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Sample records for direct thrombin inhibitor

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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

  7. [Successful direct thrombin inhibitor treatment of a left atrial appendage thrombus developed under rivaroxaban therapy].

    PubMed

    Szegedi, Nándor; Gellér, László; Tahin, Tamás; Merkely, Béla; Széplaki, Gábor

    2016-01-24

    The authors present the history of a 62-year-old man on continuous rivaroxaban therapy who was scheduled for pulmonary vein isolation due to persistent atrial fibrillation. Preoperative transesophageal echocardiography detected the presence of left atrial appendage thrombus. Thrombophilia tests showed that the patient was heterozygous carrier of the methylene-tetrahydrofolate reductase gene mutation. The authors hypothesized that a direct thrombin inhibitor might exert a more appropriate effect against thrombosis in this case and, therefore, a switch to dabigatran was performed. After two months of anticoagulation with the direct thrombin inhibitor and folic acid supplementation the thrombus resolved. The authors underline that thrombus formation may develop in atrial fibrillation even if the patient is adequately treated with rivaroxaban. This case suggests, that methylene-tetrahydrofolate reductase gene mutation may modulate the efficacy of direct Xa factor inhibitors. According to this case history, dabigatran may be an effective therapeutic option in resolving established thrombus. PMID:26772828

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. Novel anticoagulants in clinical development: focus on factor Xa and direct thrombin inhibitors.

    PubMed

    Steffel, Jan; Lüscher, Thomas F

    2009-08-01

    Vitamin K antagonists are the mainstay in the prevention and treatment of thromboembolic diseases. Although effective under optimal conditions, several drawbacks are imminent to the long-term application of these drugs due to their narrow therapeutic window, interactions with other drugs as well as the need for regular monitoring and the risk of a recurrent event versus the risk of bleeding. To overcome these downsides, novel anticoagulants are being developed; in contrast to vitamin K antagonists, these novel agents specifically and selectively block central elements of the coagulation cascade. Several clinical trials have demonstrated the efficacy and safety of selective FXa inhibitors (such as fondaparinux, rivaroxaban, apixaban) and direct thrombin inhibitors (such as lepirudin, bivalirudin, dabigatran etexilate) in the treatment of typical indications for conventional vitamin K antagonists, in particular, the prevention and treatment of venous thromboembolism. This review summarizes the results and designs of recently published and ongoing clinical trials of novel anticoagulants. PMID:19561526

  15. 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

  16. 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

  17. 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

  18. 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

  19. Meta-analysis of randomized controlled trials on risk of myocardial infarction from the use of oral direct thrombin inhibitors.

    PubMed

    Artang, Ramin; Rome, Eric; Nielsen, Jørn Dalsgaard; Vidaillet, Humberto J

    2013-12-15

    Dabigatran has been associated with greater risk of myocardial infarction (MI) than warfarin. It is unknown whether the increased risk is unique to dabigatran, an adverse effect shared by other oral direct thrombin inhibitors (DTIs), or the result of a protective effect of warfarin against MI. To address these questions, we systematically searched MEDLINE and performed a meta-analysis on randomized trials that compared oral DTIs with warfarin for any indication with end point of MIs after randomization. We furthermore performed a secondary meta-analysis on atrial fibrillation stroke prevention trials with alternative anticoagulants compared with warfarin with end point of MIs after randomization. A total of 11 trials (39,357 patients) that compared warfarin to DTIs (dabigatran, ximelagatran, and AZD0837) were identified. In these trials, patients treated with oral DTIs were more likely to experience an MI than their counterparts treated with warfarin (285 of 23,333 vs 133 of 16,024, odds ratio 1.35, 95% confidence interval 1.10 to 1.66, p = 0.005). For secondary analysis, 8 studies (69,615 patients) were identified that compared warfarin with alternative anticoagulant including factor Xa inhibitors, DTIs, aspirin, and clopidogrel. There was no significant advantage in the rate of MIs with the use of warfarin versus comparators (odds ratio 1.06, 95% confidence interval 0.85 to 1.34, p = 0.59). In conclusion, our data suggest that oral DTIs were associated with increased risk of MI. This increased risk appears to be a class effect of these agents, not a specific phenomenon unique to dabigatran or protective effect of warfarin. These findings support the need for enhanced postmarket surveillance of oral DTIs and other novel agents. PMID:24075284

  20. 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

  1. [Analysis of the Cochrane Review: Direct thrombin inhibitors versus vitamin K antagonists for preventing cerebral or systemic embolism in people with non-valvular atrial fibrillation. Cochrane Database Syst Rev. 2014,3:CD009893].

    PubMed

    Vaz Carneiro, António; Costa, João

    2014-01-01

    Ischemic stroke is one of the most important complications of lone (non-valvular) atrial fibrillation. Its prevention is usually accomplished through oral anticoagulation. Until a few years ago warfarin was the most used agent, but recently two new pharmacologic classes have been introduced for stroke prevention in these patients: oral direct thrombin inhibitors (dabigatran and ximelagatran) and oral factor Xa inhibitors (rivaroxaban, apixaban and edoxaban). In this systematic review, oral direct thrombin inhibitors were compared with warfarin for efficacy and safety. The results indicate that there is no difference in terms of efficacy (except dabigatran 150 mg BID). Oral direct thrombin inhibitors presented less hemorrhages but increased treatment withdrawal due to adverse side-effects (the authors performed post-hoc analyses excluding ximelagatran because this drug was withdrawn from the market owing to safety concerns). There was no difference in terms of mortality between the agents. PMID:24813481

  2. Ximelagatran, a new oral direct thrombin inhibitor, for the prevention of venous thromboembolic events in major elective orthopaedic surgery. Efficacy, safety and anaesthetic considerations.

    PubMed

    Rosencher, N

    2004-08-01

    The oral direct thrombin inhibitor ximelagatran shows great promise for prevention of venous thromboembolic events following major elective orthopaedic surgery. Its consistent and predictable pharmacokinetics and pharmacodynamics across a wide range of patient populations allow administration with fixed dosing and with no coagulation monitoring. In orthopaedic surgery clinical trials, ximelagatran was effective and well tolerated compared with standard therapy, with dose and timing relative to surgery important factors in determining its optimal profile. In European trials, an initial 3-mg postoperative dose of subcutaneous melagatran, the active form of ximelagatran, followed by oral ximelagatran 24 mg twice daily achieved similar efficacy and safety to enoxaparin. Although the risk of spinal haematoma following neuraxial anaesthesia is rare, it is increased by the concomitant use of anticoagulants. In orthopaedic surgery trials with ximelagatran to date, complications such as spinal haematoma have not been reported. The pharmacokinetic profile of ximelagatran suggests that concurrent use with neuraxial anaesthesia should require no further precautions than those currently necessary with low-molecular-weight heparin. PMID:15270973

  3. Budd-Chiari syndrome in very young adult patients with polycythemia vera: report of case series with good outcome with direct thrombin inhibitor treatment.

    PubMed

    Goldstein, Gal; Maor, Jacob; Kleinbaum, Yeruham; Palumbo, Michal; Sidi, Yehezkel; Salomon, Ophira

    2013-12-01

    Polycythemia vera is a Philadelphia chromosome-negative myeloproliferative disorder with incidence of 1% under the age of 25. The Budd-Chiari syndrome (BCS) is a well known complication of polycythemia vera even in children, and characterized by occlusion of hepatic outflow. A computerized archive search of medical records at Sheba Medical Center of the past three decades of patients with polycythemia vera and BCS under the age of 25 years was performed. A work-up for JAK2 V617F mutation and thrombophilia was done. Medical charts and imaging tests were carefully reviewed. Three patients under the age of 22 were finally recruited. Two of those were found in life-threatening condition and improved clinically following treatment with bivalirudin, a direct thrombin inhibitor. It is conceivable that bivalirudin contributed to a favorable outcome of those patients in comparison to historical outcome previously reported. In conclusion, polycythemia vera in the young is not a mild disease since BCS, which is one of its complication, can be fatal even in those age group unrelated to the presence of hereditary thrombophilia. Once BCS occurs, we would suggest giving a trial with bivalirudin before an invasive procedure is planned. PMID:23941968

  4. [Surgery and invasive procedures in patients on long-term treatment with oral direct thrombin or factor Xa inhibitors].

    PubMed

    Sié, P; Samama, C-M; Godier, A; Rosencher, N; Steib, A; Llau, J-V; van der Linden, P; Pernod, G; Lecompte, T; Gouin-Thibault, I; Albaladejo, P

    2011-09-01

    Direct oral anticoagulants (DOAs), inhibitors of factor IIa or Xa, are expected to replace vitamin K antagonists in most of their indications. It is likely that patients on long-term treatment with DOAs will be exposed to elective or emergency surgery or invasive procedures. Due to the present lack of experience in such conditions, we cannot make recommendations, but only propose perioperative management for optimal safety as regards the risk of bleeding and thrombosis. DOAs may increase surgical bleeding, they have no validated antagonists, they cannot be monitored by simple, standardised laboratory assays, and their pharmacokinetics vary significantly from patient to patient. Although DOAs differ in many respects, the proposals in the perioperative setting need not be specific to each. For procedures with low risk of haemorrhage, a therapeutic window of 48 h (last administration 24h before surgery, restart 24h after) is proposed. For procedures with medium or high haemorrhagic risk, we suggest stopping DOAs 5 days before surgery to ensure complete elimination of the drug in all patients. The treatment should be resumed only when the risk of bleeding has been controlled. In patients with a high risk of thrombosis (e.g. those in atrial fibrillation with an antecedent of stroke), bridging with heparin (low molecular weight, or unfractionated if the former is contraindicated) is proposed. In emergency, the procedure should be postponed for as long as possible (minimum 1-2 half-lives) and non-specific anti-haemorrhagic agents, such as recombinant human activated factor VIIa, or prothrombin concentrates, should not be given for prophylactic reversal, due to their uncertain benefit-risk. PMID:21820844

  5. 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.

  6. 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

  7. 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

  8. 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

  9. Surgery and invasive procedures in patients on long-term treatment with direct oral anticoagulants: thrombin or factor-Xa inhibitors. Recommendations of the Working Group on Perioperative Haemostasis and the French Study Group on Thrombosis and Haemostasis.

    PubMed

    Sié, Pierre; Samama, Charles M; Godier, Anne; Rosencher, Nadia; Steib, Annick; Llau, Juan V; Van der Linden, Philippe; Pernod, Gilles; Lecompte, Thomas; Gouin-Thibault, Isabelle; Albaladejo, Pierre

    2011-12-01

    Direct oral anticoagulants (DOAs)--inhibitors of thrombin or factor-Xa--are expected to replace vitamin K antagonists in most of their indications. Patients receiving long-term treatment with DOAs are likely to be exposed to elective or emergency surgery or invasive procedures. Owing to the present lack of experience in such conditions, we cannot make recommendations, but only propose perioperative management for optimal safety regarding the risk of bleeding and thrombosis. DOAs may increase surgical bleeding, they have no validated antagonists, they cannot be monitored by simple standardized laboratory assays and their pharmacokinetics vary significantly between patients. Although DOAs differ in many respects, the proposals in the perioperative setting need not be specific to each. For procedures with low haemorrhagic risk, a therapeutic window of 48 hours (last administration 24 hours before surgery, restart 24 hours after) is proposed. For procedures with medium or high haemorrhagic risk, we suggest stopping DOAs 5 days before surgery to ensure complete elimination in all patients. Treatment should be resumed only when the risk of bleeding has been controlled. In patients at high thrombotic risk (e.g. those in atrial fibrillation with a history of stroke), bridging with heparin (low molecular-weight heparin, or unfractionated heparin, if the former is contraindicated) is proposed. In an emergency, the procedure should be postponed for as long as possible (minimum 1-2 half-lives) and non-specific antihaemorrhagic agents, such as recombinant human activated factor VIIa or prothrombin complex concentrates should not be given for prophylactic reversal due to their uncertain benefit-risk. PMID:22152517

  10. 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

  11. 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

  12. Management of major bleeding complications and emergency surgery in patients on long-term treatment with direct oral anticoagulants, thrombin or factor-Xa inhibitors: proposals of the working group on perioperative haemostasis (GIHP) - March 2013.

    PubMed

    Pernod, Gilles; Albaladejo, Pierre; Godier, Anne; Samama, Charles M; Susen, Sophie; Gruel, Yves; Blais, Normand; Fontana, Pierre; Cohen, Ariel; Llau, Juan V; Rosencher, Nadia; Schved, Jean-François; de Maistre, Emmanuel; Samama, Meyer M; Mismetti, Patrick; Sié, Pierre

    2013-01-01

    Direct new oral anticoagulants (NOACs) - inhibitors of thrombin or factor Xa - are intended to be used largely in the treatment of venous thromboembolic disease or the prevention of systematic embolism in atrial fibrillation, instead of vitamin K antagonists. Like any anticoagulant treatment, they are associated with spontaneous or provoked haemorrhagic risk. Furthermore, a significant proportion of treated patients are likely to be exposed to emergency surgery or invasive procedures. Given the absence of a specific antidote, the action to be taken in these situations must be defined. The lack of data means that it is only possible to issue proposals rather than recommendations, which will evolve according to accumulated experience. The proposals presented here apply to dabigatran (Pradaxa(®)) and rivaroxaban (Xarelto(®)); data for apixaban and edoxaban are still scarce. For urgent surgery with haemorrhagic risk, the drug plasma concentration should be less or equal to 30ng/mL for dabigatran and rivaroxaban should enable surgery associated with a high bleeding risk. Beyond that, if possible, the intervention should be postponed by monitoring the drug concentration. The course to follow is then defined according to the NOAC and its concentration. If the anticoagulant dosage is not immediately available, worse propositions, based on the usual tests (prothrombin time and activated partial thromboplastin time), are presented. However, these tests do not really assess drug concentration or the risk of bleeding that depends on it. In case of serious bleeding in a critical organ, the effect of anticoagulant therapy should be reduced using a non-specific procoagulant drug as a first-line approach: activated prothrombin complex concentrate (aPCC) (FEIBA(®) 30-50U/kg) or non-activated PCC (50U/kg). In addition, for any other type of severe haemorrhage, the administration of a procoagulant drug, which is potentially thrombogenic in these patients, is discussed according

  13. 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

  14. 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

  15. 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

  16. 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

  17. 1,2-disubstituted cyclohexane derived tripeptide aldehydes as novel selective thrombin inhibitors.

    PubMed

    Harmat, N J; Di Bugno, C; Criscuoli, M; Giorgi, R; Lippi, A; Martinelli, A; Monti, S; Subissi, A

    1998-05-19

    A series of tripeptide arginine aldehydes was synthesized by replacement of proline with 1,2-disubstituted cyclohexane derivatives in the sequence of D-MePhe-Pro-Arg-H. Based on molecular modeling, further modification of the D-MePhe residue resulted in a potent and selective thrombin inhibitor. PMID:9871744

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  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. A Food Effect Study of an Oral Thrombin Inhibitor and Prodrug Approach To Mitigate It.

    PubMed

    Lee, Jihye; Kim, Bongchan; Kim, Tae Hun; Lee, Sun Hwa; Park, Hee Dong; Chung, Kyungha; Lee, Sung-Hack; Paek, Seungyup; Kim, Eunice EunKyeong; Yoon, SukKyoon; Kim, Aeri

    2016-04-01

    LB30870, a new direct thrombin inhibitor, showed 80% reduction in oral bioavailability in fed state. The present study aims to propose trypsin binding as a mechanism for such negative food effect and demonstrate a prodrug approach to mitigate food effect. Effect of food composition on fed state oral bioavailability of LB30870 was studied in dogs. Various prodrugs were synthesized, and their solubility, permeability, and trypsin binding affinity were measured. LB30870 and prodrugs were subject to cocrystallization with trypsin, and the X-ray structures of cocrystals were determined. Food effect was studied in dogs for selected prodrugs. Protein or lipid meal appeared to affect oral bioavailability of LB30870 in dogs more than carbohydrate meal. Blocking both carboxyl and amidine groups of LB30870 resulted in trypsin Ki values orders of magnitude higher than that of LB30870. Prodrugs belonged to either Biopharmaceutical Classification System I, II, or III. X-ray crystallography revealed that prodrugs did not bind to trypsin, but instead their hydrolysis product at the amidine blocking group formed cocrystal with trypsin. A prodrug with significantly less food effect than LB30870 was identified. Binding of prodrugs to food components such as dietary fiber appeared to counteract the positive effect brought with the prodrug approach. Further formulation research is warranted to enhance the oral bioavailability of prodrugs. In conclusion, this study is the first to demonstrate that the negative food effect of LB30870 can be attributed to trypsin binding. Trypsin binding study is proposed as a screening tool during lead optimization to minimize food effect. PMID:26886576

  6. 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

  7. 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

  8. 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

  9. 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

  10. [Management of major bleeding complications and emergency surgery in patients on long-term treatment with direct oral anticoagulants, thrombin or factor-Xa inhibitors. Proposals of the Working Group on Perioperative Haemostasis (GIHP) - March 2013].

    PubMed

    Pernod, G; Albaladejo, P; Godier, A; Samama, C M; Susen, S; Gruel, Y; Blais, N; Fontana, P; Cohen, A; Llau, J V; Rosencher, N; Schved, J F; de Maistre, E; Samama, M M; Mismetti, P; Sié, P

    2013-10-01

    New direct oral anticoagulants (NOAC), inhibitors of factor IIa or Xa, are expected to be widely used for the treatment of venous thromboembolic disease, or in case of atrial fibrillation. Such anticoagulant treatments are known to be associated with haemorrhagic complications. Moreover, it is likely that such patients on long-term treatment with NOAC will be exposed to emergency surgery or invasive procedures. Due to the present lack of experience in such conditions, we cannot make recommendations, but only propose management for optimal safety as regards the risk of bleeding in such emergency conditions. In this article, only dabigatran and rivaroxaban were discussed. For emergency surgery at risk of bleeding, we propose to dose the plasmatic concentration of drug. Levels inferior or equal to 30ng/mL for both dabigatran and rivaroxaban, should enable the realization of a high bleeding risk surgery. For higher concentration, it was proposed to postpone surgery by monitoring the evolution of the drug concentration. Action is then defined by the kind of NOAC and its concentration. If the dosage of the drug is not immediately available, proposals only based on the usual tests, PT and aPTT, also are presented. However, these tests do not really assess drug concentration or bleeding risk. In case of severe haemorrhage in a critical organ, it is proposed to reduce the effect of anticoagulant therapy using a nonspecific procoagulant drug (activated prothrombin concentrate, FEIBA, 30-50U/kg, or non-activated 4-factors prothrombin concentrates 50U/kg). For any other type of severe haemorrhage, the administration of such a procoagulant drug, potentially thrombogenic in these patients, will be discussed regarding concentration of NACO and possibilities for mechanical haemostasis. PMID:23993157

  11. 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

  12. 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

  13. METHODS OF TREATING OR PREVENTING DEMYELINATION USING THROMBIN INHIBITORS AND METHODS OF DETECTING DEMYELINATION USING NEUROFASCIN 155 | NCI Technology Transfer Center | TTC

    Cancer.gov

    Researchers at the Eunice Kennedy Shriver National Institute of Child Health and Human Development (“NICHD”), seek CRADA partner or collaboration for development of agents to treat multiple sclerosis or other conditions associated with myelin remodeling by administering an agent that inhibits cleavage of Neurofascin 155 or Caspr1. The agent could be a thrombin inhibitor, an agent that inhibits thrombin expression, an anti-thrombin antibody that specifically inhibits thrombin mediated cleavage of Neurofascin 155, a mutated version or fragment of Neurofascin 155 or Caspr1, or antibodies to Neurofascin 155 or Caspr1.

  14. Microfluidic Chip-Based Online Screening Coupled to Mass Spectrometry: Identification of Inhibitors of Thrombin and Factor Xa.

    PubMed

    Iyer, Janaki Krishnamoorthy; Otvos, Reka A; Kool, Jeroen; Kini, R Manjunatha

    2016-02-01

    Thrombin and factor Xa (FXa) are critical enzymes of the blood coagulation cascade and are excellent targets of anticoagulant agents. Natural sources present an array of anticoagulants that can be developed as antithrombotic drugs. High-resolution, online screening techniques have been developed for the identification of drug leads from complex mixtures. In this study, we have developed and optimized a microfluidic online screening technique coupled to nano-liquid chromatography (LC) and in parallel with a mass spectrometer for the identification of thrombin and FXa inhibitors in mixtures. Inhibitors eluting from the nano-LC were split postcolumn in a 1:1 ratio; half was fed into a mass spectrometer (where its mass is detected), and the other half was fed into a microfluidic chip (which acts as a microreactor for the online assays). With our platform, thrombin and FXa inhibitors were detected in the assay in parallel with their mass identification. These methods are suitable for the identification of inhibitors from sample amounts as low as sub-microliter volumes. PMID:26323281

  15. Direct electrochemistry and electrocatalysis of a glucose oxidase-functionalized bioconjugate as a trace label for ultrasensitive detection of thrombin.

    PubMed

    Bai, Lijuan; Yuan, Ruo; Chai, Yaqin; Yuan, Yali; Wang, Yan; Xie, Shunbi

    2012-11-18

    For the first time, a glucose oxidase-functionalized bioconjugate was prepared and served as a new trace label through its direct electrochemistry and electrocatalysis in a sandwich-type electrochemical aptasensor for ultrasensitive detection of thrombin. PMID:23032443

  16. 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

  17. 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

  18. Oral direct thrombin inhibitor AZD0837 for the prevention of stroke and systemic embolism in patients with non-valvular atrial fibrillation: a randomized dose-guiding, safety, and tolerability study of four doses of AZD0837 vs. vitamin K antagonists

    PubMed Central

    Lip, Gregory Y.H.; Rasmussen, Lars H.; Olsson, S. Bertil; Jensen, Eva C.; Persson, Anders L.; Eriksson, Ulf; Wåhlander, Karin F.C.

    2009-01-01

    Aims Oral anticoagulation with vitamin K antagonists (VKAs) for stroke prevention in atrial fibrillation (AF) is effective but has significant limitations. AZD0837, a new oral anticoagulant, is a prodrug converted to a selective and reversible direct thrombin inhibitor (AR-H067637). We report from a Phase II randomized, dose-guiding study (NCT00684307) to assess safety, tolerability, pharmacokinetics, and pharmacodynamics of extended-release AZD0837 in patients with AF. Methods and results Atrial fibrillation patients (n = 955) with ≥1 additional risk factor for stroke were randomized to receive AZD0837 (150, 300, or 450 mg once daily or 200 mg twice daily) or VKA (international normalized ratio 2–3, target 2.5) for 3–9 months. Approximately 30% of patients were naïve to VKA treatment. Total bleeding events were similar or lower in all AZD0837 groups (5.3–14.7%, mean exposure 138–145 days) vs. VKA (14.5%, mean exposure 161 days), with fewer clinically relevant bleeding events on AZD0837 150 and 300 mg once daily. Adverse events were similar between treatment groups; with AZD0837, the most common were gastrointestinal disorders (e.g. diarrhoea, flatulence, or nausea). d-Dimer, used as a biomarker of thrombogenesis, decreased in all groups in VKA-naïve subjects with treatment, whereas in VKA pre-treated patients, d-dimer levels started low and remained low in all groups. As expected, only a few strokes or systemic embolic events occurred. In the AZD0837 groups, mean S-creatinine increased by ∼10% from baseline and returned to baseline following treatment cessation. The frequency of serum alanine aminotransferase ≥3× upper limit of normal was similar for AZD0837 and VKA. Conclusion AZD0837 was generally well tolerated at all doses tested. AZD0837 treatment at an exposure corresponding to the 300 mg od dose in this study provides similar suppression of thrombogenesis at a potentially lower bleeding risk compared with dose-adjusted VKA. This study is

  19. 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

  20. 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.

  1. 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

  2. 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

  3. 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

  4. Ligand binding cooperativity: Bioisosteric replacement of CO with SO2 among thrombin inhibitors.

    PubMed

    Said, Ahmed M; Hangauer, David G

    2016-08-15

    Ligand-protein binding is a complex process that involves the formation of number of non-covalent interactions, e.g. H-bonds and hydrophobic interactions, between the ligand and the protein host. Upon binding, ligand functional groups can act synergistically (positive cooperativity) to improve the overall ligand binding affinity beyond what would be expected from their individual contributions. In this study, using thrombin as a protein model system, we evaluated the effect of the bioisosteric replacement of a carbonyl functionality with a sulphonyl functionality on positive cooperativity between their H-bonds with thrombin and hydrophobic binding in the adjacent S3 pocket. The positive cooperativity observed was greatly reduced when replacing the carbonyl group with a sulphonyl group. Evaluating how bioisosteric replacements affect cooperativity is important for making better informed ligand optimization SAR decisions. PMID:27445170

  5. The effect of a polyurethane coating incorporating both a thrombin inhibitor and nitric oxide on hemocompatibility in extracorporeal circulation

    PubMed Central

    Major, Terry C.; Brisbois, Elizabeth J.; Jones, Anna M.; Zanetti, Margaux E.; Annich, Gail M.; Bartlett, Robert H.; Handa, Hitesh

    2014-01-01

    Nitric oxide (NO) releasing (NORel) materials have been extensively investigated to create localized increases in NO concentration by the proton driven diazeniumdiolate-containing polymer coatings and demonstrated to improve extracorporeal circulation (ECC) hemocompatibility. In this work, the NORel polymeric coating composed of a diazeniumdiolated dibutylhexanediamine (DBHD-N2O2)-containing hydrophobic Elast-eon™ (E2As) polyurethane was combined with a direct thrombin inhibitor, argatroban (AG), and evaluated in a 4 h rabbit thrombogenicity model without systemic anticoagulation. In addition, the immobilizing of argatroban to E2As polymer was achieved by either a polyethylene glycol-containing (PEGDI) or hexane methylene (HMDI) diisocyanate linker. The combined polymer film was coated on the inner walls of ECC circuits to yield significantly reduced ECC thrombus formation compared to argatroban alone ECC control after 4 h blood exposure (0.6 ± 0.1 AG/HMDI/NORel vs 1.7 ± 0.2 cm2 AG/HMDI control). Platelet count (2.8 ± 0.3 AG/HMDI/NORel vs 1.9 ± 0.1 × 108/ml AG/HMDI control) and plasma fibrinogen levels were preserved after 4 h blood exposure with both the NORel/argatroban combination and the AG/HMDI control group compared to baseline. Platelet function as measured by aggregometry remained near normal in both the AG/HMDI/NORel (63 ± 5%) and AG/HMDI control (58 ± 7%) groups after 3 h compared to baseline (77 ± 1%). Platelet P-selectin mean fluorescence intensity (MFI) as measured by flow cytometry also remained near baseline levels after 4 h on ECC to ex vivo collagen stimulation (16 ± 3 AG/HMDI/NORel vs 11 ± 2 MFI baseline). These results suggest that the combined AG/HMDI/NORel polymer coating preserves platelets in blood exposure to ECCs to a better degree than AG/PEGDI/NORel, NORel alone or AG alone. These combined antithrombin, NO-mediated antiplatelet effects were shown to improve thromboresistance of the AG/HMDI/NORel polymer-coated ECCs and move

  6. 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

  7. Structure-based organic synthesis of unnatural aeruginosin hybrids as potent inhibitors of thrombin.

    PubMed

    Hanessian, Stephen; Ersmark, Karolina; Wang, Xiaotian; Del Valle, Juan R; Blomberg, Niklas; Xue, Yafeng; Fjellström, Ola

    2007-06-15

    Based on X-ray crystallographic data of complexes of chlorodysinosin A with the enzyme thrombin, a series of analogs were synthesized varying the nature of the P(1), P(2), and P(3) pharmacophoric sites and the central octahydroindole carboxyamide core. In general, introduction of a hydrophobic substituent on the d-leucine amide residue dramatically improved the inhibition of the enzyme. This is rationalized based on a better fit of the P(3) subunit in the hydrophobic S(3) enzyme site. Single digit nanomolar inhibition expressed as IC(50) was observed for several analogs. PMID:17428662

  8. 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

  9. 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

  10. Oral anticoagulation with Factor Xa and thrombin inhibitors: Is there an alternative to warfarin?

    PubMed

    Zikria, Jennifer; Ansell, Jack

    2009-12-01

    Vitamin K antagonists (VKA), such as warfarin, have been the only available oral anticoagulants despite their many limitations. The greatest medical need is to find a replacement for warfarin for long-term therapy, particularly for stroke prevention in atrial fibrillation (AF) patients. Emerging oral anticoagulants are free from many of warfarin's drawbacks and may offer a convenient alternative. Drugs in advanced development target factor Xa (rivaroxaban, apixaban) or thrombin (dabigatran etexilate). Recently, the RE-LY phase III study found dabigatran etexilate was an effective and convenient alternative to warfarin in stroke prevention for AF patients. Within the next two years, similar studies comparing rivaroxaban and apixaban versus warfarin in AF patients will become available. This paper reviews warfarin's limitations, discusses the pharmacokinetics of emerging anticoagulants in advanced development, and summarizes trials with an emphasis on head-to-head studies comparing novel anticoagulants to warfarin. PMID:20040270

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. Association between thrombin-activatable fibrinolysis inhibitor gene polymorphisms and venous thrombosis risk: a meta-analysis.

    PubMed

    Wang, Wei; Ma, He; Lu, Lili; Sun, Guixiang; Liu, Dang; Zhou, Yunti; Tong, Yue; Lu, Zhaojun

    2016-06-01

    Thrombin-activatable fibrinolysis inhibitor (TAFI) is an important antifibrinolytic factor that has been shown in increased concentrations to be associated with an increased risk for venous thrombosis. However, the effect of TAFI gene polymorphisms on the risk of venous thrombosis remains debatable. The aim of the current study was to evaluate the association of three single nucleotide polymorphisms: 505G>A (rs3742264), 1040 C>T (rs1926447) and -438G>A (rs2146881) with venous thrombosis risk using a meta-analysis. A systematic literature search for eligible studies published before 20 January 2015 was conducted in PubMed, EMBASE, Web of Science, WanFang database and Chinese National Knowledge Infrastructure. We assessed the possible association by pooled odds ratio and its 95% confidence interval. A total of 14 independent case-control studies including 2970 cases and 3049 controls were enrolled in the final meta-analysis. A significant reduction of venous thrombosis risk in the 505G>A polymorphism was observed under allele comparison, homozygote comparison and recessive models, but opposite results were seen in Asians. Likewise, there was a significant decreased susceptibility to venous thrombosis in the 1040C>T polymorphism in homozygote comparison and recessive models. In the subgroup analysis, the nonvenous thromboembolism disease group showed a significantly increased venous thrombosis risk. Pooled estimates did not show evidence of association between -438G>A and venous thrombosis risk in any genetic model. This meta-analysis suggested that although the -438G>T polymorphism is not correlated with venous thrombosis risk in all models, a trend toward reduced risk still could be observed. The A allele and AA genotype of 505G>A in whites and the TT genotype of 1040C>T were significantly associated with a decreased risk of venous thrombosis, except in the non-venous thromboembolism group. PMID:26656901

  18. 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

  19. 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 ...

  20. Dysinosin A: a novel inhibitor of Factor VIIa and thrombin from a new genus and species of Australian sponge of the family Dysideidae.

    PubMed

    Carroll, Anthony R; Pierens, Gregory K; Fechner, Greg; De Almeida Leone, Priscila; Ngo, Anna; Simpson, Moana; Hyde, Edward; Hooper, John N A; Boström, Stig-Lennart; Musil, Djordje; Quinn, Ronald J

    2002-11-13

    A new marine natural product dysinosin A 1 has been isolated from a new genus and species of sponge of the family Dysideidae found near Lizard Island, North Queensland, Australia. Dysinosin A is a potent inhibitor of the blood coagulation cascade factor VIIa and an inhibitor of the serine protease thrombin. Among the distinctive features of dysinosin A are the presence of a 5,6-dihydroxy-octahydroindole-2-carboxylic acid, 3-amino-ethyl 1-N-amidino-Delta-3-pyrroline, a sulfated glyceric acid, and d-leucine, assembled through three peptidic linkages. Dysinosin A inhibited factor VIIa at a Ki of 108 nM and thrombin at a Ki of 452 nM. The identification of the 1-N-amidino-Delta-3-pyrroline and 5,6-dihydroxy-octahydroindole-2-carboxylic acid as P1 and P2 moieties respectively, should pave the way for the design and synthesis of new structure-based inhibitors. PMID:12418859

  1. 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

  2. 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

  3. 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

  4. 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

  5. Challenges of the management of severe hemophilia A with inhibitors: two case reports emphasizing the potential interest of a high-purity human Factor VIII/von Willebrand factor concentrate and individually tailored prophylaxis guided by thrombin-generation test.

    PubMed

    Mathieu, Sophie; Crampe, Carine; Dargaud, Yesim; Lavigne-Lissalde, Géraldine; Escuriola-Ettingshausen, Carmen; Tardy, Brigitte; Meley, Roland; Thouvenin, Sandrine; Stephan, Jean L; Berger, Claire

    2015-12-01

    Severe hemophilia A is an X-linked bleeding disorder. Immune tolerance induction (ITI) is the best strategy of treatment when patients develop inhibitors. The objective is to illustrate the benefit of a high-purity human factor VIII/von Willebrand factor (VWF) concentrate (Octanate) in the management of ITI. We also wanted to raise the potential interest of laboratory assays such as thrombin-generation test (TGT) and epitope mapping. Two patients were treated during ITI, first with a recombinant FVIII and then with plasma-derived factor VIII without success, and, finally, with Octanate. Bypassing agents were used based on the results of TGT. Epitope mapping was performed during ITI therapy. These observations suggest the potential contribution of Octanate in the management of ITI in difficult cases. The use of bypassing agents can be necessary in prophylaxis or to treat bleedings, and may be guided by TGT results. Epitope mapping is used to describe the inhibitor. This article shows a decrease of the inhibitor directed against the C2 domain after initiation of Octanate. A high-purity human factor VIII/von Willebrand factor concentrate (Octanate) may be a valuable therapeutical option for ITI therapy. TGT and epitope mapping could be of help in the management of ITI. PMID:26517064

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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.

  15. Thrombin action decreases acetylcholine receptor aggregate number and stability in cultured mouse myotubes.

    PubMed

    Davenport, R W; Lanuza, M; Kim, S; Jia, M; Snyder, E; Nelson, P G

    2000-08-30

    Neurons develop and make very stable, long-term synaptic connections with other nerve cells and with muscle. Synaptic stability at the neuromuscular junction changes over development in that a proliferation of synaptic input are made to individual myotubes and synapses from all but one neuron are lost during development. In an established co-culture paradigm in which spinal motoneurons synaptically contact myotubes, thrombin and associated protease inhibitors have been shown to affect the loss of functional synaptic contacts [6]. Evidence has not been provided which clearly demonstrate whether protease/protease inhibitors affect either the pre- or postsynaptic terminal, or both. In an effort to determine whether these reagents directly affect postsynaptic receptors on myotubes, myotubes were cultured in the absence of neurons and the spontaneous presence and stability of aggregates of acetylcholine receptors (AChR) in control and thrombin-containing media were evaluated. In dishes fixed after treatment and in dishes in which individual aggregates were observed live, thrombin action appeared to increase loss of AChR aggregates over time. Hirudin, a specific inhibitor of the thrombin protease, diminished this loss. Neither reagent affected the overall incorporation or degradation of AChR; therefore, it appears these protease/protease inhibitors affect the state of AChR aggregation. PMID:10960680

  16. 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.

  17. 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

  18. 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

  19. 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

  20. New directions for protease inhibitors directed drug discovery.

    PubMed

    Hamada, Yoshio; Kiso, Yoshiaki

    2016-11-01

    Proteases play crucial roles in various biological processes, and their activities are essential for all living organisms-from viruses to humans. Since their functions are closely associated with many pathogenic mechanisms, their inhibitors or activators are important molecular targets for developing treatments for various diseases. Here, we describe drugs/drug candidates that target proteases, such as malarial plasmepsins, β-secretase, virus proteases, and dipeptidyl peptidase-4. Previously, we reported inhibitors of aspartic proteases, such as renin, human immunodeficiency virus type 1 protease, human T-lymphotropic virus type I protease, plasmepsins, and β-secretase, as drug candidates for hypertension, adult T-cell leukaemia, human T-lymphotropic virus type I-associated myelopathy, malaria, and Alzheimer's disease. Our inhibitors are also described in this review article as examples of drugs that target proteases. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 563-579, 2016. PMID:26584340

  1. The gene for the serpin thrombin inhibitor (P17), protease nexin I, is located on human chromosome 2q33-q35 and on syntenic regions in the mouse and sheep genomes

    SciTech Connect

    Carter, R.E.; Burkin, D.J.; Fournier, R.E.K.

    1995-05-01

    Protease nexin I (PNI) is the most important physiologic regulator of {alpha}-thrombin in tissues. PNI is highly expressed and developmentally regulated in the nervous system where it is concentrated at neuromuscular junctions and also central synapses in the hippocampus and striatum. Approximately 10% of identified proteins at mammalian neuromuscular junctions are serine protease inhibitors, consistent with their central role in balancing serine protease activity to develop, maintain, and remodel synapses. Southern blot hybridization of PNI cDNA to somatic cell hybrids placed the structural gene for PNI (locus PI7) on human chromosome 2q33-q35 and to syntenic chromosomes in the mouse (chromosome 1) and sheep (chromosome 2). 30 refs., 2 figs.

  2. 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

  3. Apixaban, an oral, direct inhibitor of activated Factor Xa.

    PubMed

    Shantsila, Eduard; Lip, Gregory Y H

    2008-09-01

    Apixaban is an oral, direct Factor Xa inhibitor that is being developed by Bristol-Myers Squibb Co and Pfizer Inc. Apixaban is currently undergoing phase III clinical trials for cerebrovascular ischemia, deep vein thrombosis and lung embolism, and phase II clinical trials for coronary artery disease. PMID:18729009

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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.

  13. Direct inhibitors of InhA active against Mycobacterium tuberculosis

    PubMed Central

    Manjunatha, Ujjini H.; Rao, Srinivasa P. S.; Kondreddi, Ravinder Reddy; Noble, Christian G.; Camacho, Luis R.; Tan, Bee H.; Ng, Seow H.; Ng, Pearly Shuyi; Ma, N. L.; Lakshminarayana, Suresh B.; Herve, Maxime; Barnes, S. Whitney; Yu, Weixuan; Kuhen, Kelli; Blasco, Francesca; Beer, David; Walker, John R.; Tonge, Peter J.; Glynne, Richard; Smith, Paul W.; Diagana, Thierry T.

    2015-01-01

    New chemotherapeutic agents are urgently required to combat the global spread of multi-drug resistant tuberculosis (MDR-TB). The mycobacterial enoyl reductase, InhA, is one of the few clinically-validated targets in tuberculosis drug discovery. Here, we report the identification of a new class of direct InhA inhibitors, the 4-hydroxy-2-pyridones, using phenotypic high-throughput whole-cell screening. This class of orally-active compounds showed potent bactericidal activity against common isoniazid-resistant TB clinical isolates. Biophysical studies revealed that 4-hydroxy-2-pyridones bound specifically to InhA in an NADH-dependent manner and blocked the enoyl-substrate binding pocket. The lead compound NITD-916 directly blocked InhA in a dose-dependent manner and showed in vivo efficacy in acute and established mouse models of infection by Mycobacterium tuberculosis. Collectively, our structural and biochemical data open up new avenues for rational structure-guided optimization of the 4-hydroxy-2-pyridone class of compounds for the treatment of MDR-TB. PMID:25568071

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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.

  1. Inhibitors of the Immunoproteasome: Current Status and Future Directions

    PubMed Central

    Miller, Zachary; Ao, Lin; Kim, Kyung Bo

    2013-01-01

    The ubiquitin-proteasome system (UPS) plays a vital role in maintaining protein homeostasis and regulating numerous cellular processes. The proteasome, a multi-protease complex, is the key component of the UPS and has been validated as a therapeutic target by the FDA's approval of bortezomib and carfilzomib. These proteasome inhibitor drugs have substantially improved outcomes in patients with hematological malignancies and are currently being investigated for other types of cancer as well as several other diseases. These approved proteasome inhibitors target the catalytic activity of both the constitutive proteasome and the immunoproteasome indiscriminately, and their inhibitory effects on the constitutive proteasome in normal cells are believed to contribute to unwanted side effects. In addition, selective immunoproteasome inhibition has been proposed to have unique effects on other diseases, including those involving aberrant immune function. Initially recognized for its role in the adaptive immune response, the immunoproteasome is often upregulated in disease states such as inflammatory diseases and cancer, suggesting functions beyond antigen presentation. In an effort to explore the immunoproteasome as a potential therapeutic target in these diseases, the development of immunoproteasome-specific inhibitors has become the focus of recent studies. Owing to considerable efforts by both academic and industry groups, immunoproteasome-selective inhibitors have now been identified and tested against several disease models. These inhibitors also provide a valuable set of chemical tools for investigating the biological function of the immunoproteasome. In this review, we will focus on the recent efforts towards the development of immunoproteasome-selective inhibitors. PMID:23181576

  2. 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

  3. 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

  4. Designing Allosteric Regulators of Thrombin. Exosite 2 Features Multiple Sub-Sites That Can Be Targeted By Sulfated Small Molecules for Inducing Inhibition

    PubMed Central

    Sidhu, Preetpal Singh; Abdel Aziz, May H.; Sarkar, Aurijit; Mehta, Akul Y.; Zhou, Qibing; Desai, Umesh R.

    2013-01-01

    We recently designed a group of novel exosite 2-directed, sulfated, small, allosteric inhibitors of thrombin. To develop more potent inhibitors, monosulfated benzofuran tri- and tetrameric homologs of the parent designed dimers were synthesized in 7–8 steps and found to exhibit a wide range of potencies. Among these, trimer 9a was found to be nearly 10-fold more potent than the first generation molecules. Michaelis-Menten studies indicated an allosteric mechanism of inhibition. Competitive studies using a hirudin peptide (exosite 1 ligand) and, unfractionated heparin, heparin octasaccharide and γ′-fibrinogen peptide (exosite 2 ligands), demonstrated exosite 2 recognition in a manner different from the parent dimers. Alanine scanning mutagenesis of 12 Arg/Lys residues of exosite 2 revealed a defect in 9a potency for Arg233Ala thrombin only confirming the major difference in site of recognition between the two structurally related sulfated benzofurans. The results suggest that multiple avenues are available within exosite 2 for inducing thrombin inhibition. PMID:23718540

  5. 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

  6. 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

  7. 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

  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. 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.

  10. 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.

  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. Apixaban, an oral direct Factor Xa inhibitor: awaiting the verdict.

    PubMed

    Carreiro, Jennifer; Ansell, Jack

    2008-12-01

    For the last half-century, despite its many limitations warfarin has been the mainstay of treatment for patients with venous and arterial thromboembolic disease. During the past decade, a number of new oral anticoagulant agents have been developed that may offer an alternative to warfarin. Emerging data suggest that Factor Xa may be a target for inhibition. Apixaban is one such agent. It is a potent, selective, reversible, and orally bioavailable FXa inhibitor that demonstrates antithrombotic efficacy, with a favorable pharmacokinetic profile. At present, the safety and efficacy of apixaban for the prophylaxis and treatment of venous thromboembolism is being evaluated in Phase II and Phase III trials involving nearly 25,000 patients. Trials are also underway involving over 20,000 patients for secondary prevention after acute coronary syndromes and the prevention of stroke in patients with non-valvular atrial fibrillation. This review article discusses the discovery, pharmacokinetics, attributes, and current clinical trials of this emerging drug. PMID:19012508

  13. 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

  14. Direct ribosomal binding by a cellular inhibitor of translation

    PubMed Central

    Colón-Ramos, Daniel A; Shenvi, Christina L; Weitzel, Douglas H; Gan, Eugene C; Matts, Robert; Cate, Jamie; Kornbluth, Sally

    2009-01-01

    During apoptosis and under conditions of cellular stress, several signaling pathways promote inhibition of cap-dependent translation while allowing continued translation of specific messenger RNAs encoding regulatory and stress-response proteins. We report here that the apoptotic regulator Reaper inhibits protein synthesis by binding directly to the 40S ribosomal subunit. This interaction does not affect either ribosomal association of initiation factors or formation of 43S or 48S complexes. Rather, it interferes with late initiation events upstream of 60S subunit joining, apparently modulating start-codon recognition during scanning. CrPV IRES–driven translation, involving direct ribosomal recruitment to the start site, is relatively insensitive to Reaper. Thus, Reaper is the first known cellular ribosomal binding factor with the potential to allow selective translation of mRNAs initiating at alternative start codons or from certain IRES elements. This function of Reaper may modulate gene expression programs to affect cell fate. PMID:16429152

  15. [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

  16. 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

  17. Direct oral anticoagulants and venous thromboembolism.

    PubMed

    Franchini, Massimo; Mannucci, Pier Mannuccio

    2016-09-01

    Venous thromboembolism (VTE), consisting of deep vein thrombosis and pulmonary embolism, is a major clinical concern associated with significant morbidity and mortality. The cornerstone of management of VTE is anticoagulation, and traditional anticoagulants include parenteral heparins and oral vitamin K antagonists. Recently, new oral anticoagulant drugs have been developed and licensed, including direct factor Xa inhibitors (e.g. rivaroxaban, apixaban and edoxaban) and thrombin inhibitors (e.g. dabigatran etexilate). This narrative review focusses on the characteristics of these direct anticoagulants and the main results of published clinical studies on their use in the prevention and treatment of VTE. PMID:27581829

  18. 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

    Synthetic thrombin receptor peptides (TRPs), comprising the first 6-14 amino acids of the new N-terminus tethered ligand of the thrombin receptor that is generated by thrombin's proteolytic activity, were reported to activate platelets equally with thrombin itself and are considered to be full agonists [Vu et al. (1991) Cell 64, 1057-1068]. Using aspirin plus ADP-scavengers or the ADP-receptor antagonist adenosine 5'-[alpha-thio]triphosphate to prevent the secondary effects of the potent agonists that are normally released from stimulated platelets (i.e. ADP and thromboxane A2), we assessed the direct actions of thrombin and TRPs (i.e. TRP42-47 and TRP42-55). Compared with thrombin, under these conditions, TRPs: (1) failed to aggregate platelets completely; (2) produced less activation of glycoprotein (GP)IIb-IIIa; (3) did not cause association of GPIIb and pp60c-src with the cytoskeleton; and (4) caused less alpha-granule secretion, phosphorylation of cytoplasmic phospholipase A2, arachidonic acid release and phosphatidyl inositol (PtdOH) production. Furthermore, TRPs induced transient increases in protein phosphorylation mediated by protein kinase C and protein tyrosine phosphorylation, whereas these same responses to thrombin were greater and more sustained. Hirudin added after thrombin accelerated protein dephosphorylation, thereby mimicking the rate of spontaneous dephosphorylation seen after stimulation by TRPs. Platelets totally desensitized to very high concentrations of TRPs, by prior exposure to maximally effective concentrations of the peptides, remained responsive to alpha- and gamma-thrombins. Thrombin-stimulated PtdOH production in permeabilized platelets desensitized to TRPs was abolished by guanosine 5'-[beta-thio]diphosphate (GDP[beta S]), as in normal platelets. These results are discussed in terms of the allosteric Ternary Complex Model for G-protein linked receptors [Samama et al. (1993) J. Biol. Chem. 268, 4625-4636]. We conclude that: (1) TRPs

  19. 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

  20. Inhibitors

    MedlinePlus

    ... Community Counts Blood Safety Inhibitors Articles & Key Findings Free Materials Videos Starting the Conversation Playing it Safe A Look at Hemophilia Joint Range of Motion My Story Links to Other Websites ...

  1. Effect of ketoconazole and diltiazem on the pharmacokinetics of apixaban, an oral direct factor Xa inhibitor

    PubMed Central

    Frost, Charles E; Byon, Wonkyung; Song, Yan; Wang, Jessie; Schuster, Alan E; Boyd, Rebecca A; Zhang, Donglu; Yu, Zhigang; Dias, Clapton; Shenker, Andrew; LaCreta, Frank

    2015-01-01

    Aim Apixaban is an orally active inhibitor of coagulation factor Xa and is eliminated by multiple pathways, including renal and non-renal elimination. Non-renal elimination pathways consist of metabolism by cytochrome P450 (CYP) enzymes, primarily CYP3A4, as well as direct intestinal excretion. Two single sequence studies evaluated the effect of ketoconazole (a strong dual inhibitor of CYP3A4 and P-glycoprotein [P-gp]) and diltiazem (a moderate CYP3A4 inhibitor and a P-gp inhibitor) on apixaban pharmacokinetics in healthy subjects. Method In the ketoconazole study, 18 subjects received apixaban 10 mg on days 1 and 7, and ketoconazole 400 mg once daily on days 4–9. In the diltiazem study, 18 subjects received apixaban 10 mg on days 1 and 11 and diltiazem 360 mg once daily on days 4–13. Results Apixaban maximum plasma concentration and area under the plasma concentration–time curve extrapolated to infinity increased by 62% (90% confidence interval [CI], 47, 78%) and 99% (90% CI, 81, 118%), respectively, with co-administration of ketoconazole, and by 31% (90% CI, 16, 49%) and 40% (90% CI, 23, 59%), respectively, with diltiazem. Conclusion A 2-fold and 1.4-fold increase in apixaban exposure was observed with co-administration of ketoconazole and diltiazem, respectively. PMID:25377242

  2. 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

  3. 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

  4. 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.

  5. Potent hepatitis C inhibitors bind directly to NS5A and reduce its affinity for RNA.

    PubMed

    Ascher, David B; Wielens, Jerome; Nero, Tracy L; Doughty, Larissa; Morton, Craig J; Parker, Michael W

    2014-01-01

    Hepatitis C virus (HCV) infection affects more than 170 million people. The high genetic variability of HCV and the rapid development of drug-resistant strains are driving the urgent search for new direct-acting antiviral agents. A new class of agents has recently been developed that are believed to target the HCV protein NS5A although precisely where they interact and how they affect function is unknown. Here we describe an in vitro assay based on microscale thermophoresis and demonstrate that two clinically relevant inhibitors bind tightly to NS5A domain 1 and inhibit RNA binding. Conversely, RNA binding inhibits compound binding. The compounds bind more weakly to known resistance mutants L31V and Y93H. The compounds do not affect NS5A dimerisation. We propose that current NS5A inhibitors act by favouring a dimeric structure of NS5A that does not bind RNA. PMID:24755925

  6. Potent hepatitis C inhibitors bind directly to NS5A and reduce its affinity for RNA

    PubMed Central

    Ascher, David B.; Wielens, Jerome; Nero, Tracy L.; Doughty, Larissa; Morton, Craig J.; Parker, Michael W.

    2014-01-01

    Hepatitis C virus (HCV) infection affects more than 170 million people. The high genetic variability of HCV and the rapid development of drug-resistant strains are driving the urgent search for new direct-acting antiviral agents. A new class of agents has recently been developed that are believed to target the HCV protein NS5A although precisely where they interact and how they affect function is unknown. Here we describe an in vitro assay based on microscale thermophoresis and demonstrate that two clinically relevant inhibitors bind tightly to NS5A domain 1 and inhibit RNA binding. Conversely, RNA binding inhibits compound binding. The compounds bind more weakly to known resistance mutants L31V and Y93H. The compounds do not affect NS5A dimerisation. We propose that current NS5A inhibitors act by favouring a dimeric structure of NS5A that does not bind RNA. PMID:24755925

  7. ACE inhibitors can induce circulating antibodies directed to antigens of the superficial epidermal cells.

    PubMed

    Cozzani, Emanuele; Rosa, Gian Marco; Drosera, Massimo; Intra, Chiara; Barsotti, Antonio; Parodi, Aurora

    2011-07-01

    Drug-induced pemphigus has been reported in patients receiving angiotensin-converting enzyme inhibitors. The aim of this work was to study a group of hypertensive patients without skin diseases treated with angiotensin-converting enzyme (ACE) Inhibitors (I), to verify the presence of serum circulating anti-antibodies. The indirect immunofluorescence showed that 33 sera (52.38%) presented autoantibodies directed to an antigen of the cytoplasm of the superficial epidermal keratinocytes. Two of the 33 positive sera had antibodies to Dsg1 and/or 3 in ELISA. Immunoblot analyses were negative. All the 48 control sera were found to have no circulating antibodies using the three assays. Our results would confirm that ACEI drugs may trigger the production of circulating autoantibodies also in patients without clinical manifestations of pemphigus. PMID:20563876

  8. Antitubercular drugs for an old target: GSK693 as a promising InhA direct inhibitor.

    PubMed

    Martínez-Hoyos, María; Perez-Herran, Esther; Gulten, Gulcin; Encinas, Lourdes; Álvarez-Gómez, Daniel; Alvarez, Emilio; Ferrer-Bazaga, Santiago; García-Pérez, Adolfo; Ortega, Fátima; Angulo-Barturen, Iñigo; Rullas-Trincado, Joaquin; Blanco Ruano, Delia; Torres, Pedro; Castañeda, Pablo; Huss, Sophie; Fernández Menéndez, Raquel; González Del Valle, Silvia; Ballell, Lluis; Barros, David; Modha, Sundip; Dhar, Neeraj; Signorino-Gelo, François; McKinney, John D; García-Bustos, Jose Francisco; Lavandera, Jose Luis; Sacchettini, James C; Jimenez, M Soledad; Martín-Casabona, Nuria; Castro-Pichel, Julia; Mendoza-Losana, Alfonso

    2016-06-01

    Despite being one of the first antitubercular agents identified, isoniazid (INH) is still the most prescribed drug for prophylaxis and tuberculosis (TB) treatment and, together with rifampicin, the pillars of current chemotherapy. A high percentage of isoniazid resistance is linked to mutations in the pro-drug activating enzyme KatG, so the discovery of direct inhibitors (DI) of the enoyl-ACP reductase (InhA) has been pursued by many groups leading to the identification of different enzyme inhibitors, active against Mycobacterium tuberculosis (Mtb), but with poor physicochemical properties to be considered as preclinical candidates. Here, we present a series of InhA DI active against multidrug (MDR) and extensively (XDR) drug-resistant clinical isolates as well as in TB murine models when orally dosed that can be a promising foundation for a future treatment. PMID:27428438

  9. Active-Site-Directed Inhibitors of Prolyl Oligopeptidase Abolish Its Conformational Dynamics.

    PubMed

    López, Abraham; Herranz-Trillo, Fátima; Kotev, Martin; Gairí, Margarida; Guallar, Víctor; Bernadó, Pau; Millet, Oscar; Tarragó, Teresa; Giralt, Ernest

    2016-05-17

    Deciphering conformational dynamics is crucial for understanding the biological functions of proteins and for designing compounds targeting them. In particular, providing an accurate description of microsecond-millisecond motions opens the opportunity for regulating protein-protein interactions (PPIs) by modulating the dynamics of one interacting partner. Here we analyzed the conformational dynamics of prolyl oligopeptidase (POP) and the effects of active-site-directed inhibitors on the dynamics. We used an integrated structural biology approach based on NMR spectroscopy and SAXS experiments complemented by MD simulations. We found that POP is in a slow equilibrium in solution between open and closed conformations, and that inhibitors effectively abolished this equilibrium by stabilizing the enzyme in the closed conformation. PMID:26918396

  10. Synthesis and Metabolic Studies of Host-Directed Inhibitors for Antiviral Therapy

    PubMed Central

    2013-01-01

    Targeting host cell factors required for virus replication provides an alternative to targeting pathogen components and represents a promising approach to develop broad-spectrum antiviral therapeutics. High-throughput screening (HTS) identified two classes of inhibitors (2 and 3) with broad-spectrum antiviral activity against ortho- and paramyxoviruses including influenza A virus (IAV), measles virus (MeV), respiratory syncytial virus (RSV), and human parainfluenza virus type 3 (HPIV3). Hit-to-lead optimization delivered inhibitor 28a, with EC50 values of 0.88 and 0.81 μM against IAV strain WSN and MeV strain Edmonston, respectively. It was also found that compound 28a delivers good stability in human liver S9 fractions with a half-life of 165 min. These data establish 28a as a promising lead for antiviral therapy through a host-directed mechanism. PMID:23956816

  11. 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

  12. 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

  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. 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

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. Correcting thrombin generation ex vivo using different haemostatic agents following cardiac surgery requiring the use of cardiopulmonary bypass

    PubMed Central

    Percy, Charles L.; Hartmann, Rudolf; Jones, Rhidian M.; Balachandran, Subramaniam; Mehta, Dheeraj; Dockal, Michael; Scheiflinger, Friedrich; O’Donnell, Valerie B.; Hall, Judith E.; Collins, Peter W.

    2015-01-01

    Recently, lower thrombin generation has been associated with excess bleeding post-cardiopulmonary bypass (CPB). Therefore, treatment to correct thrombin generation is a potentially important aspect of management of bleeding in this group of patients. The objective of the present study was to investigate the effects of fresh frozen plasma (FFP), recombinant factor VIIa (rFVIIa), prothrombin complex concentrate (PCC) and tissue factor pathway inhibitor (TFPI) inhibition on thrombin generation when added ex vivo to the plasma of patients who had undergone cardiac surgery requiring CPB. Patients undergoing elective cardiac surgery were recruited. Blood samples were collected before administration of heparin and 30 min after its reversal. Thrombin generation was measured in the presence and absence of different concentrations of FFP, rFVIIa, PCC and an anti-TFPI antibody. A total of 102 patients were recruited. Thrombin generation following CPB was lower compared with pre-CPB (median endogenous thrombin potential pre-CPB 339 nmol/l per min, post-CPB 155 nmol/l per min, P < 0.0001; median peak thrombin pre-CPB 35 nmol/l, post-CPB 11 nmol/l, P < 0.0001). Coagulation factors and anticoagulants decreased, apart from total TFPI, which increased (55–111 ng/ml, P < 0.0001), and VWF (144–170 IU/dl, P < 0.0001). Thrombin generation was corrected to pre-CPB levels by the equivalent of 15 ml/kg FFP, 45 μg/kg rFVIIa and 25 U/kg of PCC. Inhibition of TFPI resulted in an enhancement of thrombin generation significantly beyond pre-CPB levels. This study shows that FFP, rFVIIa, PCC and inhibition of TFPI correct thrombin generation in the plasma of patients who have undergone surgery requiring CPB. Inhibition of TFPI may be a further potential therapeutic strategy for managing bleeding in this group of patients. PMID:25928274

  2. Correcting thrombin generation ex vivo using different haemostatic agents following cardiac surgery requiring the use of cardiopulmonary bypass.

    PubMed

    Percy, Charles L; Hartmann, Rudolf; Jones, Rhidian M; Balachandran, Subramaniam; Mehta, Dheeraj; Dockal, Michael; Scheiflinger, Friedrich; O'Donnell, Valerie B; Hall, Judith E; Collins, Peter W

    2015-06-01

    Recently, lower thrombin generation has been associated with excess bleeding post-cardiopulmonary bypass (CPB). Therefore, treatment to correct thrombin generation is a potentially important aspect of management of bleeding in this group of patients. The objective of the present study was to investigate the effects of fresh frozen plasma (FFP), recombinant factor VIIa (rFVIIa), prothrombin complex concentrate (PCC) and tissue factor pathway inhibitor (TFPI) inhibition on thrombin generation when added ex vivo to the plasma of patients who had undergone cardiac surgery requiring CPB. Patients undergoing elective cardiac surgery were recruited. Blood samples were collected before administration of heparin and 30 min after its reversal. Thrombin generation was measured in the presence and absence of different concentrations of FFP, rFVIIa, PCC and an anti-TFPI antibody. A total of 102 patients were recruited. Thrombin generation following CPB was lower compared with pre-CPB (median endogenous thrombin potential pre-CPB 339 nmol/l per min, post-CPB 155 nmol/l per min, P < 0.0001; median peak thrombin pre-CPB 35 nmol/l, post-CPB 11 nmol/l, P < 0.0001). Coagulation factors and anticoagulants decreased, apart from total TFPI, which increased (55-111 ng/ml, P < 0.0001), and VWF (144-170 IU/dl, P < 0.0001). Thrombin generation was corrected to pre-CPB levels by the equivalent of 15 ml/kg FFP, 45 μg/kg rFVIIa and 25 U/kg of PCC. Inhibition of TFPI resulted in an enhancement of thrombin generation significantly beyond pre-CPB levels. This study shows that FFP, rFVIIa, PCC and inhibition of TFPI correct thrombin generation in the plasma of patients who have undergone surgery requiring CPB. Inhibition of TFPI may be a further potential therapeutic strategy for managing bleeding in this group of patients. PMID:25928274

  3. 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

  4. Potent Host-Directed Small-Molecule Inhibitors of Myxovirus RNA-Dependent RNA-Polymerases

    PubMed Central

    Krumm, Stefanie A.; Ndungu, J. Maina; Yoon, Jeong-Joong; Dochow, Melanie; Sun, Aiming; Natchus, Michael; Snyder, James P.; Plemper, Richard K.

    2011-01-01

    Therapeutic targeting of host cell factors required for virus replication rather than of pathogen components opens new perspectives to counteract virus infections. Anticipated advantages of this approach include a heightened barrier against the development of viral resistance and a broadened pathogen target spectrum. Myxoviruses are predominantly associated with acute disease and thus are particularly attractive for this approach since treatment time can be kept limited. To identify inhibitor candidates, we have analyzed hit compounds that emerged from a large-scale high-throughput screen for their ability to block replication of members of both the orthomyxovirus and paramyxovirus families. This has returned a compound class with broad anti-viral activity including potent inhibition of different influenza virus and paramyxovirus strains. After hit-to-lead chemistry, inhibitory concentrations are in the nanomolar range in the context of immortalized cell lines and human PBMCs. The compound shows high metabolic stability when exposed to human S-9 hepatocyte subcellular fractions. Antiviral activity is host-cell species specific and most pronounced in cells of higher mammalian origin, supporting a host-cell target. While the compound induces a temporary cell cycle arrest, host mRNA and protein biosynthesis are largely unaffected and treated cells maintain full metabolic activity. Viral replication is blocked at a post-entry step and resembles the inhibition profile of a known inhibitor of viral RNA-dependent RNA-polymerase (RdRp) activity. Direct assessment of RdRp activity in the presence of the reagent reveals strong inhibition both in the context of viral infection and in reporter-based minireplicon assays. In toto, we have identified a compound class with broad viral target range that blocks host factors required for viral RdRp activity. Viral adaptation attempts did not induce resistance after prolonged exposure, in contrast to rapid adaptation to a pathogen-directed

  5. 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

  6. 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

  7. The Role of Thrombin and Cell Contractility in Regulating Clustering and Collective Migration of Corneal Fibroblasts in Different ECM Environments

    PubMed Central

    Miron-Mendoza, Miguel; Graham, Eric; Kivanany, Pouriska; Quiring, Jonathan; Petroll, W. Matthew

    2015-01-01

    Purpose. We previously reported that extracellular matrix composition (fibrin versus collagen) modulates the pattern of corneal fibroblast spreading and migration in 3-D culture. In this study, we investigate the role of thrombin and cell contractility in mediating these differences in cell behavior. Methods. To assess cell spreading, corneal fibroblasts were plated on top of fibrillar collagen and fibrin matrices. To assess 3-dimensional cell migration, compacted collagen matrices seeded with corneal fibroblasts were embedded inside acellular collagen or fibrin matrices. Constructs were cultured in serum-free media containing platelet-derived growth factor (PDGF), with or without thrombin, the Rho kinase inhibitor Y-27632, and/or the myosin II inhibitor blebbistatin. We used 3-dimensional and 4-dimensional imaging to assess cell mechanical behavior, connectivity and cytoskeletal organization. Results. Thrombin stimulated increased contractility of corneal fibroblasts. Thrombin also induced Rho kinase–dependent clustering of cells plated on top of compliant collagen matrices, but not on rigid substrates. In contrast, cells on fibrin matrices coalesced into clusters even when Rho kinase was inhibited. In nested matrices, cells always migrated independently through collagen, even in the presence of thrombin. In contrast, cells migrating into fibrin formed an interconnected network. Both Y-27632 and blebbistatin reduced the migration rate in fibrin, but cells continued to migrate collectively. Conclusions. The results suggest that while thrombin-induced actomyosin contraction can induce clustering of fibroblasts plated on top of compliant collagen matrices, it does not induce collective cell migration inside 3-D collagen constructs. Furthermore, increased contractility is not required for clustering or collective migration of corneal fibroblasts interacting with fibin. PMID:25736789

  8. 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

  9. 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

  10. The non-coding RNA TERRA is a natural ligand and direct inhibitor of human telomerase

    PubMed Central

    Redon, Sophie; Reichenbach, Patrick; Lingner, Joachim

    2010-01-01

    Telomeres, the physical ends of eukaryotes chromosomes are transcribed into telomeric repeat containing RNA (TERRA), a large non-coding RNA of unknown function, which forms an integral part of telomeric heterochromatin. TERRA molecules resemble in sequence the telomeric DNA substrate as they contain 5′-UUAGGG-3′ repeats near their 3′-end which are complementary to the template sequence of telomerase RNA. Here we demonstrate that endogenous TERRA is bound to human telomerase in cell extracts. Using in vitro reconstituted telomerase and synthetic TERRA molecules we demonstrate that the 5′-UUAGGG-3′ repeats of TERRA base pair with the RNA template of the telomerase RNA moiety (TR). In addition TERRA contacts the telomerase reverse transcriptase (TERT) protein subunit independently of hTR. In vitro studies further demonstrate that TERRA is not used as a telomerase substrate. Instead, TERRA acts as a potent competitive inhibitor for telomeric DNA in addition to exerting an uncompetitive mode of inhibition. Our data identify TERRA as a telomerase ligand and natural direct inhibitor of human telomerase. Telomerase regulation by the telomere substrate may be mediated via its transcription. PMID:20460456

  11. Protein-Directed Dynamic Combinatorial Chemistry: A Guide to Protein Ligand and Inhibitor Discovery.

    PubMed

    Huang, Renjie; Leung, Ivanhoe K H

    2016-01-01

    Protein-directed dynamic combinatorial chemistry is an emerging technique for efficient discovery of novel chemical structures for binding to a target protein. Typically, this method relies on a library of small molecules that react reversibly with each other to generate a combinatorial library. The components in the combinatorial library are at equilibrium with each other under thermodynamic control. When a protein is added to the equilibrium mixture, and if the protein interacts with any components of the combinatorial library, the position of the equilibrium will shift and those components that interact with the protein will be amplified, which can then be identified by a suitable biophysical technique. Such information is useful as a starting point to guide further organic synthesis of novel protein ligands and enzyme inhibitors. This review uses literature examples to discuss the practicalities of applying this method to inhibitor discovery, in particular, the set-up of the combinatorial library, the reversible reactions that may be employed, and the choice of detection methods to screen protein ligands from a mixture of reversibly forming molecules. PMID:27438816

  12. Discovery of direct inhibitors of Keap1-Nrf2 protein-protein interaction as potential therapeutic and preventive agents.

    PubMed

    Abed, Dhulfiqar Ali; Goldstein, Melanie; Albanyan, Haifa; Jin, Huijuan; Hu, Longqin

    2015-07-01

    The Keap1-Nrf2-ARE pathway is an important antioxidant defense mechanism that protects cells from oxidative stress and the Keap1-Nrf2 protein-protein interaction (PPI) has become an important drug target to upregulate the expression of ARE-controlled cytoprotective oxidative stress response enzymes in the development of therapeutic and preventive agents for a number of diseases and conditions. However, most known Nrf2 activators/ARE inducers are indirect inhibitors of Keap1-Nrf2 PPI and they are electrophilic species that act by modifying the sulfhydryl groups of Keap1׳s cysteine residues. The electrophilicity of these indirect inhibitors may cause "off-target" side effects by reacting with cysteine residues of other important cellular proteins. Efforts have recently been focused on the development of direct inhibitors of Keap1-Nrf2 PPI. This article reviews these recent research efforts including the development of high throughput screening assays, the discovery of peptide and small molecule direct inhibitors, and the biophysical characterization of the binding of these inhibitors to the target Keap1 Kelch domain protein. These non-covalent direct inhibitors of Keap1-Nrf2 PPI could potentially be developed into effective therapeutic or preventive agents for a variety of diseases and conditions. PMID:26579458

  13. Acetylene is an active-site-directed, slow-binding, reversible inhibitor of Azotobacter vinelandii hydrogenase

    SciTech Connect

    Hyman, M.R.; Arp, D.J.

    1987-10-06

    The inhibition of purified and membrane-bound hydrogenase from Azotobacter vinelandii by dihydrogen-free acetylene was investigated. The inhibition was a time-dependent process which exhibited first-order kinetics. Both H/sub 2/ and CO protected against the inhibition by acetylene. K/sub protect(app)/ values of 0.41 and 24 ..mu..M were derived for these gases, respectively. Both H/sub 2/-oxidizing activity and the tritium exchange capacity of the purified enzyme were inhibited at the same rate by acetylene. Removal of acetylene reversed the inhibition for both the purified and the membrane-associated form of the enzyme. The purified hydrogenases from both Rhizobium japonicum and Alcaligenes eutrophus H16 were also inhibited by acetylene in a time-dependent fashion. These findings suggest that acetylene is an active-site-directed, slow-binding, reversible inhibitor of some membrane-bound hydrogenases from aerobic bacteria.

  14. 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

  15. 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.

  16. Platelet C1- inhibitor. A secreted alpha-granule protein.

    PubMed Central

    Schmaier, A H; Smith, P M; Colman, R W

    1985-01-01

    In order to characterize which proteins of the contact phase of coagulation interact with platelets, human platelets were studied immunochemically and functionally to determine if they contain C1- inhibitor. By means of monospecific antibody to C1- inhibitor, a competitive enzyme-linked immunosorbent assay (CELISA) was developed to measure directly platelet C1- inhibitor. With the CELISA, from 33 to 115 ng of C1- inhibitor antigen per 10(8) platelets from 15 normal donors was quantified in lysates of washed human platelets solubilized in nonionic detergent. The mean concentration in 10(8) platelets was 62 +/- 33 ng (SD). Plasma C1- inhibitor either in the platelet suspension medium or on the surface of the platelets could account for only from 6.5 to 16% of the total antigen measured in the solubilized platelets. Upon functional studies, platelets contained 84 +/- 36 ng (SD) of C1- inhibitor activity in 10(8) platelets. As assessed by the CELISA, platelet C1- inhibitor antigen was immunochemically identical to plasma and purified C1- inhibitor. In contrast, the mean concentration of platelet C1- inhibitor antigen in platelets from four patients with classical hereditary angioedema was 8.3 ng/10(8) platelets (range, 5.3 to 11.3 ng/10(8) platelets). 25 and 31% of the total platelet C1- inhibitor was secreted without cell lysis from normal platelets after exposure to collagen (20 micrograms/ml) and thrombin (1 U/ml), respectively, and this secretion was blocked by metabolic inhibitors. Platelet subcellular fractionation showed that platelet C1- inhibitor resided mostly in alpha-granules, similar to the location of platelet fibrinogen. Thus, human platelets contained C1- inhibitor, which became available by platelet secretion. The identification of platelet C1- inhibitor suggests that platelets may modulate the activation of the proteins of early blood coagulation and the classical complement pathways. Images PMID:3965505

  17. 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

  18. Development of Trypsin-Like Serine Protease Inhibitors as Therapeutic Agents: Opportunities, Challenges, and their Unique Structure-Based Rationales.

    PubMed

    Liang, Guyan; Bowen, J Phillip

    2016-01-01

    Xa) which prevents the conversion of prothrombin to thrombin. In addition, dabigatran etexikate (Pradaxa), the direct thrombin inhibitor (fIIa) is also now widely prescribed. PMID:26369819

  19. 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.

  20. 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.

  1. 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

  2. 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....

  3. 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....

  4. 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....

  5. 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....

  6. 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....

  7. Synergy of entry inhibitors with direct-acting antivirals uncovers novel combinations for prevention and treatment of hepatitis C

    PubMed Central

    Xiao, Fei; Fofana, Isabel; Thumann, Christine; Mailly, Laurent; Alles, Roxane; Robinet, Eric; Meyer, Nicolas; Schaeffer, Mickaël; Habersetzer, François; Doffoël, Michel; Leyssen, Pieter; Neyts, Johan; Zeisel, Mirjam B; Baumert, Thomas F

    2015-01-01

    Objective Although direct-acting antiviral agents (DAAs) have markedly improved the outcome of treatment in chronic HCV infection, there continues to be an unmet medical need for improved therapies in difficult-to-treat patients as well as liver graft infection. Viral entry is a promising target for antiviral therapy. Design Aiming to explore the role of entry inhibitors for future clinical development, we investigated the antiviral efficacy and toxicity of entry inhibitors in combination with DAAs or other host-targeting agents (HTAs). Screening a large series of combinations of entry inhibitors with DAAs or other HTAs, we uncovered novel combinations of antivirals for prevention and treatment of HCV infection. Results Combinations of DAAs or HTAs and entry inhibitors including CD81-, scavenger receptor class B type I (SR-BI)- or claudin-1 (CLDN1)-specific antibodies or small-molecule inhibitors erlotinib and dasatinib were characterised by a marked and synergistic inhibition of HCV infection over a broad range of concentrations with undetectable toxicity in experimental designs for prevention and treatment both in cell culture models and in human liver-chimeric uPA/SCID mice. Conclusions Our results provide a rationale for the development of antiviral strategies combining entry inhibitors with DAAs or HTAs by taking advantage of synergy. The uncovered combinations provide perspectives for efficient strategies to prevent liver graft infection and novel interferon-free regimens. PMID:24848265

  8. 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

  9. 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

  10. Development of inhibitor-directed enzyme prodrug therapy (IDEPT) for prostate cancer.

    PubMed

    Martin, Stacy E; Ganguly, Tanushree; Munske, Gerhard R; Fulton, Melody D; Hopkins, Mark R; Berkman, Clifford E; Black, Margaret E

    2014-10-15

    Prostate cancer (PCa) is the second most common cause of cancer death among American men after lung cancer. Unfortunately, current therapies do not provide effective treatments for patients with advanced, metastatic, or hormone refractory disease. Therefore, we seek to generate therapeutic agents for a novel PCa treatment strategy by delivering a suicide enzyme (yCDtriple) to a cell membrane bound biomarker found on PCa cells (prostate-specific membrane antigen (PSMA)). This approach has resulted in a new PCa treatment strategy reported here as inhibitor-directed enzyme prodrug therapy (IDEPT). The therapeutic agents described were generated using a click chemistry reaction between the unnatural amino acid (p-azidophenylalanine (pAzF)) incorporated into yCDtriple and the dibenzylcyclooctyne moiety of our PSMA targeting agent (DBCO-PEG4-AH2-TG97). After characterization of the therapeutic agents, we demonstrate significant PCa cell killing of PSMA-positive cells. Importantly, we demonstrate that this click chemistry approach can be used to efficiently couple a therapeutic protein to a targeting agent and may be applicable to the ablation of other types of cancers and/or malignancies. PMID:25157916

  11. Oral, direct factor Xa inhibitors in development for the prevention and treatment of thromboembolic diseases.

    PubMed

    Turpie, Alexander G G

    2007-06-01

    Anticoagulants are recommended for the prevention and treatment of a wide variety of thromboembolic events. Although existing anticoagulants are effective, their use is limited by parenteral administration or the requirement for frequent monitoring and subsequent dose adjustment. Therefore, there is an urgent need for novel, oral agents with a predictable anticoagulant action. Because of its key position in the coagulation cascade and its limited roles outside of coagulation, Factor Xa has emerged as an attractive target for novel anticoagulants. As a result, the past decade has witnessed an explosion of research into small-molecule, oral, direct Factor Xa inhibitors, and several are now in clinical development. Rivaroxaban, LY517717, YM150, apixaban, PRT054021, and DU-176b, among others, have shown considerable promise; rivaroxaban is currently furthest ahead in its developmental program, having entered phase III in 3 indications. It is hoped that, before long, these anticoagulants will allow us to enter an era of convenient, oral anticoagulation, without the need for regular monitoring or dose adjustment. PMID:17379841

  12. Rivaroxaban – an oral, direct Factor Xa inhibitor – lessons from a broad clinical study programme

    PubMed Central

    Haas, Sylvia

    2009-01-01

    Anticoagulants are recommended for the prevention and treatment of venous thromboembolism (VTE), prevention of stroke in patients with atrial fibrillation (AF) and secondary prevention in patients with acute coronary syndrome (ACS). There is a clinical need for novel anticoagulants offering improvements over current standard of care, such as fixed oral dosing and no need for routine monitoring. Rivaroxaban, an oral, once-daily, direct Factor Xa inhibitor, has recently completed the RECORD phase III programme for the prevention of VTE in patients undergoing total hip or knee replacement (THR or TKR), an indication for which it is approved in Europe and Canada. It is being investigated in large-scale phase III studies for VTE treatment and prevention of stroke in patients with AF, and phase III studies will soon commence for secondary prevention in patients with ACS. Phase I studies demonstrated that no routine anticoagulation monitoring was required, while phase II studies suggested that fixed daily doses had a wide therapeutic window. The four RECORD studies consistently showed that rivaroxaban was significantly more effective than enoxaparin in the prevention of VTE after THR and TKR, with a similar safety profile. This review describes the development of this novel anticoagulant, from bench to bedside. PMID:19187276

  13. Direct imaging of the disruption of hepatitis C virus replication complexes by inhibitors of lipid metabolism

    SciTech Connect

    Lyn, Rodney K.; Kennedy, David C.; Sagan, Selena M.; Blais, David R.; Rouleau, Yanouchka; Pegoraro, Adrian F.; Xie, X. Sunney; Stolow, Albert; Pezacki, John Paul

    2009-11-10

    Here we have simultaneously characterized the influence of inhibitors of peroxisome proliferator-activated receptor alpha (PPARalpha) and the mevalonate pathway on hepatocyte lipid metabolism and the subcellular localization of hepatitis C virus (HCV) RNA using two-photon fluorescence (TPF) and coherent anti-Stokes Raman scattering (CARS) microscopy. Using this approach, we demonstrate that modulators of PPARalpha signaling rapidly cause the dispersion of HCV RNA from replication sites and simultaneously induce lipid storage and increases in lipid droplet size. We demonstrate that reductions in the levels of cholesterol resulting from inhibition of the mevalonate pathway upregulates triglyceride levels. We also show that the rate of dispersion of HCV RNA is very rapid when using a PPARalpha antagonist. This occurs with a faster rate to that of direct inhibition of 3-hydroxy-3-methyglutaryl CoA reductase (HMG-CoA reductase) using lovastatin in living cells, demonstrating the potential therapeutic value of modulating host cell pathways as part of a strategy to eliminate chronic HCV infection.

  14. 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

  15. 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

  16. Characterization of irreversible kinase inhibitors by directly detecting covalent bond formation: a tool for dissecting kinase drug resistance.

    PubMed

    Klüter, Sabine; Simard, Jeffrey R; Rode, Haridas B; Grütter, Christian; Pawar, Vijaykumar; Raaijmakers, Hans C A; Barf, Tjeerd A; Rabiller, Matthias; van Otterlo, Willem A L; Rauh, Daniel

    2010-12-10

    Targeting protein kinases in cancer therapy with irreversible small-molecule inhibitors is moving to the forefront of kinase-inhibitor research and is thought to be an effective means of overcoming mutation-associated drug resistance in epidermal growth factor receptor kinase (EGFR). We generated a detection technique that allows direct measurements of covalent bond formation without relying on kinase activity, thereby allowing the straightforward investigation of the influence of steric clashes on covalent inhibitors in different resistant kinase mutants. The obtained results are discussed together with structural biology and biochemical studies of catalytic activity in both wild-type and gatekeeper mutated kinase variants to draw conclusions about the impact of steric hindrance and increased catalytic activity in drug-resistant kinase variants. PMID:21080395

  17. 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).

  18. 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

  19. 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.

  20. [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

  1. 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.

  2. 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

  3. 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

  4. 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

  5. Staphylococcal enterotoxin B initiates protein kinase C translocation and eicosanoid metabolism while inhibiting thrombin-induced aggregation in human platelets.

    PubMed

    Tran, Uyen; Boyle, Thomas; Shupp, Jeffrey W; Hammamieh, Rasha; Jett, Marti

    2006-08-01

    Staphylococcal enterotoxin (SE) B, a heat-stable toxin secreted by Staphylococcus aureus, has been implicated in the pathogenesis and exacerbation of several critical illnesses. It has been hypothesized that enterotoxins may interact with blood products such as platelets, in addition to T-lymphocytes and renal proximal tubule cells. The aim of this present study was to elucidate whether SEB directly alters human platelet function. Human platelet rich plasma (PRP) was pre-incubated with SEA, SEB, SEC or TSST-1, (at various concentrations and incubation times). After incubation, PRP was exposed to thrombin and aggregation was assessed. Incubation with all toxins tested resulted in decreased aggregation, specifically; exposure to 10mu g/ml of SEB for 30 min caused a 20% decrease and a 49% decrease at 90 min. A similar reduction in aggregation was seen in samples incubated with phorbol myristate acetate, a known stimulator of protein kinase C (PKC). Further, platelets exposed to SEB exhibited an increased plasma membrane PKC activity. Sphingosine, an inhibitor of PKC proved to block the SEB-induced reduction in aggregation. SEB effects on platelet metabolism were investigated using high performance liquid chromatography showing up to a 2-fold increase of active metabolites lipoxin A4 and 12-HETE, as compared to control. These data indicate that SEB is able to induce platelet dysfunction, and these effects may be mediated through activation of PKC. PMID:16550298

  6. 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

  7. 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

  8. [Direct renin inhibitor aliskiren in the treatment of cardiovascular and renal diseases].

    PubMed

    Horký, Karel

    2010-02-01

    The role of renin-angiotensin-aldosterone system (RAAS) in regulating the volume and composition of extracellular fluid, blood pressure (BP) as well as onset and progression of cardiovascular and renal diseases has been studied for more than 150 years. The compounds that block the vital stages of the RAAS cascade, such as ACE-inhibitors (ACEI), AT1-receptor blockers (ARB) and aldosterone receptor antagonists, importantly extended our treatment options. However, the positive therapeutic effects of these compounds also have certain negative consequences. Administration of ACEIs and ARBs interrupts physiological feedback for renal renin release and leads to reactive elevation of circulating active renin and greater production of angiotensin I and angiotensin II with subsequent return of aldosterone secretion to the pre-treatment levels ('escape' phenomenon). These possible adverse effects of the intermediary products of incomplete RAAS blockade leading to organ complications have facilitated the efforts to develop compounds blocking the initial stages of renin-angiotensin cascade--i.e. direct renin blockers. After several years of unsuccessful attempts, the recent years have seen development of the first non-peptide, orally long-term effective renin inhibitor, aliskiren fumarate. In monotherapy or in combination with other antihypertensives (hydrochlorothiazide, ARB, ACEI), aliskiren reduces BP in a dose-dependent manner (75-600 mg/den). Aliskiren reduces plasma renin activity (PRA) and neutralises hydrochlorothiazide-induced RAAS activation. Once daily administration of the drug leads to longer than 24-hour activity and its prolonged blocking effects on the kidneys are the basis for its renoprotectivity. In addition to the significant antihypertensive effect, clinical studies also showed a range of organoprotective properties in patients with left ventricle hypertrophy (ALLAY study), heart failure (ALOFT study) and diabetic nephropathy (AVOID study). Similar to other

  9. 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

  10. Recent findings and future directions for interpolar mitotic kinesin inhibitors in cancer therapy

    PubMed Central

    Myers, Stephanie M.; Collins, Ian

    2016-01-01

    The kinesin class of microtubule-associated motor proteins present attractive anti-cancer targets owing to their roles in key functions in dividing cells. Two interpolar mitotic kinesins Eg5 and HSET have opposing motor functions in mitotic spindle assembly with respect to microtubule movement, but both offer opportunities to develop cancer selective therapeutic agents. Here, we summarize the progress to date in developing inhibitors of Eg5 and HSET, with an emphasis on structural biology insights into the binding modes of allosteric inhibitors, compound selectivity and mechanisms of action of different chemical scaffolds. We discuss translation of preclinical studies to clinical experience with Eg5 inhibitors, recent findings on potential resistance mechanisms, and explore the implications for future anticancer drug development against these targets. PMID:26976726

  11. Recent findings and future directions for interpolar mitotic kinesin inhibitors in cancer therapy.

    PubMed

    Myers, Stephanie M; Collins, Ian

    2016-03-01

    The kinesin class of microtubule-associated motor proteins present attractive anticancer targets owing to their roles in key functions in dividing cells. Two interpolar mitotic kinesins Eg5 and HSET have opposing motor functions in mitotic spindle assembly with respect to microtubule movement, but both offer opportunities to develop cancer selective therapeutic agents. Here, we summarize the progress to date in developing inhibitors of Eg5 and HSET, with an emphasis on structural biology insights into the binding modes of allosteric inhibitors, compound selectivity and mechanisms of action of different chemical scaffolds. We discuss translation of preclinical studies to clinical experience with Eg5 inhibitors, recent findings on potential resistance mechanisms and explore the implications for future anticancer drug development against these targets. PMID:26976726

  12. 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

  13. 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

  14. Direct Effects, Compensation, and Recovery in Female Fathead Minnows Exposed to a Model Aromatase Inhibitor

    EPA Science Inventory

    The paper reports on the effects of a model aromatase inhibitor, fadrozole, on molecular and biochemical endpoints within the fathead minnow reproductive axis. Unlike previous studies, this work incorporated extensive time-course characterization over the course of an 8 d exposu...

  15. 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

  16. Posttraumatic lingual artery pseudoaneurysm treated with ultrasound-guided percutaneous thrombin injection.

    PubMed

    Masella, Pamela C; Hanson, Megan M; Hall, Brian T; Verghese, John J; Kellicut, Dwight C

    2014-07-01

    Pseudoaneurysms of the lingual artery are extremely rare and are commonly iatrogenic in nature or less frequently a result of blunt or penetrating trauma. Traditionally, these vascular abnormalities have been repaired with open or endovascular techniques. Although ultrasound-guided percutaneous thrombin injection has become a standard treatment for superficial pseudoaneurysms, there are no reports of this being used in the treatment of lingual artery pseudoaneurysms. We report the case of a 26-year-old man who suffered a penetrating head and neck injury after an improvised explosive device blast in Iraq who presented with persistent oropharyngeal swelling. Color-flow Doppler ultrasonography revealed the classic yin/yang sign of a pseudoaneurysm, and a computed tomography scan was obtained that revealed a right lingual artery pseudoaneurysm. With the lack of endovascular capabilities and the excessive risk of open surgery, thrombin was injected directly into the pseudoaneurysm under ultrasound guidance. A computed tomography scan and Doppler ultrasonography revealed complete resolution of the aneurysm. This article presents the first reported case in the English literature of a lingual artery aneurysm after penetrating trauma managed successfully with ultrasound-guided percutaneous thrombin injection. PMID:24365080

  17. Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase

    PubMed Central

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

    2016-01-01

    Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase. PMID:26867010

  18. 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

  19. 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

  20. 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

  1. Recombinant Buckwheat Trypsin Inhibitor Induces Mitophagy by Directly Targeting Mitochondria and Causes Mitochondrial Dysfunction in Hep G2 Cells.

    PubMed

    Wang, Zhuanhua; Li, Shanshan; Ren, Rong; Li, Jiao; Cui, Xiaodong

    2015-09-01

    Mitochondria are essential targets for cancer chemotherapy and other disease treatments. Recombinant buckwheat trypsin inhibitor (rBTI), a member of the potato type I proteinase inhibitor family, was derived from tartary buckwheat extracts. Our results showed that rBTI directly targeted mitochondria and induced mitochondrial fragmentation and mitophagy. This occurs through enhanced depolarization of the mitochondrial membrane potential, increasing reactive oxygen species (ROS) generation associated with the rise of the superoxide dismutase and catalase activity and glutathione peroxidase (GSH) content, and changes in the GSH/oxidized glutathione ratio. Mild and transient ROS induced by rBTI were shown to be important signaling molecules required to induce Hep G2 mitophagy to remove dysfunctional mitochondria. Furthermore, rBTI could directly induce mitochondrial fragmentation. It was also noted that rBTI highly increased colocalization of mitochondria in treated cells compared to nontreated cells. Tom 20, a subunit of the translocase of the mitochondrial outer membrane complex responsible for recognizing mitochondrial presequences, may be the direct target of rBTI. PMID:26301894

  2. A Src inhibitor regulates the cell cycle of human pluripotent stem cells and improves directed differentiation.

    PubMed

    Chetty, Sundari; Engquist, Elise N; Mehanna, Elie; Lui, Kathy O; Tsankov, Alexander M; Melton, Douglas A

    2015-09-28

    Driving human pluripotent stem cells (hPSCs) into specific lineages is an inefficient and challenging process. We show that a potent Src inhibitor, PP1, regulates expression of genes involved in the G1 to S phase transition of the cell cycle, activates proteins in the retinoblastoma family, and subsequently increases the differentiation propensities of hPSCs into all three germ layers. We further demonstrate that genetic suppression of Src regulates the activity of the retinoblastoma protein and enhances the differentiation potential of hPSCs across all germ layers. These positive effects extend beyond the initial germ layer specification and enable efficient differentiation at subsequent stages of differentiation. PMID:26416968

  3. A Src inhibitor regulates the cell cycle of human pluripotent stem cells and improves directed differentiation

    PubMed Central

    Engquist, Elise N.; Mehanna, Elie; Lui, Kathy O.; Tsankov, Alexander M.

    2015-01-01

    Driving human pluripotent stem cells (hPSCs) into specific lineages is an inefficient and challenging process. We show that a potent Src inhibitor, PP1, regulates expression of genes involved in the G1 to S phase transition of the cell cycle, activates proteins in the retinoblastoma family, and subsequently increases the differentiation propensities of hPSCs into all three germ layers. We further demonstrate that genetic suppression of Src regulates the activity of the retinoblastoma protein and enhances the differentiation potential of hPSCs across all germ layers. These positive effects extend beyond the initial germ layer specification and enable efficient differentiation at subsequent stages of differentiation. PMID:26416968

  4. 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

  5. Inhibition of Streptomyces griseus metallo-endopeptidase II (SGMPII) by active-site-directed inhibitors.

    PubMed

    Kumazaki, T; Ishii, S; Yokosawa, H

    1994-03-01

    Inactivation of Streptomyces griseus metallo-endopeptidase II (SGMPII) by ClCH2CO-DL-(N-OH)Leu-OCH3 and by ClCH2CO-DL-(N-OH)Leu-Ala-Gly-NH2 was studied kinetically. These reagents cause irreversible inhibition of the enzyme in a pseudo-first order reaction, and the inhibition reaction exhibits saturation kinetics. The second-order rate constants for inactivation of SGMPII by ClCH2CO-DL-(N-OH)Leu-OCH3 and by ClCH2CO-DL-(N-OH)Leu-Ala-Gly-NH2 were measured to be 0.12 and 8.9 M-1.s-1, respectively. The order of affinities of metallo-endopeptidases towards these irreversible inhibitors is thermolysin > SGMPII > Pseudomonas aeruginosa elastase. A competitive inhibitor of SGMPII, L-Val-L-Trp, protects the enzyme against inactivation by ClCH2CO-DL-(N-OH)Leu-Ala-Gly-NH2 in a competitive manner. Furthermore, the pH profile of the inactivation closely resembles that for the hydrolysis of synthetic peptide substrates by the enzyme. These findings suggest that these reagents bind reversibly and react irreversibly at the active site of the enzyme. PMID:8056768

  6. Inhibitor focusing: direct selection of drug targets from proteomes using activity-based probes.

    PubMed

    Nomanbhoy, Tyzoon K; Rosenblum, Jonathan; Aban, Arwin; Burbaum, Jonathan J

    2003-02-01

    In the latter stages of drug discovery and development, assays that establish drug selectivity and toxicity are important when side effects, which are often due to lack of specificity, determine drug candidate viability. There has been no comprehensive or systematic methodology to measure these factors outside of whole-animal assays, and such phenomenological assays generally fail to establish the additional targets of a given small molecule, or the molecular origin of toxicity. Consequently, small-molecule development programs destined for failure often reach advanced stages of testing, and the money and time invested in such programs could be saved if information on selectivity were available early in the process. Here, we present a methodology that utilizes chemical ABPs in combination with small-molecule inhibitors to selectively label small-molecule binding sites in whole proteomic samples. In principle, the ABP and small molecule will compete for similar binding sites, such that the small molecule will protect against modification by the ABP. Thus, after removal of the small molecule, the binding site for the ABP will be revealed, and a second probe can then be used to label the small-molecule binding sites selectively. To demonstrate this experimentally, we mapped the binding sites of the DPP4 inhibitor, IT, in a number of different tissue types. PMID:15090140

  7. 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

  8. 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

  9. 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

  10. 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

  11. RNA-seq analysis of transcriptomes in thrombin-treated and control human pulmonary microvascular endothelial cells.

    PubMed

    Cheranova, Dilyara; Gibson, Margaret; Chaudhary, Suman; Zhang, Li Qin; Heruth, Daniel P; Grigoryev, Dmitry N; Ye, Shui Qing

    2013-01-01

    thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state. PMID:23426025

  12. 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

  13. Identification of Environmental Quaternary Ammonium Compounds as Direct Inhibitors of Cholesterol Biosynthesis.

    PubMed

    Herron, Josi; Reese, Rosalyn C; Tallman, Keri A; Narayanaswamy, Rohini; Porter, Ned A; Xu, Libin

    2016-06-01

    In this study, we aim to identify environmental molecules that can inhibit cholesterol biosynthesis, potentially leading to the same biochemical defects as observed in cholesterol biosynthesis disorders, which are often characterized by congenital malformations and developmental delay. Using the Distributed Structure-Searchable Toxicity (DSSTox) Database Network developed by EPA, we first carried out in silico screening of environmental molecules that display structures similar to AY9944, a known potent inhibitor of 3β-hydroxysterol-Δ(7)-reductase (DHCR7)-the last step of cholesterol biosynthesis. Molecules that display high similarity to AY9944 were subjected to test in mouse and human neuroblastoma cells for their effectiveness in inhibiting cholesterol biosynthesis by analyzing cholesterol and its precursor using gas chromatography-mass spectrometry. We found that a common disinfectant mixture, benzalkonium chlorides (BACs), exhibits high potency in inhibiting DHCR7, as suggested by greatly elevated levels of the cholesterol precursor, 7-dehydrocholesterol (7-DHC). Subsequent structure-activity studies suggested that the potency of BACs as Dhcr7 inhibitors decrease with the length of their hydrocarbon chain: C10 > C12 ≫ C14 > C16. Real-time qPCR analysis revealed upregulation of the genes related to cholesterol biosynthesis and downregulation of the genes related to cholesterol efflux, suggesting a feedback response to the inhibition. Furthermore, an oxidative metabolite of 7-DHC that was previously identified as a biomarker in vivo was also found in cells exposed to BACs by liquid chromatography-mass spectrometry. Our findings suggest that certain environmental molecules could potently inhibit cholesterol biosynthesis, which could be a new link between environment and developmental disorders. PMID:26919959

  14. Both Cyclophilin Inhibitors and Direct-Acting Antivirals Prevent PKR Activation in HCV-Infected Cells

    PubMed Central

    Bobardt, Michael; Chatterji, Udayan; Lim, Precious; Gawlik, Katarzyna; Gallay, Philippe

    2014-01-01

    We and others demonstrated that the contact between NS5A and the host factor CypA is critical for HCV replication. CypI, by disrupting NS5A-CypA complexes, block HCV replication both in vitro and in patients. Since NS5A also binds to PKR, a central component of the IFN response, we investigated the possibility of a relationship between CypA, NS5A and PKR in the IFN response to HCV. HCV-infected cells treated with CypI, DAAs or IFN were analyzed for the expression and activation of various components of the innate response. We found that CypI (cyclosporine A, alisporivir, NIM811 and sanglifehrins), drastically prevented the activation/phosphorylation, but not the expression of IFN-induced PKR in HCV-infected cells. CypI had no effect on the expression or phosphorylation of other components of the innate response such as eiF2, NF-kB, IRF3, IRF9, STAT1 and STAT2, suggesting a specific effect on PKR. No significant activation of IFN-induced PKR was observed in the absence of HCV. Importantly, we found that several classes of DAAs such as NS3/4A protease, NS5B polymerase and NS5A inhibitors also prevented PKR activation. Furthermore, we found that PKR activation by the dsRNA mimic poly I:C cannot be prevented by CypI or DAAs. Our findings suggest that CypI do not have a unique effect on PKR activation, but rather the suppression of HCV replication by any anti-HCV inhibitor, abrogates PKR activation induced by IFN. Moreover, they suggest that the accumulation of dsRNA intermediates allows HCV to exploit the activation of PKR to counteract the IFN response. PMID:24799968

  15. 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

  16. [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

  17. 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

  18. 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

  19. 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

  20. The Rel/NF-κB Family Directly Activates Expression of the Apoptosis Inhibitor Bcl-xL

    PubMed Central

    Chen, Cailin; Edelstein, Leonard C.; Gélinas, Céline

    2000-01-01

    The transcription factors of the Rel/NF-κB family are key regulators of immune and inflammatory responses and contribute to lymphocyte proliferation, survival, and oncogenesis. The absolute correlation between the antiapoptotic and oncogenic activities of the Rel/NF-κB oncoprotein v-Rel emphasizes the importance of characterizing the death antagonists under NF-κB control. Our recent finding that the prosurvival Bcl-2 homolog Bfl-1 (also called A1) is a direct transcriptional target of NF-κB raised the issue of whether NF-κB is a specific or global regulator of death antagonists in the Bcl-2 family. Here, we demonstrate that NF-κB differentially regulates the expression of particular Bcl-2-related death inhibitors and that it directly activates the expression of Bcl-xL. While Bcl-xL was significantly upregulated by c-Rel and RelA, Bcl-2 was not. Importantly, stimuli that activate endogenous NF-κB factors also upregulated bcl-x gene expression and this effect was antagonized by an inhibitor of NF-κB activity. The expression of bcl-x suppressed apoptosis in the presence or absence of NF-κB activity. Functional analysis of the bcl-x promoter demonstrated that it is directly controlled by c-Rel. These results establish that NF-κB directly regulates the expression of distinct prosurvival factors in the Bcl-2 family, such as Bcl-xL and Bfl-1/A1. These findings raise the possibility that some of these factors may contribute to oncogenesis associated with aberrant Rel/NF-κB activity. PMID:10733571

  1. 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.

  2. Mealtime-related dosing directions for proton-pump inhibitors in gastroesophageal reflux disease: physician knowledge, patient adherence.

    PubMed

    Solem, Caitlyn; Mody, Reema; Stephens, Jennifer; Macahilig, Cynthia; Gao, Xin

    2014-01-01

    OBJECTIVE To describe physicians' knowledge, patients' adherence, and perceptions of both regarding mealtime-related dosing directions for proton-pump inhibitors (PPIs). DESIGN Chart review and survey of patients and physicians. SETTING United States, with data collected between January and July 2011. PARTICIPANTS Patients being treated for gastroesophageal reflux disease (GERD) with PPIs and their prescribing physicians. MAIN OUTCOME MEASURES Patient- and physician-reported perception of PPI mealtime-related directions as important/inconvenient (seven-point Likert scale; 7 = very important/very inconvenient); physician-reported knowledge of PPI mealtime-related dosing directions based on whether the agent is labeled to be taken 30-60 minutes before eating (DIR-esomeprazole magnesium [Nexium-AstraZeneca], lansoprazole, and omeprazole) or labeled to be taken regardless of meals (NoDIR-dexlansoprazole [Dexilant-Takeda], rabeprazole, and pantoprazole); and patient-reported PPI mealtime-related directions received and adherence to directions. RESULTS Physicians (n = 262) recruited 501 patients who had been prescribed PPIs (262 DIR/239 NoDIR; mean age 51 years, 37% men, 56% nonerosive GERD [29% undocumented]). Across PPIs, physicians frequently reported incorrect directions or "did not know directions" (29% for esomeprazole to 69% for pantoprazole). While 98% of patients reported receiving directions from their physicians and 55% from their pharmacists, only 65% of DIR patients and 18% of NoDIR received directions consistent with product labeling. Physicians perceived greater inconvenience than patients (4.4 vs. 1.6, P < 0.001) and greater importance (5.2 vs. 4.5, P < 0.001) of mealtime-related directions. Overall, 81% of patients reported taking their PPI as directed. CONCLUSION While this patient cohort was adherent to directions given, physicians' directions were often inconsistent with product labeling. Understanding physician and patient knowledge gaps may be

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. Abruption-Induced Preterm Delivery Is Associated with Thrombin-Mediated Functional Progesterone Withdrawal in Decidual Cells

    PubMed Central

    Lockwood, Charles J.; Kayisli, Umit A.; Stocco, Carlos; Murk, William; Vatandaslar, Emre; Buchwalder, Lynn F.; Schatz, Frederick

    2013-01-01

    Plasma progesterone levels remain elevated throughout human pregnancy, suggesting that reduced reproductive-tract progesterone receptor (PR) initiates labor. Placental abruption and excess thrombin generation elicit preterm delivery (PTD). PR, glucocorticoid receptor (GR), and total and p-ERK1/2 in decidual cells (DCs) and interstitial trophoblasts (IT) were assessed via immunohistochemical staining in abruption-associated PTD versus gestational-age matched control placentas, and in cultured DCs incubated with estradiol (E2) ± medroxyprogesterone acetate (MPA) ± thrombin. Immunostaining for PR was lower in DC nuclei in abruption versus control decidua and was absent from ITs; GR was higher in IT than DCs, with no abruption-related changes in either cell type; p-ERK1/2 was higher in DCs in abruption than control decidua, with total ERK 1/2 unchanged. Immunoblotting of cultured DCs demonstrated strong E2, weak MPA, and intermediate E2+MPA mediated elevation of PR-A and PR-B levels, with constitutive GR expression. In cultured DCs, thrombin inhibited PR but not GR mRNA levels, reduced PR binding to DNA and [3H]progesterone binding to PR, and enhanced phosphorylated but not total ERK1/2 levels. Coincubation with a specific p-ERK1/2 inhibitor reversed thrombin-enhanced p-ERK1/2 and lowered PR levels. Thus, abruption-associated PTD is initiated by functional progesterone withdrawal, as indicated by significantly reduced DC nuclear expression of PR-A and PR-B. Functional withdrawal of progesterone results in increased p-ERK1/2, and is thus one pathway initiating abruption-associated PTD. PMID:23058370

  8. 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

  9. Multitarget-directed tricyclic pyridazinones as G protein-coupled receptor ligands and cholinesterase inhibitors.

    PubMed

    Pau, Amedeo; Catto, Marco; Pinna, Giovanni; Frau, Simona; Murineddu, Gabriele; Asproni, Battistina; Curzu, Maria M; Pisani, Leonardo; Leonetti, Francesco; Loza, Maria Isabel; Brea, José; Pinna, Gérard A; Carotti, Angelo

    2015-06-01

    By following a multitarget ligand design approach, a library of 47 compounds was prepared, and they were tested as binders of selected G protein-coupled receptors (GPCRs) and inhibitors of acetyl and/or butyryl cholinesterase. The newly designed ligands feature pyridazinone-based tricyclic scaffolds connected through alkyl chains of variable length to proper amine moieties (e.g., substituted piperazines or piperidines) for GPCR and cholinesterase (ChE) molecular recognition. The compounds were tested at three different GPCRs, namely serotoninergic 5-HT1A, adrenergic α1A, and dopaminergic D2 receptors. Our main goal was the discovery of compounds that exhibit, in addition to ChE inhibition, antagonist activity at 5-HT1A because of its involvement in neuronal deficits typical of Alzheimer's and other neurodegenerative diseases. Ligands with nanomolar affinity for the tested GPCRs were discovered, but most of them behaved as dual antagonists of α1A and 5-HT1A receptors. Nevertheless, several compounds displaying this GPCR affinity profile also showed moderate to good inhibition of AChE and BChE, thus deserving further investigations to exploit the therapeutic potential of such unusual biological profiles. PMID:25924828

  10. Piperlongumine as a direct TrxR1 inhibitor with suppressive activity against gastric cancer.

    PubMed

    Zou, Peng; Xia, Yiqun; Ji, Jiansong; Chen, Weiqian; Zhang, Jinsan; Chen, Xi; Rajamanickam, Vinothkumar; Chen, Gaozhi; Wang, Zhe; Chen, Lingfeng; Wang, Yifeng; Yang, Shulin; Liang, Guang

    2016-05-28

    Piperlongumine (PL), a natural alkaloid isolated from the fruit of long pepper, is known to selectively kill tumor cells while sparing their normal counterparts. However, the cellular target and potent anticancer efficacy of PL in numerous types of human cancer cells have not been fully defined. We report here that PL may interact with the thioredoxin reductase 1 (TrxR1), an important selenocysteine (Sec)-containing antioxidant enzyme, to induce reactive oxygen species (ROS)-mediated apoptosis in human gastric cancer cells. By inhibiting TrxR1 activity and increasing intracellular ROS levels, PL induces a lethal endoplasmic reticulum stress and mitochondrial dysfunction in human gastric cancer cells. Importantly, knockdown of TrxR1 sensitizes cells to PL treatment, and PL displays synergistic lethality with GSH inhibitors (BSO and Erastin) against gastric cancer cells. In vivo, PL treatment markedly reduces the TrxR1 activity and tumor cell burden. Remarkably, TrxR1 was significantly overexpressed in gastric cancer cell lines and human gastric cancer tissues. Targeting TrxR1 with PL thus discloses a previously unrecognized mechanism underlying the biological activity of PL and provides an in-depth insight into the action of PL in the treatment of gastric cancer. PMID:26963494