Sample records for calibrated automated thrombin

  1. Analytical and between-subject variation of thrombin generation measured by calibrated automated thrombography on plasma samples. (United States)

    Kristensen, Anne F; Kristensen, Søren R; Falkmer, Ursula; Münster, Anna-Marie B; Pedersen, Shona


    The Calibrated Automated Thrombography (CAT) is an in vitro thrombin generation (TG) assay that holds promise as a valuable tool within clinical diagnostics. However, the technique has a considerable analytical variation, and we therefore, investigated the analytical and between-subject variation of CAT systematically. Moreover, we assess the application of an internal standard for normalization to diminish variation. 20 healthy volunteers donated one blood sample which was subsequently centrifuged, aliquoted and stored at -80 °C prior to analysis. The analytical variation was determined on eight runs, where plasma from the same seven volunteers was processed in triplicates, and for the between-subject variation, TG analysis was performed on plasma from all 20 volunteers. The trigger reagents used for the TG assays included both PPP reagent containing 5 pM tissue factor (TF) and PPPlow with 1 pM TF. Plasma, drawn from a single donor, was applied to all plates as an internal standard for each TG analysis, which subsequently was used for normalization. The total analytical variation for TG analysis performed with PPPlow reagent is 3-14% and 9-13% for PPP reagent. This variation can be minimally reduced by using an internal standard but mainly for ETP (endogenous thrombin potential). The between-subject variation is higher when using PPPlow than PPP and this variation is considerable higher than the analytical variation. TG has a rather high inherent analytical variation but considerable lower than the between-subject variation when using PPPlow as reagent.

  2. The technique of measuring thrombin generation with fluorescent substrates: 4. The H-transform, a mathematical procedure to obtain thrombin concentrations without external calibration

    NARCIS (Netherlands)

    Hemker, H.C.; Hemker, P.W.; Al Dieri, R.


    Summary In fluorogenic thrombin generation (TG) experiments, thrombin concentrations cannot be easily calculated from the rate of the fluorescent signal increase, because the calibration coefficient increases during the experiment, due to substrate consumption and quenching of the fluorescent signal

  3. Increased Automation in Stereo Camera Calibration Techniques

    Directory of Open Access Journals (Sweden)

    Brandi House


    Full Text Available Robotic vision has become a very popular field in recent years due to the numerous promising applications it may enhance. However, errors within the cameras and in their perception of their environment can cause applications in robotics to fail. To help correct these internal and external imperfections, stereo camera calibrations are performed. There are currently many accurate methods of camera calibration available; however, most or all of them are time consuming and labor intensive. This research seeks to automate the most labor intensive aspects of a popular calibration technique developed by Jean-Yves Bouguet. His process requires manual selection of the extreme corners of a checkerboard pattern. The modified process uses embedded LEDs in the checkerboard pattern to act as active fiducials. Images are captured of the checkerboard with the LEDs on and off in rapid succession. The difference of the two images automatically highlights the location of the four extreme corners, and these corner locations take the place of the manual selections. With this modification to the calibration routine, upwards of eighty mouse clicks are eliminated per stereo calibration. Preliminary test results indicate that accuracy is not substantially affected by the modified procedure. Improved automation to camera calibration procedures may finally penetrate the barriers to the use of calibration in practice.

  4. Individual differences in the calibration of trust in automation. (United States)

    Pop, Vlad L; Shrewsbury, Alex; Durso, Francis T


    The objective was to determine whether operators with an expectancy that automation is trustworthy are better at calibrating their trust to changes in the capabilities of automation, and if so, why. Studies suggest that individual differences in automation expectancy may be able to account for why changes in the capabilities of automation lead to a substantial change in trust for some, yet only a small change for others. In a baggage screening task, 225 participants searched for weapons in 200 X-ray images of luggage. Participants were assisted by an automated decision aid exhibiting different levels of reliability. Measures of expectancy that automation is trustworthy were used in conjunction with subjective measures of trust and perceived reliability to identify individual differences in trust calibration. Operators with high expectancy that automation is trustworthy were more sensitive to changes (both increases and decreases) in automation reliability. This difference was eliminated by manipulating the causal attribution of automation errors. Attributing the cause of automation errors to factors external to the automation fosters an understanding of tasks and situations in which automation differs in reliability and may lead to more appropriate trust. The development of interventions can lead to calibrated trust in automation. © 2014, Human Factors and Ergonomics Society.

  5. Automated Calibration For Numerical Models Of Riverflow (United States)

    Fernandez, Betsaida; Kopmann, Rebekka; Oladyshkin, Sergey


    Calibration of numerical models is fundamental since the beginning of all types of hydro system modeling, to approximate the parameters that can mimic the overall system behavior. Thus, an assessment of different deterministic and stochastic optimization methods is undertaken to compare their robustness, computational feasibility, and global search capacity. Also, the uncertainty of the most suitable methods is analyzed. These optimization methods minimize the objective function that comprises synthetic measurements and simulated data. Synthetic measurement data replace the observed data set to guarantee an existing parameter solution. The input data for the objective function derivate from a hydro-morphological dynamics numerical model which represents an 180-degree bend channel. The hydro- morphological numerical model shows a high level of ill-posedness in the mathematical problem. The minimization of the objective function by different candidate methods for optimization indicates a failure in some of the gradient-based methods as Newton Conjugated and BFGS. Others reveal partial convergence, such as Nelder-Mead, Polak und Ribieri, L-BFGS-B, Truncated Newton Conjugated, and Trust-Region Newton Conjugated Gradient. Further ones indicate parameter solutions that range outside the physical limits, such as Levenberg-Marquardt and LeastSquareRoot. Moreover, there is a significant computational demand for genetic optimization methods, such as Differential Evolution and Basin-Hopping, as well as for Brute Force methods. The Deterministic Sequential Least Square Programming and the scholastic Bayes Inference theory methods present the optimal optimization results. keywords: Automated calibration of hydro-morphological dynamic numerical model, Bayesian inference theory, deterministic optimization methods.

  6. Automated discrete element method calibration using genetic and optimization algorithms (United States)

    Do, Huy Q.; Aragón, Alejandro M.; Schott, Dingena L.


    This research aims at developing a universal methodology for automated calibration of microscopic properties of modelled granular materials. The proposed calibrator can be applied for different experimental set-ups. Two optimization approaches: (1) a genetic algorithm and (2) DIRECT optimization, are used to identify discrete element method input model parameters, e.g., coefficients of sliding and rolling friction. The algorithms are used to minimize the objective function characterized by the discrepancy between the experimental macroscopic properties and the associated numerical results. Two test cases highlight the robustness, stability, and reliability of the two algorithms used for automated discrete element method calibration with different set-ups.

  7. Automated system for the calibration of magnetometers

    DEFF Research Database (Denmark)

    Petrucha, Vojtech; Kaspar, Petr; Ripka, Pavel


    University. There are three axes of rotation in this design (compared to two axes in the previous version). The addition of the third axis allows us to calibrate more complex devices. An electronic compass based on a vector fluxgate magnetometer and micro electro mechanical systems (MEMS) accelerometer...... is one example. The new platform can also be used to evaluate the parameters of the compass in all possible variations in azimuth, pitch, and roll. The system is based on piezoelectric motors, which are placed on a platform made of aluminum, brass, plastic, and glass. Position sensing is accomplished...... through custom-made optical incremental sensors. The system is controlled by a microcontroller, which executes commands from a computer. The properties of the system as well as calibration and measurement results will be presented. ©2009 American Institute of Physics...

  8. Low agreement between fresh and frozen-thawed platelet-rich plasma in the calibrated automated thrombogram assay. (United States)

    Ljungkvist, M; Lövdahl, S; Zetterberg, E; Berntorp, E


    Thrombin generation tests (TGTs) are considered to give more detailed information of the overall coagulation capability of a patient than clotting-based routine assays. The TGT thrombin generation assay-calibrated automated thrombogram (TGA-CAT) uses both platelet-poor plasma (PPP) and platelet-rich plasma (PRP). Assessing PRP gives more physiological test conditions and is of great interest considering the important role platelets play in haemostasis. However, PRP needs to be assessed close after blood draw/preparation as freezing fragments the platelets. In several previous publications, the utility of frozen-thawed PRP (ft-PRP) has been promoted, and in one article, no significant difference between fresh PRP (f-PRP) and ft-PRP was reported. The aim of our study was to investigate the level of agreement between f-PRP and ft-PRP to further validate these results. Our test population contained 41 persons with haemophilia and 45 healthy subjects. We used the TGA-CAT method with a set-up according to the manufacturer of the method. The measurements showed a poor level of agreement between f-PRP and ft-PRP and differences were not systematic. Fresh and ft-PRP cannot be assumed to show equal results in the TGA-CAT assay. © 2017 John Wiley & Sons Ltd.

  9. Investigation of the thrombin-generating capacity, evaluated by thrombogram, and clot formation evaluated by thrombelastography of platelets stored in the blood bank for up to 7 days

    DEFF Research Database (Denmark)

    Johansson, Per Ingemar; Svendsen, M.S.; Salado, J.


    , in part, depend on its reflection of the dynamics of thrombin generation. MATERIAL AND METHODS: The kinetics of thrombin generation of platelets stored for 2 and 7 days, respectively, was assessed by calibrated automated thrombogram (CAT) and the lag time (min), time to peak (ttPeak; min), peak (nm.......0035). No correlation between ETP and TTG was found (P = 0.65). CONCLUSION: The kinetics of thrombin generation, as evaluated by CAT, correlates with the thrombus generation, as evaluated by thrombelastography and this may in part explain the clinical utility of the TEG in identifying clinically relevant coagulopathies...

  10. Are well-calibrated users effective users? Associations between calibration of trust and performance on an automation-aided task. (United States)

    Merritt, Stephanie M; Lee, Deborah; Unnerstall, Jennifer L; Huber, Kelli


    We present alternative operationalizations of trust calibration and examine their associations with predictors and outcomes. It is thought that trust calibration (correspondence between aid reliability and user trust in the aid) is a key to effective human-automation performance. We propose that calibration can be operationalized in three ways. Perceptual accuracy is the extent to which the user perceives the aid's reliability accurately at one point in time. Perceptual sensitivity and trust sensitivity reflect user adjustment of perceived reliability and trust as the aid's actual reliability changes over time. One hundred fifty-five students completed an X-ray screening task with an automated screener. Awareness of the aid's accuracy trajectory and error type was examined as predictors, and task performance and aid failure detection were examined as outcomes. Awareness of accuracy trajectory was significantly associated with all three operationalizations of calibration, but awareness of error type was not when considered in conjunction with accuracy trajectory. Contrary to expectations, only perceptual accuracy was significantly associated with task performance and failure detection, and combined, the three operationalizations accounted for only 9% and 4% of the variance in these outcomes, respectively. Our results suggest that the potential importance of trust calibration warrants further examination. Moderators may exist. Users who were better able to perform the task unaided were better able to identify and correct aid failure, suggesting that user task training and expertise may benefit human-automation performance.

  11. Thrombin generation assays for global evaluation of the hemostatic system: perspectives and limitations

    Directory of Open Access Journals (Sweden)

    Rita Carolina Figueiredo Duarte

    Full Text Available Abstract The existing techniques to evaluate hemostasis in clinical laboratories are not sensitive enough to detect hypercoagulable and mild hypocoagulable states. Under different experimental conditions, the thrombin generation test may meet these requirements. This technique evaluates the overall balance between procoagulant and anticoagulant forces and has provided new insights in our understanding of the coagulation cascade, as well as of the diagnosis of hypocoagulability and hypercoagulability conditions. Thrombin generated in the thrombin generation test can be quantified as platelet-rich or platelet-poor plasma using the calibrated automated thrombogram method, which monitors the cleavage of a fluorogenic substrate that is simultaneously compared to the known thrombin activity in a non-clotting plasma sample. The calibrated automated thrombogram method is an open system, in which different antibodies, proteins, enzymes and peptides can be introduced to answer specific questions regarding hemostatic processes. The thrombin generation test has great clinical potential, such as in monitoring patients taking anticoagulants and antiplatelet drugs, screening for genetic or acquired thrombotic disorders, and evaluating bleeding risk control in patients with hemophilia using bypass agents or replacement therapy. Different to conventional coagulation tests, the thrombin generation test can be used for an overall evaluation of hemostasis, the results of which can then be used to evaluate specific characteristics of hemostasis, such as prothrombin time, activated partial thromboplastin time, and levels of fibrinogen and other coagulation factors. The introduction of this method will contribute to a better understanding and evaluation of overall hemostatic processes; however, this method still requires standardization and clinical validation.

  12. A direct thrombin inhibitor suppresses protein C activation and factor Va degradation in human plasma: Possible mechanisms of paradoxical enhancement of thrombin generation. (United States)

    Kamisato, Chikako; Furugohri, Taketoshi; Morishima, Yoshiyuki


    We have demonstrated that antithrombin (AT)-independent thrombin inhibitors paradoxically increase thrombin generation (TG) in human plasma in a thrombomodulin (TM)- and protein C (PC)-dependent manner. We determined the effects of AT-independent thrombin inhibitors on the negative-feedback system, activation of PC and production and degradation of factor Va (FVa), as possible mechanisms underlying the paradoxical enhancement of TG. TG in human plasma containing 10nM TM was assayed by means of the calibrated automated thrombography. As an index of PC activation, plasma concentration of activated PC-PC inhibitor complex (aPC-PCI) was measured. The amounts of FVa heavy chain and its degradation product (FVa(307-506)) were examined by western blotting. AT-independent thrombin inhibitors, melagatran and dabigatran (both at 25-600nM) and 3-30μg/ml active site-blocked thrombin (IIai), increased peak levels of TG. Melagatran, dabigatran and IIai significantly decreased plasma concentration of aPC-PCI complex at 25nM or more, 75nM or more, and 10 and 30μg/ml, respectively. Melagatran (300nM) significantly increased FVa and decreased FVa(307-506). In contrast, a direct factor Xa inhibitor edoxaban preferentially inhibited thrombin generation (≥25nM), and higher concentrations were required to inhibit PC activation (≥150nM) and FVa degradation (300nM). The present study suggests that the inhibitions of protein C activation and subsequent degradation of FVa and increase in FVa by antithrombin-independent thrombin inhibitors may contribute to the paradoxical TG enhancement, and edoxaban may inhibit PC activation and FVa degradation as a result of TG suppression. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Experiences in Automated Calibration of a Nickel Equation of State (United States)

    Carpenter, John H.


    Wide availability of large computers has led to increasing incorporation of computational data, such as from density functional theory molecular dynamics, in the development of equation of state (EOS) models. Once a grid of computational data is available, it is usually left to an expert modeler to model the EOS using traditional techniques. One can envision the possibility of using the increasing computing resources to perform black-box calibration of EOS models, with the goal of reducing the workload on the modeler or enabling non-experts to generate good EOSs with such a tool. Progress towards building such a black-box calibration tool will be explored in the context of developing a new, wide-range EOS for nickel. While some details of the model and data will be shared, the focus will be on what was learned by automatically calibrating the model in a black-box method. Model choices and ensuring physicality will also be discussed. Sandia National Laboratories is a multi-mission laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000.

  14. Early thrombin formation capacity in trauma patients and association with venous thromboembolism. (United States)

    Voils, Stacy A; Lemon, Stephen J; Jordan, Janeen; Riley, Paul; Frye, Reginald


    Incidence of venous thromboembolism (VTE) in adult trauma patients is high despite mechanical and pharmacologic prophylaxis. We hypothesized that thrombin formation capacity as measured by calibrated automated thrombogram (CAT) is increased early in hospitalization and is associated with the development of VTE. We conducted a prospective study in adult, critically ill trauma patients. Plasma was generated from whole blood samples collected within the first 3days of hospital admission. CAT was used to determine lag time, thrombin peak, time to thrombin peak, endogenous thrombin potential (ETP), and velocity index in plasma samples from patients, and in control samples of platelet-poor, pooled normal plasma. There were 35 trauma patients and 35 controls included in this pilot analysis. Patients were a mean (SD) age of 45 (19) years, and 23 (66%) were male. The most common mechanism of injury was motor vehicle crash followed by falls, and the median (IQR) injury severity score was 17 (12-27). Three patients (8.6%) had deep vein thrombosis (DVT) confirmed by Doppler ultrasound on median hospital day 7. Compared to control samples, patients had significantly longer lag times (3.1min vs. 2.7min, p=0.02) and significantly higher ETP (1136nM∗min vs. 1019nM∗min, p=0.007), peak thrombin generation (239nM vs. 176nM, pthrombin generation between the two groups (5.5min vs. 5.7min, p=0.22). In the 3 patients with VTE compared to controls, lag times were shorter and velocity index was higher while ETP and peak thrombin generation were similar. There were no statistically significant differences in thrombin generation parameters in patients with or without VTE, but lag time was numerically shorter, and thrombin peak, time to peak and area-under-the-curve (ETP) were numerically lower in patients with DVT. We observed a thrombin generation profile in critically ill trauma patients consistent with an early hypercoagulable state; however, thrombin generation parameters did not

  15. Only minor changes in thrombin generation of children and adolescents with type 1 diabetes mellitus - A case-control study. (United States)

    Cimenti, Christina; Schlagenhauf, Axel; Leschnik, Bettina; Fröhlich-Reiterer, Elke; Jasser-Nitsche, Hildegard; Haidl, Harald; Suppan, Elisabeth; Weinhandl, Gudrun; Leberl, Maximilian; Borkenstein, Martin; Muntean, Wolfgang E


    Micro- and macrovascular diseases are frequent complications in patients with diabetes. Hypercoagulability may contribute to microvascular alterations. In this study, we investigated whether type 1 diabetes in children is associated with a hypercoagulable state by performing a global function test of coagulation - the thrombin generation assay. 75 patients with type 1 diabetes aged between 2 and 19years were compared to an age-matched healthy control group. Diabetes patients were divided into high-dose and low-dose insulin cohorts with a cut-off at 0.8Ukg(-1)d(-1). Measurements were performed with platelet poor plasma using Calibrated Automated Thrombography and 1 pM or 5 pM tissue factor. Additionally, we quantified prothrombin fragments F1+2, thrombin-antithrombin complex, prothrombin, tissue factor pathway inhibitor, and antithrombin. Patients with type 1 diabetes exhibited a significantly shorter of lag time as well as decreased thrombin peak and endogenous thrombin potential compared to control subjects with 5 pM but not with 1 pM tissue factor. In high-dose insulin patients peak thrombin generation was higher and time to peak shorter than in low-dose patients. Thrombin-antithrombin complex was decreased in patients with type 1 diabetes, whereas prothrombin fragments F1+2 was comparable in both groups. Thrombin generation parameters did not correlate with parameters of metabolic control and the duration of diabetes. Taken together, we found only minor changes of thrombin generation in children and adolescents with type 1 diabetes which - in contrast to type 2 diabetes - do not argue for a hypercoagulable state. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Automated Gravimetric Calibration to Optimize the Accuracy and Precision of TECAN Freedom EVO Liquid Handler. (United States)

    Bessemans, Laurent; Jully, Vanessa; de Raikem, Caroline; Albanese, Mathieu; Moniotte, Nicolas; Silversmet, Pascal; Lemoine, Dominique


    High-throughput screening technologies are increasingly integrated into the formulation development process of biopharmaceuticals. The performance of liquid handling systems is dependent on the ability to deliver accurate and precise volumes of specific reagents to ensure process quality. We have developed an automated gravimetric calibration procedure to adjust the accuracy and evaluate the precision of the TECAN Freedom EVO liquid handling system. Volumes from 3 to 900 µL using calibrated syringes and fixed tips were evaluated with various solutions, including aluminum hydroxide and phosphate adjuvants, β-casein, sucrose, sodium chloride, and phosphate-buffered saline. The methodology to set up liquid class pipetting parameters for each solution was to split the process in three steps: (1) screening of predefined liquid class, including different pipetting parameters; (2) adjustment of accuracy parameters based on a calibration curve; and (3) confirmation of the adjustment. The run of appropriate pipetting scripts, data acquisition, and reports until the creation of a new liquid class in EVOware was fully automated. The calibration and confirmation of the robotic system was simple, efficient, and precise and could accelerate data acquisition for a wide range of biopharmaceutical applications. © 2016 Society for Laboratory Automation and Screening.

  17. Automated calibration of TECAN genesis liquid handling workstation utilizing an online balance and density meter. (United States)

    Xie, Iris H; Wang, Michael H; Carpenter, Richard; Wu, Henry Y


    With robotics widely used in bioanalytical assays, accurate system performance is essential to ensure the quality and productivity of the robotics. In our lab, an automated calibration procedure has been developed to evaluate the precision and accuracy of the TECAN (Research Triangle Park, NC, U.S.A.) Genesis liquid handling system in a bioanalytical laboratory setting. The calibrations were performed by transferring and weighing the solvents automatically on a microbalance controlled by a Gemini program. From the data acquired, calibration reports were generated using a template. The novel aspect of this approach is the use of an on-line balance and a density meter, both of which combine to make the calibration process simple, efficient, and precise. For quantitative bioanalysis, a variety of solvents, including methanol, water, mixed solvents, and plasma, are typically used to prepare standards and unknown samples. Density information is usually unknown for the mixed solvents, and the density of plasma can vary from species to species. However, with the use of a universal density meter, the density could be obtained in seconds. The issue of solvent evaporation during the calibration process was also addressed. Calibration curves were set up for various liquid classes. Pipetting volumes ranged from 10 microL to 900 microL. Precision and accuracy results obtained from the semiannual performance evaluations showed this procedure to be reliable and user-friendly. Using the automated calibration procedure, the calibration and performance evaluation of the robotic system is considerably more efficient, and the incidence of unacceptable precision and accuracy is greatly reduced.

  18. Using Automated On-Site Monitoring to Calibrate Empirical Models of Trihalomethanes Concentrations in Drinking Water

    Directory of Open Access Journals (Sweden)

    Thomas E. Watts III


    Full Text Available An automated, on-site trihalomethanes concentration data set from a conventional water treatment plant was used to optimize powdered activated carbon and pre-chlorination doses. The trihalomethanes concentration data set was used with commonly monitored water quality parameters to improve an empirical model of trihalomethanes formation. A calibrated model was used to predict trihalomethanes concentrations the following year. The agreement between the models and measurements was evaluated. The original model predicted trihalomethanes concentrations within ~10 μg·L−1 of the measurement. Calibration improved model prediction by a factor of three to five times better than the literature model.

  19. Using Automated On-Site Monitoring to Calibrate Empirical Models of Trihalomethanes Concentrations in Drinking Water


    Thomas E. Watts III; Robyn A. Snow; Brown, Aaron W.; J. C. York; Greg Fantom; Paul S. Simone Jr.; Emmert, Gary L.


    An automated, on-site trihalomethanes concentration data set from a conventional water treatment plant was used to optimize powdered activated carbon and pre-chlorination doses. The trihalomethanes concentration data set was used with commonly monitored water quality parameters to improve an empirical model of trihalomethanes formation. A calibrated model was used to predict trihalomethanes concentrations the following year. The agreement between the models and measurements was evaluated. The...

  20. Evaluation of Automated Model Calibration Techniques for Residential Building Energy Simulation

    Energy Technology Data Exchange (ETDEWEB)

    and Ben Polly, Joseph Robertson [National Renewable Energy Lab. (NREL), Golden, CO (United States); Polly, Ben [National Renewable Energy Lab. (NREL), Golden, CO (United States); Collis, Jon [Colorado School of Mines, Golden, CO (United States)


    This simulation study adapts and applies the general framework described in BESTEST-EX (Judkoff et al 2010) for self-testing residential building energy model calibration methods. BEopt/DOE-2.2 is used to evaluate four mathematical calibration methods in the context of monthly, daily, and hourly synthetic utility data for a 1960's-era existing home in a cooling-dominated climate. The home's model inputs are assigned probability distributions representing uncertainty ranges, random selections are made from the uncertainty ranges to define "explicit" input values, and synthetic utility billing data are generated using the explicit input values. The four calibration methods evaluated in this study are: an ASHRAE 1051-RP-based approach (Reddy and Maor 2006), a simplified simulated annealing optimization approach, a regression metamodeling optimization approach, and a simple output ratio calibration approach. The calibration methods are evaluated for monthly, daily, and hourly cases; various retrofit measures are applied to the calibrated models and the methods are evaluated based on the accuracy of predicted savings, computational cost, repeatability, automation, and ease of implementation.

  1. Evaluation of Automated Model Calibration Techniques for Residential Building Energy Simulation

    Energy Technology Data Exchange (ETDEWEB)

    Robertson, J.; Polly, B.; Collis, J.


    This simulation study adapts and applies the general framework described in BESTEST-EX (Judkoff et al 2010) for self-testing residential building energy model calibration methods. BEopt/DOE-2.2 is used to evaluate four mathematical calibration methods in the context of monthly, daily, and hourly synthetic utility data for a 1960's-era existing home in a cooling-dominated climate. The home's model inputs are assigned probability distributions representing uncertainty ranges, random selections are made from the uncertainty ranges to define 'explicit' input values, and synthetic utility billing data are generated using the explicit input values. The four calibration methods evaluated in this study are: an ASHRAE 1051-RP-based approach (Reddy and Maor 2006), a simplified simulated annealing optimization approach, a regression metamodeling optimization approach, and a simple output ratio calibration approach. The calibration methods are evaluated for monthly, daily, and hourly cases; various retrofit measures are applied to the calibrated models and the methods are evaluated based on the accuracy of predicted savings, computational cost, repeatability, automation, and ease of implementation.

  2. Ground-based automated radiometric calibration system in Baotou site, China (United States)

    Wang, Ning; Li, Chuanrong; Ma, Lingling; Liu, Yaokai; Meng, Fanrong; Zhao, Yongguang; Pang, Bo; Qian, Yonggang; Li, Wei; Tang, Lingli; Wang, Dongjin


    Post-launch vicarious calibration method, as an important post launch method, not only can be used to evaluate the onboard calibrators but also can be allowed for a traceable knowledge of the absolute accuracy, although it has the drawbacks of low frequency data collections due expensive on personal and cost. To overcome the problems, CEOS Working Group on Calibration and Validation (WGCV) Infrared Visible Optical Sensors (IVOS) subgroup has proposed an Automated Radiative Calibration Network (RadCalNet) project. Baotou site is one of the four demonstration sites of RadCalNet. The superiority characteristics of Baotou site is the combination of various natural scenes and artificial targets. In each artificial target and desert, an automated spectrum measurement instrument is developed to obtain the surface reflected radiance spectra every 2 minutes with a spectrum resolution of 2nm. The aerosol optical thickness and column water vapour content are measured by an automatic sun photometer. To meet the requirement of RadCalNet, a surface reflectance spectrum retrieval method is used to generate the standard input files, with the support of surface and atmospheric measurements. Then the top of atmospheric reflectance spectra are derived from the input files. The results of the demonstration satellites, including Landsat 8, Sentinal-2A, show that there is a good agreement between observed and calculated results.

  3. Construction and calibration of a low cost and fully automated vibrating sample magnetometer

    Energy Technology Data Exchange (ETDEWEB)

    El-Alaily, T.M., E-mail: [Physics Department, Faculty of Science, Tanta University, Tanta (Egypt); El-Nimr, M.K.; Saafan, S.A.; Kamel, M.M.; Meaz, T.M. [Physics Department, Faculty of Science, Tanta University, Tanta (Egypt); Assar, S.T. [Engineering Physics and Mathematics Department, Faculty of Engineering, Tanta University, Tanta (Egypt)


    A low cost vibrating sample magnetometer (VSM) has been constructed by using an electromagnet and an audio loud speaker; where both are controlled by a data acquisition device. The constructed VSM records the magnetic hysteresis loop up to 8.3 KG at room temperature. The apparatus has been calibrated and tested by using magnetic hysteresis data of some ferrite samples measured by two scientifically calibrated magnetometers; model (Lake Shore 7410) and model (LDJ Electronics Inc. Troy, MI). Our VSM lab-built new design proved success and reliability. - Highlights: • A low cost automated vibrating sample magnetometer VSM has been constructed. • The VSM records the magnetic hysteresis loop up to 8.3 KG at room temperature. • The VSM has been calibrated and tested by using some measured ferrite samples. • Our VSM lab-built new design proved success and reliability.

  4. A comparative study using thrombin generation and three different INR methods in patients on Vitamin K antagonist treatment. (United States)

    Riva, N; Vella, K; Meli, S; Hickey, K; Zammit, D; Calamatta, C; Makris, M; Kitchen, S; Ageno, W; Gatt, A


    Vitamin K antagonist (VKA) treatment requires routine monitoring using the international normalized ratio (INR). However, different INR assays may vary in their results. The aim of this study was to assess the agreement of three different INR methods, compared with thrombin generation, in patients on VKA treatment. Sixty patients attending the Anticoagulation Clinic at Mater Dei Hospital (Msida, Malta) for VKA monitoring between August and September 2015 were enrolled. The INR was tested using a point-of-care (POC) device (CoaguChek XS Plus, Roche Diagnostics) for both capillary and venous blood samples, a photo-optical (Sysmex CS-2100i/CA-1500, Siemens) and a mechanical clot detection system (Thrombolyzer XRC, Behnk Elektronik). All assays used human recombinant thromboplastin as reagent. Thrombin generation was performed using the calibrated automated thrombogram. There was a negative curvilinear correlation between the endogenous thrombin potential and different INR assays (r≤-.75) and a strong positive linear correlation between the CoaguChek XS Plus on capillary samples and the other INR methodologies (r≥.96). All different INR assays showed good correlation with the thrombin generation potential. The POC INR showed one of the highest correlation coefficients with thrombin generation, confirming the POC devices as an accurate, valid alternative to laboratory INR in VKA patients. © 2017 John Wiley & Sons Ltd.

  5. Protein Z efficiently depletes thrombin generation in disseminated intravascular coagulation with poor prognosis. (United States)

    Lee, Nuri; Kim, Ji-Eun; Gu, Ja-Yoon; Yoo, Hyun Ju; Kim, Inho; Yoon, Sung-Soo; Park, Seonyang; Han, Kyou-Sup; Kim, Hyun Kyung


    Disseminated intravascular coagulation (DIC) is characterized by consumption of coagulation factors and anticoagulants. Thrombin generation assay (TGA) gives useful information about global hemostatic status. We developed a new TGA system that anticoagulant addition can deplete thrombin generation in plasma, which may reflect defective anticoagulant system in DIC. TGAs were measured on the calibrated automated thrombogram with and without thrombomodulin or protein Z in 152 patients who were suspected of having DIC, yielding four parameters including lag time, endogenous thrombin potential, peak thrombin and time-to-peak in each experiment. Nonsurvivors showed significantly prolonged lag time and time-to-peak in TGA-protein Z system, which was performed with added protein Z. In multivariate Cox regression analysis, lag time and time-to-peak in TGA system were significant independent prognostic factors. In TGA-protein Z system, lag time and time-to-peak were revealed as independent prognostic factors of DIC. Protein Z addition could potentiate its anticoagulant effect in DIC with poor prognosis, suggesting the presence of defective protein Z system. The prolonged lag time and time-to-peak in both TGA and TGA-protein Z systems are expected to be used as independent prognostic factors of DIC.

  6. The Antithrombotic Potential of Tinzaparin and Enoxaparin Upon Thrombin Generation Triggered In Vitro by Human Ovarian Cancer Cells IGROV1. (United States)

    Sassi, Mouna; Chakroun, Taher; Mbemba, Elisabeth; Van Dreden, Patrick; Elalamy, Ismail; Larsen, Annette K; Gerotziafas, Grigoris T


    A documented relationship between ovarian cancer and thrombosis does exist. Low-molecular-weight heparins (LMWHs) are cornerstone drugs in the primary prevention and treatment of venous thromboembolic events in patients with cancer. However, cancer cells may alter the efficiency of these antithrombotic agents. We aimed to characterize the procoagulant phenotype of human epithelial ovarian adenocarcinoma cells, IGROV1, and to compare the capacity of tinzaparin and enoxaparin to inhibit thrombin generation triggered by these cells. Thrombin generation induced by different concentrations of IGROV1 cells on platelet poor plasma (PPP) was assessed by the calibrated automated thrombogram assay. Tissue factor (TF) expression was studied using Western blot analysis. Then, the experimental model of thrombin generation was used to compare the inhibitory effect of clinically relevant concentrations of both tinzaparin and enoxaparin. The inhibitory concentration 50 (IC50) of the mean rate index and the endogenous thrombin potential and the 2-fold increase in lag time were analyzed on the basis of the anti-Xa and anti-IIa activities of the LMWHs. IGROV1 cells suspended into PPP resulted in a significant increase in thrombin generation in the absence of any exogenous source of TF and phospholipids. Tissue factor was expressed by IGROV1 cells. Tinzaparin was a more potent inhibitor of thrombin generation than enoxaparin. The inhibition of thrombin generation induced by IGROV1 cancer cells depended mainly on the anti-Xa activity of the LMWHs. This experimental study in ovarian cancer cells demonstrates that the antithrombotic activity of LMWHs is not completely predicted by the anti-Xa or anti-IIa activities measured in PPP.

  7. Automation of RELAP5 input calibration and code validation using genetic algorithm

    Energy Technology Data Exchange (ETDEWEB)

    Phung, Viet-Anh, E-mail: [Division of Nuclear Power Safety, Royal Institute of Technology, Roslagstullsbacken 21, 10691 Stockholm (Sweden); Kööp, Kaspar, E-mail: [Division of Nuclear Power Safety, Royal Institute of Technology, Roslagstullsbacken 21, 10691 Stockholm (Sweden); Grishchenko, Dmitry, E-mail: [Division of Nuclear Power Safety, Royal Institute of Technology, Roslagstullsbacken 21, 10691 Stockholm (Sweden); Vorobyev, Yury, E-mail: [National Research Center “Kurchatov Institute”, Kurchatov square 1, Moscow 123182 (Russian Federation); Kudinov, Pavel, E-mail: [Division of Nuclear Power Safety, Royal Institute of Technology, Roslagstullsbacken 21, 10691 Stockholm (Sweden)


    Highlights: • Automated input calibration and code validation using genetic algorithm is presented. • Predictions generally overlap experiments for individual system response quantities (SRQs). • It was not possible to predict simultaneously experimental maximum flow rate and oscillation period. • Simultaneous consideration of multiple SRQs is important for code validation. - Abstract: Validation of system thermal-hydraulic codes is an important step in application of the codes to reactor safety analysis. The goal of the validation process is to determine how well a code can represent physical reality. This is achieved by comparing predicted and experimental system response quantities (SRQs) taking into account experimental and modelling uncertainties. Parameters which are required for the code input but not measured directly in the experiment can become an important source of uncertainty in the code validation process. Quantification of such parameters is often called input calibration. Calibration and uncertainty quantification may become challenging tasks when the number of calibrated input parameters and SRQs is large and dependencies between them are complex. If only engineering judgment is employed in the process, the outcome can be prone to so called “user effects”. The goal of this work is to develop an automated approach to input calibration and RELAP5 code validation against data on two-phase natural circulation flow instability. Multiple SRQs are used in both calibration and validation. In the input calibration, we used genetic algorithm (GA), a heuristic global optimization method, in order to minimize the discrepancy between experimental and simulation data by identifying optimal combinations of uncertain input parameters in the calibration process. We demonstrate the importance of the proper selection of SRQs and respective normalization and weighting factors in the fitness function. In the code validation, we used maximum flow rate as the

  8. A method for automating calibration and records management for instrumentation and dosimetry

    Energy Technology Data Exchange (ETDEWEB)

    O`Brien, J.M. Jr.; Rushton, R.O.; Burns, R.E. Jr. [Atlan-Tech, Inc., Roswell, GA (United States)


    Current industry requirements are becoming more stringent on quality assurance records and documentation for calibration of instruments and dosimetry. A novel method is presented here that will allow a progressive automation scheme to be used in pursuit of that goal. This concept is based on computer-controlled irradiators that can act as stand-alone devices or be interfaced to other components via a computer local area network. In this way, complete systems can be built with modules to create a records management system to meet the needs of small laboratories or large multi-building calibration groups. Different database engines or formats can be used simply by replacing a module. Modules for temperature and pressure monitoring or shipping and receiving can be added, as well as equipment modules for direct IEEE-488 interface to electrometers and other instrumentation.

  9. The standard calibration instrument automation system for the atomic absorption spectrophotometer. Part 3: Program documentation (United States)

    Ryan, D. P.; Roth, G. S.


    Complete documentation of the 15 programs and 11 data files of the EPA Atomic Absorption Instrument Automation System is presented. The system incorporates the following major features: (1) multipoint calibration using first, second, or third degree regression or linear interpolation, (2) timely quality control assessments for spiked samples, duplicates, laboratory control standards, reagent blanks, and instrument check standards, (3) reagent blank subtraction, and (4) plotting of calibration curves and raw data peaks. The programs of this system are written in Data General Extended BASIC, Revision 4.3, as enhanced for multi-user, real-time data acquisition. They run in a Data General Nova 840 minicomputer under the operating system RDOS, Revision 6.2. There is a functional description, a symbol definitions table, a functional flowchart, a program listing, and a symbol cross reference table for each program. The structure of every data file is also detailed.

  10. Analysis and Automation of Calibration Process for Measurement Coils for Particle Accelerator Magnets

    CERN Document Server

    AUTHOR|(CDS)2226624; Azzam, Jamal; Chanthery, Elodie

    Various techniques are used to measure magnetic fields within the Magnetic Measurement (MM) section, all of which require the use of specific sensors. These sensors need to be calibrated in order to map the resulting signals to the characteristics of the magnetic field. Thus, the calibration procedure would benefit from being improved and made more stable, efficient, and technologically up-to-date. Consequently, to improve the efficiency of the calibration procedure and the work processes it was first necessary to analyze them and identify potential issues and weaknesses to be further addressed. The calibration procedure mainly suffered from too many manual operations, which were potential sources of error, and from outdated readout and data acquisition software and hardware. Firstly, a new software program had to be developed using a C++ framework called Flexible Framework for Magnetic Measurements (FFMM), that would automate the data acquisition and analysis. Next, a feasibility study had to be done ...

  11. Calibration and use of continuous heat-type automated seepage meters for submarine groundwater discharge measurements (United States)

    Mwashote, B.M.; Burnett, W.C.; Chanton, J.; Santos, I.R.; Dimova, N.; Swarzenski, P.W.


    Submarine groundwater discharge (SGD) assessments were conducted both in the laboratory and at a field site in the northeastern Gulf of Mexico, using a continuous heat-type automated seepage meter (seepmeter). The functioning of the seepmeter is based on measurements of a temperature gradient in the water between downstream and upstream positions in its flow pipe. The device has the potential of providing long-term, high-resolution measurements of SGD. Using a simple inexpensive laboratory set-up, we have shown that connecting an extension cable to the seepmeter has a negligible effect on its measuring capability. Similarly, the observed influence of very low temperature (???3 ??C) on seepmeter measurements can be accounted for by conducting calibrations at such temperatures prior to field deployments. Compared to manual volumetric measurements, calibration experiments showed that at higher water flow rates (>28 cm day-1 or cm3 cm-2 day-1) an analog flowmeter overestimated flow rates by ???7%. This was apparently due to flow resistance, turbulence and formation of air bubbles in the seepmeter water flow tubes. Salinity had no significant effect on the performance of the seepmeter. Calibration results from fresh water and sea water showed close agreement at a 95% confidence level significance between the data sets from the two media (R2 = 0.98). Comparatively, the seepmeter SGD measurements provided data that are comparable to manually-operated seepage meters, the radon geochemical tracer approach, and an electromagnetic (EM) seepage meter. ?? 2009 Elsevier Ltd.

  12. Comparatively Studied Color Correction Methods for Color Calibration of Automated Microscopy Complex of Biomedical Specimens

    Directory of Open Access Journals (Sweden)

    T. A. Kravtsova


    Full Text Available The paper considers a task of generating the requirements and creating a calibration target for automated microscopy systems (AMS of biomedical specimens to provide the invariance of algorithms and software to the hardware configuration. The required number of color fields of the calibration target and their color coordinates are mostly determined by the color correction method, for which coefficients of the equations are estimated during the calibration process. The paper analyses existing color calibration techniques for digital imaging systems using an optical microscope and shows that there is a lack of published results of comparative studies to demonstrate a particular useful color correction method for microscopic images. A comparative study of ten image color correction methods in RGB space using polynomials and combinations of color coordinate of different orders was carried out. The method of conditioned least squares to estimate the coefficients in the color correction equations using captured images of 217 color fields of the calibration target Kodak Q60-E3 was applied. The regularization parameter in this method was chosen experimentally. It was demonstrated that the best color correction quality characteristics are provided by the method that uses a combination of color coordinates of the 3rd order. The study of the influence of the number and the set of color fields included in calibration target on color correction quality for microscopic images was performed. Six train sets containing 30, 35, 40, 50, 60 and 80 color fields, and test set of 47 color fields not included in any of the train sets were formed. It was found out that the train set of 60 color fields minimizes the color correction error values for both operating modes of digital camera: using "default" color settings and with automatic white balance. At the same time it was established that the use of color fields from the widely used now Kodak Q60-E3 target does not

  13. Correlated motions and residual frustration in thrombin. (United States)

    Fuglestad, Brian; Gasper, Paul M; McCammon, J Andrew; Markwick, Phineus R L; Komives, Elizabeth A


    Thrombin is the central protease in the cascade of blood coagulation proteases. The structure of thrombin consists of a double β-barrel core surrounded by connecting loops and helices. Compared to chymotrypsin, thrombin has more extended loops that are thought to have arisen from insertions in the serine protease that evolved to impart greater specificity. Previous experiments showed thermodynamic coupling between ligand binding at the active site and distal exosites. We present a combined approach of molecular dynamics (MD), accelerated molecular dynamics (AMD), and analysis of the residual local frustration of apo-thrombin and active-site-bound (PPACK-thrombin). Community analysis of the MD ensembles identified changes upon active site occupation in groups of residues linked through correlated motions and physical contacts. AMD simulations, calibrated on measured residual dipolar couplings, reveal that upon active site ligation, correlated loop motions are quenched, but new ones connecting the active site with distal sites where allosteric regulators bind emerge. Residual local frustration analysis reveals a striking correlation between frustrated contacts and regions undergoing slow time scale dynamics. The results elucidate a motional network that probably evolved through retention of frustrated contacts to provide facile conversion between ensembles of states.

  14. Automated Energy Calibration and Fitting of LaCl3(Ce y-Spectra Using Peak Likelihood and Tabu Search

    Directory of Open Access Journals (Sweden)

    Timothy P. McClanahan


    Full Text Available An automated method for ?-emission spectrum calibration and deconvolution is presented for spaceflight applications for a Cerium doped Lanthanum Chloride, (LaCl3(Ce ?-ray detector system. This detector will be coupled with a pulsed neutron generator (PNG to induce and enhance nuclide signal quality and rates, yielding large volumes of spectral information. Automated analytical methods are required to deconvolve and quantify nuclide signals from spectra; this will both reduce human interactions in spectrum analysis and facilitate feedback to automated robotic and operations planning. Initial system tests indicate significant energy calibration drifts (>6%, that which must be mitigated for spectrum analysis. A linear energy calibration model is presently considered, with gain and zero factors. Deconvolution methods incorporate a tabu search heuristic to formulate and optimize searches using memory structures. Iterative use of a peak likelihood methodology identifies global calibration minima and peak areas. The method is compared to manual methods of calibration and indicates superior performance using tabu methods. Performance of the Tabu enhanced calibration method is superior to similar unoptimized local search. The techniques are also applicable to other emission spectroscopy, eg. X-ray and neutron.

  15. Construction and calibration of weighing lysimeters with an automated drainage system

    Directory of Open Access Journals (Sweden)

    Arthur C. Sanches

    Full Text Available ABSTRACT Quantification of the drained volume is one of the difficulties involved in using weighing lysimeters. Typically, this volume is measured by accessing a moat at the base of a lysimeter. However, it is not feasible to install the moat in small devices. Thus, the aim of this study involves developing, installing, calibrating, and checking the efficiency of small weighing lysimeters with automated drainage systems to test their functionality in field conditions. Each lysimeter is composed of a round PVC water tank with a diameter of 1.22 m and a depth of 0.58 m that is placed over a metal frame with three electronic load cells with the nominal capacity of each cell corresponding to 500 kg. The drainage system is composed of a small reservoir with a volume of 10 L, a weighing structure composed of a load cell with a nominal capacity of 30 kg, and an automatic solenoid valve driven by a device coupled to a data logger that records the data from the lysimeter and from the drainage system. Two calibrations are performed for the lysimeter as well as the drainage system to obtain equations with significant correlations (R2 > 0.9999. The drainage system was activated several times during the tests after receiving approximately 63.4 L of water from rainfall, and this in turn indicated a good performance.

  16. Automation of dosimeters calibration for radiotherapy in secondary dosimetric calibration laboratory of the CPHR; Automatizacion de la calibracion de dosimetros de radioterapia en el laboratorio secundario de calibracion dosimetrica del CPHR

    Energy Technology Data Exchange (ETDEWEB)

    Acosta, Andy L. Romero; Lores, Stefan Gutierrez, E-mail: [Centro de Proteccion e Higiene de las Radiaciones (CPHR), La Habana (Cuba)


    This paper presents the design and implementation of an automated system for measurements in the calibration of reference radiation dosimeters. It was made a software application that performs the acquisition of the measured values of electric charge, calculated calibration coefficient and automates the calibration certificate issuance. These values are stored in a log file on a PC. The use of the application improves control over the calibration process, helps to humanize the work and reduces personnel exposure. The tool developed has been applied to the calibration of dosimeters radiation patterns in the LSCD of the Centro de Proteccion e Higiene de las Radiaciones, Cuba.

  17. Reduced peak, but no diurnal variation, in thrombin generation upon melatonin supplementation in tetraplegia. A randomised, placebo-controlled study. (United States)

    Iversen, Per Ole; Dahm, Anders; Skretting, Grethe; Mowinckel, Marie-Christine; Stranda, Annicke; Østerud, Bjarne; Sandset, Per Morten; Kostovski, Emil


    Tetraplegic patients have increased risk of venous thrombosis despite anti-thrombotic prophylaxis. Moreover, they have blunted plasma variations in melatonin and altered diurnal variation of several haemostatic markers, compared with able-bodied. However, whether healthy individuals and tetraplegic patients, with or without melatonin, display abnormalities in thrombin generation during a 24-hour (h) cycle, is unknown. We therefore used the Calibrated Automated Thrombogram (CAT) assay to examine diurnal variations and the possible role of melatonin in thrombin generation. Six men with long-standing complete tetraplegia were included in a randomised placebo-controlled cross-over study with melatonin supplementation (2 mg, 4 consecutive nights), whereas six healthy, able-bodied men served as controls. Ten plasma samples were collected frequently during a 24-h awake/sleep cycle. No significant diurnal variation of any of the measured CAT indices was detected in the three study groups. Whereas endogenous thrombin potential (ETP) was independent (p > 0.05) of whether the tetraplegic men received melatonin or placebo, melatonin decreased (p = 0.005) peak values in tetraplegia compared with those given placebo. Able-bodied men had lower (p = 0.019) ETP and Lag-Time (p = 0.018) compared with tetraplegics receiving placebo. Neither the Time-to-Peak nor the Start-Tail was affected (p > 0.05) by melatonin in tetraplegia. In conclusion, indices of thrombin generation are not subjected to diurnal variation in healthy able-bodied or tetraplegia, but peak thrombin generation is reduced in tetraplegic men receiving oral melatonin.

  18. Plasma Thrombin Generation and Sensitivity to Activated Protein C Among Patients With Myeloma and Monoclonal Gammopathy of Undetermined Significance. (United States)

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


    The etiology of the prothrombotic state in myeloma has yet to be definitively characterized. Similarly, while recent evidence suggests that patients with monoclonal gammopathy of undetermined significance (MGUS) may also be at increased risk of thrombosis, the magnitude and the etiology of this risk have also yet to be defined. The present study aims to characterize patterns of plasma thrombin generation and sensitivity to the anticoagulant activity of activated protein C (APC) at the time of initial diagnosis of myeloma and in response to therapy in comparison to that observed among patients with MGUS and matched, healthy volunteers. Patients presenting with newly diagnosed/newly relapsed myeloma (n = 8), MGUS (n = 8), and matched healthy volunteers (n = 8) were recruited. Plasma thrombin generation was determined by calibrated automated thrombography. Peak thrombin generation was significantly higher in patients with myeloma (383.4 ± 33.4 nmol/L) and MGUS (353.4 ± 16.5 nmol/L) compared to healthy volunteers (276.7 ± 20.8 nmol/L; P thrombin potential was significantly lower in control plasma (228.6 ± 44.5 nmol/L × min) than in either myeloma (866.2 ± 241.3 nmol/L × min, P = .01) or MGUS plasma (627 ± 91.5 nmol/L × min, P = .003). Within the myeloma cohort, peak thrombin generation was significantly higher at diagnosis (353.2 ± 15.9 nmol/L) than following completion of the third cycle of therapy (282.1 ± 15.2 nmol/L; P < .005). Moreover, sensitivity to APC increased progressively with each cycle of chemotherapy. Further study of the etiology and evolving patterns of hypercoagulability among patients with these conditions is warranted and may have future implications for thromboprophylaxis strategies. © The Author(s) 2016.

  19. Implication of Free Fatty Acids in Thrombin Generation and Fibrinolysis in Vascular Inflammation in Zucker Rats and Evolution with Aging

    Directory of Open Access Journals (Sweden)

    Jérémy Lagrange


    Full Text Available Background: The metabolic syndrome (MetS and aging are associated with modifications in blood coagulation factors, vascular inflammation, and increased risk of thrombosis.Objectives: Our aim was to determine concomitant changes in thrombin generation in the blood compartment and at the surface of vascular smooth muscle cells (VSMCs and its interplay with adipokines, free fatty acids (FFA, and metalloproteinases (MMPs in obese Zucker rats that share features of the human MetS.Methods: Obese and age-matched lean Zucker rats were compared at 25 and 80 weeks of age. Thrombin generation was assessed by calibrated automated thrombography (CAT.Results: Endogenous thrombin potential (ETP was increased in obese rats independent of platelets and age. Clot half-lysis time was delayed with obesity and age. Interleukin (IL-1β and IL-13 were increased with obesity and age respectively. Addition of exogenous fibrinogen, leptin, linoleic, or palmitic acid increased thrombin generation in plasma whereas adiponectin had an opposite effect. ETP was increased at the surface of VSMCs from obese rats and addition of exogenous palmitic acid further enhanced ETP values. Gelatinase activity was increased in aorta at both ages in obese rats and MMP-2 activity was increased in VSMCs from obese rats.Conclusions: Our study demonstrated in MetS an early prothrombotic phenotype of the blood compartment reinforced by procoagulant properties of dedifferentiated and inflammatory VSMCs. Mechanisms involved (1 increased fibrinogen and impaired fibrinolysis and (2 increased saturated fatty acids responsible for additive procoagulant effects. Whether specifically targeting this hypercoagulability using direct thrombin inhibitors would improve outcome in MetS is worth investigating.

  20. Calibrated kallikrein generation in human plasma

    DEFF Research Database (Denmark)

    Biltoft, D; Sidelmann, J J; Olsen, L F


    OBJECTIVES: The physiological role of the contact system remains inconclusive. No obvious clinical complications have been observed for factor XII (FXII), prekallikrein (PK), or high molecular weight kininogen deficiencies even though the contact system in vitro is associated with coagulation......, fibrinolysis, and inflammation. A global generation assay measuring the initial phase of the contact system could be a valuable tool for studies of its physiological role. DESIGN AND METHODS: We investigated whether such a method could be developed using the principle of the Calibrated Automated Thrombin...

  1. Thrombin generation by hemolysis. (United States)

    Stief, Thomas W


    Hemolysis is the fragmentation of erythrocytes into microparticles (Hb-MP). Clinical hemolysis can result in a severe procoagulant state. The influence of Hb-MP on thrombin generation was quantified. Unfrozen citrated normal plasma (five donors) was supplemented with 0 or 1 g/l Hb-MP obtained through erythrocyte destruction by hypotonic lysis, freezing/thawing, or blood oxidation with 1 or 2 mmol/l chloramine-T. Pooled normal plasma was supplemented with 0-10 g/l Hb-MP and with 0-1 IU/ml low-molecular-weight heparin (dalteparin). Samples (50 microl) were tested in the recalcified coagulation activity assay. At 10 min coagulation reaction time the hypotonic lysis of erythrocytes appears to be the most procoagulant condition, followed by twice freezing/thawing, three times freezing/thawing, and once freezing/thawing. Oxidation of whole blood with 1 or 2 mmol/l chloramine-T decreases thrombin generation by about 20 or 50%, respectively. The thrombin generation in 1 mmol/l chloramine-T or 2 mmol/l oxidized plasma decreases by about 70 or 85%, respectively. The 50% inhibitory concentrations of low-molecular-weight heparin against recalcified thrombin generation are 0.01, 0.025, or 0.035 IU/ml for plasma supplemented with 0, 0.1, or 1 g/l Hb-MP, respectively. The recalcified coagulation activity assay allows one to quantify thrombin generation in critical hemolytic samples. It is suggested to find the appropriate pharmacologic dose of low-molecular-weight heparin.

  2. The effect of corn trypsin inhibitor, anti-tissue factor pathway inhibitor antibodies and phospholipids on microvesicle-associated thrombin generation in patients with pancreatic cancer and healthy controls. (United States)

    Hellum, Marit; Franco-Lie, Isabel; Øvstebø, Reidun; Hauge, Truls; Henriksson, Carola E


    Circulating microvesicles (MVs) are suggested to be important contributors to cancer-associated thrombosis due to the presence of surface-bound procoagulant molecules like tissue factor (TF) and phosphatidylserine (PS). Pancreatic cancer is considered to be one of the most prothrombotic malignancies. The aim of this study was to describe the impact of analytical variables on MV-associated thrombin generation in patients with pancreatic cancer and in healthy controls. MVs were isolated from citrated plasma and added to pooled normal plasma (PNP). Thrombin generation was measured by the calibrated automated thrombogram. The impact of corn trypsin inhibitor (CTI), anti-tissue factor pathway inhibitor (TFPI) antibodies and phospholipids was described. Antibodies against TF were used to assess TF-dependency, and MV-bound PS activity was measured with the Zymuphen MP-activity kit. MVs from the pancreatic cancer patients displayed higher thrombin generation and higher PS-activity than MVs from the healthy control group, while TF-dependency was observed in only 1 out of 13 patient samples. Adequate thrombin generation-curves were only achieved when CTI was omitted and anti-TFPI antibodies were added to PNP prepared in low contact-activating tubes. Addition of phospholipids reduced the significant differences between the two groups, and should be omitted. This modified thrombin generation assay could be useful for measurement of procoagulant circulating MVs, allowing the contribution from MVs affecting both the intrinsic and the extrinsic pathway to be measured.

  3. The effect of omega-3 polyunsaturated fatty acids on fibrin and thrombin generation in healthy subjects and subjects with cardiovascular disease. (United States)

    McEwen, Bradley J; Morel-Kopp, Marie-Christine; Tofler, Geoffrey H; Ward, Christopher M


    Hypercoagulability plays a key role in the progression of cardiovascular disease (CVD). Although omega-3 polyunsaturated fatty acid (PUFA) intake has been inversely related to the risk of cardiovascular events, the mechanisms are not fully understood. The aim of this study was to investigate the effects of omega-3 on novel markers of global coagulation. The generation of fibrin and thrombin, measured via overall hemostasis potential (OHP) assay and calibrated automated thrombography, respectively, was determined in 40 healthy subjects and 16 patients with CVD at baseline and after 4 weeks of 640 mg/day omega-3 PUFA. In healthy subjects, fibrin generation was significantly reduced, as measured by overall coagulation potential (p = 0.013), OHP (p omega-3 PUFA significantly reduced OHP and significantly increased the lag time to thrombin generation (both p omega-3 PUFA had no effect on other fibrin and thrombin generation parameters in CVD patients. Four-week omega-3 PUFA supplementation reduced thrombotic potential in healthy subjects, as shown by reduced fibrin generation and peak thrombin. There was a greater effect on fibrin generation in healthy subjects compared with those with CVD. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

  4. "Mirror image" antagonists of thrombin-induced platelet activation based on thrombin receptor structure.


    Hung, D T; Vu, T K; Wheaton, V I; Charo, I F; Nelken, N A; Esmon, N.; Esmon, C T; Coughlin, S R


    Platelet activation by thrombin plays a critical role in hemostasis and thrombosis. Based on structure-activity studies of a cloned platelet thrombin receptor, we designed two "mirror image" antagonists of thrombin and thrombin receptor function. First, "uncleavable" peptides mimicking the receptor domain postulated to interact with thrombin were found to be potent thrombin inhibitors. Second, proteolytically inactive mutant thrombins designed to bind but not cleave the thrombin receptor were...

  5. Thrombin inhibitors from different animals. (United States)

    Tanaka-Azevedo, A M; Morais-Zani, K; Torquato, R J S; Tanaka, A S


    Venous and arterial thromboembolic diseases are still the most frequent causes of death and disability in high-income countries. Clinical anticoagulants are inhibitors of enzymes involved in the coagulation pathway, such as thrombin and factor X(a). Thrombin is a key enzyme of blood coagulation system, activating the platelets, converting the fibrinogen to the fibrin net, and amplifying its self-generation by the activation of factors V, VIII, and XI. Thrombin has long been a target for the development of oral anticoagulants. Furthermore, selective inhibitors of thrombin represent a new class of antithrombotic agents. For these reasons, a number of specific thrombin inhibitors are under evaluation for possible use as antithrombotic drugs. This paper summarizes old and new interests of specific thrombin inhibitors described in different animals.

  6. Thrombin Inhibitors from Different Animals

    Directory of Open Access Journals (Sweden)

    A. M. Tanaka-Azevedo


    Full Text Available Venous and arterial thromboembolic diseases are still the most frequent causes of death and disability in high-income countries. Clinical anticoagulants are inhibitors of enzymes involved in the coagulation pathway, such as thrombin and factor Xa. Thrombin is a key enzyme of blood coagulation system, activating the platelets, converting the fibrinogen to the fibrin net, and amplifying its self-generation by the activation of factors V, VIII, and XI. Thrombin has long been a target for the development of oral anticoagulants. Furthermore, selective inhibitors of thrombin represent a new class of antithrombotic agents. For these reasons, a number of specific thrombin inhibitors are under evaluation for possible use as antithrombotic drugs. This paper summarizes old and new interests of specific thrombin inhibitors described in different animals.

  7. A hardware-software system for the automation of verification and calibration of oil metering units secondary equipment (United States)

    Boyarnikov, A. V.; Boyarnikova, L. V.; Kozhushko, A. A.; Sekachev, A. F.


    In the article the process of verification (calibration) of oil metering units secondary equipment is considered. The purpose of the work is to increase the reliability and reduce the complexity of this process by developing a software and hardware system that provides automated verification and calibration. The hardware part of this complex carries out the commutation of the measuring channels of the verified controller and the reference channels of the calibrator in accordance with the introduced algorithm. The developed software allows controlling the commutation of channels, setting values on the calibrator, reading the measured data from the controller, calculating errors and compiling protocols. This system can be used for checking the controllers of the secondary equipment of the oil metering units in the automatic verification mode (with the open communication protocol) or in the semi-automatic verification mode (without it). The peculiar feature of the approach used is the development of a universal signal switch operating under software control, which can be configured for various verification methods (calibration), which allows to cover the entire range of controllers of metering units secondary equipment. The use of automatic verification with the help of a hardware and software system allows to shorten the verification time by 5-10 times and to increase the reliability of measurements, excluding the influence of the human factor.

  8. Actions of thrombin in the interstitium. (United States)

    de Ridder, G G; Lundblad, R L; Pizzo, S V


    Thrombin is a pleiotropic enzyme best known for its contribution to fibrin formation and platelet aggregation during vascular hemostasis. There is increasing evidence to suggest a role for thrombin in the development of interstitial fibrosis, but interstitial thrombin has not been demonstrated by the direct determination of activity. Rather its presence is inferred by products of thrombin action such as fibrin and activated fibroblasts. This review will focus on possible mechanisms of thrombin formation in the interstitial space, the possible actions of thrombin, processes regulating thrombin activity in the interstitial space, and evidence supporting a role for thrombin in fibrosis. © 2015 International Society on Thrombosis and Haemostasis.

  9. Energy Modelling and Automated Calibrations of Ancient Building Simulations: A Case Study of a School in the Northwest of Spain

    Directory of Open Access Journals (Sweden)

    Ana Ogando


    Full Text Available In the present paper, the energy performance of buildings forming a school centre in the northwest of Spain was analyzed using a transient simulation of the energy model of the school, which was developed with TRNSYS, a software of proven reliability in the field of thermal simulations. A deterministic calibration approach was applied to the initial building model to adjust the predictions to the actual performance of the school, data acquired during the temperature measurement campaign. The buildings under study were in deteriorated conditions due to poor maintenance over the years, presenting a big challenge for modelling and simulating it in a reliable way. The results showed that the proposed methodology is successful for obtaining calibrated thermal models of these types of damaged buildings, as the metrics employed to verify the final error showed a reduced normalized mean bias error (NMBE of 2.73%. It was verified that a decrease of approximately 60% in NMBE and 17% in the coefficient of variation of the root mean square error (CV(RMSE was achieved due to the calibration process. Subsequent steps were performed with the aid of new software, which was developed under a European project that enabled the automated calibration of the simulations.

  10. Ground-based vicarious radiometric calibration of Landsat 7 ETM+ and Terra MODIS using an automated test site (United States)

    Czapla-Myers, J.; Leisso, N.


    The Remote Sensing Group at the University of Arizona has operated the Radiometric Calibration Test Site (RadCaTS) at Railroad Valley, Nevada, since 2004. It is an approach to ground-based vicarious calibration that does not require on-site personnel to make surface and atmospheric measurements during a satellite overpass. It was originally developed in 2002 in an attempt to increase the amount of data collected throughout the year while maintaining the accuracy of in-situ measurements. RadCaTS currently consists of four ground-viewing radiometers to measure surface reflectance, a Cimel sun photometer to make atmospheric measurements, and a weather station to measure ambient conditions. The data from these instruments are used in MODTRAN 5 to determine the top-of-atmosphere (TOA) spectral radiance for a given overpass time, and the results are compared to the sensor under test. The work presented here describes the RadCaTS instrumentation suite and automated processing scheme used to determine the surface reflectance and TOA spectral radiance. The instruments used to measure surface and atmospheric properties are presented, followed by a discussion of their spatial layout and their radiometric calibration. The RadCaTS ground-based results are compared to those from Aqua and Terra MODIS overpasses in 2008, and Landsat 7 ETM+ overpasses in 2009.

  11. Automated correction on X-rays calibration using transmission chamber and LabVIEW{sup TM}

    Energy Technology Data Exchange (ETDEWEB)

    Betti, Flavio; Potiens, Maria da Penha Albuquerque, E-mail: fbetti@ipen.b, E-mail: mppalbu@ipen.b [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)


    Uncertainties during prolonged exposure times on X-rays calibration procedures at the Instruments Calibration facilities at IPEN may suffer from efficiency (and therefore intensity) variations on the industrial X-Ray generator used. Using a transmission chamber as an online reference chamber during the whole irradiation process is proposed in order to compensate for such error source. Also temperature (and pressure) fluctuations may arise from the performance limited calibration room air conditioning system. As an open ionization chamber, that monitor chamber does require calculation of a correction factor due to the temperature and pressure effects on air density. Sending and processing data from all related instruments (electrometer, thermometer and barometer) can be more easily achieved by interfacing them to a host computer running an especially developed algorithm using LabVIEW{sup TM} environment which will not only apply the proper correction factors during runtime, but also determine the exact length of time to reach a desired condition, which can be: time period, charge collected, or air kerma, based on the previous calibration of the whole system using a reference chamber traceable to primary standard dosimetry laboratories. When performing such calibration, two temperature sensors (secondary standard thermistors) are simultaneously used, one for the transmission chamber, and other for the reference chamber. As the substitution method is used during actual customer's calibration, the readings from the second thermistor can also be used when desired for further corrections. Use of LabVIEW{sup TM} programming language allowed for a shorter development time, and it is also extremely convenient to make things easier when improvements and modifications are called for. (author)

  12. Assessing Writing in MOOCs: Automated Essay Scoring and Calibrated Peer Review™ (United States)

    Balfour, Stephen P.


    Two of the largest Massive Open Online Course (MOOC) organizations have chosen different methods for the way they will score and provide feedback on essays students submit. EdX, MIT and Harvard's non-profit MOOC federation, recently announced that they will use a machine-based Automated Essay Scoring (AES) application to assess written work in…

  13. Continuous and fast calibration of the CMS experiment: design of the automated workflows and operational experience

    CERN Document Server

    Oramus, Piotr Karol; Pfeiffer, Andreas; Franzoni, Giovanni; Govi, Giacomo Maria; Musich, Marco; Di Guida, Salvatore


    The exploitation of the full physics potential of the LHC experiments requires fast and efficient processing of the largest possible dataset with the most refined understanding of the detector conditions. To face this challenge, the CMS collaboration has setup an infrastructure for the continuous unattended computation of the alignment and calibration constants, allowing for a refined knowledge of the most time-critical parameters already a few hours after the data have been saved to disk. This is the prompt calibration framework which, since the beginning of the LHC Run-I, enables the analysis and the High Level Trigger of the experiment to consume the most up-to-date conditions optimizing the performance of the physics objects. In the Run-II this setup has been further expanded to include even more complex calibration algorithms requiring higher statistics to reach the needed precision. This imposed the introduction of a new paradigm in the creation of the calibration datasets for unattended workflows and o...

  14. Comparison of "E-Rater"[R] Automated Essay Scoring Model Calibration Methods Based on Distributional Targets (United States)

    Zhang, Mo; Williamson, David M.; Breyer, F. Jay; Trapani, Catherine


    This article describes two separate, related studies that provide insight into the effectiveness of "e-rater" score calibration methods based on different distributional targets. In the first study, we developed and evaluated a new type of "e-rater" scoring model that was cost-effective and applicable under conditions of absent human rating and…

  15. Thrombin Generation in Zebrafish Blood (United States)

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


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

  16. RADIOLOGICAL TIPS Percutaneous thrombin injection for ...

    African Journals Online (AJOL)

    seen posteriorly and is usually thin and longitudinal. A large neck diameter (e.g. >10 mm) is a relative contra-indication for thrombin injection because of a slightly higher risk of distal embolisation. There are several thrombin preparations available. In our centre, we use human thrombin from the Tisseel kit (Baxter US) where.

  17. Thrombin generation by activated factor VII on platelet activated by different agonists. Extending the cell-based model of hemostasis

    Directory of Open Access Journals (Sweden)

    Herrera Maria


    Full Text Available Abstract Background Platelet activation is crucial in normal hemostasis. Using a clotting system free of external tissue factor, we investigated whether activated Factor VII in combination with platelet agonists increased thrombin generation (TG in vitro. Methods and results TG was quantified by time parameters: lag time (LT and time to peak (TTP, and by amount of TG: peak of TG (PTG and area under thrombin formation curve after 35 minutes (AUC→35min in plasma from 29 healthy volunteers using the calibrated automated thrombography (CAT technique. TG parameters were measured at basal conditions and after platelet stimulation by sodium arachidonate (AA, ADP, and collagen (Col. In addition, the effects of recombinant activated FVII (rFVIIa alone or combined with the other platelet agonists on TG parameters were investigated. We found that LT and TTP were significantly decreased (p 35min were significantly increased (p 35min (but not PTG when compared to platelet rich plasma activated with agonists in the absence of rFVIIa. Conclusion Platelets activated by AA, ADP, Col or rFVIIa triggered TG. This effect was increased by combining rFVIIa with other agonists. Our intrinsic coagulation system produced a burst in TG independent of external tissue factor activity an apparent hemostatic effect with little thrombotic capacity. Thus we suggest a modification in the cell-based model of hemostasis.

  18. Automated in situ line of sight calibration of ASDEX Upgrade bolometers

    Energy Technology Data Exchange (ETDEWEB)

    Penzel, F., E-mail: [Max-Planck-Institute for Plasmaphysics, EURATOM Association, Garching (Germany); Meister, H.; Bernert, M.; Sehmer, T.; Trautmann, T.; Kannamüller, M.; Koll, J. [Max-Planck-Institute for Plasmaphysics, EURATOM Association, Garching (Germany); Koch, A.W. [Institute for Measurement Systems and Sensor Technology, Technische Universität München (Germany)


    The ITER Bolometer Robot Test Rig (IBOROB) is a robot-based diagnostic tool, which allows the measurement of the lines of sight (LOS) of the ITER bolometer prototypes. Up to now, it was only used as a LOS characterization device for the ITER collimator development. IBOROB was further developed and can now be operated in ASDEX Upgrade during a regular maintenance shutdown. At present, once a diagnostic like the bolometry is mounted inside the vessel, the actual LOS orientations are not measured, they are derived from CAD. The new procedure allows the fully automatic three-dimensional in situ measurement of bolometer LOS. The spatial distribution, the poloidal and toroidal alignment in the experiment coordinate system (CS), can be determined. The absolute accuracy, in reference to the tokamak CS, is provided by an additional calibration performed with a measurement arm by FARO Technologies Inc. Therefore, the amount of misalignment from the theoretical expectations can be quantified. In addition specific camera type dependencies such as internal camera reflections can be identified. Due to the high position accuracy of the robot, the LOS can be resolved with a spatial resolution of up to 0.1°. The method is explained in detail and results from two exemplary bolometer foil cameras obtained in a first set-up in ASDEX Upgrade are presented. The different steps and components needed to apply the measurements in the vessel are described with a focus on the constraints, e.g. geometrical, for an application of this method in a tokamak. Finally the consequences of the results are extrapolated to ITER and evaluated.

  19. [Preeclampsia and thrombin generation test]. (United States)

    Lattová, V; Procházka, M; Procházková, J; Ulehlová, J; Slavík, L; Lubušký, M; Brychtová, P


    Acquiring new information to allow prediction of the development of diseases associated with impaired coagulation. Design effective preventive measures most serious diseases (TEN) in the fields of gynecology and obstetrics. For pregnant women with preeclampsia, hypertension compared with women with normal pregnancies could lead to increased thrombin generation due to the synergistic effect of thrombotic risk factors. Based on the results and found statistically significant differences between the groups among pregnant can select for a higher risk of developing deep vein thrombosis. This risk group could then greatly benefit from more stringent follow-up and possible preventive treatment prophylactic doses of LMWH in reducing maternal and perinatal morbidity and mortality. Prospective study. Department of Obstetrics and Gynecology, University Hospital Olomouc. In early pregnancy - during pregnancy standard samples (up to the end of the first trimester) patients venous blood was sampled and they completed information questionnaire. A second sampling was carried out between 24 to 28 week, the third sample and between 36th to 40th week. Obtained blood samples were subsequently processed in the coagulation laboratory Hemato-Oncology Clinic and Olomouc. The blood samples were investigated protein C and S, antithrombin, FVIII level, FII, Leiden, and plasma endothelial microparticles, and lupus anticoagulant and APC resistance standardized methodologies. Thrombin generation was determined thrombin generation test. Thrombin generation was measured fully automatically using a kit (Technothrombin TGA, Technoclone, Vienna, Austria) and analyzer Ceveron Alpha (Technoclone, Vienna, Austria) with fully automatic analysis software. As the main parameter is evaluated by the maximum thrombin generation, at the same time, however, was also detected in the total amount of thrombin and the time until the beginning of the formation of thrombin. In the period 2008-2011 were analyzed

  20. 3D camera assisted fully automated calibration of scanning laser Doppler vibrometers (United States)

    Sels, Seppe; Ribbens, Bart; Mertens, Luc; Vanlanduit, Steve


    Scanning laser Doppler vibrometers (LDV) are used to measure full-field vibration shapes of products and structures. In most commercially available scanning laser Doppler vibrometer systems the user manually draws a grid of measurement locations on a 2D camera image of the product. The determination of the correct physical measurement locations can be a time consuming and diffcult task. In this paper we present a new methodology for product testing and quality control that integrates 3D imaging techniques with vibration measurements. This procedure allows to test prototypes in a shorter period because physical measurements locations will be located automatically. The proposed methodology uses a 3D time-of-flight camera to measure the location and orientation of the test-object. The 3D image of the time-of-flight camera is then matched with the 3D-CAD model of the object in which measurement locations are pre-defined. A time of flight camera operates strictly in the near infrared spectrum. To improve the signal to noise ratio in the time-of-flight measurement, a time-of-flight camera uses a band filter. As a result of this filter, the laser spot of most laser vibrometers is invisible in the time-of-flight image. Therefore a 2D RGB-camera is used to find the laser-spot of the vibrometer. The laser spot is matched to the 3D image obtained by the time-of-flight camera. Next an automatic calibration procedure is used to aim the laser at the (pre)defined locations. Another benefit from this methodology is that it incorporates automatic mapping between a CAD model and the vibration measurements. This mapping can be used to visualize measurements directly on a 3D CAD model. Secondly the orientation of the CAD model is known with respect to the laser beam. This information can be used to find the direction of the measured vibration relatively to the surface of the object. With this direction, the vibration measurements can be compared more precisely with numerical

  1. Nanostructured bioluminescent sensor for rapidly detecting thrombin. (United States)

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


    Thrombin plays a key role in thrombosis and hemostasis. The abnormal level of thrombin in body fluids may lead to different diseases, such as rheumatoid arthritis, glomerulonephritis, etc. Detection of thrombin level in blood and/or urine is one of important methods for medical diagnosis. Here, a bioluminescent sensor is developed for non-invasively and rapidly detecting thrombin in urine. The sensor is assembled through conjugating gold nanoparticles (Au NPs) and a recombinant protein containing Renilla luciferase (pRluc) by a peptide, which is thrombin specific substrate. The luciferase-catalyzed bioluminescence can be quenched by peptide-conjugating Au NPs. In the presence of thrombin, the short peptide conjugating luciferase and Au NPs is digested and cut off, which results in the recovery of bioluminescence due to the release of luciferase from Au NPs. The bioluminescence intensity at 470 nm is observed, and increases with increasing concentration of thrombin. The bioluminescence intensity of this designed sensor is significantly recovered when the thrombin digestion time lasts for 10 min. In addition, a similar linear relationship between luminescence intensity and the concentration of thrombin is found in the range of 8 nM to 8 μM in both buffer and human urine spiked samples. The limit of detection is as low as 80 pM. It is anticipated that our nanosensor could be a promising tool for clinical diagnosis of thrombin in human urine. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Increased thrombin generation in a mouse model of cancer cachexia is partially interleukin-6 dependent. (United States)

    Reddel, C J; Allen, J D; Ehteda, A; Taylor, R; Chen, V M Y; Curnow, J L; Kritharides, L; Robertson, G


    Essentials Cancer cachexia and cancer-associated thrombosis have not previously been mechanistically linked. We assessed thrombin generation and coagulation parameters in cachectic C26 tumor-bearing mice. C26 mice are hypercoagulable, partially corrected by blocking tumor derived interleukin-6. Coagulability and anti-inflammatory interventions may be clinically important in cancer cachexia. Background Cancer cachexia and cancer-associated thrombosis are potentially fatal outcomes of advanced cancer, which have not previously been mechanistically linked. The colon 26 (C26) carcinoma is a well-established mouse model of complications of advanced cancer cachexia, partially dependent on high levels of interleukin-6 (IL-6) produced by the tumor. Objectives To assess if cancer cachexia altered the coagulation state and if this was attributable to tumor IL-6 production. Methods In male BALB/c*DBA2 (F1 hybrid) mice with a C26 tumor we used modified calibrated automated thrombogram and fibrin generation (based on overall hemostatic potential) assays to assess the functional coagulation state, and also examined fibrinogen, erythrocyte sedimentation rate (ESR), platelet count, tissue factor pathway inhibitor (TFPI) and hepatic expression of coagulation factors by microarray. C26 mice were compared with non-cachectic NC26, pair-fed and sham control mice. IL-6 expression in C26 cells was knocked down by lentiviral shRNA constructs. Results C26 mice with significant weight loss and highly elevated IL-6 had elevated thrombin generation, fibrinogen, ESR, platelets and TFPI compared with all control groups. Fibrin generation was elevated compared with pair-fed and sham controls but not compared with NC26 tumor mice. Hepatic expression of coagulation factors and fibrinolytic inhibitors was increased. Silencing IL-6 in the tumor significantly, but incompletely, attenuated the increased thrombin generation, fibrinogen and TFPI. Conclusions Cachectic C26 tumor-bearing mice are in a

  3. Proinflammatory signaling functions of thrombin in cancer. (United States)

    Ebrahimi, Safieh; Rahmani, Farzad; Behnam-Rassouli, Reihane; Hoseinkhani, Fatemeh; Parizadeh, Mohammad Reza; Keramati, Mohammad Reza; Khazaie, Majid; Avan, Amir; Hassanian, Seyed Mahdi


    Thrombin-induced activation of protease-activated receptors (PARs) represents a link between inflammation and cancer. Proinflammatory signaling functions of thrombin are associated with several inflammatory diseases including neurodegenerative, cardiovascular, and of special interest in this review cancer. Thrombin-induced inflammatory responses up-regulates expression of cytokines, adhesion molecules, angiogenic factors, and matrix-degrading proteases that facilitate tumor cells proliferation, angiogenesis, invasion, and metastasis. This review summarizes the current knowledge about the mechanisms of thrombin-mediated proinflammatory responses in cancer pathology for a better understanding and hence a better management of this disease. © 2016 Wiley Periodicals, Inc.

  4. A New Colorimetrically-Calibrated Automated Video-Imaging Protocol for Day-Night Fish Counting at the OBSEA Coastal Cabled Observatory

    Directory of Open Access Journals (Sweden)

    Joaquín del Río


    Full Text Available Field measurements of the swimming activity rhythms of fishes are scant due to the difficulty of counting individuals at a high frequency over a long period of time. Cabled observatory video monitoring allows such a sampling at a high frequency over unlimited periods of time. Unfortunately, automation for the extraction of biological information (i.e., animals’ visual counts per unit of time is still a major bottleneck. In this study, we describe a new automated video-imaging protocol for the 24-h continuous counting of fishes in colorimetrically calibrated time-lapse photographic outputs, taken by a shallow water (20 m depth cabled video-platform, the OBSEA. The spectral reflectance value for each patch was measured between 400 to 700 nm and then converted into standard RGB, used as a reference for all subsequent calibrations. All the images were acquired within a standardized Region Of Interest (ROI, represented by a 2 × 2 m methacrylate panel, endowed with a 9-colour calibration chart, and calibrated using the recently implemented “3D Thin-Plate Spline” warping approach in order to numerically define color by its coordinates in n-dimensional space. That operation was repeated on a subset of images, 500 images as a training set, manually selected since acquired under optimum visibility conditions. All images plus those for the training set were ordered together through Principal Component Analysis allowing the selection of 614 images (67.6% out of 908 as a total corresponding to 18 days (at 30 min frequency. The Roberts operator (used in image processing and computer vision for edge detection was used to highlights regions of high spatial colour gradient corresponding to fishes’ bodies. Time series in manual and visual counts were compared together for efficiency evaluation. Periodogram and waveform analysis outputs provided very similar results, although quantified parameters in relation to the strength of respective rhythms were

  5. A method to measure thrombin activity in a mixture of fibrinogen and thrombin powders. (United States)

    DeAnglis, Ashley P; Nur, Israel; Gorman, Anne J; Meidler, Roberto


    Thrombin and fibrinogen powders are the active components of advanced surgical hemostasis products including the EVARREST Fibrin Sealant Patch. Measuring the enzymatic activity of thrombin in the presence of fibrinogen is challenging, as hydration of the powders in a neutral aqueous environment will cause the enzyme to rapidly react with the fibrinogen to form a fibrin clot, which in turn binds and entraps the enzyme thus preventing subsequent measurement of thrombin activity. A novel approach has been developed to overcome this challenge. After isolation of the mixture of powders, an alkaline carbonate solution is used to solubilize the proteins, while reversibly inhibiting the activity of thrombin and preventing clot formation. Once the powders have been fully solubilized, thrombin activity can be restored by neutralization in a buffered fibrinogen solution resulting in fibrin clot formulation. The rate of clot formation can be quantified in a coagulometer to determine the thrombin activity of the original powder. Samples coated with powders containing fibrinogen and varying amounts of thrombin were tested using the method described herein. The results demonstrated that the method could consistently measure the activity of (alpha) thrombin in the presence of fibrinogen over a broad range of thrombin activity levels. The test was successfully validated according to International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use Guidelines and thus is suitable for use as part of a commercial manufacturing process. A method has been developed that enables thrombin activity to be measured in a mixture of fibrinogen and thrombin powders.

  6. Aptamer Based Microsphere Biosensor for Thrombin Detection

    Directory of Open Access Journals (Sweden)

    Xudong Fan


    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.

  7. Contributions of procoagulants and anticoagulants to the international normalized ratio and thrombin generation assay in patients treated with warfarin: potential role of protein Z as a powerful determinant of coagulation assays. (United States)

    Choi, Qute; Kim, Ji-Eun; Hyun, Jungwon; Han, Kyou-Sup; Kim, Hyun Kyung


    The effects of warfarin are measured with the international normalized ratio (INR). However, the thrombin generation assay (TGA) may offer more information about global coagulation. We analyzed the monitoring performance of the TGA and INR and investigated the impact of procoagulants (fibrinogen, factor (F)II, FVII, FIX, and FX) and anticoagulants (proteins C, S, and Z) on them. The TGA was performed on a calibrated automated thrombogram, producing lag time, endogenous thrombin potential (ETP), and peak thrombin in 239 patients treated with warfarin. Pro- and anticoagulant levels were also measured. The INR was significantly and inversely correlated with ETP. The therapeutic range of ETP comparable to an INR range of 2.0-3.0 was 290.1-494.6. ETP showed comparable performance to the INR as a warfarin-monitoring parameter with respect to clinical complication rate. The median levels of FII, FVII, FIX, and FX and proteins C and Z tended to decrease gradually with increasing anticoagulation intensity according to the INR or ETP. Of note, protein Z levels decreased dramatically with increasing anticoagulation status. INRs were significantly determined by FII, FVII, and protein Z. ETP was significantly dependent on FVII, and proteins C and Z concentration. Protein Z significantly reduced the total amount of thrombin generation and prolonged PT value in vitro. The INR and ETP exhibit similar efficacy for warfarin monitoring according to the clinical complication rate. Protein Z is considered to be a significant determinant of INR and ETP in patients on warfarin therapy. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Measuring thrombin activity in frozen brain tissue. (United States)

    Reuveni, Gilad; Golderman, Valery; Shavit-Stein, Efrat; Rosman, Yossi; Shrot, Shai; Chapman, Joab; Harnof, Sagi


    Thrombin is a coagulation factor implicated in various pathological and physiological processes in the brain, exerting beneficial and deleterious effects in a concentration-dependent manner. Measurement of thrombin activity levels in pathological animal models is needed and in some cases, because of technical considerations, only frozen samples are available. In the current study, we used a quantitative method to evaluate thrombin activity in fresh and frozen brain sections of 43 male and female adult healthy mice. We stratified data per brain section, brain hemisphere, and mouse sex. We found lower thrombin activity in frozen sections compared with fresh sections, falling within levels considered central nervous system protective in previous studies. The results suggest that fresh section thrombin activity levels in healthy mice can be extrapolated from frozen brain sections. In addition, we found varying thrombin activity across the brain sections, with maximal activity in the olfactory system and hippocampus-containing sections. Thrombin activity did not vary between males and females, or between the right and the left hemispheres, in a statistically significantly manner.

  9. Effects of recombinant human prothrombin on thrombin generation in plasma from patients with hemophilia A and B. (United States)

    Hansson, K M; Gustafsson, D; Skärby, T; Frison, L; Berntorp, E


    The present study was carried out to investigate the impact of FII levels, and their increase, on the hemostatic potential in plasma from hemophilia A and B patients with and without inhibitors. Recombinant human factor (F) II (rhFII) was added ex vivo to plasma from 68 patients with hemophilia A and B, with or without inhibitors. The hemostatic potential as measured by thrombin generation (calibrated automated thrombogram [CAT]) was focused on the endogenous thrombin potential (ETP) as it has been shown to correlate with the clinical phenotype of bleeding in hemophilia patients and has also been used to guide bypassing therapy in hemophilia patients with inhibitors before elective surgery. The factor eight inhibitor bypassing agent (FEIBA(®) ) was used as a reference to the clinical situation. The study shows that rhFII concentration-dependently increased ETP by a similar magnitude in hemophilia A and B, both with and without inhibitors. Compared with FEIBA, rhFII showed a shallower concentration-response curve. In both types of hemophilia 100 mg L(-1) of rhFII roughly doubled the ETP. A corresponding response was obtained by 0.5 U mL(-1) of FEIBA. These data support the theory that FII is one of the major components responsible for the efficacy of FEIBA. The data also indicate that rhFII may be useful, alone or in combination with other coagulation factors, in some of the conditions for which FEIBA is used today, although more data are needed to substantiate this. © 2015 International Society on Thrombosis and Haemostasis.

  10. Insulin Antagonizes Thrombin-Induced Microvessel Leakage. (United States)

    Liu, Yan; Chen, Xue Lian; Wang, Lei; Martins-Green, Manuela


    Sustained increase in microvessel permeability results in cell and tissue damage. To date, it has not been possible to safely and specifically block increased microvessel permeability in vivo. We showed that insulin stimulates angiogenesis and that the new microvessels are associated with more αSMA-producing cells, suggesting greater stability. In this study, we show that local injection of insulin under the skin of mice significantly inhibits thrombin-induced microvessel permeability and that insulin improves the barrier function of primary human endothelial cells under conditions that mimic endothelium in vivo. These findings indicate that insulin antagonizes thrombin-induced microvessel permeability. At the cell and molecular levels, we show that insulin interferes with thrombin-induced VE-cadherin signaling by decreasing the ability of thrombin to induce VE-cadherin translocation to the cytoskeleton/nuclear compartment, leading to microvessel leakage. Simultaneously, the rapid activation of Src by insulin followed by the activation of Rac1, a small GTPase involved in cytoskeletal reorganization, leads to the maintenance of endothelial barrier, short-circuiting the slower thrombin-induced Src-RhoA signaling that leads to endothelial permeability. This novel mechanism by which insulin inhibits thrombin-induced permeability provides support for the use of insulin treatment in pathological conditions that involve blood-barrier dysfunction, especially as resuscitation treatment methods for extensive burns, sepsis, and other severe pathological conditions. © 2017 S. Karger AG, Basel.

  11. High precision, continuous measurements of water vapor isotopes using a field deployable analyzer with a novel automated calibration system to facilitate ecohydrological studies (United States)

    Gupta, P.; Crosson, E.; Richman, B. A.; Apodaca, R. L.; Green, I.


    The use of stable isotopic analysis techniques has proved quite valuable in establishing links between ecology and hydrology. We present an alternative and novel approach to isotope ratio mass spectrometry (IRMS) for making high-precision D/H and 18O/16O isotope ratio measurements of water vapor at a field site using wavelength-scanned cavity ring-down spectroscopy (WS-CRDS) based technology. This WS-CRDS analyzer allows continuous real-time measurements of water vapor with automated periodic calibration using liquid standards, needing no human intervention for weeks during deployment. The new automated calibration system, designed specifically for field deployment, uses syringe pumps and is robust, consistent and reliable. The advanced temperature and pressure control within the analyzer are some of the key design features that allow high precision (0.2‰ for δ18O and 1.0‰ for δD) performance at extremely low drift (physiology on the isotopic composition of water vapor in ambient air. Such measurements of water vapor, when combined with measurements of the isotopic composition of liquid water in plants, soil water and local water bodies, will close the eco-hydrological loop of any region. The ability of the WS-CRDS analyzer to make continuous, real-time measurements with a resolution on the order of a few seconds will aid in understanding the complex interdependencies between ecological and hydrological processes and will provide critical information in refining existing models of water transport in ecosystems. These studies are critical to understanding the impact of global climate change on landscapes.

  12. Thrombin has a bimodal effect on glioma cell growth.


    Schafberg, H.; Nowak, G.; Kaufmann, R.


    Using rat glioma C6 cells as a model, we have found a bimodal effect of alpha-thrombin on cell growth. In C6 cells treated with alpha-thrombin at concentrations from 0.02 nM to 1.0 nM, inhibition of cell proliferation was noted. Because the thrombin receptor agonist peptide TRAP-6 also induced inhibition of cell proliferation and the thrombin receptor antagonist peptide T1 prevented the inhibitory effect of alpha-thrombin on C6 glioma cell growth, thrombin receptor involvement in antiprolifer...

  13. Automated non-stepwise preparation of bioanalytical calibration standards and quality controls using an ultra-low volume digitizing liquid dispenser. (United States)

    Liao, Debra; Chen, Susan; Paton, Martin; Qian, Mark G


    Stepwise preparation of calibration standards and quality controls (QCs) is one of the most routine and laborious steps in bioanalysis. An alternative non-contact dispenser using low picoliter digitized dispensing technology is evaluated for its application in non-stepwise preparation of calibration curve and QCs in bioanalysis. Fluorescein was initially used to assess the accuracy and precision of dispense volumes with fluorescent measurement. Various concentrations of MX-1, an in-house proprietary small molecule compound, in neat solution and in dog plasma were prepared manually with calibrated pipettors and digitally by the digital dispenser. The plasma samples were extracted by protein precipitation. The resultant extracted samples and neat solutions of MX-1 were analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using an electrospray ionization (ESI) source in positive ion mode with selected reaction monitoring (SRM) of the mass transitions. In the three-day precision and accuracy assessment of dispensing volumes between 13 pL to 411.2 nL, the intra-day precision and accuracy ranged from 1.4% to 10.3% and -12.7% to 12.8%, respectively. The inter-day precision and accuracy ranged from 3.5% to 7.8% and -6.6% to 10.4%, respectively. For real analysis of in vivo study samples, all 49 samples analyzed showed a less than 5% difference between calibrations with digital and manual curve preparations. The resultant pharmacokinetic (PK) parameters were physiologically comparable as well. Using the digitized picoliter dispensing technology, high-speed automated precise and accurate dispense of a wide range of volumes can be achieved and tests for bioanalytical standards and QC preparations passed the stringent criteria set forth for regulated bioanalysis using LC/MS/MS-based technology. The digital dispenser has been found to be a useful tool in drug discovery for automatically preparing standards and QCs in seconds with low consumption of stock

  14. Allosteric networks in thrombin distinguish procoagulant vs. anticoagulant activities. (United States)

    Gasper, Paul M; Fuglestad, Brian; Komives, Elizabeth A; Markwick, Phineus R L; McCammon, J Andrew


    The serine protease α-thrombin is a dual-action protein that mediates the blood-clotting cascade. Thrombin alone is a procoagulant, cleaving fibrinogen to make the fibrin clot, but the thrombin-thrombomodulin (TM) complex initiates the anticoagulant pathway by cleaving protein C. A TM fragment consisting of only the fifth and sixth EGF-like domains (TM56) is sufficient to bind thrombin, but the presence of the fourth EGF-like domain (TM456) is critical to induce the anticoagulant activity of thrombin. Crystallography of the thrombin-TM456 complex revealed no significant structural changes in thrombin, suggesting that TM4 may only provide a scaffold for optimal alignment of protein C for its cleavage by thrombin. However, a variety of experimental data have suggested that the presence of TM4 may affect the dynamic properties of the active site loops. In the present work, we have used both conventional and accelerated molecular dynamics simulation to study the structural dynamic properties of thrombin, thrombin:TM56, and thrombin:TM456 across a broad range of time scales. Two distinct yet interrelated allosteric pathways are identified that mediate both the pro- and anticoagulant activities of thrombin. One allosteric pathway, which is present in both thrombin:TM56 and thrombin:TM456, directly links the TM5 domain to the thrombin active site. The other allosteric pathway, which is only present on slow time scales in the presence of the TM4 domain, involves an extended network of correlated motions linking the TM4 and TM5 domains and the active site loops of thrombin.

  15. Laboratory Monitoring of Parenteral Direct Thrombin Inhibitors. (United States)

    Van Cott, Elizabeth M; Roberts, A Joshua; Dager, William E


    Argatroban and bivalirudin are parenteral direct inhibitors of the activity of thrombin, but, unlike heparin, can inhibit both soluble as well as clot-bound thrombin. These agents do not require antithrombin as a cofactor for activity. The parenteral direct thrombin inhibitors (DTIs) can be used in a variety of settings, including heparin-induced thrombocytopenia (HIT) or an allergy to heparin, and patients requiring anticoagulation for an invasive cardiovascular intervention. Both agents have a relatively short half-life in patients without organ system failure and are typically administered by continuous infusion. Argatroban is primarily eliminated by the liver, while bivalirudin is removed by a combination of proteolytic cleavage by thrombin and renal clearance mechanisms. Several laboratory tests are available for monitoring the anticoagulant effects of the DTIs: the activated partial thromboplastin time (aPTT) and the activated clotting time (ACT) are the most commonly used assays, but on occasion, the thrombin time may be useful. Other coagulation assays such as the dilute thrombin time (dTT), chromogenic anti-IIa assays, and the ecarin clotting time (ECT) can be used. The intensity of anticoagulation with DTIs depends on the indication for use. For patients with HIT, the target aPTT is 1.5 to 3.0 and 1.5 to 2.5 times the patient's baseline value for argatroban and bivalirudin, respectively. DTI anticoagulation used during percutaneous coronary intervention can be measured using ACT. Both DTIs may cause an elevation in the international normalized ratio depending on their plasma concentration. This article will review the use of parenteral DTIs and related laboratory assays for assessing the anticoagulant effect of these drugs. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

  16. Sphingolipids as bioactive regulators of thrombin generation. (United States)

    Deguchi, Hiroshi; Yegneswaran, Subramanian; Griffin, John H


    Sphingolipids contribute to modulation of two opposing cell processes, cell growth and apoptotic cell death; ceramide and sphingosine promote the latter and sphingosine-1-phosphate triggers the former. Thrombin, a pro-inflammatory protease that is regulated by the blood coagulation cascade, exerts similar effects depending on cell type. Here we report a new mechanism for cross-talk between sphingolipid metabolism and thrombin generation. Sphingosine and sphinganine, but not ceramide or sphingosine-1-phosphate, down-regulated thrombin generation on platelet surfaces (IC(50) = 2.4 and 1.4 microm for sphingosine and sphinganine, respectively) as well as in whole plasma clotting assays. Thrombin generation was also inhibited by glucosylsphingosine, lysosphingomyelin, phytosphingosine, and primary alkylamines with >10 carbons. Acylation of the amino group ablated anticoagulant activities. Factor Va was required for the anticoagulant property of sphingosine because prothrombin activation was inhibited by sphingosine, sphinganine, and stearylamine in the presence but not in the absence of factor Va. Sphingosine did not inhibit thrombin generation when Gla-domainless factor Xa was used in prothrombinase assays, whereas sphingosine inhibited activation of Gla-domainless prothrombin by factor Xa/factor Va in the absence of phospholipids (IC(50) = 0.49 microm). Fluorescence spectroscopy studies showed that sphingosine binds to fluorescein-labeled factor Xa and that this interaction required the Gla domain. These results imply that sphingosine disrupts interactions between factor Va and the Gla domain of factor Xa in the prothrombinase complex. Thus, certain sphingolipids may be bioactive lipid mediators of thrombin generation such that certain sphingolipid metabolites may modulate proteases that affect cell growth and death, blood coagulation, and inflammation.

  17. Mechanistic Modeling of the Effects of Acidosis on Thrombin Generation. (United States)

    Mitrophanov, Alexander Y; Rosendaal, Frits R; Reifman, Jaques


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

  18. Discovery of thrombin activatable fibrinolysis inhibitor (TAFI)

    NARCIS (Netherlands)

    Bertina, R.M.; Tilburg, N.H. van; Haverkate, F.; Bouma, B.N.; Borne, P.A.K. von dem; Meijers, J.C.M.; Campbell, W.; Eaton, D.; Hendriks, D.F.; Willemse, J.L.


    CAS: blood clotting factor 11, 9013-55-2; thrombin, 9002-04-4; tissue plasminogen activator, 105913-11-9; protein C, 60202-16-6; Carboxypeptidase U,; Protein C; Tissue Plasminogen Activator,

  19. Endogenous thrombin potential in polycystic ovary syndrome

    DEFF Research Database (Denmark)

    Aziz, Mubeena; Sidelmann, Johannes J; Wissing, Marie Louise Muff


    OBJECTIVES: The objective of this study is to investigate plasma endogenous thrombin generation in four different phenotypes of polycystic ovary syndrome (PCOS) defined by Body Mass Index (BMI) and insulin resistance (IR). PCOS is diagnosed according to the Rotterdam criteria. DESIGN: Multicenter...

  20. Decreased prothrombin conversion and reduced thrombin inactivation explain rebalanced thrombin generation in liver cirrhosis.

    Directory of Open Access Journals (Sweden)

    Romy M W Kremers

    Full Text Available Impaired coagulation factor synthesis in cirrhosis causes a reduction of most pro- and anticoagulant factors. Cirrhosis patients show no clear bleeding or thrombotic phenotype, although they are at risk for both types of hemostatic event. Thrombin generation (TG is a global coagulation test and its outcome depends on underlying pro- and anticoagulant processes (prothrombin conversion and thrombin inactivation. We quantified the prothrombin conversion and thrombin inactivation during TG in 30 healthy subjects and 52 Child-Pugh (CP- A, 15 CP-B and 6 CP-C cirrhosis patients to test the hypothesis that coagulation is rebalanced in liver cirrhosis patients. Both prothrombin conversion and thrombin inactivation are reduced in cirrhosis patients. The effect on pro- and anticoagulant processes partially cancel each other out and as a result TG is comparable at 5 pM tissue factor between healthy subjects and patients. This supports the hypothesis of rebalanced hemostasis, as TG in cirrhosis patients remains within the normal range, despite large changes in prothrombin conversion and thrombin inactivation. Nevertheless, in silico analysis shows that normalization of either prothrombin conversion or thrombin inactivation to physiological levels, by for example the administration of prothrombin complex concentrates would cause an elevation of TG, whereas the normalization of both simultaneously maintains a balanced TG. Therefore, cirrhosis patients might require adapted hemostatic treatment.

  1. Decreased prothrombin conversion and reduced thrombin inactivation explain rebalanced thrombin generation in liver cirrhosis. (United States)

    Kremers, Romy M W; Kleinegris, Marie-Claire; Ninivaggi, Marisa; de Laat, Bas; Ten Cate, Hugo; Koek, Ger H; Wagenvoord, Rob J; Hemker, H Coenraad


    Impaired coagulation factor synthesis in cirrhosis causes a reduction of most pro- and anticoagulant factors. Cirrhosis patients show no clear bleeding or thrombotic phenotype, although they are at risk for both types of hemostatic event. Thrombin generation (TG) is a global coagulation test and its outcome depends on underlying pro- and anticoagulant processes (prothrombin conversion and thrombin inactivation). We quantified the prothrombin conversion and thrombin inactivation during TG in 30 healthy subjects and 52 Child-Pugh (CP-) A, 15 CP-B and 6 CP-C cirrhosis patients to test the hypothesis that coagulation is rebalanced in liver cirrhosis patients. Both prothrombin conversion and thrombin inactivation are reduced in cirrhosis patients. The effect on pro- and anticoagulant processes partially cancel each other out and as a result TG is comparable at 5 pM tissue factor between healthy subjects and patients. This supports the hypothesis of rebalanced hemostasis, as TG in cirrhosis patients remains within the normal range, despite large changes in prothrombin conversion and thrombin inactivation. Nevertheless, in silico analysis shows that normalization of either prothrombin conversion or thrombin inactivation to physiological levels, by for example the administration of prothrombin complex concentrates would cause an elevation of TG, whereas the normalization of both simultaneously maintains a balanced TG. Therefore, cirrhosis patients might require adapted hemostatic treatment.

  2. Effects of thromboprophylactic doses of apixaban and rivaroxaban on coagulation and thrombin generation in association with total hip replacement. (United States)

    Helin, Tuukka A; Virtanen, Lauri; Manninen, Mikko; Leskinen, Jarkko; Leppilahti, Juhana; Joutsi-Korhonen, Lotta; Lassila, Riitta


    Factor Xa inhibitors (FXaI) apixaban and rivaroxaban are used for thromboprophylaxis after major elective orthopaedic surgery. Because few patient sample studies exist, we postoperatively assessed patients undergoing unilateral total hip arthroplasty, including 22 treated with apixaban (2.5 mg BID) and 20 treated with rivaroxaban (10 mg OD). We collected blood samples before and 3 h after drug intake at 4 time points, preoperatively, as well as on day 1, week 1 (day 2-8) and day 28 post-operation. APTT and PT were immediately analysed. Calibrated anti-FXa activity, Russel's Viper Venom Time (RVVT) and thrombin generation (TG; Calibrated Automated Thrombogram®) captured the effects of FXaI on coagulation and TG. APTT and PT remained within the reference interval throughout, and did not correlate with FXaI levels (PT R2 = 0.44, APTT R2 = 0.07). Mean apixaban concentration at the peak varied by eightfold (19-153 ng/mL), but rivaroxaban only by 1.5-fold (111-183 ng/mL). Rivaroxaban, but not apixaban prolonged RVVT at peak levels. Both FXaIs had a prolonged lag time of TG (p < 0.001). Rivaroxaban decreased ETP peak at all time points and reached a minimum at day 28 (540 nM/min at rivaroxaban 184 ng/mL, p < 0.001), while rivaroxaban trough levels were low and ETP values normal. However, with apixaban, after an initial decrease, ETP did not differ between peak and trough levels until decreasing on day 28 at peak (990 nM/min at apixaban 112 ng/mL, p = 0.005). In conclusion, due to different dosing and pharmacology rivaroxaban and apixaban distinctly inhibited TG under postoperative conditions.

  3. Thrombin Ultrasensitive Detection Based on Chiral Supramolecular Assembly Signal-Amplified Strategy Induced by Thrombin-Binding Aptamer. (United States)

    Shen, Gang; Zhang, Hong; Yang, Chunrong; Yang, Qianfan; Tang, Yalin


    Thrombin plays a critical role in hemostasis and hemolysis. It is of high importance to develop a system toward thrombin detection with high sensitivity and high selectivity for both research and clinical diagnosis applications. In this paper, we developed a thrombin detection assay by taking advantage of the novel signal amplified strategy based on the chiral supramolecular assembly in physiological K(+) background. This assay could detect thrombin as low concentration as about 2 pM and provided a highly specific selectivity among several common interferences. Furthermore, the assay can discriminate thrombin from other nonspecific analogous proteins with high selectivity and can be used to detect thrombin in diluted real human serum samples, which suggested its great potential for rapid detection of thrombin in the clinic.

  4. The use of thrombin in the radiology department.

    LENUS (Irish Health Repository)

    Ward, E


    Thrombin is a naturally occurring coagulation protein that converts soluble fibrinogen into insoluble fibrin and plays a vital role in the coagulation cascade and in turn haemostasis. Thrombin also promotes platelet activation. In the last few years, there has been a rapid increase in the use of thrombin by radiologists in a variety of clinical circumstances. It is best known for its use in the treatment of pseudoaneurysms following angiography. However, there are now a variety of cases in the literature describing the treatment of traumatic, inflammatory and infected aneurysms with thrombin in a variety of locations within the human body. There have even been recent reports describing the use of thrombin in conventional aneurysms as well as ruptured aneurysms. Its use has also been described in the treatment of endoleaks (type II) following aneurysm repair. In nearly all of these cases, treatment with thrombin requires imaging guidance. Recently, thrombin has also been used as a topical treatment post-percutaneous intervention to reduce or stop bleeding. Most radiologists have only a limited knowledge of the pharmacodynamics of thrombin, its wide range of utilisation and its limitations. Apart from a few case reports and case series, there is little in the radiological literature encompassing the wide range of applications that thrombin may have in the radiology department. In this review article, we comprehensively describe the role and pathophysiology of thrombin, describing with examples many of its potential uses. Techniques of usage as well as pitfalls and limitations are also described.


    Brummel-Ziedins, Kathleen E.; Whelihan, Matthew F.; Gissel, Matthew; Mann, Kenneth G.; Rivard, Georges E.


    Introduction Hemophilia A displays phenotypic heterogeneity with respect to clinical severity. Aim To determine if tissue factor (TF)-initiated thrombin generation profiles in whole blood in the presence of corn trypsin inhibitor (CTI) are predictive of bleeding risk in hemophilia A. Methods We studied factor(F) VIII deficient individuals (11 mild, 4 moderate and 12 severe) with a well-characterized five-year bleeding history that included hemarthrosis, soft tissue hematoma and annual FVIII concentrate usage. This clinical information was used to generate a bleeding score. The bleeding scores (range 0–32) were separated into three groups (bleeding score groupings: 0, 0 and ≤9.6, >9.6), with the higher bleeding tendency having a higher score. Whole blood collected by phlebotomy and contact pathway suppressed by 100μg/mL CTI was stimulated to react by the addition of 5pM TF. Reactions were quenched at 20min by inhibitors. Thrombin generation, determined by ELISA for thrombin – antithrombin was evaluated in terms of clot time (CT), maximum level (MaxL) and maximum rate (MaxR) and compared to the bleeding score. Results Data are shown as the mean±SD. MaxL was significantly different (phemophilia A. PMID:19563500

  6. Effect of Electronic Polarization to Human α-Thrombin (United States)

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


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

  7. Topical thrombin preparations and their use in cardiac surgery

    Directory of Open Access Journals (Sweden)

    Brianne L Dunn


    Full Text Available Brianne L Dunn1, Walter E Uber1, John S Ikonomidis21Department of Pharmacy Services and 2Division of Cardiothoracic Surgery, Medical University of South Carolina, Charleston, South Carolina, USAAbstract: Coagulopathic bleeding may lead to increased morbidity and mortality after cardiac surgery. Topical bovine thrombin has been used to promote hemostasis after surgical procedures for over 60 years and is used frequently as a topical hemostatic agent in cardiac surgery. Recently, use of bovine thrombin has been reported to be associated with increased risk for anaphylaxis, thrombosis, and immune-mediated coagulopathy thought secondary to the production of antifactor V and antithrombin antibodies. In patients who develop bovine thrombin-induced immune-mediated coagulopathy, clinical manifestations may range from asymptomatic alterations in coagulation tests to severe hemorrhage and death. Patients undergoing cardiac surgical procedures may be at increased risk for development of antibodies to bovine thrombin products and associated complications. This adverse immunologic profile has led to the development of alternative preparations including a human and a recombinant thrombin which have been shown to be equally efficacious to bovine thrombin and have reduced antigenicity. However, the potential benefit associated with reduced antigenicity is not truly known secondary to the lack of long-term experience with these products. Given the potentially higher margin of safety and less stringent storage concerns compared to human thrombin, recombinant thrombin may be the most reasonable approach in cardiac surgery.Keywords: bovine thrombin, human thrombin, recombinant thrombin, immune-mediated coagulopathy, topical hemostatic agents, thrombin 

  8. Identification of a thrombomodulin interaction site on thrombin-activatable fibrinolysis inhibitor that mediates accelerated activation by thrombin. (United States)

    Marar, T T; Boffa, M B


    Thrombin-activatable fibrinolysis inhibitor (TAFI) is a human plasma zymogen that provides a molecular connection between coagulation and fibrinolysis. TAFI is activated through proteolytic cleavage by thrombin, thrombin in complex with the endothelial cell cofactor thrombomodulin (TM) or plasmin. Evidence from several studies suggests that TM and TAFI make direct contact at sites remote from the activating cleavage site to facilitate acceleration of thrombin-mediated TAFI activation. The elements of TAFI structure that allow accelerated activation of thrombin by TM are incompletely defined. To identify TM interaction regions on TAFI that mediate acceleration of activation by thrombin and therefore indicate TM binding sites on TAFI. We mutated selected surface-exposed charged residues on TAFI to alanine in order to identify sites that mediate acceleration of activation by TM. The kinetics of activation of the mutants by thrombin in the presence or absence of TM, as well as their thermal stabilities and antifibrinolytic potentials, were determined. TAFI variants R15A, E28A, K59A, D75A/E77A/D78A, E99A and E106A all exhibited moderately reduced catalytic efficiencies of activation by thrombin-TM. TAFI variants R377A and, particularly, R12A and R12A/R15A exhibited severely reduced activation by thrombin-TM that was not explained by differences in activation by thrombin alone. We have identified R12 as a critical residue for the activation of TAFI by thrombin-TM, extending a previous report that identified a role for this residue. R12 is likely to directly bind to TM while another key residue, R377, may affect the thrombin-TAFI interaction specifically in the presence of TM. © 2016 International Society on Thrombosis and Haemostasis.

  9. Stimulation of thrombin- and plasmin-mediated activation of thrombin-activatable fibrinolysis inhibitor by anionic molecules. (United States)

    Plug, Tom; Meijers, Joost C M


    Thrombin-activatable fibrinolysis inhibitor (TAFI) is a proenzyme that, once activated, attenuates fibrinolysis by removing C-terminal lysine residues from partially degraded fibrin. TAFI can be activated by thrombin or plasmin via a cleavage at Arg92 that removes the activation peptide from the enzyme, TAFIa. Thrombomodulin enhances thrombin-mediated TAFI activation and glycosaminoglycans enhance plasmin-mediated TAFI activation. The aim of this study was to investigate whether there are other anionic molecules that function as a cofactor for thrombin- or plasmin-mediated TAFI activation. TAFI activation by thrombin or plasmin was studied in the presence of physiological anionic molecules (polyphosphate, heparin, hyaluronan, DNA and dermatan sulfate) and the non-physiological sodium dodecyl sulfate (SDS). Additionally, the effect of these molecules on TAFIa stability and on thrombin-mediated protein C activation was determined. Unfractioned heparin, calcium-saturated polyphosphate with an average chain length of 100 monomers (Ca-PolyP100) and SDS significantly enhanced TAFI activation by thrombin and plasmin. Dermatan sulfate and polyphosphates with sodium as counter ion (Na-PolyP700, Na-PolyP100 and Na-PolyP70) enhanced plasmin-mediated but not thrombin-mediated TAFI activation. Additionally, unfractioned heparin, Ca-PolyP100 and SDS enhanced thrombin-mediated protein C activation. The different nature of anionic molecules capable of enhancing TAFI and protein C activation suggests a general mechanism. Several anionic molecules function as (potent) cofactors for thrombin- and plasmin-mediated TAFI activation and thrombin-mediated protein C activation. This may imply that thrombin and plasmin activity is regulated in the vasculature by more cofactors than currently appreciated. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Statins but not aspirin reduce thrombotic risk assessed by thrombin generation in diabetic patients without cardiovascular events: the RATIONAL trial.

    Directory of Open Access Journals (Sweden)

    Alejandro Macchia

    Full Text Available The systematic use of aspirin and statins in patients with diabetes and no previous cardiovascular events is controversial. We sought to assess the effects of aspirin and statins on the thrombotic risk assessed by thrombin generation (TG among patients with type II diabetes mellitus and no previous cardiovascular events.Prospective, randomized, open, blinded to events evaluation, controlled, 2×2 factorial clinical trial including 30 patients randomly allocated to aspirin 100 mg/d, atorvastatin 40 mg/d, both or none. Outcome measurements included changes in TG levels after treatment (8 to 10 weeks, assessed by a calibrated automated thrombogram. At baseline all groups had similar clinical and biochemical profiles, including TG levels. There was no interaction between aspirin and atorvastatin. Atorvastatin significantly reduced TG measured as peak TG with saline (85.09±55.34 nmol vs 153.26±75.55 nmol for atorvastatin and control groups, respectively; p = 0.018. On the other hand, aspirin had no effect on TG (121.51±81.83 nmol vs 116.85±67.66 nmol, for aspirin and control groups, respectively; p = 0.716. The effects of treatments on measurements of TG using other agonists were consistent.While waiting for data from ongoing large clinical randomized trials to definitively outline the role of aspirin in primary prevention, our study shows that among diabetic patients without previous vascular events, statins but not aspirin reduce thrombotic risk assessed by NCT00793754.

  11. Dabigatran abrogates brain endothelial cell permeability in response to thrombin. (United States)

    Hawkins, Brian Thomas; Gu, Yu-Huan; Izawa, Yoshikane; del Zoppo, Gregory John


    Atrial fibrillation (AF) increases the risk and severity of thromboembolic stroke. Generally, antithrombotic agents increase the hemorrhagic risk of thromboembolic stroke. However, significant reductions in thromboembolism and intracerebral hemorrhage have been shown with the antithrombin dabigatran compared with warfarin. As thrombin has been implicated in microvessel injury during cerebral ischemia, we hypothesized that dabigatran decreases the risk of intracerebral hemorrhage by direct inhibition of the thrombin-mediated increase in cerebral endothelial cell permeability. Primary murine brain endothelial cells (mBECs) were exposed to murine thrombin before measuring permeability to 4-kDa fluorescein isothiocyanate-dextran. Thrombin increased mBEC permeability in a concentration-dependent manner, without significant endothelial cell death. Pretreatment of mBECs with dabigatran completely abrogated the effect of thrombin on permeability. Neither the expressions of the endothelial cell β1-integrins nor the tight junction protein claudin-5 were affected by thrombin exposure. Oxygen-glucose deprivation (OGD) also increased permeability; this effect was abrogated by treatment with dabigatran, as was the additive effect of thrombin and OGD on permeability. Taken together, these results indicate that dabigatran could contribute to a lower risk of intracerebral hemorrhage during embolism-associated ischemia from AF by protection of the microvessel permeability barrier from local thrombin challenge.

  12. Thrombin-receptor antagonist vorapaxar in acute coronary syndromes

    DEFF Research Database (Denmark)

    Tricoci, Pierluigi; Huang, Zhen; Held, Claes


    Vorapaxar is a new oral protease-activated-receptor 1 (PAR-1) antagonist that inhibits thrombin-induced platelet activation.......Vorapaxar is a new oral protease-activated-receptor 1 (PAR-1) antagonist that inhibits thrombin-induced platelet activation....

  13. Acidosis, magnesium and acetylsalicylic acid: Effects on thrombin (United States)

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


    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.

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

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


    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 μM. 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

  15. 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)

    Leflem Lena


    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 μM. 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

  16. Bidirectional functions of thrombin on fibrinolysis: Evidence of thrombin-dependent enhancement of fibrinolysis provided by spontaneous plasma clot lysis. (United States)

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


    Besides procoagulant activity, thrombin exhibits anticoagulant and profibrinolytic activities. We demonstrated that the euglobulin clot lysis time (ECLT) was shortened by endogenously generated thrombin as a result of the inactivation of plasminogen activator inhibitor type 1 (PAI-1). In contrast, thrombin suppressed fibrinolytic activity through the activation of thrombin activatable fibrinolysis inhibitor (TAFI). Here, using three different clot lysis assays of the ECLT, the tissue plasminogen activator supplemented plasma clot lysis time (tPA-PCLT) and the spontaneous plasma clot lysis time (s-PCLT), we analyzed how the coagulation process modifies fibrinolysis. The ECLT was shortened by exogenously supplemented thrombin in a dose-dependent manner in the absence of calcium ion (Ca(++)), whereas this shortening was not observed in the presence of Ca(++) where endogenous prothrombin was effectively activated to thrombin. This shortening was also not observed for the tPA-PCLT, in which tPA is supplemented in excess and PAI-1 activity is mostly lost. On the contrary, thrombin dose-dependently prolonged the tPA-PCLT, which was mostly abolished by inhibitors of carboxypeptidase and activated FXIII, suggesting that the prolongation is TAFI- and Factor XIII-dependent. The s-PCLT was shortened when thrombin generation was boosted by supplementing tissue factor and phosphatidylserine together with Ca(++), which was more apparent in the presence of inhibitors of activated FXIII and activated TAFI. Thus, thrombin appeared to express its enhancing effect on fibrinolysis even in plasma, in addition to its inhibiting effect. These bidirectional functions of thrombin on fibrinolysis seem to take place on demand under different environments to maintain adequate vascular blood flow. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Label-free impedimetric biosensor for thrombin using the thrombin-binding aptamer as receptor (United States)

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


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

  18. An experimental model to study isolated effects of thrombin in vivo.

    NARCIS (Netherlands)

    Siller-Matula, J.M.; Bayer, G.; Bergmeister, H.; Quehenberger, P.; Petzelbauer, P.; Friedl, P.H.A.; Mesteri, I.; Jilma, B.


    BACKGROUND: In addition to a recognized role in the coagulation cascade and haemostasis, thrombin is known to have multiple functions. We hypothesized that protracted intravenous infusion of thrombin at steady state will allow to study isolated thrombin effects in vivo. METHODS: Thrombin

  19. Thrombin Preconditioning in Surgical Brain Injury in Rats. (United States)

    Benggon, Michael; Chen, Hank; Applegate, Richard L; Zhang, John


    The surgical brain injury model replicates neurosurgical brain parenchymal damage. Postsurgical brain edema correlates with postoperative neurological dysfunction. Intranasal administration is a proven method of delivering therapies to brain tissue. Thrombin preconditioning decreased brain edema and improved neurological outcomes in models of ischemic brain injury. We hypothesized thrombin preconditioning in surgical brain injury may improve postoperative brain edema and neurological outcomes. Adult male Sprague-Dawley rats (n = 78) weighing 285-355 g were randomly assigned to sham or pre-injury treatment: one-time pretreatment 1 day prior, one-time pretreatment 5 days prior, and daily preconditioning for 5 days prior. Treatment arms were divided into vehicle or thrombin therapies, and subdivided into intranasal (thrombin 5 units/50 μL 0.9 % saline) or intracerebral ventricular (thrombin 0.1 unit/10 μL 0.9 % saline) administration. Blinded observers performed neurological testing 24 h after brain injury followed immediately by measurement of brain water content. There was a significant difference in ipsilateral brain water content and neurological outcomes between all treatment groups and the sham group. However, there was no change in brain water content or neurological outcomes between thrombin- and vehicle-treated animals. Thrombin preconditioning did not significantly improve brain edema or neurological function in surgical brain injury in rats.


    Energy Technology Data Exchange (ETDEWEB)

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


    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.

  1. Label-free aptamer biosensor for selective detection of thrombin

    Energy Technology Data Exchange (ETDEWEB)

    Na, Weidan; Liu, Xiaotong; Wang, Lei; Su, Xingguang, E-mail:


    We fabricated a novel fluorescence biosensor for the selective detection of thrombin by using bovine serum albumin-capped CdS quantum dots (BSA-CdS QDs). Two kinds of designed DNA (DNA1 and DNA2) could bind to CdS QDs through the electrostatic interaction between DNA and Cd{sup 2+} on the surface of CdS QDs. The obtained DNA/BSA-CdS QDs kept stable in the solution with the fluorescence intensity obviously enhanced. Hairpin structure of DNA1contained two domains, one is the aptamer sequence of thrombin and the other is the complementary sequence of DNA2. When thrombin was added, it would bind to DNA1 and induce the hairpin structure of DNA1 changed into G-quadplex structure. Meanwhile, DNA2 would transfer from the surface of CdS QDs to DNA1 via hybridization, which resulted in the removal of DNA1 and DNA2 from the surface of CdS QDs, and led to the fluorescence intensity of CdS QDs reduced. Thus, the determination of thrombin could be achieved by monitoring the change of the fluorescence intensity of CdS QDs. The present method is simple and fast, and exhibits good selectivity for thrombin over other proteins. We have successfully detected thrombin in human serum samples with satisfactory results. - Highlights: • A novel strategy for the detection of thrombin was established based on BSA-CdS QDs. • DNA could serve as the co-ligands to stabilize CdS QDs and enhance the fluorescence intensity. • Thrombin could change the structure of DNA1 and quench the fluorescence of CdS QDs. • Thrombin in real sample was detected with satisfactory results.

  2. Defining the Boundaries of Normal Thrombin Generation: Investigations into Hemostasis (United States)

    Danforth, Christopher M.; Orfeo, Thomas; Everse, Stephen J.; Mann, Kenneth G.; Brummel-Ziedins, Kathleen E.


    In terms of its soluble precursors, the coagulation proteome varies quantitatively among apparently healthy individuals. The significance of this variability remains obscure, in part because it is the backdrop against which the hemostatic consequences of more dramatic composition differences are studied. In this study we have defined the consequences of normal range variation of components of the coagulation proteome by using a mechanism-based computational approach that translates coagulation factor concentration data into a representation of an individual's thrombin generation potential. A novel graphical method is used to integrate standard measures that characterize thrombin generation in both empirical and computational models (e.g max rate, max level, total thrombin, time to 2 nM thrombin (“clot time”)) to visualize how normal range variation in coagulation factors results in unique thrombin generation phenotypes. Unique ensembles of the 8 coagulation factors encompassing the limits of normal range variation were used as initial conditions for the computational modeling, each ensemble representing “an individual” in a theoretical healthy population. These “individuals” with unremarkable proteome composition was then compared to actual normal and “abnormal” individuals, i.e. factor ensembles measured in apparently healthy individuals, actual coagulopathic individuals or artificially constructed factor ensembles representing individuals with specific factor deficiencies. A sensitivity analysis was performed to rank either individual factors or all possible pairs of factors in terms of their contribution to the overall distribution of thrombin generation phenotypes. Key findings of these analyses include: normal range variation of coagulation factors yields thrombin generation phenotypes indistinguishable from individuals with some, but not all, coagulopathies examined; coordinate variation of certain pairs of factors within their normal ranges

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


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


    Esterolytically inactive diisopropyl fluorophosphate-conjugated thrombin (DIP-alpha-thrombin) stimulated 3H-thymidine incorporation and proliferation of growth-arrested vascular smooth muscle cells (SMCs), similar to native alpha-thrombin. Half-maximal mitogenic response of SMCs was obtained at 1 nM thrombin and was specifically blocked by the leech-derived, high-affinity thrombin inhibitor, hirudin. Native thrombin and a variety of thrombin species that were chemically modified to alter thro...

  4. Increased thrombin generation in women with polycystic ovary syndrome

    DEFF Research Database (Denmark)

    Glintborg, Dorte; Sidelmann, Johannes Jakobsen; Lambaa Altinok, Magda


    randomized to 12 months treatment with metformin, metformin + OC or OC alone. C-reactive protein (CRP), fibrinogen, total cholesterol, trunk fat mass, body mass index, estradiol, testosterone, sex hormone binding globulin (SHBG) as well as TG measures, i.e. the lag time for formation of thrombin......, the endogenous thrombin potential (ETP), peak thrombin concentration (peak) and time to peak were determined at baseline and after 12 months of treatment. Results. CRP and total testosterone were significantly higher and SHBG significantly lower in PCOS women than in controls (P=0.012, P

  5. Cytochalasins inhibit arachidonic acid metabolism in thrombin-stimulated platelets.


    Siess, W; Lapetina, E G; Cuatrecasas, P


    Low concentrations (0.5-1 microM) of cytochalasins inhibit the thrombin-stimulated polymerization of monomeric actin to filamentous actin in platelets. Similar concentrations of cytochalasin B inhibit the formation and metabolism of arachidonic acid in horse platelets stimulated by low concentrations of thrombin (0.1-0.5 unit/ml). However, the release of serotonin is not inhibited by cytochalasin B. Cytochalasins B and D (0.5-1 microM) markedly reduce, in thrombin-stimulated human or horse pl...

  6. The N-terminal thrombin receptor fragment SFLLRN, but not catalytically inactive thrombin-derived agonists, activate U937 human monocytic cells: evidence for receptor hydrolysis in thrombin-dependent signalling.


    Joseph, S.; MacDermot, J.


    It has previously been reported that murine macrophages can respond chemotactically and mitogenically to the serine proteinase thrombin. There is a similar response in these macrophages to catalytically inactivated thrombin or to peptide fragments of the thrombin B-chain [Bar-Shavit, Kahn, Mann and Wilner (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 976-980]. However, the existence of a non-proteolytic mechanism of thrombin receptor activation in mononuclear cells was not evident in the present s...

  7. Purification and Characterization of Human Thrombin Activatable Fibrinolysis Inhibitor (TAFI)

    DEFF Research Database (Denmark)

    Christensen, Trine; Skottrup, Peter Durand; Valnickova, Zuzana

    Thrombin Activatable Fibrinolysis inhibitor (TAFI) is a basic carboxypeptidase, circulating in plasma as an enzymatic inactive precursor. TAFI shares ~40% overall sequence identity with pancreas Carboxypeptidase B (PCPB) with the activation peptide being less conserved. Following activation of TA...

  8. Endovascular Thrombin Injection for a Pulmonary Artery Pseudoaneurysm: Case Report

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Jin Ho; Shin, Ji Hoon; Yoon, Hyun Ki [Dept. of Radiology and Research Institute of Radiology, Asan Medical Center, Ulsan University College of Medicine, Seoul (Korea, Republic of)


    Massive hemoptysis caused by pulmonary artery pseudoaneurysms is uncommon, and endovascular treatment such as coil embolization is the first choice for treating pulmonary artery pseudoaneurysms. Various embolic agents could be used according to the angiographic findings, yet embolization with thrombin injection is very rare. Herein, we describe a case of a pulmonary artery pseudoaneurysm that was successfully treated by endovascular thrombin injection using a microcatheter because of the difficulty in performing a coil embolization due to a short feeding artery.

  9. Thrombin Generating Capacity and Phenotypic Association in ABO Blood Groups (United States)

    Hindawi, Salwa; Hemker, H. Coenraad; de Laat, H. Bas; Huskens, Dana; Al Dieri, Raed


    Individuals with blood group O have a higher bleeding risk than non-O blood groups. This could be explained by the lower levels of FVIII and von Willebrand Factor (VWF) levels in O individuals. We investigated the relationship between blood groups, thrombin generation (TG), prothrombin activation and thrombin inactivation. Plasma levels of VWF, FVIII, antithrombin, fibrinogen, prothrombin and α2Macroglobulin (α2M) levels were determined. TG was measured in platelet rich (PRP) and platelet poor plasma (PPP) of 217 healthy donors and prothrombin conversion and thrombin inactivation were calculated. VWF and FVIII levels were lower (75% and 78%) and α2M levels were higher (125%) in the O group. TG is 10% lower in the O group in PPP and PRP. Less prothrombin was converted in the O group (86%) and the thrombin decay capacity was lower as well. In the O group, α2M plays a significantly larger role in the inhibition of thrombin (126%). In conclusion, TG is lower in the O group due to lower prothrombin conversion, and a larger contribution of α2M to thrombin inactivation. The former is unrelated to platelet function because it is similar in PRP and PPP, but can be explained by the lower levels of FVIII. PMID:26509437

  10. Thrombomodulin binding selects the catalytically active form of thrombin (United States)

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


    Human α-thrombin is a serine protease with dual functions. Thrombin acts as a procoagulant, cleaving fibrinogen to make the fibrin clot, but when bound to thrombomodulin (TM), it acts as an anticoagulant, cleaving protein C. A minimal TM fragment consisting of the 4th, 5th, and most of the 6th EGF-like domain (TM456m) has been prepared that has much improved solubility, thrombin-binding capacity, and anticoagulant activity over previous TM456 constructs. In the present work we compare backbone amide exchange of human α-thrombin in three states: apo, PPACK-bound, and TM456m-bound. Beyond causing decreased amide exchange at their binding sites, TM and PPACK both cause decreased amide exchange in regions more distant from the active site, within the γ-loop and the adjacent N-terminus of the heavy chain. The decreased amide exchange in the N-terminus of the heavy chain is consistent with the historic model of activation of serine proteases, which involves insertion of this region into the β-barrel promoting the correct conformation of the catalytic residues. Contrary to crystal structures of thrombin, HDXMS results suggest that the conformation of apo-thrombin does not yet have the N-terminus of the heavy chain properly inserted for optimal catalytic activity, and that binding of TM allosterically promotes the catalytically active conformation. PMID:26468766

  11. Thrombomodulin Binding Selects the Catalytically Active Form of Thrombin. (United States)

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


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

  12. Thrombin-induced apoptosis in neurons through activation of c-Jun-N-terminal kinase. (United States)

    Bao, Lei; Zu, Jie; He, Qianqian; Zhao, Hui; Zhou, Su; Ye, Xinchun; Yang, Xinxin; Zan, Kun; Zhang, Zuohui; Shi, Hongjuan; Cui, Guiyun


    Studies have shown that thrombin activation played a central role in cell injuries associated with intracerebral hemorrhage (ICH). Here, our study investigated the cytotoxicity of thrombin on neurons, and determined the involvement of JNK pathways in thrombin-induced neuronal apoptosis. Primary cultured neurons were treated with different doses of thrombin. Some neurons were given either SP600125 or vehicle. LDH release assay and flow cytometry were used to measure neuronal apoptosis caused by thrombin. The activation of JNK and capases-3 were measured by Western blot. Our results showed large doses of thrombin that increased the LDH release, the level of cleaved caspase-3 and apoptosis rate of neurons. JNK was activated by thrombin in a time-dependent manner. Administration of SP600125 protects neurons from thrombin-induced apoptosis. These data indicate that the activation of JNK is crucial for thrombin-induced neuronal apoptosis, and inhibition of JNK may be a potential therapeutic target for ICH.

  13. The dynamic structure of thrombin in solution. (United States)

    Fuglestad, Brian; Gasper, Paul M; Tonelli, Marco; McCammon, J Andrew; Markwick, Phineus R L; Komives, Elizabeth A


    The backbone dynamics of human α-thrombin inhibited at the active site serine were analyzed using R(1), R(2), and heteronuclear NOE experiments, variable temperature TROSY 2D [(1)H-(15)N] correlation spectra, and R(ex) measurements. The N-terminus of the heavy chain, which is formed upon zymogen activation and inserts into the protein core, is highly ordered, as is much of the double beta-barrel core. Some of the surface loops, by contrast, remain very dynamic with order parameters as low as 0.5 indicating significant motions on the ps-ns timescale. Regions of the protein that were thought to be dynamic in the zymogen and to become rigid upon activation, in particular the γ-loop, the 180s loop, and the Na(+) binding site have order parameters below 0.8. Significant R(ex) was observed in most of the γ-loop, in regions proximal to the light chain, and in the β-sheet core. Accelerated molecular dynamics simulations yielded a molecular ensemble consistent with measured residual dipolar couplings that revealed dynamic motions up to milliseconds. Several regions, including the light chain and two proximal loops, did not appear highly dynamic on the ps-ns timescale, but had significant motions on slower timescales. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  14. Intracellular Ascorbate Prevents Endothelial Barrier Permeabilization by Thrombin* (United States)

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


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

  15. Thrombin-Mediated Degradation of Human Cardiac Troponin T. (United States)

    Katrukha, Ivan A; Kogan, Alexander E; Vylegzhanina, Alexandra V; Serebryakova, Marina V; Koshkina, Ekaterina V; Bereznikova, Anastasia V; Katrukha, Alexey G


    Cardiac troponin T (cTnT) is an acknowledged biomarker of acute myocardial infarction (AMI) that is known to be prone to proteolytic degradation in serum. Such degradation is usually explained by the action of μ-calpain, although there could be other candidates for that role. In the current study, we explored the hypothesis that thrombin-mediated cTnT cleavage occurs as a result of the serum sample preparation. cTnT degradation was studied by using immunoblotting and mass spectrometry (MS) analysis. The comparison of cTnT isolated from AMI heparin plasma and serum samples showed that cTnT in the plasma samples was mainly present as the full-sized molecule (approximately 35 kDa), while in serum samples it was present as a 29-kDa fragment. The incubation of recombinant cTnT, or native ternary cardiac troponin complex with thrombin or in normal human serum (NHS), resulted in the formation of a 29-kDa product that was similar to that detected in AMI serum samples. No cTnT degradation was observed when thrombin or NHS was pretreated with hirudin, a specific inhibitor of thrombin, or during incubation of troponin in normal heparin plasma. When the products of thrombin-mediated cTnT proteolysis were analyzed by MS, 2 fragments consisting of amino acid residues (aar) 2-68 and 69-288 were identified, which suggests that thrombin cleaves cTnT between R68 and S69. The results of this study suggest that the 29-kDa fragment of cTnT in AMI serum samples mainly appears due to the cleavage by thrombin during serum sample preparation. © 2017 American Association for Clinical Chemistry.

  16. Surfactants attenuate gas embolism-induced thrombin production. (United States)

    Eckmann, David M; Diamond, Scott L


    There are no pharmacologic strategies to prevent embolism bubble-induced blood clot formation. The authors conducted experiments to measure thrombin production in sheared whole blood in the presence and absence of bubbles and three surface-active compounds. Blood samples were obtained from six volunteers seven times. The thrombin-specific substrate Boc-VPR-MCA was added to citrated blood diluted with HEPES-buffered saline. Experimental groups were as follows: sparging (air microbubble embolization) with surfactant present; sparging alone; surfactant alone; and neither surfactant nor sparging. The surfactants were Dow Corning Antifoam 1510US, Perftoran, and Pluronic F-127. Blood was sheared by a cone-plate viscometer at 100 and 500 s-1 for 5, 10, and 20 min at 37 degrees C, pipetted into excess stop buffer, and evaluated fluorimetrically. Mean values of fluorescence intensity +/- SDs for each group were compared using ANOVA. Differences were considered significant at P Surfactant addition without sparging did not change thrombin production (P > 0.05). Surfactants attenuated thrombin production in sparged samples 31.8-70.9% (P Surfactants added before sparging attenuate thrombin production. Surfactants may have a clinical application to attenuate gas embolism-induced clotting.

  17. Exploring potential anticoagulant drug formulations using thrombin generation test

    Directory of Open Access Journals (Sweden)

    Elena Zavyalova


    The thrombin generation test was used to assess the whole coagulation cascade in normal and factor-deficient human blood plasma. Potential therapeutic windows were estimated for coagulation factors, ranking them as targets for anticoagulant drugs. Thrombin and factor Xa have been revealed as the most promising targets, which fully agrees with the current drug development strategy. Inhibitors of factors Va and VIIa are expected to have narrow therapeutic windows. Inhibitors of factors VIIIa and IXa are expected to have a moderate anticoagulant effect. Factors XI and XII are poor targets for anticoagulant drugs. Compared with plasma that is deficient in factor II, the thrombin inhibitors bivalirudin and aptamer HD1 had increased activity. Both inhibitors were tested in deficient plasma providing a model of potential drug combination. The most promising combinations were anti-thrombin with anti-V/Va and also anti-thrombin with anti-IX/IXa. Each combination had an incremental dose-effect dependence that is promising from the standpoint of the therapeutic window.

  18. Aptamer-based fiber sensor for thrombin detection (United States)

    Coelho, Luís; Marques Martins de Almeida, José Manuel; Santos, José Luís; da Silva Jorge, Pedro Alberto; Martins, Maria Cristina L.; Viegas, Diana; Queirós, Raquel B.


    The detection of thrombin based on aptamer binding is studied using two different optical fiber-based configurations: long period gratings coated with a thin layer of titanium dioxide and surface plasmon resonance devices in optical fibers coated with a multilayer of gold and titanium dioxide. These structures are functionalized and the performance to detect thrombin in the range 10 to 100 nM is compared in transmission mode. The sensitivity to the surrounding refractive index (RI) of the plasmonic device is higher than 3100 nm RIU-1 in the RI range 1.335 to 1.355, a factor of 20 greater than the sensitivity of the coated grating. The detection of 10 nM of thrombin was accomplished with a wavelength shift of 3.5 nm and a resolution of 0.54 nM.

  19. Fractal gold modified electrode for ultrasensitive thrombin detection (United States)

    Xu, Li-Ping; Wang, Shuqi; Dong, Haifeng; Liu, Guodong; Wen, Yongqiang; Wang, Shutao; Zhang, Xueji


    We report a label-free and ultrasensitive aptasensor based on a fractal gold modified (FracAu) electrode for thrombin detection with a femtomolar detection limit. The FracAu electrode was prepared by electrodeposition of hydrogen tetrachloroaurate (HAuCl4) onto a bare indium tin oxide (ITO) electrode surface. After this process the electrode was characterized by SEM. A thiol-modified aptamer against thrombin was immobilized on the FracAu electrode through a self-assembling process. Upon thrombin binding, the interfacial electron transfer of the FracAu electrode was perturbed by the formation of an aptamer-thrombin complex. The concentration of thrombin in the sample solution was determined by measuring the change in the oxidation peak current of hydroxymethyl ferrocene (C11H12FeO) with differential pulse voltammetry (DPV). The current response (reduced peak current) had a linear relationship with the logarithm of thrombin concentrations in the range of 10-15 to 10-10 M with a detection limit of 5.7 fM. Furthermore, the as-prepared FracAu electrode exhibited high selectivity. The application of FracAu electrodes may be extended to prepare other types of biosensors, such as immunosensors, enzyme biosensors and DNA biosensors. These results show that FracAu electrodes have great promise for clinical diagnosis of disease-related biomarkers.We report a label-free and ultrasensitive aptasensor based on a fractal gold modified (FracAu) electrode for thrombin detection with a femtomolar detection limit. The FracAu electrode was prepared by electrodeposition of hydrogen tetrachloroaurate (HAuCl4) onto a bare indium tin oxide (ITO) electrode surface. After this process the electrode was characterized by SEM. A thiol-modified aptamer against thrombin was immobilized on the FracAu electrode through a self-assembling process. Upon thrombin binding, the interfacial electron transfer of the FracAu electrode was perturbed by the formation of an aptamer-thrombin complex. The

  20. Cross-calibration of interferometric SAR data

    DEFF Research Database (Denmark)

    Dall, Jørgen


    Generation of digital elevation models from interferometric synthetic aperture radar (SAR) data is a well established technique. Achieving a high geometric fidelity calls for a calibration accounting for inaccurate navigation data and system parameters as well as system imperfections. Fully...... automated calibration techniques are preferable, especially for operational mapping. The author presents one such technique, called cross-calibration. Though developed for single-pass interferometry, it may be applicable to multi-pass interferometry, too. Cross-calibration requires stability during mapping...

  1. New synthetic thrombin inhibitors: molecular design and experimental verification.

    Directory of Open Access Journals (Sweden)

    Elena I Sinauridze

    Full Text Available BACKGROUND: The development of new anticoagulants is an important goal for the improvement of thromboses treatments. OBJECTIVES: The design, synthesis and experimental testing of new safe and effective small molecule direct thrombin inhibitors for intravenous administration. METHODS: Computer-aided molecular design of new thrombin inhibitors was performed using our original docking program SOL, which is based on the genetic algorithm of global energy minimization in the framework of a Merck Molecular Force Field. This program takes into account the effects of solvent. The designed molecules with the best scoring functions (calculated binding energies were synthesized and their thrombin inhibitory activity evaluated experimentally in vitro using a chromogenic substrate in a buffer system and using a thrombin generation test in isolated plasma and in vivo using the newly developed model of hemodilution-induced hypercoagulation in rats. The acute toxicities of the most promising new thrombin inhibitors were evaluated in mice, and their stabilities in aqueous solutions were measured. RESULTS: New compounds that are both effective direct thrombin inhibitors (the best K(I was 1111.1 mg/kg. A plasma-substituting solution supplemented with one of the new inhibitors prevented hypercoagulation in the rat model of hemodilution-induced hypercoagulation. Activities of the best new inhibitors in physiological saline (1 µM solutions were stable after sterilization by autoclaving, and the inhibitors remained stable at long-term storage over more than 1.5 years at room temperature and at 4°C. CONCLUSIONS: The high efficacy, stability and low acute toxicity reveal that the inhibitors that were developed may be promising for potential medical applications.

  2. Cellular signaling events involved in thrombin activation of vascular endothelium

    Energy Technology Data Exchange (ETDEWEB)

    Brock, T.A.; Capasso, E.L.; Gimbrone, M.A. Jr.


    Although cytosolic calcium ((Ca))/sub I/), inositol trisphosphate (IP/sub 3/) and diacylglycerol (DAG) are important second messengers involved in stimulus-response coupling to certain hormones, little information is available regarding their role in the activation of vascular endothelial cells (EC) during coagulation. The authors have used cultured human umbilical vein EC to examine thrombin effects on (Ca)/sub I/ and on IP/sub 3/ and DAG formation. Thrombin (1 U/ml) induces a rapid (peak < 15 sec) increase in (Ca)/sub I/ (300% basal levels) in suspensions of fura-2 (fluorescent Ca/sup 2 +/ indicator)-loaded EC. In addition thrombin stimulates concentration-dependent (.001-1 U/ml) increases in calcium efflux and IP/sub 3/ formation in EC prelabeled with /sup 45/Ca or /sup 3/H-myoinositol. Peak IP/sub 3/ (182+/-14% control) is rapid (<15 sec) and temporally similar to the rise in (Ca)/sub I/. In /sup 3/H-arachidonic acid-labeled EC, thrombin increases DAG (protein kinase C activator) at 15 sec (133+/-8% control), as well as 5 min (148+/-12% control). EC preincubation with 4..beta..-phorbol 12-myristate 13-acetate (10/sup -7/M, 5 min), a potent activator of protein kinase C, blocks thrombin (1 U/ml)-induced increases in (Ca)/sub I/ and IP/sub 3/. Thus, thrombin may trigger at least two distinct signaling pathways (IP/sub 3//calcium; DAG/protein kinase C) in EC. In addition, DAG stimulation of protein kinase C may influence this mediator's action in vascular EC.

  3. Thrombin stimulates insulin secretion via protease-activated receptor-3. (United States)

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


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

  4. Aggregation of thrombin-derived C-terminal fragments as a previously undisclosed host defense mechanism

    DEFF Research Database (Denmark)

    Petrlova, Jitka; Hansen, Finja C; van der Plas, Mariena J A


    Effective control of endotoxins and bacteria is crucial for normal wound healing. During injury, the key enzyme thrombin is formed, leading to generation of fibrin. Here, we show that human neutrophil elastase cleaves thrombin, generating 11-kDa thrombin-derived C-terminal peptides (TCPs), which ...

  5. New Approaches to the Role of Thrombin in Acute Coronary Syndromes: Quo Vadis Bivalirudin, a Direct Thrombin Inhibitor?

    Directory of Open Access Journals (Sweden)

    María Asunción Esteve-Pastor


    Full Text Available The pathophysiology of acute coronary syndrome (ACS involves platelet activation and thrombus formation after the rupture of atherosclerotic plaques. Thrombin is generated at the blood-plaque interface in association with cellular membranes on cells and platelets. Thrombin also amplifies the response to the tissue injury, coagulation and platelet response, so the treatment of ACS is based on the combined use of both antiplatelet (such as aspirin, clopidogrel, prasugrel and ticagrelor and antithrombotic drugs (unfractionated heparin, enoxaparin, fondaparinux and bivalirudin. Bivalirudin competitively inhibits thrombin with high affinity, a predictable response from its linear pharmacokinetics and short action. However, a present remarkable controversy exists between the latest main Guidelines in Clinical Practice and the key trials evaluating the use of bivalirudin in ACS. The aim of this review is to update the development of bivalirudin, including pharmacological properties, obtained information from clinical trials evaluating efficacy and safety of bivalirudin in ACS; as well as the recommendations of clinical Guidelines.

  6. New Approaches to the Role of Thrombin in Acute Coronary Syndromes: Quo Vadis Bivalirudin, a Direct Thrombin Inhibitor? (United States)

    Esteve-Pastor, María Asunción; Hernández-Romero, Diana; Valdés, Mariano; Marín, Francisco


    The pathophysiology of acute coronary syndrome (ACS) involves platelet activation and thrombus formation after the rupture of atherosclerotic plaques. Thrombin is generated at the blood-plaque interface in association with cellular membranes on cells and platelets. Thrombin also amplifies the response to the tissue injury, coagulation and platelet response, so the treatment of ACS is based on the combined use of both antiplatelet (such as aspirin, clopidogrel, prasugrel and ticagrelor) and antithrombotic drugs (unfractionated heparin, enoxaparin, fondaparinux and bivalirudin). Bivalirudin competitively inhibits thrombin with high affinity, a predictable response from its linear pharmacokinetics and short action. However, a present remarkable controversy exists between the latest main Guidelines in Clinical Practice and the key trials evaluating the use of bivalirudin in ACS. The aim of this review is to update the development of bivalirudin, including pharmacological properties, obtained information from clinical trials evaluating efficacy and safety of bivalirudin in ACS; as well as the recommendations of clinical Guidelines.


    Directory of Open Access Journals (Sweden)

    L. Barazzetti


    Full Text Available In photogrammetry a camera is considered calibrated if its interior orientation parameters are known. These encompass the principal distance, the principal point position and some Additional Parameters used to model possible systematic errors. The current state of the art for automated camera calibration relies on the use of coded targets to accurately determine the image correspondences. This paper presents a new methodology for the efficient and rigorous photogrammetric calibration of digital cameras which does not require any longer the use of targets. A set of images depicting a scene with a good texture are sufficient for the extraction of natural corresponding image points. These are automatically matched with feature-based approaches and robust estimation techniques. The successive photogrammetric bundle adjustment retrieves the unknown camera parameters and their theoretical accuracies. Examples, considerations and comparisons with real data and different case studies are illustrated to show the potentialities of the proposed methodology.

  8. Crystal Structure of Thrombin in Complex with S-Variegin: Insights of a Novel Mechanism of Inhibition and Design of Tunable Thrombin Inhibitors (United States)

    Koh, Cho Yeow; Kumar, Sundramurthy; Kazimirova, Maria; Nuttall, Patricia A.; Radhakrishnan, Uvaraj P.; Kim, Seongcheol; Jagadeeswaran, Pudur; Imamura, Takayuki; Mizuguchi, Jun; Iwanaga, Sadaaki; Swaminathan, Kunchithapadam; Kini, R. Manjunatha


    The inhibition of thrombin is one of the important treatments of pathological blood clot formation. Variegin, isolated from the tropical bont tick, is a novel molecule exhibiting a unique ‘two-modes’ inhibitory property on thrombin active site (competitive before cleavage, noncompetitive after cleavage). For the better understanding of its function, we have determined the crystal structure of the human α-thrombin:synthetic-variegin complex at 2.4 Å resolution. The structure reveals a new mechanism of thrombin inhibition by disrupting the charge relay system. Based on the structure, we have designed 17 variegin variants, differing in potency, kinetics and mechanism of inhibition. The most active variant is about 70 times more potent than the FDA-approved peptidic thrombin inhibitor, hirulog-1/bivalirudin. In vivo antithrombotic effects of the variegin variants correlate well with their in vitro affinities for thrombin. Our results encourage that variegin and the variants show strong potential for the development of tunable anticoagulants. PMID:22053189

  9. Desenvolvimento e calibração de um tensiômetro eletrônico de leitura automática Development and calibration of an electronic tensiometer for automatic reading

    Directory of Open Access Journals (Sweden)

    Adunias S. Teixeira


    Full Text Available O presente trabalho apresenta o desenvolvimento e calibração de tensiômetro de leitura automática, sendo o tensiômetro de mercúrio utilizado como padrão de comparação. Os ensaios foram conduzidos no Laboratório de Mecânica e Eletrônica do Departamento de Engenharia Agrícola da UFC. Esse equipamento difere do tensiômetro tradicional por substituir o manômetro de mercúrio por sensor de pressão. Tal dispositivo gera uma saída com valor de até no máximo 4,5 volts. O tensiômetro eletrônico é conectado a uma placa de aquisição de dados (DAQ contendo um microprocessador, conversor analógico/digital (ADC e saída serial, sendo tal placa ligada a um microcomputador. A bancada de ensaio constituiu-se de três caixas plásticas preenchidas com solo de textura franco- arenosa. Os dados foram coletados durante um mês e as equações geradas foram do tipo Count = offset + apsim, em que psim representa o potencial matricial fornecido pelo tensiômetro de mercúrio (kPa e count a saída digial do ADC, com valores adimensionais de 0 a 4.095. Foram obtidos os valores máximo e mínimo de offset de 348,572 e 261,026, e de coeficiente angular de 36,675 kPa-1 e 34,421 kPa-1. Os resultados da regressão linear simples indicaram a existência de regressão a menos de 0,1% de significância e valores de coeficientes de correlação nunca inferiores a 0,9994.This paper presents the development and calibration of a tensiometer for automatic reading where mercury has been replaced by an electronic pressure sensor called the electronic tensiometer. The conventional mercury tensiometer was used as a standard for calibration. Calibration trials were conducted at the Mechanics and Electronics Laboratory of the Department of Agricultural Engineering at Ceará Federal University, Brazil. The electronic tensiometer outputs a maximum 4.5 volts and is connected to a data acquisition system (DAQ composed of a microprocessor, a 12-bit analog to digital

  10. Advances in inspection automation (United States)

    Weber, Walter H.; Mair, H. Douglas; Jansen, Dion; Lombardi, Luciano


    This new session at QNDE reflects the growing interest in inspection automation. Our paper describes a newly developed platform that makes the complex NDE automation possible without the need for software programmers. Inspection tasks that are tedious, error-prone or impossible for humans to perform can now be automated using a form of drag and drop visual scripting. Our work attempts to rectify the problem that NDE is not keeping pace with the rest of factory automation. Outside of NDE, robots routinely and autonomously machine parts, assemble components, weld structures and report progress to corporate databases. By contrast, components arriving in the NDT department typically require manual part handling, calibrations and analysis. The automation examples in this paper cover the development of robotic thickness gauging and the use of adaptive contour following on the NRU reactor inspection at Chalk River.

  11. Purification and Characterization of Human Thrombin Activatable Fibrinolysis Inhibitor (TAFI)

    DEFF Research Database (Denmark)

    Christensen, Trine; Skottrup, Peter; Valnickova, Zuzana

    Thrombin Activatable Fibrinolysis inhibitor (TAFI) is a basic carboxypeptidase, circulating in plasma as an enzymatic inactive precursor. TAFI shares ~40% overall sequence identity with pancreas Carboxypeptidase B (PCPB) with the activation peptide being less conserved. Following activation of TA...... cysteines in TAFI. In this study we have purified and characterized TAFI from human plasma....

  12. Purification and Characterization of Human Thrombin Activatable Fibrinolysis Inhibitor (TAFI)

    DEFF Research Database (Denmark)

    Christensen, Trine; Skottrup, Peter; Valnickova, Zuzana


    Thrombin Activatable Fibrinolysis inhibitor (TAFI) is a basic carboxypeptidase, circulating in plasma as an enzymatic inactive precursor. TAFI shares ~40% overall sequence identity with pancreas Carboxypeptidase B (PCPB) with the activation peptide being less conserved. Following activation of TA...... cysteines in TAFI. In this study we have purified and characterized TAFI from human plasma....

  13. 21 CFR 864.7875 - Thrombin time test. (United States)


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Thrombin time test. 864.7875 Section 864.7875 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL... fibrinogen split products for the evaluation of bleeding disorders. (b) Classification. Class II (performance...

  14. Tyrosine sulfation modulates activity of tick-derived thrombin inhibitors (United States)

    Thompson, Robert E.; Liu, Xuyu; Ripoll-Rozada, Jorge; Alonso-García, Noelia; Parker, Benjamin L.; Pereira, Pedro José Barbosa; Payne, Richard J.


    Madanin-1 and chimadanin are two small cysteine-free thrombin inhibitors that facilitate blood feeding in the tick Haemaphysalis longicornis. Here, we report a post-translational modification—tyrosine sulfation—of these two proteins that is critical for potent anti-thrombotic and anticoagulant activity. Inhibitors produced in baculovirus-infected insect cells displayed heterogeneous sulfation of two tyrosine residues within each of the proteins. One-pot ligation-desulfurization chemistry enabled access to homogeneous samples of all possible sulfated variants of the proteins. Tyrosine sulfation of madanin-1 and chimadanin proved crucial for thrombin inhibitory activity, with the doubly sulfated variants three orders of magnitude more potent than the unmodified inhibitors. The three-dimensional structure of madanin-1 in complex with thrombin revealed a unique mode of inhibition, with the sulfated tyrosine residues binding to the basic exosite II of the protease. The importance of tyrosine sulfation within this family of thrombin inhibitors, together with their unique binding mode, paves the way for the development of anti-thrombotic drug leads based on these privileged scaffolds.

  15. NMR reveals a dynamic allosteric pathway in thrombin. (United States)

    Handley, Lindsey D; Fuglestad, Brian; Stearns, Kyle; Tonelli, Marco; Fenwick, R Bryn; Markwick, Phineus R L; Komives, Elizabeth A


    Although serine proteases are found ubiquitously in both eukaryotes and prokaryotes, and they comprise the largest of all of the peptidase families, their dynamic motions remain obscure. The backbone dynamics of the coagulation serine protease, apo-thrombin (S195M-thrombin), were compared to the substrate-bound form (PPACK-thrombin). R1, R2, (15)N-{(1)H}NOEs, and relaxation dispersion NMR experiments were measured to capture motions across the ps to ms timescale. The ps-ns motions were not significantly altered upon substrate binding. The relaxation dispersion data revealed that apo-thrombin is highly dynamic, with μs-ms motions throughout the molecule. The region around the N-terminus of the heavy chain, the Na(+)-binding loop, and the 170 s loop, all of which are implicated in allosteric coupling between effector binding sites and the active site, were dynamic primarily in the apo-form. Most of the loops surrounding the active site become more ordered upon PPACK-binding, but residues in the N-terminal part of the heavy chain, the γ-loop, and anion-binding exosite 1, the main allosteric binding site, retain μs-ms motions. These residues form a dynamic allosteric pathway connecting the active site to the main allosteric site that remains in the substrate-bound form.

  16. Dabigatran reduces thrombin-induced platelet aggregation and activation in a dose-dependent manner

    DEFF Research Database (Denmark)

    Vinholt, Pernille Just; Nielsen, Christian; Söderström, Anna Cecilia


    Dabigatran is an oral anticoagulant and a reversible inhibitor of thrombin. Further, dabigatran might affect platelet function through a direct effect on platelet thrombin receptors. The aim was to investigate the effect of dabigatran on platelet activation and platelet aggregation. Healthy donor...... platelet activation and platelet thrombin receptor expression (SPAN-12 and WEDE-15 expression). Agonists were thrombin, thrombin receptor-activating peptide, protease-activated receptor-4 agonist, collagen, collagen-related peptide, arachidonic acid, and adenosine diphosphate. All concentrations...... of dabigatran fully inhibited platelet aggregation for thrombin up to 2 IU/mL, while dabigatran did not affect platelet aggregation by other agonists. Platelet activation (percentage of platelets positive for activated GPIIb/IIIa, CD63, P-selectin) was reduced after thrombin stimulation in samples...

  17. ARTIP: Automated Radio Telescope Image Processing Pipeline (United States)

    Sharma, Ravi; Gyanchandani, Dolly; Kulkarni, Sarang; Gupta, Neeraj; Pathak, Vineet; Pande, Arti; Joshi, Unmesh


    The Automated Radio Telescope Image Processing Pipeline (ARTIP) automates the entire process of flagging, calibrating, and imaging for radio-interferometric data. ARTIP starts with raw data, i.e. a measurement set and goes through multiple stages, such as flux calibration, bandpass calibration, phase calibration, and imaging to generate continuum and spectral line images. Each stage can also be run independently. The pipeline provides continuous feedback to the user through various messages, charts and logs. It is written using standard python libraries and the CASA package. The pipeline can deal with datasets with multiple spectral windows and also multiple target sources which may have arbitrary combinations of flux/bandpass/phase calibrators.

  18. Site Calibration

    DEFF Research Database (Denmark)

    Kock, Carsten Weber; Vesth, Allan

    This Site Calibration report is describing the results of a measured site calibration for a site in Denmark. The calibration is carried out by DTU Wind Energy in accordance with Ref.[3] and Ref.[4]. The measurement period is given. The site calibration is carried out before a power performance...... measurement on a given turbine to clarify the influence from the terrain on the ratio between the wind speed at the center of the turbine hub and at the met mast. The wind speed at the turbine is measured by a temporary mast placed at the foundation for the turbine. The site and measurement equipment...

  19. Calibration uncertainty

    DEFF Research Database (Denmark)

    Heydorn, Kaj; Anglov, Thomas


    Methods recommended by the International Standardization Organisation and Eurachem are not satisfactory for the correct estimation of calibration uncertainty. A novel approach is introduced and tested on actual calibration data for the determination of Pb by ICP-AES. The improved calibration...... uncertainty was verified from independent measurements of the same sample by demonstrating statistical control of analytical results and the absence of bias. The proposed method takes into account uncertainties of the measurement, as well as of the amount of calibrant. It is applicable to all types...

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

    Energy Technology Data Exchange (ETDEWEB)

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


    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.

  1. Effect of Thrombin on Human Amnion Mesenchymal Cells, Mouse Fetal Membranes, and Preterm Birth* (United States)

    Mogami, Haruta; Keller, Patrick W.; Shi, Haolin; Word, R. Ann


    Here, we investigated the effects of thrombin on matrix metalloproteinases (MMPs) and prostaglandin (PG) synthesis in fetal membranes. Thrombin activity was increased in human amnion from preterm deliveries. Treatment of mesenchymal, but not epithelial, cells with thrombin resulted in increased MMP-1 and MMP-9 mRNA and enzymatic activity. Thrombin also increased COX2 mRNA and PGE2 in these cells. Protease-activated receptor-1 (PAR-1) was localized to amnion mesenchymal and decidual cells. PAR-1-specific inhibitors and activating peptides indicated that thrombin-induced up-regulation of MMP-9 was mediated via PAR-1. In contrast, thrombin-induced up-regulation of MMP-1 and COX-2 was mediated through Toll-like receptor-4, possibly through thrombin-induced release of soluble fetal fibronectin. In vivo, thrombin-injected pregnant mice delivered preterm. Mmp8, Mmp9, and Mmp13, and PGE2 content was increased significantly in fetal membranes from thrombin-injected animals. These results indicate that thrombin acts through multiple mechanisms to activate MMPs and PGE2 synthesis in amnion. PMID:24652285

  2. Thrombin-Induced Podocyte Injury Is Protease-Activated Receptor Dependent. (United States)

    Sharma, Ruchika; Waller, Amanda P; Agrawal, Shipra; Wolfgang, Katelyn J; Luu, Hiep; Shahzad, Khurrum; Isermann, Berend; Smoyer, William E; Nieman, Marvin T; Kerlin, Bryce A


    Nephrotic syndrome is characterized by massive proteinuria and injury of specialized glomerular epithelial cells called podocytes. Studies have shown that, whereas low-concentration thrombin may be cytoprotective, higher thrombin concentrations may contribute to podocyte injury. We and others have demonstrated that ex vivo plasma thrombin generation is enhanced during nephrosis, suggesting that thrombin may contribute to nephrotic progression. Moreover, nonspecific thrombin inhibition has been shown to decrease proteinuria in nephrotic animal models. We thus hypothesized that thrombin contributes to podocyte injury in a protease-activated receptor-specific manner during nephrosis. Here, we show that specific inhibition of thrombin with hirudin reduced proteinuria in two rat nephrosis models, and thrombin colocalized with a podocyte-specific marker in rat glomeruli. Furthermore, flow cytometry immunophenotyping revealed that rat podocytes express the protease-activated receptor family of coagulation receptors in vivo High-concentration thrombin directly injured conditionally immortalized human and rat podocytes. Using receptor-blocking antibodies and activation peptides, we determined that thrombin-mediated injury depended upon interactions between protease-activated receptor 3 and protease-activated receptor 4 in human podocytes, and between protease-activated receptor 1 and protease-activated receptor 4 in rat podocytes. Proximity ligation and coimmunoprecipitation assays confirmed thrombin-dependent interactions between human protease-activated receptor 3 and protease-activated receptor 4, and between rat protease-activated receptor 1 and protease-activated receptor 4 in cultured podocytes. Collectively, these data implicate thrombinuria as a contributor to podocyte injury during nephrosis, and suggest that thrombin and/or podocyte-expressed thrombin receptors may be novel therapeutic targets for nephrotic syndrome. Copyright © 2017 by the American Society of

  3. GPI Calibrations (United States)

    Rantakyrö, Fredrik T.


    "The Gemini Planet Imager requires a large set of Calibrations. These can be split into two major sets, one set associated with each observation and one set related to biweekly calibrations. The observation set is to optimize the correction of miscroshifts in the IFU spectra and the latter set is for correction of detector and instrument cosmetics."

  4. Reversible thrombin detection by aptamer functionalized STING sensors (United States)

    Actis, Paolo; Rogers, Adam; Nivala, Jeff; Vilozny, Boaz; Seger, R. Adam; Jejelowo, Olufisayo; Pourmand, Nader


    Signal Transduction by Ion NanoGating (STING) is a label-free technology based on functionalized quartz nanopipettes. The nanopipette pore can be decorated with a variety of recognition elements and the molecular interaction is transduced via a simple electrochemical system. A STING sensor can be easily and reproducibly fabricated and tailored at the bench starting from inexpensive quartz capillaries. The analytical application of this new biosensing platform, however, was limited due to the difficult correlation between the measured ionic current and the analyte concentration in solution. Here we show that STING sensors functionalized with aptamers allow the quantitative detection of thrombin. The binding of thrombin generates a signal that can be directly correlated to its concentration in the bulk solution. PMID:21636261

  5. Structure-function relationships in thrombin-activatable fibrinolysis inhibitor. (United States)

    Plug, T; Meijers, J C M


    Thrombin-activatable fibrinolysis inhibitor (TAFI) is an important regulator in the balance of coagulation and fibrinolysis. TAFI is a metallocarboxypeptidase that circulates in plasma as zymogen. Activated TAFI (TAFIa) cleaves C-terminal lysine or arginine residues from peptide substrates. The removal of C-terminal lysine residues from partially degraded fibrin leads to reduced plasmin formation and thus attenuation of fibrinolysis. TAFI also plays a role in inflammatory processes via the removal of C-terminal arginine or lysine residues from bradykinin, thrombin-cleaved osteopontin, C3a, C5a and chemerin. TAFI has been studied extensively over the past three decades and recent publications provide a wealth of information, including crystal structures, mutants and structural data obtained with antibodies and peptides. In this review, we combined and compared available data on structure/function relationships of TAFI. © 2016 International Society on Thrombosis and Haemostasis.

  6. Thrombin aptasensing with inherently electroactive graphene oxide nanoplatelets as labels (United States)

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


    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 μM, 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.

  7. Mechanistic Modeling of the Effects of Acidosis on Thrombin Generation (United States)


    trick, MD. The Leiden Thrombophilia Study, completed previously (FRR), was funded by the Netherlands Heart Foundation (89-063). The authors declare...TAT) data on induced acidosis for an in vivo porcine model; the data values are des- ignated with square markers and were extracted from Figure 5 in...2, C and D). These model predictions were consistent with experi- mental results for a porcine model of acidosis (Fig. 2E).9 Figure 6. Thrombin

  8. Combined thrombin and autologous blood for repair of lumbar durotomy. (United States)

    Moussa, Wael Mohamed Mohamed; Aboul-Enein, Hisham A


    Lumbar durotomy can be intended or unintended and can result in persistent cerebrospinal fluid (CSF) leak. Several methods are used to manage this complication including bed rest and CSF diversion. In this study, we theorize that the use of thrombin-soaked gel foam together with autologous blood laid on the sutured dural tear can prevent persistent CSF leak. A retrospective review of the records of patients who underwent lumbar surgery and had an unintended dural tear with CSF leak, comparing the outcome of patients who were submitted to thrombin-soaked gel foam together with autologous blood (group A) to patients treated by subfacial drain, tight bandage, and bed rest (group B). A total of 1371 patients had lumbar surgery, of whom 131 had dural tear. Group A included 62 patients, while group B included 69 patients. 8.1 % of group A patients had CSF leak as compared to 17.4 % of group B patients at postoperative day 14. The incidence of postoperative CSF leak and duration of postoperative hospital stay were statistically lower in group A than in group B (p thrombin and autologous blood for repair of lumbar durotomy is an effective and a relatively cheap way to decrease CSF leak in the early postoperative period as well as decreasing postoperative hospital stay. It also resulted in decreased complications rate in the late postoperative period.

  9. Metabolic Plasticity in Resting and Thrombin Activated Platelets (United States)

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


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

  10. Metabolic plasticity in resting and thrombin activated platelets.

    Directory of Open Access Journals (Sweden)

    Saranya Ravi

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

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

    Directory of Open Access Journals (Sweden)

    Marina eBen Shimon


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

  12. Thrombin preferentially induces autophagy in glia cells in the rat central nervous system. (United States)

    Hu, Shukun; Wu, Gang; Ding, Xin; Zhang, Yi


    Autophagy widely occurs after intracerebral hemorrhage (ICH). In our previous study, we demonstrated that thrombin, a serine protease produced after hematoma, contributes to ICH-induced autophagy. However, whether thrombin plays a neuronal and/or astrocytic role in autophagy induction is largely unknown. Here, we examined the autophagic role of thrombin on neurons and glia cells, respectively. In vivo, we found that intracaudate injection of thrombin specifically elevated the astrocytic expression of beclin-1 and LC3, two autophagic markers, and promoted the formation of autophagic vacuoles within astrocytes rather than neurons in the ipsilateral basal ganglia. Consistent with this, thrombin enhanced the LC3-II level and increased the number of MDC-labeled autophagic vacuoles in cultured astrocytes. These results indicated that thrombin preferentially activated astrocytic autophagy after ICH, and therefore provided novel insights into the pathophysiological mechanisms and therapeutic targets for hemorrhage stroke and brain trauma. Copyright © 2016. Published by Elsevier Ireland Ltd.

  13. An aptamer assay using rolling circle amplification coupled with thrombin catalysis for protein detection. (United States)

    Guo, Limin; Hao, Lihua; Zhao, Qiang


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

  14. Binding modes of thrombin binding aptamers investigated by simulations and experiments (United States)

    Trapaidze, A.; Bancaud, A.; Brut, M.


    Thrombin binding aptamers HD1 and HD22 are the most studied aptamers, both for therapeutic and sensing purposes. Yet, there is still no commercialized aptamer-based sensor device for thrombin detection, suggesting that the binding modes of these aptamers remain to be precisely described. Here, we investigate thrombin-aptamer interactions with molecular dynamics simulations, and show that the different solved structures of HD1-thrombin complex are energetically similar and consequently possibly co-existing. Conversely, HD22 folding is much more stable, and its binding energy with thrombin is significantly larger than that of HD1 complexes. These results are confronted to experiments, which consist in monitoring aggregation of aptamer-functionalized gold nanoparticles triggered by thrombin. HD1 alone, but not HD22, can trigger aggregation, meaning that this aptamer has multiple sites of interactions with thrombin. Furthermore, pre-incubation of HD22 with thrombin impedes HD1 aggregation, suggesting that HD1 and HD22 have competing affinities for the same binding site. Altogether, this study shows that the characterization of aptamer-thrombin interactions by structural and kinetic experiments joined to simulations is necessary for the development of biosensors.

  15. Low Anticoagulant Heparin Blocks Thrombin-Induced Endothelial Permeability in a PAR-Dependent Manner (United States)

    Gonzales, Joyce N.; Kim, Kyung-mi; Zemskova, Marina A.; Rafikov, Ruslan; Heeke, Brenten; Varn, Matthew N.; Black, Stephen; Kennedy, Thomas P.; Verin, Alexander D.; Zemskov, Evgeny A.


    Acute lung injury and acute respiratory distress syndrome are accompanied by thrombin activation and fibrin deposition that enhances lung inflammation, activates endothelial cells and disrupts lung paracellular permeability. Heparin possesses anti-inflammatory properties but its clinical use is limited by hemorrhage and heparin induced thrombocytopenia. We studied the effects of heparin and low anticoagulant 2-O, 3-O desulfated heparin (ODSH) on thrombin-induced increases in paracellular permeability of cultured human pulmonary endothelial cells (EC). Pretreatment with heparin or ODSH blocked thrombin-induced decrease in the EC transendothelial electrical resistance (TER), attenuated thrombin-stimulated paracellular gap formation and actin cytoskeletal rearrangement. Our data demonstrated that heparin and ODSH had inhibitory effects on thrombin-induced RhoA activation and intracellular calcium elevation. Thrombin-stimulated phosphorylation of the cytoskeletal regulatory proteins, myosin light chain and ezrin/radixin/moesin, were also reduced. In these effects, low anticoagulant ODSH was more potent than heparin. Heparin or ODSH alone produced decreases in the EC TER that were abolished by siRNA-mediated depletion of the thrombin receptor, PAR-1. We also demonstrated that, in contrast to heparin, ODSH did not possess thrombin-binding activity. Results suggest that heparin and low anticoagulant ODSH, can interfere with thrombin-activated signaling. PMID:24469066

  16. Thrombomodulin tightens the thrombin active site loops to promote protein C activation. (United States)

    Koeppe, Julia R; Seitova, Almagoul; Mather, Timothy; Komives, Elizabeth A


    Thrombomodulin (TM) forms a 1:1 complex with thrombin. Whereas thrombin alone cleaves fibrinogen to make the fibrin clot, the thrombin-TM complex cleaves protein C to initiate the anticoagulant pathway. Crystallographic investigations of the complex between thrombin and TMEGF456 did not show any changes in the thrombin active site. Therefore, research has focused recently on how TM may provide a docking site for the protein C substrate. Previous work, however, showed that when the thrombin active site was occupied with substrate analogues labeled with fluorophores, the fluorophores responded differently to active (TMEGF1-6) versus inactive (TMEGF56) fragments of TM. To investigate this further, we have carried out amide H/(2)H exchange experiments on thrombin in the presence of active (TMEGF45) and inactive (TMEGF56) fragments of TM. Both on-exchange and off-exchange experiments show changes in the thrombin active site loops, some of which are observed only when the active TM fragment is bound. These results are consistent with the previously observed fluorescence changes and point to a mechanism by which TM changes the thrombin substrate specificity in favor of protein C cleavage.

  17. Identification of the primary structural defect in the dysthrombin thrombin Quick I: substitution of cysteine for arginine-382. (United States)

    Henriksen, R A; Mann, K G


    A congenitally dysfunctional form of prothrombin, prothrombin Quick, was isolated from the plasma of an individual with less than 2% of normal prothrombin activity. Following activation of prothrombin Quick, two dysfunctional thrombins, thrombin Quick I and thrombin Quick II, were isolated. Functional characterization of thrombin Quick I indicated an increase in KM and a decrease in kcat, relative to thrombin, for release of fibrinopeptide A. Comparison of kcat/KM for thrombin Quick I to the value obtained for thrombin yielded a relative catalytic efficiency of 0.012 for thrombin Quick I [Henriksen, R. A., & Owen, W. G. (1987) J. Biol. Chem. 262, 4664-4669]. Lysyl endopeptidase digestor of reduced and S-carboxymethylated thrombin and thrombin Quick I has resulted in the identification of an altered peptide in this dysthrombin. Edman degradation of the isolated peptide has shown that the altered residue in this protein is Arg-382 which is replaced by Cys. This could result from a point mutation in the Arg codon, CGC, to yield TGC. Together, these results indicate that Arg-382 is a critical residue in determining the specificity of thrombin toward fibrinogen. Similar relative activities for thrombin Quick I in stimulating platelet aggregation, in the release of prostacyclin from human umbilical vein endothelium, and in the release of fibrinopeptide A suggest that these activities of thrombin share the same specificity determinants.

  18. Geometric calibration of ERS satellite SAR images

    DEFF Research Database (Denmark)

    Mohr, Johan Jacob; Madsen, Søren Nørvang


    Geometric calibration of the European Remote Sensing (ERS) Satellite synthetic aperture radar (SAR) slant range images is important in relation to mapping areas without ground reference points and also in relation to automated processing. The relevant SAR system parameters are discussed and calib......Geometric calibration of the European Remote Sensing (ERS) Satellite synthetic aperture radar (SAR) slant range images is important in relation to mapping areas without ground reference points and also in relation to automated processing. The relevant SAR system parameters are discussed...... on a seven-year ERS-1 and a four-year ERS-2 time series, the long term stability is found to be sufficient to allow a single calibration covering the entire mission period. A descending and an ascending orbit tandem pair of the ESA calibration site on Flevoland, suitable for calibration of ERS SAR processors...

  19. Exosite 2-Directed Ligands Attenuate Protein C Activation by the Thrombin-Thrombomodulin Complex. (United States)

    Chen, Kai; Stafford, Alan R; Wu, Chengliang; Yeh, Calvin H; Kim, Paul Y; Fredenburgh, James C; Weitz, Jeffrey I


    Thrombin activity, inhibition, and localization are regulated by two exosites that flank the active site. Substrates, cofactors, and inhibitors bind to exosite 1 to promote active site access, whereas exosite 2 interactions hold thrombin on cells, platelets, and proteins. The exosites also serve allosteric roles, whereby ligand binding alters thrombin activity. Previously, we showed that ligands that bind exosite 2 attenuate the exosite 1-mediated interaction of thrombin with fibrin, demonstrating allosteric connection between the exosites. To determine the functional consequences of these inter-exosite interactions, we examined the effect of exosite 2 ligands on thrombin's interaction with thrombomodulin, a key cofactor that binds exosite 1 and redirects thrombin activity to the anticoagulant protein C pathway. Exosite 2-directed ligands, which included the HD22 aptamer, glycoprotein 1bα-derived peptide, and fibrinogen γ'-chain peptide, reduced the level of exosite 1-mediated thrombin binding to the thrombomodulin peptide consisting of the fourth, fifth, and sixth epidermal-like growth factor-like domains, decreasing affinity by >10-fold, and attenuated thrombomodulin-dependent activation of protein C by 60-80%. The ligands had similar effects on thrombin-mediated protein C activation with intact soluble thrombomodulin and with thrombomodulin on the surface of cultured endothelial cells. Their activity was exosite 2-specific because it was attenuated when RA-thrombin, a variant lacking exosite 2, was used in place of thrombin. These results indicate that additional reactions mediated by exosite 1 are amenable to regulation by exosite 2 ligation, providing further evidence of inter-exosite allosteric regulation of thrombin activity.

  20. Amide H/2H exchange reveals a mechanism of thrombin activation. (United States)

    Koeppe, Julia R; Komives, Elizabeth A


    Thrombin is a dual action serine protease in the blood clotting cascade. Similar to other clotting factors, thrombin is mainly present in the blood in a zymogen form, prothrombin. Although the two cleavage events required to activate thrombin are well-known, little is known about why the thrombin precursors are inactive proteases. Although prothrombin is much larger than thrombin, prethrombin-2, which contains all of the same amino acids as thrombin, but has not yet been cleaved between Arg320 and Ile321, remains inactive. Crystal structures of both prethrombin-2 and thrombin are available and show almost no differences in the active site conformations. Slight differences were, however, seen in the loops surrounding the active site, which are larger in thrombin than in most other trypsin-like proteases, and have been shown to be important for substrate specificity. To explore whether the dynamics of the active site loops were different in the various zymogen forms of thrombin, we employed amide H/(2)H exchange experiments to compare the exchange rates of regions of thrombin with the same regions of prothrombin, prethrombin-2, and meizothrombin. Many of the surface loops showed less exchange in the zymogen forms, including the large loop corresponding to anion binding exosite 1. Conversely, the autolysis loop and sodium-binding site exchanged more readily in the zymogen forms. Prothrombin and prethrombin-2 gave nearly identical results while meizothrombin in some regions more closely resembled active thrombin. Thus, cleavage of the Arg320-Ile321 peptide bond is the key to formation of the active enzyme, which involves increased dynamics of the substrate-binding loops and decreased dynamics of the catalytic site.

  1. Thrombin-inhibitory activity of whale heparin oligosaccharides. (United States)

    Ototani, N; Kodama, C; Kikuchi, M; Yosizawa, Z


    Whale heparin was partially digested with a purified heparinase and the oligosaccharide fractions with 8-20 monosaccharide units were isolated from the digest by gel filtration on Sephadex G-50, followed by affinity chromatography on a column of antithrombin III immobilized on Sepharose 4B. A marked difference in the inhibitory activity for thrombin in the presence of antithrombin III was observed between the high-affinity fractions for antithrombin III of octasaccharide approximately hexadecasaccharide and those of octadecasaccharide approximately eicosasaccharide. The disaccharide compositions of these hexadeca-, octadeca-, and eicosasaccharides were analyzed by high-performance liquid chromatography after digestion with a mixture of purified heparitinases 1 and 2 and heparinase. The analytical data indicated that the proportions of trisulfated disaccharide (IdUA(2S)alpha 1----4GlcNS(6S)) and disulfated disaccharide (UA1----4GlcNS(6S)) increased with the manifestation of high thrombin-inhibitory activity, while that of monosulfated disaccharide (UA1----4GlcNS) decreased. The present observations, together with those so far reported, suggest that the presence of the former structural elements, specifically IdUA(2S)alpha 1----4GlcNS(6S), as well as the antithrombin III-binding pentasaccharide at the proper positions in the molecules of whale heparin oligosaccharides is essential for the manifestation of high inhibitory activity for thrombin in the presence of antithrombin III. The structural bases for the manifestation of the anticoagulant activity of whale and porcine heparins and their oligosaccharides are also discussed.

  2. RNAi targeting embryonic myosin heavy chain isoform inhibited bound thrombin-induced migration of vascular smooth muscle cells

    National Research Council Canada - National Science Library

    Sunagawa, Masanori; Shimada, Seiji; Nakamura, Mariko; Kosugi, Tadayoshi


    To investigate the effect of bound thrombin, a complex of alpha-thrombin with fibrin fragments derived from clots, on proliferation and migration of cultured rabbit vascular smooth muscle cells, cell...

  3. Site calibration

    DEFF Research Database (Denmark)

    Gómez Arranz, Paula; Georgieva Yankova, Ginka

    The report describes site calibration measurements carried out on a site in Denmark. The measurements are carried out in accordance to Ref. [1]. The site calibration is carried out before a power performance measurement on a given turbine to clarify the influence from the terrain on the ratio...... between the wind speed at the center of the turbine hub and at the met mast. The wind speed at the turbine is measured by a temporary mast placed at the foundation for the turbine. The site and measurement equipment is detailed described in [2]. The possible measurement sector for power performance...... according to [1] is also described in [2] and no results from the site calibration have shown any necessary exclusion from this sector. All parts of the sensors and the measurement system have been installed by DTU....

  4. Three different signal amplification strategies for the impedimetric sandwich detection of thrombin

    Energy Technology Data Exchange (ETDEWEB)

    Ocaña, Cristina; Valle, Manel del, E-mail:


    In this work, we report a comparative study on three highly specific amplification strategies for the ultrasensitive detection of thrombin with the use of aptamer sandwich protocol. The protocol consisted on the use of a first thrombin aptamer immobilized on the electrode surface, the recognition of thrombin protein, and the reaction with a second biotinylated thrombin aptamer forming the sandwich. Through the exposed biotin end, three variants have been tested to amplify the electrochemical impedance signal. The strategies included (a) silver enhancement treatment, (b) gold enhancement treatment and (c) insoluble product produced by the combination of the enzyme horseradish peroxidase (HRP) and 3-amino-9-ethylcarbazole (AEC). The properties of the sensing surface were probed by electrochemical impedance measurements in the presence of the ferrocyanide/ferricyanide redox marker. Insoluble product strategy and silver enhancement treatment resulted in the lowest detection limit (0.3 pM), while gold enhancement method resulted in the highest reproducibility, 8.8% RSD at the pM thrombin concentration levels. Results of silver and gold enhancement treatment also permitted direct inspection by scanning electron microscopy (SEM). - Highlights: • Aptasensor to detect thrombin reaching the femtomolar level. • Biosensing protocol employs two thrombin aptamers in a sandwich capture scheme. • Use of second biotinylated aptamer allows many amplification and detection variants. • Precipitation reaction provides the highest signal amplification of ca. 3 times. • Double recognition event improves remarkably selectivity for thrombin detection.

  5. Mechanisms of modulation of brain microvascular endothelial cells function by thrombin. (United States)

    Brailoiu, Eugen; Shipsky, Megan M; Yan, Guang; Abood, Mary E; Brailoiu, G Cristina


    Brain microvascular endothelial cells are a critical component of the blood-brain barrier. They form a tight monolayer which is essential for maintaining the brain homeostasis. Blood-derived proteases such as thrombin may enter the brain during pathological conditions like trauma, stroke, and inflammation and further disrupts the permeability of the blood-brain barrier, via incompletely characterized mechanisms. We examined the underlying mechanisms evoked by thrombin in rat brain microvascular endothelial cells (RBMVEC). Our results indicate that thrombin, acting on protease-activated receptor 1 (PAR1) increases cytosolic Ca(2+) concentration in RBMVEC via Ca(2+) release from endoplasmic reticulum through inositol 1,4,5-trisphosphate receptors and Ca(2+) influx from extracellular space. Thrombin increases nitric oxide production; the effect is abolished by inhibition of the nitric oxide synthase or by antagonism of PAR1 receptors. In addition, thrombin increases mitochondrial and cytosolic reactive oxygen species production via PAR1-dependent mechanisms. Immunocytochemistry studies indicate that thrombin increases F-actin stress fibers, and disrupts the tight junctions. Thrombin increased the RBMVEC permeability assessed by a fluorescent flux assay. Taken together, our results indicate multiple mechanisms by which thrombin modulates the function of RBMVEC and may contribute to the blood-brain barrier dysfunction. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Glycoprotein Ib activation by thrombin stimulates the energy metabolism in human platelets. (United States)

    Corona de la Peña, Norma; Gutiérrez-Aguilar, Manuel; Hernández-Reséndiz, Ileana; Marín-Hernández, Álvaro; Rodríguez-Enríquez, Sara


    Thrombin-induced platelet activation requires substantial amounts of ATP. However, the specific contribution of each ATP-generating pathway i.e., oxidative phosphorylation (OxPhos) versus glycolysis and the biochemical mechanisms involved in the thrombin-induced activation of energy metabolism remain unclear. Here we report an integral analysis on the role of both energy pathways in human platelets activated by several agonists, and the signal transducing mechanisms associated with such activation. We found that thrombin, Trap-6, arachidonic acid, collagen, A23187, epinephrine and ADP significantly increased glycolytic flux (3-38 times vs. non-activated platelets) whereas ristocetin was ineffective. OxPhos (33 times) and mitochondrial transmembrane potential (88%) were increased only by thrombin. OxPhos was the main source of ATP in thrombin-activated platelets, whereas in platelets activated by any of the other agonists, glycolysis was the principal ATP supplier. In order to establish the biochemical mechanisms involved in the thrombin-induced OxPhos activation in platelets, several signaling pathways associated with mitochondrial activation were analyzed. Wortmannin and LY294002 (PI3K/Akt pathway inhibitors), ristocetin and heparin (GPIb inhibitors) as well as resveratrol, ATP (calcium-release inhibitors) and PP1 (Tyr-phosphorylation inhibitor) prevented the thrombin-induced platelet activation. These results suggest that thrombin activates OxPhos and glycolysis through GPIb-dependent signaling involving PI3K and Akt activation, calcium mobilization and protein phosphorylation.

  7. Glycoprotein Ib activation by thrombin stimulates the energy metabolism in human platelets (United States)

    Corona de la Peña, Norma; Gutiérrez-Aguilar, Manuel; Hernández-Reséndiz, Ileana; Marín-Hernández, Álvaro


    Thrombin-induced platelet activation requires substantial amounts of ATP. However, the specific contribution of each ATP-generating pathway i.e., oxidative phosphorylation (OxPhos) versus glycolysis and the biochemical mechanisms involved in the thrombin-induced activation of energy metabolism remain unclear. Here we report an integral analysis on the role of both energy pathways in human platelets activated by several agonists, and the signal transducing mechanisms associated with such activation. We found that thrombin, Trap-6, arachidonic acid, collagen, A23187, epinephrine and ADP significantly increased glycolytic flux (3–38 times vs. non-activated platelets) whereas ristocetin was ineffective. OxPhos (33 times) and mitochondrial transmembrane potential (88%) were increased only by thrombin. OxPhos was the main source of ATP in thrombin-activated platelets, whereas in platelets activated by any of the other agonists, glycolysis was the principal ATP supplier. In order to establish the biochemical mechanisms involved in the thrombin-induced OxPhos activation in platelets, several signaling pathways associated with mitochondrial activation were analyzed. Wortmannin and LY294002 (PI3K/Akt pathway inhibitors), ristocetin and heparin (GPIb inhibitors) as well as resveratrol, ATP (calcium-release inhibitors) and PP1 (Tyr-phosphorylation inhibitor) prevented the thrombin-induced platelet activation. These results suggest that thrombin activates OxPhos and glycolysis through GPIb-dependent signaling involving PI3K and Akt activation, calcium mobilization and protein phosphorylation. PMID:28817667

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

    Directory of Open Access Journals (Sweden)

    Caroline Fabri Bittencourt Rodrigues


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

  9. Changes in thrombin generation in children after cardiac surgery and ex-vivo response to blood products and haemostatic agents

    DEFF Research Database (Denmark)

    Andreasen, Jo B; Ravn, Hanne B; Hvas, Anne-Mette


    to test the hypothesis that thrombin generation reveals an impaired haemostasis after paediatric cardiac surgery and that ex-vivo addition of platelet concentrate and haemostatic agents improves thrombin generation. The study comprised 29 children with congenital heart disease, who underwent corrective...... thrombin generation significantly (all P Changes were most pronounced after addition of platelet concentrate. The present study showed that thrombin generation was significantly reduced after cardiopulmonary bypass in children, both when analysed in platelet-poor and platelet-rich plasma...

  10. Insights into thrombin activatable fibrinolysis inhibitor function and regulation. (United States)

    Foley, J H; Kim, P Y; Mutch, N J; Gils, A


    Fibrinolysis is initiated when the zymogen plasminogen is converted to plasmin via the action of plasminogen activators. Proteolytic cleavage of fibrin by plasmin generates C-terminal lysine residues capable of binding both plasminogen and the plasminogen activator, thereby stimulating plasminogen activator-mediated plasminogen activation and propagating fibrinolysis. This positive feedback mechanism is regulated by activated thrombin activatable fibrinolysis inhibitor (TAFIa), which cleaves C-terminal lysine residues from the fibrin surface, thereby decreasing its cofactor activity. TAFI can be activated by thrombin alone, but the rate of activation is accelerated when in complex with thrombomodulin. Plasmin is also known to activate TAFI. TAFIa has no known physiologic inhibitors and consequently, its primary regulatory mechanism involves its intrinsic thermal instability. The rate of TAFI activation and stability of the active form, TAFIa, function in maintaining its concentration above the threshold value required to down-regulate fibrinolysis. Although all methods to quantify TAFI or TAFIa have their limitations, epidemiologic studies have indicated that elevated TAFI levels are correlated with an increased risk of venous thrombosis. Major efforts have been made to develop TAFI inhibitors that can either directly interfere with TAFIa activity or impair its activation. However, the anti-inflammatory properties of TAFIa might complicate the development and application of a TAFIa inhibitor that aims to increase the efficiency of thrombolytic therapy. © 2013 International Society on Thrombosis and Haemostasis.

  11. WEDGE: An anticoagulant thrombin mutant produced by autoactivation (United States)

    Wood, D.C.; Pelc, L.A.; Pozzi, N.; Wallisch, M.; Verbout, N.G.; Tucker, E.I.; Gruber, A.; Di Cera, E.


    Summary Background Production of therapeutically relevant proteases typically involves activation of a zymogen precursor by external enzymes, which may raise regulatory issues about availability and purity. Recent studies of thrombin precursors have shown how to engineer constructs that spontaneously convert to the mature protease by autoactivation, without the need of external enzymes. Objectives Autoactivation is an innovative strategy that promises to simplify the production of proteases of therapeutic relevance, but has not been tested in practical applications. This study aims to provide a direct test of this strategy. Methods An autoactivating version of the thrombin mutant W215A/E217A (WE), currently in pre-clinical development as an anticoagulant, is engineered. Results and Conclusions The autoactivating version of WE can be produced in large quantities, like WE made in BHK cells or E. coli, and retains all significant functional properties in vitro and in vivo. The results serve as proof of principle that autoactivation is an innovative and effective strategy for production of trypsin-like proteases of therapeutic relevance. PMID:25369995

  12. Contact system activation and high thrombin generation in hyperthyroidism. (United States)

    Kim, Namhee; Gu, Ja-Yoon; Yoo, Hyun Ju; Han, Se Eun; Kim, Young Il; Nam-Goong, Il Sung; Kim, Eun Sook; Kim, Hyun Kyung


    Hyperthyroidism is associated with increased thrombotic risk. As contact system activation through formation of neutrophil extracellular traps (NET) has emerged as an important trigger of thrombosis, we hypothesized that the contact system is activated along with active NET formation in hyperthyroidism and that their markers correlate with disease severity. In 61 patients with hyperthyroidism and 40 normal controls, the levels of coagulation factors (fibrinogen, and factor VII, VIII, IX, XI and XII), D-dimer, thrombin generation assay (TGA) markers, NET formation markers (histone-DNA complex, double-stranded DNA and neutrophil elastase) and contact system markers (activated factor XII (XIIa), high-molecular-weight kininogen (HMWK), prekallikrein and bradykinin) were measured. Patients with hyperthyroidism showed higher levels of fibrinogen (median (interquartile range), 315 (280-344) vs 262 (223-300), P = 0.001), D-dimer (103.8 (64.8-151.5) vs 50.7 (37.4-76.0), P hyperthyroidism's contribution to coagulation and contact system activation. Free T4 was significantly correlated with factors VIII and IX, D-dimer, double-stranded DNA and bradykinin. This study demonstrated that contact system activation and abundant NET formation occurred in the high thrombin generation state in hyperthyroidism and were correlated with free T4 level. © 2017 European Society of Endocrinology.

  13. Molecular dynamics simulations of aptamer-binding reveal generalized allostery in thrombin. (United States)

    Xiao, Jiajie; Salsbury, Freddie R


    Thrombin is an attractive target for antithrombotic therapy due to its central role in thrombosis and hemostasis as well as its role in inducing tumor growth, metastasis, and tumor invasion. The thrombin-binding DNA aptamer (TBA), is under investigation for anticoagulant drugs. Although aptamer binding experiments have been revealed various effects on thrombin's enzymatic activities, the detailed picture of the thrombin's allostery from TBA binding is still unclear. To investigate thrombin's response to the aptamer-binding at the molecular level, we compare the mechanical properties and free energy landscapes of the free and aptamer-bound thrombin using microsecond-scale all-atom GPU-based molecular dynamics simulations. Our calculations on residue fluctuations and coupling illustrate the allosteric effects of aptamer-binding at the atomic level, highlighting the exosite II, 60s, γ and the sodium loops, and the alpha helix region in the light chains involved in the allosteric changes. This level of details clarifies the mechanisms of previous experimentally demonstrated phenomena, and provides a prediction of the reduced autolysis rate after aptamer-binding. The shifts in thrombin's ensemble of conformations and free energy surfaces after aptamer-binding demonstrate that the presence of bound-aptamer restricts the conformational freedom of thrombin suggesting that conformational selection, i.e. generalized allostery, is the dominant mechanism of thrombin-aptamer binding. The profound perturbation on thrombin's mechanical and thermodynamic properties due to the aptamer-binding, which was revealed comprehensively as a generalized allostery in this work, may be exploited in further drug discovery and development.

  14. Batroxobin Binds Fibrin with Higher Affinity and Promotes Clot Expansion to a Greater Extent than Thrombin* (United States)

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


    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 Aα- and Bβ-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 γA/γA isoform of fibrin(ogen) and the γA/γ′ variant with an extended γ-chain. Thrombin binds to the γ′-chain and forms a higher affinity interaction with γA/γ′-fibrin(ogen) than γA/γA-fibrin(ogen). In contrast, batroxobin binds both fibrin(ogen) isoforms with similar high affinity (Kd values of about 0.5 μm) even though it does not interact with the γ′-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 γA/γA-fibrinogen with batroxobin. Furthermore, although both thrombin and batroxobin bind to the central E-region of fibrinogen with a Kd value of 2–5 μm, the α(17–51) and Bβ(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. PMID:23612970

  15. Astrid-2 EMMA Magnetic Calibration

    DEFF Research Database (Denmark)

    Merayo, José M.G.; Brauer, Peter; Risbo, Torben


    of the magnetometer readings in each position were related to the field magnitudes from the Observatory, and a least squares fit for the 9 magnetometer calibration parameters was performed (3 offsets, 3 scale values and 3 inter-axes angles). After corrections for the magnetometer digital-to-analogue converters...... experiment built as a collaboration between the DTU, Department of Automation and the Department of Plasma Physics, The Alfvenlaboratory, Royal Institute of Technology (RIT), Stockholm. The final magnetic calibration of the Astrid-2 satellite was done at the Lovoe Magnetic Observatory under the Geological...... Survey of Sweden near Stockholm on the night of May 15.-16., 1997. The magnetic calibration and the intercalibration between the star camera and the magnetic sensor was performed by measuring the Earth's magnetic field and simultaneously observing the star sky with the camera. The rotation matrix between...

  16. The Organophosphate Paraoxon and Its Antidote Obidoxime Inhibit Thrombin Activity and Affect Coagulation In Vitro.

    Directory of Open Access Journals (Sweden)

    Valery Golderman

    Full Text Available Organophosphates (OPs are potentially able to affect serine proteases by reacting with their active site. The potential effects of OPs on coagulation factors such as thrombin and on coagulation tests have been only partially characterized and potential interactions with OPs antidotes such as oximes and muscarinic blockers have not been addressed. In the current study, we investigated the in vitro interactions between coagulation, thrombin, the OP paraoxon, and its antidotes obidoxime and atropine. The effects of these substances on thrombin activity were measured in a fluorescent substrate and on coagulation by standard tests. Both paraoxon and obidoxime but not atropine significantly inhibited thrombin activity, and prolonged prothrombin time, thrombin time, and partial thromboplastin time. When paraoxon and obidoxime were combined, a significant synergistic effect was found on both thrombin activity and coagulation tests. In conclusion, paraoxon and obidoxime affect thrombin activity and consequently alter the function of the coagulation system. Similar interactions may be clinically relevant for coagulation pathways in the blood and possibly in the brain.

  17. Protamine sulfate down-regulates thrombin generation by inhibiting factor V activation.

    LENUS (Irish Health Repository)

    Ni Ainle, Fionnuala


    Protamine sulfate is a positively charged polypeptide widely used to reverse heparin-induced anticoagulation. Paradoxically, prospective randomized trials have shown that protamine administration for heparin neutralization is associated with increased bleeding, particularly after cardiothoracic surgery with cardiopulmonary bypass. The molecular mechanism(s) through which protamine mediates this anticoagulant effect has not been defined. In vivo administration of pharmacologic doses of protamine to BALB\\/c mice significantly reduced plasma thrombin generation and prolonged tail-bleeding time (from 120 to 199 seconds). Similarly, in pooled normal human plasma, protamine caused significant dose-dependent prolongations of both prothrombin time and activated partial thromboplastin time. Protamine also markedly attenuated tissue factor-initiated thrombin generation in human plasma, causing a significant decrease in endogenous thrombin potential (41% +\\/- 7%). As expected, low-dose protamine effectively reversed the anticoagulant activity of unfractionated heparin in plasma. However, elevated protamine concentrations were associated with progressive dose-dependent reduction in thrombin generation. To assess the mechanism by which protamine mediates down-regulation of thrombin generation, the effect of protamine on factor V activation was assessed. Protamine was found to significantly reduce the rate of factor V activation by both thrombin and factor Xa. Protamine mediates its anticoagulant activity in plasma by down-regulation of thrombin generation via a novel mechanism, specifically inhibition of factor V activation.

  18. A new type of thrombin inhibitor, noncytotoxic phospholipase A2, from the Naja haje cobra venom. (United States)

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


    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. Copyright 2009 Elsevier Ltd. All rights reserved.

  19. Thrombin-unique coagulation system protein with multifaceted impacts on cancer and metastasis. (United States)

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


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

  20. The Organophosphate Paraoxon and Its Antidote Obidoxime Inhibit Thrombin Activity and Affect Coagulation In Vitro (United States)

    Golderman, Valery; Shavit-Stein, Efrat; Tamarin, Ilia; Rosman, Yossi; Shrot, Shai; Rosenberg, Nurit


    Organophosphates (OPs) are potentially able to affect serine proteases by reacting with their active site. The potential effects of OPs on coagulation factors such as thrombin and on coagulation tests have been only partially characterized and potential interactions with OPs antidotes such as oximes and muscarinic blockers have not been addressed. In the current study, we investigated the in vitro interactions between coagulation, thrombin, the OP paraoxon, and its antidotes obidoxime and atropine. The effects of these substances on thrombin activity were measured in a fluorescent substrate and on coagulation by standard tests. Both paraoxon and obidoxime but not atropine significantly inhibited thrombin activity, and prolonged prothrombin time, thrombin time, and partial thromboplastin time. When paraoxon and obidoxime were combined, a significant synergistic effect was found on both thrombin activity and coagulation tests. In conclusion, paraoxon and obidoxime affect thrombin activity and consequently alter the function of the coagulation system. Similar interactions may be clinically relevant for coagulation pathways in the blood and possibly in the brain. PMID:27689805

  1. TRAP Induces More Intense Tyrosine Phosphorylation than Thrombin with Differential Ultrastructural Features (United States)

    Fusté, Berta; Díaz-Ricart, Maribel; Jensen, Morten Krogh; Ordinas, Antonio; Escolar, Ginés; White, James G.


    We have analyzed modifications on platelet ultrastructural morphology, cytoskeletal assembly, and tyrosine phosphorylation developing in platelets activated by both thrombin and the thrombin receptor-activating peptide (TRAP). Washed platelets exposed to various concentrations of thrombin or TRAP, for different periods, were: fixed and examined by electron microscopy, or lysed and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under similar activating conditions, thrombin and TRAP induced different sequences of activation causing distinctive morphological and biochemical changes. Platelets exposed to thrombin showed centralized organelles encircled by constricted microtubule coils and granules secreting their contents through narrow channels of the open canalicular system. In contrast, activation by TRAP induced swelling of the open canalicular system with organelles remaining randomly dispersed and microtubules peripherally distributed. Compared to thrombin activation, TRAP induced higher rates of actin polymerization; increased association of actin-binding protein, myosin, and α-actinin; and higher association of tyrosine-phosphorylated proteins with the insoluble cytoskeletal fraction. Secretion of intragranule substances, measured as expression of P-selectin and lysosomal integral membrane protein at the surface level, were similar for both agonists at equivalent concentrations. Our biochemical observations indicate that TRAP causes more intense changes in signaling through tyrosine phosphorylation of proteins associated with the cytoskeletal fraction than thrombin. However, as derived from ultrastructural observations, TRAP seems to be less efficient in triggering cytoskeletal assembly and internal contraction in an organized manner in contrast with the natural protease. PMID:12057926

  2. Thrombin-inhibiting nanoparticles rapidly constitute versatile and detectable anticlotting surfaces (United States)

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


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

  3. Thrombin Induces Inositol Trisphosphate-Mediated Spatially Extensive Responses in Lung Microvessels. (United States)

    Escue, Rachel; Kandasamy, Kathirvel; Parthasarathi, Kaushik


    Activation of plasma membrane receptors initiates compartmentalized second messenger signaling. Whether this compartmentalization facilitates the preferential intercellular diffusion of specific second messengers is unclear. Toward this, the receptor-mediated agonist, thrombin, was instilled into microvessels in a restricted region of isolated blood-perfused mouse lungs. Subsequently, the thrombin-induced increase in endothelial F-actin was determined using confocal fluorescence microscopy. Increased F-actin was evident in microvessels directly treated with thrombin and in those located in adjoining thrombin-free regions. This increase was abrogated by inhibiting inositol trisphosphate-mediated calcium release with Xestospongin C (XeC). XeC also inhibited the thrombin-induced increase in the amplitude of endothelial cytosolic Ca(2+) oscillations. Instillation of thrombin and XeC into adjacent restricted regions increased F-actin in microvessels in the thrombin-treated and adjacent regions but not in those in the XeC-treated region. Thus, inositol trisphosphate, and not calcium, diffused interendothelially to the spatially remote thrombin-free microvessels. Thus, activation of plasma membrane receptors increased the ambit of inflammatory responses via a second messenger different from that used by stimuli that induce cell-wide increases in second messengers. Thrombin however failed to induce the spatially extensive response in microvessels of mice lacking endothelial connexin43, suggesting a role for connexin43 gap junctions. Compartmental second messenger signaling and interendothelial communication define the specific second messenger involved in exacerbating proinflammatory responses to receptor-mediated agonists. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  4. Synergism between thrombin and adrenaline (epinephrine) in human platelets. Marked potentiation of inositol phospholipid metabolism. (United States)

    Steen, V M; Tysnes, O B; Holmsen, H


    We have studied synergism between adrenaline (epinephrine) and low concentrations of thrombin in gel-filtered human platelets prelabelled with [32P]Pi. Suspensions of platelets, which did not contain added fibrinogen, were incubated at 37 degrees C to measure changes in the levels of 32P-labelled phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP) and phosphatidate (PA), aggregation and dense-granule secretion after stimulation. Adrenaline alone (3.5-4.0 microM) did not cause a change in any parameter (phosphoinositide metabolism, aggregation and dense-granule secretion), but markedly enhanced the thrombin-induced responses over a narrow range of thrombin concentrations (0.03-0.08 units/ml). The thrombin-induced hydrolysis of inositol phospholipids by phospholipase C, which was measured as the formation of [32P]PA, was potentiated by adrenaline, as was the increase in the levels of [32P]PIP2 and [32P]PIP. The presence of adrenaline caused a shift to the left for the thrombin-induced changes in the phosphoinositide metabolism, without affecting the maximal levels of 32P-labelled compounds obtained. A similar shift by adrenaline in the dose-response relationship was previously demonstrated for thrombin-induced aggregation and dense-granule secretion. Also, the narrow range of concentrations of thrombin over which adrenaline potentiates thrombin-induced platelet responses is the same for changes in phosphoinositide metabolism and physiological responses (aggregation and dense-granule secretion). Our observations clearly indicate that adrenaline directly or indirectly influences thrombin-induced changes in phosphoinositide metabolism. PMID:2845924

  5. The oral thrombin inhibitor dabigatran: strengths and weaknesses. (United States)

    Schulman, Sam; Majeed, Ammar


    Since the quest for a better replacement of warfarin started several decades ago and new compounds were brought forward to clinical trials, the concept of an ideal anticoagulant frequently became presented in lectures and articles. We have here reviewed strengths and weaknesses of the oral thrombin inhibitor dabigatran in terms of pharmacokinetics and clinical data. When strengths clearly exceed the weaknesses for any characteristic, the drug fits into the concept of an ideal anticoagulant in that domain. It is evident that dabigatran does not accomplish that concept for all characteristics but it reaches well above warfarin. We believe it is unlikely that any drug will fulfill all criteria for the ideal anticoagulant. Laboratory testing for dabigatran will not be discussed in any detail in this article, which is instead the focus of other articles from this issue of Seminars in Thrombosis & Hemostasis. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

  6. Loop Electrostatics Asymmetry Modulates the Preexisting Conformational Equilibrium in Thrombin. (United States)

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


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

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

    DEFF Research Database (Denmark)

    Pasternak, Anna; Hernandez, Frank J; Rasmussen, Lars Melholt


    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...... 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...

  8. Reactivity of mouse antibodies against bromelain-treated mouse erythrocytes with thrombin-treated mouse platelets. (United States)

    Kawaguchi, S


    The reactivity of mouse antibodies against bromelain-treated mouse erythrocytes (BrMRBC) with mouse platelets before and after thrombin treatment was assessed by flow cytometry. Anti-BrMRBC antibodies could bind to thrombin-treated platelets, although normal platelets were also weakly reactive with the antibodies. The binding of anti-BrMRBC antibodies to platelets was confirmed by complement-dependent lysis. It is suggested that thrombin-activated platelets may be a real target for anti-BrMRBC antibodies. PMID:2467876

  9. Thrombin contributes to protective immunity in pneumonia-derived sepsis via fibrin polymerization and platelet-neutrophil interactions. (United States)

    Claushuis, T A M; de Stoppelaar, S F; Stroo, I; Roelofs, J J T H; Ottenhoff, R; van der Poll, T; Van't Veer, C


    Essentials Immunity and coagulation are linked during sepsis but the role of thrombin is not fully elucidated. We investigated the effect of thrombin inhibition on murine Klebsiella pneumosepsis outcome. Thrombin is crucial for survival and limiting bacterial growth in pneumonia derived sepsis. Thrombin improves host defense via fibrin and enhancement of platelet-neutrophil interactions. Background Innate immunity and coagulation are closely linked during sepsis. Their interaction can be detrimental to the outcome because of microvascular failure but can also enhance host defense. The role of thrombin therein has not been fully elucidated. Objective We aimed to investigate the contribution of thrombin to the host response during pneumonia-derived sepsis. Methods Mice treated with the specific thrombin inhibitor dabigatran or control chow were infected with the common human sepsis pathogen Klebsiella (K.) pneumoniae via the airways. In subsequent infection experiments, mice were additionally treated with ancrod to deplete fibrinogen. Ex vivo Klebsiella growth was assessed by incubating human whole blood or specific blood components in various conditions with Klebsiella. Results Thrombin inhibition by dabigatran enhanced bacterial outgrowth and spreading, and accelerated mortality. Thrombin inhibition did not influence neutrophil recruitment to the lung or activation or neutrophil extracellular trap formation. Dabigatran reduced D-dimer formation and fibrin deposition in the lung. Fibrin depletion also enhanced bacterial outgrowth and spreading, and thrombin inhibition had no additional effect. Both thrombin and fibrin polymerization inhibited ex vivo Klebsiella outgrowth in human whole blood, which was neutrophil dependent, and the effect of thrombin required the presence of platelets and platelet protease activated receptor-1. In vivo thrombin inhibition reduced platelet-neutrophil complex formation and endothelial cell activation, but did not prevent sepsis

  10. Thrombin Generation Measurements in Patients Scheduled for Laparoscopic Bariatric Surgery. (United States)

    Thereaux, Jérémie; Mingant, Fanny; Roche, Charles; Galinat, Hubert; Couturaud, Francis; Lacut, Karine


    Obese patients are known to be in an in vitro hypercoagulable state relative to normal-weight patients. Our study aimed to identify markers of enhanced coagulability (endogenous thrombin potential (ETP)) in morbidly obese patients using the thrombin generation (TG) test. All patients scheduled for laparoscopic bariatric surgery (LBS) between September 1, 2014 and January 31, 2016 were eligible for our prospective study. We used logistic regression to compute the odds ratio (OR) across ETP quartile distributions to evaluate the risk of enhanced TG. We studied 102 patients, 77.5% were female, mean age was 41.2 ± 12.1 years, and mean BMI was 45.5 ± 7.0 k/m(2). Total cholesterol and fibrinogen levels were found to be independent risk factors for patients in the 4th quartile distribution of the ETP distribution (OR (95% CI)) 2.6 (1.2 to 5.4) (P = 0.01) and 2.2 (1.1 to 4.5 (P = 0.03). Patients in the 4th quartile of the ETP distribution had a lower ETP 1 month after surgery (157 (144-196) vs. 120 (98-140); P < 0.001) in parallel with a trend toward lower total cholesterol levels (5.0 ± 0.9 vs. 4.4 ± 1.0; P = 0.06). Fibrinogen levels were stable (4.5 ± 1.0 vs. 4.4 ± 0.9); P = 0.7). Our study highlights the role of total cholesterol and blood inflammatory marker levels in enhancing ETP in morbidly obese patients. Further studies are necessary to confirm the decreased ETP following LBS with the expected reduced inflammatory marker and total cholesterol levels.

  11. Use of a robotic manipulator in the simulation of the automation of a calibration process of dosemeters; Uso de un manipulador robotico en la simulacion de la automatizacion de un proceso de calibracion de dosimetros

    Energy Technology Data Exchange (ETDEWEB)

    Benitez R, J.S.; Najera H, M.C. [Instituto Nacional de Investigaciones Nucleares, A.P. 18-1027, 11801 Mexico D.F. (Mexico)


    The development of a system based in a manipulative robot which simulates the operative sequence in a calibration process of dosemeters is presented. In this process it is performed the monitoring of the dosemeter positions and the calibrator by mean of an arm of articulated robot which develops the movement sequences and the taking a decision based on the information coming from the external sensors. (Author)

  12. Interferometric Calibration with Natural Distributed Targets

    DEFF Research Database (Denmark)

    Dall, Jørgen; Christensen, Erik Lintz


    Cross-calibration is a fully automated algorithm for calibration of interferometric synthetic aperture radar (IFSAR) data. It has been developed for single-pass interferometry, but the principles may be applicable to multi-pass interferometry, too. The algorithm is based on natural distributed ta....... The algorithm appears to be fairly robust with respect to the terrain type. However, the result of the calibration may deteriorate if the terrain elevation, as measured with the SAR, changes systematically with the incidence angle or the aspect angle....

  13. Reduced activity of TAFI (thrombin-activatable fibrinolysis inhibitor) in acute promyelocytic leukaemia

    NARCIS (Netherlands)

    Meijers, JCM; Oudijk, EJD; Mosnier, LO; Nieuwenhuis, HK; Fijnheer, R; Bouma, Bonno N.; Bos, R

    Acute promyelocytic leukaemia (APL) is a disease that is distinguished from other leukaemias by the high potential for early haemorrhagic death. Several processes are involved, such as disseminated intravascular coagulation and hyperfibrinolysis. Recently, TAFI (thrombin-activatable fibrinolysis

  14. Reversal of trauma-induced amnesia in mice by a thrombin receptor antagonist. (United States)

    Itzekson, Zeev; Maggio, Nicola; Milman, Anat; Shavit, Efrat; Pick, Chaim G; Chapman, Joab


    Minimal traumatic brain injury (mTBI) is associated with the existence of retrograde amnesia and microscopic bleeds containing activated coagulation factors. In an mTBI model, we report that thrombin induces amnesia through its receptor protease-activated receptor 1 (PAR-1). Thrombin activity was significantly elevated (32 %, p Amnesia was assessed by the novel object recognition test in mTBI animals and in animals injected intracerebroventricularly (ICV) with either thrombin or a PAR-1 agonist 1 h after the acquisition phase. Saline-injected controls had a preference index of over 0.3 while mTBI animals and those injected with thrombin or the PAR-1 agonist spent equal time with both objects indicating no recall of the object presented to them 24 h previously (p amnesia following mTBI, revealing a novel therapeutic target for the cognitive effects of brain trauma.

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

    DEFF Research Database (Denmark)

    Rangnekar, Abhijit; Zhang, Alex M.; Shiyuan Li, Susan


    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...

  16. Comparing thrombin generation in patients with hemophilia A and patients on vitamin K antagonists

    NARCIS (Netherlands)

    de Koning, M L Y; Fischer, K; de Laat, B; Huisman, A; Ninivaggi, M; Schutgens, R E G

    Essentials: It is unknown if hemophilia patients with atrial fibrillation need anticoagulation. Endogenous thrombin potentials (ETP) in hemophilia patients and patients on coumarins were compared. Severe hemophilia patients had comparable ETP to therapeutic international normalized ratio (INR). In

  17. Treatment of accidental perianal injection of topical thrombin with intravenous antithrombin. (United States)

    Nielsen, Vance G; Paidy, Samata R; McLeod, Whitney; Fox, Alexandra; Nfonsam, Valentine N


    While topical thrombin application can markedly improve surgical hemostasis, rapid absorption of thrombin can result in pulmonary embolism and death. We report a case of accidental interstitial infiltration of topical thrombin after hemorrhoidectomy that was treated with administration of human antithrombin and heparin anticoagulation. Except for a marked decrease in antithrombin activity from super normal to normal values, the patient exhibited no laboratory or clinical signs of pulmonary embolism, thrombin mediated consumptive loss of procoagulants, or regional thrombosis. The patient had an uncomplicated recovery without sign of thrombotic morbidity. While it is hoped that such a medical misadventure should not occur, our case may serve as a reference to guide anticoagulant therapy if such a clinical scenario arises.

  18. Normal to increased thrombin generation in patients undergoing liver transplantation despite prolonged conventional coagulation tests

    NARCIS (Netherlands)

    Lisman, Ton; Bakhtiari, Kamran; Pereboom, Ilona T. A.; Hendriks, Herman G. D.; Meijers, Joost C. M.; Porte, Robert J.

    Background & Aims: Patients with liver disease often show substantial changes in their hemostatic system, which may aggravate further during liver transplantation. Recently, thrombin generation in patients with stable disease was shown to be indistinguishable from controls provided thrombomodulin,

  19. Hypercoagulability following major partial liver resection - detected by thrombomodulin-modified thrombin generation testing

    NARCIS (Netherlands)

    Potze, W.; Alkozai, E. M.; Adelmeijer, J.; Porte, R. J.; Lisman, T.

    BackgroundConventional coagulation tests are frequently prolonged after liver surgery, suggesting a post-operative hypocoagulability. However, these tests are unreliable for assessment of the haemostatic status in these patients. In contrast, thrombin generation testing measures the true balance

  20. Active but inoperable thrombin is accumulated in a plasma protein layer surrounding Streptococcus pyogenes

    NARCIS (Netherlands)

    Naudin, Clément; Hurley, Sinead M.; Malmström, Erik; Plug, Tom; Shannon, Oonagh; Meijers, Joost C. M.; Mörgelin, Matthias; Björck, Lars; Herwald, Heiko


    Activation of thrombin is a critical determinant in many physiological and pathological processes including haemostasis and inflammation. Under physiological conditions many of these functions are involved in wound healing or eradication of an invading pathogen. However, when activated systemically,

  1. Thrombin Cleavage of Plasmodium falciparum Erythrocyte Membrane Protein 1 Inhibits Cytoadherence. (United States)

    Gillrie, Mark R; Renaux, Bernard; Russell-Goldman, Eleanor; Avril, Marion; Brazier, Andrew J; Mihara, Koichiro; Di Cera, Enrico; Milner, Danny A; Hollenberg, Morley D; Smith, Joseph D; Ho, May


    Plasmodium falciparum malaria remains one of the most deadly infections worldwide. The pathogenesis of the infection results from the sequestration of infected erythrocytes (IRBC) in vital organs, including the brain, with resulting impairment of blood flow, hypoxia, and lactic acidosis. Sequestration occurs through the adhesion of IRBC to host receptors on microvascular endothelium by Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a large family of variant surface antigens, each with up to seven extracellular domains that can bind to multiple host receptors. Consequently, antiadhesive therapies directed at single endothelial adhesion molecules may not be effective. In this study, we demonstrated that the serine protease thrombin, which is pivotal in the activation of the coagulation cascade, cleaved the major parasite adhesin on the surface of IRBC. As a result, adhesion under flow was dramatically reduced, and already adherent IRBC were detached. Thrombin cleavage sites were mapped to the Duffy binding-like δ1 (DBLδ1) domain and interdomains 1 and 2 in the PfEMP1 of the parasite line IT4var19. Furthermore, we observed an inverse correlation between the presence of thrombin and IRBC in cerebral malaria autopsies of children. We investigated a modified (R67A) thrombin and thrombin inhibitor, hirugen, both of which inhibit the binding of substrates to exosite I, thereby reducing its proinflammatory properties. Both approaches reduced the barrier dysfunction induced by thrombin without affecting its proteolytic activity on PfEMP1, raising the possibility that thrombin cleavage of variant PfEMP1 may be exploited as a broadly inhibitory antiadhesive therapy. Plasmodium falciparum malaria is the third leading cause of mortality due to a pathogen, with 214 million people infected and 438,000 deaths annually. The adhesion of Plasmodium falciparum-infected erythrocytes (IRBC) to microvascular endothelium is a major pathological process in severe malaria

  2. Thrombin-linked aptamer assay for detection of platelet derived growth factor BB on magnetic beads in a sandwich format. (United States)

    Guo, Limin; Zhao, Qiang


    Here we describe a thrombin-linked aptamer assay (TLAA) for protein by using thrombin as an enzyme label, harnessing enzyme activity of thrombin and aptamer affinity binding. TLAA converts detection of specific target proteins to the detection of thrombin by using a DNA sequence that consists of two aptamers with the first aptamer binding to the specific target protein and the second aptamer binding to thrombin. Through the affinity binding, the thrombin enzyme is labeled on the protein target, and thrombin catalyzes the hydrolysis of small peptide substrate into product, generating signals for quantification. As a proof of principle, we show a sandwich TLAA for platelet derived growth factor BB (PDGF-BB) by using anti-PDGF-BB antibody coated on magnetic beads and an oligonucleotide containing the aptamer for PDGF-BB and the aptamer for thrombin. The binding of PDGF-BB to both the antibody and the aptamer results in labeling the complex with thrombin. We achieved detection of PDGF-BB at 16 pM. This TLAA contributes a new application of thrombin and its aptamer in bioanalysis, and shows potentials in assay developments. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Identification of berberine as a direct thrombin inhibitor from traditional Chinese medicine through structural, functional and binding studies (United States)

    Wang, Xing; Zhang, Yuxin; Yang, Ying; Wu, Xia; Fan, Hantian; Qiao, Yanjiang


    Thrombin acts as a key enzyme in the blood coagulation cascade and represents a potential drug target for the treatment of several cardiovascular diseases. The aim of this study was to identify small-molecule direct thrombin inhibitors from herbs used in traditional Chinese medicine (TCM). A pharmacophore model and molecular docking were utilized to virtually screen a library of chemicals contained in compositions of traditional Chinese herbs, and these analyses were followed by in vitro bioassay validation and binding studies. Berberine (BBR) was first confirmed as a thrombin inhibitor using an enzymatic assay. The BBR IC50 value for thrombin inhibition was 2.92 μM. Direct binding studies using surface plasmon resonance demonstrated that BBR directly interacted with thrombin with a KD value of 16.39 μM. Competitive binding assay indicated that BBR could bind to the same argartroban/thrombin interaction site. A platelet aggregation assay demonstrated that BBR had the ability to inhibit thrombin-induced platelet aggregation in washed platelets samples. This study proved that BBR is a direct thrombin inhibitor that has activity in inhibiting thrombin-induced platelet aggregation. BBR may be a potential candidate for the development of safe and effective thrombin-inhibiting drugs.

  4. Dynamics of Thrombin Generation and Flux from Clots during Whole Human Blood Flow over Collagen/Tissue Factor Surfaces. (United States)

    Zhu, Shu; Lu, Yichen; Sinno, Talid; Diamond, Scott L


    Coagulation kinetics are well established for purified blood proteases or human plasma clotting isotropically. However, less is known about thrombin generation kinetics and transport within blood clots formed under hemodynamic flow. Using microfluidic perfusion (wall shear rate, 200 s(-1)) of corn trypsin inhibitor-treated whole blood over a 250-μm long patch of type I fibrillar collagen/lipidated tissue factor (TF; ∼1 TF molecule/μm(2)), we measured thrombin released from clots using thrombin-antithrombin immunoassay. The majority (>85%) of generated thrombin was captured by intrathrombus fibrin as thrombin-antithrombin was largely undetectable in the effluent unless Gly-Pro-Arg-Pro (GPRP) was added to block fibrin polymerization. With GPRP present, the flux of thrombin increased to ∼0.5 × 10(-12) nmol/μm(2)-s over the first 500 s of perfusion and then further increased by ∼2-3-fold over the next 300 s. The increased thrombin flux after 500 s was blocked by anti-FXIa antibody (O1A6), consistent with thrombin-feedback activation of FXI. Over the first 500 s, ∼92,000 molecules of thrombin were generated per surface TF molecule for the 250-μm-long coating. A single layer of platelets (obtained with αIIbβ3 antagonism preventing continued platelet deposition) was largely sufficient for thrombin production. Also, the overall thrombin-generating potential of a 1000-μm-long coating became less efficient on a per μm(2) basis, likely due to distal boundary layer depletion of platelets. Overall, thrombin is robustly generated within clots by the extrinsic pathway followed by late-stage FXIa contributions, with fibrin localizing thrombin via its antithrombin-I activity as a potentially self-limiting hemostatic mechanism. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Inactivation of single-chain urokinase-type plasminogen activator by thrombin in human subjects. (United States)

    Braat, E A; Levi, M; Bos, R; Haverkate, F; Lassen, M R; de Maat, M P; Rijken, D C


    Thrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) into a virtually inactive two-chain form (tcu-PA/T), a process that may protect a blood clot from early fibrinolysis. It is not known under what circumstances tcu-PA/T can be generated in vivo. We have studied the occurrence of tcu-PA/T in human subjects with a varying degree of hypercoagulability. tcu-PA/T was assessed in the plasma of patients with disseminated intravascular coagulation (DIC), endotoxin-treated volunteers, patients with unstable angina pectoris, and patients selected for hip replacement. Relationships between tcu-PA/T and several markers reflecting thrombin generation were examined. tcu-PA/T was observed only in the plasma of patients with DIC and was associated with all thrombin markers and with scu-PA and urokinase antigen. Prothrombin fragment 1 + 2 and urokinase antigen were independent predictors of tcu-PA/T. The fact that tcu-PA/T could not be detected in the other three groups was explained by a lower extent of thrombin generation, a greater inhibition of thrombin by antithrombin, or less available urokinase antigen in these groups. The contribution of scu-PA to total urokinase antigen was decreased in the patients with DIC because of inactivation by thrombin, which may be an additional explanation for the inadequate fibrinolysis observed in these patients. These findings show that scu-PA can be inactivated in the circulation under severe pathophysiologic circumstances and that the process of inactivation depends not only on the generation of thrombin but also on the control of thrombin activity by its inhibitor antithrombin.

  6. Ezrin/radixin/moesin proteins differentially regulate endothelial hyperpermeability after thrombin (United States)

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


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

  7. Percutaneous ultrasound-guided thrombin injection for the treatment of iatrogenic femoral artery pseudoaneurysms

    Energy Technology Data Exchange (ETDEWEB)

    Vlachou, Paraskevi A. [Department of Radiology, Leicester Royal Infirmary, Leicester (United Kingdom); Karkos, Christos D., E-mail: [Department of Vascular and Endovascular Surgery, Leicester Royal Infirmary, Leicester (United Kingdom); Bains, Salena; McCarthy, Mark J. [Department of Vascular and Endovascular Surgery, Leicester Royal Infirmary, Leicester (United Kingdom); Fishwick, Guy; Bolia, Amman [Department of Radiology, Leicester Royal Infirmary, Leicester (United Kingdom)


    Purpose: To audit our experience with ultrasound-guided thrombin injection for the treatment of iatrogenic femoral artery pseudoaneurysms. Methods: A retrospective study of 85 consecutive patients undergoing percutaneous ultrasound-guided thrombin injection of post-catheterization femoral pseudoaneurysms during the period January 2002 to May 2007. Results: Pseudoaneurysms had a mean maximum diameter of 3.3 cm (range 1.0-7.6 cm) and a mean neck width of 3.4 mm (range 1.0-7.0 mm). No statistically significant correlation existed between maximum diameter and neck width (Kendall's rank correlation tau b = -0.09, p = 0.5). The median dose of thrombin injected was 425 U (range 100-1500 U). The procedure resulted in complete sac thrombosis in 81 (95%) patients. Seventy-nine pseudoaneurysms thrombosed immediately after one injection, whereas two required a second thrombin injection. There were no procedural complications. The maximum diameter of the pseudoaneurysm was predictive of procedural success (Wilcoxon's rank sum test, p = 0.001) and of the 5 patients with a pseudoaneurysm measuring {>=}6 cm, ultrasound-guided thrombin injection was unsuccessful in 4 (4/5 versus 0/80, p < 0.0001, Fisher's exact test). Three of these necessitated implantation of a stent-graft, whereas one required repeated thrombin injection and coil placement. In contrast, the pseudoaneurysm neck width did not seem to relate to the success of the procedure. Conclusion: Percutaneous ultrasound-guided thrombin injection of is a quick, effective and safe treatment for iatrogenic femoral pseudoaneurysms. For larger pseudoaneurysms, although it is worth attempting more than one thrombin injection, endovascular repair may eventually be required.

  8. Novel Role for p21-activated Kinase 2 in Thrombin-induced Monocyte Migration* (United States)

    Gadepalli, Ravisekhar; Kotla, Sivareddy; Heckle, Mark R.; Verma, Shailendra K.; Singh, Nikhlesh K.; Rao, Gadiparthi N.


    To understand the role of thrombin in inflammation, we tested its effects on migration of THP-1 cells, a human monocytic cell line. Thrombin induced THP-1 cell migration in a dose-dependent manner. Thrombin induced tyrosine phosphorylation of Pyk2, Gab1, and p115 RhoGEF, leading to Rac1- and RhoA-dependent Pak2 activation. Downstream to Pyk2, Gab1 formed a complex with p115 RhoGEF involving their pleckstrin homology domains. Furthermore, inhibition or depletion of Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, or Pak2 levels substantially attenuated thrombin-induced THP-1 cell F-actin cytoskeletal remodeling and migration. Inhibition or depletion of PAR1 also blocked thrombin-induced activation of Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, and Pak2, resulting in diminished THP-1 cell F-actin cytoskeletal remodeling and migration. Similarly, depletion of Gα12 negated thrombin-induced Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, and Pak2 activation, leading to attenuation of THP-1 cell F-actin cytoskeletal remodeling and migration. These novel observations reveal that thrombin induces monocyte/macrophage migration via PAR1-Gα12-dependent Pyk2-mediated Gab1 and p115 RhoGEF interactions, leading to Rac1- and RhoA-targeted Pak2 activation. Thus, these findings provide mechanistic evidence for the role of thrombin and its receptor PAR1 in inflammation. PMID:24025335

  9. The impact of the endothelial protein C receptor on thrombin generation and clot lysis. (United States)

    Pepler, Laura; Wu, Chengliang; Dwivedi, Dhruva J; Wu, Cynthia; Kim, Paul Y; Liaw, Patricia C


    When thrombin is bound to thrombomodulin (TM), it becomes a potent activator of protein C (PC) and thrombin-activable fibrinolysis inhibitor (TAFI). Activation of PC is enhanced when PC is bound to the endothelial protein C receptor (EPCR). Activated protein C (APC) inhibits thrombin generation while activated TAFI (TAFIa) attenuates fibrinolysis. To determine the impact of diminished EPCR function on thrombin generation and fibrinolysis we generated cells that expressed TM and a variant of EPCR (R96C) that does not bind PC. To determine the impact of EPCR on the generation of APC and TAFIa and how this affects thrombin generation and fibrinolysis we performed thrombin generation and clot lysis assays in the presence of cells expressing wild-type TM and EPCR (WT cells) or wild-type TM and the R96C variant of EPCR (R96C cells). In the presence of R96C cells, thrombin generation in normal plasma is increased, as a result of impaired PC activation when compared to WT cells. In addition, clot lysis is delayed in normal plasma in the presence of R96C cells, despite no increase in TAFI activation. In PC deficient plasma, clot lysis is delayed in the presence of WT and R96C cells as a result of increased TAFI activation. We demonstrate that impaired EPCR function can be detected by thrombin generation and clot lysis assays on cells expressing TM and EPCR. We also demonstrated that deficiency in EPCR has procoagulant effects that lead to a delay in clot lysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Identification and Mechanistic Analysis of a Novel Tick-Derived Inhibitor of Thrombin (United States)

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


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

  11. Thrombin Production and Human Neutrophil Elastase Sequestration by Modified Cellulosic Dressings and Their Electrokinetic Analysis (United States)

    Edwards, Judson Vincent; Prevost, Nicolette


    Wound healing is a complex series of biochemical and cellular events. Optimally, functional material design addresses the overlapping acute and inflammatory stages of wound healing based on molecular, cellular, and bio-compatibility issues. In this paper the issues addressed are uncontrolled hemostasis and inflammation which can interfere with the orderly flow of wound healing. In this regard, we review the serine proteases thrombin and elastase relative to dressing functionality that improves wound healing and examine the effects of charge in cotton/cellulosic dressing design on thrombin production and elastase sequestration (uptake by the wound dressing). Thrombin is central to the initiation and propagation of coagulation, and elastase is released from neutrophils that can function detrimentally in a stalled inflammatory phase characteristic of chronic wounds. Electrokinetic fiber surface properties of the biomaterials of this study were determined to correlate material charge and polarity with function relative to thrombin production and elastase sequestration. Human neutrophil elastase sequestration was assessed with an assay representative of chronic wound concentration with cotton gauze cross-linked with three types of polycarboxylic acids and one phosphorylation finish; thrombin production, which was assessed in a plasma-based assay via a fluorogenic peptide substrate, was determined for cotton, cotton-grafted chitosan, chitosan, rayon/polyester, and two kaolin-treated materials including a commercial hemorrhage control dressing (QuickClot Combat Gauze). A correlation in thrombin production to zeta potential was found. Two polycarboxylic acid cross linked and a phosphorylated cotton dressing gave high elastase sequestration. PMID:24956451

  12. Thrombin generation - a potentially useful biomarker of thrombotic risk in Philadelphia-negative myeloproliferative neoplasms. (United States)

    Mihaila, Romeo-Gabriel


    The diagnosis of essential thrombocythemia and polycythemia vera is often made during a thrombotic event which can be serious. Philadelphia-negative chronic myeloproliferative neoplasia patients have an increased thrombotic risk. This is assessed using various scoring systems but these are far from ideal and individual risk. The currend trend to personalised medicine requires finding the most useful thrombotic risk biomarker in these patients. Routine tests for coagulation do not take account of both pro- and anti-coagulant factors which is why these tests are not useful in patients with Philadelphia-negative myeloproliferative neoplasms. Thrombin generation reflects more accurately the balance between pro- and anti-coagulant factors. Some parameters of thrombin generation such as the endogenous thrombin potential are higher in Philadelphia-negative myeloproliferative neoplasm patients, especially in JAK2 V617F carriers than in healthy controls. They are even higher in those with reactive thrombocytosis. The JAK2 V617F allele burden correlates more with a higher thrombin generation potential in patients who are not treated with hydroxycarbamidum. Instead, JAK2 V617F-positive patients with Philadelphia-negative myeloproliferative neoplasms were the most sensitive to hydroxycarbamidum, as was reflected in lower values of platelet thrombin generation potential. The use of thrombin generation examination in these patients would enable detection of imminent thrombosis and personalised prophylactic management.

  13. Low thrombin generation predicts poor prognosis in ischemic stroke patients after thrombolysis. (United States)

    Hudák, Renáta; Székely, Edina G; Kovács, Katalin R; Nagy, Attila; Hofgárt, Gergely; Berényi, Ervin; Csiba, László; Kappelmayer, János; Bagoly, Zsuzsa


    Thrombolysis by intravenous recombinant tissue plasminogen activator (rt-PA) is an effective therapy in acute ischemic stroke (AIS). Thrombin generation test (TGT) is a global hemostasis test providing information about the speed and amount of generated thrombin in plasma. Here we aimed to find out whether results of this test before the initiation of thrombolysis might predict outcomes. Study population included 120 consecutive AIS patients, all within 4.5 hours of their symptom onset, who underwent thrombolysis by rt-PA. Blood samples were collected from all patients upon admission and TGT was performed using platelet poor plasma. Clinical data of patients including the NIHSS were registered at admission, day 1 and 7 after therapy. The ASPECT score was assessed using CT images taken before and 24 hours after thrombolysis. Long-term functional outcome was defined 3 months after the event by the modified Rankin Scale. Endogenous Thrombin Potential (ETP) and Peak Thrombin were significantly lower in patients with cardioembolic IS. Symptomatic intracranial hemorrhage (SICH) was found in 6 patients and was significantly associated with low ETP and Peak Thrombin levels. A multiple logistic regression model revealed that an ETP result in the lower quartile is an independent predictor of mortality within the first two weeks (OR: 6.03; 95%CI: 1.2-30.16, pThrombin parameters increase the risk of therapy associated SICH. A low ETP result is an independent predictor of short- and long-term mortality following thrombolysis.

  14. Cardiac troponin T degradation in serum is catalysed by human thrombin. (United States)

    Streng, Alexander S; de Boer, Douwe; van Doorn, William P T M; Kocken, Jordy M M; Bekers, Otto; Wodzig, Will K W H


    Cardiac troponin T (cTnT) has been shown to be present in fragmented forms in human serum after acute myocardial infarction (AMI). While calpain-1 and caspase-3 have been identified as intracellular proteases able to cleave the N-terminus of cTnT, it is still unclear which proteases are responsible for the extensive and progressive cTnT fragmentation observed in serum of AMI-patients. In this pilot study we have investigated the possibility that human thrombin may be involved in this process. Purified human cTnT was spiked in unprocessed and deproteinated serum in the presence or absence of either purified human thrombin or PPACK thrombin inhibitor. After immunoprecipitation, SDS-PAGE and Western blotting we observed an increase in cTnT fragmentation when purified thrombin was added to deproteinated serum. Consequently, the addition of thrombin inhibitor to unprocessed serum resulted in a decrease of cTnT fragmentation. Our results suggest that multiple enzymes are involved in cTnT degradation, and that thrombin plays an important role. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. A reusable aptasensor of thrombin based on DNA machine employing resonance light scattering technique. (United States)

    Hou, Yining; Liu, Jifeng; Hong, Min; Li, Xia; Ma, Yanhua; Yue, Qiaoli; Li, Chen-Zhong


    The design of molecular nanodevices attracted great interest in these years. Herein, a reusable, sensitive and specific aptasensor was constructed based on an extension-contraction movement of DNA interconversion for the application of human thrombin detection. The present biosensor was based on resonance light scattering (RLS) using magnetic nanoparticles (MNPs) as the RLS probe. MNPs coated with streptavidin can combine with biotin labeled thrombin aptamers. The combined nanoparticles composite is monodispersed in aqueous medium. When thrombin was added a sandwich structure can form on the surface of MNPs, which induced MNPs aggregation. RLS signal was therefore enhanced, and there is a linear relationship between RLS increment and thrombin concentration in the range of 60pM-6.0nM with a limit of detection at 3.5pM (3.29SB/m, according to the recent recommendation of IUPAC). The present aptasensor can be repeatedly used for at least 6 cycling times by heat to transfer G-quadruplex conformation to single strand of DNA sequence and release thrombin. MNPs can be captured by applying the external magnetic field. Furthermore, the proposed biosensor was successfully applied to detect thrombin in human plasma. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. The Phosphatase Inhibitor Calyculin-A Impairs Clot Retraction, Platelet Activation, and Thrombin Generation

    Directory of Open Access Journals (Sweden)

    Renáta Hudák


    Full Text Available The aim of this study was to investigate the effect of the serine/threonine protein phosphatase inhibitor, calyculin-A (CLA, on clot formation and on the procoagulant activity of human platelets. Platelet-rich plasma (PRP samples were preincubated with buffer or CLA and subsequently platelets were activated by the protease-activated receptor 1 (PAR-1 activator, thrombin receptor activating peptide (TRAP. Clot retraction was detected by observing clot morphology up to 1 hour, phosphatidylserine- (PS- expression was studied by flow cytometry, and thrombin generation was measured by a fluorimetric assay. For the intracellular Ca2+ assay, platelets were loaded with calcium-indicator dyes and the measurements were carried out using a ratiometric method with real-time confocal microscopy. CLA preincubation inhibited clot retraction, PS-expression, and thrombin formation. TRAP activation elicited Ca2+ response and PS-expression in a subset of platelets. The activated PRP displayed significantly faster and enhanced thrombin generation compared to nonactivated samples. CLA pretreatment abrogated PS-exposure and clot retraction also in TRAP-activated samples. As a consequence of the inhibitory effect on calcium elevation and PS-expression, CLA significantly downregulated thrombin generation in PRP. Our results show that CLA pretreatment may be a useful tool to investigate platelet activation mechanisms that contribute to clot formation and thrombin generation.

  17. Electrical Stimulus Controlled Binding/Unbinding of Human Thrombin-Aptamer Complex (United States)

    Gosai, Agnivo; Ma, Xiao; Balasubramanian, Ganesh; Shrotriya, Pranav


    The binding/unbinding of the human thrombin and its 15-mer single stranded DNA aptamer, under the application of external stimulus in the form of electrostatic potential/electric field, is investigated by a combination of continuum analysis and atomistic molecular dynamics simulation. In agreement with the experiments that demonstrate the influence of electrostatic potential on the thrombin/aptamer complex, our computations show that the application of positive electric field successfully unbinds the thrombin from the aptamer. Results from umbrella sampling simulations reveal that there is a decrease in the free energy of binding between the thrombin and aptamer in presence of positive electric fields. Hydrogen bonding and non-bonded interaction energies, and hence the free energy of binding, between the thrombin and its aptamer reduce as the applied electric field is shifted from negative to positive values. Our analyses demonstrate that application of electrical stimulus modifies the molecular interactions within the complex and consequently, electrical field can be used to modulate the association between the thrombin and its aptamer.

  18. Targeting thrombin long-term after an acute coronary syndrome: Opportunities and challenges. (United States)

    De Caterina, Raffaele; Goto, Shinya


    Patients after an acute coronary syndrome (ACS) are at increased risk of recurrent thrombotic events, justifying the search for additional antithrombotic treatments. The pathophysiology of ACS involves arterial thrombus formation, in turn occurring because of a combination of platelet activation and fibrin formation, with thrombin playing a key role in both. Antiplatelet therapy, targeting the thromboxane pathway and the ADP P2Y12 receptor has been widely accepted for secondary prevention after an ACS. Now, data from recent clinical trials in such patients also encourage the pursuit of inhibiting thrombin formation or thrombin-mediated platelet activation in addition to antiplatelet therapy. This "triple pathway inhibition", including inhibition of thrombin activity or thrombin receptor(s), is currently an option in pure ACS, but already a must in the setting of ACS accompanied by atrial fibrillation (AF), where anticoagulants have been shown to be much more effective than antiplatelet agents in preventing stroke. We here discuss the challenges of managing combined thrombin activity or receptor inhibition and antiplatelet therapy in all such patients. Translating this into practice still requires further studies and patient tailoring to fully exploit its potential. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Use of gelatin-thrombin matrix haemostatic sealant in neurosurgery: Anaesthetic implications and review of literature

    Directory of Open Access Journals (Sweden)

    Puneet Khanna


    Full Text Available The topical haemostatic agents have been developed to be used as adjunctive measures to promote haemostasis. These include bone wax, absorbable gel sponges, microfibrillar collagen, oxidised regenerated cellulose, gelatin sponges with thrombin, gelatin-thrombin matrix sealant or fibrin sealants. Gelatin-thrombin matrix sealant is a mixture of a bovine-derived gelatin matrix and human-derived thrombin component that are mixed together at the time of use. This agent has been found to be more effective haemostat than thrombin-soaked gelatine sponges. The possible adverse effects of this can be transmission of diseases from human or bovine sources, allergic reactions, thromboembolism, disseminated intravascular coagulopathy (DIC, perilesional oedema, and compression of neural tissue. Although it is used routinely in the operating room, there is little literature available on the perioperative implications with use of intraoperative gelatin-thrombin matrix sealant. Here, we present clinical report of 20 neurosurgical patients where the sealant was used and literature in view of current evidence has been reviewed.

  20. Active but inoperable thrombin is accumulated in a plasma protein layer surrounding Streptococcus pyogenes. (United States)

    Naudin, Clément; Hurley, Sinead M; Malmström, Erik; Plug, Tom; Shannon, Oonagh; Meijers, Joost C M; Mörgelin, Matthias; Björck, Lars; Herwald, Heiko


    Activation of thrombin is a critical determinant in many physiological and pathological processes including haemostasis and inflammation. Under physiological conditions many of these functions are involved in wound healing or eradication of an invading pathogen. However, when activated systemically, thrombin can contribute to severe and life-threatening conditions by causing complications such as multiple multi-organ failure and disseminated intravascular coagulation. In the present study we investigated how the activity of thrombin is modulated when it is bound to the surface of Streptococcus pyogenes. Our data show that S. pyogenes bacteria become covered with a proteinaceous layer when incubated with human plasma, and that thrombin is a constituent of this layer. Though the coagulation factor is found attached to the bacteria with a functional active site, thrombin has lost its capacity to interact with its natural substrates and inhibitors. Thus, the interaction of bacteria with human plasma renders thrombin completely inoperable at the streptococcal surface. This could represent a host defense mechanism to avoid systemic activation of coagulation which could be otherwise induced when bacteria enter the circulation and cause systemic infection.

  1. The Ca2+-mobilizing potency of alpha-thrombin and thrombin-receptor-activating peptide on human platelets -- concentration and time effects of thrombin-induced Ca2+ signaling.

    NARCIS (Netherlands)

    Heemskerk, J.W.M.; Feijge, M.A.H.; Henneman, L.; Rosing, J.; Hemker, H.C.


    Department of Biochemistry, Maastricht University, The Netherlands. In single platelets and in suspensions of platelets, alpha-thrombin evokes dose-dependent, transient increases in cytosolic Ca2+ concentration, [Ca2+]i, which are more prolonged than the [Ca2+]i

  2. Automation of interferometric observations (United States)

    Bester, M.; Degiacomi, C. G.; Danchi, W. C.; Greenhill, L. J.; Townes, C. H.

    The Infrared Spatial Interferometer (ISI) is a heterodyne interferometer that operates in the 9-12 micron atmospheric window. It is located at Mount Wilson and consists of two 1.65-m Pfund-type telescopes. Presently baselines range up to 35 m. Lately the performance of the ISI was improved significantly, providing higher quality interferometric data. The improvements include all-reflective front-end optics, larger bandwidth and higher quantum efficiency heterodyne detectors, a fringe calibration system, a CCD autoguiding system, and a more advanced computer control system. The newly developed control software allows the observations to be largely automated.

  3. Suomi NPP VIIRS Reflective Solar Bands Operational Calibration Reprocessing

    Directory of Open Access Journals (Sweden)

    Slawomir Blonski


    Full Text Available Radiometric calibration coefficients for the VIIRS (Visible Infrared Imaging Radiometer Suite reflective solar bands have been reprocessed from the beginning of the Suomi NPP (National Polar-orbiting Partnership mission until present. An automated calibration procedure, implemented in the NOAA (National Oceanic and Atmospheric Administration JPSS (Joint Polar Satellite System operational data production system, was applied to reprocess onboard solar calibration data and solar diffuser degradation measurements. The latest processing parameters from the operational system were used to include corrected solar vectors, optimized directional dependence of attenuation screens transmittance and solar diffuser reflectance, updated prelaunch calibration coefficients without an offset term, and optimized Robust Holt-Winters filter parameters. The parameters were consistently used to generate a complete set of the radiometric calibration coefficients for the entire duration of the Suomi NPP mission. The reprocessing has demonstrated that the automated calibration procedure can be successfully applied to all solar measurements acquired from the beginning of the mission until the full deployment of the automated procedure in the operational processing system. The reprocessed calibration coefficients can be further used to reprocess VIIRS SDR (Sensor Data Record and other data products. The reprocessing has also demonstrated how the automated calibration procedure can be used during activation of the VIIRS instruments on the future JPSS satellites.

  4. Can the diagnostic reliability of the thrombin generation test as a global haemostasis assay be improved? The impact of calcium chloride concentration. (United States)

    Parunov, L A; Surov, S S; Liang, Y; Lee, T K; Ovanesov, M V


    Thrombin generation test (TGT) is a global haemostasis assay with a potential to predict bleeding tendencies and treatment effects in patients with haemophilia. Despite 15 years of clinical research, the diagnostic value of TGT remains controversial, possibly due to suboptimal sensitivity to coagulation deficiencies, robustness and reproducibility. The goal of this study was to explore the effect of calcium chloride (CaCl2 ) concentration on the TGT's response to intrinsic coagulation factors (F) VIII, IX and XIa. Normal and factor-deficient plasmas supplemented with lacking coagulation factor and different CaCl2 levels were tested by calibrated thrombinography assay. Thrombin peak height (TPH) was strongly CaCl2 dependent, increasing sharply from no TG at 5 mm to a peak at 13.8 mm of CaCl2 (95% confidence interval [CI]: 13.0, 14.5) in normal and normalized deficient plasmas and at 11.9 mm (CI: 9.7, 14.2) in deficient plasmas, and then decreasing slowly to a complete inhibition at 30-40 mm. In contrast, TG lag time, time to peak and endogenous thrombin potential were nearly insensitive to CaCl2 concentrations between 10 and 20 mm. The maximal difference between the TPH in deficient and supplemented plasmas was observed at 15.5 mm (CI: 12.8, 18.1). Variations in CaCl2 concentration in the assay mixture and sodium citrate concentrations in patient plasma samples may affect TGT responses, sensitivity and result in increased inter- and intra-laboratory variance. Implementation of TGT by clinical and quality control laboratories may require optimization of CaCl2 concentration. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  5. Library Automation


    Dhakne, B. N.; Giri, V. V; Waghmode, S. S.


    New technologies library provides several new materials, media and mode of storing and communicating the information. Library Automation reduces the drudgery of repeated manual efforts in library routine. By use of library automation collection, Storage, Administration, Processing, Preservation and communication etc.

  6. A colorimetric aptamer biosensor based on cationic polymer and gold nanoparticles for the ultrasensitive detection of thrombin. (United States)

    Chen, Zhengbo; Tan, Yuan; Zhang, Chenmeng; Yin, Lu; Ma, He; Ye, Nengsheng; Qiang, Hong; Lin, Yuqing


    A colorimetric assay for the ultrasensitive determination of thrombin based on cationic polymer and gold nanoparticles was presented, in which unmodified gold nanoparticles (AuNPs) was used as probes and 21-mer thrombin-binding aptamer (TBA) as sensing elements. Upon the addition of thrombin, TBA interacted specifically with thrombin to form a G-quadruplex structure. As a result, the conformation change facilitated the cationic polymer, poly(diallyldimethylammonium chloride) (PDDA)-induced AuNP aggregation. Thus, the visible change in color from wine-red to blue-purple was readily seen by the naked eye. The colorimetric sensor could detect thrombin down to 1 pM with high selectivity in the presence of other interferring proteins. Furthermore, the assay was successfully employed to determine thrombin in human serum sample, which suggested its great potential for diagnostic purposes. Copyright © 2014 Elsevier B.V. All rights reserved.


    Kovach, Ildiko M.; Kakalis, Lazaros; Jordan, Frank; Zhang, Daoning


    Thrombin is the pivotal serine protease enzyme in the blood cascade system and thus a target of drug design for control of its activity. The most efficient non-physiologic inhibitor of thrombin is hirudin, a naturally occurring small protein. Hirudin and its synthetic mimics employ a range of hydrogen bonding, salt bridging and hydrophobic interactions with thrombin to achieve tight binding with Ki values in the nano- to femtomolar range. The one-dimensional 1H NMR spectrum carried out at 600 MHz reveals a resonance at 15.33 ppm downfield from silanes in complexes between human α-thrombin and r-hirudin in pH 5.6-8.8 buffers and between 5 and 35 °C. There is also a resonance between 15.17 and 15.54 ppm seen in human α-thrombin complexes with hirunorm IV, hirunorm V, an Nα(Me)Arg-peptide, RGD-hirudin and Nα-2-naphthylsulfonyl-glycyl-DL-4-amidinophenylalanyl-piperidide acetate salt (NAPAP), while there is no such low-field resonance observed in a complex of porcine trypsin and NAPAP. The chemical shifts suggest that these resonances represent H-bonded environments. H-donor acceptor distances in the corresponding H-bonds are estimated to be thrombin with r-hirudin results in an additional signal at 18.03 ppm, which is 0.10 ppm upfield from one observed (Kovach, I. M. et al. Biochemistry 2009, 48, 7296–7304) for thrombin covalently modified with PPACK. In contrast, the peak at 15.33 ppm remains unchanged. The fractionation factors for the thrombin-hirudin type complexes are near 1.0 within 20% error. The most likely site of the short H-bond in thrombin complexes with the hirudin family of inhibitors is in the hydrophobic patch of the C-terminus of hirudin where Glu57’ and Glu58’ are embedded and interact with Arg75 and Arg77 and their solvate water (on thrombin). Glu57’ and Glu58’ present in the hirudin family of inhibitors is a key binding epitope of fibrinogen, thrombin’s prime substrate, which lends substantial interest to the SHB as a binding

  8. Thrombin induced by the extrinsic pathway and PAR-1 regulated inflammation at the site of fracture repair. (United States)

    Sato, Nobutaka; Ichikawa, Jiro; Wako, Masanori; Ohba, Tetsuro; Saito, Masanori; Sato, Hironao; Koyama, Kensuke; Hagino, Tetsuo; Schoenecker, Jonathan G; Ando, Takashi; Haro, Hirotaka


    Thrombin (coagulation factor IIa) is a serine protease encoded by the F2 gene. Pro-thrombin (coagulation factor II) is cut to generate thrombin in the coagulation cascade that results in a reduction of blood loss. Procoagulant states that lead to activation of thrombin are common in bone fracture sites. However, its physiological roles and relationship with osteoblasts in bone fractures are largely unknown. We herein report various effects of thrombin on mouse osteoblastic MC3T3-E1 cells. MC3T3-E1 cells expressed proteinase-activated receptor 1 (PAR1), also known as the coagulation factor II receptor. They also produced monocyte chemoattractant protein (MCP-1), tissue factor (TF), MCSF and IL-6 upon thrombin stimulation through the PI3K-Akt and MEK-Erk1/2 pathways. Furthermore, MCP-1 obtained from thrombin-stimulated MC3T3-E1 cells induced migration by macrophage RAW264 cells. All these effects of thrombin on MC3T3-E1 cells were abolished by the selective non-peptide thrombin receptor inhibitor SCH79797. We also found that thrombin, PAR-1, MCP-1, TF as well as phosphorylated AKT and p42/44 were significantly expressed at the fracture site of mouse femoral bone. Collectively, thrombin/PAR-1 interaction regulated MCP-1, TF, MCSF and IL-6 production by MC3T3-E1 cells. Furthermore, MCP-1 induced RAW264 cell migration. Thrombin may thus be a novel cytokine that regulates several aspects of osteoblast function and fracture healing. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Victor Constantin Diculescu


    Full Text Available 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.

  10. An electrochemical label-free and sensitive thrombin aptasensor based on graphene oxide modified pencil graphite electrode. (United States)

    Ahour, F; Ahsani, M K


    In this work, we tactfully constructed a novel label-free electrochemical aptasensor for rapid and facile detection of thrombin using graphene oxide (GO) and thrombin binding aptamer (TBA). The strategy relies on the preferential adsorption of single-stranded DNA (ssDNA) to GO over aptamer-target complexes. The TBA-thrombin complex formation was monitored by differential pulse voltammetry (DPV) using the guanine oxidation signal. In the absence of thrombin, the aptamers adsorbed onto the surface of GO leading to a strong background guanine oxidation signal. Conversely, in the presence of thrombin, the conformational transformation of TBA after incubating with the thrombin solution and formation of the aptamer-thrombin complexes which had weak binding ability to GO, leads to the desorption of TBA-thrombin complex from electrode surface and significant oxidation signal decrease. The selectivity of the biosensor was studied using other biological substances. The biosensor's signal was proportional to the thrombin concentration from 0.1 to 10nM with a detection limit of 0.07nM. Particularly, the proposed method could be widely applied to the aptamer-based determination of other target analytes. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Heparin Binds Lamprey Angiotensinogen and Promotes Thrombin Inhibition through a Template Mechanism. (United States)

    Wei, Hudie; Cai, Haiyan; Wu, Jiawei; Wei, Zhenquan; Zhang, Fei; Huang, Xin; Ma, Lina; Feng, Lingling; Zhang, Ruoxi; Wang, Yunjie; Ragg, Hermann; Zheng, Ying; Zhou, Aiwu


    Lamprey angiotensinogen (l-ANT) is a hormone carrier in the regulation of blood pressure, but it is also a heparin-dependent thrombin inhibitor in lamprey blood coagulation system. The detailed mechanisms on how angiotensin is carried by l-ANT and how heparin binds l-ANT and mediates thrombin inhibition are unclear. Here we have solved the crystal structure of cleaved l-ANT at 2.7 Å resolution and characterized its properties in heparin binding and protease inhibition. The structure reveals that l-ANT has a conserved serpin fold with a labile N-terminal angiotensin peptide and undergoes a typical stressed-to-relaxed conformational change when the reactive center loop is cleaved. Heparin binds l-ANT tightly with a dissociation constant of ∼10 nm involving ∼8 monosaccharides and ∼6 ionic interactions. The heparin binding site is located in an extensive positively charged surface area around helix D involving residues Lys-148, Lys-151, Arg-155, and Arg-380. Although l-ANT by itself is a poor thrombin inhibitor with a second order rate constant of 500 m(-1) s(-1), its interaction with thrombin is accelerated 90-fold by high molecular weight heparin following a bell-shaped dose-dependent curve. Short heparin chains of 6-20 monosaccharide units are insufficient to promote thrombin inhibition. Furthermore, an l-ANT mutant with the P1 Ile mutated to Arg inhibits thrombin nearly 1500-fold faster than the wild type, which is further accelerated by high molecular weight heparin. Taken together, these results suggest that heparin binds l-ANT at a conserved heparin binding site around helix D and promotes the interaction between l-ANT and thrombin through a template mechanism conserved in vertebrates. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. FAK phosphorylation plays a central role in thrombin-induced RPE cell migration. (United States)

    Aguilar-Solis, E D; Lee-Rivera, I; Álvarez-Arce, A; López, E; López-Colomé, A M


    The migration of retinal pigment epithelial (RPE) cells is an important step in various pathologic conditions including subretinal neovascularization (SRN), proliferative vitreoretinopathy (PVR) and, importantly, as a consequence of retinal surgery. Therefore, the elucidation of the mechanisms underlying RPE trans-differentiation and migration is essential for devising effective treatments aimed to the prevention of these disorders. A common event in these pathologies is the alteration of the blood-retina barrier (BRB), which allows the interaction of RPE cells with thrombin, a pro-inflammatory protease contained in serum. Our previous work has demonstrated that thrombin induces RPE cell cytoskeletal remodeling and migration, hallmark processes in the development of PVR; however, the molecular mechanisms involved are still unclear. Cell migration requires the disassembly of focal adhesions induced by Focal Adhesion Kinase (FAK) phosphorylation, together with the formation of actin stress fibers. The aim of the present work was to identify thrombin-activated signaling pathways leading to FAK phosphorylation and to determine FAK participation in thrombin-induced RPE cell migration. Results demonstrate that the activation of PAR1 by thrombin induces FAK autophosphorylation at Y397 and the subsequent phosphorylation of Y576/577 within the activation loop. FAK phosphorylation was shown to be under the control of c/nPKC and PI3K/PKC-ζ, as well as by Rho/ROCK, since the inhibition of these pathways prevented thrombin-induced FAK phosphorylation and the consequent disassembly of focal adhesions, in parallel to FAK-dependent actin stress fiber formation and RPE cell migration. These findings demonstrate, for the first time, that thrombin stimulation of RPE cell transformation and migration are regulated by FAK tyrosine phosphorylation. Thus, targeting FAK phosphorylation may provide a strategical basis for PVR treatment. Copyright © 2017. Published by Elsevier Inc.

  13. Traceable Pyrgeometer Calibrations

    Energy Technology Data Exchange (ETDEWEB)

    Dooraghi, Mike; Kutchenreiter, Mark; Reda, Ibrahim; Habte, Aron; Sengupta, Manajit; Andreas, Afshin; Newman, Martina


    This poster presents the development, implementation, and operation of the Broadband Outdoor Radiometer Calibrations (BORCAL) Longwave (LW) system at the Southern Great Plains Radiometric Calibration Facility for the calibration of pyrgeometers that provide traceability to the World Infrared Standard Group.

  14. Marketing automation

    National Research Council Canada - National Science Library

    Raluca Dania Todor


      The automation of the marketing process seems to be nowadays, the only solution to face the major changes brought by the fast evolution of technology and the continuous increase in supply and demand...

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

    Directory of Open Access Journals (Sweden)

    Jayavel Arumugam

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

  16. Recovery from trauma induced amnesia correlates with normalization of thrombin activity in the mouse hippocampus. (United States)

    Ben Shimon, Marina; Zeimer, Talya; Shavit Stein, Efrat; Artan-Furman, Avital; Harnof, Sagi; Chapman, Joab; Eisenkraft, Arik; Pick, Chaim G; Maggio, Nicola


    Transient amnesia is a common consequence of minimal traumatic brain injury (mTBI). However, while recent findings have addressed the mechanisms involved in its onset, the processes contributing to its recovery have not yet been addressed. Recently, we have found that thrombin is detected at high concentrations in the brain of mice after exposure to mTBI and that in such settings amnesia is rescued by either inhibiting thrombin activity or by blockade of PAR1. Here, we report that mice spontaneously recover from amnesia after two weeks from mTBI exposure. At this time point, long term potentiation was equally evoked in injured vs. control animals with thrombin concentration in the brain being normalized at this stage. These findings, which refer to the specific aspect of memory retrieval upon mTBI, together with our previous work, hint to a strong correlation between cognitive defects in the context of mTBI and thrombin concentrations in the brain. This may suggest that a possible scavenging of thrombin in the brain at early phases following mTBI may improve memory function.

  17. A sensitive nanoporous gold-based electrochemical aptasensor for thrombin detection. (United States)

    Qiu, Huajun; Sun, Yanli; Huang, Xirong; Qu, Yinbo


    An attempt was made in the present paper to develop a nanoporous gold (NPG)-based electrochemical aptasensor for thrombin detection. The substrate electrode NPG was in situ fabricated by a facile one-step square wave potential pulse (SWPP) treatment. The treatment involved repeated gold oxidation-reduction and intensive H(2) bubbles evolution. After 100min treatment, the active surface area of Au increased greatly (34 times). The electrochemical aptasensor was fabricated using a layer-by-layer assembling strategy. A "sandwich" structure was formed via thrombin connecting the aptamer-modified NPG and the aptamer-modified Au nanoparticles (AuNPs). The AuNPs was modified with two kinds of single strand DNA (ssDNA). One was aptamer of thrombin, but the other was not, reducing the cross-reaction between thrombin and its aptamer on the same AuNP. The electrochemical signal produced by the [Ru(NH(3))(6)](3+) bound to ssDNA via electrostatic interaction was measured by chronocoulometry. Due to the amplification effects of both NPG and AuNPs, this novel NPG-based aptasensor could detect thrombin quantitatively in the range of 0.01-22nM with a detection limit as low as 30fM. The present aptasensor also exhibited excellent selectivity, stability and reusability. Copyright 2010 Elsevier B.V. All rights reserved.

  18. Elevated thrombin-forming capacity of tissue factor-activated cord compared with adult plasma. (United States)

    Cvirn, G; Gallistl, S; Rehak, T; Jürgens, G; Muntean, W


    Clinically observed excellent hemostasis in neonates despite low levels of clotting factors is not completely understood so far. Therefore, we investigated whether physiological low levels of the inhibitor protein C (PC) facilitate thrombin formation in tissue factor (TF)-activated plasma samples. PC was activated by endogenously generated thrombin after addition of soluble thrombomodulin (TM). The capability of activated PC (APC) to suppress thrombin formation was significantly more pronounced in adult than in cord plasma. Addition of 4 nm of TM decreased the thrombin potential (TP) in cord plasma by 10%, and in adult plasma by 52% in the presence of 5 pm TF. We demonstrate that this low anticoagulant action of PC is attributable to the low levels of tissue factor pathway inhibitor (TFPI) and antithrombin (AT) physiologically present in cord plasma. Addition of 4 nm TM decreased the TP by 58% in cord plasma adjusted to contain TFPI and AT at adult levels in the presence of 5 pm TF. Thus, the combined low anticoagulant action of the three inhibitors APC, TFPI, and AT in cord plasma allows enhanced thrombin formation associated with shorter clotting times compared with adult plasma when low amounts of TF are applied to initiate clot formation. Although our laboratory experiments do not allow definite conclusions for various clinical situations, our data might contribute to explain excellent hemostasis in neonates despite low levels of procoagulants.

  19. Bufadienolides from Kalanchoe daigremontiana as thrombin inhibitors-In vitro and in silico study. (United States)

    Kolodziejczyk-Czepas, Joanna; Sieradzka, Malgorzata; Moniuszko-Szajwaj, Barbara; Pecio, Łukasz; Ponczek, Michal B; Nowak, Pawel; Stochmal, Anna


    Thrombin is an active plasma coagulation factor II, critical for the formation of fibrin clot during blood coagulation. For that reason, this protein is also a crucial target for different anti-thrombotic therapies. The work is based on in vitro evaluation of the inhibitory effect of bufadienolide-rich fraction, isolated from roots of Kalanchoe daigremontiana (1-50μg/ml) on enzymatic properties of a serine proteinase - thrombin. The efficacy of the inhibition of amidolytic activity of thrombin (measured as a hydrolysis of the chromogenic substrate S-2238, Chromogenix) attained about 10 and 66%, respectively. The IC50, established for the examined bufadienolide fraction was 2.79μg/ml, while the IC50 calculated for argatroban (reference compound) was 0.78μg/ml. Linearization conducted using Lineweaver-Burk plot indicated that the K. daigremontiana fraction contains compounds that are uncompetitive inhibitors of thrombin. K. daigremontiana fraction was also able to reduce the proteolytic activity of thrombin towards its physiological substrate, i.e. fibrinogen. Additionally, this study is supported by in silico analysis of interactions of the most common compounds, identified in the examined in Kalanchoe extract to crystal structure of this enzyme. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Simulated Thrombin Generation in the Presence of Surface-Bound Heparin and Circulating Tissue Factor. (United States)

    Dydek, E Victoria; Chaikof, Elliot L


    An expanded computational model of surface induced thrombin generation was developed that includes hemodynamic effects, 22 biochemical reactions and 44 distinct chemical species. Surface binding of factors V, VIII, IX, and X was included in order to more accurately simulate the formation of the surface complexes tenase and prothrombinase. In order to model these reactions, the non-activated, activated and inactivated forms were all considered. This model was used to investigate the impact of surface bound heparin on thrombin generation with and without the additive effects of thrombomodulin (TM). In total, 104 heparin/TM pairings were evaluated (52 under venous conditions, 52 under arterial conditions), the results demonstrating the synergistic ability of heparin and TM to reduce thrombin generation. Additionally, the role of circulating tissue factor (TF(p)) was investigated and compared to that of surface-bound tissue factor (TF(s)). The numerical results suggest that circulating TF has the power to amplify thrombin generation once the coagulation cascade is already initiated by surface-bound TF. TF(p) concentrations as low as 0.01 nM were found to have a significant impact on total thrombin generation.

  1. Thrombin promotes diet-induced obesity through fibrin-driven inflammation. (United States)

    Kopec, Anna K; Abrahams, Sara R; Thornton, Sherry; Palumbo, Joseph S; Mullins, Eric S; Divanovic, Senad; Weiler, Hartmut; Owens, A Phillip; Mackman, Nigel; Goss, Ashley; van Ryn, Joanne; Luyendyk, James P; Flick, Matthew J


    Obesity promotes a chronic inflammatory and hypercoagulable state that drives cardiovascular disease, type 2 diabetes, fatty liver disease, and several cancers. Elevated thrombin activity underlies obesity-linked thromboembolic events, but the mechanistic links between the thrombin/fibrin(ogen) axis and obesity-associated pathologies are incompletely understood. In this work, immunohistochemical studies identified extravascular fibrin deposits within white adipose tissue and liver as distinct features of mice fed a high-fat diet (HFD) as well as obese patients. Fibγ390-396A mice carrying a mutant form of fibrinogen incapable of binding leukocyte αMβ2-integrin were protected from HFD-induced weight gain and elevated adiposity. Fibγ390-396A mice had markedly diminished systemic, adipose, and hepatic inflammation with reduced macrophage counts within white adipose tissue, as well as near-complete protection from development of fatty liver disease and glucose dysmetabolism. Homozygous thrombomodulin-mutant ThbdPro mice, which have elevated thrombin procoagulant function, gained more weight and developed exacerbated fatty liver disease when fed a HFD compared with WT mice. In contrast, treatment with dabigatran, a direct thrombin inhibitor, limited HFD-induced obesity development and suppressed progression of sequelae in mice with established obesity. Collectively, these data provide proof of concept that targeting thrombin or fibrin(ogen) may limit pathologies in obese patients.

  2. Marine Diterpenes: Molecular Modeling of Thrombin Inhibitors with Potential Biotechnological Application as an Antithrombotic

    Directory of Open Access Journals (Sweden)

    Rebeca Cristina Costa Pereira


    Full Text Available Thrombosis related diseases are among the main causes of death and incapacity in the world. Despite the existence of antithrombotic agents available for therapy, they still present adverse effects like hemorrhagic risks which justify the search for new options. Recently, pachydictyol A, isopachydictyol A, and dichotomanol, three diterpenes isolated from Brazilian marine brown alga Dictyota menstrualis were identified as potent antithrombotic molecules through inhibition of thrombin, a key enzyme of coagulation cascade and a platelet agonist. Due to the biotechnological potential of these marine metabolites, in this work we evaluated their binding mode to thrombin in silico and identified structural features related to the activity in order to characterize their molecular mechanism. According to our theoretical studies including structure-activity relationship and molecular docking analysis, the highest dipole moment, polar surface area, and lowest electronic density of dichotomanol are probably involved in its higher inhibition percentage towards thrombin catalytic activity compared to pachydictyol A and isopachydictyol A. Interestingly, the molecular docking studies also revealed a good shape complementarity of pachydictyol A and isopachydictyol A and interactions with important residues and regions (e.g., H57, S195, W215, G216, and loop-60, which probably justify their thrombin inhibitor effects demonstrated in vitro. Finally, this study explored the structural features and binding mode of these three diterpenes in thrombin which reinforced their potential to be further explored and may help in the design of new antithrombotic agents.

  3. Platelet-derived microparticles regulates thrombin generation via phophatidylserine in abdominal sepsis. (United States)

    Wang, Yongzhi; Zhang, Su; Luo, Lingtao; Norström, Eva; Braun, Oscar Ö; Mörgelin, Matthias; Thorlacius, Henrik


    Sepsis is associated with dysfunctional coagulation. Recent data suggest that platelets play a role in sepsis by promoting neutrophil accumulation. Herein, we show that cecal ligation and puncture (CLP) triggered systemic inflammation, which is characterized by formation of IL-6 and CXC chemokines as well as neutrophil accumulation in the lung. Platelet depletion decreased neutrophil accumulation, IL-6, and CXC chemokines formation in septic lungs. Depletion of platelets increased peak thrombin formation and total thrombin generation (TG) in plasma from septic animals. CLP elevated circulating levels of platelet-derived microparticles (PMPs). In vitro generated PMPs were a potent inducer of TG. Interestingly, in vitro wild-type recombinant annexin V abolished PMP-induced thrombin formation whereas a mutant annexin V protein, which does not bind to phosphatidylserine (PS), had no effect. Administration of wild-type, but not mutant annexin V, significantly inhibited thrombin formation in septic animals. Moreover, CLP-induced formation of thrombin-antithrombin complexes were reduced in platelet-depleted mice and in animals pretreated with annexin V. PMP-induced TG attenuated in FXII- and FVII-deficient plasma. These findings suggest that sepsis-induced TG is dependent on platelets. Moreover, PMPs formed in sepsis are a potent inducer of TG via PS exposure, and activation of both the intrinsic and extrinsic pathway of coagulation. In conclusion, these observations suggest that PMPs and PS play an important role in dysfunctional coagulation in abdominal sepsis. © 2017 Wiley Periodicals, Inc.

  4. In vivo-generated thrombin and plasmin do not activate the complement system in baboons. (United States)

    Keshari, Ravi S; Silasi, Robert; Lupu, Cristina; Taylor, Fletcher B; Lupu, Florea


    Sepsis concurrently activates both coagulation and complement systems. Although complement activation by bacteria is well documented, work in mice and in vitro suggests that coagulation proteases can directly cleave complement proteins. We aimed to determine whether generation of coagulation proteases in vivo can activate the complement cascade in 2 highly coagulopathic models. We compared temporal changes in activation biomarkers of coagulation (thrombin-antithrombin [TAT]), fibrinolysis (plasmin-antiplasmin [PAP]), and complement (C3b, C5a, C5b-9) in baboons infused with factor Xa (FXa) and phospholipids (FXa/phosphatidylcholine-phosphatidylserine [PCPS]) vs LD100 Escherichia coli We found that, albeit with different timing, both FXa/PCPS and E coli infusion led to robust thrombin and plasmin generation. Conversely, only E coli challenge activated the complement system, reaching a maximum at 2 hours postchallenge during the peaks of lipopolysaccharide and bacteremia but not of TAT and PAP. Despite inducing a strong burst of thrombin and plasmin, FXa/PCPS infusion did not produce measurable levels of complement activation in vivo. Similarly, ex vivo incubation of baboon serum with thrombin, plasmin, or FXa did not show noticeable complement cleavage unless supraphysiologic amounts of enzymes were used. Our results suggest that in vivo-generated thrombin and plasmin do not directly activate the complement in nonhuman primates. © 2017 by The American Society of Hematology.

  5. Allosteric changes in solvent accessibility observed in thrombin upon active site occupation. (United States)

    Croy, Carrie Hughes; Koeppe, Julia R; Bergqvist, Simon; Komives, Elizabeth A


    The solvent accessibility of thrombin in its substrate-free and substrate-bound forms has been compared by amide hydrogen/deuterium (H/(2)H) exchange. The optimized inhibitor peptide dPhe-Pro-Arg chloromethyl ketone (PPACK) was used to simulate the substrate-bound form of thrombin. These studies were motivated by the lack of observed changes in the active site of thrombin in the crystal structure of the thrombin-thrombomodulin complex. This result appeared to contradict amide exchange studies on the thrombin-thrombomodulin complex that suggested subtle changes occur in the active site loops upon thrombomodulin binding. Our results show that two active site loops, residues 214-222 and residues 126-132, undergo decreases in solvent accessibility due to steric contacts with PPACK substrate. However, we also observe two regions outside the active site undergoing solvent protection upon substrate binding. The first region corresponds to anion binding exosite 1, and the second is a beta-strand-containing loop which runs through the core of the molecule and contains Trp141 which makes critical contacts with anion binding exosite 1. These results indicate two pathways of allosteric change that connect the active site to the distal anion binding exosite 1.

  6. Thrombin-Activatable Fibrinolysis Inhibitor in Chronic Thromboembolic Pulmonary Hypertension. (United States)

    Yaoita, Nobuhiro; Satoh, Kimio; Satoh, Taijyu; Sugimura, Koichiro; Tatebe, Shunsuke; Yamamoto, Saori; Aoki, Tatsuo; Miura, Masanobu; Miyata, Satoshi; Kawamura, Takeshi; Horiuchi, Hisanori; Fukumoto, Yoshihiro; Shimokawa, Hiroaki


    The pathogenesis of chronic thromboembolic pulmonary hypertension (CTEPH) remains to be elucidated. Thrombin-activatable fibrinolysis inhibitor (TAFI) inhibits fibrinolysis. It remains to be elucidated whether TAFI is directly involved in the pathogenesis of CTEPH. We examined potential involvement of TAFI in the pathogenesis of CTEPH in humans. We enrolled 68 consecutive patients undergoing right heart catheterization in our hospital, including those with CTEPH (n=27), those with pulmonary arterial hypertension (n=22), and controls (non-pulmonary hypertension, n=19). Whole blood clot lysis assay showed that the extent of clot remaining after 4 hours was significantly higher in CTEPH compared with pulmonary arterial hypertension or controls (41.9 versus 26.5 and 24.6%, both P<0.01). Moreover, plasma levels of TAFI were significantly higher in CTEPH than in pulmonary arterial hypertension or controls (19.4±4.2 versus 16.1±4.5 or 16.3±3.3 μg/mL, both P<0.05), which remained unchanged even after hemodynamic improvement by percutaneous transluminal pulmonary angioplasty. Furthermore, the extent of clot remaining after 4 hours was significantly improved with CPI-2KR (an inhibitor of activated TAFI) or prostaglandin E1 (an inhibitor of activation of platelets). Importantly, plasma levels of TAFI were significantly correlated with the extent of clot remaining after 4 hours. In addition, the extent of clot remaining after 4 hours was improved with an activated TAFI inhibitor. These results indicate that plasma levels of TAFI are elevated in patients with CTEPH and are correlated with resistance to clot lysis in those patients. © 2016 American Heart Association, Inc.

  7. Parameterizations for reducing camera reprojection error for robot-world hand-eye calibration (United States)

    Accurate robot-world, hand-eye calibration is crucial to automation tasks. In this paper, we discuss the robot-world, hand-eye calibration problem which has been modeled as the linear relationship AX equals ZB, where X and Z are the unknown calibration matrices composed of rotation and translation ...

  8. A global model for residential energy use: Uncertainty in calibration to regional data

    NARCIS (Netherlands)

    van Ruijven, B.; de Vries, B.|info:eu-repo/dai/nl/068361599; van Vuuren, D.P.; van der Sluijs, J.P.|info:eu-repo/dai/nl/073427489


    Uncertainties in energy demand modelling allow for the development of different models, but also leave room for different calibrations of a single model. We apply an automated model calibration procedure to analyse calibration uncertainty of residential sector energy use modelling in the TIMER 2.0

  9. Endothelial Angiogenesis and Barrier Function in Response to Thrombin Require Ca2+ Influx through the Na+/Ca2+ Exchanger* (United States)

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


    Thrombin acts on the endothelium by activating protease-activated receptors (PARs). The endothelial thrombin-PAR system becomes deregulated during pathological conditions resulting in loss of barrier function and a pro-inflammatory and pro-angiogenic endothelial phenotype. We reported recently that the ion transporter Na+/Ca2+ exchanger (NCX) operating in the Ca2+-influx (reverse) mode promoted ERK1/2 activation and angiogenesis in vascular endothelial growth factor-stimulated primary human vascular endothelial cells. Here, we investigated whether Ca2+ influx through NCX was involved in ERK1/2 activation, angiogenesis, and endothelial barrier dysfunction in response to thrombin. Reverse-mode NCX inhibitors and RNAi-mediated NCX1 knockdown attenuated ERK1/2 phosphorylation in response to thrombin or an agonist of PAR-1, the main endothelial thrombin receptor. Conversely, promoting reverse-mode NCX by suppressing Na+-K+-ATPase activity enhanced ERK1/2 activation. Reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced primary human vascular endothelial cell angiogenesis, quantified as proliferation and tubular differentiation. Reverse-mode NCX inhibitors or NCX1 knockdown preserved barrier integrity upon thrombin stimulation in vitro. Moreover, the reverse-mode NCX inhibitor SEA0400 suppressed Evans' blue albumin extravasation to the lung and kidneys and attenuated edema formation and ERK1/2 activation in the lungs of mice challenged with a peptide activator of PAR-1. Mechanistically, thrombin-induced ERK1/2 activation required NADPH oxidase 2-mediated reactive oxygen species (ROS) production, and reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced ROS production. We propose that reverse-mode NCX is a novel mechanism contributing to thrombin-induced angiogenesis and hyperpermeability by mediating ERK1/2 activation in a ROS-dependent manner. Targeting reverse-mode NCX could be beneficial in pathological conditions involving

  10. Thrombin induces heme oxygenase-1 expression in human synovial fibroblasts through protease-activated receptor signaling pathways (United States)


    Introduction Thrombin is a key factor in the stimulation of fibrin deposition, angiogenesis, and proinflammatory processes. Abnormalities in these processes are primary features of osteoarthritis (OA). Heme oxygenase (HO)-1 is a stress-inducible rate-limiting enzyme in heme degradation that confers cytoprotection against oxidative injury. Here, we investigated the intracellular signaling pathways involved in thrombin-induced HO-1 expression in human synovial fibroblasts (SFs). Methods Thrombin-mediated HO-1 expression was assessed with quantitative real-time (q)PCR. The mechanisms of action of thrombin in different signaling pathways were studied by using Western blotting. Knockdown of protease-activated receptor (PAR) proteins was achieved by transfection with siRNA. Chromatin immunoprecipitation assays were used to study in vivo binding of Nrf2 to the HO-1 promoter. Transient transfection was used to examine HO-1 activity. Results Osteoarthritis synovial fibroblasts (OASFs) showed significant expression of thrombin, and expression was higher than in normal SFs. OASFs stimulation with thrombin induced concentration- and time-dependent increases in HO-1 expression. Pharmacologic inhibitors or activators and genetic inhibition by siRNA of protease-activated receptors (PARs) revealed that the PAR1 and PAR3 receptors, but not the PAR4 receptor, are involved in thrombin-mediated upregulation of HO-1. Thrombin-mediated HO-1 expression was attenuated by thrombin inhibitor (PPACK), PKCδ inhibitor (rottlerin), or c-Src inhibitor (PP2). Stimulation of cells with thrombin increased PKCδ, c-Src, and Nrf2 activation. Conclusion Our results suggest that the interaction between thrombin and PAR1/PAR3 increases HO-1 expression in human synovial fibroblasts through the PKCδ, c-Src, and Nrf2 signaling pathways. PMID:22541814

  11. Thrombin impairs human endometrial endothelial angiogenesis; implications for progestin-only contraceptive-induced abnormal uterine bleeding. (United States)

    Shapiro, John P; Guzeloglu-Kayisli, Ozlem; Kayisli, Umit A; Semerci, Nihan; Huang, S Joseph; Arlier, Sefa; Larsen, Kellie; Fadda, Paolo; Schatz, Frederick; Lockwood, Charles J


    Progestin-only contraceptives induce abnormal uterine bleeding, accompanied by prothrombin leakage from dilated endometrial microvessels and increased thrombin generation by human endometrial stromal cell (HESC)-expressed tissue factor. Initial studies of the thrombin-treated HESC secretome identified elevated levels of cleaved chondroitin sulfate proteoglycan 4 (CSPG4), impairing pericyte-endothelial interactions. Thus, we investigated direct and CSPG4-mediated effects of thrombin in eliciting abnormal uterine bleeding by disrupting endometrial angiogenesis. Liquid chromatography/tandem mass spectrometry, enzyme-linked immunosorbent assay (ELISA) and quantitative real-time-polymerase chain reaction (PCR) evaluated conditioned medium supernatant and cell lysates from control versus thrombin-treated HESCs. Pre- and post-Depo medroxyprogesterone acetate (DMPA)-administered endometria were immunostained for CSPG4. Proliferation, apoptosis and tube formation were assessed in human endometrial endothelial cells (HEECs) incubated with recombinant human (rh)-CSPG4 or thrombin or both. Thrombin induced CSPG4 protein expression in cultured HESCs as detected by mass spectrometry and ELISA (p<.02, n=3). Compared to pre-DMPA endometria (n=5), stromal cells in post-DMPA endometria (n=5) displayed stronger CSPG4 immunostaining. In HEEC cultures (n=3), total tube-formed mesh area was significantly higher in rh-CSPG4 versus control (p<.05). However, thrombin disrupted HEEC tube formation by a concentration- and time-dependent reduction of angiogenic parameters (p<.05), whereas CSPG4 co-treatment did not reverse these thrombin-mediated effects. These results suggest that disruption of HEEC tube formation by thrombin induces aberrant angiogenesis and abnormal uterine bleeding in DMPA users. Mass spectrometry analysis identified several HESC-secreted proteins regulated by thrombin. Therapeutic agents blocking angiogenic effects of thrombin in HESCs can prevent or minimize progestin

  12. Discovery and evaluation of potent P1 aryl heterocycle-based thrombin inhibitors. (United States)

    Young, Mary Beth; Barrow, James C; Glass, Kristen L; Lundell, George F; Newton, Christina L; Pellicore, Janetta M; Rittle, Kenneth E; Selnick, Harold G; Stauffer, Kenneth J; Vacca, Joseph P; Williams, Peter D; Bohn, Dennis; Clayton, Franklin C; Cook, Jacquelynn J; Krueger, Julie A; Kuo, Lawrence C; Lewis, S Dale; Lucas, Bobby J; McMasters, Daniel R; Miller-Stein, Cynthia; Pietrak, Beth L; Wallace, Audrey A; White, Rebecca B; Wong, Bradley; Yan, Youwei; Nantermet, Philippe G


    In an effort to discover potent, clinically useful thrombin inhibitors, a rapid analogue synthetic approach was used to explore the P(1) region. Various benzylamines were coupled to a pyridine/pyrazinone P(2)-P(3) template. One compound with an o-thiadiazole benzylic substitution was found to have a thrombin K(i) of 0.84 nM. A study of ortho-substituted five-membered-ring heterocycles was undertaken and subsequently demonstrated that the o-triazole and tetrazole rings were optimal. Combination of these potent P(1) aryl heterocycles with a variety of P(2)-P(3) groups produced a compound with an extraordinary thrombin inhibitory activity of 1.4 pM. It is hoped that this potency enhancement in P(1) will allow for more diversification in the P(2)-P(3) region to ultimately address additional pharmacological concerns.

  13. Modeling the microscopic electrical properties of thrombin binding aptamer (TBA) for label-free biosensors (United States)

    Alfinito, Eleonora; Reggiani, Lino; Cataldo, Rosella; De Nunzio, Giorgio; Giotta, Livia; Guascito, Maria Rachele


    Aptamers are chemically produced oligonucleotides, able to bind a variety of targets such as drugs, proteins and pathogens with high sensitivity and selectivity. Therefore, aptamers are largely employed for producing label-free biosensors (aptasensors), with significant applications in diagnostics and drug delivery. In particular, the anti-thrombin aptamers are biomolecules of high interest for clinical use, because of their ability to recognize and bind the thrombin enzyme. Among them, the DNA 15-mer aptamer (TBA), has been widely explored around the possibility of using it in aptasensors. This paper proposes a microscopic model of the electrical properties of TBA and of the aptamer-thrombin complex, combining information from both structure and function, following the issues addressed in an emerging branch of electronics known as proteotronics. The theoretical results are compared and validated with measurements reported in the literature. Finally, the model suggests resistance measurements as a novel tool for testing aptamer-target affinity.

  14. A Universal Base in a Specific Role: Tuning up a Thrombin Aptamer with 5-Nitroindole (United States)

    Tsvetkov, Vladimir B.; Varizhuk, Anna M.; Pozmogova, Galina E.; Smirnov, Igor P.; Kolganova, Natalia A.; Timofeev, Edward N.


    In this study we describe new modified analogs of the thrombin binding aptamer (TBA) containing 5-nitroindole residues. It has been shown that all modified TBAs form an anti-parallel G-quadruplex structure and retain the ability to inhibit thrombin. The most advanced TBA variant (TBA-N8) has a substantially increased clotting time and two-fold lower IC50 value compared to the unmodified prototype. Molecular modelling studies suggest that the improved anticoagulant properties of TBA-N8 result from changes in the binding mode of the analog. A modified central loop in TBA-N8 is presumed to participate in the binding of the target protein. Studies of FAM labelled TBA and TBA-N8 showed an improved binding affinity of the modified aptamer and provided evidence of a direct interaction between the modified central loop and thrombin. Our findings have implications for the design of new aptamers with improved binding affinities.

  15. Unravelling the mechanism and significance of thrombin binding to platelet glycoprotein Ib. (United States)

    Ruggeri, Zaverio M; Zarpellon, Alessandro; Roberts, James R; Mc Clintock, Richard A; Jing, Hua; Mendolicchio, G Loredana


    The main question concerning the mechanism of a-thrombin binding to platelet membrane glycoprotein (GP)Ib is whether it involves both thrombin exosite I and exosite II. The solution of two independent crystal structures suggests alternative explanations that may actually reflect different modes of binding with distinct pathophysiological significance. With respect to function, it is still unclear whether thrombin binding to GPIb promotes procoagulant and prothrombotic pathways of response to vascular injury or limits such responses by sequestering, at least temporarily, the active enzyme. We review here published information on these topics and touch upon ongoing studies aimed at finding definitive answers to outstanding questions relevant for a better understanding of thrombosis and haemostasis.

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

    Akkaya, Alper; Pazarlioglu, Nurdan


    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.

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

    DEFF Research Database (Denmark)

    van der Plas, Mariena J A; Bhongir, Ravi K V; Kjellström, Sven


    Pseudomonas aeruginosa is an opportunistic pathogen known for its immune evasive abilities amongst others by degradation of a large variety of host proteins. Here we show that digestion of thrombin by P. aeruginosa elastase leads to the release of the C-terminal thrombin-derived peptide FYT21...

  18. Salmon-derived thrombin inhibits development of chronic pain through an endothelial barrier protective mechanism dependent on APC. (United States)

    Smith, Jenell R; Galie, Peter A; Slochower, David R; Weisshaar, Christine L; Janmey, Paul A; Winkelstein, Beth A


    Many neurological disorders are initiated by blood-brain barrier breakdown, which potentiates spinal neuroinflammation and neurodegeneration. Peripheral neuropathic injuries are known to disrupt the blood-spinal cord barrier (BSCB) and to potentiate inflammation. But, it is not known whether BSCB breakdown facilitates pain development. In this study, a neural compression model in the rat was used to evaluate relationships among BSCB permeability, inflammation and pain-related behaviors. BSCB permeability increases transiently only after injury that induces mechanical hyperalgesia, which correlates with serum concentrations of pro-inflammatory cytokines, IL-7, IL-12, IL-1α and TNF-α. Mammalian thrombin dually regulates vascular permeability through PAR1 and activated protein C (APC). Since thrombin protects vascular integrity through APC, directing its affinity towards protein C, while still promoting coagulation, might be an ideal treatment for BSCB-disrupting disorders. Salmon thrombin, which prevents the development of mechanical allodynia, also prevents BSCB breakdown after neural injury and actively inhibits TNF-α-induced endothelial permeability in vitro, which is not evident the case for human thrombin. Salmon thrombin's production of APC faster than human thrombin is confirmed using a fluorogenic assay and APC is shown to inhibit BSCB breakdown and pain-related behaviors similar to salmon thrombin. Together, these studies highlight the impact of BSCB on pain and establish salmon thrombin as an effective blocker of BSCB, and resulting nociception, through its preferential affinity for protein C. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Acceleration of the thrombin inactivation of single chain urokinase-type plasminogen activator (pro-urokinase) by thrombomodulin

    NARCIS (Netherlands)

    Munk, G.A.W. de; Groeneveld, E.; Rijken, D.C.


    The in vitro effects of thrombomodulin on the inactivation of single chain urokinase-type plasminogen activator (scu-PA) by thrombin were investigated by incubating scu-PA with varying concentrations of human thrombin, in both the absence and presence of soluble rabbit thrombomodulin. 50%

  20. The suicide substrate reaction between plasminogen activator inhibitor 1 acid thrombin is regulated by the cofactors vitronectin and heparin

    NARCIS (Netherlands)

    van Meijer, M.; Smilde, A.; Tans, G.; Nesheim, M. E.; Pannekoek, H.; Horrevoets, A. J.


    The interaction of thrombin with plasminogen activator inhibitor 1 (PAI-1) is shown to result in the simultaneous formation of both cleaved PAI-1 and a sodium dodecyl sulfate-stable thrombin-PAI-1 complex. The kinetics of this reaction can be described by a ''suicide substrate'' mechanism that

  1. Mutations in the fourth EGF-like domain affect thrombomodulin-induced changes in the active site of thrombin. (United States)

    Koeppe, Julia R; Beach, Muneera A; Baerga-Ortiz, Abel; Kerns, S Jordan; Komives, Elizabeth A


    A number of alanine and more conservative mutants of residues in the fourth domain of thrombomodulin (TM) were prepared and assayed for protein C activation and for thrombin binding. Several of the alanine mutations appeared to cause misfolding or structural defects as assessed by poor expression and/or NMR HSQC experiments, while more conservative mutations at the same site appeared to allow correct folding and preserved activity. Several of the conservative mutants bound more weakly to thrombin despite the fact that the fourth domain does not directly contact thrombin in the crystal structure of the thrombin-TM complex. A few of the mutant TM fragments bound thrombin with an affinity similar to that of the wild type but exhibited decreases in k cat for protein C activation. These mutants were also less able to cause a change in the steady state fluorescence of fluorescein-EGR-chloromethylketone bound to the active site of thrombin. These results suggest that some residues within the fourth domain of TM may primarily interact with protein C but others are functionally important for altering the way TM interacts with thrombin. Residues in the fourth domain that primarily affect k cat for protein C activation may do this by changing the active site of thrombin.

  2. Real-time detection of α-thrombin binding to single-strand DNA aptamers by a highly sensitive Si-based waveguide SPR biosensor (United States)

    Huang, Chi-Chieh; Hsu, Hsin-Feng; Chen, Sz-Hau; Tsai, Kun-Yu; Huang, Yang-Tung; Lin, Chih-Sheng; Hsu, Shih-Hsin


    αIn this paper, real-time characterization of α-thrombin binding to single-strand DNA (ssDNA) aptamers by novel Si-based waveguide SPR biosensors has been investigated. The gold nanoparticles (AuNPs) modified with anti-thrombin antibodies were employed to bind with α-thrombin via strong antibody/antigen affinity for SPR signal amplification. The detection limit of 1 pM for -thrombin detection was achieved.

  3. Distinct PKC isoforms mediate cell survival and DNA synthesis in thrombin-induced myofibroblasts. (United States)

    Bogatkevich, Galina S; Gustilo, Estella; Oates, Jim C; Feghali-Bostwick, Carol; Harley, Russell A; Silver, Richard M; Ludwicka-Bradley, Anna


    Thrombin activates protease-activated receptor (PAR)-1 and induces a myofibroblast phenotype in normal lung fibroblasts that resembles the phenotype of scleroderma lung fibroblasts. We now demonstrate that PAR-1 expression is dramatically increased in lung tissue from scleroderma patients, where it is associated with inflammatory and fibroproliferative foci. We also observe that thrombin induces resistance to apoptosis in normal lung fibroblasts, and this process is regulated by protein kinase C (PKC)-epsilon but not by PKC-alpha. Overexpression of a constitutively active (c-a) form of PAR-1 or PKC-epsilon significantly inhibits Fas ligand-induced apoptosis in lung fibroblasts, whereas scleroderma lung fibroblasts are resistant to apoptosis de novo. Thrombin translocates p21Cip1/WAF1, a signaling molecule downstream of PKC, from the nucleus to cytoplasm in normal lung fibroblasts mimicking the localization of p21Cip1/WAF1 in scleroderma lung fibroblasts. Overexpression of c-a PKC-alpha or PKC-epsilon results in accumulation of p21Cip1/WAF1 in the cytoplasm. Depletion of PKC-alpha or inhibition of mitogen-activated protein kinase (MAPK) blocks thrombin-induced DNA synthesis in lung fibroblasts. Inhibition of PKC by calphostin or PKC-alpha, but not PKC-epsilon, by antisense oligonucleotides prevents thrombin-induced MAPK phosphorylation and accumulation of G(1) phase regulatory protein cyclin D1, suggesting that PKC-alpha, MAPK, and cyclin D1 mediate lung fibroblast proliferation. These data demonstrate that two distinct PKC isoforms mediate thrombin-induced resistance to apoptosis and proliferation and suggest that p21Cip1/WAF1 promotes both phenomena.

  4. Evaluation of antithrombotic activity of thrombin DNA aptamers by a murine thrombosis model.

    Directory of Open Access Journals (Sweden)

    Elena Zavyalova

    Full Text Available Aptamers are nucleic acid based molecular recognition elements with a high potential for the theranostics. Some of the aptamers are under development for therapeutic applications as promising antithrombotic agents; and G-quadruplex DNA aptamers, which directly inhibit the thrombin activity, are among them. RA-36, the 31-meric DNA aptamer, consists of two thrombin binding pharmacophores joined with the thymine linker. It has been shown earlier that RA-36 directly inhibits thrombin in the reaction of fibrinogen hydrolysis, and also it inhibits plasma and blood coagulation. Studies of both inhibitory and anticoagulation effects had indicated rather high species specificity of the aptamer. Further R&D of RA-36 requires exploring its efficiency in vivo. Therefore the development of a robust and adequate animal model for effective physiological studies of aptamers is in high current demand. This work is devoted to in vivo study of the antithrombotic effect of RA-36 aptamer. A murine model of thrombosis has been applied to reveal a lag and even prevention of thrombus formation when RA-36 was intravenous bolus injected in high doses of 1.4-7.1 µmol/kg (14-70 mg/kg. A comparative study of RA-36 aptamer and bivalirudin reveals that both direct thrombin inhibitors have similar antithrombotic effects for the murine model of thrombosis; though in vitro bivalirudin has anticoagulation activity several times higher compared to RA-36. The results indicate that both RA-36 aptamer and bivalirudin are direct thrombin inhibitors of different potency, but possible interactions of the thrombin-inhibitor complex with other components of blood coagulation cascade level the physiological effects for both inhibitors.

  5. Thrombin like activity of Asclepias curassavica L. latex: action of cysteine proteases. (United States)

    Shivaprasad, H V; Rajesh, R; Nanda, B L; Dharmappa, K K; Vishwanath, B S


    To validate the scientific basis of plant latex to stop bleeding on fresh cuts. Cysteine protease(s) from Asclepias curassavica (Asclepiadaceae) plant latex was assessed for pro-coagulant and thrombin like activities. A waxy material from the latex of Asclepias curassavica latex was removed by freezing and thawing. The resulted latex enzyme fraction was assayed for proteolytic activity using denatured casein as substrate. Its coagulant activity and thrombin like activity were determined using citrated plasma and pure fibrinogen, respectively. Inhibition studies were performed using specific protease inhibitors to know the type of protease. The latex enzyme fraction exhibited strong proteolytic activity when compared to trypsin and exerted pro-coagulant action by reducing plasma clotting time from 195 to 58 s whereas trypsin reduced clotting time marginally from 195 to 155 s. The pro-coagulant activity of this enzyme fraction was exerted by selectively hydrolyzing A alpha and B beta subunits of fibrinogen to form fibrin clot when pure fibrinogen was used as substrate as assessed by fibrinogen-agarose plate method and fibrinogen polymerization assay. Trypsin failed to induce any fibrin clot under similar conditions. The electrophoretic pattern of latex enzyme fraction-induced fibrin clot was very much similar to that of thrombin-induced fibrin clot and mimic thrombin like action. The proteolytic activity including thrombin like activity of Asclepias curassavica latex enzyme fraction was completely inhibited by iodoaceticacid (IAA). Cysteine proteases from Asclepias curassavica latex exhibited strong pro-coagulant action and were found to be specific in its action (Thrombin like). This could be the basis for the use of plant latex in pharmacological applications that justify their use as folk medicine.

  6. Thrombin generation assay: interactions between chronic inflammation and haemostasis in patients with autoimmune diseases. (United States)

    Solfietti, Laura; Binello, Giovanni B; Stella, Stefania; Bazzan, Mario; Salierno, Milena; Roccatello, Dario


    Growing evidences show a direct link between inflammation and activation of haemostasis. That could increase thrombotic and cardiovascular risk in patients with active autoimmune diseases such as rheumatoid arthritis (RA) and systemic sclerosis (SSc). The aim of this study was to evaluate a possible hypercoagulable condition in RA and SSc patients, using the thrombin generation assay (TGA). TGA was assessed in 44 RA [33 with active disease (actRA) and 11 inactive (non-actRA)], 25 SSc patients and 41 healthy controls using a fluorimetric technique and the TGA RB Low reagent. The Lag time (tLag), the time to thrombin peak (tPeak), the maximal concentration of formed thrombin (Peak), the velocity of thrombin generation (velocity) and the total amount of thrombin generated (AUC) were determined. As compared to the control group, tLag was found to be significantly reduced both in patients with actRA (p=0.0001) and non-actRA (p=0.01); tPeak was found to be reduced in actRA patients (p=0.0002). Similarly, as compared to healthy subjects, Peak and AUC were found to be increased in actRA patients (p=0.01; p=0.002), as well as D-dimer (p=0.01). Analysing SSc vs RA, a higher Peak and AUC were detected in RA patients. The TGA profile identified in actRA patients (decreased tLag and tPeak combined with higher thrombin peak and greater AUC) reflects a hypercoagulable state that could make patients more susceptible to develop a cardiovascular disease.

  7. Ligand Binding to Anion-binding Exosites Regulates Conformational Properties of Thrombin* (United States)

    Malovichko, Marina V.; Sabo, T. Michael; Maurer, Muriel C.


    Thrombin participates in coagulation, anticoagulation, and initiation of platelet activation. To fulfill its diverse roles and maintain hemostasis, this serine protease is regulated via the extended active site region and anion-binding exosites (ABEs) I and II. For the current project, amide proton hydrogen-deuterium exchange coupled with MALDI-TOF mass spectrometry was used to characterize ligand binding to individual exosites and to investigate the presence of exosite-active site and exosite-exosite interactions. PAR3(44–56) and PAR1(49–62) were observed to bind to thrombin ABE I and then to exhibit long range effects over to ABE II. By contrast, Hirudin(54–65) focused more on ABE I and did not transmit influences over to ABE II. Although these three ligands were each directed to ABE I, they did not promote the same conformational consequences. d-Phe-Pro-Arg-chloromethyl ketone inhibition at the thrombin active site led to further local and long range consequences to thrombin-ABE I ligand complexes with the autolysis loop often most affected. When Hirudin(54–65) was bound to ABE I, it was still possible to bind GpIbα(269–286) or fibrinogen γ′(410–427) to ABE II. Each ligand exerted its predominant influences on thrombin and also allowed interexosite communication. The results obtained support the proposal that thrombin is a highly dynamic protein. The transmission of ligand-specific local and long range conformational events is proposed to help regulate this multifunctional enzyme. PMID:23378535

  8. Catheter-directed endovascular application of thrombin: Report of 3 cases and review of the literature. (United States)

    Maybody, Majid; Madoff, David C; Thornton, Raymond H; Morales, Steven A; Moskowitz, Chaya S; Hsu, Meier; Brody, Lynn A; Brown, Karen T; Covey, Anne M

    To report 3 new cases of catheter-directed endovascular application of thrombin and explore trends by analysis of published case series. Institutional Review Board approved this retrospective study. All cases of non-tumoral arterial embolization performed from January 2003 to January 2015 at our institution were retrospectively reviewed. Thrombin was used in 7 of 589 cases. In 3 cases intra arterial thrombin was injected via catheter to treat active hemorrhage. Four cases were excluded due to percutaneous injection into visceral pseudoaneurysms (n=3) and making ex vivo autologous clot to be injected via catheter (n=1). Fisher's exact and the Wilcoxon rank sum tests were used to assess for association with acute nontarget thrombosis. Catheter-directed thrombin was used in 3/589 (0.5%) cases at our institution. All three cases were technically successful with no further bleeding (100%). Nontarget thrombosis of proximal branches occurred in 2 patients (67%) with no significant clinical consequences. Including our 3 cases, a total of 28 cases were reviewed. Of the variables examined-location (p=0.99), size (p=0.66) and etiology of vascular lesion (p=0.92), pseudoaneurysm neck anatomy (p=0.14), thrombin units (p=0.47), volume (p=0.76) or technique of use of small doses (p=0.99), use of other embolic material (p=0.67) and use of adjunct techniques (p=0.99)-none were found to be significantly associated with acute nontarget thrombosis. Technical success was 96% with no reports of reperfusion after treatment. Catheter-directed endovascular thrombin can be an additional tool to treat pseudoaneurysms not amenable to conventional embolization. Further studies are required to optimize technique and outcomes. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Systems biology of coagulation initiation: kinetics of thrombin generation in resting and activated human blood. (United States)

    Chatterjee, Manash S; Denney, William S; Jing, Huiyan; Diamond, Scott L


    Blood function defines bleeding and clotting risks and dictates approaches for clinical intervention. Independent of adding exogenous tissue factor (TF), human blood treated in vitro with corn trypsin inhibitor (CTI, to block Factor XIIa) will generate thrombin after an initiation time (T(i)) of 1 to 2 hours (depending on donor), while activation of platelets with the GPVI-activator convulxin reduces T(i) to ∼20 minutes. Since current kinetic models fail to generate thrombin in the absence of added TF, we implemented a Platelet-Plasma ODE model accounting for: the Hockin-Mann protease reaction network, thrombin-dependent display of platelet phosphatidylserine, VIIa function on activated platelets, XIIa and XIa generation and function, competitive thrombin substrates (fluorogenic detector and fibrinogen), and thrombin consumption during fibrin polymerization. The kinetic model consisting of 76 ordinary differential equations (76 species, 57 reactions, 105 kinetic parameters) predicted the clotting of resting and convulxin-activated human blood as well as predicted T(i) of human blood under 50 different initial conditions that titrated increasing levels of TF, Xa, Va, XIa, IXa, and VIIa. Experiments with combined anti-XI and anti-XII antibodies prevented thrombin production, demonstrating that a leak of XIIa past saturating amounts of CTI (and not "blood-borne TF" alone) was responsible for in vitro initiation without added TF. Clotting was not blocked by antibodies used individually against TF, VII/VIIa, P-selectin, GPIb, protein disulfide isomerase, cathepsin G, nor blocked by the ribosome inhibitor puromycin, the Clk1 kinase inhibitor Tg003, or inhibited VIIa (VIIai). This is the first model to predict the observed behavior of CTI-treated human blood, either resting or stimulated with platelet activators. CTI-treated human blood will clot in vitro due to the combined activity of XIIa and XIa, a process enhanced by platelet activators and which proceeds in the

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


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


    Blood coagulation factor VIII (fVIII) is a plasma protein that is decreased or absent in hemophilia A. It is isolated as a mixture of heterodimers that contain a variably sized heavy chain and a common light chain. Thrombin catalyzes the activation of fVIII in a reaction that is associated with cleavages in both types of chain. We isolated a serine protease from Bothrops jararacussu snake venom that catalyzes thrombin-like heavy-chain cleavage but not light-chain cleavage in porcine fVIII as ...

  11. Traumatic axillary artery pseudoaneurysm treated with intravascular balloon occlusion and percutaneous thrombin injection

    Directory of Open Access Journals (Sweden)

    Maria Carratola, BS


    Full Text Available Axillary artery pseudoaneurysms are relatively rare, with few reported cases found in the literature. Furthermore, treatment with percutaneous thrombin injection has not yet been reported. We report the case of a 59-year-old man with a large (10 cm post-traumatic pseudoaneurysm of the left axillary artery found five weeks after a motorcycle crash. The patient sustained multiple injuries, including fractures of the left scapula and clavicle. Edema was observed at the time of diagnosis. Arteriography with successful ultrasound-guided percutaneous thrombin injection was undertaken. The patient experienced no complications after the procedure.

  12. Automated Wormscan (United States)

    Puckering, Timothy; Thompson, Jake; Sathyamurthy, Sushruth; Sukumar, Sinduja; Shapira, Tirosh; Ebert, Paul


    There has been a recent surge of interest in computer-aided rapid data acquisition to increase the potential throughput and reduce the labour costs of large scale Caenorhabditis elegans studies. We present Automated WormScan, a low-cost, high-throughput automated system using commercial photo scanners, which is extremely easy to implement and use, capable of scoring tens of thousands of organisms per hour with minimal operator input, and is scalable. The method does not rely on software training for image recognition, but uses the generation of difference images from sequential scans to identify moving objects. This approach results in robust identification of worms with little computational demand. We demonstrate the utility of the system by conducting toxicity, growth and fecundity assays, which demonstrate the consistency of our automated system, the quality of the data relative to manual scoring methods and congruity with previously published results. PMID:28413617

  13. Structural Characterization of a Thrombin-Aptamer Complex by High Resolution Native Top-Down Mass Spectrometry (United States)

    Zhang, Jiang; Loo, Rachel R. Ogorzalek; Loo, Joseph A.


    Native mass spectrometry (MS) with electrospray ionization (ESI) has evolved as an invaluable tool for the characterization of intact native proteins and non-covalently bound protein complexes. Here we report the structural characterization by high resolution native top-down MS of human thrombin and its complex with the Bock thrombin binding aptamer (TBA), a 15-nucleotide DNA with high specificity and affinity for thrombin. Accurate mass measurements revealed that the predominant form of native human α-thrombin contains a glycosylation mass of 2205 Da, corresponding to a sialylated symmetric biantennary oligosaccharide structure without fucosylation. Native MS showed that thrombin and TBA predominantly form a 1:1 complex under near physiological conditions (pH 6.8, 200 mM NH4OAc), but the binding stoichiometry is influenced by the solution ionic strength. In 20 mM ammonium acetate solution, up to two TBAs were bound to thrombin, whereas increasing the solution ionic strength destabilized the thrombin-TBA complex and 1 M NH4OAc nearly completely dissociated the complex. This observation is consistent with the mediation of thrombin-aptamer binding through electrostatic interactions and it is further consistent with the human thrombin structure that contains two anion binding sites on the surface. Electron capture dissociation (ECD) top-down MS of the thrombin-TBA complex performed with a high resolution 15 Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer showed the primary binding site to be at exosite I located near the N-terminal sequence of the heavy chain, consistent with crystallographic data. High resolution native top-down MS is complementary to traditional structural biology methods for structurally characterizing native proteins and protein-DNA complexes. [Figure not available: see fulltext.

  14. Longitudinal assessment of thrombin generation potential in response to alteration of antiplatelet therapy after TIA or ischaemic stroke.

    LENUS (Irish Health Repository)

    Tobin, W O


    The impact of changing antiplatelet therapy on thrombin generation potential in patients with ischaemic cerebrovascular disease (CVD) is unclear. We assessed patients within 4 weeks of TIA or ischaemic stroke (baseline), and then 14 days (14d) and >90 days (90d) after altering antiplatelet therapy. Thrombin generation was assessed in platelet poor plasma. Ninety-one patients were recruited. Twenty-four were initially assessed on no antiplatelet therapy, and then after 14d (N = 23) and 90d (N = 8) on aspirin monotherapy; 52 were assessed on aspirin monotherapy, and after 14 and 90 days on aspirin and dipyridamole combination therapy; 21 patients were assessed on aspirin and after 14 days (N = 21) and 90 days (N = 19) on clopidogrel. Peak thrombin generation and endogenous thrombin potential were reduced at 14 and 90 days (p ≤ 0.04) in the overall cohort. We assessed the impact of individual antiplatelet regimens on thrombin generation parameters to investigate the cause of this effect. Lag time and time-to-peak thrombin generation were unchanged at 14 days, but reduced 90 days after commencing aspirin (p ≤ 0.009). Lag time, peak thrombin generation and endogenous thrombin potential were reduced at both 14 and 90 days after adding dipyridamole to aspirin (p ≤ 0.01). Lag time was reduced 14 days after changing from aspirin to clopidogrel (p = 0.045), but this effect was not maintained at 90 days (p = 0.2). This pilot study did not show any consistent effects of commencing aspirin, or of changing from aspirin to clopidogrel on thrombin generation potential during follow-up. The addition of dipyridamole to aspirin led to a persistent reduction in peak and total thrombin generation ex vivo, and illustrates the diverse, potentially beneficial, newly recognised \\'anti-coagulant\\' effects of dipyridamole in ischaemic CVD.

  15. (-)-Epigallocatechin-3-gallate decreases thrombin/paclitaxel-induced endothelial tissue factor expression via the inhibition of c-Jun terminal NH2 kinase phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Huang-Joe [Institute of Biotechnology, National Tsing Hua University, No. 101, Section 2, Kuang Fu Road, Hsinchu 30013, Taiwan (China); Division of Cardiology, Department of Medicine, China Medical University Hospital, No. 2, Yuh-Der Road, Taichung 40447, Taiwan (China); Lo, Wan-Yu [Department of Medical Research, China Medical University Hospital, No. 2, Yuh-Der Road, Taichung 40447, Taiwan (China); Graduate Integration of Chinese and Western Medicine, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan (China); Lu, Te-Ling [School of Pharmacy, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan (China); Huang, Haimei, E-mail: [Institute of Biotechnology, National Tsing Hua University, No. 101, Section 2, Kuang Fu Road, Hsinchu 30013, Taiwan (China)


    Patients with paclitaxel-eluting stents are concerned with stent thrombosis caused by premature discontinuation of dual antiplatelet therapy or clopidogrel resistance. This study investigates the effect of (-)-epigallocatechin-3-gallate (EGCG) on the expression of thrombin/paclitaxel-induced endothelial tissue factor (TF) expressions in human aortic endothelial cells (HAECs). EGCG was nontoxic to HAECs at 6 h up to a concentration of 25 {mu}mol/L. At a concentration of 25 {mu}mol/L, EGCG pretreatment potently inhibited both thrombin-stimulated and thrombin/paclitaxel-stimulated endothelial TF protein expression. Thrombin and thrombin/paclitaxel-induced 2.6-fold and 2.9-fold increases in TF activity compared with the control. EGCG pretreatment caused a 29% and 38% decrease in TF activity on thrombin and thrombin/paclitaxel treatment, respectively. Real-time polymerase chain reaction (PCR) showed that thrombin and thrombin/paclitaxel-induced 3.0-fold and 4.6-fold TF mRNA expressions compared with the control. EGCG pretreatment caused an 82% and 72% decrease in TF mRNA expression on thrombin and thrombin/paclitaxel treatment, respectively. The c-Jun terminal NH2 kinase (JNK) inhibitor SP600125 reduced thrombin/paclitaxel-induced TF expression. Furthermore, EGCG significantly inhibited the phosphorylation of JNK to 49% of thrombin/paclitaxel-stimulated HAECs at 60 min. Immunofluorescence assay did not show an inhibitory effect of EGCG on P65 NF-{kappa}B nuclear translocation in the thrombin/paclitaxel-stimulated endothelial cells. In conclusion, EGCG can inhibit TF expression in thrombin/paclitaxel-stimulated endothelial cells via the inhibition of JNK phosphorylation. The unique property of EGCG may be used to develop a new drug-eluting stent by co-coating EGCG and paclitaxel.

  16. Library Automation. (United States)

    Husby, Ole


    The challenges and potential benefits of automating university libraries are reviewed, with special attention given to cooperative systems. Aspects discussed include database size, the role of the university computer center, storage modes, multi-institutional systems, resource sharing, cooperative system management, networking, and intelligent…

  17. Thrombin Generation Assay in Hospitalized Nonsurgical Patients: A New Tool to Assess Venous Thromboembolism Risk? (United States)

    Espitia, Olivier; Fouassier, Marc; Hardouin, Jean-Benoît; Pistorius, Marc-Antoine; Agard, Christian; Planchon, Bernard; Trossaert, Marc; Pottier, Pierre


    Assessment of venous thromboembolism (VTE) risk is important to determine optimal primary prophylaxis in hospitalized patients. The Padua score helps to recognize patients with high VTE risk, but quantifying a VTE risk is often challenging in medical patients. Thrombin generation assay (TGA) reflects the pro-/anticoagulant balance and thus could help to better quantify VTE risk in medical hospitalized patients. To analyze the relation between TGA and VTE risk according to Padua score in medical hospitalized patients. Between May and October 2013, 105 patients were included in an unselected cohort group of patients admitted to an internal medicine department in a large, university hospital. Within the 36 hours after admission and before any anticoagulant therapy, Padua score was calculated and sample for TGA was collected for each patient. Thrombin generation assay (velocity, peak, and endogenous thrombin potential [ETP]) was performed with 1 and 5 picomol/l (pM) tissue factor (TF) reagent. In patients with high Padua score (n = 29), velocity, peak, and ETP differed from patients with low Padua score. This difference was present at 1 and 5 pM TF, in ETP (P older age, higher body mass index, myocardial infarction, C-reactive protein >5 mg/L, reduced mobility with bed rest significantly increased velocity 1 pM TF value. Single thrombin generation measurement could help to identify patients at risk of VTE in medical hospitalized patients. © The Author(s) 2015.

  18. Role of zinc ions in activation and inactivation of thrombin-activatable fibrinolysis inhibitor

    NARCIS (Netherlands)

    Marx, Pauline F.; Bouma, Bonno N.; Meijers, Joost C. M.


    Thrombin-activatable fibrinolysis inhibitor (TAFI) circulates as an inactive proenzyme of a carboxypeptidase B-like enzyme (TAFIa). It functions by removing C-terminal lysine residues from partially degraded fibrin that are important in tissue-type plasminogen activator mediated plasmin formation.

  19. Risk of acute coronary artery disease associated with functional thrombin activatable fibrinolysis inhibitor plasma level. (United States)

    Santamaría, Amparo; Martínez-Rubio, Antonio; Borrell, Montserrat; Mateo, José; Ortín, Rosa; Fontcuberta, Jordi


    To our knowledge, there is little information about functional thrombin activatable fibrinolysis inhibitor (TAFI) levels and the risk of acute coronary artery disease (CAD). We investigated the risk of acute CAD related to plasma levels of functional TAFI. We found that functional TAFI levels in plasma (above 126%), increased the risk of acute CAD almost 4-fold.

  20. Percutaneous thrombin injection for the treatment of a post-pancreatitis pseudoaneurysm

    Energy Technology Data Exchange (ETDEWEB)

    Puri, S.; Nicholson, A.A.; Breen, D.J. [Dept. of Radiology, Hull Royal Infirmary, Hull (United Kingdom)


    Visceral artery pseudoaneurysms are often treated surgically or by transcatheter embolisation. We report a case of a pseudoaneurysm in a patient with chronic pancreatitis, which was successfully occluded by percutaneous injection of thrombin into the pseudoaneurysmal sac as a first-line management. (orig.)

  1. A sensitive bioimmunoassay for thrombin-cleaved two-chain urokinase-type plasminogen activator (abstract)

    NARCIS (Netherlands)

    Braat, E.A.M.; Nauland, U.; Dooijewaard, G.; Rijken, P.C.


    Thrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) into a virtually inactive two-chain form (tcu-PA/T). Little is known about the physiological importance of tcu-PA/T. To examine the occurrence of tcu-PA/T in vivo, we developed a sensitive and specific bioimmunoassay (BIA)

  2. Interactions between thrombin and natural products of Millettia speciosa Champ. using capillary zone electrophoresis. (United States)

    Zhang, Shuyu; Yin, Ting; Ling, Xiaomei; Liang, Hong; Zhao, Yuying


    In Chinese medicine Suberect Spatholobus Stem is used to treat menoxenia, blood deficiency, numb paralyses, and so on. In folk, Millettia speciosa is often used as a substitute for Suberect Spatholobus Stem in some areas but it has not been reported whether M. speciosa is the eligible substitute for Suberect Spatholobus Stem or not till now. In this study, a capillary zone electrophoretic method was applied to determinate the interactions between natural products isolated from M. speciosa Champ. and thrombin for the first time. Both qualitative and quantitative characterizations of the molecule-enzyme binding were determined. Twenty ingredients were isolated from M. speciosa Champ. and the results showed that compared with positive and negative control, the compounds YT-1, YT-2, YT-3, YT-8, YT-9, YT-10, YT-11, YT-12, YT-14, YT-15, YT-16, and YT-20 interacted with thrombin while the other eight had no binding to thrombin. The binding constants of the interaction between compounds and thrombin were calculated by the Scatchard analysis formula. Because M. speciosa contains these compounds which have different levels of anticoagulant activity, it may be the eligible substitute for Suberect Spatholobus Stem.

  3. Allosteric Partial Inhibition of Monomeric Proteases. Sulfated Coumarins Induce Regulation, not just Inhibition, of Thrombin (United States)

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


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

  4. Polyphosphate is a cofactor for the activation of factor XI by thrombin (United States)

    Choi, Sharon H.; Smith, Stephanie A.


    Factor XI deficiency is associated with a bleeding diathesis, but factor XII deficiency is not, indicating that, in normal hemostasis, factor XI must be activated in vivo by a protease other than factor XIIa. Several groups have identified thrombin as the most likely activator of factor XI, although this reaction is slow in solution. Although certain nonphysiologic anionic polymers and surfaces have been shown to enhance factor XI activation by thrombin, the physiologic cofactor for this reaction is uncertain. Activated platelets secrete the highly anionic polymer polyphosphate, and our previous studies have shown that polyphosphate has potent procoagulant activity. We now report that polyphosphate potently accelerates factor XI activation by α-thrombin, β-thrombin, and factor XIa and that these reactions are supported by polyphosphate polymers of the size secreted by activated human platelets. We therefore propose that polyphosphate is a natural cofactor for factor XI activation in plasma that may help explain the role of factor XI in hemostasis and thrombosis. PMID:21976677

  5. Inhibition of Src family kinases improves cognitive function after intraventricular hemorrhage or intraventricular thrombin. (United States)

    Liu, Da Zhi; Waldau, Ben; Ander, Bradley P; Zhan, Xinhua; Stamova, Boryana; Jickling, Glen C; Lyeth, Bruce G; Sharp, Frank R


    Intraventricular hemorrhage causes spatial memory loss, but the mechanism remains unknown. Our recent studies demonstrated that traumatic brain injury activates Src family kinases, which cause spatial memory loss. To test whether the spatial memory loss was due to blood in the ventricles, which activated Src family kinases, we infused autologous whole blood or thrombin into the lateral ventricles of adult rats to model non-traumatic intraventricular hemorrhage. Hippocampal neuron loss was examined 1 day to 5 weeks later. Spatial memory function was assessed 29 to 33 days later using the Morris water maze. Five weeks after the ventricular injections of blood or thrombin, there was death of most hippocampal neurons and significant memory deficits compared with sham operated controls. These data show that intraventricular thrombin is sufficient to kill hippocampal neurons and produce spatial memory loss. In addition, systemic administration of the non-specific Src family kinase inhibitor PP2 or intraventricular injection of siRNA-Fyn, a Src family kinase family member, prevented hippocampal neuronal loss and spatial memory deficits following intraventricular hemorrhage. The data support the conclusions that thrombin mediates the hippocampal neuronal cell death and spatial memory deficits produced by intraventricular blood and that these can be blocked by non-specific inhibition of Src family kinases or by inhibiting Fyn.

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

    Energy Technology Data Exchange (ETDEWEB)

    Pozzi, Nicola; Chen, Raymond; Chen, Zhiwei; Bah, Alaji; Di Cera, Enrico (St. Louis-MED)


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

  7. Thrombin detection in murine plasma using engineered fluorescence resonance energy transfer aptadimers (United States)

    Trapaidze, Ana; Brut, Marie; Mazères, Serge; Estève, Daniel; Gué, Anne-Marie; Bancaud, Aurélien


    Biodetection strategies, in which two sides of one target protein are targeted simultaneously, have been shown to increase specificity, selectivity, and affinity, and it has been suggested that they constitute excellent candidates for protein sensing in complex media. In this study we propose a method to engineer the sequence of a DNA construct dedicated to reversible thrombin detection. This construct, called Fluorescence Resonance Energy Transfer (FRET) aptadimer, is assembled with two aptamers, which target different epitopes of thrombin, interconnected with a DNA linker that contains a FRET couple and a reversible double helix stem. In the absence of target, the stem is stable maintaining a FRET couple in close proximity, and fluorescence is unquenched upon thrombin addition due to the dehybridization of the stem. We define design rules for the conception of FRET aptadimers, and develop a software to optimize their functionality. One engineered FRET aptadimer sequence is subsequently characterized experimentally by temperature scanning fluorimetry, demonstrating the relevance of our technology for thrombin sensing in bulk and diluted murine plasma.

  8. The Tick-Derived Anticoagulant Madanin Is Processed by Thrombin and Factor Xa (United States)

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


    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 α-thrombin. We hereby demonstrate that competitive inhibition of α-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 α-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. PMID:23951260

  9. Carbon monoxide releasing molecule-2 inhibition of snake venom thrombin-like activity: novel biochemical "brake"? (United States)

    Nielsen, Vance G; Bazzell, Charles M


    A complication of defibrinogenation therapy with snake venom enzymes such as ancrod is hypofibrinogenemia associated bleeding secondary to no human-derived inhibitor being available to inactivate or diminish the activity of such enzymes. Of interest, ancrod contains a critical histidine residue without which enzymatic activity is inhibited, and carbon monoxide has been demonstrated to inhibit biomolecular function by interacting with histidine moieties in ion channels. We tested the hypothesis that exposure of three different snake venoms containing serine proteases with thrombin-like activity (which included ancrod) to carbon monoxide derived from carbon monoxide releasing molecule-2 would diminish their effects on plasmatic coagulation as assessed by thrombelastography. In the case of the Malayan pit viper and Eastern diamondback rattlesnake venoms, carbon monoxide diminished the effects of thrombin-like activity. In contrast, timber rattlesnake venom demonstrated enhancement of "thrombin-generating" activity with simultaneous loss of thrombin-like activity in response to carbon monoxide exposure. These findings may serve as the rational basis for not just continuing to investigate the potential of snake venom enzymes as clinical defibrinogenating agents, but to also to assess the potential to stop such agents from becoming a catalytic "runaway train" by judicious application of a biochemical "brake" such as carbon monoxide.

  10. Protease inhibitors, part 13: Specific, weakly basic thrombin inhibitors incorporating sulfonyl dicyandiamide moieties in their structure. (United States)

    Clare, B W; Scozzafava, A; Supuran, C T


    A series of compounds has been prepared by reaction of dicyandiamide with alkyl/arylsulfonyl halides as well as arylsulfonylisocyanates to locate a lead for obtaining weakly basic thrombin inhibitors with sulfonyldicyandiamide moieties as the S1 anchoring group. The detected lead was sulfanilyl-dicyandiamide (K1 of 3 microM against thrombin, and 15 microM against trypsin), which has been further derivatized at the 4-amino group by incorporating arylsulfonylureido as well as amino acyl/dipeptidyl groups protected at the amino terminal moiety with benzyloxycarbonyl or tosylureido moieties. The best compound obtained (ts-D-Phe-Pro-sulfanilyl-dicyandiamide) showed inhibition constants of 9 nM against thrombin and 1400 nM against trypsin. pKa measurements showed that the new derivatives reported here do indeed possess a reduced basicity, with the pKa of the modified guanidine moieties in the range 7.9-8.3 pKa units. Molecular mechanics calculations showed that the preferred tautomeric form of these compounds is of the type ArSO2N=C(NH2) NH-CN, probably allowing for the formation of favorable interaction between this new anchoring group and the active site amino acid residue Asp 189, critical for substrate/inhibitor binding to this type of serine protease. Thus, the main finding of the present paper is that the sulfonyldicyandiamide group may constitute an interesting alternative for obtaining weakly basic, potent thrombin inhibitors, which bind with less affinity to trypsin.

  11. Antithrombotic action of annexin V proved as efficient as direct inhibition of tissue factor or thrombin.

    NARCIS (Netherlands)

    Galan, A.; Heerde, W.L. van; Escolar, G.; Ordinas, A.; Sixma, J.J.; Groot, P.G. de


    The role of phospholipid platelet membrane and tissue factor in thrombin generation and thrombus formation is accepted. In the present study we have explored antithrombotic action of strategies aimed to block exposure of negatively charged phospholipids and we compared effects with those obtained

  12. Gelatin-thrombin hemostatic matrix in the management of placental site postpartum hemorrhage: a case report. (United States)

    Wohlmuth, Cinna T; Dela Merced, Jacqueline


    Gelatin-thrombin matrix (FloSeal, Baxter Healthcare Corporation, Fremont, California), a biodegradable hemostatic sealant, has been shown to control bleeding in multiple intraoperative scenarios and surgical disciplines. However, limited data is available regarding its use in obstetrics. A case of placental-site obstetric hemorrhage controlled with gelatin-thrombin matrix at time of cesarean delivery is presented. A 28-year-old woman underwent a tertiary repeat cesarean delivery for complete placenta previa. The placenta was removed without evidence of placenta accreta. Profuse bleeding occurred over the placental implantation site, resulting in hemorrhage, uncontrolled by conservative uterine-sparing methods. Upon preparation for emergency hysterectomy, gelatin-thrombin matrix was applied, resulting in rapid control of hemorrhage. Abnormal placentation is a predisposing factor of postpartum hemorrhage. In the absence of placenta accreta, profuse hemorrhage can occur from large vascular sinuses associated with a denuded implantation site in the poorly contractile lower uterine segment. In the event of unsuccessful hemorrhage control with conservative techniques, emergency hysterectomy is performed as a life-saving procedure. Multiple unit blood transfusion is often encountered. In the case presented, the use of gelatin-thrombin matrix gel for placental site hemorrhage management at the time of cesarean delivery resulted in rapid control of hemorrhage, uterine preservation and avoidance of massive blood transfusion.

  13. Two different proteins that compete for binding to thrombin have opposite kinetic and thermodynamic profiles. (United States)

    Baerga-Ortiz, Abel; Bergqvist, Simon; Mandell, Jeffrey G; Komives, Elizabeth A


    Thrombin binds thrombomodulin (TM) at anion binding exosite 1, an allosteric site far from the thrombin active site. A monoclonal antibody (mAb) has been isolated that competes with TM for binding to thrombin. Complete binding kinetic and thermodynamic profiles for these two protein-protein interactions have been generated. Binding kinetics were measured by Biacore. Although both interactions have similar K(D)s, TM binding is rapid and reversible while binding of the mAb is slow and nearly irreversible. The enthalpic contribution to the DeltaG(bind) was measured by isothermal titration calorimetry and van't Hoff analysis. The contribution to the DeltaG(bind) from electrostatic steering was assessed from the dependence of the k(a) on ionic strength. Release of solvent H(2)O molecules from the interface was assessed by monitoring the decrease in amide solvent accessibility at the interface upon protein-protein binding. The mAb binding is enthalpy driven and has a slow k(d). TM binding appears to be entropy driven and has a fast k(a). The favorable entropy of the thrombin-TM interaction seems to be derived from electrostatic steering and a contribution from solvent release. The two interactions have remarkably different thermodynamic driving forces for competing reactions. The possibility that optimization of binding kinetics for a particular function may be reflected in different thermodynamic driving forces is discussed.

  14. A Reusable Impedimetric Aptasensor for Detection of Thrombin Employing a Graphite-Epoxy Composite Electrode (United States)

    Ocaña, Cristina; Pacios, Mercè; del Valle, Manel


    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. PMID:22736991

  15. Discovery and characterization of an antibody directed against exosite I of thrombin. (United States)

    Baglin, T P; Langdown, J; Frasson, R; Huntington, J A


    ESSENTIALS: An IgA paraprotein with anti-thrombin activity was not associated with a severe bleeding phenotype. This observation challenges the paradigm that anticoagulant therapy necessarily increases bleeding risk. Characterization of the antibody showed that it specifically binds to thrombin exosite I. A therapeutic drug with the properties of this antibody might be an antithrombotic that doesn't cause bleeding. We report the case of a 54-year-old female who presented with a traumatic subdural hemorrhage. Coagulation tests were markedly prolonged due to the presence of an anti-thrombin IgA paraprotein at 3 g L(-1) . The patient made a complete recovery and has had no abnormal bleeding during a 7-year follow-up, despite the persistence of the paraprotein. To determine how the paraprotein prolonged clotting tests by defining its target and its epitope. The paraprotein was purified and added to normal pooled plasma for in vitro clotting assays. Binding studies were conducted to determine the affinity of the IgA for thrombin. The Fab was isolated and crystallized with thrombin. The purified IgA was sufficient to confer the patient's in vitro coagulation profile in normal pooled plasma, and was found to bind specifically and with high affinity to thrombin. A crystal structure of the Fab fragment in complex with thrombin revealed an exosite I interaction involving CDRH3 of the antibody. Although the patient originally presented with a subdural bleed, the hematoma resolved without intervention, and no other bleeding event occurred during the subsequent 7 years. During this period, the patient's IgA paraprotein levels have remained constant at 3 g L(-1) , suggesting that the presence of a high-affinity, exosite I-directed antibody is consistent with normal hemostasis. A therapeutic derivative of this antibody might therefore permit antithrombotic dose escalation without an associated increase in the risk of bleeding. © 2015 The Authors. Journal of Thrombosis and



    Pasquini, C.; CUNHA, IBS


    The high-performance microcomputer controlled gravimetric-burette described in Part 1 has been employed to automate some routine analytical procedures, A direct potentiometric determination of fluoride ions in drinking water was developed to include an automatic calibration step, matrix adjustment and sample determination. Also the complex and cumbersome titrimetric procedure far determination of the kappa number (lignin content) in paper pulp, has been automated by using the gravimetric unit...

  17. Gemini Planet Imager observational calibrations XI: pipeline improvements and enhanced calibrations after two years on sky (United States)

    Perrin, Marshall D.; Ingraham, Patrick; Follette, Katherine B.; Maire, Jérôme; Wang, Jason J.; Savransky, Dmitry; Arriaga, Pauline; Bailey, Vanessa P.; Bruzzone, Sebastian; Chilcote, Jeffrey K.; De Rosa, Robert J.; Draper, Zachary H.; Fitzgerald, Michael P.; Greenbaum, Alexandra Z.; Hung, Li-Wei; Konopacky, Quinn; Macintosh, Bruce; Marchis, Franck; Marois, Christian; Millar-Blanchaer, Maxwell A.; Nielsen, Eric; Rajan, Abhijith; Rameau, Julien; Rantakyro, Fredrik T.; Ruffio, Jean-Baptiste; Ward-Duong, Kimberly; Wolff, Schuyler G.; Zalesky, Joseph


    The Gemini Planet Imager has been successfully obtaining images and spectra of exoplanets, brown dwarfs, and debris and protoplanetary circumstellar disks using its integral field spectrograph and polarimeter. GPI observations are transformed from raw data into high-quality astrometrically and photometrically calibrated datacubes using the GPI Data Reduction Pipeline, an open-source software framework continuously developed by our team and available to the community. It uses a flexible system of reduction recipes composed of individual primitive steps, allowing substantial customization of processing depending upon science goals. This paper provides a broad overview of the GPI pipeline, summarizes key lessons learned, and describes improved calibration methods and new capabilities available in the latest version. Enhanced automation better supports observations at the telescope with streamlined and rapid data processing, for instance through real-time assessments of contrast performance and more automated calibration file processing. We have also incorporated the GPI Data Reduction Pipeline as one component in a larger automated data system to support the GPI Exoplanet Survey campaign, while retaining its flexibility and stand-alone capabilities to support the broader GPI observer community. Several accompanying papers describe in more detail specific aspects of the calibration of GPI data in both spectral and polarimetric modes.

  18. Affinity capillary electrophoresis with laser induced fluorescence detection for thrombin analysis using nuclease-resistant RNA aptamers. (United States)

    Hao, Lihua; Bai, Yunlong; Wang, Hailin; Zhao, Qiang


    Aptamer affinity capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF) combines the advantages of affinity aptamer, rapid CE separation, and high sensitivity detection. Here we reported an affinity CE-LIF assay for thrombin by using a fluorophore-labeled RNA aptamer containing 2'-fluoro modification in sugar rings of pyrimidine nucleotides (C and U) as affinity ligand. This RNA aptamer has high binding affinity, specificity and biostability. Thrombin at 0.2nM was successfully detected. This RNA aptamer allowed for the detection of thrombin spiked in diluted human serum sample due to the nuclease resistance. The RNA aptamer has comparable binding affinity to a 29-mer DNA aptamer for thrombin, and the binding site of the RNA aptamer on thrombin partially overlaps with the binding site of the 29-mer DNA aptamer on thrombin. It shows the nuclease-resistant RNA aptamers are promising in assays for thrombin. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Thrombin generation capacity of prothrombin complex concentrate in an in vitro dilutional model.

    Directory of Open Access Journals (Sweden)

    Oliver Grottke

    Full Text Available BACKGROUND: The use of PCC for the treatment of trauma-induced coagulopathy potentially increase the risk of thromboembolism and disseminated intravascular coagulation, which is addressed to an imbalance of both pro- and anticoagulants. As PCCs differ in composition, we used an in vitro dilutional approach to assess the overall thrombin generation of five different PCCs through various laboratory assays. METHODS: The vitamin K-dependent coagulation factors, heparin, and antithrombin were assessed in five commercially available PCCs. The procoagulant potential of the PCCs was assessed in plasma and whole blood from 4 healthy donors by means of classical coagulation assays, thrombin generation assay and thromboelastometry. In order to reflect coagulopathy, whole blood was diluted to 80, 60, 40, and 20% with Ringer's lactate solution. RESULTS: The five different PCCs were characterised by comparable levels of factors II, VII, IX and X (all around 20-30 IU/mL, whereas the heparin (0 to 17.6 IU/mL and antithrombin (0.06 to 1.29 IU/mL levels were remarkably different between manufactures. In vitro dilution of blood induced a prolongation of the PT and aPTT, and attenuation of thrombin generation and ExTem induced thromboelastometry. Overall, non- or low-heparin containing PCCs restored the in vitro dilutional coagulopathy, whereas PCCs containing heparin have an anticoagulant effect. The thrombin generation assay showed to be the most sensitive method for assessment of PCC effects. CONCLUSIONS: This study shows that most available PCCs are not balanced regarding their pro- and anticoagulants. The effect of measured differences in thrombin generation among different PCCs requires further investigations to elaborate the clinical meaning of this finding in the treatment of trauma induced coagulopathy.

  20. Synthesis Polarimetry Calibration (United States)

    Moellenbrock, George


    Synthesis instrumental polarization calibration fundamentals for both linear (ALMA) and circular (EVLA) feed bases are reviewed, with special attention to the calibration heuristics supported in CASA. Practical problems affecting modern instruments are also discussed.

  1. RNA-seq reveals novel transcriptome of genes and their isoforms in human pulmonary microvascular endothelial cells treated with thrombin.

    Directory of Open Access Journals (Sweden)

    Li Qin Zhang

    Full Text Available The dysregulation of vascular endothelial cells by thrombin has been implicated in the development of a number of pathologic disorders such as inflammatory conditions, cancer, diabetes, coronary heart disease. However, transcriptional regulation of vascular endothelial cells by thrombin is not completely understood. In the present study, Illumina RNA-seq was used to profile the transcriptome in human pulmonary microvascular endothelial cells (HMVEC-L treated with thrombin for 6 h to gain insight into thrombin's direct effects on the endothelial function. Out of 100 million total reads from a paired end sequencing assay, 91-94% of the reads were aligned to over 16,000 genes in the reference human genome. Thrombin upregulated 150 known genes and 480 known isoforms, and downregulated 2,190 known genes and 3,574 known isoforms by at least 2 fold. Of note, thrombin upregulated 1,775 previously unknown isoforms and downregulated 12,202 previously unknown isoforms by at least 2 fold. Many genes displayed isoform specific differential expression levels and different usage of transcriptional start sites after the thrombin treatment. The cross comparisons between our RNA-seq data and those of DNA microarray analysis of either 6 h thrombin treated HUVEC or 5 h TNFα treated HMVEC have provided a significant overlapping list of differentially expressed genes, supporting the robust utility of our dataset. Further in-depth follow-up analysis of the transcriptional regulation reported in this study may shed light on molecular pathogenic mechanisms underlying thrombin mediated endothelial dysfunction in various diseases and provide new leads of potential therapeutic targets.

  2. RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin (United States)

    Zhang, Li Qin; Cheranova, Dilyara; Gibson, Margaret; Ding, Shinghua; Heruth, Daniel P.; Fang, Deyu; Ye, Shui Qing


    The dysregulation of vascular endothelial cells by thrombin has been implicated in the development of a number of pathologic disorders such as inflammatory conditions, cancer, diabetes, coronary heart disease. However, transcriptional regulation of vascular endothelial cells by thrombin is not completely understood. In the present study, Illumina RNA-seq was used to profile the transcriptome in human pulmonary microvascular endothelial cells (HMVEC-L) treated with thrombin for 6 h to gain insight into thrombin's direct effects on the endothelial function. Out of 100 million total reads from a paired end sequencing assay, 91–94% of the reads were aligned to over 16,000 genes in the reference human genome. Thrombin upregulated 150 known genes and 480 known isoforms, and downregulated 2,190 known genes and 3,574 known isoforms by at least 2 fold. Of note, thrombin upregulated 1,775 previously unknown isoforms and downregulated 12,202 previously unknown isoforms by at least 2 fold. Many genes displayed isoform specific differential expression levels and different usage of transcriptional start sites after the thrombin treatment. The cross comparisons between our RNA-seq data and those of DNA microarray analysis of either 6 h thrombin treated HUVEC or 5 h TNFα treated HMVEC have provided a significant overlapping list of differentially expressed genes, supporting the robust utility of our dataset. Further in-depth follow-up analysis of the transcriptional regulation reported in this study may shed light on molecular pathogenic mechanisms underlying thrombin mediated endothelial dysfunction in various diseases and provide new leads of potential therapeutic targets. PMID:22359579

  3. Characterization of Thrombin-Bound Dabigatran Effects on Protease-Activated Receptor-1 Expression and Signaling In Vitro (United States)

    Chen, Buxin; Soto, Antonio G.; Coronel, Luisa J.; Goss, Ashley; van Ryn, Joanne


    Thrombin, the key effector protease of the coagulation cascade, drives fibrin deposition and activates human platelets through protease-activated receptor-1 (PAR1). These processes are critical to the progression of thrombotic diseases. Thrombin is the main target of anticoagulant therapy, and major efforts have led to the discovery of new oral direct inhibitors of thrombin. Dabigatran is the first oral anticoagulant licensed for the prevention of thromboembolisms associated with orthopedic surgery and stroke prevention in atrial fibrillation. Dabigatran is a direct thrombin inhibitor that effectively blocks thrombin’s catalytic activity but does not preclude thrombin’s exosites and binding to fibrinogen. Thus, we hypothesized that catalytically inactive thrombin retains the capacity to bind to PAR1 through exosite-I and may modulate its function independent of receptor cleavage and activation. Here, we report that dabigatran at clinically relevant concentrations is an effective and acute inhibitor of thrombin-induced PAR1 cleavage, activation, internalization, and β-arrestin recruitment in vitro. Interestingly, prolonged exposure to catalytic inactive thrombin incubated with dabigatran at 20-fold higher therapeutic concentration resulted in increased PAR1 cell-surface expression, which correlated with higher detectable levels of ubiquitinated receptor. These findings are consistent with ubiquitin function as a negative regulator of PAR1 constitutive internalization. Increased PAR1 expression also enhanced agonist-induced phosphoinositide hydrolysis and endothelial barrier permeability. Thus, catalytically inactive thrombin appears to modulate PAR1 function in vitro by stabilizing receptor cell-surface expression; but given the high clearance rate of thrombin, the high concentration of dabigatran required to achieve this effect the in vivo physiologic relevance is unknown. PMID:25934730

  4. Upregulation of Thrombin/Matrix Metalloproteinase-1/Protease-Activated Receptor-1 Chain in Proliferative Diabetic Retinopathy. (United States)

    Abu El-Asrar, Ahmed M; Alam, Kaiser; Nawaz, Mohd Imtiaz; Mohammad, Ghulam; Van den Eynde, Kathleen; Siddiquei, Mohammad Mairaj; Mousa, Ahmed; De Hertogh, Gert; Opdenakker, Ghislain


    Selective proteolytic activation of protease-activated receptor-1 (PAR1) by thrombin and matrix metalloproteinase-1 (MMP-1) plays a central role in enhancing angiogenesis. We investigated the expression levels of thrombin, MMP-1, and PAR1 and correlated these levels with vascular endothelial growth factor (VEGF) in proliferative diabetic retinopathy (PDR). In addition, we examined the expression of PAR1 and thrombin in the retinas of diabetic rats and PAR1 in human retinal microvascular endothelial cells (HRMEC) following exposure to high-glucose, the proinflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and the hypoxia mimetic agent cobalt chloride (CoCl2). Vitreous samples from 32 PDR and 23 nondiabetic patients, epiretinal membranes from 10 patients with PDR, retinas of rats, and HRMEC were studied by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and Western blot analysis. An assay for in vitro cell migration angiogenesis was performed in HRMEC. In epiretinal membranes, PAR1 was expressed in vascular endothelial cells, CD45-expressing leukocytes, and myofibroblasts. ELISA and Western blot assays revealed significant increases in the expression levels of thrombin, MMP-1, and VEGF in vitreous samples from PDR patients compared to nondiabetic controls. Significant positive correlations were found between the levels of VEGF and the levels of thrombin (r = 0.41; p = 0.006) and MMP-1 (r = 0.66; p thrombin (approximately 50 kDa) were detected in rat retinas after induction of diabetes. The proinflammatory cytokines IL-1β and TNF-α, but not high-glucose and CoCl2, induced upregulation of cleaved PAR1 (approximately 30 kDa) in HRMEC. In addition, thrombin and MMP-1 induced VEGF in HRMEC and vorapaxar, a PAR1 inhibitor, inhibited thrombin-induced migration in HRMEC. Interactions among thrombin, MMP-1, PAR1, and VEGF might facilitate angiogenesis in PDR.

  5. Design of Factor XIII V34X Activation Peptides to Control Ability to Interact With Thrombin Mutants (United States)

    Jadhav, Madhavi A.; Lucas, R. Cory; Goldsberry, Whitney N.; Maurer, Muriel C.


    Thrombin helps to activate Factor XIII (FXIII) by hydrolyzing the R37-G38 peptide bond. The resultant transglutaminase introduces cross-links into the fibrin clot. With the development of therapeutic coagulation factors, there is a need to better understand interactions involving FXIII. Such knowledge will help predict ability to activate FXIII and thus ability to promote/hinder the generation of transglutaminase activity. Kinetic parameters have been determined for a series of thrombin species hydrolyzing the FXIII (28′41) V34X activation peptides (V34, V34L, V34F, and V34P). The V34P substitution introduces PAR4 character into the FXIII, and the V34F exhibits important similarities to the cardioprotective V34L. FXIII activation peptides containing V34, V34L, or V34P could each be accommodated by alanine mutants of thrombin lacking either the W60d or Y60a residue in the 60-insertion loop. By contrast, FXIII V34F AP could be cleaved by thrombin W60dA but not by Y60aA. FXIII V34P is highly reliant on the thrombin W215 platform for its strong substrate properties whereas FXIII V34F AP becomes the first segment that can maintain its Km upon loss of the critical thrombin W215 residue. Interestingly, FXIII V34F AP could also be readily accommodated by thrombin L99A and E217A. Hydrolysis of FXIII V34F AP by thrombin W217A/E217A (WE) was similar to that of FXIII V34L AP whereas WE could not effectively cleave FXIII V34P AP. FXIII V34F and V34P AP show promise for designing FXIII activation systems that are either tolerant of or greatly hindered by the presence of anticoagulant thrombins. PMID:21798378

  6. The influence of the sequence of nanoparticles injection to solution on the rate of fibrinogen-thrombin reaction (United States)

    Kirichenko, M. N.; Krivokhiza, S. V.; Chaikov, L. L.; Bulychev, N. A.


    The influence of Fe2O3 nanoparticles on the rate of fibrinogen-thrombin reaction is studied. The nanoparticles were obtained in acoustoplasma discharge with cavitation. The sequence of nanoparticles injection appeared to change dramatically the rate and result of enzymatic reaction. In case of nanoparticles injection to fibrinogen before thrombin addition, enzymatic reaction practically stopped at the first stage. The mixing of nanoparticles with thrombin before its addition to fibrinogen leads to acceleration of gel formation in comparison with reaction without nanoparticles. We believe that Fe2O3 nanoparticles can modify the rate of enzymatic reaction, in one case acting as inhibitors of the reaction and as activators in other.

  7. Inositol cyclic triphosphate [inositol 1,2-(cyclic)-4,5-triphosphate] is formed upon thrombin stimulation of human platelets.


    Ishii, H; Connolly, T M; Bross, T E; Majerus, P W


    Cleavage of polyphosphoinositides in vitro by phospholipase C results in formation of both cyclic and noncyclic inositol phosphates. We have now isolated the cyclic product of phosphatidylinositol 4,5-bisphosphate cleavage, inositol 1,2(cyclic)-4,5-triphosphate [cIns(1:2,4,5)P3], from thrombin-treated platelets. We found 0.2-0.4 nmol of cIns-(1:2,4,5)P3 per 10(9) platelets at 10 sec after thrombin; none was found in unstimulated platelets or in platelets 10 min after thrombin addition. We con...

  8. Salmon and Human Thrombin Differentially Regulate Radicular Pain, Glial-Induced Inflammation and Spinal Neuronal Excitability through Protease-Activated Receptor-1 (United States)

    Smith, Jenell R.; Syre, Peter P.; Oake, Shaina A.; Nicholson, Kristen J.; Weisshaar, Christine L.; Cruz, Katrina; Bucki, Robert; Baumann, Bethany C.; Janmey, Paul A.; Winkelstein, Beth A.


    Chronic neck pain is a major problem with common causes including disc herniation and spondylosis that compress the spinal nerve roots. Cervical nerve root compression in the rat produces sustained behavioral hypersensitivity, due in part to the early upregulation of pro-inflammatory cytokines, the sustained hyperexcitability of neurons in the spinal cord and degeneration in the injured nerve root. Through its activation of the protease-activated receptor-1 (PAR1), mammalian thrombin can enhance pain and inflammation; yet at lower concentrations it is also capable of transiently attenuating pain which suggests that PAR1 activation rate may affect pain maintenance. Interestingly, salmon-derived fibrin, which contains salmon thrombin, attenuates nerve root-induced pain and inflammation, but the mechanisms of action leading to its analgesia are unknown. This study evaluates the effects of salmon thrombin on nerve root-mediated pain, axonal degeneration in the root, spinal neuronal hyperexcitability and inflammation compared to its human counterpart in the context of their enzymatic capabilities towards coagulation substrates and PAR1. Salmon thrombin significantly reduces behavioral sensitivity, preserves neuronal myelination, reduces macrophage infiltration in the injured nerve root and significantly decreases spinal neuronal hyperexcitability after painful root compression in the rat; whereas human thrombin has no effect. Unlike salmon thrombin, human thrombin upregulates the transcription of IL-1β and TNF-α and the secretion of IL-6 by cortical cultures. Salmon and human thrombins cleave human fibrinogen-derived peptides and form clots with fibrinogen with similar enzymatic activities, but salmon thrombin retains a higher enzymatic activity towards coagulation substrates in the presence of antithrombin III and hirudin compared to human thrombin. Conversely, salmon thrombin activates a PAR1-derived peptide more weakly than human thrombin. These results are the

  9. A study on the sensitivity of photogrammetric camera calibration and stitching

    CSIR Research Space (South Africa)



    Full Text Available This paper presents a detailed simulation study of an automated robotic photogrammetric camera calibration system. The system performance was tested for sensitivity with regard to noise in the robot movement, camera mounting and image processing...

  10. Calibration of Geodetic Instruments

    Directory of Open Access Journals (Sweden)

    Marek Bajtala


    Full Text Available The problem of metrology and security systems of unification, correctness and standard reproducibilities belong to the preferred requirements of theory and technical practice in geodesy. Requirements on the control and verification of measured instruments and equipments increase and the importance and up-to-date of calibration get into the foreground. Calibration possibilities of length-scales (of electronic rangefinders and angle-scales (of horizontal circles of geodetic instruments. Calibration of electronic rangefinders on the linear comparative baseline in terrain. Primary standard of planar angle – optical traverse and its exploitation for calibration of the horizontal circles of theodolites. The calibration equipment of the Institute of Slovak Metrology in Bratislava. The Calibration process and results from the calibration of horizontal circles of selected geodetic instruments.

  11. Factor Xa generation by computational modeling: an additional discriminator to thrombin generation evaluation.

    Directory of Open Access Journals (Sweden)

    Kathleen E Brummel-Ziedins

    Full Text Available Factor (fXa is a critical enzyme in blood coagulation that is responsible for the initiation and propagation of thrombin generation. Previously we have shown that analysis of computationally generated thrombin profiles is a tool to investigate hemostasis in various populations. In this study, we evaluate the potential of computationally derived time courses of fXa generation as another approach for investigating thrombotic risk. Utilizing the case (n = 473 and control (n = 426 population from the Leiden Thrombophilia Study and each individual's plasma protein factor composition for fII, fV, fVII, fVIII, fIX, fX, antithrombin and tissue factor pathway inhibitor, tissue factor-initiated total active fXa generation was assessed using a mathematical model. FXa generation was evaluated by the area under the curve (AUC, the maximum rate (MaxR and level (MaxL and the time to reach these, TMaxR and TMaxL, respectively. FXa generation was analyzed in the entire populations and in defined subgroups (by sex, age, body mass index, oral contraceptive use. The maximum rates and levels of fXa generation occur over a 10- to 12- fold range in both cases and controls. This variation is larger than that observed with thrombin (3-6 fold in the same population. The greatest risk association was obtained using either MaxR or MaxL of fXa generation; with an ∼2.2 fold increased risk for individuals exceeding the 90(th percentile. This risk was similar to that of thrombin generation(MaxR OR 2.6. Grouping defined by oral contraceptive (OC use in the control population showed the biggest differences in fXa generation; a >60% increase in the MaxR upon OC use. FXa generation can distinguish between a subset of individuals characterized by overlapping thrombin generation profiles. Analysis of fXa generation is a phenotypic characteristic which may prove to be a more sensitive discriminator than thrombin generation among all individuals.


    NARCIS (Netherlands)


    Platelets activated with thrombin release bactericidal factors. We studied the role of the susceptibility of viridans streptococci to these bactericidal factors in the development of infective endocarditis (IE). By using the experimental endocarditis rabbit model, the initial adherence and the

  13. Thrombin-dependent intravascular leukocyte trafficking regulated by fibrin and the platelet receptors GPIb and PAR4. (United States)

    Kaplan, Zane S; Zarpellon, Alessandro; Alwis, Imala; Yuan, Yuping; McFadyen, James; Ghasemzadeh, Mehran; Schoenwaelder, Simone M; Ruggeri, Zaverio M; Jackson, Shaun P


    Thrombin is a central regulator of leukocyte recruitment and inflammation at sites of vascular injury, a function thought to involve primarily endothelial PAR cleavage. Here we demonstrate the existence of a distinct leukocyte-trafficking mechanism regulated by components of the haemostatic system, including platelet PAR4, GPIbα and fibrin. Utilizing a mouse endothelial injury model we show that thrombin cleavage of platelet PAR4 promotes leukocyte recruitment to sites of vascular injury. This process is negatively regulated by GPIbα, as seen in mice with abrogated thrombin-platelet GPIbα binding (hGPIbα(D277N)). In addition, we demonstrate that fibrin limits leukocyte trafficking by forming a physical barrier to intravascular leukocyte migration. These studies demonstrate a distinct 'checkpoint' mechanism of leukocyte trafficking involving balanced thrombin interactions with PAR4, GPIbα and fibrin. Dysregulation of this checkpoint mechanism is likely to contribute to the development of thromboinflammatory disorders.

  14. Low thrombin activatable fibrinolysis inhibitor activity levels are associated with an increased risk of a first myocardial infarction in men

    NARCIS (Netherlands)

    Meltzer, Mirjam E.; Doggen, Carine J. M.; de Groot, Philip G.; Meijers, Joost C. M.; Rosendaal, Frits R.; Lisman, Ton

    Background Studies on the relation between thrombin activatable fibrinolysis inhibitor (TAFI) and arterial thrombosis have produced conflicting results. TAFI regulates fibrinolysis, but other roles of this inhibitor, including anti-inflammatory properties, have also been demonstrated. Design and

  15. Balloon-assisted ultrasound-guided thrombin injection of a pseudoaneurysm in the posterior tibial artery: A case report

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Taeg Ki; Jeon, Yong Sun; Hong, Kee Chun; Cho, Soon Gu; Kim, Eu Gene [Inha University School of Medicine, Incheon (Korea, Republic of)


    An ultrasound-guided direct injection of thrombin is currently the first choice of treatment for the postcatheterization pseudoaneurysm, mainly in the femoral artery. A pseudoaneurysm in the posterior tibial artery is very rare, so there are not enough reports about proper treatment yet. We report a case of a balloon-assisted injection of thrombin under ultrasonography-guidance to manage a pseudoaneurysm in the posterior tibial artery and concurrently to prevent a distal artery embolization.

  16. Avathrin: a novel thrombin inhibitor derived from a multicopy precursor in the salivary glands of the ixodid tick, Amblyomma variegatum. (United States)

    Iyer, Janaki Krishnamoorthy; Koh, Cho Yeow; Kazimirova, Maria; Roller, Ladislav; Jobichen, Chacko; Swaminathan, Kunchithapadam; Mizuguchi, Jun; Iwanaga, Sadaaki; Nuttall, Patricia A; Chan, Mark Y; Kini, R Manjunatha


    Tick saliva is a rich source of antihemostatic compounds. We amplified a cDNA from the salivary glands of the tropical bont tick (Amblyomma variegatum) using primers based on the variegin sequence, which we previously identified as a novel thrombin inhibitor from the same tick species. The transcript encodes a precursor protein comprising a signal peptide and 5 repeats of variegin-like sequences that could be processed into multiple short peptides. These peptides share 31 to 34% identity with variegin. Here, we structurally and functionally characterized one of these peptides named "avathrin." Avathrin is a fast, tight binding competitive inhibitor with an affinity of 545 pM for thrombin and is 4 orders of magnitude more selective towards thrombin than to the other serine proteases of the coagulation cascade. The crystal structure of thrombin-avathrin complex at 2.09 Å revealed that avathrin interacts with the thrombin active site and exosite-I. Although avathrin is cleaved by thrombin, the C-terminal cleavage product continues to exert prolonged inhibition. Avathrin is more potent than hirulog-1 in a murine carotid artery thrombosis model. Such precursor proteins that could be processed into multiple thrombin inhibiting peptides appear to be widespread among Amblyomminae, providing an enormous library of molecules for development as potent antithrombotics.-Iyer, J. K., Koh, C. Y., Kazimirova, M., Roller, L., Jobichen, C., Swaminathan, K., Mizuguchi, J., Iwanaga, S., Nuttall, P. A., Chan, M. Y., Kini, R. M. Avathrin: a novel thrombin inhibitor derived from a multicopy precursor in the salivary glands of the ixodid tick, Amblyomma variegatum. © FASEB.

  17. Designing allosteric regulators of thrombin. Monosulfated benzofuran dimers selectively interact with Arg173 of exosite 2 to induce inhibition. (United States)

    Abdel Aziz, May H; Sidhu, Preetpal Singh; Liang, Aiye; Kim, Ji Yeong; Mosier, Philip D; Zhou, Qibing; Farrell, David H; Desai, Umesh R


    Earlier, we reported on the design of sulfated benzofuran dimers (SBDs) as allosteric inhibitors of thrombin (Sidhu et al. J. Med. Chem.201154 5522-5531). To identify the site of binding of SBDs, we studied thrombin inhibition in the presence of exosite 1 and 2 ligands. Whereas hirudin peptide and heparin octasaccharide did not affect the IC(50) of thrombin inhibition by a high affinity SBD, the presence of full-length heparin reduced inhibition potency by 4-fold. The presence of γ' fibrinogen peptide, which recognizes Arg93, Arg97, Arg173, Arg175, and other residues, resulted in a loss of affinity that correlated with the ideal Dixon-Webb competitive profile. Replacement of several arginines and lysines of exosite 2 with alanine did not affect thrombin inhibition potency, except for Arg173, which displayed a 22-fold reduction in IC(50). Docking studies suggested a hydrophobic patch around Arg173 as a plausible site of SBD binding to thrombin. The absence of the Arg173-like residue in factor Xa supported the observed selectivity of inhibition by SBDs. Cellular toxicity studies indicated that SBDs are essentially nontoxic to cells at concentrations as high as 250 mg/kg. Overall, the work presents the localization of the SBD binding site, which could lead to allosteric modulators of thrombin that are completely different from all clinically used anticoagulants.

  18. Simultaneous thrombin and plasmin generation capacities in normal and abnormal states of coagulation and fibrinolysis in children and adults. (United States)

    Simpson, Mindy L; Goldenberg, Neil A; Jacobson, Linda J; Bombardier, Christopher G; Hathaway, William E; Manco-Johnson, Marilyn J


    Thrombin and plasmin are the key enzymes involved in coagulation and fibrinolysis, respectively. Plasma coagulative and fibrinolytic potentials in normal children and adults, and in representative pathologically altered hemostatic states, were evaluated via simultaneous assessment of thrombin and plasmin generation. An assay of Simultaneous Thrombin and Plasmin generation (STP) was developed to measure thrombin and plasmin in plasma using individual fluorometric substrates. Coagulation is initiated with dilute tissue factor, phospholipid, and calcium in platelet-poor plasma; fibrinolysis is accelerated via tissue plasminogen activator (tPA). Abnormal states of hemostasis were investigated. STP assay reproducibility and normal adult and pediatric values for measured and calculated parameters have been established. Onset of both thrombin and plasmin generation was significantly delayed in children relative to adults (pacid. Plasmin generation was greatly enhanced by alpha-2-antiplasmin deficiency and excess tPA reagent. Simultaneous assessment of thrombin and plasmin generation in plasma shows promise for affording an enhanced understanding of overall coagulative and fibrinolytic functions in physiological and pathologically altered states of hemostasis in children and adults. Copyright © 2010 Elsevier Ltd. All rights reserved.

  19. Inhibition of Thrombin With PPACK-Nanoparticles Restores Disrupted Endothelial Barriers and Attenuates Thrombotic Risk in Experimental Atherosclerosis. (United States)

    Palekar, Rohun U; Jallouk, Andrew P; Myerson, Jacob W; Pan, Hua; Wickline, Samuel A


    A role for thrombin in the pathogenesis of atherosclerosis has been suggested through clinical and experimental studies revealing a critical link between the coagulation system and inflammation. Although approved drugs for inhibition of thrombin and thrombin-related signaling have demonstrated efficacy, their clinical application to this end may be limited because of significant potential for bleeding side effects. Thus, we sought to implement a plaque-localizing nanoparticle-based approach to interdict thrombin-induced inflammation and hypercoagulability in atherosclerosis. We deployed a novel magnetic resonance spectroscopic method to quantify the severity of endothelial damage for correlation with traditional metrics of vessel procoagulant activity after dye-laser injury in fat-fed apolipoprotein E-null mice. We demonstrate that a 1-month course of treatment with antithrombin nanoparticles carrying the potent thrombin inhibitor PPACK (d-phenylalanyl-l-prolyl-l-arginyl chloromethylketone) nanoparticle (1) reduces the expression and secretion of proinflammatory and procoagulant molecules, (2) diminishes plaque procoagulant activity without the need for systemic anticoagulation, (3) rapidly restores disrupted vascular endothelial barriers, and (4) retards plaque progression in lesion-prone areas. These observations illustrate the role of thrombin as a pleiotropic atherogenic molecule under conditions of hypercholesterolemia and suggest the utility of its inhibition with locally acting antithrombin nanoparticle therapeutics as a rapid-acting anti-inflammatory strategy in atherosclerosis to reduce thrombotic risk. © 2016 American Heart Association, Inc.

  20. Targeting the GPIbα Binding Site of Thrombin To Simultaneously Induce Dual Anticoagulant and Antiplatelet Effects (United States)


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

  1. Calibration of areal surface topography measuring instruments (United States)

    Seewig, J.; Eifler, M.


    The ISO standards which are related to the calibration of areal surface topography measuring instruments are the ISO 25178-6xx series which defines the relevant metrological characteristics for the calibration of different measuring principles and the ISO 25178-7xx series which defines the actual calibration procedures. As the field of areal measurement is however not yet fully standardized, there are still open questions to be addressed which are subject to current research. Based on this, selected research results of the authors in this area are presented. This includes the design and fabrication of areal material measures. For this topic, two examples are presented with the direct laser writing of a stepless material measure for the calibration of the height axis which is based on the Abbott- Curve and the manufacturing of a Siemens star for the determination of the lateral resolution limit. Based on these results, as well a new definition for the resolution criterion, the small scale fidelity, which is still under discussion, is presented. Additionally, a software solution for automated calibration procedures is outlined.

  2. Addition of thrombin reduces the recovery of extracellular vesicles from blood plasma (United States)

    Arakelyan, Anush; Fitzgerald, Wendy; Vagida, Murad; Vasilieva, Elena; Grivel, Jean-Charles


    Extracellular vesicles (EVs) are widely studied as a system of intercellular communication, as markers of various diseases, as well as a vehicle for delivery of various bioactive molecules to various cells. Investigation of EVs’ structure and function requires their isolation and precise quantification. However, in the current literature, there are significant discrepancies in the estimated numbers of EVs in different body fluids. In part, this discrepancy is due to the difference in EVs isolation protocols used by different investigators. A common protocol that includes ExoQuick™ is often used to isolate EVs from body fluids and culture medium. Here, we show that in the case of isolation of EVs from blood, thrombin should be omitted from the protocol as clots formed due to the thrombin-triggered coagulation may entrap many EVs thus leading to the underestimation of their numbers. PMID:28936260

  3. Addition of thrombin reduces the recovery of extracellular vesicles from blood plasma

    Directory of Open Access Journals (Sweden)

    Anush Arakelyan


    Full Text Available Extracellular vesicles (EVs are widely studied as a system of intercellular communication, as markers of various diseases, as well as a vehicle for delivery of various bioactive molecules to various cells. Investigation of EVs’ structure and function requires their isolation and precise quantification. However, in the current literature, there are significant discrepancies in the estimated numbers of EVs in different body fluids. In part, this discrepancy is due to the difference in EVs isolation protocols used by different investigators. A common protocol that includes ExoQuick ™ is often used to isolate EVs from body fluids and culture medium. Here, we show that in the case of isolation of EVs from blood, thrombin should be omitted from the protocol as clots formed due to the thrombin-triggered coagulation may entrap many EVs thus leading to the underestimation of their numbers.

  4. Activity of thrombin-activatable fibrinolysis inhibitor in the plasma of patients with abdominal aortic aneurysm. (United States)

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


    Patients with abdominal aortic aneurysm (AAA) experience impaired balance between fibrinolysis and coagulation, manifested by increased prothrombotic tendency and intensified inflammatory processes. The aim of this study was to evaluate the TAFI activity level (thrombin activatable fibrinolysis inhibitor) in the plasma of AAA patients. Plasma levels of PAI-1 (plasminogen activator inhibitor type 1), urokinase-type plasminogen activator and uPAR (urokinase-type plasminogen activator receptor) were measured as markers of fibrinolytic activity. The study showed that the activity of the thrombin-activatable fibrinolysis inhibitor in the plasma of AAA patients was significantly lower than in the plasma of the control individuals (64.6 ± 10.1 vs. 54.2 ± 10.9%, P fibrinolysis inhibitor TAFI may heighten the blood fibrinolytic potential in AAA patients and contribute to the development of comorbidities. Therefore, TAFI participation in AAA pathogenesis cannot be excluded.

  5. Anti-Helicobacter pylori and thrombin inhibitory components from Chinese dragon's blood, Dracaena cochinchinensis. (United States)

    Zhu, Yingdong; Zhang, Ping; Yu, Haiping; Li, Jia; Wang, Ming-Wei; Zhao, Weimin


    Chemical studies on the constituents of Dracaena cochinchinensis led to the discovery of eight new flavonoid derivatives ( 1- 8) along with 14 known compounds ( 9- 22). The identification and structural elucidation of these isolates were based on spectral analyses. All isolates were tested for antibacterial activities against Helicobacter pylori (ATCC43504) and thrombin inhibitory effects. As a result, new flavonoid derivatives 6 and 7 and (2 S)-4',7-dihydroxy-8-methylflavan ( 11) were found to be most efficacious against H. pylori (ATCC43504) with MIC values of 29.5, 29.5, and 31.3 microM, respectively, and the seven new flavonoid derivatives ( 1- 7) and one known biflavonoid ( 9) were observed to exhibit moderate thrombin inhibitory activity.

  6. Trypsin, Tryptase, and Thrombin Polarize Macrophages towards a Pro-Fibrotic M2a Phenotype.

    Directory of Open Access Journals (Sweden)

    Michael J V White

    Full Text Available For both wound healing and the formation of a fibrotic lesion, circulating monocytes enter the tissue and differentiate into fibroblast-like cells called fibrocytes and pro-fibrotic M2a macrophages, which together with fibroblasts form scar tissue. Monocytes can also differentiate into classically activated M1 macrophages and alternatively activated M2 macrophages. The proteases thrombin, which is activated during blood clotting, and tryptase, which is released by activated mast cells, potentiate fibroblast proliferation and fibrocyte differentiation, but their effect on macrophages is unknown. Here we report that thrombin, tryptase, and the protease trypsin bias human macrophage differentiation towards a pro-fibrotic M2a phenotype expressing high levels of galectin-3 from unpolarized monocytes, or from M1 and M2 macrophages, and that these effects appear to operate through protease-activated receptors. These results suggest that proteases can initiate scar tissue formation by affecting fibroblasts, fibrocytes, and macrophages.

  7. Gelatin-thrombin hemostatic matrix injection to salvage refractory post-renal graft biopsy bleed

    Directory of Open Access Journals (Sweden)

    V Jain


    Full Text Available Post-renal biopsy bleeding refractory to angioembolization usually requires graft nephrectomy as a life-saving measure. Gelatin-thrombin hemostatic matrix injection in the needle tract is a novel attempt to control bleeding in such cases and to salvage the allograft. We hereby describe two cases of post-graft biopsy bleed. Both these patients continued to bleed even after angioembolization. They were shifted to the operating room upon developing hypotension, having received multiple blood transfusions with the intention of performing graft nephrectomy to save their lives. However, bleeding was successfully controlled by using Gelatin-thrombin hemostatic matrix injection in the biopsy needle tract. Patients improved hemodynamically after the procedure. Graft function returned to normal in both the cases. At an average follow-up of 10.4 months, both the patients have shown stable graft functions.

  8. Aptamer/Protein Proximity Binding-Triggered Molecular Machine for Amplified Electrochemical Sensing of Thrombin. (United States)

    Yang, Jianmei; Dou, Baoting; Yuan, Ruo; Xiang, Yun


    The development of convenient and sensitive methods without involving any enzymes or complex nanomaterials for the monitoring of proteins is of great significance in disease diagnostics. In this work, we describe the validation of a new aptamer/protein proximity binding-triggered molecular machinery amplification strategy for sensitive electrochemical assay of thrombin in complex serum samples. The sensing interface is prepared by self-assembly of three-stranded DNA complexes on the gold electrode. The association of two distinct functional aptamers with different sites of thrombin triggers proximity binding-induced displacement of one of the short single-stranded DNAs (ssDNAs) from the surface-immobilized three-stranded DNA complexes, exposing a prelocked toehold domain to hybridize with a methylene blue (MB)-tagged fuel ssDNA strand (MB-DNA). Subsequent toehold-mediated strand displacement by the MB-DNA leads to the release and recycling of the aptamer/protein complexes and the function of the molecular machine. Eventually, a large number of MB-DNA strands are captured by the sensor surface, generating drastically amplified electrochemical responses from the MB tags for sensitive detection of thrombin. Our signal amplified sensor is completely enzyme-free and shows a dynamic range from 5 pM to 1 nM with a detection limit of 1.7 pM. Such sensor also has a high specificity for thrombin assay in serum samples. By changing the affinity probe pairs, the developed sensor can be readily expanded as a more general platform for sensitive detection of different types of proteins.

  9. Simultaneous measurement of thrombin and plasmin generation to assess the interplay between coagulation and fibrinolysis. (United States)

    Matsumoto, Tomoko; Nogami, Keiji; Shima, Midori


    Normal haemostasis is maintained by a controlled balance between coagulation and fibrinolysis, involving thrombin and plasmin the respective key enzymes. Simultaneous evaluation of both enzymes facilitates, therefore, an overall understanding of normal and pathological haemostasis. Combined thrombin and plasmin generation (T/P-G) assays have been recently described, and we have adapted the technique to investigate the interplay between coagulation and fibrinolysis in patients with various haemostatic disorders. Our modified T/P-G was initiated by the addition of a mixture of optimised lower concentrations of tissue factor and tissue-type plasminogen activator. Thrombin generation (TG) and plasmin generation (PG) were monitored simultaneously using individual fluorescent substrates in separate microtitre wells. The relationship between coagulation and fibrinolysis was demonstrated by analysing the effects of thrombin inhibitors, activated protein C and thrombomodulin. The most evident impairments in TG were observed with plasma samples deficient of coagulation factors participating in the prothrombinase complex. Defects in PG were observed with deficiencies of factor (F)V, FX, fibrinogen, and plasminogen. TG appeared to be a prerequisite for the initiation of PG, and overall PG was governed by fibrinogen concentration. TG in patients with haemophilia A correlated with levels of FVIII activity, but there was no significant relationship between PG and FVIII:C, confirming that the abnormal haemostasis in haemophilia A results in a severe imbalance between coagulation and fibrinolysis. The findings demonstrate that global haemostasis depends on a sensitive balance between coagulation and fibrinolysis, and that the modified T/P-G assay could provide an enhanced understanding of haemorrhage and thrombosis in clinical practice.

  10. Successful endovascular treatment of a hemodialysis graft pseudoaneurysm by covered stent and direct percutaneous thrombin injection.

    LENUS (Irish Health Repository)

    Keeling, Aoife N


    Vascular access for hemodialysis remains a challenge for nephrologists, vascular surgeons, and interventional radiologists alike. Arteriovenous fistula and synthetic grafts remain the access of choice for long-term hemodialysis; however, they are subject to complications from infection and repeated needle cannulation. Pseudoaneurysms are an increasingly recognized adverse event. At present, there are many minimally invasive methods to repair these wall defects. We present a graft pseudoaneurysm, which required a combination of endovascular stent graft placement and percutaneous thrombin injection for successful occlusion.

  11. Spontaneous pseudoaneurysm of the uterine artery during pregnancy treated by direct thrombin injection: A case report

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Jung Hee; Kim, See Hyung; Kim, Young Hwan [Dept. Radiology, Keimyung University School of Medicine, Dongsan Medical Center, Daegu (Korea, Republic of)


    Pseudoaneurysm of uterine artery during pregnancy is a very rare disease. It is mostly associated with uterine artery injury, usually occurring after proceeding conditions such as history of gynecologic operation and infection. However, the best treatment modality has not been established yet. Herein, we reported a case of spontaneous formation of uterine artery pseudoaneurysm during pregnancy treated by direct thrombin injection without any complication or recurrence.

  12. Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin. (United States)

    Treuheit, Nicholas A; Beach, Muneera A; Komives, Elizabeth A


    Several lines of experimental evidence including amide exchange and NMR suggest that ligands binding to thrombin cause reduced backbone dynamics. Binding of the covalent inhibitor dPhe-Pro-Arg chloromethyl ketone to the active site serine, as well as noncovalent binding of a fragment of the regulatory protein, thrombomodulin, to exosite 1 on the back side of the thrombin molecule both cause reduced dynamics. However, the reduced dynamics do not appear to be accompanied by significant conformational changes. In addition, binding of ligands to the active site does not change the affinity of thrombomodulin fragments binding to exosite 1; however, the thermodynamic coupling between exosite 1 and the active site has not been fully explored. We present isothermal titration calorimetry experiments that probe changes in enthalpy and entropy upon formation of binary ligand complexes. The approach relies on stringent thrombin preparation methods and on the use of dansyl-l-arginine-(3-methyl-1,5-pantanediyl)amide and a DNA aptamer as ligands with ideal thermodynamic signatures for binding to the active site and to exosite 1. Using this approach, the binding thermodynamic signatures of each ligand alone as well as the binding signatures of each ligand when the other binding site was occupied were measured. Different exosite 1 ligands with widely varied thermodynamic signatures cause a similar reduction in ΔH and a concomitantly lower entropy cost upon DAPA binding at the active site. The results suggest a general phenomenon of enthalpy-entropy compensation consistent with reduction of dynamics/increased folding of thrombin upon ligand binding to either the active site or exosite 1.

  13. Nanoparticles That Sense Thrombin Activity As Synthetic Urinary Biomarkers of Thrombosis (United States)


    Thrombin is a serine protease and regulator of hemostasis that plays a critical role in the formation of obstructive blood clots, or thrombosis, that is a life-threatening condition associated with numerous diseases such as atherosclerosis and stroke. To detect thrombi in living animals, we design and conjugate thrombin-sensitive peptide substrates to the surface of nanoparticles. Following intravenous infusion, these “synthetic biomarkers” survey the host vasculature for coagulation and, in response to substrate cleavage by thrombin, release ligand-encoded reporters into the host urine. To detect the urinary reporters, we develop a companion 96-well immunoassay that utilizes antibodies to bind specifically to the ligands, thus capturing the reporters for quantification. Using a thromboplastin-induced mouse model of pulmonary embolism, we show that urinary biomarker levels differentiate between healthy and thrombotic states and correlate closely with the aggregate burden of clots formed in the lungs. Our results demonstrate that synthetic biomarkers can be engineered to sense vascular diseases remotely from the urine and may allow applications in point-of-care diagnostics. PMID:24015809

  14. Design, synthesis and antithrombotic evaluation of novel non-peptide thrombin inhibitors. (United States)

    Chen, Dongxing; Shi, Jinyu; Liu, Jing; Zhang, Xueying; Deng, Xiaoying; Yang, Yanyan; Cui, Shuang; Zhu, Qihua; Gong, Guoqing; Xu, Yungen


    Ten derivatives of 4-((1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-1-yl)methyl)benzimida-mide (I-1∼I-2, II-1∼II-8) were designed, synthesized and evaluated for their inhibitory effect on human thrombin. Compound II-7 (IC50=82.8nM), which showed the strongest thrombin inhibitory activity among the tested compounds, was chosen as the lead compound, and ten carbamate derivatives (II-9a∼II-13a, II-9b∼II-12b, II-14) were prepared and evaluated for their anticoagulant activity. The results indicate that most of the tested compounds exhibit a certain degree of inhibitory effect on thrombin-induced platelet aggregation, among which compounds II-11a (IC50=8.16μM) and II-14 (IC50=1.95μM) show better anti-platelet aggregation activity than the others. The in vivo experimental results in rat venous thrombosis model also demonstrate compounds II-11a and II-14 can significantly reduce thrombosis in a dose-response manner. It is worth pointing out that the enhanced potency of compound II-14 may be the synergetic effect of 2-hydroxymethyl-3,5,6-trimethylpyrazine (HTMP) and II-7 which are generated by hydrolysis in vivo. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Electrochemical Raduction Potential Shifts of Graphene Oxide Employed in Thrombin Detection

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Hanall; Hwang, Shinjae; Kim, Kyuwon [Incheon National Univ., Incheon (Korea, Republic of)


    We have reported a feasibility study on thrombin detection by the measurement of peak potentials shifts for the electrochemical reduction of GO. Our novel strategy has demonstrated that the shifts are fairly dependent upon the concentration of target thrombin. We are extending this result to determine quantitatively various target material such as proteins, DNA, metal ions. The development of electrochemical biosensor is currently under the intense investigation owing to their great promise for rapid, convenience and low-cost detection of analytes. Especially, graphene oxide (GO), an oxygen-rich carbonaceous layered material, has attracted strong interest as an substrate material because of its own unique characteristics. Based on recent studies GO consists of intact graphitic regions interspersed with sp{sup 3}-hybridized carbons containing hydroxyl and epoxy functional groups on the top and bottom surfaces of each sheet and sp{sup 2}-hybridized carbons containing carboxylate and carbonyl groups principally at the sheet edges. The intrinsic oxygen-containing functional groups have been used as initial sites for deposition of biomolecules such as DNA and proteins on the GO sheets and provide the possibility to be an ideal platform for detecting of target materials. In addition, GO is electrochemically reducible, which enables it as an indicator or label for electrochemical sensor applications. Several studies have employed the GO-indicator to determine DNA, Hg{sup 2+}, and thrombin.

  16. [Heparin cofactor II, a thrombin inhibitor with a still not clarified physiologic role]. (United States)

    Rossi, E B; Duboscq, C L; Kordich, L C


    Heparin Cofactor II (HCII) is a glycoprotein in human plasma which inactivates thrombin rapidly in the presence of dermatan sulfate. Inhibition occurs by formation of a stable equimolar complex between HCII and thrombin. HCII association with thrombotic events has not always been observed, thus decreased HCII does not appear to be a strong risk factor for thromboembolic events. Reduced HCII levels have been detected in different clinical conditions, such as hepatic failure, disseminated intravascular coagulation, thalasemina, sickle cell anemia. Increased physiological levels have been found in pregnant women and oral contraception. In our laboratory, we measured HCII plasmatic levels in the normal Buenos Aires city population and in patients under different clinical conditions, such as sepsis, diabetis, burns, oral anticoagulation and in patients treated with heparin, hyperhomcysteinemia in whom septic and diabetic patients showed decreased values. HCII thrombin inhibition possibly takes place in extravascular sites where dermatan sulfate is present. HCII activity would be important in the regulation of wound healing, inflammation, or neuronal development.

  17. Contributions of thrombin targets to tissue factor-dependent metastasis in hyperthrombotic mice. (United States)

    Yokota, N; Zarpellon, A; Chakrabarty, S; Bogdanov, V Y; Gruber, A; Castellino, F J; Mackman, N; Ellies, L G; Weiler, H; Ruggeri, Z M; Ruf, W


    Tumor cell tissue factor (TF)-initiated coagulation supports hematogenous metastasis by fibrin formation, platelet activation and monocyte/macrophage recruitment. Recent studies identified host anticoagulant mechanisms as a major impediment to successful hematogenous tumor cell metastasis. Here we address mechanisms that contribute to enhanced metastasis in hyperthrombotic mice with functional thrombomodulin deficiency (TM(Pro) mice). Pharmacological and genetic approaches were combined to characterize relevant thrombin targets in a mouse model of experimental hematogenous metastasis. TF-dependent, but contact pathway-independent, syngeneic breast cancer metastasis was associated with marked platelet hyperreactivity and formation of leukocyte-platelet aggregates in immune-competent TM(Pro) mice. Blockade of CD11b or genetic deletion of platelet glycoprotein Ibα excluded contributions of these receptors to enhanced platelet-dependent metastasis in hyperthrombotic mice. Mice with very low levels of the endothelial protein C receptor (EPCR) did not phenocopy the enhanced metastasis seen in TM(Pro) mice. Genetic deletion of the thrombin receptor PAR1 or endothelial thrombin signaling targets alone did not diminish enhanced metastasis in TM(Pro) mice. Combined deficiency of PAR1 on tumor cells and the host reduced metastasis in TM(Pro) mice. Metastasis in the hyperthrombotic TM(Pro) mouse model is mediated by platelet hyperreactivity and contributions of PAR1 signaling on tumor and host cells. © 2013 International Society on Thrombosis and Haemostasis.

  18. CMS silicon strip tracker calibration work-flow and tools

    CERN Document Server

    Lampen, Tapio


    The Silicon Strip Tracker of CMS is by far the biggest detector of its kind ever operated. Its 15 000 detector modules and 9 million readout channels are individually calibrated in order to achieve the optimal data quality for the experiment. Software tools were designed to automate the operations and reduce the need for maintenance as much as possible, taking care to use pre-existing software frameworks, when possible. The calibration software implements a dedicated scheme of event building, on-line distributed analysis, storage management, data analysis and configuration archival. Dedicated user interfaces and web-applications were developed to ease the operations, speed-up the calibration process, monitor its quality, track the problems. A complete set of monitoring analyses are also performed online during data taking to validate the data acquired. The calibration parameters are measured continuously, and the values are fed back to the calibration database in order to refine the on-line event reconstructi...

  19. Site Calibration, FGW

    DEFF Research Database (Denmark)

    Kock, Carsten Weber; Vesth, Allan

    This Site Calibration report is describing the results of a measured site calibration for a site in Denmark. The calibration is carried out by DTU Wind Energy in accordance with Ref.[3] and Ref.[4]. The measurement period is given. The site calibration is carried out before a power performance...... measurement on a given turbine to clarify the influence from the terrain on the ratio between the wind speed at the center of the turbine hub and at the met mast. The wind speed at the turbine is measured by a temporary mast placed at the foundation for the turbine. The site and measurement equipment...

  20. Automated Budget System - (United States)

    Department of Transportation — The Automated Budget System (ABS) automates management and planning of the Mike Monroney Aeronautical Center (MMAC) budget by providing enhanced capability to plan,...

  1. Automation 2017

    CERN Document Server

    Zieliński, Cezary; Kaliczyńska, Małgorzata


    This book consists of papers presented at Automation 2017, an international conference held in Warsaw from March 15 to 17, 2017. It discusses research findings associated with the concepts behind INDUSTRY 4.0, with a focus on offering a better understanding of and promoting participation in the Fourth Industrial Revolution. Each chapter presents a detailed analysis of a specific technical problem, in most cases followed by a numerical analysis, simulation and description of the results of implementing the solution in a real-world context. The theoretical results, practical solutions and guidelines presented are valuable for both researchers working in the area of engineering sciences and practitioners looking for solutions to industrial problems. .

  2. Marketing automation

    Directory of Open Access Journals (Sweden)

    TODOR Raluca Dania


    Full Text Available The automation of the marketing process seems to be nowadays, the only solution to face the major changes brought by the fast evolution of technology and the continuous increase in supply and demand. In order to achieve the desired marketing results, businessis have to employ digital marketing and communication services. These services are efficient and measurable thanks to the marketing technology used to track, score and implement each campaign. Due to the technical progress, the marketing fragmentation, demand for customized products and services on one side and the need to achieve constructive dialogue with the customers, immediate and flexible response and the necessity to measure the investments and the results on the other side, the classical marketing approached had changed continue to improve substantially.

  3. South African initiative for pre-flight radiometric calibration of satellite imagers

    CSIR Research Space (South Africa)

    Griffith, D


    Full Text Available or the UUT to the collimated beam coming from the monochromator. m) A mounting region for the UUT. Calibration measurements are automated using LabView. Figure 1: Schematic of Spectral Calibration Bench 3.2. Radiometric Modeling The relative spectral...

  4. Structure-activity analysis of synthetic alpha-thrombin-receptor-activating peptides. (United States)

    Van Obberghen-Schilling, E; Rasmussen, U B; Vouret-Craviari, V; Lentes, K U; Pavirani, A; Pouysségur, J


    alpha-Thrombin stimulates G-protein-coupled effectors leading to secretion and aggregation in human platelets, and to a mitogenic response in CCL39 hamster fibroblasts. alpha-Thrombin receptors can be activated by synthetic peptides corresponding to the receptor sequence starting with serine-42, at the proposed cleavage site. We have previously determined that the agonist domain of receptor-activating peptides resides within the five N-terminal residues [Vouret-Craviari, Van Obberghen-Schilling, Rasmussen, Pavirani, Lecocq and Pouysségur (1992) Mol. Biol. Cell. 3, 95-102], although the 7-residue peptide (SFFLRNP) corresponding to the hamster alpha-thrombin receptor was 10 times more potent than the 5-residue peptide for activation of human platelets. In the present study we have analysed the role of individual amino acids in receptor activation by using a series of modified hexa- or hepta-peptides derived from the human alpha-thrombin-receptor sequence. Cellular events examined here include phospholipase C activation, adenylyl cyclase inhibition and DNA synthesis stimulation in non-transformed CCL39 fibroblasts and a tumorigenic variant of that line (A71 cells). Modification of the peptide sequence had similar functional consequence for each of the assays described, indicating that either a unique receptor or pharmacologically indistinguishable receptor subtypes activate distinct G-protein signalling pathways. Furthermore, we found that: (1) the N-terminal serine can be replaced by small or intermediately sized amino acids (+/- hydroxyl groups) without loss of activity. However, its replacement by an aromatic side-chain or omission of the N-terminal amino group severely reduces activity. (2) An aromatic side-chain on the penultimate N-terminal residue appears to play a critical role since phenylalanine in this position can be substituted by tyrosine without complete loss of activity whereas an alanine in its place is not tolerated. (3) Deletion of the first

  5. SMAP RADAR Calibration and Validation (United States)

    West, R. D.; Jaruwatanadilok, S.; Chaubel, J.


    The Soil Moisture Active Passive (SMAP) mission is planned to launch on Jan 8, 2015. The mission employs L-band radar and radiometer measurements to estimate soil moisture with 4% volumetric accuracy at a resolution of 10 km, and freeze-thaw state at a resolution of 1-3 km. Immediately following launch, there will be a 3 month instrument checkout period, followed by 6 months of level 1 (L1) calibration and validation. In this presentation, we will discuss the plans and preparations for the calibration and validation of L1 radar data from SMAP. At the start of the L1 cal/val period, we will validate the operation of the instrument and of the ground processing using tools that look at readily identifiable surface features such as coast lines and corner reflectors. Geometric biases will be fit and removed. Radiometric cross-calibration with PALSAR and Aquarius will also be performed using target regions in the Amazon rain forest selected for their stability and uniformity. As the L1 cal/val period progresses, the performance of the automated short and long term calibration modules in ground processing will be tracked and verified using data from stable reference targets such as the wind corrected ocean and selected areas of rain forest that have shown good temporal stability. The performance of the radio frequency interference (RFI) removal algorithm will be validated by processing data with the algorithm turned on and off, and using different parameter settings. Additional information on the extent of RFI will be obtained from a special RFI survey conducted early in the L1 cal/val period. Radar transmissions are turned off during the RFI survey, and receive only data are collected over a variety of operating frequencies. The model based Faraday rotation corrections will also be checked during the L1 cal/val by comparing the model Faraday rotation with the measured Faraday rotation obtained by the SMAP Radiometer. This work is supported by the SMAP project at the Jet

  6. Site Calibration report

    DEFF Research Database (Denmark)

    Gómez Arranz, Paula; Vesth, Allan

    This report describes the site calibration carried out at Østerild, during a given period. The site calibration was performed with two Windcube WLS7 (v1) lidars at ten measurements heights. The lidar is not a sensor approved by the current version of the IEC 61400-12-1 [1] and therefore the site...

  7. TWSTFT Link Calibration Report (United States)


    Washington Headquarters Services , Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington VA 22202-4302...and Bauch A (2014) THE EUROPEAN TW CALIBRATION CAMPAIGN 2014 IN THE SCOPE OF GALILEO (TGVF- FOC), An opportunity to update, TW link calibrations in

  8. Lidar to lidar calibration

    DEFF Research Database (Denmark)

    Fernandez Garcia, Sergio; Villanueva, Héctor

    This report presents the result of the lidar to lidar calibration performed for ground-based lidar. Calibration is here understood as the establishment of a relation between the reference lidar wind speed measurements with measurement uncertainties provided by measurement standard and correspondi...

  9. β2-Glycoprotein I binds factor XI and inhibits its activation by thrombin and factor XIIa: Loss of inhibition by clipped β2-glycoprotein I


    Shi, Tong; Iverson, G. Michael; Qi, Jian C.; Cockerill, Keith A.; Linnik, Matthew D.; Konecny, Pamela; Krilis, Steven A.


    Activation of factor XI (FXI) by thrombin in vivo plays a role in coagulation by providing an important positive feedback mechanism for additional thrombin generation. FXI is activated in vitro by thrombin, or FXIIa in the presence of dextran sulfate. In this report, we investigated the effect of β2-glycoprotein I (β2GPI) on the activation of FXI. β2GPI bound FXI in vitro and inhibited its activation to FXIa by thrombin and FXIIa. The affinity of the interaction between β2GPI and FXI was equi...


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

  11. Sandia WIPP calibration traceability

    Energy Technology Data Exchange (ETDEWEB)

    Schuhen, M.D. [Sandia National Labs., Albuquerque, NM (United States); Dean, T.A. [RE/SPEC, Inc., Albuquerque, NM (United States)


    This report summarizes the work performed to establish calibration traceability for the instrumentation used by Sandia National Laboratories at the Waste Isolation Pilot Plant (WIPP) during testing from 1980-1985. Identifying the calibration traceability is an important part of establishing a pedigree for the data and is part of the qualification of existing data. In general, the requirement states that the calibration of Measuring and Test equipment must have a valid relationship to nationally recognized standards or the basis for the calibration must be documented. Sandia recognized that just establishing calibration traceability would not necessarily mean that all QA requirements were met during the certification of test instrumentation. To address this concern, the assessment was expanded to include various activities.

  12. A label-free and high sensitive aptamer biosensor based on hyperbranched polyester microspheres for thrombin detection

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Chong [Jiangsu Key Laboratory of Biofunctional Materials, Biomedical Functional Materials Collaborative Innovation Center, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023 (China); Institute of Agricultural Products Processing, Jiangsu Academy of Agricultural Sciences, Nanjing 210014 (China); Han, Qiaorong [Jiangsu Key Laboratory of Biofunctional Materials, Biomedical Functional Materials Collaborative Innovation Center, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023 (China); Wang, Daoying; Xu, Weimin [Institute of Agricultural Products Processing, Jiangsu Academy of Agricultural Sciences, Nanjing 210014 (China); Wang, Weijuan [Jiangsu Key Laboratory of Biofunctional Materials, Biomedical Functional Materials Collaborative Innovation Center, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023 (China); Zhao, Wenbo, E-mail: [Jiangsu Key Laboratory of Biofunctional Materials, Biomedical Functional Materials Collaborative Innovation Center, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023 (China); Zhou, Min, E-mail: [Department of Vascular Surgery, the Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008 (China)


    Highlights: • A label-free thrombin aptamer biosensor applied in whole blood has been developed. • The aptamer biosensor showed a wide detection range and a low detection limit. • The antibiofouling idea utilized for biosensor is significant for diagnostics. - Abstract: In this paper, we have synthesized hyperbranched polyester microspheres with carboxylic acid functional groups (HBPE-CA) and developed a label-free electrochemical aptamer biosensor using thrombin-binding aptamer (TBA) as receptor for the measurement of thrombin in whole blood. The indium tin oxide (ITO) electrode surface modified with HBPE-CA microspheres was grafted with TBA, which has excellent binding affinity and selectivity for thrombin. Binding of the thrombin at the modified ITO electrode surface greatly restrained access of electrons for a redox probe of [Fe(CN){sub 6}]{sup 3−/4−}. Moreover, the aptamer biosensor could be used for detection of thrombin in whole blood, a wide detection range (10 fM–100 nM) and a detection limit on the order of 0.90 fM were demonstrated. Control experiments were also carried out by using bull serum albumin (BSA) and lysozyme in the absence of thrombin. The good stability and repeatability of this aptamer biosensor were also proved. We expect that this demonstration will lead to the development of highly sensitive label-free sensors based on aptamer with lower cost than current technology. The integration of the technologies, which include anticoagulant, sensor and nanoscience, will bring significant input to high-performance biosensors relevant to diagnostics and therapy of interest for human health.

  13. Anti-inflammatory and anti-fibrinolytic effects of thrombomodulin alfa through carboxypeptidase B2 in the presence of thrombin. (United States)

    Tawara, Shunsuke; Sakai, Takumi; Matsuzaki, Osamu


    Thrombomodulin (TM) alfa, a recombinant human soluble TM, enhances activation of pro-carboxypeptidase B2 (pro-CPB2) by thrombin. Activated pro-CPB2 (CPB2) exerts anti-inflammatory and anti-fibrinolytic activities. Therefore, TM alfa may also have anti-inflammatory and anti-fibrinolytic effects through CPB2. However, these effects of TM alfa have not been elucidated. In the present study, we investigated the effects of TM alfa on inactivation of complement component C5a as an anti-inflammatory effect and prolongation of clot lysis time as an anti-fibrinolytic effect via CPB2 in vitro. CPB2 activity and tissue factor-induced thrombin generation was examined by a chromogenic assay. C5a inactivation was evaluated by C-terminal cleavage of C5a and inhibition of C5a-induced human neutrophil migration. Clot lysis time prolongation was examined by a tissue-type plasminogen activator-induced clot lysis assay. CPB2 activity in human plasma was increased by TM alfa and thrombin in a concentration-dependent manner. TM alfa inhibited tissue factor-induced thrombin generation and enhanced pro-CPB2 activation in human plasma simultaneously. The mass spectrum of C5a treated with TM alfa, thrombin, and pro-CPB2 was decreased at 156m/z, indicating that TM alfa enhanced the processing of C5a to C-terminal-cleaved C5a, an inactive form of C5a. C5a-induced human neutrophil migration was decreased after C5a treatment with TM alfa, thrombin, and pro-CPB2. TM alfa prolonged the clot lysis time in human plasma, and this effect was completely abolished by addition of a CPB2 inhibitor. TM alfa exerts anti-inflammatory and anti-fibrinolytic effects through CPB2 in the presence of thrombin in vitro. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Thrombin Cleavage of Inter-α-inhibitor Heavy Chain 1 Regulates Leukocyte Binding to an Inflammatory Hyaluronan Matrix. (United States)

    Petrey, Aaron C; de la Motte, Carol A


    Dynamic alterations of the extracellular matrix in response to injury directly modulate inflammation and consequently the promotion and resolution of disease. During inflammation, hyaluronan (HA) is increased at sites of inflammation where it may be covalently modified with the heavy chains (HC) of inter-α-trypsin inhibitor. Deposition of this unique, pathological form of HA (HC-HA) leads to the formation of cable-like structures that promote adhesion of leukocytes. Naive mononuclear leukocytes bind specifically to inflammation-associated HA matrices but do not adhere to HA constitutively expressed under homeostatic conditions. In this study, we have directly investigated a role for the blood-coagulation protease thrombin in regulating the adhesion of monocytic cells to smooth muscle cells producing an inflammatory matrix. Our data demonstrate that the proteolytic activity of thrombin negatively regulates the adhesion of monocytes to an inflammatory HC-HA complex. This effect is independent of protease-activated receptor activation but requires proteolytic activity toward a novel substrate. Components of HC-HA complexes were predicted to contain conserved thrombin-susceptible cleavage sites based on sequence analysis, and heavy chain 1 (HC1) was confirmed to be a substrate of thrombin. Thrombin treatment is sufficient to cleave HC1 associated with either cell-surface HA or serum inter-α-trypsin inhibitor. Furthermore, thrombin treatment of the inflammatory matrix leads to dissolution of HC-HA cable structures and abolishes leukocyte adhesion. These data establish a novel mechanism whereby thrombin cleavage of HC1 regulates the adhesive properties of an inflammatory HA matrix. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. New insights into conformational and functional stability of human alpha-thrombin probed by high hydrostatic pressure. (United States)

    Lima, Luis Mauricio T R; Zingali, Russolina B; Foguel, Débora; Monteiro, Robson Q


    The effects of high hydrostatic pressure (HHP) and urea on conformational transitions of human alpha-thrombin structure were studied by fluorescence spectroscopy and by measuring the catalytic activity of the enzyme. Treatment of thrombin with urea produced a progressive red shift in the center of mass of the intrinsic fluorescence emission spectrum, with a maximum displacement of 650 cm(-1). HHP (270 MPa) shifted the centre of mass by only 370 cm(-1). HHP combined with a subdenaturing urea concentration (1.5 m) displaced the centre of mass by approximately 750 cm(-1). The binding of the fluorescent probe bis(8-anilinonaphthalene-1-sulfonate) to thrombin was increased by 1.8-, 4.0-, and 2.7-fold after treatment with high urea concentration, HHP or HHP combined with urea, respectively, thus suggesting that all treatments convert the enzyme to partially folded intermediates with exposed hydrophobic regions. On the other hand, treatment of thrombin with urea (but not HHP) combined with dithiothreitol progressively displaced the fluorescent probe, thus suggesting that this condition converts the enzyme to a completely unfolded state. Urea and HHP also led to different conformations when changes in the thrombin catalytic site environment were assessed using the fluorescence emission of fluorescein-d-Phe-Pro-Arg-cloromethylketone-alpha-thrombin: addition of urea up to 2 m gradually decreased the fluorescence emission of the probe to 65% of the initial intensity, whereas HHP caused a progressive increase in fluorescence. Hydrolysis of the synthetic substrate S-2238 was enhanced (35%) in 2 m urea and gradually abolished at higher concentrations, while HHP (270 MPa) inhibited the enzyme's catalytic activity by 45% and abolished it when 1.5 m urea was also present. Altogether, analysis of urea and HHP effects on thrombin structure and activity indicates the formation of dissimilar intermediate states during denaturation by these agents.

  16. The Calibration Home Base for Imaging Spectrometers

    Directory of Open Access Journals (Sweden)

    Johannes Felix Simon Brachmann


    Full Text Available The Calibration Home Base (CHB is an optical laboratory designed for the calibration of imaging spectrometers for the VNIR/SWIR wavelength range. Radiometric, spectral and geometric calibration as well as the characterization of sensor signal dependency on polarization are realized in a precise and highly automated fashion. This allows to carry out a wide range of time consuming measurements in an ecient way. The implementation of ISO 9001 standards in all procedures ensures a traceable quality of results. Spectral measurements in the wavelength range 380–1000 nm are performed to a wavelength uncertainty of +- 0.1 nm, while an uncertainty of +-0.2 nm is reached in the wavelength range 1000 – 2500 nm. Geometric measurements are performed at increments of 1.7 µrad across track and 7.6 µrad along track. Radiometric measurements reach an absolute uncertainty of +-3% (k=1. Sensor artifacts, such as caused by stray light will be characterizable and correctable in the near future. For now, the CHB is suitable for the characterization of pushbroom sensors, spectrometers and cameras. However, it is planned to extend the CHBs capabilities in the near future such that snapshot hyperspectral imagers can be characterized as well. The calibration services of the CHB are open to third party customers from research institutes as well as industry.

  17. Soil moisture calibration of TDR multilevel probes

    Directory of Open Access Journals (Sweden)

    Serrarens Daniel


    Full Text Available Time domain reflectometry (TDR probes are increasingly used for field estimation of soil water content. The objective of this study was to evaluate the accuracy of the multilevel TDR probe under field conditions. For this purpose, eight such TDR probes were installed in small plots that were seeded with beans and sorghum. Data collection from the probes was such that soil moisture readings were automated and logged using a standalone field unit. Neutron probe measurements were used to calibrate the TDR probes. Soil-probe contact and soil compaction were critical to the accuracy of the TDR, especially when a number of TDR probes are combined for a single calibration curve. If each probe is calibrated individually, approximate measurement errors were between 0.005 and 0.015 m³ m-3. However, measurement errors doubled to approximately 0.025 to 0.03 m³ m-3, when TDR probes were combined to yield a single calibration curve.

  18. Effects of thrombin, PAR-1 activating peptide and a PAR-1 antagonist on umbilical artery resistance in vitro

    Directory of Open Access Journals (Sweden)

    Elliott John T


    Full Text Available Abstract Background The non-thrombotic effects of thrombin in cardiovascular tissues, as mediated via the protease activated receptors (PARs, and particularly PAR-1, have been the focus of much recent research. The aims of this study were to evaluate the effects of thrombin, a specific PAR-1 activating peptide (PAR1-AP, and a PAR-1 antagonist on human umbilical artery tone in vitro. Methods Human umbilical artery samples were obtained from 17 women at term. Arterial rings were suspended under physiologic conditions for isometric recording. The in vitro effects of thrombin (0.5 units/mL to 3 units/mL, PAR1-AP TFLLR-NH2 [10(-9 to 10(-6 M], and PAR-1 antagonist (N-trans cinnamoyl- p-fluoroPhe-p-guanidinoPhe-Leu-Arg-Orn-NH2 [10(-9 M to 10(-5 M] on umbilical artery tone were measured. Results Both thrombin and TFLLR-NH2 exerted a potent cumulative vasodilatory effect on human umbilical artery resistance (P 0.05. Conclusion These findings highlight a potential role for thrombin and PAR-1 receptors in vascular regulation of feto-placental blood flow in normal pregnancy, and in association with the vascular lesions associated with IUGR and pre-eclampsia.

  19. Signal amplification aptamer biosensor for thrombin based on a glassy carbon electrode modified with graphene, quantum dots and gold nanoparticles (United States)

    Xie, Lingling; You, Liqin; Cao, Xiaoyu


    A novel electrogenerated chemiluminescence (ECL) assay for sensitive determination of thrombin is designed employing CdSe/ZnS quantum dots served as an ECL label. This ECL sensor is fabricated on graphene modified glassy carbon electrode which is then covered with a low surface coverage of gold nanoparticles (AuNPs). An aptamer is used to selectively recognize the target. The thiol-terminated aptamer is first immobilized on AuNPs/graphene modified electrode, and then thrombin is imported to form the aptamer-thrombin complexes. After blocking the nonspecifically bound oligonucleotides with MCH solution, another CdSe/ZnS quantum dots modified aptamer is hybridized with the free thiol-terminated aptamer to form a DNA complexe. A decreased ECL signal is observed upon recognition of the target thrombin. The integrated ECL intensity versus the concentration of thrombin is linear in the range from 0.01 to 50 nM. The detection limit is 10 fM. The present aptasensor also exhibits excellent selectivity, stability and reusability. This sensing system can provide a promising label-free model for aptamer-based compounds sensitive detection.

  20. Apoferritin Protein Nanoparticles Dually labeled with Aptamer and Horseradish Peroxidase as a Sensing Probe for Thrombin Detection

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Jie; Liu, Meiling; Zhang, Youyu; Li, Haitao; Lin, Yuehe; Yao, Shouzhuo


    A sandwich-type electrochemical aptasensor has been developed for the detection of thrombin, based on dual signal-amplification using HRP and apoferritin. Aptamer1 (Apt1) loaded on core/shell Fe3O4/Au magnetic nanoparticle (AuMNP) was used as recognition elements, and apoferritin dually labeled with Aptamer2 (Apt2) and HRP was used as a detection probe. Sandwich-type complex, Apt1/thrombin/Apt2–apoferritin NPs–HRP was formed by the affinity reactions between AuMNPs–Apt1, thrombin, and Apt2–apoferritin–HRP. The complex was anchored on a screen-printed carbon electrode (SPCE). Differential pulse voltammetry (DPV) was used to monitor the electrode response. The proposed aptasensor yielded a linear current response to thrombin concentrations over a broad range of 0.5 pM to 100 pM with a detection limit of 0.07 pM (S/N = 3). The detection signal was amplified by using apoferritin and HRP. This nanoparticle-based aptasensor offers a new method for rapid, sensitive, selective, and inexpensive quantification of thrombin, and offers a promising potential in biomarker detection and disease diagnosis. --------------------------------------------------------------------------------

  1. Magnetic relaxation switch and colorimetric detection of thrombin using aptamer-functionalized gold-coated iron oxide nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Liang Guohai; Cai Shaoyu; Zhang Peng [Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433 (China); Peng Youyuan [Department of Chemistry, Quanzhou Normal University, Quanzhou 362000 (China); Chen Hui; Zhang Song [Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433 (China); Kong Jilie, E-mail: [Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433 (China)


    We describe a sensitive biosensing system combining magnetic relaxation switch diagnosis and colorimetric detection of human {alpha}-thrombin, based on the aptamer-protein interaction induced aggregation of Fe{sub 3}O{sub 4}-Au nanoparticles. To demonstrate the concept, gold-coated iron oxide nanoparticle was synthesized by iterative reduction of HAuCl{sub 4} onto the dextran-coated Fe{sub 3}O{sub 4} nanoparticles. The resulting core-shell structure had a flowerlike shape with pretty narrow size distribution (referred to as 'nanorose'). The two aptamers corresponding to human {alpha}-thrombin were conjugated separately to two distinct nanorose populations. Once a solution containing human {alpha}-thrombin was introduced, the nanoroses switched from a well dispersed state to an aggregated one, leading to a change in the spin-spin relaxation time (T{sub 2}) as well as the UV-Vis absorption spectra of the solution. Thus the qualitative and quantitative detection method for human {alpha}-thrombin was established. The dual-mode detection is clearly advantageous in obtaining a more reliable result; the detection range is widened as well. By using the dual-mode detection method, a detectable T{sub 2} change is observed with 1.0 nM human {alpha}-thrombin, and the detection range is from 1.6 nM to 30.4 nM.

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

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


    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 PKCα/β, δ, and ε 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 PKCα/β, δ, and ε might stop the fibrotic processes in human idiopathic pulmonary fibrosis by preventing thrombin-induced EMT. PMID:23919450

  3. A novel nanosensor composed of aptamer bio-dots and gold nanoparticles for determination of thrombin with multiple signals. (United States)

    Kuang, Lan; Cao, Shu-Ping; Zhang, Li; Li, Qiu-Hong; Liu, Zhi-Chao; Liang, Ru-Ping; Qiu, Jian-Ding


    Thrombin is a crucial multifunctional enzyme involved in many physiological and pathological processes. Its detection is of great importance. In this work, a novel bio-dots nanosensor for detection of thrombin with colorimetric, fluorometric and light-scattering signals is developed. This nanosensor is composed of thrombin-binding aptamer bio-dots (TBA-dots) and gold nanoparticles (AuNPs), where TBA-dots serve as fluorometric reporter and AuNPs function as multiple roles as colorimetric reporter, light scattering reporter and fluorescence quencher. TBA-dots retain inherent structures of aptamer to specifically interact with thrombin and simultaneously induce the aggregation of AuNPs. The mechanism of the sensing system is based on distance-dependent aggregation of AuNPs and fluorescence resonance energy transfer (FRET). The nanosensor needs no further surface functionalization required for the as-prepared bio-dots and AuNPs, which provides a sensitive method for the selective detection of thrombin with a detection limit as low as 0.59nM. In addition, it provides a brand new perspective for bio-dots and its potential use in bioanalysis. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. A colorimetric aptamer biosensor based on cationic polythiophene derivative as peroxidase mimetics for the ultrasensitive detection of thrombin. (United States)

    Liu, Mei; Li, Jiao; Li, Baoxin


    A colorimetric assay for the ultrasensitive determination of thrombin was presented, in which the cationic polythiophene derivative was used as catalyst of the 3,3',5,5'-tetramethylbenzidine (TMB)-H 2 O 2 reaction and the thrombin-binding aptamer (TBA) was used as inducing polymer's different conformation elements. It was found the cationic polythiophene derivative, poly[3-(3'-N,N,N-triethylamino-1'-propyloxy)-4-methyl-2,5-t-hiophene hydrochloride] (PMNT), can catalyze the oxidation reaction of TMB in the presence of H 2 O 2 to produce a blue color solution. The catalytic activity of PMNT on the TMB-H 2 O 2 reaction was closely relevant to the conformation of PMNT. The absorbance of TMB-H 2 O 2 was distinctly increased in the presence of TBA. With the addition of thrombin, TBA interacted with thrombin to form a G-quadruplex structure. The conformational change weakened the catalytic activity of PMNT and resulted in a decrease in the absorbance. The colorimetric sensor could detect thrombin down to 4pM with high selectivity against other interfering proteins. This work is not only of importance for a better understanding of the unique properties of cationic polythiophenes derivative but also have great potential for medical diagnostics and therapy for human health. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Rapid and ultrasensitive detection of active thrombin based on the Vmh2 hydrophobin fused to a Green Fluorescent Protein. (United States)

    Piscitelli, Alessandra; Pennacchio, Anna; Cicatiello, Paola; Politi, Jane; De Stefano, Luca; Giardina, Paola


    A fusion protein designed in order to combine the fluorescence emission of the Green Fluorescent Protein (GFP) with the adhesion ability of the class I hydrophobin Vmh2 was heterologously produced in the yeast Pichia pastoris. The Vmh2-GFP fusion protein has proven to be a smart and effective tool for the study of Vmh2 self-assembling. Since the two proteins were linked by the specific cutting site of the thrombin, the fusion protein was used as the active biological element in the realization of a thrombin biosensor. When the thrombin present in the target solution specifically hydrolyzed its cleavage sequence, a consequent decrease in the fluorescence intensity of the sample could be observed. The Vmh2-GFP based assay allowed quantification of thrombin in solution with a detection limit of 2.27aM. The specificity of the assay with respect to other proteases and proteins granted the measurement of thrombin added to healthy human plasma with same high sensitivity and a limit of detection of 2.3aM. Further advantages of the developed biosensor are the simplicity of its design and preparation, and the low requirements in terms of samples, reagents and time. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Fibrinogen and thrombin concentrations are critical for fibrin glue adherence in rat high-risk colon anastomoses

    Directory of Open Access Journals (Sweden)

    Eliseo Portilla-de Buen


    Full Text Available OBJECTIVE: Fibrin glues have not been consistently successful in preventing the dehiscence of high-risk colonic anastomoses. Fibrinogen and thrombin concentrations in glues determine their ability to function as sealants, healers, and/or adhesives. The objective of the current study was to compare the effects of different concentrations of fibrinogen and thrombin on bursting pressure, leaks, dehiscence, and morphology of high-risk ischemic colonic anastomoses using fibrin glue in rats. METHODS: Colonic anastomoses in adult female Sprague-Dawley rats (weight, 250-350 g treated with fibrin glue containing different concentrations of fibrinogen and thrombin were evaluated at post-operative day 5. The interventions were low-risk (normal or high-risk (ischemic end-to-end colonic anastomoses using polypropylene sutures and topical application of fibrinogen at high (120 mg/mL or low (40 mg/mL concentrations and thrombin at high (1000 IU/mL or low (500 IU/mL concentrations. RESULTS: Ischemia alone, anastomosis alone, or both together reduced the bursting pressure. Glues containing a low fibrinogen concentration improved this parameter in all cases. High thrombin in combination with low fibrinogen also improved adherence exclusively in low-risk anastomoses. No differences were detected with respect to macroscopic parameters, histopathology, or hydroxyproline content at 5 days post-anastomosis. CONCLUSIONS: Fibrin glue with a low fibrinogen content normalizes the bursting pressure of high-risk ischemic left-colon anastomoses in rats at day 5 after surgery.

  7. Lidar to lidar calibration

    DEFF Research Database (Denmark)

    Georgieva Yankova, Ginka; Courtney, Michael

    This report presents the result of the lidar to lidar calibration performed for ground-based lidar. Calibration is here understood as the establishment of a relation between the reference lidar wind speed measurements with measurement uncertainties provided by measurement standard and corresponding...... lidar wind speed indications with associated measurement uncertainties. The lidar calibration concerns the 10 minute mean wind speed measurements. The comparison of the lidar measurements of the wind direction with that from the reference lidar measurements are given for information only....

  8. Automated Laser Seeker Performance Evaluation System (ALSPES) (United States)

    Martin, Randal G.; Robinson, Elisa L.


    The Automated Laser Seeker Performance Evaluation System (ALSPES), which supports the Hellfire missile and Copperhead projectile laser seekers, is discussed. The ALSPES capabilities in manual and automatic operation are described, and the ALSPES test hardware is examined, including the computer system, the laser/attenuator, optics systems, seeker test fixture, and the measurement and test equipment. The calibration of laser energy and test signals in ALSPES is considered.

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

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


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

  10. The amplitude of coagulation curves from thrombin time tests allows dysfibrinogenemia caused by the common mutation FGG-Arg301 to be distinguished from hypofibrinogenemia. (United States)

    Jacquemin, M; Vanlinthout, I; Van Horenbeeck, I; Debasse, M; Toelen, J; Schoeters, J; Lavend'homme, R; Freson, K; Peerlinck, K


    Thrombin time (TT) tests are useful for diagnosing coagulation disorders involving abnormal fibrinogen but do not allow us to distinguish between qualitative and quantitative defects. However, with the widening availability of optical coagulation automates, more information about the coagulation process is becoming increasingly accessible. In this study, we compared the coagulation curves of TT tests carried out with plasma from healthy donors with those from patients with acquired low Clauss fibrinogen levels or with dysfibrinogenemia caused by a heterozygous point mutation in the fibrinogen γ-chain that results in a p.Arg301(275)Cys substitution. The functional fibrinogen levels of these three groups of samples were also measured with the Clauss method, and their fibrinogen protein levels were determined by ELISA. Our data indicate that the amplitude and maximal velocity of coagulation curves from plasma samples from FGG p.Arg301(275)Cys dysfibrinogenemic patients were comparable to those from plasma samples with fibrinogen in the normal range, whereas the amplitude of coagulation curves from patients with acquired low fibrinogen levels was lower. Examination of the amplitude of coagulation curves generated during TT tests may provide additional information to enable the differential diagnoses of diseases following a low fibrinogen measurement by the Clauss method. © 2017 John Wiley & Sons Ltd.

  11. Laser guided automated calibrating system for accurate bracket ...

    African Journals Online (AJOL)

    Background: The basic premise of preadjusted bracket system is accurate bracket positioning. It is widely recognized that accurate bracket placement is of critical importance in the efficient application of biomechanics and in realizing the full potential of a preadjusted edgewise appliance. Aim: The purpose of this study was ...

  12. Laser Guided Automated Calibrating System for Accurate Bracket ...

    African Journals Online (AJOL)

    It is widely recognized that accurate bracket placement is of critical importance in the efficient application of biomechanics and in realizing the full potential of a preadjusted edgewise appliance. When bands were used originally, Angle[1] thought the best position to place the bracket was at the center of the tooth.

  13. Using ARUCO coded targets for camera calibration automation


    Alves Da Silva, Sergio Leandro [UNESP; Garcia Tommaselli, Antonio Maria; Artero, Almir Olivette


    Este trabalho apresenta uma proposta para a automatização do processo de calibração de câmaras usando a localização e medição de pontos em alvos codificados, com precisão subpixel, o que contribui para minimizar os erros de localização, independente da orientação da câmara e da escala da imagem. Para alcançar este objetivo foram analisados os alvos codificados mais relevantes na literatura, sendo escolhido o padrão ARUCO, devido à sua flexibilidade, capacidade para representar até 1.024 alvos...

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

    Energy Technology Data Exchange (ETDEWEB)

    Hill-Eubanks, D.C.; Parker, C.G.; Lollar, P. (Univ. of Vermont, Burlington (USA))


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

  15. Renal function and plasma dabigatran level measured at trough by diluted thrombin time assay

    Directory of Open Access Journals (Sweden)

    Marta E. Martinuzzo


    Full Text Available Dabigatran etexilate (direct thrombin inhibitor is effective in preventing embolic stroke in patients with atrial fibrillation. It does not require laboratory control, but given the high renal elimination, its measurement in plasma is important in renal failure. The objectives of the study were to verify the analytical quality of the diluted thrombin time assay for measurement of dabigatran plasma concentration (cc, correlate cc with classic coagulation assays, prothrombin time (PT and activated partial thromboplastin time (APTT, and evaluate them according to the creatinine clearance (CLCr. Forty plasma samples of patients (34 consecutive and 6 suspected of drug accumulation receiving dabigatran at 150 (n = 19 or 110 (n = 21 mg/12 hours were collected. Blood samples were drawn at 10-14 hours of the last intake. Dabigatran concentration was determined by diluted thrombin time (HemosIl DTI, Instrumentation Laboratory (IL. PT and APTT (IL were performed on two fotooptical coagulometers, ACL TOP 300 and 500 (IL. DTI presented intra-assay coefficient of variation < 5.4% and inter-assay < 6%, linearity range 0-493 ng/ml. Patients' cc: median 83 (4-945 ng/ml. Individuals with CLCr in the lowest tertile (22.6-46.1 ml/min showed significantly higher median cc: 308 (49-945, compared to the average 72 (12-190 and highest tertile, 60 (4-118 ng/ml. Correlation between cc and APTT or PT were moderate, r2 = 0.59 and -0.66, p < 0.0001, respectively. DTI test allowed us to quantify plasma dabigatran levels, both in patients with normal or altered renal function, representing a useful tool in clinical situations such as renal failure, pre surgery or emergencies

  16. Thrombelastographic characterization of the thrombin-like activity of Crotalus simus and Bothrops asper venoms. (United States)

    Nielsen, Vance G; Boyer, Leslie V; Redford, Daniel T; Ford, Paul


    : Annually, thousands suffer venomous snake-bite from Crotalus simus and Bothrops asper vipers in central and South America. The goals of the present study were to generally characterize the thrombin-like effects of venom from these snakes in human plasma with viscoelastic methods. Human plasma was exposed to the venom of three different C. simus subspecies and venoms obtained from B. asper vipers located in three different locations in Mexico. To characterize the factor X-activating and thrombin-like activity of these venoms, plasma (normal or factor XIII deficient) was pretreated with a variety of additives (e.g., heparin) in the absence or presence of calcium prior to exposure to 2.0 μg/ml of each viper's venom. These profiles were compared with plasma without venom that had contact activation of coagulation. Coagulation kinetics were determined with thrombelastography. All venoms had thrombin-like activity, with C. s. simus creating a slow growing, weak clot that was likely mediated by metalloproteinases. In contrast, B. asper venoms had rapid onset of coagulation and a high velocity of thrombus growth. Further, B. asper venom activity was calcium-independent, activated prothrombin, activated factor XIII, and independently polymerized fibrinogen. The viscoelastic methods used were able to differentiate subspecies of C. simus and specimens of B. asper, and provide insight into the mechanisms by which the venoms acted on plasma. These methods may be useful in the profiling of similar venoms and perhaps can assist in the assessment of interventions designed to treat envenomation (e.g., antivenom).

  17. Thrombin-activatable fibrinolysis inhibitor influences disease severity in humans and mice with pneumococcal meningitis. (United States)

    Mook-Kanamori, B B; Valls Serón, M; Geldhoff, M; Havik, S R; van der Ende, A; Baas, F; van der Poll, T; Meijers, J C M; P Morgan, B; Brouwer, M C; van de Beek, D


    Mortality and morbidity in patients with bacterial meningitis result from the proinflammatory response and dysregulation of coagulation and fibrinolysis. Thrombin-activatable fibrinolysis inhibitor (TAFI) is activated by free thrombin or thrombin in complex with thrombomodulin, and plays an antifibrinolytic role during fibrin clot degradation, but also has an anti-inflammatory role by inactivating proinflammatory mediators, such as complement activation products. To assess the role of TAFI in pneumococcal meningitis. We performed a prospective nationwide genetic association study in patients with bacterial meningitis, determined TAFI and complement levels in cerebrospinal fluid (CSF), and assessed the function of TAFI in a pneumococcal meningitis mouse model by using Cpb2 (TAFI) knockout mice. Polymorphisms (reference sequences: rs1926447 and rs3742264) in the CPB2 gene, coding for TAFI, were related to the development of systemic complications in patients with pneumococcal meningitis. Higher protein levels of TAFI in CSF were significantly associated with CSF complement levels (C3a, iC3b, and C5b-9) and with more systemic complications in patients with bacterial meningitis. The risk allele of rs1926447 (TT) was associated with higher levels of TAFI in CSF. In the murine model, consistent with the human data, Cpb2-deficient mice had decreased disease severity, as reflected by lower mortality, and attenuated cytokine levels and bacterial outgrowth in the systemic compartment during disease, without differences in the brain compartment, as compared with wild-type mice. These findings suggest that TAFI plays an important role during pneumococcal meningitis, which is likely to be mediated through inhibition of the complement system, and influences the occurrence of systemic complications and inflammation. © 2015 International Society on Thrombosis and Haemostasis.

  18. Silk fibroin based carrier system for delivery of fibrinogen and thrombin as coagulant supplements. (United States)

    Teuschl, Andreas H; Zipperle, Johannes; Huber-Gries, Carina; Kaplan, David L


    The control of bleeding is one of the most important interventions after a traumatic injury. Hemostatic devices delivering blood clotting accelerating agents such as fibrinogen are increasingly used due to their efficacy and their ease of application. In the present study, we describe a method to incorporate the coagulant supplements fibrinogen and thrombin in silk protein sponges by mixing the coagulants with an aqueous silk solution, followed by molding, freeze-drying, and water annealing. In this combination system, we demonstrate the delivery of fibrinogen while maintaining its hemostatic potential. Concentration ratios of silk to fibrinogen of 1.0%/2.8%, 2.3%/1.5%, and 3.0%/0.8% were used. The thrombin-induced fibrin polymeric network filled the space in and next to the silk spongy structure but also remained interconnected to the silk, providing an intact network. The mechanical characterization of the fibrinogen-releasing silk sponges before and after the induction of the fibrinogen polymerization demonstrated that the fibrin network resulted in reduced permanent deformation from 21.1% to 6.5%, 19.6% to 5.7%, and 12.7% to 9.4% for the 2.8%, 1.5%, and 0.8% fibrinogen-containing silk sponges, respectively. Moreover, the fibrin formation lead to a more linear elastic behavior over longer strain ranges. In combination, the Calcein-AM/PI staining and MTT assay results indicate uniform cell adhesion on the surface and cytocompatibility of the silk/fibrin sponges, respectively. Moreover, the co-delivery of thrombin with fibrinogen via silk as carrier material is described, offering a more mechanically robust and durable system while preserving hemostatic features of the coagulant substances for the generation of hemostatic devices. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 687-696, 2017. © 2016 Wiley Periodicals, Inc.

  19. Approximation Behooves Calibration

    DEFF Research Database (Denmark)

    da Silva Ribeiro, André Manuel; Poulsen, Rolf


    Calibration based on an expansion approximation for option prices in the Heston stochastic volatility model gives stable, accurate, and fast results for S&P500-index option data over the period 2005–2009....

  20. SRHA calibration curve (United States)

    U.S. Environmental Protection Agency — an UV calibration curve for SRHA quantitation. This dataset is associated with the following publication: Chang, X., and D. Bouchard. Surfactant-Wrapped Multiwalled...

  1. Air Data Calibration Facility (United States)

    Federal Laboratory Consortium — This facility is for low altitude subsonic altimeter system calibrations of air vehicles. Mission is a direct support of the AFFTC mission. Postflight data merge is...

  2. SPOTS Calibration Example

    Directory of Open Access Journals (Sweden)

    Patterson E.


    Full Text Available The results are presented using the procedure outlined by the Standardisation Project for Optical Techniques of Strain measurement to calibrate a digital image correlation system. The process involves comparing the experimental data obtained with the optical measurement system to the theoretical values for a specially designed specimen. The standard states the criteria which must be met in order to achieve successful calibration, in addition to quantifying the measurement uncertainty in the system. The system was evaluated at three different displacement load levels, generating strain ranges from 289 µstrain to 2110 µstrain. At the 289 µstrain range, the calibration uncertainty was found to be 14.1 µstrain, and at the 2110 µstrain range it was found to be 28.9 µstrain. This calibration procedure was performed without painting a speckle pattern on the surface of the metal. Instead, the specimen surface was prepared using different grades of grit paper to produce the desired texture.

  3. Ames Balance Calibration Laboratory (United States)

    Federal Laboratory Consortium — Operations at the lab include calibrating balances for the Ames Wind Tunnels as well as for approved outside projects. Ames has a large inventory of TASK multi-piece...

  4. Traceable Pyrgeometer Calibrations

    Energy Technology Data Exchange (ETDEWEB)

    Dooraghi, Mike; Kutchenreiter, Mark; Reda, Ibrahim; Habte, Aron; Sengupta, Manajit; Andreas, Afshin; Newman, Martina; Webb, Craig


    This presentation provides a high-level overview of the progress on the Broadband Outdoor Radiometer Calibrations for all shortwave and longwave radiometers that are deployed by the Atmospheric Radiation Measurement program.

  5. Automated Gravimetric Management Of Solutions Part 2.* Automated Gravimetric Approach To Direct Potentiometry And Kappa Number Determination


    Pasquini C.; Cunha I.B.S.


    The high-performance microcomputer controlled gravimetric burette described in Part 1 has been employed to automate some routine analytical procedures. A direct potentiometric determination of fluoride ions in drinking water was developed to include an automatic calibration step, matrix adjustment and sample determination. Also the complex and cumbersome titrimetric procedure for determination of the kappa number (lignin content) in paper pulp, has been automated by using the gravimetric unit...

  6. The Automated Bicron Tester: Automated electronic instrument diagnostic, testing, and alignment system with records generation

    Energy Technology Data Exchange (ETDEWEB)

    Rao, G.S.; Maddox, S.R.; Turner, G.W.; Vandermolen, R.I.


    The Bicron Surveyor MX is a portable radiation monitoring instrument used by the Office of Radiation Protection at Oak Ridge National Laboratory. This instrument must be calibrated in order to assure reliable operation. A manual calibration procedure was developed, but it was time consuming and repetitive. Therefore, an automated tester station that would allow the technicians to calibrate the instruments faster and more reliably was developed. With the automated tester station, calibration records and accountability could be generated and maintained automatically. This allows the technicians to concentrate on repairing defective units. The Automated Bicron Tester consists of an operator interface, an analog board, and a digital controller board. The panel is the user interface that allows the technician to communicate with the tester. The analog board has an analog-to-digital converter (ADC) that converts the signals from the instrument into digital data that the tester can manipulate. The digital controller board contains the circuitry to perform the test and to communicate the results to the host personal computer (PC). The tester station is connected to the unit under test through a special test harness that attaches to a header on the Bicron. The tester sends pulse trains to the Bicron and measures the resulting meter output. This is done to determine if the unit is functioning properly. The testers are connected to the host PC through an RS-485 serial line. The host PC polls all the tester stations that are connected to it and collects data from those that have completed a calibration. It logs these data and stores the record in a format ready for export to the Maintenance, Accountability, Jobs, and Inventory Control (MAJIC) database. It also prints a report. The programs for the Automated Bicron Tester and the host are written in the C language.

  7. Thrombin activation of protein C requires prior processing by a liver proprotein convertase. (United States)

    Essalmani, Rachid; Susan-Resiga, Delia; Guillemot, Johann; Kim, Woojin; Sachan, Vatsal; Awan, Zuhier; Chamberland, Ann; Asselin, Marie-Claude; Ly, Kévin; Desjardins, Roxane; Day, Robert; Prat, Annik; Seidah, Nabil G


    Protein C, a secretory vitamin K-dependent anticoagulant serine protease, inactivates factors Va/VIIIa. It is exclusively synthesized in liver hepatocytes as an inactive zymogen (proprotein C). In humans, thrombin cleavage of the propeptide at PR(221)↓ results in activated protein C (APC; residues 222-461). However, the propeptide is also cleaved by a furin-like proprotein convertase(s) (PCs) at KKRSHLKR(199)↓ (underlined basic residues critical for the recognition by PCs), but the order of cleavage is unknown. Herein, we present evidence that at the surface of COS-1 cells, mouse proprotein C is first cleaved by the convertases furin, PC5/6A, and PACE4. In mice, this cleavage occurs at the equivalent site, KKRKILKR(198)↓, and requires the presence of Arg(198) at P1 and a combination of two other basic residues at either P2 (Lys(197)), P6 (Arg(193)), or P8 (Lys(191)) positions. Notably, the thrombin-resistant R221A mutant is still cleaved by these PCs, revealing that convertase cleavage can precede thrombin activation. This conclusion was supported by the fact that the APC-specific activity in the medium of COS-1 cells is exclusively dependent on prior cleavage by the convertases, because both R198A and R221A lack protein C activity. Primary cultures of hepatocytes derived from wild-type or hepatocyte-specific furin, PC5/6, or complete PACE4 knock-out mice suggested that the cleavage of overexpressed proprotein C is predominantly performed by furin intracellularly and by all three proprotein convertases at the cell surface. Indeed, plasma analyses of single-proprotein convertase-knock-out mice showed that loss of the convertase furin or PC5/6 in hepatocytes results in a ∼30% decrease in APC levels, with no significant contribution from PACE4. We conclude that prior convertase cleavage of protein C in hepatocytes is critical for its thrombin activation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Post-traumatic hepatic artery pseudoaneurysm treated with endovascular embolization and thrombin injection. (United States)

    Francisco, Lloret Estañ; Asunción, López Conesa; Antonio, Capel Alemán; Ricardo, Robles Campos; Manuel, Reus Pintado; Caridad, Marín Hernández


    Post-traumatic hepatic artery pseudoaneurysm is uncommon, appearing in approximately 1% of hepatic trauma cases. Most are extrahepatic (80%) and have a late onset. Although they are usually asymptomatic, they should always be treated becasue of the high risk of complications, especially breakage. Currently the treatment of choice is endovascular embolization with coils or the exclusion of the pseudoaneurysm using other intravascular devices. Recently there have been accounts of a treatment that combines embolization with coils and image-guided percutaneous human thrombin injection. We present a case of post-traumatic hepatic artery pseudoaneurysm that was successfully treated using this combined technique.

  9. Jet Calibration at ATLAS

    CERN Document Server

    Camacho, R; The ATLAS collaboration


    The accurate measurement of jets at high transverse momentum produced in proton proton collision at a centre of mass energy at \\sqrt(s)=7 TeV is important in many physics analysis at LHC. Due to the non-compensating nature of the ATLAS calorimeter, signal losses due to noise thresholds and in dead material the jet energy needs to be calibrated. Presently, the ATLAS experiment derives the jet calibration from Monte Carlo simulation using a simple correction that relates the true and the reconstructed jet energy. The jet energy scale and its uncertainty are derived from in-situ measurements and variation in the Monte Carlo simulation. Other calibration schemes have been also developed, they use hadronic cell calibrations or the topology of the jet constituents to reduce hadronic fluctuations in the jet response, improving in that way the jet resolution. The performances of the various calibration schemes using data and simulation, the evaluation of the modelling of the properties used to derive each calibration...

  10. Calibrating nacelle lidars

    Energy Technology Data Exchange (ETDEWEB)

    Courtney, M.


    Nacelle mounted, forward looking wind lidars are beginning to be used to provide reference wind speed measurements for the power performance testing of wind turbines. In such applications, a formal calibration procedure with a corresponding uncertainty assessment will be necessary. This report presents four concepts for performing such a nacelle lidar calibration. Of the four methods, two are found to be immediately relevant and are pursued in some detail. The first of these is a line of sight calibration method in which both lines of sight (for a two beam lidar) are individually calibrated by accurately aligning the beam to pass close to a reference wind speed sensor. A testing procedure is presented, reporting requirements outlined and the uncertainty of the method analysed. It is seen that the main limitation of the line of sight calibration method is the time required to obtain a representative distribution of radial wind speeds. An alternative method is to place the nacelle lidar on the ground and incline the beams upwards to bisect a mast equipped with reference instrumentation at a known height and range. This method will be easier and faster to implement and execute but the beam inclination introduces extra uncertainties. A procedure for conducting such a calibration is presented and initial indications of the uncertainties given. A discussion of the merits and weaknesses of the two methods is given together with some proposals for the next important steps to be taken in this work. (Author)

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

    Energy Technology Data Exchange (ETDEWEB)

    Gomez, M.L.; Tellez-Inon, M.T. (Instituto de Ingenieria Genetica y Biologia Molecular, Buenos Aires (Argentina)); Medrano, E.E.; Cafferatta, E.G.A. (Instituto de Investigaciones Bioquimicas Fundacion Campomar, Buenos Aires (Argentina))


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

  12. Measurement quality assurance for beta particle calibrations at NIST

    Energy Technology Data Exchange (ETDEWEB)

    Soares, C.G.; Pruitt, J.S. [National Institute of Standards and Technology, Gaithersburg, MD (United States)


    Standardized beta-particle fields have been established in an international standard and have been adopted for use in several U.S. dosimeter and instrument testing standards. Calibration methods and measurement quality assurance procedures employed at the National Institute of Standards and Technology (NIST) for beta-particle calibrations in these reference fields are discussed. The calibration facility including the NIST-automated extrapolation ionization chamber is described, and some sample results of calibrations are shown. Methods for establishing and maintaining traceability to NIST of secondary laboratories are discussed. Currently, there are problems in finding a good method for routine testing of traceability to NIST. Some examples of past testing methods are given and solutions to this problem are proposed.

  13. Gulf of Mexico Climate-History Calibration Study (United States)

    Spear, Jessica W.; Poore, Richard Z.


    Reliable instrumental records of past climate are available for about the last 150 years only. To supplement the instrumental record, reconstructions of past climate are made from natural recorders such as trees, ice, corals, and microfossils preserved in sediments. These proxy records provide information on the rate and magnitude of past climate variability, factors that are critical to distinguishing between natural and human-induced climate change in the present. However, the value of proxy records is heavily dependent on calibration between the chemistry of the natural recorder and of the modern environmental conditions. The Gulf of Mexico Climate and Environmental History Project is currently undertaking a climate-history calibration study with material collected from an automated sediment trap. The primary focus of the calibration study is to provide a better calibration of low-latitude environmental conditions and shell chemistry of calcareous microfossils, such as planktic Foraminifera.

  14. The protective role of (−)-epigallocatechin-3-gallate in thrombin-induced neuronal cell apoptosis and JNK-MAPK activation (United States)

    He, Qianqian; Bao, Lei; Zimering, Jeffrey; Zan, Kun; Zhang, Zuohui; Shi, Hongjuan; Zu, Jie; Yang, Xinxin; Hua, Fang; Ye, Xinchun


    (−)-Epigallocatechin-3-gallate (EGCG), the major polyphenolic component of green tea, has anti-inflammatory and antioxidant properties and provides neuroprotection against central nervous system diseases. Yet, it is not known whether EGCG may be neuroprotective against intracerebral hemorrhage. In this study, we used a simplified in-vitro model of thrombin neurotoxicity to test whether EGCG provides neuroprotection against thrombin-associated toxicity. Exposure of primary cortical neurons to thrombin (100 U/ml) caused dose-dependent and time-dependent cytotoxicity. Cell Counting Kit 8 and lactate dehydrogenase were used to monitor cell viability after exposure of neurons to thrombin or EGCG and after EGCG pretreatment. Flow cytometric analysis and western blotting demonstrated that thrombin-induced neuron degeneration occurs through apoptosis. A concentration of 25 μM EGCG significantly abolished thrombin-induced toxicity and prevented apoptosis by suppressing c-Jun-N-terminal kinase (JNK) phosphorylation, and the JNK inhibitor SP600125 reduced thrombin-induced caspase 3 activation and apoptosis. These data suggest that EGCG may have protective effects against thrombin-induced neuroapoptosis by inhibiting the activation of JNK, leading to caspase 3 cleavage. EGCG is a novel candidate neuroprotective agent against intracerebral hemorrhage-induced neurotoxicity. PMID:25839175

  15. Inactivation of active thrombin-activable fibrinolysis inhibitor takes place by a process that involves conformational instability rather than proteolytic cleavage

    NARCIS (Netherlands)

    Marx, PF; Hackeng, TM; Dawson, PE; Griffin, JH; Meijers, JCM; Bouma, Bonno N.


    Thrombin-activable fibrinolysis inhibitor (TAFI) is present in the circulation as an inactive zymogen. Thrombin converts TAFI to a carboxypeptidase B-like enzyme (TAFIa) by cleaving at Arg(92) in a process accelerated by the cofactor, thrombomodulin. TAFIa attenuates fibrinolysis. TAFIa can be

  16. A thrombin generation assay may reduce the need for compression ultrasonography for the exclusion of deep venous thrombosis in the elderly

    NARCIS (Netherlands)

    Haas, F.J.L.M.; Schutgens, R.E.G.; Kluft, C.; Biesma, D.H.


    Introduction. The exclusion of deep venous thrombosis (DVT) by pre-test probability (PTP) and D-dimer in the elderly is safe, but has a low specificity. There is increasing interest in the diagnostic role of thrombin generation in patients with thrombosis. We evaluated the value of thrombin

  17. Thermodynamic and biological evaluation of a thrombin binding aptamer modified with several unlocked nucleic acid (UNA) monomers and a 2′-C-piperazino-UNA monomer

    DEFF Research Database (Denmark)

    Jensen, Troels B.; Henriksen, Jonas Rosager; Rasmussen, Bjarne E.


    Thrombin binding aptamer is a DNA 15-mer which forms a G-quadruplex structure and possess promising anticoagulant properties due to specific interactions with thrombin. Herein we present the influence of a single 2′-C-piperazino-UNA residue and UNA residues incorporated in several positions on th...

  18. Manufacturing and automation

    Directory of Open Access Journals (Sweden)

    Ernesto Córdoba Nieto


    Full Text Available The article presents concepts and definitions from different sources concerning automation. The work approaches automation by virtue of the author’s experience in manufacturing production; why and how automation prolects are embarked upon is considered. Technological reflection regarding the progressive advances or stages of automation in the production area is stressed. Coriat and Freyssenet’s thoughts about and approaches to the problem of automation and its current state are taken and examined, especially that referring to the problem’s relationship with reconciling the level of automation with the flexibility and productivity demanded by competitive, worldwide manufacturing.

  19. Radiation Dose Estimation by Automated Cytogenetic Biodosimetry. (United States)

    Rogan, Peter K; Li, Yanxin; Wilkins, Ruth C; Flegal, Farrah N; Knoll, Joan H M


    The dose from ionizing radiation exposure can be interpolated from a calibration curve fit to the frequency of dicentric chromosomes (DCs) at multiple doses. As DC counts are manually determined, there is an acute need for accurate, fully automated biodosimetry calibration curve generation and analysis of exposed samples. Software, the Automated Dicentric Chromosome Identifier (ADCI), is presented which detects and discriminates DCs from monocentric chromosomes, computes biodosimetry calibration curves and estimates radiation dose. Images of metaphase cells from samples, exposed at 1.4-3.4 Gy, that had been manually scored by two reference laboratories were reanalyzed with ADCI. This resulted in estimated exposures within 0.4-1.1 Gy of the physical dose. Therefore, ADCI can determine radiation dose with accuracies comparable to standard triage biodosimetry. Calibration curves were generated from metaphase images in ~10 h, and dose estimations required ~0.8 h per 500 image sample. Running multiple instances of ADCI may be an effective response to a mass casualty radiation event. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email:

  20. Modulation of human uterine smooth muscle cell collagen contractility by thrombin, Y-27632, TNF alpha and indomethacin

    Directory of Open Access Journals (Sweden)

    Smith Terry J


    Full Text Available Abstract Background Preterm labour occurs in approximately 10% of pregnancies and is a major cause of infant morbidity and mortality. However, the pathways involved in regulating contractility in normal and preterm labour are not fully elucidated. Our aim was to utilise a human myometrial contractility model to investigate the effect of a number of uterine specific contractility agents in this system. Therefore, we investigated the contractile response of human primary uterine smooth muscle cells or immortalised myometrial smooth muscle cells cultured within collagen lattices, to known mediators of uterine contractility, which included thrombin, the ROCK-1 inhibitor Y-27632, tumour necrosis factor alpha (TNF alpha and the non-steroidal anti-inflammatory indomethacin. Methods Cell contractility was calculated over time, with the collagen gel contraction assay, utilising human primary uterine smooth muscle cells (hUtSMCs and immortalised myometrial smooth muscle cells (hTERT-HM: a decrease in collagen gel area equated to an increase in contractility. RNA was isolated from collagen embedded cells and gene expression changes were analysed by real time fluorescence reverse transcription polymerase chain reaction. Scanning electron and fluorescence microscopy were employed to observe cell morphology and cell collagen gel interactions. Statistical analysis was performed using ANOVA followed by Tukey's post hoc tests. Results TNF alpha increased collagen contractility in comparison to the un-stimulated collagen embedded hUtSMC cells, which was inhibited by indomethacin, while indomethacin alone significantly inhibited contraction. Thrombin augmented the contractility of uterine smooth muscle cell and hTERT-HM collagen gels, this effect was inhibited by the thrombin specific inhibitor, hirudin. Y-27632 decreased both basal and thrombin-induced collagen contractility in the hTERT-HM embedded gels. mRNA expression of the thrombin receptor, F2R was up

  1. Three months of strictly controlled daily endurance exercise reduces thrombin generation and fibrinolytic risk markers in younger moderately overweight men

    DEFF Research Database (Denmark)

    Gram, Anne Sofie; Bladbjerg, Else-Marie; Skov, Jane


    of 600 kcal/day (HIGH), 300 kcal/day (MOD), or to maintain their habitual lifestyle (CON). Fasting blood samples were collected before and after the intervention and analysed for thrombin generation (endogenous thrombin potential, ETP) and concentrations of tissue-type plasminogen activator (t......-PA), plasminogen activator inhibitor type 1 (PAI-1), and von Willebrand factor (vWF). RESULTS: We observed significant within-group decreases in ETP (MOD 7 %; HIGH 6 %) and in t-PA (MOD 22 %; HIGH 21 %) and PAI-1 (MOD 16 %; HIGH 32 %) in both training groups, and no changes in the CON group. At 3 months, between...

  2. Secretory products from thrombin-stimulated human platelets exert an inhibitory effect on NK-cytotoxic activity

    DEFF Research Database (Denmark)

    Skov Madsen, P; Hokland, P; Hokland, M


    We have investigated the interaction between human platelets and the NK-system, with special emphasis on the action of secretory products from platelets in an NK assay with 51Cr-labelled K562 as target cells. Supernatants from thrombin-stimulated platelets added to the NK assay consistently...... with thrombin, are capable of secreting different, yet undefined factors, which significantly inhibit NK activity in vitro. The results also suggest that the role of products from contaminating in vitro activated platelets should be borne in mind when performing conventional NK assays. Udgivelsesdato: 1986-Oct...

  3. Computational study of some benzamidine-based inhibitors of thrombin-like snake venom proteinases (United States)

    Henriques, Elsa S.; Nascimento, Marco A. C.; Ramos, Maria João

    Pit viper venoms contain a number of serine proteinases that, despite their observed coagulant thrombin-like action in vitro, exhibit a paradoxical benign defibrinogenating (anticoagulant) action in vivo, with clinical applications in preventing thrombi and improved blood circulation. Considering that several benzamidine-based inhibitors, some highly selective to thrombin, also inhibit the enzymatic activity of such venombins, the modeling of their enzyme-inhibitor interactions could provide valuable information on the topological factors that determine the divergences in activity. The first step, and the object of the present study, was to derive the necessary set of parameters, consistent with the CHARMM force field, and to perform molecular dynamics (MD) simulations on a few selected representatives of the inhibitors in question under physiological conditions. Bonding and van der Waals parameters were derived by analogy to similar ones in the existing force field. Net atomic charges were obtained with a restrained fitting to the molecular electrostatic potential generated at B3LYP/6-31G(d) level. The parameters were refined to reproduce the available experimental geometries and crystal data, and the MD simulations of the free inhibitors in aqueous solution at 298 K provided an insightful description of their available conformational space.

  4. Investigation of the phenotype heterogeneity in severe hemophilia A using thromboelastography, thrombin generation, and thrombodynamics. (United States)

    Tarandovskiy, Ivan D; Balandina, Anna N; Kopylov, Konstantine G; Konyashina, Nadezhda I; Kumskova, Maria A; Panteleev, Mikhail A; Ataullakhanov, Fazoil I


    Hemophilia A (HA) patients with similar factor VIII levels can demonstrate varying bleeding tendencies. In particular, 10-15% of all severe HA patients (FVIII:Cinvestigated the coagulation status of different bleeding phenotypes using various types of global coagulation assays. Ten HA patients with severe phenotype and eleven patients with mild phenotypes were included in the study. For each patient, thromboelastography (TE), thrombodynamics (TD), and kaolin- or tissue factor-induced thrombin generation (TG) were measured. TG in platelet-rich plasma (PRP) was investigated using our original modification when the thrombin generation curve showed two peaks, previously shown to depend on platelet activity. We also utilized TG and TD with the addition of thrombomodulin. The second peak amplitude and ETP of PRP TG were the only parameters that were significantly higher in mild bleeders (peak 41.6 ± 3.5 nM, ETP 1966 ± 169 nM*min) than in patients with severe bleeding (peak 28.3 ± 3.3 nM, ETP 1359 ± 130 nM*min). Our results suggest that severe and mild HA phenotypes could be distiguished by TG assay in PRP suggesting that difference in platelet activity can be involved in the phenotype formation. According to our previous results we can suppose that the mechanism of the phenotypic heterogeneity is linked with TG mediated by PS-expressing platelets. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Biocompatibility and Effectiveness Evaluation of a New Hemostatic Embolization Agent: Thrombin Loaded Alginate Calcium Microsphere

    Directory of Open Access Journals (Sweden)

    Fengqi Xuan


    Full Text Available Background. Until now, there has been no ideal embolization agent for hemorrhage in interventional treatment. In this study, the thrombin was encapsulated in alginate calcium microsphere using electrostatic droplet technique to produce new embolization agent: thrombin loaded alginate calcium microspheres (TACMs. Objectives. The present work was to evaluate the biocompatibility and hemostatic efficiency of TACMs. Methods. Cell cytotoxicity, hemolysis, and superselective embolization of dog liver arteries were performed to investigate the biocompatibility of TACMs. To clarify the embolic effect of TACMs mixed thrombus in vivo, hepatic artery injury animal model of 6 beagles was established and transcatheter artery embolization for bleeding was performed. Results. Coculture with VECs revealed the noncytotoxicity of TACMs, and the hemolysis experiment was negligible. Moreover, the histological study of TACMs in liver blood vessel showed signs of a slight inflammatory reaction. The results of transcatheter application of TACMs mixed thrombus for bleeding showed that the blood flow was shut down completely after the TACMs mixed thrombus was delivered and the postprocedural survival rate of animal models at 12 weeks was 100%. Conclusions. With their good biocompatibility and superior hemostatic efficiency, TACMs might be a promising new hemostatic agent with a wide range of potential applications.

  6. A sensitive HIV-1 envelope induced fusion assay identifies fusion enhancement of thrombin

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, De-Chun; Zhong, Guo-Cai; Su, Ju-Xiang [Department of Microbiology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081 (China); Liu, Yan-Hong [Second Affiliated Hospital of Harbin Medical University, 246 Xuefu Road, Harbin, Heilongjiang 150081 (China); Li, Yan; Wang, Jia-Ye [Department of Microbiology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081 (China); Hattori, Toshio [Department of Emerging Infectious Diseases, Division of Internal Medicine, Graduate School of Medicine, Tohoku University, Sendai 9808574 (Japan); Ling, Hong, E-mail: [Department of Microbiology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081 (China); Department of Parasitology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081 (China); Key Lab of Heilongjiang Province for Infection and Immunity, Key Lab of Heilongjiang Province Education Bureau for Etiology, Harbin, Heilongjiang 150081 (China); Zhang, Feng-Min, E-mail: [Department of Microbiology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081 (China); Key Lab of Heilongjiang Province for Infection and Immunity, Key Lab of Heilongjiang Province Education Bureau for Etiology, Harbin, Heilongjiang 150081 (China)


    To evaluate the interaction between HIV-1 envelope glycoprotein (Env) and target cell receptors, various cell-cell-fusion assays have been developed. In the present study, we established a novel fusion system. In this system, the expression of the sensitive reporter gene, firefly luciferase (FL) gene, in the target cells was used to evaluate cell fusion event. Simultaneously, constitutively expressed Renilla luciferase (RL) gene was used to monitor effector cell number and viability. FL gave a wider dynamic range than other known reporters and the introduction of RL made the assay accurate and reproducible. This system is especially beneficial for investigation of potential entry-influencing agents, for its power of ruling out the false inhibition or enhancement caused by the artificial cell-number variation. As a case study, we applied this fusion system to observe the effect of a serine protease, thrombin, on HIV Env-mediated cell-cell fusion and have found the fusion enhancement activity of thrombin over two R5-tropic HIV strains.

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

    Directory of Open Access Journals (Sweden)

    Kristensen Torsten


    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.

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

    DEFF Research Database (Denmark)

    Valnickova, Zuzana; Thaysen-Andersen, Morten; Højrup, Peter


    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 fib......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......PA-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....

  9. Cysteine proteases from the Asclepiadaceae plants latex exhibited thrombin and plasmin like activities. (United States)

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


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

  10. Correlation between Interleukin-6 and Thrombin-Antithrombin III Complex Levels in Retinal Diseases. (United States)

    Ehrlich, Rita; Zahavi, Alon; Axer-Siegel, Ruth; Budnik, Ivan; Dreznik, Ayelet; Dahbash, Mor; Nisgav, Yael; Megiddo, Elinor; Kenet, Gili; Weinberger, Dov; Livnat, Tami


    This study aims to evaluate and correlate the levels of interleukin-6 (IL-6) and thrombin-antithrombin III complex (TAT) in the vitreous of patients with different vitreoretinal pathologies. Vitreous samples were collected from 78 patients scheduled for pars plana vitrectomy at a tertiary medical center. Patients were divided by the underlying vitreoretinal pathophysiology, as follows: macular hole (MH)/epiretinal membrane (ERM) (n = 26); rhegmatogenous retinal detachment (RRD) (n = 32); and proliferative diabetic retinopathy (PDR) (n = 20). Levels of IL-6 and TAT were measured by enzyme-linked immunosorbent assay and compared among the groups. A significant difference was found in the vitreal IL-6 and TAT levels between the MH/ERM group and both the PDR and RRD groups (P < 0.001 for all). Diabetes was associated with higher IL-6 levels in the RRD group. Different relationships between the IL-6 and TAT levels were revealed in patients with different ocular pathologies. Our results imply that variations in vitreal TAT level may be attributable not only to an inflammatory reaction or blood-retinal barrier breakdown, but also to intraocular tissue-dependent regulation of thrombin.

  11. FVIIa-sTF and Thrombin Inhibitory Activities of Compounds Isolated from Microcystis aeruginosa K-139

    Directory of Open Access Journals (Sweden)

    Andrea Roxanne J. Anas


    Full Text Available The rise of bleeding and bleeding complications caused by oral anticoagulant use are serious problems nowadays. Strategies that block the initiation step in blood coagulation involving activated factor VII-tissue factor (fVIIa-TF have been considered. This study explores toxic Microcystis aeruginosa K-139, from Lake Kasumigaura, Ibaraki, Japan, as a promising cyanobacterium for isolation of fVIIa-sTF inhibitors. M. aeruginosa K-139 underwent reversed-phase solid-phase extraction (ODS-SPE from 20% MeOH to MeOH elution with 40%-MeOH increments, which afforded aeruginosin K-139 in the 60% MeOH fraction; micropeptin K-139 and microviridin B in the MeOH fraction. Aeruginosin K-139 displayed an fVIIa-sTF inhibitory activity of ~166 µM, within a 95% confidence interval. Micropeptin K-139 inhibited fVIIa-sTF with EC50 10.62 µM, which was more efficient than thrombin inhibition of EC50 26.94 µM. The thrombin/fVIIa-sTF ratio of 2.54 in micropeptin K-139 is higher than those in 4-amidinophenylmethane sulfonyl fluoride (APMSF and leupeptin, when used as positive controls. This study proves that M. aeruginosa K-139 is a new source of fVIIa-sTF inhibitors. It also opens a new avenue for micropeptin K-139 and related depsipeptides as fVIIa-sTF inhibitors.

  12. Collagen, fibrinogen and thrombin biological addesive is effective in treating experimental liver injuries

    Directory of Open Access Journals (Sweden)


    Full Text Available ABSTRACT Objective : to evaluate the effectiveness of a collagen-based adhesive associated with fibrinogen and thrombin in experimental liver injury in rats. Methods : the study included 30 Wistar rats randomly divided into three groups: A, B and C. All underwent standard liver traumatic injury. In group A the lesion was treated with the adhesive; in group B, with conventional absorbable suture; and in group C, there was no treatment. We analyzed the time of hemostasis, mortality, occurrence of adhesions and any histological changes. Results : there was no statistical difference in relation to mortality (p = 0.5820. The group treated with the adhesive showed the lowest hemostasis times (p = 0.0573, odds ratio 13.5 and lower incidence of adhesions (p = 0.0119. Microscopic histological alterations of Groups A and B were similar, with foreign body granuloma formation separating the adhesive material or the suture from the hepatic stroma. Conclusion : the adhesive of collagen associated with fibrinogen and thrombin was effective in the treatment of experimental hepatic injury, providing a lower incidence of adhesions between the liver and surrounding structures.

  13. The collagen, fibrinogen and thrombin biological adhesive is effective in treating experimental liver injuries

    Directory of Open Access Journals (Sweden)

    Frederico Michelino de Oliveira

    Full Text Available ABSTRACT Objective: to evaluate the effectiveness of an collagen-based adhesive associated with fibrinogen and thrombin in experimental liver injuries in rats. Methods: we randomly divided 30 Wistar rats into three groups: A, B and C. All underwent a standard liver traumatic injury. In group A, the lesion was treated with the adhesive; in group B, with conventional, absorbable suture; group C received no treatment. We analyzed the time of hemostasis, mortality, occurrence of adhesions and any histological changes. Results: there was no statistical difference in relation to mortality (p=0.5820. The adhesive treated group showed the lowest hemostasis times (p=0.0573, odds ratio 13.5 and lower incidence of adhesions (p=0.0119. The histological alterations of the Groups A and B were similar, with foreign body granuloma formation separating the adhesive material and the hepatic stroma suture. Conclusion: the collagen adhesive associated with fibrinogen and thrombin was effective in treating experimental hepatic injury, providing a lower incidence of adhesions between the liver and surrounding structures.

  14. FVIIa-sTF and Thrombin Inhibitory Activities of Compounds Isolated from Microcystis aeruginosa K-139. (United States)

    Anas, Andrea Roxanne J; Mori, Akane; Tone, Mineka; Naruse, Chiaki; Nakajima, Anna; Asukabe, Hirohiko; Takaya, Yoshiaki; Imanishi, Susumu Y; Nishizawa, Tomoyasu; Shirai, Makoto; Harada, Ken-Ichi


    The rise of bleeding and bleeding complications caused by oral anticoagulant use are serious problems nowadays. Strategies that block the initiation step in blood coagulation involving activated factor VII-tissue factor (fVIIa-TF) have been considered. This study explores toxic Microcystis aeruginosa K-139, from Lake Kasumigaura, Ibaraki, Japan, as a promising cyanobacterium for isolation of fVIIa-sTF inhibitors. M. aeruginosa K-139 underwent reversed-phase solid-phase extraction (ODS-SPE) from 20% MeOH to MeOH elution with 40%-MeOH increments, which afforded aeruginosin K-139 in the 60% MeOH fraction; micropeptin K-139 and microviridin B in the MeOH fraction. Aeruginosin K-139 displayed an fVIIa-sTF inhibitory activity of ~166 µM, within a 95% confidence interval. Micropeptin K-139 inhibited fVIIa-sTF with EC50 10.62 µM, which was more efficient than thrombin inhibition of EC50 26.94 µM. The thrombin/fVIIa-sTF ratio of 2.54 in micropeptin K-139 is higher than those in 4-amidinophenylmethane sulfonyl fluoride (APMSF) and leupeptin, when used as positive controls. This study proves that M. aeruginosa K-139 is a new source of fVIIa-sTF inhibitors. It also opens a new avenue for micropeptin K-139 and related depsipeptides as fVIIa-sTF inhibitors.

  15. A systems approach to hemostasis: 3. Thrombus consolidation regulates intrathrombus solute transport and local thrombin activity (United States)

    Welsh, John D.; Tomaiuolo, Maurizio; Wu, Jie; Colace, Thomas V.; Diamond, Scott L.


    Hemostatic thrombi formed after a penetrating injury have a distinctive structure in which a core of highly activated, closely packed platelets is covered by a shell of less-activated, loosely packed platelets. We have shown that differences in intrathrombus molecular transport emerge in parallel with regional differences in platelet packing density and predicted that these differences affect thrombus growth and stability. Here we test that prediction in a mouse vascular injury model. The studies use a novel method for measuring thrombus contraction in vivo and a previously characterized mouse line with a defect in integrin αIIbβ3 outside-in signaling that affects clot retraction ex vivo. The results show that the mutant mice have a defect in thrombus consolidation following vascular injury, resulting in an increase in intrathrombus transport rates and, as predicted by computational modeling, a decrease in thrombin activity and platelet activation in the thrombus core. Collectively, these data (1) demonstrate that in addition to the activation state of individual platelets, the physical properties of the accumulated mass of adherent platelets is critical in determining intrathrombus agonist distribution and platelet activation and (2) define a novel role for integrin signaling in the regulation of intrathrombus transport rates and localization of thrombin activity. PMID:24951426

  16. Calibration of a DG–model for fluorescence microscopy

    DEFF Research Database (Denmark)

    Hansen, Christian Valdemar

    ) is an impor- tant and widely used microscopy method for visualization of molecular transport processes in living cells. Thus, the motivation for making an automated reliable analysis of the image data is high. In this contribution, we present and comment on the calibration of a Discontinuous......–Galerkin simulator [3, 4] on segmented cell images. The cell geometry is extracted from FLIP images using the Chan– Vese active contours algorithm [1] while the DG simulator is implemented in FEniCS [5]. Simulated FLIP sequences based on optimal parameters from the PDE model are presented, with an overall goal...... of making an automated analysis tool for FLIP images....

  17. Calibration Under Uncertainty.

    Energy Technology Data Exchange (ETDEWEB)

    Swiler, Laura Painton; Trucano, Timothy Guy


    This report is a white paper summarizing the literature and different approaches to the problem of calibrating computer model parameters in the face of model uncertainty. Model calibration is often formulated as finding the parameters that minimize the squared difference between the model-computed data (the predicted data) and the actual experimental data. This approach does not allow for explicit treatment of uncertainty or error in the model itself: the model is considered the %22true%22 deterministic representation of reality. While this approach does have utility, it is far from an accurate mathematical treatment of the true model calibration problem in which both the computed data and experimental data have error bars. This year, we examined methods to perform calibration accounting for the error in both the computer model and the data, as well as improving our understanding of its meaning for model predictability. We call this approach Calibration under Uncertainty (CUU). This talk presents our current thinking on CUU. We outline some current approaches in the literature, and discuss the Bayesian approach to CUU in detail.

  18. Configuration Management Automation (CMA) - (United States)

    Department of Transportation — Configuration Management Automation (CMA) will provide an automated, integrated enterprise solution to support CM of FAA NAS and Non-NAS assets and investments. CMA...

  19. Autonomy and Automation (United States)

    Shively, Jay


    A significant level of debate and confusion has surrounded the meaning of the terms autonomy and automation. Automation is a multi-dimensional concept, and we propose that Remotely Piloted Aircraft Systems (RPAS) automation should be described with reference to the specific system and task that has been automated, the context in which the automation functions, and other relevant dimensions. In this paper, we present definitions of automation, pilot in the loop, pilot on the loop and pilot out of the loop. We further propose that in future, the International Civil Aviation Organization (ICAO) RPAS Panel avoids the use of the terms autonomy and autonomous when referring to automated systems on board RPA. Work Group 7 proposes to develop, in consultation with other workgroups, a taxonomy of Levels of Automation for RPAS.

  20. WFIRST WFI Calibration Requirements (United States)

    Scolnic, Daniel; Casertano, Stephano; WFIRST Calibration Group


    The Wide Field InfraRed Survey Telescope (WFIRST), with a planned launch in the mid-2020’s, will enable multiple generation-defining measurements in astrophysics and cosmology. One of the key goals of the mission is to limit calibration uncertainties in order to enable a wide range of experiments. Here we present the work of the WFIRST WFI Calibration Working Group, which has compiled a comprehensive set of calibration needs derived from the Mission science requirements, and has outlined a plan toachieve them. In many areas, the accuracy required has yet to be reached in any comparable mission or project. We present here the various plans of on-ground characterization, pre-launch data; internal measurements and observations in orbit; and external observations.

  1. Site Calibration report

    DEFF Research Database (Denmark)

    Yordanova, Ginka; Vesth, Allan

    The report describes site calibration measurements carried out on a site in Denmark. The measurements are carried out in accordance to Ref. [1]. The site calibration is carried out before a power performance measurement on a given turbine to clarify the influence from the terrain on the ratio...... between the wind speed at the center of the turbine hub and at the met mast. The wind speed at the turbine is measured by a temporary mast placed at the foundation for the turbine. The site and measurement equipment is detailed described in [2]. The possible measurement sector for power performance...... according to [1] is also described in [2] and no results from the site calibration have shown any necessary exclusion from this sector. All parts of the sensors and the measurement system have been installed by DTU....

  2. Calibrating the Athena telescope (United States)

    de Bruijne, J.; Guainazzi, M.; den Herder, J.; Bavdaz, M.; Burwitz, V.; Ferrando, P.; Lumb, D.; Natalucci, L.; Pajot, F.; Pareschi, G.


    Athena is ESA's upcoming X-ray mission, currently set for launch in 2028. With two nationally-funded, state-of-the-art instruments (a high-resolution spectrograph named X-IFU and a wide-field imager named WFI), and a telescope collecting area of 1.4-2 m^2 at 1 keV, the calibration of the spacecraft is a challenge in itself. This poster presents the current (spring 2017) plan of how to calibrate the Athena telescope. It is based on a hybrid approach, using bulk manufacturing and integration data as well as dedicated calibration measurements combined with a refined software model to simulate the full response of the optics.

  3. Workflow automation architecture standard

    Energy Technology Data Exchange (ETDEWEB)

    Moshofsky, R.P.; Rohen, W.T. [Boeing Computer Services Co., Richland, WA (United States)


    This document presents an architectural standard for application of workflow automation technology. The standard includes a functional architecture, process for developing an automated workflow system for a work group, functional and collateral specifications for workflow automation, and results of a proof of concept prototype.

  4. Deep Impact instrument calibration (United States)

    Klaasen, K.; A'Hearn, M. F.; Baca, M.; Delamere, A.; Desnoyer, M.; Farnham, T.; Groussin, O.; Hampton, D.; Ipatov, S.; Li, J.-Y.; Lisse, C.; Mastrodemos, N.; McLaughlin, S.; Sunshine, J.; Thomas, P.; Wellnitz, D.


    Calibration of NASA's Deep Impact spacecraft instruments allows reliable scientific interpretation of the images and spectra returned from comet Tempel 1. Calibrations of the four onboard remote sensing imaging instruments have been performed in the areas of geometric calibration, spatial resolution, spectral resolution, and radiometric response. Error sources such as noise (random, coherent, encoding, data compression), detector readout artifacts, scattered light, and radiation interactions have been quantified. The point spread functions (PSFs) of the medium resolution instrument and its twin impactor targeting sensor are near the theoretical minimum [~1.7 pixels full width at half maximum (FWHM)]. However, the high resolution instrument camera was found to be out of focus with a PSF FWHM of ~9 pixels. The charge coupled device (CCD) read noise is ~1 DN. Electrical cross-talk between the CCD detector quadrants is correctable to <2 DN. The IR spectrometer response nonlinearity is correctable to ~1%. Spectrometer read noise is ~2 DN. The variation in zero-exposure signal level with time and spectrometer temperature is not fully characterized; currently corrections are good to ~10 DN at best. Wavelength mapping onto the detector is known within 1 pixel; spectral lines have a FWHM of ~2 pixels. About 1% of the IR detector pixels behave badly and remain uncalibrated. The spectrometer exhibits a faint ghost image from reflection off a beamsplitter. Instrument absolute radiometric calibration accuracies were determined generally to <10% using star imaging. Flat-field calibration reduces pixel-to-pixel response differences to ~0.5% for the cameras and <2% for the spectrometer. A standard calibration image processing pipeline is used to produce archival image files for analysis by researchers.

  5. Calibration of scanning Lidar

    DEFF Research Database (Denmark)

    Gómez Arranz, Paula; Courtney, Michael

    This report describes the tests carried out on a scanning lidar at the DTU Test Station for large wind turbines, Høvsøre. The tests were divided in two parts. In the first part, the purpose was to obtain wind speed calibrations at two heights against two cup anemometers mounted on a mast. Additio......This report describes the tests carried out on a scanning lidar at the DTU Test Station for large wind turbines, Høvsøre. The tests were divided in two parts. In the first part, the purpose was to obtain wind speed calibrations at two heights against two cup anemometers mounted on a mast...

  6. Calibrated entanglement entropy (United States)

    Bakhmatov, I.; Deger, N. S.; Gutowski, J.; Colgáin, E. Ó.; Yavartanoo, H.


    The Ryu-Takayanagi prescription reduces the problem of calculating entanglement entropy in CFTs to the determination of minimal surfaces in a dual anti-de Sitter geometry. For 3D gravity theories and BTZ black holes, we identify the minimal surfaces as special Lagrangian cycles calibrated by the real part of the holomorphic one-form of a spacelike hypersurface. We show that (generalised) calibrations provide a unified way to determine holographic entanglement entropy from minimal surfaces, which is applicable to warped AdS3 geometries. We briefly discuss generalisations to higher dimensions.

  7. Calibrating Legal Judgments

    Directory of Open Access Journals (Sweden)

    Frederick Schauer


    Full Text Available Objective to study the notion and essence of legal judgments calibration the possibilities of using it in the lawenforcement activity to explore the expenses and advantages of using it. Methods dialectic approach to the cognition of social phenomena which enables to analyze them in historical development and functioning in the context of the integrity of objective and subjective factors it determined the choice of the following research methods formallegal comparative legal sociological methods of cognitive psychology and philosophy. Results In ordinary life people who assess other peoplersaquos judgments typically take into account the other judgments of those they are assessing in order to calibrate the judgment presently being assessed. The restaurant and hotel rating website TripAdvisor is exemplary because it facilitates calibration by providing access to a raterrsaquos previous ratings. Such information allows a user to see whether a particular rating comes from a rater who is enthusiastic about every place she patronizes or instead from someone who is incessantly hard to please. And even when less systematized as in assessing a letter of recommendation or college transcript calibration by recourse to the decisional history of those whose judgments are being assessed is ubiquitous. Yet despite the ubiquity and utility of such calibration the legal system seems perversely to reject it. Appellate courts do not openly adjust their standard of review based on the previous judgments of the judge whose decision they are reviewing nor do judges in reviewing legislative or administrative decisions magistrates in evaluating search warrant representations or jurors in assessing witness perception. In most legal domains calibration by reference to the prior decisions of the reviewee is invisible either because it does not exist or because reviewing bodies are unwilling to admit using what they in fact know and employ. Scientific novelty for the first

  8. Calibration with Absolute Shrinkage

    DEFF Research Database (Denmark)

    Øjelund, Henrik; Madsen, Henrik; Thyregod, Poul


    is suggested to cope with the singular design matrix most often seen in chemometric calibration. Furthermore, the proposed algorithm may be generalized to all convex norms like Sigma/beta (j)/(gamma) where gamma greater than or equal to 1, i.e. a method that continuously varies from ridge regression...... to the lasso. The lasso is applied both directly as a calibration method and as a method to select important variables/wave lengths. It is demonstrated that the lasso algorithm, in general, leads to parameter estimates of which some are zero while others are quite large (compared to e.g. the traditional PLS...

  9. Thrombin Time (United States)

    ... eGFR) Estrogen/Progesterone Receptor Status Estrogens Ethanol Extractable Nuclear Antigen Antibodies (ENA) Panel Factor V Leiden Mutation ... and Acute Coronary Syndrome Heart Disease Hemochromatosis Hemoglobin Abnormalities Hepatitis HIV Infection and AIDS Huntington Disease Hypertension ...

  10. Thrombin Time (United States)

    ... Glance What is being tested? Common Questions Related Content View Sources ... part of an investigation of excessive bleeding or inappropriate blood clot formation ( thrombotic episode ), particularly to evaluate ...

  11. A critical comparison of systematic calibration protocols for activated sludge models: a SWOT analysis. (United States)

    Sin, Gürkan; Van Hulle, Stijn W H; De Pauw, Dirk J W; van Griensven, Ann; Vanrolleghem, Peter A


    Modelling activated sludge systems has gained an increasing momentum after the introduction of activated sludge models (ASMs) in 1987. Application of dynamic models for full-scale systems requires essentially a calibration of the chosen ASM to the case under study. Numerous full-scale model applications have been performed so far which were mostly based on ad hoc approaches and expert knowledge. Further, each modelling study has followed a different calibration approach: e.g. different influent wastewater characterization methods, different kinetic parameter estimation methods, different selection of parameters to be calibrated, different priorities within the calibration steps, etc. In short, there was no standard approach in performing the calibration study, which makes it difficult, if not impossible, to (1) compare different calibrations of ASMs with each other and (2) perform internal quality checks for each calibration study. To address these concerns, systematic calibration protocols have recently been proposed to bring guidance to the modeling of activated sludge systems and in particular to the calibration of full-scale models. In this contribution four existing calibration approaches (BIOMATH, HSG, STOWA and WERF) will be critically discussed using a SWOT (Strengths, Weaknesses, Opportunities, Threats) analysis. It will also be assessed in what way these approaches can be further developed in view of further improving the quality of ASM calibration. In this respect, the potential of automating some steps of the calibration procedure by use of mathematical algorithms is highlighted.

  12. An automated data collection system for a Charpy impact tester (United States)

    Weigman, Bernard J.; Spiegel, F. Xavier


    A method for automated data collection has been developed for a Charpy impact tester. A potentiometer is connected to the pivot point of the hammer and measures the angular displacement of the hammer. This data is collected with a computer and, through appropriate software, accurately records the energy absorbed by the specimen. The device can be easily calibrated with minimal effort.

  13. Aptamer-based organic-silica hybrid affinity monolith prepared via "thiol-ene" click reaction for extraction of thrombin. (United States)

    Wang, Zheng; Zhao, Jin-cheng; Lian, Hong-zhen; Chen, Hong-yuan


    A novel strategy for preparing aptamer-based organic-silica hybrid monolithic column was developed via "thiol-ene" click chemistry. Due to the large specific surface area of the hybrid matrix and the simplicity, rapidness and high efficiency of "thiol-ene" click reaction, the average coverage density of aptamer on the organic-silica hybrid monolith reached 420 pmol μL(-1). Human α-thrombin can be captured on the prepared affinity monolithic column with high specificity and eluted by NaClO4 solution. N-p-tosyl-Gly-Pro-Arg p-nitroanilide acetate was used as the sensitive chromogenic substrate of thrombin. The thrombin enriched by this affinity column was detected with a detection of limit of 0.01 μM by spectrophotometry. Furthermore, the extraction recovery of thrombin at 0.15 μM in human serum was 91.8% with a relative standard deviation of 4.0%. These results indicated that "thiol-ene" click chemistry provided a promising technique to immobilize aptamer on organic-inorganic hybrid monolith and the easily-assembled affinity monolithic material could be used to realize highly selective recognition of trace proteins. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Genetic variation in thrombin-activatable fibrinolysis inhibitor (TAFI) is associated with the risk of splanchnic vein thrombosis

    NARCIS (Netherlands)

    de Bruijne, Emile L. E.; Darwish Murad, Sarwa; de Maat, Moniek P. M.; Tanck, Michael W. T.; Haagsma, Elizabeth B.; van Hoek, Bart; Rosendaal, Frits R.; Janssen, Harry L. A.; Leebeek, Frank W. G.


    Splanchnic vein thrombosis (SVT) has been associated with a hypercoagulable state. Thrombin-activatable fibrinolysis inhibitor (TAFI) may contribute to a hypercoagulable state, and therefore we were interested in the role of TAFI in SVT. Since the disease is frequently associated with liver

  15. Genetic variation in thrombin-activatable fibrinolysis inhibitor (TAR) is associated with the risk of splanchnic vein thrombosis

    NARCIS (Netherlands)

    de Bruijne, Emile L. E.; Murad, Sarwa Darwish; de Maat, Moniek P. M.; Tanck, Michael W. T.; Haagsma, Elizabeth B.; van Hoeks, Bart; Rosendaal, Frits R.; Janssen, Harry L. A.; Leebeek, Frank W. G.

    Splanchnic vein thrombosis (SVT) has been associated with a hypercoagulable state. Thrombin-activatable fibrinolysis inhibitor (TAFI) may contribute to a hypercoagulable state, and therefore we were interested in the role of TAR in SVT. Since the disease is frequently associated with liver

  16. In vitro effect of hemodilution on activated clotting time and high-dose thrombin time during cardiopulmonary bypass

    NARCIS (Netherlands)

    Huyzen, RJ; vanOeveren, W; Wei, FY; Stellingwerf, P; Boonstra, PW; Gu, YJ

    Background. Extreme dilution of clotting factors, as may occur during pediatric or neonatal cardiopulmonary bypass, often leads to inadequate monitoring of anticoagulation with activated dotting time (ACT). In this study we postulate that the high-dose thrombin time (HiTT) is less influenced by

  17. Development of an Efficient G-Quadruplex-Stabilised Thrombin-Binding Aptamer Containing a Three-Carbon Spacer Molecule

    DEFF Research Database (Denmark)

    Aaldering, Lukas J.; Poongavanam, Vasanthanathan; Langkjær, Niels


    The thrombin-binding aptamer (TBA), which shows anticoagulant properties, is one of the most studied G-quadruplex-forming aptamers. In this study, we investigated the impact of different chemical modifications such as a three-carbon spacer (spacer-C3), unlocked nucleic acid (UNA) and 3′-amino...

  18. Different Potential of Extracellular Vesicles to Support Thrombin Generation: Contributions of Phosphatidylserine, Tissue Factor, and Cellular Origin. (United States)

    Tripisciano, Carla; Weiss, René; Eichhorn, Tanja; Spittler, Andreas; Heuser, Thomas; Fischer, Michael Bernhard; Weber, Viktoria


    Cells release diverse types of vesicles constitutively or in response to proliferation, injury, inflammation, or stress. Extracellular vesicles (EVs) are crucial in intercellular communication, and there is emerging evidence for their roles in inflammation, cancer, and thrombosis. We investigated the thrombogenicity of platelet-derived EVs, which constitute the majority of circulating EVs in human blood, and assessed the contributions of phosphatidylserine and tissue factor exposure on thrombin generation. Addition of platelet EVs to vesicle-free human plasma induced thrombin generation in a dose-dependent manner, which was efficiently inhibited by annexin V, but not by anti-tissue factor antibodies, indicating that it was primarily due to the exposure of phosphatidylserine on platelet EVs. Platelet EVs exhibited higher thrombogenicity than EVs from unstimulated monocytic THP-1 cells, but blockade of contact activation significantly reduced thrombin generation by platelet EVs. Stimulation of monocytic cells with lipopolysaccharide enhanced their thrombogenicity both in the presence and in the absence of contact activation, and thrombin generation was efficiently blocked by anti-tissue factor antibodies. Our study provides evidence that irrespective of their cellular origin, EVs support the propagation of coagulation via the exposure of phosphatidylserine, while the expression of functional tissue factor on EVs appears to be limited to pathological conditions.

  19. A sensitive bioimmunoassay for thrombin-cleaved two-chain urokinase-type plasminogen activator in human body fluids

    NARCIS (Netherlands)

    Braat, E.A.M.; Nauland, U.; Dooijewaard, G.; Rijken, D.C.


    Thrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) into a two-chain form (tcu-PA/T), which is virtually inactive in plasminogen activator assays. Little is known about the physiological importance of tcu-PA/T. To examine the occurrence of tcu-PA/T in vivo, we developed a

  20. Alkylation of phosphorothioated thrombin binding aptamers improves the selectivity of inhibition of tumor cell proliferation upon anticoagulation. (United States)

    Yang, Xiantao; Zhu, Yuejie; Wang, Chao; Guan, Zhu; Zhang, Lihe; Yang, Zhenjun


    Recently, aptamers have been extensively researched for therapy and diagnostic applications. Thrombin-binding aptamer is a 15nt deoxyribonucleic acid screened by SELEX, it can specifically bind to thrombin and inhibit blood coagulation. Since it is also endowed with excellent antitumor activity, the intrinsic anticoagulation advantage converted to a main potential side effect for its further application in antiproliferative therapy. Site-specific alkylation was conducted through nucleophilic reaction of phosphorothioated TBAs using bromide reagents. Circular dichroism (CD) spectroscopy and surface plasmon resonance (SPR) measurements were used to evaluate anticoagulation activity, and a CCK-8 assay was used to determine cell proliferation activity. The CD spectra of the modified TBAs were weakened, and their affinity for thrombin was dramatically reduced, as reflected by the KD values. On the other hand, their inhibition of A549 cells was retained. Incorporation of different alkyls apparently disrupted the binding of TBA to thrombin while maintaining the antitumor activity. A new modification strategy was established for the use of TBA as a more selective antitumor agent. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. ROS/TXNIP pathway contributes to thrombin induced NLRP3 inflammasome activation and cell apoptosis in microglia. (United States)

    Ye, Xinchun; Zuo, Dandan; Yu, Lu; Zhang, Liang; Tang, Jiao; Cui, Chengcheng; Bao, Lei; Zan, Kun; Zhang, Zuohui; Yang, Xinxin; Chen, Hao; Tang, Hai; Zu, Jie; Shi, Hongjuan; Cui, Guiyun


    There is no effective therapy for intracerebral hemorrhage (ICH) because of poor understanding of the mechanisms of brain injury after hemorrhage. The NLRP3 inflammasome, as a vital component of innate immune system, which is associated with a wide range of human CNS disorders, including ICH. But its detailed mechanisms in ICH remain mainly unclear. In this study, BV2 cells with thrombin exposure were used to investigate the role of NLRP3 inflammasome in thrombin-induced brain injury. We used western blot to detect NLRP3 inflammasome activation and the expression of thioredoxin binding protein (TXNIP), DCFH-DA to investigate intracellular reactive oxygen species (ROS), flow cytometry to analyze apoptosis. Our results showed that ROS inhibitor N-acetyl-l-cysteine (NAC) suppressed the upregulation of intracellular ROS and TXNIP expression. Furthermore, the cell apoptosis and expression of apoptotic protein were significantly attenuated after treatment of thrombin with NAC or NLRP3 antagonist (MCC950). Thrombin activates ROS/TXNIP/NLRP3 signaling in BV2 cells, which may indicate a mechanism that pro-inflammatory and pro-apoptotic contributes to the development of ICH. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Host defense peptides of thrombin modulate inflammation and coagulation in endotoxin-mediated shock and Pseudomonas aeruginosa sepsis

    DEFF Research Database (Denmark)

    Kalle, Martina; Papareddy, Praveen; Kasetty, Gopinath


    present a new treatment concept for sepsis and endotoxin-mediated shock, based on host defense peptides from the C-terminal part of human thrombin, found to have a broad and inhibitory effect on multiple sepsis pathologies. Thus, the peptides abrogate pro-inflammatory cytokine responses to endotoxin...

  3. Binding characteristics of thrombin-activatable fibrinolysis inhibitor to streptococcal surface collagen-like proteins A and B

    NARCIS (Netherlands)

    Seron, Mercedes Valls; Plug, Tom; Marquart, J. Arnoud; Marx, Pauline F.; Herwald, Heiko; de Groot, Philip G.; Meijers, Joost C. M.


    Streptococcus pyogenes is the causative agent in a wide range of diseases in humans. Thrombin-activatable fibrinolysis inhibitor (TAFI) binds to collagen-like proteins ScIA and ScIB at the surface of S. pyogenes. Activation of TAFI at this surface redirects inflammation from a transient to chronic

  4. Thrombin-Derived Host-Defense Peptides Modulate Monocyte/Macrophage Inflammatory Responses to Gram-Negative Bacteria

    DEFF Research Database (Denmark)

    Hansen, Finja C; Strömdahl, Ann-Charlotte; Mörgelin, Matthias


    , the effect of GKY25 on the interaction of monocytes/macrophages with Gram-negative bacteria was explored. Electron microscopy analysis showed that fibrin slough from non-healing wounds, colonized with Staphylococcus aureus and Pseudomonas aeruginosa, contains C-terminal thrombin epitopes associated...

  5. Automation in Clinical Microbiology (United States)

    Ledeboer, Nathan A.


    Historically, the trend toward automation in clinical pathology laboratories has largely bypassed the clinical microbiology laboratory. In this article, we review the historical impediments to automation in the microbiology laboratory and offer insight into the reasons why we believe that we are on the cusp of a dramatic change that will sweep a wave of automation into clinical microbiology laboratories. We review the currently available specimen-processing instruments as well as the total laboratory automation solutions. Lastly, we outline the types of studies that will need to be performed to fully assess the benefits of automation in microbiology laboratories. PMID:23515547


    NARCIS (Netherlands)

    Kazemi, S.; Yatawatta, S.; Zaroubi, S.


    This paper introduces an amendment to radio interferometric calibration of sources below the noise level. The main idea is to employ the information of the stronger sources' measured signals as a plug-in criterion to solve for the weaker ones. For this purpose, we construct a number of source

  7. NVLAP calibration laboratory program

    Energy Technology Data Exchange (ETDEWEB)

    Cigler, J.L.


    This paper presents an overview of the progress up to April 1993 in the development of the Calibration Laboratories Accreditation Program within the framework of the National Voluntary Laboratory Accreditation Program (NVLAP) at the National Institute of Standards and Technology (NIST).

  8. Calibrating Communication Competencies (United States)

    Surges Tatum, Donna


    The Many-faceted Rasch measurement model is used in the creation of a diagnostic instrument by which communication competencies can be calibrated, the severity of observers/raters can be determined, the ability of speakers measured, and comparisons made between various groups.

  9. ECAL Energy Flow Calibration

    CERN Multimedia

    CERN. Geneva


    My talk will be covering my work as a whole over the course of the semester. The focus will be on using energy flow calibration in ECAL to check the precision of the corrections made by the light monitoring system used to account for transparency loss within ECAL crystals due to radiation damage over time.

  10. Entropic calibration revisited

    Energy Technology Data Exchange (ETDEWEB)

    Brody, Dorje C. [Blackett Laboratory, Imperial College, London SW7 2BZ (United Kingdom)]. E-mail:; Buckley, Ian R.C. [Centre for Quantitative Finance, Imperial College, London SW7 2AZ (United Kingdom); Constantinou, Irene C. [Blackett Laboratory, Imperial College, London SW7 2BZ (United Kingdom); Meister, Bernhard K. [Blackett Laboratory, Imperial College, London SW7 2BZ (United Kingdom)


    The entropic calibration of the risk-neutral density function is effective in recovering the strike dependence of options, but encounters difficulties in determining the relevant greeks. By use of put-call reversal we apply the entropic method to the time reversed economy, which allows us to obtain the spot price dependence of options and the relevant greeks.

  11. Measurement System & Calibration report

    DEFF Research Database (Denmark)

    Gómez Arranz, Paula; Villanueva, Héctor

    This Measurement System & Calibration report is describing DTU’s measurement system installed at a specific wind turbine. A part of the sensors has been installed by others, the rest of the sensors have been installed by DTU. The results of the measurements, described in this report, are only val...


    Directory of Open Access Journals (Sweden)

    G. Kemper


    step with the help of the nadir camera and the GPS/IMU data, an initial orientation correction and radial correction were calculated. With this approach, the whole project was calculated and calibrated in one step. During the iteration process the radial and tangential parameters were switched on individually for the camera heads and after that the camera constants and principal point positions were checked and finally calibrated. Besides that, the bore side calibration can be performed either on basis of the nadir camera and their offsets, or independently for each camera without correlation to the others. This must be performed in a complete mission anyway to get stability between the single camera heads. Determining the lever arms of the nodal-points to the IMU centre needs more caution than for a single camera especially due to the strong tilt angle. Prepared all these previous steps, you get a highly accurate sensor that enables a fully automated data extraction with a rapid update of you existing data. Frequently monitoring urban dynamics is then possible in fully 3D environment.

  13. Calibration Procedures on Oblique Camera Setups (United States)

    Kemper, G.; Melykuti, B.; Yu, C.


    the nadir camera and the GPS/IMU data, an initial orientation correction and radial correction were calculated. With this approach, the whole project was calculated and calibrated in one step. During the iteration process the radial and tangential parameters were switched on individually for the camera heads and after that the camera constants and principal point positions were checked and finally calibrated. Besides that, the bore side calibration can be performed either on basis of the nadir camera and their offsets, or independently for each camera without correlation to the others. This must be performed in a complete mission anyway to get stability between the single camera heads. Determining the lever arms of the nodal-points to the IMU centre needs more caution than for a single camera especially due to the strong tilt angle. Prepared all these previous steps, you get a highly accurate sensor that enables a fully automated data extraction with a rapid update of you existing data. Frequently monitoring urban dynamics is then possible in fully 3D environment.

  14. Effect of Locked-Nucleic Acid on a Biologically Active G-Quadruplex. A Structure-Activity Relationship of the Thrombin Aptamer

    Directory of Open Access Journals (Sweden)

    Michael B. Jarstfer


    Full Text Available Here we tested the ability to augment the biological activity of the thrombin aptamer, d(GGTTGGTGTGGTTGG, by using locked nucleic acid (LNA to influence its G-quadruplex structure. Compared to un-substituted control aptamer, LNA-containing aptamers displayed varying degrees of thrombin inhibition. Aptamers with LNA substituted in either positions G5, T7, or G8 showed decreased thrombin inhibition, whereas LNA at position G2 displayed activity comparable to un-substituted control aptamer. Interestingly, the thermal stability of the substituted aptamers does not correlate to activity – the more stable aptamers with LNA in position G5, T7, or G8 showed the least thrombin inhibition, while a less stable aptamer with LNA at G2 was as active as the un-substituted aptamer. These results suggest that LNA substitution at sites G5, T7, and G8 directly perturbs aptamer-thrombin affinity. This further implies that for the thrombin aptamer, activity is not dictated solely by the stability of the G-quadruplex structure, but by specific interactions between the central TGT loop and thrombin and that LNA can be tolerated in a biologically active nucleic acid structure albeit in a position dependent fashion.

  15. Mercury CEM Calibration

    Energy Technology Data Exchange (ETDEWEB)

    John F. Schabron; Joseph F. Rovani; Susan S. Sorini


    The Clean Air Mercury Rule (CAMR) which was published in the Federal Register on May 18, 2005, requires that calibration of mercury continuous emissions monitors (CEMs) be performed with NIST-traceable standards. Western Research Institute (WRI) is working closely with the Electric Power Research Institute (EPRI), the National Institute of Standards and Technology (NIST), and the Environmental Protection Agency (EPA) to facilitate the development of the experimental criteria for a NIST traceability protocol for dynamic elemental mercury vapor generators. The traceability protocol will be written by EPA. Traceability will be based on the actual analysis of the output of each calibration unit at several concentration levels ranging from about 2-40 ug/m{sup 3}, and this analysis will be directly traceable to analyses by NIST using isotope dilution inductively coupled plasma/mass spectrometry (ID ICP/MS) through a chain of analyses linking the calibration unit in the power plant to the NIST ID ICP/MS. Prior to this project, NIST did not provide a recommended mercury vapor pressure equation or list mercury vapor pressure in its vapor pressure database. The NIST Physical and Chemical Properties Division in Boulder, Colorado was subcontracted under this project to study the issue in detail and to recommend a mercury vapor pressure equation that the vendors of mercury vapor pressure calibration units can use to calculate the elemental mercury vapor concentration in an equilibrium chamber at a particular temperature. As part of this study, a preliminary evaluation of calibration units from five vendors was made. The work was performed by NIST in Gaithersburg, MD and Joe Rovani from WRI who traveled to NIST as a Visiting Scientist.

  16. Rapid Upregulation of Orai1 Abundance in the Plasma Membrane of Platelets Following Activation with Thrombin and Collagen Related Peptide

    Directory of Open Access Journals (Sweden)

    Guilai Liu


    Full Text Available Background: Blood platelets accomplish primary hemostasis following vascular injury and contribute to the orchestration of occlusive vascular disease. Platelets are activated by an increase of cytosolic Ca2+-activity ([Ca2+]i, which is accomplished by Ca2+-release from intracellular stores and subsequent store operated Ca2+ entry (SOCE through Ca2+ release activated Ca2+ channel moiety Orai1. Powerful activators of platelets include thrombin and collagen related peptide (CRP, which are in part effective by activation of small G- protein Rac1. The present study explored the influence of thrombin and CRP on Orai1 protein abundance and cytosolic Ca2+-activity ([Ca2+]i in platelets drawn from wild type mice. Methods: Orai1 protein surface abundance was quantified utilizing CF™488A conjugated antibodies, and [Ca2+]i was determined with Fluo3-fluorescence. Results: In resting platelets, Orai1 protein abundance and [Ca2+]i were low. Thrombin (0.02 U/ml and CRP (5ug/ml within 2 min increased [Ca2+]i and Orai1 protein abundance at the platelet surface. [Ca2+]i was further increased by Ca2+ ionophore ionomycin (1 µM and by store depletion with the sarcoendoplasmatic Ca2+ ATPase inhibitor thapsigargin (1 µM. However, Orai1 protein abundance at the platelet surface was not significantly affected by ionomycin and only slightly increased by thapsigargin. The effect of thrombin and CRP on Orai1 abundance and [Ca2+]i was significantly blunted by Rac1 inhibitor NSC23766 (50 µM. Conclusion: The increase of [Ca2+]i following stimulation of platelets with thrombin and collagen related peptide is potentiated by ultrarapid Rac1 sensitive translocation of Orai1 into the cell membrane.

  17. Reduced Requirement for Prothrombin Complex Concentrate for the Restoration of Thrombin Generation in Plasma From Liver Transplant Recipients. (United States)

    Abuelkasem, Ezeldeen; Hasan, Shaheer; Mazzeffi, Michael A; Planinsic, Raymond M; Sakai, Tetsuro; Tanaka, Kenichi A


    Plasma transfusion remains the mainstay hemostatic therapy during liver transplantation (LT) in most countries. However, a large volume is required for plasma to achieve clinically relevant factor increases. Prothrombin complex concentrate (PCC) is a low-volume alternative to plasma in warfarin reversal, but its efficacy has not been well studied in LT. Blood samples were collected from 28 LT patients at baseline (T0) and 30 minutes after graft reperfusion (T1). Factor X and antithrombin levels were measured. Ex vivo effects of PCC (0.2 and 0.4 IU/mL) and 10% volume replacement with normal plasma were compared in LT and warfarin plasma by measuring lag time, thrombin peak, and endogenous thrombin potential (ETP) using thrombin generation (TG) assay. Coagulation status was worsened at T1 as international normalized ratio increased from 1.7 to 3.0, and factor X was decreased from 49% to 28%. TG measurements showed normal lag time and ETP at T0 and T1, but low-normal peak at T0, and below-normal peak at T1. Both doses of PCC increased peak and ETP, while 10% volume plasma had minimal effects on TG. Thrombin inhibition appears to be very slow after adding 0.4 IU/mL of PCC in LT plasma due to low antithrombin. The same doses of PCC and plasma were insufficient for warfarin reversal. Reduced TG in LT can be more effectively restored by using PCC rather than plasma. The required doses of PCC for LT patients seem to be lower than warfarin reversal due to slow thrombin inhibition.

  18. Stimulus-response click chemistry based aptamer-functionalized mesoporous silica nanoparticles for fluorescence detection of thrombin. (United States)

    Chen, Zhonghui; Sun, Mi; Luo, Fang; Xu, Kefeng; Lin, Zhenyu; Zhang, Lan


    In most aptamer based stimulus response mesoporous silica nanoparticles (MSN) systems, the aptamer is modified on the MSN via electrostatic interaction, however leakage might exist after a certain time in the system and hence the stability is not good. In this study, the pores of MSN were capped by aptamer through click chemistry reaction for the first time and the system was then employed to develop a fluorescence biosensor. Specifically, the aptamer of the target (thrombin in this study) was hybridized with its complementary DNA (which was initially modified with alkyne at the terminal) to form a double strand DNA (dsDNA) firstly, and then this dsDNA was modified on N 3 modified MSN via Cu(I) catalyzed alkyne-azide cycloaddition reaction. The guest molecules (fluorescein) were blocked in the pores of the MSN with high efficiency and nearly no leakage was detected. Upon the introduction of thrombin, thrombin specifically recognized its aptamer, so aptamer released from the MSN; and the single strand DNA(ssDNA) left could not cap the pores of the MSN efficiently and hence caused the releasing of fluorescein into the solution. The enhanced fluorescence intensity of the system has a good linear relationship with the thrombin concentration in the range of 50-1000ngmL -1 with a detection limit of 28.46ngmL -1 . The proposed biosensor has been successfully applied to detect thrombin in serum samples with high selectivity. The same strategy can be applied to develop biosensors for different targets by changing the adopted aptamer. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Field calibration of cup anemometers

    DEFF Research Database (Denmark)

    Schmidt Paulsen, Uwe; Mortensen, Niels Gylling; Hansen, Jens Carsten


    A field calibration method and results are described along with the experience gained with the method. The cup anemometers to be calibrated are mounted in a row on a 10-m high rig and calibrated in the free wind against a reference cup anemometer. The method has been reported [1] to improve...... the statistical bias on the data relative to calibrations carried out in a wind tunnel. The methodology is sufficiently accurate for calibration of cup anemometers used for wind resource assessments and provides a simple, reliable and cost-effective solution to cup anemometer calibration, especially suited...

  20. Enhancing the Affinity of Anti-Human α-Thrombin 15-mer DNA Aptamer and Anti-Immunoglobulin E Aptamer by PolyT Extension. (United States)

    Bai, Yunlong; Li, Yapiao; Zhang, Dapeng; Wang, Hailin; Zhao, Qiang


    Aptamer affinity capillary electrophoresis-laser-induced fluorescence (CE-LIF) for protein detection takes advantage of aptamers for their ease of synthesis and labeling, small size, and having many negative charges. Its success relies on the high binding affinity of aptamers. One 15-mer DNA aptamer (5'-GGT TGG TGT GGT TGG-3', Apt15) shows desirable specificity for human α-thrombin, an important enzyme with multiple functions in blood. However, Apt15 has weak binding affinity, and the use of Apt15 in affinity CE-LIF analysis remains challenging. Here we reported that extension of Apt15 at the 3'-end with a polyT tail having length of 18 T or longer significantly enhanced its affinity and enabled a well-isolated and stable peak for thrombin-aptamer complex in affinity CE. It was likely that the improvement of binding affinity resulted from double binding, an additional interaction of the polyT tail with thrombin in addition to the Apt15 section binding to thrombin. With dye-labeled Apt15 having a T25 tail, we achieved detection of thrombin at concentrations as low as 0.1 nM by affinity CE-LIF. This aptamer probe specifically bound to human α-thrombin, showing negligible affinity for human β- and γ-thrombin, which are proteolyzed derivatives of human alpha α-thrombin and share similar structure. This strategy of adding a polyT extension also enhanced the binding affinity of anti-immunoglobulin E aptamer in CE-LIF analysis, showing that the affinity enhancement approach is not limited to the thrombin-binding aptamer and has potential for more applications in bioanalysis.

  1. Contributions of Protease-Activated Receptors PAR1 and PAR4 to Thrombin-Induced GPIIbIIIa Activation in Human Platelets. (United States)

    Duvernay, Matthew T; Temple, Kayla J; Maeng, Jae G; Blobaum, Anna L; Stauffer, Shaun R; Lindsley, Craig W; Hamm, Heidi E


    Human platelets display a unique dual receptor system for responding to its primary endogenous activator, α-thrombin. Because of the lack of efficacious antagonists, the field has relied on synthetic peptides and pepducins to describe protease-activated receptor PAR1 and PAR4 signaling. The precise contributions of each receptor have not been established in the context of thrombin. We took advantage of newly discovered PAR antagonists to contrast the contribution of PAR1 and PAR4 to thrombin-mediated activation of the platelet fibrin receptor (GPIIbIIIa). PAR1 is required for platelet activation at low but not high concentrations of thrombin, and maximal platelet activation at high concentrations of thrombin requires PAR4. As the concentration of thrombin is increased, PAR1 signaling is quickly overcome by PAR4 signaling, leaving a narrow window of low thrombin concentrations that exclusively engage PAR1. PAR4 antagonism reduces the maximum thrombin response by over 50%. Thus, although the PAR1 response still active at higher concentrations of thrombin, this response is superseded by PAR4. Truncation of a known PAR4 antagonist and identification of the minimum pharmacophore converted the mechanism of inhibition from noncompetitive to competitive, such that the antagonist could be outcompeted by increasing doses of the ligand. Fragments retained efficacy against both soluble and tethered ligands with lower cLogP values and an increased free fraction in plasma. These reversible, competitive compounds represent a route toward potentially safer PAR4 antagonists for clinical utility and the development of tools such as radioligands and positron emission tomography tracers that are not currently available to the field for this target. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  2. Thrombin-induced reactive oxygen species generation in platelets: A novel role for protease-activated receptor 4 and GPIbα. (United States)

    Carrim, Naadiya; Arthur, Jane F; Hamilton, Justin R; Gardiner, Elizabeth E; Andrews, Robert K; Moran, Niamh; Berndt, Michael C; Metharom, Pat


    Platelets are essential for maintaining haemostasis and play a key role in the pathogenesis of cardiovascular disease. Upon ligation of platelet receptors through subendothelial matrix proteins, intracellular reactive oxygen species (ROS) are generated, further amplifying the platelet activation response. Thrombin, a potent platelet activator, can signal through GPIbα and protease-activated receptor (PAR) 1 and PAR4 on human platelets, and recently has been implicated in the generation of ROS. While ROS are known to have key roles in intra-platelet signalling and subsequent platelet activation, the precise receptors and signalling pathways involved in thrombin-induced ROS generation have yet to be fully elucidated. To investigate the relative contribution of platelet GPIbα and PARs to thrombin-induced reactive oxygen species (ROS) generation. Highly specific antagonists targeting PAR1 and PAR4, and the GPIbα-cleaving enzyme, Naja kaouthia (Nk) protease, were used in quantitative flow cytometry assays of thrombin-induced ROS production. Antagonists of PAR4 but not PAR1, inhibited thrombin-derived ROS generation. Removal of the GPIbα ligand binding region attenuated PAR4-induced and completely inhibited thrombin-induced ROS formation. Similarly, PAR4 deficiency in mice abolished thrombin-induced ROS generation. Additionally, GPIbα and PAR4-dependent ROS formation were shown to be mediated through focal adhesion kinase (FAK) and NADPH oxidase 1 (NOX1) proteins. Both GPIbα and PAR4 are required for thrombin-induced ROS formation, suggesting a novel functional cooperation between GPIbα and PAR4. Our study identifies a novel role for PAR4 in mediating thrombin-induced ROS production that was not shared by PAR1. This suggests an independent signalling pathway in platelet activation that may be targeted therapeutically. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  3. Conformational states of vitronectin: preferential expression of an antigenic epitope when vitronectin is covalently and noncovalently complexed with thrombin-antithrombin III or treated with urea. (United States)

    Tomasini, B R; Mosher, D F


    A difference in recognition of the adhesive glycoprotein vitronectin (also called S-protein, serum spreading factor, and epibolin) by monoclonal antibody 8E6 (Hayman EG, et al, Proc Natl Acad Sci USA 80:4003, 1983) was investigated using a competitive enzyme-immunosorbent assay and immunoaffinity chromatography. Recognition of vitronectin in serum was approximately 50-fold greater than recognition of vitronectin in plasma. Recognition of vitronectin incubated with heparin, thrombin-antithrombin III complex, or heparin and thrombin-antithrombin III complex together was 2.5-, 7-, or 32-fold greater, respectively, than recognition of vitronectin alone. Thrombin or antithrombin III by itself did not induce the antigenic change. Factor Xa-antithrombin III was less effective than thrombin-antithrombin III in induction of the change. Dextran sulfate and fucoidan were more potent than heparin in induction of the antigenic change, whereas dermatan sulfate, hyaluronic acid, heparan sulfate, chondroitin sulfate, or keratan sulfate were less effective. Immunoblotting analysis of serum and of vitronectin incubated with thrombin and antithrombin III demonstrated the presence of complexes composed of vitronectin and thrombin-antithrombin III that could only be dissociated with reducing agent. N-ethylmaleimide completely blocked the formation of the presumably disulfide-bonded complexes and partially blocked the antigenic change. Both non-disulfide-bonded and disulfide-bonded vitronectin bound to antibody-Sepharose from a mixture of vitronectin and thrombin-antithrombin III. Treatment of vitronectin with 8 mol/L urea resulted in enhanced recognition by the monoclonal antibody. Thus, the 8E6 antibody reacts with an epitope that is preferentially expressed by noncovalently and covalently linked vitronectin/thrombin-antithrombin III complexes and by urea-treated vitronectin. The change in vitronectin induced by thrombin-antithrombin III, therefore, is a physiological correlate of

  4. Mercury Calibration System

    Energy Technology Data Exchange (ETDEWEB)

    John Schabron; Eric Kalberer; Joseph Rovani; Mark Sanderson; Ryan Boysen; William Schuster


    U.S. Environmental Protection Agency (EPA) Performance Specification 12 in the Clean Air Mercury Rule (CAMR) states that a mercury CEM must be calibrated with National Institute for Standards and Technology (NIST)-traceable standards. In early 2009, a NIST traceable standard for elemental mercury CEM calibration still does not exist. Despite the vacature of CAMR by a Federal appeals court in early 2008, a NIST traceable standard is still needed for whatever regulation is implemented in the future. Thermo Fisher is a major vendor providing complete integrated mercury continuous emissions monitoring (CEM) systems to the industry. WRI is participating with EPA, EPRI, NIST, and Thermo Fisher towards the development of the criteria that will be used in the traceability protocols to be issued by EPA. An initial draft of an elemental mercury calibration traceability protocol was distributed for comment to the participating research groups and vendors on a limited basis in early May 2007. In August 2007, EPA issued an interim traceability protocol for elemental mercury calibrators. Various working drafts of the new interim traceability protocols were distributed in late 2008 and early 2009 to participants in the Mercury Standards Working Committee project. The protocols include sections on qualification and certification. The qualification section describes in general terms tests that must be conducted by the calibrator vendors to demonstrate that their calibration equipment meets the minimum requirements to be established by EPA for use in CAMR monitoring. Variables to be examined include linearity, ambient temperature, back pressure, ambient pressure, line voltage, and effects of shipping. None of the procedures were described in detail in the draft interim documents; however they describe what EPA would like to eventually develop. WRI is providing the data and results to EPA for use in developing revised experimental procedures and realistic acceptance criteria based on

  5. Lithography process calibration with applications in defect printability analysis (United States)

    Wu, Shao-Po; Liu, Hua-Yu; Chang, Fang C.; Karklin, Linard


    Lithography process simulation has proven to be a useful and effective tool for process characterization, namely, properly characterize critical dimension (CD) variations from the design that are caused by proximity effects and distortions introduced by the patterning tool, reticle, resist processing and etching. Accurate lithography process simulator further enables process engineers to automate the tasks of advanced mask design, verification and inspection that are used in deep-sub-micron semiconductor manufacturing. However, to get the most benefit from process simulations, we should properly calibrate the simulation model according to the process to be characterized. That is, given a representative set of CD measurements obtained from the process, we fine-tune the process model parameters so that the simulated/predicted CDs well match the measured CDs. By doing so, we can ensure to some extent that process simulations give sensible results to be used in the design analysis, verification and inspection applications. In this paper, we would like to demonstrate the possibility of obtaining an accurate process model for lithography process simulations via model calibration. We will also demonstrate the accuracy of calibrated process simulations by applying the calibrated model in mask defect printability analysis. For simplicity, the process model and the algorithms used in model calibration will not be discussed in this article but in our future publications. In Section 2, we present the characterization and calibration of a 0.18 micrometer DUV lithography process using positive chemically amplified resist (APEX-E) as an example. We describe the test pattern selections, the calibration process, and the performance of the calibrated model in terms of predicting the CD measurements given test patterns. In Section 3, we briefly describe the technology of defect printability analysis based on process simulations. We will demonstrate that with the help of calibrated

  6. Thrombin-activatable fibrinolysis inhibitor activity in healthy and diseased dogs

    DEFF Research Database (Denmark)

    Jessen, Lisbeth Rem; Wiinberg, Bo; Kjelgaard-Hansen, Mads


    Background: In people, increased thrombin-activatable fibrinolysis inhibitor (TAFI) antigen has been associated with increased risk of thrombosis, and decreased TAFI may contribute to bleeding diathesis. TAFI activity in dogs has been described in experimental models, but not in dogs...... with spontaneous disease. Objective: The aim of this study was to compare TAFI activity in healthy dogs with TAFI activity in dogs with spontaneous disease. Methods: Plasma samples from 20 clinically healthy Beagles and from 35 dogs with various diseases were analyzed using a commercial chromogenic assay...... that measured TAFI activity relative to activity in standardized pooled human plasma. Results: Median TAFI activity for the 20 Beagles was 46.1% (range 32.2-70.8%) compared with 62.6% (29.1-250%) for the 35 diseased dogs, and 14/35 (40%) had TAFI activities >the upper limit for controls. The highest individual...

  7. Proteolytic signatures define unique thrombin-derived peptides present in human wound fluid in vivo

    DEFF Research Database (Denmark)

    Saravanan, Rathi; Adav, Sunil S; Choong, Yeu Khai


    The disease burden of failing skin repair and non-healing ulcers is extensive. There is an unmet need for new diagnostic approaches to better predict healing activity and wound infection. Uncontrolled and excessive protease activity, of endogenous or bacterial origin, has been described as a major...... contributor to wound healing impairments. Proteolytic peptide patterns could therefore correlate and "report" healing activity and infection. This work describes a proof of principle delineating a strategy by which peptides from a selected protein, human thrombin, are detected and attributed to proteolytic...... aureolysin, respectively. Corresponding peptide sequences were identified in wound fluids from acute and non-healing ulcers, and notably, one peptide, FYT21 (FYTHVFRLKKWIQKVIDQFGE), was only present in wound fluid from non-healing ulcers colonized by P. aeruginosa and S. aureus. Our result is a proof...

  8. Ultrasensitive electrochemiluminescence detection of thrombin based on aptamer and cystamine modified gold nanoparticle probe (United States)

    Duan, Ruixue; Zhou, Xiaoming


    Recently, our group showed that one can detect specific oligonucleotides at low femtomolar levels with the electrochemiluminescence (ECL) biobarcode approach based on tris-(2, 2'-bipyridyl) ruthenium (TBR)-labeled cysteamine. It would be a significant advance to use the cysteamine assisted ECL biobarcode assay to detect protein targets in addition to DNA targets. Taking advantage of sandwich binding of two affinity aptamers for increased specificity, TBR-cysteamine as biobarcode for signal amplification and magnetic beads based ECL technology for rapid detection, a promising assay for thrombin quantification is developed. The sandwich complex could be selectively captured by micromagnetic particles and then quantified by ECL signals. Current cysteamine-Gold nanoparticle (GNP) conjugates based ECL biobarcode assay is expected to become a powerful tool for protein analysis.

  9. Oral, direct thrombin and factor Xa inhibitors: the replacement for warfarin, leeches, and pig intestines? (United States)

    Straub, Alexander; Roehrig, Susanne; Hillisch, Alexander


    To prevent thromboses after surgery, patients have until now had to inject themselves daily with heparin. For stroke prophylaxis in atrial fibrillation, patients take vitamin K antagonists of the coumarin type, which have a narrow therapeutic window and whose dosage must be regularly monitored. In order to improve the standard of therapy in thromboembolic diseases such as deep-vein thrombosis, pulmonary embolism, and stroke in atrial fibrillation, intensive research has been carried out over the last decade in the search for new, orally active thrombin and factor Xa inhibitors. A number of these compounds are already on the market or are in advanced clinical development; they could revolutionize the anticoagulant market. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. A study of the conditions and accuracy of the thrombin time assay of plasma fibrinogen

    DEFF Research Database (Denmark)

    Jespersen, J; Sidelmann, Johannes Jakobsen


    The conditions, accuracy, precision and possible error of the thrombin time assay of plasma fibrinogen are determined. Comparison with an estimation of clottable protein by absorbance at 280 nm gave a correlation coefficient of 0.96 and the regression line y = 1.00 x + 0.56 (n = 34). Comparison...... with a radial immunodiffusion method yielded the correlation coefficient 0.97 and the regression line y = 1.18 x = 2.47 (n = 26). The presence of heparin in clinically applied concentrations produced a slight shortening of the clotting times. The resulting error in the estimated concentrations of fibrinogen...... was too small to affect the clinical usefulness of the determinations. The influence of fibrin(ogen) degradation products was significant only in excessive amounts in samples containing low levels of fibrinogen....

  11. Thrombin inhibition with melagatran does not attenuate renal ischaemia-reperfusion injury in rats

    DEFF Research Database (Denmark)

    Nitescu, Nicoletta; Grimberg, Elisabeth; Ricksten, Sven-Erik


    BACKGROUND: Renal ischaemia-reperfusion (IR) is associated with activation of the coagulation system and inflammation within the kidney. The aim of the present study was to examine the effects of selective thrombin inhibition with melagatran on kidney morphology and function in rats subjected...... of saline vehicle or melagatran [0.5 mumol/kg, subcutaneously (s.c.)] followed by a continuous infusion throughout (0.08 micromol/kg/h, s.c.). Forty-eight hours after IR, renal function was assessed in anaesthetized animals and kidney histology was analysed semi-quantitatively. RESULTS: Rats in group IR...... characterized by tubular necrosis and atrophy, tubular cast formation and interstitial inflammation. In addition, there was significant vascular congestion in the inner stripe of the outer medullary zone. Melagatran treatment had no significant effects on any of the abnormalities in kidney morphology...

  12. Thrombin induces Egr-1 expression in fibroblasts involving elevation of the intracellular Ca2+ concentration, phosphorylation of ERK and activation of ternary complex factor

    Directory of Open Access Journals (Sweden)

    Thiel Gerald


    Full Text Available Abstract Background The serine protease thrombin catalyzes fibrin clot formation by converting fibrinogen into fibrin. Additionally, thrombin stimulation leads to an activation of stimulus-responsive transcription factors in different cell types, indicating that the gene expression pattern is changed in thrombin-stimulated cells. The objective of this study was to analyze the signaling cascade leading to the expression of the zinc finger transcription factor Egr-1 in thrombin-stimulated lung fibroblasts. Results Stimulation of 39M1-81 fibroblasts with thrombin induced a robust and transient biosynthesis of Egr-1. Reporter gene analysis revealed that the newly synthesized Egr-1 was biologically active. The signaling cascade connecting thrombin stimulation with Egr-1 gene expression required elevated levels of cytosolic Ca2+, the activation of diacylgycerol-dependent protein kinase C isoenzymes, and the activation of extracellular signal-regulated protein kinase (ERK. Stimulation of the cells with thrombin triggered the phosphorylation of the transcription factor Elk-1. Expression of a dominant-negative mutant of Elk-1 completely prevented Egr-1 expression in stimulated 39M1-81 cells, indicating that Elk-1 or related ternary complex factors connect the intracellular signaling cascade elicited by activation of protease-activated receptors with transcription of the Egr-1 gene. Lentiviral-mediated expression of MAP kinase phosphatase-1, a dual-specific phosphatase that dephosphorylates and inactivates ERK in the nucleus, prevented Elk-1 phosphorylation and Egr-1 biosynthesis in thrombin stimulated 39M1-81 cells, confirming the importance of nuclear ERK and Elk-1 for the upregulation of Egr-1 expression in thrombin-stimulated lung fibroblasts. 39M1-81 cells additionally express M1 muscarinic acetylcholine receptors. A comparison between the signaling cascades induced by thrombin or carbachol showed no differences, except that signal transduction via M

  13. Building a framework to manage trust in automation (United States)

    Metcalfe, J. S.; Marathe, A. R.; Haynes, B.; Paul, V. J.; Gremillion, G. M.; Drnec, K.; Atwater, C.; Estepp, J. R.; Lukos, J. R.; Carter, E. C.; Nothwang, W. D.


    All automations must, at some point in their lifecycle, interface with one or more humans. Whether operators, end-users, or bystanders, human responses can determine the perceived utility and acceptance of an automation. It has been long believed that human trust is a primary determinant of human-automation interactions and further presumed that calibrating trust can lead to appropriate choices regarding automation use. However, attempts to improve joint system performance by calibrating trust have not yet provided a generalizable solution. To address this, we identified several factors limiting the direct integration of trust, or metrics thereof, into an active mitigation strategy. The present paper outlines our approach to addressing this important issue, its conceptual underpinnings, and practical challenges encountered in execution. Among the most critical outcomes has been a shift in focus from trust to basic interaction behaviors and their antecedent decisions. This change in focus inspired the development of a testbed and paradigm that was deployed in two experiments of human interactions with driving automation that were executed in an immersive, full-motion simulation environment. Moreover, by integrating a behavior and physiology-based predictor within a novel consequence-based control system, we demonstrated that it is possible to anticipate particular interaction behaviors and influence humans towards more optimal choices about automation use in real time. Importantly, this research provides a fertile foundation for the development and integration of advanced, wearable technologies for sensing and inferring critical state variables for better integration of human elements into otherwise fully autonomous systems.

  14. Automated DNA Sequencing System

    Energy Technology Data Exchange (ETDEWEB)

    Armstrong, G.A.; Ekkebus, C.P.; Hauser, L.J.; Kress, R.L.; Mural, R.J.


    Oak Ridge National Laboratory (ORNL) is developing a core DNA sequencing facility to support biological research endeavors at ORNL and to conduct basic sequencing automation research. This facility is novel because its development is based on existing standard biology laboratory equipment; thus, the development process is of interest to the many small laboratories trying to use automation to control costs and increase throughput. Before automation, biology Laboratory personnel purified DNA, completed cycle sequencing, and prepared 96-well sample plates with commercially available hardware designed specifically for each step in the process. Following purification and thermal cycling, an automated sequencing machine was used for the sequencing. A technician handled all movement of the 96-well sample plates between machines. To automate the process, ORNL is adding a CRS Robotics A- 465 arm, ABI 377 sequencing machine, automated centrifuge, automated refrigerator, and possibly an automated SpeedVac. The entire system will be integrated with one central controller that will direct each machine and the robot. The goal of this system is to completely automate the sequencing procedure from bacterial cell samples through ready-to-be-sequenced DNA and ultimately to completed sequence. The system will be flexible and will accommodate different chemistries than existing automated sequencing lines. The system will be expanded in the future to include colony picking and/or actual sequencing. This discrete event, DNA sequencing system will demonstrate that smaller sequencing labs can achieve cost-effective the laboratory grow.

  15. UV irradiance radiometers calibration procedure


    Doctorovich I. V.; Butenko V. K.; Hodovaniouk V. N.; Fodchuk I. M.; Yuriev V. G.


    The paper deals with the problems arising at calibration of narrow-band spectral-sensitive radiometers. The procedure of irradiance unit transfer to UV radiometers — UV radiometers calibration procedure — is presented.

  16. The SLAC Vertical Comparator for the Calibration of Digital Levels

    Energy Technology Data Exchange (ETDEWEB)

    Woschitz, Helmut; /Graz U.; Gassner, Georg; Ruland, Robert; /SLAC


    Digital levels replaced spirit levels in most fields of precise height measurements because of the automation of the height readings. Three manufacturers offer digital levels with a single reading resolution of 10 {micro}m, and for all of them systematic effects are known. In Europe several facilities for system calibration of digital levels using vertical comparators were established within the last decade. However, there still was no system calibration facility in North America. In order to guarantee the accuracy required for the alignment of experiments at the Stanford Linear Accelerator Center (SLAC) a calibration facility for the system calibration of digital levels was built. In this paper the setup of the SLAC vertical comparator is described in detail and its standard uncertainty is derived. In order to perform traditional rod calibration of conventional line-scaled rods, a CCD camera was integrated into the SLAC comparator. The CCD camera setup is also briefly described. To demonstrate the capabilities of the comparator, results of system and rod calibration are shown.

  17. Calibration of robotic drilling systems with a moving rail

    Directory of Open Access Journals (Sweden)

    Tian Wei


    Full Text Available Industrial robots are widely used in aircraft assembly systems such as robotic drilling systems. It is necessary to expand a robot’s working range with a moving rail. A method for improving the position accuracy of an automated assembly system with an industrial robot mounted on a moving rail is proposed. A multi-station method is used to control the robot in this study. The robot only works at stations which are certain positions defined on the moving rail. The calibration of the robot system is composed by the calibration of the robot and the calibration of the stations. The calibration of the robot is based on error similarity and inverse distance weighted interpolation. The calibration of the stations is based on a magnetic strip and a magnetic sensor. Validation tests were performed in this study, which showed that the accuracy of the robot system gained significant improvement using the proposed method. The absolute position errors were reduced by about 85% to less than 0.3 mm compared with the maximum nearly 2 mm before calibration.

  18. Automatic 3D ultrasound calibration for image guided therapy using intramodality image registration. (United States)

    Schlosser, Jeffrey; Kirmizibayrak, Can; Shamdasani, Vijay; Metz, Steve; Hristov, Dimitre


    Many real time ultrasound (US) guided therapies can benefit from management of motion-induced anatomical changes with respect to a previously acquired computerized anatomy model. Spatial calibration is a prerequisite to transforming US image information to the reference frame of the anatomy model. We present a new method for calibrating 3D US volumes using intramodality image registration, derived from the 'hand-eye' calibration technique. The method is fully automated by implementing data rejection based on sensor displacements, automatic registration over overlapping image regions, and a self-consistency error metric evaluated continuously during calibration. We also present a novel method for validating US calibrations based on measurement of physical phantom displacements within US images. Both calibration and validation can be performed on arbitrary phantoms. Results indicate that normalized mutual information and localized cross correlation produce the most accurate 3D US registrations for calibration. Volumetric image alignment is more accurate and reproducible than point selection for validating the calibrations, yielding <1.5 mm root mean square error, a significant improvement relative to previously reported hand-eye US calibration results. Comparison of two different phantoms for calibration and for validation revealed significant differences for validation (p = 0.003) but not for calibration (p = 0.795).

  19. Calibrating animal-borne proximity loggers. (United States)

    Rutz, Christian; Morrissey, Michael B; Burns, Zackory T; Burt, John; Otis, Brian; St Clair, James J H; James, Richard


    Growing interest in the structure and dynamics of animal social networks has stimulated efforts to develop automated tracking technologies that can reliably record encounters in free-ranging subjects. A particularly promising approach is the use of animal-attached 'proximity loggers', which collect data on the incidence, duration and proximity of spatial associations through inter-logger radio communication. While proximity logging is based on a straightforward physical principle - the attenuation of propagating radio waves with distance - calibrating systems for field deployment is challenging, since most study species roam across complex, heterogeneous environments.In this study, we calibrated a recently developed digital proximity-logging system ('Encounternet') for deployment on a wild population of New Caledonian crows Corvus moneduloides. Our principal objective was to establish a quantitative model that enables robust post hoc estimation of logger-to-logger (and, hence, crow-to-crow) distances from logger-recorded signal-strength values. To achieve an accurate description of the radio communication between crow-borne loggers, we conducted a calibration exercise that combines theoretical analyses, field experiments, statistical modelling, behavioural observations, and computer simulations.We show that, using signal-strength information only, it is possible to assign crow encounters reliably to predefined distance classes, enabling powerful analyses of social dynamics. For example, raw data sets from field-deployed loggers can be filtered at the analysis stage to include predominantly encounters where crows would have come to within a few metres of each other, and could therefore have socially learned new behaviours through direct observation. One of the main challenges for improving data classification further is the fact that crows - like most other study species - associate across a wide variety of habitats and behavioural contexts, with different signal

  20. Lidar calibration experiments

    DEFF Research Database (Denmark)

    Ejsing Jørgensen, Hans; Mikkelsen, T.; Streicher, J.


    A series of atmospheric aerosol diffusion experiments combined with lidar detection was conducted to evaluate and calibrate an existing retrieval algorithm for aerosol backscatter lidar systems. The calibration experiments made use of two (almost) identical mini-lidar systems for aerosol cloud...... detection to test the reproducibility and uncertainty of lidars. Lidar data were obtained from both single-ended and double-ended Lidar configurations. A backstop was introduced in one of the experiments and a new method was developed where information obtained from the backstop can be used in the inversion...... algorithm. Independent in-situ aerosol plume concentrations were obtained from a simultaneous tracer gas experiment with SF6, and comparisons with the two lidars were made. The study shows that the reproducibility of the lidars is within 15%, including measurements from both sides of a plume...

  1. Calibrated vapor generator source (United States)

    Davies, J.P.; Larson, R.A.; Goodrich, L.D.; Hall, H.J.; Stoddard, B.D.; Davis, S.G.; Kaser, T.G.; Conrad, F.J.


    A portable vapor generator is disclosed that can provide a controlled source of chemical vapors, such as, narcotic or explosive vapors. This source can be used to test and calibrate various types of vapor detection systems by providing a known amount of vapors to the system. The vapor generator is calibrated using a reference ion mobility spectrometer. A method of providing this vapor is described, as follows: explosive or narcotic is deposited on quartz wool, placed in a chamber that can be heated or cooled (depending on the vapor pressure of the material) to control the concentration of vapors in the reservoir. A controlled flow of air is pulsed over the quartz wool releasing a preset quantity of vapors at the outlet. 10 figs.

  2. Accurate borehole probe calibration

    Energy Technology Data Exchange (ETDEWEB)

    Tchen, T.; Eisler, P. (CSIRO, Mount Waverley, Vic. (Australia). Division of Geomechanics)

    The In Situ Minerals Analysis Group in the CSIRO Division of Geomechanics has developed quantitative borehole logging techniques applicable to iron-ore and coal deposits. They are used currently to determine the formation density, either the iron-ore grades or the raw coal-ash contents, as appropriate, and the borehole diameter. The in-situ analyses depend on probe-calibration equations which were formulated by linear regression analysis that related the probe's spectral outputs with the required geological variable. Calibration equations consisting of a linear combination of first-order terms gave excellent assaying accuracy. The group achieved further improvements in assaying accuracy by developing a more generalised calibration model based on second-order terms and cross-product terms of the probe's spectral parameters. The logging data used for the statistical analysis were recorded in mine development boreholes at three Pilbara iron-ore mines and at a Queensland coal mine. Application of the generalised model, in place of the first-order model, resulted in a reduction of the root mean square (RMS) deviation between assays obtained in the laboratory and by logging, of about 15% relative for iron-ore grades and of about 8% relative for raw coal-ash content. The study also shows that the accuracy obtained using the conventional, non-spectrometric calibration model is inferior to that obtained by using either of the two spectrometric models, where the comparisons made are based on the same set of logging data. 8 refs., 6 figs., 3 tabs.

  3. Calibrated Properties Model

    Energy Technology Data Exchange (ETDEWEB)

    H. H. Liu


    This report has documented the methodologies and the data used for developing rock property sets for three infiltration maps. Model calibration is necessary to obtain parameter values appropriate for the scale of the process being modeled. Although some hydrogeologic property data (prior information) are available, these data cannot be directly used to predict flow and transport processes because they were measured on scales smaller than those characterizing property distributions in models used for the prediction. Since model calibrations were done directly on the scales of interest, the upscaling issue was automatically considered. On the other hand, joint use of data and the prior information in inversions can further increase the reliability of the developed parameters compared with those for the prior information. Rock parameter sets were developed for both the mountain and drift scales because of the scale-dependent behavior of fracture permeability. Note that these parameter sets, except those for faults, were determined using the 1-D simulations. Therefore, they cannot be directly used for modeling lateral flow because of perched water in the unsaturated zone (UZ) of Yucca Mountain. Further calibration may be needed for two- and three-dimensional modeling studies. As discussed above in Section 6.4, uncertainties for these calibrated properties are difficult to accurately determine, because of the inaccuracy of simplified methods for this complex problem or the extremely large computational expense of more rigorous methods. One estimate of uncertainty that may be useful to investigators using these properties is the uncertainty used for the prior information. In most cases, the inversions did not change the properties very much with respect to the prior information. The Output DTNs (including the input and output files for all runs) from this study are given in Section 9.4.

  4. A mathematical model for camera calibration based on straight lines

    Directory of Open Access Journals (Sweden)

    Antonio M. G. Tommaselli


    Full Text Available In other to facilitate the automation of camera calibration process, a mathematical model using straight lines was developed, which is based on the equivalent planes mathematical model. Parameter estimation of the developed model is achieved by the Least Squares Method with Conditions and Observations. The same method of adjustment was used to implement camera calibration with bundles, which is based on points. Experiments using simulated and real data have shown that the developed model based on straight lines gives results comparable to the conventional method with points. Details concerning the mathematical development of the model and experiments with simulated and real data will be presented and the results with both methods of camera calibration, with straight lines and with points, will be compared.

  5. Fuzzy Interpolation and Other Interpolation Methods Used in Robot Calibrations

    Directory of Open Access Journals (Sweden)

    Ying Bai


    Full Text Available A novel interpolation algorithm, fuzzy interpolation, is presented and compared with other popular interpolation methods widely implemented in industrial robots calibrations and manufacturing applications. Different interpolation algorithms have been developed, reported, and implemented in many industrial robot calibrations and manufacturing processes in recent years. Most of them are based on looking for the optimal interpolation trajectories based on some known values on given points around a workspace. However, it is rare to build an optimal interpolation results based on some random noises, and this is one of the most popular topics in industrial testing and measurement applications. The fuzzy interpolation algorithm (FIA reported in this paper provides a convenient and simple way to solve this problem and offers more accurate interpolation results based on given position or orientation errors that are randomly distributed in real time. This method can be implemented in many industrial applications, such as manipulators measurements and calibrations, industrial automations, and semiconductor manufacturing processes.

  6. Automated stopcock actuator


    Vandehey, N. T.; O\\'Neil, J. P.


    Introduction We have developed a low-cost stopcock valve actuator for radiochemistry automation built using a stepper motor and an Arduino, an open-source single-board microcontroller. The con-troller hardware can be programmed to run by serial communication or via two 5–24 V digital lines for simple integration into any automation control system. This valve actuator allows for automated use of a single, disposable stopcock, providing a number of advantages over stopcock manifold systems ...

  7. Laboratory Automation and Middleware. (United States)

    Riben, Michael


    The practice of surgical pathology is under constant pressure to deliver the highest quality of service, reduce errors, increase throughput, and decrease turnaround time while at the same time dealing with an aging workforce, increasing financial constraints, and economic uncertainty. Although not able to implement total laboratory automation, great progress continues to be made in workstation automation in all areas of the pathology laboratory. This report highlights the benefits and challenges of pathology automation, reviews middleware and its use to facilitate automation, and reviews the progress so far in the anatomic pathology laboratory. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Complacency and Automation Bias in the Use of Imperfect Automation. (United States)

    Wickens, Christopher D; Clegg, Benjamin A; Vieane, Alex Z; Sebok, Angelia L


    We examine the effects of two different kinds of decision-aiding automation errors on human-automation interaction (HAI), occurring at the first failure following repeated exposure to correctly functioning automation. The two errors are incorrect advice, triggering the automation bias, and missing advice, reflecting complacency. Contrasts between analogous automation errors in alerting systems, rather than decision aiding, have revealed that alerting false alarms are more problematic to HAI than alerting misses are. Prior research in decision aiding, although contrasting the two aiding errors (incorrect vs. missing), has confounded error expectancy. Participants performed an environmental process control simulation with and without decision aiding. For those with the aid, automation dependence was created through several trials of perfect aiding performance, and an unexpected automation error was then imposed in which automation was either gone (one group) or wrong (a second group). A control group received no automation support. The correct aid supported faster and more accurate diagnosis and lower workload. The aid failure degraded all three variables, but "automation wrong" had a much greater effect on accuracy, reflecting the automation bias, than did "automation gone," reflecting the impact of complacency. Some complacency was manifested for automation gone, by a longer latency and more modest reduction in accuracy. Automation wrong, creating the automation bias, appears to be a more problematic form of automation error than automation gone, reflecting complacency. Decision-aiding automation should indicate its lower degree of confidence in uncertain environments to avoid the automation bias. © 2015, Human Factors and Ergonomics Society.

  9. In vitro anti-inflammatory and anti-coagulant effects of antibiotics towards Platelet Activating Factor and thrombin

    Directory of Open Access Journals (Sweden)

    Demopoulos Constantinos A


    Full Text Available Abstract Background Sepsis is characterized as a systemic inflammatory response that results from the inability of the immune system to limit bacterial spread during an ongoing infection. In this condition the significant mediator of inflammation Platelet Activating Factor (PAF and the coagulant factor thrombin are implicated. In animal models, treatment with PAF-antagonists or co-administration of antibiotics with recombinant-PAF-Acetylhydrolase (rPAF-AH have exhibited promising results. In order to examine the putative anti-inflammatory and/or antithrombotic interactions between antibiotic treatment used in sepsis with PAF and/or thrombin, we studied the in vitro effects of these compounds towards PAF or/and thrombin related activities and towards PAF basic metabolic enzymes. Methods We assessed the inhibitory effect of these drugs against PAF or thrombin induced aggregation on washed rabbit platelets (WRPs or rabbit Platelet Reach Plasma (rPRP by evaluating their IC50 values. We also studied their effect on Cholinephosphotransferase of PAF (PAF-CPT/Lyso-PAF-Acetyltransferase (Lyso-PAF-AT of rabbit leukocytes (RLs, as well as on rabbit plasma-PAF-AH, the key enzymes of both de novo/remodelling PAF biosynthesis and PAF degradation, respectively. Results Several antibiotics inhibited PAF-induced platelet aggregation of both WRPs and rPRP in a concentration-depended manner, with clarithromycin, azithromycin and amikacin exhibiting the higher inhibitory effect, while when combined they synergistically inhibited PAF. Higher concentrations of all antibiotics tested were needed in order to inhibit PAF induced aggregation of rPRP, but also to inhibit thrombin induced aggregation of WRPs. Concentrations of these drugs similar to their IC50 values against PAF activity in WRPs, inhibited also in vitro PAF-CPT and Lyso-PAF-AT activities of rabbit leukocytes, while only clarithromycin and azithromycin increased rabbit plasma-PAF-AH activity. Conclusions

  10. In vitro anti-inflammatory and anti-coagulant effects of antibiotics towards Platelet Activating Factor and thrombin. (United States)

    Tsoupras, Alexandros B; Chini, Maria; Tsogas, Nickolaos; Lioni, Athina; Tsekes, George; Demopoulos, Constantinos A; Lazanas, Marios C


    Sepsis is characterized as a systemic inflammatory response that results from the inability of the immune system to limit bacterial spread during an ongoing infection. In this condition the significant mediator of inflammation Platelet Activating Factor (PAF) and the coagulant factor thrombin are implicated. In animal models, treatment with PAF-antagonists or co-administration of antibiotics with recombinant-PAF-Acetylhydrolase (rPAF-AH) have exhibited promising results. In order to examine the putative anti-inflammatory and/or antithrombotic interactions between antibiotic treatment used in sepsis with PAF and/or thrombin, we studied the in vitro effects of these compounds towards PAF or/and thrombin related activities and towards PAF basic metabolic enzymes. We assessed the inhibitory effect of these drugs against PAF or thrombin induced aggregation on washed rabbit platelets (WRPs) or rabbit Platelet Reach Plasma (rPRP) by evaluating their IC50 values. We also studied their effect on Cholinephosphotransferase of PAF (PAF-CPT)/Lyso-PAF-Acetyltransferase (Lyso-PAF-AT) of rabbit leukocytes (RLs), as well as on rabbit plasma-PAF-AH, the key enzymes of both de novo/remodelling PAF biosynthesis and PAF degradation, respectively. Several antibiotics inhibited PAF-induced platelet aggregation of both WRPs and rPRP in a concentration-depended manner, with clarithromycin, azithromycin and amikacin exhibiting the higher inhibitory effect, while when combined they synergistically inhibited PAF. Higher concentrations of all antibiotics tested were needed in order to inhibit PAF induced aggregation of rPRP, but also to inhibit thrombin induced aggregation of WRPs. Concentrations of these drugs similar to their IC50 values against PAF activity in WRPs, inhibited also in vitro PAF-CPT and Lyso-PAF-AT activities of rabbit leukocytes, while only clarithromycin and azithromycin increased rabbit plasma-PAF-AH activity. These newly found properties of antibiotics used in sepsis

  11. In vitro anti-inflammatory and anti-coagulant effects of antibiotics towards Platelet Activating Factor and thrombin (United States)


    Background Sepsis is characterized as a systemic inflammatory response that results from the inability of the immune system to limit bacterial spread during an ongoing infection. In this condition the significant mediator of inflammation Platelet Activating Factor (PAF) and the coagulant factor thrombin are implicated. In animal models, treatment with PAF-antagonists or co-administration of antibiotics with recombinant-PAF-Acetylhydrolase (rPAF-AH) have exhibited promising results. In order to examine the putative anti-inflammatory and/or antithrombotic interactions between antibiotic treatment used in sepsis with PAF and/or thrombin, we studied the in vitro effects of these compounds towards PAF or/and thrombin related activities and towards PAF basic metabolic enzymes. Methods We assessed the inhibitory effect of these drugs against PAF or thrombin induced aggregation on washed rabbit platelets (WRPs) or rabbit Platelet Reach Plasma (rPRP) by evaluating their IC50 values. We also studied their effect on Cholinephosphotransferase of PAF (PAF-CPT)/Lyso-PAF-Acetyltransferase (Lyso-PAF-AT) of rabbit leukocytes (RLs), as well as on rabbit plasma-PAF-AH, the key enzymes of both de novo/remodelling PAF biosynthesis and PAF degradation, respectively. Results Several antibiotics inhibited PAF-induced platelet aggregation of both WRPs and rPRP in a concentration-depended manner, with clarithromycin, azithromycin and amikacin exhibiting the higher inhibitory effect, while when combined they synergistically inhibited PAF. Higher concentrations of all antibiotics tested were needed in order to inhibit PAF induced aggregation of rPRP, but also to inhibit thrombin induced aggregation of WRPs. Concentrations of these drugs similar to their IC50 values against PAF activity in WRPs, inhibited also in vitro PAF-CPT and Lyso-PAF-AT activities of rabbit leukocytes, while only clarithromycin and azithromycin increased rabbit plasma-PAF-AH activity. Conclusions These newly found

  12. Potentiation of thrombin generation in hemophilia A plasma by coagulation factor VIII and characterization of antibody-specific inhibition.

    Directory of Open Access Journals (Sweden)

    Bhavya S Doshi

    Full Text Available Development of inhibitory antibodies to coagulation factor VIII (fVIII is the primary obstacle to the treatment of hemophilia A in the developed world. This adverse reaction occurs in 20-30% of persons with severe hemophilia A treated with fVIII-replacement products and is characterized by the development of a humoral and neutralizing immune response to fVIII. Patients with inhibitory anti-fVIII antibodies are treated with bypassing agents including recombinant factor VIIa (rfVIIa. However, some patients display poor hemostatic response to bypass therapy and improved treatment options are needed. Recently, we demonstrated that fVIII inhibitors display widely variable kinetics of inhibition that correlate with their respective target epitopes. Thus, it was hypothesized that for antibodies that display slow rates of inhibition, supplementation of rfVIIa with fVIII would result in improved thrombin generation and be predictive of clinical responses to this novel treatment regimen. In order to test this hypothesis, 10 murine monoclonal antibodies (MAbs with non-overlapping epitopes spanning fVIII, differential inhibition titers, and inhibition kinetics were studied using a thrombin generation assay. Of the 3 MAbs with high inhibitory titers, only the one with fast and complete (classically defined as "type I" kinetics displayed significant inhibition of thrombin generation with no improvement upon supplementation of rfVIIa with fVIII. The other two MAbs that displayed incomplete (classically defined as "type II" inhibition did not suppress the potentiation of thrombin generation by fVIII. All antibodies that did not completely inhibit fVIII activity demonstrated potentiation of thrombin generation by the addition of fVIII as compared to rfVIIa alone. In conclusion, fVIII alone or in combination with rfVIIa corrects the thrombin generation defect produced by the majority of anti-fVIII MAbs better than single agent rfVIIa. Therefore, combined f

  13. Will Automated Vehicles Negatively Impact Traffic Flow?

    Directory of Open Access Journals (Sweden)

    S. C. Calvert


    Full Text Available With low-level vehicle automation already available, there is a necessity to estimate its effects on traffic flow, especially if these could be negative. A long gradual transition will occur from manual driving to automated driving, in which many yet unknown traffic flow dynamics will be present. These effects have the potential to increasingly aid or cripple current road networks. In this contribution, we investigate these effects using an empirically calibrated and validated simulation experiment, backed up with findings from literature. We found that low-level automated vehicles in mixed traffic will initially have a small negative effect on traffic flow and road capacities. The experiment further showed that any improvement in traffic flow will only be seen at penetration rates above 70%. Also, the capacity drop appeared to be slightly higher with the presence of low-level automated vehicles. The experiment further investigated the effect of bottleneck severity and truck shares on traffic flow. Improvements to current traffic models are recommended and should include a greater detail and understanding of driver-vehicle interaction, both in conventional and in mixed traffic flow. Further research into behavioural shifts in driving is also recommended due to limited data and knowledge of these dynamics.

  14. Thrombin decreases expression of the glutamate transporter GLAST and inhibits glutamate uptake in primary cortical astrocytes via the Rho kinase pathway. (United States)

    Piao, Chunshu; Ralay Ranaivo, Hantamalala; Rusie, Allison; Wadhwani, Nitin; Koh, Sookyong; Wainwright, Mark S


    Astrocyte glutamate transporters GLAST and GLT1 play a key role in regulating neuronal excitation and their levels are altered in patients with epilepsy, and after traumatic brain injury. The mechanisms which regulate their expression are not well understood. We tested the hypothesis that exposure of astrocytes to high levels of thrombin, as may occur after a compromise of the blood-brain barrier, would reduce astrocyte glutamate transporter levels. In isolated rat cortical astrocytes we examined the effects of thrombin on the expression and function of glutamate transporters, and the signaling pathways involved in these responses by using Western blotting and selective inhibitors. Thrombin induced a selective decrease in the expression of GLAST but not GLT1, with a corresponding decrease in the capacity of astrocytes to take up glutamate. Activation of the thrombin receptor PAR-1 with an activating peptide induced a similar decrease in the expression of GLAST and compromise of glutamate uptake. The downregulation of GLAST induced by thrombin was mediated by the mitogen activated protein kinases p38 MAPK, ERK and JNK, but inhibition of these kinases did not prevent the decrease in glutamate uptake induced by thrombin. In contrast, inhibition of the Rho kinase pathway using the specific inhibitor, Y27632, suppressed both the decrease in the expression of GLAST and the decrease in glutamate uptake induced by thrombin. In hippocampal astrocyte cultures, thrombin caused a decrease in both GLAST and GLT1. In tissue resected from brains of children with intractable epilepsy, we found a decrease in the integrity of the blood-brain barrier along with a reduction in immunoreactivity for both transporters which was associated with an increase in cleaved thrombin and reactive astrogliosis. The in vitro results suggest a specific mechanism by which thrombin may lead to a compromise of astrocyte function and enhanced synaptic excitability after the blood-brain barrier is

  15. Thrombin mediates migration of rat brain astrocytes via PLC, Ca²⁺, CaMKII, PKCα, and AP-1-dependent matrix metalloproteinase-9 expression. (United States)

    Lin, Chih-Chung; Lee, I-Ta; Wu, Wen-Bin; Liu, Chiung-Ju; Hsieh, Hsi-Lung; Hsiao, Li-Der; Yang, Chien-Chung; Yang, Chuen-Mao


    Matrix metalloproteinase-9 (MMP-9) plays a crucial role in pathological processes of brain inflammation, injury, and neurodegeneration. Thrombin has been known as a regulator of MMP-9 expression and cells migration. However, the mechanisms underlying thrombin-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells) remain unclear. Here, we demonstrated that thrombin induced the expression of pro-form MMP-9 and migration of RBA-1 cells, which were inhibited by pretreatment with the inhibitor of Gq-coupled receptor (GPAnt2A), Gi/o-coupled receptor (GPAnt2), PC-PLC (D609), PI-PLC (U73122), Ca(2+)-ATPase (thapsigargin, TG), calmodulin (CaMI), CaMKII (KN62), PKC (Gö6976 or GF109203X), MEK1/2 (PD98059), p38 MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) or the intracellular calcium chelator (BAPTA/AM) and transfection with siRNA of PKCα, Erk2, JNK1, p38 MAPK, c-Jun, or c-Fos. In addition, thrombin-induced elevation of intracellular Ca(2+) concentration was attenuated by PPACK (a thrombin inhibitor). Thrombin further induced CaMKII phosphorylation and PKCα translocation, which were inhibited by U73122, D609, KN62, TG, or BAPTA/AM. Thrombin also induced PKCα-dependent p42/p44 MAPK and JNK1/2, but not p38 MAPK activation. Finally, we showed that thrombin enhanced c-Fos expression and c-Jun phosphorylation. c-Fos mRNA levels induced by thrombin were reduced by PD98059, SP600125, and Gö6976, but not SB202190. Thrombin stimulated in vivo binding of c-Fos to the MMP-9 promoter, which was reduced by pretreatment with SP600125 or PD98059, but not SB202190. These results concluded that thrombin activated a PLC/Ca(2+)/CaMKII/PKCα/p42/p44 MAPK and JNK1/2 pathway, which in turn triggered AP-1 activation and ultimately induced MMP-9 expression in RBA-1 cells.

  16. p300 and C/EBPβ-regulated IKKβ expression are involved in thrombin-induced IL-8/CXCL8 expression in human lung epithelial cells. (United States)

    Huang, Zheng-Wei; Lien, Gi-Shih; Lin, Chien-Huang; Jiang, Chun-Ping; Chen, Bing-Chang


    Asthma and chronic obstructive pulmonary disease (COPD) are common chronic lung inflammatory diseases. Thrombin and interleukin (IL)-8/C-X-C chemokine ligand 8 (CXCL8) play critical roles in lung inflammation. Our previous study showed that c-Src-dependent IκB kinase (IKK)/IκBα/nuclear factor (NF)-κB and mitogen-activated protein kinase kinase kinase 1 (MEKK1)/extracellular signal-regulated kinase (ERK)/ribosomal S6 protein kinase (RSK)-dependent CAAT/enhancer-binding protein β (C/EBPβ) activation are involved in thrombin-induced IL-8/CXCL8 expression in human lung epithelial cells. In this study, we aimed to investigate the roles of p300 and C/EBPβ-reliant IKKβ expression in thrombin-induced IL-8/CXCL8 expression. Thrombin-induced increases in IL-8/CXCL8-luciferase activity and IL-8/CXCL8 release were inhibited by p300 small interfering (siRNA). Thrombin-caused histone H3 acetylation was attenuated by p300 siRNA. Stimulation of cells with thrombin for 12h resulted in increases in IKKβ expression and phosphorylation in human lung epithelial cells. However, thrombin did not affect p65 expression. Moreover, 12h of thrombin stimulation produced increases in IKKβ expression and phosphorylation, and IκBα phosphorylation, which were inhibited by C/EBPβ siRNA. Finally, treatment of cells with thrombin caused increases in p300 and C/EBPβ complex formation, p65 and C/EBPβ complex formation, and recruitment of p300, p65, and C/EBPβ to the IL-8/CXCL8 promoter. These results imply that p300-dependent histone H3 acetylation and C/EBPβ-regulated IKKβ expression contribute to thrombin-induced IL-8/CXCL8 expression in human lung epithelial cells. Results of this study will help clarify C/EBPβ signaling pathways involved in thrombin-induced IL-8/CXCL8 expression in human lung epithelial cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Impact of data quality and quantity and the calibration procedure on crop growth model calibration (United States)

    Seidel, Sabine J.; Werisch, Stefan


    , biomass partitioning, LAI, plant height, rooting depth and duration of growing period, as well as an (3) automated calibration using the AMALGAM optimization algorithm and Pareto front analysis based on the data listed in (2). Three different calibration strategies have been applied for the estimation of the parameters of the soil hydraulic property functions: (1) using pedotransfer functions based on soil texture data derived from soil sampling (2) using a laboratory evaporation method for the determination of pF-curves and unsaturated hydraulic conductivity (HYPROP), and (3) inverse estimation by multiobjective optimization and Pareto front analysis using the AMALGAM algorithm based on time series of soil moisture in three soil depths. The results show that simulations of yield and soil water dynamics can simultaneously be improved if the data quantity used for calibration increases (from strategy 1 to 3). The study quantifies the impacts of different model calibration procedures and data input on the modeling results. Even though parameter estimation using an multiobjective optimization algorithm is computationally demanding, it enhances the accuracy of model predictions and thus the overall reliability of the modeling results. To estimate climate change impacts based on crop growth modeling, we suggest a proper model calibration based on the simultaneous estimation of soil hydraulic parameters, crop phenology, growth and yield-related parameters using comprehensive experimental data.

  18. Dual-colored graphene quantum dots-labeled nanoprobes/graphene oxide: functional carbon materials for respective and simultaneous detection of DNA