romero, Fernando; Mercado, José-Ángel; Pliego-Alfaro, Fernando; Muñoz-Blanco, Juan; Caballero, José L

    2016-01-01

    Understanding the nature of pathogen host interaction may help improve strawberry (Fragaria × ananassa) cultivars. Plant resistance to pathogenic agents usually operates through a complex network of defense mechanisms mediated by a diverse array of signaling molecules. In strawberry, resistance to a variety of pathogens has been reported to be mostly polygenic and quantitatively inherited, making it difficult to associate molecular markers with disease resistance genes. Colletotrichum acutatum spp. is a major strawberry pathogen, and completely resistant cultivars have not been reported. Moreover, strawberry defense network components and mechanisms remain largely unknown and poorly understood. Assessment of the strawberry response to C. acutatum included a global transcript analysis, and acidic hormones SA and JA measurements were analyzed after challenge with the pathogen. Induction of transcripts corresponding to the SA and JA signaling pathways and key genes controlling major steps within these defense pathways was detected. Accordingly, SA and JA accumulated in strawberry after infection. Contrastingly, induction of several important SA, JA, and oxidative stress-responsive defense genes, including FaPR1-1, FaLOX2, FaJAR1, FaPDF1, and FaGST1, was not detected, which suggests that specific branches in these defense pathways (those leading to FaPR1-2, FaPR2-1, FaPR2-2, FaAOS, FaPR5, and FaPR10) were activated. Our results reveal that specific aspects in SA and JA dependent signaling pathways are activated in strawberry upon interaction with C. acutatum. Certain described defense-associated transcripts related to these two known signaling pathways do not increase in abundance following infection. This finding suggests new insight into a specific putative molecular strategy for defense against this pathogen. PMID:27471515

  19. Partial Activation of SA- and JA-Defensive Pathways in Strawberry upon Colletotrichum acutatum Interaction

    PubMed Central

    Amil-Ruiz, Francisco; Garrido-Gala, José; Gadea, José; Blanco-Portales, Rosario; Muñoz-Mérida, Antonio; Trelles, Oswaldo; de los Santos, Berta; Arroyo, Francisco T.; Aguado-Puig, Ana; Romero, Fernando; Mercado, José-Ángel; Pliego-Alfaro, Fernando; Muñoz-Blanco, Juan; Caballero, José L.

    2016-01-01

    Understanding the nature of pathogen host interaction may help improve strawberry (Fragaria × ananassa) cultivars. Plant resistance to pathogenic agents usually operates through a complex network of defense mechanisms mediated by a diverse array of signaling molecules. In strawberry, resistance to a variety of pathogens has been reported to be mostly polygenic and quantitatively inherited, making it difficult to associate molecular markers with disease resistance genes. Colletotrichum acutatum spp. is a major strawberry pathogen, and completely resistant cultivars have not been reported. Moreover, strawberry defense network components and mechanisms remain largely unknown and poorly understood. Assessment of the strawberry response to C. acutatum included a global transcript analysis, and acidic hormones SA and JA measurements were analyzed after challenge with the pathogen. Induction of transcripts corresponding to the SA and JA signaling pathways and key genes controlling major steps within these defense pathways was detected. Accordingly, SA and JA accumulated in strawberry after infection. Contrastingly, induction of several important SA, JA, and oxidative stress-responsive defense genes, including FaPR1-1, FaLOX2, FaJAR1, FaPDF1, and FaGST1, was not detected, which suggests that specific branches in these defense pathways (those leading to FaPR1-2, FaPR2-1, FaPR2-2, FaAOS, FaPR5, and FaPR10) were activated. Our results reveal that specific aspects in SA and JA dependent signaling pathways are activated in strawberry upon interaction with C. acutatum. Certain described defense-associated transcripts related to these two known signaling pathways do not increase in abundance following infection. This finding suggests new insight into a specific putative molecular strategy for defense against this pathogen. PMID:27471515

  20. Multigrid block preconditioning for a coupled system of partial differential equations modeling the electrical activity in the heart.

    PubMed

    Sundnes, J; Lines, G T; Mardal, K A; Tveito, A

    2002-12-01

    The electrical activity of the heart may be modeled with a system of partial differential equations (PDEs) known as the bidomain model. Computer simulations based on these equations may become a helpful tool to understand the relationship between changes in the electrical field and various heart diseases. Because of the rapid variations in the electrical field, sufficiently accurate simulations require a fine-scale discretization of the equations. For realistic geometries this leads to a large number of grid points and consequently large linear systems to be solved for each time step. In this paper, we present a fully coupled discretization of the bidomain model, leading to a block structured linear system. We take advantage of the block structure to construct an efficient preconditioner for the linear system, by combining multigrid with an operator splitting technique. PMID:12468421

  1. Rare earth oxide-doped titania nanocomposites with enhanced photocatalytic activity towards the degradation of partially hydrolysis polyacrylamide

    NASA Astrophysics Data System (ADS)

    Li, Jinhuan; Yang, Xia; Yu, Xiaodan; Xu, Leilei; Kang, Wanli; Yan, Wenhua; Gao, Hongfeng; Liu, Zhonghe; Guo, Yihang

    2009-01-01

    Rare-earth oxide-doped titania nanocomposites (RE 3+/TiO 2, where RE = Eu 3+, Pr 3+, Gd 3+, Nd 3+, and Y 3+) were prepared by a one-step sol-gel-solvothermal method. The products exhibited anatase phase structure, mesoporosity, and interesting surface compositions with three oxygen species and two titanium species. The products were used as the photocatalysts to degrade a partially hydrolysis polyacrylamide (HPAM) under UV-light irradiation, a very useful polymer in oil recovery. For comparison, Degussa P25 and as-prepared pure TiO 2 were also tested under the same conditions. The enhanced photocatalytic activity was obtained on as-prepared Eu 3+ (Gd 3+, Pr 3+)/TiO 2 composites, and the reasons were explained. Finally, the degradation pathway of HPAM over the RE 3+/TiO 2 composite was put forward based on the intermediates produced during the photocatalysis procedure.

  2. The role of chordin fragments generated by partial tolloid cleavage in regulating BMP activity.

    PubMed

    Troilo, Helen; Barrett, Anne L; Wohl, Alexander P; Jowitt, Thomas A; Collins, Richard F; Bayley, Christopher P; Zuk, Alexandra V; Sengle, Gerhard; Baldock, Clair

    2015-10-01

    Chordin-mediated regulation of bone morphogenetic protein (BMP) family growth factors is essential in early embryogenesis and adult homoeostasis. Chordin binds to BMPs through cysteine-rich von Willebrand factor type C (vWC) homology domains and blocks them from interacting with their cell surface receptors. These domains also self-associate and enable chordin to target related proteins to fine-tune BMP regulation. The chordin-BMP inhibitory complex is strengthened by the secreted glycoprotein twisted gastrulation (Tsg); however, inhibition is relieved by cleavage of chordin at two specific sites by tolloid family metalloproteases. As Tsg enhances this cleavage process, it serves a dual role as both promoter and inhibitor of BMP signalling. Recent developments in chordin research suggest that rather than simply being by-products, the cleavage fragments of chordin continue to play a role in BMP regulation. In particular, chordin cleavage at the C-terminus potentiates its anti-BMP activity in a type-specific manner. PMID:26517884

  3. The role of chordin fragments generated by partial tolloid cleavage in regulating BMP activity

    PubMed Central

    Troilo, Helen; Barrett, Anne L.; Wohl, Alexander P.; Jowitt, Thomas A.; Collins, Richard F.; Bayley, Christopher P.; Zuk, Alexandra V.; Sengle, Gerhard; Baldock, Clair

    2015-01-01

    Chordin-mediated regulation of bone morphogenetic protein (BMP) family growth factors is essential in early embryogenesis and adult homoeostasis. Chordin binds to BMPs through cysteine-rich von Willebrand factor type C (vWC) homology domains and blocks them from interacting with their cell surface receptors. These domains also self-associate and enable chordin to target related proteins to fine-tune BMP regulation. The chordin–BMP inhibitory complex is strengthened by the secreted glycoprotein twisted gastrulation (Tsg); however, inhibition is relieved by cleavage of chordin at two specific sites by tolloid family metalloproteases. As Tsg enhances this cleavage process, it serves a dual role as both promoter and inhibitor of BMP signalling. Recent developments in chordin research suggest that rather than simply being by-products, the cleavage fragments of chordin continue to play a role in BMP regulation. In particular, chordin cleavage at the C-terminus potentiates its anti-BMP activity in a type-specific manner. PMID:26517884

  4. Local electronic structure and photoelectrochemical activity of partial chemically etched Ti-doped hematite

    NASA Astrophysics Data System (ADS)

    Rioult, Maxime; Belkhou, Rachid; Magnan, Hélène; Stanescu, Dana; Stanescu, Stefan; Maccherozzi, Francesco; Rountree, Cindy; Barbier, Antoine

    2015-11-01

    The direct conversion of solar light into chemical energy or fuel through photoelectrochemical water splitting is promising as a clean hydrogen production solution. Ti-doped hematite (Ti:α-Fe2O3) is a potential key photoanode material, which despite its optimal band gap, excellent chemical stability, abundance, non-toxicity and low cost, still has to be improved. Here we give evidence of a drastic improvement of the water splitting performances of Ti-doped hematite photoanodes upon a HCl wet-etching. In addition to the topography investigation by atomic force microscopy, a detailed determination of the local electronic structure has been carried out in order to understand the phenomenon and to provide new insights in the understanding of solar water splitting. Using synchrotron radiation based spectromicroscopy (X-PEEM), we investigated the X-ray absorption spectral features at the L3 Fe edge of the as grown surface and of the wet-etched surface on the very same sample thanks to patterning. We show that HCl wet etching leads to substantial surface modifications of the oxide layer including increased roughness and chemical reduction (presence of Fe2 +) without changing the band gap. We demonstrate that these changes are profitable and correlated to the drastic changes of the photocatalytic activity.

  5. Partial characterization of cold active amylases and proteases of Streptomyces sp. from Antarctica

    PubMed Central

    Cotârleţ, Mihaela; Negoiţă, Teodor Gh.; Bahrim, Gabriela E.; Stougaard, Peter

    2011-01-01

    The aim of this study was to isolate novel enzyme-producing bacteria from vegetation samples from East Antarctica and also to characterize them genetically and biochemically in order to establish their phylogeny. The ability to grow at low temperature and to produce amylases and proteases cold-active was also tested. The results of the 16S rRNA gene sequence analysis showed that the 4 Alga rRNA was 100% identical to the sequences of Streptomyces sp. rRNA from Norway and from the Solomon Islands. The Streptomyces grew well in submerged system at 20°C, cells multiplication up to stationary phase being drastically increased after 120 h of submerged cultivation. The beta-amylase production reached a maximum peak after seven days, while alpha-amylase and proteases were performing biosynthesis after nine days of submerged cultivation at 20°C. Newly Streptomyces were able to produce amylase and proteases in a cold environment. The ability to adapt to low temperature of these enzymes could make them valuable ingredients for detergents, the food industry and bioremediation processes which require low temperatures. PMID:24031702

  6. C-Terminal extension of a plant cysteine protease modulates proteolytic activity through a partial inhibitory mechanism.

    PubMed

    Dutta, Sruti; Choudhury, Debi; Dattagupta, Jiban K; Biswas, Sampa

    2011-09-01

    The amino acid sequence of ervatamin-C, a thermostable cysteine protease from a tropical plant, revealed an additional 24-amino-acid extension at its C-terminus (CT). The role of this extension peptide in zymogen activation, catalytic activity, folding and stability of the protease is reported. For this study, we expressed two recombinant forms of the protease in Escherichia coli, one retaining the CT-extension and the other with it truncated. The enzyme with the extension shows autocatalytic zymogen activation at a higher pH of 8.0, whereas deletion of the extension results in a more active form of the enzyme. This CT-extension was not found to be cleaved during autocatalysis or by limited proteolysis by different external proteases. Molecular modeling and simulation studies revealed that the CT-extension blocks some of the substrate-binding unprimed subsites including the specificity-determining subsite (S2) of the enzyme and thereby partially occludes accessibility of the substrates to the active site, which also corroborates the experimental observations. The CT-extension in the model structure shows tight packing with the catalytic domain of the enzyme, mediated by strong hydrophobic and H-bond interactions, thus restricting accessibility of its cleavage sites to the protease itself or to the external proteases. Kinetic stability analyses (T(50) and t(1/2) ) and refolding experiments show similar thermal stability and refolding efficiency for both forms. These data suggest that the CT-extension has an inhibitory role in the proteolytic activity of ervatamin-C but does not have a major role either in stabilizing the enzyme or in its folding mechanism. PMID:21707922

  7. Steviol and steviol-glycoside: glucosyltransferase activities in Stevia rebaudiana Bertoni--purification and partial characterization.

    PubMed

    Shibata, H; Sawa, Y; Oka, T; Sonoke, S; Kim, K K; Yoshioka, M

    1995-08-20

    The leaves of Stevia rebaudiana Bertoni contain sweet compounds which are glycosides of diterpene derivative steviol (ent-13-hydroxykaur-16-en-19-oic acid). Its main constituents are stevioside (triglucosylated steviol; 13-O-beta-sophorosyl-19-O-beta-glucosyl-steviol) and rebaudioside-A (tetraglucosylated steviol; 2'-O-beta-glucosyl-13-O-beta-sophorosyl-19-O-beta-glucosyl-stev iol). From the extracts of S. rebaudiana Bertoni, two glucosyltransferases (GTases I and IIB) acting on steviol and steviol-glycosides were isolated, and another distinct activity (GTase IIA) acting on steviol was detected. Purified GTase I (subunit M(r) 24,600) catalyzed glucose transfer from UDP-glucose to steviol and steviolmonoside (steviol-13-O-glucopyranoside), but not to other steviol-glycosides. Apparent Km values were 71.4 microM for steviol and 360 microM for UDP-glucose. GTase IIB (subunit M(r) 30,700) showed a broad substrate specificity, acting on steviol, steviolmonoside, steviolbioside (13-O-beta-sophorosyl-steviol), and stevioside. Apparent Km values were 182 microM for steviol, 44 microM for steviolbioside, 95 microM for stevioside, and 385 microM for UDP-glucose. The two enzymes had a similar optimum pH at 6.5. They also acted effectively on ubiquitous flavonol aglycones, quercetin, and kaempferol and utilized kaempferol at a higher rate than steviol and steviol-glycosides. The apparent Km values of GTase I and IIB for kaempferol were 12 and 31 microM, respectively. PMID:7646064

  8. Metabolism of 1-alkyl-2-acyl-GPC in human platelets in response to stimulation by thrombin

    SciTech Connect

    Alam, I.; Silver, M.J.

    1987-02-15

    Washed human platelets were incubated with radioactive 1-(/sup 3/H)alkyl-2-hydroxyglycero-3-phosphocholine (lyso-PAF) at 37 degrees C. (/sup 3/H)lyso-PAF was converted by platelets into (/sup 3/H)alkylacyl-GPC which was incorporated. Incorporation of radioactivity was time dependent and reached a maximum of 57 percent in one h. This formation and incorporation of (/sup 3/H)alkylacyl-GPC was inhibited (50%) by extracellular calcium (1.3 mM). Labeled platelets were treated for 5 min with either thrombin (2.5 U/ml) or saline solution. While there was no change in the saline control, thrombin induced a reduction in the content of (/sup 3/H)alkylacyl-GPC, accompanied by an increase in (3H)lyso-PAF presumably by stimulation of phospholipase A2. There was no apparent increase in radioactivity comigrating with PAF. This was probably due to the overwhelming dilution of the radioactive alkylacyl-GPC by the endogenous nonradioactive compound (ratio-1/3200). These studies suggest that human platelets can take up lyso-PAF and acylate it to alkylacyl-GPC which is susceptible to phospholipase A2 activity.

  9. Inhibition of Cellular Adhesion by Immunological Targeting of Osteopontin Neoepitopes Generated through Matrix Metalloproteinase and Thrombin Cleavage

    PubMed Central

    Jürets, Alexander; Le Bras, Marie; Staffler, Günther; Stein, Gesine; Leitner, Lukas; Neuhofer, Angelika; Tardelli, Matteo; Turkof, Edvin; Zeyda, Maximilian; Stulnig, Thomas M.

    2016-01-01

    Osteopontin (OPN), a secreted protein involved in inflammatory processes and cancer, induces cell adhesion, migration, and activation of inflammatory pathways in various cell types. Cells bind OPN via integrins at a canonical RGD region in the full length form as well as to a contiguous cryptic site that some have shown is unmasked upon thrombin or matrix metalloproteinase cleavage. Thus, the adhesive capacity of osteopontin is enhanced by proteolytic cleavage that may occur in inflammatory conditions such as obesity, atherosclerosis, rheumatoid arthritis, tumor growth and metastasis. Our aim was to inhibit cellular adhesion to recombinant truncated proteins that correspond to the N-terminal cleavage products of thrombin- or matrix metalloproteinase-cleaved OPN in vitro. We specifically targeted the cryptic integrin binding site with monoclonal antibodies and antisera induced by peptide immunization of mice. HEK 293 cells adhered markedly stronger to truncated OPN proteins than to full length OPN. Without affecting cell binding to the full length form, the raised monoclonal antibodies specifically impeded cellular adhesion to the OPN fragments. Moreover, we show that the peptides used for immunization were able to induce antisera, which impeded adhesion either to all OPN forms, including the full-length form, or selectively to the corresponding truncated recombinant proteins. In conclusion, we developed immunological tools to selectively target functional properties of protease-cleaved OPN forms, which could find applications in treatment and prevention of various inflammatory diseases and cancers. PMID:26840958

  10. Partial Antiviral Activities Detection of Chicken Mx Jointing with Neuraminidase Gene (NA) against Newcastle Disease Virus

    PubMed Central

    Zhang, Yani; Fu, Dezhi; Chen, Hao; Zhang, Zhentao; Shi, Qingqing; Elsayed, Ahmed Kamel; Li, Bichun

    2013-01-01

    As an attempt to increase the resistance to Newcastle Disease Virus (NDV) and so further reduction of its risk on the poultry industry. This work aimed to build the eukaryotic gene co-expression plasmid of neuraminidase (NA) gene and myxo-virus resistance (Mx) and detect the gene expression in transfected mouse fibroblasts (NIH-3T3) cells, it is most important to investigate the influence of the recombinant plasmid on the chicken embryonic fibroblasts (CEF) cells. cDNA fragment of NA and mutant Mx gene were derived from pcDNA3.0-NA and pcDNA3.0-Mx plasmid via PCR, respectively, then NA and Mx cDNA fragment were inserted into the multiple cloning sites of pVITRO2 to generate the eukaryotic co-expression plasmid pVITRO2-Mx-NA. The recombinant plasmid was confirmed by restriction endonuclease treatment and sequencing, and it was transfected into the mouse fibroblasts (NIH-3T3) cells. The expression of genes in pVITRO2-Mx-NA were measured by RT-PCR and indirect immunofluorescence assay (IFA). The recombinant plasmid was transfected into CEF cells then RT-PCR and the micro-cell inhibition tests were used to test the antiviral activity for NDV. Our results showed that co-expression vector pVITRO2-Mx-NA was constructed successfully; the expression of Mx and NA could be detected in both NIH-3T3 and CEF cells. The recombinant proteins of Mx and NA protect CEF cells from NDV infection until after 72 h of incubation but the individually mutagenic Mx protein or NA protein protects CEF cells from NDV infection till 48 h post-infection, and co-transfection group decreased significantly NDV infection compared with single-gene transfection group (P<0. 05), indicating that Mx-NA jointing contributed to delaying the infection of NDV in single-cell level and the co-transfection of the jointed genes was more powerful than single one due to their synergistic effects. PMID:23977111

  11. Apoptin T108 phosphorylation is not required for its tumor-specific nuclear localization but partially affects its apoptotic activity

    SciTech Connect

    Lee, Y.-H.; Cheng, C.-M.; Chang, Y.-F.; Wang, T.-Y.; Yuo, C.-Y.; E-mail: m815006@kmu.edu.tw

    2007-03-09

    Apoptin, a chicken anemia virus-encoded protein, induces apoptosis in human tumor cells but not in normal cells. In addition, Apoptin also exhibits tumor-specific nuclear localization and tumor-specific phosphorylation on threonine 108 (T108). Here, we studied the effects of T108 phosphorylation on the tumor-specific nuclear localization and apoptotic activity of Apoptin. We first showed that a hemagglutinin (HA)-tagged Apoptin, but not the green fluorescent protein-fused Apoptin used in many previous studies, exhibited the same intracellular distribution pattern as native Apoptin. We then made and analyzed an HA-Apoptin mutant with its T108 phosphorylation site abolished. We found that Apoptin T108 phosphorylation is not required for its tumor-specific nuclear localization and abolishing the T108 phosphorylation of Apoptin does affect its apoptotic activity in tumor cells but only partially. Our results support the previous finding that Apoptin contains two distinct apoptosis domains located separately at the N- and C-terminal regions and suggest that the T108 phosphorylation may only be required for the apoptotic activity mediated through the C-terminal apoptosis domain.

  12. Novel thrombolytic protease from edible and medicinal plant Aster yomena (Kitam.) Honda with anticoagulant activity: purification and partial characterization.

    PubMed

    Choi, Jun-Hui; Kim, Dae-Won; Park, Se-Eun; Choi, Bong-Suk; Sapkota, Kumar; Kim, Seung; Kim, Sung-Jun

    2014-10-01

    A thrombolytic protease named kitamase possessing anticoagulant property was purified from edible and medicinal plant Aster yomena (Kitam.) Honda. Kitamase showed a molecular weight of 50 kDa by SDS-PAGE and displayed a strong fibrin zymogram lysis band corresponding to the similar molecular mass. The enzyme was active at high temperatures (50°C). The fibrinolytic activity of kitamase was strongly inhibited by EDTA, EGTA, TPCK and PMSF, inhibited by Zn(2+). The Km and Vmax values for substrate S-2251 were determined as 4.31 mM and 23.81 mM/mg respectively. It dissolved fibrin clot directly and specifically cleaved the α, Aα and γ-γ chains of fibrin and fibrinogen. In addition, kitamase delayed the coagulation time and increased activated partial thromboplastin time and prothrombin time. Kitamase exerted a significant protective effect against collagen and epinephrine induced pulmonary thromboembolism in mice. These results suggest that kitamase may have the property of metallo-protease like enzyme, novel fibrino(geno)lytic enzyme and a potential to be a therapeutic agent for thrombosis. PMID:24746735

  13. Radiation inactivation analysis of assimilatory NADH:nitrate reductase. Apparent functional sizes of partial activities associated with intact and proteolytically modified enzyme

    SciTech Connect

    Solomonson, L.P.; McCreery, M.J.; Kay, C.J.; Barber, M.J.

    1987-06-25

    Recently we demonstrated that target sizes for the partial activities of nitrate reductase were considerably smaller than the 100-kDa subunit which corresponded to the target size of the full (physiologic) activity NADH:nitrate reductase. These results suggested that the partial activities resided on functionally independent domains and that radiation inactivation may be due to localized rather than extensive damage to protein structure. The present study extends these observations and addresses several associated questions. Monophasic plots were observed over a wide range of radiation doses, suggesting a single activity component in each case. No apparent differences were observed over a 10-fold range of concentration for each substrate, suggesting that the observed slopes were not due to marked changes in Km values. Apparent target sizes estimated for partial activities associated with native enzyme and with limited proteolysis products of native enzyme suggested that the functional size obtained by radiation inactivation analysis is independent of the size of the polypeptide chain. The presence of free radical scavengers during irradiation reduced the apparent target size of both the physiologic and partial activities by an amount ranging from 24 to 43%, suggesting that a free radical mechanism is at least partially responsible for the inactivation. Immunoblot analysis of nitrate reductase irradiated in the presence of free radical scavengers revealed formation of distinct bands at 90, 75, and 40 kDa with increasing doses of irradiation rather than complete destruction of the polypeptide chain.

  14. Infestin, a thrombin inhibitor presents in Triatoma infestans midgut, a Chagas' disease vector: gene cloning, expression and characterization of the inhibitor.

    PubMed

    Campos, I T N; Amino, R; Sampaio, C A M; Auerswald, E A; Friedrich, T; Lemaire, H-G; Schenkman, S; Tanaka, A S

    2002-09-01

    This work describes the purification, gene cloning and expression of infestin, a thrombin inhibitor from midguts of Triatoma infestans. Infestin is located in the midgut and its purification was performed by anion-exchange and affinity chromatographies. The N-terminal sequence and the sequence of tryptic peptides were determined. Using RT-PCR, total RNA and infestin cDNA information, a DNA fragment was cloned which encodes a multi non-classical Kazal-type serine protease inhibitor. Isolated native infestin has two non-classical Kazal-type domains and shows an apparent molecular mass of 13 kDa, while its gene codes for a protein with four non-classical Kazal-type domains corresponding to an apparent molecular mass of 22 kDa. Two recombinant infestins, r-infestin 1-2 and r-infestin 1-4, were constructed using the vector pVT102U/alpha and expressed in S. cerevisiae. Native and r-infestin 1-2 showed very similar inhibitory activities towards thrombin and trypsin with dissociation constants of 43.5 and 25 pM for thrombin and 2.0 and 3.1 nM for trypsin, respectively. No other serine protease of the blood coagulation cascade was inhibited by the r-infestin 1-2. Surprisingly, r-infestin 1-4 inhibited not only thrombin and trypsin (K(i) of 0.8 and 5.2 nM, respectively), but also factor XIIa, factor Xa and plasmin (K(i) of 78 pM, 59.2 and 1.1 nM, respectively). PMID:12213235

  15. Inhibition of the effects of thrombin on guinea pig platelets by the diacylglycerol lipase inhibitor RHC 80267

    SciTech Connect

    Amin, D.; Sutherland, C.A.; Khandwala, A.S.; Jamall, I.S.; Kapoor, A.L.

    1986-10-01

    Phospholipase C (PLC) and diacylglycerol lipase (DGL) activities were found in guinea pig platelet microsome preparations. No phospholipase A2 (PLA2) activity was detected. RHC 80267 (1,6-di (0-(carbamoyl) cyclohexanone oxime)hexane) inhibited DGL activity (IC50 = 4 uM) from guinea pig platelet microsomes but had no effect on PLC. RHC 80267 inhibited platelet aggregation (IC50 = 11 uM), release of arachidonic acid (AA), its metabolites, and ATP (IC50 = 4.5 uM) when guinea pig platelets were challenged with a low concentration of thrombin. We propose that PLC-DGL is an important enzymatic pathway for the release of AA in guinea pig platelets.

  16. Telmisartan protects against diabetic vascular complications in a mouse model of obesity and type 2 diabetes, partially through peroxisome proliferator activated receptor-{gamma}-dependent activity

    SciTech Connect

    Toyama, Kensuke; Nakamura, Taishi; Kataoka, Keiichiro; Yasuda, Osamu; Fukuda, Masaya; Tokutomi, Yoshiko; Dong, Yi-Fei; Ogawa, Hisao; Kim-Mitsuyama, Shokei

    2011-07-08

    Highlights: {yields} Telmisartan, an angiotensin receptor blocker, acts as a partial PPAR{gamma} agonist. {yields} The protective effects of telmisartan against diabetic vascular injury were associated with attenuation of vascular NF{kappa}B activation and TNF {alpha}. {yields} PPAR{gamma} activity of telmisartan was involved in the normalization of vascular PPAR{gamma} downregulation in diabetic mice. {yields} We provided the first evidence indicating that PPAR{gamma} activity of telmisartan contributed to the protective effects of telmisartan against diabetic vascular complication. -- Abstract: Experimental and clinical data support the notion that peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) activation is associated with anti-atherosclerosis as well as anti-diabetic effect. Telmisartan, an angiotensin receptor blocker (ARB), acts as a partial PPAR{gamma} agonist. We hypothesized that telmisartan protects against diabetic vascular complications, through PPAR{gamma} activation. We compared the effects of telmisartan, telmisartan combined with GW9662 (a PPAR{gamma} antagonist), and losartan with no PPAR{gamma} activity on vascular injury in obese type 2 diabetic db/db mice. Compared to losartan, telmisartan significantly ameliorated vascular endothelial dysfunction, downregulation of phospho-eNOS, and coronary arterial remodeling in db/db mice. More vascular protective effects of telmisartan than losartan were associated with greater anti-inflammatory effects of telmisartan, as shown by attenuation of vascular nuclear factor kappa B (NF{kappa}B) activation and tumor necrosis factor {alpha}. Coadministration of GW9662 with telmisartan abolished the above mentioned greater protective effects of telmisartan against vascular injury than losartan in db/db mice. Thus, PPAR{gamma} activity appears to be involved in the vascular protective effects of telmisartan in db/db mice. Moreover, telmisartan, but not losartan, prevented the downregulation of

  17. Influence of factor VIII:C and factor IX activity in plasmas of haemophilic dogs on the activated partial thromboplastin time measured with two commercial reagents.

    PubMed

    Mischke, R

    2000-05-01

    The present study is based on 145 plasma samples with a reduced activity of factor VIII:C (range: 0.009-0.62 IU mL-1) and 28 samples with a reduced factor IX activity (range: 0.035-0.55 IU mL-1). The samples were collected from dogs with haemophilia A (n=22) or haemophilia B (n=3), some of these during substitution therapy. For all samples the activated partial thromboplastin time (APTT) was measured with two commercial reagents containing kaolin as a contact activator. In each case, the deficiency of factor VIII:C or IX was reflected in abnormal results of the APTT. This was true for both reagents. A significant correlation (P < 0.001) was found between factor VIII:C activity and APTT (reagent 1, Pathromtin(R); Spearman's rank correlation coefficient, rS=-0.731, reagent 2, PTT-Reagenz; rS=-0.875) as well as between factor IX activity and APTT (reagent 1, rS=-0.819; reagent 2, rS=-0.955]. In each case, the relationship between coagulation factor activity and APTT could be proven most precisely by geometric regression. The results of this study illustrate the applicability of commercial APTT test kits as a sensitive screening test of factor VIII:C and IX deficiencies in canine plasma. PMID:10792470

  18. Thrombin Injection for Treatment of Brachial Artery Pseudoaneurysm at the Site of a Hemodialysis Fistula: Report of Two Patients

    SciTech Connect

    Clark, Timothy W.I.; Abraham, Robert J.

    2000-09-15

    We report two patients with arteriovenous hemodialysis fistulas that were complicated by brachial artery pseudoaneurysms. Each pseudoanerysm was percutaneously thrombosed with an injection of thrombin, using techniques to prevent escape of thrombin into the native brachial artery. In one patient, an angioplasty balloon was inflated across the neck of the aneurysm during thrombin injection. In the second patient, thrombin was injected during ultrasound-guided compression of the neck of the pseudoaneurysm. Complete thrombosis of each pseudoaneurysm was achieved within 30 sec. No ischemic or embolic events occurred. This technique may be useful in treating pseudoaneurysms of smaller peripheral arteries.

  19. Partial Sleep Restriction Activates Immune Response-Related Gene Expression Pathways: Experimental and Epidemiological Studies in Humans

    PubMed Central

    Rantanen, Ville; Kronholm, Erkki; Surakka, Ida; van Leeuwen, Wessel M. A.; Lehto, Maili; Matikainen, Sampsa; Ripatti, Samuli; Härmä, Mikko; Sallinen, Mikael; Salomaa, Veikko; Jauhiainen, Matti; Alenius, Harri; Paunio, Tiina; Porkka-Heiskanen, Tarja

    2013-01-01

    Epidemiological studies have shown that short or insufficient sleep is associated with increased risk for metabolic diseases and mortality. To elucidate mechanisms behind this connection, we aimed to identify genes and pathways affected by experimentally induced, partial sleep restriction and to verify their connection to insufficient sleep at population level. The experimental design simulated sleep restriction during a working week: sleep of healthy men (N = 9) was restricted to 4 h/night for five nights. The control subjects (N = 4) spent 8 h/night in bed. Leukocyte RNA expression was analyzed at baseline, after sleep restriction, and after recovery using whole genome microarrays complemented with pathway and transcription factor analysis. Expression levels of the ten most up-regulated and ten most down-regulated transcripts were correlated with subjective assessment of insufficient sleep in a population cohort (N = 472). Experimental sleep restriction altered the expression of 117 genes. Eight of the 25 most up-regulated transcripts were related to immune function. Accordingly, fifteen of the 25 most up-regulated Gene Ontology pathways were also related to immune function, including those for B cell activation, interleukin 8 production, and NF-κB signaling (P<0.005). Of the ten most up-regulated genes, expression of STX16 correlated negatively with self-reported insufficient sleep in a population sample, while three other genes showed tendency for positive correlation. Of the ten most down-regulated genes, TBX21 and LGR6 correlated negatively and TGFBR3 positively with insufficient sleep. Partial sleep restriction affects the regulation of signaling pathways related to the immune system. Some of these changes appear to be long-lasting and may at least partly explain how prolonged sleep restriction can contribute to inflammation-associated pathological states, such as cardiometabolic diseases. PMID:24194869

  20. Construction of photoelectrochemical thrombin aptasensor via assembling multilayer of graphene-CdS nanocomposites.

    PubMed

    Shangguan, Li; Zhu, Wei; Xue, Yanchun; Liu, Songqin

    2015-02-15

    A photoelectrochemical (PEC) aptasensor for highly sensitive and specific detection of thrombin was developed by using graphene–CdS nanocomposites multilayer as photoactive species and electroactive mediator hexaammineruthenium(III) chloride (Ru(NH(3))(6)(3+)) as signal enhancer. Graphene–CdS nanocomposites (G–CdS) were synthesized by one-pot reduction of oxide graphene and CdCl2 with thioacetamide. The photoactive multilayer was prepared by alternative assembly of the negatively charged 3-mercaptopropionic acid modified graphene–CdS nanocomposites (MPA-G–CdS) and the positively charged polyethylenimine (PEI) on ITO electrode. This layer-by-layer assembly method enhanced the stability and homogeneity of the photocurrent readout of G–CdS. Thrombin aptamer was covalently bound to the multilayer by using glutaraldehyde as cross-linking. Electroactive mediator (Ru(NH(3))(6)(3+)) could interact with the DNA phosphate backbone and thus facilitated the electron transfer between G–CdS multilayer and electrode and enhanced the photocurrent. Hybridizing of a long complementary DNA with thrombin aptamer could increase the adsorption amount of (Ru(NH(3))(6)(3+)), which in turn boosted the signal readout. In the presence of target thrombin, the affinity interaction between thrombin and its aptamer resulted in the long complementary DNA releasing from the G–CdS multilayer and decreasing of photocurrent signal. On the basis of G–CdS multilayer as the photoactive species, (Ru (NH(3))(6)(3+)) as an electroactive mediator, and aptamer as a recognition module, a high sensitive PEC aptasensor for thrombin detection was proposed. The thrombin aptasensor displayed a linear range from 2.0 pM to 600.0 pM and a detection limit of 1.0 pM. The present strategy provided a promising ideology for the future development of PEC biosensor. PMID:25314620

  1. Egg yolks inhibit activation of NF-κB and expression of its target genes in adipocytes after partial delipidation

    PubMed Central

    Shen, Qiwen; Riedl, Ken M.; Cole, Rachel M.; Lehman, Christopher; Xu, Lu; Alder, Hansjuerg; Belury, Martha A.; Schwartz, Steven J.; Ziouzenkova, Ouliana

    2015-01-01

    How composition of egg yolk (EY) influences NF-κB, a key transcription pathway in inflammation, remains unclear. We performed partial delipidation of EY that removed 20–30% of cholesterol and triglycerides. The resulting polar and non-polar fractions were termed EY-P and EY-NP. NF-κB activation in response to EY from different suppliers and their fractions was examined in 3T3-L1 adipocytes using a NF-κB response element reporter assay and by analyzing expression of 248 inflammatory genes. Although EY-P and EY contained similar level of vitamins, carotenoids, and fatty acids, only delipidated EY-P fraction suppressed NF-κB via down-regulation of toll like receptor-2 and up-regulation of inhibitory toll interacting protein (Tollip) and lymphocyte antigen 96 (Ly96). Our data suggest that anti-inflammatory activity of lutein and retinol were blunted by non-polar lipids in EY likely via crosstalk between SREBP and NF-κB pathways in adipocytes. Thus, moderate delipidation may improve their beneficial properties of regular eggs. PMID:25620076

  2. Vibration reduction in helicopter rotors using an actively controlled partial span trailing edge flap located on the blade

    NASA Technical Reports Server (NTRS)

    Millott, T. A.; Friedmann, P. P.

    1994-01-01

    This report describes an analytical study of vibration reduction in a four-bladed helicopter rotor using an actively controlled, partial span, trailing edge flap located on the blade. The vibration reduction produced by the actively controlled flap (ACF) is compared with that obtained using individual blade control (IBC), in which the entire blade is oscillated in pitch. For both cases a deterministic feedback controller is implemented to reduce the 4/rev hub loads. For all cases considered, the ACF produced vibration reduction comparable with that obtained using IBC, but consumed only 10-30% of the power required to implement IBC. A careful parametric study is conducted to determine the influence of blade torsional stiffness, spanwise location of the control flap, and hinge moment correction on the vibration reduction characteristics of the ACF. The results clearly demonstrate the feasibility of this new approach to vibration reduction. It should be emphasized than the ACF, used together with a conventional swashplate, is completely decoupled from the primary flight control system and thus it has no influence on the airworthiness of the helicopter. This attribute is potentially a significant advantage when compared to IBC.

  3. A novel perovskite based catalyst with high selectivity and activity for partial oxidation of methane for fuel cell applications.

    PubMed

    Staniforth, J; Evans, S E; Good, O J; Darton, R J; Ormerod, R M

    2014-10-28

    Solid oxide fuel cells (SOFCs) have the potential to revolutionise the present fuel economy due to their higher fuel conversion efficiency compared with standard heat engines and the possibility of utilizing the heat produced in a combined heat and power system. One of the reasons they have yet to fulfil this potential is that the conventional anode material of choice, a nickel/yttria-stabilised zirconia cermet, requires a high temperature production process and under operating conditions is susceptible to carbon and sulphur poisoning. Perovskite-based materials have been proposed as potential anode materials for SOFCs due to their potentially high electronic conductivity and catalytic properties. One of the problems in realizing this potential has been their low catalytic activity towards methane reforming compared to conventional nickel based cermet materials. A nickel doped strontium zirconate material produced by low temperature hydrothermal synthesis is described which has high activity for methane reforming and high selectivity towards partial oxidation of methane as opposed to total oxidation products. Initial studies show a very low level of carbon formation which does not increase over time. PMID:25069597

  4. Cardiovascular protection and antioxidant activity of the extracts from the mycelia of Cordyceps sinensis act partially via adenosine receptors.

    PubMed

    Yan, Xiao-Feng; Zhang, Zhong-Miao; Yao, Hong-Yi; Guan, Yan; Zhu, Jian-Ping; Zhang, Lin-Hui; Jia, Yong-Liang; Wang, Ru-Wei

    2013-11-01

    Mycelia of cultured Cordyceps sinensis (CS) is one of the most common substitutes for natural CS and was approved for arrhythmia in China. However, the role of CS in ameliorating injury during ischemia-reperfusion (I/R) is still unclear. We examined effects of extracts from CS on I/R and investigated the possible mechanisms. Post-ischemic coronary perfusion pressure, ventricular function, and coronary flow were measured using the Langendorff mouse heart model. Oxidative stress of cardiac homogenates was performed using an ELISA. Our results indicate that CS affords cardioprotection possibly through enhanced adenosine receptor activation. Cardioprotection was demonstrated by reduced post-ischemic diastolic dysfunction and improved recovery of pressure development and coronary flow. Treatment with CS largely abrogates oxidative stress and damage in glucose- or pyruvate-perfused hearts. Importantly, observed reductions in oxidative stress [glutathione disulfide (GSSG)]/[GSSG + glutathione] and [malondialdehyde (MDA)]/[superoxide dismutase + MDA] ratios as well as the resultant damage upon CS treatment correlate with functional markers of post-ischemic myocardial outcome. These effects of CS were partially blocked by 8-ρ-sulfophenyltheophylline, an adenosine receptor antagonist. Our results demonstrate a suppressive role of CS in ischemic contracture. Meanwhile, the results also suggest pre-ischemic adenosine receptor activation may be involved in reducing contracture in hearts pretreated with CS. PMID:23192916

  5. Acetyl-L-carnitine restores choline acetyltransferase activity in the hippocampus of rats with partial unilateral fimbria-fornix transection.

    PubMed

    Piovesan, P; Quatrini, G; Pacifici, L; Taglialatela, G; Angelucci, L

    1995-02-01

    Transection of the fimbria-fornix bundle in adult rats results in degeneration of the septohippocampal cholinergic pathway, reminiscent of that occurring in aging as well as Alzheimer disease. We report here a study of the effect of a treatment with acetyl-L-carnitine (ALCAR) in three-month-old Fischer 344 rats bearing a partial unilateral fimbria-fornix transection. ALCAR is known to ameliorate some morphological and functional disturbances in the aged central nervous system (CNS). We used choline acetyltransferase (ChAT) and acetyl cholinesterase (AChE) as markers of central cholinergic function, and nerve growth factor (NGF) levels as indicative of the trophic regulation of the medio-septal cholinergic system. ChAT and AChE activities were significantly reduced in the hippocampus (HIPP) ipsilateral to the lesion as compared to the contralateral one, while no changes were observed in the septum (SPT), nucleus basalis magnocellularis (NBM) or frontal cortex (FCX). ALCAR treatment restored ChAT activity in the ipsilateral HIPP, while AChE levels were not different from those of untreated animals, and did not affect NGF content in either SPT or HIPP. PMID:7793306

  6. Evaluation of Prothrombin Time and Activated Partial Thromboplastin Time in Hypertensive Patients Attending a Tertiary Hospital in Calabar, Nigeria

    PubMed Central

    Nnenna Adaeze, Nnamani; Uchenna Emeribe, Anthony; Abdullahi Nasiru, Idris; Babayo, Adamu; Uko, Emmanuel K.

    2014-01-01

    Introduction. Several biomedical findings have established the effects of hypertension on haemostasis and roles of blood coagulation products in the clinical course of hypertension. Methods. This cross-sectional study aimed at determining effects of hypertension on prothrombin time (PT) and activated partial thromboplastin time (APTT) in hypertensive patients in comparison with normotensive subjects attending a tertiary hospital in Calabar. Forty-two (42) hypertensive patients and thirty-nine (39) normotensive control subjects were investigated for PT and APTT using Quick one-stage methods. Results. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) correlated positively with APTT (r = 0.3072, r = 0.4988; P < 0.05) in hypertensive patients. DBP, SBP, PT, and APTT were significantly higher in hypertensive patients when compared to normotensive subjects (P < 0.05). DBP correlated negatively with duration of illness (r = −0.3097; P < 0.05) in hypertensive patients and positively with age of normotensive subjects (r = 0.3523; P < 0.05). Conclusion. The results obtained indicated that measurements of PT and APTT may serve as indices for evaluating hemostatic abnormalities in hypertensive patients and guide for antihypertensive therapy. However, to have better understanding of hemostatic activities in hypertension, it is recommended to conduct D-dimer, platelet factors, and protein assays. PMID:25477963

  7. Plasma membrane associated phospholipase C from human platelets: Synergistic stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis by thrombin and guanosine 5 prime -O-(3-thiotriphosphate)

    SciTech Connect

    Baldassare, J.J.; Henderson, P.A.; Fisher, G.J. )

    1989-01-10

    The effects of thrombin and GTP{gamma}S on the hydrolysis of phosphoinositides by membrane-associated phospholipase C (PLC) from human platelets were examined with endogenous ({sup 3}H)inositol-labeled membranes or with lipid vesicles containing either ({sup 3}H)phosphatidylinositol or ({sup 3}H)phosphatidylinositol 4,5-bisphosphate. GTP{gamma}S (1 {mu}M) or thrombin (1 unit/mL) did not stimulate release of inositol trisphosphate (IP{sub 3}), inositol bisphosphate (IP{sub 2}), or inositol phosphate (IP) from ({sup 3}H)inositol-labeled membranes. IP{sub 2} and IP{sub 3}, but not IP, from ({sup 3}H)inositol-labeled membranes were, however, stimulated 3-fold by GTP{gamma}S (1 {mu}M) plus thrombin (1 unit/mL). A higher concentration of GTP{gamma}S (100 {mu}M) alone also stimulated IP{sub 2} and IP{sub 3}, but not IP, release. In the presence of 1 mM calcium, release of IP{sub 2} and IP{sub 3} was increased 6-fold over basal levels; however, formation of IP was not observed. At submicromolar calcium concentration, hydrolysis of exogenous phosphatidylinositol 4,5-bisphosphate (PIP{sub 2}) by platelet membrane associated PLC was also markedly enhanced by GTP{gamma}S (100 {mu}M) or GTP{gamma}S (1 {mu}M) plus thrombin (1 unit/mL). Under identical conditions, exogenous phosphatidylinositol (PI) was not hydrolyzed. The same substrate specificity was observed when the membrane-associated PLC was activated with 1 mM calcium. Thrombin-induced hydrolysis of PIP{sub 2} was inhibited by treatment of the membranes with pertussis toxin or pretreatment of intact platelets with 12-O-tetradecanoyl-13-acetate (TPA) prior to preparation of membranes. Pertussis toxin did not inhibit GTP{gamma}S (100 {mu}M) or calcium (1 mM) dependent PIP{sub 2} breakdown, while TPA inhibited GTP{gamma}S-dependent but not calcium-dependent phospholipase C activity.

  8. Parasympathetic activity and blood catecholamine responses following a single partial-body cryostimulation and a whole-body cryostimulation.

    PubMed

    Hausswirth, Christophe; Schaal, Karine; Le Meur, Yann; Bieuzen, François; Filliard, Jean-Robert; Volondat, Marielle; Louis, Julien

    2013-01-01

    The aim of this study was to compare the effects of a single whole-body cryostimulation (WBC) and a partial-body cryostimulation (PBC) (i.e., not exposing the head to cold) on indices of parasympathetic activity and blood catecholamines. Two groups of 15 participants were assigned either to a 3-min WBC or PBC session, while 10 participants constituted a control group (CON) not receiving any cryostimulation. Changes in thermal, physiological and subjective variables were recorded before and during the 20-min after each cryostimulation. According to a qualitative statistical analysis, an almost certain decrease in skin temperature was reported for all body regions immediately after the WBC (mean decrease±90% CL, -13.7±0.7°C) and PBC (-8.3±0.3°C), which persisted up to 20-min after the session. The tympanic temperature almost certainly decreased only after the WBC session (-0.32±0.04°C). Systolic and diastolic blood pressures were very likely increased after the WBC session, whereas these changes were trivial in the other groups. In addition, heart rate almost certainly decreased after PBC (-10.9%) and WBC (-15.2%) sessions, in a likely greater proportion for WBC compared to PBC. Resting vagal-related heart rate variability indices (the root-mean square difference of successive normal R-R intervals, RMSSD, and high frequency band, HF) were very likely increased after PBC (RMSSD: +54.4%, HF: +138%) and WBC (RMSSD: +85.2%, HF: +632%) sessions without any marked difference between groups. Plasma norepinephrine concentrations were likely to very likely increased after PBC (+57.4%) and WBC (+76.2%), respectively. Finally, cold and comfort sensations were almost certainly altered after WBC and PBC, sensation of discomfort being likely more pronounced after WBC than PBC. Both acute cryostimulation techniques effectively stimulated the autonomic nervous system (ANS), with a predominance of parasympathetic tone activation. The results of this study also suggest that a

  9. Parasympathetic Activity and Blood Catecholamine Responses Following a Single Partial-Body Cryostimulation and a Whole-Body Cryostimulation

    PubMed Central

    Hausswirth, Christophe; Schaal, Karine; Le Meur, Yann; Bieuzen, François; Filliard, Jean-Robert; Volondat, Marielle; Louis, Julien

    2013-01-01

    The aim of this study was to compare the effects of a single whole-body cryostimulation (WBC) and a partial-body cryostimulation (PBC) (i.e., not exposing the head to cold) on indices of parasympathetic activity and blood catecholamines. Two groups of 15 participants were assigned either to a 3-min WBC or PBC session, while 10 participants constituted a control group (CON) not receiving any cryostimulation. Changes in thermal, physiological and subjective variables were recorded before and during the 20-min after each cryostimulation. According to a qualitative statistical analysis, an almost certain decrease in skin temperature was reported for all body regions immediately after the WBC (mean decrease±90% CL, -13.7±0.7°C) and PBC (-8.3±0.3°C), which persisted up to 20-min after the session. The tympanic temperature almost certainly decreased only after the WBC session (-0.32±0.04°C). Systolic and diastolic blood pressures were very likely increased after the WBC session, whereas these changes were trivial in the other groups. In addition, heart rate almost certainly decreased after PBC (-10.9%) and WBC (-15.2%) sessions, in a likely greater proportion for WBC compared to PBC. Resting vagal-related heart rate variability indices (the root-mean square difference of successive normal R-R intervals, RMSSD, and high frequency band, HF) were very likely increased after PBC (RMSSD: +54.4%, HF: +138%) and WBC (RMSSD: +85.2%, HF: +632%) sessions without any marked difference between groups. Plasma norepinephrine concentrations were likely to very likely increased after PBC (+57.4%) and WBC (+76.2%), respectively. Finally, cold and comfort sensations were almost certainly altered after WBC and PBC, sensation of discomfort being likely more pronounced after WBC than PBC. Both acute cryostimulation techniques effectively stimulated the autonomic nervous system (ANS), with a predominance of parasympathetic tone activation. The results of this study also suggest that a

  10. The emerging role of the thrombin receptor (PAR-1) in melanoma metastasis--a possible therapeutic target.

    PubMed

    Villares, Gabriel J; Zigler, Maya; Bar-Eli, Menashe

    2011-01-01

    Melanoma remains as the deadliest form of skin cancer with limited and inefficient treatment options available for patients with metastatic disease. Within the last decade, the thrombin receptor, Protease Activated Receptor-1, has been described as an essential gene involved in the progression of human melanoma. PAR-1 is known to activate adhesive, invasive and angiogenic factors to promote melanoma metastasis. It is overexpressed not only in metastatic melanoma cell lines but is also highly expressed in metastatic lesions as compared to primary nevi and normal skin. Recently, PAR-1 has been described to regulate the gap junction protein Connexin 43 and the tumor suppressor gene Maspin to promote the metastatic melanoma phenotype. Herein, we review the role of PAR-1 in the progression of melanoma as well as utilizing PAR-1-regulated genes as potential therapeutic targets for melanoma treatment. PMID:21378407

  11. Intrabronchial Infusion of Autologous Blood Plus Thrombin for Intractable Pneumothorax After Bronchial Occlusion Using Silicon Spigots

    PubMed Central

    Nakahara, Yasuharu; Kawamura, Tetsuji; Sasaki, Shin; Tsukamoto, Hiroaki; Mochiduki, Yoshiro

    2016-01-01

    Background: Bronchial occlusion therapy using silicon spigots is effective for intractable pneumothorax. However, sometimes the pneumothorax is refractory to bronchial occlusion because of collateral ventilation. For such difficult pneumothoraces, we attempted an intrabronchial infusion of autologous blood plus thrombin to control collateral ventilation and stop air leaks. Methods: We performed bronchial occlusions using silicon spigots in patients with spontaneous pneumothorax secondary to emphysema and refractory to chest drainage, but which was inoperable owing to each patient’s poor surgical candidacy and poor overall health condition. When bronchial occlusion proved ineffective, we undertook intrabronchial infusion of autologous blood plus thrombin, 2 to 4 days after bronchial occlusion. A catheter was inserted into the subpleural area, through a gap between the silicon spigot and the bronchial wall, using a flexible bronchoscope under fluoroscopic guidance. Autologous blood, followed by a thrombin solution, was infused using the catheter. We repeated the same infusion a total of 4 to 6 times while changing the target bronchi. All interventions were performed under local anesthesia. Results: The subjects were 9 men, aged from 61 to 88 years, with smoking histories. Three patients also had interstitial pneumonia, and 6 patients had undergone pleurodesis in vain before bronchial occlusion. For 4of the 9 patients, autologous blood plus thrombin infusions successfully stopped air leaks, and in 3 patients, intrabronchial infusions and pleurodesis halted leaks altogether. Conclusion: Intrabronchial infusion of autologous blood plus thrombin was effective for intractable pneumothoraces that could not be clinically managed, even by bronchial occlusion using silicon spigots. PMID:27454474

  12. Fluorescent measurement of affinity binding between thrombin and its aptamers using on-chip affinity monoliths.

    PubMed

    Gao, Changlu; Sun, Xiuhua; Woolley, Adam T

    2013-05-24

    A microfluidic chip with integrated 2mm long monoliths incorporated with poly(ethylene glycol) (PEG) groups was developed for thrombin-aptamer interaction study. The non-G quartet forming oligonucleotide coated monoliths was compared to a 15 mer thrombin-binding aptamer, in which affinity binding and elution processes were real-time monitored fluorescently. The results showed that the fluorescence intensity of aptamer stationary phase is approximately 10 times higher than that of the control column, which is probably due to the successful suppression of nonspecific adsorption between thrombin and aptamers/monoliths by using PEG-monolith. The experiment was repeated using human serum albumin (HSA) and green fluorescence protein (GFP) as interferences, it was double confirmed that thrombin was selectively retained by PEG-monolith. An elution efficiency of 75% was achieved with an elute of 200mM acetic acid and 2M NaCI, and the eluted thrombin was successfully separated in an ionic buffer system of 20mM NaHCO3 (pH 9.5) with 3% PEG. The hydrophilic and antifouling properties of PEG-monolith greatly decrease nonspecific adsorption and enhance detection sensitivity, which provided an alternative method to perform on-chip fluorescent measurement of bioaffinity binding. PMID:23587316

  13. Dual aptamer-immobilized surfaces for improved affinity through multiple target binding in potentiometric thrombin biosensing.

    PubMed

    Goda, Tatsuro; Higashi, Daiki; Matsumoto, Akira; Hoshi, Toru; Sawaguchi, Takashi; Miyahara, Yuji

    2015-11-15

    We developed a label-free and reagent-less potentiometric biosensor with improved affinity for thrombin. Two different oligomeric DNA aptamers that can recognize different epitopes in thrombin were introduced in parallel or serial manners on the sensing surface to capture the target via multiple contacts as found in many biological systems. The spacer and linker in the aptamer probes were optimized for exerting the best performance in molecular recognition. To gain the specificity of the sensor to the target, an antifouling molecule, sulfobeaine-3-undecanethiol (SB), was introduced on the sensor to form a self-assembled monolayer (SAM). Surface characterization revealed that the aptamer probe density was comparable to the distance of the two epitopes in thrombin, while the backfilling SB SAM was tightly aligned on the surface to resist nonspecific adsorption. The apparent binding parameters were obtained by thrombin sensing in potentiometry using the 1:1 Langmuir adsorption model, showing the improved dissociation constants (Kd) with the limit of detection of 5.5 nM on the dual aptamer-immobilized surfaces compared with single aptamer-immobilized ones. A fine control of spacer and linker length in the aptamer ligand was essential to realize the multivalent binding of thrombin on the sensor surface. The findings reported herein are effective for improving the sensitivity of potentiometric biosensor in an affordable way towards detection of tiny amount of biomolecules. PMID:26067329

  14. Ethanol interferes with thrombin mediated changes in the morphology and cytoskeleton of human vascular endothelial cells

    SciTech Connect

    Pratt, K.J.; Rubin, R.; Hoek, J.; Williams, S.K. )

    1991-03-15

    The effect of physiological concentrations of ethanol (EtOH) on the response of human vascular endothelial cells (EC) to thrombin was examined Treatment of EC with EtOH concentrations of 20-85mM for 2-10 min. produced no significant changes in the morphology of 3- and 4-day monolayers established on fibronectin coated polystyrene. When examined immunofluorescently no significantly changes in the microfilament or microtubule structures were seen. Exposure of EC monolayers to 0.5 and 1 U/ml of thrombin for 1-60 minutes causes a concentration and time dependent monolayer retraction, evidenced by a general decrease in cell size, increase in visible gaps in the monolayer and redistribution of the microtubule and microfilament networks. Pretreatment of EC monolayers with EtOH for 3-5 minutes prior to addition of thrombin prevents the changes seen with thrombin alone. Immunofluorescent examination of the microfilament and microtubule structures suggests than EtOH may act in part via the microtubule network, which appears to be disorganized/disrupted when the EC are exposed to EtOH and then thrombin. Colchicine studies show that EC which have been pretreated with EtOH respond to colchicine differently then cells which have not previously seen EtOH. These data suggest that EtOH may alter EC monolayer responsiveness either by indirect changes which are reflected in cytoskeletal disorganization or possibly by direct influence on the cytoskeleton.

  15. Three different signal amplification strategies for the impedimetric sandwich detection of thrombin.

    PubMed

    Ocaña, Cristina; del Valle, Manel

    2016-03-17

    In this work, we report a comparative study on three highly specific amplification strategies for the ultrasensitive detection of thrombin with the use of aptamer sandwich protocol. The protocol consisted on the use of a first thrombin aptamer immobilized on the electrode surface, the recognition of thrombin protein, and the reaction with a second biotinylated thrombin aptamer forming the sandwich. Through the exposed biotin end, three variants have been tested to amplify the electrochemical impedance signal. The strategies included (a) silver enhancement treatment, (b) gold enhancement treatment and (c) insoluble product produced by the combination of the enzyme horseradish peroxidase (HRP) and 3-amino-9-ethylcarbazole (AEC). The properties of the sensing surface were probed by electrochemical impedance measurements in the presence of the ferrocyanide/ferricyanide redox marker. Insoluble product strategy and silver enhancement treatment resulted in the lowest detection limit (0.3 pM), while gold enhancement method resulted in the highest reproducibility, 8.8% RSD at the pM thrombin concentration levels. Results of silver and gold enhancement treatment also permitted direct inspection by scanning electron microscopy (SEM). PMID:26920780

  16. Genetic Determinants of Thrombin Generation and Their Relation to Venous Thrombosis: Results from the GAIT-2 Project

    PubMed Central

    Martin-Fernandez, Laura; Ziyatdinov, Andrey; Carrasco, Marina; Millon, Juan Antonio; Martinez-Perez, Angel; Vilalta, Noelia; Brunel, Helena; Font, Montserrat; Hamsten, Anders; Souto, Juan Carlos; Soria, José Manuel

    2016-01-01

    Background Venous thromboembolism (VTE) is a common disease where known genetic risk factors explain only a small portion of the genetic variance. Then, the analysis of intermediate phenotypes, such as thrombin generation assay, can be used to identify novel genetic risk factors that contribute to VTE. Objectives To investigate the genetic basis of distinct quantitative phenotypes of thrombin generation and its relationship to the risk of VTE. Patients/Methods Lag time, thrombin peak and endogenous thrombin potential (ETP) were measured in the families of the Genetic Analysis of Idiopathic Thrombophilia 2 (GAIT-2) Project. This sample consisted of 935 individuals in 35 extended families selected through a proband with idiopathic thrombophilia. We performed also genome wide association studies (GWAS) with thrombin generation phenotypes. Results The results showed that 67% of the variation in the risk of VTE is attributable to genetic factors. The heritabilities of lag time, thrombin peak and ETP were 49%, 54% and 52%, respectively. More importantly, we demonstrated also the existence of positive genetic correlations between thrombin peak or ETP and the risk of VTE. Moreover, the major genetic determinant of thrombin generation was the F2 gene. However, other suggestive signals were observed. Conclusions The thrombin generation phenotypes are strongly genetically determined. The thrombin peak and ETP are significantly genetically correlated with the risk of VTE. In addition, F2 was identified as a major determinant of thrombin generation. We reported suggestive signals that might increase our knowledge to explain the variability of this important phenotype. Validation and functional studies are required to confirm GWAS results. PMID:26784699

  17. The dimeric structure of factor XI and zymogen activation

    PubMed Central

    Geng, Yipeng; Verhamme, Ingrid M.; Smith, Stephen B.; Sun, Mao-fu; Matafonov, Anton; Cheng, Qiufang; Smith, Stephanie A.; Morrissey, James H.

    2013-01-01

    Factor XI (fXI) is a homodimeric zymogen that is converted to a protease with 1 (1/2-fXIa) or 2 (fXIa) active subunits by factor XIIa (fXIIa) or thrombin. It has been proposed that the dimeric structure is required for normal fXI activation. Consistent with this premise, fXI monomers do not reconstitute fXI-deficient mice in a fXIIa-dependent thrombosis model. FXI activation by fXIIa or thrombin is a slow reaction that can be accelerated by polyanions. Phosphate polymers released from platelets (poly-P) can enhance fXI activation by thrombin and promote fXI autoactivation. Poly-P increased initial rates of fXI activation 30- and 3000-fold for fXIIa and thrombin, respectively. FXI monomers were activated more slowly than dimers by fXIIa in the presence of poly-P. However, this defect was not observed when thrombin was the activating protease, nor during fXI autoactivation. The data suggest that fXIIa and thrombin activate fXI by different mechanisms. FXIIa may activate fXI through a trans-activation mechanism in which the protease binds to 1 subunit of the dimer, while activating the other subunit. For activation by thrombin, or during autoactivation, the data support a cis-activation mechanism in which the activating protease binds to and activates the same fXI subunit. PMID:23515926

  18. Inter-costal Liver Ablation Under Real Time MR-Thermometry With Partial Activation Of A HIFU Phased Array Transducer

    NASA Astrophysics Data System (ADS)

    Quesson, Bruno; Merle, Mathilde; Köhler, Max; Mougenot, Charles; Roujol, Sebastien; de Senneville, Baudouin Denis; Moonen, Chrit

    2010-03-01

    HIFU ablation of tumours located inside the liver is hampered by the rib cage, which partially obstructs the beam path and may create adverse effects such as skin burns. This study presents a method for selectively deactivating the transducer elements causing undesired temperature increases near the bones. A manual segmentation of the bones visualized on 3D anatomical MR images acquired prior to sonication was performed to identify the beam obstruction. The resulting mask was projected (ray tracing starting from the focal point) on the transducer and elements with more than 50% obstruction of their active surface were deactivated. The effectiveness of the method for HIFU ablations is demonstrated ex vivo and in vivo in the liver of pigs with real-time MR thermometry, using the proton resonant frequency (PRF) method. For both ex vivo and in vivo experiments, the temperature increase near the bones was significantly reduced when the elements located in front of the ribs were deactivated. The temperature evolution at the focal point were similar, indicative of the absence of loss of heating efficacy when the elements were deactivated. This method is simple, rapid and reliable and allows to perform intercostal MRgHIFU ablation of the liver while sparing the ribs.

  19. Highly efficient active optical interconnect incorporating a partially chlorinated ribbon POF in conjunction with a visible VCSEL.

    PubMed

    Lee, Hak-Soon; Lee, Sang-Shin; Kim, Bong-Seok; Son, Yung-Sung

    2014-05-19

    A low-loss 4-ch active optical interconnect (AOI) enabling passive alignment was proposed and built resorting to a transmitter (Tx) incorporating a red 680-nm VCSEL, which is linked to a receiver (Rx) module via a partially chlorinated ribbon POF. The POF was observed to exhibit an extremely low loss of ~0.24 dB/m at λ = 680 nm, in comparison to ~1.29 dB/m at λ = 850 nm, and a large numerical aperture of ~0.42. Both the Tx and Rx, which taps into a beam router based on collimated beam optics involving a pair of spherical lenses, were meant to be substantially alignment tolerant and compact. The achieved tolerance for the constructed modules was beyond 40 μm in terms of the positioning of VCSEL and photodetector. The proposed AOI was completed by linking the Tx with the Rx via a 3-m long ribbon POF, incurring a transmission loss of as small as 3.2 dB. The AOI was practically assessed in terms of a high-speed data transmission over a wide range of temperatures and then exploited to convey full HD video signals. PMID:24921299

  20. New mutations of Saccharomyces cerevisiae that partially relieve both glucose and galactose repression activate the protein kinase Snf1.

    PubMed

    Rodríguez, Cristina; Sanz, Pascual; Gancedo, Carlos

    2003-03-01

    We isolated from Saccharomyces cerevisiae two mutants, esc1-1 and ESC3-1, in which genes FBP1, ICL1 or GDH2 were partially derepressed during growth in glucose or galactose. The isolation was done starting with a triple mutant pyc1 pyc2 mth1 unable to grow in glucose-ammonium medium and selecting for mutants able to grow in the non-permissive medium. HXT1 and HXT2 which encode glucose transporters were expressed at high glucose concentrations in both esc1-1 and ESC3-1 mutants, while derepression of invertase at low glucose concentrations was impaired. REG1, cloned as a suppressor of ESC3-1, was not allelic to ESC3-1. Two-hybrid analysis showed an increased interaction of the protein kinase Snf1 with Snf4 in the ESC3-1 mutant; this was not due to mutations in SNF1 or SNF4. ESC3-1 did not bypass the requirement of Snf1 for derepression. We hypothesize that ESC3-1 either facilitates activation of Snf1 or interferes with its glucose-dependent inactivation. PMID:12702249

  1. Antibacterial activity and mechanical properties of partially hydrolyzed sago starch-alginate edible film containing lemongrass oil.

    PubMed

    Maizura, M; Fazilah, A; Norziah, M H; Karim, A A

    2007-08-01

    Edible films were prepared from a mixture of partially hydrolyzed sago starch and alginate (SA). Lemongrass oil (0.1% to 0.4%, v/w) and glycerol (0% and 20%, w/w) were incorporated in the films to act as natural antimicrobial agent and plasticizer, respectively. The films were characterized for antimicrobial activity, water vapor permeability (WVP), tensile strength (TS), percent elongation at break (%E), and water solubility (WS). Fourier transform infrared (FTIR) spectroscopy was conducted to determine functional group interactions between the matrix and lemongrass oil. The zone of inhibition was increased significantly (P < 0.05) by addition of lemongrass oil at all levels in the presence and the absence of glycerol. This indicates that the film containing lemongrass oil was effective against Escherichia coli O157:H7 at all levels. In the absence of glycerol, the tensile strength of film decreased as the oil content increased, but there was no significant (P > 0.05) difference in percent elongation. The percent elongation at break and WVP values for film with 20% glycerol was found to be increased significantly (P < 0.05) with an increase in lemongrass oil content. Addition of lemongrass oil did not have any interaction with the functional groups of films as measured by FTIR. PMID:17995673

  2. Crustal differentiation due to partial melting of granitic rocks in an active continental margin, the Ryoke Belt, Southwest Japan

    NASA Astrophysics Data System (ADS)

    Akasaki, Eri; Owada, Masaaki; Kamei, Atsushi

    2015-08-01

    The continental margin of Pacific Asia is dominated by the voluminous Cretaceous to Paleogene granitic rocks. The Ryoke granitoids that occur in the Ryoke Belt in the Southwest Japan Arc are divided into the older and younger granites. The high-K Kibe Granite represents the younger granitic intrusion and is exposed in the Yanai area in the western part of Ryoke Belt. The Kibe Granite is associated with the coeval Himurodake Quartz Diorite and their intrusive age is 91 Ma. However, the Gamano-Obatake Granodiorite, the older granite, intruded the host Ryoke gneisses at 95 Ma. The Gamano-Obatake Granodiorite is characterized by the localized development of migmatitic structure attributed to the intrusion of the Himurodake Quartz Diorite into the granodiorite. Leucocratic pools and patches occur in the granodiorite in the vicinity of the quartz diorite. The Sr and Nd isotopic compositions of the Gamano-Obatake Granodiorite corrected to 91 Ma are plotted within those of the Kibe Granite. Geochemical modeling suggests that partial melting took place in the Gamano-Obatake Granodiorite and resulted in the formation of the Kibe Granite magma. The Himurodake Quartz Diorite is believed to be a heat source for this event. This can be considered as an essential process for the formation of the evolved younger Ryoke granite and for the crustal differentiation in the active continental margin.

  3. Effect of temperature on AOB activity of a partial nitritation SBR treating landfill leachate with extremely high nitrogen concentration.

    PubMed

    Gabarró, J; Ganigué, R; Gich, F; Ruscalleda, M; Balaguer, M D; Colprim, J

    2012-12-01

    This study investigates the effects of temperature on ammonia oxidizing bacteria activity in a partial nitritation (PN) sequencing batch reactor. Stable PN was achieved in a 250 L SBR with a minimum operating volume of 111L treating mature landfill leachate containing an ammonium concentration of around 6000 mg N-NH(4)(+)L(-1) at both 25 and 35 °C. A suitable influent to feed an anammox reactor was achieved in both cases. A kinetic model was applied to study the influence of free ammonia (FA), the free nitrous acid (FNA) inhibition, and the inorganic carbon (IC) limitation. NH(4)(+) and NO(2)(-) concentrations were similar at 25 and 35 °C experiments (about 2500 mg N-NH(4)(+)L(-1) and 3500 mg N-NO(2)(-)L(-1)), FA and FNA concentrations differed due to the strong temperature dependence. FNA was the main source of inhibition at 25 °C, while at 35 °C combined FA and FNA inhibition occurred. DGGE results demonstrated that PN-SBR sludge was enriched on the same AOB phylotypes in both experiments. PMID:23079415

  4. Sulfated Polysaccharides Isolated from Cloned Grateloupia filicina and Their Anticoagulant Activity

    PubMed Central

    Chen, Xiaolin; Yang, Shengfeng; Wang, Jinxia; Song, Lin; Xing, Ronge; Liu, Song; Yu, Huahua; Li, Pengcheng

    2015-01-01

    Sulfated polysaccharides (GSP) were isolated from the cloned Grateloupia filicina which was cultured in Jiaozhou Bay, Qingdao, China. The yield of GSP was 15.75%. The total sugar and sulfate were 40.90 and 19.89%, respectively. And the average molecular weight was 11.7 KDa. The results of neutral sugar analysis showed that GSP was mainly sulfated polysaccharides of galactose. The experiments for activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) anticoagulant assays in vitro indicated that GSP was a good potential anticoagulant. Therefore, this study supplied new thought for the cloned Grateloupia filicina exploitation of high-value products. PMID:25945340

  5. Determining the effect of storage conditions on prothrombin time, activated partial thromboplastin time and fibrinogen concentration in rat plasma samples.

    PubMed

    Goyal, Vinod Kumar; Kakade, Somesh; Pandey, Santosh Kumar; Gothi, Anil Kalidas; Nirogi, Ramakrishna

    2015-10-01

    Coagulation parameters are usually included in clinical and preclinical safety studies to evaluate the effect of xenobiotics on the extrinsic or intrinsic pathways of coagulation. The analysis is generally performed at the time of terminal sacrifice where many activities are scheduled. Chances of delay in analysis are likely particularly when blood is collected for coagulation via the abdominal vena cava. This experiment was planned to assess the variations in coagulation parameters caused by delay in analysis as well as by storage conditions. Blood was collected from the posterior vena cava under isoflurane anesthesia, and the plasma was separated immediately. Coagulation parameters were evaluated at 0, 6, 24 and 48 h from the plasma stored at room temperature, as well as plasma stored under refrigerated and freezing conditions. Stability of the analytes in blood was also evaluated under refrigerated conditions for 6 h. All parameters were analyzed using a semi-automated coagulometer. Prothrombin time (PT) was stable under all three storage conditions for up to 6 h. Although statistically significant differences were observed for activated partial thromboplastin time (APTT) at room and refrigeration temperatures for up to 6 h, the difference was clinically non-relevant. Fibrinogen was found to be the most stable parameter that showed consistency in results even up to 48 h under all three storage conditions. Plasma for PT can be stored and analyzed without any significant changes for up to 6 h from the actual blood collection, while fibrinogen level testing can be extended for up to 48 h after collection under any storage condition. For reliable APTT results, plasma samples should be run immediately after collection. PMID:26206586

  6. A Universal Base in a Specific Role: Tuning up a Thrombin Aptamer with 5-Nitroindole

    PubMed Central

    Tsvetkov, Vladimir B.; Varizhuk, Anna M.; Pozmogova, Galina E.; Smirnov, Igor P.; Kolganova, Natalia A.; Timofeev, Edward N.

    2015-01-01

    In this study we describe new modified analogs of the thrombin binding aptamer (TBA) containing 5-nitroindole residues. It has been shown that all modified TBAs form an anti-parallel G-quadruplex structure and retain the ability to inhibit thrombin. The most advanced TBA variant (TBA-N8) has a substantially increased clotting time and two-fold lower IC50 value compared to the unmodified prototype. Molecular modelling studies suggest that the improved anticoagulant properties of TBA-N8 result from changes in the binding mode of the analog. A modified central loop in TBA-N8 is presumed to participate in the binding of the target protein. Studies of FAM labelled TBA and TBA-N8 showed an improved binding affinity of the modified aptamer and provided evidence of a direct interaction between the modified central loop and thrombin. Our findings have implications for the design of new aptamers with improved binding affinities.

  7. A Universal Base in a Specific Role: Tuning up a Thrombin Aptamer with 5-Nitroindole

    NASA Astrophysics Data System (ADS)

    Tsvetkov, Vladimir B.; Varizhuk, Anna M.; Pozmogova, Galina E.; Smirnov, Igor P.; Kolganova, Natalia A.; Timofeev, Edward N.

    2015-11-01

    In this study we describe new modified analogs of the thrombin binding aptamer (TBA) containing 5-nitroindole residues. It has been shown that all modified TBAs form an anti-parallel G-quadruplex structure and retain the ability to inhibit thrombin. The most advanced TBA variant (TBA-N8) has a substantially increased clotting time and two-fold lower IC50 value compared to the unmodified prototype. Molecular modelling studies suggest that the improved anticoagulant properties of TBA-N8 result from changes in the binding mode of the analog. A modified central loop in TBA-N8 is presumed to participate in the binding of the target protein. Studies of FAM labelled TBA and TBA-N8 showed an improved binding affinity of the modified aptamer and provided evidence of a direct interaction between the modified central loop and thrombin. Our findings have implications for the design of new aptamers with improved binding affinities.

  8. Thrombin products: economic impact of immune-mediated coagulopathies and practical formulary considerations.

    PubMed

    Voils, Stacy A

    2009-07-01

    Thrombin has demonstrated utility in aiding surgical hemostasis since its introduction more than 60 years ago. It is used across a wide variety of surgical procedures by virtually every specialty. Only recently have new equally effective and safe products entered the market, causing decision makers to evaluate formulary selection among products with otherwise modest differences. This evaluation includes identifying costs beyond those of acquisition and storage, as well as indirect factors such as monitoring or specialized distribution requirements. One factor to consider specifically in selection of topical thrombin products is the potential for patients to develop an immune-mediated coagulopathy (IMC) after exposure to bovine-derived thrombin. Costs due to adverse drug events fall into the category of indirect costs and, in some instances, can be substantial if bleeding due to IMC occurs. PMID:19558281

  9. Effect of Na+ binding on the conformation, stability and molecular recognition properties of thrombin

    PubMed Central

    De Filippis, Vincenzo; De Dea, Elisa; Lucatello, Filippo; Frasson, Roberta

    2005-01-01

    In the present work, the effect of Na+ binding on the conformational, stability and molecular recognition properties of thrombin was investigated. The binding of Na+ reduces the CD signal in the far-UV region, while increasing the intensity of the near-UV CD and fluorescence spectra. These spectroscopic changes have been assigned to perturbations in the environment of aromatic residues at the level of the S2 and S3 sites, as a result of global rigidification of the thrombin molecule. Indeed, the Na+-bound form is more stable to urea denaturation than the Na+-free form by ∼2 kcal/mol (1 cal≡4.184 J). Notably, the effects of cation binding on thrombin conformation and stability are specific to Na+ and parallel the affinity order of univalent cations for the enzyme. The Na+-bound form is even more resistant to limited proteolysis by subtilisin, at the level of the 148-loop, which is suggestive of the more rigid conformation this segment assumes in the ‘fast’ form. Finally, we have used hirudin fragment 1–47 as a molecular probe of the conformation of thrombin recognition sites in the fast and ‘slow’ form. From the effects of amino acid substitutions on the affinity of fragment 1–47 for the enzyme allosteric forms, we concluded that the specificity sites of thrombin in the Na+-bound form are in a more open and permissible conformation, compared with the more closed structure they assume in the slow form. Taken together, our results indicate that the binding of Na+ to thrombin serves to stabilize the enzyme into a more open and rigid conformation. PMID:15971999

  10. Dabigatran is Less Effective Than Warfarin at Attenuating Mechanical Heart Valve-Induced Thrombin Generation

    PubMed Central

    Jaffer, Iqbal H; Stafford, Alan R; Fredenburgh, James C; Whitlock, Richard P; Chan, Noel C; Weitz, Jeffrey I

    2015-01-01

    Background Patients with mechanical heart valves (MHV) require warfarin to prevent thromboembolism. Although dabigatran was as effective as warfarin for stroke prevention in atrial fibrillation when compared with warfarin in patients with MHV, the study was stopped early because of more strokes and bleeding with dabigatran. To determine why dabigatran was less effective than warfarin, we compared their effects on thrombin generation induced by MHV. Methods and Results Thrombin generation in the absence or presence of valve leaflets or sewing ring segments (SRS) was quantified. Studies were done in control plasma, plasma depleted of factors (F) XII, XI, or VII, plasma containing varying concentrations of dabigatran, or plasma from patients on dabigatran or warfarin with varying dabigatran concentrations or international normalized ratio (INR) values. Mean endogenous thrombin potential (ETP) increased 1.2-, 1.5-, and 1.8-fold in the presence of leaflets, Teflon SRS, and Dacron SRS, respectively. Whereas ETP in FVII-depleted and control plasma was similar, ETP was reduced to background levels in FXII-depleted plasma and abrogated in FXI-depleted plasma. Dabigatran had little effect on ETP at concentrations below 400 ng/mL, whereas in plasma from warfarin-treated patients, ETP was suppressed with INR values over 1.5. Conclusions MHV induce thrombin generation via the intrinsic pathway and generate sufficient thrombin to overwhelm clinically relevant dabigatran concentrations. In contrast, warfarin is more effective than dabigatran at suppressing MHV-induced thrombin generation. These data explain why dabigatran failed in MHV patients and suggest that strategies targeting FXII or FXI may suppress the root cause of thrombosis in such patients. PMID:26304938

  11. Traumatic Inferior Gluteal Artery Aneurysm Managed with Emergency Transcatheter Thrombin Injection

    SciTech Connect

    Juszkat, Robert; Zielinski, Maciej; Wykretowicz, Mateusz; Piekarek, Alina; Majewski, Waclaw

    2010-06-15

    Pseudoaneurysms of the inferior gluteal artery (IGA) are rare and are often caused by trauma. Treatment options vary and include surgery, ultrasound-guided percutaneous thrombin injection, and endovascular procedures such as stent-graft placement, coil embolization, and glue injection. We report a 70-year-old male who presented to the hospital after a road accident with a posttraumatic pseudoaneurysm that was treated by endovascular thrombin embolization. To the best of our knowledge, this is the first reported case of inferior gluteal artery false aneurysm treated by this method.

  12. Percutaneous Thrombin Injection to Complete SMA Pseudoaneurysm Exclusion After Failing of Endograft Placement

    SciTech Connect

    Szopinski, Piotr Ciostek, Piotr; Pleban, Eliza; Iwanowski, Jaroslaw; Krol, Malgorzata Serafin-; Marianowska, Agnieszka; Noszczyk, Wojciech

    2005-05-15

    Visceral aneurysms are potentially life-threatening vascular lesions. Superior mesenteric artery (SMA) pseudoaneurysms are a rare but well-recognized complication of chronic pancreatitis. Open surgical repair of such an aneurysm, especially in patients after previous surgical treatment, might be dangerous and risky. Stent graft implantation makes SMA pseudoaneurysm exclusion possible and therefore avoids a major abdominal operation. Percutaneous direct thrombin injection is also one of the methods of treating aneurysms in this area. We report a first case of percutaneous ultrasound-guided thrombin injection to complete SMA pseudoaneurysm exclusion after an unsuccessful endograft placement. Six-month follow-up did not demonstrate any signs of aneurysm recurrence.

  13. Thrombin-thrombomodulin interaction: energetics and potential role of water as an allosteric effector.

    PubMed

    De Cristofaro, R; Picozzi, M; De Candia, E; Rocca, B; Landolfi, R

    1995-08-15

    The interaction of rabbit lung thrombomodulin (TM) and C-terminal hirudin 54-65 fragment (Hir54-65) with human alpha-thrombin were investigated by exploiting their competitive inhibition of thrombin-fibrinogen interaction. Measurements of Ki values for TM and Hir54-65 interactions with human alpha-thrombin performed over a temperature range spanning from 10 to 40 degrees C showed a constant enthalpy for both ligands. The enthalpic and entropic contributions to the free energy of binding, however, are different for TM and the hirudin peptide. The calculated values of delta H and delta S, in fact, were -47.3 +/- 2.51 kJ (-11.3 +/- 0.6 kcal)/mol and -42.7 +/- 7.9 J (-10.2 +/- 1.9 cal)/mol.K for the hirudin peptide, while being -22.9 +/- 2.09 kJ (-5.47 +/- 0.5 kcal)/mol and 102.50 +/- 6.69 J (24.5 +/- 1.6 cal)/mol.K respectively for TM binding. These findings indicate that the interaction between thrombin and Hir54-65 is largely driven by the enthalpic contribution, whereas the positive entropy change is the driving force for the formation of the thrombin-TM complex. In other experiments performed in the presence of various concentrations of either sorbitol or sucrose it could be demonstrated that the value of the equilibrium association constant for thrombin-TM interaction increases as a function of the osmotic pressure, while the thrombin-Hir54-65 interaction was not affected by the same conditions. Moreover, control experiments showed that no major conformational changes are produced on TM by osmotic pressures used in the present study. From these experiments it was calculated that roughly 35 water molecules are released into the bulk water upon TM binding. Such a phenomenon, which is likely to be responsible for the entropic change described above, indicates the relevance of hydration processes for the formation of the thrombin-TM adduct. PMID:7646471

  14. Computer based screening of compound databases: 1. Preselection of benzamidine-based thrombin inhibitors.

    PubMed

    Fox, T; Haaksma, E E

    2000-07-01

    We present a computational protocol which uses the known three-dimensional structure of a target enzyme to identify possible ligands from databases of compounds with low molecular weight. This is accomplished by first mapping the essential interactions in the binding site with the program GRID. The resulting regions of favorable interaction between target and ligand are translated into a database query, and with UNITY a flexible 3D database search is performed. The feasibility of this approach is calibrated with thrombin as the target. Our results show that the resulting hit lists are enriched with thrombin inhibitors compared to the total database. PMID:10896314

  15. Liquid-Crystal Biosensor Based on Nickel-Nanosphere-Induced Homeotropic Alignment for the Amplified Detection of Thrombin.

    PubMed

    Zhao, Dongyu; Peng, Yi; Xu, Lihong; Zhou, Wei; Wang, Qian; Guo, Lin

    2015-10-28

    A new liquid-crystal (LC)-based sensor operated by nickel nanosphere (NiNS)-induced homeotropic alignment for the label-free monitoring of thrombin was reported. When doped with NiNSs, a uniform vertical orientation of 4-cyano-4'-pentylbiphenyl (5CB) was easily obtained. A sandwich system of aptamer/thrombin/aptamer-functionalized gold nanoparticles (AuNPs) was fabricated, and AuNPs-aptamer conjugation caused the disruption of the 5CB orientation, leading to an obvious change of the optical appearance from a dark to a bright response to thrombin concentrations from 0.1 to 100 nM. This design also allowed quantitative detection of the thrombin concentration. This distinctive and sensitive thrombin LC sensor provides a new principle for building LC-sensing systems. PMID:26458050

  16. Differential pathway coupling efficiency of the activated insulin receptor drives signaling selectivity by XMetA, an allosteric partial agonist antibody

    Technology Transfer Automated Retrieval System (TEKTRAN)

    XMetA, an anti-insulin receptor (IR) monoclonal antibody, is an allosteric partial agonist of the IR. We have previously reported that XMetA activates the “metabolic-biased” Akt kinase signaling pathway while having little or no effect on the “mitogenic” MAPK signaling pathwayof ERK 1/2. To inves...

  17. HIV-induced T-cell activation/exhaustion in rectal mucosa is controlled only partially by antiretroviral treatment.

    PubMed

    Rueda, Cesar Mauricio; Velilla, Paula Andrea; Chougnet, Claire A; Montoya, Carlos Julio; Rugeles, Maria Teresa

    2012-01-01

    Peripheral blood T-cells from untreated HIV-1-infected patients exhibit reduced immune responses, usually associated with a hyperactivated/exhausted phenotype compared to HAART treated patients. However, it is not clear whether HAART ameliorates this altered phenotype of T-cells in the gastrointestinal-associated lymphoid tissue (GALT), the main site for viral replication. Here, we compared T-cells from peripheral blood and GALT of two groups of chronically HIV-1-infected patients: untreated patients with active viral replication, and patients on suppressive HAART. We characterized the T-cell phenotype by measuring PD-1, CTLA-4, HLA-DR, CD25, Foxp3 and granzyme A expression by flow cytometry; mRNA expression of T-bet, GATA-3, ROR-γt and Foxp3, and was also evaluated in peripheral blood mononuclear cells and rectal lymphoid cells. In HIV-1+ patients, the frequency of PD-1(+) and CTLA-4(+) T-cells (both CD4+ and CD8+ T cells) was higher in the GALT than in the blood. The expression of PD-1 by T-cells from GALT was higher in HIV-1-infected subjects with active viral replication compared to controls. Moreover, the expression per cell of PD-1 and CTLA-4 in CD4(+) T-cells from blood and GALT was positively correlated with viral load. HAART treatment decreased the expression of CTLA-4 in CD8(+) T cells from blood and GALT to levels similar as those observed in controls. Frequency of Granzyme A(+) CD8(+) T-cells in both tissues was low in the untreated group, compared to controls and HAART-treated patients. Finally, a switch towards Treg polarization was found in untreated patients, in both tissues. Together, these findings suggest that chronic HIV-1 infection results in an activated/exhausted T-cell phenotype, despite T-cell polarization towards a regulatory profile; these alterations are more pronounced in the GALT compared to peripheral blood, and are only partiality modulated by HAART. PMID:22276176

  18. Characterization of CM572, a Selective Irreversible Partial Agonist of the Sigma-2 Receptor with Antitumor Activity.

    PubMed

    Nicholson, Hilary; Comeau, Anthony; Mesangeau, Christophe; McCurdy, Christopher R; Bowen, Wayne D

    2015-08-01

    The sigma-2 receptors are promising therapeutic targets because of their significant upregulation in tumor cells compared with normal tissue. Here, we characterize CM572 [3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)-6-isothiocyanatobenzo[d]oxazol-2(3H)-one] (sigma-1 Ki ≥ 10 µM, sigma-2 Ki = 14.6 ± 6.9 nM), a novel isothiocyanate derivative of the putative sigma-2 antagonist, SN79 [6-acetyl-3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)benzo[d]oxazol-2(3H)-one]. CM572 bound irreversibly to sigma-2 receptors by virtue of the isothiocyanate moiety but not to sigma-1. Studies in human SK-N