WorldWideScience
 
 
1

Topical use of recombinant human thrombin for operative hemostasis.  

UK PubMed Central (United Kingdom)

BACKGROUND: The topical use of thrombin has a long history in surgery as an adjunct for achieving operative hemostasis. Until recently the majority of thrombin used topically was derived from bovine plasma. This preparation has been proven to be immunogenic and has led to safety concerns in recent years. Recombinant human thrombin (rhThrombin) has recently been developed as an alternative for topical use for surgical hemostasis. OBJECTIVE: To review the clinical safety and efficacy data relating to rhThrombin using bovine-derived thrombin as a comparative standard. METHODS: This review summaries recent literature regarding topical use of rhThrombin using bovine thrombin as the 'gold standard' for topical surgical hemostasis. CONCLUSIONS: The data indicates that topical rhThrombin is as effective as bovine thrombin for hemostasis and significantly less immunogenic.

Anderson CD; Bowman LJ; Chapman WC

2009-01-01

2

Recent developments in topical thrombins.  

Science.gov (United States)

Managing blood loss is part of the surgeon's responsibility during surgical procedures, and a variety of therapeutic strategies are available to help accomplish this. Topical haemostatic agents are among the agents used to control surgical bleeding and locally arrest blood flow. Bovine thrombin is a commonly used topical haemostatic agent; however, its use has been associated with potential risks, including well-documented cases of antibody-mediated coagulopathy. This coagulopathy develops as a consequence of antibody formation directed against bovine thrombin, other bovine coagulation proteins, and their human orthologs. The fact that a coagulopathy can result in association with the use of bovine plasma-derived thrombin preparations prompted the FDA to require pharmaceutical companies to place a black-box warning in their prescribing information for products containing bovine plasma-derived thrombin. Recently, human plasma-derived thrombin and recombinant human thrombin have been approved by the FDA with the expectation that they will be less immunogenic than the bovine-derived product. In clinical studies, purified human plasma-derived thrombin and recombinant thrombin have demonstrated equivalent efficacy and safety, with improved immunogenicity profiles compared with bovine-derived thrombin agents. Well-designed and adequately powered clinical trials should be conducted to indicate whether human thrombin products would improve the risk-benefit and cost-benefit profiles for surgeries complicated by excessive bleeding. PMID:19572062

Kessler, Craig M; Ortel, Thomas L

2009-07-01

3

Recent developments in topical thrombins.  

UK PubMed Central (United Kingdom)

Managing blood loss is part of the surgeon's responsibility during surgical procedures, and a variety of therapeutic strategies are available to help accomplish this. Topical haemostatic agents are among the agents used to control surgical bleeding and locally arrest blood flow. Bovine thrombin is a commonly used topical haemostatic agent; however, its use has been associated with potential risks, including well-documented cases of antibody-mediated coagulopathy. This coagulopathy develops as a consequence of antibody formation directed against bovine thrombin, other bovine coagulation proteins, and their human orthologs. The fact that a coagulopathy can result in association with the use of bovine plasma-derived thrombin preparations prompted the FDA to require pharmaceutical companies to place a black-box warning in their prescribing information for products containing bovine plasma-derived thrombin. Recently, human plasma-derived thrombin and recombinant human thrombin have been approved by the FDA with the expectation that they will be less immunogenic than the bovine-derived product. In clinical studies, purified human plasma-derived thrombin and recombinant thrombin have demonstrated equivalent efficacy and safety, with improved immunogenicity profiles compared with bovine-derived thrombin agents. Well-designed and adequately powered clinical trials should be conducted to indicate whether human thrombin products would improve the risk-benefit and cost-benefit profiles for surgeries complicated by excessive bleeding.

Kessler CM; Ortel TL

2009-07-01

4

Preclinical safety of recombinant human thrombin.  

Science.gov (United States)

Recombinant human thrombin (rhThrombin) is being developed as an alternative to thrombin products purified from pooled human or bovine plasma, which are currently marketed for topical hemostasis. Preclinical studies of rhThrombin were conducted prior to its evaluation as a topical adjunct to surgical hemostasis in clinical trials. No overt clinical pathology or signs were observed in cynomolgus monkeys following implantation of a gelatin sponge containing either rhThrombin or bovine thrombin to a surgical liver wound, and similar gross and microscopic wound healing characteristics were observed over an eight-week recovery period with either compound. Repeated subcutaneous injections of rhThrombin or bovine thrombin to cynomolgus monkeys produced no treatment-related effects. Whereas no monkeys demonstrated anti-rhThrombin antibody seroconversion, specific anti-bovine antibodies were detected in all tested monkeys exposed to bovine thrombin. Addition of rhThrombin or bovine thrombin to mouse fibroblast cells resulted in expected detachment and shape change. Topical application of rhThrombin to rabbits did not cause irritation to the eye, normal skin, or abraded skin. These studies showed that topical, subcutaneous, or implanted rhThrombin was minimally immunogenic, safe, and well tolerated in nonclinical models, and supported the clinical evaluation of rhThrombin in a variety of surgical settings. PMID:16971028

Heffernan, Jane K; Ponce, Rafael A; Zuckerman, Linda A; Volpone, John P; Visich, Jennifer; Giste, Erika E; Jenkins, Nancy; Boster, Dan; Pederson, Susan; Knitter, Glenn; Palmer, Thomas; Wills, Margaret; Early, Richard J; Rogge, Mark C

2006-09-12

5

A review of three stand-alone topical thrombins for surgical hemostasis.  

UK PubMed Central (United Kingdom)

BACKGROUND: Topical thrombins are active hemostatic agents that can be used to minimize blood loss during surgery. Before 2007, the only topical thrombins available were derived from bovine plasma. Antibody formation to bovine thrombin and/or factor V, with subsequent risk of cross-reactivity with human factor V, and hemorrhagic complications associated with human factor-V deficiencies have been described in case reports of surgeries in which bovine thrombins were used. This risk is now included in the boxed warning section of the bovine thrombin prescribing information. In 2007 and 2008, 2 new topical thrombins from nonbovine sources received approval for use from the US Food and Drug Administration. The 3 active topical thrombins that are currently marketed are bovine plasma-derived thrombin, human plasma-derived thrombin, and human recombinant thrombin. OBJECTIVE: The purpose of this review was to evaluate the literature on the efficacy and safety of topical thrombins and discuss the pharmacoeconomic considerations associated with their use. METHODS: PubMed, EMBASE, and International Pharmaceutical Abstracts were searched for relevant papers published in English through October 10,2008, using the terms thrombin, human recombinant thrombin, bovine thrombin, plasma derived thrombin, and topical thrombin. Manufacturer-provided materials were also reviewed. Abstracts and unpublished data, as well as evaluations of sealants, adhesives, glues, and other hemostats that contain thrombin mixed with fibrinogen and other clotting factors, were excluded. RESULTS: Four randomized, double-blind studies involving the active, stand-alone topical thrombins were found. The bovine thrombin involved in these studies was the predecessor to the currently marketed, highly purified bovine formulation. No studies comparing the human products, studies involving the highly purified bovine preparation, or placebo-controlled studies involving bovine thrombin were found. In a Phase III comparison of human recombinant thrombin and bovine thrombin, the percentages of patients who achieved hemostasis within 10 minutes of topical thrombin application were 95.4% and 95.1%, respectively (95% CI, -3.7 to 5.0). The incidence of hemostasis within 10 minutes was also similar in a Phase III comparison of human plasma-derived thrombin and bovine thrombin (both, 97.4% [95% CI, 0.96 to 1.05]). In the study that compared human recombinant and bovine thrombin, the incidence of antiproduct antibody formation was 21.5% (43/200) in the bovine thrombin group and 1.5% (3/198) in the human recombinant thrombin group (P < 0.001); patients with antibodies to bovine thrombin had numerically higher incidences of bleeding or thromboembolic events than did patients without these antibodies (19% vs 13%; P value not reported). Human plasma-derived thrombin is available as a frozen sterile solution that must be thawed before application, whereas the human recombinant and bovine plasma-derived products are supplied as unrefrigerated sterile powders that must be reconstituted before use. The human thrombins are more costly than bovine thrombin on a per-vial basis. The average wholesale prices (US $, 2008) for 5000-IU vials of bovine thrombin and human recombinant thrombin were $87.85 and $103.20, respectively; the average wholesale price for a 4000- to 6000-IU vial of human plasma-derived thrombin was $96.00. CONCLUSIONS: Topical thrombins vary in the ways in which they are manufactured and their safety profiles, storage requirements, and costs. Human recombinant thrombin and human plasma-derived thrombin have each been shown to have hemostatic efficacy comparable to that of bovine thrombin. Bovine thrombin carries the risk of formation of cross-reactive antibodies to bovine thrombin, factor V, and other impurities that may be present in these formulations. Immunogenicity data for the currently marketed, highly purified bovine thrombin relative to older formulations of bovine thrombin could not be found. Whether the potential safety advantage justifies the added cost of the hu

Cheng CM; Meyer-Massetti C; Kayser SR

2009-01-01

6

Development of antibodies to thrombin and factor V with recurrent bleeding in a patient exposed to topical bovine thrombin.  

UK PubMed Central (United Kingdom)

A 65 year old patient who was exposed to topical bovine thrombin during cardiac surgery developed markedly prolonged clotting times and a severe bleeding diathesis. Mixing studies with normal plasma failed to correct the clotting times. Platelet transfusions, immunosuppressive and immunomodulatory therapies were ineffective, but plasmapheresis was effective in decreasing clotting times and in the resolution of clinical bleeding events. The patient's purified IgG reacted with bovine thrombin by immunoblotting and enzyme-linked immunosorbent assay (ELISA). However, the IgG reacted minimally with human thrombin. In view of the severe bleeding, a coexisting inhibitor was sought. The patient's factor V activity was 1% of normal and was not corrected by mixing with normal plasma, demonstrating the presence of an inhibitor against factor V. The patient's IgG reacted with both bovine and human factor V. Immunoblotting localized the site of antibody binding to the light chain of activated bovine factor V. Detectable amounts of bovine factor V were found in commercial bovine thrombin preparations by ELISA. The data suggest that patients exposed to topical bovine thrombin may develop antibodies to thrombin and factor V. Anti-thrombin antibodies may mask coexisting factor V inhibitors responsible for clinical bleeding.

Zehnder JL; Leung LL

1990-11-01

7

Topical bovine thrombin: a 21-year review of topical bovine thrombin spontaneous case safety reports submitted to FDA's Adverse Event Reporting System.  

UK PubMed Central (United Kingdom)

PURPOSE: To review topical bovine thrombin spontaneous adverse event (AE) reports that were forwarded to the US Food and Drug Administration's (FDA) Adverse Event Reporting System (AERS) between January 1986 and December 2006. METHODS: Forty-one spontaneous AE reports were summarized for reported AE profile and chronological reporting patterns. Each AE report was adjudicated by a hematologist for the topical bovine thrombin product that was given and the AE(s) that were reported. AEs were grouped as allergic, coagulopathy/bleeding, and all other AEs combined. Grouped AE serial analyses were carried out using successive 3-year time increments between 1986 (the year an AE report was first noted for a bovine thrombin product) and 2006 (the first full year that was available at the time of initiation of the data summary). MAIN OUTCOME MEASURES: The primary outcome measures were every 3-year trend lines for all-AE reports, all reporters, and topical bovine thrombin brand mentions for 2 AE groups of interest (allergic events and coagulopathy/bleeding events). RESULTS: The all-AE spontaneous reporter trend showed a downward appearance for AE reporting activity that started in 1995-1998 and continued through 2004-2006. The all-AE reports trend showed two potential safety signals that could be identified serially: (1) a prominent 1989-1991 peak that was attributable to allergic events (in particular, anaphylaxis), and (2) a small 1995-2000 broad peak that was attributable in part to coagulopathy/bleeding events. Allergic events were predominantly reported with products approved prior to 1995, were not temporally associated with prior medical literature case reports, and continued to be forwarded to the FDA at low levels up to the end of this study in 2006. Coagulopathy/bleeding events were reported only with products approved prior to 1995, were temporally associated with medical literature case reports, and were not forwarded to the FDA after 2000. CONCLUSIONS: Overall, spontaneous AE reporting for topical bovine thrombin occurs at very low levels, and appears to have been decreasing since 1995. The serial reporting patterns for topical bovine thrombin are best explained as a strong safety signal for allergic events with ongoing, low level reporting, and a weak safety signal for coagulopathy/bleeding events that ceased on or before 2000. Although this descriptive trend analysis cannot measure associations or causation, the coagulopathy/bleeding signal may have been prompted by multiple, antecedent published case reports. The subsequent diminishment of signal attributed to thrombin likewise may coincide with lack of such reporting in larger follow-up clinical trials or, alternatively, in the introduction and growing market share of thrombin brands of greater purity. Currently marketed topical bovine thrombin formulations are rarely volunteered as possible causes of adverse events.

Clark JA; Humphries JE; Crean S; Reynolds MW

2010-02-01

8

The role of platelets and recombinant factor VIIa on thrombin generation, platelet activation and clot formation.  

Science.gov (United States)

In the present study we assessed the effect of platelet counts and rFVIIa on thrombin generation, platelet activation and clot formation after tissue factor pathway activation in human plasma aiming to investigate the mechanism by which rFVIIa induces haemostasis in patients with severe thrombocytopenia. Plasma samples with platelet counts from 5 x 10(9)/l to 150 x 10(9)/l were spiked with rFVIIa (1 micro g/ml) or buffer. Clotting was initiated in the presence of diluted thromboplastin. Thrombin generation was assessed using the Thrombogram-Thrombinoscope trade mark assay. The kinetics of platelet activation was assessed using flow cytometry to measure the expression the P-selectin on platelet membrane of washed platelets suspended in defibrinated homologous PPP. Thromboelastography was used to evaluate the effect of platelets and rFVIIa on the kinetics of clot formation and clot's firmness. In the presence of low platelet counts the endogenous thrombin potential (ETP) and the maximum concentration of generated thrombin (Cmax) were reduced by 60%-70%. The lag-time of thrombin generation and the time required to reach the Cmax (Tmax) were prolonged, the velocity of platelet activation was decreased and thrombus formation was delayed. Recombinant FVIIa accelerated thrombin generation and platelet activation but it did not significantly modify ETP or Cmax. Recombinant FVIIa enhanced platelet activation in a TF and thrombin dependent manner since its effect on the studied parameters was abolished when TF was omitted or when hirudin was added into the experimental system respectively. Recombinant FVIIa normalized the velocity of clot formation but it did not modify clot firmness, which depended mainly on platelets' count. In conclusion, in experimental conditions simulating severe thrombocytopenia rFVIIa in the presence of low amounts of TF, accelerates thrombin generation, without increasing the maximum amount of generated thrombin, thus leading in enhanced platelet activation and rapid clot formation. PMID:15116259

Gerotziafas, Grigoris T; Chakroun, Tahar; Depasse, François; Arzoglou, Pantelis; Samama, Meyer M; Elalamy, Ismail

2004-05-01

9

The role of platelets and recombinant factor VIIa on thrombin generation, platelet activation and clot formation.  

UK PubMed Central (United Kingdom)

In the present study we assessed the effect of platelet counts and rFVIIa on thrombin generation, platelet activation and clot formation after tissue factor pathway activation in human plasma aiming to investigate the mechanism by which rFVIIa induces haemostasis in patients with severe thrombocytopenia. Plasma samples with platelet counts from 5 x 10(9)/l to 150 x 10(9)/l were spiked with rFVIIa (1 micro g/ml) or buffer. Clotting was initiated in the presence of diluted thromboplastin. Thrombin generation was assessed using the Thrombogram-Thrombinoscope trade mark assay. The kinetics of platelet activation was assessed using flow cytometry to measure the expression the P-selectin on platelet membrane of washed platelets suspended in defibrinated homologous PPP. Thromboelastography was used to evaluate the effect of platelets and rFVIIa on the kinetics of clot formation and clot's firmness. In the presence of low platelet counts the endogenous thrombin potential (ETP) and the maximum concentration of generated thrombin (Cmax) were reduced by 60%-70%. The lag-time of thrombin generation and the time required to reach the Cmax (Tmax) were prolonged, the velocity of platelet activation was decreased and thrombus formation was delayed. Recombinant FVIIa accelerated thrombin generation and platelet activation but it did not significantly modify ETP or Cmax. Recombinant FVIIa enhanced platelet activation in a TF and thrombin dependent manner since its effect on the studied parameters was abolished when TF was omitted or when hirudin was added into the experimental system respectively. Recombinant FVIIa normalized the velocity of clot formation but it did not modify clot firmness, which depended mainly on platelets' count. In conclusion, in experimental conditions simulating severe thrombocytopenia rFVIIa in the presence of low amounts of TF, accelerates thrombin generation, without increasing the maximum amount of generated thrombin, thus leading in enhanced platelet activation and rapid clot formation.

Gerotziafas GT; Chakroun T; Depasse F; Arzoglou P; Samama MM; Elalamy I

2004-05-01

10

Thrombin cleaves recombinant human thrombopoietin: One of the proteolytic events that generates truncated forms of?thrombopoietin  

Digital Repository Infrastructure Vision for European Research (DRIVER)

A heterogeneity in the molecular weight (Mr) of thrombopoietin (TPO) has been reported. We found several thrombin cleavage sites in human, rat, murine, and canine TPOs, and also found that human TPO undergoes selective proteolysis by thrombin. Recombinant human TPO (rhTPO) was incubated with human p...

Kato, Takashi; Oda, Atsushi; Inagaki, Yoshimasa; Ohashi, Hideya; Matsumoto, Atsushi; Ozaki, Katsutoshi; Miyakawa, Yoshitaka

11

In vitro effects of recombinant activated factor VII on thrombin generation and coagulation following inhibition of platelet procoagulant activity by prasugrel.  

UK PubMed Central (United Kingdom)

INTRODUCTION: Prasugrel is a thienopyridyl P2Y12 antagonist with potent antiplatelet effects. At present, little is known about its effects on thrombin generation or what strategies may emergently reverse its anticoagulant effects. In the current study we evaluated whether recombinant activated factor VII may reverse prasugrel induced effects and increase thrombin generation in an in vitro model. METHODS: The effect of prasugrel active metabolite, PAM (R-138727), was evaluated on platelet aggregation, thrombin generation, and rotational thromboelastometry parameters using blood from 20 healthy volunteers. Additionally, we evaluated the effects of adenosine diphosphate (ADP) and recombinant activated factor VII on restoring these parameters towards baseline values. RESULTS: PAM reduced maximum platelet aggregation and led to platelet disaggregation. It also decreased peak thrombin, increased lag time, and increased time to peak thrombin. Treatment with recombinant activated factor VII restored all three parameters of thrombin generation towards baseline. ADP decreased lag time and time to peak thrombin, but had no effect on peak thrombin. When recombinant activated factor VII and ADP were combined they had a greater effect on thrombin parameters than either drug alone. PAM also increased thromboelastometric clotting time and clot formation time, but had no effect on maximum clot firmness. Treatment with either recombinant activated factor VII or ADP restored these values towards baseline. CONCLUSIONS: Recombinant activated factor VII restores thrombin generation in the presence of PAM. In patients taking prasugrel with life-threatening refractory bleeding it has the potential to be a useful therapeutic approach. Additional clinical studies are needed to validate our findings.

Mazzeffi M; Szlam F; Jakubowski JA; Tanaka KA; Sugidachi A; Levy JH

2013-07-01

12

Assessment of thrombus imaging potency of thrombin-targeting recombinant hirudin in vitro and in vivo  

International Nuclear Information System (INIS)

[en] The purpose of this study is to evaluate the effect of recombinant hirudin HV2 (rHHV2) as a thrombus imaging agent. 125I-rHHV2 and 125I-Th were prepared with Chloramine method, the labeling rate were 86.64% and 62.20%, with the radioactive purity of 89.70% and 91.22%, with the specific activity of 22.4 TBq/mmol and 94.43 TBq/mmol respectively. The competitive radioassay showed that the Th-fibrin complex formation did not affect the ability of rHHV2 binding with Th. In the complex, the molecular binding ratio of rHHV2 to Th and fibrinogen was 14:14:1. 99mTc-rHHV2 was prepared by 2-iminothiolane modified method, the labeled rate was 94%, with the radioactive purity of 93.90%, with the specific activity of 2.30 TBq/mmol. It was used to image fresh thrombi on arteries and veins of dog or rabbit (30 ?g/kg). In SPECT images, all thrombin were clearly visible, arterial thrombosis imaging can be seen clearly within 45 min after injection and fade away slowly, venous thrombosis imaging also can be seen within 30 min after injection and quantitative imaging ratios between the thrombus and opposite vessel increased following the time. Biodistribution studies in mouse demonstrated that rHHV2 was excreted from kidneys. These data indicate that Th in Th-fibrin complex could be a potent target for diagnosis of thrombus and 99mTc-rHHV2 could be a new thrombotic imaging agent. (authors)

2003-01-01

13

Thrombin generation and fibrinolysis in anti-factor IX treated blood and plasma spiked with factor VIII inhibitor bypassing activity or recombinant factor VIIa.  

UK PubMed Central (United Kingdom)

Activated prothrombin complex concentrates (aPCC) and recombinant activated factor VIIa (rFVIIa) are two important therapies in haemophilia patients with inhibitors and improve clot stability. We hypothesized that potential differences in procoagulant and fibrinolytic actions of aPCC and rFVIIa may lie in the clot stability against fibrinolytic activation. We used thrombin generation, fluorescence detection and thromboelastometry in anti-factor IXa (FIXa) aptamer-treated whole blood (WB) and plasma to evaluate: (i) generation of thrombin and activated factor X (FXa) and (ii) viscoelastic properties of blood clots in the presence of tissue plasminogen activator (tPA) after addition of aPCC (0.4 U mL(-1)) or rFVIIa (60 nm). Peak thrombin generation increased from 85 +/- 19 nm in aptamer-treated plasma to 276 +/- 83 nm and 119 +/- 22 nm after addition of aPCC and rFVIIa respectively (P < 0.001). FXa activity increased within 20 min by 87 +/- 6% and by 660 +/- 97% after addition of aPCC and rFVIIa respectively (P < 0.001). TPA-induced lysis time increased from 458 +/- 378 s in aptamer-treated WB to 1597 +/- 366 s (P = 0.001) and 1132 +/- 214 s (P = 0.075), after addition of aPCC and rFVIIa respectively. In this haemophilia model using the anti-FIXa aptamer, the larger amount of thrombin was generated with aPCC compared with rFVIIa, while FXa generation was more rapidly increased in the presence of rFVIIa. Furthermore, clot formation in anti-FIXa aptamer-treated WB was less susceptible to tPA-induced fibrinolysis after adding aPCC compared with rFVIIa.

Bolliger D; Szlam F; Molinaro RJ; Escobar MA; Levy JH; Tanaka KA

2010-05-01

14

Triabin, a highly potent exosite inhibitor of thrombin.  

Science.gov (United States)

Triabin, a new thrombin inhibitor, has been purified from the saliva of Triatoma pallidipennis, a blood-sucking triatomine bug. It forms a noncovalent complex with thrombin at a molar ratio of 1:1, inhibits thrombin-induced platelet aggregation, and prolongs thrombin clotting time and activated partial thromboplastin time. However, it only minimally suppresses the amidolytic activity of thrombin, as measured by a chromogenic peptide substrate assay. It completely blocks trypsin-catalyzed cleavage of thrombin, probably via protection of the anion-binding exosite and inhibits the effect of thrombomodulin on thrombin in a dose-dependent fashion. These results indicate that the inhibitor is directed toward the anion-binding exosite of thrombin. The protein was partially sequenced and the information used to isolate cDNA clones from a T. pallidipennis salivary gland library. Four slightly polymorphic variants coding for mature proteins of 142 amino acids preceded by a putative leader sequence were obtained. The recombinant protein expressed in the periplasmic space of Escherichia coli has a biological activity similar to that of salivary triabin, as tested in a thrombin-induced platelet aggregation assay. In addition, recombinant triabin inhibits thrombin-catalyzed hydrolysis of fibrinogen with a Ki of about 3 pM. PMID:7499380

Noeske-Jungblut, C; Haendler, B; Donner, P; Alagon, A; Possani, L; Schleuning, W D

1995-12-01

15

[New anticoagulants - direct thrombin inhibitors].  

UK PubMed Central (United Kingdom)

Direct thrombin-inhibitors inactivate not only free but also fibrin-bound thrombin. The group of parenteral direct thrombin-inhibitors includes the recombinant hirudins lepirudin and desirudin, the synthetic hirudin bivalirudin, and the small molecule argatroban. All these compounds do not interact with PF4/heparin-antibodies. Therefore, argatroban as well as bivalirudin are currently used to treat heparin-induced thrombocytopenia (HIT). The oral direct thrombin-inhibitor dabigatran etexilate is already licensed in many countries for the treatment of non-valvular atrial fibrillation. Dabigatran etexilate reveals a stable and predictable effect that allows a medication without dose adjustment or monitoring. The substance shows only few interactions with other drugs but strong inhibitors of p-glycoprotein can increase plasma levels of dabigatran substantially. After oral intake, the prodrug dabigatran etexilate is cleaved by esterase-mediated hydrolyses to the active compound dabigatran. Elimination of dabigatran is predominantly renal. Safety and efficacy of dabigatran etexilate were tested in an extensive clinical study program. Non-inferiority compared to current standard treatments was shown for prophylaxis of venous thromboembolic events after total knee and hip replacement, for stroke prevention in atrial fibrillation, and for treatment of acute venous thromboembolism. In daily practice, Dabigatran etexilate competes against the new direct factor Xa-inhibitors. In the absence of direct comparative clinical trials, it is not yet clear if one class of substances has distinct advantages over the other.

Brand B; Graf L

2012-11-01

16

[New anticoagulants - direct thrombin inhibitors].  

Science.gov (United States)

Direct thrombin-inhibitors inactivate not only free but also fibrin-bound thrombin. The group of parenteral direct thrombin-inhibitors includes the recombinant hirudins lepirudin and desirudin, the synthetic hirudin bivalirudin, and the small molecule argatroban. All these compounds do not interact with PF4/heparin-antibodies. Therefore, argatroban as well as bivalirudin are currently used to treat heparin-induced thrombocytopenia (HIT). The oral direct thrombin-inhibitor dabigatran etexilate is already licensed in many countries for the treatment of non-valvular atrial fibrillation. Dabigatran etexilate reveals a stable and predictable effect that allows a medication without dose adjustment or monitoring. The substance shows only few interactions with other drugs but strong inhibitors of p-glycoprotein can increase plasma levels of dabigatran substantially. After oral intake, the prodrug dabigatran etexilate is cleaved by esterase-mediated hydrolyses to the active compound dabigatran. Elimination of dabigatran is predominantly renal. Safety and efficacy of dabigatran etexilate were tested in an extensive clinical study program. Non-inferiority compared to current standard treatments was shown for prophylaxis of venous thromboembolic events after total knee and hip replacement, for stroke prevention in atrial fibrillation, and for treatment of acute venous thromboembolism. In daily practice, Dabigatran etexilate competes against the new direct factor Xa-inhibitors. In the absence of direct comparative clinical trials, it is not yet clear if one class of substances has distinct advantages over the other. PMID:23117667

Brand, B; Graf, L

2012-11-01

17

Thrombin activity resides on LVAD Dacron inflow and outflow grafts.  

UK PubMed Central (United Kingdom)

Patients on left ventricular assist devices (LVADs) are at increased risk for thromboembolism, and they experience elevations in platelet release and thrombin activity indices during device implantation. The Dacron grafts that lead from the ventricle to the LVAD and back to the aorta may harbor thrombin activity and protect this thrombin from anticoagulant action. To investigate this possibility, specimens isolated from LVAD grafts at the time of device explantation or implantation were incubated with citrated platelet poor plasma (cPPP), cPPP and 2 U/ml of heparin, cPPP and 2 U/ml of recombinant hirudin (r-hirudin), or cPPP and 50 U/ml of heparin. Thrombin activity was measured by the increase in incubated cPPP fibrinopeptide A (FPA) concentrations over cPPP FPA levels, without material contact. A chromogenic substrate for thrombin verified the effects seen. Thrombin activity was found at comparable levels on both the inflow and outflow LVAD grafts at explantation. This activity was not inhibited by low concentrations of heparin, but 50 U/ml of heparin and 2 U/ml of r-hirudin reduced the activity. At graft implantation (after preclotting), thrombin activity was higher than at explantation, but susceptibility to inhibition by heparin was also significantly greater. These results confirm that the LVAD Dacron grafts harbor surface thrombin activity resistant to anticoagulation that may be a primary source for LVAD thrombogenicity.

Wagner WR; Johnson PC; Heil BV; Thompson KA; Kormos RL; Griffith BP

1992-07-01

18

Thrombin activity resides on LVAD Dacron inflow and outflow grafts.  

Science.gov (United States)

Patients on left ventricular assist devices (LVADs) are at increased risk for thromboembolism, and they experience elevations in platelet release and thrombin activity indices during device implantation. The Dacron grafts that lead from the ventricle to the LVAD and back to the aorta may harbor thrombin activity and protect this thrombin from anticoagulant action. To investigate this possibility, specimens isolated from LVAD grafts at the time of device explantation or implantation were incubated with citrated platelet poor plasma (cPPP), cPPP and 2 U/ml of heparin, cPPP and 2 U/ml of recombinant hirudin (r-hirudin), or cPPP and 50 U/ml of heparin. Thrombin activity was measured by the increase in incubated cPPP fibrinopeptide A (FPA) concentrations over cPPP FPA levels, without material contact. A chromogenic substrate for thrombin verified the effects seen. Thrombin activity was found at comparable levels on both the inflow and outflow LVAD grafts at explantation. This activity was not inhibited by low concentrations of heparin, but 50 U/ml of heparin and 2 U/ml of r-hirudin reduced the activity. At graft implantation (after preclotting), thrombin activity was higher than at explantation, but susceptibility to inhibition by heparin was also significantly greater. These results confirm that the LVAD Dacron grafts harbor surface thrombin activity resistant to anticoagulation that may be a primary source for LVAD thrombogenicity. PMID:1457938

Wagner, W R; Johnson, P C; Heil, B V; Thompson, K A; Kormos, R L; Griffith, B P

19

Leech thrombin inhibitors.  

UK PubMed Central (United Kingdom)

Serine proteases (SP), such as thrombin, factor Xa, elastase, trypsin are implicated in many clinical disorders such as emphysema, arthritis and cardiovascular diseases. These enzymes, in normal physiological conditions, are regulated by naturally occurring serine protease inhibitors, such as anti-thrombin III involved in thromb in inhibition. Primitive parasitic invertebrates have co-evolved highly specific mechanisms to communicate with their hosts for survival purposes, by blocking host processes such as blood coagulation. Thus a battery of new powerful molecules from blood-sucker animals acting at different points of the coagulation cascade such like factor Xa, thrombin, platelets aggregation inhibitors have been isolated and are now at a clinical level. In this review, we focus our attention on thrombin inhitors.

Salzet M

2002-01-01

20

Leech thrombin inhibitors.  

Science.gov (United States)

Serine proteases (SP), such as thrombin, factor Xa, elastase, trypsin are implicated in many clinical disorders such as emphysema, arthritis and cardiovascular diseases. These enzymes, in normal physiological conditions, are regulated by naturally occurring serine protease inhibitors, such as anti-thrombin III involved in thromb in inhibition. Primitive parasitic invertebrates have co-evolved highly specific mechanisms to communicate with their hosts for survival purposes, by blocking host processes such as blood coagulation. Thus a battery of new powerful molecules from blood-sucker animals acting at different points of the coagulation cascade such like factor Xa, thrombin, platelets aggregation inhibitors have been isolated and are now at a clinical level. In this review, we focus our attention on thrombin inhitors. PMID:11945154

Salzet, Michel

2002-01-01

 
 
 
 
21

Thrombin clotting time.  

UK PubMed Central (United Kingdom)

Thrombin clotting time (TCT) is a coagulation assay used to diagnose congenital and acquired fibrinogen deficiency (Adcock et al., Coagulation handbook, Esoterix Coagulation, Austin, TX, 2002), as well as to identify contamination by heparin, prior to performing additional coagulation assays. This test is based on the principle that in citrated plasma, the addition of Thrombin allows for formation of a stable clot. The time required for the formation of a stable clot is recorded in seconds and represents the actual TCT result.

Ignjatovic V

2013-01-01

22

Effects of topical ocular administration of high doses of human recombinant interferon alpha-2b and feline recombinant interferon omega on naturally occurring viral keratoconjunctivitis in cats.  

UK PubMed Central (United Kingdom)

OBJECTIVE: To determine whether 14-day topical ocular administration of high doses of feline recombinant interferon omega (FelFN) or human recombinant interferon alpha-2b (HulFN) solution improves clinical disease and decreases virus shedding in cats with naturally acquired viral keratoconjunctivitis. ANIMALS: 36 cats with upper respiratory tract disease and ocular involvement. PROCEDURES: Cats received 1 drop of FelFN solution (1 × 10(6) U/mL), HulFN solution (1 × 10(6) U/mL), or saline (0.9% NaCl) solution (12 cats/group) in each eye twice daily for 14 days (beginning day 1). Oropharyngeal and conjunctival swab samples were collected from each cat before (day 0) and on day 14 of treatment for virus isolation (VI) and real-time quantitative PCR (RT-qPCR) testing to detect feline herpesvirus-1 and feline calicivirus. Subjective clinical scores were recorded on days 0, 3, 7, 10, and 14. RESULTS: The number of cats for which feline herpesvirus-1 was detected via VI or RT-qPCR assay was generally (albeit not always significantly) lower on day 14, compared with day 0 findings; however, findings on days 0 or 14 did not differ among groups. The number of cats for which feline calicivirus was detected via VI or RT-qPCR assay did not differ significantly between days 0 and 14 for any group. Clinical scores significantly decreased over the 14-day period but did not differ among groups. CONCLUSIONS AND CLINICAL RELEVANCE: In cats with naturally occurring viral keratoconjunctivitis, bilateral ocular administration of high doses of FelFN or HulFN twice daily for 14 days did not improve clinical disease or virus shedding, compared with treatment with saline solution.

Slack JM; Stiles J; Leutenegger CM; Moore GE; Pogranichniy RM

2013-02-01

23

Thrombin clotting time.  

Science.gov (United States)

Thrombin clotting time (TCT) is a coagulation assay used to diagnose congenital and acquired fibrinogen deficiency (Adcock et al., Coagulation handbook, Esoterix Coagulation, Austin, TX, 2002), as well as to identify contamination by heparin, prior to performing additional coagulation assays. This test is based on the principle that in citrated plasma, the addition of Thrombin allows for formation of a stable clot. The time required for the formation of a stable clot is recorded in seconds and represents the actual TCT result. PMID:23546710

Ignjatovic, Vera

2013-01-01

24

Recombiner  

International Nuclear Information System (INIS)

Burnable gases in a reactor container are delivered by a blower, and oxygen and hydrogen in the burnable gases are heated by a heater. Oxygen and hydrogen are combined by a recombiner into steams, and a gas/liquid two phase flow during the steam formation is cooled by a cooler. Then, the gas/liquid two phase flow is separated by a gas/liquid separator, and liquefied water is introduced into a suppression chamber to control the concentration of the burnable gases released into the container. In this case, dynamic equipments such as a blower, a cooler and a gas/liquid separator in the recombiner are disposed fixedly, and static equipments, i.e., only the portions of the heater and the recombiner are combined integrally so as to be portable. With such a constitution, complexity due to common use of the portion is mitigated without degradation of reliability, and operation for the maintenance and handling can be conducted flexibly. (T.M.).

1990-12-25

25

Autoactivation of thrombin precursors.  

UK PubMed Central (United Kingdom)

Trypsin-like proteases are synthesized as inactive zymogens and convert to the mature form upon activation by specific enzymes, often assisted by cofactors. Central to this paradigm is that the zymogen does not convert spontaneously to the mature enzyme, which in turn does not feed back to activate its zymogen form. In the blood, the zymogens prothrombin and prethrombin-2 require the prothrombinase complex to be converted to the mature protease thrombin, which is unable to activate prothrombin or prethrombin-2. Here, we show that replacement of key residues within the activation domain causes these zymogens to spontaneously convert to thrombin. The conversion is started by the zymogen itself, which is capable of binding ligands at the active site, and is abrogated by inactivation of the catalytic residue Ser-195. The product of autoactivation is functionally and structurally equivalent to wild-type thrombin. Zymogen autoactivation is explained by conformational selection, a basic property of the trypsin fold uncovered by structural and rapid kinetics studies. Both the zymogen and protease undergo a pre-existing equilibrium between active and inactive forms. The equilibrium regulates catalytic activity in the protease and has the potential to unleash activity in the zymogen to produce autoactivation. A new strategy emerges for the facile production of enzymes through zymogen autoactivation that is broadly applicable to trypsin-like proteases of biotechnological and clinical interest.

Pozzi N; Chen Z; Zapata F; Niu W; Barranco-Medina S; Pelc LA; Di Cera E

2013-04-01

26

Autoactivation of thrombin precursors.  

Science.gov (United States)

Trypsin-like proteases are synthesized as inactive zymogens and convert to the mature form upon activation by specific enzymes, often assisted by cofactors. Central to this paradigm is that the zymogen does not convert spontaneously to the mature enzyme, which in turn does not feed back to activate its zymogen form. In the blood, the zymogens prothrombin and prethrombin-2 require the prothrombinase complex to be converted to the mature protease thrombin, which is unable to activate prothrombin or prethrombin-2. Here, we show that replacement of key residues within the activation domain causes these zymogens to spontaneously convert to thrombin. The conversion is started by the zymogen itself, which is capable of binding ligands at the active site, and is abrogated by inactivation of the catalytic residue Ser-195. The product of autoactivation is functionally and structurally equivalent to wild-type thrombin. Zymogen autoactivation is explained by conformational selection, a basic property of the trypsin fold uncovered by structural and rapid kinetics studies. Both the zymogen and protease undergo a pre-existing equilibrium between active and inactive forms. The equilibrium regulates catalytic activity in the protease and has the potential to unleash activity in the zymogen to produce autoactivation. A new strategy emerges for the facile production of enzymes through zymogen autoactivation that is broadly applicable to trypsin-like proteases of biotechnological and clinical interest. PMID:23467412

Pozzi, Nicola; Chen, Zhiwei; Zapata, Fatima; Niu, Weiling; Barranco-Medina, Sergio; Pelc, Leslie A; Di Cera, Enrico

2013-03-06

27

Ristocetin and the thrombin clotting time.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The addition of the antibiotic ristocetin to plasma accelerated the thrombin clotting time (TCT) in 20 out of 22 subjects. Prior incubation of ristocetin with thrombin or plasma did not alter its effect on the TCT. Ristocetin accelerated clotting greatly at low but not at high levels of thrombin. A ...

Aronstam, A; Dennis, B; Friesen, M N; Clark, W F; Linton, A L; Lindsay, R M

28

Recombinant snake venom prothrombin activators.  

UK PubMed Central (United Kingdom)

Three prothrombin activators; ecarin, which was originally isolated from the venom of the saw-scaled viper Echis carinatus, trocarin from the rough-scaled snake Tropidechis carinatus, and oscutarin from the Taipan snake Oxyuranus scutellatus, were expressed in mammalian cells with the purpose to obtain recombinant prothrombin activators that could be used to convert prothrombin to thrombin. We have previously reported that recombinant ecarin can efficiently generate thrombin without the need for additional cofactors, but does not discriminate non-carboxylated prothrombin from biologically active ?-carboxylated prothrombin. Here we report that recombinant trocarin and oscutarin could not efficiently generate thrombin without additional protein co-factors. We confirm that both trocarin and oscutarin are similar to human coagulation Factor X (FX), explaining the need for additional cofactors. Sequencing of a genomic fragment containing 7 out of the 8 exons coding for oscutarin further confirmed the similarity to human FX.

Lövgren A

2013-05-01

29

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

UK PubMed Central (United Kingdom)

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 ?M. 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 ?M for apixaban; 0.049 ± 0.007, 0.070 ± 0.009, 0.43 ± 0.07, 0.048 ± 0.008, and 0.022 ± 0.005 ?M for rivaroxaban; and 0.063 ± 0.019, 0.18 ± 0.06, 0.50 ± 0.08, 0.55 ± 0.06, and 0.57 ± 0.27 ?M 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 ?g/mL reversed the anticoagulant effects of apixaban more at 0.2 ?M than at 2 ?M. Our study suggests that the CAT method is a sensitive assay to monitor the pharmacodynamic and pharmacokinetic properties of apixaban, rivaroxaban, and dabigatran, and may provide insight into the mechanism of action of these inhibitors. Recombinant activated factor VII may have some potential to reverse the anticoagulant effects of apixaban in vitro.

Wong PC; White A; Luettgen J

2013-02-01

30

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

Science.gov (United States)

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 ?M. 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 ?M for apixaban; 0.049 ± 0.007, 0.070 ± 0.009, 0.43 ± 0.07, 0.048 ± 0.008, and 0.022 ± 0.005 ?M for rivaroxaban; and 0.063 ± 0.019, 0.18 ± 0.06, 0.50 ± 0.08, 0.55 ± 0.06, and 0.57 ± 0.27 ?M 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 ?g/mL reversed the anticoagulant effects of apixaban more at 0.2 ?M than at 2 ?M. Our study suggests that the CAT method is a sensitive assay to monitor the pharmacodynamic and pharmacokinetic properties of apixaban, rivaroxaban, and dabigatran, and may provide insight into the mechanism of action of these inhibitors. Recombinant activated factor VII may have some potential to reverse the anticoagulant effects of apixaban in vitro. PMID:23466964

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

2013-02-01

31

Zinc modulates thrombin adsorption to fibrin  

International Nuclear Information System (INIS)

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.

1990-01-01

32

Direct thrombin inhibitors in cardiovascular disease.  

UK PubMed Central (United Kingdom)

Limitations of commonly used anticoagulants, unfractionated heparin, low-molecular-weight heparin, and oral vitamin K antagonists have prompted the development of alternative therapies. Direct thrombin inhibitors are a new class of anticoagulants that bind directly to thrombin and inhibit its interaction with substrates. In this Review, we critically examine the evidence from randomized controlled trials for the efficacy and safety of the parenteral direct thrombin inhibitors bivalirudin and argatroban, and the novel oral direct thrombin inhibitor dabigatran etexilate, in cardiovascular and thrombotic disease.

Arsenault KA; Hirsh J; Whitlock RP; Eikelboom JW

2012-07-01

33

THERAPEUTIC GRADE THROMBIN PRODUCTION AND PRODUCTS  

UK PubMed Central (United Kingdom)

The invention provides an improved process for the large-scale production of therapeutic grade thrombin of excellent viral safety and storage-stability, comprising purification of viricide treated crude thrombin by ion-exchange chromatography on a single column using a sulfalkyl-activated polysaccharide, particularly sulfopropyl-Spherodex, as the ion exchange medium and increasing concentrations of phosphate buffer for elution. After recovery of thrombin in the final eluate, the phosphate buffer is exchanged for a stabilizing formulation buffer, and the stabilized thrombin is subjected to viral filtration and optional dry heat treatment for further viral inactivation. The final product has high specific activity and is obtained in good yield.

PROBA Zbigniew; BRODNIEWICZ Teresa; DUPUIS Nicolas; BUI-KHAC Trung

34

Aptamer Based Microsphere Biosensor for Thrombin Detection  

Directory of Open Access Journals (Sweden)

Full Text Available We have developed an optical microsphere resonator biosensor using aptamer asreceptor for the measurement of the important biomolecule thrombin. The sphere surface ismodified with anti-thrombin aptamer, which has excellent binding affinity and selectivityfor thrombin. Binding of the thrombin at the sphere surface is monitored by the spectralposition of the microsphere’s whispering gallery mode resonances. A detection limit on theorder of 1 NIH Unit/mL is demonstrated. Control experiments with non-aptameroligonucleotide and BSA are also carried out to confirm the specific binding betweenaptamer and thrombin. We expect that this demonstration will lead to the development ofhighly sensitive biomarker sensors based on aptamer with lower cost and higher throughputthan current technology.

Hongying Zhu; Jonathan D. Suter; Ian M. White; Xudong Fan

2006-01-01

35

[Direct oral thrombin inhibitor, "dabigatran"].  

UK PubMed Central (United Kingdom)

Dabigatran is an oral, direct, and competitive inhibitor of thrombin, which is administered to patients with non-valvular atrial fibrillation for prevention of stroke at a dose of 110 mg twice daily or 150 mg twice daily. Anticoagulation by dabigatran is "hybrid anticoagulation", consisting of action of both dabigatran and physiological coagulation inhibitors because warfarin inhibits production of protein C and protein S but dabigatran does not. Management of dabigatran is easier than that of warfarin because food restriction is unnecessary, drug interaction is small, and absorption time is short and serum concentration corresponds to the anticoagulatory effect in dabigatran treatment. The RE-LY trial confirmed effectiveness and safety of both doses of dabigatran for prevention of stroke and both doses of dabigatran had much lower risks of intracranial bleeding compared with warfarin. Compliance to guidance of dabigatran treatment is essential for avoidance of severe hemorrhagic complications.

Yasaka M

2013-01-01

36

The unresolved safety concerns of bovine thrombin  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract A recent review has suggested that bovine thrombin is not associated with an increased risk of bleeding in surgical populations. In spite of extremely limited evidence available, many valuable resources (e.g. safety surveillance and post-marketing programs, case reports) were excluded in reaching this conclusion. While waiting for the adequately powered, controlled clinical trials to address the effects of bovine thrombin on bleeding and thrombotic events, the potential risk cannot be simply ignored. Rather, continued vigilance in the post-surgical setting for bleeding events that may be associated with the development of acquired coagulation factor inhibitors following bovine thrombin administration is warranted.

Shander Aryeh; Javidroozi Mazyar

2008-01-01

37

Therapeutic grade thrombin produced by chromatography  

UK PubMed Central (United Kingdom)

A process for the large-scale production of therapeutic grade thrombin of excellent viral safety and storage-stability is carried out by purification of viricide treated crude thrombin by ion-exchange chromatography on a single column using a sulfalkyl-activated polysaccharide, particularly a non-compressible composite medium of sulfoalkyl-activated dextran and silica particles, as the ion exchange medium and using increasing concentrations of phosphate buffer for elution. After recovery of thrombin in the final eluate, the phosphate buffer is exchanged for a stabilizing formulation buffer, and the stabilized thrombin is subjected to viral filtration and optional dry heat treatment for further viral inactivation. The final product has high specific activity and is obtained in good yield.

PROBA ZBIGNIEW; BRODNIEWICZ TERESA

38

Low endogenous thrombin potential in trained subjects.  

UK PubMed Central (United Kingdom)

INTRODUCTION: A paradox seems to exist: exercising leads to clotting activation in conventional clotting tests, but exercising persons have a low risk of thrombosis. In this study we tried to evaluate the effect of exercise performance status on in vitro plasma thrombin generation, which represents an overall function test of hemostasis. MATERIALS AND METHODS: We compared 56 trained subjects to 98 healthy age matched sedentary volunteers. Blood samples were analyzed for thrombin generation using calibrated automated thrombography. Microparticles were quantified using ELISA. Additionally prothrombin fragments 1 + 2, thrombin-antithrombin complex, tissue factor pathway inhibitor, antithrombin and prothrombin were measured. The group of the trained subjects performed an incremental cycle-ergometer exercise test after taking the blood sample. RESULTS: A significantly lower endogenous thrombin potential was observed in the group of the trained subjects compared to the sedentary individuals (p = 0.007). Microparticles (ELISA) were significantly lower in the trained subjects compared to the sedentary subjects (p = 0.001). Prothrombin fragments 1 + 2 (p < 0.001) and thrombin-antithrombin complex (p = 0.01) were significant higher in the trained subjects and antithrombin (p = 0.02) as well as prothrombin (p < 0.0001) were significantly lower in this group, whereas tissue factor pathway inhibitor values did not show significant differences. Both maximal and submaximal power output was significantly negatively related to endogenous thrombin potential (r = -0.43, r = -0.45) and thrombin peak (r = -0.44, r = -0.42). CONCLUSIONS: Trained subjects have a lower endogenous thrombin potential than sedentary subjects possibly explaining the lower incidence of thrombosis in this group despite a higher acute clotting activation during strenuous exercise.

Cimenti C; Schlagenhauf A; Leschnik B; Schretter M; Tschakert G; Gröschl W; Seibert FJ; Hofmann P; Muntean WE

2013-06-01

39

Thrombin-aptamer recognition: a revealed ambiguity.  

UK PubMed Central (United Kingdom)

Aptamers are structured oligonucleotides that recognize molecular targets and can function as direct protein inhibitors. The best-known example is the thrombin-binding aptamer, TBA, a single-stranded 15-mer DNA that inhibits the activity of thrombin, the key enzyme of coagulation cascade. TBA folds as a G-quadruplex structure, as proved by its NMR structure. The X-ray structure of the complex between TBA and human ?-thrombin was solved at 2.9-Å resolution, but did not provide details of the aptamer conformation and the interactions with the protein molecule. TBA is rapidly processed by nucleases. To improve the properties of TBA, a number of modified analogs have been produced. In particular, a modified TBA containing a 5'-5' polarity inversion site, mTBA, has higher stability and higher affinity toward thrombin with respect to TBA, although it has a lower inhibitory activity. We present the crystal structure of the thrombin-mTBA complex at 2.15-Å resolution; the resulting model eventually provides a clear picture of thrombin-aptamers interaction, and also highlights the structural bases of the different properties of TBA and mTBA. Our findings open the way for a rational design of modified aptamers with improved potency as anticoagulant drugs.

Russo Krauss I; Merlino A; Giancola C; Randazzo A; Mazzarella L; Sica F

2011-09-01

40

Thrombin-aptamer recognition: a revealed ambiguity.  

Science.gov (United States)

Aptamers are structured oligonucleotides that recognize molecular targets and can function as direct protein inhibitors. The best-known example is the thrombin-binding aptamer, TBA, a single-stranded 15-mer DNA that inhibits the activity of thrombin, the key enzyme of coagulation cascade. TBA folds as a G-quadruplex structure, as proved by its NMR structure. The X-ray structure of the complex between TBA and human ?-thrombin was solved at 2.9-Å resolution, but did not provide details of the aptamer conformation and the interactions with the protein molecule. TBA is rapidly processed by nucleases. To improve the properties of TBA, a number of modified analogs have been produced. In particular, a modified TBA containing a 5'-5' polarity inversion site, mTBA, has higher stability and higher affinity toward thrombin with respect to TBA, although it has a lower inhibitory activity. We present the crystal structure of the thrombin-mTBA complex at 2.15-Å resolution; the resulting model eventually provides a clear picture of thrombin-aptamers interaction, and also highlights the structural bases of the different properties of TBA and mTBA. Our findings open the way for a rational design of modified aptamers with improved potency as anticoagulant drugs. PMID:21715374

Russo Krauss, Irene; Merlino, Antonello; Giancola, Concetta; Randazzo, Antonio; Mazzarella, Lelio; Sica, Filomena

2011-06-28

 
 
 
 
41

A high affinity, antidote-controllable prothrombin and thrombin-binding RNA aptamer inhibits thrombin generation and thrombin activity.  

UK PubMed Central (United Kingdom)

BACKGROUND: The conversion of prothrombin to thrombin is one of two non-duplicated enzymatic reactions during coagulation. Thrombin has long been considered an optimal anticoagulant target because it plays a crucial role in fibrin clot formation by catalyzing the cleavage of fibrinogen, upstream coagulation cofactors and platelet receptors. Although a number of anti-thrombin therapeutics exist, it is challenging to use them clinically due to their propensity to induce bleeding. Previously, we isolated a modified RNA aptamer (R9D-14) that binds prothrombin with high affinity and is a potent anticoagulant in vitro. OBJECTIVES: We sought to explore the structure of R9D-14 and elucidate its anticoagulant mechanism(s). In addition to designing an optimized aptamer (RNA(R9D-14T)), we also explored whether complementary antidote oligonucleotides can rapidly modulate the optimized aptamer's anticoagulant activity. METHODS AND RESULTS: RNA(R9D-14T) binds prothrombin and thrombin pro/exosite I with high affinity and inhibits both thrombin generation and thrombin exosite I-mediated activity (i.e. fibrin clot formation, feedback activity and platelet activation). RNA(R9D-14T) significantly prolongs the aPTT, PT and TCT clotting assays, and is a more potent inhibitor than the thrombin exosite I DNA aptamer ARC-183. Moreover, a complementary oligonucleotide antidote can rapidly (< 2 min) and durably (>2 h) reverse RNA(R9D-14T) anticoagulation in vitro. CONCLUSIONS: Powerful anticoagulation, in conjunction with antidote reversibility, suggests that RNA(R9D-14T) may be ideal for clinical anticoagulation in settings that require rapid and robust anticoagulation, such as cardiopulmonary bypass, deep vein thrombosis, stroke or percutaneous coronary intervention.

Bompiani KM; Monroe DM; Church FC; Sullenger BA

2012-05-01

42

Thrombin activity associated with neuronal damage during acute focal ischemia.  

UK PubMed Central (United Kingdom)

Mechanisms of ischemic neuronal and vascular injury remain obscure. Here we test the hypothesis that thrombin, a blood-borne coagulation factor, contributes to neurovascular injury during acute focal ischemia. Stroke was induced in adult Sprague Dawley rats by occluding the middle cerebral artery. Intra-arterial thrombin infusion during ischemia significantly increased vascular disruption and cellular injury. Intravenous infusion of argatroban, a direct thrombin inhibitor, alleviated neurovascular injury. Immunostaining showed thrombin on neurons in the ischemic core. Using an activatable cell-penetrating peptide engineered to detect thrombin activity, we discovered that thrombin proteolytic activity was specifically associated with neuronal damage during ischemia. Protease activated receptor-1, the presumptive thrombin receptor, appeared to mediate ischemic neurovascular injury. Furthermore, rats receiving thrombin during ischemia showed cognitive deficit, whereas rats receiving argatroban retained intact learning and memory. These results suggest a potential role for thrombin contributing to neurovascular injury and several potential avenues for neuroprotection.

Chen B; Friedman B; Whitney MA; Winkle JA; Lei IF; Olson ES; Cheng Q; Pereira B; Zhao L; Tsien RY; Lyden PD

2012-05-01

43

A monoclonal antibody against bovine thrombin reacting to the C-terminal side of thrombin.  

UK PubMed Central (United Kingdom)

We succeeded in producing a monoclonal antibody (MAb) against bovine thrombin. The MAb belonged to mouse IgG(1), and its light chain consisted of kappa-chain. The MAb reacted with bovine and human thrombins, which were coated by coupling to poly-lysine-coated wells with glutaraldehyde, but did not react with the thrombin-like enzyme, habutobin. Furthermore, the MAb did not react with thrombin which was coated to plates without poly-lysine and glutaraldehyde. The concentration of thrombin in ovalbumin solution (10 mg/mL) could be measured by means of the enzyme-linked immunosorbent assay (ELISA) double sandwich method using the MAb and polyclonal antibody. Thrombin added to defibrinated plasma could not be detected by means of the ELISA double sandwich method using the present MAb, and this may be due to the AT-III activity in the defibrinated plasma. Postclotting thrombin could be detected by means of the ELISA-double sandwich method using the MAb. It is suggested, from the results of our experiments, that the MAb obtained reacted in a limited fashion to the C-terminal of bovine thrombin.

Moriyama T; Nakamura M; Kinjoh K; Tanaka T; Kosugi T

2001-01-01

44

Thrombin inhibition profiles in healthy individuals and thrombophilic patients.  

UK PubMed Central (United Kingdom)

Inhibition of thrombin by endogenous inhibitors plays a central role in the spatiotemporal control of clot formation. A failure to adequately inactivate thrombin such as in antithrombin deficiency generates a strong prothrombotic phenotype. To study if and to what extent delayed thrombin inactivation rates beyond antithrombin deficiency contribute to the prothrombotic phenotype we measured thrombin inhibition profiles in plasma samples obtained from 16 healthy individuals and 39 thrombophilic patients, including 17 patients diagnosed positive for anti-prothrombin/phospholipid antibodies. To test thrombin inhibition, thrombin was added to plasma, and endogenous thrombin inhibition stopped by addition of the reversible thrombin inhibitor argatroban. Subsequently, the amount of argatroban-complexed thrombin was measured using an oligonucleotide-based enzyme capture assay. In normal human plasma thrombin at concentrations up to 4 ng/ml (109 pM) became inactivated with an average half-life time of 56.4 ± 4.7 seconds (s). In antithrombin-deficient plasma the thrombin half-life was prolonged to 168.2 ± 14.9 s. Among the thrombophilic patients, only one with mild antithrombin deficiency showed impaired thrombin inactivation rates, whereas all other patients including the antiphospholipid positive patients showed thrombin inhibiting capacities within the normal range. We conclude that thrombin added to normal human plasma at subthreshold levels of ~100 pM or below becomes inactivated with a half-life time below 1 minute. Antiphospholipid antibodies do not prolong thrombin half-life times, making it unlikely that delayed thrombin inactivation contributes to the thrombotic phenotype of the antiphospholipid syndrome. In contrast, plasma levels of antithrombin falling below 80% of normal markedly prolong the thrombin half-life.

Rühl H; Müller J; Harbrecht U; Fimmers R; Oldenburg J; Mayer G; Pötzsch B

2012-05-01

45

Characteristics of a new DNA aptamer, direct inhibitor of thrombin.  

UK PubMed Central (United Kingdom)

Characteristics of a new antithrombin DNA-aptamer RE31 were studied. This aptamer inhibited thrombin formation in human plasma catalyzed by exogenous (lengthening of thrombin time) and endogenous thrombin (lengthening of partial prothrombin time and activated partial thromboplastin time). In addition, the aptamer completely suppressed thrombin-induced aggregation of human platelets. On the other hand, RE31 did not reduce amidolytic activity of thrombin towards the short peptide substrate, in other words, did not modify the state of enzyme active center. By the capacity to inhibit clotting reactions, RE31 was superior to the previously described highly effective 31-component antithrombin aptamer 31TBA (thrombin binding aptamer, TBA). The effect of RE31 was species-specific: it inhibited human thrombin activity more effectively than activities of rat and rabbit thrombins.

Mazurov AV; Titaeva EV; Khaspekova SG; Storojilova AN; Spiridonova VA; Kopylov AM; Dobrovolsky AB

2011-02-01

46

Topical Use of Recombinant Human Epidermal Growth Factor (EGF)-Based Cream to Prevent Radiation Dermatitis in Breast Cancer Patients: a Single-Blind Randomized Preliminary Study.  

UK PubMed Central (United Kingdom)

Background: The purpose of this study was to assess the effectiveness of a recombinant human epidermal growth factor (EGF)-based cream for the prevention of acute radiation dermatitis in breast cancer patients receiving radiotherapy (RT). Materials and Methods: Between December 2012 and April 2013, 40 breast cancer patients who received postoperative RT were prospectively enrolled in this study and randomly assigned to receive human recombinant EGF-based cream (intervention group) or general supportive skin care (control group). The grade of radiation dermatitis and pain score were examined at weekly intervals during RT and 6 weeks after RT completion. Results: All patients completed the planned RT and complied well with instructions for applying the study cream and general supportive skin care. In the intervention group, radiation dermatitis of maximum grade 3, 2, and 1 developed in 3 (15%), 11 (55%), and 6 patients (30%), respectively. In comparison, in the control group, radiation dermatitis of maximum grade 3, 2, and 1 developed in 8 (40%), 10 (50%), and 2 patients (10%), respectively. The intervention group showed lower incidence of grade 3 radiation dermatitis than the control group (p=0.068 in univariate analysis and p=0.035 in multivariate analysis). There was no statistically significant difference in the maximal pain score between the two groups (p=0.934). Conclusions: This single-blind randomized preliminary study showed that recombinant human EGF-based cream can have a beneficial role in preventing or minimizing radiation dermatitis in breast cancer patients. To confirm the results of our study, additional studies with a large sample size are required.

Kong M; Hong SE

2013-01-01

47

Acyclovir Topical  

Science.gov (United States)

local topical ... Topical acyclovir comes as a cream and an ointment to apply to the skin. Acyclovir cream is ... explain any part you do not understand. Use topical acyclovir exactly as directed. Do not use more ...

48

Bexarotene Topical  

Science.gov (United States)

Targretin® Topical Gel ... Topical bexarotene is used to treat cutaneous T-cell lymphoma (CTCL, a type of skin cancer) that ... Topical bexarotene comes as a gel to apply to the skin. It is usually applied once every ...

49

Increased thrombin generation after acute versus chronic coronary disease as assessed by the thrombin generation test.  

Science.gov (United States)

Atherosclerosis is the most common pathophysiologic substrate of coronary artery disease (CAD). Whereas plaque progression and arterial remodeling are critical components in chronic CAD, intracoronary thrombosis over plaque disruption is causally related to acute CAD. It was the objective of this study to investigate the differences between prior acute CAD and chronic CAD by a simple global coagulation assay measuring thrombin generation. A cross-sectional study involving 15 healthy controls, 35 patients with chronic stable CAD, and 60 patients after an episode of acute myocardial infarction (AMI) was performed. Thrombin generation was measured between three and 11 months after the initial diagnosis (mean 6 months) by a commercially available fluorogenic assay (Technothrombin TGA). In each patient the lag phase, velocity index and peak thrombin were obtained from the thrombogram profile. Traditional cardiovascular risk factors were recorded, and the inflammatory markers, fibrinogen and hs-C-reactive protein were determined. Compared with stable CAD patients, showing normal thrombograms, those with previous AMI showed earlier lag phase (p < 0.05) and significant increase of both the velocity index (p < 0.001) and peak thrombin (p < 0.05), indicating faster and higher thrombin generation in the AMI group. Differences in thrombin generation between stable and acute CAD patients remained significant (p < 0.001) after adjusting for conventional CAD risk factors (age, gender, diabetes, hypertension, smoking, and hypercholesterolemia). In conclusion, patients with a previous history of acute CAD showed earlier, faster and higher thrombin generation than stable chronic CAD patients. The thrombin generation test may be of clinical value to monitor hypercoagulable/vulnerable blood and/or guide therapy in CAD. PMID:18278189

Orbe, Josune; Zudaire, Maite; Serrano, Rosario; Coma-Canella, Isabel; Martínez de Sizarrondo, Sara; Rodríguez, Jose A; Páramo, Jose A

2008-02-01

50

Label-free sensing of thrombin based on quantum dots and thrombin binding aptamer.  

UK PubMed Central (United Kingdom)

A facile and sensitive label-free approach for detection of thrombin based on CdTe quantum dots (QDs) and thrombin binding aptamer (TBA) is presented. The crude QDs can be "activated" with fluorescence enhancement by adding extra Cd(2+) to the solution in basic medium. As a result, the positively charged Cd(2+)-activating CdTe QDs could interact with the negatively charged TBA, leading to fluorescence quenching. When thrombin was added, TBA was induced to form a G-quadruplex structure and combined specifically with its target, releasing the QDs with a recovery of the fluorescence intensity. The sensing approach is based on the strongly specific interactions between TBA and thrombin over the electrostatic interactions between TBA and positively charged QDs. Based on the fluorescence enhancement of QDs, selective detection of thrombin was successfully achieved. A linear response for thrombin was observed in the range from 1.4 nM to 21 nM with a detection limit of 0.70 nM.

Zhang X; Hu R; Shao N

2013-03-01

51

Thrombin in myocardial ischemia-reperfusion during cardiac surgery.  

Science.gov (United States)

Thrombin is a multifunctional protease with procoagulant, pro-inflammatory, and pro-apoptotic effects. Thrombin has direct potentially adverse effects on the endothelium and on cardiomyocytes, which are independent of its procoagulant effects, and it has emerged as a possible mediator of ischemia-reperfusion injury. Several lines of experimental evidence specifically implicate thrombin to be involved in myocardial ischemia-reperfusion injury. Cardiopulmonary bypass increases thrombin generation progressively, but reperfusion after myocardial ischemia induces an additional distinct and rapid increase in thrombin generation. Clinical studies have shown that thrombin formation during cardiac surgery, especially during myocardial reperfusion, is involved with myocardial damage and impaired hemodynamic recovery. Therefore, strategies to improve thrombin control during cardiopulmonary bypass might be beneficial. PMID:19559265

Raivio, Peter; Lassila, Riitta; Petäjä, Jari

2009-07-01

52

Inhibition of thrombin activity with DNA-aptamers.  

UK PubMed Central (United Kingdom)

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.

Dobrovolsky AB; Titaeva EV; Khaspekova SG; Spiridonova VA; Kopylov AM; Mazurov AV

2009-07-01

53

Hemalin, a thrombin inhibitor isolated from a midgut cDNA library from the hard tick Haemaphysalis longicornis.  

UK PubMed Central (United Kingdom)

A full-length sequence of a thrombin inhibitor (designated as hemalin) from the midgut of parthenogenetic Haemaphysalis longicornis has been identified. Sequence analysis shows that this gene belongs to the Kunitz-type family, containing two Kunitz domains with high homology to boophilin, the thrombin inhibitor from Rhipicephalus (Boophilus) microplus. The recombinant protein expressed in insect cells delayed bovine plasma clotting time and inhibited both thrombin-induced fibrinogen clotting and platelet aggregation. A 20-kDa protein was detected from the midgut lysate with antiserum against recombinant hemalin. The gene is expressed at all stages of the tick except for the egg stage, and hemalin mRNA mainly in the midgut of the female adult tick. Real-time PCR analysis shows that this gene has a distinctly high expression level in the rapid bloodsucking period of the larvae, nymphs, and adults. Disruption of the hemalin gene by RNA interference led to a 2-day extension of the tick blood feeding period, and 27.7% of the RNA-treated ticks did not successfully complete the blood feeding. These findings indicate that the newly identified thrombin inhibitor from the midgut of H. longicornis might play an important role in tick blood feeding.

Liao M; Zhou J; Gong H; Boldbaatar D; Shirafuji R; Battur B; Nishikawa Y; Fujisaki K

2009-02-01

54

Hemalin, a thrombin inhibitor isolated from a midgut cDNA library from the hard tick Haemaphysalis longicornis.  

Science.gov (United States)

A full-length sequence of a thrombin inhibitor (designated as hemalin) from the midgut of parthenogenetic Haemaphysalis longicornis has been identified. Sequence analysis shows that this gene belongs to the Kunitz-type family, containing two Kunitz domains with high homology to boophilin, the thrombin inhibitor from Rhipicephalus (Boophilus) microplus. The recombinant protein expressed in insect cells delayed bovine plasma clotting time and inhibited both thrombin-induced fibrinogen clotting and platelet aggregation. A 20-kDa protein was detected from the midgut lysate with antiserum against recombinant hemalin. The gene is expressed at all stages of the tick except for the egg stage, and hemalin mRNA mainly in the midgut of the female adult tick. Real-time PCR analysis shows that this gene has a distinctly high expression level in the rapid bloodsucking period of the larvae, nymphs, and adults. Disruption of the hemalin gene by RNA interference led to a 2-day extension of the tick blood feeding period, and 27.7% of the RNA-treated ticks did not successfully complete the blood feeding. These findings indicate that the newly identified thrombin inhibitor from the midgut of H. longicornis might play an important role in tick blood feeding. PMID:19061894

Liao, Min; Zhou, Jinlin; Gong, Haiyan; Boldbaatar, Damdinsuren; Shirafuji, Rika; Battur, Banzragch; Nishikawa, Yoshifumi; Fujisaki, Kozo

2008-12-25

55

A thrombin inhibitor from the gut of Boophilus microplus ticks.  

Science.gov (United States)

A thrombin inhibitor was identified for the first time in the gut of the cattle tick Boophilus microplus. Here we present the partial purification and characterization of this new molecule, which was purified from the gut extract by three chromatographic steps: ion-exchange, gel filtration and affinity chromatography in a thrombin-Sepharose resin. In SDS-PAGE the inhibitor showed an apparent molecular mass of circa 26 kDa, which is different from the two thrombin inhibitors present in the saliva of this tick. The new inhibitor delays bovine plasma clotting time and inhibits both thrombin induced fibrinogen clotting and thrombin induced platelet aggregation. However, it does not interfere with thrombin amidolytic activity upon a small substrate (H-D-Phe-Pip-Arg-para-nitroanilide), which does not require binding to thrombin exosites. Therefore, the inhibitor does not block thrombin active site, although it must interfere with one of the thrombin exosites. B. microplus gut thrombin inhibitor (BmGTI) is also capable of enhancing activated protein C (APC) activity upon its specific substrate (H-D-Glu-Pro-Arg-para-nitroanilide), an activity never described before among B. microplus molecules. PMID:17710557

Ricci, Clarisse Gravina; Pinto, Antônio Frederico Michel; Berger, Markus; Termignoni, Carlos

2007-08-21

56

Bimatoprost Topical  

Science.gov (United States)

Topical bimatoprost is used to treat hypotrichosis (less than the normal amount of hair) of the eyelashes ... the growth of longer, thicker, and darker lashes. Topical bimatoprost is in a class of medications called ...

57

Clindamycin Topical  

Science.gov (United States)

Topical clindamycin is used to treat acne. Clindamycin is in a class of medications called lincomycin antibiotics. ... Topical clindamycin comes as a foam, a gel, a solution (liquid), a lotion, and a pledget (swab) ...

58

Estradiol Topical  

Science.gov (United States)

Estradiol topical gel and emulsion (lotion type mixture) are used to treat and prevent hot flushes (hot flashes; sudden ... life; the end of monthly menstrual periods). Estradiol topical gel is also used to treat vaginal dryness, ...

59

Testosterone Topical  

Science.gov (United States)

Testosterone topical gel is used to treat the symptoms of low testosterone in men who do not produce enough ... Topical testosterone comes as a gel to apply to the skin. It is usually applied once a ...

60

Oxybutynin Topical  

Science.gov (United States)

Oxybutynin topical gel is used to control frequent urination, urgent need to urinate, and urge urinary incontinence (sudden strong ... Topical oxybutynin comes as a gel to apply to the skin. It is usually applied once a ...

 
 
 
 
61

Fluorouracil Topical  

Science.gov (United States)

Fluorouracil cream and topical solution are used to treat actinic or solar keratoses (scaly or crusted lesions [ ... by years of too much exposure to sunlight). Fluorouracil cream and topical solution are also used to ...

62

Tretinoin Topical  

Science.gov (United States)

local topical ... Tretinoin comes in topical liquid, cream, and gel. Tretinoin usually is used daily at bedtime or once every 2 or 3 days. Follow ... nonmedicated cosmetics on cleansed skin. Do not use topical preparations with a lot of alcohol, menthol, spices, ...

63

Aptamer/thrombin/aptamer-AuNPs sandwich enhanced surface plasmon resonance sensor for the detection of subnanomolar thrombin.  

Science.gov (United States)

A sensitive and selective aptamer/thrombin/aptamer-AuNPs sandwich enhanced surface plasmon resonance (SPR) sensor has been developed for real-time detection of subnanomolar thrombin. In this protocol, one thiol-modified thrombin aptamer (TBA29) was immobilized on gold nanoparticles (AuNPs) via Au-S bonding. The other biotinylated thrombin aptamer (TBA15) was grafted onto streptavidin pretreated SPR gold film through biotin-streptavidin recognition. The presence of thrombin would then induce the formation of a double aptamer sandwich structure on the SPR gold film and results in obvious enhancement of SPR signal, which was proportional to the concentration of thrombin. This proposed assay took advantage of sandwich binding of two affinity aptamers for increased specificity, AuNPs for signal enhancement, as well as SPR signal readout for real-time detection. The SPR signal had a good linear relationship with thrombin concentration in the range of 0.1-75nM, and the detection limit for thrombin was determined to be as low as 0.1nM. It was found that aptamer functionalized AuNPs enhanced the signal of SPR response and thus increased the limit of detection 4-fold and 5-fold compared to direct detection format without AuNPs. This sensor also showed good selectivity for thrombin without being affected by some other proteins, such as BSA and lysozyme. Furthermore, this proposed SPR sensing platform was successfully applied to thrombin analysis in diluted human serum samples. PMID:23584389

Bai, Yunfeng; Feng, Feng; Zhao, Lu; Wang, Chunyu; Wang, Haiyan; Tian, Maozhong; Qin, Jun; Duan, Yali; He, Xiaoxiao

2013-03-20

64

Aptamer/thrombin/aptamer-AuNPs sandwich enhanced surface plasmon resonance sensor for the detection of subnanomolar thrombin.  

UK PubMed Central (United Kingdom)

A sensitive and selective aptamer/thrombin/aptamer-AuNPs sandwich enhanced surface plasmon resonance (SPR) sensor has been developed for real-time detection of subnanomolar thrombin. In this protocol, one thiol-modified thrombin aptamer (TBA29) was immobilized on gold nanoparticles (AuNPs) via Au-S bonding. The other biotinylated thrombin aptamer (TBA15) was grafted onto streptavidin pretreated SPR gold film through biotin-streptavidin recognition. The presence of thrombin would then induce the formation of a double aptamer sandwich structure on the SPR gold film and results in obvious enhancement of SPR signal, which was proportional to the concentration of thrombin. This proposed assay took advantage of sandwich binding of two affinity aptamers for increased specificity, AuNPs for signal enhancement, as well as SPR signal readout for real-time detection. The SPR signal had a good linear relationship with thrombin concentration in the range of 0.1-75nM, and the detection limit for thrombin was determined to be as low as 0.1nM. It was found that aptamer functionalized AuNPs enhanced the signal of SPR response and thus increased the limit of detection 4-fold and 5-fold compared to direct detection format without AuNPs. This sensor also showed good selectivity for thrombin without being affected by some other proteins, such as BSA and lysozyme. Furthermore, this proposed SPR sensing platform was successfully applied to thrombin analysis in diluted human serum samples.

Bai Y; Feng F; Zhao L; Wang C; Wang H; Tian M; Qin J; Duan Y; He X

2013-09-01

65

ROCK mediates the inflammatory response in thrombin induced microglia.  

UK PubMed Central (United Kingdom)

To investigate whether the ROCK pathway is involved in thrombin-induced microglial inflammatory response, thrombin-induced microglia were pretreated with the thrombin inhibitor argatroban or a ROCK inhibitor Y-27632. Microglial inflammatory response was evaluated by phagocytosis of fluorescein labeled latex beads analyses and inflammatory mediators' expression such as nitric oxide (NO) and tumor necrosis factor-alpha (TNF-?). Compared to non-induced microglia, thrombin-induced microglia show significantly enhanced phagocytotic capacity and increased ROCK, NO and TNF-? expression. Pretreatment of thrombin-induced microglia with argatroban or Y-27632 significantly decreased phagocytotic capacity and reduced ROCK, NO and TNF-? expression. Therefore, the ROCK pathway may play a vital role in the mechanisms by which thrombin induces microglia in the inflammatory response.

Cui G; Zuo T; Zhao Q; Hu J; Jin P; Zhao H; Jing J; Zhu J; Chen H; Liu B; Hua F; Ye X

2013-09-01

66

Calibrated automated thrombin generation in frozen-thawed platelet-rich plasma to detect hypercoagulability.  

UK PubMed Central (United Kingdom)

To enhance the practical applicability of the calibrated automated thrombogram (CAT) we investigated whether frozen-thawed platelet-rich plasma (ft-PRP) can be used to assess the function of the protein C inhibitory pathway, while preserving the natural phospholipid composition. Recalcified ft-PRP triggered with 0.5 pM recombinant human tissue factor shows a median thrombin potential of 1,779 nM x min, against 1,576 nM x min for fresh PRP. To obtain approximately 70% inhibition, 6.7 nM activated protein C (APC) has to be added, instead of 25 nM in fresh PRP; so the relative APC resistance of PRP appears to depend upon the presence of intact platelets. Factor VIII, added to normal ft-PRP to obtain a concentration of 3.3 U/ml, increases the thrombin potential in the presence of APC 1.5-fold, from 524 to 808 nM x min, in keeping with previously published increases in thrombotic risk in patients with high factor VIII levels. We conclude that thrombography in ft-PRP, with and without added APC, can be used to assess known risk factors for thrombosis, which allows the design of large clinical studies aimed at proving the relationship between thrombin potential and clinical outcome.

Regnault V; Béguin S; Lecompte T

2003-01-01

67

Calibrated automated thrombin generation in frozen-thawed platelet-rich plasma to detect hypercoagulability.  

Science.gov (United States)

To enhance the practical applicability of the calibrated automated thrombogram (CAT) we investigated whether frozen-thawed platelet-rich plasma (ft-PRP) can be used to assess the function of the protein C inhibitory pathway, while preserving the natural phospholipid composition. Recalcified ft-PRP triggered with 0.5 pM recombinant human tissue factor shows a median thrombin potential of 1,779 nM x min, against 1,576 nM x min for fresh PRP. To obtain approximately 70% inhibition, 6.7 nM activated protein C (APC) has to be added, instead of 25 nM in fresh PRP; so the relative APC resistance of PRP appears to depend upon the presence of intact platelets. Factor VIII, added to normal ft-PRP to obtain a concentration of 3.3 U/ml, increases the thrombin potential in the presence of APC 1.5-fold, from 524 to 808 nM x min, in keeping with previously published increases in thrombotic risk in patients with high factor VIII levels. We conclude that thrombography in ft-PRP, with and without added APC, can be used to assess known risk factors for thrombosis, which allows the design of large clinical studies aimed at proving the relationship between thrombin potential and clinical outcome. PMID:12853709

Regnault, Véronique; Béguin, Suzette; Lecompte, Thomas

2003-01-01

68

Hypersensitivity to thrombin of platelets from hypercholesterolemic rats  

Energy Technology Data Exchange (ETDEWEB)

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.

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

1986-03-01

69

The thrombin inhibitor, argatroban, inhibits breast cancer metastasis to bone.  

UK PubMed Central (United Kingdom)

BACKGROUND: Breast cancer has the potential to metastasize to bone, causing debilitating symptoms. Although many tumor cells have thrombin-generating systems originating from tissue factor (TF), therapy in terms of the coagulation system is not well established. To elucidate the efficacy of the thrombin inhibitor, argatroban, on bone metastasis, we investigated TF activation and vascular endothelial growth factor (VEGF) secretion on treatment with thrombin and argatroban. METHODS: MDA-231 breast cancer cells were treated with thrombin in presence or absence of argatroban, and TF activity was measured in the form of activated factor X. Enzyme-linked immunosorbent assay (ELISA) was used to measure VEGF concentrations in the medium. MDA-231 cells were injected into the left heart ventricle of mice, and then argatroban or saline was administered intraperitoneally for 28 days. After 28 days, incidence of bone metastasis was evaluated in the limbs by radiography. RESULTS: TF activity and VEGF secretion were upregulated by thrombin. Argatroban inhibited the enhancement of TF activity and VEGF secretion induced by thrombin. In vivo analysis revealed that the number of metastasized limbs in the argatroban group was significantly lower compared with the saline group (P < 0.05). CONCLUSIONS: Thrombin not only enhances VEGF secretion but also has a positive feedback mechanism to reexpress TF. These results indicate that inhibition of thrombin is of great value in suppression of tumor metastasis. Argatroban is a noteworthy and useful thrombin inhibitor because it has already been used in the clinical setting and has antimetastatic effects in vivo.

Asanuma K; Wakabayashi H; Okamoto T; Asanuma Y; Akita N; Yoshikawa T; Hayashi T; Matsumine A; Uchida A; Sudo A

2013-07-01

70

The role of the red cell membrane in thrombin generation.  

UK PubMed Central (United Kingdom)

Red blood cells have historically been viewed as innocent bystanders in the process of blood coagulation and thrombin generation; however a century of clinical evidence linking red blood cells to thrombosis suggests the contrary. In this brief review, the biochemical evidence for red blood cell involvement in thrombin generation is evaluated. It is concluded that in addition to platelets, red blood cells actively participate in thrombin generation. A sub-fraction of red blood cells express phosphatidylserine on their surface and unlike platelets, red blood cells produce thrombin through the meizothrombin pathway, which has interesting consequences in the context of clot formation and stabilization.

Whelihan MF; Mann KG

2013-05-01

71

Matrix metalloproteinase-10 is upregulated by thrombin in endothelial cells and increased in patients with enhanced thrombin generation.  

UK PubMed Central (United Kingdom)

OBJECTIVE: Thrombin is a multifunctional serine protease that promotes vascular proinflammatory responses whose effect on endothelial MMP-10 expression has not previously been evaluated. METHODS AND RESULTS: Thrombin induced endothelial MMP-10 mRNA and protein levels, through a protease-activated receptor-1 (PAR-1)-dependent mechanism, in a dose- and time-dependent manner. This effect was mimicked by a PAR-1 agonist peptide (TRAP-1) and antagonized by an anti-PAR-1 blocking antibody. MMP-10 induction was dependent on extracellular regulated kinase1/2 (ERK1/2) and c-jun N-terminal kinase (JNK) pathways. By serial deletion analysis, site-directed mutagenesis and electrophoretic mobility shift assay an AP-1 site in the proximal region of MMP-10 promoter was found to be critical for thrombin-induced MMP-10 transcriptional activity. Thrombin and TRAP-1 upregulated MMP-10 in murine endothelial cells in culture and in vivo in mouse aorta. This effect of thrombin was not observed in PAR-1-deficient mice. Interestingly, circulating MMP-10 levels (P<0.01) were augmented in patients with endothelial activation associated with high (disseminated intravascular coagulation) and moderate (previous acute myocardial infarction) systemic thrombin generation. CONCLUSIONS: Thrombin induces MMP-10 through a PAR-1-dependent mechanism mediated by ERK1/2, JNK, and AP-1 activation. Endothelial MMP-10 upregulation could be regarded as a new proinflammatory effect of thrombin whose pathological consequences in thrombin-related disorders and plaque stability deserve further investigation.

Orbe J; Rodríguez JA; Calvayrac O; Rodríguez-Calvo R; Rodríguez C; Roncal C; Martínez de Lizarrondo S; Barrenetxe J; Reverter JC; Martínez-González J; Páramo JA

2009-12-01

72

Thrombin generation and heparin-induced thrombocytopenia.  

UK PubMed Central (United Kingdom)

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a severe complication of heparin therapy. IgG antibodies targeting the platelet factor 4-heparin complex activate platelets and generate microparticles with procoagulant activity. OBJECTIVES: To determine whether the thrombin generation assay is capable of detecting procoagulant activity induced by patient platelet-poor plasma (PPP) in donor platelet-rich plasma (PRP). PATIENTS AND METHODS: We explored two groups of patients; group 1 (n = 23): patients with a positive clinical and biological diagnosis of HIT; group 2 (n = 25): patients with a negative clinical and biological diagnosis of HIT. Mixtures of donor PRP and patient PPP (1:1) were incubated either with unfractionated heparin 0.2 U mL(-1) or with physiological saline. Thrombin generation was assessed by calibrated thrombinography. The effect of heparin on the mixtures was evaluated according to the ratio of the values with and without heparin (wH/woH) of the five thrombogram parameters. RESULTS: With low heparin concentrations, plasma of group 1 activates donor platelets and generates procoagulant activity. A set of three ratios outside the cut-off values corresponds to the 'HIT thrombogram profile', characterized by a highly specific aspect of the thrombogram wH in relation to the thrombogram woH. None of the group 2 patients presented a HIT thrombogram profile. The results of thrombinography correlate well with the results of the platelet aggregation test. CONCLUSION: Our studies illustrate the central paradox of HIT, namely enhancement of thrombin generation in the presence of heparin. The HIT thrombogram profile as it is defined in this study can detect the procoagulant activity of HIT IgG antibodies.

Tardy-Poncet B; Piot M; Chapelle C; France G; Campos L; Garraud O; Decousus H; Mismetti P; Tardy B

2009-09-01

73

Topical product  

UK PubMed Central (United Kingdom)

A topical product is provided, which contains supercritical birch bark extract as an active component. The supercritical birch bark extract contains betulin and lupeol as main components. The topical product may further include auxiliary active components, such as tea tree oil, olive oil, camomile oil, allantoin and lanolin. It has a strong antiseptic, healing and regeneration effects.

SAREK JAN; VLK MARTIN

74

AI Topics  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The items in this collage were selected from the AI TOPICS Web site's "AI in the News" collection that can be found -- complete with links to the item's source and related AI TOPICS pages -- at www. aaai.org/aitopics/html/current.php. Please note that: (1) an excerpt may not reflect the overall teno...

Glick, Jonathan

75

AI Topics  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The debut of the AI in the News column elsewhere in this issue of AI Magazine created a good opportunity to introduce the professional community to the AI Topics web site, home of the AI in the news virtual page. Although AI Topics is designed for the lay public, it serves a much larger audience.

Buchanan, Bruce G.; Glick, Jonathan

76

Development and Optimization of a Thrombin Sandwich Aptamer Microarray  

Digital Repository Infrastructure Vision for European Research (DRIVER)

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...

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

77

Acidosis, magnesium and acetylsalicylic acid: Effects on thrombin  

Science.gov (United States)

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.

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

2013-03-01

78

Alternative monitoring of argatroban using plasma-diluted thrombin time.  

UK PubMed Central (United Kingdom)

OBJECTIVE: To report a case of heparin-induced thrombocytopenia (HIT) in a patient with concurrent liver dysfunction and a prolonged baseline activated partial thromboplastin time (aPTT) in whom argatroban therapy was monitored with aPTT and a novel plasma-diluted thrombin assay. CASE SUMMARY: An 80-year-old man with HIT and liver dysfunction was treated with argatroban, which was initiated at a dose of 0.5 ?g/kg/min and gradually decreased to 0.09 ?g/kg/min. The patient had a mildly prolonged aPTT at baseline (37.5 seconds). He was concurrently monitored with aPTT, per institution protocol, and plasma-diluted thrombin time. Plasma-diluted thrombin times were consistently lower than aPTTs, but mirrored the trend of the aPTTs. Eleven hours after argatroban was stopped, the aPTT remained elevated (53.9 seconds), while the plasma-diluted thrombin time returned to normal range (26.4 seconds). The patient's therapy was transitioned to warfarin and he had a hospital course with no thrombotic or bleeding complications. DISCUSSION: Plasma-diluted thrombin time is a novel laboratory test consisting of 1 part patient plasma diluted with 3 parts normal plasma. Plasma-diluted thrombin time has been shown to blunt the sensitivity of the thrombin time and may be more accurate for drug monitoring. A MEDLINE search revealed 2 studies using the plasma-diluted thrombin time assay. The first study compared aPTT and plasma-diluted thrombin times in blood samples mixed with argatroban, bivalirudin, or lepirudin at 3 different concentrations. Blood samples contained lupus inhibitors, vitamin k deficiency, or normal baseline aPTTs. The aPTT overestimated drug concentrations in all samples with lupus anticoagulant and vitamin k deficiency, while the plasma-diluted thrombin time correctly estimated drug concentrations in nearly all samples. The second study looked at monitoring dabigatran with plasma-diluted thrombin time and found a linear relationship between the plasma-diluted thrombin time and the dabigatran dose-response curve. CONCLUSIONS: Plasma-diluted thrombin time may be an alternative for direct thrombin inhibitor monitoring in patients with elevated aPTT values at baseline. Further randomized control trials are needed to determine its applicability in clinical practice.

Wanat MA; Hart SR; Putney D; Liebl MG; Chandler W

2013-04-01

79

Topical analgesics.  

UK PubMed Central (United Kingdom)

Historically, analgesics were applied by the topical route of administration. With the advent of oral formulations of drugs, topical application became less popular among physicians, although patients still rated this method of drug delivery as efficacious and practical. We now appreciate that peripheral mechanisms of actions of a variety of preparations rationalizes their topical application and gives further opportunity to target peripheral receptors and neural pathways that previously required systemic administration to achieve therapeutic effect. Therefore, a peripheral effect can be generated by using locally applied drug and, consequently, systemic concentrations of that drug may not reach the level at which systemic side effects can occur.

McCleane G

2007-12-01

80

Towards a standardization of thrombin generation assessment: The influence of tissue factor, platelets and phospholipids concentration on the normal values of Thrombogram-Thrombinoscope assay  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Thrombin generation assay was developed several years ago to mimic physiological coagulation mechanisms but it had important limitations. Thrombogram-Thrombinoscope assay using a fluorogenic substrate, allows obtaining thrombin generation curves in non-defibrinated platelet rich plasma (PRP) in a fully automated manner. Methods We standardised the methodology of Thrombogram-Thrombinoscope and we evaluated the precision of thrombin generation parameters (lag-time, maximum concentration of thrombin [Cmax], time required to reach Cmax [Tmax] and endogenous thrombin potential ETP) using different concentrations of recombinant human tissue factor, platelets or phospholipids. Normal values of thrombin generation assay were established in optimal experimental conditions. Results In the presence of low TF concentrations (final dilution of thromboplastin in plasma: 1/1000–1/2000) the Thrombogram assay showed intra-assay and inter-assay coefficients of variation lower than 9%. Thrombin generation parameters showed an important inter-individual variability and the coefficients of variation ranged from 18% to 50%. In PRP the lag-time, Cmax and Tmax but not the ETP, were influenced by TF concentration. Thrombin generation parameters were not influenced by variations of platelet concentration from 50 × 109/l to 400 × 109/l. The addition of synthetic procoagulant phospholipids in PPP strongly influenced all the parameters of thrombogram. For all the parameters of thrombogram a threshold effect was observed in the presence of phspholipid concentrations equal or higher to 4 ?M. In frozen-thawed PRP the lag-time and the Tmax were significantly reduced and the Cmax was increased compared to the fresh PRP, but the ETP, the intra assay and the inter-assay coefficients of variation were similar in both test-systems. Conclusion Thrombogram-Thrombinoscope assay performed in fresh or in frozen-thawed PRP has an acceptable precision, with low inter-assay and intra-assay coefficient of variations. The concentration of TF is determinant for the normal values of the studied parameters of thrombin generation. When the assay is performed in PPP, thrombin generation parameters are influenced by the concentration of procoagulant synthetic phospholipids. The optimal experimental conditions were obtained in the presence of 1/1000 final dilution of thromboplastin, a platelet count higher than 50 × 109/l and a synthetic phospholipid concentration higher than 4 ?M.

Gerotziafas Grigoris T; Depasse François; Busson Joël; Leflem Lena; Elalamy Ismail; Samama Meyer M

2005-01-01

 
 
 
 
81

Towards a standardization of thrombin generation assessment: the influence of tissue factor, platelets and phospholipids concentration on the normal values of Thrombogram-Thrombinoscope assay.  

UK PubMed Central (United Kingdom)

BACKGROUND: Thrombin generation assay was developed several years ago to mimic physiological coagulation mechanisms but it had important limitations. Thrombogram-Thrombinoscope assay using a fluorogenic substrate, allows obtaining thrombin generation curves in non-defibrinated platelet rich plasma (PRP) in a fully automated manner. METHODS: We standardised the methodology of Thrombogram-Thrombinoscope and we evaluated the precision of thrombin generation parameters (lag-time, maximum concentration of thrombin [Cmax], time required to reach Cmax [Tmax] and endogenous thrombin potential ETP) using different concentrations of recombinant human tissue factor, platelets or phospholipids. Normal values of thrombin generation assay were established in optimal experimental conditions. RESULTS: In the presence of low TF concentrations (final dilution of thromboplastin in plasma: 1/1000-1/2000) the Thrombogram assay showed intra-assay and inter-assay coefficients of variation lower than 9%. Thrombin generation parameters showed an important inter-individual variability and the coefficients of variation ranged from 18% to 50%. In PRP the lag-time, Cmax and Tmax but not the ETP, were influenced by TF concentration. Thrombin generation parameters were not influenced by variations of platelet concentration from 50 x 10(9)/l to 400 x 10(9)/l. The addition of synthetic procoagulant phospholipids in PPP strongly influenced all the parameters of thrombogram. For all the parameters of thrombogram a threshold effect was observed in the presence of phospholipid concentrations equal or higher to 4 microM. In frozen-thawed PRP the lag-time and the Tmax were significantly reduced and the Cmax was increased compared to the fresh PRP, but the ETP, the intra assay and the inter-assay coefficients of variation were similar in both test-systems. CONCLUSION: Thrombogram-Thrombinoscope assay performed in fresh or in frozen-thawed PRP has an acceptable precision, with low inter-assay and intra-assay coefficient of variations. The concentration of TF is determinant for the normal values of the studied parameters of thrombin generation. When the assay is performed in PPP, thrombin generation parameters are influenced by the concentration of procoagulant synthetic phospholipids. The optimal experimental conditions were obtained in the presence of 1/1000 final dilution of thromboplastin, a platelet count higher than 50 x 10(9)/l and a synthetic phospholipid concentration higher than 4 microM.

Gerotziafas GT; Depasse F; Busson J; Leflem L; Elalamy I; Samama MM

2005-10-01

82

Crystallization and preliminary X-ray analysis of the complex of human ?-thrombin with a modified thrombin-binding aptamer  

Science.gov (United States)

The thrombin-binding aptamer (TBA) is a consensus DNA 15-mer that binds specifically to human ?-thrombin at nanomolar concentrations and inhibits its procoagulant functions. Recently, a modified TBA (mTBA) containing a 5?–5? inversion-of-polarity site has been shown to be more stable and to possess a higher thrombin affinity than its unmodified counterpart. The structure of the thrombin–TBA complex has previously been determined at low resolution, but did not provide a detailed picture of the aptamer conformation or of the protein–DNA assembly, while that of the complex with mTBA is unknown. Crystallographic analysis of the thrombin–mTBA complex has been attempted. The crystals diffracted to 2.15?Å resolution and belonged to space group I222.

Russo Krauss, Irene; Merlino, Antonello; Randazzo, Antonio; Mazzarella, Lelio; Sica, Filomena

2010-01-01

83

Tacrolimus Topical  

Science.gov (United States)

Tacrolimus ointment is used to treat the symptoms of eczema (atopic dermatitis; a skin disease that causes ... whose eczema has not responded to another medication. Tacrolimus is in a class of medications called topical ...

84

Functional phage display of leech-derived tryptase inhibitor (LDTI): construction of a library and selection of thrombin inhibitors.  

Science.gov (United States)

The recombinant phage antibody system pCANTAB 5E has been used to display functionally active leech-derived tryptase inhibitor (LDTI) on the tip of the filamentous M13 phage. A limited combinatorial library of 5.2 x 10(4) mutants was created with a synthetic LDTI gene, using a degenerated oligonucleotide and the pCANTAB 5E phagemid. The mutations were restricted to the P1-P4' positions of the reactive site. Fusion phages and appropriate host strains containing the phagemids were selected after binding to thrombin and DNA sequencing. The variants LDTI-2T (K8R, I9V, S10, K11W, P12A), LDTI-5T (K8R, I9V, S10, K11S, P12L) and LDTI-10T (K8R, I9L, S10, K11D, P12I) were produced with a Saccharomyces cerevisiae expression system. The new inhibitors, LDTI-2T and -5T, prolong the blood clotting time, inhibit thrombin (Ki 302 nM and 28 nM) and trypsin (Ki 6.4 nM and 2.1 nM) but not factor Xa, plasma kallikrein or neutrophil elastase. The variant LDTI-10T binds to thrombin but does not inhibit it. The relevant reactive site sequences of the thrombin inhibiting variants showed a strong preference for arginine in position P1 (K8R) and for valine in P1' (I9V). The data indicate further that LDTI-5T might be a model candidate for generation of active-site directed thrombin inhibitors and that LDTI in general may be useful to generate specific inhibitors suitable for a better understanding of enzyme-inhibitor interactions. PMID:10518924

Tanaka, A S; Silva, M M; Torquato, R J; Noguti, M A; Sampaio, C A; Fritz, H; Auerswald, E A

1999-09-10

85

Functional phage display of leech-derived tryptase inhibitor (LDTI): construction of a library and selection of thrombin inhibitors.  

UK PubMed Central (United Kingdom)

The recombinant phage antibody system pCANTAB 5E has been used to display functionally active leech-derived tryptase inhibitor (LDTI) on the tip of the filamentous M13 phage. A limited combinatorial library of 5.2 x 10(4) mutants was created with a synthetic LDTI gene, using a degenerated oligonucleotide and the pCANTAB 5E phagemid. The mutations were restricted to the P1-P4' positions of the reactive site. Fusion phages and appropriate host strains containing the phagemids were selected after binding to thrombin and DNA sequencing. The variants LDTI-2T (K8R, I9V, S10, K11W, P12A), LDTI-5T (K8R, I9V, S10, K11S, P12L) and LDTI-10T (K8R, I9L, S10, K11D, P12I) were produced with a Saccharomyces cerevisiae expression system. The new inhibitors, LDTI-2T and -5T, prolong the blood clotting time, inhibit thrombin (Ki 302 nM and 28 nM) and trypsin (Ki 6.4 nM and 2.1 nM) but not factor Xa, plasma kallikrein or neutrophil elastase. The variant LDTI-10T binds to thrombin but does not inhibit it. The relevant reactive site sequences of the thrombin inhibiting variants showed a strong preference for arginine in position P1 (K8R) and for valine in P1' (I9V). The data indicate further that LDTI-5T might be a model candidate for generation of active-site directed thrombin inhibitors and that LDTI in general may be useful to generate specific inhibitors suitable for a better understanding of enzyme-inhibitor interactions.

Tanaka AS; Silva MM; Torquato RJ; Noguti MA; Sampaio CA; Fritz H; Auerswald EA

1999-09-01

86

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

Science.gov (United States)

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).

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

2013-04-01

87

Strategies and progress towards the ideal orally active thrombin inhibitor.  

UK PubMed Central (United Kingdom)

Thrombin plays a key role in the control of thrombus formation, for which reason its inhibition has become a target for new antithrombotics. Important issues in the profile of the ideal thrombin inhibitor are: potency, selectivity, oral bioavailability, half-life in the circulatory system and safety. Although many potent direct inhibitors of thrombin have been discovered, most of these inhibitors lack sufficient oral bioavailability. This is often associated with the presence of highly basic functionalities such as guanidine or amidine. These basic functionalities in the P1 moiety are preferred by thrombin and are present in the first generation of thrombin inhibitors. Recently, several orally active direct thrombin inhibitors have been disclosed. Most of these inhibitors originate from leads of the first generation. Two major optimization strategies could be identified to further improve these leads: A: maintain the highly basic P1 moiety and compensate its negative effects, and B: reduce the basicity of the P1 moiety and compensate for the decrease in inhibitory activity. The progress made using these strategies is evaluated. In addition, screening large sets of compounds yielded new structures that provide useful starting points for optimization. The optimization strategy used to convert leads from screening into potent orally active thrombin inhibitors is also be evaluated.

Rewinkel JB; Adang AE

1999-12-01

88

APTAMER-BASED SERRS SENSOR FOR THROMBIN DETECTION  

Energy Technology Data Exchange (ETDEWEB)

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.

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

89

Topical analgesics.  

UK PubMed Central (United Kingdom)

Our knowledge and understanding of the pathophysiology and treatment of pain is increasing; however, we should not lose sight of the simple opportunities that exist for intercepting pain at peripheral targets. Although systemic medication often has peripheral and central modes of action, the appeal for provision of medication close to where these peripheral targets exist should be high. If these sites can be attacked with relatively high concentrations of active drug while keeping systemic levels of that drug below the level at which systemic side effects become apparent, then this should lead to desirable outcomes. Even though the number of true topical agents with an indication for this use is small, a number of other topical agents are available that evidence suggests have the possibility of being effective. Given the increased understanding of pain, the likelihood of further topical agents becoming available is high.

McCleane G

2007-01-01

90

Topical analgesics.  

UK PubMed Central (United Kingdom)

BACKGROUND AND OBJECTIVES: Pain treatment involves the usage of common and opioid analgesics, nonsteroidal anti-inflammatory drugs (NSAIDs) and adjuvant analgesics. Traditionally, these drugs are administered systemically or into the neuraxis. However, when analgesics are applied through these pathways, they are associated with significant side effects, which can hinder its use. Topical administration of analgesics is an alternative. The objective of this paper is to discuss topical analgesics, the mechanisms of action and clinical efficacy. CONTENT: This is a review paper addressing the usage of the topical local anesthetics: capsaicin, clonidine, tricyclic antidepressants, ketamine, opioids and cannabinoids, discussing mechanism of action and effectiveness. CONCLUSIONS: Topical analgesics are promising as a strategy for pain treatment, as they are associated with lower incidence of side effects. The benefit of local anesthetics, NSAID's and capsaicin is well established. However, the efficacy of clonidine, tricyclic antidepressants, ketamine, opioids and cannabinoids is still questionable. Studies have shown that the multimodal approach is an alternative, but studies are needed to confirm this hypothesis.

Flores MP; Castro AP; Nascimento Jdos S

2012-03-01

91

The effect of thrombin activated factor XIII, thrombin and plasmin on the chemiluminescence produced by human neutrophils stimulated by opsonized zymosan (Mannozym)  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Coagulation factor XIII formed by thrombin activation from zymogen factor XIII decreases the chemiluminescence (CL) of human neutrophils stimulated by opsonized zymosan (Mannozym). At high concentrations, thrombin and plasmin also decreased the CL induced by opsonized zymosan. The inhibitory effect ...

Sipka S; Abel G; Czirják L; Csongor J; Szegedi G; Fachet J; Sipka Sándor (1945-) (laboratóriumi szakorvos, laboratóriumi hemato lógus és immunológus, klinikai farmakológus szakorvos)

92

Bifunctional combined aptamer for simultaneous separation and detection of thrombin.  

UK PubMed Central (United Kingdom)

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.

Bing T; Liu X; Cheng X; Cao Z; Shangguan D

2010-02-01

93

Bifunctional combined aptamer for simultaneous separation and detection of thrombin.  

Science.gov (United States)

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

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

2009-11-11

94

Thrombin stimulates activation of the cerebral 5-lipoxygenase pathway during blood-brain cell contact.  

UK PubMed Central (United Kingdom)

The purpose of this study was to identity the trigger mechanism activating the 5-lipoxygenase pathway during blood-brain cell contact and to estimate the contribution of blood and brain cells to the cysteinyl-leukotriene (LT) biosynthesis observed under these conditions. Incubation of dissociated rat brain cells in Krebs-Henseleit solution for up to 60 min did not stimulate any detectable cysteinyl-LT biosynthesis. Incubation of recalcified rat whole blood in vitro for up to 60 min led to release of only small amounts of cysteinyl-LT into the serum samples. However, coincubation of dissociated rat brain cells with physiologically recalcified autologous whole blood triggered a time-dependent release of large amounts of immunoreactive cysteinyl-LT into the serum samples. By reverse-phase HPLC, immunoreactive cysteinyl-LT was identified as a mixture of LTC4, LTD4, and LTE4. The extent of the 5-lipoxygenase stimulation depended on the amount of autologous blood coincubated with the dissociated brain cells. Activation of the 5-lipoxygenase pathway also occurred with coincubation of dissociated rat brain cells with recalcified autologous plasma. Stimulation of cysteinyl-LT biosynthesis during blood-brain cell contact remained unaffected by aprotinin, but concentration-dependent inhibition by the structurally and functionally unrelated thrombin inhibitors D-Phe-Pro-Arg-CH2Cl and recombinant hirudin was seen. Finally, when dissociated rat brain cells were incubated in Krebs-Henseleit solution in the presence of human alpha-thrombin, a concentration-dependent release of cysteinyl-LT into the buffer samples was observed. These data demonstrate that, in rats, during blood-brain cell contact, stimulation of the 5-lipoxygenase pathway in brain cells proceeds via alpha-thrombin as effector molecule.

Winking M; Heldt RM; Simmet T

1996-07-01

95

Cleavage of a specific bond in troponin C by thrombin.  

UK PubMed Central (United Kingdom)

Limited proteolysis of rabbit skeletal troponin C with bovine thrombin yielded two fragments, TH1 (Mr = 11000) containing Ca2+ binding regions I--III and TH2 (Mr = 6000) containing region IV. Determination of the partial sequences of the fragments established the site of cleavage at Arg120-Ala121. Secondary cleavage by thrombin at other arginyl or lysyl residues in troponin C was ruled out by the sequence data and by the amino acid compositions of the two fragments.

Leavis PC; Rosenfeld S; Lu RC

1978-08-01

96

[Direct inhibitors of thrombin, hirudin, bivalirudin, and dabigatran etexilate].  

Science.gov (United States)

Thrombin inhibition is an important objective in the prevention and treatment of thrombosis. A new molecule, dabigatran etexilate or Pradaxa(®), has been recently licensed for thromboprophylaxis in major orthopedic surgery in several countries but not in the USA. In contrast, the FDA has approved it for prevention in patients with non-valvular atrial fibrillation. This new orally active anticoagulant is being developed for the treatment of venous thromboembolism and acute coronary syndromes in patients with non-valvular atrial fibrillation. Dabigatran is a reversible inhibitor of free thrombin and clot-bound thrombin. An oral thrombin inhibitor melagatran is no longer available due to hepatic toxicity. Several other thrombin inhibitors are used via parenteral administration: lepirudine and desirudine, bivalirudine and argatroban. They are mostly given to patients with heparin-induced thrombocytopenia (HIT). Bivalirudine is used for acute coronary syndrome in patients undergoing percutaneous interventions. The main pharmacologic characteristics of thrombin inhibitor agents are presented focusing on dabigatran etexilate and including the main results of clinical trials. PMID:21239127

Meddahi, S; Samama, M M

2011-01-15

97

[Direct inhibitors of thrombin, hirudin, bivalirudin, and dabigatran etexilate].  

UK PubMed Central (United Kingdom)

Thrombin inhibition is an important objective in the prevention and treatment of thrombosis. A new molecule, dabigatran etexilate or Pradaxa(®), has been recently licensed for thromboprophylaxis in major orthopedic surgery in several countries but not in the USA. In contrast, the FDA has approved it for prevention in patients with non-valvular atrial fibrillation. This new orally active anticoagulant is being developed for the treatment of venous thromboembolism and acute coronary syndromes in patients with non-valvular atrial fibrillation. Dabigatran is a reversible inhibitor of free thrombin and clot-bound thrombin. An oral thrombin inhibitor melagatran is no longer available due to hepatic toxicity. Several other thrombin inhibitors are used via parenteral administration: lepirudine and desirudine, bivalirudine and argatroban. They are mostly given to patients with heparin-induced thrombocytopenia (HIT). Bivalirudine is used for acute coronary syndrome in patients undergoing percutaneous interventions. The main pharmacologic characteristics of thrombin inhibitor agents are presented focusing on dabigatran etexilate and including the main results of clinical trials.

Meddahi S; Samama MM

2011-02-01

98

Biochemical characterization of bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI)  

DEFF Research Database (Denmark)

BACKGROUND: TAFI is a plasma protein assumed to be an important link between coagulation and fibrinolysis. The three-dimensional crystal structures of authentic mature bovine TAFI (TAFIa) in complex with tick carboxypeptidase inhibitor, authentic full lenght bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI), and recombinant human TAFI have recently been solved. In light of these recent advances, we have characterized authentic bovine TAFI biochemically and compared it to human TAFI. RESULTS: The four N-linked glycosylation sequons within the activation peptide were all occupied in bovine TAFI, similar to human TAFI, while the sequon located within the enzyme moiety of the bovine protein was non-glycosylated. The enzymatic stability and the kinetic constants of TAFIa differed somewhat between the two proteins, as did the isoelectric point of TAFI, but not TAFIa. Equivalent to human TAFI, bovine TAFI was a substrate for transglutaminases and could be proteolytically cleaved by trypsin or thrombin/solulin complex, although small differences in the fragmentation patterns were observed. Furthermore, bovine TAFI exhibited intrinsic activity and TAFIa attenuated tPA-mediated fibrinolysis similar to the human protein. CONCLUSION: The findings presented here suggest that the properties of these two orthologous proteins are similar and that conclusions reached using the bovine TAFI may be extrapolated to the human protein.

Valnickova, Zuzana; Thaysen-Andersen, Morten

2009-01-01

99

Biochemical characterization of bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI)  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background TAFI is a plasma protein assumed to be an important link between coagulation and fibrinolysis. The three-dimensional crystal structures of authentic mature bovine TAFI (TAFIa) in complex with tick carboxypeptidase inhibitor, authentic full lenght bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI), and recombinant human TAFI have recently been solved. In light of these recent advances, we have characterized authentic bovine TAFI biochemically and compared it to human TAFI. Results The four N-linked glycosylation sequons within the activation peptide were all occupied in bovine TAFI, similar to human TAFI, while the sequon located within the enzyme moiety of the bovine protein was non-glycosylated. The enzymatic stability and the kinetic constants of TAFIa differed somewhat between the two proteins, as did the isoelectric point of TAFI, but not TAFIa. Equivalent to human TAFI, bovine TAFI was a substrate for transglutaminases and could be proteolytically cleaved by trypsin or thrombin/solulin complex, although small differences in the fragmentation patterns were observed. Furthermore, bovine TAFI exhibited intrinsic activity and TAFIa attenuated tPA-mediated fibrinolysis similar to the human protein. Conclusion The findings presented here suggest that the properties of these two orthologous proteins are similar and that conclusions reached using the bovine TAFI may be extrapolated to the human protein.

Valnickova Zuzana; Thaysen-Andersen Morten; Højrup Peter; Christensen Trine; Sanggaard Kristian W; Kristensen Torsten; Enghild Jan J

2009-01-01

100

Biochemical characterization of bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI).  

UK PubMed Central (United Kingdom)

BACKGROUND: TAFI is a plasma protein assumed to be an important link between coagulation and fibrinolysis. The three-dimensional crystal structures of authentic mature bovine TAFI (TAFIa) in complex with tick carboxypeptidase inhibitor, authentic full lenght bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI), and recombinant human TAFI have recently been solved. In light of these recent advances, we have characterized authentic bovine TAFI biochemically and compared it to human TAFI. RESULTS: The four N-linked glycosylation sequons within the activation peptide were all occupied in bovine TAFI, similar to human TAFI, while the sequon located within the enzyme moiety of the bovine protein was non-glycosylated. The enzymatic stability and the kinetic constants of TAFIa differed somewhat between the two proteins, as did the isoelectric point of TAFI, but not TAFIa. Equivalent to human TAFI, bovine TAFI was a substrate for transglutaminases and could be proteolytically cleaved by trypsin or thrombin/solulin complex, although small differences in the fragmentation patterns were observed. Furthermore, bovine TAFI exhibited intrinsic activity and TAFIa attenuated tPA-mediated fibrinolysis similar to the human protein. CONCLUSION: The findings presented here suggest that the properties of these two orthologous proteins are similar and that conclusions reached using the bovine TAFI may be extrapolated to the human protein.

Valnickova Z; Thaysen-Andersen M; Højrup P; Christensen T; Sanggaard KW; Kristensen T; Enghild JJ

2009-01-01

 
 
 
 
101

Comparison of natural and recombinant tissue factor proteins: new insights.  

UK PubMed Central (United Kingdom)

Tissue factor (TF), an initiator of blood coagulation in vivo, is expressed in a variety of cells. Sufficient natural TF has been isolated to clone and express recombinant proteins ranging from full-length TF to its extracellular domain. Because of the limited availability of natural TF, recombinant proteins have been used as surrogates. Despite the differences in their post-translational modifications, it has been accepted that membrane-anchored recombinant TFs are quite similar to the natural TF. Recent studies, however, have shown that post-translational modifications play an important role in TF-triggered thrombin generation.

Butenas S

2013-07-01

102

Feasibility of using thrombin generation assay (TGA) for monitoring bypassing agent therapy in patients with hemophilia having inhibitors.  

UK PubMed Central (United Kingdom)

BACKGROUND: Monitoring bypassing agent therapy and observing concordance with clinical hemostasis is crucial in vital hemorrhages and major surgeries in patients with hemophilia having inhibitor. OBJECTIVE: We aimed to investigate the value of the thrombin generation assay (TGA) and thromboelastography (TEG) for monitoring hemostasis in patients with hemophilia having inhibitor, during supplementation therapy with bypassing agents. PATIENTS AND METHODS: The study group consisted of 7 patients with hemophilia having factor VIII inhibitor. All patients were male. The median age of the participants was 10 years. Age range was 6 to 32 years. The median inhibitor level was 10 Bethesda units (BU), with a range of 5 to 32 BU. A total of 17 bleeding episodes were evaluated. Both TEG and TGA tests were assessed in addition to clinical responses. Assessments were made prior to bypass agent therapy such as recombinant factor VIIa (rFVIIa) or activated prothrombin complex concentrate (aPCC) for bleeding episodes, during the first hour and 24 hours after either intervention in patients. RESULTS: No relation between clinical response and TGA or TEG parameters was found in patients. There was no difference between clinical responses after rFVIIa and aPCC treatments. However, after aPCC treatment, endogenous thrombin potential and peak thrombin levels and also TEG R, K, and alpha angle degrees were significantly higher. CONCLUSIONS: In conclusion, we found that the clinical effectiveness of bypass therapy in hemophilia cannot be assessed by TGA and TEG.

Ay Y; Balkan C; Karapinar DY; Akin M; Bilenoglu B; Kavakli K

2013-07-01

103

Thrombin, a mediator of cerebrovascular inflammation in AD and hypoxia.  

UK PubMed Central (United Kingdom)

Considerable evidence implicates hypoxia and vascular inflammation in Alzheimer's disease (AD). Thrombin, a multifunctional inflammatory mediator, is demonstrable in the brains of AD patients both in the vessel walls and senile plaques. Hypoxia-inducible factor 1? (HIF-1?), a key regulator of the cellular response to hypoxia, is also upregulated in the vasculature of human AD brains. The objective of this study is to investigate inflammatory protein expression in the cerebrovasculature of transgenic AD mice and to explore the role of thrombin as a mediator of cerebrovascular inflammation and oxidative stress in AD and in hypoxia-induced changes in brain endothelial cells. Immunofluorescent analysis of the cerebrovasculature in AD mice demonstrates significant (p < 0.01-0.001) increases in thrombin, HIF-1?, interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinases (MMPs), and reactive oxygen species (ROS) compared to controls. Administration of the thrombin inhibitor dabigatran (100 mg/kg) to AD mice for 34 weeks significantly decreases expression of inflammatory proteins and ROS. Exposure of cultured brain endothelial cells to hypoxia for 6 h causes an upregulation of thrombin, HIF-1?, MCP-1, IL-6, and MMP2 and ROS. Treatment of endothelial cells with the dabigatran (1 nM) reduces ROS generation and inflammatory protein expression (p < 0.01-0.001). The data demonstrate that inhibition of thrombin in culture blocks the increase in inflammatory protein expression and ROS generation evoked by hypoxia. Also, administration of dabigatran to transgenic AD mice diminishes ROS levels in brain and reduces cerebrovascular expression of inflammatory proteins. Taken together, these results suggest that inhibiting thrombin generation could have therapeutic value in AD and other disorders where hypoxia, inflammation, and oxidative stress are involved.

Tripathy D; Sanchez A; Yin X; Luo J; Martinez J; Grammas P

2013-01-01

104

Thrombin, a mediator of cerebrovascular inflammation in AD and hypoxia  

Science.gov (United States)

Considerable evidence implicates hypoxia and vascular inflammation in Alzheimer's disease (AD). Thrombin, a multifunctional inflammatory mediator, is demonstrable in the brains of AD patients both in the vessel walls and senile plaques. Hypoxia-inducible factor 1? (HIF-1?), a key regulator of the cellular response to hypoxia, is also upregulated in the vasculature of human AD brains. The objective of this study is to investigate inflammatory protein expression in the cerebrovasculature of transgenic AD mice and to explore the role of thrombin as a mediator of cerebrovascular inflammation and oxidative stress in AD and in hypoxia-induced changes in brain endothelial cells. Immunofluorescent analysis of the cerebrovasculature in AD mice demonstrates significant (p < 0.01–0.001) increases in thrombin, HIF-1?, interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinases (MMPs), and reactive oxygen species (ROS) compared to controls. Administration of the thrombin inhibitor dabigatran (100 mg/kg) to AD mice for 34 weeks significantly decreases expression of inflammatory proteins and ROS. Exposure of cultured brain endothelial cells to hypoxia for 6 h causes an upregulation of thrombin, HIF-1?, MCP-1, IL-6, and MMP2 and ROS. Treatment of endothelial cells with the dabigatran (1 nM) reduces ROS generation and inflammatory protein expression (p < 0.01–0.001). The data demonstrate that inhibition of thrombin in culture blocks the increase in inflammatory protein expression and ROS generation evoked by hypoxia. Also, administration of dabigatran to transgenic AD mice diminishes ROS levels in brain and reduces cerebrovascular expression of inflammatory proteins. Taken together, these results suggest that inhibiting thrombin generation could have therapeutic value in AD and other disorders where hypoxia, inflammation, and oxidative stress are involved.

Tripathy, Debjani; Sanchez, Alma; Yin, Xiangling; Luo, Jinhua; Martinez, Joseph; Grammas, Paula

2013-01-01

105

Thrombin generation in patients after acute deep-vein thrombosis.  

Science.gov (United States)

Thrombin generation measurement may be of value for assessing the risk of venous thromboembolism, but its long term profile has not been assessed in patients. We evaluated thrombin generation by Calibrated Automated Thrombogram (CAT) in plasma during follow up of 104 consecutive patients after an acute episode of deep venous thrombosis. Blood was drawn three times over the course of 24 months. Thrombin generation was measured in absence and presence of thrombomodulin and compared to a reference range derived from thrombin generation curves in 137 healthy volunteers. Thrombin generation of patients showed significantly higher endogenous thrombin potential (ETP) and peak height compared to the reference population. Differences were more pronounced in assays triggered with 1 pM TF. Inhibition by thrombomodulin was attenuated in patients off anticoagulants as compared to the reference population (21% vs. 42.2%, p < 0.0001); inhibition in patients on anticoagulant treatment was less pronounced (9.7%, p < 0.0001). Protein C activity, protein S antigen as well as free protein S showed highly negative correlation with ETP in all patients. A significant negative relation was found between FVIII levels and thrombomodulin induced reduction of ETP and peak height. In conclusion, thrombin generation by CAT reflects changes in coagulation status in patients following a thromboembolic event and is most sensitive at CAT analysis triggered with 1 pM TF. A role for factor VIII as an important attributable cause of hypercoagulability is reflected by the reduced inhibitory effect of thrombomodulin at high factor VIII levels. PMID:18690343

ten Cate-Hoek, Arina J; Dielis, Arne W J H; Spronk, Henri M H; van Oerle, René; Hamulyák, Karly; Prins, Martin H; ten Cate, Hugo

2008-08-01

106

Gelatin-Thrombin Matrix for Intraoperative Hemostasis in Abdomino-Pelvic Surgery: A Systematic Review.  

Science.gov (United States)

Different hemostatic methods are available for mild to moderate intraoperative bleeding during open and laparoscopic abdomino-pelvic surgery, but topical hemostats have gained popularity. We sought to review evidence on the use of a gelatin-thrombin matrix (FloSeal®) in elective abdominal and pelvic surgery. A systematic search of PubMed, EMBASE, and Cochrane databases was conducted. The primary endpoints were intraoperative bleeding and number of transfusions. Secondary endpoints included operative time, postoperative complications, re-operation for bleeding, mortality, and duration of hospitalization. Of five controlled trials, only three were prospective, randomized-controlled studies. The first, in open myomectomy, showed that hemostatic matrix dramatically reduced intraoperative bleeding and transfusion rates compared with conventional hemostatic measures. Hemostatic matrix also reduced postoperative stay. Similar results were obtained in a trial comparing FloSeal versus infrared-sapphire coagulator during open renal tumor enucleation. In the third, FloSeal was equally as effective as conventional suture methods in preventing staple-line bleeding after sleeve gastrectomy. Data were not pooled because of the heterogeneity in design. There is insufficient evidence that FloSeal provides better results than conventional hemostasis in abdominal and pelvic surgery, except for open myomectomy. Well-designed randomized trials are needed to evaluate the use of gelatin-thrombin matrix in elective abdomino-pelvic surgery outcomes. PMID:23700183

Mayol, Julio M; Zapata, Carolina

2013-05-22

107

Gelatin-Thrombin Matrix for Intraoperative Hemostasis in Abdomino-Pelvic Surgery: A Systematic Review.  

UK PubMed Central (United Kingdom)

Different hemostatic methods are available for mild to moderate intraoperative bleeding during open and laparoscopic abdomino-pelvic surgery, but topical hemostats have gained popularity. We sought to review evidence on the use of a gelatin-thrombin matrix (FloSeal®) in elective abdominal and pelvic surgery. A systematic search of PubMed, EMBASE, and Cochrane databases was conducted. The primary endpoints were intraoperative bleeding and number of transfusions. Secondary endpoints included operative time, postoperative complications, re-operation for bleeding, mortality, and duration of hospitalization. Of five controlled trials, only three were prospective, randomized-controlled studies. The first, in open myomectomy, showed that hemostatic matrix dramatically reduced intraoperative bleeding and transfusion rates compared with conventional hemostatic measures. Hemostatic matrix also reduced postoperative stay. Similar results were obtained in a trial comparing FloSeal versus infrared-sapphire coagulator during open renal tumor enucleation. In the third, FloSeal was equally as effective as conventional suture methods in preventing staple-line bleeding after sleeve gastrectomy. Data were not pooled because of the heterogeneity in design. There is insufficient evidence that FloSeal provides better results than conventional hemostasis in abdominal and pelvic surgery, except for open myomectomy. Well-designed randomized trials are needed to evaluate the use of gelatin-thrombin matrix in elective abdomino-pelvic surgery outcomes.

Mayol JM; Zapata C

2013-05-01

108

Recombinant Programming  

Digital Repository Infrastructure Vision for European Research (DRIVER)

This research report presents a promising new approach to computation called Recombinant Programming. The novelty of our approach is that it separates the program into two layers of computation: the recombination and the interpretation layer. The recombination layer takes sequences as inputs and all...

Pawlak, Renaud; Cuesta, Carlos; Younessi, Houman

109

Fluorogenic peptide-based substrates for monitoring thrombin activity.  

UK PubMed Central (United Kingdom)

The synthesis of a series of peptides containing C-terminal 7-amino-4-methylcoumarin (AMC) for use in the thrombin generation test (TGT) is described. The lead structure in this project was H-Gly-Gly-Arg-AMC, of which the water solubility and kinetic parameters (K(M) and k(cat)) are greatly improved over those of the substrate in current use in the TGT: Cbz-Gly-Gly-Arg-AMC. A series of N-terminally substituted Gly-Gly-Arg-AMC derivatives were synthesized, as well as implementation of structural changes at either the P(2) or P(3) position of the peptide backbone. Furthermore, two substrates were synthesized that have structural similarities to the chromogenic thrombin substrate SQ68 or that contain a 1,2,3-triazole moiety in the peptide chain, mimicking an amide bond. To determine the applicability of newly synthesized fluorogenic substrates for monitoring continuous thrombin generation, the K(M) and k(cat) values of the conversion of these fluorogenic substrates by thrombin (FIIa) and factor?Xa (FXa) were quantified. An initial selection was made on basis of these data, and suitable substrates were further evaluated as substrates in the thrombin generation assay. Assessment of the acquired data showed that several substrates, including the SQ68 derivative Et-malonate-Gly-Arg-AMC and N-functionalized Gly-Gly-Arg-AMC derivatives, are suitable candidates for replacement of the substrate currently in use.

van Berkel SS; van der Lee B; van Delft FL; Wagenvoord R; Hemker HC; Rutjes FP

2012-04-01

110

Development and Optimization of a Thrombin Sandwich Aptamer Microarray  

Directory of Open Access Journals (Sweden)

Full Text Available 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.

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

2012-01-01

111

Levels of thrombin activatable fibrinolysis inhibitor in gestational diabetes mellitus.  

UK PubMed Central (United Kingdom)

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a procarboxypeptidase, which is synthesised in liver and activated by thrombin and the thrombin-thrombomodulin complex. TAFI suppresses fibrinolysis by removing carboxy-terminal lysine residues from partially degraded fibrin. In this study we aimed to assess the circulating levels of TAFI antigen, 'a fibrinolytic parameter' in women with gestational diabetes (GDM). Thirty-four pregnant women with GDM and 50 pregnant women with normal glucose tolerance were included in the study. Plasma TAFI antigen levels were significantly higher in pregnant women with GDM when compared with controls. Increased TAFI levels may contribute to the decreased fibrinolytic potency, causing a thrombophilic state. GDM is regarded as a specific form of diabetes, and it could in addition be a predictor of type 2 diabetes mellitus in the future and the risk of complications due to hypercoagulability increases in this disease. Increased TAFI levels may also have a role in increased risk of hypercoagulability.

Gumus II; Kargili A; Karakurt F; Kasapoglu B; Derbent A; Kaygusuz I; Koca C; Sevgili S

2013-04-01

112

Monitoring unfractionated heparin therapy in dogs by measuring thrombin generation.  

UK PubMed Central (United Kingdom)

BACKGROUND: The calibrated automated thrombogram (CAT), an assay that permits measurement of thrombin generation in plasma, may be useful in studying hemostatic disorders and anticoagulant therapy in animals. OBJECTIVES: The aims of the study were to measure thrombin generation in healthy Beagle dogs and to evaluate the potential use of the CAT assay for monitoring therapy with unfractionated heparin (UFH). Methods: Individual platelet-poor plasma samples and a plasma pool from 20 healthy adult Beagles were prepared. Serial UFH plasma dilutions were used to establish an in vitro heparin-sensitivity curve. The pharmacodynamic effects of heparin in vivo were evaluated in Beagles using the CAT assay to measure thrombin generation with tissue factor at a concentration of 5 pM for initiation. RESULTS: In healthy Beagles, the range of endogenous thrombin potential (ETP) was 238.7-414.0 nM/min (mean ± SD, 340.4 ± 63.1 nM/min). ETP intra-assay and interassay variations were 7.1% and 12.9%, respectively. In vitro, a UFH concentration ?0.4 U/mL resulted in total inhibition of thrombin generation. In vivo, the maximal effect of UFH on ETP was observed at 170 ± 36 minutes (range, 120-210 minutes) and resulted in a decrease in ETP of 38.5 ± 7.8% (range, 26.5-50.3%). In 210-420 minutes, ETP returned to baseline in 5 dogs. CONCLUSION: Our study demonstrates that thrombin generation can be measured in canine plasma and may be useful in assessing the degree of anticoagulation provided by UFH.

Allegret V; Dunn M; Bédard C

2011-03-01

113

Highly efficient control of thrombin activity by multivalent nanoparticles.  

Science.gov (United States)

We have demonstrated that the incorporation of sulfated galactose acid (sulf-Gal) into thrombin-binding-aptamer (TBA)-conjugated gold nanoparticles (TBA-AuNPs) enables highly effective inhibition of thrombin activity toward fibrinogen. AuNP bioconjugates (TBA(15)/TBA(29)/sulf-Gal-AuNPs) were prepared from 13 nm AuNPs, 15-mer thrombin-binding aptamer (TBA(15)), 29-mer thrombin-binding aptamer (TBA(29)), and sulf-Gal. The numbers of TBA and sulf-Gal molecules per AuNP proved to have a strong impact on inhibitory potency. The best results were observed for 15-TBA(15)/TBA(29)/sulf-Gal-AuNPs (with 15 TBA(15) and 15 TBA(29) molecules per AuNP), which, because of their particularly flexible conformation and multivalency, exhibited ultrahigh binding affinity toward thrombin (K(d)=3.4×10(-12) M) and thus extremely high anticoagulant (inhibitory) potency. Compared to the case without inhibitors (the "normal" value), their measured thrombin clotting time (TCT) was 91 times longer, whereas for TBA(15) alone it was only 7.2 times longer. Their anticoagulant activity was suppressed by TBA-complementary-sequence (cTBA)-modified AuNPs (cTBA(15)/cTBA(29)-AuNPs) at a rate that was 20 times faster than that of free cTBA(15)/cTBA(29). Thus, easily prepared, low-cost, multivalent AuNPs show great potential for biomedical control of blood clotting. PMID:21850727

Hsu, Chia-Lun; Chang, Huan-Tsung; Chen, Chao-Tsen; Wei, Shih-Chun; Shiang, Yen-Chun; Huang, Chih-Ching

2011-08-17

114

Highly efficient control of thrombin activity by multivalent nanoparticles.  

UK PubMed Central (United Kingdom)

We have demonstrated that the incorporation of sulfated galactose acid (sulf-Gal) into thrombin-binding-aptamer (TBA)-conjugated gold nanoparticles (TBA-AuNPs) enables highly effective inhibition of thrombin activity toward fibrinogen. AuNP bioconjugates (TBA(15)/TBA(29)/sulf-Gal-AuNPs) were prepared from 13 nm AuNPs, 15-mer thrombin-binding aptamer (TBA(15)), 29-mer thrombin-binding aptamer (TBA(29)), and sulf-Gal. The numbers of TBA and sulf-Gal molecules per AuNP proved to have a strong impact on inhibitory potency. The best results were observed for 15-TBA(15)/TBA(29)/sulf-Gal-AuNPs (with 15 TBA(15) and 15 TBA(29) molecules per AuNP), which, because of their particularly flexible conformation and multivalency, exhibited ultrahigh binding affinity toward thrombin (K(d)=3.4×10(-12) M) and thus extremely high anticoagulant (inhibitory) potency. Compared to the case without inhibitors (the "normal" value), their measured thrombin clotting time (TCT) was 91 times longer, whereas for TBA(15) alone it was only 7.2 times longer. Their anticoagulant activity was suppressed by TBA-complementary-sequence (cTBA)-modified AuNPs (cTBA(15)/cTBA(29)-AuNPs) at a rate that was 20 times faster than that of free cTBA(15)/cTBA(29). Thus, easily prepared, low-cost, multivalent AuNPs show great potential for biomedical control of blood clotting.

Hsu CL; Chang HT; Chen CT; Wei SC; Shiang YC; Huang CC

2011-09-01

115

G-quadruplex-based DNAzyme for facile colorimetric detection of thrombin.  

Science.gov (United States)

Thrombin-binding aptamer is found to bind hemin to form a catalytic complex whose activity is significantly promoted by the addition of thrombin, which enables the colorimetric detection of thrombin with high specificity and sensitivity in a facile way. PMID:18665289

Li, Tao; Wang, Erkang; Dong, Shaojun

2008-06-09

116

Thrombin promotes epithelial ovarian cancer cell invasion by inducing epithelial-mesenchymal transition  

Science.gov (United States)

Objective Over-expression of thrombin in ovarian cancer cells is associated with poor prognosis. In this study, we investigated the role of thrombin in inducing epithelial-mesenchymal transition (EMT) in SKOV3 epithelial ovarian cancer cells. Methods After thrombin treatment SKOV3 cells were subjected to western blots, reverse-transcription PCR, and enzyme-linked immunosorbent assay to quantify EMT-related proteins, mRNA expression of SMAD2, DKK1, and sFRP1, and the secretion of matrix metalloproteinases (MMPs) and cytokines. Meanwhile, invasion ability was evaluated using transwell assays. Results The results indicated a dose- and time-dependent down-regulation of E-cadherin and upregulation of N-cadherin and vimentin in thrombin-treated SKOV3 cells, compared with the thrombin-free control group (psFRP1 mRNA in thrombin-treated SKOV3 cells compared to control cells (p<0.05). Thrombin-treated SKOV3 cells exhibited increased secretion of MMP-9, MMP-2, interleukin (IL)-8, and IL-6 and increased invasion compared to untreated cells (p<0.05). Thrombin altered the morphology of SKOV3 cells to a spindle-like phenotype. Addition of hirudin to thrombin-treated cells reversed the effects of thrombin. Conclusion Thrombin induced EMT and promoted the invasion of SKOV3 cells, possibly via distinct signaling pathways. Hirudin inhibited the effects of thrombin, suggesting that anticoagulant therapy could be a novel therapeutic strategy for ovarian carcinoma.

Zhong, Yi-Cun; Zhang, Ting; Di, Wen

2013-01-01

117

Differential profiles of thrombin inhibitors (heparin, hirudin, bivalirudin, and dabigatran) in the thrombin generation assay and thromboelastography in vitro.  

UK PubMed Central (United Kingdom)

Thrombin is a central enzyme in hemostasis and thrombosis, and a proven target for anticoagulant therapies. We compared four marketed and representative thrombin inhibitors, heparin, hirudin, bivalirudin, and dabigatran, in in-vitro spike-in assays that covered their therapeutic ranges. The assays employed were low tissue factor (1 pmol/l)-triggered thrombin generation assay (TGA) with plasma and 1:8000 Recombiplastin-triggered thromboelastography (TEG) with whole blood, with or without tissue plasminogen activator (tPA)-induced fibrinolysis. The three direct thrombin inhibitors (DTIs) prolonged TGA lag time and TEG clotting time (R) with a potency stack-ranking of hirudin>dabigatran approximately equal to bivalirudin. Heparin had the most steep concentration-response curve for both parameters. In TGA, 1-2 ?mol/l dabigatran or hirudin resulted in complete inhibition on peak, slope, and endogenous thrombin potential, whereas bivalirudin had no effect on these parameters up to 10 ?mol/l. All three DTIs, but not heparin, displayed a paradoxical increase in peak and slope in the low concentration range. In TEG, whereas all four agents reduced clot strength (maximal amplitude) in synergy with tPA, hirudin was the only DTI that reduced maximal amplitude appreciably without tPA. Dabigatran had the strongest potentiating effect on tPA-induced fibrinolytic activity (Ly30). With regard to the effects on coagulation and clot strength (lag time, R, and maximal amplitude) in the respective therapeutic range, dabigatran elicited the most modest changes. In summary, our observations highlight the distinct features of each agent in thrombin generation, coagulation, and fibrinolysis. The contrasts between the agents are consistent with their known properties and are informative on efforts to define the optimal profiles of new anticoagulants.

Xu Y; Wu W; Wang L; Chintala M; Plump AS; Ogletree ML; Chen Z

2013-04-01

118

Thrombin regulates components of the fibrinolytic system in human mesangial cells  

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Besides its procoagulant activity, thrombin has been shown to stimulate cell proliferation and to regulate the fibrinolytic pathway. We report here the effect of purified human alpha thrombin on the synthesis of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) by cultured human mesangial cells. Thrombin (0 to 2.5 U/ml) increased in a time- and dose-dependent manner the production of t-PA and PAI-1 (2- to 3-fold increase of secreted t-PA and PAI-1 release during a 24 hour incubation). This effect was associated with a twofold increase in DNA synthesis measured by 3H-thymidine incorporation. Zymographic analysis and reverse fibrin autography showed that thrombin also increased the level of the 110 Kd t-PA-PAI-1 complex, whereas PAI-1 was present as a free 50 Kd form in the culture medium conditioned by unstimulated and thrombin-stimulated cells. Free t-PA was never observed. Both membrane binding and catalytic activity of thrombin were required since the effects of 1 U/ml thrombin were inhibited by addition 2 U/ml hirudin, which inhibits the membrane binding and catalytic activity of thrombin, and since DFP-inactivated thrombin, which has the ability to bind but which has no enzymatic activity, did not induce t-PA or PAI-1. Gamma thrombin, which does not bind to thrombin receptor, did not increase t-PA and PAI-1 releases. The effects of thrombin were probably mediated by protein kinase C activation since H7, an inhibitor of protein kinases, inhibited significantly thrombin effects on t-PA and PAI-1 production, and since addition of an activator of protein kinase A, 8-bromocyclic AMP (100 microM), induced a significant inhibition of the thrombin effect. The effects of thrombin were also suppressed by 1.25 micrograms/ml alpha amanitin, suggesting a requirement of de novo RNA synthesis.

Villamediana, L.M.; Rondeau, E.; He, C.J.; Medcalf, R.L.; Peraldi, M.N.; Lacave, R.; Delarue, F.; Sraer, J.D. (INSERM Unite 64, Paris (France))

1990-11-01

119

Thrombin regulates components of the fibrinolytic system in human mesangial cells  

International Nuclear Information System (INIS)

Besides its procoagulant activity, thrombin has been shown to stimulate cell proliferation and to regulate the fibrinolytic pathway. We report here the effect of purified human alpha thrombin on the synthesis of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) by cultured human mesangial cells. Thrombin (0 to 2.5 U/ml) increased in a time- and dose-dependent manner the production of t-PA and PAI-1 (2- to 3-fold increase of secreted t-PA and PAI-1 release during a 24 hour incubation). This effect was associated with a twofold increase in DNA synthesis measured by 3H-thymidine incorporation. Zymographic analysis and reverse fibrin autography showed that thrombin also increased the level of the 110 Kd t-PA-PAI-1 complex, whereas PAI-1 was present as a free 50 Kd form in the culture medium conditioned by unstimulated and thrombin-stimulated cells. Free t-PA was never observed. Both membrane binding and catalytic activity of thrombin were required since the effects of 1 U/ml thrombin were inhibited by addition 2 U/ml hirudin, which inhibits the membrane binding and catalytic activity of thrombin, and since DFP-inactivated thrombin, which has the ability to bind but which has no enzymatic activity, did not induce t-PA or PAI-1. Gamma thrombin, which does not bind to thrombin receptor, did not increase t-PA and PAI-1 releases. The effects of thrombin were probably mediated by protein kinase C activation since H7, an inhibitor of protein kinases, inhibited significantly thrombin effects on t-PA and PAI-1 production, and since addition of an activator of protein kinase A, 8-bromocyclic AMP (100 microM), induced a significant inhibition of the thrombin effect. The effects of thrombin were also suppressed by 1.25 micrograms/ml alpha amanitin, suggesting a requirement of de novo RNA synthesis.

1990-01-01

120

The NMR solution structure of recombinant RGD-hirudin  

International Nuclear Information System (INIS)

The solution structure of a new recombinant RGD-hirudin, which has the activities of anti-thrombin and anti-platelet aggregation, was determined by 1H nuclear magnetic resonance spectroscopy and compared with the conformations of recombinant wild-type hirudin and hirudin (variant 2, Lys47) of the hirudin thrombin complex. On the basis of total 1284 distance and dihedral angle constraints derived from a series of NMR spectra, 20 conformers were computed with ARIA/CNS programs. The structure of residues 3-30 and 37-48 form a molecular core with two antiparallel ?-sheets as the other two hirudins. However, significant differences were found in the surface electrostatic charge distributions among the three hirudins, especially in the RGD segment of recombinant RGD-hirudin. This difference may be greatly beneficial to its additional function of anti-platelet aggregation. The difference in extended C-terminal makes its both ionic and hydrophobic interactions with the fibrinogen recognition exosite of thrombin more effective.

2000-01-00

 
 
 
 
121

A Guided Mode Resonance Aptasensor for Thrombin Detection  

Directory of Open Access Journals (Sweden)

Full Text Available Recent developments in aptamers have led to their widespread use in analytical and diagnostic applications, particularly for biosensing. Previous studies have combined aptamers as ligands with various sensors for numerous applications. However, merging the aptamer developments with guided mode resonance (GMR) devices has not been attempted. This study reports an aptasensor based home built GMR device. The 29-mer thrombin aptamer was immobilized on the surface of a GMR device as a recognizing ligand for thrombin detection. The sensitivity reported in this first trial study is 0.04 nm/?M for thrombin detection in the concentration range from 0.25 to 1 ?M and the limit of detection (LOD) is 0.19 ?M. Furthermore, the binding affinity constant (Ka) measured is in the range of 106 M?1. The investigation has demonstrated that such a GMR aptasensor has the required sensitivity for the real time, label-free, in situ detection of thrombin and provides kinetic information related to the binding.

Sheng-Fu Lin; Ting-Jou Ding; Jen-Tsai Liu; Chien-Chieh Lee; Tsung-Hsun Yang; Wen-Yih Chen; Jenq-Yang Chang

2011-01-01

122

Thrombin in the management of full thickness macular holes.  

UK PubMed Central (United Kingdom)

PURPOSE: As thrombin is a known stimulator of retinal glial and pigment epithelial cells, we performed a pilot study to evaluate the use of thrombin as adjunctive mitogen therapy in vitreous surgery for full-thickness macular holes. METHODS: Macular hole surgery was performed on 25 eyes of 24 consecutive patients with stage II, III, or IV macular holes. After removal of the posterior hyaloid, peeling of epiretinal membranes, and fluid-air exchange, 0.05 mL or 0.1 mL of thrombin (100 units/ mL) was placed over the macular hole. After infusion of a gas tamponade, the patient's head was kept in a supine position for 1 hour, and then was kept in a prone position for approximately 2 weeks. RESULTS: Closure of the macular hole with one procedure was achieved in 80% of the eyes. Sixty-five percent of the eyes with a closed macular hole achieved a visual acuity of 20/40 or better. Postoperative inflammation was present in all eyes, and a small hypopyon developed in 28% of the eyes. CONCLUSION: Thrombin therapy failed to markedly increase the success rate of macular hole surgery.

Vine AK; Johnson MW

1996-01-01

123

Mutant N143P Reveals How Na+ Activates Thrombin*  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The molecular mechanism of thrombin activation by Na+ remains elusive. Its kinetic formulation requires extension of the classical Botts-Morales theory for the action of a modifier on an enzyme to correctly account for the contribution of the E*, E, and E:Na+ forms. The extended scheme establishes t...

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

124

Translational success stories: development of direct thrombin inhibitors.  

UK PubMed Central (United Kingdom)

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.

Coppens M; Eikelboom JW; Gustafsson D; Weitz JI; Hirsh J

2012-09-01

125

Thrombin induces an inflammatory phenotype in a human brain endothelial cell line.  

Science.gov (United States)

In this study, we utilized the human brain endothelial cell line, hCMEC/D3, to determine the effects of the coagulation factor, thrombin, on the human blood-brain barrier (BBB). We show that thrombin increased the mRNA and cell surface levels of ICAM-1 and VCAM-1 in hCMEC/D3 cells. Thrombin similarly upregulated several chemokines implicated in human neurological conditions. Additionally, the paracellular permeability of the human BBB in vitro was also increased following thrombin treatment. Overall, this study demonstrates that thrombin can effectively induce an inflamed phenotype in an in vitro human BBB. PMID:22381244

Alabanza, Leah M; Bynoe, Margaret S

2012-02-29

126

Thrombin induces an inflammatory phenotype in a human brain endothelial cell line.  

UK PubMed Central (United Kingdom)

In this study, we utilized the human brain endothelial cell line, hCMEC/D3, to determine the effects of the coagulation factor, thrombin, on the human blood-brain barrier (BBB). We show that thrombin increased the mRNA and cell surface levels of ICAM-1 and VCAM-1 in hCMEC/D3 cells. Thrombin similarly upregulated several chemokines implicated in human neurological conditions. Additionally, the paracellular permeability of the human BBB in vitro was also increased following thrombin treatment. Overall, this study demonstrates that thrombin can effectively induce an inflamed phenotype in an in vitro human BBB.

Alabanza LM; Bynoe MS

2012-04-01

127

An affinity capture involved enzymatic assay for thrombin by using peptide aptamers as affinity ligands on magnetic beads.  

UK PubMed Central (United Kingdom)

Here we present a sensitive and specific assay for thrombin through the affinity capture of thrombin with mRNA display generated peptide aptamers on magnetic beads and the subsequent thrombin-involved enzymatic reaction. Thrombin at 50 fM can be detected.

Zhao Q; Gao J

2013-09-01

128

Therapeutic thrombin injection of pseudoaneurysms: a multicenter experience.  

UK PubMed Central (United Kingdom)

The standard non-invasive treatment of pseudoaneurysms has been ultrasound-guided compression (UGC). Problems with UGC include pain at the site of compression, long compression times and incomplete closure. Each of these difficulties is exacerbated with large pseudoaneurysms. Recently, ultrasound-guided injection of pseudoaneurysms with thrombin has gained popularity. The goal of this study was to report a multicenter registry using this technique and in so doing detail the clinical utility and safety of this emerging procedure. The medical records of all patients diagnosed with pseudoaneurysm in the vascular laboratory who underwent thrombin injection over the past year were reviewed for patient characteristics and clinical outcome. There were 91 patients (55 male) with a mean age of 69 years. Three patients also had an arteriovenous fistula. The majority of patients were receiving one or more antiplatelet agents and/or anticoagulants. All patients underwent pseudoaneurysm injection with bovine thrombin. The mean aneurysm diameter was 3.3 cm, with a range of 1.5-6.3 cm. Successful thrombosis of the pseudoaneurysm was achieved in 89/91 (98%) of cases. Anticoagulation with heparin was used in one of the unsuccessful cases. In two cases, UGC was used to close a small active region that did not completely thrombose after thrombin injection. There were two patients who had recurrence of pseudoaneurysm the day after successful injection and thrombosis of the pseudoaneurysm. There were no local complications after injection; however, one patient suffered a pulmonary embolus that was thought to be unrelated to the procedure. In conclusion, thrombin injection for the treatment of pseudoaneurysms is safe and effective, even in patients receiving anticoagulation. This procedure should be considered as the initial therapeutic approach for peripheral pseudoaneurysms.

Mohler ER 3rd; Mitchell ME; Carpenter JP; Strandness DE Jr; Jaff MR; Beckman JA; Gerhard-Herman M

2001-11-01

129

Evaluation of DNA aptamers directed to thrombin as potential thrombus imaging agents  

Energy Technology Data Exchange (ETDEWEB)

Two DNA aptamers directed against two separate exosites on human {alpha}-thrombin were evaluated for thrombus-imaging potential. Aptamer ODN 1 is directed to the thrombin substrate binding site (exosite 1). Our finding that ODN 1 competes with fibrin for binding to exosite 1 on thrombin suggests that ODN 1 will not be useful for thrombus imaging. Aptamer ODN 2 is directed against the thrombin heparin binding site (exosite 2). ODN 2 bound to model thrombi that were formed either by clotting purified fibrinogen with thrombin, or by recalcifying citrated plasma. As the thrombin content of thrombi was increased the rate of ODN 2 uptake into preformed thrombi increased, whereas the rate of release of ODN 2 out of preformed thrombi decreased. This in vitro data suggested that ODN 2 might be useful for thrombus imaging because it can bind to exosite 2 on fibrin-bound thrombin. However, in a rabbit jugular vein model using thrombus supplemented with human thrombin, ODN 2 uptake was equal to the ovalbumin control, and did not reflect thrombin content. While the in vitro results with ODN 2 were consistent with thrombus imaging, the rapid clearance of ODN 2 from circulation, combined with slow mass transfer in the clot, seem to work against in vivo thrombin-dependent imaging or washout analysis.

Dougan, Hayes E-mail: dougan@triumf.ca; Weitz, Jeffrey I.; Stafford, Alan R.; Gillespie, Kris D.; Klement, Petr; Hobbs, John B.; Lyster, Donald M

2003-01-01

130

Stimulation of phosphoinositide hydrolysis by thrombin in embryonic chick heart cells  

International Nuclear Information System (INIS)

[en] The authors have shown that muscarinic receptor stimulation results in phosphoinositide (PI) hydrolysis in dissociated embryonic chick heart cells, with maximal stimulation produced by 1 mM carbachol. In an effort to identify other stimulators of PI turnover, they studied the effects of thrombin in primary cultures of 13-day embryonic chick heart cells. Thrombin increased PI hydrolysis, as measured by the accumulation of [3H]inositol 1-phosphate in the presence of 10 mM LiCl. Stimulation occurred in a dose-dependent manner with half-maximal stimulation obtained at 0.07 U/ml and maximal stimulation at 0.3 U/ml thrombin. The effects of maximal doses of carbachol and thrombin were additive, suggesting different mechanisms of action or different populations of cells. Effects of both hormones were inhibited by pretreatment of the monolayer cultures with 4?-phorbol 12 ?-myristate 13?-acetate. The simultaneous addition of hirudin with thrombin blocked thrombin-stimulated PI turnover. An active-site-blocked derivative of thrombin was ineffective in stimulating IP formation. These data indicate a proteolytic action of thrombin in stimulation of the PI response. To their knowledge, these are the first data suggesting that there may be thrombin receptors or effects of thrombin on heart cells

1986-03-05

131

Stimulation of phosphoinositide hydrolysis by thrombin in embryonic chick heart cells  

Energy Technology Data Exchange (ETDEWEB)

The authors have shown that muscarinic receptor stimulation results in phosphoinositide (PI) hydrolysis in dissociated embryonic chick heart cells, with maximal stimulation produced by 1 mM carbachol. In an effort to identify other stimulators of PI turnover, they studied the effects of thrombin in primary cultures of 13-day embryonic chick heart cells. Thrombin increased PI hydrolysis, as measured by the accumulation of (/sup 3/H)inositol 1-phosphate in the presence of 10 mM LiCl. Stimulation occurred in a dose-dependent manner with half-maximal stimulation obtained at 0.07 U/ml and maximal stimulation at 0.3 U/ml thrombin. The effects of maximal doses of carbachol and thrombin were additive, suggesting different mechanisms of action or different populations of cells. Effects of both hormones were inhibited by pretreatment of the monolayer cultures with 4..beta..-phorbol 12 ..beta..-myristate 13..cap alpha..-acetate. The simultaneous addition of hirudin with thrombin blocked thrombin-stimulated PI turnover. An active-site-blocked derivative of thrombin was ineffective in stimulating IP formation. These data indicate a proteolytic action of thrombin in stimulation of the PI response. To their knowledge, these are the first data suggesting that there may be thrombin receptors or effects of thrombin on heart cells.

Jones, L.G.; Brown, J.H.

1986-03-05

132

Evaluation of DNA aptamers directed to thrombin as potential thrombus imaging agents  

International Nuclear Information System (INIS)

Two DNA aptamers directed against two separate exosites on human ?-thrombin were evaluated for thrombus-imaging potential. Aptamer ODN 1 is directed to the thrombin substrate binding site (exosite 1). Our finding that ODN 1 competes with fibrin for binding to exosite 1 on thrombin suggests that ODN 1 will not be useful for thrombus imaging. Aptamer ODN 2 is directed against the thrombin heparin binding site (exosite 2). ODN 2 bound to model thrombi that were formed either by clotting purified fibrinogen with thrombin, or by recalcifying citrated plasma. As the thrombin content of thrombi was increased the rate of ODN 2 uptake into preformed thrombi increased, whereas the rate of release of ODN 2 out of preformed thrombi decreased. This in vitro data suggested that ODN 2 might be useful for thrombus imaging because it can bind to exosite 2 on fibrin-bound thrombin. However, in a rabbit jugular vein model using thrombus supplemented with human thrombin, ODN 2 uptake was equal to the ovalbumin control, and did not reflect thrombin content. While the in vitro results with ODN 2 were consistent with thrombus imaging, the rapid clearance of ODN 2 from circulation, combined with slow mass transfer in the clot, seem to work against in vivo thrombin-dependent imaging or washout analysis.

2003-01-01

133

Mechanism of thrombin-induced arachidonic acid release in osteoblast-like cells.  

UK PubMed Central (United Kingdom)

In a previous study, we have reported that thrombin stimulates phosphatidylcholine hydrolysis by phospholipase (PL) D, but has little effect on phosphoinositide hydrolysis by PLC in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the mechanism of the thrombin-induced arachidonic acid (AA) release in MC3T3-E1 cells. Thrombin stimulated AA release dose dependently in the range between 0.1 and 1 U/ml. Quinacrine, a PLA2 inhibitor, suppressed the thrombin-induced AA release. In addition, quinacrine also suppressed the thrombin-induced prostaglandin E2 synthesis in these cells. On the other hand, propranolol, which is known to inhibit phosphatidic acid phosphohydrolase, did not affect the thrombin-induced AA release. 1(6-((17beta-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H- pyrrole-2,5-dione (U-73122), a PLC inhibitor, had no effect on the AA release by thrombin. In addition, 1,6-bis-(cyclohexyloximinocarbonylamino)-hexane (RHC-80267), a selective inhibitor of diacylglycerol lipase, had little effect on the thrombin-induced AA release. Neither propranolol, U-73122 nor RHC-80267 affect the thrombin-induced prostaglandin E2 synthesis. These results strongly suggest that thrombin induces AA release not by phosphatidylcholine hydrolysis by PLD nor phosphoinositide hydrolysis by PLC but mainly by PLA2 in osteoblast-like cells.

Suzuki A; Kozawa O; Shinoda J; Watanabe-Tomita Y; Saito H; Oiso Y

1997-06-01

134

Roles of low specificity and cofactor interaction sites on thrombin during factor XIII activation. Competition for cofactor sites on thrombin determines its fate.  

Science.gov (United States)

Factor XIII is activated by thrombin, and this reaction is enhanced by the presence of fibrin(ogen). Using a substrate-based screening assay for factor XIII activity complemented by kinetic analysis of activation peptide cleavage, we show by using thrombin mutants of surface-exposed residues that Arg-178, Arg-180, Asp-183, Glu-229, Arg-233, and Trp-50 of thrombin are necessary for direct activation of factor XIII. These residues define a low specificity site known to be important also for both protein C activation and for inhibition of thrombin by antithrombin. The enhancing effect of fibrinogen occurs as a consequence of its conversion to fibrin and subsequent polymerization. Surface residues of thrombin further involved in high specificity fibrin-enhanced factor XIII activation were identified as His-66, Tyr-71, and Asn-74. These residues represent a distinct interaction site on thrombin (within exosite I) also employed by thrombomodulin in its cofactor-enhanced activation of protein C. In competition experiments, thrombomodulin inhibited fibrin-enhanced factor XIII activation. Based upon these and prior published results, we propose that the polymerization process forms a fibrin cofactor that acts to approximate thrombin and factor XIII bound to separate and complementary domains of fibrinogen. This enables enhanced factor XIII activation to be localized around the fibrin clot. We also conclude that proximity to and competition for cofactor interaction sites primarily directs the fate of thrombin. PMID:12794066

Philippou, Helen; Rance, James; Myles, Timothy; Hall, Scott W; Ariens, Robert A; Grant, Peter J; Leung, Lawrence; Lane, David A

2003-06-06

135

Roles of low specificity and cofactor interaction sites on thrombin during factor XIII activation. Competition for cofactor sites on thrombin determines its fate.  

UK PubMed Central (United Kingdom)

Factor XIII is activated by thrombin, and this reaction is enhanced by the presence of fibrin(ogen). Using a substrate-based screening assay for factor XIII activity complemented by kinetic analysis of activation peptide cleavage, we show by using thrombin mutants of surface-exposed residues that Arg-178, Arg-180, Asp-183, Glu-229, Arg-233, and Trp-50 of thrombin are necessary for direct activation of factor XIII. These residues define a low specificity site known to be important also for both protein C activation and for inhibition of thrombin by antithrombin. The enhancing effect of fibrinogen occurs as a consequence of its conversion to fibrin and subsequent polymerization. Surface residues of thrombin further involved in high specificity fibrin-enhanced factor XIII activation were identified as His-66, Tyr-71, and Asn-74. These residues represent a distinct interaction site on thrombin (within exosite I) also employed by thrombomodulin in its cofactor-enhanced activation of protein C. In competition experiments, thrombomodulin inhibited fibrin-enhanced factor XIII activation. Based upon these and prior published results, we propose that the polymerization process forms a fibrin cofactor that acts to approximate thrombin and factor XIII bound to separate and complementary domains of fibrinogen. This enables enhanced factor XIII activation to be localized around the fibrin clot. We also conclude that proximity to and competition for cofactor interaction sites primarily directs the fate of thrombin.

Philippou H; Rance J; Myles T; Hall SW; Ariens RA; Grant PJ; Leung L; Lane DA

2003-08-01

136

Crystallization and preliminary X-ray analysis of the complex of human alpha-thrombin with a modified thrombin-binding aptamer.  

Science.gov (United States)

The thrombin-binding aptamer (TBA) is a consensus DNA 15-mer that binds specifically to human alpha-thrombin at nanomolar concentrations and inhibits its procoagulant functions. Recently, a modified TBA (mTBA) containing a 5'-5' inversion-of-polarity site has been shown to be more stable and to possess a higher thrombin affinity than its unmodified counterpart. The structure of the thrombin-TBA complex has previously been determined at low resolution, but did not provide a detailed picture of the aptamer conformation or of the protein-DNA assembly, while that of the complex with mTBA is unknown. Crystallographic analysis of the thrombin-mTBA complex has been attempted. The crystals diffracted to 2.15 A resolution and belonged to space group I222. PMID:20693681

Russo Krauss, Irene; Merlino, Antonello; Randazzo, Antonio; Mazzarella, Lelio; Sica, Filomena

2010-07-29

137

Crystallization and preliminary X-ray analysis of the complex of human alpha-thrombin with a modified thrombin-binding aptamer.  

UK PubMed Central (United Kingdom)

The thrombin-binding aptamer (TBA) is a consensus DNA 15-mer that binds specifically to human alpha-thrombin at nanomolar concentrations and inhibits its procoagulant functions. Recently, a modified TBA (mTBA) containing a 5'-5' inversion-of-polarity site has been shown to be more stable and to possess a higher thrombin affinity than its unmodified counterpart. The structure of the thrombin-TBA complex has previously been determined at low resolution, but did not provide a detailed picture of the aptamer conformation or of the protein-DNA assembly, while that of the complex with mTBA is unknown. Crystallographic analysis of the thrombin-mTBA complex has been attempted. The crystals diffracted to 2.15 A resolution and belonged to space group I222.

Russo Krauss I; Merlino A; Randazzo A; Mazzarella L; Sica F

2010-08-01

138

Aptamer-based surface plasmon fibre sensor for thrombin detection  

Science.gov (United States)

A series of surface plasmonic fibre devices were fabricated using multiple coatings deposited on a lapped section of a single mode fibre and post-fabrication UV laser irradiation processing with a phase mask, producing a surface relief grating structure. These devices showed high spectral sensitivity in the aqueous index regime ranging up to 4000 nm/RIU for wavelength and 800 dB/RIU for intensity. The devices were then coated with human thrombin binding aptamer. Several concentrations of thrombin in buffer solution were made, ranging from 1nM to 1?M. All the concentrations were detectable by the devices demonstrating that sub-nM concentrations may be monitored.

Allsop, T.; Nagel, D.; Neal, R.; Davies, E. M.; Mou, C.; Bond, P.; Rehman, S.; Kalli, K.; Webb, D. J.; Calverhouse, P.; Mascini, M.; Bennion, I.

2010-04-01

139

Thrombin aptasensing with inherently electroactive graphene oxide nanoplatelets as labels.  

UK PubMed Central (United Kingdom)

Graphene and its associated materials are commonly used as the transducing platform in biosensing. We propose a different approach for the application of graphene in biosensing. Here, we utilized graphene oxide nanoplatelets as the inherently electroactive labels for the aptasensing of thrombin. The basis of detection lies in the ability of graphene oxide to be electrochemically reduced, thereby providing a well-defined reduction wave; one graphene oxide nanoplatelet of dimension 50 × 50 nm can provide a reduction signal by accepting ~22,000 electrons. We demonstrate that by using graphene oxide nanoplatelets as an inherently electroactive label, we can detect thrombin in the concentration range of 3 pM-0.3 ?M, with good selectivity of the aptamer towards interferences by bovine serum albumin, immunoglobulin G and avidin. Therefore, the inherently electroactive graphene oxide nanoplatelets are a material which can serve as an electroactive label, in a manner similar to metallic nanoparticles.

Loo AH; Bonanni A; Pumera M

2013-06-01

140

Thrombin aptasensing with inherently electroactive graphene oxide nanoplatelets as labels  

Science.gov (United States)

Graphene and its associated materials are commonly used as the transducing platform in biosensing. We propose a different approach for the application of graphene in biosensing. Here, we utilized graphene oxide nanoplatelets as the inherently electroactive labels for the aptasensing of thrombin. The basis of detection lies in the ability of graphene oxide to be electrochemically reduced, thereby providing a well-defined reduction wave; one graphene oxide nanoplatelet of dimension 50 × 50 nm can provide a reduction signal by accepting ~22 000 electrons. We demonstrate that by using graphene oxide nanoplatelets as an inherently electroactive label, we can detect thrombin in the concentration range of 3 pM-0.3 ?M, with good selectivity of the aptamer towards interferences by bovine serum albumin, immunoglobulin G and avidin. Therefore, the inherently electroactive graphene oxide nanoplatelets are a material which can serve as an electroactive label, in a manner similar to metallic nanoparticles.

Loo, Adeline Huiling; Bonanni, Alessandra; Pumera, Martin

2013-05-01

 
 
 
 
141

Tissue factor-driven thrombin generation and inflammation in atherosclerosis.  

UK PubMed Central (United Kingdom)

The transmembrane receptor tissue factor is a prominent protein expressed at macrophages and smooth muscle cells within human atherosclerotic lesions. While many coagulation proteins are detectable in atherosclerosis, a locally active thrombin and fibrin generating molecular machinery may be instrumental in manipulating cellular functions involved in atherogenesis. These include inflammation, angiogenesis and cell proliferation. Indeed, many experimental studies in mice show a correlation between hypercoagulability and increased atherosclerosis. In mice, the amount of atherosclerosis and/or the plaque phenotype, appear to be modifiable by specific anticoagulant interventions. While attempts to vary tissue factor level in the vasculature does not directly reduce plaque burden, the overexpression of tissue factor pathway inhibitor attenuates thrombogenicity and neo intima formation in mice. Moreover, inhibition of factor Xa or thrombin with novel selective agents, including rivaroxaban and dabigatran, inhibits inflammation associated with atherosclerosis in apoE(-/-) mice. The potential to modify a complex chronic disease like atherosclerosis with novel selective anticoagulants merits further clinical study.

ten Cate H

2012-05-01

142

RAPIDLY DISINTEGRATING LYOPHILIZED ORAL FORMULATIONS OF A THROMBIN RECEPTOR ANTAGONIST  

UK PubMed Central (United Kingdom)

Disclosed is a lyophilized rapidly disintegrating solid dosage form, one embodiment of which comprises a thrombin receptor antagonist such as, formul a (A), or a pharmaceutically acceptable salt or hydrate thereof, a polymer s uch as gelatin, and a matrix forming agent such as mannitol. Systems for eff ectively buffering the pre-lyophilized suspension are taught, along with met hods of treating patients at risk for acute coronary syndrome by administeri ng such a rapidly disintegrating solid dosage form.

PAVLOVSKY ANASTASIA; MONTEITH DAVID; DUGGIRALA SRINIVAS; ERBEY JOHN R II; FENG KUNG-I; VELTRI ENRICO P; CHAWDRY SULIMAN; FALVO MICHAEL ANGELO

143

The thrombogram: monitoring thrombin generation in platelet-rich plasma.  

UK PubMed Central (United Kingdom)

A method is described in which thrombin activity in clotting plasma can be monitored through the continuous measurement of the fluorescent split-product of the substrate Z-Gly-Gly-Arg-AMC. The signal is not impaired by turbidity; therefore proper measurement is not disturbed by the occurrence of a clot or the presence of platelets and direct measurement in platelet rich plasma is possible.

Hemker HC; Giesen PL; Ramjee M; Wagenvoord R; Béguin S

2000-04-01

144

Topical Anesthetics - Full Version  

Science.gov (United States)

... Topical Anesthetics - Full Version. ... Without this supervision, patients may apply large amounts of topical anesthetics to their skin. ... More results from www.fda.gov/drugs/drugsafety/drugsafetypodcasts

145

Stabilization of the E* form turns thrombin into an anticoagulant.  

Science.gov (United States)

Previous studies have shown that deletion of nine residues in the autolysis loop of thrombin produces a mutant with an anticoagulant propensity of potential clinical relevance, but the molecular origin of the effect has remained unresolved. The x-ray crystal structure of this mutant solved in the free form at 1.55 A resolution reveals an inactive conformation that is practically identical (root mean square deviation of 0.154 A) to the recently identified E* form. The side chain of Trp(215) collapses into the active site by shifting > 10 A from its position in the active E form, and the oxyanion hole is disrupted by a flip of the Glu(192)-Gly(193) peptide bond. This finding confirms the existence of the inactive form E* in essentially the same incarnation as first identified in the structure of the thrombin mutant D102N. In addition, it demonstrates that the anticoagulant profile often caused by a mutation of the thrombin scaffold finds its likely molecular origin in the stabilization of the inactive E* form that is selectively shifted to the active E form upon thrombomodulin and protein C binding. PMID:19473969

Bah, Alaji; Carrell, Christopher J; Chen, Zhiwei; Gandhi, Prafull S; Di Cera, Enrico

2009-05-27

146

Stabilization of the E* Form Turns Thrombin into an Anticoagulant  

Energy Technology Data Exchange (ETDEWEB)

Previous studies have shown that deletion of nine residues in the autolysis loop of thrombin produces a mutant with an anticoagulant propensity of potential clinical relevance, but the molecular origin of the effect has remained unresolved. The x-ray crystal structure of this mutant solved in the free form at 1.55 {angstrom} resolution reveals an inactive conformation that is practically identical (root mean square deviation of 0.154 {angstrom}) to the recently identified E* form. The side chain of Trp215 collapses into the active site by shifting >10 {angstrom} from its position in the active E form, and the oxyanion hole is disrupted by a flip of the Glu192-Gly193 peptide bond. This finding confirms the existence of the inactive form E* in essentially the same incarnation as first identified in the structure of the thrombin mutant D102N. In addition, it demonstrates that the anticoagulant profile often caused by a mutation of the thrombin scaffold finds its likely molecular origin in the stabilization of the inactive E* form that is selectively shifted to the active E form upon thrombomodulin and protein C binding.

Bah, Alaji; Carrell, Christopher J.; Chen, Zhiwei; Gandhi, Prafull S.; Di Cera, Enrico; (WU-MED)

2009-07-31

147

Stabilization of the E* form turns thrombin into an anticoagulant.  

UK PubMed Central (United Kingdom)

Previous studies have shown that deletion of nine residues in the autolysis loop of thrombin produces a mutant with an anticoagulant propensity of potential clinical relevance, but the molecular origin of the effect has remained unresolved. The x-ray crystal structure of this mutant solved in the free form at 1.55 A resolution reveals an inactive conformation that is practically identical (root mean square deviation of 0.154 A) to the recently identified E* form. The side chain of Trp(215) collapses into the active site by shifting > 10 A from its position in the active E form, and the oxyanion hole is disrupted by a flip of the Glu(192)-Gly(193) peptide bond. This finding confirms the existence of the inactive form E* in essentially the same incarnation as first identified in the structure of the thrombin mutant D102N. In addition, it demonstrates that the anticoagulant profile often caused by a mutation of the thrombin scaffold finds its likely molecular origin in the stabilization of the inactive E* form that is selectively shifted to the active E form upon thrombomodulin and protein C binding.

Bah A; Carrell CJ; Chen Z; Gandhi PS; Di Cera E

2009-07-01

148

Stabilization of the E* Form Turns Thrombin into an Anticoagulant*  

Science.gov (United States)

Previous studies have shown that deletion of nine residues in the autolysis loop of thrombin produces a mutant with an anticoagulant propensity of potential clinical relevance, but the molecular origin of the effect has remained unresolved. The x-ray crystal structure of this mutant solved in the free form at 1.55 ? resolution reveals an inactive conformation that is practically identical (root mean square deviation of 0.154 ?) to the recently identified E* form. The side chain of Trp215 collapses into the active site by shifting >10 ? from its position in the active E form, and the oxyanion hole is disrupted by a flip of the Glu192–Gly193 peptide bond. This finding confirms the existence of the inactive form E* in essentially the same incarnation as first identified in the structure of the thrombin mutant D102N. In addition, it demonstrates that the anticoagulant profile often caused by a mutation of the thrombin scaffold finds its likely molecular origin in the stabilization of the inactive E* form that is selectively shifted to the active E form upon thrombomodulin and protein C binding.

Bah, Alaji; Carrell, Christopher J.; Chen, Zhiwei; Gandhi, Prafull S.; Di Cera, Enrico

2009-01-01

149

Rational design of hirulog-type inhibitors of thrombin.  

Science.gov (United States)

The two crystal structures of thrombin complexed with its most potent natural inhibitor hirudin and with the active-site inhibitor D-Phe-Pro-Arg-CH2Cl [Rydel, T.J. et al., J. Mol. Biol., 221 (1991) 583; Bode, W. et al., EMBO J., 8 (1989) 3467] were used as a basis to design a new inhibitor, combining the high specificity of the polypeptide hirudin with the simpler chemistry of an organic compound. In the new inhibitor, the C-terminal amino acid residues 53-65 of hirudin are linked by a spacer peptide of four glycines to the active-site inhibitor NAPAP (N alpha-(2-naphthyl-sulfonyl-glycyl)-DL-p-amidinophenylalanyl-piperi dine). Energy minimization techniques served as a tool to determine the preferred configuration at the amidinophenylalanine and the modified piperidine moiety of the inhibitor. The predictions are supported by the interaction energies determined for D- and L-NAPAP in complex with thrombin, which are in good agreement with experimentally determined dissociation constants. The conformational flexibility of the linker peptide in the new inhibitors was investigated with molecular dynamics techniques. A correlation between the Pl' position and the interactions of the linker peptide with the protein is suggested. Modifications of the linker peptide are proposed based on the distribution of its main-chain torsion angles in order to enhance its binding to thrombin. PMID:7876896

Egner, U; Hoyer, G A; Schleuning, W D

1994-10-01

150

A novel hemostatic sealant composed of gelatin, transglutaminase and thrombin effectively controls liver trauma-induced bleeding in dogs.  

UK PubMed Central (United Kingdom)

AIM: novel hemostatic sealant based on the in situ gel formation of gelatin catalyzed by thrombin and crosslinked by transglutaminase was designed. The aim of this study was to investigate the efficacy of the hemostatic sealant in control of bleeding caused by liver trauma in dogs. METHODS: Hepatic trauma that mimicked the grade III-IV rupture of liver was made in 20 dogs. The traumatic lesion was topically administered the hemostatic sealant (treatment group, n=10), or a thrombin solution (control group, n=10). The time to achieve hemostasis and the blood loss were measured. Contrast-enhanced ultrasound (CEUS) examination was performed in each animal on d 3, d 7, and d 10 d postoperatively to study the healing of the lesions. RESULTS: The mean time to achieve hemostasis in the treatment group was significantly shorter than that in the control group (1.20±0.33 vs 6.70±0.64 min, P<0.05). The mean blood loss in the treatment group was significantly less than that in the control group (47.22±8.61 vs 79.29±11.97 mL, P<0.05). In CEUS examination, the traumatic lesions in the treatment group became significantly smaller on d 3, and disappeared on d 7, whereas the lesions in the control group still existed on d 10. Ascites were never found during 10 d follow-up in the treatment group but were observed on d 3 and d 7 in the control group. CONCLUSION: Compared with thrombin, the novel hemostatic sealant shows much better efficacy in hemostasis and may promote wound healing in dog liver trauma.

Xie X; Tian JK; Lv FQ; Wu R; Tang WB; Luo YK; Huang YQ; Tang J

2013-07-01

151

Topical cannabinoid enhances topical morphine antinociception.  

UK PubMed Central (United Kingdom)

Opioids and cannabinoids produce antinociception through both spinal and supraspinal action. Both opioids and cannabinoids also have important peripheral action. Many previous studies indicate that systemically administered cannabinoids enhance antinociceptive properties of opioids. Experiments were conducted to test the hypothesis that topical cannabinoids would enhance the topical antinociceptive effects of morphine. Antinociception was measured in the radiant tail-flick test after immersion of the tail of mice into a solution of dimethyl sulfoxide (DMSO) containing WIN 55, 212-2, a cannabinoid agonist and morphine, an opioid agonist. Morphine and WIN 55, 212-2 produce time dependent topical analgesic effects limited to the portion of the tail exposed to drugs. WIN 55, 212-2 had a potency lower than that of morphine. The topical antinociceptive effects of WIN 55, 212-2 were blocked by systemic pretreatment of cannabinoid CB1 receptor selective antagonist, AM 251. This suggests that topical antinociceptive effects of WIN 55, 212-2 involve CB1 receptors. Combination of topical WIN 55, 212-2 with topical morphine yielded significantly greater analgesic effects than that of topical morphine alone. The ability of the CB1 receptor antagonist AM 251 to antagonize the enhancement of antinociception of morphine by WIN 55, 212-2 indicates that WIN 55, 212-2 acts through a CB1 receptor to enhance the potency of topical morphine. Additionally, spinally administered ineffective doses of WIN 55, 212-2 potentiated the antinociceptive effects of topical morphine. These results demonstrate an antinociceptive interaction between topical opioids with topical, and spinal cannabinoids. These observations are significant in using of topical combination of cannabinoid and morphine in the management of pain.

Yesilyurt O; Dogrul A; Gul H; Seyrek M; Kusmez O; Ozkan Y; Yildiz O

2003-09-01

152

Topical cannabinoid enhances topical morphine antinociception.  

Science.gov (United States)

Opioids and cannabinoids produce antinociception through both spinal and supraspinal action. Both opioids and cannabinoids also have important peripheral action. Many previous studies indicate that systemically administered cannabinoids enhance antinociceptive properties of opioids. Experiments were conducted to test the hypothesis that topical cannabinoids would enhance the topical antinociceptive effects of morphine. Antinociception was measured in the radiant tail-flick test after immersion of the tail of mice into a solution of dimethyl sulfoxide (DMSO) containing WIN 55, 212-2, a cannabinoid agonist and morphine, an opioid agonist. Morphine and WIN 55, 212-2 produce time dependent topical analgesic effects limited to the portion of the tail exposed to drugs. WIN 55, 212-2 had a potency lower than that of morphine. The topical antinociceptive effects of WIN 55, 212-2 were blocked by systemic pretreatment of cannabinoid CB1 receptor selective antagonist, AM 251. This suggests that topical antinociceptive effects of WIN 55, 212-2 involve CB1 receptors. Combination of topical WIN 55, 212-2 with topical morphine yielded significantly greater analgesic effects than that of topical morphine alone. The ability of the CB1 receptor antagonist AM 251 to antagonize the enhancement of antinociception of morphine by WIN 55, 212-2 indicates that WIN 55, 212-2 acts through a CB1 receptor to enhance the potency of topical morphine. Additionally, spinally administered ineffective doses of WIN 55, 212-2 potentiated the antinociceptive effects of topical morphine. These results demonstrate an antinociceptive interaction between topical opioids with topical, and spinal cannabinoids. These observations are significant in using of topical combination of cannabinoid and morphine in the management of pain. PMID:14499448

Yesilyurt, Ozgur; Dogrul, Ahmet; Gul, Husamettin; Seyrek, Melik; Kusmez, Ozkan; Ozkan, Yalcin; Yildiz, Oguzhan

2003-09-01

153

Thrombin promotes epithelial ovarian cancer cell invasion by inducing epithelial-mesenchymal transition.  

UK PubMed Central (United Kingdom)

OBJECTIVE: Over-expression of thrombin in ovarian cancer cells is associated with poor prognosis. In this study, we investigated the role of thrombin in inducing epithelial-mesenchymal transition (EMT) in SKOV3 epithelial ovarian cancer cells. METHODS: After thrombin treatment SKOV3 cells were subjected to western blots, reverse-transcription PCR, and enzyme-linked immunosorbent assay to quantify EMT-related proteins, mRNA expression of SMAD2, DKK1, and sFRP1, and the secretion of matrix metalloproteinases (MMPs) and cytokines. Meanwhile, invasion ability was evaluated using transwell assays. RESULTS: The results indicated a dose- and time-dependent down-regulation of E-cadherin and upregulation of N-cadherin and vimentin in thrombin-treated SKOV3 cells, compared with the thrombin-free control group (p<0.05). There was a dose- and time-dependent increase in the levels of SMAD2 and DKK1 mRNAs and a decrease in the levels of sFRP1 mRNA in thrombin-treated SKOV3 cells compared to control cells (p<0.05). Thrombin-treated SKOV3 cells exhibited increased secretion of MMP-9, MMP-2, interleukin (IL)-8, and IL-6 and increased invasion compared to untreated cells (p<0.05). Thrombin altered the morphology of SKOV3 cells to a spindle-like phenotype. Addition of hirudin to thrombin-treated cells reversed the effects of thrombin. CONCLUSION: Thrombin induced EMT and promoted the invasion of SKOV3 cells, possibly via distinct signaling pathways. Hirudin inhibited the effects of thrombin, suggesting that anticoagulant therapy could be a novel therapeutic strategy for ovarian carcinoma.

Zhong YC; Zhang T; Di W; Li WP

2013-07-01

154

Self-assembled DNA-based giant thrombin nanoparticles for controlled release.  

UK PubMed Central (United Kingdom)

Protein-aptamer interactions have been used in a wide range of fields, including medical diagnosis and protein delivery. Herein, we report a method for thrombin delivery with thrombin-binding aptamer (TBA), which is one of the well-known aptamers for thrombin, by generating giant thrombin nanoparticles (GTNPs). GTNPs can be synthesized by crosslinking thrombin with DNA nanostructures that possess several TBA molecules. To generate GTNPs, two different DNA nanostructures were used. Y-shaped DNA with TBA and X-shaped DNA with TBA were used for 250 and 650 nm GTNPs, respectively. Controlled release of thrombin from GTNPs was performed by adding complementary DNA (cDNA) to TBA. To investigate thrombin release from GTNPs, the sizes of the GTNPs were measured using dynamic light scattering, atomic force microscopy (AFM), and scanning electron microscopy (SEM). We confirmed a decrease in the size of GTNPs with various concentrations of cDNA, suggesting the release of thrombin. Based on these results, we expect that our method can be used to control the amount of thrombin released effectively. Our method is also widely applicable for effective protein delivery.

Sung JH; Han D; Lee JB

2013-02-01

155

Self-assembled DNA-based giant thrombin nanoparticles for controlled release.  

Science.gov (United States)

Protein-aptamer interactions have been used in a wide range of fields, including medical diagnosis and protein delivery. Herein, we report a method for thrombin delivery with thrombin-binding aptamer (TBA), which is one of the well-known aptamers for thrombin, by generating giant thrombin nanoparticles (GTNPs). GTNPs can be synthesized by crosslinking thrombin with DNA nanostructures that possess several TBA molecules. To generate GTNPs, two different DNA nanostructures were used. Y-shaped DNA with TBA and X-shaped DNA with TBA were used for 250 and 650 nm GTNPs, respectively. Controlled release of thrombin from GTNPs was performed by adding complementary DNA (cDNA) to TBA. To investigate thrombin release from GTNPs, the sizes of the GTNPs were measured using dynamic light scattering, atomic force microscopy (AFM), and scanning electron microscopy (SEM). We confirmed a decrease in the size of GTNPs with various concentrations of cDNA, suggesting the release of thrombin. Based on these results, we expect that our method can be used to control the amount of thrombin released effectively. Our method is also widely applicable for effective protein delivery. PMID:23297045

Sung, Jong Hwan; Han, Daehoon; Lee, Jong Bum

2013-01-07

156

Thrombin-induced shape changes of cultured endothelial cells: metabolic and functional observations.  

Science.gov (United States)

Cultured human umbilical vein endothelial cells (EC) incubated in the presence of 2-deoxy-D-glucose (20 mmol/l) or at 4 degrees C lost their ability to undergo shape changes when exposed to thrombin (1 N.I.H. u/ml). Drugs blocking Ca++-flux (verapamil and nifedipin), microfilament disrupting agents (cytochalasin B and D) and microtubule disrupting agents (colchicine and colcemid) did not prevent thrombin-induced shape changes. None of the agents tested inhibited the accelerated thrombin-induced 51Cr-release from the cells. Pretreatment of EC with thrombin did not influence their ability to mediate clot retraction. PMID:6686347

Galdal, K S; Evensen, S A; Nilsen, E

1983-10-01

157

Thrombin-induced shape changes of cultured endothelial cells: metabolic and functional observations.  

UK PubMed Central (United Kingdom)

Cultured human umbilical vein endothelial cells (EC) incubated in the presence of 2-deoxy-D-glucose (20 mmol/l) or at 4 degrees C lost their ability to undergo shape changes when exposed to thrombin (1 N.I.H. u/ml). Drugs blocking Ca++-flux (verapamil and nifedipin), microfilament disrupting agents (cytochalasin B and D) and microtubule disrupting agents (colchicine and colcemid) did not prevent thrombin-induced shape changes. None of the agents tested inhibited the accelerated thrombin-induced 51Cr-release from the cells. Pretreatment of EC with thrombin did not influence their ability to mediate clot retraction.

Galdal KS; Evensen SA; Nilsen E

1983-10-01

158

Dose-dependent differential effects of thrombin in allergic bronchial asthma.  

UK PubMed Central (United Kingdom)

BACKGROUND: Apart from its role in the coagulation system, thrombin plays an important role in the inflammatory response through its protease-activated receptor(PAR)s. However, the role of thrombin in the immune response is not clear. OBJECTIVE: We evaluated if thrombin has a modulatory role in allergic bronchial asthma. METHODS: Bronchial asthma was induced in mice by intraperitoneal sensitization and inhalation challenge with ovalbumin. Thrombin or its inhibitors were administered by inhalation before each allergen challenge. RESULTS: Mice with low but sustained coagulation activation had reduced allergic inflammation and allergic asthma was inhibited by low doses but worsened by high doses of thrombin. Allergic asthma was worsened by antithrombin, argatroban, hirudin and anti-thrombomodulin antibody. Mice with an increased concentration of an inhibitor of both thrombin and activated protein C had worsened disease. Heterozygous PAR-1 mice had less allergic inflammation but PAR-1 agonist worsened it. Allergic bronchial inflammation was worsened in mice that received adoptive transfer of PAR-1 agonist-treated Th2 cells compared to controls. Low concentrations of thrombin suppressed but high-dose of it enhanced maturation and secretion of cytokines in dendritic cells. CONCLUSIONS: The effects of thrombin on allergic asthma are dose-dependent with detrimental effects at high dose and protective ones at low dose. These data demonstrate that thrombin modulates the outcome in allergic bronchial asthma. This article is protected by copyright. All rights reserved.

Miyake Y; D'Alessandro-Gabazza CN; Takagi T; Naito M; Hataji O; Nakahara H; Yuda H; Fujimoto H; Kobayashi H; Yasuma T; Toda M; Kobayashi T; Yano Y; Morser J; Taguchi O; Gabazza EC

2013-08-01

159

Real time monitoring of thrombin interactions with its aptamers: insights into the sandwich complex formation.  

UK PubMed Central (United Kingdom)

Aptamers are raising an increasing interest for biosensor applications as replacements for antibodies due to their high stability and low cost. Thrombin, a key enzyme in the coagulation cascade, is an archetypical target against which two different aptamers, binding to two different exosites, have been selected. Recent studies dedicated to thrombin monitoring applications of biosensors have taken advantage of a potential sandwich-like structure between thrombin and these two aptamers for amplification purposes. However, in most cases, only end-point analysis was observed as a result of labeling requirements, thus preventing access to the kinetics of the complex formation. By using Surface Plasmon Resonance (SPR) imaging of aptamer-functionalized biosensors, we followed the binding of thrombin on the sensor and its interaction with a second reporter aptamer in real-time and in a label-free manner. Surprisingly, we showed that the injection of a second unlabeled-aptamer following the previous thrombin injection destabilized the thrombin-aptamer complex formed on the sensor surface, thus limiting any further amplification. However, the direct co-injection of thrombin, pre-complexed with a biotinylated aptamer bound to streptavidin efficiently increased the SPR signal by comparison to single thrombin detection. The various injection sequences performed may be rationalized considering a poor selectivity of one of the aptamers towards its exosite and a further negative allosteric effect upon sandwich complexation of the thrombin with its aptamers.

Daniel C; Mélaïne F; Roupioz Y; Livache T; Buhot A

2013-02-01

160

Topic: A Literature Review  

Digital Repository Infrastructure Vision for European Research (DRIVER)

There is a wide discussion for Chinese topic structure and topic-sentence acquisition in Second Language Acquisition since Li & Thompson (1976). This paper reviews the contribution made by Li & Thompson on topic and later researches on the basis of them. The relationship between subject and topic al...

Dandan Zhang

 
 
 
 
161

Topic: A Literature Review  

Directory of Open Access Journals (Sweden)

Full Text Available There is a wide discussion for Chinese topic structure and topic-sentence acquisition in Second Language Acquisition since Li & Thompson (1976). This paper reviews the contribution made by Li & Thompson on topic and later researches on the basis of them. The relationship between subject and topic also is concentrated.

Dandan Zhang

2009-01-01

162

Matrix metalloproteinase-10 effectively reduces infarct size in experimental stroke by enhancing fibrinolysis via a thrombin-activatable fibrinolysis inhibitor-mediated mechanism.  

UK PubMed Central (United Kingdom)

BACKGROUND: The fibrinolytic and matrix metalloproteinase (MMP) systems cooperate in thrombus dissolution and extracellular matrix proteolysis. The plasminogen/plasmin system activates MMPs, and some MMPs have been involved in the dissolution of fibrin by targeting fibrin(ogen) directly or by collaborating with plasmin. MMP-10 has been implicated in inflammatory/thrombotic processes and vascular integrity, but whether MMP-10 could have a profibrinolytic effect and represent a promising thrombolytic agent is unknown. METHODS AND RESULTS: The effect of MMP-10 on fibrinolysis was studied in vitro and in vivo, in MMP-10-null mice (Mmp10(-/-)), with the use of 2 different murine models of arterial thrombosis: laser-induced carotid injury and ischemic stroke. In vitro, we showed that MMP-10 was capable of enhancing tissue plasminogen activator-induced fibrinolysis via a thrombin-activatable fibrinolysis inhibitor inactivation-mediated mechanism. In vivo, delayed fibrinolysis observed after photochemical carotid injury in Mmp10(-/-) mice was reversed by active recombinant human MMP-10. In a thrombin-induced stroke model, the reperfusion and the infarct size in sham or tissue plasminogen activator-treated animals were severely impaired in Mmp10(-/-) mice. In this model, administration of active MMP-10 to wild-type animals significantly reduced blood reperfusion time and infarct size to the same extent as tissue plasminogen activator and was associated with shorter bleeding time and no intracranial hemorrhage. This effect was not observed in thrombin-activatable fibrinolysis inhibitor-deficient mice, suggesting thrombin-activatable fibrinolysis inhibitor inactivation as one of the mechanisms involved in the MMP-10 profibrinolytic effect. CONCLUSIONS: A novel profibrinolytic role for MMP-10 in experimental ischemic stroke is described, opening new pathways for innovative fibrinolytic strategies in arterial thrombosis.

Orbe J; Barrenetxe J; Rodriguez JA; Vivien D; Orset C; Parks WC; Birkland TP; Serrano R; Purroy A; Martinez de Lizarrondo S; Angles-Cano E; Páramo JA

2011-12-01

163

[Factors influencing thrombin generation measured as thrombin-antithrombin complexes levels and using calibrated automated thrombogram in patients with advanced coronary artery disease].  

UK PubMed Central (United Kingdom)

INTRODUCTION: Haemostatic factors play an important role in atherothrombosis. Thrombin generation is a crucial stage of blood coagulation. OBJECTIVES: Comparison of different thrombin generation markers: thrombin-antithrombin complex (TAT) generation and calibrated automated thrombogram method (CAT). Identification of factors influencing thrombin generation in patients with stable angina (SA) enrolled to the coronary artery bypass grafting (CABG) surgery. Analysis of traditional (age, gender, hypertension and diabetes) and novel (fibrinogen and C-reactive protein [CRP]) risk factors and the antiplatelet therapy (aspirin 75-150 mg/d) in relation to coagulation. PATIENTS AND METHODS: In 135 SA patients with left main coronary artery stenosis (> 50%) or major epicardial artery stenosis (> 70%), plasma TAT levels, maximal thrombin concentration (C(max)) and endogenous thrombin potential (ETP) were determined. A marker of the platelet activation (beta-thromboglobulin) was also measured. RESULTS: No correlations among TAT, C(max), ETP, risk factors and beta-thromboglobulin were observed. Linear regression model showed that independent predictors of TAT levels were age (beta = 0.5; p = < 0.0001), male gender and diabetes (beta = 0.36; p = 0.02). CRP independently predicted TAT and ETP (beta = -0.24 and beta = 0.22; p < 0.05, respectively), while fibrinogen predicted C(max) (beta = 0.21; p < 0.05). Independent predictors of beta-thromboglobulin were a male gender and aspirin use cessation (beta = 0.46; p = 0.01). Aspirin treatment had no effect on thrombin generation. CONCLUSIONS: Age, higher fibrinogen, CRP, diabetes and male gender influence thrombin generation and/or coagulation activation in SA patients. Plasma levels of thrombin-antithrombin complexes do not correlate with the parameters obtained using the calibrated automated thrombogram method (C(max), ETP).

Stepie? E; Plicner D; Branicka A; Stankiewicz E; Pazdan A; Sniezek-Maciejewska M; Górkiewicz I; Kapelak B; Sadowski J

2007-07-01

164

Topical report review status  

Energy Technology Data Exchange (ETDEWEB)

This report provides industry with procedures for submitting topical reports, guidance on how the U.S. Nuclear Regulatory Commission (NRC) processes and responds to topical report submittals, and an accounting, with review schedules, of all topical reports currently accepted for review schedules, of all topical reports currently accepted for review by the NRC. This report will be published annually. Each sponsoring organization with one or more topical reports accepted for review copies.

NONE

1997-08-01

165

Topical report review status  

International Nuclear Information System (INIS)

This report provides industry with procedures for submitting topical reports, guidance on how the U.S. Nuclear Regulatory Commission (NRC) processes and responds to topical report submittals, and an accounting, with review schedules, of all topical reports currently accepted for review schedules, of all topical reports currently accepted for review by the NRC. This report will be published annually. Each sponsoring organization with one or more topical reports accepted for review copies.

1997-01-01

166

Sustained heparin effect contributes to reduced plasma thrombin generation capacity early after cardiac surgery.  

UK PubMed Central (United Kingdom)

INTRODUCTION: Thrombin is a key component in the coagulation cascade, and impaired thrombin generation has been linked to increased bleeding after surgical procedures. The aim was to evaluate postoperative thrombin generation capacity in plasma after cardiac surgery, and its potential associations to activity of individual coagulation factors and heparin. MATERIAL AND METHODS: Forty-eight coronary artery bypass grafting patients were included in a prospective observational cohort study. Thrombin generation capacity was analysed in plasma with calibrated automated thrombogram with tissue factor as activator before (baseline), and 2 h and 24 h after surgery. In addition, plasma activity of coagulation factors II, V, VII, VIII, IX, X, XI, XIII, were determined. Heparin effect was assessed by anti-Xa activity, APTT and thrombin time. RESULTS: Thrombin generation was markedly reduced 2h after surgery compared to baseline. Peak levels decreased with median 74% (interquartile range 52-90), p<0.001, and endogenous thrombin generation potential decreased with 65% (43-86), p<0.001. Postoperative changes in endogenous thrombin generation potential correlated inversely to changes in anti-Xa activity (r=-0.51, p=0.010) and to changes in thrombin time (r=-0.51, p=0.009), but there were no correlations to changes in individual coagulation factor activity. CONCLUSIONS: A marked reduction in thrombin generation potential was observed in the early postoperative phase after cardiac surgery. The decrease was independent of reductions in individual coagulation factor activity but correlated to heparin effects. The results indicate that a sustained heparin effect contributes to the postoperative reduction in thrombin generation capacity.

Radulovic V; Hyllner M; Ternström L; Karlsson M; Bylock A; Hansson KM; Baghaei F; Jeppsson A

2012-11-01

167

Inhibition of thrombin-mediated factor V activation contributes to the anticoagulant activity of fibrinogen ?'.  

UK PubMed Central (United Kingdom)

BACKGROUND: Besides its role in blood clotting, fibrinogen exerts a poorly understood anticoagulant function by binding thrombin and modulating its activity. In particular, the ?A/?' fibrinogen isoform binds with high affinity to thrombin exosite II through the anionic carboxyl-terminal end of the ?' chain. This interaction down-regulates thrombin-mediated factor VIII (FVIII) activation, but its effect on FV activation is unknown. OBJECTIVES: To investigate the overall anticoagulant activity of fibrinogen and particularly of fibrinogen ?' in plasma, and to verify whether the fibrinogen ?' carboxyl-terminal peptide affects thrombin-mediated FV activation. METHODS: Thrombin generation was measured by calibrated automated thrombography in whole and defibrinated plasma and in plasma supplemented with the (sulfated) fibrinogen ?' carboxyl-terminal peptide (0-500 ?mol L(-1) ). The effect of the peptide on thrombin-mediated FV activation was studied in model systems and in plasma. RESULTS: Total fibrinogen prolonged the lag time of thrombin generation at low tissue factor (TF) concentrations. The fibrinogen ?' peptide dose-dependently prolonged the lag time and decreased the peak height of thrombin generation at low TF, whereas a scrambled control peptide was ineffective. These effects persisted in the presence of an anti-FVIII antibody, suggesting that the peptide may also inhibit thrombin-mediated activation of FV. This was confirmed in model systems and in plasma. CONCLUSIONS: Total fibrinogen and the fibrinogen ?' peptide have an overall anticoagulant effect on thrombin generation determined at low TF. Inhibition of thrombin-mediated FV activation by the fibrinogen ?' peptide is a novel mechanism of the anticoagulant activity of fibrinogen ?'.

Omarova F; Uitte De Willige S; Ariëns RA; Rosing J; Bertina RM; Castoldi E

2013-09-01

168

The modulation of gene expression in osteoblasts by thrombin coated on biphasic calcium phosphate ceramic.  

Science.gov (United States)

For many years, fibrin sealants were associated with bone substitutes to promote bone healing. However, the osteoblastic response to fibrin sealant components remains poorly documented. In this study, MC3T3-E1 osteoblastic cells were cultured on biphasic calcium phosphate ceramic (MBCP) coated with Tissucol components (thrombin and fibrinogen). Analysis of osteoblastic differentiation markers by RT-PCR revealed that MBCP coated with Tissucol stimulated mRNA levels for osteocalcin and alkaline phosphatase (ALP). Of all the components of Tissucol, thrombin has been reported to affect osteoblastic behavior. Our results demonstrated that low thrombin concentrations (0.5-5 U/ml) stimulated mRNA levels for ALP, whereas high thrombin concentrations (50-100 U/ml) decreased mRNA levels for ALP and PTH/PTHrP receptor and also increased mRNA level for the osteoclastogenesis inhibitor OPG. As thrombin stimulated angiogenesis, we then wondered whether thrombin could influence the expression of angiogenic factors. Low thrombin concentrations were shown to up-regulate mRNA levels for VEGF-B and VEGF-R1, suggesting an autocrine/paracrine role for VEGF-B. Higher thrombin concentrations also up-regulated mRNA for VEGF-A and neuropilin-1. In conclusion, the association of MBCP with thrombin and fibrinogen appears to be a convenient scaffold for bone cell differentiation. Thrombin could also acts at the cellular level by increasing the angiogenic potential of osteoblasts as well as their responsiveness to thrombin and VEGF. PMID:16436294

Bluteau, Gilles; Pilet, Paul; Bourges, Xavier; Bilban, Melitta; Spaethe, Reiner; Daculsi, Guy; Guicheux, Jérôme

2006-01-24

169

Topical treatments for pain.  

Science.gov (United States)

Topical analgesics exert their analgesic benefit locally and without significant systemic absorption. The mechanism of the topical analgesic is unique to the specific medication. Key differences between topical and transdermal analgesics are discussed in this article. A new term, targeted peripheral analgesics, has been suggested to replace the term topical analgesics, but is not in widespread use. Topical analgesics have been studied in an increasing number of painful clinical conditions; the results of many of these studies are summarized in this review. Recent data suggest that at least one topical analgesic, although applied peripherally, may result in central nervous system alterations of pain processing. PMID:15228883

Argoff, Charles E

2004-08-01

170

Topical treatments for pain.  

UK PubMed Central (United Kingdom)

Topical analgesics exert their analgesic benefit locally and without significant systemic absorption. The mechanism of the topical analgesic is unique to the specific medication. Key differences between topical and transdermal analgesics are discussed in this article. A new term, targeted peripheral analgesics, has been suggested to replace the term topical analgesics, but is not in widespread use. Topical analgesics have been studied in an increasing number of painful clinical conditions; the results of many of these studies are summarized in this review. Recent data suggest that at least one topical analgesic, although applied peripherally, may result in central nervous system alterations of pain processing.

Argoff CE

2004-08-01

171

Superactivated platelets: thrombus regulators, thrombin generators, and potential clinical targets.  

UK PubMed Central (United Kingdom)

Platelets contribute to hemostasis by forming the platelet plug and then contributing to coagulation by providing a catalytic surface where thrombin generation occurs efficiently. This catalytic activity, known as the platelet procoagulant response, is being recognized as a nuanced response. This review examines platelets' response to strong stimuli, which results in the formation of a platelet subpopulation (superactivated platelets) with several unique properties, including enhanced procoagulant activity. These platelets contribute uniquely to thrombus architecture and seem to have thrombus regulatory activity. Superactivated platelets' role in diseases of thrombosis and hemostasis, as either potentiating or mitigating factors, is not currently known, but may be an important pharmacological target.

Mazepa M; Hoffman M; Monroe D

2013-08-01

172

Low molecular weight heparin inhibits plasma thrombin generation via direct targeting of factor IXa: contribution of the serpin-independent mechanism.  

UK PubMed Central (United Kingdom)

BACKGROUND: Although heparin possesses multiple mechanisms of action, enhanced factor Xa inhibition by antithrombin is accepted as the predominant therapeutic mechanism. The contribution of FIXa inhibition to heparin activity in human plasma remains incompletely defined. OBJECTIVES: To determine the relevance of FIXa as a therapeutic target for heparins, particularly serpin-independent inhibition of intrinsic tenase (FIXa-FVIIIa) activity. PATIENTS/METHODS: Thrombin generation was detected by fluorogenic substrate cleavage. The inhibitory potencies (EC(50) s) of low molecular weight heparin (LMWH), super-sulfated LMWH (ssLMWH), fondaparinux and unfractionated heparin (UFH) were determined by plotting concentration vs. relative velocity index (ratio ± heparin). Inhibition was compared under FIX-dependent and FIX-independent conditions (0.2 or 4 pm tissue factor [TF], respectively) in normal plasma, and in mock-depleted or antithrombin/FIX-depleted plasma supplemented with recombinant FIX. RESULTS: UFH and fondaparinux demonstrated similar potency under FIX-dependent and FIX-independent conditions, whereas LMWH (2.9-fold) and ssLMWH (5.1-fold) demonstrated increased potency with limiting TF. UFH (62-fold) and fondaparinux (42-fold) demonstrated markedly increased EC(50) values in antithrombin-depleted plasma, whereas LMWH (9.4-fold) and ssLMWH (two-fold) were less affected, with an EC(50) within the therapeutic range for LMWH. The molecular target for LMWH/ssLMWH was confirmed by supplementing FIX/antithrombin-depleted plasma with 90 nm recombinant FIX possessing mutations in the heparin-binding exosite. Mutated FIX demonstrated resistance to inhibition of thrombin generation by LMWH and ssLMWH that paralleled the effect of these mutations on intrinsic tenase inhibition. CONCLUSIONS: Therapeutic LMWH concentrations inhibit plasma thrombin generation via antithrombin-independent interaction with the FIXa heparin-binding exosite.

Buyue Y; Misenheimer TM; Sheehan JP

2012-10-01

173

Synergistic effect of aptamers that inhibit exosites 1 and 2 on thrombin.  

Science.gov (United States)

Thrombin is a multifunctional protease that plays a key role in hemostasis, thrombosis, and inflammation. Most thrombin inhibitors currently used as antithrombotic agents target thrombin's active site and inhibit all of its myriad of activities. Exosites 1 and 2 are distinct regions on the surface of thrombin that provide specificity to its proteolytic activity by mediating binding to substrates, receptors, and cofactors. Exosite 1 mediates binding and cleavage of fibrinogen, proteolytically activated receptors, and some coagulation factors, while exosite 2 mediates binding to heparin and to platelet receptor GPIb-IX-V. The crystal structures of two nucleic acid ligands bound to thrombin have been solved. Previously Padmanabhan and colleagues solved the structure of a DNA aptamer bound to exosite 1 and we reported the structure of an RNA aptamer bound to exosite 2 on thrombin. Based upon these structural studies we speculated that the two aptamers would not compete for binding to thrombin. We observe that simultaneously blocking both exosites with the aptamers leads to synergistic inhibition of thrombin-dependent platelet activation and procoagulant activity. This combination of exosite 1 and exosite 2 inhibitors may provide a particularly effective antithrombotic approach. PMID:19846574

Nimjee, Shahid M; Oney, Sabah; Volovyk, Zoya; Bompiani, Kristin M; Long, Steve B; Hoffman, Maureane; Sullenger, Bruce A

2009-10-21

174

Covalent binding of human thrombin to a human endothelial cell-associated protein  

Energy Technology Data Exchange (ETDEWEB)

Binding of {sup 125}I-thrombin to endothelial cells derived from human umbilical vein was studied in tissue culture. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography revealed covalent binding of thrombin in a 72-kDa complex. Some characteristics of the 72-kDa complex were compared to those of the complexes formed between thrombin and protease nexin originating from fibroblasts or platelets: (1) its electrophoretic mobility on SDS-PAGE is identical to that of the thrombin-platelet protease nexin complex, (2) heparin prevents the appearance of the complex on the cell surface, (3) plasmin in a 100-fold molar excess prevents the covalent linkage of thrombin, suggesting that the protease specificity of the endothelial component involved in the complex might not be restricted to thrombin. Yet no release, nor any secretion of the endothelial protein, could be detected. These results indicate that active thrombin binds covalently to a specific endothelial protein that is in several respects similar to fibroblasts or platelet protease nexin and provides a thrombin binding site distinct from thrombomodulin and glycosaminoglycans.

Leroy-Viard, K.; Jandrot-Perrus, M.; Tobelem, G.; Guillin, M.C. (Univ. Paris 7 (France))

1989-03-01

175

Combined thrombin-collagen injection for the management of an iatrogenic pseudoanuerysm in the popliteal region  

Science.gov (United States)

Pseudoaneurysm formation is a rare but recognized complication of total knee arthroplasty (TKR), with fewer than 20 cases described in the literature. Multiple management techniques have been described for such pseudoaneurysms. As thrombin injection is an established technique, we report a case of a post-TKR tibioperoneal pseudoaneurysm successfully occluded via percutaneous injection of a dual thrombin-collagen preparation.

McCarthy, Eoghan; O'Mahony, Niamh; Ryan, Mark; Guiney, Michael

2012-01-01

176

Topical anesthesia in phacoemulsification  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Purpose : To evaluate the efficacy of topical anesthesia; topical Benoxinate 0.4% (Oxybuprocaine) and Xylocaine (Lidocaine) gel, in selected cataract patients as an alternative to peribulbar or retrobulbar block anesthesia during cataract surgery. Materials and Methods : Prospe...

Waheeb Saad

177

Ciclopirox Topical Solution  

Science.gov (United States)

Ciclopirox topical solution is used along with regular nail trimming to treat fungal infections of the fingernails and toenails ( ... using ciclopirox without talking to your doctor.Ciclopirox topical solution will work best if you trim your ...

178

Expression and functional characterization of chitribrisin, a thrombin-like enzyme, in the venom of the Chinese green pit viper (Trimeresurus albolabris).  

Science.gov (United States)

In the present study, functionally active, recombinant chitribrisin, which is a thrombin-like enzyme in the venom of the Chinese green pit viper (Trimeresurus albolabris), was expressed and purified using a prokaryotic system. The fusion protein of chitribrisin, together with TrxA and 6x His via an E.coli expression vector pET-32a(+), was successfully expressed in E.coli BL21(DE3) cells. After the fusion protein was isolated and purified by chelated Ni(2+) resin and specifically cleaved by enterokinase, the recombinant chitribrisin showed a strong fibrinogenolytic activity against the alpha and beta chains of human plasminogen-free fibrinogen and weak fibrinogen clotting activity. In addition, multiple sequence alignment revealed that the expressed chitribrisin was homologous to GPV-TL1 and GPV-TL2 from the snake venom of T. albolabris from central Thailand in terms of the amino acid sequence identities. However, there were some differences in the amino acid sequences of the proteins from the same species from different geographical locations. The causes for the geographical variation in TELs in the same species remain to be investigated. Mutagenesis of chitribrisin should be performed in future studies to study the structural and functional relationship and to identify the critical residues responsible for the properties of the thrombin-like enzyme. PMID:19303445

Lin, Yixin; Yu, Xiaodong; He, Qiyi; Li, Heng; Li, Dehua; Song, Xixun; Wang, Yusheng; Wen, Haoping; Deng, Huanhuan; Deng, Jiangyu

2009-03-19

179

Expression and functional characterization of chitribrisin, a thrombin-like enzyme, in the venom of the Chinese green pit viper (Trimeresurus albolabris).  

UK PubMed Central (United Kingdom)

In the present study, functionally active, recombinant chitribrisin, which is a thrombin-like enzyme in the venom of the Chinese green pit viper (Trimeresurus albolabris), was expressed and purified using a prokaryotic system. The fusion protein of chitribrisin, together with TrxA and 6x His via an E.coli expression vector pET-32a(+), was successfully expressed in E.coli BL21(DE3) cells. After the fusion protein was isolated and purified by chelated Ni(2+) resin and specifically cleaved by enterokinase, the recombinant chitribrisin showed a strong fibrinogenolytic activity against the alpha and beta chains of human plasminogen-free fibrinogen and weak fibrinogen clotting activity. In addition, multiple sequence alignment revealed that the expressed chitribrisin was homologous to GPV-TL1 and GPV-TL2 from the snake venom of T. albolabris from central Thailand in terms of the amino acid sequence identities. However, there were some differences in the amino acid sequences of the proteins from the same species from different geographical locations. The causes for the geographical variation in TELs in the same species remain to be investigated. Mutagenesis of chitribrisin should be performed in future studies to study the structural and functional relationship and to identify the critical residues responsible for the properties of the thrombin-like enzyme.

Lin Y; Yu X; He Q; Li H; Li D; Song X; Wang Y; Wen H; Deng H; Deng J

2009-09-01

180

In vitro studies using a global hemostasis assay to examine the anticoagulation effects in plasma by the direct thrombin inhibitors: dabigatran and argatroban.  

UK PubMed Central (United Kingdom)

This study aimed to assess whether a global hemostatic assay we developed can measure the anticoagulant effects of the direct thrombin inhibitors (DTIs)--dabigatran and argatroban. A normal plasma pool (NPP) spiked with one of the DTIs and five plasma samples from patients with coronary heart disease spiked with dabigatran were examined. Fibrin formation and fibrin degradation were initiated by adding recombinant tissue factor (together with washed-frozen-thawed platelets and CaCl(2)) and recombinant tissue plasminogen activator. Fibrin optical density (OD) was recorded, based on which coagulation activation profile (Cp) and fibrinolysis activation profile (Fp) were determined. Moreover, the sum of OD values registered over time (fibrin OD-sum) was calculated to reflect the capacity of fibrin formation under the general effect by Cp and Fp. The endogenous thrombin potential (ETP) and the standard clotting markers i.e., activated partial thromboplastin time (APTT) and prothrombin time expressed as International Normalized Ratio (INR) were also analyzed. Results demonstrated that APTT, INR and ETP could detect the effects of the DTIs except for INR in NPP containing dabigatran. In our global assay, the DTIs depressed the fibrin formation (shown as decreased fibrin OD-sum value) by leading to decrease of Cp and increase of Fp. Thus, our global assay which examines both fibrin formation and degradation seems more advantageous than the other methods mentioned above, as regards the possibility of being a laboratory tool to monitor the antithrombotic therapy with DTIs.

He S; Wallèn H; Bark N; Blombäck M

2013-02-01

 
 
 
 
181

In vitro studies using a global hemostasis assay to examine the anticoagulation effects in plasma by the direct thrombin inhibitors: dabigatran and argatroban.  

Science.gov (United States)

This study aimed to assess whether a global hemostatic assay we developed can measure the anticoagulant effects of the direct thrombin inhibitors (DTIs)--dabigatran and argatroban. A normal plasma pool (NPP) spiked with one of the DTIs and five plasma samples from patients with coronary heart disease spiked with dabigatran were examined. Fibrin formation and fibrin degradation were initiated by adding recombinant tissue factor (together with washed-frozen-thawed platelets and CaCl(2)) and recombinant tissue plasminogen activator. Fibrin optical density (OD) was recorded, based on which coagulation activation profile (Cp) and fibrinolysis activation profile (Fp) were determined. Moreover, the sum of OD values registered over time (fibrin OD-sum) was calculated to reflect the capacity of fibrin formation under the general effect by Cp and Fp. The endogenous thrombin potential (ETP) and the standard clotting markers i.e., activated partial thromboplastin time (APTT) and prothrombin time expressed as International Normalized Ratio (INR) were also analyzed. Results demonstrated that APTT, INR and ETP could detect the effects of the DTIs except for INR in NPP containing dabigatran. In our global assay, the DTIs depressed the fibrin formation (shown as decreased fibrin OD-sum value) by leading to decrease of Cp and increase of Fp. Thus, our global assay which examines both fibrin formation and degradation seems more advantageous than the other methods mentioned above, as regards the possibility of being a laboratory tool to monitor the antithrombotic therapy with DTIs. PMID:22843196

He, Shu; Wallèn, Håkan; Bark, Niklas; Blombäck, Margareta

2013-02-01

182

Detection of thrombin using an excimer aptamer switch labeled with dual pyrene molecules.  

UK PubMed Central (United Kingdom)

We constructed an excimer aptamer probe containing one pyrene molecule at each end of a DNA aptamer to achieve the detection of thrombin, which binds to the heparin-binding site of thrombin with high binding affinity. The specific binding of thrombin to the excimer aptamer probe brought the two pyrene molecules at the termini of the duplex of the aptamer into close proximity, generating an excimer. The excimer emitted a distinct fluorescence peak, and fluorometric measurement of excimer allowed the sensitive detection of thrombin. The effects of experimental conditions like pH, ionic strength, and cations were investigated and optimized. The detection limit for thrombin was about 42 pM. This aptamer switch has potential in the study of molecular interactions and protein sensing with other switch-based detection strategy.

Zhao Q; Cheng L

2013-10-01

183

Detection of thrombin using an excimer aptamer switch labeled with dual pyrene molecules.  

Science.gov (United States)

We constructed an excimer aptamer probe containing one pyrene molecule at each end of a DNA aptamer to achieve the detection of thrombin, which binds to the heparin-binding site of thrombin with high binding affinity. The specific binding of thrombin to the excimer aptamer probe brought the two pyrene molecules at the termini of the duplex of the aptamer into close proximity, generating an excimer. The excimer emitted a distinct fluorescence peak, and fluorometric measurement of excimer allowed the sensitive detection of thrombin. The effects of experimental conditions like pH, ionic strength, and cations were investigated and optimized. The detection limit for thrombin was about 42 pM. This aptamer switch has potential in the study of molecular interactions and protein sensing with other switch-based detection strategy. PMID:23912830

Zhao, Qiang; Cheng, Lin

2013-08-04

184

Kinetic characterization of inhibition of human thrombin with DNA aptamers by turbidimetric assay.  

Science.gov (United States)

A sensitive turbidimetric method for detecting fibrin association was used to study the kinetics of fibrinogen hydrolysis with thrombin. The data were complemented by high-performance liquid chromatography (HPLC) measurements of the peptide products, fibrinopeptides released during hydrolysis. Atomic force microscopy (AFM) data showed that the fibril diameter is the main geometric parameter influencing the turbidity. The turbidimetric assay was validated using thrombin with the standard activity. To study thrombin inhibitors, a kinetic model that allows estimating the inhibition constants and the type of inhibition was proposed. The kinetic model was used to study the inhibitory activity of the two DNA aptamers 15-TBA (thrombin-binding aptamer) and 31-TBA, which bind to thrombin exosites. For the first time, 31-TBA was shown to possess the competitive inhibition type, whereas the shortened aptamer 15-TBA has the noncompetitive inhibition type. PMID:22056408

Zavyalova, Elena G; Protopopova, Anna D; Yaminsky, Igor V; Kopylov, Aleksey M

2011-10-15

185

Kinetic characterization of inhibition of human thrombin with DNA aptamers by turbidimetric assay.  

UK PubMed Central (United Kingdom)

A sensitive turbidimetric method for detecting fibrin association was used to study the kinetics of fibrinogen hydrolysis with thrombin. The data were complemented by high-performance liquid chromatography (HPLC) measurements of the peptide products, fibrinopeptides released during hydrolysis. Atomic force microscopy (AFM) data showed that the fibril diameter is the main geometric parameter influencing the turbidity. The turbidimetric assay was validated using thrombin with the standard activity. To study thrombin inhibitors, a kinetic model that allows estimating the inhibition constants and the type of inhibition was proposed. The kinetic model was used to study the inhibitory activity of the two DNA aptamers 15-TBA (thrombin-binding aptamer) and 31-TBA, which bind to thrombin exosites. For the first time, 31-TBA was shown to possess the competitive inhibition type, whereas the shortened aptamer 15-TBA has the noncompetitive inhibition type.

Zavyalova EG; Protopopova AD; Yaminsky IV; Kopylov AM

2012-02-01

186

Specificity of the thrombin receptor for agonist peptide is defined by its extracellular surface  

Science.gov (United States)

G-PROTEIN-COUPLED receptors for catecholamines and some other small ligands are activated when agonists bind to the transmem-brane region of the receptor1. The docking interactions through which peptide agonists activate their receptors are less well characterized2-7. The thrombin receptor is a specialized peptide receptor. It is activated by binding its tethered ligand domain, which is unmasked upon receptor cleavage by thrombin8,9. Human and Xenopus thrombin receptor homologues are each selectively activated by the agonist peptide representing their respective tethered ligand domains. Here we identify receptor domains that confer this agonist specificity by replacing the Xenopus receptor's amino-terminal exodomain and three extracellular loops with the corresponding human structures. This switches receptor specificity from Xenopus to human. The specificity of these thrombin receptors for their respective peptide agonists is thus determined by their extracellular surfaces. Our results indicate that agonist interaction with extracellular domains is important for thrombin receptor activation.

Gerszten, Robert E.; Chen, Ji; Ishli, Maki; Ishil, Kenji; Wang, Ling; Nanevicz, Tania; Turck, Christoph W.; Vu, Thien-Khai H.; Coughlin, Shaun R.

1994-04-01

187

High-resolution structures of two complexes between thrombin and thrombin-binding aptamer shed light on the role of cations in the aptamer inhibitory activity.  

Science.gov (United States)

The G-quadruplex architecture is a peculiar structure adopted by guanine-rich oligonucleotidic sequences, and, in particular, by several aptamers, including the thrombin-binding aptamer (TBA) that has the highest inhibitory activity against human ?-thrombin. A crucial role in determining structure, stability and biological properties of G-quadruplexes is played by ions. In the case of TBA, K(+) ions cause an enhancement of the aptamer clotting inhibitory activity. A detailed picture of the interactions of TBA with the protein and with the ions is still lacking, despite the importance of this aptamer in biomedical field for detection and inhibition of ?-thrombin. Here, we fill this gap by presenting a high-resolution crystallographic structural characterization of the thrombin-TBA complex formed in the presence of Na(+) or K(+) and a circular dichroism study of the structural stability of the aptamer both free and complexed with ?-thrombin, in the presence of the two ionic species. The results indicate that the different effects exerted by Na(+) and K(+) on the inhibitory activity of TBA are related to a subtle perturbation of a few key interactions at the protein-aptamer interface. The present data, in combination with those previously obtained on the complex between ?-thrombin and a modified aptamer, may allow the design of new TBA variants with a pharmacological performance enhancement. PMID:22669903

Russo Krauss, Irene; Merlino, Antonello; Randazzo, Antonio; Novellino, Ettore; Mazzarella, Lelio; Sica, Filomena

2012-06-04

188

High-resolution structures of two complexes between thrombin and thrombin-binding aptamer shed light on the role of cations in the aptamer inhibitory activity.  

UK PubMed Central (United Kingdom)

The G-quadruplex architecture is a peculiar structure adopted by guanine-rich oligonucleotidic sequences, and, in particular, by several aptamers, including the thrombin-binding aptamer (TBA) that has the highest inhibitory activity against human ?-thrombin. A crucial role in determining structure, stability and biological properties of G-quadruplexes is played by ions. In the case of TBA, K(+) ions cause an enhancement of the aptamer clotting inhibitory activity. A detailed picture of the interactions of TBA with the protein and with the ions is still lacking, despite the importance of this aptamer in biomedical field for detection and inhibition of ?-thrombin. Here, we fill this gap by presenting a high-resolution crystallographic structural characterization of the thrombin-TBA complex formed in the presence of Na(+) or K(+) and a circular dichroism study of the structural stability of the aptamer both free and complexed with ?-thrombin, in the presence of the two ionic species. The results indicate that the different effects exerted by Na(+) and K(+) on the inhibitory activity of TBA are related to a subtle perturbation of a few key interactions at the protein-aptamer interface. The present data, in combination with those previously obtained on the complex between ?-thrombin and a modified aptamer, may allow the design of new TBA variants with a pharmacological performance enhancement.

Russo Krauss I; Merlino A; Randazzo A; Novellino E; Mazzarella L; Sica F

2012-09-01

189

Myocardial Topical Negative Pressure  

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The aim of this research was to find out if it is possible to increase myocardial microvasular blood flow by applying a topical negative pressure source directly onto the myocardium. Topical negative pressure is a relatively new wound healing technique. When used in wound therapy, topical negative ...

Lindstedt, Sandra

190

Platelet activation and endogenous thrombin potential in pre-eclampsia.  

UK PubMed Central (United Kingdom)

INTRODUCTION: Platelets and the coagulation system may be involved in the pathogenesis of pre-eclampsia. We investigated whether platelet and coagulation activation markers, are elevated in pre-eclampsia. MATERIALS/METHODS: Case-control study in which activated platelets, platelet-monocyte/ neutrophil aggregates, platelet microparticles (measured by flow cytometry) and four markers of thrombin generation capacity (endogenous thrombin potential (ETP), peak height, lag time and time to peak) using the Calibrated Automated Thrombogram system were assessed in pregnant women of similar gestational age with (n=46) and without (n=46) pre-eclampsia, and in healthy non-pregnant women (n=42). RESULTS: The percentage of, CD62P+ platelets (p=0.013), CD62P+ platelet microparticles (p=0.029) and platelet-monocyte aggregates (p=0.019) were significantly higher in women with pre-eclampsia than the pregnant controls. Both groups of pregnant women had significantly higher ETP and peak height (p <0.001) than the healthy non pregnant group and the women with pre-eclampsia had significantly higher ETP and peak height (p<0.001) than the normotensive pregnant controls. CONCLUSION: In the most comprehensive laboratory analysis to date, we found evidence of both platelet and coagulation activation in women with pre-eclampsia.

Macey MG; Bevan S; Alam S; Verghese L; Agrawal S; Beski S; Thuraisingham R; MacCallum PK

2010-03-01

191

Crystal structure of an RNA aptamer bound to thrombin.  

Science.gov (United States)

Aptamers, an emerging class of therapeutics, are DNA or RNA molecules that are selected to bind molecular targets that range from small organic compounds to large proteins. All of the determined structures of aptamers in complex with small molecule targets show that aptamers cage such ligands. In structures of aptamers in complex with proteins that naturally bind nucleic acid, the aptamers occupy the nucleic acid binding site and often mimic the natural interactions. Here we present a crystal structure of an RNA aptamer bound to human thrombin, a protein that does not naturally bind nucleic acid, at 1.9 A resolution. The aptamer, which adheres to thrombin at the binding site for heparin, presents an extended molecular surface that is complementary to the protein. Protein recognition involves the stacking of single-stranded adenine bases at the core of the tertiary fold with arginine side chains. These results exemplify how RNA aptamers can fold into intricate conformations that allow them to interact closely with extended surfaces on non-RNA binding proteins. PMID:18971322

Long, Stephen B; Long, Meredith B; White, Rebekah R; Sullenger, Bruce A

2008-10-29

192

Thrombin-specific inactivation of endothelial cell derived plasminogen activator  

Energy Technology Data Exchange (ETDEWEB)

Although thrombin (T) has diverse functions in the overall hemostatic mechanism, relatively little is known about its direct effect on components of the fibrinolytic enzyme system. The authors have investigated the interaction of T with plasminogen activators (PA) derived from bovine aortic endothelial cells (EC) in culture (2-5th passage, preconfluent monolayers). Varying concentrations of purified bovine or human thrombin were added to EC-conditioned media (CM). CM + T mixtures were assayed at various times for PA activity using purified plasminogen and a sensitive /sup 125/I-fibrinogenolytic or caseinolytic assay. T (5 nM), but not plasmin or trypsin at equivalent concentrations, resulted in a time-dependent inhibition of the PA activity in CM. T had no effect on the PA activity of urokinase, streptokinase or preformed plasmin. The ability of T to inactivate the EC-derived PA was abolished by prior treatment of T with active site-directed reagents. SDS-PAGE and zymography with copolymerized fibrinogen and plasminogen revealed further specificity in that only one of the multiple-molecular weight forms of PA present in EC-CM was inactivated by T. The authors conclude that in a highly specific fashion, T inactivates the predominant PA present in EC-CM by limited proteolysis. Thus, another potentially important function of T is suggested which may have particular significance in the temporal regulation of coagulation and fibrinolysis at the blood-endothelium interface.

Highsmith, R.F.; Gallaher, M.J.

1986-03-05

193

Thrombin-specific inactivation of endothelial cell derived plasminogen activator  

International Nuclear Information System (INIS)

Although thrombin (T) has diverse functions in the overall hemostatic mechanism, relatively little is known about its direct effect on components of the fibrinolytic enzyme system. The authors have investigated the interaction of T with plasminogen activators (PA) derived from bovine aortic endothelial cells (EC) in culture (2-5th passage, preconfluent monolayers). Varying concentrations of purified bovine or human thrombin were added to EC-conditioned media (CM). CM + T mixtures were assayed at various times for PA activity using purified plasminogen and a sensitive 125I-fibrinogenolytic or caseinolytic assay. T (5 nM), but not plasmin or trypsin at equivalent concentrations, resulted in a time-dependent inhibition of the PA activity in CM. T had no effect on the PA activity of urokinase, streptokinase or preformed plasmin. The ability of T to inactivate the EC-derived PA was abolished by prior treatment of T with active site-directed reagents. SDS-PAGE and zymography with copolymerized fibrinogen and plasminogen revealed further specificity in that only one of the multiple-molecular weight forms of PA present in EC-CM was inactivated by T. The authors conclude that in a highly specific fashion, T inactivates the predominant PA present in EC-CM by limited proteolysis. Thus, another potentially important function of T is suggested which may have particular significance in the temporal regulation of coagulation and fibrinolysis at the blood-endothelium interface.

1986-01-01

194

Batroxobin binds fibrin with higher affinity and promotes clot expansion to a greater extent than thrombin.  

UK PubMed Central (United Kingdom)

Batroxobin is a thrombin-like serine protease from the venom of Bothrops atrox moojeni that clots fibrinogen. In contrast to thrombin, which releases fibrinopeptide A and B from the NH2-terminal domains of the A?- and B?-chains of fibrinogen, respectively, batroxobin only releases fibrinopeptide A. Because the mechanism responsible for these differences is unknown, we compared the interactions of batroxobin and thrombin with the predominant ?A/?A isoform of fibrin(ogen) and the ?A/?' variant with an extended ?-chain. Thrombin binds to the ?'-chain and forms a higher affinity interaction with ?A/?'-fibrin(ogen) than ?A/?A-fibrin(ogen). In contrast, batroxobin binds both fibrin(ogen) isoforms with similar high affinity (Kd values of about 0.5 ?M) even though it does not interact with the ?'-chain. The batroxobin-binding sites on fibrin(ogen) only partially overlap with those of thrombin because thrombin attenuates, but does not abrogate, the interaction of ?A/?A-fibrinogen with batroxobin. Furthermore, although both thrombin and batroxobin bind to the central E-region of fibrinogen with a Kd value of 2-5 ?M, the ?(17-51) and B?(1-42) regions bind thrombin but not batroxobin. Once bound to fibrin, the capacity of batroxobin to promote fibrin accretion is 18-fold greater than that of thrombin, a finding that may explain the microvascular thrombosis that complicates envenomation by B. atrox moojeni. Therefore, batroxobin binds fibrin(ogen) in a manner distinct from thrombin, which may contribute to its higher affinity interaction, selective fibrinopeptide A release, and prothrombotic properties.

Vu TT; Stafford AR; Leslie BA; Kim PY; Fredenburgh JC; Weitz JI

2013-06-01

195

Topical treatment of ichthyoses.  

UK PubMed Central (United Kingdom)

Management of ichthyoses is a complex and continuously dynamic process. Primary treatments of ichthyosis are by means of topical moisturizers and topical medications. Patients and families need to have reasonable and realistic expectations when it comes to topical therapy. Topical medications cannot cure the scaling, but can gradually reduce it and thus improve their condition. No one treatment regimen works for everyone, and the best topical therapy for each patient may be the result of months (or years) of painstaking effort on both the physician's and the patient's behalf. As patients get older and their activities and lifestyles change, so should their topical treatment regimen. Bear in mind that the more complex the skin care regimen and costly the topical treatments, the less likely a patient and their family will be compliant.

Fleckman P; Newell BD; van Steensel MA; Yan AC

2013-01-01

196

The influence of tissue factor and tissue factor pathway inhibitor polymorphisms on thrombin generation in stable coronary artery disease.  

Science.gov (United States)

In patients with stable coronary heart disease (n = 1,001) we investigated the influence of tissue factor (TF) and TF pathway inhibitor (TFPI) polymorphisms on thrombin generation in vivo, measured by prothrombin fragment (F) 1 and 2, and the potential to generate thrombin ex vivo, measured by the calibrated automated thrombogram assay. Additionally, circulating levels of TF and TFPI were correlated to the different parameters of thrombin generation. The TF 5466 and TFPI -399 polymorphisms associated with higher thrombin generation in vivo, the latter also with a prolonged lag time of the thrombin generation ex vivo(p thrombogram parameters were observed. PMID:21555871

Opstad, Trine B; Pettersen, Alf-Aage R; Bratseth, Vibeke; Arnesen, Harald; Seljeflot, Ingebjørg

2011-05-10

197

Increased thrombin generation and fibrinogen level after therapeutic plasma transfusion: relation to bleeding.  

UK PubMed Central (United Kingdom)

In a clinical setting, fresh frozen plasma (FFP) is transfused to diluted patients with complicated surgery or trauma, as guided by prolonged conventional coagulation times or low fibrinogen levels. However, the limited sensitivity of these coagulation tests may restrict their use in measuring the effect of transfusion and hence predicting the risk of perioperative bleeding. We used the more sensitive, calibrated automated thrombogram (CAT) method to evaluate the result of therapeutic FFP transfusion to 51 patients with dilutional coagulopathy. Thrombin generation was measured in pre- and post-transfusion plasma samples in the presence of either platelets or phospholipids. For all patients, the transfusion led to higher plasma coagulation factor levels, a shortened activated partial thromboplastin time, and a significant increase in thrombin generation (peak height and endogenous thrombin potential). Interestingly, thrombin generation parameters and fibrinogen levels were higher in post-transfusion plasmas from patients who stopped bleeding (n = 32) than for patients with ongoing bleeding (n = 19). Plasmas from 15 of the 19 patients with ongoing bleeding were markedly low in either thrombin generation or fibrinogen level. We conclude that the thrombin generation method detects improved haemostatic activity after plasma transfusion. Furthermore, the data suggest that thrombin generation and fibrinogen are independent determinants of the risk of perioperative bleeding in this patient group.

Schols SE; van der Meijden PE; van Oerle R; Curvers J; Heemskerk JW; van Pampus EC

2008-01-01

198

Increased thrombin generation and fibrinogen level after therapeutic plasma transfusion: relation to bleeding.  

Science.gov (United States)

In a clinical setting, fresh frozen plasma (FFP) is transfused to diluted patients with complicated surgery or trauma, as guided by prolonged conventional coagulation times or low fibrinogen levels. However, the limited sensitivity of these coagulation tests may restrict their use in measuring the effect of transfusion and hence predicting the risk of perioperative bleeding. We used the more sensitive, calibrated automated thrombogram (CAT) method to evaluate the result of therapeutic FFP transfusion to 51 patients with dilutional coagulopathy. Thrombin generation was measured in pre- and post-transfusion plasma samples in the presence of either platelets or phospholipids. For all patients, the transfusion led to higher plasma coagulation factor levels, a shortened activated partial thromboplastin time, and a significant increase in thrombin generation (peak height and endogenous thrombin potential). Interestingly, thrombin generation parameters and fibrinogen levels were higher in post-transfusion plasmas from patients who stopped bleeding (n = 32) than for patients with ongoing bleeding (n = 19). Plasmas from 15 of the 19 patients with ongoing bleeding were markedly low in either thrombin generation or fibrinogen level. We conclude that the thrombin generation method detects improved haemostatic activity after plasma transfusion. Furthermore, the data suggest that thrombin generation and fibrinogen are independent determinants of the risk of perioperative bleeding in this patient group. PMID:18217136

Schols, Saskia E M; van der Meijden, Paola E J; van Oerle, René; Curvers, Joyce; Heemskerk, Johan W M; van Pampus, Elisabeth C M

2008-01-01

199

Selective plasmalogen substrate utilization by thrombin-stimulated Ca(2+)-independent PLA(2) in cardiomyocytes.  

Science.gov (United States)

Thrombin stimulation of rabbit ventricular myocytes activates a membrane-associated, Ca(2+)-independent phospholipase A(2) (PLA(2)) capable of hydrolyzing plasmenylcholine (choline plasmalogen), plasmanylcholine (alkylacyl choline phospholipid), and phosphatidylcholine substrates. To identify the endogenous phospholipid substrates, we quantified the effects of thrombin stimulation on diradyl phospholipid mass and arachidonic acid and lysophospholipid production. Thrombin stimulation resulted in a selective decrease in arachidonylated plasmenylcholine, with no change in arachidonylated phosphatidylcholine. The decrease in arachidonylated plasmenylcholine was accompanied by an increase in plasmenylcholine species containing linoleic and linolenic acids at the sn-2 position. A decrease in arachidonylated plasmenylethanolamine was also observed after thrombin stimulation, with no concomitant change in arachidonylated phosphatidylethanolamine. Thrombin stimulation resulted in the selective production of lysoplasmenylcholine, with no increase in lysophosphatidylcholine content. There was no evidence for significant acetylation of lysophospholipids to form platelet-activating factor. Arachidonic acid released after thrombin stimulation was rapidly oxidized to prostacyclin. Thus thrombin-stimulated Ca(2+)-independent PLA(2) selectively hydrolyzes arachidonylated plasmalogen substrates, resulting in production of lysoplasmalogens and prostacyclin as the principal bioactive products. PMID:10843891

McHowat, J; Creer, M H

2000-06-01

200

Selective plasmalogen substrate utilization by thrombin-stimulated Ca(2+)-independent PLA(2) in cardiomyocytes.  

UK PubMed Central (United Kingdom)

Thrombin stimulation of rabbit ventricular myocytes activates a membrane-associated, Ca(2+)-independent phospholipase A(2) (PLA(2)) capable of hydrolyzing plasmenylcholine (choline plasmalogen), plasmanylcholine (alkylacyl choline phospholipid), and phosphatidylcholine substrates. To identify the endogenous phospholipid substrates, we quantified the effects of thrombin stimulation on diradyl phospholipid mass and arachidonic acid and lysophospholipid production. Thrombin stimulation resulted in a selective decrease in arachidonylated plasmenylcholine, with no change in arachidonylated phosphatidylcholine. The decrease in arachidonylated plasmenylcholine was accompanied by an increase in plasmenylcholine species containing linoleic and linolenic acids at the sn-2 position. A decrease in arachidonylated plasmenylethanolamine was also observed after thrombin stimulation, with no concomitant change in arachidonylated phosphatidylethanolamine. Thrombin stimulation resulted in the selective production of lysoplasmenylcholine, with no increase in lysophosphatidylcholine content. There was no evidence for significant acetylation of lysophospholipids to form platelet-activating factor. Arachidonic acid released after thrombin stimulation was rapidly oxidized to prostacyclin. Thus thrombin-stimulated Ca(2+)-independent PLA(2) selectively hydrolyzes arachidonylated plasmalogen substrates, resulting in production of lysoplasmalogens and prostacyclin as the principal bioactive products.

McHowat J; Creer MH

2000-06-01

 
 
 
 
201

Label-free fluorescent detection of thrombin using G-quadruplex-based DNAzyme as sensing platform.  

Science.gov (United States)

We report herein a label-free and sensitive fluorescent method for detection of thrombin using a G-quadruplex-based DNAzyme as the sensing platform. The thrombin-binding aptamer (TBA) is able to bind hemin to form the G-quadruplex-based DNAzyme, and thrombin can significantly enhance the activity of the G-quadruplex-based DNAzyme. The G-quadruplex-based DNAzyme is found to effectively catalyze the H(2)O(2)-mediated oxidation of thiamine, giving rise to fluorescence emission. This allows us to utilize the H(2)O(2)-thiamine fluorescent system for the quantitative analysis of thrombin. The assay shows a linear toward thrombin concentration in the range of 0.01-0.12 nM. The present limit of detection for thrombin is 1 pM, and the sensitivity for analyzing thrombin is improved by about 10,000-fold as compared with the reported colorimetric counterpart. The work also demonstrates that thiamine is an excellent substrate for the fluorescence assay using the G-quadruplex-based DNAzyme as the sensing platform. PMID:21725571

Zhang, Yuanfu; Li, Baoxin; Jin, Yan

2011-07-04

202

Label-free fluorescent detection of thrombin using G-quadruplex-based DNAzyme as sensing platform.  

UK PubMed Central (United Kingdom)

We report herein a label-free and sensitive fluorescent method for detection of thrombin using a G-quadruplex-based DNAzyme as the sensing platform. The thrombin-binding aptamer (TBA) is able to bind hemin to form the G-quadruplex-based DNAzyme, and thrombin can significantly enhance the activity of the G-quadruplex-based DNAzyme. The G-quadruplex-based DNAzyme is found to effectively catalyze the H(2)O(2)-mediated oxidation of thiamine, giving rise to fluorescence emission. This allows us to utilize the H(2)O(2)-thiamine fluorescent system for the quantitative analysis of thrombin. The assay shows a linear toward thrombin concentration in the range of 0.01-0.12 nM. The present limit of detection for thrombin is 1 pM, and the sensitivity for analyzing thrombin is improved by about 10,000-fold as compared with the reported colorimetric counterpart. The work also demonstrates that thiamine is an excellent substrate for the fluorescence assay using the G-quadruplex-based DNAzyme as the sensing platform.

Zhang Y; Li B; Jin Y

2011-08-01

203

OLIGONUCLEOTIDE-HIRULOG18 CONJUGATED BY CLICK CHEMISTRY AND ITS INHIBITION ON THROMBIN  

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Full Text Available We have shown previously that oligonucleotide conjugated hirulog (bivalirudin) has a higher inhibitory activityagainst thrombin compared with hirulog. The negative charged oligonucleotide was considered to be responsible for theadditive activity. To further investigate the related effect of oligonucleotide, the oligonucleotide conjugated hirulog18 wasprepared in the present work and the activity of the conjugate against thrombin was measured. Hirulog18, a peptide lack oftwo N-terminal amino acid residues of hirulog, has little inhibitory activity against thrombin compared with hirulog. Theconjugate oligonucleotide-hiruglog18 was successfully synthesized using click chemistry and validated by MALDI-MS andgel electrophoresis. The activity of the conjugate against thrombin was measured by the chromogenic assay usingChromozym TH as substrate. It was found surprisingly that oligonucleotide-hirulog18 had an inhibitory effect better thanhirulog18 and hirulog. Strong negative charged heparin was used to study the binding mode betweenoligonucleotide-hirulog18 and thrombin. The results suggested that the negative charged oligonucleotide could be helpful forthe conjugate’s binding to the anion-binding exosite of thrombin via the Coulomb force. The highly inhibitory effect of theoligonucleotide-hirulog18 conjugate against thrombin may present a new strategy to generate a novel class of directthrombin inhibitors.

JIE CHAO, JIAN-NING LIU?SHOU-JUN XIAO AND YAN-CHUN TANG

2011-01-01

204

Isoproterenol attenuates the thrombin-induced increase in pulmonary endothelial permeability  

Energy Technology Data Exchange (ETDEWEB)

The proposed permeability-decreasing effect of the beta-agonist, isoproterenol, was tested by measurement of the clearance rate of /sup 125/I-albumin across bovine pulmonary artery endothelial cell monolayers. These cells were seeded on gelatinized polycarbonate filters and mounted in a modified Boyden chamber with Dulbecco's Modified Eagle Medium (DMEM) and 1% BSA in the luminal and abluminal chambers. Baseline clearance rates were measured for an initial 30 min with the cells incubated at 37/sup 0/C in DMEM. At 30 min, 70 ul of either DMEM, ..cap alpha..-thrombin (10/sup -6/M), ..cap alpha..-thrombin and isoproterenol (10/sup -6/M) or isoproterenol alone was added to the luminal chamber. Clearance rates were determined at 30-60 min in each group and normalized to the baseline clearance rates of each (n = 8) cell monolayer. The experimental/baseline values were 0.98 +/- 0.08 (mean +/- SEM) for DMEM, 1.90 +/- 0.10 for thrombin, 1.52 +/- 0.08 for thrombin and isoproterenol, and 0.83 +/- 0.10 for isoproterenol alone. Thrombin increased (p < 0.05) the clearance rate from the control DMEM value and isoproterenol reduced (p < 0.05) the response to thrombin. Therefore, isoproterenol attenuates the thrombin-induced increase in pulmonary endothelial permeability.

Minnear, F.L.; Hill, L.A.

1986-03-01

205

A new type of thrombin inhibitor, noncytotoxic phospholipase A2, from the Naja haje cobra venom.  

Science.gov (United States)

Thrombin is a key enzyme in the blood coagulation cascade and is also involved in carcinogenesis; therefore, its inhibitors are of fundamental and clinical importance. Snake venoms are widely used as sources of proteins that affect blood coagulation. We have isolated a new protein, called TI-Nh, from the Naja haje cobra venom. TI-Nh is a mixed-type inhibitor of thrombin (K(i) of 72.8 nM for a synthetic peptide substrate) and effectively inhibits thrombin-induced platelet aggregation with an IC(50) value of 0.2 nM. At concentrations up to approximately 50 nM, at which the thrombin-clotting time is substantially prolonged, TI-Nh exerts no detectable effects on both the intrinsic and extrinsic pathways of the coagulation cascade. It does not hydrolyze either fibrinogen or thrombin. Although TI-Nh bears structural features typical of group IB phospholipases A(2) (PLA(2)s), it possesses relatively weak enzymatic activity and is nontoxic to PC12 cells at concentrations up to 15 microM. Nevertheless, TI-Nh evokes neurite outgrowth in these cells at a concentration of approximately 1 microM, similar to cytotoxic snake PLA(2)s with strong enzymatic activity. TI-Nh is the first thrombin inhibitor found in the venom of the Elapidae snake family, and it is the first phospholipase shown to inhibit thrombin. PMID:19622365

Osipov, Alexey V; Filkin, Sergey Yu; Makarova, Yana V; Tsetlin, Victor I; Utkin, Yuri N

2009-07-19

206

An Immunohistochemical Analysis of Tissue Thrombin Expression in the Human Atria  

Science.gov (United States)

Objective Thrombin, the final coagulation product of the coagulation cascade, has been demonstrated to have many physiological effects, including pro-fibrotic actions via protease-activated receptor (PAR)-1. Recent investigations have demonstrated that activation of the cardiac local coagulation system was associated with atrial fibrillation. However, the distribution of thrombin in the heart, especially difference between the atria and the ventricle, remains to be clarified. We herein investigated the expression of thrombin and other related proteins, as well as tissue fibrosis, in the human left atria and left ventricle. Methods We examined the expression of thrombin and other related molecules in the autopsied hearts of patients with and without atrial fibrillation. An immunohistochemical analysis was performed in the left atria and the left ventricle. Results The thrombin was immunohistologically detected in both the left atria and the left ventricles. Other than in the myocardium, the expression of thrombin was observed in the endocardium and the subendocardium of the left atrium. Thrombin was more highly expressed in the left atrium compared to the left ventricle, which was concomitant with more tissue fibrosis and inflammation, as detected by CD68 expression, in the left atrium. We also confirmed the expression of prothrombin in the left atrium. The expression of PAR-1 was observed in the endocardium, subendocardium and myocardium in the left atrium. In patients with atrial fibrillation, strong thrombin expression was observed in the left atrium. Conclusions The strong expression levels of thrombin, prothrombin and PAR-1 were demonstrated in the atrial tissues of human autopsied hearts.

Ito, Keiichi; Date, Taro; Ikegami, Masahiro; Hongo, Kenichi; Fujisaki, Masami; Katoh, Daisuke; Yoshino, Takuya; Anzawa, Ryuko; Nagoshi, Tomohisa; Yamashita, Seigo; Inada, Keiichi; Matsuo, Seiichiro; Yamane, Teiichi; Yoshimura, Michihiro

2013-01-01

207

Thrombostatin FM compounds: direct thrombin inhibitors ? mechanism of action in vitro and in vivo  

Energy Technology Data Exchange (ETDEWEB)

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 {mu}m, respectively. They competitively inhibit {alpha}-thrombin-induced cleavage of a chromogenic substrate at 4.4--8.2 {mu}m. 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 {alpha}-thrombin-induced calcium flux in fibroblasts with an IC{sub 50} of 6.9 {+-} 1.2 {mu}m. FM19 achieved 100% inhibition of threshold {alpha}- or {gamma}-thrombin-induced platelet aggregation at 8.4 {+-} 4.7 {mu}m and 16 {+-} 4 {mu}m, 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.

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. (Case Western); (Michigan); (TSRL); (WU-MED)

2008-04-29

208

Impact of experimental haemodilution on platelet function, thrombin generation and clot firmness: effects of different coagulation factor concentrates.  

UK PubMed Central (United Kingdom)

BACKGROUND: Haemodilution during resuscitation after massive haemorrhage may worsen the coagulopathy and perpetuate bleeding. MATERIALS AND METHODS: Blood samples from healthy donors were diluted (30 and-60%) using crystalloids (saline, Ringer's lactate, Plasmalyte(TM)) or colloids (6% hydroxyethylstarch [HES130/0.4], 5% human albumin, and gelatin). The effects of haemodilution on platelet adhesion (Impact R), thrombin generation (TG), and thromboelastometry (TEM) parameters were analysed as were the effects of fibrinogen, prothrombin complex concentrates (PCC), activated recombinant factor VII (FVIIa), and cryoprecipates on haemodilution. RESULTS: Platelet interactions was already significantly reduced at 30% haemodilution. Platelet reactivity was not improved by addition of any of the concentrates tested. A decrease in TG and marked alterations of TEM parameters were noted at 60% haemodilution. HES130/0.4 was the expander with the most deleterious action. TG was significantly enhanced by PCC whereas rFVIIa only caused a mild acceleration of TG initiation. Fibrinogen restored the alterations of TEM parameters caused by haemodilution including those caused by HES 130/0.4. Cryoprecipitates significantly improved the alterations caused by haemodilution on TG and TEM parameters; the effects on TG disappeared after ultracentrifugation of the cryoprecipitates. DISCUSSION: The haemostatic alterations caused by haemodilution are multifactorial and affect both blood cells and coagulation. In our in vitro approach, HES 130/0.4 had the most deleterious effect on haemostasis parameters. Coagulation factor concentrates did not improve platelet interactions in the Impact R, but did have favourable effects on coagulation parameters measured by TG and TEM. Fibrinogen notably improved TEM parameters without increasing thrombin generation, suggesting that this concentrate may help to preserve blood clotting abilities during haemodilution without enhancing the prothrombotic risk.

Caballo C; Escolar G; Diaz-Ricart M; Lopez-Vílchez I; Lozano M; Cid J; Pino M; Beltrán J; Basora M; Pereira A; Galan AM

2013-07-01

209

Impact of experimental haemodilution on platelet function, thrombin generation and clot firmness: effects of different coagulation factor concentrates  

Science.gov (United States)

Background Haemodilution during resuscitation after massive haemorrhage may worsen the coagulopathy and perpetuate bleeding. Materials and methods Blood samples from healthy donors were diluted (30 and-60%) using crystalloids (saline, Ringer’s lactate, PlasmalyteTM) or colloids (6% hydroxyethylstarch [HES130/0.4], 5% human albumin, and gelatin). The effects of haemodilution on platelet adhesion (Impact R), thrombin generation (TG), and thromboelastometry (TEM) parameters were analysed as were the effects of fibrinogen, prothrombin complex concentrates (PCC), activated recombinant factor VII (FVIIa), and cryoprecipates on haemodilution. Results Platelet interactions was already significantly reduced at 30% haemodilution. Platelet reactivity was not improved by addition of any of the concentrates tested. A decrease in TG and marked alterations of TEM parameters were noted at 60% haemodilution. HES130/0.4 was the expander with the most deleterious action. TG was significantly enhanced by PCC whereas rFVIIa only caused a mild acceleration of TG initiation. Fibrinogen restored the alterations of TEM parameters caused by haemodilution including those caused by HES 130/0.4. Cryoprecipitates significantly improved the alterations caused by haemodilution on TG and TEM parameters; the effects on TG disappeared after ultracentrifugation of the cryoprecipitates. Discussion The haemostatic alterations caused by haemodilution are multifactorial and affect both blood cells and coagulation. In our in vitro approach, HES 130/0.4 had the most deleterious effect on haemostasis parameters. Coagulation factor concentrates did not improve platelet interactions in the Impact R, but did have favourable effects on coagulation parameters measured by TG and TEM. Fibrinogen notably improved TEM parameters without increasing thrombin generation, suggesting that this concentrate may help to preserve blood clotting abilities during haemodilution without enhancing the prothrombotic risk.

Caballo, Carolina; Escolar, Gines; Diaz-Ricart, Maribel; Lopez-Vilchez, Irene; Lozano, Miguel; Cid, Joan; Pino, Marcos; Beltran, Joan; Basora, Misericordia; Pereira, Arturo; Galan, Ana M.

2013-01-01

210

Topical report review status  

International Nuclear Information System (INIS)

[en] A Topical Report Review Status is scheduled to be published semi-annually. The primary purpose of this document is to provide periodic progress reports of on-going topical report reviews, to identify those topical reports for which the Nuclear Regulatory Commission (NRC) staff review has been completed and, to the extent practicable, to provide NRC management with sufficient information regarding the conduct of the topical report program to permit taking whatever actions deemed necessary or appropriate. This document is also intended to be a source of information to NRC Licensing Project Managers and other NRC personnel regarding the status of topical reports which may be referenced in applications for which they have responsibility. This status report is published primarily for internal NRC use in managing the topical report program, but is also used by NRC to advise the industry of report review status

1982-01-01

211

[Ethyl esters of N-amidinobenzoyl aminoacids: inhibitory effect on thrombin, blood coagulation and platelet aggregation  

UK PubMed Central (United Kingdom)

In a previous paper we described the synthesis and the antiproteolytic activity of N-(3- and N-(4-amidinobenzoyl)-L-amino acids. As an extension of these studies we examined the possible inhibitory effect of these compounds on thrombin, blood coagulation and platelet aggregation. Values of Ki for the binding to thrombin are independent of the nature of the amino acids side chain and superimposable with those obtained for the formation of benzamidine-thrombin adduct. These data suggest slight effect on blood coagulation. The benzamidine derivatives are more active on platelets.

Ferroni R; Orlandini P; Guarneri M; Taddeo U; Franzè D; Bardi A

1987-10-01

212

Combinatorial synthesis of thrombin-binding aptamers containing iso-guanine.  

UK PubMed Central (United Kingdom)

A library of all possible substitutions of guanine by iso-guanine (iG) in the thrombin aptamer was prepared by split and mix synthesis. A colorimetric assay was used to screen for functional oligomers in the library. Colorimetrically active oligonucleotides were selected and sequenced by the Maxam-Gilbert method. The sequenced oligonucleotides were individually resynthesized, and their affinities for thrombin were assayed by isothermal titration calorimetry. Three aptamer sequences containing iG were found to have enhanced binding activity to human alpha-thrombin compared to the parent aptamer.

Nallagatla SR; Heuberger B; Haque A; Switzer C

2009-05-01

213

Construction and characterization of a novel staphylokinase variant with thrombin-inhibitory activity.  

UK PubMed Central (United Kingdom)

Staphylokinase (SAK) is an effective thrombolysis agent for therapy of myocardial infarction. We have constructed a fusion SAK variant (SAK-HV) with a thrombin-binding domain composed of 12 amino acids from hirudin and expressed it in Escherichia coli and purified the resultant protein. SAK-HV maintained fibrinolytic activity similar to SAK and had anticoagulant activity attributable to its hirudin segment. Measurement of thrombin-binding activity in vitro demonstrated that SAK-HV possessed binding activity with thrombin while SAK did not. SAK-HV might thus be a more potent thrombolytic agent with anticoagulation property than SAK.

Wang M; Wang Y; Wang J; Zou M; Liu S; Xu T; Cai X; Wu C; Wang J; Xu D

2009-12-01

214

Construction and characterization of a novel staphylokinase variant with thrombin-inhibitory activity  

UK PubMed Central (United Kingdom)

Staphylokinase (SAK) is an effective thrombolysis agent for therapy of myocardial infarction. We have constructed a fusion SAK variant (SAK-HV) with a thrombin-binding domain composed of 12 amino acids from hirudin and expressed it in Escherichia coli and purified the resultant protein. SAK-HV maintained fibrinolytic activity similar to SAK and had anticoagulant activity attributable to its hirudin segment. Measurement of thrombin-binding activity in vitro demonstrated that SAK-HV possessed binding activity with thrombin while SAK did not. SAK-HV might thus be a more potent thrombolytic agent with anticoagulation property than SAK.

Wang Min; Wang Yuanyuan; Wang Jinfeng; Zou Minji; Liu Shen; Xu Tao; Cai Xin; Wu Chen; Wang Jiaxi; Xu Donggang

2009-12-01

215

Construction and characterization of a novel staphylokinase variant with thrombin-inhibitory activity.  

Science.gov (United States)

Staphylokinase (SAK) is an effective thrombolysis agent for therapy of myocardial infarction. We have constructed a fusion SAK variant (SAK-HV) with a thrombin-binding domain composed of 12 amino acids from hirudin and expressed it in Escherichia coli and purified the resultant protein. SAK-HV maintained fibrinolytic activity similar to SAK and had anticoagulant activity attributable to its hirudin segment. Measurement of thrombin-binding activity in vitro demonstrated that SAK-HV possessed binding activity with thrombin while SAK did not. SAK-HV might thus be a more potent thrombolytic agent with anticoagulation property than SAK. PMID:19685208

Wang, Min; Wang, Yuanyuan; Wang, Jinfeng; Zou, Minji; Liu, Shen; Xu, Tao; Cai, Xin; Wu, Chen; Wang, Jiaxi; Xu, Donggang

2009-08-15

216

[Direct thrombin inhibitors in coronary angioplasty. Value of bivalirudin ].  

Science.gov (United States)

During coronary angioplasty, the association of platelet inhibitors and antithrombin agents is required to prevent myocardial infarction. Bivalirudine, a synthetic direct thrombin inhibitor, has been widely validated in this context and has shown its efficacy and safety in several comparative studies. It is officially recommended as a replacement of NFH and LMWH associated or not with anti-GPIIb/IIIa agents because at comparable efficacy it causes fewer bleeding complications. In acute coronary syndromes without ST elevation, anti GPIIb/IIIa agents reduce angioplasty-related complications and mortality, especially in high risk patients in salvage situations. In the REPLACE-2 trial the clinical efficacy of bivalirudine (associated only when necessary with anti-GPIIb/IIIa agents) was no less than that of NFH associated systematically with anti-GPIIb/IIIa agents at the time of intervention. The incidents of serious adverse events at 30 days (death, infarctus, emergency revascularisation, major bleeding) in the bivalirudine group was 9.2% versus 10.2% in the NFH group. In a retrospective analysis, these results did not seem to be influenced by the prior administration of clopidogrel. Finally, the one year follow-up results showed a lower mortality in patients treated with bivalirudine (1.9% versus 2.5%), essentially in the high risk sub-groups such as the elderly, the diabetic or the renal failure patients. Clinical trials are underway (ACUITY) to study the interaction of anti GPIIb/IIIa agents with bivalirudine in the first hours of acute coronary syndromes and should confirm a major role of direct anti-thrombin drugs in the safety of angioplasty. PMID:16553238

Coste, P; Labèque, J N; Leroux, L; Laplace, G; Jaïs, C; Gerbaud, E; Dos Santos, P

2006-02-01

217

Topical Dosage Form Questions  

Science.gov (United States)

... Topical Dosage Form Questions. Introduction: ANDA Number. Date of Submission. Applicant's Name. Established Name of the Drug Product. ... More results from www.fda.gov/drugs/developmentapprovalprocess/howdrugsaredevelopedandapproved

218

Changes in amniotic fluid concentration of thrombin-antithrombin III complexes in patients with preterm labor: evidence of an increased thrombin generation  

Science.gov (United States)

Objective Preterm labor is associated with excessive maternal thrombin generation as evidenced by increased circulating thrombin–antithrombin (TAT) III complexes concentration. In addition to its hemostatic functions, thrombin has uterotonic properties that may participate in the mechanism leading to preterm birth in cases of intrauterine bleeding. Thrombin also has a proinflammatory role, and inflammation is associated with increased thrombin generation. The aim of this study was to determine whether intra-amniotic infection/inflammation (IAI) is associated with increased amniotic fluid (AF) thrombin generation in women with preterm and term deliveries. Study design This cross-sectional study included the following groups: 1) mid-trimester (n=74); 2) term not in labor (n=39); 3) term in labor (n=25); 4) term in labor with IAI (n=22); 5) spontaneous preterm labor (PTL) who delivered at term (n=62); 6) PTL without IAI who delivered preterm (n=59); 7) PTL with IAI (n=71). The AF TAT III complexes concentration was measured by ELISA. Non-parametric statistics were used for analysis. Results 1) TAT III complexes were identified in all AF samples; 2) patients with PTL who delivered preterm, with and without IAI, had a significantly higher median AF TAT III complexes concentration than those with an episode of PTL who delivered at term (p<0.001, p=0.03, respectively); 3) among patients with preterm labor without IAI, elevated AF TAT III complexes concentration were independently associated with a shorter amniocentesis-to-delivery interval (hazard ratio- 1.5, 95%CI, 1.07–2.1); 4) among patients at term, those with IAI had a higher median AF TAT III complexes concentration than those without IAI, whether in labor or not in labor (p=0.02); 5) there was no significant difference between the median AF TAT III complexes concentration of patients at term with and without labor; and 6) patients who had a mid-trimester amniocentesis had a lower median AF TAT III complexes concentration than that of patients at term not in labor (p<0.001). Conclusions We present herein a distinct difference in the pattern of intra-amniotic thrombin generation between term and preterm parturition. Preterm labor leading to preterm delivery is associated with an increased intra-amniotic thrombin generation, regardless of the presence of IAI. In contrast, term delivery is associated with an increased intra-amniotic thrombin generation only in patients with IAI.

Erez, Offer; Romero, Roberto; Vaisbuch, Edi; Chaiworapongsa, Tinnakorn; Kusanovic, Juan Pedro; Mazaki-Tovi, Shali; Gotsch, Francesca; Gomez, Ricardo; Maymon, Eli; Pacora, Percy; Edwin, Samuel S.; Kim, Chong Jai; Than, Nandor Gabor; Mittal, Pooja; Yeo, Lami; Dong, Zhong; Yoon, Bo Hyun; Hassan, Sonia S; Mazor, Moshe

2012-01-01

219

Valve regulated lead acid recombinant batteries SLA  

Energy Technology Data Exchange (ETDEWEB)

Over the past several months, a number of papers have appeared concerning possible flows in the mechanism and operation of lead-acid recombinant batteries, otherwise known as valve regulated batteries or sealed lead-acid (SLA) batteries. Topic discussed in this short review include proper charging or recharging, heat management, and premature failures.

James, J.; Aidman, E.; Aidman, G.; Orsino, J.

1993-05-01

220

Topics in meson spectroscopy  

International Nuclear Information System (INIS)

[en] In this mini-review I discuss three topics in meson spectroscopy. The production of heavy quarkonium states, S-wave scattering below 1 GeV, and exotic hybrid meson production. This is not intended to be a comprehensive review, just an overview of several topics of current interest

2003-01-01

 
 
 
 
221

Topics in meson spectroscopy  

International Nuclear Information System (INIS)

[en] In this mini-review I discuss three topics in meson spectroscopy. The production of heavy quarkonium states, S-wave scattering below 1 GeV, and exotic hybrid meson production. This is not intended to be a comprehensive review, just an overview of several topics of current interest. (orig.)

2002-01-01

222

A fluorescent sandwich assay for thrombin using aptamer modified magnetic beads and quantum dots  

International Nuclear Information System (INIS)

We describe an aptamer-based sandwich assay for thrombin by using a pair of thrombin-binding aptamers, namely one 15-mer aptamer (denoted as Apt15) and one 29-mer aptamer (denoted as Apt29). Either Apt29 or Apt15 can be used as capture aptamers on magnetic beads or reporter aptamers on the quantum dots to form the sandwich complex. Detection of thrombin is achieved by the fluorescent measurement of quantum dots in the sandwich complex. The choice of capture aptamers and reporter aptamers, and the effect of the addition order of the aptamers modified magnetic beads and the aptamers modified quantum dots were investigated. Detection of 0.05 nM thrombin was accomplished. The proteins hemoglobin, lysozyme, and transferrin did not interfere in this assay. (author)

2012-01-01

223

Human Thrombin Detection Through a Sandwich Aptamer Microarray: Interaction Analysis in Solution and in Solid Phase  

Directory of Open Access Journals (Sweden)

Full Text Available We have developed an aptamer-based microarray for human thrombin detection exploiting two non-overlapping DNA thrombin aptamers recognizing different exosites of the target protein. The 15-mer aptamer (TBA1) binds the fibrinogen-binding site, whereas the 29-mer aptamer (TBA2) binds the heparin binding domain. Extensive analysis on the complex formation between human thrombin and modified aptamers was performed by Electrophoresis Mobility Shift Assay (EMSA), in order to verify in solution whether the chemical modifications introduced would affect aptamers/protein recognition. The validated system was then applied to the aptamer microarray, using the solid phase system devised by the solution studies. Finally, the best procedure for Sandwich Aptamer Microarray (SAM) and the specificity of the sandwich formation for the developed aptasensor for human thrombin were optimized.

Alice Sosic; Anna Meneghello; Erica Cretaio; Barbara Gatto

2011-01-01

224

The Tick-Derived Anticoagulant Madanin Is Processed by Thrombin and Factor Xa  

Science.gov (United States)

The cysteine-less peptidic anticoagulants madanin-1 and madanin-2 from the bush tick Haemaphysalis longicornis are the founding members of the MEROPS inhibitor family I53. It has been previously suggested that madanins exert their functional activity by competing with physiological substrates for binding to the positively charged exosite I (fibrinogen-binding exosite) of ?-thrombin. We hereby demonstrate that competitive inhibition of ?-thrombin by madanin-1 or madanin-2 involves binding to the enzyme's active site. Moreover, the blood coagulation factors IIa and Xa are shown to hydrolyze both inhibitors at different, although partially overlapping cleavage sites. Finally, the three-dimensional structure of the complex formed between human ?-thrombin and a proteolytic fragment of madanin-1, determined by X-ray crystallography, elucidates the molecular details of madanin-1 recognition and processing by the proteinase. Taken together, the current findings establish the mechanism of action of madanins, natural anticoagulants that behave as cleavable competitive inhibitors of thrombin.

Figueiredo, Ana C.; de Sanctis, Daniele; Pereira, Pedro Jose Barbosa

2013-01-01

225

The tick-derived anticoagulant madanin is processed by thrombin and factor xa.  

UK PubMed Central (United Kingdom)

The cysteine-less peptidic anticoagulants madanin-1 and madanin-2 from the bush tick Haemaphysalis longicornis are the founding members of the MEROPS inhibitor family I53. It has been previously suggested that madanins exert their functional activity by competing with physiological substrates for binding to the positively charged exosite I (fibrinogen-binding exosite) of ?-thrombin. We hereby demonstrate that competitive inhibition of ?-thrombin by madanin-1 or madanin-2 involves binding to the enzyme's active site. Moreover, the blood coagulation factors IIa and Xa are shown to hydrolyze both inhibitors at different, although partially overlapping cleavage sites. Finally, the three-dimensional structure of the complex formed between human ?-thrombin and a proteolytic fragment of madanin-1, determined by X-ray crystallography, elucidates the molecular details of madanin-1 recognition and processing by the proteinase. Taken together, the current findings establish the mechanism of action of madanins, natural anticoagulants that behave as cleavable competitive inhibitors of thrombin.

Figueiredo AC; de Sanctis D; Pereira PJ

2013-01-01

226

Prolyl endopeptidase and thrombin inhibitory diterpenoids from the bark of Xylopia aethiopica.  

UK PubMed Central (United Kingdom)

The inhibitory effects of seven diterpenes, belonging to three different structural classes and isolated from the bark of Xylopia aethiopica, were investigated against the enzymes prolyl endopeptidase (PEP) and alpha-thrombin. Five compounds exhibited inhibitory activity against them.

Diderot NT; Silvere N; Yasin A; Zareen S; Fabien Z; Etienne T; Choudhary MI; Atta-Ur-Rahman

2005-09-01

227

Increased anticoagulant activity of thrombin-binding DNA aptamers by nanoscale organization on DNA nanostructures  

DEFF Research Database (Denmark)

Control over thrombin activity is much desired to regulate blood clotting in surgical and therapeutic situations. Thrombin-binding RNA and DNA aptamers have been used to inhibit thrombin activity and thus the coagulation cascade. Soluble DNA aptamers, as well as two different aptamers tethered by a flexible single-strand linker, have been shown to possess anticoagulant activity. Here, we link multiple aptamers at programmed positions on DNA nanostructures to optimize spacing and orientation of the aptamers and thereby to maximize anticoagulant activity in functional assays. By judicious engineering of the DNA nanostructures, we have created a novel, functional DNA nanostructure, which is a multi-aptamer inhibitor with activity eightfold higher than free aptamer. Reversal of the thrombin inhibition was also achieved by the use of single-stranded DNA antidotes, thus enabling significant control over blood coagulation.

Rangnekar, Abhijit; Zhang, Alex M.

2012-01-01

228

Direct detection of aptamer-thrombin binding via surface-enhanced Raman spectroscopy.  

UK PubMed Central (United Kingdom)

In this study, we exploit the sensitivity offered by surface-enhanced Raman scattering (SERS) for the direct detection of thrombin using the thrombin-binding aptamer (TBA) as molecular receptor. The technique utilizes immobilized silver nanoparticles that are functionalized with thiolated thrombin-specific binding aptamer, a 15-mer (5'-GGTTGGTGTGGTTGG-3') quadruplex forming oligonucleotide. In addition to the Raman vibrational bands corresponding to the aptamer and blocking agent, new peaks (mainly at 1140, 1540, and 1635 cm(-1)) that are characteristic of the protein are observed upon binding of thrombin. These spectral changes are not observed when the aptamer-nanoparticle assembly is exposed to a nonbinding protein such as bovine serum albumin (BSA). This methodology could be further used for the development of label-free biosensors for direct detection of proteins and other molecules of interest for which aptamers are available.

Pagba CV; Lane SM; Cho H; Wachsmann-Hogiu S

2010-07-01

229

Direct detection of aptamer-thrombin binding via surface-enhanced Raman spectroscopy  

Science.gov (United States)

In this study, we exploit the sensitivity offered by surface-enhanced Raman scattering (SERS) for the direct detection of thrombin using the thrombin-binding aptamer (TBA) as molecular receptor. The technique utilizes immobilized silver nanoparticles that are functionalized with thiolated thrombin-specific binding aptamer, a 15-mer (5'-GGTTGGTGTGGTTGG-3') quadruplex forming oligonucleotide. In addition to the Raman vibrational bands corresponding to the aptamer and blocking agent, new peaks (mainly at 1140, 1540, and 1635 cm-1) that are characteristic of the protein are observed upon binding of thrombin. These spectral changes are not observed when the aptamer-nanoparticle assembly is exposed to a nonbinding protein such as bovine serum albumin (BSA). This methodology could be further used for the development of label-free biosensors for direct detection of proteins and other molecules of interest for which aptamers are available.

Pagba, Cynthia V.; Lane, Stephen M.; Cho, Hansang; Wachsmann-Hogiu, Sebastian

2010-07-01

230

Fibrinolytic action of an enzyme preparation covalently bound with modified thrombin  

Energy Technology Data Exchange (ETDEWEB)

It was pointed out previously that modification of thrombin at the tryptophan, tyrosine, arginine and lysine residues impairs its affinity for fibrinogen. Since a long binding site of macromolecular substrate in the thrombin molecule is responsible for binding of the enzyme with the platelet membrane also, it is probably preferable to carry out the modification at other amino acid residues. The purpose of this paper was to confirm experimentally the validity of this approach to the targeted modification of alpha-thrombin in order to obtain a protein polymer matrix with affinity for centers of thrombus formation. Technetium 99m was used as a label in assessing the ability of the modified thrombin to destroy the fibrin clot.

Maksimenko, A.V.; Rusetskii, A.N.; Torchilin, V.P.

1987-06-01

231

Pseudoaneurysm After Spontaneous Rupture of Renal Angiomyolipoma in Tuberous Sclerosis: Successful Treatment with Percutaneous Thrombin Injection  

International Nuclear Information System (INIS)

We report a case of a large perinephric pseudoaneurysm due to spontaneous rupture of renal angiomyolipoma, occluded by percutaneous thrombin injection under ultrasound guidance in a young woman affected by tuberous sclerosis.

2005-01-01

232

Genetic and pharmacological modifications of thrombin formation in apolipoprotein e-deficient mice determine atherosclerosis severity and atherothrombosis onset in a neutrophil-dependent manner.  

UK PubMed Central (United Kingdom)

BACKGROUND: Variations in the blood coagulation activity, determined genetically or by medication, may alter atherosclerotic plaque progression, by influencing pleiotropic effects of coagulation proteases. Published experimental studies have yielded contradictory findings on the role of hypercoagulability in atherogenesis. We therefore sought to address this matter by extensively investigating the in vivo significance of genetic alterations and pharmacologic inhibition of thrombin formation for the onset and progression of atherosclerosis, and plaque phenotype determination. METHODOLOGY/PRINCIPAL FINDINGS: We generated transgenic atherosclerosis-prone mice with diminished coagulant or hypercoagulable phenotype and employed two distinct models of atherosclerosis. Gene-targeted 50% reduction in prothrombin (FII(-/WT):ApoE(-/-)) was remarkably effective in limiting disease compared to control ApoE(-/-) mice, associated with significant qualitative benefits, including diminished leukocyte infiltration, altered collagen and vascular smooth muscle cell content. Genetically-imposed hypercoagulability in TM(Pro/Pro):ApoE(-/-) mice resulted in severe atherosclerosis, plaque vulnerability and spontaneous atherothrombosis. Hypercoagulability was associated with a pronounced neutrophilia, neutrophil hyper-reactivity, markedly increased oxidative stress, neutrophil intraplaque infiltration and apoptosis. Administration of either the synthetic specific thrombin inhibitor Dabigatran etexilate, or recombinant activated protein C (APC), counteracted the pro-inflammatory and pro-atherogenic phenotype of pro-thrombotic TM(Pro/Pro):ApoE(-/-) mice. CONCLUSIONS/SIGNIFICANCE: We provide new evidence highlighting the importance of neutrophils in the coagulation-inflammation interplay during atherogenesis. Our findings reveal that thrombin-mediated proteolysis is an unexpectedly powerful determinant of atherosclerosis in multiple distinct settings. These studies suggest that selective anticoagulants employed to prevent thrombotic events may also be remarkably effective in clinically impeding the onset and progression of cardiovascular disease.

Borissoff JI; Otten JJ; Heeneman S; Leenders P; van Oerle R; Soehnlein O; Loubele ST; Hamulyák K; Hackeng TM; Daemen MJ; Degen JL; Weiler H; Esmon CT; van Ryn J; Biessen EA; Spronk HM; ten Cate H

2013-01-01

233

Mapping Topics and Topic Bursts in PNAS  

CERN Multimedia

Scientific research is highly dynamic. New areas of science continually evolve;others gain or lose importance, merge or split. Due to the steady increase in the number of scientific publications it is hard to keep an overview of the structure and dynamic development of one's own field of science, much less all scientific domains. However, knowledge of hot topics, emergent research frontiers, or change of focus in certain areas is a critical component of resource allocation decisions in research labs, governmental institutions, and corporations. This paper demonstrates the utilization of Kleinberg's burst detection algorithm, co-word occurrence analysis, and graph layout techniques to generate maps that support the identification of major research topics and trends. The approach was applied to analyze and map the complete set of papers published in the Proceedings of the National Academy of Sciences (PNAS) in the years 1982-2001. Six domain experts examined and commented on the resulting maps in an attempt to ...

Mane, K; Mane, Ketan; B\\"orner, Katy

2004-01-01

234

Implicit stage topics  

Directory of Open Access Journals (Sweden)

Full Text Available Il a souvent été proposé que les éléments spatio-temporels en position initiale de phrase spécifient le cadre de l’événement dénoté par la proposition et ont une interprétation thématique ou topicale. Alors que les topiques spatio-temporels explicites ont souvent été étudiés, Erteschik-Schir (1997, 1999) propose l’idée que les topiques spatio-temporels, ou topiques scéniques (stage topics) peuvent aussi être implicites.Dans cet article, nous offrons des arguments en faveur de la notion de topique scénique implicite. Nous montrons qu’un certain nombre de cas d’inversion nominale en français, une configuration syntaxique qui est favorisée par la présence d’un topique scénique explicite, s’expliquent par la présence d’un topique scénique implicite. Le fait que les topiques scéniques implicites interagissent avec la structure syntaxique de la même façon que les topiques scéniques explicites constitue un argument empirique en faveur de leur existence.It has often been proposed that sentence-initial spatio-temporal elements specify the frame in which the whole proposition takes place and are topical (i.e. thematic). Whereas considerable attention has been paid to explicit spatio-temporal topics, Erteschik-Shir (1997, 1999) argues that spatio-temporal topics, or stage topics, can also be implicit.In this article we provide evidence in favour of the notion of implicit stage topic. We show that a certain number of nominal inversion cases in French, a syntactic configuration which is triggered by the presence of an explicit stage topic, are explained by the presence of an implicit stage topic. The fact that implicit stage topics interact with syntactic structure the same way explicit stage topics do constitutes a strong empirical argument in favour of their existence.

Karen Lahousse

2008-01-01

235

Molecularly imprinted aptamers of gold nanoparticles for the enzymatic inhibition and detection of thrombin.  

Science.gov (United States)

We prepared thrombin-binding aptamer-conjugated gold nanoparticles (TBA-Au NPs) through a molecularly imprinted (MP) approach, which provide highly efficient inhibition activity toward the polymerization of fibrinogen. Au NPs (diameter, 13 nm), 15-mer thrombin-binding aptamer (TBA(15)) with different thymidine linkers, and 29-mer thrombin-binding aptamer (TBA(29)) with different thymidine linkers (Tn) in the presence of thrombin (Thr) as a template were used to prepare MP-Thr-TBA(15)/TBA(29)-Tn-Au NPs. Thrombin molecules were then removed from Au NPs surfaces by treating with 100 mM Tris-NaOH (pH ca. 13.0) to form MP-TBA(15)/TBA(29)-Tn-Au NPs. The length of the thymidine linkers and TBA density on Au NPs surfaces have strong impact on the orientation, flexibility, and stability of MP-TBA(15)/TBA(29)-Tn-Au NPs, leading to their stronger binding strength with thrombin. MP-TBA(15)/TBA(29)-T(15)-Au NPs (ca. 42 TBA(15) and 42 TBA(29) molecules per Au NP; 15-mer thymidine on aptamer terminal) provided the highest binding affinity toward thrombin with a dissociation constant of 5.2 × 10(-11) M. As a result, they had 8 times higher anticoagulant (inhibitory) potency relative to TBA(15)/TBA(29)-T(15)-Au NPs (prepared in the absence of thrombin). We further conducted thrombin clotting time (TCT) measurements in plasma samples and found that MP-TBA(15)/TBA(29)-T(15)-Au NPs had greater anticoagulation activity relative to four commercial drugs (heparin, argatroban, hirudin, and warfarin). In addition, we demonstrated that thrombin induced the formation of aggregates from MP-TBA(15)-T(15)-Au NPs and MP-TBA(29)-T(15)-Au NPs, thereby allowing the colorimetric detection of thrombin at the nanomolar level in serum samples. Our result demonstrates that our simple molecularly imprinted approach can be applied for preparing various functional nanomaterials to control enzyme activity and targeting important proteins. PMID:22300379

Liao, Yu-Ju; Shiang, Yen-Chun; Huang, Chih-Ching; Chang, Huan-Tsung

2012-02-23

236

Molecularly imprinted aptamers of gold nanoparticles for the enzymatic inhibition and detection of thrombin.  

UK PubMed Central (United Kingdom)

We prepared thrombin-binding aptamer-conjugated gold nanoparticles (TBA-Au NPs) through a molecularly imprinted (MP) approach, which provide highly efficient inhibition activity toward the polymerization of fibrinogen. Au NPs (diameter, 13 nm), 15-mer thrombin-binding aptamer (TBA(15)) with different thymidine linkers, and 29-mer thrombin-binding aptamer (TBA(29)) with different thymidine linkers (Tn) in the presence of thrombin (Thr) as a template were used to prepare MP-Thr-TBA(15)/TBA(29)-Tn-Au NPs. Thrombin molecules were then removed from Au NPs surfaces by treating with 100 mM Tris-NaOH (pH ca. 13.0) to form MP-TBA(15)/TBA(29)-Tn-Au NPs. The length of the thymidine linkers and TBA density on Au NPs surfaces have strong impact on the orientation, flexibility, and stability of MP-TBA(15)/TBA(29)-Tn-Au NPs, leading to their stronger binding strength with thrombin. MP-TBA(15)/TBA(29)-T(15)-Au NPs (ca. 42 TBA(15) and 42 TBA(29) molecules per Au NP; 15-mer thymidine on aptamer terminal) provided the highest binding affinity toward thrombin with a dissociation constant of 5.2 × 10(-11) M. As a result, they had 8 times higher anticoagulant (inhibitory) potency relative to TBA(15)/TBA(29)-T(15)-Au NPs (prepared in the absence of thrombin). We further conducted thrombin clotting time (TCT) measurements in plasma samples and found that MP-TBA(15)/TBA(29)-T(15)-Au NPs had greater anticoagulation activity relative to four commercial drugs (heparin, argatroban, hirudin, and warfarin). In addition, we demonstrated that thrombin induced the formation of aggregates from MP-TBA(15)-T(15)-Au NPs and MP-TBA(29)-T(15)-Au NPs, thereby allowing the colorimetric detection of thrombin at the nanomolar level in serum samples. Our result demonstrates that our simple molecularly imprinted approach can be applied for preparing various functional nanomaterials to control enzyme activity and targeting important proteins.

Liao YJ; Shiang YC; Huang CC; Chang HT

2012-06-01

237

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

International Nuclear Information System (INIS)

[en] 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 125I-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

1988-10-18

238

Different approaches for the detection of thrombin by an electrochemical aptamer-based assay coupled to magnetic beads.  

Science.gov (United States)

Different assay formats based on the coupling of magnetic beads with electrochemical transduction were compared here for the detection of thrombin by using a thrombin specific aptamer. By using the thrombin-binding aptamer, a direct and an indirect competitive assay for thrombin have been developed by immobilising the aptamer or the protein, respectively. Moreover, another strategy was based on the direct measurement of the enzymatic product of thrombin captured by the immobilised aptamer. All the assays were developed by coupling the electrochemical transduction with the innovative and advantageous use of magnetic beads. The assays based on the immobilisation of the protein were not successful since no binding was recorded between thrombin and its aptamer. With the direct competitive assay, when the aptamer was immobilised onto the magnetic beads, a detection limit of 430nM for thrombin was achieved. A lower detection limit for the protein (175nM) was instead obtained by detecting the product of the enzymatic reaction catalysed by thrombin. All these assays were finally compared with a sandwich assay which reached a detection limit of 0.45nM of thrombin demonstrating the best analytical performances. With this comparison the importance of a deep study on the different analytical approaches for thrombin detection to reach the performances of the best assay configuration has been demonstrated. PMID:18313283

Centi, S; Messina, G; Tombelli, S; Palchetti, I; Mascini, M

2008-01-29

239

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

Energy Technology Data Exchange (ETDEWEB)

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.

Mao, S.J.T.; Yates, M.T.; Owen, T.J.; Krstenansky, J.L. (Merrell Dow Research Institute, Cincinnati, OH (USA))

1988-10-18

240

Obesity and thrombin-generation profiles in women with venous thromboembolism.  

Science.gov (United States)

Obesity is a known risk factor for venous and arterial thrombosis but the mechanisms are still unclear. In women, obesity is correlated with low-grade inflammation and recent data show that BMI is positively associated with thrombin generation. We explored the correlations between obesity, inflammation and thrombin generation in women with increased thrombotic risk by looking at a cohort of women with prior venous thrombosis. One hundred and fifty-six women age 18-65 years were enrolled at diagnosis of first venous thromboembolism (VTE). Plasma samples were obtained at least 3 weeks after cessation of anticoagulant treatment. Thrombin generation was determined with the calibrated automated thrombography (CAT) assay and the Innovance ETP assay. Thrombin generation started later but was more pronounced with higher endogenous thrombin generation potential (ETP) determined with CAT in patients with obesity. The Innovance ETP assay showed results consistent with CAT. Furthermore, patients with obesity had significantly higher levels of fibrinogen, C-reactive protein and plasminogen activator inhibitor-I (PAI-I) than patients without obesity. Increased levels of fibrinogen were the main determinant of the prolonged lag-time in patients with obesity whereas higher levels of prothrombin could account for the difference in the ETP between the groups. We found an association between BMI and ETP values using two different methods to measure thrombin generation. Obesity correlated with increased thrombin generation in women with VTE and the main determinants of this hypercoagulable state were increased levels of fibrinogen and prothrombin. This shows a possible link between obesity, low-grade inflammation and increased thrombin generation in women at increased risk for future thrombosis. PMID:23470648

Sonnevi, Kristina; Tchaikovski, Svetlana N; Holmström, Margareta; Antovic, Jovan P; Bremme, Katarina; Rosing, Jan; Lärfars, Gerd

2013-07-01

 
 
 
 
241

Whole-blood thrombin generation monitored with a calibrated automated thrombogram-based assay.  

UK PubMed Central (United Kingdom)

BACKGROUND: The calibrated automated thrombogram (CAT) assay in plasma is a versatile tool to investigate patients with hypo- or hypercoagulable phenotypes. The objective was to make this method applicable for whole blood measurements. METHODS: Thin-layer technology and the use of a rhodamine 110-based thrombin substrate appear to be essential for a reliable thrombin generation (TG) assay in whole blood. Using this knowledge we developed a whole blood CAT-based assay. RESULTS: We demonstrated that the whole blood CAT-based assay is a sensitive and rapid screening test to assess function of the hemostatic system under more nearly physiological conditions than the TG assay in plasma. Under conditions of low tissue factor concentration (0.5 pmol/L) and 50% diluted blood, the intraassay CV of the thrombogram parameters, endogenous thrombin potential and thrombin peak height, were 6.7% and 6.5%, respectively. The respective interassay CVs were 12% and 11%. The mean interindividual variation (SD) of 40 healthy volunteers was 633 (146) nmol · min/L for the endogenous thrombin potential and 128 (23) nmol/L for the thrombin peak. Surprisingly, erythrocytes contributed more than platelets to the procoagulant blood cell membranes necessary for optimal TG. Statistically significant (P < 0.001) and potentially clinically significant correlations were observed between circulating factor-VIII concentrations in blood of hemophilia A patients and endogenous thrombin potential (r = 0.62) and thrombin peak height (r = 0.58). CONCLUSIONS: We have developed a reliable method to measure TG in whole blood. The assay can be performed with a drop of blood and may provide a useful measurement of TG under more physiological conditions than plasma.

Ninivaggi M; Apitz-Castro R; Dargaud Y; de Laat B; Hemker HC; Lindhout T

2012-08-01

242

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

UK PubMed Central (United Kingdom)

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

Rahman MA; Son JI; Won MS; Shim YB

2009-08-01

243

Behaviour of homologous 125I fibrinogen after thrombin and ancrod infusion in rabbits  

International Nuclear Information System (INIS)

[en] The behaviour of radioactively labelled fibrinogen after infusion of thrombin or ancrod is investigated. Common factors and differences in the behaviour of fibrinogen after infusion of these two enzymes, which act proteolytically on the fibrinogen, are dealt with. Rabbits received an i.v. injection of homologous 125I-fibrinogen 3 days before ancrod or thrombin infusion. On the day of the experiments, one group of animals received an ancrod infusion (1.5 U/kg body weight for 30 minutes), the other a thrombin infusion (600 U/kg body weight for 60 minutes). Intravenous ancrod and thrombin infusions lowered the fibrinogen level to 30% or 50% of the initial value due to intravascular coagulation. About 50% of the 125I fibrinogen was transformed after ancrod exposure into a non-coagulating fraction of fibrinogen derivatives which produces no fibrinolytic decomposition products. (orig./AJ)[de] Das Verhalten von radioaktivmarkiertem Fibrinogen nach Infusion von Thrombin bzw. Ancrod wird untersucht. Gemeinsamkeiten und Unterschiede in dem Verhalten von Fibrinogen nach Infusion dieser beiden am Fibrinogen proteolytisch angreifenden Enzyme werden herausgestellt. Kaninchen wurde 3 Tage vor Ancord- bzw. Thrombin-Infusion homologenes 125J-Fibrinogen intravenoes injiziert. Das Verhalten dieses Fibrinogens wurde ueber die naechsten drei Tage verfolgt. Am Versuchstag wurde einer Gruppe von Kaninchen Ancrod (1,5 U/kg Koerpergewicht ueber 30 Min.) und einer zweiten Gruppe Thrombin (600 U/kg Koerpergewicht ueber 60 Min.) infundiert. Die intravenoese Ancrod- bzw. Thrombin-Infusion liess den Fibrinogenspiegel durch intravaskulaere Gerinnung auf 30% bzw. 50% des Ausgangswertes absinken. Etwa 50% des 125J-Fibrinogens wurde nach Ancrodeinwirkung in eine nicht gerinnbare Fraktion von Fibrinogenderivaten ueberfuehrt, die keine fibrinolytischen Abbauprodukte darstellt. (orig./AJ)

1977-01-01

244

The production of phosphatidylinositol trisphosphate is stimulated by thrombin in human platelets  

Energy Technology Data Exchange (ETDEWEB)

Untreated human platelets labeled to equilibrium with 32Pi contained undetectable levels of 3-phosphorylated phosphoinositides. Stimulation of platelets with thrombin for 5 min resulted in an enormous increase in the amount of phosphatidylinositol 3,4-bisphosphate. We now report that the levels of phosphatidylinositol 3,4,5-trisphosphate are greatly elevated within 90 s of treatment of platelets with thrombin. Phosphatidylinositol 3,4,5-phosphate might have an important role in platelet aggregation.

Nolan, R.D.; Lapetina, E.G. (Burroughs Wellcome Co., Research Triangle Park, NC (USA))

1991-01-31

245

Sensitive and antifouling impedimetric aptasensor for the determination of thrombin in undiluted serum sample.  

UK PubMed Central (United Kingdom)

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.

Qi H; Shangguan L; Li C; Li X; Gao Q; Zhang C

2013-01-01

246

Pulsatile equibiaxial stretch inhibits thrombin-induced RhoA and NF-?B activation  

International Nuclear Information System (INIS)

[en] This study investigated interactions between the effects of mechanical stretch and thrombin on RhoA activation in rat aortic smooth muscle cells (RASMC). Equibiaxial, pulsatile stretch, or thrombin produced a significant increase in RhoA activation. Surprisingly, in combination, 30 min of stretch inhibited the ability of thrombin to activate RhoA. NO donors and 8-bromo-cGMP significantly inhibited thrombin-induced RhoA activation. Interestingly, the nitric oxide synthase (NOS) inhibitor L-NAME increased basal RhoA activity, suggesting that NOS activity exerts a tonic inhibition on RhoA. Stretching RASMC increases nitrite production, consistent with the idea that NO contributes to the inhibitory effects of stretch. Thrombin stimulates MAP kinase and NF-?B pathways through Rho and these responses were blocked by 8-bromo-cGMP or stretch and restored by L-NAME. These data suggest that stretch, acting through NO and cGMP, can prevent the ability of thrombin to stimulate Rho signaling pathways that contribute to pathophysiological proliferative and inflammatory responses

2008-07-18

247

Is fibrin formation and thrombin generation increased during and after an acute coronary syndrome?  

UK PubMed Central (United Kingdom)

INTRODUCTION AND METHODS: In order to study coagulation and fibrinolysis in acute coronary syndrome (ACS) we used a recently developed assay, called OH-index, which provides simultaneous measurements of fibrin formation and fibrinolysis (fibrin degradation) in the patients' plasma. We also investigated thrombin generation using the calibrated automated thrombogram (CAT), and assessed thrombin generation in vivo by measuring F1+2 plasma concentrations. In addition, to better characterize the patients we also assessed markers of inflammation and endothelial function. Eighty-seven ACS patients were sampled at admission, within 24 hours during treatment with low molecular weight heparin (LMH), and 6 months later; 65 healthy controls were also sampled. RESULTS: As assessed by OH-index fibrin formation was slightly depressed at admission, profoundly depressed during LMH treatment and comparable to controls at 6 months, whereas fibrin degradation was elevated, particularly during LMH treatment. F1+2 levels decreased during LMH treatment but did not deviate significantly from controls at admission or in convalescence. CAT data showed that peak thrombin was higher at admission and after 6 months compared to controls, whereas the endogenous thrombin potential only tended to be elevated. Both variables were strongly reduced during LMH treatment. Patients had elevated levels of markers of inflammation and endothelial function as expected. CONCLUSION: ACS-patients have an increased capacity to generate thrombin and an enhanced capacity to degrade fibrin in the acute phase. Increased thrombin generation persists also 6 months after the event.

Skeppholm M; Kallner A; Malmqvist K; Blombäck M; Wallén H

2011-11-01

248

Persistent high factor VIII activity leading to increased thrombin generation - a prospective cohort study.  

UK PubMed Central (United Kingdom)

INTRODUCTION: A persistently elevated level of factor VIII (FVIII) is an independent risk factor for venous thromboembolism (VTE). Although the pathophysiology of VTE is unclear, the involvement of thrombin generation (TG) has been postulated. Consequently this study was designed to (i) investigate the relationships between FVIII, Thrombin generation test (TGT) parameters and D-dimer in VTE patients, (ii) determine whether elevated levels of FVIII and increased TG in these patients are transient or sustained. PATIENTS AND METHODS: After an initial period of anticoagulation had been completed 91 VTE patients and 52 healthy controls were recruited. FVIII levels were determined by one-stage clotting (FVIII:C) and chromogenic (FVIII:Ch) assays. The potential to generate thrombin was measured using the Calibrated Automated Thrombogram (CAT) and D-Dimer was by immuno-turbidometric assay. RESULTS: Patients' FVIII:C levels and FVIII:Ch, exhibited good agreement (rs=0.94; p<0.0001), although FVIII:C exhibited a mean bias of -6%. FVIII:Ch show a significant correlation with TGT Peak Thrombin (rs=0.30; p=0.004) and Peak Thrombin was found to be significantly higher (p=0.04) in patients with FVIII>200 iu/dL. Furthermore elevated levels of FVIII and increased thrombin generation parameters appeared to be consistent over time. CONCLUSION: Our data suggests that high FVIII leading to increased TG confers a significant risk of recurrent VTE and therefore we speculate that these patients may benefit from prolonged anticoagulation therapy.

Ryland JK; Lawrie AS; Mackie IJ; Machin SJ

2012-04-01

249

Thrombin generation in acute cardioembolic and non-cardioembolic ischemic stroke.  

UK PubMed Central (United Kingdom)

Objective. Increased thrombin generation, as measured by the Calibrated Automated Thrombogram (CAT), has recently been reported to predict ischemic stroke, especially stroke with a cardioembolic source. However, there are few studies on thrombin generation using CAT in patients with manifest ischemic stroke, particularly in patients with cardioembolic stroke not yet on anticoagulation. Materials and methods. Therefore, a prospective cohort study of 205 stroke patients > 45 years of age was performed. They were recruited during their hospital stay or shortly thereafter. Inclusion criteria were ischemic stroke or TIA within two weeks and no atrial fibrillation (AF) in the history or at inclusion. Patients received a thumb ECG device in order to detect silent AF. Blood samples were collected at inclusion and after 1 month. Thrombin generation in plasma after addition of tissue factor was assessed in patients and in healthy controls. Results. Mean age of patients was 72 ± 7 years and 43% were females. Peak thrombin concentrations were variable among stroke patients but overall significantly higher at both time points (p < 0.0001) compared to controls, and tended to be highest in patients in whom paroxysmal atrial fibrillation was subsequently documented. Conclusion. Thrombin generation in patients with acute cardioembolic and non-cardioembolic schemic stroke/TIA is variable but overall higher compared to healthy subjects. The long-term prognostic value of thrombin generation in patients with a recent ischemic stroke deserves further investigation.

Rooth E; Sobocinski-Doliwa P; Antovic J; Frykman Kull V; Von Arbin M; Rosenqvist M; Wallén H

2013-09-01

250

Thrombin stimulates MMP-9 mRNA expression through AP-1 pathway in human mesangial cells  

UK PubMed Central (United Kingdom)

AIM: To investigate the thrombin mediated induction of gelatinase B (MMP-9) in mesangial cells (MC) and the underlying role of activator protein?1 (AP-1). METHODS: Cultured human mesangial cells were exposed to thrombin in the presence or absence of hirudin, curcumin, and c-fos antisense or sense oligonucleotides. Northern hybridization was employed to assess MMP-9 mRNA expression, and electrophoretic mobility shift assay (EMSA) for AP-1 DNA binding activity. RESULTS: The levels of MMP-9 mRNA in the cell treated with different doses of thrombin (500, 1500, and 4500 u/L, respectively) were 1.1, 3.3, and 4.8 times higher than that in the control, respectively. There was also an increase in AP-1 binding activity (3.5, 5.9, and 7.1 fold than that of the control) in accordance with MMP-9 mRNA levels in the presence of thrombin. Hirudin, curcumin, and c-fos antisense oligonucleotides could block thrombin-induced expression of MMP-9 mRNA as well as AP-1 binding activity. CONCLUSION: Thrombin is a potent stimulator of MMP-9 gene expression in human mesangial cells, and the underlying intracellular events are mediated, at least partly, by AP-1 pathway.

LIU Wen-Hu; CHEN Xiang-Mei; FU Bo

2000-01-01

251

Highly sensitive optical biosensor for thrombin based on structure switching aptamer-luminescent silica nanoparticles.  

UK PubMed Central (United Kingdom)

We describe here the construction of a sensitive and selective optical sensor system for the detection of human ?-thrombin. The surface functionalized luminescent [Ru(dpsphen)(3)](4-) (dpsphen-4,7-diphenyl-1,10-phenanthroline disulfonate) ion doped silica nanoparticles (SiNPs) with a size ~70 nm have been prepared. The DABCYL (2-(4-dimethylaminophenyl)diazenyl-benzoic acid) quencher labeled thrombin binding aptamer is conjugated to the surface of SiNPs using BS(3) (bis(sulfosuccinimidyl) suberate) as a cross-linker, resulting in the conformational change of aptamer to form G-quadruplex structure upon the addition of thrombin. The binding event is translated into a change in the luminescence intensity of Ru(II) complex via FRET mechanism, due to the close proximity of DABCYL quencher with SiNPs. The selective detection of thrombin using the SiNPs-aptamer system up to 4 nM is confirmed by comparing its sensitivity towards other proteins. This work demonstrates the application of simple aptamer-SiNPs conjugate as a highly sensitive system for the detection of thrombin and also it is highly sensitive towards thrombin in the presence of other proteins and complex medium such as BSA.

Babu E; Mareeswaran PM; Rajagopal S

2013-01-01

252

Electrochemical impedance spectroscopy for study of aptamer-thrombin interfacial interactions.  

Science.gov (United States)

A simple and highly sensitive electrochemical impedance spectroscopy (EIS) biosensor based on a thrombin-binding aptamer as molecular recognition element was developed for the determination of thrombin. The signal enhancement was achieved by using gold nanoparticles (GNPs), which was electrodeposited onto a glassy carbon electrode (GCE), as a platform for the immobilization of the thiolated aptamer. In the measurement of thrombin, the change in interfacial electron transfer resistance of the biosensor using a redox couple of [Fe(CN)(6)](3-/4-) as the probe was monitored. The increase of the electron transfer resistance of the biosensor is linear with the concentration of thrombin in the range from 0.12nM to 30nM. The association and dissociation rate constants of the immobilized aptamer-thrombin complex were 6.7x10(3)M(-1)s(-1) and 1.0x10(-4)s(-1), respectively. The association and dissociation constants of three different immobilized aptamers binding with thrombin were measured and the difference of the dissociation constants obtained was discussed. This work demonstrates that GNPs electrodeposited on GCE used as a platform for the immobilization of the thiolated aptamer can improve the sensitivity of an EIS biosensor for the determination of protein. This work also demonstrates that EIS method is an efficient method for the determination of association and dissociation constants on GNPs modified GCE. PMID:18339536

Li, Xiaoxia; Shen, Lihua; Zhang, Dongdong; Qi, Honglan; Gao, Qiang; Ma, Fen; Zhang, Chengxiao

2008-02-08

253

Identification of a thrombin sequence with growth factor activity on macrophages  

International Nuclear Information System (INIS)

In contrast to fibroblasts, the exposure of G0/G1-arrested J774 cells, a murine macrophage-like tumor cell line, with either active or esterolytically inactive diisopropyl phosphorofluoridate-conjugated ?-thrombin results in a mitogenic response as measured by increased [3H]thymidine incorporation. This response to thrombin is optimal at 10 nM and is specifically blocked by hirudin, a high-affinity thrombin inhibitor. When prethrombin 1 is cleaved with cyanogen bromide, a fragment (peptide CB67-129) is produced that, like the parent thrombin molecule, is mitogenic for J774 cells but not for fibroblasts. Limited tryptic digests of this fragment retain the ability to stimulate macrophages - a function that can be mimicked by a synthetic tetradecapeptide homologue of CB67-129 but not by any of a series of well-known growth promoters. The mitogenic effects of this peptide are not limited to J774 cells but can be expressed in other macrophage-like tumor cells lines. In addition to increased [3H]thymidine incorporation, the synthetic B chain peptide stimulates cell proliferation as evidenced by a dose-dependent increase in total protein per culture well and cell number. The authors conclude that the thrombin molecule contains a macrophage growth factor domain that is separate and distinct from its active center. Thus, thrombin, in addition to its major role in hemostasis and thrombosis, may also have important functions in such basic processes as the inflammatory response and monocytopoiesis

1986-01-01

254

Recombinant Technology and Probiotics  

Directory of Open Access Journals (Sweden)

Full Text Available Recombinant technology has led the way to monumental advances in the development of useful molecules, including the development of safe probiotics. The development of novel approaches using recombinant technology and probiotics that allow accurate targeting of therapeutics to the mucosa is an interesting area of research. The creation and use of recombinant probiotics expressing recombinantovalbumin, recombinant ovalbumin mutants and yet-to-be-designed recombinant hypo/non-allergenic molecules offer the opportunity to further investigate their effects for food, nutrition, environment andhealth. This review highlights advances in native probiotics and recombinant probiotics expressing native and recombinant molecules for food, nutrition, environment and health.

Icy D’Silva

2011-01-01

255

TOPICAL GEL COMPOSITION  

UK PubMed Central (United Kingdom)

Improved topical gel compositions, such as those containing brimonidine for the treatment of skin disorders are described. The gel compositions contain carbomer and paraben, and are substantially free of paraben crystalline particles after an extended period of storage.

BUGE JEAN-CHRISTOPHE; NADAU FOURCADE KARINE; MEUNIER CYRIL

256

Topical photodynamic therapy  

Directory of Open Access Journals (Sweden)

Full Text Available Topical photodynamic therapy is a therapeutic modality in development, thus arises grate interest among dermatologists worldwide. It is an effective therapy for actinic keratosis, superficial BCC and Bowenos disease. Treatment efficacy, good cosmetics, low risk of skin cancer, low invasiveness, low rate of adverse events, facility for treating multiple or large lesions, especially in poor healing sites and, for penile, digital and facial involvement, low general toxicity and possibility of repeating the treatments with the same efficiency, enable topical photodynamic therapy to become increasingly practiced treatment modality. Researching aimed topical photodynamic therapy to prove as a treatment modality for clinical use in other dermatoses, is in experimental phase. To answer the question when dermatologist should consider using topical photodynamic therapy treatment modatility, we are present available date.

Polja?ki Mirjana; Jovanovi? Marina; Matovi? Ljubinka; Lugonja Branislava; Gaji? Branislava; Roš Tatjana

2006-01-01

257

Topical Gel : A Review  

Directory of Open Access Journals (Sweden)

Full Text Available Topical drug administration is a localized drug delivery system anywhere in the body through ophthalmic, rectal, vaginal and skin as topical routes. Skin is one of the most readily accessible organs on human body for topical administration and is main route of topical drug delivery system.The skin of an average adult body covers a surface area approximately 2m2 and receives about one third of the blood circulating through the body. An average human skin surface is known to contain, on the average 40-70 hair follicles and 200-300 sweat ducts on every square centimeter of the skin. Although skin has been divided histologically into the stratum corneum, the living epidermis and the dermis, collectively it can be considered a laminate of barrier, permeation of this laminate can occur by diffusion via:

Ms. Rashmi; Mr. Rajeev Garg; Mr. Sandeep Kumar; Dr. G. D.Gupta

2008-01-01

258

Topical Anesthetics - Overview  

Science.gov (United States)

... health advisory to alert you to the potential hazards of using skin-numbing products, also known as topical anesthetics, for cosmetic procedures. ... More results from www.fda.gov/drugs/drugsafety/drugsafetypodcasts

259

Topics in Elementary Geometry  

CERN Multimedia

Presents classical results from geometry, such as Pythagoras' theorem, the nine-point circle, Morley's triangle, and Poncelet's polygons. This book contains geometric theorems and covers a range of topics in elementary plane Euclidean geometry

Bottema, O

2009-01-01

260

Thrombin generation in chronic obstructive pulmonary disease: dependence on plasma factor composition.  

UK PubMed Central (United Kingdom)

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is associated with an increased risk for thromboembolic events. We investigated thrombin generation profiles in COPD patients and their dependence on plasma factor/inhibitor composition. METHODS: Factors (f) (fII, fV, fVII, fVIII, fIX, fX), antithrombin, protein C (PC) and free tissue factor pathway inhibitor (fTFPI) from 60 COPD patients (aged 64.2 ± 10.1 years; a mean forced expiratory volume in 1 second [FEV(1)], 55.6 ± 15.8% of predicted values) were compared with those for 43 controls matched for age, sex, weight and smoking. Patients receiving anticoagulation were excluded. Using each individual's plasma coagulation protein composition, tissue factor-initiated thrombin generation was assessed computationally. RESULTS: COPD patients had higher fII (115 ± 16 vs 102 ± 10%, p < 0.0001), fV (114 ± 19 vs 102 ± 12%, p = 0.0002), fVII (111 ± 15 vs 102 ± 17%, p = 0.002), fVIII (170 ± 34 vs 115 ± 27%, p < 0.0001), and fIX (119 ± 21 vs 107 ± 17%, p = 0.003), and lower fTFPI (17.7 ± 3.2 vs 18.9 ± 3.2 ng/ml, p = 0.047) compared with controls, while fX, antithrombin, and PC were similar in both groups. Computational thrombin generation profiles showed that compared with controls, COPD patients had higher maximum thrombin levels (+28.3%, p < 0.0001), rates of thrombin generation (+46.1%, p < 0.0001) and total thrombin formation (+14.4%, p < 0.001), together with shorter initiation phase of thrombin generation (p < 0.0001) and the time to maximum thrombin levels (p < 0.0001). Thrombin generation profiles in COPD patients can be normalized via correction of fII, fVIII , fIX and TFPI. The severity of COPD and inflammatory markers were not associated with thrombin generation profiles. CONCLUSIONS: Prothrombotic phenotype in COPD patients is largely driven by increased prothrombin, fVIII, fIX, and lower fTFPI.

Undas A; Jankowski M; Kaczmarek P; Sladek K; Brummel-Ziedins K

2011-10-01

 
 
 
 
261

Topical treatment of melasma  

Directory of Open Access Journals (Sweden)

Full Text Available Melasma is a common hypermelanotic disorder affecting the face that is associated with considerable psychological impacts. The management of melasma is challenging and requires a long-term treatment plan. In addition to avoidance of aggravating factors like oral pills and ultraviolet exposure, topical therapy has remained the mainstay of treatment. Multiple options for topical treatment are available, of which hydroquinone (HQ) is the most commonly prescribed agent. Besides HQ, other topical agents for which varying degrees of evidence for clinical efficacy exist include azelaic acid, kojic acid, retinoids, topical steroids, glycolic acid, mequinol, and arbutin. Topical medications modify various stages of melanogenesis, the most common mode of action being inhibition of the enzyme, tyrosinase. Combination therapy is the preferred mode of treatment for the synergism and reduction of untoward effects. The most popular combination consists of HQ, a topical steroid, and retinoic acid. Prolonged HQ usage may lead to untoward effects like depigmentation and exogenous ochronosis. The search for safer alternatives has given rise to the development of many newer agents, several of them from natural sources. Well-designed controlled clinical trials are needed to clarify their role in the routine management of melasma.

Bandyopadhyay Debabrata

2009-01-01

262

Thrombin detection using a piezoelectric aptamer-linked immunosorbent assay.  

Science.gov (United States)

The development of diagnostic assays using highly targeted specific aptamers with existing detection platforms has been an endeavor with few opportunities until now. Many current commercially available diagnostic platforms make use of detection systems employing capture agents composed of modified antigen-specific antibodies coupled with a variety of detection modalities, including radioimmunoassays, fluorescence-based detection assays, electro/chemiluminescence assays, and immunoradiometric assays. In the studies presented here, a novel frequency-modulating technology from BioScale called Acoustic Membrane MicroParticle (AMMP) detection was used to demonstrate a sensitive and reproducible method of incorporating aptamers as capture and detection agents. The method provides a robust and rapid detection of thrombin in human serum while also eliminating the labor-intensive efforts of Western blot analysis and is not affected by the interfering substances found in serum that often affect optical-based detection systems. In addition, we have demonstrated, for the first time, the adaptation of the AMMP platform to exploit aptamers against a clinically relevant target. The AMMP platform is an ideal medium for using aptamers in commercial assay development for application in a clinical setting. PMID:23994562

Collins, Cheryl M; Yui, Samuel; Roberts, Charles E S; Kojic, Igor

2013-08-28

263

Thrombin-activatable fibrinolysis inhibitor in hypothyroidism and hyperthyroxinaemia.  

UK PubMed Central (United Kingdom)

Endocrine disorders affect both the coagulation and fibrinolytic systems, and have been associated with the development of cardiovascular diseases. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a link between coagulation and the fibrinolytic system. The aim of this study was to determine the effect of thyroid hormone excess and deficiency on TAFI levels and function. The effect of hyperthyroxinemia on TAFI was studied in healthy volunteers who were randomised to receive levothyroxine or no medication for 14 days in a crossover design. The effect of hypothyroidism on TAFI was studied in a multicentre observational cohort study. Blood was drawn before treatment of patients with newly diagnosed hypothyroidism and when euthyroidism was achieved. Plasma clot-lysis times, activated TAFI (TAFIa)-dependent prolongation of clot-lysis and TAFI levels were measured. Thyroid hormone excess resulted in a hypofibrinolytic condition and in an enhanced TAFIa-dependent prolongation of clot lysis. A trend towards decreased plasma TAFI levels was observed in healthy volunteers who used levothyroxine. Hypothyroidism resulted in hyperfibrinolysis and a reduced TAFIa-dependent prolongation of clot lysis. In conclusion, alterations of TAFIa-dependent prolongation of clot lysis in patients with thyroid disorders may cause an impaired haemostatic balance. The disturbed haemostatic balance in patients with hyperthyroidism might make them prone to thrombosis, while the risk for bleeding may increase in patients with hypothyroidism.

Verkleij CJ; Stuijver DJ; van Zaane B; Squizzato A; Brandjes DP; Büller HR; Meijers JC; Gerdes VE

2013-02-01

264

Thrombin-Binding Aptamer Quadruplex Formation: AFM and Voltammetric Characterization.  

Science.gov (United States)

The adsorption and the redox behaviour of thrombin-binding aptamer (TBA) and extended TBA (eTBA) were studied using atomic force microscopy and voltammetry at highly oriented pyrolytic graphite and glassy carbon. The different adsorption patterns and degree of surface coverage were correlated with the sequence base composition, presence/absence of K(+), and voltammetric behaviour of TBA and eTBA. In the presence of K(+), only a few single-stranded sequences present adsorption, while the majority of the molecules forms stable and rigid quadruplexes with no adsorption. Both TBA and eTBA are oxidized and the only anodic peak corresponds to guanine oxidation. Upon addition of K(+) ions, TBA and eTBA fold into a quadruplex, causing the decrease of guanine oxidation peak and occurrence of a new peak at a higher potential due to the oxidation of G-quartets. The higher oxidation potential of G-quartets is due to the greater difficulty of electron transfer from the inside of the quadruplex to the electrode surface than electron transfer from the more flexible single strands. PMID:20798847

Diculescu, Victor Constantin; Chiorcea-Paquim, Ana-Maria; Eritja, Ramon; Oliveira-Brett, Ana Maria

2010-06-16

265

Thrombin-Binding Aptamer Quadruplex Formation: AFM and Voltammetric Characterization.  

UK PubMed Central (United Kingdom)

The adsorption and the redox behaviour of thrombin-binding aptamer (TBA) and extended TBA (eTBA) were studied using atomic force microscopy and voltammetry at highly oriented pyrolytic graphite and glassy carbon. The different adsorption patterns and degree of surface coverage were correlated with the sequence base composition, presence/absence of K(+), and voltammetric behaviour of TBA and eTBA. In the presence of K(+), only a few single-stranded sequences present adsorption, while the majority of the molecules forms stable and rigid quadruplexes with no adsorption. Both TBA and eTBA are oxidized and the only anodic peak corresponds to guanine oxidation. Upon addition of K(+) ions, TBA and eTBA fold into a quadruplex, causing the decrease of guanine oxidation peak and occurrence of a new peak at a higher potential due to the oxidation of G-quartets. The higher oxidation potential of G-quartets is due to the greater difficulty of electron transfer from the inside of the quadruplex to the electrode surface than electron transfer from the more flexible single strands.

Diculescu VC; Chiorcea-Paquim AM; Eritja R; Oliveira-Brett AM

2010-01-01

266

Therapeutic correction of thrombin generation in dilution-induced coagulopathy: computational analysis based on a data set of healthy subjects.  

UK PubMed Central (United Kingdom)

BACKGROUND: Prothrombin complex concentrates (PCCs), which contain different coagulation proteins, are attractive alternatives to the standard methods to treat dilution-induced (and, generally, traumatic) coagulopathy. We investigated the ability of a novel PCC composition to restore normal thrombin generation in diluted blood. The performance of the proposed PCC composition (coagulation factors [F] II, IX, and X and the anticoagulant antithrombin), designated PCC-AT, was compared with that of FVIIa and PCC-FVII, which is the PCC composition containing FII, FVII, FIX, and FX (main components of most PCCs). METHODS: We used a thoroughly validated computational model to simulate thrombin generation in normal and diluted blood for 472 healthy subjects in the control group of the Leiden Thrombophilia Study. For every simulated thrombin curve, we calculated and analyzed five standard thrombin generation parameters. RESULTS: The three therapeutic agents (FVIIa, PCC-FVII, and PCC-AT) caused statistically significant changes in each of the five thrombin generation parameters in diluted blood. Factor VIIa tended to primarily impact clotting time, thrombin peak time, and maximum slope of the thrombin curve, whereas in the case of PCC-FVII, thrombin peak height and the area under the thrombin curve were affected particularly strongly. As a result, these two therapeutics tended to push those respective parameters outside their normal ranges. PCC-AT significantly outperformed both FVIIa and PCC-FVII in its ability to normalize individual thrombin generation parameters in diluted blood. Furthermore, PCC-AT could simultaneously restore all five thrombin generation parameters to their normal levels in every subject in the study group. CONCLUSIONS: Our computational results suggest that PCC-AT may demonstrate a superior ability to restore normal thrombin generation compared with FVIIa and PCC-FVII.

Mitrophanov AY; Rosendaal FR; Reifman J

2012-08-01

267

Preanalytic variables of thrombin generation: towards a standard procedure and validation of the method.  

UK PubMed Central (United Kingdom)

BACKGROUND: Thrombin generation assays are sensitive methods for assessment of the overall clotting potential of plasma, but, despite their common use in thrombosis research, standardization of preanalytic conditions is lacking. In order to set up a standardized protocol, we analyzed different preanalytic variables and validated the calibrated automated thrombogram method. METHODS AND RESULTS: Thrombin generation was assessed with 0, 1 and 5 pm tissue factor (TF). Variations in thrombin generation were mostly attributable to the type of collection tube, mainly because of variations in contact activation. The collection tube also determined the influence of other preanalytic variables on thrombin generation, e.g. the need for a discard tube, the storage of whole blood, and the centrifugation method. Regarding the collection system, blood drawn through intravenous catheters or butterfly needles showed significantly more hemolysis than blood obtained with venipuncture using conventional needles. The results showed that a discard tube is still needed for thrombin generation measurements. After blood collection, whole blood is best centrifuged immediately, to prevent activation or degradation of coagulation proteins, and a second centrifugation step at 10,000 × g is recommended. After thawing, plasma is best analyzed immediately, as storage resulted in thrombin generation results outside the 10% range of the reference sample. On the basis of these results, we set up an in-house standardized protocol, which was used for validation, resulting in coefficients of variations of < 15% for all derived parameters with both the 1 and 5 pm TF triggers. CONCLUSION: Thrombin generation was greatly influenced by preanalytic conditions, demonstrating the need for an international standardized protocol.

Loeffen R; Kleinegris MC; Loubele ST; Pluijmen PH; Fens D; van Oerle R; ten Cate H; Spronk HM

2012-12-01

268

Thrombin generation post elective caesarean section: effect of low molecular weight heparin.  

UK PubMed Central (United Kingdom)

INTRODUCTION: Caesarean section (CS) is a significant risk factor for venous thromboembolism.. Low molecular weight heparin (LMWH) is commonly used for thromboprophylaxis post emergency caesarean delivery. However, no consensus exists regarding LMWH thromboprophylaxis following elective caesarean section. Measures of thrombin formation may indicate the full anticoagulant activity of LMWH in this setting. MATERIALS AND METHODS: Anti-Xa, tissue factor pathway inhibitor (TFPI), thrombin anti-thrombin complex (TAT) and endogenous thrombin potential (ETP) were measured in twenty healthy women who received 4,500 IU tinzaparin 6 hours post CS (CS1), twenty women who received 4,500 IU tinzaparin at 10-12 hours post delivery (CS2) and twenty women post spontaneous vaginal delivery (SVD). RESULTS: Prior to initiation of LMWH, TAT levels at 6 hours post delivery were significantly higher in the CS1 and CS2 groups than the SVD group (P<0.002); TAT levels were significantly reduced up to 24 hours post LMWH treatment despite declining anti-Xa levels (P<0.001). In CS1, peak thrombin and ETP were significantly reduced following LMWH prophylaxis (P<0.0001; P<0.002) and reverted to pre-delivery levels 10 hours post LMWH. TFPI levels mirror anti-Xa levels during the 24 hours following LMWH treatment in CS1 group with peak levels coinciding with peak anti-Xa levels 4 hours post injection. CONCLUSION: In women post caesarean section, anti-Xa levels do not reflect the full anticoagulant effects of LMWH. In-vivo thrombin production (TAT) is effectively reduced even when anti-Xa levels are negligible. LMWH thromboprophylaxis in this healthy cohort of patients appears to have a sustained effect in reducing excess thrombin production post elective caesarean section.

Ismail SK; Norris L; Muttukrishna S; Higgins JR

2012-11-01

269

Studies of thrombin-induced proteoglycan release in the degradation of human and bovine cartilage.  

Science.gov (United States)

Because fibrin is commonly observed within arthritic joints, studies were undertaken to determine whether purified coagulation and fibrinolytic proteases degrade cartilage in vitro and to seek evidence for the activation of coagulation in arthritic joints through measurements of the levels of inhibitor-enzyme complexes and several other proteins associated with coagulation and fibrinolysis. The concentrations of 13 plasma proteins and complexes of thrombin and Factor Xa with antithrombin III were measured in synovial fluids recovered at the time of knee replacement surgery. All zymogens necessary to constitute the coagulation cascade were present. Thrombin and the combination of prothrombin plus prothrombinase induced proteoglycan release from both normal and arthritic cartilages. Factor Xa and plasmin induced release from diseased cartilage only, and urokinase, tissue plasminogen activator, and activated protein C were without effect at the levels used. At saturating levels of thrombin (> or = 2.0 microM) 80% of the proteoglycan content of normal cartilage was released within 24 h. Thrombin, which is cationic, reversibly binds cartilage with Kd = 7.0 +/- 1.0 microM and Bmax = 820 +/- 70 ng/mg of human cartilage. Levels of thrombin-antithrombin III complexes in synovial fluids and arthritis were 4-fold higher in osteo (OA) and 43-fold higher in rheumatoid (RA) than in controls (0.98 nM). Factor Xa-antithrombin III complex levels were threefold lower in OA and fivefold higher in RA than in controls (0.24 nM). These elevated levels of enzyme-inhibitor complexes imply a history of activation of coagulation within the joint, especially in RA. Since thrombin degrades cartilage in vitro and had been generated in vivo, as inferred by the existence of thrombin-antithrombin III complexes, intraarticular activation of coagulation may both contribute to the pathology of arthritis and comprise a target for therapy and diagnosis. PMID:8040300

Furmaniak-Kazmierczak, E; Cooke, T D; Manuel, R; Scudamore, A; Hoogendorn, H; Giles, A R; Nesheim, M

1994-08-01

270

Nanomaterial-amplified "signal off/on" electrogenerated chemiluminescence aptasensors for the detection of thrombin.  

UK PubMed Central (United Kingdom)

Two electrogenerated chemiluminescence (ECL) aptasensors for the detection of thrombin were developed using the thrombin binding aptamer (TBA) taken as a molecular recognition element and nanomaterial as a carrier of the ECL capture/signal probe. In the "signal off" aptasensor, the thiolated capture probe (ss-DNA, 12-mer) was self-assembled on the gold nanoparticles (GNPs) which were self-assembled on the surface of gold electrode, and hybridized with six-base segment of the ss-DNA sequence (Tgt-aptamer, 21-mer) containing TBA-I (ss-DNA, 15-mer) tagged with ruthenium complex, producing a high ECL intensity. Introduction of the analyte thrombin triggered the dissociation of the Tgt-aptamer tagged with ruthenium complex from the aptasensors, led to significantly decrease in ECL intensity. The decreased ECL intensity was in proportion to the concentration of thrombin in a range from 2.7×10(-12) to 2.7×10(-9) M with a detection limit of 8×10(-13) M. In the "signal on" aptasensor, the thiolated TBA-I was self-assembled on the gold electrode for capturing thrombin onto the electrode and then the TBA-II (ss-DNA, 29-mer) labeled with single-walled carbon-nanotubes (SWNT)-ECL tag was bound with epitope of thrombin, producing a high ECL intensity. The increased ECL intensity was linearly with the concentration of thrombin from 1.0×10(-14) M to 1.0×10(-11) M with a detection limit of 3×10(-15) M. The present work demonstrates that using nanomaterial as a carrier for capture probe and signal probe is a promising way to amplify the ECL signal and to improve the sensitivity of the aptasensors.

Li Y; Qi H; Gao Q; Yang J; Zhang C

2010-10-01

271

The variable detergent sensitivity of proteases that are utilized for recombinant protein affinity tag removal.  

UK PubMed Central (United Kingdom)

Recombinant proteins typically include one or more affinity tags to facilitate purification and/or detection. Expression constructs with affinity tags often include an engineered protease site for tag removal. Like other enzymes, the activities of proteases can be affected by buffer conditions. The buffers used for integral membrane proteins contain detergents, which are required to maintain protein solubility. We examined the detergent sensitivity of six commonly-used proteases (enterokinase, factor Xa, human rhinovirus 3C protease, SUMOstar, tobacco etch virus protease, and thrombin) by use of a panel of 94 individual detergents. Thrombin activity was insensitive to the entire panel of detergents, thus suggesting it as the optimal choice for use with membrane proteins. Enterokinase and factor Xa were only affected by a small number of detergents, making them good choices as well.

Vergis JM; Wiener MC

2011-08-01

272

The variable detergent sensitivity of proteases that are utilized for recombinant protein affinity tag removal.  

Science.gov (United States)

Recombinant proteins typically include one or more affinity tags to facilitate purification and/or detection. Expression constructs with affinity tags often include an engineered protease site for tag removal. Like other enzymes, the activities of proteases can be affected by buffer conditions. The buffers used for integral membrane proteins contain detergents, which are required to maintain protein solubility. We examined the detergent sensitivity of six commonly-used proteases (enterokinase, factor Xa, human rhinovirus 3C protease, SUMOstar, tobacco etch virus protease, and thrombin) by use of a panel of 94 individual detergents. Thrombin activity was insensitive to the entire panel of detergents, thus suggesting it as the optimal choice for use with membrane proteins. Enterokinase and factor Xa were only affected by a small number of detergents, making them good choices as well. PMID:21539919

Vergis, James M; Wiener, Michael C

2011-04-24

273

Fluorescent measurement of affinity binding between thrombin and its aptamers using on-chip affinity monoliths.  

UK PubMed Central (United Kingdom)

A microfluidic chip with integrated 2mm long monoliths incorporated with poly(ethylene glycol) (PEG) groups was developed for thrombin-aptamer interaction study. The non-G quartet forming oligonucleotide coated monoliths was compared to a 15 mer thrombin-binding aptamer, in which affinity binding and elution processes were real-time monitored fluorescently. The results showed that the fluorescence intensity of aptamer stationary phase is approximately 10 times higher than that of the control column, which is probably due to the successful suppression of nonspecific adsorption between thrombin and aptamers/monoliths by using PEG-monolith. The experiment was repeated using human serum albumin (HSA) and green fluorescence protein (GFP) as interferences, it was double confirmed that thrombin was selectively retained by PEG-monolith. An elution efficiency of 75% was achieved with an elute of 200mM acetic acid and 2M NaCI, and the eluted thrombin was successfully separated in an ionic buffer system of 20mM NaHCO3 (pH 9.5) with 3% PEG. The hydrophilic and antifouling properties of PEG-monolith greatly decrease nonspecific adsorption and enhance detection sensitivity, which provided an alternative method to perform on-chip fluorescent measurement of bioaffinity binding.

Gao C; Sun X; Woolley AT

2013-05-01

274

Body composition as determinant of thrombin generation in plasma: the Hoorn study.  

UK PubMed Central (United Kingdom)

OBJECTIVE: The association between obesity and cardiovascular disease and venous thromboembolism might, at least partially, be explained by a hypercoagulable state. The extent to which body fat mass and its distribution contribute to a hypercoagulable state is unknown. In this study, we investigated the association between body composition and thrombin generation and evaluated the potential mediating role of low-grade inflammation. METHODS AND RESULTS: We studied 586 individuals from the Hoorn Study (mean age, 69.7 ± 6.5 years, 298 women) in whom body composition was assessed by whole body dual-energy absorptiometry. Thrombin generation was measured using the calibrated automated thrombogram. Multiple regression analyses showed a positive association between total body fat and thrombin generation in women but not in men. In addition, detailed analyses of regional body composition showed that central but not peripheral fat mass was associated with greater thrombin generation and that there was a trend toward an inverse association with peripheral lean mass. The reported positive associations were partially attenuated by low-grade inflammation, however. CONCLUSIONS: Body fat mass, in particular a central pattern of fat distribution, is associated with higher levels of thrombin generation in elderly women but not in men. This association may partially be explained by adiposity-related low-grade inflammation, but this hypothesis needs to be further investigated in mechanistic/prospective studies.

Beijers HJ; Ferreira I; Spronk HM; Bravenboer B; Dekker JM; Nijpels G; ten Cate H; Stehouwer CD

2010-12-01

275

Evaluation of the hemostatic potential including thrombin generation of three different therapeutic pathogen-reduced plasmas.  

UK PubMed Central (United Kingdom)

BACKGROUND AND OBJECTIVES: Several pathogen inactivation methods currently applied to therapeutic plasma may result in products with different hemostatic properties. This study aims at evaluating and comparing the hemostatic potential of different therapeutic plasma preparations currently available in France. MATERIALS AND METHODS: We studied three types of pathogen-reduced plasma for transfusion (MB/light, Amotosalen/UVA, industrial S/D plasma). Quarantine, non-pathogen-reduced plasma, was used as a control. This study compared more specifically the content in FVIII, fibrinogen (clottable and antigen assays) and ADAMTS-13 and evaluated the intrinsic hemostatic properties using a thrombin generation test [Calibrated Automated Thrombogram (CAT)] at high and low concentrations of tissue factor to assess the maximum quantity of thrombin generated or the contribution of FVIII and FIX in the amplification phase of thrombin generation, respectively. RESULTS: The median FVIII concentration was >70 IU/dl for each preparation. Endogenous thrombin potential values were significantly different among the methods of plasma preparation (P<0·001) but were all in the range of the values measured in donors' plasma. Control by the thrombomodulin-activated protein C system was preserved in all preparations (>50% inhibition of endogenous thrombin potential). Fibrinogen concentrations were all within normal range but fibrinogen levels were lower in the plasmas treated with photochemical methods. ADAMTS-13 levels were preserved. CONCLUSION: The hemostatic potential appears well preserved in all therapeutic plasmas tested but there are some differences between preparations, the clinical relevance of which remains to be elucidated.

Hacquard M; Lecompte T; Belcour B; Geschier C; Jacquot C; Jacquot E; Schneider T

2012-05-01

276

Nanochannels for diagnostic of thrombin-related diseases in human blood.  

UK PubMed Central (United Kingdom)

A high sensitive voltammetric method for rapid determination of thrombin spiked in whole blood by taking advantage of both aptamer-based recognition and the use of a nanoporous membrane has been developed. The nanoporous membrane not only acts as platform for the thrombin recognition but also as filter of the micrometric components such as white and red blood cells, consequently minimizing matrix effects. The protocol involves a sandwich format in the inner walls (200 nm diameter) of an anodized alumina oxide filter membrane (AAO). The analytical signal, by DPV oxidation of [Fe(CN)(6)](4-), is based on the blockage in the pores which affects the diffusion of [Fe(CN)(6)](4-) to the screen-printed carbon electrotransducer (SPCEs) modified with the membrane. By labeling the anti-thrombin IgG with AuNPs followed by silver enhancement a greater passive signal enhancement in comparison to the membrane blockage has been observed. The contribution of both electrostatic/steric effects in this blockage due to the subsequent formation of the aptamer-thrombin complex and the final sandwich assay is investigated. The efficiency of the system is also monitored by microscopic techniques. The resulted biosensing system allows detecting thrombin spiked in whole blood at very low levels (LOD 1.8 ng mL(-1)) which are within the range of clinical interest for the diagnostic of coagulation abnormalities as well as pulmonary metastasis.

de la Escosura-Muñiz A; Chunglok W; Surareungchai W; Merkoçi A

2013-02-01

277

Thrombin generation and procoagulant phospholipids in patients with essential thrombocythemia and reactive thrombocytosis.  

UK PubMed Central (United Kingdom)

Thrombocytosis is a commonly encountered clinical scenario and can be either a secondary process (reactive thrombocytosis), or due to clonal disorder (ie, essential thrombocythemia). This distinction is important as it carries implications for evaluation, prognosis and treatment. In this study we compared procoagulant potential in essential thrombocythemia and reactive thrombocytosis by measuring the thrombin generation and the level of circulating procoagulant phospholipids with functional tests. Twenty nine patients with essential thrombocythemia and 24 with reactive thrombocytosis were studied. Thrombin generation was determined by calibrated automated thrombography. Procoagulant phospholipids were detected by a chronometric standardised method (STA-Procoag-PPL). Patients with reactive thrombocytosis had a longer lag time, higher endogenous thrombin potential, peak of thrombin generation and velocity index than patients with essential thrombocythemia. The level of circulating procoagulant phospholipids was increased in patients with essential thrombocythemia as observed with the procoagulant phospholipids assay. Each parameter was analysed using ROC curves. Highest areas under the curve (AUC) were found for lag time and procoagulant phospholipids ratio (0.817 and 0.853 respectively), associated with high negative predictive value for ET (92.3% and 80 % respectively). In conclusion, patients with essential thrombocythemia and reactive thrombocytosis displayed significant differences in terms of thrombin generation and levels of procoagulant phospholipids. Among these parameters, lag time and procoagulant phospholipids ratio could help to differentiate between reactive thrombocytosis and essential thrombocythemia patients.

Mignon I; Grand F; Boyer F; Hunault-Berger M; Hamel JF; Macchi L

2013-07-01

278

Carbon nanotube-enhanced electrochemical aptasensor for the detection of thrombin.  

Science.gov (United States)

A novel electrochemical aptasensor for the detection of thrombin was developed on basis of the thrombin-binding aptamer (TBA) as a molecular recognition element and multi-walled carbon nanotubes (MWCNTs) as a carrier of the electrochemical capture probe. Amine-modified capture probe (12-mer) was covalently conjugated to the MWCNTs modified glassy carbon electrode (GCE). The target aptamer probe (21-mer) contains TBA (15-mer) labeled with ferrocene (Fc), which is designed to hybridize with capture probe and specifically recognize thrombin, is immobilized on the electrode surface by hybridization reaction. Introduction of the analyte thrombin triggered the dissociation of the aptamer probe labeled with Fc from the biosensors, led to a significant decrease in peak current intensity. Differential pulse voltammetry (DPV) was employed to detect the target analyte with different concentrations. The decreased peak current was in proportion to the concentration of thrombin in a range from 1.0x10(-12) to 5.0x10(-10)M with a detection limit of 5x10(-13)M. The present work demonstrates that using MWCNTs as a carrier for electrochemical capture probe is a promising way to amplify the electrochemical signal and to improve the sensitivity of the electrochemical aptasensor. PMID:20441948

Liu, Xiaorong; Li, Yan; Zheng, Jianbin; Zhang, Juncai; Sheng, Qinglin

2010-03-19

279

Fluorescent measurement of affinity binding between thrombin and its aptamers using on-chip affinity monoliths.  

Science.gov (United States)

A microfluidic chip with integrated 2mm long monoliths incorporated with poly(ethylene glycol) (PEG) groups was developed for thrombin-aptamer interaction study. The non-G quartet forming oligonucleotide coated monoliths was compared to a 15 mer thrombin-binding aptamer, in which affinity binding and elution processes were real-time monitored fluorescently. The results showed that the fluorescence intensity of aptamer stationary phase is approximately 10 times higher than that of the control column, which is probably due to the successful suppression of nonspecific adsorption between thrombin and aptamers/monoliths by using PEG-monolith. The experiment was repeated using human serum albumin (HSA) and green fluorescence protein (GFP) as interferences, it was double confirmed that thrombin was selectively retained by PEG-monolith. An elution efficiency of 75% was achieved with an elute of 200mM acetic acid and 2M NaCI, and the eluted thrombin was successfully separated in an ionic buffer system of 20mM NaHCO3 (pH 9.5) with 3% PEG. The hydrophilic and antifouling properties of PEG-monolith greatly decrease nonspecific adsorption and enhance detection sensitivity, which provided an alternative method to perform on-chip fluorescent measurement of bioaffinity binding. PMID:23587316

Gao, Changlu; Sun, Xiuhua; Woolley, Adam T

2013-03-29

280

Carbon nanotube-enhanced electrochemical aptasensor for the detection of thrombin.  

UK PubMed Central (United Kingdom)

A novel electrochemical aptasensor for the detection of thrombin was developed on basis of the thrombin-binding aptamer (TBA) as a molecular recognition element and multi-walled carbon nanotubes (MWCNTs) as a carrier of the electrochemical capture probe. Amine-modified capture probe (12-mer) was covalently conjugated to the MWCNTs modified glassy carbon electrode (GCE). The target aptamer probe (21-mer) contains TBA (15-mer) labeled with ferrocene (Fc), which is designed to hybridize with capture probe and specifically recognize thrombin, is immobilized on the electrode surface by hybridization reaction. Introduction of the analyte thrombin triggered the dissociation of the aptamer probe labeled with Fc from the biosensors, led to a significant decrease in peak current intensity. Differential pulse voltammetry (DPV) was employed to detect the target analyte with different concentrations. The decreased peak current was in proportion to the concentration of thrombin in a range from 1.0x10(-12) to 5.0x10(-10)M with a detection limit of 5x10(-13)M. The present work demonstrates that using MWCNTs as a carrier for electrochemical capture probe is a promising way to amplify the electrochemical signal and to improve the sensitivity of the electrochemical aptasensor.

Liu X; Li Y; Zheng J; Zhang J; Sheng Q

2010-06-01

 
 
 
 
281

Phospholipase abolishes the effect of stimulated platelets on the thrombin activation of factor VIII.  

UK PubMed Central (United Kingdom)

Factor VIII functions as a cofactor in the intrinsic coagulation pathway and must first be activated to function optimally in this capacity. Low concentrations of thrombin activate factor VIII, and the presence of stimulated platelets is known to enhance the activation of factor VIII complexed to von Willebrand factor. The current studies show that platelets stimulated by thrombin, collagen, or calcium ionophore will increase the activation of isolated factor VIII by thrombin. Ongoing platelet release is not necessary for the enhanced factor VIII activation, nor is platelet von Willebrand factor or platelet membrane glycoproteins Ib or IIb/IIIa. Platelet membrane phospholipids, on the other hand, are important for the enhanced activation of factor VIII by thrombin because the effect of stimulated platelets is abolished by incubation of the stimulated platelets with phospholipases. These results suggest that the enhanced activation of factor VIII by thrombin in the presence of stimulated platelets may be mediated by factor VIII binding to platelet phospholipid or to a receptor whose functional integrity is dependent on surrounding membrane phospholipid.

Rick ME; Krizek DM

1988-01-01

282

Phospholipase abolishes the effect of stimulated platelets on the thrombin activation of factor VIII.  

Science.gov (United States)

Factor VIII functions as a cofactor in the intrinsic coagulation pathway and must first be activated to function optimally in this capacity. Low concentrations of thrombin activate factor VIII, and the presence of stimulated platelets is known to enhance the activation of factor VIII complexed to von Willebrand factor. The current studies show that platelets stimulated by thrombin, collagen, or calcium ionophore will increase the activation of isolated factor VIII by thrombin. Ongoing platelet release is not necessary for the enhanced factor VIII activation, nor is platelet von Willebrand factor or platelet membrane glycoproteins Ib or IIb/IIIa. Platelet membrane phospholipids, on the other hand, are important for the enhanced activation of factor VIII by thrombin because the effect of stimulated platelets is abolished by incubation of the stimulated platelets with phospholipases. These results suggest that the enhanced activation of factor VIII by thrombin in the presence of stimulated platelets may be mediated by factor VIII binding to platelet phospholipid or to a receptor whose functional integrity is dependent on surrounding membrane phospholipid. PMID:3120821

Rick, M E; Krizek, D M

1988-01-01

283

In vivo fluorescence imaging of atherosclerotic plaques with activatable cell-penetrating peptides targeting thrombin activity.  

UK PubMed Central (United Kingdom)

Thrombin and other coagulation enzymes have been shown to be important during atherosclerotic disease development. Study of these proteases is currently limited because of lack of robust molecular imaging agents for imaging protease activity in vivo. Activatable cell penetrating peptides (ACPPs) have been used to monitor MMP activity in tumors and, in principle, can be modified to detect other proteases. We have developed a probe that incorporates the peptide sequence DPRSFL from the proteinase activated receptor 1 (PAR-1) into an ACPP and shown that it is preferentially cleaved by purified thrombin. Active thrombin in serum cleaves DPRSFL-ACPP with >90% inhibition by lepirudin or argatroban. The DPRSFL-ACPP cleavage product accumulated in advanced atherosclerotic lesions in living mice, with 85% reduction in retention upon pre-injection of mice with hirudin. Uptake of the ACPP cleavage product was highest in plaques with histological features associated with more severe disease. Freshly resected human atheromas bathed in DPRSFL-ACPP retained 63% greater cleavage product compared to control ACPP. In conclusion, DPRSFL-ACPP can be used to study thrombin activity in coagulation and atherosclerosis with good spatial and temporal resolution. Thrombin-sensitive ACPPs may be developed into probes for early detection and intraoperative imaging of high risk atherosclerotic plaques.

Olson ES; Whitney MA; Friedman B; Aguilera TA; Crisp JL; Baik FM; Jiang T; Baird SM; Tsimikas S; Tsien RY; Nguyen QT

2012-06-01

284

Highly sensitive detection of thrombin using SERS-based magnetic aptasensors.  

Science.gov (United States)

This paper reports a method of highly sensitive detection of thrombin using a surface-enhanced Raman scattering (SERS)-based magnetic aptasensor. Magnetic beads and gold nanoparticles (Au NPs) were used as supporting substrates and sensing probes, respectively. For this purpose, 15-mer thrombin-binding aptamers (TBA15) were immobilized onto the surface of magnetic beads, and then thrombin antigens and 29-mer thrombin-binding aptamer (TBA29)-conjugated Au NPs were sequentially added for the formation of sandwich aptamer complexes. Quantitative analysis was performed by monitoring the intensity variation of a characteristic SERS signal of Raman reporter molecules. Because all of the reactions occur in solution, this SERS-based immunoassay technique can solve the diffusion-limited kinetic problems on a solid substrate. The limit of detection (LOD) of thrombin, determined by the SERS-based aptasensor, was estimated to be 0.27pM. The proposed method is expected to be a good clinical tool for the diagnosis of a thrombotic disease. PMID:23557978

Yoon, Jiyeon; Choi, Namhyun; Ko, Juhui; Kim, Kihyung; Lee, Sangyeop; Choo, Jaebum

2013-03-14

285

Highly sensitive detection of thrombin using SERS-based magnetic aptasensors.  

UK PubMed Central (United Kingdom)

This paper reports a method of highly sensitive detection of thrombin using a surface-enhanced Raman scattering (SERS)-based magnetic aptasensor. Magnetic beads and gold nanoparticles (Au NPs) were used as supporting substrates and sensing probes, respectively. For this purpose, 15-mer thrombin-binding aptamers (TBA15) were immobilized onto the surface of magnetic beads, and then thrombin antigens and 29-mer thrombin-binding aptamer (TBA29)-conjugated Au NPs were sequentially added for the formation of sandwich aptamer complexes. Quantitative analysis was performed by monitoring the intensity variation of a characteristic SERS signal of Raman reporter molecules. Because all of the reactions occur in solution, this SERS-based immunoassay technique can solve the diffusion-limited kinetic problems on a solid substrate. The limit of detection (LOD) of thrombin, determined by the SERS-based aptasensor, was estimated to be 0.27pM. The proposed method is expected to be a good clinical tool for the diagnosis of a thrombotic disease.

Yoon J; Choi N; Ko J; Kim K; Lee S; Choo J

2013-09-01

286

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

UK PubMed Central (United Kingdom)

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

Chuang YC; Lin YS; Liu HS; Wang JR; Yeh TM

2013-07-01

287

Increased procoagulant function of microparticles in pediatric inflammatory bowel disease: role in increased thrombin generation.  

UK PubMed Central (United Kingdom)

OBJECTIVES: Patients with inflammatory bowel disease (IBD) have a higher risk for venous thromboembolism compared with non-IBD subjects. The pathogenic mechanisms of the thrombotic events are not fully understood. We investigated levels of circulating microparticles and their influence on thrombin generation in pediatric patients with IBD during active and quiescent disease compared with healthy controls. METHODS: Plasma samples were collected from 33 pediatric patients with Crohn disease (CD), 20 pediatric patients with ulcerative colitis (UC), and 60 healthy controls. Microparticles' procoagulant activity was measured by enzyme-linked immunosorbent assay, and the dependency of thrombin generation on microparticles-derived tissue factor was determined by means of calibrated automated thrombography. RESULTS: The procoagulant function of microparticles was significantly increased in patients with active and inactive CD, and active UC compared with controls. Endogenous thrombin potential was significantly higher in patients with CD and UC compared with controls. A minor influence of microparticles on thrombin generation was only observed for patients with active UC. CONCLUSIONS: Our study shows increased procoagulant function of microparticles in pediatric patients with active and quiescent CD and active UC compared with controls, but demonstrates that they are not a major cause for the higher thrombin generation in pediatric patients with IBD.

Deutschmann A; Schlagenhauf A; Leschnik B; Hoffmann KM; Hauer A; Muntean W

2013-04-01

288

Isoproterenol attenuates the thrombin-induced increase in pulmonary endothelial permeability  

International Nuclear Information System (INIS)

The proposed permeability-decreasing effect of the beta-agonist, isoproterenol, was tested by measurement of the clearance rate of 125I-albumin across bovine pulmonary artery endothelial cell monolayers. These cells were seeded on gelatinized polycarbonate filters and mounted in a modified Boyden chamber with Dulbecco's Modified Eagle Medium (DMEM) and 1% BSA in the luminal and abluminal chambers. Baseline clearance rates were measured for an initial 30 min with the cells incubated at 370C in DMEM. At 30 min, 70 ul of either DMEM, ?-thrombin (10-6M), ?-thrombin and isoproterenol (10-6M) or isoproterenol alone was added to the luminal chamber. Clearance rates were determined at 30-60 min in each group and normalized to the baseline clearance rates of each (n = 8) cell monolayer. The experimental/baseline values were 0.98 +/- 0.08 (mean +/- SEM) for DMEM, 1.90 +/- 0.10 for thrombin, 1.52 +/- 0.08 for thrombin and isoproterenol, and 0.83 +/- 0.10 for isoproterenol alone. Thrombin increased (p

1986-01-01

289

Topics in Physical Mathematics  

CERN Multimedia

This title adopts the view that physics is the primary driving force behind a number of developments in mathematics. Previously, science and mathematics were part of natural philosophy and many mathematical theories arose as a result of trying to understand natural phenomena. This situation changed at the beginning of last century as science and mathematics diverged. These two fields are collaborating once again; 'Topics in Mathematical Physics' takes the reader through this journey. The author discusses topics where the interaction of physical and mathematical theories has led to new points o

Marathe, Kishore

2010-01-01

290

Profile of thrombin generation in children with acute lymphoblastic leukemia treated by Berlin-Frankfurt-Münster (BFM) protocols.  

Science.gov (United States)

Treatment with L-asparaginase is associated with coagulation disturbances with deep venous thrombosis being the most common clinical consequence. Use of the calibrated automated thrombogram allows precise estimation of thrombin generated in vitro. We show the first data on thrombin generation, measured by calibrated automated thrombography (CAT), in children with acute lymphoblastic leukemia treated with L-asparaginase. Thrombin generation was measured by means of CAT in 23 children treated for acute lymphoblastic leukemia. Samples were obtained at predefined time points during the induction and reinduction phase of acute lymphoblastic leukemia-intercontinental Berlin-Frankfurt-Münster (BFM) 2000 or Associazione Italiana Ematologica Oncologia Pedaitrica Interim BFM 2000 protocols. Antihrombin and fibrinogen were measured on the same sample. Twenty-eight sets of thrombin generation measurements were collected from 23 patients. We observed no significant effect of antithrombin deficiency and/or hypofibrinogenemia on thrombin generation. Endogenous thrombin generation and peak thrombin were significantly higher during induction than in the reinduction phase (P?thrombin generation, reaching maximum in a median of 7.5 days after the onset of infection. Two of those patients developed deep venous thrombosis at the time of peaked endogenous thrombin generation. Thrombin generation in children with acute lymphoblastic leukemia treated according to BFM protocols is significantly higher during the induction phase compared with reinduction and is not substantially affected by hypofibrinogenemia and/or antithrombin deficiency. Severe infection during the induction phase enhances thrombin generation with subsequent risk of thrombosis. PMID:22227959

Lejhancova-Tousovska, Katerina; Zapletal, Ondrej; Vytiskova, Sona; Strbackova, Petra; Sterba, Jaroslav

2012-03-01

291

Profile of thrombin generation in children with acute lymphoblastic leukemia treated by Berlin-Frankfurt-Munster (BFM) protocols.  

UK PubMed Central (United Kingdom)

Treatment with L-asparaginase is associated with coagulation disturbances with deep venous thrombosis being the most common clinical consequence. Use of the calibrated automated thrombogram allows precise estimation of thrombin generated in vitro. We show the first data on thrombin generation, measured by calibrated automated thrombography (CAT), in children with acute lymphoblastic leukemia treated with L-asparaginase. Thrombin generation was measured by means of CAT in 23 children treated for acute lymphoblastic leukemia. Samples were obtained at predefined time points during the induction and reinduction phase of acute lymphoblastic leukemia-intercontinental Berlin-Frankfurt-Münster (BFM) 2000 or Associazione Italiana Ematologica Oncologia Pedaitrica Interim BFM 2000 protocols. Antihrombin and fibrinogen were measured on the same sample. Twenty-eight sets of thrombin generation measurements were collected from 23 patients. We observed no significant effect of antithrombin deficiency and/or hypofibrinogenemia on thrombin generation. Endogenous thrombin generation and peak thrombin were significantly higher during induction than in the reinduction phase (P?thrombin generation, reaching maximum in a median of 7.5 days after the onset of infection. Two of those patients developed deep venous thrombosis at the time of peaked endogenous thrombin generation. Thrombin generation in children with acute lymphoblastic leukemia treated according to BFM protocols is significantly higher during the induction phase compared with reinduction and is not substantially affected by hypofibrinogenemia and/or antithrombin deficiency. Severe infection during the induction phase enhances thrombin generation with subsequent risk of thrombosis.

Lejhancova-Tousovska K; Zapletal O; Vytiskova S; Strbackova P; Sterba J

2012-03-01

292

Recombination and functional studies of a dual-action peptide for diabetes.  

UK PubMed Central (United Kingdom)

OBJECTIVE: To study a recombined chimeric peptide consisting of lysozyme N-terminal sequence and exendin-4 (shortly LYZ(N)-EX4) as a dual-action peptide for diabetes. METHODS: LYZ(N)-EX4 was recombined into plasmid pET-32a(+) and expressed in Escherichia coli. The fusion protein was separated by affinity chromatography and hydrolyzed by enterokinase to prepare LYZ(N)-EX4. The chimeric peptide was digested by thrombin and the digests were analyzed by HPLC. The secondary peptides were identified by mass spectrometry. Biological activities of the thrombin digests were determined in vitro, using NIT-1 cells for insulin promoting action and using human white blood cells (WBC) for anti-AGEs action. RESULTS: The fusion protein was highly expressed in E. coli and LYZ(N)-EX4 was obtained via hydrolysis of the fusion protein. The thrombin digests of LYZ(N)-EX4 were separated by HPLC into two peaks, which were identified as LYZ(N) and EX4 by mass spectrametry. Functional studies found that the digests were able to antagonize the effects of AGEs on expression of RAGE mRNA in WBC, promote cell activity, stimulate PDX-1 mRNA expression and increase insulin secretion by NIT-1 cells, suggesting the actions of LYZ(N) and EX4 on the cells. CONCLUSIONS: LYZ(N)-EX4 was sensitive to thrombin digestion, and the secondary peptides LYZ(N) and EX4 could function as anti-AGEs and insulin-promoting peptides, respectively.

Zheng J; Zhu Y; Yan Q; Zhong M; Zhao S; Liu Y

2013-05-01

293

Towards a standardization of thrombin generation assessment: The influence of tissue factor, platelets and phospholipids concentration on the normal values of Thrombogram-Thrombinoscope assay  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Abstract Background Thrombin generation assay was developed several years ago to mimic physiological coagulation mechanisms but it had important limitations. Thrombogram-Thrombinoscope assay using a fluorogenic substrate, allows obtaining thrombin generation curves in non-defibrinat...

Gerotziafas Grigoris T; Depasse François; Busson Joël; Leflem Lena; Elalamy Ismail; Samama Meyer M

294

Advanced Topics in Aerodynamics  

DEFF Research Database (Denmark)

"Advanced Topics in Aerodynamics" is a comprehensive electronic guide to aerodynamics,computational fluid dynamics, aeronautics, aerospace propulsion systems, design and relatedtechnology. We report data, tables, graphics, sketches,examples, results, photos, technical andscientific literature, for higher education, learning, reference, research and engineering services.

Filippone, Antonino

1999-01-01

295

Topical Therapeutic Delivery System  

UK PubMed Central (United Kingdom)

The present invention relates to an oil-in-water emulsion topical delivery system comprising an oil phase an aqueous phase phenoxyethanol an effective exfoliatingamount of a hydrophobic hydroxycarboxylic acid a non-ionic emulsifier having an HLB of from about 7 to about 10 and at least one skin-supporting ordermatopharmaceutically active agent.

MURAD HOWARD

296

TOPICAL TREATMENT OF MELASMA  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Melasma is a common hypermelanotic disorder affecting the face that is associated with considerable psychological impacts. The management of melasma is challenging and requires a long-term treatment plan. In addition to avoidance of aggravating factors like oral pills and ultraviolet exposure, topic...

Bandyopadhyay, Debabrata

297

Topical treatment of melasma  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Melasma is a common hypermelanotic disorder affecting the face that is associated with considerable psychological impacts. The management of melasma is challenging and requires a long-term treatment plan. In addition to avoidance of aggravating factors like oral pills and ultraviolet exposure, topic...

Bandyopadhyay Debabrata

298

TOPICAL SKIN CARE FORMULATIONS  

UK PubMed Central (United Kingdom)

Disclosed is a topical skin care composition comprising: (a) a combination of the following extracts: Malpighia punicifolia (acerola) extract Argania spinosa (argan) extract Myrciaria dubia (camu camu) extract Punica granatum (pomegrannate) extract Pinus sylvestris extract Terminalia ferdinandiana (kakadu plum) extract Linum usitatissimum (linseed) extract Ribes nigrum (black current) extract Secale cereale (rye) extract algae extract and yeast extract and (b) a dermatologically acceptable vehicle.

HINES MICHELLE; FLORENCE TIFFANY

299

Advances in topical analgesics.  

UK PubMed Central (United Kingdom)

SUMMARY: This review will allow physicians to understand the role of topical agents in the treatment of intractable pain syndromes. Increasing medical providers' familiarity with these agents will allow their incorporation as part of a complex analgesic regimen for an improved pain management plan benefiting the patient population at large.

Anitescu M; Benzon HT; Argoff CE

2013-08-01

300

Topical immunomodulators in dermatology  

Directory of Open Access Journals (Sweden)

Full Text Available Topical immunomodulators are agents that regulate the local immune response of the skin. They are now emerging as the therapy of choice for several immune-mediated dermatoses such as atopic dermatitis, contact allergic dermatitis, alopecia areata, psoriasis, vitiligo, connective tissue disorders such as morphea and lupus erythematosus, disorders of keratinization and several benign and malignant skin tumours, because of their comparable efficacy, ease of application and greater safety than their systemic counterparts. They can be used on a domiciliary basis for longer periods without aggressive monitoring. In this article, we have discussed the mechanism of action, common indications and side-effects of the commonly used topical immunomodulators, excluding topical steroids. Moreover, newer agents, which are still in the experimental stages, have also been described. A MEDLINE search was undertaken using the key words "topical immunomodulators, dermatology" and related articles were also searched. In addition, a manual search for many Indian articles, which are not indexed, was also carried out. Wherever possible, the full article was reviewed. If the full article could not be traced, the abstract was used.

Khandpur Sujay; Sharma V; Sumanth K

2004-01-01

 
 
 
 
301

Topics in CP violation  

International Nuclear Information System (INIS)

[en] This presentation explores a variety of topics related to the general theme of CP violation. The presentation begins by reviewing the experimental status of evidence for CP violation in particle processes. There is only one system where this has been observed, and that is in the decays of neutral K mesons. 17 refs., 1 tab

1993-01-01

302

Topical anesthesia in phacoemulsification  

Directory of Open Access Journals (Sweden)

Full Text Available Purpose : To evaluate the efficacy of topical anesthesia; topical Benoxinate 0.4% (Oxybuprocaine) and Xylocaine (Lidocaine) gel, in selected cataract patients as an alternative to peribulbar or retrobulbar block anesthesia during cataract surgery. Materials and Methods : Prospective non-comparative evaluation of patients? and surgeon?s satisfaction at the end of the procedure. Three hundred patients (300 eyes) were included in the study. The procedure was explained to patients with details regarding what will happen and what to expect during surgery. All patients received topical anesthesia with Benoxinate 0.4% eye drops and Xylocaine gel 2%. All surgeries were done by the same surgeon using the same machine (updated LEGACY phacoemulsifier, Alcon) and approach (clear corneal incision) and followed by a foldable intraocular lens (IOL) implantation. Results : None of the patients had severe pain during the procedure; only 2% (six of 300) required use of intravenous sedation (Propofol), both the surgeon?s and the patients? satisfaction were high. Eye movements and blepharospasm were not significant problems, and no serious complications occurred. Rate of vitreous loss due to posterior capsule tear/rupture was within literature reported range and not different from our previous experience. Conclusion : Topical anesthesia is a satisfactory and safe alternative to retrobulbar and peribulbar anesthesia for clear corneal phacoemulsification and intraocular lens implantation in selected cataract patients in the hands of experienced cataract surgeon.

Waheeb Saad

2010-01-01

303

Selected topics in magnetism  

CERN Multimedia

Part of the ""Frontiers in Solid State Sciences"" series, this volume presents essays on such topics as spin fluctuations in Heisenberg magnets, quenching of spin fluctuations by high magnetic fields, and kondo effect and heavy fermions in rare earths amongst others.

Gupta, L C

1993-01-01

304

Thrombin binding aptamer, more than a simple aptamer: chemically modified derivatives and biomedical applications.  

UK PubMed Central (United Kingdom)

The thrombin binding aptamer (TBA) is a well characterized chair-like, antiparallel quadruplex structure that binds specifically to thrombin at nanomolar concentrations and therefore it has interesting anticoagulant properties. In this article we review the research involved in the development of new TBA derivatives with improved anticoagulant properties as well as the use of the TBA as a model compound for the study of quadruplex structures. Specifically, we describe the impact of modified nucleosides and non-natural backbones in the guanine tetrads or in the loops and the introduction of pendant groups at the 3' or 5'-ends. The modified oligonucleotides are shown to be excellent tools for the understanding of the molecular structure of the TBA and its folding properties. Finally, we review the use of the TBA-Thrombin recognition system for the development of analytical tools based on the TBA folding.

Avino A; Fabrega C; Tintore M; Eritja R

2012-01-01

305

Thrombin immobilization to methacrylic acid grafted poly(3-hydroxybutyrate) and its in vitro application.  

UK PubMed Central (United Kingdom)

Poly(3-hydroxybutyrate) is nontoxic and biodegradable, with good biocompatibility and potential support for long-term implants. For this reason, it is a good support for enzyme immobilization. Enzyme immobilization could not be done directly because poly(3-hydroxybutyrate) has no functional groups. Therefore, modification should be done for enzyme immobilization. In this study, methacrylic acid was graft polymerized to poly(3-hydroxybutyrate) and thrombin was immobilized to polymethacrylic acid grafted poly(3-hydroxybutyrate). In fact, graft polymerization of methacrylic acid to poly(3-hydroxybutyrate) and thrombin immobilization was a model study. Biomolecule immobilized poly(3-hydroxybutyrate) could be used as an implant. Thrombin was selected as a biomolecule for this model study and it was immobilized to methacrylic acid grafted poly(3-hydroxybutyrate). Then the developed product was used to stop bleeding.

Akkaya A; Pazarlioglu N

2013-01-01

306

Allosteric activation of human ?-thrombin through exosite 2 by suramin analogs.  

UK PubMed Central (United Kingdom)

Thrombin is a serine protease that plays fundamental roles in hemostasis. We have recently elucidated the crystal structure of thrombin in complex with suramin, evidencing the interaction through the anion binding exosite 2. Here, we show that the activity of thrombin toward natural and synthetic substrates is enhanced by suramin as well as analogs of suramin at a low micromolar range prior to an inhibitory component at higher concentrations. Suramin analogs substituted by phenyl and chlorine instead of methyl were the most efficient in promoting allosteric activation, with an enhancement of enzymatic activity of 250% and 630% respectively. We discuss the importance of exosite 2 as a regulatory site for ligands in both the procoagulant and inhibitory scenarios.

Cargnelutti MT; Marques AF; Esser D; Monteiro RQ; Kassack MU; Lima LM

2012-04-01

307

Poliovirus RNA recombination.  

UK PubMed Central (United Kingdom)

We are developing an in vitro system for poliovirus RNA recombination. In this system, two mutant RNAs are replicated with poliovirus RNA-dependent RNA polymerase. Recombination will produce RNAs containing neither mutation and will be the only progeny RNAs that are infectious. We will use this system to determine what proteins and reaction conditions are required for recombination and to study the details of the mechanism of recombination.

Pata J; Kirkegaard K

1991-01-01

308

Concentration dependent anti-inflammatory effects thrombin on polyphosphate-mediated inflammatory responses in vitro and in vivo.  

UK PubMed Central (United Kingdom)

AIM AND OBJECTIVE: Recent results indicate that polyphosphate (polyP) released by human endothelial cells can function as a pro-inflammatory mediator, and it has been reported that low thrombin concentrations mediate anti-inflammatory activities. This study was undertaken to investigate whether low thrombin concentrations can modulate polyP-mediated inflammatory responses in human umbilical vein endothelial cells (HUVECs) and in mice. METHODS: Concentration dependent anti-inflammatory effects of thrombin such as barrier protection, inhibition of cell adhesion molecule expression and inhibition of monocytes adhesion and migration toward human endothelial cells against polyP-mediated pro-inflammatory activities were tested in vitro and in vivo. The concentration-dependent effects of thrombin on polyP-induced nuclear factor (NF)-?B activation and the production of tumor necrosis factor (TNF)-? and interleukin (IL)-6 were also tested. RESULTS: We found that at low concentrations (25-75 pM), thrombin inhibits polyP-mediated barrier disruption, the expressions of cell adhesion molecules, and leukocyte to HUVEC adhesion/migration. Interestingly, polyP-induced NF-?B activation and the production of TNF-? and IL-6 were inhibited by low thrombin concentrations in HUVECs. These anti-inflammatory functions of thrombin were confirmed in polyP-injected mice. CONCLUSION: These results suggest that thrombin at 25-75 pM may have therapeutic potential for various systemic inflammatory diseases.

Ku SK; Bae JS

2013-06-01

309

Thrombogenicity of small-diameter prosthetic grafts: relative contributions of graft-associated thrombin and factor Xa.  

UK PubMed Central (United Kingdom)

PURPOSE: We evaluated the contributions of coagulation factors IIa (thrombin) and Xa to small-diameter prosthetic graft thrombogenicity in vivo. METHODS: Preclotted and nonpreclotted (collagen-coated) polyester grafts were studied before and 24 hours after implantation into pig femoral arteries. After incubation of explanted grafts was performed with plasma depleted of vitamin K-dependent coagulation factors by barium chloride adsorption (Ba-plasma), graft-associated thrombin activity was determined by radioimmunoassay for fibrinopeptide A. Fibrinopeptide A levels reflect thrombin-mediated fibrin formation. Factor Xa activity was characterized by measuring activation of prothrombin added to Ba-plasma. RESULTS: Thrombin and factor Xa were associated with the luminal surfaces of preclotted grafts before and 24 hours after implantation. Nonpreclotted grafts had negligible procoagulant activity before implantation. After 24 hours in vivo graft-associated factor Xa activity was similar in both nonpreclotted and preclotted grafts; however, more thrombin was bound to nonpreclotted coated grafts (p < 0.01). CONCLUSIONS: The procoagulant activity of small-diameter prosthetic grafts persists for 24 hours after implantation and is attributable not only to graft-associated thrombin but also to de novo thrombin elaboration induced by factor Xa. Moreover, graft-associated procoagulant activity is not dependent on preclotting because it develops on nonpreclotted, collagen-coated grafts as well. Treatment strategies to attenuate graft thrombosis may require the inhibition of both thrombin and factor Xa.

Toursarkissian B; Eisenberg PR; Abendschein DR; Rubin BG

1997-04-01

310

Is thrombin generation the new rapid, reliable and relevant pharmacological tool for the development of anticoagulant drugs?  

Science.gov (United States)

The ex vivo testing emerges as an essential and critical step for the selection of the most promising prospective anticoagulant agents. The aim of the present study was to validate the thrombin generation assay as an ex vivo pharmacological screening test for measuring the anticoagulant behaviour and potency of molecules. The effects of six thrombin and/or factor Xa (FXa) inhibitors (argatroban, lepirudin, PPACK, enoxaparin, ZK-807834, fondaparinux) were investigated on the time course of thrombin catalytic activity triggered by the tissue factor pathway in platelet-poor plasma (PPP) of male healthy volunteers using the Calibrated Automated Thrombogram((R)) (CAT) method. In the presence of the anticoagulant drugs, the thrombin activity profiles were dose-dependently modified according to their specific enzyme inhibitory activity. ZK-807834 was the most potent drug for reducing the C(max) and the V(max) but also for prolonging the T(max). Lepirudin most efficiently delayed the lag time whereas enoxaparin was the most powerfully drug for diminishing the endogenous thrombin potential (ETP). In conclusion, the thrombin activity profile performed with the CAT method is a very rapid, suitable and reliable pharmacological tool for screening thrombin and/or FXa inhibitors whatever their inhibition mode. It consists of a powerful alternative for the classical PT clotting assay, especially regarding to the time course and the total amount of active thrombin generated. Last but not least, it provides insight into the mechanism of action of the compounds. PMID:19124079

Robert, Séverine; Ghiotto, Jérémie; Pirotte, Bernard; David, Jean-Louis; Masereel, Bernard; Pochet, Lionel; Dogné, Jean-Michel

2008-12-24

311

Is thrombin generation the new rapid, reliable and relevant pharmacological tool for the development of anticoagulant drugs?  

UK PubMed Central (United Kingdom)

The ex vivo testing emerges as an essential and critical step for the selection of the most promising prospective anticoagulant agents. The aim of the present study was to validate the thrombin generation assay as an ex vivo pharmacological screening test for measuring the anticoagulant behaviour and potency of molecules. The effects of six thrombin and/or factor Xa (FXa) inhibitors (argatroban, lepirudin, PPACK, enoxaparin, ZK-807834, fondaparinux) were investigated on the time course of thrombin catalytic activity triggered by the tissue factor pathway in platelet-poor plasma (PPP) of male healthy volunteers using the Calibrated Automated Thrombogram((R)) (CAT) method. In the presence of the anticoagulant drugs, the thrombin activity profiles were dose-dependently modified according to their specific enzyme inhibitory activity. ZK-807834 was the most potent drug for reducing the C(max) and the V(max) but also for prolonging the T(max). Lepirudin most efficiently delayed the lag time whereas enoxaparin was the most powerfully drug for diminishing the endogenous thrombin potential (ETP). In conclusion, the thrombin activity profile performed with the CAT method is a very rapid, suitable and reliable pharmacological tool for screening thrombin and/or FXa inhibitors whatever their inhibition mode. It consists of a powerful alternative for the classical PT clotting assay, especially regarding to the time course and the total amount of active thrombin generated. Last but not least, it provides insight into the mechanism of action of the compounds.

Robert S; Ghiotto J; Pirotte B; David JL; Masereel B; Pochet L; Dogné JM

2009-03-01

312

Studies of the binding mechanism between aptamers and thrombin by circular dichroism, surface plasmon resonance and isothermal titration calorimetry.  

UK PubMed Central (United Kingdom)

Thrombin, a multifunctional serine protease, has both procoagulant and anticoagulant functions in human blood. Thrombin has two electropositive exosites. One is the fibrinogen-binding site and the other is the heparin-binding site. Over the past decade, two thrombin-binding aptamers (15-mer and 29-mer) were reported by SELEX technique. Recently, many studies examined the interactions between the 15-mer aptamer and thrombin extensively, but the data on the difference of these two aptamers binding to thrombin are still lacking and worth investigating for fundamental understanding. In the present study, we combined conformational data from circular dichroism (CD), kinetics and thermodynamics information from surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) to compare the binding mechanism between the two aptamers with thrombin. Special attentions were paid to the formation of G-quadruplex and the effects of ions on the aptamer conformation on the binding and the kinetics discrimination between specific and nonspecific interactions of the binding. The results indicated reasonably that the 15-mer aptamer bound to fibrinogen-binding site of thrombin using a G-quadruplex structure and was dominated by electrostatic interactions, while the 29-mer aptamer bound to heparin-binding site thrombin using a duplex structure and was driven mainly by hydrophobic effects.

Lin PH; Chen RH; Lee CH; Chang Y; Chen CS; Chen WY

2011-12-01

313

Stent graft exclusion of pseudo-aneurysm arising from PTFE hemodialysis graft after recurrence following ultrasound guided thrombin injection.  

UK PubMed Central (United Kingdom)

There are various non-invasive or minimally invasive techniques for management of pseudoaneurysms including ultrasound guided compression, ultrasound guided thrombin injection and covered stent placement. We report a case where a covered stent graft was successfully used for the treatment of a pseudoaneurysm directly arising from a PTFE graft which recurred 3 months following treatment with ultrasound guided thrombin injection.

Ananthakrishnan G; Bhat R; Severn A; Chakraverty S

2008-10-01

314

Bovine thrombin induces an acquired coagulopathy in sensitized patients undergoing revision spinal surgery: a report of two cases.  

UK PubMed Central (United Kingdom)

STUDY DESIGN: A report of two cases is presented. OBJECTIVE: To raise awareness of bovine thrombin-induced factor V deficiency. SUMMARY OF BACKGROUND DATA: Bovine thrombin is a frequently used hemostatic agent in spinal surgery. Current preparations contain clotting factors in addition to thrombin, particularly factor V, which are immunogenic. Re-exposure of sensitized patients to bovine thrombin products during subsequent surgery may lead to the formation of antibodies that cross-react with human clotting factors, most commonly against factor V. Hemorrhagic complications have been reported in nonspinal patients due to a bovine thrombin-induced factor V deficiency. METHODS: Two spinal cases are reported, and the literature is reviewed. RESULTS: In the cases outlined, both patients underwent revision spinal surgery, with re-exposure to bovine thrombin. Both patients developed abnormal coagulation profiles, with an acquired factor V deficiency. No hemorrhagic complications occurred; however, second-stage surgery was delayed in one patient and not undertaken in the other. In both patients, the coagulopathy resolved spontaneously. CONCLUSIONS: Bovine thrombin-induced coagulopathy is well recognized in cardiac surgery but has not been reported in spinal surgical patients. Data available from cardiac surgical patients suggests that those who are sensitized to two or more bovine clotting factors are at greatest risk of hemorrhagic complications. The cases we present demonstrate that this phenomenon occurs in spinal surgical patients and serve to raise awareness of the potential danger of bovine thrombin in sensitized patients.

Poynton AR; Nelson MC; McCance SE; Levine RL; O'Leary PF

2003-06-01

315

An integrated mathematical model of thrombin-, histamine-and VEGF-mediated signalling in endothelial permeability.  

UK PubMed Central (United Kingdom)

BACKGROUND: Endothelial permeability is involved in injury, inflammation, diabetes and cancer. It is partly regulated by the thrombin-, histamine-, and VEGF-mediated myosin-light-chain (MLC) activation pathways. While these pathways have been investigated, questions such as temporal effects and the dynamics of multi-mediator regulation remain to be fully studied. Mathematical modeling of these pathways facilitates such studies. Based on the published ordinary differential equation models of the pathway components, we developed an integrated model of thrombin-, histamine-, and VEGF-mediated MLC activation pathways. RESULTS: Our model was validated against experimental data for calcium release and thrombin-, histamine-, and VEGF-mediated MLC activation. The simulated effects of PAR-1, Rho GTPase, ROCK, VEGF and VEGFR2 over-expression on MLC activation, and the collective modulation by thrombin and histamine are consistent with experimental findings. Our model was used to predict enhanced MLC activation by CPI-17 over-expression and by synergistic action of thrombin and VEGF at low mediator levels. These may have impact in endothelial permeability and metastasis in cancer patients with blood coagulation. CONCLUSION: Our model was validated against a number of experimental findings and the observed synergistic effects of low concentrations of thrombin and histamine in mediating the activation of MLC. It can be used to predict the effects of altered pathway components, collective actions of multiple mediators and the potential impact to various diseases. Similar to the published models of other pathways, our model can potentially be used to identify important disease genes through sensitivity analysis of signalling components.

Wei XN; Han BC; Zhang JX; Liu XH; Tan CY; Jiang YY; Low BC; Tidor B; Chen YZ

2011-01-01

316

An Integrated Mathematical Model of Thrombin-, Histamine-and VEGF-Mediated Signalling in Endothelial Permeability  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Endothelial permeability is involved in injury, inflammation, diabetes and cancer. It is partly regulated by the thrombin-, histamine-, and VEGF-mediated myosin-light-chain (MLC) activation pathways. While these pathways have been investigated, questions such as temporal effects and the dynamics of multi-mediator regulation remain to be fully studied. Mathematical modeling of these pathways facilitates such studies. Based on the published ordinary differential equation models of the pathway components, we developed an integrated model of thrombin-, histamine-, and VEGF-mediated MLC activation pathways. Results Our model was validated against experimental data for calcium release and thrombin-, histamine-, and VEGF-mediated MLC activation. The simulated effects of PAR-1, Rho GTPase, ROCK, VEGF and VEGFR2 over-expression on MLC activation, and the collective modulation by thrombin and histamine are consistent with experimental findings. Our model was used to predict enhanced MLC activation by CPI-17 over-expression and by synergistic action of thrombin and VEGF at low mediator levels. These may have impact in endothelial permeability and metastasis in cancer patients with blood coagulation. Conclusion Our model was validated against a number of experimental findings and the observed synergistic effects of low concentrations of thrombin and histamine in mediating the activation of MLC. It can be used to predict the effects of altered pathway components, collective actions of multiple mediators and the potential impact to various diseases. Similar to the published models of other pathways, our model can potentially be used to identify important disease genes through sensitivity analysis of signalling components.

Wei XN; Han BC; Zhang JX; Liu XH; Tan CY; Jiang YY; Low BC; Tidor B; Chen YZ

2011-01-01

317

Thrombin receptor levels in platelet concentrates during storage and their impact on platelet functionality.  

UK PubMed Central (United Kingdom)

BACKGROUND: Quality control of platelet (PLT) concentrates is challenging, due to PLT lesions, which are difficult to detect with routine methods. The search for reliable PLT lesion biomarkers is focused on the role of PLTs in primary hemostasis. PLT transfusions also have a significant impact on secondary hemostasis. In this phase, responsiveness of PLTs to small amounts of thrombin is crucial. PAR1 and PAR4 are protease-activated receptors and are responsible for thrombin reactivity of human PLTs. This study should elucidate if levels of those two receptors are changing in PLT concentrates during storage and if those changes have an impact on PLT aggregation and support of thrombin generation. STUDY DESIGN AND METHODS: PLT concentrates from buffy coat preparations were stored in SSP+ solution for 9 days at 22±2°C on a horizontal flatbed agitator, and samples were taken daily for analysis. PAR1 and PAR4 levels were evaluated using Western blot analysis. PLT aggregation was measured using Born aggregometry and specific PAR1 or PAR4 agonists. Thrombin generation was measured using calibrated automated thrombography. RESULTS: Levels of both receptors (PAR1 and PAR4) started to decrease after 5 days of storage. PAR1-mediated PLT aggregation remained constant, whereas PAR4-mediated PLT aggregation decreased with storage time. Rate of thrombin generation was accelerated after 5 days of storage. CONCLUSION: Decreasing levels of PARs in PLT concentrates after 5 days of storage influenced PAR4-mediated, but not PAR1-mediated, aggregation. Thrombin generation with senescent PLTs was increased, which may be attributed to other mechanisms promoting increased phosphatidylserine exposure.

Schlagenhauf A; Kozma N; Leschnik B; Wagner T; Muntean W

2012-06-01

318

Colorimetric detection of DNA by modulation of thrombin activity on gold nanoparticles.  

Science.gov (United States)

A colorimetric, non-cross-linking aggregation-based gold-nanoparticle (AuNP) probe has been developed for the detection of DNA and the analysis of single-nucleotide polymorphism (SNP). The probe acts by modulating the enzyme activity of thrombin relative to fibrinogen. A thrombin-binding aptamer with a 29-base-long oligonucleotide (TBA(29)) assembled on the nanoparticles (TBA(29)-AuNPs) through sandwich DNA hybridization was found to possess ultra-high anticoagulant potency. The enzyme inhibition of thrombin was determined by thrombin-induced aggregation of fibrinogen-functionalized 56 nm AuNPs (Fib-AuNPs). The potency of the inhibition of TBA(29)-AuNPs relative to thrombin--and thus the degree of aggregation of the Fib-AuNPs--is highly dependent on the concentration of perfectly matched DNA (DNA(pm)). Under optimal conditions [Tris-HCl (20 mM, pH 7.4), KCl (5 mM), MgCl(2) (1 mM), CaCl(2) (1 mM), NaCl (150 mM), thrombin (10 pM), and TBA(29)-AuNPs (20 pM)], the new TBA(29)-AuNP/Fib-AuNP probe shows linear sensitivity to DNA(pm) in the concentration range 20-500 pM with a correlation coefficient of 0.96. The limit of detection for DNA(pm) was experimentally determined to be 12 pM, based on a signal-to-noise ratio (S/N) of 3. The new probe was successfully applied to the analysis of an SNP that is responsible for sickle cell anemia. Relative to conventional molecular-beacon-based probes, the new probe offers the advantages of higher sensitivity and selectivity towards DNA and lower cost, showing its great potential for practical studies of SNPs. PMID:21287648

Jian, Jyun-Wei; Huang, Chih-Ching

2011-02-01

319

Colorimetric detection of DNA by modulation of thrombin activity on gold nanoparticles.  

UK PubMed Central (United Kingdom)

A colorimetric, non-cross-linking aggregation-based gold-nanoparticle (AuNP) probe has been developed for the detection of DNA and the analysis of single-nucleotide polymorphism (SNP). The probe acts by modulating the enzyme activity of thrombin relative to fibrinogen. A thrombin-binding aptamer with a 29-base-long oligonucleotide (TBA(29)) assembled on the nanoparticles (TBA(29)-AuNPs) through sandwich DNA hybridization was found to possess ultra-high anticoagulant potency. The enzyme inhibition of thrombin was determined by thrombin-induced aggregation of fibrinogen-functionalized 56 nm AuNPs (Fib-AuNPs). The potency of the inhibition of TBA(29)-AuNPs relative to thrombin--and thus the degree of aggregation of the Fib-AuNPs--is highly dependent on the concentration of perfectly matched DNA (DNA(pm)). Under optimal conditions [Tris-HCl (20 mM, pH 7.4), KCl (5 mM), MgCl(2) (1 mM), CaCl(2) (1 mM), NaCl (150 mM), thrombin (10 pM), and TBA(29)-AuNPs (20 pM)], the new TBA(29)-AuNP/Fib-AuNP probe shows linear sensitivity to DNA(pm) in the concentration range 20-500 pM with a correlation coefficient of 0.96. The limit of detection for DNA(pm) was experimentally determined to be 12 pM, based on a signal-to-noise ratio (S/N) of 3. The new probe was successfully applied to the analysis of an SNP that is responsible for sickle cell anemia. Relative to conventional molecular-beacon-based probes, the new probe offers the advantages of higher sensitivity and selectivity towards DNA and lower cost, showing its great potential for practical studies of SNPs.

Jian JW; Huang CC

2011-02-01

320

Thrombin stimulates stress fiber assembly in RPE cells by PKC/CPI-17-mediated MLCP inactivation.  

UK PubMed Central (United Kingdom)

Most retinal proliferative diseases involve blood-retinal barrier (BRB) breakdown, exposing the retinal pigment epithelium (RPE) to thrombin, which triggers cell transformation, proliferation and migration through the activation of PAR-1. These processes require the assembly of contractile stress fibers containing actin and non-muscle myosin II, which allow cell movement upon phosphorylation of the myosin light chains (MLCs). PKC family of kinases promotes agonist-mediated contraction in smooth muscle and endothelial cells through the activation of its downstream target, the PKC-potentiated inhibitory protein of 17 kDa (CPI-17), which specifically inhibits MLC phosphatase. Although the participation of PKC in RPE cell transdifferentiation has been suggested, the role of PKC/CPI-17 signaling has not been investigated. The purpose of this study was to analyze the involvement of specific PKC isoenzymes and their effector protein CPI-17 in thrombin-induced MLC phosphorylation and actin stress fiber assembly in RPE cells. Rat RPE cells in primary culture were shown to respond to thrombin stimulation by activation of conventional, novel and atypical PKC isoforms and the downstream phosphorylation of CPI-17 and MLC, which in turn promoted actin stress fiber assembly. These effects were prevented by the pharmacological inhibition of conventional PKC isoenzymes (Ro-32-0432) and novel PKC? (rottlerin and ?V1-1 antagonist peptide), as well as by myristoylated pseudosubstrates specifically directed to conventional and atypical PKC isoforms. Thrombin effects were mimicked by phorbol 12-myristate 13-acetate (PMA), further confirming the involvement of diacylglycerol (DAG)-sensitive classical and novel PKC isoforms in thrombin-induced actin cytoskeleton modification. The present work shows, for the first time, the functional expression of the oncoprotein CPI-17 in RPE cells and suggests that PKC/CPI-17 signaling is involved in the control of actin cytoskeletal remodeling leading to cell motility in RPE cells exposed to thrombin, and hence could contribute to the development of proliferative eye diseases.

Ruiz-Loredo AY; López E; López-Colomé AM

2012-03-01

 
 
 
 
321

Topical Treatment of Allergic Rhinitis  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Topical medications have dramatically changed the treatment of rhinitis. While systemic treatment is often more potent, topical treatment has fewer side effects. However, topical preparations also have side effects which should be considered when treating rhinitis. Topical steroids are potent anti-i...

Greenbaum, Joseph

322

Recombineering homologous recombination constructs in Drosophila.  

UK PubMed Central (United Kingdom)

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Carreira-Rosario A; Scoggin S; Shalaby NA; Williams ND; Hiesinger PR; Buszczak M

2013-01-01

323

Characterization of the thrombin generation potential of leukemic and solid tumor cells by calibrated automated thrombography.  

UK PubMed Central (United Kingdom)

BACKGROUND: Thrombin, the final enzyme of blood coagulation, is a multifunctional serine protease also involved in the progression of cancer. Tumor cells may activate blood coagulation proteases through the expression of procoagulant activities. However, specific information about the thrombin generation potential of malignant tissues is lacking. In this study we applied a single global coagulation test, the calibrated automated thrombogram assay, to characterize the specific procoagulant phenotypes of different tumor cells. DESIGN AND METHODS: Malignant hematologic cells (i.e. NB4, HEL, and K562) or solid tumor cells (i.e. MCF-7 breast cancer and H69 small cell lung cells) were selected for the study. The calibrated automated thrombo-gram assay was performed in normal plasma and in plasma samples selectively deficient in factor VII, XII, IX or X, in the absence or presence of a specific anti-tissue factor antibody. Furthermore, cell tissue factor levels were characterized by measuring antigen, activity and mRNA expression. RESULTS: In normal plasma, NB4 induced the highest thrombin generation, followed by MCF-7, H69, HEL, and K562 cells. The anti-tissue factor antibody, as well as deficiencies of factors VII, IX and XII affected the thrombin generation potential of malignant cells to different degrees, allowing differentiation of the two different pathways of blood clotting activation - by tissue factor or contact activation. The thrombin generation capacity of NB4 and MCF-7 cells was tissue factor-dependent, as it was highly sensitive to inhibition by anti-tissue factor antibody and factor VII deficiency, while the thrombin generation capacity of H69, HEL and K562 was contact activation-dependent, as no thrombin was generated by these cells in factor XII-deficient plasma. CONCLUSIONS: This study demonstrates that the calibrated automated thrombogram assay is capable of quantifying, characterizing, and comparing the thrombin generation capacity of different tumor cells. This provides a useful tool for understanding the key factors determining the global pro-coagulant profile of tumors, which is important for addressing specific targeted therapy for the prevention of thrombosis and for cancer.

Marchetti M; Diani E; ten Cate H; Falanga A

2012-08-01

324

Saccular Aneurysm of Superior Vena Cava Treated with Percutaneous, Transcatheter Thrombin Injection.  

UK PubMed Central (United Kingdom)

We report the case of successful endovascular treatment of large saccular aneurysm of SVC in a patient with vascular malformation of right hand and chest. Considering the high risk of surgery, the patient was referred for percutaneous intervention. Venography showed communication between the aneurysm and SVC, just below brachiocephalic confluence. That is why the decision of balloon-protected transcatheter thrombin injection was made. Selective catheter was placed in the aneurysm and balloon occlusion catheter in SVC. Both catheters were withdrawn right after thrombin injection. During follow-up, aneurysm slightly enlarged in early observation and after a year shrinkage was observed.

Jargiello T; Durakiewicz M; Sojka M; Czekajska-Chehab E; Szczerbo-Trojanowska M

2013-06-01

325

RNA aptamer to thrombin binds anion-binding exosite-2 and alters protease inhibition by heparin-binding serpins.  

Science.gov (United States)

We studied the RNA aptamer Toggle-25/thrombin interaction during inhibition by antithrombin (AT), heparin cofactor II (HCII) and protein C inhibitor (PCI). Thrombin inhibition was reduced 3-fold by Toggle-25 for AT and HCII, but it was slightly enhanced for PCI. In the presence of glycosaminoglycans, AT and PCI had significantly reduced thrombin inhibition with Toggle-25, but it was only reduced 3-fold for HCII. This suggested that the primary effect of aptamer binding was through the heparin-binding site of thrombin, anion-binding exosite-2 (exosite-2). We localized the Toggle-25 binding site to Arg 98, Glu 169, Lys 174, Asp 175, Arg 245, and Lys 248 of exosite-2. We conclude that a RNA aptamer to thrombin exosite-2 might provide an effective clinical reagent to control heparin's anticoagulant action. PMID:15196911

Jeter, Martha L; Ly, Linda V; Fortenberry, Yolanda M; Whinna, Herbert C; White, Rebekah R; Rusconi, Christopher P; Sullenger, Bruce A; Church, Frank C

2004-06-18

326

Thrombin induces epithelial-mesenchymal transition via PAR-1, PKC, and ERK1/2 pathways in A549 cells.  

Science.gov (United States)

ABSTRACT Thrombin activates protease-activated receptor (PAR)-1 and induces a myofibroblast phenotype in normal lung fibroblasts. The origins of myofibroblasts are resident fibroblasts, fibrocytes, and epithelial-mesenchymal transition (EMT). We investigated the effects of thrombin, an important mediator of interstitial lung fibrosis, on EMT in A549 human alveolar epithelial cells. We show that thrombin induced EMT and collagen I secretion through the activation of PAR-1, and PKC and ERK1/2 phosphorylation in A549 cells. These effects were largely prevented by a specific PAR-1 antagonist, short interfering RNA (siRNA) directed against PAR-1, or specific PKC?/?, ?, and ? inhibitors. These data indicated that interaction with thrombin and alveolar epithelial cells might directly contribute to the pathogenesis of pulmonary fibrosis through EMT. Targeting PAR-1 on the pulmonary epithelium or specific inhibitors to PKC?/?, ?, and ? might stop the fibrotic processes in human idiopathic pulmonary fibrosis by preventing thrombin-induced EMT. PMID:23919450

Song, Jeong Sup; Kang, Chun Mi; Park, Chan Kwon; Yoon, Hyung Kyu

2013-08-06

327

Topical Methotrexate In Localized Psoriasis  

Directory of Open Access Journals (Sweden)

Full Text Available A comparative study of topical methotrexate and topical tar in eighteen patients of localized psoriasis with bilateral involvement of both lower legs, equal in area and disease severity was carried out. The patients were asked to apply topical methorexate 0.25% in a hydrophilic gel twice daily on right leg. The test sites were score, before therapy, after one month and after two months. The result with topical methotrexate preparation was promising but was comparable to topical tar formulation.

Rath Namita; Kar Hemant Kumar

2004-01-01

328

New topical antifungal drugs.  

UK PubMed Central (United Kingdom)

The new antifungal drugs used for topical treatment of superficial, skin and mucosal mycoses are reviewed. Amorolfine and allylamines (naftifine and terbinafine) are promising original molecules with new and different modes of action against fungi. Rilopirox is a new pyridone derivative under study. A great number of azole derivatives, such as oxiconazole, isoconazole, sulconazole, and terconazole, are used as topical antifungals. Three of them are synthesized in Barcelona by pharmaceutical laboratories: sertaconazole, flutrimazole and eberconazole. All of them are now in the register process for commercialization. The combination of antifungals with active products, such as keratoplastics, is used mainly for the treatment of onychomycoses; 40% urea associated with 1% bifonazole has shown high efficacy for this indication.

Torres-Rodríguez JM

1993-01-01

329

New topical antifungal drugs.  

Science.gov (United States)

The new antifungal drugs used for topical treatment of superficial, skin and mucosal mycoses are reviewed. Amorolfine and allylamines (naftifine and terbinafine) are promising original molecules with new and different modes of action against fungi. Rilopirox is a new pyridone derivative under study. A great number of azole derivatives, such as oxiconazole, isoconazole, sulconazole, and terconazole, are used as topical antifungals. Three of them are synthesized in Barcelona by pharmaceutical laboratories: sertaconazole, flutrimazole and eberconazole. All of them are now in the register process for commercialization. The combination of antifungals with active products, such as keratoplastics, is used mainly for the treatment of onychomycoses; 40% urea associated with 1% bifonazole has shown high efficacy for this indication. PMID:8118161

Torres-Rodríguez, J M

1993-01-01

330

Superconductivity elementary topics  

CERN Document Server

This book describes the elementary concepts of superconductivity and discusses the topics of flux-lattice melting, magnetization including the para-Meissner effect, microwave absorption, a.c. resistivity along with the London penetration depth, the Mössbauer effect, levitation, fractals and nuclear magnetic resonance. There are appendices covering superconducting compounds, the isotope effect, symmetries, the pseudogap, relativistic superconductivity, the Cherenkov effect and soft vortices. Also included is an appendix on the quantum Hall effect. In all of the chapters, the theoretical descrip

Shrivastava, KN

2000-01-01

331

Topic in Depth - Bioinformatics  

Science.gov (United States)

Computational molecular biology, which now is commonly called bioinformatics, draws on mathematics and computer science to inform research in biology. In this folder, you'll find informational and educational sites alike to explore the topic of bioinformatics. This evolving area of research advances our knowledge of biological systems and contributes to medical research, but also raises ethical issues and demands increased collaboration among scientists.

2010-09-10

332

Topical skin care composition  

UK PubMed Central (United Kingdom)

A topical composition comprising: (a) petroselinic acid and/or derivatives thereof (b) a retinoid and/or a LRAT/ARAT inhibitor and (c) a dermatologically acceptable vehicle. Such skin care compositions are useful for treating and/or preventing normal, but undesirable, skin conditions selected from the group consisting of wrinkling, sagging, photodamaged skin, dry skin and age spots and soothing sensitive skin.

BARRETT KAREN ELIZABETH; GREEN MARTIN RICHARD; RAWLINGS ANTHONY VINCENT

333

Topic in Depth - Chlorine  

Science.gov (United States)

Chlorine, a chemical element whose name means âÂÂpale green,â is explored from a number of angles in this informative Topic in Depth.WeâÂÂve all heard of chlorine being used in swimming pools and drinking water, but this jack-of-all-trades chemical element is also used in making everything from plastics and dry cleaning products to insecticides and pharmaceuticals.

2010-09-15

334

Topics in industrial mathematics  

International Nuclear Information System (INIS)

Mathematical methods are widely used to solve practical problems arising in modern industry. This article outlines some of the topics relevant to AECL programmes. This covers the applications of transmission and neutron transport tomography to determine density distributions in rocks and two phase flow situations. Another example covered is the use of variational methods to solve the problems of aerosol migration and control theory. (author). 7 refs.

1992-01-01

335

An Integrated Mathematical Model of Thrombin-, Histamine-and VEGF-Mediated Signalling in Endothelial Permeability  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Background: Endothelial permeability is involved in injury, inflammation, diabetes and cancer. It is partly regulated by the thrombin-, histamine-, and VEGF-mediated myosin-light-chain (MLC) activation pathways. While these pathways have been investigated, questions such as temporal effects and the ...

Wei, X.n.; Han, B.c.; Zhang, J.x.; Liu, X.h.; Tan, C.y.; Jiang, Y.y.; Low, Boon Chuan; Chen, Yu Zong; Tidor, Bruce

336

Inflammation and Coagulation Activity in Unstable Coronary Artery Disease and the Influences of Thrombin Inhibition  

Digital Repository Infrastructure Vision for European Research (DRIVER)

In patients with unstable coronary artery disease, this study evaluated the degree of inflammation and coagulation activity, relations to myocardial cell damage, prognosis, and influences of randomisation to 72 h infusion with three different doses of inogatran, a direct thrombin inhibitor (n=904...

Oldgren, Jonas

337

In vivo catabolism of human heparin cofactor II and its complex with thrombin  

Energy Technology Data Exchange (ETDEWEB)

The plasma clearance of human heparin cofactor II (HC) and its complex with thrombin (HC-T) was studied in mice and compared to the clearance of two other plasma proteinase inhibitor complexes: antithrombin III-thrombin (AT-T) and ..cap alpha.. 1-proteinase inhibitor-elastase (..cap alpha.. /sub 1/PI-E). Purified HC was labelled with /sup 125/I without loss of activity as assessed by kinetic analysis of thrombin inhibition and by ability to form a detergent-resistant complex with thrombin. /sup 125/I-HC cleared from the circulation with a t1/2 of 80 min; /sup 125/I-HC-T cleared with a t1/2 of 10 min. When coinjected, a 2500-fold molar excess of unlabelled HC-T partially blocked the clearance of /sup 125/I-HC-T (t1/2 of 53 min), suggesting the involvement of a receptor-mediated process in the catabolism of HC-T. Coinjection of a 1500-fold molar excess of AT-T also slowed the clearance of /sup 125/I-HC-T (t1/2 of 29 min); a 2000-fold molar excess of ..cap alpha.. /sub 1/PI-E further competed for /sup 125/I-HC-T recovered at autopsy was localized to the liver; similar results have been obtained for AT-T and ..cap alpha.. /sub 1/PI-trypsin.

Pratt, C.W.; Church, F.C.; Pizzo, S.V.

1987-05-01

338

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

DEFF Research Database (Denmark)

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 T(m) 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.

Pasternak, Anna; Hernandez, Frank J

2011-01-01

339

Development of a Multiplex Sandwich Aptamer Microarray for the Detection of VEGF165 and Thrombin  

Directory of Open Access Journals (Sweden)

Full Text Available In this work we have developed a multiplex microarray system capable of detecting VEGF165 and thrombin. We recently described a Sandwich Aptamer Microarray (SAM) for thrombin detection feasible for use in multiplex microarrays; here we describe a new aptasensor for VEGF165 detection employing Vap7 and VEa5, two DNA aptamers recognizing different sites of the protein. The aptamers were modified to be adapted to the solid phase platform of SAM and their capability to simultaneously recognize VEGF165 by forming a ternary complex was analyzed in solution. Having so defined the best tandem arrangement of modified aptamers, we set up the aptasensor for VEGF165, and finally analyzed the multiplex system with the two aptasensors for the simultaneous detection of VEGF165 and thrombin. The results indicate that each sandwich is specific, even when the two proteins are mixed. The system performance is consistent with the behavior evidenced by the biochemical analysis, which proves to be valuable to drive the evaluation and refinement of aptamers prior to or along the development of a detection platform. Since thrombin upregulates VEGF expression, the simultaneous recognition of these two proteins could be useful in the analysis of biomarkers in pathologies characterized by neo-angiogenesis.

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

2013-01-01

340

Increased thrombin generation measured in the presence of thrombomodulin in women with early pregnancy loss.  

Science.gov (United States)

Compared with 537 parous controls with no history of pregnancy loss, a lower thrombomodulin-related inhibition of the endogenous thrombin potential was measured in 264 cases with previous unexplained pregnancy loss, especially when losses occurred between 9 and 12 weeks of gestation. Adjusting age, protein S, factor VIII, factor V Leiden, and prothrombin G20210A did not change the results. PMID:21130429

de Saint Martin, Luc; Duchemin, Jérôme; Bohec, Caroline; Couturaud, Francis; Mottier, Dominique; Collet, Michel; Blouch, Marie-Thérèse; Pasquier, Elisabeth

2010-12-03

 
 
 
 
341

Increased thrombin generation measured in the presence of thrombomodulin in women with early pregnancy loss.  

UK PubMed Central (United Kingdom)

Compared with 537 parous controls with no history of pregnancy loss, a lower thrombomodulin-related inhibition of the endogenous thrombin potential was measured in 264 cases with previous unexplained pregnancy loss, especially when losses occurred between 9 and 12 weeks of gestation. Adjusting age, protein S, factor VIII, factor V Leiden, and prothrombin G20210A did not change the results.

de Saint Martin L; Duchemin J; Bohec C; Couturaud F; Mottier D; Collet M; Blouch MT; Pasquier E

2011-04-01

342

Neurovascular unit and the effects of dosage in VEGF toxicity: role for oxidative stress and thrombin.  

UK PubMed Central (United Kingdom)

Bidirectional communication between neurons and vascular cells is important to the maintenance of the central nervous system (CNS) milieu. Vascular endothelial growth factor (VEGF), through its ability to affect both vascular and neuronal cells, is likely a key protein in this process. Despite considerable literature documenting a neuroprotective function for VEGF, overexpression of this protein has also been shown in a wide variety of CNS diseases, including Alzheimer's disease (AD). Increased oxidative stress and elevated thrombin levels have also been documented in AD, specifically in the microvasculature. The aim of the current study is to examine endothelial cells and neurons in vitro to determine the effects of oxidative stress and thrombin on VEGF release as well as the effects of low and high dose VEGF on neuronal viability. The data show that microvessels isolated from AD patients secrete significantly higher levels of VEGF compared to control-derived vessels. Exposure of brain endothelial cells to oxidative stress (sodium nitroprusside, menadione, or hydrogen peroxide) or thrombin significantly increases VEGF expression. Exposure of cultured neurons to oxidative stress increases expression of thrombin. Treating rat cortical neurons with high dose VEGF (?500 ng/ml) decreases neuronal survival and expression of the anti-apoptotic protein Bcl-2 while increasing proapoptic proteins caspase 3 and phosphorylated p38 MAPK. High dose VEGF also negates the decrease in amyloid-? evoked by low dose VEGF. These results suggest that despite literature supporting neuroprotective effects of this protein, caution is warranted prior to implementation of VEGF as a therapeutic in the brain.

Sanchez A; Tripathy D; Luo J; Yin X; Martinez J; Grammas P

2013-01-01

343

Thrombin inhibition attenuates neurodegeneration and cerebral edema formation following transient forebrain ischemia.  

UK PubMed Central (United Kingdom)

The disturbance of microcirculation following cerebral ischemia leads to an enlargement of cerebral infarct volume. Endogenous thrombin may play a role in this disturbance of microcirculation following cerebral ischemia. Therefore, the inhibition of thrombin may improve neurodegeneration and the accumulation of cerebral edema following cerebral ischemia in gerbils. The effects of thrombin inhibitor (argatroban) on cerebral ischemia were investigated in comparison with thromboxane A2 synthase inhibitor (ozagrel) and cyclooxygenase inhibitor (aspirin) following bilateral common carotid artery occlusion and reperfusion (CCA:O/R) in male Mongolian gerbils. This study consisted of three experiments: (1) morbidity and survival ratio (n=40 for each), (2) histopathology (n=12 for each), and (3) mean arterial blood pressure, local cerebral blood flow (CBF), and cerebral specific gravity (n=8 for each). Argatroban treatment improved survival ratio and stroke index, and decreased ischemically injured cell numbers in cortex and hippocampus and cerebral edema in cortex compared with aspirin and saline, in concert with the fast recovery of local CBF without reactive hyperemia following bilateral CCA:O/R. Ozagrel treatment also improved those factors compared with saline, in concert with the fast recovery of local CBF with reactive hyperemia. Aspirin treatment improved survival ratio and stroke index, and decreased ischemically injured cell numbers in cortex. Thrombin inhibition with argatroban decreases neurodegeneration and cerebral edema following bilateral CCA:O/R in gerbils.

Ohyama H; Hosomi N; Takahashi T; Mizushige K; Kohno M

2001-06-01

344

Thrombin inhibition attenuates neurodegeneration and cerebral edema formation following transient forebrain ischemia.  

Science.gov (United States)

The disturbance of microcirculation following cerebral ischemia leads to an enlargement of cerebral infarct volume. Endogenous thrombin may play a role in this disturbance of microcirculation following cerebral ischemia. Therefore, the inhibition of thrombin may improve neurodegeneration and the accumulation of cerebral edema following cerebral ischemia in gerbils. The effects of thrombin inhibitor (argatroban) on cerebral ischemia were investigated in comparison with thromboxane A2 synthase inhibitor (ozagrel) and cyclooxygenase inhibitor (aspirin) following bilateral common carotid artery occlusion and reperfusion (CCA:O/R) in male Mongolian gerbils. This study consisted of three experiments: (1) morbidity and survival ratio (n=40 for each), (2) histopathology (n=12 for each), and (3) mean arterial blood pressure, local cerebral blood flow (CBF), and cerebral specific gravity (n=8 for each). Argatroban treatment improved survival ratio and stroke index, and decreased ischemically injured cell numbers in cortex and hippocampus and cerebral edema in cortex compared with aspirin and saline, in concert with the fast recovery of local CBF without reactive hyperemia following bilateral CCA:O/R. Ozagrel treatment also improved those factors compared with saline, in concert with the fast recovery of local CBF with reactive hyperemia. Aspirin treatment improved survival ratio and stroke index, and decreased ischemically injured cell numbers in cortex. Thrombin inhibition with argatroban decreases neurodegeneration and cerebral edema following bilateral CCA:O/R in gerbils. PMID:11384620

Ohyama, H; Hosomi, N; Takahashi, T; Mizushige, K; Kohno, M

2001-06-01

345

Biochemical characterization of bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI)  

Digital Repository Infrastructure Vision for European Research (DRIVER)

BACKGROUND: TAFI is a plasma protein assumed to be an important link between coagulation and fibrinolysis. The three-dimensional crystal structures of authentic mature bovine TAFI (TAFIa) in complex with tick carboxypeptidase inhibitor, authentic full lenght bovine plasma thrombin-activatable fibrin...

Valnickova, Zuzana; Thaysen-Andersen, Morten; Højrup, Peter; Christensen, Trine; Sanggaard, Kristian W; Kristensen, Torsten

346

Role of thrombin in the proliferative response of T-47D mammary tumor cells  

International Nuclear Information System (INIS)

The growth of the human metastatic cell line (T-47D) in a chemically defined medium (DM) is shown to be dependent on the presence of three traditional growth factors: epidermal growth factor, insulin, and transferrin. The addition of thrombin further stimulates its growth. The mitogenic action on a human mammary tumor cell lines from epithelial origin is a novel action of thrombin. Cells in the DM show striking morphological changes which are dramatically enhanced by the addition of thrombin. These observations are part of a pleiotropic response to the growth factors: the protein content of the cells increases in the defined medium; the 2DG gels of the 35S- and 35P-labeled proteins show important changes in spots, several of which are probably of cytoskeletal origin. It is also shown that cells in a semisolid growth factor-supplemented medium have growth advantages over their counterparts grown with serum. All the phenotypic changes mentioned above reveal the important role of growth factors in the growth and behavior of this mammary cell line. The results obtained with thrombin indicate a new site of action of this enzyme which may be important in the metastatic spread of human mammary tumor cells

1987-01-01

347

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

UK PubMed Central (United Kingdom)

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 T(m) 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.

Pasternak A; Hernandez FJ; Rasmussen LM; Vester B; Wengel J

2011-02-01

348

Thrombin binds to a high-affinity approximately 900,000-dalton site on human platelets  

Energy Technology Data Exchange (ETDEWEB)

The functional sizes of the binding sites for thrombin on human platelets and isolated membranes have been determined by the technique of radiation inactivation: similar results were obtained. Independent studies using different radiation doses (0, 3, and 48 Mrad) and different thrombin concentrations (10(-10), 10(-8), and 10(-6) M) confirmed the presence of three binding sites with functional sizes of 900,000, 30,000, and 4000 daltons. The binding site of lowest apparent size (4000 daltons) probably corresponds to what has been termed nonspecific binding since its dissociation constant (2900 nM) is well outside the physiological range. The site of intermediate size (30,000 daltons) is also probably not involved in platelet activation since its dissociation constant (11 nM) is also beyond the concentration range required for activation, although it may be involved in other aspects of platelet-thrombin interaction. The sites with the largest functional size are probably important in platelet function since their dissociation constant (0.3 nM) is in the range required for platelet activation. The functional size of these sites (900,000 daltons) suggests that the high-affinity site for thrombin binding to platelets may involve a multimolecular complex of membrane components.

Harmon, J.T.; Jamieson, G.A.

1985-01-01

349

Pharmacophore design, virtual screening, molecular docking and optimization approaches to discover potent thrombin inhibitors.  

Science.gov (United States)

Thrombin plays a key role in the regulation of hemostasis and thrombosis. Inhibition of thrombin is therefore an effective therapeutic target to prevent the formation of blood clots and related thromboembolism disorders. Hence, we have developed chemical feature based pharmacophore models of thrombin inhibitors. The best hypothesis, Hypo1, is characterized with two hydrogen bond acceptors (A), one hydrophobic (H) and one ring aromatic (R) feature. Hypo1 was cross validated using several techniques to prove its validity and statistical significance. The well validated model Hypo1 was used as a 3D query to perform virtual screening. The scores obtained from virtual screening were sorted by applying drug-like filters and molecular docking studies. Finally, 4 compounds were obtained as drug-like leads based on scoring functions, binding modes and molecular interactions at the active site. These 4 molecules were further optimized by adding different substitutions in their side chains. When compared to the original database hits, optimized molecules showed high scoring function, good binding modes and molecular interactions. Hence, we suggest that, upon optimization, these four database hits can act as potential virtual leads to design novel thrombin inhibitors. Also, our model could be useful to retrieve the structurally diverse compounds from various databases. PMID:23713461

Loganathan, Chandrasekaran; Sakkiah, Sugunadevi; Lee, Keun Woo; Kabilan, Senthamaraikannan; Meganathan, Chandrasekaran

2013-11-01

350

The direct thrombin inhibitor argatroban: a review of its use in patients with and without HIT  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Argatroban is a synthetic direct thrombin inhibitor with a relative short elimination half-life of 45 minutes and elimination which is predominantly performed via hepatic metabolism. Argatroban anticoagulation has been systematically studied in patients exhibiting the heparin-induced thrombocytopeni...

Koster, Andreas; Fischer, Karl-Georg; Harder, Sebastian; Mertzlufft, Fritz

351

In vivo catabolism of human heparin cofactor II and its complex with thrombin  

International Nuclear Information System (INIS)

[en] The plasma clearance of human heparin cofactor II (HC) and its complex with thrombin (HC-T) was studied in mice and compared to the clearance of two other plasma proteinase inhibitor complexes: antithrombin III-thrombin (AT-T) and ? 1-proteinase inhibitor-elastase (? 1PI-E). Purified HC was labelled with 125I without loss of activity as assessed by kinetic analysis of thrombin inhibition and by ability to form a detergent-resistant complex with thrombin. 125I-HC cleared from the circulation with a t1/2 of 80 min; 125I-HC-T cleared with a t1/2 of 10 min. When coinjected, a 2500-fold molar excess of unlabelled HC-T partially blocked the clearance of 125I-HC-T (t1/2 of 53 min), suggesting the involvement of a receptor-mediated process in the catabolism of HC-T. Coinjection of a 1500-fold molar excess of AT-T also slowed the clearance of 125I-HC-T (t1/2 of 29 min); a 2000-fold molar excess of ? 1PI-E further competed for 125I-HC-T recovered at autopsy was localized to the liver; similar results have been obtained for AT-T and ? 1PI-trypsin

1987-05-01

352

Impairment of thrombin generation in the early phases of the host response of sepsis.  

UK PubMed Central (United Kingdom)

PURPOSE: The purpose was to investigate the presence of hypercoagulability in the very early phase of the host response to an infection in the clinical course of sepsis and septic shock. MATERIAL AND METHODS: Twenty-four patients with chemotherapy-associated febrile neutropenia were evaluated at baseline, at the time of fever onset, and 48 hours thereafter using the thrombin generation test, a more physiological and global assay of hemostasis. RESULTS: The rate of thrombin generation was decreased and no signals of systemic hypercoagulability could be observed during the first 48 hours of sepsis. Moreover, patients that evolved to septic shock presented a more significant impairment in thrombin generation than those with noncomplicated sepsis. CONCLUSIONS: Patients with sepsis and febrile neutropenia present an impairment in thrombin generation from very early stages of their disease course. These results suggest that the procoagulant in vitro alterations described during sepsis do not necessarily translate into a clinically relevant systemic hypercoagulable state. These findings could help explain why treatment with systemic anticoagulants did not translate to clinical benefits in human sepsis and highlight the need for a better understanding of the hemostatic alterations in sepsis before new treatments targeting coagulation activation are developed.

Picoli-Quaino SK; Alves BE; Faiotto VB; Montalvao SA; De Souza CA; Annichino-Bizzacchi JM; De Paula EV

2013-09-01

353

Prolyl endopeptidase and thrombin inhibitory diterpenoids from the bark of Xylopia aethiopica.  

Science.gov (United States)

The inhibitory effects of seven diterpenes, belonging to three different structural classes and isolated from the bark of Xylopia aethiopica, were investigated against the enzymes prolyl endopeptidase (PEP) and alpha-thrombin. Five compounds exhibited inhibitory activity against them. PMID:16195597

Diderot, Noungoue Tchamo; Silvere, Ngouela; Yasin, Amsha; Zareen, Seema; Fabien, Zelefack; Etienne, Tsamo; Choudhary, M Iqbal; Atta-Ur-Rahman

2005-09-01

354

Thrombin binds to a high-affinity approximately 900,000-dalton site on human platelets  

International Nuclear Information System (INIS)

The functional sizes of the binding sites for thrombin on human platelets and isolated membranes have been determined by the technique of radiation inactivation: similar results were obtained. Independent studies using different radiation doses (0, 3, and 48 Mrad) and different thrombin concentrations (10(-10), 10(-8), and 10(-6) M) confirmed the presence of three binding sites with functional sizes of 900,000, 30,000, and 4000 daltons. The binding site of lowest apparent size (4000 daltons) probably corresponds to what has been termed nonspecific binding since its dissociation constant (2900 nM) is well outside the physiological range. The site of intermediate size (30,000 daltons) is also probably not involved in platelet activation since its dissociation constant (11 nM) is also beyond the concentration range required for activation, although it may be involved in other aspects of platelet-thrombin interaction. The sites with the largest functional size are probably important in platelet function since their dissociation constant (0.3 nM) is in the range required for platelet activation. The functional size of these sites (900,000 daltons) suggests that the high-affinity site for thrombin binding to platelets may involve a multimolecular complex of membrane components

1985-01-01

355

Interactions of bovine thrombin and plasma albumin with low-energy surfaces.  

UK PubMed Central (United Kingdom)

Surface configurations are vessels fabricated from tubing and plate, films deposited on the surface of vessels, and beads confined in vessels. The average association constant between thrombin and sites on commercial poly(methyl methacrylate) surface (Lucite) is near 4 X 10(8) liters/mole at 22 degrees C, pH 7.0, and ionic strength 0.15. Depending on Lucite composition, average adsorption U, in molecules/cm2 of apparent solution-surface interface, ranges from 0.7 to 8.8 X 10(11). Analysis based on the assumptions that solution dimensions are preserved, adsorption is random, and surface rearrangement is negligible indicates a paucity of surface sites. Plasma albumin competes with thrombin for surface sites. Attempts to detect, by thrombin adsorption, the presence of free sites at 4.5 X 10-9M albumin or the displacement of bound albumin indicate an albumin-site association contrast greater than 1.6 X 10(9). Cross-linked poly(methyl acrylate) bead surface has U less than 5 X 10(10). In contrast to acrylic resins are silicone gum, polypropylene, and polyisobutylene, for which U ranges from 15 to 20 X 10(11). Analysis as above indicates that sites are of frequent occurrence. Material composition suggests that thrombin can interact with nonpolar groups. Further characteristics of low-energy surfaces are that progressive surface denaturation is small and there is a large variance between nominally equivalent configurations.

Waugh DF; Lippe JA; Freund YR

1978-09-01

356

A reusable impedimetric aptasensor for detection of thrombin employing a graphite-epoxy composite electrode.  

UK PubMed Central (United Kingdom)

Here, we report the application of a label-free electrochemical aptasensor based on a graphite-epoxy composite electrode for the detection of thrombin; in this work, aptamers were immobilized onto the electrodes surface using wet physical adsorption. The detection principle is based on the changes of the interfacial properties of the electrode; these were probed in the presence of the reversible redox couple [Fe(CN)(6)](3-)/[Fe(CN)(6)](4-) using impedance measurements. The electrode surface was partially blocked due to formation of aptamer-thrombin complex, resulting in an increase of the interfacial electron-transfer resistance detected by Electrochemical Impedance Spectroscopy (EIS). The aptasensor showed a linear response for thrombin in the range of 7.5 pM to 75 pM and a detection limit of 4.5 pM. The aptasensor was regenerated by breaking the complex formed between the aptamer and thrombin using 2.0 M NaCl solution at 42 °C, showing its operation for different cycles. The interference response caused by main proteins in serum has been characterized.

Ocaña C; Pacios M; del Valle M

2012-01-01

357

A Reusable Impedimetric Aptasensor for Detection of Thrombin Employing a Graphite-Epoxy Composite Electrode  

Directory of Open Access Journals (Sweden)

Full Text Available Here, we report the application of a label-free electrochemical aptasensor based on a graphite-epoxy composite electrode for the detection of thrombin; in this work, aptamers were immobilized onto the electrodes surface using wet physical adsorption. The detection principle is based on the changes of the interfacial properties of the electrode; these were probed in the presence of the reversible redox couple [Fe(CN)6]3?/[Fe(CN)6]4? using impedance measurements. The electrode surface was partially blocked due to formation of aptamer-thrombin complex, resulting in an increase of the interfacial electron-transfer resistance detected by Electrochemical Impedance Spectroscopy (EIS). The aptasensor showed a linear response for thrombin in the range of 7.5 pM to 75 pM and a detection limit of 4.5 pM. The aptasensor was regenerated by breaking the complex formed between the aptamer and thrombin using 2.0 M NaCl solution at 42 °C, showing its operation for different cycles. The interference response caused by main proteins in serum has been characterized.

Cristina Ocaña; Mercè Pacios; Manel del Valle

2012-01-01

358

Thrombin activation and increased fibrinolysis in patients with chronic liver disease  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The respective roles of intravascular coagulation (DIC) and fibrinolysis were assessed in severe chronic liver disease by measuring thrombin-antithrombin (TAT) complexes, tissue-type plasminogen activator antigen (tPA Ag) and fibrinogen and fibrin degradation products (FgDP and FbDP respectively) in...

359

Treatment of a Splenic Artery Pseudoaneurysm by Endoscopic Ultrasound-Guided Thrombin Injection  

International Nuclear Information System (INIS)

We present a case of a splenic artery pseudoaneurysm secondary to pancreatitis that was successfully treated by transgastric injection of thrombin under endoscopic ultrasound guidance. There has been no recurrence on follow-up CT angiography, and thus complex surgery or endovascular intervention has been avoided.

2007-01-01

360

Structure/function aspects of neutral P1 residue peptide inhibitors of thrombin.  

UK PubMed Central (United Kingdom)

Control of thrombin by its inhibition in indications such as myocardial infarction, unstable angina or stroke has been demonstrated to be therapeutically valuable. However restoration of hemostasis by targeting thrombin while avoiding its fellow serine proteinases, (e.g. plasmin, trypsin), remains a challenge of medicinal chemistry. Tripeptide-boronates and -phosphonates with neutral P1 side chains meet these criteria. Development of novel, high yielding chemical routes furnishes a wide range of un-natural P1 functionalities, demonstrating that this indeed is a class effect with selectivity conferred by the uncharged P1 residue. For example N-benzyloxycarbonyl-D-phenylalanylprolyl-1- (3-methoxypropyl) boroglycine ester (1) has a Ki value for thrombin of 7 nM and greater than two order of magnitude higher with all other serine proteinases tested. The ester group determines the kinetics of inhibition by tripeptide phosphonates, with diphenylphosphonates being slow tight binding inhibitors, showing 50% reversibility of inhibition. Therefore this design of inhibitors offers a facile strategic approach to development as thrombin specific pharmaceutical agents.

Deadman J; Claeson G; Scully MF

1995-01-01

 
 
 
 
361

Interactions between thrombin and natural products of Millettia nitita var. hirsutissima using capillary zone electrophoresis.  

Science.gov (United States)

A sensitive and selective high-performance analytical method based on capillary zone electrophoresis (CZE) was developed for investigating interactions between natural products isolated from Millettia nitita var. hirsutissima and thrombin qualitatively and quantitatively for the first time. The results showed that, compared with positive and negative control, the compounds ZYY-5 (genistein-8-C-beta-d-apiofuranosyl-(1-->6)-O-beta-d-glucopyranoside), ZYY-6 (calycosin), ZYY-8 (isoliquiritigenin), ZYY-9 (formononetin), ZYY-12 (gliricidin), ZYY-13 (8-O-methylretusin), FJ-2 (dihydrokaempferol), FJ-3 (biochanin), FJ-5 (afromosin) and XC-2 (hirsutissimiside F) interacted with thrombin, while ZYY-1 (sphaerobioside), ZYY-2 (formononetin-7-O-beta-d-apiofuranosyl-(1-->6)-O-beta-d-glucopyranoside), ZYY-3 (genistein-5-methylether-7-O-alpha-l-rhamnopyranosyl-(1-->6)-O-beta-d-glucopyranoside), ZYY-4 (retusin-7,8-O-beta-d-diglucopyranoside), ZYY-7 (symplocoside), ZYY-10 (ononin), ZYY-11 (genistin), ZYY-14 (afromosin-7-O-beta-d-glucopyranoside), ZYY-15 (lanceolarin), FJ-1 (liquiritigenin), FJ-4 (7,2-dihydroxy,4-methoxyisoflavan) and XC-1 (sphaerobioside) had no binding to thrombin. This indicated that the reported CZE method for the determination of compound-thrombin interactions is powerful, sensitive and fast, and requires less amounts of reagents, and further, it can be employed as a reliable alternative to other methods. PMID:19931496

Zhang, Shuyu; Cheng, Jun; Chen, Wenjing; Ling, Xiaomei; Zhao, Yuying; Feng, Jie; Xiang, Cheng; Liang, Hong

2009-11-10

362

Interactions between thrombin and natural products of Millettia nitita var. hirsutissima using capillary zone electrophoresis.  

UK PubMed Central (United Kingdom)

A sensitive and selective high-performance analytical method based on capillary zone electrophoresis (CZE) was developed for investigating interactions between natural products isolated from Millettia nitita var. hirsutissima and thrombin qualitatively and quantitatively for the first time. The results showed that, compared with positive and negative control, the compounds ZYY-5 (genistein-8-C-beta-d-apiofuranosyl-(1-->6)-O-beta-d-glucopyranoside), ZYY-6 (calycosin), ZYY-8 (isoliquiritigenin), ZYY-9 (formononetin), ZYY-12 (gliricidin), ZYY-13 (8-O-methylretusin), FJ-2 (dihydrokaempferol), FJ-3 (biochanin), FJ-5 (afromosin) and XC-2 (hirsutissimiside F) interacted with thrombin, while ZYY-1 (sphaerobioside), ZYY-2 (formononetin-7-O-beta-d-apiofuranosyl-(1-->6)-O-beta-d-glucopyranoside), ZYY-3 (genistein-5-methylether-7-O-alpha-l-rhamnopyranosyl-(1-->6)-O-beta-d-glucopyranoside), ZYY-4 (retusin-7,8-O-beta-d-diglucopyranoside), ZYY-7 (symplocoside), ZYY-10 (ononin), ZYY-11 (genistin), ZYY-14 (afromosin-7-O-beta-d-glucopyranoside), ZYY-15 (lanceolarin), FJ-1 (liquiritigenin), FJ-4 (7,2-dihydroxy,4-methoxyisoflavan) and XC-1 (sphaerobioside) had no binding to thrombin. This indicated that the reported CZE method for the determination of compound-thrombin interactions is powerful, sensitive and fast, and requires less amounts of reagents, and further, it can be employed as a reliable alternative to other methods.

Zhang S; Cheng J; Chen W; Ling X; Zhao Y; Feng J; Xiang C; Liang H

2009-12-01

363

Topics in CP violation  

International Nuclear Information System (INIS)

Given the varied backgrounds of the members of this audience this talk will be a grab bag of topics related to the general theme of CP Violation. I do not have time to dwell in detail on any of them. First, for the astronomers and astrophysicists among you, I want to begin by reviewing the experimental status of evidence for CP violation in particle processes. There is only one system where this has been observed, and that is in the decays of neutral K mesons.

1993-01-01

364

Topics in CP violation  

Energy Technology Data Exchange (ETDEWEB)

Given the varied backgrounds of the members of this audience this talk will be a grab bag of topics related to the general theme of CP Violation. I do not have time to dwell in detail on any of them. First, for the astronomers and astrophysicists among you, I want to begin by reviewing the experimental status of evidence for CP violation in particle processes. There is only one system where this has been observed, and that is in the decays of neutral K mesons.

Quinn, H.R.

1993-02-01

365

Topics in Operator Theory  

CERN Document Server

This is the first volume of a collection of original and review articles on recent advances and new directions in a multifaceted and interconnected area of mathematics and its applications. It encompasses many topics in theoretical developments in operator theory and its diverse applications in applied mathematics, physics, engineering, and other disciplines. The purpose is to bring in one volume many important original results of cutting edge research as well as authoritative review of recent achievements, challenges, and future directions in the area of operator theory and its applications.

Ball, Joseph A; Helton, JWilliam; Rodman, Leiba; Spitkovsky, Iiya

2010-01-01

366

Effect of BAX499 aptamer on tissue factor pathway inhibitor function and thrombin generation in models of hemophilia.  

UK PubMed Central (United Kingdom)

INTRODUCTION: In hemophilia, thrombin generation is significantly suppressed due to decreased factor (F)X activation. Clinical studies and experiments with transgenic mice have suggested that the severity of hemophilia is substantially reduced by tissue factor pathway inhibitor (TFPI) deficiency. METHODS: We evaluated the effect of TFPI antagonist aptamer BAX499 (formerly ARC19499) on TFPI function in purified systems and on thrombin generation and clot formation in plasma and blood. RESULTS: BAX499 effectively neutralized TFPI inhibition of FXa and FXa dependent inhibition of TF/FVIIa by TFPI. BAX499 did not inhibit FXa or TF/FVIIa when used up to 500 nM. In the synthetic coagulation proteome with TFPI at its mean physiologic concentration, BAX499 at 1 - 10nM increased thrombin generation triggered with 5 pM relipidated TF in a concentration-dependent manner. In severe hemophilia A or B models using the synthetic coagulation proteome, the addition of BAX499 at 5 nM increased thrombin generation to the levels observed in normal control. Thrombin generation measured in induced hemophilia B plasma required ~100nM BAX499 to restore thrombin levels to those seen in untreated plasma. In induced hemophilia B whole blood, BAX499 repaired the clotting time but failed to appreciably impact the propagation phase of thrombin generation. CONCLUSION: These data suggest that inhibition of TFPI by BAX499 may have potential for hemophilia treatment but requires further study in blood-based hemophilia systems.

Gissel M; Orfeo T; Foley JH; Butenas S

2012-12-01

367

The influence of tissue factor and tissue factor pathway inhibitor polymorphisms on thrombin generation in stable coronary artery disease.  

UK PubMed Central (United Kingdom)

In patients with stable coronary heart disease (n = 1,001) we investigated the influence of tissue factor (TF) and TF pathway inhibitor (TFPI) polymorphisms on thrombin generation in vivo, measured by prothrombin fragment (F) 1 and 2, and the potential to generate thrombin ex vivo, measured by the calibrated automated thrombogram assay. Additionally, circulating levels of TF and TFPI were correlated to the different parameters of thrombin generation. The TF 5466 and TFPI -399 polymorphisms associated with higher thrombin generation in vivo, the latter also with a prolonged lag time of the thrombin generation ex vivo(p < 0.05 for all).The TF -1812 TT and the TF -603 GG genotypes were associated with lower peak thrombin and a decreased average net rate of thrombin activation during the propagation phases (p ? 0.05), and the TFPI -33 TC genotype with prolonged lag time (p < 0.05) and additionally time to peak (p = 0.06). Strong correlations between TFPI levels, prothrombin fragment 1 and 2 as well as calibrated automated thrombogram parameters were observed.

Opstad TB; Pettersen AA; Bratseth V; Arnesen H; Seljeflot I

2010-01-01

368

Thrombin mitogenic responses and protein phosphorylation are different in cultured human endothelial cells derived from large and microvessels  

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It is well established that thrombin induces various biological responses in endothelial cells derived from large vessels. However, little is known about the effects of thrombin on the microvasculature. Protein phosphorylation may be one of the mechanisms by which an extracellular stimulus initiates cellular events like proliferation. Therefore, we have compared the effects of either human alpha-thrombin or phorbol esters (TPA) on the proliferation or protein phosphorylation in endothelial cells derived from large vessels (umbilical vein, HUVEC) or microvessels (omental tissue, HOMEC). In HOMEC, thrombin did not stimulate cell proliferation and protein phosphorylation while TPA slightly reduced the cell proliferation and induced the phosphorylation of a 27-kDa protein. In contrast, in HUVEC, thrombin or TPA markedly enhanced the cell proliferation and stimulated the phosphorylation of a 59-kDa protein. These data indicate that endothelial cells from large and small vessels respond differently to thrombin and there is a complex and as yet unclear relationship between the proliferation and the protein phosphorylation induced by thrombin.

Dupuy, E.; Bikfalvi, A.; Rendu, F.; Toledano, S.L.; Tobelem, G. (INSERM U 150, Paris (France))

1989-12-01

369

Expression of a fibrinogen fusion peptide in Escherichia coli: a model thrombin substrate for structure/function analysis.  

Science.gov (United States)

The initial event in fibrin clot formation is the thrombin-catalyzed cleavage of the A alpha chain of human fibrinogen. Most of the information required for thrombin recognition and cleavage of the A alpha chain lies in the amino terminal 51 residue CNBr fragment. By selective modification of residues in this region, we probed the features that participate in thrombin interactions. We constructed a vector which expressed a tripartite protein (tribrid) consisting of amino acids 1 to 50 of the A alpha chain followed by 60 amino acids of chicken collagen and the beta-galactosidase protein from Escherichia coli. Cell lysates run on NaDodSO4-polyacrylamide gels contained the predicted band of molecular weight (mol wt) 125,000. The tribrid reacted with a monoclonal antibody, Mab-Y18, which recognizes the amino terminus of the A alpha chain. When cell lysates were incubated with thrombin, FPA was released. By including one heterogeneous oligonucleotide in the construction, we generated plasmids that encoded three specific amino acid substitutions. Surprisingly, changing Gly14 to Val did not alter thrombin cleavage, although recognition by Mab-Y18 was lost. Substitution of lie for Arg23 did not alter either thrombin cleavage or monoclonal recognition. Substitution of Leu for Arg 16 altered thrombin cleavage; unexpectedly, recognition by Mab-Y18 was not changed. PMID:2642712

Lord, S T; Fowlkes, D M

1989-01-01

370

Expression of a fibrinogen fusion peptide in Escherichia coli: a model thrombin substrate for structure/function analysis.  

UK PubMed Central (United Kingdom)

The initial event in fibrin clot formation is the thrombin-catalyzed cleavage of the A alpha chain of human fibrinogen. Most of the information required for thrombin recognition and cleavage of the A alpha chain lies in the amino terminal 51 residue CNBr fragment. By selective modification of residues in this region, we probed the features that participate in thrombin interactions. We constructed a vector which expressed a tripartite protein (tribrid) consisting of amino acids 1 to 50 of the A alpha chain followed by 60 amino acids of chicken collagen and the beta-galactosidase protein from Escherichia coli. Cell lysates run on NaDodSO4-polyacrylamide gels contained the predicted band of molecular weight (mol wt) 125,000. The tribrid reacted with a monoclonal antibody, Mab-Y18, which recognizes the amino terminus of the A alpha chain. When cell lysates were incubated with thrombin, FPA was released. By including one heterogeneous oligonucleotide in the construction, we generated plasmids that encoded three specific amino acid substitutions. Surprisingly, changing Gly14 to Val did not alter thrombin cleavage, although recognition by Mab-Y18 was lost. Substitution of lie for Arg23 did not alter either thrombin cleavage or monoclonal recognition. Substitution of Leu for Arg 16 altered thrombin cleavage; unexpectedly, recognition by Mab-Y18 was not changed.

Lord ST; Fowlkes DM

1989-01-01

371

Characterization of a modified gold platform for the development of a label-free anti-thrombin aptasensor.  

UK PubMed Central (United Kingdom)

This work reports the characterization of a modified gold surface as a platform for the development of a label free aptasensor for thrombin detection. The biorecognition platform was obtained by the self-assembly of 4-mercaptobenzoic acid onto a gold surface, covalent attachment of streptavidin and further immobilization of the biotinylated anti-thrombin aptamer. The biosensing platform was characterized by cyclic voltammetry, electrochemical impedance spectroscopy, surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring. The biorecognition event aptamer-thrombin was detected from changes in the SPR angle produced as a consequence of the molecular interaction between the aptasensor and the target protein. The biosensing platform demonstrated to be highly selective for human thrombin even in the presence of large excess of bovine thrombin, bovine serum albumin, cytochrome C, lysozyme and myoglobin. The relationship between the changes in the SPR angle and thrombin concentration was linear up to 0.19 ?mol L(-1) (R(2)=0.992) while the detection limit was of 12.0 nmol L(-1) (240 fmol in the sample). This new sensing approach represents an interesting and promising alternative for the SPR-based quantification of thrombin.

Jalit Y; Gutierrez FA; Dubacheva G; Goyer C; Coche-Guerente L; Defrancq E; Labbé P; Rivas GA; Rodríguez MC

2013-03-01

372

Cationic transfersomes based topical genetic vaccine against hepatitis B.  

Science.gov (United States)

DNA vaccines have been shown to elicit both cellular and humoral immune responses and to be effective in a variety of preclinical bacterial, viral, and parasitic animal models. We have recently described a needle-free method of vaccination, transcutaneous immunization, based on topical application of vaccine antigens on intact skin using a novel carrier system, namely transfersomes. In the present study, a novel modified version of transfersomes, i.e., cationic transfersomes for topical DNA vaccine delivery has been developed. Cationic transfersomes composed of cationic lipid DOTMA and sodium deoxycholate as constitutive lipids were prepared and optimized for their size, shape, zeta potentials, deformability and loading efficiency. Plasmid DNA encoding hepatitis B surface antigen (HBsAg) was loaded in the cationic transfersomes using charge neutralization method. The immune stimulating activity was studied by measuring serum anti-HBsAg titer and cytokines level (IL-2 and IFN-gamma) following topical applications of plasmid DNA loaded cationic transfersomes in Balb/c mice and results were compared with naked DNA applied topically as well as naked DNA and pure recombinant HBsAg administered intramuscularly. Results revealed that DNA loaded cationic transfersomes elicited significantly (*Ptransfersomes elicited a comparable serum antibody titer and endogenous cytokines levels as produced after intramuscular recombinant HBsAg administration. The study signifies the potential of cationic transfersomes as DNA vaccine carriers for effective topical immunization. PMID:17446015

Mahor, Sunil; Rawat, Amit; Dubey, Praveen K; Gupta, Prem N; Khatri, Kapil; Goyal, Amit K; Vyas, S P

2007-03-12

373

Effects of fibrin, thrombin, and blood on breast capsule formation in a preclinical model.  

UK PubMed Central (United Kingdom)

BACKGROUND: The root cause of capsular contracture (CC) associated with breast implants is unknown. Recent evidence points to the possible role of fibrin and bacteria in CC formation. OBJECTIVES: The authors sought to determine whether fibrin, thrombin, and blood modulated the histological and microbiological outcomes of breast implant capsule formation in a rabbit model. METHODS: The authors carried out a case-control study to assess the influence of fibrin, thrombin, and blood on capsule wound healing in a rabbit model. Eighteen New Zealand white rabbits received four tissue expanders. One expander acted as a control, whereas the other expander pockets received one of the following: fibrin glue, rabbit blood, or thrombin sealant. Intracapsular pressure/volume curves were compared among the groups, and histological and microbiological evaluations were performed (capsules, tissue expanders, rabbit skin, and air). The rabbits were euthanized at two or four weeks. RESULTS: At four weeks, the fibrin and thrombin expanders demonstrated significantly decreased intracapsular pressure compared to the control group. In the control and fibrin groups, mixed inflammation correlated with decreased intracapsular pressure, whereas mononuclear inflammation correlated with increased intracapsular pressure. The predominant isolate in the capsules, tissue expanders, and rabbit skin was coagulase-negative staphylococci. For fibrin and thrombin, both cultures that showed an organism other than staphylococci and cultures that were negative were associated with decreased intracapsular pressure, whereas cultures positive for staphylococci were associated with increased intracapsular pressure. CONCLUSIONS: Fibrin application during breast implantation may reduce rates of CC, but the presence of staphylococci is associated with increased capsule pressure even in the presence of fibrin, so care should be taken to avoid bacterial contamination.

Marques M; Brown SA; Cordeiro ND; Rodrigues-Pereira P; Cobrado ML; Morales-Helguera A; Lima N; Luís A; Mendanha M; Gonçalves-Rodrigues A; Amarante J

2011-03-01

374

Thrombin generation in plasma of healthy adults and children: chromogenic versus fluorogenic thrombogram analysis.  

Science.gov (United States)

Coagulation tests and coagulation factor assays have been complemented recently with experimental tests to measure the total amount of thrombin formed. We have presently analyzed thrombin generation of healthy adult and paediatric plasma samples via a fluorogenic and a chromogenic method. The chromogenic method was performed on the fully automated Behring Coagulation System (BCS) and fluorogenic assays via Calibrated Automated Thrombography (CAT), after coagulation induction by various tissue factor (TF) concentrations. Sample distribution and variability were analyzed for the four main coagulation parameters, derived via computerized curve analysis in each method. Results for both methods were correlated. At the recommended TF concentration (300 pM), thrombin generation via BCS was less variable than via CAT (1-6 pM), but at comparable TF concentrations (1-6 pM), the CAT sensitivity was higher than that of BCS. Inhibition of intrinsic coagulation with the anti-factor VIII antibody BO2C11 revealed that the BCS detected extrinsic coagulation exclusively, at all TF concentrations tested. In contrast, at low TF concentrations (1 and 2.5 pM), via CAT, intrinsic coagulation pathway amplification was measured. At standardized TF concentrations (300 pM in BCS vs. 2.5 pM in CAT), different reference values between adults and children were found, for all parameters, except Tmax. In adult samples, the best correlation between both methods was observed for ETP(CAT) versus ETP(BCS) and for Peak height(CAT) versus Cmax(BCS), when thrombin generation was exclusively extrinsic (300 pM in BCS vs. 6 pM in CAT). In conclusion, differential thrombin generation characteristics in BCS and CAT are relevant for their clinical applicability. PMID:17849049

Devreese, Katrien; Wijns, Walter; Combes, Isabelle; Van kerckhoven, Soetkin; Hoylaerts, Marc F

2007-09-01

375

Thrombin generation in Cushing's Syndrome: do the conventional clotting indices tell the whole truth?  

Science.gov (United States)

Cushing's Syndrome (CS) is associated with an increased mortality, where hypercoagulability seems to have a crucial role in both arterial and venous thrombosis. Parameters of in vitro thrombin generation (TG) such as lag time, peak thrombin and endogenous thrombin potential (ETP), that describe the time until thrombin burst, the peak amount of TG and the total amount of thrombin generated, respectively as well as classical clotting markers were evaluated in 33 CS patients compared to both a group of 28 patients matched for the features of Metabolic Syndrome (MetS) and 31 healthy individuals. CS and MetS patients had shorter lag time (p PTT) was shorter (p PTT correlated inversely to urinary free cortisol (r = -0.45; p = 0.009). BMI correlated negatively to lag time (r = -0.40; p = 0.0001) and positively to peak and ETP (r = + 0.34; p = 0.001, r = + 0.28; p = 0.008, respectively). Obese and diabetic patients had shorter lag time (p = 0.0005; p = 0.0002, respectively), higher ETP (p = 0.0006; p = 0.007, respectively) and peak (p = 0.0003; p = 0.0005, respectively) as well as a more prolonged PT (p = 0.04; p = 0.009, respectively). Hypertensive individuals had higher ETP (p = 0.004), peak (p = 0.0008) and FVIII (p = 0.001). Our findings confirm a prothrombotic state in both CS and MetS patients, though lag time was less shortened in CS. The high levels of endogenous physiological anticoagulants, could possibly represent a protective mechanism against hypercoagulability seen in CS patients. PMID:23408210

Koutroumpi, S; Spiezia, L; Albiger, N; Barbot, M; Bon, M; Maggiolo, S; Gavasso, S; Simioni, P; Frigo, A; Mantero, F; Scaroni, C

2013-02-14

376

Elevated levels of thrombin-generating microparticles in stored red blood cells.  

UK PubMed Central (United Kingdom)

BACKGROUND: During storage, red blood cells (RBCs) lose their membrane stability, leading to haemolysis and microparticle (MP) formation. The use of RBCs stored for more than 28 days has been associated with an increased incidence of deep vein thrombosis. However, the exact mechanism by which coagulation activation is enhanced in stored RBCs is still unknown. OBJECTIVES: To investigate the relevant potential procoagulant activities of MPs and study the relative procoagulant factors for initiating the coagulation on MPs in stored RBCs. STUDY DESIGN AND METHODS: MPs were isolated from the plasma of RBC units stored in citrate-phosphate-dextrose-adenine. At seven storage time-points (d0, d7, d14, d21, d28, d35 and d42), MPs were morphologically observed, quantified and analysed for tissue factor, factor XI (FXI) and their thrombin-generating potential. RESULTS: MPs were observed using electron microscopy. The size of the MPs ranged from 0·272 ?m to 0·973 ?m in diameter. During the storage of RBCs in plastic bags, the MP concentration increased from 3389 ± 218/?l at day 0 to 61 586 ± 2237/?l at d42. Thrombin generation was dependent on the total number of MPs (r = 0·987). Anti-human FXI antibody inhibited thrombin concentrations by 50·3% compared with control plasma, whereas antitissue factor and antitissue factor pathway inhibitor failed to reduce thrombin concentrations. CONCLUSIONS: Our study provides evidence that MP formation due to RBC storage might propagate coagulation not only by exposing phosphatidylserine, but also by initiating thrombin generation independently of tissue factor in a FXI -dependent manner.

Gao Y; Lv L; Liu S; Ma G; Su Y

2013-07-01

377

Diclofenac 1% Topical (osteoarthritis pain)  

Science.gov (United States)

... topical gel is used to relieve pain from osteoarthritis (arthritis caused by a breakdown of the lining ... Diclofenac 1% topical liquid is used to relieve osteoarthritis pain in the knees. Diclofenac is in a ...

378

Topical tretinoin in acanthosis nigricans  

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Efficacy of topical tretinoin was assessed in 30 cases of idiopathic acanthosis nigricans which were recalcitrant to conventional modalities of treatment. Topical tretinoin once at night application was found to be very effective both clinically and histologically.

Lahiri Koushik; Malakar Subrata

379

Topical tretinoin in acanthosis nigricans  

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Full Text Available Efficacy of topical tretinoin was assessed in 30 cases of idiopathic acanthosis nigricans which were recalcitrant to conventional modalities of treatment. Topical tretinoin once at night application was found to be very effective both clinically and histologically.

Lahiri Koushik; Malakar Subrata

1996-01-01

380

Evaluation of a gelatin matrix as a topical hemostatic agent for hepatic bleeding in the dog.  

UK PubMed Central (United Kingdom)

New generation topical hemostatic agents containing thrombin have been developed for use in surgical procedures when control of bleeding by conventional methods is either ineffective or impractical. The authors compared the safety, hemostatic efficacy, and handling characteristics of a thrombin-containing topical surgical hemostatic agent (a gelatin matrix) to a hemostatic gelatin sponge for treatment of parenchymal bleeding after liver biopsy. Fourteen dogs were enrolled in this prospective clinical study. Paired 1.5 cm × 1.5 cm and 0.5 cm deep liver biopsies were obtained via laparotomy for each dog. One bleeding liver biopsy lesion was treated with the gelatin matrix and the other with a gelatin sponge. The treated liver biopsy sites were compared for bleeding severity, time to hemostasis, cumulative blood loss, and hemostatic agent handling characteristics. Median time to hemostasis was significantly shorter (P = 0.034) and median cumulative blood loss was significantly lower (P = 0.033) for the lesions treated with the gelatin matrix than the gelatin sponge. Adverse reactions were not observed within the first 24 hr postoperatively. When used to control parenchymal bleeding from liver biopsy sites in the dog, the evaluated gelatin matrix was safe and more effective than the gelatin sponge.

Polidoro DP; Kass PH

2013-09-01

 
 
 
 
381

Evaluation of a gelatin matrix as a topical hemostatic agent for hepatic bleeding in the dog.  

Science.gov (United States)

New generation topical hemostatic agents containing thrombin have been developed for use in surgical procedures when control of bleeding by conventional methods is either ineffective or impractical. The authors compared the safety, hemostatic efficacy, and handling characteristics of a thrombin-containing topical surgical hemostatic agent (a gelatin matrix) to a hemostatic gelatin sponge for treatment of parenchymal bleeding after liver biopsy. Fourteen dogs were enrolled in this prospective clinical study. Paired 1.5 cm × 1.5 cm and 0.5 cm deep liver biopsies were obtained via laparotomy for each dog. One bleeding liver biopsy lesion was treated with the gelatin matrix and the other with a gelatin sponge. The treated liver biopsy sites were compared for bleeding severity, time to hemostasis, cumulative blood loss, and hemostatic agent handling characteristics. Median time to hemostasis was significantly shorter (P = 0.034) and median cumulative blood loss was significantly lower (P = 0.033) for the lesions treated with the gelatin matrix than the gelatin sponge. Adverse reactions were not observed within the first 24 hr postoperatively. When used to control parenchymal bleeding from liver biopsy sites in the dog, the evaluated gelatin matrix was safe and more effective than the gelatin sponge. PMID:23861265

Polidoro, Daniel P; Kass, Philip H

2013-07-16

382

Atomic Processes in Plasmas: Eleventh APS Topical Conference. Proceedings  

International Nuclear Information System (INIS)

These proceedings represent papers presented at the Eleventh American Physical Society Topical Conference on Atomic Processes in Plasmas, held in Alabama, in March, 1998. The topics discussed included atomic structure, photoionization, atomic processes in magnetic fusion plasmas, and in laser plasmas, excitation of atomic ions, spectroscopy and diagnostics. There were sessions on ionization and recombination, atomic processes in inertial fusion plasmas and in industrial plasmas. The Office of Fusion Energy of the U.S. Department of Energy provided the financial support towards the publication of these proceedings. There were 32 papers presented at the conference,out of which 11 have been abstracted for the Energy,Science and Technology database

1998-01-01

383