A sensitive sandwich-type electrochemical aptasensor for thrombin detection based on platinum nanoparticles decorated carbon nanocages as signal labels.

PubMed

Gao, Fenglei; Du, Lili; Zhang, Yu; Zhou, Fuyi; Tang, Daoquan

2016-12-15

In this work, a novel and sensitive sandwich-type electrochemical aptasensor has been developed for thrombin detection based on platinum nanoparticles (Pt NPs) decorated carbon nanocages (CNCs) as signal tags. The morphological and compositional of the Pt NPs/CNCs were examined using transmission electron microscopy, X-ray diffraction, and Raman spectroscopy. The results showed that the Pt NPs with about 3-5nm in diameter were well dispersed on the surface of CNCs. The thiolated aptamer was firstly immobilized on the gold electrode to capture the thrombin molecules, and then aptamer functionalized Pt NPs/CNCs nanocomposites were used to fabricate a sandwich sensing platform. Then, the high-content Pt NPs on carbon nanocages acting as hydrogen peroxide-mimicking enzyme catalyzed the reduction of H2O2, resulting in significant electrochemical signal amplification. Differential pulse voltammetry is employed to detect thrombin with different concentrations. Under optimized conditions, the approach provided a good linear response range from 0.05 pM to 20nM with a low detection limit of 10fM. This Pt NPs/CNCs-based aptasensor shows good precision, acceptable stability and reproducibility, which provided a promising strategy for electrochemical aptamer-based detection of other biomolecules. Copyright © 2016 Elsevier B.V. All rights reserved.

A Novel Photoelectrochemical Biosensor for Tyrosinase and Thrombin Detection

PubMed Central

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

2016-01-01

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

Fluorescence detection of thrombin using autocatalytic strand displacement cycle reaction and a dual-aptamer DNA sandwich assay.

PubMed

Niu, Shuyan; Qu, Lijing; Zhang, Qing; Lin, Jiehua

2012-02-15

A sensitive and specific sandwich assay for the detection of thrombin is described. Two affiliative aptamers were used to increase the assay specificity through sandwich recognition. Recognition DNA loaded on gold nanoparticles (AuNPs) partially hybridized with the initiator DNA, which was displaced by surviving DNA. After the initiator DNA was released into the solution, one hairpin structure was opened, which in turn opened another hairpin structure. The initiator DNA was displaced and released into the solution again by another hairpin structure because of the hybridized reaction. Then the released initiator DNA initiated another autocatalytic strand displacement reaction. A sophisticated network of three such duplex formation cycles was designed to amplify the fluorescence signal. Other proteins, such as bovine serum albumin and lysozyme, did not interfere with the detection of thrombin. This approach enables rapid and specific thrombin detection with reduced costs and minimized material consumption compared with traditional assay processes. The detection limit of thrombin was as low as 4.3 × 10⁻¹³ M based on the AuNP amplification and the autocatalytic strand displacement cycle reaction. This method could be used in biological samples with excellent selectivity. Copyright © 2011 Elsevier Inc. All rights reserved.

Rapid and simple G-quadruplex DNA aptasensor with guanine chemiluminescence detection.

PubMed

Cho, Sandy; Park, Lucienne; Chong, Richard; Kim, Young Teck; Lee, Ji Hoon

2014-02-15

Cost-effective and sensitive aptasensor with guanine chemiluminescence detection capable of simply quantifying thrombin in human serum was developed using thrombin aptamer (TBA), one of the G-quadruplex DNA aptamers, without expensive nanoparticles and complicated procedures. Guanines of G-quadruplex TBA-conjugated carboxyfluorescein (6-FAM) bound with thrombin do not react with 3,4,5-trimethoxylphenylglyoxal (TMPG) in the presence of tetra-n-propylammonium hydroxide (TPA), whereas guanines of free TBA- and TBA-conjugated 6-FAM immobilized on the surface of graphene oxide rapidly react with TMPG to emit light. Thus, guanine chemiluminescence in 5% human serum with thrombin was lower than that without thrombin when TBA-conjugated 6-FAM was added in two samples and incubated for 20 min. In other words, the brightness of guanine chemiluminescence was quenched due to the formation of G-quadruplex TBA-conjugated 6-FAM bound with thrombin in a sample. High-energy intermediate, capable of emitting dim light by itself, formed from the reaction between guanines of TBA and TMPG in the presence of TPA, transfers energy to 6-FAM to emit bright light based on the principle of chemiluminescence energy transfer (CRET). G-quadruplex TBA aptasensor devised using the rapid interaction between TBA-conjugated 6-FAM and thrombin quantified trace levels of thrombin without complicated procedures. The limit of detection (LOD = background + 3 × standard deviation) of G-quadruplex TBA aptasensor with good linear calibration curve, accuracy, precision, and recovery was as low as 12.3 nM in 5% human serum. Using the technology reported in this research, we expect that various types of G-quadruplex DNA aptasensors capable of specifically sensing a target molecule such as ATP, HIV, ochratoxin, potassium ions, and thrombin can be developed. © 2013 Elsevier B.V. All rights reserved.

Automatic and integrated micro-enzyme assay (AIμEA) platform for highly sensitive thrombin analysis via an engineered fluorescence protein-functionalized monolithic capillary column.

PubMed

Lin, Lihua; Liu, Shengquan; Nie, Zhou; Chen, Yingzhuang; Lei, Chunyang; Wang, Zhen; Yin, Chao; Hu, Huiping; Huang, Yan; Yao, Shouzhuo

2015-04-21

Nowadays, large-scale screening for enzyme discovery, engineering, and drug discovery processes require simple, fast, and sensitive enzyme activity assay platforms with high integration and potential for high-throughput detection. Herein, a novel automatic and integrated micro-enzyme assay (AIμEA) platform was proposed based on a unique microreaction system fabricated by a engineered green fluorescence protein (GFP)-functionalized monolithic capillary column, with thrombin as an example. The recombinant GFP probe was rationally engineered to possess a His-tag and a substrate sequence of thrombin, which enable it to be immobilized on the monolith via metal affinity binding, and to be released after thrombin digestion. Combined with capillary electrophoresis-laser-induced fluorescence (CE-LIF), all the procedures, including thrombin injection, online enzymatic digestion in the microreaction system, and label-free detection of the released GFP, were integrated in a single electrophoretic process. By taking advantage of the ultrahigh loading capacity of the AIμEA platform and the CE automatic programming setup, one microreaction column was sufficient for many times digestion without replacement. The novel microreaction system showed significantly enhanced catalytic efficiency, about 30 fold higher than that of the equivalent bulk reaction. Accordingly, the AIμEA platform was highly sensitive with a limit of detection down to 1 pM of thrombin. Moreover, the AIμEA platform was robust and reliable to detect thrombin in human serum samples and its inhibition by hirudin. Hence, this AIμEA platform exhibits great potential for high-throughput analysis in future biological application, disease diagnostics, and drug screening.

An ultrasensitive chemiluminescence aptasensor for thrombin detection based on iron porphyrin catalyzing luminescence desorbed from chitosan modified magnetic oxide graphene composite.

PubMed

Sun, Yuanling; Wang, Yanhui; Li, Jianbo; Ding, Chaofan; Lin, Yanna; Sun, Weiyan; Luo, Chuannan

2017-11-01

In this work, an ultrasensitive chemiluminescence (CL) aptasensor was prepared for thrombin detection based on iron porphyrin catalyzing luminol - hydrogen peroxide luminescence under alkaline conditions, and iron porphyrin was desorbed from chitosan modified magnetic oxide graphene composite (CS@Fe 3 O 4 @GO). Firstly, CS@Fe 3 O 4 @GO was prepared. CS@Fe 3 O 4 @GO has advantages of the good biocompatibility and positively charged on its surface of CS, the large specific surface area of GO and the easy separation characteristics of Fe 3 O 4 . GO, Fe 3 O 4 and CS@Fe 3 O 4 @GO were confirmed by transmission electron microscopy (TEM), scanning electron microscope (SEM), fourier transform infrared (FTIR) and X-ray powder diffraction (XRD). Then, thrombin aptamer (T-Apt) and hemin (HM, an iron porphyrin) were sequentially modified on the surface of CS@Fe 3 O 4 @GO to form CS@Fe 3 O 4 @GO@T-Apt@HM. The immobilization properties of CS@Fe 3 O 4 @GO to T-Apt and adsorption properties of CS@Fe 3 O 4 @GO@T-Apt to HM were sequentially researched through the curves of kinetics and the curves of thermodynamics. When thrombin existed in solutions, HM was desorbed from the surface of CS@Fe 3 O 4 @GO@T-Apt@HM owing to the strong specific recognition ability between thrombin and T-Apt, causing the changes of CL signal. Under optimized CL conditions, thrombin could be measured with the linear concentration range of 5.0×10 -15 -2.5×10 -10 mol/L. The detection limit was 1.5×10 -15 mol/L (3δ) while the relative standard deviation (RSD) was 3.2%. Finally, the CS@Fe 3 O 4 @GO@T-Apt@HM-CL aptasensor was used for the determination of thrombin in practical serum samples and recoveries ranged from 95% to 103%. Those satisfactory results revealed potential application of the CS@Fe 3 O 4 @GO@T-Apt@HM-CL aptasensor for thrombin detection in monitoring and diagnosis of human blood diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

An electrochemical aptasensor for thrombin detection based on direct electrochemistry of glucose oxidase using a functionalized graphene hybrid for amplification.

PubMed

Bai, Lijuan; Yan, Bin; Chai, Yaqin; Yuan, Ruo; Yuan, Yali; Xie, Shunbi; Jiang, Liping; He, Ying

2013-11-07

In this work, we reported a new label-free electrochemical aptasensor for highly sensitive detection of thrombin using direct electron transfer of glucose oxidase (GOD) as a redox probe and a gold nanoparticle-polyaniline-graphene (Au-PANI-Gra) hybrid for amplification. The Au-PANI-Gra hybrid with large surface area provided a biocompatible sensing platform for the immobilization of GOD. GOD was encapsulated into the three-dimensional netlike (3-mercaptopropyl)trimethoxysilane (MPTS) to form the MPTS-GOD biocomposite, which not only retained the native functions and properties, but also exhibited tunable porosity, high thermal stability, and chemical inertness. With abundant thiol tail groups on MPTS, MPTS-GOD was able to chemisorb onto the surface of the Au-PANI-Gra modified electrode through the strong affinity of the Au-S bond. The electrochemical signal originated from GOD, avoiding the addition or labeling of other redox mediators. After immobilizing the thiolated thrombin binding aptamer through gold nanoparticles (AuNPs), GOD as a blocking reagent was employed to block the remaining active sites of the AuNPs and avoid the nonspecific adsorption. The proposed method avoided the labeling process of redox probes and increased the amount of electroactive GOD. The concentration of thrombin was monitored based on the decrease of current response through cyclic voltammetry (CV) in 0.1 M PBS (pH 7.4). With the excellent direct electron transfer of double layer GOD membranes, the resulting aptasensor exhibited high sensitivity for detection of thrombin with a wide linear range from 1.0 × 10(-12) to 3.0 × 10(-8) M. The proposed aptasensor also showed good stability, satisfactory reproducibility and high specificity, which provided a promising strategy for electrochemical aptamer-based detection of other biomolecules.

Ultrasensitive aptamer-based protein detection via a dual amplified biocatalytic strategy

PubMed Central

Xiang, Yun; Zhang, Yuyong; Qian, Xiaoqing; Chai, Yaqin; Wang, Joseph; Yuan, Ruo

2010-01-01

We present an ultrasensitive aptasensor for electronic monitoring of proteins through a dual amplified strategy in this paper. The target protein thrombin is sandwiched between an electrode surface confined aptamer and an aptamer-enzyme-carbon nanotube bioconjugate. The analytical signal amplification is achieved by coupling the signal amplification nature of multiple enzymes with the biocatalytic signal enhancement of redox-recycling. Our novel dramatic signal amplification strategy, with a detection limit of 8.3 fM, shows about 4 orders of magnitude improvement in sensitivity for thrombin detection compared to other universal single enzyme-based assay. This makes our approach an attractive alternative to other common PCR-based signal amplification in ultralow level of protein detection. PMID:20452761

Reversible thrombin detection by aptamer functionalized STING sensors

PubMed Central

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

2011-01-01

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

Platelet-targeting sensor reveals thrombin gradients within blood clots forming in microfluidic assays and in mouse.

PubMed

Welsh, J D; Colace, T V; Muthard, R W; Stalker, T J; Brass, L F; Diamond, S L

2012-11-01

Thrombin undergoes convective and diffusive transport, making it difficult to visualize during thrombosis. We developed the first sensor capable of revealing inner clot thrombin dynamics. An N-terminal-azido thrombin-sensitive fluorescent peptide (ThS-P) with a thrombin-releasable quencher was linked to anti-CD41 using click chemistry to generate a thrombin-sensitive platelet binding sensor (ThS-Ab). Rapid thrombin cleavage of ThS-P (K(m) = 40.3 μm, k(cat) = 1.5 s(-1) ) allowed thrombin monitoring by ThS-P or ThS-Ab in blood treated with 2-25 pm tissue factor (TF). Individual platelets had > 20-fold more ThS-Ab fluorescence after clotting. In a microfluidic assay of whole blood perfusion over collagen ± linked TF (wall shear rate = 100 s(-1) ), ThS-Ab fluorescence increased between 90 and 450 s for 0.1-1 molecule-TF μm(-2) and co-localized with platelets near fibrin. Without TF, neither thrombin nor fibrin was detected on the platelet deposits by 450 s. Using a microfluidic device to control the pressure drop across a thrombus forming on a porous collagen/TF plug (521 s(-1) ), thrombin and fibrin were detected at the thrombus-collagen interface at a zero pressure drop, whereas 80% less thrombin was detected at 3200 Pa in concert with fibrin polymerizing within the collagen. With anti-mouse CD41 ThS-Ab deployed in a mouse laser injury model, the highest levels of thrombin arose between 40 and 160 s nearest the injury site where fibrin co-localized and where the thrombus was most mechanically stable. ThS-Ab reveals thrombin locality, which depends on surface TF, flow and intrathrombus pressure gradients. © 2012 International Society on Thrombosis and Haemostasis.

A novel "signal-on/off" sensing platform for selective detection of thrombin based on target-induced ratiometric electrochemical biosensing and bio-bar-coded nanoprobe amplification strategy.

PubMed

Wang, Lanlan; Ma, Rongna; Jiang, Liushan; Jia, Liping; Jia, Wenli; Wang, Huaisheng

2017-06-15

A novel dual-signal ratiometric electrochemical aptasensor for highly sensitive and selective detection of thrombin has been designed on the basis of signal-on and signal-off strategy. Ferrocene labeled hairpin probe (Fc-HP), thrombin aptamer and methyl blue labeled bio-bar-coded AuNPs (MB-P3-AuNPs) were rationally introduced for the construction of the assay platform, which combined the advantages of the recognition of aptamer, the amplification of bio-bar-coded nanoprobe, and the ratiometric signaling readout. In the presence of thrombin, the interaction between thrombin and the aptamer leads to the departure of MB-P3-AuNPs from the sensing interface, and the conformation of the single stranded Fc-HP to a hairpin structure to take the Fc confined near the electrode surface. Such conformational changes resulted in the oxidation current of Fc increased and that of MB decreased. Therefore, the recognition event of the target can be dual-signal ratiometric electrochemical readout in both the "signal-off" of MB and the "signal-on" of Fc. The proposed strategy showed a wide linear detection range from 0.003 to 30nM with a detection limit of 1.1 pM. Moreover, it exhibits good performance of excellent selectivity, good stability, and acceptable fabrication reproducibility. By changing the recognition probe, this protocol could be easily expanded into the detection of other targets, showing promising potential applications in disease diagnostics and bioanalysis. Copyright © 2016. Published by Elsevier B.V.

Electrochemical aptasensor based on the dual-amplification of G-quadruplex horseradish peroxidase-mimicking DNAzyme and blocking reagent-horseradish peroxidase.

PubMed

Yuan, Yali; Gou, Xuxu; Yuan, Ruo; Chai, Yaqin; Zhuo, Ying; Mao, Li; Gan, Xianxue

2011-06-15

A simple electrochemical aptasensor for sensitive detection of thrombin was fabricated with G-quadruplex horseradish peroxidase-mimicking DNAzyme (hemin/G-quadruplex system) and blocking reagent-horseradish peroxidase as dual signal-amplification scheme. Gold nanoparticles (nano-Au) were firstly electrodeposited onto single wall nanotube (SWNT)-graphene modified electrode surface for the immobilization of electrochemical probe of nickel hexacyanoferrates nanoparticles (NiHCFNPs). Subsequently, another nano-Au layer was electrodeposited for further immobilization of thrombin aptamer (TBA), which later formed hemin/G-quadruplex system with hemin. Horseradish peroxidases (HRP) then served as blocking reagent to block possible remaining active sites and avoided the non-specific adsorption. In the presence of thrombin, the TBA binded to thrombin and the hemin released from the hemin/G-quadruplex electrocatalytic structure, increasing steric hindrance of the aptasensor and decomposing hemin/G-quadruplex electrocatalytic structure, which finally decreased the electrocatalytic efficiency of aptasensor toward H(2)O(2) in the presence of NiHCFNPs with a decreased electrochemical signal. On the basis of the synergistic amplifying action, a detection limit as low as 2 pM for thrombin was obtained. Copyright © 2011 Elsevier B.V. All rights reserved.

Real-time measurement of free thrombin: evaluation of the usability of a new thrombin assay for coagulation monitoring during extracorporeal circulation.

PubMed

Krajewski, Stefanie; Krauss, Sabrina; Kurz, Julia; Neumann, Bernd; Schlensak, Christian; Wendel, Hans P

2014-03-01

In patients undergoing cardiac surgery with heart-lung machine support, adequate anticoagulation to mitigate blood clotting caused by the artificial surfaces of the extracorporeal circulation (ECC) system is essential. These patients routinely receive heparin, whose effectiveness is monitored by measurements of the activated clotting time (ACT). However, ACT values only poorly correlate with the actual hemostatic status. The aim of our study was to evaluate the detection of free thrombin in heparinized human blood as a monitor of anticoagulation during ECC. Human whole blood was anticoagulated with different concentrations of heparin (0.75, 1, 2 or 3 IU/ml) and circulated in the Chandler-loop model for up to 240 min at 37 °C. Next to ACT, ECC-mediated changes in free active thrombin, prothrombin fragment 1+2 (F1+2) and thrombin-antithrombin-III (TAT) levels were measured before and during circulation. Platelet activation and cell count parameters were further investigated. Our study shows that detection of ECC-mediated changes in free thrombin is possible in blood anticoagulated with 0.75 or 1 IU/ml heparin, whereas no thrombin was detectable at higher heparin concentrations. Thrombin generation during 240 min of ECC is comparable to F 1+2 and TAT plasma levels during ECC. Thrombin is the key enzyme in the coagulation cascade and hence represents a promising marker for monitoring the coagulation status of patients. Although detection of free thrombin was not feasible at high heparin concentrations, the employed test represents an additional test to current laboratory methods investigating blood coagulation at low heparin concentrations. Copyright © 2013 Elsevier Ltd. All rights reserved.

Reversible thrombin detection by aptamer functionalized STING sensors.

PubMed

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

2011-07-15

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. Copyright © 2011 Elsevier B.V. All rights reserved.

Discovering the enzyme mimetic activity of metal-organic framework (MOF) for label-free and colorimetric sensing of biomolecules.

PubMed

Wang, Ying; Zhu, Yingjing; Binyam, Atsebeha; Liu, Misha; Wu, Yinan; Li, Fengting

2016-12-15

A label-free sensing strategy based on the enzyme-mimicking activity of MOF was demonstrated for colorimetric detection of biomolecules. Firstly obvious blue color was observed due to the high efficiency of peroxidase-like catalytic activity of Fe-MIL-88A (an ion-based MOF material) toward 3,3',5,5'-tetramethylbenzidine (TMB). Then in the presence of target biomolecule and corresponding aptamer, the mimetic activity of Fe-MIL-88A can be strongly inhibited and used directly to realize the colorimetric detection. On the basis of the interesting findings, we designed a straightforward, label-free and sensitive colorimetric method for biomolecule detection by using the enzyme mimetic property of MOF coupling with molecular recognition element. Compared with the existed publications, our work breaks the routine way by setting up an inorganic-organic MOF-aptamer hybrid platform for colorimetric determination of biomolecules, expanding the targets scope from H2O2 or glucose to biomolecules. As a proof of concept, thrombin and thrombin aptamer was used as a model analyte. The limit of detection of 10nM can be achieved with naked eyes and ultrahigh selectivity of thrombin toward numerous interfering substances with 10-fold concentration was demonstrated significantly. Of note, the method was further applied for the detection of thrombin in human serum samples, showing the results in agreement with those values obtained in an immobilization buffer by the colorimetric method. This inorganic-organic MOF-aptamer sensing strategy may in principle be universally applicable for the detection of a range of environmental or biomedical molecules of interests. Copyright © 2016 Elsevier B.V. All rights reserved.

Chemiluminescence and chemiluminescence resonance energy transfer (CRET) aptamer sensors using catalytic hemin/G-quadruplexes.

PubMed

Liu, Xiaoqing; Freeman, Ronit; Golub, Eyal; Willner, Itamar

2011-09-27

The incorporation of hemin into the thrombin/G-quadruplex aptamer assembly or into the ATP/G-quadruplex nanostructure yields active DNAzymes that catalyze the generation of chemiluminescence. These catalytic processes enable the detection of thrombin and ATP with detection limits corresponding to 200 pM and 10 μM, respectively. The conjugation of the antithrombin or anti-ATP aptamers to CdSe/ZnS semiconductor quantum dots (QDs) allowed the detection of thrombin or ATP through the luminescence of the QDs that is powered by a chemiluminescence resonance energy-transfer (CRET) process stimulated by the hemin/G-quadruplex/thrombin complex or the hemin/G-quadruplex/ATP nanostructure, in the presence of luminol/H(2)O(2). The advantages of applying the CRET process for the detection of thrombin or ATP, by the resulting hemin/G-quadruplex DNAzyme structures, are reflected by low background signals and the possibility to develop multiplexed aptasensor assays using different sized QDs. © 2011 American Chemical Society

Diluted thrombin time reliably measures low to intermediate plasma dabigatran concentrations.

PubMed

Božič-Mijovski, Mojca; Malmström, Rickard E; Malovrh, Petra; Antovic, Jovan P; Vene, Nina; Šinigoj, Petra; Mavri, Alenka

2016-07-01

Direct oral anticoagulant dabigatran was first introduced as a fixed-dose drug without routine coagulation monitoring, but current recommendations suggest that diluted thrombin time can be used to estimate plasma drug level. The aim of this study was to assess a diluted thrombin time assay based on the same thrombin reagent already used for traditional thrombin time measurements that reliably measure low to intermediate plasma dabigatran levels. We included 44 patients with atrial fibrillation who started treatment with dabigatran 150 mg (23 patients) or 110 mg (21 patients) twice a day. Blood samples were collected at baseline (no dabigatran) and 2-4 weeks after the beginning of dabigatran therapy at trough and at peak. Plasma dabigatran levels were measured with diluted thrombin time and compared to liquid chromatography with tandem mass spectrometry as the reference method. The performance of the diluted thrombin time was compared to Hemoclot® Thrombin Inhibitor and Ecarin Chromogenic Assay. In ex vivo plasma samples, diluted thrombin time highly correlated with the liquid chromatography with tandem mass spectrometry (Pearson's R = 0.9799). In the low to intermediate range (dabigatran concentration ≤ 100 µg/L) diluted thrombin time correlated significantly more closely to the liquid chromatography with tandem mass spectrometry (R = 0.964) than Hemoclot® Thrombin Inhibitor (R = 0.935, p = 0.05) or Ecarin Chromogenic Assay (R = 0.915, p < 0.01). It was also the only functional assay without any significant bias in the low to intermediate range. Both trough and peak diluted thrombin time values were similar to liquid chromatography with tandem mass spectrometry. We conclude that the diluted thrombin time assay presented in this study reliably detects dabigatran and that it is superior to the Hemoclot® Thrombin Inhibitor assay in the low to intermediate range. © The Author(s) 2015.

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

PubMed

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

2016-04-21

Although thrombin has an important role in both central and peripheral nerve diseases, characterization of the anatomical distribution of its proteolytic activity has been limited by available methods. This study presents the development, challenges, validation and implementation of a novel histochemical method for visualization of thrombin activity in the nervous system. The method is based on the cleavage of the substrate, Boc-Asp(OBzl)-Pro-Arg-4MβNA by thrombin to liberate free 4-methoxy-2-naphthylamine (4MβNA). In the presence of 5-nitrosalicylaldehyde, free 4MβNA is captured, yielding an insoluble yellow fluorescent precipitate which marks the site of thrombin activity. The sensitivity of the method was determined in vitro using known concentrations of thrombin while the specificity was verified using a highly specific thrombin inhibitor. Using this method we determined the spatial distribution of thrombin activity in mouse brain following transient middle cerebral artery occlusion (tMCAo) and in mouse sciatic nerve following crush injury. Fluorescence microscopy revealed well-defined thrombin activity localized to the right ischemic hemisphere in cortical areas and in the striatum compared to negligible thrombin activity contralaterally. The histochemical localization of thrombin activity following tMCAo was in good correlation with the infarct areas per triphenyltetrazolium chloride staining and to thrombin activity measured biochemically in tissue punches (85 ± 35 and 20 ± 3 mU/ml, in the cortical and striatum areas respectively, compared to 7 ± 2 and 13 ± 2 mU/ml, in the corresponding contralateral areas; mean ± SEM; p<0.05). In addition, 24 h following crush injury, focal areas of highly elevated thrombin activity were detected in teased sciatic fibers. This observation was supported by the biochemical assay and western blot technique. The histochemical method developed in this study can serve as an important tool for studying the role of thrombin in physiological and pathological conditions. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

A quantum dot-aptamer beacon using a DNA intercalating dye as the FRET reporter: application to label-free thrombin detection.

PubMed

Chi, Chun-Wei; Lao, Yeh-Hsing; Li, Yi-Shan; Chen, Lin-Chi

2011-03-15

A new quantum dot (QD)-aptamer (apt) beacon that acts by folding-induced dissociation of a DNA intercalating dye, BOBO-3(B), is demonstrated with label-free thrombin detection. The beacon, denoted as QD-apt:B, is constructed by (1) coupling of a single-stranded thrombin aptamer to Qdot 565 via EDC/Sulfo-NHS chemistry and (2) staining the duplex regions of the aptamer on QD with excess BOBO-3 before thrombin binding. When mixing a thrombin sample with QD-apt:B, BOBO-3 is competed away from the beacon due to target-induced aptamer folding, which then causes a decrease in QD fluorescence resonance energy transfer (FRET)-mediated BOBO-3 emission and achieves thrombin quantitation. In this work, the effects of Mg(2+), coupling time, and aptamer type on the beacon's performances are investigated and discussed thoroughly with various methods, including transmission electron microscopy (TEM), dynamic light scattering (DLS), and two-color differential gel electrophoresis. Using the best aptamer beacon (HTQ37), we attain highly specific and wide-range detection (from nM to μM) of thrombin in buffer, and the beacon can sense nM-range thrombin in 15% diluted serum. Compared to the reported QD aptamer assays, our method is advantageous from the aspect of using a simple sensory unit design without losing the detection sensitivity. Therefore, we consider the QD-apt:B beacon a potential alternative to immuno-reagents and an effective tool to study nucleic acid folding on QD as well. Copyright © 2011 Elsevier B.V. All rights reserved.

Homologous desensitization of HEL cell thrombin receptors. Distinguishable roles for proteolysis and phosphorylation.

PubMed

Brass, L F

1992-03-25

Loss of sensitivity to thrombin following an initial response is characteristic of a number of cell types, including platelets. It has recently been proposed that thrombin receptors resemble other G protein-coupled receptors, but that activation involves a novel mechanism in which thrombin cleaves the receptor, exposing a new N terminus that serves as the ligand for the receptor. Based upon this model, we have examined the mechanism of thrombin receptor desensitization by comparing the effects of thrombin with those of a peptide corresponding to the N-terminal sequence of the receptor following proteolysis by thrombin: SFLLRNPNDKYEPF or TRP42/55. Like thrombin, TRP42/55 stimulated pertussis toxin-sensitive inositol 1,4,5-trisphosphate formation, raised cytosolic Ca2+, and inhibited cAMP formation in the megakaryoblastic HEL cell line. Exposure to either thrombin or TRP42/55 desensitized the cells to both, but not to a third agonist, neuropeptide Y. The rate of recovery after desensitization depended upon the order of agonist addition. Resensitization of the cell to thrombin following a brief exposure to thrombin required up to 24 h and could be inhibited with cycloheximide. Resensitization to TRP42/55 after exposure to thrombin, or to thrombin after exposure to TRP42/55, on the other hand, was detectable within 30 min and could be inhibited by serine/threonine phosphatase inhibitors, but not by cycloheximide. Loss of responsiveness to thrombin and TRP42/55 was also observed following addition of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). However, while the protein kinase inhibitor staurosporine completely prevented the desensitization caused by TPA, it had only a limited effect on the desensitization caused by TRP42/55. These results demonstrate that the G protein-mediated effects of thrombin can be reproduced by a receptor-derived peptide and suggest that desensitization occurs by at least two mechanisms. The first, which is seen with thrombin, but not TRP42/55, involves proteolysis and requires protein synthesis for recovery. The second, which occurs with TRP42/55 and TPA, as well as with thrombin, involves phosphorylation, possibly of the receptor itself. Although protien kinase C is activated by thrombin and is presumably responsible for the desensitization caused by TPA, it does not appear to play a major role in receptor desensitization caused by thrombin and TRP42/55. This suggests that other kinases, such as those which inactivate adrenergic receptors and rhodopsin, are involved in the down-regulation of thrombin receptor function.

A cascade signal amplification strategy for surface enhanced Raman spectroscopy detection of thrombin based on DNAzyme assistant DNA recycling and rolling circle amplification.

PubMed

Gao, Fenglei; Du, Lili; Tang, Daoquan; Lu, Yao; Zhang, Yanzhuo; Zhang, Lixian

2015-04-15

A sensitive protocol for surface enhanced Raman spectroscopy (SERS) detection of thrombin is designed with R6G-Ag NPs as a signal tag by combining DNAzyme assistant DNA recycling and rolling circle amplification (RCA). Molecular beacon (MB) as recognition probe immobilizes on the glass slides and performs the amplification procedure. After thrombin-induced structure-switching DNA hairpins of probe 1, the DNAzyme is liberated from the caged structure, which hybridizes with the MB for cleavage of the MB in the presence of cofactor Zn(2+) and initiates the DNA recycling process, leading to the cleavage of a large number of MB and the generation of numerous primers for triggering RCA reaction. The long amplified RCA product which contained hundreds of tandem-repeat sequences, which can bind with oligonucleotide functionalized Ag NPs reporters. The attached signal tags can be easily read out by SERS. Because of the cascade signal amplification, these newly designed protocols provides a sensitive SERS detection of thrombin down to the femolar level (2.3fM) with a linear range of 5 orders of magnitude (from 10(-14) to 10(-9)M) and have high selectivity toward its target protein. The proposed method is expected to be a good clinical tool for the diagnosis of a thrombotic disease. Copyright © 2014 Elsevier B.V. All rights reserved.

Aptamer-crosslinked microbubbles: smart contrast agents for thrombin-activated ultrasound imaging.

PubMed

Nakatsuka, Matthew A; Mattrey, Robert F; Esener, Sadik C; Cha, Jennifer N; Goodwin, Andrew P

2012-11-27

Thrombosis, or malignant blood clotting, is associated with numerous cardiovascular diseases and cancers. A microbubble contrast agent is presented that produces ultrasound harmonic signal only when exposed to elevated thrombin levels. Initially silent microbubbles are activated in the presence of both thrombin-spiked and freshly clotting blood in three minutes with detection limits of 20 nM thrombin and 2 aM microbubbles. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Homogenous assay for protein detection based on proximity DNA hybridization and isothermal circular strand displacement amplification reaction.

PubMed

Zhang, Manjun; Li, Ruimin; Ling, Liansheng

2017-06-01

This work proposed a homogenous fluorescence assay for proteins, based on the target-triggered proximity DNA hybridization in combination with strand displacement amplification (SDA). It benefited from target-triggered proximity DNA hybridization to specifically recognize the target and SDA making recycling signal amplification. The system included a molecular beacon (MB), an extended probe (EP), and an assistant probe (AP), which were not self-assembly in the absence of target proteins, due to the short length of the designed complementary sequence among MB, EP, and AP. Upon addition of the target proteins, EP and AP are bound to the target proteins, which induced the occurrence of proximity hybridization between MB, EP, and AP and followed by strand displacement amplification. Through the primer extension, a tripartite complex of probes and target was displaced and recycled to hybridize with another MB, and the more opened MB enabled the detection signal to amplify. Under optimum conditions, it was used for the detection of streptavidin and thrombin. Fluorescence intensity was proportional to the concentration of streptavidin and thrombin in the range of 0.2-30 and 0.2-35 nmol/L, respectively. Furthermore, this fluorescent method has a good selectivity, in which the fluorescence intensity of thrombin was ~37-fold or even larger than that of the other proteins at the same concentration. It is a new and simple method for SDA-involved target protein detection and possesses a great potential for other protein detection in the future. Graphical abstract A homogenous assay for protein detection is based on proximity DNA hybridization and strand displacement amplification reaction.

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

PubMed

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

2012-11-18

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

Immunization by bovine thrombin used with fibrin glue during cardiovascular operations. Development of thrombin and factor V inhibitors.

PubMed

Berruyer, M; Amiral, J; Ffrench, P; Belleville, J; Bastien, O; Clerc, J; Kassir, A; Estanove, S; Dechavanne, M

1993-05-01

Brief case histories of three patients aged 58, 38, and 44 years are reported. All underwent cardiovascular operations. Subsequently hemostasis test abnormalities developed between the seventh and eighth postoperative days after exposure to bovine thrombin used with fibrin glue. These were characterized by an increased activated partial thromboplastin time (64 to 147 seconds), prothrombin time (19 to 24 seconds), bovine thrombin time (> 120 seconds) and a markedly reduced factor V level (< 10% in two patients and 16% in the third patient). A patient plasma dilution of 1 in 200 with a normal plasma pool was necessary to correct bovine thrombin time. No fast-acting or progressive inhibitor against factor V could be detected by coagulation tests, and fresh frozen plasma perfusion had no effect. Plasmapheresis was performed preventatively to avoid bleeding, and factor V levels stabilized at around 50% after two to four exchanges. Immunologic studies showed that the inhibitors were directed not only against bovine factors but also against human ones. Therefore factor V decrease could have been the result of rapid clearance from the circulation of complexes formed with a nonneutralizing inhibitor that is not detected by clotting tests. These antibodies were purified by standard methods and immunoaffinity. Fast immunization could be explained by a prior sensitization to bovine thrombin exposure during previous operations. It is suggested that bovine thrombin used with fibrin glue contains small amounts of factor V and may be responsible for these abnormalities. This is in agreement with previous literature reports. However, these described neutralizing factor V inhibitors, which were easily detected.

Biobar-coded gold nanoparticles and DNAzyme-based dual signal amplification strategy for ultrasensitive detection of protein by electrochemiluminescence.

PubMed

Xia, Hui; Li, Lingling; Yin, Zhouyang; Hou, Xiandeng; Zhu, Jun-Jie

2015-01-14

A dual signal amplification strategy for electrochemiluminescence (ECL) aptasensor was designed based on biobar-coded gold nanoparticles (Au NPs) and DNAzyme. CdSeTe@ZnS quantum dots (QDs) were chosen as the ECL signal probes. To verify the proposed ultrasensitive ECL aptasensor for biomolecules, we detected thrombin (Tb) as a proof-of-principle analyte. The hairpin DNA designed for the recognition of protein consists of two parts: the sequences of catalytical 8-17 DNAzyme and thrombin aptamer. Only in the presence of thrombin could the hairpin DNA be opened, followed by a recycling cleavage of excess substrates by catalytic core of the DNAzyme to induce the first-step amplification. One part of the fragments was captured to open the capture DNA modified on the Au electrode, which further connected with the prepared biobar-coded Au NPs-CdSeTe@ZnS QDs to get the final dual-amplified ECL signal. The limit of detection for Tb was 0.28 fM with excellent selectivity, and this proposed method possessed good performance in real sample analysis. This design introduces the new concept of dual-signal amplification by a biobar-coded system and DNAzyme recycling into ECL determination, and it is promising to be extended to provide a highly sensitive platform for various target biomolecules.

Enzyme-guided plasmonic biosensor based on dual-functional nanohybrid for sensitive detection of thrombin.

PubMed

Yan, Jing; Wang, Lida; Tang, Longhua; Lin, Lei; Liu, Yang; Li, Jinghong

2015-08-15

Rapid and sensitive methodologies for the detection of protein are in urgent requirement for clinic diagnostics. Localized surface plasmon resonance (LSPR) of metal nanostructures has the potential to circumvent this problem due to its sensitive optical properties and strong electromagnetic near-field enhancements. In this work, an enzyme mediated plasmonic biosensor on the basis of a dual-functional nanohybrid was developed for the detection of thrombin. By utilizing LSPR-responsive nanohybrid and anaptamer-enzyme conjugated reporting probe, the sensing platform brings enhanced signal, stability as well as simplicity. Enzymatic reaction catalyzed the reduction of Au(3+) to Au° in situ, further leading to the rapid crystal growth of gold nanoparticles (AuNPs). The LSPR absorbance band and color changed company with the nanoparticle generation, which can be real-time monitoring by UV-visible spectrophotometer and naked eye. Nanohybrid constructed by gold and magnetic nanoparticles acts as a dual functional plasmonic unit, which not only plays the role of signal production, but also endows the sensor with the function of magnetic separation. Simultaneously, the introduction of enzyme effectively regulates the programming crystal growth of AuNPs. In addition, enzyme also serves as signal amplifier owing to its high catalysis efficiency. The response of the plasmonic sensor varies linearly with the logarithmic thrombin concentration up to 10nM with a limit of detection of 200 pM. The as-proposed strategy shows good analytical performance for thrombin determination. This simple, disposable method is promising in developing universal platforms for protein monitoring, drug discovery and point-of-care diagnostics. Copyright © 2015 Elsevier B.V. All rights reserved.

Construction of photoelectrochemical thrombin aptasensor via assembling multilayer of graphene-CdS nanocomposites.

PubMed

Shangguan, Li; Zhu, Wei; Xue, Yanchun; Liu, Songqin

2015-02-15

A photoelectrochemical (PEC) aptasensor for highly sensitive and specific detection of thrombin was developed by using graphene–CdS nanocomposites multilayer as photoactive species and electroactive mediator hexaammineruthenium(III) chloride (Ru(NH(3))(6)(3+)) as signal enhancer. Graphene–CdS nanocomposites (G–CdS) were synthesized by one-pot reduction of oxide graphene and CdCl2 with thioacetamide. The photoactive multilayer was prepared by alternative assembly of the negatively charged 3-mercaptopropionic acid modified graphene–CdS nanocomposites (MPA-G–CdS) and the positively charged polyethylenimine (PEI) on ITO electrode. This layer-by-layer assembly method enhanced the stability and homogeneity of the photocurrent readout of G–CdS. Thrombin aptamer was covalently bound to the multilayer by using glutaraldehyde as cross-linking. Electroactive mediator (Ru(NH(3))(6)(3+)) could interact with the DNA phosphate backbone and thus facilitated the electron transfer between G–CdS multilayer and electrode and enhanced the photocurrent. Hybridizing of a long complementary DNA with thrombin aptamer could increase the adsorption amount of (Ru(NH(3))(6)(3+)), which in turn boosted the signal readout. In the presence of target thrombin, the affinity interaction between thrombin and its aptamer resulted in the long complementary DNA releasing from the G–CdS multilayer and decreasing of photocurrent signal. On the basis of G–CdS multilayer as the photoactive species, (Ru (NH(3))(6)(3+)) as an electroactive mediator, and aptamer as a recognition module, a high sensitive PEC aptasensor for thrombin detection was proposed. The thrombin aptasensor displayed a linear range from 2.0 pM to 600.0 pM and a detection limit of 1.0 pM. The present strategy provided a promising ideology for the future development of PEC biosensor. Copyright © 2014 Elsevier B.V. All rights reserved.

Modeling thrombin generation: plasma composition based approach.

PubMed

Brummel-Ziedins, Kathleen E; Everse, Stephen J; Mann, Kenneth G; Orfeo, Thomas

2014-01-01

Thrombin has multiple functions in blood coagulation and its regulation is central to maintaining the balance between hemorrhage and thrombosis. Empirical and computational methods that capture thrombin generation can provide advancements to current clinical screening of the hemostatic balance at the level of the individual. In any individual, procoagulant and anticoagulant factor levels together act to generate a unique coagulation phenotype (net balance) that is reflective of the sum of its developmental, environmental, genetic, nutritional and pharmacological influences. Defining such thrombin phenotypes may provide a means to track disease progression pre-crisis. In this review we briefly describe thrombin function, methods for assessing thrombin dynamics as a phenotypic marker, computationally derived thrombin phenotypes versus determined clinical phenotypes, the boundaries of normal range thrombin generation using plasma composition based approaches and the feasibility of these approaches for predicting risk.

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

PubMed Central

Olson, Emilia S.; Whitney, Michael A.; Friedman, Beth; Aguilera, Todd A.; Crisp, Jessica L.; Baik, Fred M.; Jiang, Tao; Baird, Stephen M.; Tsimikas, Sotirios; Tsien, Roger Y.

2012-01-01

Thrombin and other coagulation enzymes have been shown to be important during atherosclerotic disease development. Study of these proteases is currently limited because of lack of robust molecular imaging agents for imaging protease activity in vivo. Activatable cell penetrating peptides (ACPPs) have been used to monitor MMP activity in tumors and, in principle, can be modified to detect other proteases. We have developed a probe that incorporates the peptide sequence DPRSFL from the proteinase activated receptor 1 (PAR-1) into an ACPP and shown that it is preferentially cleaved by purified thrombin. Active thrombin in serum cleaves DPRSFL–ACPP with >90% inhibition by lepirudin or argatroban. The DPRSFL–ACPP cleavage product accumulated in advanced atherosclerotic lesions in living mice, with 85% reduction in retention upon pre-injection of mice with hirudin. Uptake of the ACPP cleavage product was highest in plaques with histological features associated with more severe disease. Freshly resected human atheromas bathed in DPRSFL–ACPP retained 63% greater cleavage product compared to control ACPP. In conclusion, DPRSFL–ACPP can be used to study thrombin activity in coagulation and atherosclerosis with good spatial and temporal resolution. Thrombin-sensitive ACPPs may be developed into probes for early detection and intraoperative imaging of high risk atherosclerotic plaques. PMID:22534729

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

PubMed

Olson, Emilia S; Whitney, Michael A; Friedman, Beth; Aguilera, Todd A; Crisp, Jessica L; Baik, Fred M; Jiang, Tao; Baird, Stephen M; Tsimikas, Sotirios; Tsien, Roger Y; Nguyen, Quyen T

2012-06-01

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

A sensitive gold nanoparticles sensing platform based on resonance energy transfer for chemiluminescence light on detection of biomolecules.

PubMed

Qin, Guoxing; Zhao, Shulin; Huang, Yong; Jiang, Jing; Liu, Yi-Ming

2013-08-15

In this article, we report a gold nanoparticles (AuNPs) sensing platform based on chemiluminescence resonance energy transfer (CRET) for light on detection of biomolecules. In designing such a CRET-based biosensing platform, the aptamer was first covalently labeled with a chemiluminescent reagent, N-(4-aminobutyl)-N-ethylisoluminol (ABEI). The ABEI labeled aptamer was then hybridized with AuNPs functionalized ssDNA which was complementary to the aptamer, obtaining the aptasensor. The CRET between ABEI and AuNPs in the aptasensor led to the CL quenching of ABEI. In the presence of a target analyte, it formed a complex with aptamer, and released ABEI-aptamer from AuNPs surface that resulted in CL recovery of ABEI. To test this design, a thrombin (used as a model analyte) aptasensor was prepared and evaluated. The results indicate that the proposed approach is simple and provided a linear range of 50-550 pM for thrombin detection with a detection limit of 15 pM. This new methodology can be easily extended to assay other biomolecules by simply changing the recognition sequence with the substrate aptamer. Copyright © 2013 Elsevier B.V. All rights reserved.

Global measurement of coagulation in plasma from normal and haemophilia dogs using a novel modified thrombin generation test – Demonstrated in vitro and ex vivo

PubMed Central

Madsen, Daniel Elenius; Nichols, Timothy C.; Merricks, Elizabeth P.; Waters, Emily K.; Wiinberg, Bo

2017-01-01

Introduction Canine models of severe haemophilia resemble their human equivalents both regarding clinical bleeding phenotype and response to treatment. Therefore pre-clinical studies in haemophilia dogs have allowed researchers to make valuable translational predictions regarding the potency and efficacy of new anti-haemophilia drugs (AHDs) in humans. To refine in vivo experiments and reduce number of animals, such translational studies are ideally preceded by in vitro prediction of compound efficacy using a plasma based global coagulation method. One such widely used method is the thrombin generation test (TGT). Unfortunately, commercially available TGTs are incapable of distinguishing between normal and haemophilia canine plasma, and therefore in vitro prediction using TGT has so far not been possible in canine plasma material. Aim Establish a modified TGT capable of: 1) distinguishing between normal and haemophilia canine plasma, 2) monitoring correlation between canine plasma levels of coagulation factor VIII (FVIII) and IX (FIX) and thrombin generation, 3) assessing for agreement between compound activity and thrombin generation in ex vivo samples. Methods A modified TGT assay was established where coagulation was triggered using a commercially available activated partial thromboplastin time reagent. Results With the modified TGT a significant difference was observed in thrombin generation between normal and haemophilia canine plasma. A dose dependent thrombin generation was observed when assessing haemophilia A and B plasma spiked with dilution series of FVIII and FIX, respectively. Correlation between FVIII activity and thrombin generation was observed when analyzing samples from haemophilia A dogs dosed with canine FVIII. Limit of detection was 0.1% (v/v) FVIII or FIX. Conclusion A novel modified TGT suitable for monitoring and prediction of replacement therapy efficacy in plasma from haemophilia A and B dogs was established. PMID:28384182

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

PubMed Central

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

2011-01-01

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

A post-labeling strategy based on dye-induced peeling of the aptamer off single-walled carbon nanotubes for electrochemical aptasensing.

PubMed

Fu, Yingchun; Wang, Ting; Bu, Lijuan; Xie, Qingji; Li, Penghao; Chen, Jinhua; Yao, Shouzhuo

2011-03-07

A simple and efficient post-labeling strategy based on dye-induced peeling of the aptamer molecules off single-walled carbon nanotubes was developed for electrochemical aptasensing of thrombin with a detection limit down to 3 pM.

Optimisation and Characterisation of Anti-Fouling Ternary SAM Layers for Impedance-Based Aptasensors

PubMed Central

Miodek, Anna; Regan, Edward M.; Bhalla, Nikhil; Hopkins, Neal A.E.; Goodchild, Sarah A.; Estrela, Pedro

2015-01-01

An aptasensor with enhanced anti-fouling properties has been developed. As a case study, the aptasensor was designed with specificity for human thrombin. The sensing platform was developed on screen printed electrodes and is composed of a self-assembled monolayer made from a ternary mixture of 15-base thiolated DNA aptamers specific for human thrombin co-immobilised with 1,6-hexanedithiol (HDT) and further passivated with 1-mercapto-6-hexanol (MCH). HDT binds to the surface by two of its thiol groups forming alkyl chain bridges and this architecture protects from non-specific attachment of molecules to the electrode surface. Using Electrochemical Impedance Spectroscopy (EIS), the aptasensor is able to detect human thrombin as variations in charge transfer resistance (Rct) upon protein binding. After exposure to a high concentration of non-specific Bovine Serum Albumin (BSA) solution, no changes in the Rct value were observed, highlighting the bio-fouling resistance of the surface generated. In this paper, we present the optimisation and characterisation of the aptasensor based on the ternary self-assembled monolayer (SAM) layer. We show that anti-fouling properties depend on the type of gold surface used for biosensor construction, which was also confirmed by contact angle measurements. We further studied the ratio between aptamers and HDT, which can determine the specificity and selectivity of the sensing layer. We also report the influence of buffer pH and temperature used for incubation of electrodes with proteins on detection and anti-fouling properties. Finally, the stability of the aptasensor was studied by storage of modified electrodes for up to 28 days in different buffers and atmospheric conditions. Aptasensors based on ternary SAM layers are highly promising for clinical applications for detection of a range of proteins in real biological samples. PMID:26426017

Optimisation and Characterisation of Anti-Fouling Ternary SAM Layers for Impedance-Based Aptasensors.

PubMed

Miodek, Anna; Regan, Edward M; Bhalla, Nikhil; Hopkins, Neal A E; Goodchild, Sarah A; Estrela, Pedro

2015-09-29

An aptasensor with enhanced anti-fouling properties has been developed. As a case study, the aptasensor was designed with specificity for human thrombin. The sensing platform was developed on screen printed electrodes and is composed of a self-assembled monolayer made from a ternary mixture of 15-base thiolated DNA aptamers specific for human thrombin co-immobilised with 1,6-hexanedithiol (HDT) and further passivated with 1-mercapto-6-hexanol (MCH). HDT binds to the surface by two of its thiol groups forming alkyl chain bridges and this architecture protects from non-specific attachment of molecules to the electrode surface. Using Electrochemical Impedance Spectroscopy (EIS), the aptasensor is able to detect human thrombin as variations in charge transfer resistance (Rct) upon protein binding. After exposure to a high concentration of non-specific Bovine Serum Albumin (BSA) solution, no changes in the Rct value were observed, highlighting the bio-fouling resistance of the surface generated. In this paper, we present the optimisation and characterisation of the aptasensor based on the ternary self-assembled monolayer (SAM) layer. We show that anti-fouling properties depend on the type of gold surface used for biosensor construction, which was also confirmed by contact angle measurements. We further studied the ratio between aptamers and HDT, which can determine the specificity and selectivity of the sensing layer. We also report the influence of buffer pH and temperature used for incubation of electrodes with proteins on detection and anti-fouling properties. Finally, the stability of the aptasensor was studied by storage of modified electrodes for up to 28 days in different buffers and atmospheric conditions. Aptasensors based on ternary SAM layers are highly promising for clinical applications for detection of a range of proteins in real biological samples.

Thrombin impairs human endometrial endothelial angiogenesis; implications for progestin-only contraceptive-induced abnormal uterine bleeding.

PubMed

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

2017-06-01

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-only contraceptive-induced abnormal uterine bleeding. Copyright © 2017. Published by Elsevier Inc.

Ultrasensitive thrombin detection based on direct electrochemistry of highly loaded hemoglobin spheres-encapsulated platinum nanoparticles as labels and electrocatalysts.

PubMed

Wu, Yongmei; Xu, Wenju; Bai, Lijuan; Yuan, Yali; Yi, Huayu; Chai, Yaqin; Yuan, Ruo

2013-12-15

For the first time, a sandwich-type electrochemical method was proposed for ultrasensitive thrombin (TB) detection based on direct electrochemistry of highly loaded hemoglobin spheres-encapsulated platinum nanoparticles (PtNPs@Hb) as labels and electrocatalysts. The prepared PtNPs@Hb not only exhibited good biocompatibility, excellent electrocatalytic activity, but also presented redox activity of Hb. Thus, it was employed for the fabrication of aptasensor without any extraneous redox mediators, leading to a simple preparation process for the aptasensor. The high loading of Hb spheres as redox mediators could enhance the electrochemical signal. Importantly, the synergetic electrocatalytic behavior of Hb and PtNPs toward H2O2 reduction greatly amplified the electrochemical signal, resulting in the high sensitivity of aptasensor. Consequently, under optimal conditions, the designed aptasensor exhibited a lower detection limit of 0.05 pM and wide dynamic linear range from 0.15 pM to 40 nM for TB detection. Additionally, the proposed mediator-free and signal-amplified electrochemical aptasensor showed great potential in portable and cost-effective TB sensing devices. Copyright © 2013 Elsevier B.V. All rights reserved.

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

PubMed Central

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

2015-01-01

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

Enhancement of aptamer immobilization using egg shell-derived nano-sized spherical hydroxyapatite for thrombin detection in neuroclinic.

PubMed

Derkus, Burak; Arslan, Yavuz Emre; Emregul, Kaan C; Emregul, Emel

2016-09-01

In the present study, we describe the sonochemical isolation of nano-sized spherical hydroxyapatite (nHA) from egg shell and application towards thrombin aptasensing. In addition to the sonochemical method, two conventional methods present in literature were carried out to perform a comparative study. Various analysis methods including Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR), X-Ray Diffraction (XRD), Energy-Dispersive Analysis of X-Rays (EDAX), and Thermal Gravimetric Analysis (TGA) have been applied for the characterization of nHA and its nanocomposite with marine-derived collagen isolated from Rhizostoma pulmo jellyfish. TEM micrographs revealed the sonochemically synthesized nHA nanoparticles to have a unique porous spherical shape with a diameter of approximately 60-80nm when compared to hydroxyapatite nanoparticles synthesized using the other two methods which had a typical needle shaped morphology. EDAX, XRD and FTIR results demonstrated that the obtained patterns belonged to hydroxyapatite. Electrochemical impedance spectroscopy (EIS) is the main analyzing technique of the developed thrombin aptasensor. The proposed aptasensor has a detection limit of 0.25nM thrombin. For clinical application of the developed aptasensor, thrombin levels in blood and cerebrospinal fluid (CSF) samples obtained from patients with Multiple Sclerosis, Myastenia Gravis, Epilepsy, Parkinson, polyneuropathy and healthy donors were analyzed using both the aptasensor and commercial ELISA kit. The results showed that the proposed system is a promising candidate for clinical analysis of thrombin. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular sensing with magnetic nanoparticles using magnetic spectroscopy of nanoparticle Brownian motion.

PubMed

Zhang, Xiaojuan; Reeves, Daniel B; Perreard, Irina M; Kett, Warren C; Griswold, Karl E; Gimi, Barjor; Weaver, John B

2013-12-15

Functionalized magnetic nanoparticles (mNPs) have shown promise in biosensing and other biomedical applications. Here we use functionalized mNPs to develop a highly sensitive, versatile sensing strategy required in practical biological assays and potentially in vivo analysis. We demonstrate a new sensing scheme based on magnetic spectroscopy of nanoparticle Brownian motion (MSB) to quantitatively detect molecular targets. MSB uses the harmonics of oscillating mNPs as a metric for the freedom of rotational motion, thus reflecting the bound state of the mNP. The harmonics can be detected in vivo from nanogram quantities of iron within 5s. Using a streptavidin-biotin binding system, we show that the detection limit of the current MSB technique is lower than 150 pM (0.075 pmole), which is much more sensitive than previously reported techniques based on mNP detection. Using mNPs conjugated with two anti-thrombin DNA aptamers, we show that thrombin can be detected with high sensitivity (4 nM or 2 pmole). A DNA-DNA interaction was also investigated. The results demonstrated that sequence selective DNA detection can be achieved with 100 pM (0.05 pmole) sensitivity. The results of using MSB to sense these interactions, show that the MSB based sensing technique can achieve rapid measurement (within 10s), and is suitable for detecting and quantifying a wide range of biomarkers or analytes. It has the potential to be applied in variety of biomedical applications or diagnostic analyses. © 2013 Elsevier B.V. All rights reserved.

Impact of Hormone-Associated Resistance to Activated Protein C on the Thrombotic Potential of Oral Contraceptives: A Prospective Observational Study

PubMed Central

Müller, Jens; Sukhitashvili, Shorena; Welz, Julia; Kuhn, Walther C.; Oldenburg, Johannes; Rudlowski, Christian; Pötzsch, Bernd

2014-01-01

Introduction The increased thrombotic risk of oral contraceptives (OC) has been attributed to various alterations of the hemostatic system, including acquired resistance to activated protein C (APC). To evaluate to what extent OC-associated APC resistance induces a prothrombotic state we monitored plasma levels of thrombin and molecular markers specific for thrombin formation in women starting OC use. Elevated plasma levels of thrombin have been reported to characterize situations of high thrombotic risk such as trauma-induced hypercoagulability, but have not yet been studied during OC use. Patients and Methods Blood samples were collected prospectively from healthy women (n = 21) before and during three menstruation cycles after start of OC. APC resistance was evaluated using a thrombin generation-based assay. Plasma levels of thrombin and APC were directly measured using highly sensitive oligonucleotide-based enzyme capture assay (OECA) technology. Thrombin generation markers and other hemostasis parameters were measured additionally. Results All women developed APC resistance as indicated by an increased APC sensitivity ratio compared with baseline after start of OC (p = 0.0003). Simultaneously, plasma levels of thrombin, prothrombin fragment 1+2, and of thrombin-antithrombin complexes did not change, ruling out increased thrombin formation. APC plasma levels were also not influenced by OC use, giving further evidence that increased thrombin formation did not occur. Conclusions In the majority of OC users no enhanced thrombin formation occurs despite the development of APC resistance. It cannot be ruled out, however, that thrombin formation might occur to a greater extent in the presence of additional risk factors. If this were the case, endogenous thrombin levels might be a potential biomarker candidate to identify women at high thrombotic risk during OC treatment. Large-scale studies are required to assess the value of plasma levels of thrombin as predictors of OC-associated thrombotic risk. PMID:25121606

Gold-Coated Superparamagnetic Nanoparticles for Single Methyl Discrimination in DNA Aptamers

PubMed Central

Tintoré, Maria; Mazzini, Stefania; Polito, Laura; Marelli, Marcello; Latorre, Alfonso; Somoza, Álvaro; Aviñó, Anna; Fàbrega, Carme; Eritja, Ramon

2015-01-01

Au- and iron-based magnetic nanoparticles (NPs) are promising NPs for biomedical applications due to their unique properties. The combination of a gold coating over a magnetic core puts together the benefits from adding the magnetic properties to the robust chemistry provided by the thiol functionalization of gold. Here, the use of Au-coated magnetic NPs for molecular detection of a single methylation in DNA aptamer is described. Binding of α-thrombin to two aptamers conjugated to these NPs causes aggregation, a phenomenon that can be observed by UV, DLS and MRI. These techniques discriminate a single methylation in one of the aptamers, preventing aggregation due to the inability of α-thrombin to recognize it. A parallel study with gold and ferromagnetic NPs is detailed, concluding that the Au coating of FexOy NP does not affect their performance and that they are suitable as complex biosensors. These results prove the high detection potency of Au-coated SPIONs for biomedical applications especially for DNA repair detection. PMID:26593913

Tetra(p-tolyl)borate-functionalized solvent polymeric membrane: a facile and sensitive sensing platform for peroxidase and peroxidase mimetics.

PubMed

Wang, Xuewei; Qin, Wei

2013-07-22

The determination of peroxidase activities is the basis for enzyme-labeled bioaffinity assays, peroxidase-mimicking DNAzymes- and nanoparticles-based assays, and characterization of the catalytic functions of peroxidase mimetics. Here, a facile, sensitive, and cost-effective solvent polymeric membrane-based peroxidase detection platform is described that utilizes reaction intermediates with different pKa values from those of substrates and final products. Several key but long-debated intermediates in the peroxidative oxidation of o-phenylenediamine (o-PD) have been identified and their charge states have been estimated. By using a solvent polymeric membrane functionalized by an appropriate substituted tetraphenylborate as a receptor, those cationic intermediates could be transferred into the membrane from the aqueous phase to induce a large cationic potential response. Thus, the potentiometric indication of the o-PD oxidation catalyzed by peroxidase or its mimetics can be fulfilled. Horseradish peroxidase has been detected with a detection limit at least two orders of magnitude lower than those obtained by spectrophotometric techniques and traditional membrane-based methods. As an example of peroxidase mimetics, G-quadruplex DNAzymes were probed by the intermediate-sensitive membrane and a label-free thrombin detection protocol was developed based on the catalytic activity of the thrombin-binding G-quadruplex aptamer. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

3D DNA origami as programmable anchoring points for bioreceptors in fiber optic surface plasmon resonance biosensing.

PubMed

Daems, Devin; Pfeifer, Wolfgang; Rutten, Iene; Sacca, Barbara; Spasic, Dragana; Lammertyn, Jeroen

2018-06-27

Many challenges in biosensing originate from the fact that the all-important nano-architecture of the biosensor's surface, including precise density and orientation of bioreceptors, is not entirely comprehended. Here we introduced a 3D DNA origami as bioreceptor carrier to functionalize the fiber optic surface plasmon resonance (FO-SPR) sensor with nanoscale precision. Starting from a 24-helix bundle, two distinct DNA origami structures were designed to position thrombin-specific aptamers with different density and distance (27 and 113 nm) from the FO-SPR surface. The origami-based biosensors proved to be not only capable of reproducible, label-free thrombin detection, but revealed also valuable innovative features: (1) a significantly better performance in the absence of backfilling, known as essential in biosensing field, suggesting improved bioreceptor orientation and accessibility and (2) a wider linear range compared to previously reported thrombin biosensors. We envisage that our method will be beneficial both for scientists and clinicians looking for new surface (bio)chemistry and improved diagnostics.

Thrombin-inhibiting nanoparticles rapidly constitute versatile and detectable anticlotting surfaces

NASA Astrophysics Data System (ADS)

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

2014-09-01

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

Concizumab, an anti-tissue factor pathway inhibitor antibody, induces increased thrombin generation in plasma from haemophilia patients and healthy subjects measured by the thrombin generation assay.

PubMed

Waters, E K; Sigh, J; Friedrich, U; Hilden, I; Sørensen, B B

2017-09-01

Concizumab, a humanized monoclonal antibody against tissue factor pathway inhibitor (TFPI), is being developed as a subcutaneously (s.c.) administered treatment for haemophilia. It demonstrated a concentration-dependent procoagulant effect in functional TFPI assays; however, global haemostatic assays, such as the thrombin generation assay (TGA), offer a more complete picture of coagulation. We investigated how concizumab affects thrombin generation following ex vivo spiking in plasma from haemophilia patients using the TGA, and if the assay can detect the effect of multiple s.c. concizumab doses in healthy subjects. For the ex vivo spiking study, platelet-poor plasma (PPP) from 18 patients with severe haemophilia was spiked with 0.001-500 nm concizumab. For the multiple-dosing study, four healthy males received concizumab 250 μg kg -1 s.c. every other day for eight doses; blood was collected before and after dosing and processed into PPP. In both studies, thrombin generation was measured using a Calibrated Automated Thrombogram ® system with 1 pm tissue factor. In spiked samples from haemophilia patients, peak thrombin and endogenous thrombin potential (ETP) increased concentration dependently, reaching near-normal levels at concizumab concentrations >10 nm. Repeated s.c. doses of concizumab in healthy subjects increased both peak thrombin and ETP; these effects were sustained throughout the dosing interval. Thrombin generation assay demonstrated increased thrombin generation with concizumab after ex vivo spiking of haemophilia plasma and multiple s.c. doses in healthy subjects, supporting both the utility of the TGA in evaluating concizumab treatment and the potential of s.c. concizumab as a novel haemophilia therapy. © 2017 The Authors. Haemophilia Published by John Wiley & Sons Ltd.

Heparin Cofactor II in Atherosclerotic Lesions from the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) Study

PubMed Central

Rau, Jill C.; Deans, Carolyn; Hoffman, Maureane R.; Thomas, David B.; Malcom, Gray T.; Zieske, Arthur W.; Strong, Jack P.; Koch, Gary G.; Church, Frank C.

2009-01-01

Heparin cofactor II (HCII) is a serine protease inhibitor (serpin) that has been shown to be a predictor of decreased atherosclerosis in the elderly and protective against atherosclerosis in mice. HCII inhibits thrombin in vitro and HCII-thrombin complexes have been detected in human plasma. Moreover, the mechanism of protection against atherosclerosis in mice was determined to be the inhibition of thrombin. Despite this evidence, the presence of HCII in human atherosclerotic tissue has not been reported. In this study, using samples of coronary arteries obtained from the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study, we explore the local relationship between HCII and (pro)thrombin in atherosclerosis. We found that HCII and (pro)thrombin are co-localized in the lipid-rich necrotic core of atheromas. A significant positive correlation between each protein and the severity of the atherosclerotic lesion was present. These results suggest that HCII is in a position to inhibit thrombin in atherosclerotic lesions where thrombin can exert a proatherogenic inflammatory response. However, these results should be tempered by the additional findings from this, and other studies, that indicate the presence of other plasma proteins (antithrombin, albumin, and α1-protease inhibitor) in the same localized region of the atheroma. PMID:19747479

Screening of benzamidine-based thrombin inhibitors via a linear interaction energy in continuum electrostatics model

NASA Astrophysics Data System (ADS)

Nicolotti, Orazio; Giangreco, Ilenia; Miscioscia, Teresa Fabiola; Convertino, Marino; Leonetti, Francesco; Pisani, Leonardo; Carotti, Angelo

2010-02-01

A series of 27 benzamidine inhibitors covering a wide range of biological activity and chemical diversity was analysed to derive a Linear Interaction Energy in Continuum Electrostatics (LIECE) model for analysing the thrombin inhibitory activity. The main interactions occurring at the thrombin binding site and the preferred binding conformations of inhibitors were explicitly biased by including into the LIECE model 10 compounds extracted from X-ray solved thrombin-inhibitor complexes available from the Protein Data Bank (PDB). Supported by a robust statistics ( r 2 = 0.698; q 2 = 0.662), the LIECE model was successful in predicting the inhibitory activity for about 76% of compounds ( r ext 2 ≥ 0.600) from a larger external test set encompassing 88 known thrombin inhibitors and, more importantly, in retrieving, at high sensitivity and with better performance than docking and shape-based methods, active compounds from a thrombin combinatorial library of 10240 mimetic chemical products. The herein proposed LIECE model has the potential for successfully driving the design of novel thrombin inhibitors with benzamidine and/or benzamidine-like chemical structure.

An amplified electrochemiluminescent aptasensor using Au nanoparticles capped by 3,4,9,10-perylene tetracarboxylic acid-thiosemicarbazide functionalized C60 nanocomposites as a signal enhancement tag

NASA Astrophysics Data System (ADS)

Ma, Meng-Nan; Zhang, Xia; Zhuo, Ying; Chai, Ya-Qin; Yuan, Ruo

2015-01-01

A novel electrochemiluminescent (ECL) signal tag of Au nanoparticles capped by 3,4,9,10-perylene tetracarboxylic acid-thiosemicarbazide functionalized C60 nanocomposites (AuNPs/TSC-PTC/C60NPs) was developed for thrombin (TB) aptasensor construction based on the peroxydisulfate/oxygen (S2O82-/O2) system. For signal tag fabrication, the C60 nanoparticles (C60NPs) were prepared and then coated with 3,4,9,10-perylene tetracarboxylic acid (PTCA) by π-π stacking interactions. Afterwards, thiosemicarbazide (TSC) was linked with PTCA functionalized C60NPs via amidation for further assembling Au nanoparticles (AuNPs). Finally, detection aptamer of thrombin (TBA 2) was labeled on the ECL signal amplification tag of AuNPs/TSC-PTC/C60NPs. Herein, TSC, with the active groups of -NH2 and -SH, was selected and introduced into the ECL S2O82-/O2 system for the first time, which could not only offer the active groups of -SH to absorb AuNPs for TBA 2 anchoring but also remarkably enhance the ECL signal of the S2O82-/O2 system by the formation of TSC-PTC/C60NPs for signal amplification. Meanwhile, the sensing interface of a glassy carbon electrode (GCE) was modified by AuNPs/graphene (AuNPs/GR) nanocomposites with the large specific surface area and the active sites, followed by immobilization of thiol-terminated thrombin capture aptamer (TBA 1). With the formation of the sandwich-type structure of TBA 1, TB, and TBA 2 signal probes, a desirable enhanced ECL signal was measured in the testing buffer of an S2O82-/O2 solution for detecting TB. The aptasensor exhibited a good linear relationship for TB detection in the range of 1 × 10-5-10 nM with a detection limit of 3.3 fM.A novel electrochemiluminescent (ECL) signal tag of Au nanoparticles capped by 3,4,9,10-perylene tetracarboxylic acid-thiosemicarbazide functionalized C60 nanocomposites (AuNPs/TSC-PTC/C60NPs) was developed for thrombin (TB) aptasensor construction based on the peroxydisulfate/oxygen (S2O82-/O2) system. For signal tag fabrication, the C60 nanoparticles (C60NPs) were prepared and then coated with 3,4,9,10-perylene tetracarboxylic acid (PTCA) by π-π stacking interactions. Afterwards, thiosemicarbazide (TSC) was linked with PTCA functionalized C60NPs via amidation for further assembling Au nanoparticles (AuNPs). Finally, detection aptamer of thrombin (TBA 2) was labeled on the ECL signal amplification tag of AuNPs/TSC-PTC/C60NPs. Herein, TSC, with the active groups of -NH2 and -SH, was selected and introduced into the ECL S2O82-/O2 system for the first time, which could not only offer the active groups of -SH to absorb AuNPs for TBA 2 anchoring but also remarkably enhance the ECL signal of the S2O82-/O2 system by the formation of TSC-PTC/C60NPs for signal amplification. Meanwhile, the sensing interface of a glassy carbon electrode (GCE) was modified by AuNPs/graphene (AuNPs/GR) nanocomposites with the large specific surface area and the active sites, followed by immobilization of thiol-terminated thrombin capture aptamer (TBA 1). With the formation of the sandwich-type structure of TBA 1, TB, and TBA 2 signal probes, a desirable enhanced ECL signal was measured in the testing buffer of an S2O82-/O2 solution for detecting TB. The aptasensor exhibited a good linear relationship for TB detection in the range of 1 × 10-5-10 nM with a detection limit of 3.3 fM. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr05918b

Correction of microplate location effects improves performance of the thrombin generation test

PubMed Central

2013-01-01

Background Microplate-based thrombin generation test (TGT) is widely used as clinical measure of global hemostatic potential and it becomes a useful tool for control of drug potency and quality by drug manufactures. However, the convenience of the microtiter plate technology can be deceiving: microplate assays are prone to location-based variability in different parts of the microtiter plate. Methods In this report, we evaluated the well-to-well consistency of the TGT variant specifically applied to the quantitative detection of the thrombogenic substances in the immune globulin product. We also studied the utility of previously described microplate layout designs in the TGT experiment. Results Location of the sample on the microplate (location effect) contributes to the variability of TGT measurements. Use of manual pipetting techniques and applications of the TGT to the evaluation of procoagulant enzymatic substances are especially sensitive. The effects were not sensitive to temperature or choice of microplate reader. Smallest location effects were observed with automated dispenser-based calibrated thrombogram instrument. Even for an automated instrument, the use of calibration curve resulted in up to 30% bias in thrombogenic potency assignment. Conclusions Use of symmetrical version of the strip-plot layout was demonstrated to help to minimize location artifacts even under the worst-case conditions. Strip-plot layouts are required for quantitative thrombin-generation based bioassays used in the biotechnological field. PMID:23829491

Correction of microplate location effects improves performance of the thrombin generation test.

PubMed

Liang, Yideng; Woodle, Samuel A; Shibeko, Alexey M; Lee, Timothy K; Ovanesov, Mikhail V

2013-07-05

Microplate-based thrombin generation test (TGT) is widely used as clinical measure of global hemostatic potential and it becomes a useful tool for control of drug potency and quality by drug manufactures. However, the convenience of the microtiter plate technology can be deceiving: microplate assays are prone to location-based variability in different parts of the microtiter plate. In this report, we evaluated the well-to-well consistency of the TGT variant specifically applied to the quantitative detection of the thrombogenic substances in the immune globulin product. We also studied the utility of previously described microplate layout designs in the TGT experiment. Location of the sample on the microplate (location effect) contributes to the variability of TGT measurements. Use of manual pipetting techniques and applications of the TGT to the evaluation of procoagulant enzymatic substances are especially sensitive. The effects were not sensitive to temperature or choice of microplate reader. Smallest location effects were observed with automated dispenser-based calibrated thrombogram instrument. Even for an automated instrument, the use of calibration curve resulted in up to 30% bias in thrombogenic potency assignment. Use of symmetrical version of the strip-plot layout was demonstrated to help to minimize location artifacts even under the worst-case conditions. Strip-plot layouts are required for quantitative thrombin-generation based bioassays used in the biotechnological field.

Dynamic Consequences of Mutation of Tryptophan 215 in Thrombin.

PubMed

Peacock, Riley B; Davis, Jessie R; Markwick, Phineus R L; Komives, Elizabeth A

2018-05-08

Thrombin normally cleaves fibrinogen to promote coagulation; however, binding of thrombomodulin to thrombin switches the specificity of thrombin toward protein C, triggering the anticoagulation pathway. The W215A thrombin mutant was reported to have decreased activity toward fibrinogen without significant loss of activity toward protein C. To understand how mutation of Trp215 may alter thrombin specificity, hydrogen-deuterium exchange experiments (HDXMS), accelerated molecular dynamics (AMD) simulations, and activity assays were carried out to compare the dynamics of Trp215 mutants with those of wild type (WT) thrombin. Variation in NaCl concentration had no detectable effect on the sodium-binding (220s CT ) loop, but appeared to affect other surface loops. Trp215 mutants showed significant increases in amide exchange in the 170s CT loop consistent with a loss of H-bonding in this loop identified by the AMD simulations. The W215A thrombin showed increased amide exchange in the 220s CT loop and in the N-terminus of the heavy chain. The AMD simulations showed that a transient conformation of the W215A thrombin has a distorted catalytic triad. HDXMS experiments revealed that mutation of Phe227, which engages in a π-stacking interaction with Trp215, also caused significantly increased amide exchange in the 170s CT loop. Activity assays showed that only the F227V mutant had wild type catalytic activity, whereas all other mutants showed markedly lower activity. Taken together, the results explain the reduced pro-coagulant activity of the W215A mutant and demonstrate the allosteric connection between Trp215, the sodium-binding loop, and the active site.

Diagnosing human blood clotting deficiency.

PubMed

Ong, Chong Cheen; Gopinath, Subash C B; Rebecca, Leong Wei Xian; Perumal, Veeradasan; Lakshmipriya, Thangavel; Saheed, Mohamed Shuaib Mohamed

2018-05-15

There are different clotting factors present in blood, carries the clotting cascade and excessive bleeding may cause a deficiency in the clotting Diagnosis of this deficiency in clotting drastically reduces the potential fatality. For enabling a sensor to detect the clotting factors, suitable probes such as antibody and aptamer have been used to capture these targets on the sensing surface. Two major clotting factors were widely studied for the diagnosis of clotting deficiency, which includes factor IX and thrombin. In addition, factor IX is considered as the substitute for heparin and the prothrombotic associated with the increased thrombin generation are taking into account their prevalence. The biosensors, surface plasmon resonance, evanescent-field-coupled waveguide-mode sensor, metal-enhanced PicoGreen fluorescence and electrochemical aptasensor were well-documented and improvements have been made for high-performance sensing. We overviewed detecting factor IX and thrombin using these biosensors, for the potential application in medical diagnosis. Copyright © 2018 Elsevier B.V. All rights reserved.

Activation of PAR-1/NADPH oxidase/ROS signaling pathways is crucial for the thrombin-induced sFlt-1 production in extravillous trophoblasts: possible involvement in the pathogenesis of preeclampsia.

PubMed

Huang, Qi-Tao; Chen, Jian-Hong; Hang, Li-Lin; Liu, Shi-San; Zhong, Mei

2015-01-01

Preeclampsia was characterized by excessive thrombin generation in placentas and previous researches showed that thrombin could enhance soluble Fms-like tyrosine kinase 1 (sFlt-1) expression in first trimester trophoblasts. However, the detailed mechanism for the sFlt-1 over-production induced by thrombin was largely unknown. The purpose of this study was to explore the possible signaling pathway of thrombin-induced sFlt-1 production in extravillous trophoblasts (EVT). An EVT cell line (HRT-8/SVneo) was treated with various concentrations of thrombin. The mRNA expression and protein secretion of sFlt-1 in EVT were detected with real-time polymerase chain reaction and ELISA, respectively. The levels of intracellular reactive oxygen species (ROS) production were determined by DCFH-DA. Exposure of EVT to thrombin induced increased intracellular ROS generation and overexpression of sFlt-1 at both mRNA and protein levels in a dose dependent manner. Short interfering RNA (siRNA) directed against PAR-1 or apocynin (an inhibitor of NADPH oxidase) could decrease the intracellular ROS generation and subsequently suppressed the production of sFlt-1 at mRNA and protein levels. Our results suggested that thrombin increased sFlt-1 production in EVT via the PAR-1 /NADPH oxidase /ROS signaling pathway. This also highlights the PAR-1 / NADPH oxidase / ROS pathway might be a potential therapeutic target for the prevention of preeclampsia in the future. © 2015 S. Karger AG, Basel.

Therapeutic Correction of Thrombin Generation in Dilution-Induced Coagulopathy: Computational Analysis Based on a Data Set of Healthy Subjects

DTIC Science & Technology

2012-01-01

Factor VIIa tended to primarily impact clotting time, thrombin peak time, and maximum slope of the thrombin curve, whereas in the case of PCC- FVII ...constituents of existing PCCs are the four coagulation factors (F) II (prothrombin), FVII , FIX, and FX.3 Notably, FVII inhibits thrombin generation by...proposed PCC composition (coagulation factors [F] II, IX, and X and the anticoagulant antithrombin), designated PCC-AT, was compared with that of

Polyethylenimine-coated Fe3O4 nanoparticles effectively quench fluorescent DNA, which can be developed as a novel platform for protein detection.

PubMed

Ma, Long; Sun, Nana; Zhang, Jinyan; Tu, Chunhao; Cao, Xiuqi; Duan, Demin; Diao, Aipo; Man, Shuli

2017-11-23

We report a novel assembly of polyethyleneimine (PEI)-coated Fe 3 O 4 nanoparticles (NPs) with single-stranded DNA (ssDNA), and the fluorescence of the dye labeled in the DNA is remarkably quenched. In the presence of a target protein, the protein-DNA aptamer mutual interaction releases the ssDNA from this assembly and hence restores the fluorescence. This feature could be adopted to develop an aptasensor for protein detection. As a proof-of-concept, for the first time, we have used this proposed sensing strategy to detect thrombin selectively and sensitively. Furthermore, simultaneous multiple detection of thrombin and lysozyme in a complex protein mixture has been proven to be possible.

Application of 2-dimensional difference gel electrophoresis (2D-DIGE) to the study of thrombin-activated human platelet secretome.

PubMed

Della Corte, Anna; Maugeri, Norma; Pampuch, Agnieszka; Cerletti, Chiara; de Gaetano, Giovanni; Rotilio, Domenico

2008-02-01

Thrombin is an agonist inducing platelet activation. We combined two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MALDI-TOF MS) to analyse differentially expressed proteins secreted from thrombin-stimulated platelets. Human washed platelets, from healthy volunteers, were stimulated with thrombin 0.5 U/ml at 37 degrees C without stirring and the secreted proteins were resolved by 2D-DIGE. By image analysis, 1094 spots were detected in the 2D gel. The spots whose mean intensity showed at least a five-fold change intensity increase or decrease in the thrombin-activated platelet gel in comparison with unstimulated control were digested by trypsin and subjected to MALDI-TOF MS analysis. Peptides from mass spectra of in-gel digest samples were matched against available databases, using the Mascot search engine (Matrix Science) for peptide mass fingerprint. In the activated platelet secretome, transferrin, glutathione-transferase, WD repeat protein, ER-60, thrombospondin-1 precursor and thrombospondin were the most abundant. Also lamin A, a nuclear protein, not previously identified in platelets, appeared to be released. The novel strategy to combine 2D-DIGE with MALDI-TOF MS is a useful approach for a quantitative analysis of the effect of thrombin on the secretome profile of human platelets.

Non-invasive imaging and cellular tracking of pulmonary emboli by near-infrared fluorescence and positron-emission tomography

PubMed Central

Page, Michael J.; Lourenço, André L.; David, Tovo; LeBeau, Aaron M.; Cattaruzza, Fiore; Castro, Helena C.; VanBrocklin, Henry F.; Coughlin, Shaun R.; Craik, Charles S.

2015-01-01

Functional imaging of proteolytic activity is an emerging strategy to quantify disease and response to therapy at the molecular level. We present a new peptide-based imaging probe technology that advances these goals by exploiting enzymatic activity to deposit probes labelled with near-infrared (NIR) fluorophores or radioisotopes in cell membranes of disease-associated proteolysis. This strategy allows for non-invasive detection of protease activity in vivo and ex vivo by tracking deposited probes in tissues. We demonstrate non-invasive detection of thrombin generation in a murine model of pulmonary embolism using our protease-activated peptide probes in microscopic clots within the lungs with NIR fluorescence optical imaging and positron-emission tomography. Thrombin activity is imaged deep in tissue and tracked predominantly to platelets within the lumen of blood vessels. The modular design of our probes allows for facile investigation of other proteases, and their contributions to disease by tailoring the protease activation and cell-binding elements. PMID:26423607

Investigation of the heparin-thrombin interaction by dynamic force spectroscopy.

PubMed

Wang, Congzhou; Jin, Yingzi; Desai, Umesh R; Yadavalli, Vamsi K

2015-06-01

The interaction between heparin and thrombin is a vital step in the blood (anti)coagulation process. Unraveling the molecular basis of the interactions is therefore extremely important in understanding the mechanisms of this complex biological process. In this study, we use a combination of an efficient thiolation chemistry of heparin, a self-assembled monolayer-based single molecule platform, and a dynamic force spectroscopy to provide new insights into the heparin-thrombin interaction from an energy viewpoint at the molecular scale. Well-separated single molecules of heparin covalently attached to mixed self-assembled monolayers are demonstrated, whereby interaction forces with thrombin can be measured via atomic force microscopy-based spectroscopy. Further these interactions are studied at different loading rates and salt concentrations to directly obtain kinetic parameters. An increase in the loading rate shows a higher interaction force between the heparin and thrombin, which can be directly linked to the kinetic dissociation rate constant (koff). The stability of the heparin/thrombin complex decreased with increasing NaCl concentration such that the off-rate was found to be driven primarily by non-ionic forces. These results contribute to understanding the role of specific and nonspecific forces that drive heparin-thrombin interactions under applied force or flow conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

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

PubMed Central

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

2012-01-01

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

Structure of a retro-binding peptide inhibitor complexed with human alpha-thrombin.

PubMed

Tabernero, L; Chang, C Y; Ohringer, S L; Lau, W F; Iwanowicz, E J; Han, W C; Wang, T C; Seiler, S M; Roberts, D G; Sack, J S

1995-02-10

The crystallographic structure of the ternary complex between human alpha-thrombin, hirugen and the peptidyl inhibitor Phe-alloThr-Phe-O-CH3, which is acylated at its N terminus with 4-guanidino butanoic acid (BMS-183507), has been determined at 2.6 A resolution. The structure reveals a unique "retro-binding" mode for this tripeptide active site inhibitor. The inhibitor binds with its alkyl-guanidine moiety in the primary specificity pocket and its two phenyl rings occupying the hydrophobic proximal and distal pockets of the thrombin active site. In this arrangement the backbone of the tripeptide forms a parallel beta-strand to the thrombin main-chain at the binding site. This is opposite to the orientation of the natural substrate, fibrinogen, and all the small active site-directed thrombin inhibitors whose bound structures have been previously reported. BMS-183507 is the first synthetic inhibitor proved to bind in a retro-binding fashion to thrombin, in a fashion similar to that of the N-terminal residues of the natural inhibitor hirudin. Furthermore, this new potent thrombin inhibitor (Ki = 17.2 nM) is selective for thrombin over other serine proteases tested and may be a template to be considered in designing hirudin-based thrombin inhibitors with interactions at the specificity pocket.

Phage display of the serpin alpha-1 proteinase inhibitor randomized at consecutive residues in the reactive centre loop and biopanned with or without thrombin.

PubMed

Scott, Benjamin M; Matochko, Wadim L; Gierczak, Richard F; Bhakta, Varsha; Derda, Ratmir; Sheffield, William P

2014-01-01

In spite of the power of phage display technology to identify variant proteins with novel properties in large libraries, it has only been previously applied to one member of the serpin superfamily. Here we describe phage display of human alpha-1 proteinase inhibitor (API) in a T7 bacteriophage system. API M358R fused to the C-terminus of T7 capsid protein 10B was directly shown to form denaturation-resistant complexes with thrombin by electrophoresis and immunoblotting following exposure of intact phages to thrombin. We therefore developed a biopanning protocol in which thrombin-reactive phages were selected using biotinylated anti-thrombin antibodies and streptavidin-coated magnetic beads. A library consisting of displayed API randomized at residues 357 and 358 (P2-P1) yielded predominantly Pro-Arg at these positions after five rounds of thrombin selection; in contrast the same degree of mock selection yielded only non-functional variants. A more diverse library of API M358R randomized at residues 352-356 (P7-P3) was also probed, yielding numerous variants fitting a loose consensus of DLTVS as judged by sequencing of the inserts of plaque-purified phages. The thrombin-selected sequences were transferred en masse into bacterial expression plasmids, and lysates from individual colonies were screening for API-thrombin complexing. The most active candidates from this sixth round of screening contained DITMA and AAFVS at P7-P3 and inhibited thrombin 2.1-fold more rapidly than API M358R with no change in reaction stoichiometry. Deep sequencing using the Ion Torrent platform confirmed that over 800 sequences were significantly enriched in the thrombin-panned versus naïve phage display library, including some detected using the combined phage display/bacterial lysate screening approach. Our results show that API joins Plasminogen Activator Inhibitor-1 (PAI-1) as a serpin amenable to phage display and suggest the utility of this approach for the selection of "designer serpins" with novel reactivity and/or specificity.

Phage Display of the Serpin Alpha-1 Proteinase Inhibitor Randomized at Consecutive Residues in the Reactive Centre Loop and Biopanned with or without Thrombin

PubMed Central

Scott, Benjamin M.; Matochko, Wadim L.; Gierczak, Richard F.; Bhakta, Varsha; Derda, Ratmir; Sheffield, William P.

2014-01-01

In spite of the power of phage display technology to identify variant proteins with novel properties in large libraries, it has only been previously applied to one member of the serpin superfamily. Here we describe phage display of human alpha-1 proteinase inhibitor (API) in a T7 bacteriophage system. API M358R fused to the C-terminus of T7 capsid protein 10B was directly shown to form denaturation-resistant complexes with thrombin by electrophoresis and immunoblotting following exposure of intact phages to thrombin. We therefore developed a biopanning protocol in which thrombin-reactive phages were selected using biotinylated anti-thrombin antibodies and streptavidin-coated magnetic beads. A library consisting of displayed API randomized at residues 357 and 358 (P2–P1) yielded predominantly Pro-Arg at these positions after five rounds of thrombin selection; in contrast the same degree of mock selection yielded only non-functional variants. A more diverse library of API M358R randomized at residues 352–356 (P7–P3) was also probed, yielding numerous variants fitting a loose consensus of DLTVS as judged by sequencing of the inserts of plaque-purified phages. The thrombin-selected sequences were transferred en masse into bacterial expression plasmids, and lysates from individual colonies were screening for API-thrombin complexing. The most active candidates from this sixth round of screening contained DITMA and AAFVS at P7–P3 and inhibited thrombin 2.1-fold more rapidly than API M358R with no change in reaction stoichiometry. Deep sequencing using the Ion Torrent platform confirmed that over 800 sequences were significantly enriched in the thrombin-panned versus naïve phage display library, including some detected using the combined phage display/bacterial lysate screening approach. Our results show that API joins Plasminogen Activator Inhibitor-1 (PAI-1) as a serpin amenable to phage display and suggest the utility of this approach for the selection of “designer serpins” with novel reactivity and/or specificity. PMID:24427287

Porous platinum nanotubes labeled with hemin/G-quadruplex based electrochemical aptasensor for sensitive thrombin analysis via the cascade signal amplification.

PubMed

Sun, Aili; Qi, Qingan; Wang, Xuannian; Bie, Ping

2014-07-15

For the first time, a sensitive electrochemical aptasensor for thrombin (TB) was developed by using porous platinum nanotubes (PtNTs) labeled with hemin/G-quadruplex and glucose dehydrogenase (GDH) as labels. Porous PtNTs with large surface area exhibited the peroxidase-like activity. Coupling with GDH and hemin/G-quadruplex as NADH oxidase and HRP-mimicking DNAzyme, the cascade signal amplification was achieved by the following ways: in the presence of glucose and NAD(+) in the working buffer, GDH electrocatalyzed the oxidation of glucose with the production of NADH. Then, hemin/G-quadruplex as NADH oxidase catalyzed the oxidation of NADH to in situ generate H2O2. Based on the corporate electrocatalysis of PtNTs and hemin/G-quadruplex toward H2O2, the electrochemical signal was significantly amplified, allowing the detection limit of TB down to 0.15 pM level. Moreover, the proposed strategy was simple because the intercalated hemin offered the readout signal, avoiding the adding of additional redox mediator as signal donator. Such an electrochemical aptasensor is highly promising for sensitive detection of other proteins in clinical diagnostics. Copyright © 2014 Elsevier B.V. All rights reserved.

Electrospray ionisation mass spectrometry facilitates detection of fibrinogen (Bbeta 14 Arg --> Cys) mutation in a family with thrombosis.

PubMed

Brennan, S O; Hammonds, B; Spearing, R; George, P M

1997-12-01

We report the first direct detection of a fibrinogen mutation by electrospray ionisation mass spectrometry. The propositus, from a family with a history of thrombosis, came to attention after a pulmonary embolism subsequent to a spontaneous abortion. Prolonged thrombin (41 s) and reptilase times (26 s) together with an impairment of fibrinopeptide B release suggested a mutation at the thrombin cleavage site of the Bbeta chain. Direct mass analysis of purified fibrin chains from a thrombin induced clot showed that 50% of the Bbeta chains remained uncleaved. The measured mass of the mono sialo isoform of this uncleaved chain was 54150 Da, compared to a value of 54198 Da for normal Bbeta chains. This decrease of 48 Da in the intact protein is indicative of either a Bbeta 14 Arg to Cys, or Arg to Leu substitution. Heterozygosity for the Bbeta 14 Arg --> Cys mutation was verified by PCR amplification and DNA sequence analysis.

Fibrinogen Bastia (gamma 318 Asp-->Tyr) a novel abnormal fibrinogen characterized by defective fibrin polymerization.

PubMed

Lounes, K C; Soria, C; Valognes, A; Turchini, M F; Soria, J; Koopman, J

1999-12-01

A new congenital dysfibrinogen, Fibrinogen Bastia, was discovered in a 20-year-old woman with no clinical symptoms. The plasma thrombin-clotting time was severely prolonged. The functional plasma fibrinogen concentration was low (0.2 mg/ml), whereas the immunological concentration was normal (2.9 mg/ml). Purified fibrinogen Bastia displayed a markedly prolonged thrombin-clotting time related to a delayed thrombin-induced fibrin polymerization. Both the thrombin-clotting time and the fibrin polymerization were partially corrected by the addition of calcium ions. The anomaly of fibrinogen Bastia was found to be located in the gamma-chain since by SDS-PAGE performed according to the method of Laemmli two gamma-chains were detected, one normal and one with an apparently lower molecular weight. Furthermore, analysis of plasmin degradation products demonstrated that calcium ions only partially protect fibrinogen Bastia gamma-chain against plasmin digestion, suggesting that the anomaly is located in the C-terminal part of the gamma-chain. Sequence analysis of PCR-amplified genomic DNA fragments of the propositus demonstrated a single base substitution (G-->T) in the exon VIII of the gamma chain gene, resulting in the amino acid substitution 318 Asp (GAC)-->Tyr (TAC). The PCR clones were recloned and 50% of them contained the mutation, indicating that the patient was heterozygous. These data indicate that residue Asp 318 is important for normal fibrin polymerization and the protective effect of calcium ions against plasmin degradation of the C-terminal part of the gamma-chain.

Comparison of sea turtle thrombocyte aggregation to human platelet aggregation in whole blood.

PubMed

Soslau, Gerald; Prest, Phillip J; Class, Reiner; George, Robert; Paladino, Frank; Violetta, Gary

2005-11-01

The endangered sea turtles are living "fossils" that afford us an opportunity to study the hemostatic process as it likely existed millions of years ago. There are essentially no data about turtle thrombocyte aggregation prior to our studies. Thrombocytes are nucleated cells that serve the same hemostatic functions as the anucleated mammalian platelet. Sea turtle thrombocytes aggregate in response to collagen and beta-thrombin. Ristocetin induces an agglutination/aggregation response indicating the presence of a von Willebrand-like receptor, GPIb, found in all mammalian platelets. Samples treated with alpha-thrombin plus gamma-thrombin followed by ristocetin results in a rapid, stronger response than ristocetin alone. These responses are inhibited by the RGDS peptide that blocks fibrinogen cross-linking of mammalian platelets via the fibrinogen receptor, GPIIb/IIIa. Three platelet-like proteins, GPIb, GPIIb/IIIa and P-selection are detected in sea turtle thrombocytes by fluorescence activated cell sorting. Turtle thrombocytes do not respond to ADP, epinephrine, serotonin, thromboxane A2 mimetic, U46619, trypsin, or alpha-thrombin and gamma-thrombin added alone. Comparison of hemostasis in sea turtles to other vertebrates could provide a framework for understanding the structure/function and evolution of these pathways and their individual components.

DNA sensors and aptasensors based on the hemin/G-quadruplex-controlled aggregation of Au NPs in the presence of L-cysteine.

PubMed

Niazov-Elkan, Angelica; Golub, Eyal; Sharon, Etery; Balogh, Dora; Willner, Itamar

2014-07-23

L-cysteine induces the aggregation of Au nanoparticles (NPs), resulting in a color transition from red to blue due to interparticle plasmonic coupling in the aggregated structure. The hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme catalyzes the aerobic oxidation of L-cysteine to cystine, a process that inhibits the aggregation of the NPs. The degree of inhibition of the aggregation process is controlled by the concentration of the DNAzyme in the system. These functions are implemented to develop sensing platforms for the detection of a target DNA, for the analysis of aptamer-substrate complexes, and for the analysis of L-cysteine in human urine samples. A hairpin DNA structure that includes a recognition site for the DNA analyte and a caged G-quadruplex sequence, is opened in the presence of the target DNA. The resulting self-assembled hemin/G-quadruplex acts as catalyst that controls the aggregation of the Au NPs. Also, the thrombin-binding aptamer folds into a G-quadruplex nanostructure upon binding to thrombin. The association of hemin to the resulting G-quadruplex aptamer-thrombin complex leads to a catalytic label that controls the L-cysteine-mediated aggregation of the Au NPs. The hemin/G-qaudruplex-controlled aggregation of Au NPs process is further implemented for visual and spectroscopic detection of L-cysteine concentration in urine samples. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An electrochemical aptasensor for thrombin using synergetic catalysis of enzyme and porous Au@Pd core-shell nanostructures for signal amplification.

PubMed

Xu, Wenju; Yi, Huayu; Yuan, Yali; Jing, Pei; Chai, Yaqin; Yuan, Ruo; Wilson, George S

2015-02-15

In this work, a sensitive electrochemical aptasensor for thrombin (TB) based on synergetic catalysis of enzyme and porous Au@Pd core-shell nanostructure has been constructed. With the advantages of large surface area and outstanding catalytic performance, porous Au@Pd core-shell nanostructures were firstly employed as the nanocarrier for the immobilization of electroactive toluidine blue (Tb), hemin/G-quadruplex formed by intercalating hemin into the TB aptamer (TBA) and glucose oxidase (GOx). As a certain amount of glucose was added into the detection cell, GOx rapidly catalyzed the oxidation of glucose, coupling with the local generation of H2O2 in the presence of dissolved O2. Then, porous Au@Pd nanoparticles and hemin/G-quadruplex as the peroxidase mimics efficiently catalyzed the reduction of H2O2, amplifying the electrochemical signal and improving the sensitivity. Finally, a detection limit of 0.037pM for TB was achieved. The excellent performance of the aptasensor indicated its promising prospect as a valuable tool in simple and cost-effective TB detection in clinical application. Copyright © 2014 Elsevier B.V. All rights reserved.

Triple Quenching of a Novel Self-Enhanced Ru(II) Complex by Hemin/G-Quadruplex DNAzymes and Its Potential Application to Quantitative Protein Detection.

PubMed

Zhao, Min; Liao, Ni; Zhuo, Ying; Chai, Ya-Qin; Wang, Ji-Peng; Yuan, Ruo

2015-08-04

Herein, a novel "on-off" electrochemiluminescence (ECL) aptasensor for highly sensitive determination of thrombin has been constructed based on the triple quenching of the effect of hemin/G-quadruplex DNAzymes upon the Ru(II) complex-based ECL system. First, a strong initial ECL signal was achieved by the dual amplification strategies of (i) intramolecular coreaction of a self-enhanced Ru(II)-based molecule (PTCA-PEI-Ru(II)) and (ii) intermolecular coreaction between PTCA-PEI-Ru(II) and nicotinamide adenine dinucleotide (NADH), which was named the signal-on state. Then, a novel triple quenching of the effect of multifunctional hemin/G-quadruplex DNAzymes upon the Ru(II) complex-based ECL system was designed to realize the desirable signal-off state, which was outlined as follows: (i) the hemin/G-quadruplex DNAzymes mimicked NADH oxidase to oxidize NADH and in situ generate the H2O2, consuming the coreactant of NADH; (ii) its active center of hemin could oxidize the excited state PTCA-PEI-Ru(II)* to PTCA-PEI-Ru(III), making the energy and electron transfer quench; (iii) it also acted as horseradish peroxidase (HRP) to catalyze the H2O2 for in situ producing the quencher of O2. Based on triple quenching of the effect of hemin/G-quadruplex DNAzymes, the highly sensitive "on-off" thrombin aptasensor was developed with a wide linear detection range of 1.0 × 10(-14) M to 1.0 × 10(-10) M and a detection limit down to the femtomolar level.

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

NASA Astrophysics Data System (ADS)

Yu, Johnson Chung Sing

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

Improvement of Aptamer Affinity by Dimerization

PubMed Central

Hasegawa, Hijiri; Taira, Ken-ichi; Sode, Koji; Ikebukuro, Kazunori

2008-01-01

To increase the affinities of aptamers for their targets, we designed an aptamer dimer for thrombin and VEGF. This design is based on the avidity of the antibody, which enables the aptamer to connect easily since it is a single-strand nucleic acid. In this study, we connected a 15-mer thrombin-binding aptamer with a 29-mer thrombin-binding aptamer. Each aptamer recognizes a different part of the thrombin molecule, and the aptamer dimer has a Kd value which is 1/10 of that of the monomers from which it is composed. Also, the designed aptamer dimer has higher inhibitory activity than the reported (15-mer) thrombin-inhibiting aptamer. Additionally, we connected together two identical aptamers against vascular endothelial growth factor (VEGF165), which is a homodimeric protein. As in the case of the anti-thrombin aptamer, the dimeric anti-VEGF aptamer had a much lower Kd value than that of the monomer. This study demonstrated that the dimerization of aptamers effectively improves the affinities of those aptamers for their targets. PMID:27879754

Highly sensitive detection for proteins using graphene oxide-aptamer based sensors.

PubMed

Gao, Li; Li, Qin; Li, Raoqi; Yan, Lirong; Zhou, Yang; Chen, Keping; Shi, Haixia

2015-07-07

In recent years, the detection of proteins by using bare graphene oxide (GO) to quench the fluorescence of fluorescein-labeled aptamers has been reported. However, the proteins can be adsorbed on the surface of bare GO to prevent the sensitivity from further being improved. In order to solve this problem, polyethylene glycol (PEG)-protected GO was used to prevent the proteins using thrombin as an example from nonspecific binding. The detection limit was improved compared to bare GO under the optimized ratio of GO to PEG concentration. The results show that our method is a promising technique for the detection of proteins.

Ce(III, IV)-MOF electrocatalyst as signal-amplifying tag for sensitive electrochemical aptasensing.

PubMed

Yu, Hua; Han, Jing; An, Shangjie; Xie, Gang; Chen, Sanping

2018-06-30

Metal-organic frameworks (MOFs) as a new class of porous materials have attracted increasing attention in the field of biomimetic catalysis. This study firstly reports a mixed valence state Ce-MOF possessing intrinsic catalytic activity towards thionine (Thi), and its application in constructing an amplified electrochemical aptasensor for thrombin detection. As noticed, the novel catalytic process combines the advantages of 3D infinite extension of the Ce(III, IV)-MOF skeleton containing large amounts of catalytic sites and spontaneous recycling of the Ce(III)/Ce(IV) for electrochemical reduction of Thi, thereby presenting amplified electrochemical signals. To further improve the aptasensor performance, the high selectivity of proximity binding-induced DNA strand displacement and high efficiency of exonuclease III-assisted recycling amplification were incorporated into the assay. The aptasensor was employed to detect thrombin in complex serum samples, which shows high sensitivity, specificity, stability and reproducibility. This work offers an opportunity to develop MOF-based electrocatalyst as signal-amplifying tag for versatile bioassays and catalytic applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Agonists of proteinase-activated receptor 1 induce plasma extravasation by a neurogenic mechanism.

PubMed

de Garavilla, L; Vergnolle, N; Young, S H; Ennes, H; Steinhoff, M; Ossovskaya, V S; D'Andrea, M R; Mayer, E A; Wallace, J L; Hollenberg, M D; Andrade-Gordon, P; Bunnett, N W

2001-08-01

Thrombin, generated in the circulation during injury, cleaves proteinase-activated receptor 1 (PAR1) to stimulate plasma extravasation and granulocyte infiltration. However, the mechanism of thrombin-induced inflammation in intact tissues is unknown. We hypothesized that thrombin cleaves PAR1 on sensory nerves to release substance P (SP), which interacts with the neurokinin 1 receptor (NK1R) on endothelial cells to cause plasma extravasation. PAR1 was detected in small diameter neurons known to contain SP in rat dorsal root ganglia by immunohistochemistry and in situ hybridization. Thrombin and the PAR1 agonist TFLLR-NH(2) (TF-NH(2)) increased [Ca(2+)](i) >50% of cultured neurons (EC(50)s 24 mu ml(-1) and 1.9 microM, respectively), assessed using Fura-2 AM. The PAR1 agonist completely desensitized responses to thrombin, indicating that thrombin stimulates neurons through PAR1. Injection of TF-NH(2) into the rat paw stimulated a marked and sustained oedema. An NK1R antagonist and ablation of sensory nerves with capsaicin inhibited oedema by 44% at 1 h and completely by 5 h. In wild-type but not PAR1(-/-) mice, TF-NH(2) stimulated Evans blue extravasation in the bladder, oesophagus, stomach, intestine and pancreas by 2 - 8 fold. Extravasation in the bladder, oesophagus and stomach was abolished by an NK1R antagonist. Thus, thrombin cleaves PAR1 on primary spinal afferent neurons to release SP, which activates the NK1R on endothelial cells to stimulate gap formation, extravasation of plasma proteins, and oedema. In intact tissues, neurogenic mechanisms are predominantly responsible for PAR1-induced oedema.

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

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

Nieman, M T; Burke, F; Warnock, M

2008-04-29

Novel pentapeptides called Thrombostatin FM compounds consisting mostly of D-isomers and unusual amino acids were prepared based upon the stable angiotensin converting enzyme breakdown product of bradykinin - RPPGF. These peptides are direct thrombin inhibitors prolonging the thrombin clotting time, activated partial thromboplastin time, and prothrombin time at ≥0.78, 1.6, and 1.6 μm, respectively. They competitively inhibit α-thrombin-induced cleavage of a chromogenic substrate at 4.4--8.2 μm. They do not significantly inhibit plasma kallikrein, factor (F) XIIa, FXIa, FIXa, FVIIa-TF, FXa, plasmin or cathepsin G. One form, FM19 [rOicPaF(p-Me)], blocks α-thrombin-induced calcium flux in fibroblasts with an IC 50 of 6.9more » ± 1.2 μm. FM19 achieved 100% inhibition of threshold α- or γ-thrombin-induced platelet aggregation at 8.4 ± 4.7 μm and 16 ± 4 μm, respectively. The crystal structure of thrombin in complex with FM19 shows that the N-terminal D-Arg retrobinds into the S1 pocket, its second residue Oic interacts with His-57, Tyr-60a and Trp-60d, and its C-terminal p-methyl Phe engages thrombin's aryl binding site composed of Ile-174, Trp-215, and Leu-99. When administered intraperitoneal, intraduodenal, or orally to mice, FM19 prolongs thrombin clotting times and delays carotid artery thrombosis. FM19, a low affinity reversible direct thrombin inhibitor, might be useful as an add-on agent to address an unmet need in platelet inhibition in acute coronary syndromes in diabetics and others who with all current antiplatelet therapy still have reactive platelets.« less

G quadruplex-based FRET probes with the thrombin-binding aptamer (TBA) sequence designed for the efficient fluorometric detection of the potassium ion.

PubMed

Nagatoishi, Satoru; Nojima, Takahiko; Galezowska, Elzbieta; Juskowiak, Bernard; Takenaka, Shigeori

2006-11-01

The dual-labeled oligonucleotide derivative, FAT-0, carrying 6- carboxyfluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA) labels at the 5' and 3' termini of the thrombin-binding aptamer (TBA) sequence 5'-GGT TGG TGT GGT TGG-3', and its derivatives, FAT-n (n=3, 5, and 7) with a spacer at the 5'-end of a TBA sequence of T(m)A (m=2, 4, and 6) have been designed and synthesized. These fluorescent probes were developed for monitoring K(+) concentrations in living organisms. Circular dichroism, UV-visible absorption, and fluorescence studies revealed that all FAT-n probes could form intramolecular tetraplex structures after binding K(+). Fluorescence resonance energy transfer and quenching results are discussed taking into account dye-dye contact interactions. The relationship between the fluorescence behavior of the probes and the spacer length in FAT-n was studied in detail and is discussed.

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

PubMed

Cheng, Christine M; Meyer-Massetti, Carla; Kayser, Steven R

2009-01-01

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

Acquired Dysfibrinogenemia Caused by Autoantibody Inhibiting Fibrin Polymerization in a Patient with MELAS Syndrome and Bleeding Tendency.

PubMed

Lee, Nuri; Kim, Ji-Eun; Yoo, Hyun Ju; Gu, JaYoon; Kim, Hyori; Chung, Junho; Koh, Youngil; Kim, Hyun Kyung

2016-12-01

We present a case of acquired dysfibrinogenemia caused by an autoantibody that inhibited fibrin polymerization in a patient previously diagnosed with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, stroke-like episodes). The patient showed prolonged PT, aPTT, and thrombin time. There was no factor deficiency but fibrinogen antigen and activity were decreased. ELISA for detection of fibrinogen antibodies were performed and IgG purified from the patient's plasma bound to fibrinogen more strongly than did control IgG, indicating the presence of a fibrinogen-specific antibody. Thrombin-mediated fibrin polymerization was severely impaired in the patient, although thrombin-induced fibrinopeptide A release was normal. Scanning electron microscopy was used to investigate the structure of fibrin clots and revealed many pores on the surface of patient's fibrin clots. Since MELAS is often associated with autoimmune disorders, a work-up for the presence of anti-fibrinogen antibody is necessary when bleeding tendency occurs in MELAS patients along with prolonged thrombin time. © 2016 by the Association of Clinical Scientists, Inc.

A nonenzymatic DNA nanomachine for biomolecular detection by target recycling of hairpin DNA cascade amplification.

PubMed

Zheng, Jiao; Li, Ningxing; Li, Chunrong; Wang, Xinxin; Liu, Yucheng; Mao, Guobin; Ji, Xinghu; He, Zhike

2018-06-01

Synthetic enzyme-free DNA nanomachine performs quasi-mechanical movements in response to external intervention, suggesting the promise of constructing sensitive and specific biosensors. Herein, a smart DNA nanomachine biosensor for biomolecule (such as nucleic acid, thrombin and adenosine) detection is developed by target-assisted enzyme-free hairpin DNA cascade amplifier. The whole DNA nanomachine system is constructed on gold nanoparticle which decorated with hundreds of locked hairpin substrate strands serving as DNA tracks, and the DNA nanomachine could be activated by target molecule toehold-mediated exchange on gold nanoparticle surface, resulted in the fluorescence recovery of fluorophore. The process is repeated so that each copy of the target can open multiplex fluorophore-labeled hairpin substrate strands, resulted in amplification of the fluorescence signal. Compared with the conventional biosensors of catalytic hairpin assembly (CHA) without substrate in solution, the DNA nanomachine could generate 2-3 orders of magnitude higher fluorescence signal. Furthermore, the DNA nanomachine could be used for nucleic acid, thrombin and adenosine highly sensitive specific detection based on isothermal, and homogeneous hairpin DNA cascade signal amplification in both buffer and a complicated biomatrix, and this kind of DNA nanomachine could be efficiently applied in the field of biomedical analysis. Copyright © 2018 Elsevier B.V. All rights reserved.

In vivo ultrasound visualization of non-occlusive blood clots with thrombin-sensitive contrast agents.

PubMed

Nakatsuka, Matthew A; Barback, Christopher V; Fitch, Kirsten R; Farwell, Alexander R; Esener, Sadik C; Mattrey, Robert F; Cha, Jennifer N; Goodwin, Andrew P

2013-12-01

The use of microbubbles as ultrasound contrast agents is one of the primary methods to diagnose deep venous thrombosis. However, current microbubble imaging strategies require either a clot sufficiently large to produce a circulation filling defect or a clot with sufficient vascularization to allow for targeted accumulation of contrast agents. Previously, we reported the design of a microbubble formulation that modulated its ability to generate ultrasound contrast from interaction with thrombin through incorporation of aptamer-containing DNA crosslinks in the encapsulating shell, enabling the measurement of a local chemical environment by changes in acoustic activity. However, this contrast agent lacked sufficient stability and lifetime in blood to be used as a diagnostic tool. Here we describe a PEG-stabilized, thrombin-activated microbubble (PSTA-MB) with sufficient stability to be used in vivo in circulation with no change in biomarker sensitivity. In the presence of actively clotting blood, PSTA-MBs showed a 5-fold increase in acoustic activity. Specificity for the presence of thrombin and stability under constant shear flow were demonstrated in a home-built in vitro model. Finally, PSTA-MBs were able to detect the presence of an active clot within the vena cava of a rabbit sufficiently small as to not be visible by current non-specific contrast agents. By activating in non-occlusive environments, these contrast agents will be able to detect clots not diagnosable by current contrast agents. Copyright © 2013 Elsevier Ltd. All rights reserved.

Does a thrombin-based topical haemostatic agent reduce blood loss and transfusion requirements after total knee revision surgery? A randomized, controlled trial.

PubMed

Romanò, Carlo L; Monti, Lorenzo; Logoluso, Nicola; Romanò, Delia; Drago, Lorenzo

2015-11-01

The aim of the present study was to assess the efficacy of a thrombin-based topical haemostatic in reducing blood requirements after total knee replacement (TKR) revision surgery. This prospective, randomized, controlled study was designed to evaluate the haemostatic efficacy and safety of a thrombin-based topical haemostatic (Floseal) versus standard treatment in patients receiving total knee revision arthroplasty. The decrease in haemoglobin values postsurgery and the blood units transfused were recorded. The decision to transfuse was made by a surgeon blinded to the patient's group allocation. Forty-eight patients were enroled in the study; twenty-four patients each were randomized to the treatment and control groups, respectively. The median decrease in haemoglobin concentration on the first postoperative day was 2.2 g/dL in the treatment group and 2.7 g/dL in the control group. A significant reduction in units of blood transfused was also observed in the treatment group compared with the control group [1.1 ± 1.13 (range 0-4) vs. 1.9 ± 1.41 (range 0-5) blood units; P = 0.04]. No major treatment-related adverse events were recorded in the study. This study shows that a thrombin-based topical haemostatic reduces the need for blood transfusion in TKR revision surgery. A thrombin-based topical haemostatic agent can be an appropriate solution to enhance haemostasis and vessel sealing at the operative site in TKR revision surgery, in order to reduce the need for blood transfusion after surgery. II.

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

PubMed

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

2016-01-01

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

Gold nanoparticles-based protease assay

PubMed Central

Guarise, Cristian; Pasquato, Lucia; De Filippis, Vincenzo; Scrimin, Paolo

2006-01-01

We describe here a simple assay that allows the visual detection of a protease. The method takes advantage of the high molar absorptivity of the plasmon band of gold colloids and is based on the color change of their solution when treated with dithiols. We used C- and N-terminal cysteinyl derivatives of a peptide substrate exploiting its selective recognition and cleavage by a specific protease. Contrary to the native ones, cleaved peptides are unable to induce nanoparticles aggregation; hence, the color of the solution does not change. The detection of two proteases is reported: thrombin (involved in blood coagulation and thrombosis) and lethal factor (an enzyme component of the toxin produced by Bacillus anthracis). The sensitivity of this nanoparticle-based assay is in the low nanomolar range. PMID:16537471

Gold nanoparticles-based protease assay.

PubMed

Guarise, Cristian; Pasquato, Lucia; De Filippis, Vincenzo; Scrimin, Paolo

2006-03-14

We describe here a simple assay that allows the visual detection of a protease. The method takes advantage of the high molar absorptivity of the plasmon band of gold colloids and is based on the color change of their solution when treated with dithiols. We used C- and N-terminal cysteinyl derivatives of a peptide substrate exploiting its selective recognition and cleavage by a specific protease. Contrary to the native ones, cleaved peptides are unable to induce nanoparticles aggregation; hence, the color of the solution does not change. The detection of two proteases is reported: thrombin (involved in blood coagulation and thrombosis) and lethal factor (an enzyme component of the toxin produced by Bacillus anthracis). The sensitivity of this nanoparticle-based assay is in the low nanomolar range.

Tyrosine sulfation modulates activity of tick-derived thrombin inhibitors

NASA Astrophysics Data System (ADS)

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

2017-09-01

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.

Bovine thrombin safety reporting: an example of study design and publication bias.

PubMed

Crean, Sheila; Michels, Shannon L; Moschella, Kevin; Reynolds, Matthew W

2010-01-01

Bovine thrombin, a popular hemostat and sealant since 1945, has recently been subjected to clinical trial testing due to reformulations in 1998. We sought to compare adverse event rates of early observational studies with those of later interventional trials. A MEDLINE-based literature search in publications that report safety in bovine thrombin exposed surgical patients was extracted and reviewed. In 38 studies, about half were case reports and 31.5% were interventional trials. In case reports, 41% of authors reported severe coagulopathic adverse events. In contrast, whereas blood complications were common in large trials, no association of harm was established for bovine thrombin product exposure and/or immunization. In this review, later clinical trials failed to reproduce the common and severe coagulopathy predicted by earlier observational studies in bovine exposed patients. This example illustrates that perceptions of safety can change as a function of study design, even for a widely adopted, well established biologic such as thrombin. Caution must be exercised in interpreting evidence from observational studies alone.

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

PubMed Central

Gissel, Matthew; Orfeo, Thomas; Foley, Jonathan H; Butenas, Saulius

2012-01-01

Summary Introduction In hemophilia, thrombin generation is significantly suppressed due to decreased factor (F)X activation. Clinical studies and experiments with transgenic mice have suggested that the severity of hemophilia is substantially reduced by tissue factor pathway inhibitor (TFPI) deficiency. Methods We evaluated the effect of TFPI antagonist aptamer BAX499 (formerly ARC19499) on TFPI function in purified systems and on thrombin generation and clot formation in plasma and blood. Results BAX499 effectively neutralized TFPI inhibition of FXa and FXa dependent inhibition of TF/FVIIa by TFPI. BAX499 did not inhibit FXa or TF/FVIIa when used up to 500 nM. In the synthetic coagulation proteome with TFPI at its mean physiologic concentration, BAX499 at 1 – 10 nM increased thrombin generation triggered with 5 pM relipidated TF in a concentration-dependent manner. In severe hemophilia A or B models using the synthetic coagulation proteome, the addition of BAX499 at 5 nM increased thrombin generation to the levels observed in normal control. Thrombin generation measured in induced hemophilia B plasma required ~100 nM BAX499 to restore thrombin levels to those seen in untreated plasma. In induced hemophilia B whole blood, BAX499 repaired the clotting time but failed to appreciably impact the propagation phase of thrombin generation. Conclusion These data suggest that inhibition of TFPI by BAX499 may have potential for hemophilia treatment but requires further study in blood-based hemophilia systems. PMID:22951415

Role of the thrombin receptor in restenosis and atherosclerosis.

PubMed

Baykal, D; Schmedtje, J F; Runge, M S

1995-02-23

Thrombus generation is central to thrombosis at vascular lesion sites, including post-PCTA acute reocclusion and chronic restenosis. Thrombin stimulates platelet activation, monocyte and neutrophil chemotaxis, and endothelial production of prothrombotic factors. The varied physiologic effects of thrombin are due to the widespread presence of thrombin receptors in many cell types. The receptor is uniquely activated: thrombin binds to the receptor at the thrombin anion-binding exosite, the receptor ligand ("tethered ligand") apparently being a sequence of 6 amino acids (SFLLRN). Thus, peptides corresponding to the sequence of the tethered ligand can stimulate almost all functions of native thrombin itself. Several intracellular signaling pathways have been identified as important in the restenosis process: the G protein-related pathway, cyclic adenosine monophosphate (cAMP) mediator pathway, and tyrosine kinase activation pathway. In situ hybridization has demonstrated an increase in thrombin receptor mRNA throughout the period of neointimal and vascular lesion development. The mechanism of this increase is unknown, but may be mediated by multiple inflammatory modulators. Several strategies have been tested in animal models for inhibiting thrombin: (1) Hirudin not only prevents thrombin from cleaving fibrinogen, but also prevents thrombin receptor activation. (2) Thrombin receptor antagonist peptides block platelet aggregation effects of thrombin. (3) Mono- and polyclonal antibodies inhibit thrombin receptor activation. (4) Antisense oligonucleotides block thrombin receptor expression.

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

PubMed Central

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

2013-01-01

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

The Effects of Thrombin on Adenyl Cyclase Activity and a Membrane Protein from Human Platelets

PubMed Central

Brodie, G. N.; Baenziger, Nancy Lewis; Chase, Lewis R.; Majerus, Philip W.

1972-01-01

Washed human platelets were incubated with 0.1-1.0 U/ml human thrombin and the effects on adenyl cyclase activity and on a platelet membrane protein (designated thrombin-sensitive protein) were studied. Adenyl cyclase activity was decreased 70-90% when intact platelets were incubated with thrombin. The T½ for loss of adenyl cyclase activity was less than 15 sec at 1 U/ml thrombin. There was no decrease of adenyl cyclase activity when sonicated platelets or isolated membranes were incubated with these concentrations of thrombin. Loss of adenyl cyclase activity was relatively specific since the activities of other platelet membrane enzymes were unaffected by thrombin. Prior incubation of platelets with dibutyryl cyclic adenosine monophosphate (AMP), prostaglandin E1, or theophylline protected adenyl cyclase from inhibition by thrombin. Incubation of intact but not disrupted platelets with thrombin resulted in the release of thrombin-sensitive protein from the platelet membrane. The rapid release of this protein (T½ < 15 sec) at low concentrations of thrombin suggested that removal of thrombin-sensitive protein from the platelet membrane is an integral part of the platelet release reaction. This hypothesis is supported by the parallel effects of thrombin on adenyl cyclase activity and thrombin-sensitive protein release in the presence of dibutyryl cyclic AMP, prostaglandin E1, and theophylline at varying concentrations of thrombin. Images PMID:4331802

Thrombin-induced glucose transport via Src–p38 MAPK pathway in vascular smooth muscle cells

PubMed Central

Kanda, Yasunari; Watanabe, Yasuhiro

2005-01-01

Thrombin is a mitogen for vascular smooth muscle cells (VSMC) and has been implicated in the development in atherosclerosis. However, little is known about the role of thrombin in glucose transport in VSMC. In this study, we examined the effect of thrombin on glucose uptake in rat A10 VSMC. We found that thrombin induced glucose uptake in a dose-dependent manner while hirudin, a potent thrombin inhibitor, prevented glucose uptake in the cells. PP2, a selective inhibitor of Src, prevented the thrombin-induced glucose uptake, but did not affect insulin-induced uptake. We also examined whether mitogen-activated protein kinase (MAPK) inhibitors influenced thrombin-induced glucose uptake. The p38 MAPK inhibitor (SB203580) inhibited thrombin-induced glucose uptake, but the MEK inhibitor (PD98059) did not. In contrast to thrombin, SB203580 did not affect insulin-induced glucose uptake. Furthermore, thrombin failed to translocate the insulin-sensitive glucose transporter GLUT4. These findings suggest that thrombin stimulates glucose transport via Src and subsequent p38 MAPK activation in VSMC. PMID:15951827

Anticoagulants and the propagation phase of thrombin generation.

PubMed

Orfeo, Thomas; Gissel, Matthew; Butenas, Saulius; Undas, Anetta; Brummel-Ziedins, Kathleen E; Mann, Kenneth G

2011-01-01

The view that clot time-based assays do not provide a sufficient assessment of an individual's hemostatic competence, especially in the context of anticoagulant therapy, has provoked a search for new metrics, with significant focus directed at techniques that define the propagation phase of thrombin generation. Here we use our deterministic mathematical model of tissue-factor initiated thrombin generation in combination with reconstructions using purified protein components to characterize how the interplay between anticoagulant mechanisms and variable composition of the coagulation proteome result in differential regulation of the propagation phase of thrombin generation. Thrombin parameters were extracted from computationally derived thrombin generation profiles generated using coagulation proteome factor data from warfarin-treated individuals (N = 54) and matching groups of control individuals (N = 37). A computational clot time prolongation value (cINR) was devised that correlated with their actual International Normalized Ratio (INR) values, with differences between individual INR and cINR values shown to derive from the insensitivity of the INR to tissue factor pathway inhibitor (TFPI). The analysis suggests that normal range variation in TFPI levels could be an important contributor to the failure of the INR to adequately reflect the anticoagulated state in some individuals. Warfarin-induced changes in thrombin propagation phase parameters were then compared to those induced by unfractionated heparin, fondaparinux, rivaroxaban, and a reversible thrombin inhibitor. Anticoagulants were assessed at concentrations yielding equivalent cINR values, with each anticoagulant evaluated using 32 unique coagulation proteome compositions. The analyses showed that no anticoagulant recapitulated all features of warfarin propagation phase dynamics; differences in propagation phase effects suggest that anticoagulants that selectively target fXa or thrombin may provoke fewer bleeding episodes. More generally, the study shows that computational modeling of the response of core elements of the coagulation proteome to a physiologically relevant tissue factor stimulus may improve the monitoring of a broad range of anticoagulants.

Protein sensing by nanofluidic crystal and its signal enhancement

PubMed Central

Sang, Jianming; Du, Hongtan; Wang, Wei; Chu, Ming; Wang, Yuedan; Li, Haichao; Alice Zhang, Haixia; Wu, Wengang; Li, Zhihong

2013-01-01

Nanofluidics has a unique property that ionic conductance across a nanometer-sized confined space is strongly affected by the space surface charge density, which can be utilized to construct electrical read-out biosensor. Based on this principle, this work demonstrated a novel protein sensor along with a sandwich signal enhancement approach. Nanoparticles with designed aptamer onside are assembled in a suspended micropore to form a 3-dimensional network of nanometer-sized interstices, named as nanofluidic crystal hereafter, as the basic sensing unit. Proteins captured by aptamers will change the surface charge density of nanoparticles and thereby can be detected by monitoring the ionic conductance across this nanofluidic crystal. Another aptamer can further enlarge the variations of the surface charge density by forming a sandwich structure (capturing aptamer/protein/signal enhancement aptamer) and the read-out conductance as well. The preliminary experimental results indicated that human α-thrombin was successfully detected by the corresponding aptamer modified nanofluidic crystal with the limit of detection of 5 nM (0.18 μg/ml) and the read-out signal was enhanced up to 3 folds by using another thrombin aptamer. Being easy to graft probe, facile and low-cost to prepare the nano-device, and having an electrical read-out, the present nanofluidic crystal scheme is a promising and universal strategy for protein sensing. PMID:24404017

Characterization of the influence of external stimulus on protein-nucleic acid complex through multiscale computations

NASA Astrophysics Data System (ADS)

Gosai, Agnivo

The concomitant detection, monitoring and analysis of biomolecules have assumed utmost importance in the field of medical diagnostics as well as in different spheres of biotechnology research such as drug development, environmental hazard detection and biodefense. There is an increased demand for the modulation of the biological response for such detection / sensing schemes which will be facilitated by the sensitive and controllable transmission of external stimuli. Electrostatic actuation for the controlled release/capture of biomolecules through conformational transformations of bioreceptors provides an efficient and feasible mechanism to modulate biological response. In addition, electrostatic actuation mechanism has the advantage of allowing massively parallel schemes and measurement capabilities that could ultimately be essential for biomedical applications. Experiments have previously demonstrated the unbinding of thrombin from its aptamer in presence of small positive electrode potential whereas the complex remained associated in presence of small negative potentials / zero potential. However, the nanoscale physics/chemistry involved in this process is not clearly understood. In this thesis a combination of continuum mechanics based modeling and a variety of atomistic simulation techniques have been utilized to corroborate the aforementioned experimental observations. It is found that the computational approach can satisfactorily predict the dynamics of the electrically excited aptamer-thrombin complex as well as provide an analytical model to characterize the forced binding of the complex.

A multifunctional hemin@metal-organic framework and its application to construct an electrochemical aptasensor for thrombin detection.

PubMed

Xie, Shunbi; Ye, Jiawei; Yuan, Yali; Chai, Yaqin; Yuan, Ruo

2015-11-21

A new type of multifunctional metal-organic framework (MOF) has been synthesized by encapsulating hemin into the nano-sized Fe-MIL-88 MOFs (hemin@MOFs) and first applied in an electrochemical aptasensor to detect thrombin (TB) with the aid of an enzyme for signal amplification. The gold nanoparticle functionalized hemin@MOFs (Au/hemin@MOFs) have not only simultaneously served as redox mediators and solid electrocatalysts, but have also been utilized as an ideal loading platform to immobilize a large number of biomolecules. In this aptasensor, Au/hemin@MOFs conjugated with glucose oxidase (GOD) and thrombin binding aptamer (TBA II) were used as the secondary aptamer bioconjugates (Au/hemin@MOF-TBA II-GOD bioconjugates), and TB was sandwiched between Au/hemin@MOF-TBA II-GOD bioconjugates and the amino-terminated TBA I which was self-assembled on the gold nanoparticle (AuNP) modified electrode. The GOD could oxidize glucose into gluconic acid accompanied by the generation of H2O2. The generated H2O2 on the electrode surface was further electrocatalyzed by hemin@MOFs to amplify the electrochemical signal of hemin contained in hemin@MOFs. Therefore, the synthesized hemin@MOFs represented a new paradigm for multifunctional materials since it combined three different functions including serving as catalysts, redox mediators and loading platforms within a single material. With such an ingenious design, a wide linear range of 0.0001 nM to 30 nM was acquired with a relatively low detection limit of 0.068 pM for TB detection.

Aptamer-based potentiometric measurements of proteins using ion-selective microelectrodes.

PubMed

Numnuam, Apon; Chumbimuni-Torres, Karin Y; Xiang, Yun; Bash, Ralph; Thavarungkul, Panote; Kanatharana, Proespichaya; Pretsch, Ernö; Wang, Joseph; Bakker, Eric

2008-02-01

We here report on the first example of an aptamer-based potentiometric sandwich assay of proteins. The measurements are based on CdS quantum dot labels of the secondary aptamer, which were determined with a novel solid-contact Cd2+-selective polymer membrane electrode after dissolution with hydrogen peroxide. The electrode exhibited cadmium ion detection limits of 100 pM in 100 mL samples and of 1 nM in 200 microL microwells, using a calcium-selective electrode as a pseudoreference electrode. As a prototype example, thrombin was measured in 200 microL samples with a lower detection limit of 0.14 nM corresponding to 28 fmol of analyte. The results show great promise for the potentiometric determination of proteins at very low concentrations in microliter samples.

Effect of Thrombin on Human Amnion Mesenchymal Cells, Mouse Fetal Membranes, and Preterm Birth*

PubMed Central

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

2014-01-01

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

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

Wang, Huang-Joe; Division of Cardiology, Department of Medicine, China Medical University Hospital, No. 2, Yuh-Der Road, Taichung 40447, Taiwan; Lo, Wan-Yu

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 inmore » 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.« less

Thrombin-induced apoptosis in neurons through activation of c-Jun-N-terminal kinase.

PubMed

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

2017-01-01

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.

Imbalance in the pro-hepatocyte growth factor activation system in bleomycin-induced lung fibrosis in mice.

PubMed

Phin, Sophie; Marchand-Adam, Sylvain; Fabre, Aurélie; Marchal-Somme, Joëlle; Bantsimba-Malanda, Claudie; Kataoka, Hiroaki; Soler, Paul; Crestani, Bruno

2010-03-01

Hepatocyte growth factor (HGF) is a growth factor for alveolar epithelial cells. Activation of pro-HGF to HGF is regulated by the HGF activator (HGFA), a serine protease, and a specific inhibitor (HGFA inhibitor-1, HAI-1). An imbalance in the HGFA/HAI-1 system might contribute to lung fibrosis. Pro-HGF activation capacity from bronchoalveolar lavage (BAL) fluid was evaluated 3, 7, and 14 days after the intratracheal bleomycin injection (Bleo) in mice with or without thrombin. BAL fluid from naïve mice was used as control. HGFA and HAI-1 mRNA were evaluated by QPCR in the whole lung or by Western blot in BAL fluid. BAL fluid from control mice and Bleo mice activated pro-HGF in vitro at a similar degree. Thrombin accelerated proHGF activation by Bleo BAL on Day 3 and Day 7, but not on Day 14, or in control BAL. Incubation of pro-HGF with BAL from Bleo Day 3 and Day 7 mice increased phosphorylation of HGFR on A549 cells. Thrombin-induced pro-HGF activation was inhibited by an anti-HGFA antibody and accelerated by an anti-HAI-1 antibody. Active HGFA was not detected in control BAL and was strongly induced in Bleo BAL. HGFA concentrations were higher on Day 3 and Day 7 than on Day 14. HAI-1 was detected at low levels in control BAL and increased strongly by Day 3 with stable concentrations until Day 14. By demonstrating an imbalance between HGFA and HAI-1 expression in BAL fluid, our results highlight a defective thrombin-dependent proHGF activation system at the fibrotic phase of bleomycin-induced pulmonary fibrosis.

Inhibition of thrombin by functionalized C60 nanoparticles revealed via in vitro assays and in silico studies.

PubMed

Liu, Yanyan; Fu, Jianjie; Pan, Wenxiao; Xue, Qiao; Liu, Xian; Zhang, Aiqian

2018-01-01

The studies on the human toxicity of nanoparticles (NPs) are far behind the rapid development of engineered functionalized NPs. Fullerene has been widely used as drug carrier skeleton due to its reported low risk. However, different from other kinds of NPs, fullerene-based NPs (C 60 NPs) have been found to have an anticoagulation effect, although the potential target is still unknown. In the study, both experimental and computational methods were adopted to gain mechanistic insight into the modulation of thrombin activity by nine kinds of C 60 NPs with diverse surface chemistry properties. In vitro enzyme activity assays showed that all tested surface-modified C 60 NPs exhibited thrombin inhibition ability. Kinetic studies coupled with competitive testing using 3 known inhibitors indicated that six of the C 60 NPs, of greater hydrophobicity and hydrogen bond (HB) donor acidity or acceptor basicity, acted as competitive inhibitors of thrombin by directly interacting with the active site of thrombin. A simple quantitative nanostructure-activity relationship model relating the surface substituent properties to the inhibition potential was then established for the six competitive inhibitors. Molecular docking analysis revealed that the intermolecular HB interactions were important for the specific binding of C 60 NPs to the active site canyon, while the additional stability provided by the surface groups through van der Waals interaction also play a key role in the thrombin binding affinity of the NPs. Our results suggest that thrombin is a possible target of the surface-functionalized C 60 NPs relevant to their anticoagulation effect. Copyright © 2017. Published by Elsevier B.V.

Incidence of thromboembolic events after use of gelatin-thrombin-based hemostatic matrix during intracranial tumor surgery.

PubMed

Gazzeri, Roberto; Galarza, Marcelo; Conti, Carlo; De Bonis, Costanzo

2018-01-01

Association between the use of hemostatic agents made from collagen/gelatin mixed with thrombin and thromboembolic events in patients undergoing tumor resection has been suggested. This study evaluates the relationship between flowable hemostatic matrix and deep vein thrombosis in a large cohort of patients treated for brain tumor removal. The authors conducted a retrospective, multicenter, clinical review of all craniotomies for tumor removal performed between 2013 and 2014. Patients were classified in three groups: group I (flowable gelatin hemostatic matrix with thrombin), group II (gelatin hemostatic without thrombin), and group III (classical hemostatic). A total of 932 patients were selected: tumor pathology included 441 gliomas, 296 meningiomas, and 195 metastases. Thromboembolic events were identified in 4.7% of patients in which gelatin matrix with thrombin was applied, in 8.4% of patients with gelatin matrix without thrombin, and in 3.6% of cases with classical methods of hemostasis. Patients with venous thromboembolism had an increased proportion of high-grade gliomas (7.2%). Patients receiving a greater dose than 10 ml gelatin hemostatic had a higher rate of thromboembolic events. Intracranial hematoma requiring reintervention occurred in 19 cases: 4.5% of cases of group III, while reoperation was performed in 1.3 and 1.6% of patients in which gelatin matrix with or without thrombin was applied. Gelatin matrix hemostat is an efficacious tool for neurosurgeons in cases of difficult intraoperative bleeding during cranial tumor surgery. This study may help to identify those patients at high risk for developing thromboembolism and to treat them accordingly.

The effect of cigarette smoke extract on thrombomodulin-thrombin binding: an atomic force microscopy study.

PubMed

Wei, Yujie; Zhang, Xuejie; Xu, Li; Yi, Shaoqiong; Li, Yi; Fang, Xiaohong; Liu, Huiliang

2012-10-01

Cigarette smoking is a well-known risk factor for cardiovascular disease. Smoking can cause vascular endothelial dysfunction and consequently trigger haemostatic activation and thrombosis. However, the mechanism of how smoking promotes thrombosis is not fully understood. Thrombosis is associated with the imbalance of the coagulant system due to endothelial dysfunction. As a vital anticoagulation cofactor, thrombomodulin (TM) located on the endothelial cell surface is able to regulate intravascular coagulation by binding to thrombin, and the binding results in thrombosis inhibition. This work focused on the effects of cigarette smoke extract (CSE) on TM-thrombin binding by atomic force microscopy (AFM) based single-molecule force spectroscopy. The results from both in vitro and live-cell experiments indicated that CSE could notably reduce the binding probability of TM and thrombin. This study provided a new approach and new evidence for studying the mechanism of thrombosis triggered by cigarette smoking.

A multifunctional hemin@metal-organic framework and its application to construct an electrochemical aptasensor for thrombin detection

NASA Astrophysics Data System (ADS)

Xie, Shunbi; Ye, Jiawei; Yuan, Yali; Chai, Yaqin; Yuan, Ruo

2015-10-01

A new type of multifunctional metal-organic framework (MOF) has been synthesized by encapsulating hemin into the nano-sized Fe-MIL-88 MOFs (hemin@MOFs) and first applied in an electrochemical aptasensor to detect thrombin (TB) with the aid of an enzyme for signal amplification. The gold nanoparticle functionalized hemin@MOFs (Au/hemin@MOFs) have not only simultaneously served as redox mediators and solid electrocatalysts, but have also been utilized as an ideal loading platform to immobilize a large number of biomolecules. In this aptasensor, Au/hemin@MOFs conjugated with glucose oxidase (GOD) and thrombin binding aptamer (TBA II) were used as the secondary aptamer bioconjugates (Au/hemin@MOF-TBA II-GOD bioconjugates), and TB was sandwiched between Au/hemin@MOF-TBA II-GOD bioconjugates and the amino-terminated TBA I which was self-assembled on the gold nanoparticle (AuNP) modified electrode. The GOD could oxidize glucose into gluconic acid accompanied by the generation of H2O2. The generated H2O2 on the electrode surface was further electrocatalyzed by hemin@MOFs to amplify the electrochemical signal of hemin contained in hemin@MOFs. Therefore, the synthesized hemin@MOFs represented a new paradigm for multifunctional materials since it combined three different functions including serving as catalysts, redox mediators and loading platforms within a single material. With such an ingenious design, a wide linear range of 0.0001 nM to 30 nM was acquired with a relatively low detection limit of 0.068 pM for TB detection.A new type of multifunctional metal-organic framework (MOF) has been synthesized by encapsulating hemin into the nano-sized Fe-MIL-88 MOFs (hemin@MOFs) and first applied in an electrochemical aptasensor to detect thrombin (TB) with the aid of an enzyme for signal amplification. The gold nanoparticle functionalized hemin@MOFs (Au/hemin@MOFs) have not only simultaneously served as redox mediators and solid electrocatalysts, but have also been utilized as an ideal loading platform to immobilize a large number of biomolecules. In this aptasensor, Au/hemin@MOFs conjugated with glucose oxidase (GOD) and thrombin binding aptamer (TBA II) were used as the secondary aptamer bioconjugates (Au/hemin@MOF-TBA II-GOD bioconjugates), and TB was sandwiched between Au/hemin@MOF-TBA II-GOD bioconjugates and the amino-terminated TBA I which was self-assembled on the gold nanoparticle (AuNP) modified electrode. The GOD could oxidize glucose into gluconic acid accompanied by the generation of H2O2. The generated H2O2 on the electrode surface was further electrocatalyzed by hemin@MOFs to amplify the electrochemical signal of hemin contained in hemin@MOFs. Therefore, the synthesized hemin@MOFs represented a new paradigm for multifunctional materials since it combined three different functions including serving as catalysts, redox mediators and loading platforms within a single material. With such an ingenious design, a wide linear range of 0.0001 nM to 30 nM was acquired with a relatively low detection limit of 0.068 pM for TB detection. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr04532k

Nanopore Force Spectroscopy of Aptamer–Ligand Complexes

PubMed Central

Arnaut, Vera; Langecker, Martin; Simmel, Friedrich C.

2013-01-01

The stability of aptamer–ligand complexes is probed in nanopore-based dynamic force spectroscopy experiments. Specifically, the ATP-binding aptamer is investigated using a backward translocation technique, in which the molecules are initially pulled through an α-hemolysin nanopore from the cis to the trans side of a lipid bilayer membrane, allowed to refold and interact with their target, and then translocated back in the trans–cis direction. From these experiments, the distribution of bound and unbound complexes is determined, which in turn allows determination of the dissociation constant Kd ≈ 0.1 mM of the aptamer and of voltage-dependent unfolding rates. The experiments also reveal differences in binding of the aptamer to AMP, ADP, or ATP ligands. Investigation of an aptamer variant with a stabilized ATP-binding site indicates fast conformational switching of the original aptamer before ATP binding. Nanopore force spectroscopy is also used to study binding of the thrombin-binding aptamer to its target. To detect aptamer–target interactions in this case, the stability of the ligand-free aptamer—containing G-quadruplexes—is tuned via the potassium content of the buffer. Although the presence of thrombin was detected, limitations of the method for aptamers with strong secondary structures and complexes with nanomolar Kd were identified. PMID:24010663

THROMBIN GENERATION AND BLEEDING IN HEMOPHILIA A

PubMed Central

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

2012-01-01

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 (p<0.001) between the groups: 504±114nM, 315±117nM, and 194±91nM; with higher thrombin concentrations in the groups with lower bleeding scores. MaxR was higher in the groups with a lower bleeding score; 97±51nM/min, 86±60nM/min and 39±16nM/min (p=0.09). No significant difference was detected in CT among the groups, 5.6±1.3min, 4.7±0.7min, 5.6±1.3min. Conclusions Our empirical study in CTI-inhibited whole blood shows that the MaxL of thrombin generation appears to correlate with the bleeding phenotype of hemophilia A. PMID:19563500

OVER-EXPRESSION OF THE THROMBIN RECEPTOR (PAR-1) IN THE PLACENTA IN PREECLAMPSIA: A MECHANISM FOR THE INTERSECTION OF COAGULATION AND INFLAMMATION

PubMed Central

EREZ, OFFER; ROMERO, ROBERTO; KIM, SUNG-SU; KIM, JUNG-SUN; KIM, YEON MEE; WILDMAN, DEREK E; THAN, NANDOR GABOR; MAZAKI-TOVI, SHALI; GOTSCH, FRANCESCA; PINELES, BETH; KUSANOVIC, JUAN PEDRO; ESPINOZA, JIMMY; MITTAL, POOJA; MAZOR, MOSHE; HASSAN, SONIA S.; KIM, CHONG JAI

2008-01-01

Objective Preeclampsia (PE) is characterized by excessive thrombin generation that has been implicated in the multiple organ damage associated with the disease. The biological effects of thrombin on coagulation and inflammation are mediated by protease activated receptor-1 (PAR-1), a G-protein coupled receptor. The aim of this study was to determine whether preterm preeclampsia (PE) is associated with changes in placental expression of PAR-1. Study design This cross-sectional study included two groups matched for gestational age at delivery: 1) patients with preterm PE (<37 weeks of gestation; n=26) and 2) a control group of patients with preterm labor without intraamniotic infection (n=26). Placental tissue microarrays were immunostained for PAR-1. Immunoreactivity of PAR-1 in the villous trophoblasts was graded as negative, weak-positive, or strong-positive. Results 1) The proportion of cases with strong PAR-1 immunoreactivity was significantly higher in placentas of patients with preeclampsia than in placentas from the control group [37.5% (9/24) vs. 8.7% (2/23); p=0.036, respectively]. 2) PAR-1 immunoreactivity was found in the cellular compartments of the placental villous tree, mainly in villous trophoblasts and stromal endothelial cells. 3) PAR-1 was detected in 92.3% (24/26) of the placentas of women with preeclampsia and in 88.5% (23/26) of the placentas from the control group (p=1). Conclusion Placentas from pregnancies complicated by preterm PE had a significantly higher frequency of strong PAR-1 expression than placentas from women with spontaneous PTL. This observation is consistent with a role for PAR-1 as a mediator of the effect of thrombin on coagulation and inflammation in preeclampsia. We propose that the effects of thrombin in PE are due to increased thrombin generation and higher expression of PAR-1, the major receptor for this enzyme. PMID:18570113

Retro-binding thrombin active site inhibitors: identification of an orally active inhibitor of thrombin catalytic activity.

PubMed

Iwanowicz, Edwin J; Kimball, S David; Lin, James; Lau, Wan; Han, W-C; Wang, Tammy C; Roberts, Daniel G M; Schumacher, W A; Ogletree, Martin L; Seiler, Steven M

2002-11-04

A series of retro-binding inhibitors of human alpha-thrombin was prepared to elucidate structure-activity relationships (SAR) and optimize in vivo performance. Compounds 9 and 11, orally active inhibitors of thrombin catalytic activity, were identified to be efficacious in a thrombin-induced lethality model in mice.

Multiplexed evaluation of capture agent binding kinetics using arrays of silicon photonic microring resonators.

PubMed

Byeon, Ji-Yeon; Bailey, Ryan C

2011-09-07

High affinity capture agents recognizing biomolecular targets are essential in the performance of many proteomic detection methods. Herein, we report the application of a label-free silicon photonic biomolecular analysis platform for simultaneously determining kinetic association and dissociation constants for two representative protein capture agents: a thrombin-binding DNA aptamer and an anti-thrombin monoclonal antibody. The scalability and inherent multiplexing capability of the technology make it an attractive platform for simultaneously evaluating the binding characteristics of multiple capture agents recognizing the same target antigen, and thus a tool complementary to emerging high-throughput capture agent generation strategies.

New synthetic thrombin inhibitors: molecular design and experimental verification.

PubMed

Sinauridze, Elena I; Romanov, Alexey N; Gribkova, Irina V; Kondakova, Olga A; Surov, Stepan S; Gorbatenko, Aleksander S; Butylin, Andrey A; Monakov, Mikhail Yu; Bogolyubov, Alexey A; Kuznetsov, Yuryi V; Sulimov, Vladimir B; Ataullakhanov, Fazoyl I

2011-01-01

The development of new anticoagulants is an important goal for the improvement of thromboses treatments. The design, synthesis and experimental testing of new safe and effective small molecule direct thrombin inhibitors for intravenous administration. 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. New compounds that are both effective direct thrombin inhibitors (the best K(I) was <1 nM) and strong anticoagulants in plasma (an IC(50) in the thrombin generation assay of approximately 100 nM) were discovered. These compounds contain one of the following new residues as the basic fragment: isothiuronium, 4-aminopyridinium, or 2-aminothiazolinium. LD(50) values for the best new inhibitors ranged from 166.7 to >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. The high efficacy, stability and low acute toxicity reveal that the inhibitors that were developed may be promising for potential medical applications.

[Concepts in anticoagulant therapy - past, present, and future].

PubMed

Graf, L

2012-11-01

The understanding of the clotting system emerged in parallel to the development of anticoagulants. In contrast to vitamin K-antagonists and heparins that where discovered by chance, new anticoagulants have been systematically designed to specifically inhibit single clotting factors. Both clotting factors Xa (FXa) and thrombin play a crucial role within the new cell-based model of hemostasis. Thus it is obvious that FXa and thrombin turned out to be ideal targets for anticoagulation. The proof of the concept of selective inhibition of thrombin and FXa has been provided by hirudin and fondaparinux, respectively. By now, a whole group of new oral anticoagulants has been licensed: the direct FXa-inhibitors rivaroxaban, apixaban, and edoxaban as well as the direct thrombin dabigatran etexilate. Furthermore, a bundle of FXa- and thrombin-inhibitors that differ from the so far licensed products mainly in pharmacokinetics are in an advanced phase of development. A further innovative concept of anticoagulation that entered its clinical phase of development is the inhibition of factor VIII. Other new concepts such as inhibition of initiation of coagulation by blocking factor VIIa, inhibition of contact factor XII, or inhibition of factor IX are in an early phase of development.

Dual inhibition of HY023016 based on binding properties of platelet membrane receptor subunit glycoprotein Ibα and thrombin exosites.

PubMed

Chen, Qiu-Fang; Cui, Shuang; Shen, Hui-Liang; Chen, Xiang; Li, Yun-Zhan; Wu, Qian; Xu, Yun-Gen; Gong, Guo-Qing

2018-03-05

Thrombin has long been suggested as a desirable antithrombotic target, but anti-thrombin therapy without anti-platelet thereby has never achieved the ideal effect. HY023016 is a novel compound, in our previous study, it exerted better anti-thrombotic than dabigatran etexilate. The present study aims to illustrate the excess anti-thrombotic molecular mechanisms of HY023016 through thrombin anion exosites and the platelet membrane receptor subunit glycoprotein Ibα (GPIbα). HY023016 strongly inhibited the conversion of fibrinogen to fibrous may via blocking thrombin exosite I. We also discovered that HY023016 remarkably inhibited exosite II by a loss of affinity for the γ'-peptide of fibrinogen and for heparin. Furthermore, a solid phase binding assay revealed that HY023016 inhibited ristocetin-induced washed platelets bind to von Willebrand factor (vWF). In GST pull-down assay, HY023016 decreased the binding of recombinant vWF-A1 to GPIbα N-terminal. Thus, HY023016 provides an innovative idea for designing multi-targeted anti-thrombotic drugs and laying a scientific foundation for reducing "total thrombosis risk" in a clinical drug treatment. Copyright © 2018 Elsevier B.V. All rights reserved.

Activated factor XI and tissue factor in aortic stenosis: Links with thrombin generation

PubMed Central

Luszczak, Joanna; Undas, Anetta; Gissel, Matthew; Olszowska, Maria; Butenas, Saulius

2011-01-01

Introduction In our previous studies we showed that a significant proportion of patients with various cardiovascular diseases have active tissue factor (TF) and factor (F)XIa in their plasma. Objective To evaluate these two proteins in plasma from patients with aortic stenosis (AS) and established their relationship with the severity of the disease. Methods Fifty-four consecutive patients with AS, including 38 (70.4%) severe AS patients, were studied. Plasma FXIa and TF activity were determined in clotting assays by measuring the response to inhibitory monoclonal antibodies. Results TF activity was detectible in plasma from 14 of 54 patients (25.9%), including 13 of 38 with severe AS (34.2%) and 1 of 16 (6.25%) with moderate AS (p=0.052). FXIa activity was found in 12 (22.2%) patients, mostly in individuals with severe AS (11 of 38, 28.9%, p=0.067). All 12 patients with circulating FXIa had active TF in their plasma as well. Severe AS patients with detectable TF had higher maximal (111±20 vs 97±16 mm Hg, p=0.02) and mean (61±12 vs 53±8 mm Hg, p=0.02) transvalvular gradient, compared with those without such activity in plasma. In severe AS patients with detectable active TF, prothrombin fragment 1.2, a thrombin generation marker, was higher than in patients without TF (375±122 vs. 207±64 pM, p<0.001). Conclusions Detectable FXIa and TF activity was observed for the first time in AS patients, primarily in severe ones. This activity correlates with thrombin generation in those patients. PMID:21519234

Identification of berberine as a direct thrombin inhibitor from traditional Chinese medicine through structural, functional and binding studies

NASA Astrophysics Data System (ADS)

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

2017-03-01

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.

Thrombin enhances herpes simplex virus infection of cells involving protease-activated receptor 1.

PubMed

Sutherland, M R; Friedman, H M; Pryzdial, E L G

2007-05-01

We have previously shown that the surface of purified herpes family viruses can initiate thrombin production by expressing host-encoded and virus-encoded procoagulant factors. These enable the virus to bypass the normal cell-regulated mechanisms for initiating coagulation, and provide a link between infection and vascular disease. In the current study we investigated why these viruses may have evolved to generate thrombin. Using cytolytic viral plaque assays, the current study examines the effect of thrombin on human umbilical vein endothelial cell (HUVEC) or human foreskin fibroblast (HFF) infection by purified herpes simplex virus type 1 (HSV1) and type 2 (HSV2). Demonstrating that the availability of thrombin is an advantage to the virus, purified thrombin added to serum-free inoculation media resulted in up to a 3-fold enhancement of infection depending on the virus strain and cell type. The effect of thrombin on HUVEC infection was generally greater than its effect on HFF. To illustrate the involvement of thrombin produced during inoculation, hirudin was shown to inhibit the infection of each HSV strain, but only when serum containing clotting factors for thrombin production was present in media. The involvement of protease-activated receptor 1 (PAR1) was supported using PAR1-activating peptides in place of thrombin and PAR1-specific antibodies to inhibit the effects of thrombin. These data show that HSV1 and HSV2 initiate thrombin production to increase the susceptibility of cells to infection through a mechanism involving PAR1-mediated cell modulation.

Near-infrared photoluminescence biosensing platform with gold nanorods-over-gallium arsenide nanohorn array.

PubMed

Zhang, Yiming; Jiang, Tao; Tang, Longhua

2017-11-15

The near-infrared (NIR) optical detection of biomolecules with high sensitivity and reliability have been expected, however, it is still a challenge. In this work, we present a gold nanorods (AuNRs)-over-gallium arsenide nanohorn-like array (GaAs NHA) system that can be used for the ultrasensitive and specific NIR photoluminescence (PL) detection of DNA and proteins. The fabrication of GaAs NHA involved the technique of colloidal lithography and inductively coupled plasma dry etching, yielding large-area and well-defined nanostructural array, and exhibiting an improved PL emission compared to the planar GaAs substrate. Importantly, we found that the DNA-bridged AuNRs attachment on NHA could further improve the PL intensity from GaAs, and thereby provide the basis for the NIR optical sensing of biological analytes. We demonstrated that DNA and thrombin could be sensitively and specifically detected, with the detection limit of 1 pM for target DNA and 10 pM for thrombin. Such ultrasensitive NIR optical platform can extend to the detection of other biomarkers and is promising for clinical diagnostics. Copyright © 2017 Elsevier B.V. All rights reserved.

Thrombin-induced Migration and Matrix Metalloproteinase-9 Expression Are Regulated by MAPK and PI3K Pathways in C6 Glioma Cells

PubMed Central

Kim, Jiyoung; Lee, Jae-Won; Kim, Song-In; Choi, Yong-Joon; Lee, Won-Ki; Jeong, Myung-Ja; Cha, Sang-Hoon; Lee, Hee Jae; Chun, Wanjoo

2011-01-01

Glioblastoma multiforme is one of the most common and aggressive tumors in central nervous system. It often possesses characteristic necrotic lesions with hemorrhages, which increase the chances of exposure to thrombin. Thrombin has been known as a regulator of MMP-9 expression and cancer cell migration. However, the effects of thrombin on glioma cells have not been clearly understood. In the present study, influences of thrombin on glioma cell migration were examined using Boyden chamber migration assay and thrombin-induced changes in MMP-9 expression were measured using zymography, semi-quantitative RT-PCR, and Western blotting. Furthermore, underlying signaling pathways by which thrombin induces MMP-9 expression were examined. Thrombin-induced migration and MMP-9 expression were significantly potentiated in the presence of wortmannin, a PI3K inhibitor, whereas MAPK inhibitors suppressed thrombin-induced migration and MMP-9 expression in C6 glioma cells. The present data strongly demonstrate that MAPK and PI3K pathways evidently regulate thrombin-induced migration and MMP-9 expression of C6 glioma cells. Therefore, the control of these pathways might be a beneficial therapeutic strategy for treatment of invasive glioblastoma multiforme. PMID:21994479

Tissue thrombin is associated with the pathogenesis of dilated cardiomyopathy.

PubMed

Ito, Keiichi; Hongo, Kenichi; Date, Taro; Ikegami, Masahiro; Hano, Hiroshi; Owada, Mamiko; Morimoto, Satoshi; Kashiwagi, Yusuke; Katoh, Daisuke; Yoshino, Takuya; Yoshii, Akira; Kimura, Haruka; Nagoshi, Tomohisa; Kajimura, Ichige; Kusakari, Yoichiro; Akaike, Toru; Minamisawa, Susumu; Ogawa, Kazuo; Minai, Kosuke; Ogawa, Takayuki; Kawai, Makoto; Yajima, Junji; Matsuo, Seiichiro; Yamane, Teiichi; Taniguchi, Ikuo; Morimoto, Sachio; Yoshimura, Michihiro

2017-02-01

Thrombin is a serine protease known to be the final product of the coagulation cascade. However, thrombin plays other physiological roles in processes such as gastric contractions and vessel wound healing, and a state of coagulability is increased in patients with dilated cardiomyopathy (DCM). In this study, we investigate the role of thrombin in the pathogenesis of DCM. The purpose of this study is to clarify the role of thrombin in the pathogenesis of DCM and investigate the possibility of treatment against DCM by thrombin inhibition. We investigated the expression of thrombin in the left ventricles of five patients with DCM who underwent the Batista operation and four patients without heart disease. Furthermore, we investigated the involvement of thrombin in the development of DCM using knock-in mice with a deletion mutation of cardiac troponin T that causes human DCM (∆K210 knock-in mouse) (B6;129-Tnnt2 tm2Mmto ) and assessed the effects of a direct thrombin inhibitor, dabigatran on ∆K210 knock-in mice using echocardiographic examinations, the Kaplan-Meier method and Western blotting. The immunohistochemical analysis showed a strong thrombin expression in the DCM patients compared to the patients without heart disease. In immunohistochemical analysis, a strong thrombin expression was observed in the heart tissues analysis in the ∆K210 knock-in mice. Dabigatran administration significantly improved fractional shortening according to the echocardiographic examination and the survival outcomes in ∆K210 knock-in mice. Tissue thrombin is involved in the pathogenesis of DCM and thrombin inhibition can be beneficial for the treatment of DCM. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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

PubMed

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

2016-10-28

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 α IIb β 3 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.

Phloretin suppresses thrombin-mediated leukocyte-platelet-endothelial interactions.

PubMed

Kim, Min Soo; Park, Sin-Hye; Han, Seon-Young; Kim, Yun-Ho; Lee, Eun-Jung; Yoon Park, Jung Han; Kang, Young-Hee

2014-04-01

Thrombin playing a pivotal role in coagulation cascade may influence the onset and progression of atherosclerosis as a pro-inflammatory mediator. This study investigated whether phloretin found in apple tree leaves, severed a linkage between thrombosis and atherosclerosis by thrombin. Human endothelial cells were pre-treated with 1-20 μM phloretin and stimulated with 10 U/mL thrombin. Phloretin attenuated adhesion of THP-1 monocytes and platelets to thrombin-inflamed endothelial cells with concurrent inhibition of protease-activated receptor (PAR-1) induction. The thrombin induction of endothelial CD40, endothelial integrin β3 and P-selectin, and monocytic CD40L was dampened by phloretin. Additionally, phloretin inhibited monocyte secretion of MCP-1, IL-6 and IL-8 responsible for pro-inflammatory activity of thrombin inducing endothelial CD40. The monocyte COX-2 induction and PGE2 secretion due to thrombin were down-regulated by phloretin, deterring endothelial CD40 expression. Thrombin promoted production of PAI-1 and tissue factor in monocytes was attenuated by phloretin through blocking PAR-1 and CD40. Thrombin up-regulated the induction of endothelial connective tissue growth factor independent of PAR-1 activation, which was reversed by phloretin. Phloretin disturbed tethering and stable adhesion of monocytes and platelets onto endothelium during increased thrombosis by thrombin. Phloretin would be a potent agent preventing thrombosis and atherosclerosis. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The monoclonal antibody that recognizes an epitope in the C-terminal region of the fibrinogen alpha-chain reacts with soluble fibrin and fibrin monomer generated by thrombin but not with those formed as plasmin degradation products.

PubMed

Suzuki, Akiko; Ebinuma, Hiroyuki; Matsuo, Masanao; Miyazaki, Osamu; Yago, Hirokazu

2007-01-01

The presence of soluble fibrin (SF) provides evidence of thrombin activation in the blood; therefore, SF is a useful marker for diagnosing blood coagulation diseases such as disseminated intravascular coagulation (DIC). The antibody that specifically detects SF could be a useful tool for diagnosing thrombotic diseases. By using an acid-solubilized desAA-FM (fibrin monomer) as an immunogen, we developed a monoclonal antibody, namely J2-23, which specifically reacts with SF and FM. We examined the specificity of J2-23 by ELISA and immunoblotting and confirmed the reactivity of J2-23 with SF and FM by gel filtration. J2-23 specifically reacted with SF, but not with fibrinogen or plasmic fibrinogen-derived Fbg-X, Fbg-Y, Fbg-E, and D; thrombin-treated Fbn-X, Fbn-Y, and Fbn-E; and plasmic cross-linked fibrin (DD, XDP). The epitope recognized by J2-23 was located within the Aalpha 502-521 region on the C-terminal of the fibrinogen alpha-chain. The reactivity of J2-23 disappeared following the action of the fibrinolytic enzyme plasmin. Furthermore, J2-23 reacted not only with SF but also with FM in plasma from DIC patients. This indicated that J2-23 specifically detected coagulation without reflecting the plasmin action. We demonstrated the potential of J2-23 as a useful antibody for detecting SF for diagnosing blood coagulation.

Genetic Determinants of Thrombin Generation and Their Relation to Venous Thrombosis: Results from the GAIT-2 Project

PubMed Central

Martin-Fernandez, Laura; Ziyatdinov, Andrey; Carrasco, Marina; Millon, Juan Antonio; Martinez-Perez, Angel; Vilalta, Noelia; Brunel, Helena; Font, Montserrat; Hamsten, Anders; Souto, Juan Carlos; Soria, José Manuel

2016-01-01

Background Venous thromboembolism (VTE) is a common disease where known genetic risk factors explain only a small portion of the genetic variance. Then, the analysis of intermediate phenotypes, such as thrombin generation assay, can be used to identify novel genetic risk factors that contribute to VTE. Objectives To investigate the genetic basis of distinct quantitative phenotypes of thrombin generation and its relationship to the risk of VTE. Patients/Methods Lag time, thrombin peak and endogenous thrombin potential (ETP) were measured in the families of the Genetic Analysis of Idiopathic Thrombophilia 2 (GAIT-2) Project. This sample consisted of 935 individuals in 35 extended families selected through a proband with idiopathic thrombophilia. We performed also genome wide association studies (GWAS) with thrombin generation phenotypes. Results The results showed that 67% of the variation in the risk of VTE is attributable to genetic factors. The heritabilities of lag time, thrombin peak and ETP were 49%, 54% and 52%, respectively. More importantly, we demonstrated also the existence of positive genetic correlations between thrombin peak or ETP and the risk of VTE. Moreover, the major genetic determinant of thrombin generation was the F2 gene. However, other suggestive signals were observed. Conclusions The thrombin generation phenotypes are strongly genetically determined. The thrombin peak and ETP are significantly genetically correlated with the risk of VTE. In addition, F2 was identified as a major determinant of thrombin generation. We reported suggestive signals that might increase our knowledge to explain the variability of this important phenotype. Validation and functional studies are required to confirm GWAS results. PMID:26784699

Kinetic modeling sheds light on the mode of action of recombinant factor VIIa on thrombin generation.

PubMed

Mitrophanov, Alexander Y; Reifman, Jaques

2011-10-01

The therapeutic potential of a hemostatic agent can be assessed by investigating its effects on the quantitative parameters of thrombin generation. For recombinant activated factor VII (rFVIIa)--a promising hemostasis-inducing biologic--experimental studies addressing its effects on thrombin generation yielded disparate results. To elucidate the inherent ability of rFVIIa to modulate thrombin production, it is necessary to identify rFVIIa-induced effects that are compatible with the available biochemical knowledge about thrombin generation mechanisms. The existing body of knowledge about coagulation biochemistry can be rigorously represented by a computational model that incorporates the known reactions and parameter values constituting the biochemical network. We used a thoroughly validated numerical model to generate activated factor VII (FVIIa) titration curves in the cases of normal blood composition, hemophilia A and B blood, blood lacking factor VII, blood lacking tissue factor pathway inhibitor, and diluted blood. We utilized the generated curves to perform systematic fold-change analyses for five quantitative parameters characterizing thrombin accumulation. The largest fold changes induced by increasing FVIIa concentration were observed for clotting time, thrombin peak time, and maximum slope of the thrombin curve. By contrast, thrombin peak height was much less affected by FVIIa titrations, and the area under the thrombin curve stayed practically unchanged. Comparisons with experimental data demonstrated that the computationally derived patterns can be observed in vitro. rFVIIa modulates thrombin generation primarily by accelerating the process, without significantly affecting the total amount of generated thrombin. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

PubMed Central

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

1994-01-01

Synthetic thrombin receptor peptides (TRPs), comprising the first 6-14 amino acids of the new N-terminus tethered ligand of the thrombin receptor that is generated by thrombin's proteolytic activity, were reported to activate platelets equally with thrombin itself and are considered to be full agonists [Vu et al. (1991) Cell 64, 1057-1068]. Using aspirin plus ADP-scavengers or the ADP-receptor antagonist adenosine 5'-[alpha-thio]triphosphate to prevent the secondary effects of the potent agonists that are normally released from stimulated platelets (i.e. ADP and thromboxane A2), we assessed the direct actions of thrombin and TRPs (i.e. TRP42-47 and TRP42-55). Compared with thrombin, under these conditions, TRPs: (1) failed to aggregate platelets completely; (2) produced less activation of glycoprotein (GP)IIb-IIIa; (3) did not cause association of GPIIb and pp60c-src with the cytoskeleton; and (4) caused less alpha-granule secretion, phosphorylation of cytoplasmic phospholipase A2, arachidonic acid release and phosphatidyl inositol (PtdOH) production. Furthermore, TRPs induced transient increases in protein phosphorylation mediated by protein kinase C and protein tyrosine phosphorylation, whereas these same responses to thrombin were greater and more sustained. Hirudin added after thrombin accelerated protein dephosphorylation, thereby mimicking the rate of spontaneous dephosphorylation seen after stimulation by TRPs. Platelets totally desensitized to very high concentrations of TRPs, by prior exposure to maximally effective concentrations of the peptides, remained responsive to alpha- and gamma-thrombins. Thrombin-stimulated PtdOH production in permeabilized platelets desensitized to TRPs was abolished by guanosine 5'-[beta-thio]diphosphate (GDP[beta S]), as in normal platelets. These results are discussed in terms of the allosteric Ternary Complex Model for G-protein linked receptors [Samama et al. (1993) J. Biol. Chem. 268, 4625-4636]. We conclude that: (1) TRPs are partial agonists for the thrombin receptor and produce incomplete receptor desensitization in keeping with their lower intrinsic activity; (2) thrombin's effects in platelets, even in TRP-desensitized platelets, are entirely mediated through the recently cloned G-protein linked receptor, and (3) thrombin's ability to produce sustained signals, compared with TRPs, may require the continued progressive proteolytic activation of naive thrombin receptors. Images Figure 3 PMID:7526841

Identification of protein–protein interfaces by decreased amide proton solvent accessibility

PubMed Central

Mandell, Jeffrey G.; Falick, Arnold M.; Komives, Elizabeth A.

1998-01-01

Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry was used to identify peptic fragments from protein complexes that retained deuterium under hydrogen exchange conditions due to decreased solvent accessibility at the interface of the complex. Short deuteration times allowed preferential labeling of rapidly exchanging surface amides so that primarily solvent accessibility changes and not conformational changes were detected. A single mass spectrum of the peptic digest mixture was analyzed to determine the deuterium content of all proteolytic fragments of the protein. The protein–protein interface was reliably indicated by those peptides that retained more deuterons in the complex compared with control experiments in which only one protein was present. The method was used to identify the kinase inhibitor [PKI(5–24)] and ATP-binding sites in the cyclic-AMP-dependent protein kinase. Three overlapping peptides identified the ATP-binding site, three overlapping peptides identified the glycine-rich loop, and two peptides identified the PKI(5–24)-binding site. A complex of unknown structure also was analyzed, human α-thrombin bound to an 83-aa fragment of human thrombomodulin [TMEGF(4–5)]. Five peptides from thrombin showed significantly decreased solvent accessibility in the complex. Three peptides identified the anion-binding exosite I, confirming ligand competition experiments. Two peptides identified a new region of thrombin near the active site providing a potential mechanism of how thrombomodulin alters thrombin substrate specificity. PMID:9843953

A Universal Base in a Specific Role: Tuning up a Thrombin Aptamer with 5-Nitroindole

NASA Astrophysics Data System (ADS)

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

2015-11-01

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

A Universal Base in a Specific Role: Tuning up a Thrombin Aptamer with 5-Nitroindole

PubMed Central

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

2015-01-01

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

Thrombin mediated transcriptional regulation using DNA aptamers in DNA based cell free protein synthesis

PubMed Central

Iyer, Sukanya

2013-01-01

Realizing the potential of cell free systems will require development of ligand sensitive gene promoters that control gene expression in response to a ligand of interest. Here, we describe an approach to designing ligand sensitive transcriptional control in cell free systems that is based on the combination of a DNA aptamer that binds thrombin and the T7 bacteriophage promoter. Placement of the aptamer near the T7 promoter, and using a primarily single stranded template, results in up to a five-fold change in gene expression in a ligand concentration dependent manner. We further demonstrate that the sensitivity to thrombin concentration and the fold change in expression can be tuned by altering the position of the aptamer. The results described here pave the way for the use of DNA aptamers to achieve modular regulation of transcription in response to a wide variety of ligands in cell free systems. PMID:24059754

Protein kinase C and P2Y12 take center stage in thrombin-mediated activation of mammalian target of rapamycin complex 1 in human platelets.

PubMed

Moore, S F; Hunter, R W; Hers, I

2014-05-01

Rapamycin, an inhibitor of mammalian target of rapamycin complex-1 (mTORC1), reduces platelet spreading, thrombus stability, and clot retraction. Despite an important role of mTORC1 in platelet function, little is known about how it is regulated. The objective of this study was to determine the signaling pathways that regulate mTORC1 in human platelets. Mammalian target of rapamycin complex-1 activation was assessed by measuring the phosphorylation of its downstream substrate ribosomal S6 kinase 1 (p70S6K). Thrombin or the protein kinase C (PKC) activator phorbal 12-myristate 13-acetate stimulated activation of mTORC1 in a PKC-dependent, Akt-independent manner that correlated with phosphorylation of tuberin/tuberous sclerosis 2 (TSC2) (Ser939 and Thr1462). In contrast, insulin-like growth factor 1 (IGF-1)-stimulated TSC2 phosphorylation was completely dependent on phosphoinositide 3 kinase (PI3 kinase)/Akt but did not result in any detectable mTORC1 activation. Early (Ser939 and Thr1462) and late (Thr1462) TSC2 phosphorylation in response to thrombin were directly PKC dependent, whereas later TSC2 (Ser939) and p70S6K phosphorylation were largely dependent on paracrine signaling through P2Y(12). PKC-mediated adenosine diphosphate (ADP) secretion was essential for thrombin-stimulated mTORC1 activation, as (i) ADP rescued p70S6K phosphorylation in the presence of a PKC inhibitor and (ii) P2Y(12) antagonism prevented thrombin-mediated mTORC1 activation. Rescue of mTORC1 activation with exogenous ADP was completely dependent on the Src family kinases but independent of PI3 kinase/Akt. Interestingly, although inhibition of Src blocked the ADP rescue, it had little effect on thrombin-stimulated p70S6K phosphorylation under conditions where PKC was not inhibited. These results demonstrate that thrombin activates the mTORC1 pathway in human platelets through PKC-mediated ADP secretion and subsequent activation of P2Y(12), in a manner largely independent of the canonical PI3 kinase/Akt pathway. © 2014 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.

Protein kinase C and P2Y12 take center stage in thrombin-mediated activation of mammalian target of rapamycin complex 1 in human platelets

PubMed Central

Moore, S F; Hunter, R W; Hers, I

2014-01-01

Background Rapamycin, an inhibitor of mammalian target of rapamycin complex-1 (mTORC1), reduces platelet spreading, thrombus stability, and clot retraction. Despite an important role of mTORC1 in platelet function, little is known about how it is regulated. The objective of this study was to determine the signaling pathways that regulate mTORC1 in human platelets. Methods Mammalian target of rapamycin complex-1 activation was assessed by measuring the phosphorylation of its downstream substrate ribosomal S6 kinase 1 (p70S6K). Results Thrombin or the protein kinase C (PKC) activator phorbal 12-myristate 13-acetate stimulated activation of mTORC1 in a PKC-dependent, Akt-independent manner that correlated with phosphorylation of tuberin/tuberous sclerosis 2 (TSC2) (Ser939 and Thr1462). In contrast, insulin-like growth factor 1 (IGF-1)–stimulated TSC2 phosphorylation was completely dependent on phosphoinositide 3 kinase (PI3 kinase)/Akt but did not result in any detectable mTORC1 activation. Early (Ser939 and Thr1462) and late (Thr1462) TSC2 phosphorylation in response to thrombin were directly PKC dependent, whereas later TSC2 (Ser939) and p70S6K phosphorylation were largely dependent on paracrine signaling through P2Y12. PKC-mediated adenosine diphosphate (ADP) secretion was essential for thrombin-stimulated mTORC1 activation, as (i) ADP rescued p70S6K phosphorylation in the presence of a PKC inhibitor and (ii) P2Y12 antagonism prevented thrombin-mediated mTORC1 activation. Rescue of mTORC1 activation with exogenous ADP was completely dependent on the Src family kinases but independent of PI3 kinase/Akt. Interestingly, although inhibition of Src blocked the ADP rescue, it had little effect on thrombin-stimulated p70S6K phosphorylation under conditions where PKC was not inhibited. Conclusion These results demonstrate that thrombin activates the mTORC1 pathway in human platelets through PKC-mediated ADP secretion and subsequent activation of P2Y12, in a manner largely independent of the canonical PI3 kinase/Akt pathway. PMID:24612393

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

PubMed

Orbe, Josune; Rodríguez, José A; Calvayrac, Olivier; Rodríguez-Calvo, Ricardo; Rodríguez, Cristina; Roncal, Carmen; Martínez de Lizarrondo, Sara; Barrenetxe, Jaione; Reverter, Juan C; Martínez-González, José; Páramo, José A

2009-12-01

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

Thrombin-induced p38 mitogen-activated protein kinase activation is mediated by epidermal growth factor receptor transactivation pathway

PubMed Central

Kanda, Yasunari; Mizuno, Katsushige; Kuroki, Yasutomi; Watanabe, Yasuhiro

2001-01-01

Thrombin is a potent mitogen for vascular smooth muscle cells (VSMC) and has been implicated its pathogenic role in vascular remodelling. However, the signalling pathways by which thrombin mediates its mitogenic response are not fully understood.We have previously reported that thrombin activates p38 mitogen-activated protein kinase (p38 MAPK) by a tyrosine kinase-dependent mechanism, and that p38 MAPK has a role in thrombin-induced mitogenic response in rat VSMC.In the present study, we examine the involvement of epidermal growth factor (EGF) receptor in thrombin-induced p38 MAPK activation. We found that thrombin induced EGF receptor tyrosine phosphorylation (transactivation) in A10 cells, a clonal VSMC cell line. A selective inhibitor of EGF receptor kinase (AG1478) inhibited the p38 MAPK activation in a dose-dependent manner, whereas it had no effect on the response to platelet-derived growth factor (PDGF). EGF receptor phosphorylation induced by thrombin was inhibited by BAPTA-AM and GF109203X, which suggest a requirement for intracellular Ca2+ increase and protein kinase C.We next examined the effect of AG1478 on thrombin-induced DNA synthesis. AG1478 inhibited thrombin-induced DNA synthesis in a dose-dependent manner. In contrast, PDGF-induced DNA synthesis was not affected by AG1478.In conclusion, these data suggest that the EGF receptor transactivation and subsequent p38 MAPK activation is required for thrombin-induced proliferation of VSMC. PMID:11309236

The protease thrombin is an endogenous mediator of hippocampal neuroprotection against ischemia at low concentrations but causes degeneration at high concentrations

NASA Astrophysics Data System (ADS)

Striggow, Frank; Riek, Monika; Breder, Jörg; Henrich-Noack, Petra; Reymann, Klaus G.; Reiser, Georg

2000-02-01

We have considered the extracellular serine protease thrombin and its receptor as endogenous mediators of neuronal protection against brain ischemia. Exposure of gerbils to prior mild ischemic insults, here two relatively short-lasting occlusions (2 min) of both common carotid arteries applied at 1-day intervals 2 days before a severe occlusion (6 min), caused a robust ischemic tolerance of hippocampal CA1 neurons. This resistance was impaired if the specific thrombin inhibitor hirudin was injected intracerebroventricularly before each short-lasting insult. Thus, efficient native neuroprotective mechanisms exist and endogenous thrombin seems to be involved therein. In vitro experiments using organotypic slice cultures of rat hippocampus revealed that thrombin can have protective but also deleterious effects on hippocampal CA1 neurons. Low concentrations of thrombin (50 pM, 0.01 unit/ml) or of a synthetic thrombin receptor agonist (10 μM) induced significant neuroprotection against experimental ischemia. In contrast, 50 nM (10 units/ml) thrombin decreased further the reduced neuronal survival that follows the deprivation of oxygen and glucose, and 500 nM even caused neuronal cell death by itself. Degenerative thrombin actions also might be relevant in vivo, because hirudin increased the number of surviving neurons when applied before a 6-min occlusion. Among the thrombin concentrations tested, 50 pM induced intracellular Ca2+ spikes in fura-2-loaded CA1 neurons whereas higher concentrations caused a sustained Ca2+ elevation. Thus, distinct Ca2+ signals may define whether or not thrombin initiates protection. Taken together, in vivo and in vitro data suggest that thrombin can determine neuronal cell death or survival after brain ischemia.

Differential inhibitory action of apixaban on platelet and fibrin components of forming thrombi: Studies with circulating blood and in a platelet-based model of thrombin generation.

PubMed

Pujadas-Mestres, Lluis; Lopez-Vilchez, Irene; Arellano-Rodrigo, Eduardo; Reverter, Joan Carles; Lopez-Farre, Antonio; Diaz-Ricart, Maribel; Badimon, Juan Jose; Escolar, Gines

2017-01-01

Mechanisms of action of direct oral anticoagulants (DOAC) suggest a potential therapeutic use in the prevention of thrombotic complications in arterial territories. However, effects of DOACs on platelet activation and aggregation have not been explored in detail. We have investigated the effects of apixaban on platelet and fibrin components of thrombus formation under static and flow conditions. We assessed the effects of apixaban (10, 40 and 160 ng/mL) on: 1) platelet deposition and fibrin formation onto a thrombogenic surface, with blood circulating at arterial shear-rates; 2) viscoelastic properties of forming clots, and 3) thrombin generation in a cell-model of coagulation primed by platelets. In studies with flowing blood, only the highest concentration of apixaban, equivalent to the therapeutic Cmax, was capable to significantly reduce thrombus formation, fibrin association and platelet-aggregate formation. Apixaban significantly prolonged thromboelastometry parameters, but did not affect clot firmness. Interestingly, results in a platelet-based model of thrombin generation under more static conditions, revealed a dose dependent persistent inhibitory action by apixaban, with concentrations 4 to 16 times below the therapeutic Cmax significantly prolonging kinetic parameters and reducing the total amount of thrombin generated. Our studies demonstrate the critical impact of rheological conditions on the antithrombotic effects of apixaban. Studies under flow conditions combined with modified thrombin generation assays could help discriminating concentrations of apixaban that prevent excessive platelet accumulation, from those that deeply impair fibrin formation and may unnecessarily compromise hemostasis.

Differential inhibitory action of apixaban on platelet and fibrin components of forming thrombi: Studies with circulating blood and in a platelet-based model of thrombin generation

PubMed Central

Arellano-Rodrigo, Eduardo; Reverter, Joan Carles; Lopez-Farre, Antonio; Diaz-Ricart, Maribel; Badimon, Juan Jose; Escolar, Gines

2017-01-01

Introduction Mechanisms of action of direct oral anticoagulants (DOAC) suggest a potential therapeutic use in the prevention of thrombotic complications in arterial territories. However, effects of DOACs on platelet activation and aggregation have not been explored in detail. We have investigated the effects of apixaban on platelet and fibrin components of thrombus formation under static and flow conditions. Methods We assessed the effects of apixaban (10, 40 and 160 ng/mL) on: 1) platelet deposition and fibrin formation onto a thrombogenic surface, with blood circulating at arterial shear-rates; 2) viscoelastic properties of forming clots, and 3) thrombin generation in a cell-model of coagulation primed by platelets. Results In studies with flowing blood, only the highest concentration of apixaban, equivalent to the therapeutic Cmax, was capable to significantly reduce thrombus formation, fibrin association and platelet-aggregate formation. Apixaban significantly prolonged thromboelastometry parameters, but did not affect clot firmness. Interestingly, results in a platelet-based model of thrombin generation under more static conditions, revealed a dose dependent persistent inhibitory action by apixaban, with concentrations 4 to 16 times below the therapeutic Cmax significantly prolonging kinetic parameters and reducing the total amount of thrombin generated. Conclusions Our studies demonstrate the critical impact of rheological conditions on the antithrombotic effects of apixaban. Studies under flow conditions combined with modified thrombin generation assays could help discriminating concentrations of apixaban that prevent excessive platelet accumulation, from those that deeply impair fibrin formation and may unnecessarily compromise hemostasis. PMID:28192448

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

NASA Technical Reports Server (NTRS)

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

1981-01-01

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

An ultrasensitive universal detector based on neutralizer displacement

NASA Astrophysics Data System (ADS)

Das, Jagotamoy; Cederquist, Kristin B.; Zaragoza, Alexandre A.; Lee, Paul E.; Sargent, Edward H.; Kelley, Shana O.

2012-08-01

Diagnostic technologies that can provide the simultaneous detection of nucleic acids for gene expression, proteins for host response and small molecules for profiling the human metabolome will have a significant advantage in providing comprehensive patient monitoring. Molecular sensors that report changes in the electrostatics of a sensor's surface on analyte binding have shown unprecedented sensitivity in the detection of charged biomolecules, but do not lend themselves to the detection of small molecules, which do not carry significant charge. Here, we introduce the neutralizer displacement assay that allows charge-based sensing to be applied to any class of molecule irrespective of the analyte charge. The neutralizer displacement assay starts with an aptamer probe bound to a neutralizer. When analyte binding occurs the neutralizer is displaced, which results in a dramatic change in the surface charge for all types of analytes. We have tested the sensitivity, speed and specificity of this system in the detection of a panel of molecules: (deoxy)ribonucleic acid, ribonucleic acid, cocaine, adenosine triphosphate and thrombin.

Screening cleavage of Factor XIII V34X Activation Peptides by thrombin mutants: A strategy for controlling fibrin architecture.

PubMed

Jadhav, Madhavi A; Goldsberry, Whitney N; Zink, Sara E; Lamb, Kelsey N; Simmons, Katelyn E; Riposo, Carmela M; Anokhin, Boris A; Maurer, Muriel C

2017-10-01

In blood coagulation, thrombin converts fibrinogen into fibrin monomers that polymerize into a clot network. Thrombin also activates Factor XIII by cleaving the R37-G38 peptide bond of the Activation Peptide (AP) segment. The resultant transglutaminase introduces covalent crosslinks into the fibrin clot. A strategy to modify clot architecture would be to design FXIII AP sequences that are easier or more difficult to be thrombin-cleaved thus controlling initiation of crosslinking. To aid in this design process, FXIII V34X (28-41) Activation Peptides were kinetically ranked for cleavage by wild-type thrombin and several anticoagulant mutants. Thrombin-catalyzed hydrolysis of aromatic FXIII F34, W34, and Y34 APs was compared with V34 and L34. Cardioprotective FXIII L34 remained the variant most readily cleaved by wild-type thrombin. The potent anticoagulant thrombins W215A and W215A/E217A (missing a key substrate platform for binding fibrinogen) were best able to hydrolyze FXIII F34 and W34 APs. Thrombin I174A and L99A could effectively accommodate FXIII W34 and Y34 APs yielding kinetic parameters comparable to FXIII AP L34 with wild-type thrombin. None of the aromatic FXIII V34X APs could be hydrolyzed by thrombin Y60aA. FXIII F34 and W34 are promising candidates for FXIII - anticoagulant thrombin systems that could permit FXIII-catalyzed crosslinking in the presence of reduced fibrin formation. By contrast, FXIII Y34 with thrombin (Y60aA or W215A/E217A) could help assure that both fibrin clot formation and protein crosslinking are hindered. Regulating the activation of FXIII is predicted to be a strategy for helping to control fibrin clot architecture and its neighboring environments. Copyright © 2017 Elsevier B.V. All rights reserved.

Thrombin specificity. Requirement for apolar amino acids adjacent to the thrombin cleavage site of polypeptide substrate.

PubMed

Chang, J Y

1985-09-02

alpha-Thrombin cleavage of 30 polypeptide hormones and their derivatives were analysed by quantitative amino-terminal analysis. The polypeptides included secretin, vasoactive intestinal polypeptide, cholecystokinin fragment, dynorphin A, somatostatins, gastrin-releasing peptide, calcitonins and human parathyroid hormone fragment. Most of them were selected mainly on the ground that they contain sequence structures homologous to the well known tripeptide substrates of alpha-thrombin. All selected polypeptides have one single major cleavage site and both Arg-Xaa and Lys-Xaa bonds were found to be selectively cleaved by alpha-thrombin. Under fixed conditions (1 nmol polypeptide/0.5 NIH unit alpha-thrombin in 20 microliters of 50 mM ammonium bicarbonate at 25 degrees C), the time required for 50% cleavage ranges from less than 1 min to longer than 24 h. Heparin invariably enhanced thrombin cleavage on all polypeptide analysed. The optimum cleavage site for alpha-thrombin has the structures of (a) P4-P3-Pro-Arg-P1'-P2', where P3 and P4 are hydrophobic amino acid and P1', P2' are nonacidic amino acids and (b) P2-Arg-P1', where P2 or P1' are Gly. The requirement for hydrophobic P3 and P4 was further demonstrated by the drastic decrease of thrombin cleavage rates in both gastrin-releasing peptide and calcitonins after chemical removal of hydrophobic P3 and P4 residues. The requirement for nonacidic P1' and P2' residues was demonstrated by the drastic increase of thrombin cleavage rates in both calcitonin and parathyroid hormone fragments, after specific chemical modification of acidic P1' and P2' residues. These findings confirm the importance of hydrophobic P2-P4 residues for thrombin specificity and provide new evidence to indicate that apolar P1' and P2' residues are also crucial for thrombin specificity. It is concluded that specific cleavage of polypeptides by alpha-thrombin can be reasonably predicted and that chemical modification can be a useful tool in enhancing thrombin cleavage.

Role of thrombin signalling in platelets in haemostasis and thrombosis

NASA Astrophysics Data System (ADS)

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

2001-09-01

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

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

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

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

2010-05-11

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

DETECTION OF TWO ISOMERIC BINDING CONFIGURATIONS IN A PROTEIN-APTAMER COMPLEX WITH A BIOLOGICAL NANOPORE

PubMed Central

Van Meervelt, Veerle; Soskine, Misha; Maglia, Giovanni

2015-01-01

Protein-DNA interactions play critical roles in biological systems, and they often involve complex mechanisms and dynamics that are not easily measured by ensemble experiments. Recently, we have shown that folded proteins can be internalised inside ClyA nanopores and studied by ionic current recordings at the single-molecule level. Here, we use ClyA nanopores to sample the interaction between the G-quadruplex fold of the thrombin binding aptamer (TBA) and human thrombin (HT). Surprisingly, the internalisation of the HT:TBA complex inside the nanopore induced two types of current blockades with distinguished residual current and lifetime. Using single nucleobase substitutions to TBA we showed that these two types of blockades originate from TBA binding to thrombin with two isomeric orientations. Voltage dependencies and the use of ClyA nanopores with two different diameters allowed assessing the effect of the applied potential and confinement, and revealed that the two binding configurations of TBA to HT display different lifetimes. These results show that the ClyA nanopores might provide a new approach to probe conformational heterogeneity in protein:DNA interactions. PMID:25493908

A sensitive HPLC-MS/MS method for the simultaneous detection of microbial transglutaminase, and bovine and porcine fibrinogen/thrombin in restructured meat.

PubMed

Jira, Wolfgang; Schwägele, Fredi

2017-12-15

A sensitive HPLC-MS/MS method for the simultaneous detection of microbial transglutaminase (TG) from Streptomyces mobaraensis, and bovine and porcine fibrinogen/thrombin in restructured meat was developed using tryptic marker peptides of TG (five markers), and bovine and porcine fibrinogen (six markers each). Meat binding experiments with beef and pork were performed using a technical TG mixture (Activa, Ajinomoto), and bovine and porcine plasmapowder FG (PPFG; Sonac B.V.). The method developed allows the simultaneous detection of the use of these cold-set binders in raw and heated samples. The peak areas of the fibrinogen marker peptides were increased by a factor of about 100, compared to blank values originating from the occurrence of residual blood in meat, using a concentration of 0.6% bovine and porcine PPFG. A differentiation between the use of blood plasma powder and PPFG using the ratios of fibrinogen to serotransferrin peptide peak areas seems to be possible. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comparison of 10 Different Hemostatic Dressings in an Aortic Injury

DTIC Science & Technology

2003-02-01

lateral peritoneal recesses of the abdomen bilaterally. The rate of bleeding was quantified (grams accumulated every 10 sec- onds) in the suction...used frequently for hemostasis in dental procedures19 D5 TachoComb S Nycomed Amersham, Linz, Austria Collagen -based ( collagen from horse tendons...product with human fibrinogen and thrombin Collagen attracts and activates platelets to achieve hemostasis. D5 also had fibrinogen and thrombin, but at a

Reproducibility, stability, and biological variability of thrombin generation using calibrated automated thrombography in healthy dogs.

PubMed

Cuq, Benoît; Blois, Shauna L; Wood, R Darren; Monteith, Gabrielle; Abrams-Ogg, Anthony C; Bédard, Christian; Wood, Geoffrey A

2018-06-01

Thrombin plays a central role in hemostasis and thrombosis. Calibrated automated thrombography (CAT), a thrombin generation assay, may be a useful test for hemostatic disorders in dogs. To describe CAT results in a group of healthy dogs, and assess preanalytical variables and biological variability. Forty healthy dogs were enrolled. Lag time (Lag), time to peak (ttpeak), peak thrombin generation (peak), and endogenous thrombin potential (ETP) were measured. Direct jugular venipuncture and winged-needle catheter-assisted saphenous venipuncture were used to collect samples from each dog, and results were compared between methods. Sample stability at -80°C was assessed over 12 months in a subset of samples. Biological variability of CAT was assessed via nested ANOVA using samples obtained weekly from a subset of 9 dogs for 4 consecutive weeks. Samples for CAT were stable at -80°C over 12 months of storage. Samples collected via winged-needle catheter venipuncture showed poor repeatability compared to direct venipuncture samples; there was also poor agreement between the 2 sampling methods. Intra-individual variability of CAT parameters was below 25%; inter-individual variability ranged from 36.9% to 78.5%. Measurement of thrombin generation using CAT appears to be repeatable in healthy dogs, and samples are stable for at least 12 months when stored at -80°C. Direct venipuncture sampling is recommended for CAT. Low indices of individuality suggest that subject-based reference intervals are more suitable when interpreting CAT results. © 2018 American Society for Veterinary Clinical Pathology.

DNA-Aptamer optical biosensors based on a LPG-SPR optical fiber platform for point-of-care diagnostic

NASA Astrophysics Data System (ADS)

Coelho, L.; Queirós, R. B.; Santos, J. L.; Martins, M. Cristina L.; Viegas, D.; Jorge, P. A. S.

2014-03-01

Surface Plasmon Resonance (SPR) is the base for some of the most sensitive label free optical fiber biosensors. However, most solutions presented to date require the use of fragile fiber optic structure such as adiabatic tapers or side polished fibers. On the other hand, long-period fiber gratings (LPG) present themselves as an interesting solution to attain an evanescent wave refractive index sensor platform while preserving the optical fiber integrity. The combination of these two approaches constitute a powerful platform that can potentially reach the highest sensitivities as it was recently demonstrated by detailed theoretical study [1, 2]. In this work, a LPG-SPR platform is explored in different configurations (metal coating between two LPG - symmetric and asymmetric) operating in the telecom band (around 1550 nm). For this purpose LPGs with period of 396 μm are combined with tailor made metallic thin films. In particular, the sensing regions were coated with 2 nm of chromium to improve the adhesion to the fiber and 16 nm of gold followed by a 100 nm thick layer of TiO2 dielectric material strategically chosen to attain plasmon resonance in the desired wavelength range. The obtained refractometric platforms were then validated as a biosensor. For this purpose the detection of thrombin using an aptamer based probe was used as a model system for protein detection. The surface of the sensing fibers were cleaned with isopropanol and dried with N2 and then the aminated thrombin aptamer (5'-[NH2]- GGTTGGTGTGGTTGG-3') was immobilized by physisorption using Poly-L-Lysine (PLL) as cationic polymer. Preliminary results indicate the viability of the LPFG-SPR-APTAMER as a flexible platforms point of care diagnostic biosensors.

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

PubMed Central

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

2016-01-01

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

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

PubMed Central

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

2015-01-01

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

Dabigatran abrogates brain endothelial cell permeability in response to thrombin

PubMed Central

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

2015-01-01

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. PMID:25669912

Histamine and thrombin modulate endothelial focal adhesion through centripetal and centrifugal forces.

PubMed Central

Moy, A B; Van Engelenhoven, J; Bodmer, J; Kamath, J; Keese, C; Giaever, I; Shasby, S; Shasby, D M

1996-01-01

We examined the contribution of actin-myosin contraction to the modulation of human umbilical vein endothelial cell focal adhesion caused by histamine and thrombin. Focal adhesion was measured as the electrical resistance across a cultured monolayer grown on a microelectrode. Actin-myosin contraction was measured as isometric tension of cultured monolayers grown on a collagen gel. Histamine immediately decreased electrical resistance but returned to basal levels within 3-5 min. Histamine did not increase isometric tension. Thrombin also immediately decreased electrical resistance, but, however, resistance did not return to basal levels for 40-60 min. Thrombin also increased isometric tension, ML-7, an inhibitor of myosin light chain kinase, prevented increases in myosin light chain phosphorylation and increases in tension development in cells exposed to thrombin. ML-7 did not prevent a decline in electrical resistance in cells exposed to thrombin. Instead, ML-7 restored the electrical resistance to basal levels in a shorter period of time (20 min) than cells exposed to thrombin alone. Also, histamine subsequently increased electrical resistance to above basal levels, and thrombin initiated an increase in resistance during the time of peak tension development. Hence, histamine and thrombin modulate endothelial cell focal adhesion through centripetal and centrifugal forces. PMID:8613524

Localization and characterization of an alpha-thrombin-binding site on platelet glycoprotein Ib alpha.

PubMed

De Marco, L; Mazzucato, M; Masotti, A; Ruggeri, Z M

1994-03-04

Glycoprotein (GP) Ib alpha is required for expression of the highest affinity alpha-thrombin-binding site on platelets, possibly contributing to platelet activation through a pathway involving cleavage of a specific receptor. This function may be important for the initiation of hemostasis and may also play a role in the development of pathological vascular occlusion. We have now identified a discrete sequence in the extracytoplasmic domain of GP Ib alpha, including residues 271-284 of the mature protein, which appears to be part of the high affinity alpha-thrombin-binding site. Synthetic peptidyl mimetics of this sequence inhibit alpha-thrombin binding to GP Ib as well as platelet activation and aggregation induced by subnanomolar concentrations of the agonist; they also inhibit alpha-thrombin binding to purified glycocalicin, the isolated extracytoplasmic portion of GP Ib alpha. The inhibitory peptides interfere with the clotting of fibrinogen by alpha-thrombin but not with the amidolytic activity of the enzyme on a small synthetic substrate, a finding compatible with the concept that the identified GP Ib alpha sequence interacts with the anion-binding exosite of alpha-thrombin but not with its active proteolytic site. The crucial structural elements of this sequence necessary for thrombin binding appear to be a cluster of negatively charged residues as well as three tyrosine residues that, in the native protein, may be sulfated. GP Ib alpha has no significant overall sequence homology with the thrombin inhibitor, hirudin, nor with the specific thrombin receptor on platelets; all three molecules, however, possess a distinct region rich in negatively charged residues that appear to be involved in thrombin binding. This may represent a case of convergent evolution of unrelated proteins for high affinity interaction with the same ligand.

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

PubMed

Mazzeffi, Michael; Szlam, Fania; Jakubowski, Joseph A; Tanaka, Kenichi A; Sugidachi, Atsuhiro; Levy, Jerrold H

2013-07-01

Prasugrel is a thienopyridyl P2Y12 antagonist with potent antiplatelet effects. At present, little is known about its effects on thrombin generation or what strategies may emergently reverse its anticoagulant effects. In the current study we evaluated whether recombinant activated factor VII may reverse prasugrel induced effects and increase thrombin generation in an in vitro model. The effect of prasugrel active metabolite, PAM (R-138727), was evaluated on platelet aggregation, thrombin generation, and rotational thromboelastometry parameters using blood from 20 healthy volunteers. Additionally, we evaluated the effects of adenosine diphosphate (ADP) and recombinant activated factor VII on restoring these parameters towards baseline values. PAM reduced maximum platelet aggregation and led to platelet disaggregation. It also decreased peak thrombin, increased lag time, and increased time to peak thrombin. Treatment with recombinant activated factor VII restored all three parameters of thrombin generation towards baseline. ADP decreased lag time and time to peak thrombin, but had no effect on peak thrombin. When recombinant activated factor VII and ADP were combined they had a greater effect on thrombin parameters than either drug alone. PAM also increased thromboelastometric clotting time and clot formation time, but had no effect on maximum clot firmness. Treatment with either recombinant activated factor VII or ADP restored these values towards baseline. Recombinant activated factor VII restores thrombin generation in the presence of PAM. In patients taking prasugrel with life-threatening refractory bleeding it has the potential to be a useful therapeutic approach. Additional clinical studies are needed to validate our findings. Copyright © 2013 Elsevier Ltd. All rights reserved.

Structural Characterization of a Thrombin-Aptamer Complex by High Resolution Native Top-Down Mass Spectrometry

NASA Astrophysics Data System (ADS)

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

2017-09-01

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.

The pharmacological modulation of thrombin-induced cerebral thromboembolism in the rabbit.

PubMed Central

May, G. R.; Paul, W.; Crook, P.; Butler, K. D.; Page, C. P.

1992-01-01

1. Intracarotid (i.c.) administration of thrombin induced a marked accumulation of 111indium-labelled platelets and 125I-labelled fibrinogen within the cranial vasculature of anaesthetized rabbits. 2. Thrombin (100 iu kg-1, i.c.) - induced platelet accumulation was completely abolished by pretreatment with desulphatohirudin (CGP 39393; 1 mg kg-1 i.c., 1 min prior to thrombin). Administration of CGP 39393 1 or 20 min after thrombin produced a significant reduction in platelet accumulation. 3. Intravenous (i.v.) administration of the platelet activating factor (PAF) receptor antagonist BN 52021 (10 mg kg-1) 5 min prior to thrombin (100 iu kg-1, i.c.) had no effect on platelet accumulation. 4. An inhibitor of NO biosynthesis, L-NG-nitro arginine methyl ester (L-NAME; 100 mg kg-1, i.c.), had no significant effect on the cranial platelet accumulation response to thrombin (10 iu kg-1, i.c.) when administered 5 min prior to thrombin. 5. Defibrotide (32 or 64 mg kg-1 bolus i.c. followed by 32 or 64 mg kg-1 h-1, i.c., infusion for 45 min) treatment begun 20 min after thrombin (100 iu kg-1, i.c.) did not significantly modify the cranial platelet accumulation response. 6. Cranial platelet accumulation induced by thrombin (100 iu kg-1, i.c.) was significantly reversed by the fibrinolytic drugs urokinase (20 iu kg-1, i.c., infusion for 45 min), anisoylated plasminogen streptokinase activator complex (APSAC) (200 micrograms kg-1, i.v. bolus) or recombinant tissue plasminogen activator (rt-PA; 100 micrograms kg-1, i.c. bolus followed by 20 micrograms kg-1 min-1, i.c., infusion for 45 min) administered 20 min after thrombin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1504722

Comparison of fibrinogen- and collagen-based treatments for penetrating wounds with comminuted femur fractures in a Swine model.

PubMed

Rothwell, Stephen W; Sawyer, Evelyn; Lombardini, Eric; Royal, Joseph; Tang, Haiyin; Selwyn, Reed; Bodo, Michael; Settle, Timothy L

2013-01-01

Military servicemembers in combat operations often sustain injuries to the extremities from highspeed projectiles, resulting in bleeding and comminuted open fractures. Severe injury with bone fragmentation can result in limb amputation. Surgical treatment options include materials that promote osteogenesis and bone proliferation, such as growth hormones, stem cells, or mineralized matrix adjuncts. However, none of these are amenable to use by the first responder, nor do they address the question of hemorrhage control, which is a common problem in traumatic injuries. Our hypothesis was that treatment with a fibrinogen-based protein mixture at the time of the bone injury will provide both hemostasis and a supportive environment for preservation of injured bone. A comminuted femur fracture was produced in 28 female Yorkshire swine, and one of four treatments was instilled into the wound immediately after injury. Each animal was evaluated for the following parameters: inflammation, new bone growth, osteoclast proliferation, callus formation, and femur wound cavity fill, using post-mortem computed tomography and analysis of histological sections. Overall, salmon fibrinogen?thrombin and porcine fibrinogen?thrombin showed a trend for improved healing based on bone filling and calcification. However, statistically significant differences could not be established between treatment groups. These findings indicate that a fibrinogen?thrombin matrix may be a useful as an immediate response product to enhance fracture healing. Salmon fibrinogen?thrombin has the advantages of cost and a pathogen profile compared to mammalian fibrinogens. 2013.

Prediction of enzyme binding: human thrombin inhibition study by quantum chemical and artificial intelligence methods based on X-ray structures.

PubMed

Mlinsek, G; Novic, M; Hodoscek, M; Solmajer, T

2001-01-01

Thrombin is a serine protease which plays important roles in the human body, the key one being the control of thrombus formation. The inhibition of thrombin has become a target for new antithrombotics. The aim of our work was to (i) construct a model which would enable us to predict Ki values for the binding of an inhibitor into the active site of thrombin based on a database of known X-ray structures of inhibitor-enzyme complexes and (ii) to identify the structural and electrostatic characteristics of inhibitor molecules crucially important to their effective binding. To retain as much of the 3D structural information of the bound inhibitor as possible, we implemented the quantum mechanical/molecular mechanical (QM/MM) procedure for calculating the molecular electrostatic potential (MEP) at the van der Waals surfaces of atoms in the protein's active site. The inhibitor was treated quantum mechanically, while the rest of the complex was treated by classical means. The obtained MEP values served as inputs into the counter-propagation artificial neural network (CP-ANN), and a genetic algorithm was subsequently used to search for the combination of atoms that predominantly influences the binding. The constructed CP-ANN model yielded Ki values predictions with a correlation coefficient of 0.96, with Ki values extended over 7 orders of magnitude. Our approach also shows the relative importance of the various amino acid residues present in the active site of the enzyme for inhibitor binding. The list of residues selected by our automatic procedure is in good correlation with the current consensus regarding the importance of certain crucial residues in thrombin's active site.

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

NASA Astrophysics Data System (ADS)

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

1994-09-01

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

Evaluating antithrombotic activity of HY023016 on rat hypercoagulable model.

PubMed

Chen, Qiu-Fang; Li, Yun-Zhan; Wang, Xin-Hui; Su, You-Rui; Cui, Shuang; Miao, Ming-Xing; Jiang, Zhen-Zhou; Jiang, Mei-Ling; Jiang, Ai-Dou; Chen, Xiang; Xu, Yun-Gen; Gong, Guo-Qing

2016-06-15

The generation of thrombus is not considered as an isolated progression without other pathologic processes, which may also enhance procoagulant state. The purpose of this study was to assess whether HY023016, a novel dabigatran prodrug and an oral direct thrombin inhibitor, or dabigatran etexilate, another thrombin inhibitor can improve the state of whole blood hypercoagulability in vitro/vivo. By using whole blood flow cytometry we explored the effects of HY023016 and dabigatran etexilate on thrombin and ADP-induced human platelet-leukocyte aggregation generated in vitro. With the method of continuous infusion of thrombin intravenous, we successfully established a rat hypercoagulable model and evaluated the effect of HY023016 or dabigatran etexilate in vivo. HY023016 was able to inhibit thrombin- or ADP-induced platelet P-selectin or CD40L expression, leukocyte CD11b expression and formation of platelet-leukocyte aggregates in dose-dependent manner. Dabigatran etexilate was unable to affect ADP-induced platelet P-selectin or CD40L expression, leukocyte CD11b expression and formation of platelet-leukocyte aggregates. Based on rat hypercoagulable model, dabigatran etexilate could reverse thrombin-induced circulatory system hypercoagulable state in a concentration-dependent manner. Dabigatran etexilate also inhibited electrical stimulation induced formation of arterial thrombus in rat under hypercoagulable state, and extracorporal circulation-induced formation of thrombus in dose-dependent manner. Compared with dabigatran etexilate, HY023016 showed nearly equal or even better antithrombotic activity, regardless of reversing the cycle of rat hypercoagulable state or inhibiting platelet-leukocyte aggregation. In surrmary, HY023016 could effectively improve hypercoagulable state of circulatory system. Copyright © 2016. Published by Elsevier B.V.

Thrombin Induces Inositol Trisphosphate-Mediated Spatially Extensive Responses in Lung Microvessels.

PubMed

Escue, Rachel; Kandasamy, Kathirvel; Parthasarathi, Kaushik

2017-04-01

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.

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

PubMed Central

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

1988-01-01

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

Formation of PI 3-kinase products in platelets by thrombin, but not collagen, is dependent on synergistic autocrine stimulation, particularly through secreted ADP.

PubMed

Selheim, F; Idsøe, R; Fukami, M H; Holmsen, H; Vassbotn, F S

1999-10-05

Platelet activation by thrombin or collagen results in secretion and synthesis of several platelet agonists that enhance the responses to the primary agonists (autocrine stimulation). To disclose the effects of thrombin and collagen on the phosphorylation of 3-phosphoinositides per se we incubated platelets with five inhibitors of platelet autocrine stimulation (IAS) that act extracellularly. We found that IAS almost totally blocked thrombin-induced production of phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P(2)] and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]. In contrast, collagen induced massive production of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) in the presence of IAS. When testing the effect of each inhibitor individually we found the strongest inhibition of thrombin-induced PtdIns(3,4)P(2) production with the ADP scavenger system CP/CPK. Furthermore, we found a strong synergistic effect between exogenously added ADP and thrombin on production of PtdIns(3,4)P(2). In contrast to the results from 3-phosphorylated phosphoinositides, CP/CPK had little effect on thrombin-induced protein tyrosine phosphorylation. Our results show the importance of autocrine stimulation in thrombin-induced accumulation of 3-phosphorylated phosphoinositides and raise the question as to whether thrombin by itself is capable of inducing PI 3-K activation. In marked contrast to thrombin, collagen per se appears to be able to trigger increased production of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3). Copyright 1999 Academic Press.

Detection of Subclinical Arthritis in Mice by a Thrombin Receptor-Derived Imaging Agent.

PubMed

Friedman, Beth; Whitney, Michael A; Savariar, Elamprakash N; Caneda, Christa; Steinbach, Paul; Xiong, Qing; Hingorani, Dina V; Crisp, Jessica; Adams, Stephen R; Kenner, Michael; Lippert, Csilla N; Nguyen, Quyen T; Guma, Monica; Tsien, Roger Y; Corr, Maripat

2018-01-01

Functional imaging of synovitis could improve both early detection of rheumatoid arthritis (RA) and long-term outcomes. Given the intersection of inflammation with coagulation protease activation, this study was undertaken to examine coagulation protease activities in arthritic mice with a dual-fluorescence ratiometric activatable cell-penetrating peptide (RACPP) that has a linker, norleucine (Nle)-TPRSFL, with a cleavage site for thrombin. K/BxN-transgenic mice with chronic arthritis and mice with day 1 passive serum-transfer arthritis were imaged in vivo for Cy5:Cy7 emission ratiometric fluorescence from proteolytic cleavage and activation of RACPP NleTPRSFL . Joint thickness in mice with serum-transfer arthritis was measured from days 0 to 10. The cleavage-evoked release of Cy5-tagged tissue-adhesive fragments enabled microscopic correlation with immunohistochemistry for inflammatory markers. Thrombin dependence of ratiometric fluorescence was tested by ex vivo application of RACPP NleTPRSFL and argatroban to cryosections obtained from mouse hind paws on day 1 of serum-transfer arthritis. In chronic arthritis, RACPP NleTPRSFL fluorescence ratios of Cy5:Cy7 emission were significantly higher in diseased swollen ankles of K/BxN-transgenic mice than in normal mouse ankles. A high ratio of RACPP NleTPRSFL fluorescence in mouse ankles and toes on day 1 of serum-transfer arthritis correlated with subsequent joint swelling. Foci of high ratiometric fluorescence localized to inflammation, as demarcated by immune reactivity for citrullinated histones, macrophages, mast cells, and neutrophils, in soft tissue on day 1 of serum-transfer arthritis. Ex vivo application of RACPP NleTPRSFL to cryosections obtained from mice on day 1 of serum-transfer arthritis produced ratiometric fluorescence that was inhibited by argatroban. RACPP NleTPRSFL activation detects established experimental arthritis, and the detection of inflammation by RACPP NleTPRSFL on day 1 of serum-transfer arthritis correlates with disease progression. © 2017, American College of Rheumatology.

Glycoprotein Ib activation by thrombin stimulates the energy metabolism in human platelets

PubMed Central

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

2017-01-01

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

Thrombin generation correlates with disease duration in multiple sclerosis (MS): Novel insights into the MS-associated prothrombotic state.

PubMed

Parsons, Martin Em; O'Connell, Karen; Allen, Seamus; Egan, Karl; Szklanna, Paulina B; McGuigan, Christopher; Ní Áinle, Fionnuala; Maguire, Patricia B

2017-01-01

Thrombin is well recognised for its role in the coagulation cascade but it also plays a role in inflammation, with enhanced thrombin generation observed in several inflammatory disorders. Although patients with multiple sclerosis (MS) have a higher incidence of thrombotic disease, thrombin generation has not been studied to date. The aim of this study was to characterise calibrated automated thrombography parameters in patients with relapsing-remitting MS (RRMS) and primary progressive MS (PPMS) in comparison to healthy controls (HCs). Calibrated automated thrombography was performed on platelet poor plasma from 15 patients with RRMS, 15 with PPMS and 19 HCs. We found that patients with RRMS generate thrombin at a significantly faster rate than the less inflammatory subtype, PPMS or HCs. In addition, the speed of thrombin generation was significantly correlated with time from clinical diagnosis in both subtypes. However, in RRMS the rate of thrombin generation was increased with increased time from clinical diagnosis, while in PPMS the rate of thrombin generation decreased with increased time from clinical diagnosis. These data likely reflect the differential active proinflammatory states in each MS subtype and provide novel mechanistic insights into the clinically relevant prothrombotic state observed in these patients.

Methods of Treating or Preventing Demyelation Using Thrombin Inhibitors | NCI Technology Transfer Center | TTC

Cancer.gov

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

The complete N-terminal extension of heparin cofactor II is required for maximal effectiveness as a thrombin exosite 1 ligand

PubMed Central

2013-01-01

Background Heparin cofactor II (HCII) is a circulating protease inhibitor, one which contains an N-terminal acidic extension (HCII 1-75) unique within the serpin superfamily. Deletion of HCII 1-75 greatly reduces the ability of glycosaminoglycans (GAGs) to accelerate the inhibition of thrombin, and abrogates HCII binding to thrombin exosite 1. While a minor portion of HCII 1-75 can be visualized in a crystallized HCII-thrombin S195A complex, the role of the rest of the extension is not well understood and the affinity of the HCII 1-75 interaction has not been quantitatively characterized. To address these issues, we expressed HCII 1-75 as a small, N-terminally hexahistidine-tagged polypeptide in E. coli. Results Immobilized purified HCII 1-75 bound active α-thrombin and active-site inhibited FPR-ck- or S195A-thrombin, but not exosite-1-disrupted γT-thrombin, in microtiter plate assays. Biotinylated HCII 1-75 immobilized on streptavidin chips bound α-thrombin and FPR-ck-thrombin with similar KD values of 330-340 nM. HCII 1-75 competed thrombin binding to chip-immobilized HCII 1-75 more effectively than HCII 54-75 but less effectively than the C-terminal dodecapeptide of hirudin (mean Ki values of 2.6, 8.5, and 0.29 μM, respectively). This superiority over HCII 54-75 was also demonstrated in plasma clotting assays and in competing the heparin-catalysed inhibition of thrombin by plasma-derived HCII; HCII 1-53 had no effect in either assay. Molecular modelling of HCII 1-75 correctly predicted those portions of the acidic extension that had been previously visualized in crystal structures, and suggested that an α-helix found between residues 26 and 36 stabilizes one found between residues 61-67. The latter region has been previously shown by deletion mutagenesis and crystallography to play a crucial role in the binding of HCII to thrombin exosite 1. Conclusions Assuming that the KD value for HCII 1-75 of 330-340 nM faithfully predicts that of this region in intact HCII, and that 1-75 binding to exosite 1 is GAG-dependent, our results support a model in which thrombin first binds to GAGs, followed by HCII addition to the ternary complex and release of HCII 1-75 for exosite 1 binding and serpin mechanism inhibition. They further suggest that, in isolated or transferred form, the entire HCII 1-75 region is required to ensure maximal binding of thrombin exosite 1. PMID:23496873

Risk of bleeding in surgical patients treated with topical bovine thrombin sealants: a review of the literature

PubMed Central

Reynolds, Matthew W; Clark, John; Crean, Sheila; Samudrala, Srinath

2008-01-01

Background One of the most anticipated, but potentially serious complications during or after surgery are bleeding events. Among the many potential factors associated with bleeding complications in surgery, the use of bovine thrombin has been anecdotally identified as a possible cause of increased bleeding risk. Most of these reports of bleeding events in association with the use of topical bovine thrombin have been limited to case reports lacking clear cause and effect relationship determination. Recent studies have failed to establish significant differences in the rates of bleeding events between those treated with bovine thrombin and those treated with either human or recombinant thrombin. Methods We conducted a search of MEDLINE for the most recent past 10 years (1997–2007) and identified all published studies that reported a study of surgical patients with a clear objective to examine the risk of bleeding events in surgical patients. We also specifically noted the reporting of any topical bovine thrombin used during surgical procedures. We aimed to examine whether there were any differences in the risk of bleeds in general surgical populations as compared to those studies that reported exposure to topical bovine thrombin. Results We identified 21 clinical studies that addressed the risk of bleeding in surgery. Of these, 5 studies analyzed the use of bovine thrombin sealants in surgical patients. There were no standardized definitions for bleeding events employed across these studies. The rates of bleeds in the general surgery studies ranged from 0.1%–20.2%, with most studies reporting rates between 2.6%–4%. The rates of bleeding events ranged from 0.0%–13% in the bovine thrombin studies with most studies reporting between a 2%–3% rate. Conclusion The risk of bleeds was not clearly different in those studies reporting use of bovine thrombin in all patients compared to the other surgical populations studied. A well-designed and well-controlled study is needed to accurately examine the bleeding risks in surgical patients treated and unexposed to topical bovine thrombin, and to evaluate the independent risk associated with topical bovine thrombin as well as other risk factors. PMID:18348725

Bi-enzyme functionlized hollow PtCo nanochains as labels for an electrochemical aptasensor.

PubMed

Bai, Lijuan; Yuan, Ruo; Chai, Yaqin; Yuan, Yali; Zhuo, Ying; Mao, Li

2011-07-15

In this work, a new signal amplification strategy based on hollow PtCo nanochains (HPtCoNCs) functionalized by bi-enzyme-horseradish peroxidase mimicking DNAzyme (HRP-DNAzyme) and glucose oxidase (GOD), as well as ferrocene-labeled secondary thrombin aptamer (Fc-TBA 2), is developed to construct a highly sensitive electrochemical aptasensor. The HRP-DNAzyme contains a special G-quadruplex structure with an intercalated hemin. With the surface area enlarged by HPtCoNCs, the amount of immobilized Fc-TBA 2, hemin and GOD can be enhanced. Under the enzyme catalysis of GOD, d-glucose is rapidly oxidized into gluconic acid accompanying with the generation of H₂O₂, which is further electrocatalyzed by Pt nanoparticles and HPR-DNAzyme to improve the electrochemical signal of Fc. With several amplification factors mentioned above, a wide linear ranged from 0.001 to 30 nM is acquired with a relatively low detection limit of 0.39 pM for thrombin. The present work demonstrates that using HPtCoNCs as labels is a promising way to amplify the analysis signal and improve the sensitivity of aptasensors. Copyright © 2011 Elsevier B.V. All rights reserved.

A Linear Temporal Increase in Thrombin Activity and Loss of Its Receptor in Mouse Brain following Ischemic Stroke.

PubMed

Bushi, Doron; Stein, Efrat Shavit; Golderman, Valery; Feingold, Ekaterina; Gera, Orna; Chapman, Joab; Tanne, David

2017-01-01

Brain thrombin activity is increased following acute ischemic stroke and may play a pathogenic role through the protease-activated receptor 1 (PAR1). In order to better assess these factors, we obtained a novel detailed temporal and spatial profile of thrombin activity in a mouse model of permanent middle cerebral artery occlusion (pMCAo). Thrombin activity was measured by fluorescence spectroscopy on coronal slices taken from the ipsilateral and contralateral hemispheres 2, 5, and 24 h following pMCAo ( n  = 5, 6, 5 mice, respectively). Its spatial distribution was determined by punch samples taken from the ischemic core and penumbra and further confirmed using an enzyme histochemistry technique ( n  = 4). Levels of PAR1 were determined using western blot. Two hours following pMCAo, thrombin activity in the stroke core was already significantly higher than the contralateral area (11 ± 5 vs. 2 ± 1 mU/ml). At 5 and 24 h, thrombin activity continued to rise linearly ( r  = 0.998, p  = 0.001) and to expand in the ischemic hemisphere beyond the ischemic core reaching deleterious levels of 271 ± 117 and 123 ± 14 mU/ml (mean ± SEM) in the basal ganglia and ischemic cortex, respectively. The peak elevation of thrombin activity in the ischemic core that was confirmed by fluorescence histochemistry was in good correlation with the infarcts areas. PAR1 levels in the ischemic core decreased as stroke progressed and thrombin activity increased. In conclusion, there is a time- and space-related increase in brain thrombin activity in acute ischemic stroke that is closely related to the progression of brain damage. These results may be useful in the development of therapeutic strategies for ischemic stroke that involve the thrombin-PAR1 pathway in order to prevent secondary thrombin related brain damage.

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

PubMed

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

2015-06-01

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

Antiphospholipid antibody profiles in lupus nephritis with glomerular microthrombosis: a prospective study of 124 cases.

PubMed

Zheng, Hui; Chen, Yi; Ao, Wen; Shen, Yan; Chen, Xiao-wei; Dai, Min; Wang, Xiao-dong; Yan, Yu-cheng; Yang, Cheng-de

2009-01-01

Glomerular microthrombosis (GMT) is a common vascular change in patients with lupus nephritis (LN). The mechanism underlying GMT is largely unknown. Although several studies have reported the association of antiphospholipid antibodies (aPL) with GMT, the relation between GMT and aPL remains controversial. Previous studies have demonstrated that some aPL could bind to several hemostatic and fibrinolytic proteases that share homologous enzymatic domains. Of the protease-reactive aPL, some can inhibit the anticoagulant activity of activated protein C and the fibrinolytic function of plasmin, and hinder the antithrombin inactivation of thrombin. The purpose of this study was to investigate the prevalence of GMT in LN patients and examine the relation between the aPL profiles (including some protease-reactive aPL) and GMT. Renal biopsy specimens were examined for the presence of glomerular microthrombi. Plasma samples from 25 LN patients with GMT (LN-GMT group) and 99 LN patients without GMT (LN-non-GMT group) were tested for lupus anticoagulant and antibodies against cardiolipin, beta2 glycoprotein I, plasmin, thrombin, tissue plasminogen activator, and annexin II. The prevalence of GMT in LN patients was 20.2%. Compared with the LN-non-GMT group, the LN-GMT group had an elevated systemic lupus erythematosus disease activity index; elevated renal tissue injury activity and chronicity indices; elevated serum creatinine, blood urea nitrogen, and proteinuria levels; a lower serum C3 level and much intense glomerular C3, C1q staining; and a higher frequency of hypertension (P < 0.05 for all). Additionally, the detection rate of lupus anticoagulant, immunoglobulin G (IgG) anti-beta2 glycoprotein I and anti-thrombin antibodies were higher in the LN-GMT group than in the LN-non-GMT group (P < 0.05 for all). No statistical differences were found in the detection rates of IgG anti-cardiolipin, plasmin, tissue plasminogen activator, or annexin II antibodies (P > 0.05 for all). No detectable difference in IgM autoantibodies to the above antigens was observed between the two groups. GMT occurs in approximately 20.2% of LN patients. Patients with GMT have severer renal tissue injuries and poorer renal functions than patients without GMT. The lupus anticoagulant and antibodies against beta2 glycoprotein I and thrombin may play a role in GMT.

Antiphospholipid antibody profiles in lupus nephritis with glomerular microthrombosis: a prospective study of 124 cases

PubMed Central

Zheng, Hui; Chen, Yi; Ao, Wen; Shen, Yan; Chen, Xiao-wei; Dai, Min; Wang, Xiao-dong; Yan, Yu-cheng; Yang, Cheng-de

2009-01-01

Introduction Glomerular microthrombosis (GMT) is a common vascular change in patients with lupus nephritis (LN). The mechanism underlying GMT is largely unknown. Although several studies have reported the association of antiphospholipid antibodies (aPL) with GMT, the relation between GMT and aPL remains controversial. Previous studies have demonstrated that some aPL could bind to several hemostatic and fibrinolytic proteases that share homologous enzymatic domains. Of the protease-reactive aPL, some can inhibit the anticoagulant activity of activated protein C and the fibrinolytic function of plasmin, and hinder the antithrombin inactivation of thrombin. The purpose of this study was to investigate the prevalence of GMT in LN patients and examine the relation between the aPL profiles (including some protease-reactive aPL) and GMT. Methods Renal biopsy specimens were examined for the presence of glomerular microthrombi. Plasma samples from 25 LN patients with GMT (LN-GMT group) and 99 LN patients without GMT (LN-non-GMT group) were tested for lupus anticoagulant and antibodies against cardiolipin, β2 glycoprotein I, plasmin, thrombin, tissue plasminogen activator, and annexin II. Results The prevalence of GMT in LN patients was 20.2%. Compared with the LN-non-GMT group, the LN-GMT group had an elevated systemic lupus erythematosus disease activity index; elevated renal tissue injury activity and chronicity indices; elevated serum creatinine, blood urea nitrogen, and proteinuria levels; a lower serum C3 level and much intense glomerular C3, C1q staining; and a higher frequency of hypertension (P < 0.05 for all). Additionally, the detection rate of lupus anticoagulant, immunoglobulin G (IgG) anti-β2 glycoprotein I and anti-thrombin antibodies were higher in the LN-GMT group than in the LN-non-GMT group (P < 0.05 for all). No statistical differences were found in the detection rates of IgG anti-cardiolipin, plasmin, tissue plasminogen activator, or annexin II antibodies (P > 0.05 for all). No detectable difference in IgM autoantibodies to the above antigens was observed between the two groups. Conclusions GMT occurs in approximately 20.2% of LN patients. Patients with GMT have severer renal tissue injuries and poorer renal functions than patients without GMT. The lupus anticoagulant and antibodies against β2 glycoprotein I and thrombin may play a role in GMT. PMID:19545416

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

PubMed

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

2008-02-01

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

Binding-induced DNA walker for signal amplification in highly selective electrochemical detection of protein.

PubMed

Ji, Yuhang; Zhang, Lei; Zhu, Longyi; Lei, Jianping; Wu, Jie; Ju, Huangxian

2017-10-15

A binding-induced DNA walker-assisted signal amplification was developed for highly selective electrochemical detection of protein. Firstly, the track of DNA walker was constructed by self-assembly of the high density ferrocene (Fc)-labeled anchor DNA and aptamer 1 on the gold electrode surface. Sequentially, a long swing-arm chain containing aptamer 2 and walking strand DNA was introduced onto gold electrode through aptamers-target specific recognition, and thus initiated walker strand sequences to hybridize with anchor DNA. Then, the DNA walker was activated by the stepwise cleavage of the hybridized anchor DNA by nicking endonuclease to release multiple Fc molecules for signal amplification. Taking thrombin as the model target, the Fc-generated electrochemical signal decreased linearly with logarithm value of thrombin concentration ranging from 10pM to 100nM with a detection limit of 2.5pM under the optimal conditions. By integrating the specific recognition of aptamers to target with the enzymatic cleavage of nicking endonuclease, the aptasensor showed the high selectivity. The binding-induced DNA walker provides a promising strategy for signal amplification in electrochemical biosensor, and has the extensive applications in sensitive and selective detection of the various targets. Copyright © 2017 Elsevier B.V. All rights reserved.

Grape intake reduces thrombin generation and enhances plasma fibrinolysis. Potential role of circulating procoagulant microparticles.

PubMed

Ammollo, Concetta T; Semeraro, Fabrizio; Milella, Rosa Anna; Antonacci, Donato; Semeraro, Nicola; Colucci, Mario

2017-12-01

Phytochemicals contained in grapes down-regulate several prothrombotic pathways in vitro. We evaluated the effect of grape consumption on coagulation and fibrinolysis in healthy volunteers. Thirty subjects were enrolled: 20 were given grape (5 g/kg body weight/day for 3 weeks), while 10 served as controls. Blood samples were taken at baseline (T0), at the end of the grape diet (T1) and after 4-week wash-out (T2). Grape intake caused a significant decrease of the procoagulant and inflammatory responses of whole blood and/or mononuclear cells to bacterial lipopolysaccharide at both T1 and T2. At plasma level, grape diet decreased thrombin generation at T1 and T2, largely through a reduction in the number and/or activity of procoagulant microparticles. This anticoagulant effect resulted in the formation of clots that were more susceptible to fibrinolysis, mainly because of a lesser activation of thrombin activatable fibrinolysis inhibitor. No difference in any variables was detected in controls at the time points considered. In conclusion, chronic grape consumption induces sustained anticoagulant and profibrinolytic effects with potential benefits for human health. Copyright © 2017 Elsevier Inc. All rights reserved.

Selective activation of heparin cofactor II by a sulfated polysaccharide isolated from the leaves of Artemisia princeps.

PubMed

Hayakawa, Y; Hayashi, T; Hayashi, T; Niiya, K; Sakuragawa, N

1995-10-01

While checking anticoagulant activities in crude fractions from Wakan-Yakus (traditional herbal drugs), we detected antithrombin activity in the polysaccharide fraction of the leaves of Artemisia princeps Pamp. A sulfated polysaccharide purified from the crude fractions by ion-exchange chromatography on DEAE-cellulose and gel filtration on Sepharose 6B potentiated the heparin cofactor II (HC II)-dependent antithrombin activity but not the antithrombin activity of antithrombin III (AT III). The polysaccharide enhanced the HC II-thrombin reaction more than 6000-fold. The apparent second-order rate constant of thrombin inhibition by HC II increased from 3.8 x 10(4) (in the absence of the polysaccharide) to 2.5 x 10(8) M-1 min-1 in the presence of 25-125 micrograms/ml of the polysaccharide. In human plasma, the polysaccharide accelerated the formation of thrombin-HC II complex. The stimulating effect on HC II-dependent antithrombin activity was almost totally abolished by treatment with chondroitinase AC I, heparinase or heparitinase, while chondroitinase ABC or chondroitinase AC II had little or no effect. These results suggest that the polysaccharide is a glycosaminoglycan-like material with properties that are quite distinct from heparin or dermatan sulfate.

Adrenaline potentiates PI 3-kinase in platelets stimulated with thrombin and SFRLLN: role of secreted ADP.

PubMed

Selheim, F; Frøyset, A K; Strand, I; Vassbotn, F S; Holmsen, H

2000-11-17

Adrenaline significantly potentiated late thrombin- and SFRLLN-induced PtdIns(3,4)P(2) production. Furthermore, the potentiating effect of adrenaline on thrombin-induced PtdIns(3, 4)P(2) production was independent on secreted ADP, whereas, the effect of adrenaline on SFRLLN-induced PtdIns(3,4)P(2) production was completely dependent of secreted ADP. However, the ADP-dependent accumulation of PtdIns(3,4)P(2) was not required for irreversible platelet aggregation induced by SFRLLN in the presence of adrenaline. It is concluded that adrenaline can replace secreted ADP to potentiate PtdIns(3,4)P(2) production in thrombin-stimulated but not in SFRLLN-stimulated platelets, thus demonstrating a qualitative difference between platelet stimulation by thrombin and the thrombin receptor activating peptide SFRLLN.

Novel Role for p21-activated Kinase 2 in Thrombin-induced Monocyte Migration*

PubMed Central

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

2013-01-01

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

Performance of coagulation tests in patients on therapeutic doses of dabigatran: a cross-sectional pharmacodynamic study based on peak and trough plasma levels.

PubMed

Hawes, E M; Deal, A M; Funk-Adcock, D; Gosselin, R; Jeanneret, C; Cook, A M; Taylor, J M; Whinna, H C; Winkler, A M; Moll, S

2013-08-01

Knowledge of anticoagulation status during dabigatran therapy may be desirable in certain clinical situations. To determine the coagulation tests that are most useful for assessing dabigatran's anticoagulant effect. Peak and trough blood samples from 35 patients taking dabigatran 150 mg twice daily, and one sample each from 30 non-anticoagulated individuals, were collected. Mass spectrometry and various coagulation assays were performed. 'Therapeutic range' was defined as the range of plasma dabigatran concentrations determined by mass spectrometry between the 2.5th and 97.5th percentiles of all values. The therapeutic range was 27-411 ng mL(-1) . The prothrombin time (PT) and activated partial thromboplastin time (APTT), determined with multiple reagents, and activated clotting time (ACT) were insensitive to therapeutic dabigatran: 29%, 18% and 40% of samples had a normal PT, APTT, and ACT, respectively. However, normal PT, ACT and APTT ruled out dabigatran levels above the 75th percentile. The thrombin clotting time (TCT) correlated well and linearly with dabigatran levels below the 50th percentile, but was unmeasurable above it. The dilute thrombin time, ecarin clotting time and ecarin chromogenic assay showed linear correlations with dabigatran levels over a broad range, and identified therapeutic and supratherapeutic levels. The prothrombin time, APTT and ACT are often normal in spite of therapeutic dabigatran plasma levels. The TCT is useful for detecting minimal dabigatran levels. The dilute thrombin time and chromogenic and clotting ecarin assays accurately identify therapeutic and supratherapeutic dabigatran levels. This trial is registered at www.clinicaltrials.gov (#NCT01588327). © 2013 International Society on Thrombosis and Haemostasis.

Kinetic Modeling Sheds Light on the Mode of Action of Recombinant Factor VIIa on Thrombin Generation

DTIC Science & Technology

2011-01-01

Regular Article Kinetic modeling sheds light on the mode of action of recombinant factor VIIa on thrombin generation Alexander Y. Mitrophanov...its effects on the quantitative parameters of thrombin generation. For recombinant activated factor VII (rFVIIa) ― a promising hemostasis-inducing...modulate thrombin production , it is necessary to identify rFVIIa-induced effects that are compatible with the available biochemical knowledge about

Effect of Locked-Nucleic Acid on a Biologically Active G-Quadruplex. A Structure-Activity Relationship of the Thrombin Aptamer

PubMed Central

Bonifacio, Laura; Church, Frank C.; Jarstfer, Michael B.

2008-01-01

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. PMID:19325759

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

PubMed

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

1995-11-01

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

Macrophage Migration Inhibitory Factor-Induced Autophagy Contributes to Thrombin-Triggered Endothelial Hyperpermeability in Sepsis.

PubMed

Chao, Chiao-Hsuan; Chen, Hong-Ru; Chuang, Yung-Chun; Yeh, Trai-Ming

2018-07-01

Vascular leakage contributes to the high morbidity and mortality associated with sepsis. Exposure of the endothelium to inflammatory mediators, such as thrombin and cytokines, during sepsis leads to hyperpermeability. We recently observed that autophagy, a cellular process for protein turnover, is involved in macrophage migration inhibitory factor (MIF)-induced endothelial hyperpermeability. Even though it is known that thrombin induces endothelial cells to secrete MIF and to increase vascular permeability, the possible role of autophagy in this process is unknown. In this study, we proposed and tested the hypothesis that MIF-induced autophagy plays an important role in thrombin-induced endothelial hyperpermeability. We evaluated the effects of thrombin on endothelial permeability, autophagy induction, and MIF secretion in vitro using the human microvascular endothelial cell line-1 and human umbilical vein endothelial cells. Several mechanisms/read outs of endothelial permeability and autophagy formation were examined. We observed that blocking autophagy attenuated thrombin-induced endothelial hyperpermeability. Furthermore, thrombin-induced MIF secretion was involved in this process because MIF inhibition reduced thrombin-induced autophagy and hyperpermeability. Finally, we showed that blocking MIF or autophagy effectively alleviated vascular leakage and mortality in endotoxemic mice. Thus, MIF-induced autophagy may represent a common mechanism causing vascular leakage in sepsis.

Pulsatile equibiaxial stretch inhibits thrombin-induced RhoA and NF-{kappa}B activation

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

Haga, Jason H.; Kaunas, Roland; Radeff-Huang, Julie

2008-07-18

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

Duplex/quadruplex oligonucleotides: Role of the duplex domain in the stabilization of a new generation of highly effective anti-thrombin aptamers.

PubMed

Russo Krauss, Irene; Napolitano, Valeria; Petraccone, Luigi; Troisi, Romualdo; Spiridonova, Vera; Mattia, Carlo Andrea; Sica, Filomena

2018-02-01

Recently, mixed duplex/quadruplex oligonucleotides have attracted great interest for use as biomedical aptamers. In the case of anti-thrombin aptamers, the addition of duplex-forming sequences to a G-quadruplex module identical or very similar to the best-known G-quadruplex of the Thrombin Binding Aptamer (HD1) results in new or improved biological properties, such as higher activity or different recognition properties with respect to HD1. Remarkably, this bimodular fold was hypothesized, based on its sequence, for the only anti-thrombin aptamer in advanced clinical trial, NU172. Whereas cation modulation of G-quadruplex conformation and stability is well characterized, only few data from similar analysis on duplex/quadruplex oligonucleotides exist. Here we have performed a characterization of structure and stability of four different duplex/quadruplex anti-thrombin aptamers, including NU172, in the presence of different cations and in physiological-mimicking conditions in comparison to HD1, by means of spectroscopic techniques (UV and circular dichroism) and differential scanning calorimetry. Our data show a strong reciprocal influence of each domain on the stability of the other and in particular suggest a stabilizing effect of the duplex region in the presence of solutions mimicking the physiological conditions, strengthening the idea that bimodular aptamers present better therapeutic potentialities than those containing a single G-quadruplex domain. Copyright © 2017 Elsevier B.V. All rights reserved.

Robust and specific ratiometric biosensing using a copper-free clicked quantum dot-DNA aptamer sensor

NASA Astrophysics Data System (ADS)

Zhang, Haiyan; Feng, Guoqiang; Guo, Yuan; Zhou, Dejian

2013-10-01

We report herein the successful preparation of a compact and functional CdSe-ZnS core-shell quantum dot (QD)-DNA conjugate via highly efficient copper-free ``click chemistry'' (CFCC) between a dihydro-lipoic acid-polyethylene glycol-azide (DHLA-PEG-N3) capped QD and a cyclooctyne modified DNA. This represents an excellent balance between the requirements of high sensitivity, robustness and specificity for the QD-FRET (Förster resonance energy transfer) based sensor as confirmed by a detailed FRET analysis on the QD-DNA conjugate, yielding a relatively short donor-acceptor distance of ~5.8 nm. We show that this CFCC clicked QD-DNA conjugate is not only able to retain the native fluorescence quantum yield (QY) of the parent DHLA-PEG-N3 capped QD, but also well-suited for robust and specific biosensing; it can directly quantitate, at the pM level, both labelled and unlabelled complementary DNA probes with a good SNP (single-nucleotide polymorphism) discrimination ability in complex media, e.g. 10% human serum via target-binding induced FRET changes between the QD donor and the dye acceptor. Furthermore, this sensor has also been successfully exploited for the detection, at the pM level, of a specific protein target (thrombin) via the encoded anti-thrombin aptamer sequence in the QD-DNA conjugate.We report herein the successful preparation of a compact and functional CdSe-ZnS core-shell quantum dot (QD)-DNA conjugate via highly efficient copper-free ``click chemistry'' (CFCC) between a dihydro-lipoic acid-polyethylene glycol-azide (DHLA-PEG-N3) capped QD and a cyclooctyne modified DNA. This represents an excellent balance between the requirements of high sensitivity, robustness and specificity for the QD-FRET (Förster resonance energy transfer) based sensor as confirmed by a detailed FRET analysis on the QD-DNA conjugate, yielding a relatively short donor-acceptor distance of ~5.8 nm. We show that this CFCC clicked QD-DNA conjugate is not only able to retain the native fluorescence quantum yield (QY) of the parent DHLA-PEG-N3 capped QD, but also well-suited for robust and specific biosensing; it can directly quantitate, at the pM level, both labelled and unlabelled complementary DNA probes with a good SNP (single-nucleotide polymorphism) discrimination ability in complex media, e.g. 10% human serum via target-binding induced FRET changes between the QD donor and the dye acceptor. Furthermore, this sensor has also been successfully exploited for the detection, at the pM level, of a specific protein target (thrombin) via the encoded anti-thrombin aptamer sequence in the QD-DNA conjugate. Electronic supplementary information (ESI) available: Details on the synthesis, purification and characterisation of the DHLA-PEG600-N3, cyclooctyne-DNA, and QD-TBA20 conjugates as well as all supporting figures and tables. See DOI: 10.1039/c3nr02897f

Graphene oxide and DNA aptamer based sub-nanomolar potassium detecting optical nanosensor

NASA Astrophysics Data System (ADS)

Datta, Debopam; Sarkar, Ketaki; Mukherjee, Souvik; Meshik, Xenia; Stroscio, Michael A.; Dutta, Mitra

2017-08-01

Quantum-dot (QD) based nanosensors are frequently used by researchers to detect small molecules, ions and different biomolecules. In this article, we present a sensor complex/system comprised of deoxyribonucleic acid (DNA) aptamer, gold nanoparticle and semiconductor QD, attached to a graphene oxide (GO) flake for detection of potassium. As reported herein, it is demonstrated that QD-aptamer-quencher nanosensor functions even when tethered to GO, opening the way to future applications where sensing can be accomplished simultaneously with other previously demonstrated applications of GO such as serving as a nanocarrier for drug delivery. Herein, it is demonstrated that the DNA based thrombin binding aptamer used in this study undergoes the conformational change needed for sensing even when the nanosensor complex is anchored to the GO. Analysis with the Hill equation indicates the interaction between aptamer and potassium follows sigmoidal Hill kinetics. It is found that the quenching efficiency of the optical sensor is linear with the logarithm of concentration from 1 pM to 100 nM and decreases for higher concentration due to unavailability of aptamer binding sites. Such a simple and sensitive optical aptasensor with minimum detection capability of 1.96 pM for potassium ion can also be employed in-vitro detection of different physiological ions, pathogens and disease detection methods.

Engineering multi-functional bacterial outer membrane vesicles as modular nanodevices for biosensing and bioimaging.

PubMed

Chen, Qi; Rozovsky, Sharon; Chen, Wilfred

2017-07-04

Outer membrane vesicles (OMVs) are proteoliposomes derived from the outer membrane and periplasmic space of many Gram-negative bacteria including E. coli as part of their natural growth cycle. Inspired by the natural ability of E. coli to sort proteins to both the exterior and interior of OMVs, we reported here a one-pot synthesis approach to engineer multi-functionalized OMV-based sensors for both antigen binding and signal generation. SlyB, a native lipoprotein, was used a fusion partner to package nanoluciferase (Nluc) within OMVs, while a previously developed INP-Scaf3 surface scaffold was fused to the Z-domain for antibody recruiting. The multi-functionalized OMVs were used for thrombin detection with a detection limit of 0.5 nM, comparable to other detection methods. Using the cohesin domains inserted between the Z-domain and INP, these engineered OMVs were further functionalized with a dockerin-tagged GFP for cancer cell imaging.

Boolean Logic Tree of Label-Free Dual-Signal Electrochemical Aptasensor System for Biosensing, Three-State Logic Computation, and Keypad Lock Security Operation.

PubMed

Lu, Jiao Yang; Zhang, Xin Xing; Huang, Wei Tao; Zhu, Qiu Yan; Ding, Xue Zhi; Xia, Li Qiu; Luo, Hong Qun; Li, Nian Bing

2017-09-19

The most serious and yet unsolved problems of molecular logic computing consist in how to connect molecular events in complex systems into a usable device with specific functions and how to selectively control branchy logic processes from the cascading logic systems. This report demonstrates that a Boolean logic tree is utilized to organize and connect "plug and play" chemical events DNA, nanomaterials, organic dye, biomolecule, and denaturant for developing the dual-signal electrochemical evolution aptasensor system with good resettability for amplification detection of thrombin, controllable and selectable three-state logic computation, and keypad lock security operation. The aptasensor system combines the merits of DNA-functionalized nanoamplification architecture and simple dual-signal electroactive dye brilliant cresyl blue for sensitive and selective detection of thrombin with a wide linear response range of 0.02-100 nM and a detection limit of 1.92 pM. By using these aforementioned chemical events as inputs and the differential pulse voltammetry current changes at different voltages as dual outputs, a resettable three-input biomolecular keypad lock based on sequential logic is established. Moreover, the first example of controllable and selectable three-state molecular logic computation with active-high and active-low logic functions can be implemented and allows the output ports to assume a high impediment or nothing (Z) state in addition to the 0 and 1 logic levels, effectively controlling subsequent branchy logic computation processes. Our approach is helpful in developing the advanced controllable and selectable logic computing and sensing system in large-scale integration circuits for application in biomedical engineering, intelligent sensing, and control.

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

PubMed Central

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

2015-01-01

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

Evidence for a hyperglycaemia-dependent decrease of antithrombin III-thrombin complex formation in humans.

PubMed

Ceriello, A; Giugliano, D; Quatraro, A; Marchi, E; Barbanti, M; Lefèbvre, P

1990-03-01

In the presence of increased levels of fibrinopeptide A, decreased antithrombin III biological activity, and thrombin-antithrombin III complex levels are seen in diabetic patients. Induced-hyperglycaemia in diabetic and normal subjects decreased antithrombin III activity and thrombin-antithrombin III levels, and increased fibrinopeptide A plasma levels, while antithrombin III concentration did not change; heparin was shown to reduced these phenomena. In diabetic patients, euglycaemia induced by insulin infusion restored antithrombin III activity, thrombin-antithrombin III complex and fibrinopeptide A concentrations; heparin administration had the same effects. These data stress the role of a hyperglycaemia-dependent decrease of antithrombin III activity in precipitating thrombin hyperactivity in diabetes mellitus.

Sequence and structure insights of kazal type thrombin inhibitor protein: Studied with phylogeny, homology modeling and dynamic MM/GBSA studies.

PubMed

Jadhav, Aparna; Dash, RadhaCharan; Hirwani, Raj; Abdin, Malik

2018-03-01

Despite the wide medical importance of serine protease inhibitors, many of kazal type proteins are still to be explored. These thrombin inhibiting proteins are found in the digestive system of hematophagous organisms mainly Arthropods. We studied one of such protein i.e. Kazal type-1 protein from sand-fly Phlebotomus papatasi as its structure and interaction with thrombin is unclear. Initially, Dipetalin a kazal-follistasin domain protein was run through PSI-BLAST to retrieve related sequences. Using this set of sequence a phylogenetic tree was constructed, which identified a distantly related kazal type-1 protein. A three-dimensional structure was predicted for this protein and was aligned with Rhodniin for further evaluation. To have a comparative understanding of it's binding at the thrombin active site, the aligned kazal model-thrombin and rhodniin-thrombin complexes were subjected to molecular dynamics simulations. Dynamics analysis with reference to main chain RMSD, H-chain residue RMSF and total energy showed rhodniin-thrombin complex as a more stable system. Further, the MM/GBSA method was applied that calculated the binding free energy (ΔG binding ) for rhodniin and kazal model as -220.32kcal/Mol and -90.70kcal/Mol, respectively. Thus, it shows that kazal model has weaker bonding with thrombin, unlike rhodniin. Copyright © 2017 Elsevier B.V. All rights reserved.

Gelatin-thrombin hemostatic matrix in neurosurgical procedures: hemostasis effectiveness and economic value of clinical and surgical procedure-related benefits.

PubMed

Esposito, Felice; Cappabianca, Paolo; Angileri, Filippo F; Cavallo, Luigi M; Priola, Stefano M; Crimi, Salvatore; Solari, Domenico; Germanò, Antonino F; Tomasello, Francesco

2016-07-26

Gelatin-thrombin hemostatic matrix (FloSeal®) use is associated with shorter surgical times and less blood loss, parameters that are highly valued in neurosurgical procedures. We aimed to assess the effectiveness of gelatin-thrombin in neurosurgical procedures and estimate its economic value. In a 6-month retrospective evaluation at 2 hospitals, intraoperative and postoperative information were collected from patients undergoing neurosurgical procedures where bleeding was controlled with gelatin-thrombin matrix or according to local bleeding control guidelines (control group). Study endpoints were: length of surgery, estimated blood loss, hospitalization duration, blood units utilized, intensive care unit days, postoperative complications, and time-to-recovery. Statistical methods compared endpoints between the gelatin-thrombin and control groups and resource utilization costs were estimated. Seventy-eight patients (38 gelatin-thrombin; 40 control) were included. Gelatin-thrombin was associated with a shorter surgery duration than control 166±40 versus 185±55, p=0.0839); a lower estimated blood loss (185±80 versus 250±95ml; p=0.0017); a shorter hospital stay (10±3 versus 13±3 days; p<0.001); fewer intensive care unit days (10 days/3 patients and 20 days/4 patients); and shorter time-to-recovery (3±2.2 versus 4±2.8 weeks; p=0861). Fewer gelatin-thrombin patients experienced postoperative complications (3 minor) than the control group (5 minor; 3 major). No gelatin-thrombin patient required blood transfusion; 5 units were administered in the control group. The cost of gelatin-thrombin (€268.40/unit) was offset by the shorter surgery duration (difference of 19 minutes at €858 per hour) and the economic value of improved the other endpoint outcomes (ie, shorter hospital stay, less blood loss/lack of need for transfusion, fewer intensive care unit days, and complications). Gelatin-thrombin hemostatic matrix use in patients undergoing neurosurgical procedures was associated with better intra- and post-operative parameters than conventional hemostasis methods, with these parameters having substantial economic benefits.

Effects of Aerobic Capacity on Thrombin-Induced Hydrocephalus and White Matter Injury.

PubMed

Ni, Wei; Gao, Feng; Zheng, Mingzhe; Koch, Lauren G; Britton, Steven L; Keep, Richard F; Xi, Guohua; Hua, Ya

2016-01-01

We have previously shown that intracerebral hemorrhage-induced brain injury is less in rats bred for high aerobic capacity (high capacity runners; HCR) compared with those bred for low aerobic capacity (low capacity runners; LCRs). Thrombin, an essential component in the coagulation cascade, is produced after cerebral hemorrhage. Intraventricular injection of thrombin causes significant hydrocephalus and white matter damage. In the present study, we examined the effect of exercise capacity on thrombin-induced hydrocephalus and white matter damage. Mid-aged (13-month-old) female LCRs (n = 13) and HCRs (n = 12) rats were used in this study. Rats received an intraventricular injection of thrombin (3 U, 50 μl). All rats underwent magnetic resonance imaging (MRI) at 24 h and were then euthanized for brain histology and Western blot. The mortalities were 20 % in LCRs and 33 % in HCRs after thrombin injection (p > 0.05). No rats died after saline injection. Intraventricular thrombin injection resulted in hydrocephalus and periventricular white matter damage as determined on MRI. In LCR rats, thrombin induced significant ventricle enlargement (23.0 ± 2.3 vs12.8 ± 1.9 mm(3) in LCR saline group; p < 0.01) and white matter lesion (9.3 ± 7.6 vs 0.6 ± 0.5 mm(3) in LCR saline group, p < 0.05). In comparison, in HCR rats thrombin induced less ventricular enlargement (17.3 ± 3.9 vs 23.0 ± 2.3 mm(3) in LCRs, p < 0.01) and smaller white matter lesions (2.6 ± 1.2 mm(3) vs 9.3 ± 7.6 mm(3) in LCRs, p < 0.05). In LCR rats, there was also upregulation of heat shock protein-32, a stress marker, and microglial activation in the periventricular white matter. These changes were significantly reduced in HCR rats. Intraventricular injection of thrombin caused more white matter damage and hydrocephalus in rats with low aerobic capacity. A differential effect of thrombin may contribute to differences in the effects of cerebral hemorrhage with aerobic capacity.

Fusion of the C-terminal triskaidecapeptide of hirudin variant 3 to alpha1-proteinase inhibitor M358R increases the serpin-mediated rate of thrombin inhibition

PubMed Central

2013-01-01

Background Alpha-1 proteinase inhibitor (API) is a plasma serpin superfamily member that inhibits neutrophil elastase; variant API M358R inhibits thrombin and activated protein C (APC). Fusing residues 1-75 of another serpin, heparin cofactor II (HCII), to API M358R (in HAPI M358R) was previously shown to accelerate thrombin inhibition over API M358R by conferring thrombin exosite 1 binding properties. We hypothesized that replacing HCII 1-75 region with the 13 C-terminal residues (triskaidecapeptide) of hirudin variant 3 (HV354-66) would further enhance the inhibitory potency of API M358R fusion proteins. We therefore expressed HV3API M358R (HV354-66 fused to API M358R) and HV3API RCL5 (HV354-66 fused to API F352A/L353V/E354V/A355I/I356A/I460L/M358R) API M358R) as N-terminally hexahistidine-tagged polypeptides in E. coli. Results HV3API M358R inhibited thrombin 3.3-fold more rapidly than API M358R; for HV3API RCL5 the rate enhancement was 1.9-fold versus API RCL5; neither protein inhibited thrombin as rapidly as HAPI M358R. While the thrombin/Activated Protein C rate constant ratio was 77-fold higher for HV3API RCL5 than for HV3API M358R, most of the increased specificity derived from the API F352A/L353V/E354V/A355I/I356A/I460L API RCL 5 mutations, since API RCL5 remained 3-fold more specific than HV3API RCL5. An HV3 54-66 peptide doubled the Thrombin Clotting Time (TCT) and halved the binding of thrombin to immobilized HCII 1-75 at lower concentrations than free HCII 1-75. HV3API RCL5 bound active site-inhibited FPR-chloromethyl ketone-thrombin more effectively than HAPI RCL5. Transferring the position of the fused HV3 triskaidecapeptide to the C-terminus of API M358R decreased the rate of thrombin inhibition relative to that mediated by HV3API M358R by 11-to 14-fold. Conclusions Fusing the C-terminal triskaidecapeptide of HV3 to API M358R-containing serpins significantly increased their effectiveness as thrombin inhibitors, but the enhancement was less than that seen in HCII 1-75–API M358R fusion proteins. HCII 1-75 was a superior fusion partner, in spite of the greater affinity of the HV3 triskaidecapeptide, manifested both in isolated and API-fused form, for thrombin exosite 1. Our results suggest that HCII 1-75 binds thrombin exosite 1 and orients the attached serpin scaffold for more efficient interaction with the active site of thrombin than the HV3 triskaidecapeptide. PMID:24215622

Reactivity of mouse antibodies against bromelain-treated mouse erythrocytes with thrombin-treated mouse platelets.

PubMed Central

Kawaguchi, S

1989-01-01

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

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

PubMed

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

2016-12-01

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.

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

PubMed

Kamisato, Chikako; Furugohri, Taketoshi; Morishima, Yoshiyuki

2016-05-01

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

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

PubMed

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

2016-01-01

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

Thrombin-induced activation of RhoA in platelet shape change.

PubMed

Bodie, S L; Ford, I; Greaves, M; Nixon, G F

2001-09-14

Thrombin-induced activation of RhoA and its involvement in the regulation of myosin II light chain(20) phosphorylation (MLC-P) in alpha-toxin permeabilized platelets was investigated. Permeabilized platelets, expressing normal levels of P-selectin, displayed a Ca(2+)-dependent increase in shape change and MLC-P. Thrombin activated RhoA as measured by a rhotekin-binding assay within 30 s of stimulation under conditions of constant [Ca(2+)](i). Under the same conditions and timecourse, thrombin or GTPgammaS induced an increase in MLC-P and platelet shape change which was not dependent on an increase in [Ca(2+)](i). The thrombin- and GTPgammaS-induced MLC-P in constant [Ca(2+)](i) was inhibited by the addition of Y27632, a Rho-kinase inhibitor. This study directly demonstrates that thrombin can activate RhoA in platelets in a timecourse compatible with a role in increasing MLC-P and shape change (not involving an increase in [Ca(2+)](i)). This is also Rho-kinase-dependent. Copyright 2001 Academic Press.

Sulfated Low Molecular Weight Lignins, Allosteric Inhibitors of Coagulation Proteinases via the Heparin Binding Site, Significantly Alter the Active Site of Thrombin and Factor Xa Compared to Heparin

PubMed Central

Henry, Brian L.; Desai, Umesh R.

2014-01-01

Sulfated low molecular weight lignins (LMWLs) have been found to bind in the heparin binding sites of coagulation proteinases. LMWLs represent a library of diverse non-carbohydrate, aromatic molecules which are structures different from heparin, but still potently inhibit thrombin and factor Xa. To better understand their mechanism of action, we studied the effects of three sulfated LMWLs (CDSO3, FDSO3, and SDSO3) on the active sites of thrombin and factor Xa. LMWLs were found to uniformly inhibit the catalytic activity of thrombin and factor Xa, regardless of the substrate used. Michaelis-Menten kinetic studies indicate that maximal velocity of hydrolysis of each chromogenic substrate decreases significantly in the presence of sulfated LMWLs, while the effect on Michaelis constant is dependent on the nature of the substrate. These studies indicate that LMWLs inhibit thrombin and factor Xa through allosteric disruption of the catalytic apparatus, specifically through the catalytic step. As opposed to heparin, LMWLs significantly alter the binding of the active site fluorescent ligand p-aminobenzamidine. LMWLs also had a greater effect on the molecular orientation of fluorescein-labeled His 57 than heparin. The molecular geometry surrounding the most important catalytic amino acid, Ser 195, was significantly altered by the binding of LMWLs while heparin had no measurable effect on Ser 195. These results further advance the concept of sulfated LMWLs as heparin mimics and will aid the design of anticoagulants based on their novel scaffold. PMID:25242245

Sulfated low molecular weight lignins, allosteric inhibitors of coagulation proteinases via the heparin binding site, significantly alter the active site of thrombin and factor xa compared to heparin.

PubMed

Henry, Brian L; Desai, Umesh R

2014-11-01

Sulfated low molecular weight lignins (LMWLs) have been found to bind in the heparin binding sites of coagulation proteinases. LMWLs represent a library of diverse non-carbohydrate, aromatic molecules which are structures different from heparin, but still potently inhibit thrombin and factor Xa. To better understand their mechanism of action, we studied the effects of three sulfated LMWLs (CDSO3, FDSO3, and SDSO3) on the active sites of thrombin and factor Xa. LMWLs were found to uniformly inhibit the catalytic activity of thrombin and factor Xa, regardless of the substrate used. Michaelis-Menten kinetic studies indicate that maximal velocity of hydrolysis of each chromogenic substrate decreases significantly in the presence of sulfated LMWLs, while the effect on Michaelis constant is dependent on the nature of the substrate. These studies indicate that LMWLs inhibit thrombin and factor Xa through allosteric disruption of the catalytic apparatus, specifically through the catalytic step. As opposed to heparin, LMWLs significantly alter the binding of the active site fluorescent ligand p-aminobenzamidine. LMWLs also had a greater effect on the molecular orientation of fluorescein-labeled His 57 than heparin. The molecular geometry surrounding the most important catalytic amino acid, Ser 195, was significantly altered by the binding of LMWLs while heparin had no measurable effect on Ser 195. These results further advance the concept of sulfated LMWLs as heparin mimics and will aid the design of anticoagulants based on their novel scaffold. Copyright © 2014 Elsevier Ltd. All rights reserved.

Reducing duplex examinations in patients with iatrogenic pseudoaneurysms.

PubMed

Stone, Patrick A; Aburahma, Ali F; Flaherty, Sarah K

2006-06-01

Ultrasound-guided thrombin injection has become the initial treatment of choice for femoral access-related pseudoaneurysms. Patients typically undergo serial duplex examinations to assess for spontaneous resolution of small iatrogenic pseudoaneurysms (IPSAs) (<2.5 cm), or may require repeated diagnostic, therapeutic, and follow-up studies for larger IPSAs (>2.5 cm). We evaluated the impact of a revised treatment algorithm that includes primary treatment of both small (<2.5 cm) and larger pseudoaneurysms (>2.5 cm), rather than observation of smaller ones, and attempts to establish a single duplex examination via a point-of-care treatment strategy. We reviewed 105 consecutive patients treated with ultrasound-guided thrombin injection from July 2001 through September 2004. Patient, IPSAs, characteristics, and treatment methods were examined. The number of duplex examinations per patient was evaluated over the treatment interval. Also, published cost data were used to compare primary treatment of small ISPAs vs observation with serial duplex examinations. Successful thrombosis occurred in 103 (98.1%) of 105 treated pseudoaneurysms. No minor or major complications occurred after thrombin injection in either small or large ISPAs, and both failures requiring operation were in the large aneurysm group. The recurrence rate for the series was 1.9% (2/105), and both recurrences were successfully treated with an additional thrombin injection. A single injection was successful in treating 43 (97.7%) of 44 small (<2.5 cm) IPSAs, and one required a second injection. Patients had an average of 3.3 duplex examinations in our first year of treatment experience, which declined to 1.5 by our third year with the institution of a point-of-care service model for all pseudoaneurysms. Based on this decreased use of duplex examination and an average treatment cohort of 35 IPSA patients per year our institution, we determined this results in a reduction of 35 hours of laboratory time and nearly 70 ultrasounds per year. Similarly for small pseudoaneurysms, a point-of-service primary treatment program rather than observation results in an estimated cost savings of $12,000, based on treating 15 small IPSAs per year. Ultrasound-guided thrombin injection is safe and effective for the treatment of nearly all iatrogenic pseudoaneurysms. We recommend primary treatment of small pseudoaneurysms by ultrasound-guided thrombin injection rather than observation with serial duplex scans. A point-of-care treatment algorithm can result in cost savings by reducing the number of necessary duplex examinations.

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

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

Oberdisse, E.; Lapetina, E.G.

1987-05-14

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

Induction of apoptosis by thrombin in the cultured neurons of dorsal motor nucleus of the vagus.

PubMed

Wu, X; Zhang, W; Li, J-Y; Chai, B-X; Peng, J; Wang, H; Mulholland, M W

2011-03-01

A previous study demonstrated the presence of protease-activated receptor (PAR) 1 and 2 in the dorsal motor nucleus of vagus (DMV). The aim of this study is to characterize the effect of thrombin on the apoptosis of DMV neurons. The dorsal motor nucleus of vagus neurons were isolated from neonatal rat brainstems using micro-dissection and enzymatic digestion and cultured. Apoptosis of DMV neurons were examined in cultured neurons. Apoptotic neuron was examined by TUNEL and ELISA. Data were analyzed using anova and Student's t-test. Exposure of cultured DMV neurons to thrombin (0.1 to 10 U mL(-1)) for 24 h significantly increased apoptosis. Pretreatment of DMV neurons with hirudin attenuated the apoptotic effect of thrombin. Similar induction of apoptosis was observed for the PAR1 receptor agonist SFLLR, but not for the PAR3 agonist TFRGAP, nor for the PAR4 agonist YAPGKF. Protease-activated receptors 1 receptor antagonist Mpr(Cha) abolished the apoptotic effect of thrombin, while YPGKF, a specific antagonist for PAR4, demonstrated no effect. After administration of thrombin, phosphorylation of JNK and P38 occurred as early as 15 min, and remained elevated for up to 45 min. Pretreatment of DMV neurons with SP600125, a specific inhibitor for JNK, or SB203580, a specific inhibitor for P38, significantly inhibited apoptosis induced by thrombin. Thrombin induces apoptosis in DMV neurons through a mechanism involving the JNK and P38 signaling pathways. © 2010 Blackwell Publishing Ltd.

Comparative biochemical studies of fresh frozen plasma and pooled solvent/detergent-treated plasma (octaplasLG® ) with focus on protein S and its impact in different thrombin generation assay set-ups.

PubMed

Heger, A; Janisch, S; Pock, K; Römisch, J

2016-10-01

The solvent/detergent treatment enables effective and robust inactivation of all lipid-enveloped viruses, but also inactivates partly sensitive plasma proteins such as protein S. The aim of this study was to investigate the thrombin generation capacity of octaplasLG ® , in particular focusing on the function of protein S in thrombin generation assay and the impact of assay settings. Sixteen octaplasLG ® batches and 32 units of single donor fresh frozen plasma (FFP) were investigated. For protein S, both functional activity and free antigen levels were measured. Thrombin generation assay was performed using two fluorogenic tests with different triggers. Finally, rotational thromboelastometry was performed. Mean protein S levels were lower in octaplasLG ® , but a wider range of values was found for FFP. Clotting parameters and thrombin generation capacities overlapped between the two plasma groups as demonstrated using both thrombin generation assays and different triggers. Spiking studies with protein S-depleted plasma, human purified protein S or antibodies against protein S confirmed a correlation between protein S and thrombin generation capacity under specific assay conditions, especially in an assay with low tissue factor concentration. Correlation between protein S and thrombin generation capacity was demonstrated in the TGA. Due to higher variability in protein S content in the FFP group, overlapping haemostatic potentials of the two plasma groups were found. © 2016 International Society of Blood Transfusion.

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

PubMed Central

Vergis, James M.; Wiener, Michael C.

2011-01-01

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

21 CFR 864.7875 - Thrombin time test.

Code of Federal Regulations, 2010 CFR

2010-04-01

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

21 CFR 864.7875 - Thrombin time test.

Code of Federal Regulations, 2014 CFR

2014-04-01

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

21 CFR 864.7875 - Thrombin time test.

Code of Federal Regulations, 2011 CFR

2011-04-01

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

21 CFR 864.7875 - Thrombin time test.

Code of Federal Regulations, 2012 CFR

2012-04-01

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

21 CFR 864.7875 - Thrombin time test.

Code of Federal Regulations, 2013 CFR

2013-04-01

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

Thrombin-induced microglial activation impairs hippocampal neurogenesis and spatial memory ability in mice.

PubMed

Yang, Yuan; Zhang, Meikui; Kang, Xiaoni; Jiang, Chen; Zhang, Huan; Wang, Pei; Li, Jingjing

2015-09-26

To investigate the effects of microglia/macrophages activation induced by intrastriatal thrombin injection on dentate gyrus neurogenesis and spatial memory ability in mice. The male C57BL/6 mice were divided into 4 groups of 10: sham, intracerebral hemorrhage (ICH), ICH + hirudin (thrombin inhibitor), and ICH + indometacin (Indo, an anti-inflammation drug). ICH model was created by intrastriatal thrombin (1U) injection. BrdU (50 mg/kg) was administrated on the same day after surgery for 6 consecutive days. Motor functions were evaluated with rotarod and beam walking tests. The spatial memory deficit was measured with Morris water maze (MWM). Cell quantification was performed for doublecortin (DCX, immature neuron), BrdU (S-phase proliferating cell population) and CD68 (activated microglia/macrophage) immune-reactive cells. Microglia/macrophages activation induced by intrastriatal thrombin injection reduced hippocampal neurogenesis and impaired spatial memory ability, but did not affect the motor function at 3 and 5 days post-injury. Both hirudin and indometacin reduced microglia/macrophages activation, enhanced hippocampal neurogenesis, and improved spatial memory ability in mice. Microglia/macrophages activation induced by intrastriatal thrombin injection might be responsible for the spatial memory deficit. Targeting both thrombin and inflammation systems in acute phase of ICH might be important in alleviating the significant spatial memory deficits.

Activation of platelet-rich plasma using thrombin receptor agonist peptide.

PubMed

Landesberg, Regina; Burke, Andrea; Pinsky, David; Katz, Ronald; Vo, Jennifer; Eisig, Sidney B; Lu, Helen H

2005-04-01

This study proposes an alternative preparation method of platelet-rich plasma (PRP). Specifically, we compare the use of thrombin receptor agonist peptide-6 (TRAP) and bovine thrombin as a clotting agent in the preparation of PRP. PRP was prepared by centrifugation and clotted with thrombin or TRAP. In vitro clotting times were monitored as a function of TRAP concentration, and clot retraction was determined by measuring clot diameter over time. Following the optimization of TRAP concentration, experiments were repeated with the addition of several commercially available bone substitutes. The release of PRP-relevant growth factors as a function of PRP preparation was also determined. The most rapid polymerization of PRP takes place with the addition of thrombin, followed by TRAP/Allogro (Ceramed, Lakewood, CO), TRAP/BioGlass (Mo-Sci, Rolla, MN), TRAP/BioOss (Osteohealth, Shirley, NY), and TRAP alone. Thrombin caused considerable clot retraction (43%), whereas TRAP alone resulted in only 15% retraction. TRAP/Allogro, TRAP/BioOss, and TRAP/BioGlass all exhibited minimal retraction (8%). The use of TRAP to activate clot formation in the preparation of PRP may be a safe alternative to bovine thrombin. It results in an excellent working time and significantly less clot retraction than the currently available methods of PRP production.

Flow cytometry analysis reveals different activation profiles of thrombin- or TRAP-stimulated platelets in db/db mice. The regulatory role of PAR-3.

PubMed

Kassassir, Hassan; Siewiera, Karolina; Talar, Marcin; Przygodzki, Tomasz; Watala, Cezary

2017-06-01

Recent studies have shown that it may be the concentration of thrombin, which is discriminative in determining of the mechanism of platelet activation via protease activated receptors (PARs). Whether the observed phenomenon of differentiated responses of mouse platelets to various thrombin concentrations in non-diabetic db/+ and diabetic db/db mice depends upon the concerted action of various PARs, remains to be established. We found elevated reactivity of platelets, as well as the enhanced PAR-3 expression in response to both the used concentrations of AYPGKF in db/db mice, as compared to db/+ heterozygotes. At low concentration of thrombin platelets from diabetic mice demonstrated hyperreactivity, reflected by higher expression of PAR-3. For higher thrombin concentration, blood platelets from db/db mice appeared hyporeactive, compared to db/+ animals, while no significant differences in PAR-3 expression were observed between diabetic and non-diabetic mice. The novel and previously unreported finding resulting from our study is that the increased expression of PAR-3 in response to either TRAP for PAR-4 or low thrombin (when PAR-4 is not the efficient thrombin receptor) may be one of the key events contributing to higher reactivity of platelets in db/db mice. Copyright © 2017 Elsevier Inc. All rights reserved.

Thrombin Generating Capacity and Phenotypic Association in ABO Blood Groups.

PubMed

Kremers, Romy M W; Mohamed, Abdulrahman B O; Pelkmans, Leonie; Hindawi, Salwa; Hemker, H Coenraad; de Laat, H Bas; Huskens, Dana; Al Dieri, Raed

2015-01-01

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.

Thrombin Generating Capacity and Phenotypic Association in ABO Blood Groups

PubMed Central

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

2015-01-01

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

MECHANISMS INVOLVED IN FIBRIN FORMATION

PubMed Central

Boyles, Paul W.; Ferguson, John H.; Muehlke, Paul H.

1951-01-01

That the role of thrombin in the conversion of fibrinogen to fibrin is essentially enzymatic, is established not only by the minute amounts of thrombin which are effective but also by the complete independence of fibrin yields and thrombin concentrations over a very wide range of thrombin dilutions and clotting times. The thrombin-fibrinogen reaction, in the phase beyond the "latent period" at least, seems fundamentally "first order." Technical requirements of the experiments leading to these conclusions include: (1) a highly purified (e.g. 97 per cent "clottable") fibrinogen, (2) absence of traces of thrombic impurities in the fibrinogen, (3) absence of fibrinolytic protease contaminant of the thrombin and the fibrinogen, and (4) sufficient stability of the thrombin even at very high dilutions. Four conditions affecting thrombin stability have been investigated. Fibrin yields are not significantly modified by numerous experimental circumstances that influence the clotting time, such as (1) temperature, (2) pH, (3) non-specific salt action due to electrical (ionic) charges, which alter the Coulomb forces involved in the fibrillar aggregation, (4) specific ion effects, whether clot-accelerating (e.g. Ca++) or clot-inhibitory (e.g. Fe(CN)6''''), (5) occluding (adsorptive) colloids, which have a "fibrinoplastic" action, e.g. (a) acacia and probably (b) fibrinogen which has been mildly "denatured" by salt-heating, acidification, etc. The data with which several European workers have attempted to substantiate the idea of a two-stage thrombin-fibrinogen reaction with an intermediary "profibrin" (allegedly partly "denatured") have been reanalyzed with controls which lead us to very different conclusions, viz. (1) denaturation and fibrin formation are independent; (2) partial denaturation is "fibrinoplastic" (see above); and (3) conditions of strong salinity and acid pH (5.1) usually do not completely prevent the thrombin-fibrinogen reaction but merely prolong the "latent" phase and lessen the time required for completion of essentially the same reaction (fibrin polymerization) when more favorable clotting conditions are restored. Thus, our experiments advance the modern concepts concerning the coagulation mechanisms along lines that, for the most part, agree with those of the Harvard physical chemists, and we oppose the European views concerning a two-stage reaction, "profibrin," and "the denaturase theory" of clotting. PMID:14832433

The Effects of Steroids on Coagulation Dysfunction Induced by Cardiopulmonary Bypass: A Steroids in Cardiac Surgery (SIRS) Trial Substudy.

PubMed

Paparella, Domenico; Parolari, Alessandro; Rotunno, Crescenzia; Vincent, Jessica; Myasoedova, Veronica; Guida, Pietro; De Palo, Micaela; Margari, Vito; Devereaux, Philip J; Lamy, Andre; Alamanni, Francesco; Yusuf, Salim; Whitlock, Richard

2017-01-01

Cardiopulmonary bypass (CPB) surgery, despite heparin administration, elicits activation of coagulation system resulting in coagulopathy. Anti-inflammatory effects of steroid treatment have been demonstrated, but its effects on coagulation system are unknown. The primary objective of this study is to assess the effects of methylprednisolone on coagulation function by evaluating thrombin generation, fibrinolysis, and platelet activation in high-risk patients undergoing cardiac surgery with CPB. The Steroids In caRdiac Surgery study is a double-blind, randomized, controlled trial performed on 7507 patients worldwide who were randomized to receive either intravenous methylprednisolone, 250 mg at anesthetic induction and 250 mg at initiation of CPB (n = 3755), or placebo (n = 3752). A substudy was conducted in 2 sites to collect blood samples perioperatively to measure prothrombin fragment 1.2 (PF1+2, thrombin generation), plasmin-antiplasmin complex (PAP, fibrinolysis), platelet factor 4 (PF4 platelet activation), and fibrinogen. Eighty-one patients were enrolled in the substudy (37 placebo vs 44 in treatment group). No difference in clinical outcome was detected, including postoperative bleeding and need for blood products transfusion. All patients showed changes of all plasma biomarkers with greater values than baseline in both groups. This reaction was attenuated significantly in the treatment group for PF1.2 (P = 0.040) and PAP (P = 0.042) values at the first intraoperative measurement. No difference between groups was detected for PF4. Methylprednisolone treatment attenuates activation of coagulation system in high-risk patients undergoing CPB surgery. Reduction of thrombin generation and fibrinolysis activation may lead to reduced blood loss after surgery. Copyright © 2017 Elsevier Inc. All rights reserved.

Human plasma-derived immunoglobulin G fractionated by an aqueous two-phase system, caprylic acid precipitation, and membrane chromatography has a high purity level and is free of detectable in vitro thrombogenic activity.

PubMed

Vargas, M; Segura, Á; Wu, Y-W; Herrera, M; Chou, M-L; Villalta, M; León, G; Burnouf, T

2015-02-01

Instituto Clodomiro Picado has developed an immunoglobulin G (IgG) plasma fractionation process combining a polyethylene glycol/phosphate aqueous two-phase system (ATPS), caprylic acid precipitation and anion-exchange membrane chromatography. We evaluated the purity and in vitro thrombogenicity of such IgG, in line with current international requirements. Contributions of the different production steps to reduce thrombogenicity were assessed at 0·2 l-scale, and then the methodology was scaled-up to a 10 l-scale and final products (n = 3) were analysed. Purity, immunoglobulin composition, and subclass distribution were determined by electrophoretic and immunochemical methods. The in vitro thrombogenic potential was determined by a thrombin generation assay (TGA) using a Technothrombin fluorogenic substrate. Prekallikrein activator (PKA), plasmin, factor Xa, thrombin and thrombin-like activities were assessed using S-2302, S-2251, S-2222, S-2238 and S-2288 chromogenic substrates, respectively, and FXI by an ELISA. The thrombogenicity markers were reduced mostly during the ATPS step and were found to segregate mostly into the discarded liquid upper phase. The caprylic acid precipitation eliminated the residual procoagulant activity. The IgG preparations made from the 10 l-batches contained 100% gamma proteins, low residual IgA and undetectable IgM. The IgG subclass distribution was not substantially affected by the process. TGA and amidolytic activities revealed an undetectable in vitro thrombogenic risk and the absence of proteolytic enzymes in the final product. Fractionating human plasma by an ATPS combined with caprylic acid and membrane chromatography resulted in an IgG preparation of high purity and free of a detectable in vitro thrombogenic risk. © 2014 International Society of Blood Transfusion.

Methodical aspects of blood coagulation measurements in birds applying commercial reagents--a pilot study.

PubMed

Guddorf, Vanessa; Kummerfeld, Norbert; Mischke, Reinhard

2014-01-01

The aim of this study was to examine the suitability of commercially available reagents for measurements of coagulation parameters in citrated plasma from birds. Therefore, plasma samples of 17 healthy donor birds of different species were used to determine prothrombin time (PT), activated partial thromboplastin time (aPTT) and thrombin time (TT) applying various commercial reagents which are routinely used in coagulation diagnostics in humans and mammals. A PT reagent based on human placental thromboplastin yielded not only shorter clotting times than a reagent containing recombinant human tissue factor (median 49 vs. 84 s), but also showed a minor range of distribution of values (43-55 s vs. 30-147 s, minimum-maximum, n = 5 turkeys). An aPTT reagent containing kaolin and phospholipids of animal origin delivered the shortest clotting times and the lowest range of variation in comparison to three other reagents of different composition. However, even when this reagent was used, aPTTs were partially extremely long (> 200 s). Thrombin time was 38 s (28-57 s, n = 5 chicken) when measured with bovine thrombin at a final concentration of 2 IU thrombin/ ml. Coefficients of variation for within-run precision analysis (20 repetitions) of PT was 8.0% and 4.7% for aPTT measurements using selected reagents of mammalian origin. In conclusion, of the commercially available reagents tested, a PT reagent based on human placental thromboplastin and an aPTT reagent including rabbit brain phospholipid and kaolin, show some promise for potential use in birds.

Dabigatran Etexilate Reduces Thrombin-Induced Inflammation and Thrombus Formation in Experimental Ischemic Stroke.

PubMed

Dittmeier, Melanie; Wassmuth, Kathrin; Schuhmann, Michael K; Kraft, Peter; Kleinschnitz, Christoph; Fluri, Felix

2016-01-01

Dabigatran etexilate (DE), a direct-acting, oral inhibitor of thrombin, significantly reduces the risk of stroke compared with traditional anticoagulants, without increasing the risk of major bleeding. However, studies on the fate of cerebral tissue after ischemic stroke in patients receiving DE are sparse and the role of dabigatran-mediated reduction of thrombin in this context has not yet been investigated. Here, we investigated whether pretreatment with DE reduces thrombin-mediated pro-inflammatory mechanisms and leakage of the blood-brain barrier (BBB) following ischemic stroke in rats. Male Wistar rats received DE (15 mg/kg) or a vehicle solution 1 hour before transient middle cerebral artery occlusion (tMCAO) for 90 minutes. Infarct volume, neurologic outcome and intracranial hemorrhage (ICH) were determined after tMCAO. Thrombin generation was indirectly assessed by measuring thrombin/antithrombin III complex. Microvascular patency was evaluated histologically. Cytokine expression and immunoreactivity of cluster of differentiation (CD) 68 were examined to characterize inflammatory processes after pretreatment with DE. BBB integrity was examined by quantifying brain edema. Rats given DE revealed a significant reduction in infarct size without an increase in ICH and significant recovery of neurologic deficits compared to controls. Administration of DE decreased thrombin generation and thrombus formation, dampened the CD68-immunoreactivity and attenuated pro-inflammatory cytokine expression in the cerebral parenchyma ipsilateral to the ischemic lesion. BBB permeability was unaltered following treatment with DE. In summary, prophylactic anticoagulation with DE improves stroke outcome by reducing thrombin-induced inflammation and thrombus formation without increasing the rate of ICH.

Ethanol inhibits thrombin-induced secretion by human platelets at a site distinct from phospholipase C or protein kinase C.

PubMed Central

Benistant, C; Rubin, R

1990-01-01

Ethanol is known to inhibit the activation of platelets in response to several physiological agonists, but the mechanism of this action is unclear. The addition of physiologically relevant concentrations of ethanol (25-150 mM) to suspensions of washed human platelets resulted in the inhibition of thrombin-induced secretion of 5-hydroxy[14C]tryptamine. Indomethacin was included in the incubation buffer to prevent feedback amplification by arachidonic acid metabolites. Ethanol had no effect on the activation of phospholipase C by thrombin, as determined by the formation of inositol phosphates and the mobilization of intracellular Ca2+. Moreover, ethanol did not interfere with the thrombin-induced formation of diacylglycerol or phosphatidic acid. Stimulation of platelets with phorbol ester (5-50 nM) resulted in 5-hydroxy[14C]tryptamine release comparable with those with threshold doses of thrombin. However, ethanol did not inhibit phorbol-ester-induced secretion. Ethanol also did not interfere with thrombin- or phorbol-ester-induced phosphorylation of myosin light chain (20 kDa) or a 47 kDa protein, a known substrate for protein kinase C. By electron microscopy, ethanol had no effect on thrombin-induced shape change and pseudopod formation, but prevented granule centralization and fusion. The results indicate that ethanol does not inhibit platelet secretion by interfering with the activation of phosphoinositide-specific phospholipase C or protein kinase C by thrombin. Rather, the data demonstrate an inhibition of a Ca2(+)-mediated event such as granule centralization. Images p495-a PMID:2117442

Thrombin increases hyposmotic taurine efflux and accelerates ICI-swell and RVD in 3T3 fibroblasts by a src-dependent EGFR transactivation.

PubMed

Vázquez-Juárez, E; Ramos-Mandujano, G; Lezama, R A; Cruz-Rangel, S; Islas, L D; Pasantes-Morales, H

2008-02-01

The present study in Swiss3T3 fibroblasts examines the effect of thrombin on hyposmolarity-induced osmolyte fluxes and RVD, and the contribution of the src/EGFR pathway. Thrombin (5 U/ml) added to a 30% hyposmotic medium markedly increased hyposmotic 3H-taurine efflux (285%), accelerated the volume-sensitive Cl- current (ICI-swell) and increased RVD rate. These effects were reduced (50-65%) by preventing the thrombin-induced intracellular Ca2+ [Ca2+]i rise with EGTA-AM, or with the phospholipase C (PLC) blocker U73122. Ca2+calmodulin (CaM) and calmodulin kinase II (CaMKII) also participate in this Ca2+-dependent pathway. Thrombin plus hyposmolarity increased src and EGFR phosphorylation, whose blockade by PP2 and AG1478, decreased by 30-50%, respectively, the thrombin effects on hyposmotic taurine efflux, ICI-swell and RVD. Ca2+- and src/EGFR-mediated pathways operate independently as shown by (1) the persistence of src and EGFR activation when [Ca2+]i rise is prevented and (2) the additive effect on taurine efflux, ICI-swell or RVD by simultaneous inhibition of the two pathways, which essentially suppressed these events. PLC-Ca2+- and src/EGFR-signaling pathways operate in the hyposmotic condition and because thrombin per se failed to increase taurine efflux and ICI-swell under isosmotic condition it seems that it is merely amplifying these previously activated mechanisms. The study shows that thrombin potentiates hyposmolarity-induced osmolyte fluxes and RVD by increasing src/EGFR-dependent signaling, in addition to the Ca2+-dependent pathway.

Thrombin Cleavage of Plasmodium falciparum Erythrocyte Membrane Protein 1 Inhibits Cytoadherence

PubMed Central

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.

2016-01-01

ABSTRACT 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. PMID:27624125

Intracellular Ascorbate Prevents Endothelial Barrier Permeabilization by Thrombin*

PubMed Central

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

2015-01-01

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

Thrombin Receptors and Protease-Activated Receptor-2 in Human Placentation

PubMed Central

O’Brien, Peter J.; Koi, Hideki; Parry, Samuel; Brass, Lawrence F.; Strauss, Jerome F.; Wang, Li-Peng; Tomaszewski, John E.; Christenson, Lane K.

2003-01-01

Proteolysis of the thrombin receptor, protease activated receptor-1 (PAR1), may enhance normal and pathological cellular invasion, and indirect evidence suggests that activation of PAR1 expressed by invasive extravillous trophoblasts (EVTs) influences human placentation. Here we describe PAR1, PAR2, and PAR3 protein distribution in the developing human placenta and implicate PAR1 and PAR2 activation in functions central to EVT invasion. PAR1, PAR2, and PAR3 are expressed in cultured 8- to 13-week-old EVTs, and in situ in 18- to 20-week-old placental syncytiotrophoblasts and invasive trophoblasts. Thrombin, but not the PAR2 agonist peptide SLIGKV, inhibited proliferation in cultured EVTs, although both agonists stimulated phosphoinositide hydrolysis and EVT invasion through Matrigel barriers. Thrombin-induced phosphoinositide hydrolysis was completely inhibited and the thrombin effect on proliferation was prevented when PAR1 cleavage was first blocked with specific monoclonal antibodies, indicating that PAR1 is the predominant thrombin receptor on EVTs. Together these results support a role for PAR1, and potentially PAR2 and PAR3 in the invasive phase of human placentation. PMID:14507634

Inhibition of thrombin action ameliorates insulin resistance in type 2 diabetic db/db mice.

PubMed

Mihara, Masatomo; Aihara, Ken-ichi; Ikeda, Yasumasa; Yoshida, Sumiko; Kinouchi, Mizuho; Kurahashi, Kiyoe; Fujinaka, Yuichi; Akaike, Masashi; Matsumoto, Toshio

2010-02-01

The binding of thrombin to its receptor stimulates inflammatory cytokines including IL-6 and monocyte chemoattractant protein-1 (MCP-1); both are associated with the development of insulin resistance. Because increased adiposity enhanced the expression of coagulation factor VII that stimulates the coagulation pathway in adipose tissue, we tested whether the inhibition of thrombin action ameliorates insulin resistance in obese diabetic (Lpr(-/-):db/db) mice. The 4-wk administration of argatroban, a selective thrombin inhibitor, reduced fasting plasma glucose and ameliorated insulin resistance in these mice. It also reduced adipocyte size and macrophage infiltration into adipose tissue. The aberrant gene expression of MCP-1, IL-6, adiponectin, and factor VII and suppressed insulin receptor substrate-1-Akt signaling in adipose tissue of db/db mice were reversed by argatroban treatment. These results demonstrate that increased adiposity enhances the production of thrombin in adipose tissue by stimulating factor VII expression and suggest that increased thrombin activity in adipose tissue plays an important role in the development of insulin resistance via enhancing MCP-1 production, leading to macrophage infiltration and insulin receptor substrate-1-Akt pathway inactivation.

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

PubMed Central

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

2010-01-01

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

Reduced thrombin formation and excessive fibrinolysis are associated with bleeding complications in patients with dengue fever: a case–control study comparing dengue fever patients with and without bleeding manifestations

PubMed Central

2013-01-01

Background Dengue cases have been classified according to disease severity into dengue fever (DF) and dengue hemorrhagic fever (DHF). Although DF is considered a non-severe manifestation of dengue, it has been recently demonstrated that DF represents a heterogeneous group of patients with varied clinical complications and grades of severity. Particularly, bleeding complications, commonly associated to DHF, can be detected in half of the patients with DF. Although a frequent complication, the causes of bleedings in DF have not been fully addressed. Thus, the aim of this study was to perform a comprehensive evaluation of possible pathophysiological mechanisms that could contribute to the bleeding tendency observed in patients with DF. Methods This is a case–control study that enrolled adults with DF without bleeding and adults with DF and bleeding complications during the defervescence period. Healthy controls were also included. Peripheral blood counts, inflammatory, fibrinolysis and endothelial cell activation markers, and thrombin generation were evaluated in patients and controls. Results We included 33 adults with DF without complications, 26 adults with DF and bleeding and 67 healthy controls. Bleeding episodes were mild in 15 (57.6%) and moderate in 11 (42.4%) patients, 8 (30.7%) patients had bleedings in multiple sites. Patients with DF and bleedings had lower platelet counts than DF without bleeding (median = 19,500 vs. 203,500/mm3, P < 0,0001). Levels of TNF-α, thrombomodulin and VWF were significantly increased in the two dengue groups than in healthy controls, but similar between patients with and without bleedings. Plasma levels of tPA and D-dimer were significantly increased in patients with bleedings (median tPA levels were 4.5, 5.2, 11.7 ng/ml, P < 0.0001 and median D-dimer levels were 515.5, 1028 and 1927 ng/ml, P < 0.0001). The thrombin generation test showed that patients with bleeding complications had reduced thrombin formation (total thrombin generated were 3753.4 in controls, 3367.5 in non-bleeding and 2274.5nM in bleeding patients, P < 0.002). Conclusions DF can manifest with spontaneous bleedings, which are associated with specific coagulation and fibrinolysis profiles that are not significantly present in DF without this complication. Particularly, thrombocytopenia, excessive fibrinolysis and reduced thrombin formation may contribute to the bleeding manifestations in DF. PMID:23890510

Reduced thrombin formation and excessive fibrinolysis are associated with bleeding complications in patients with dengue fever: a case-control study comparing dengue fever patients with and without bleeding manifestations.

PubMed

Orsi, Fernanda A; Angerami, Rodrigo N; Mazetto, Bruna M; Quaino, Susan K P; Santiago-Bassora, Fernanda; Castro, Vagner; de Paula, Erich V; Annichino-Bizzacchi, Joyce M

2013-07-28

Dengue cases have been classified according to disease severity into dengue fever (DF) and dengue hemorrhagic fever (DHF). Although DF is considered a non-severe manifestation of dengue, it has been recently demonstrated that DF represents a heterogeneous group of patients with varied clinical complications and grades of severity. Particularly, bleeding complications, commonly associated to DHF, can be detected in half of the patients with DF. Although a frequent complication, the causes of bleedings in DF have not been fully addressed. Thus, the aim of this study was to perform a comprehensive evaluation of possible pathophysiological mechanisms that could contribute to the bleeding tendency observed in patients with DF. This is a case-control study that enrolled adults with DF without bleeding and adults with DF and bleeding complications during the defervescence period. Healthy controls were also included. Peripheral blood counts, inflammatory, fibrinolysis and endothelial cell activation markers, and thrombin generation were evaluated in patients and controls. We included 33 adults with DF without complications, 26 adults with DF and bleeding and 67 healthy controls. Bleeding episodes were mild in 15 (57.6%) and moderate in 11 (42.4%) patients, 8 (30.7%) patients had bleedings in multiple sites. Patients with DF and bleedings had lower platelet counts than DF without bleeding (median = 19,500 vs. 203,500/mm3, P < 0,0001). Levels of TNF-α, thrombomodulin and VWF were significantly increased in the two dengue groups than in healthy controls, but similar between patients with and without bleedings. Plasma levels of tPA and D-dimer were significantly increased in patients with bleedings (median tPA levels were 4.5, 5.2, 11.7 ng/ml, P < 0.0001 and median D-dimer levels were 515.5, 1028 and 1927 ng/ml, P < 0.0001). The thrombin generation test showed that patients with bleeding complications had reduced thrombin formation (total thrombin generated were 3753.4 in controls, 3367.5 in non-bleeding and 2274.5nM in bleeding patients, P < 0.002). DF can manifest with spontaneous bleedings, which are associated with specific coagulation and fibrinolysis profiles that are not significantly present in DF without this complication. Particularly, thrombocytopenia, excessive fibrinolysis and reduced thrombin formation may contribute to the bleeding manifestations in DF.

Thrombin/Matrix Metalloproteinase-9-Dependent SK-N-SH Cell Migration is Mediated Through a PLC/PKC/MAPKs/NF-κB Cascade.

PubMed

Yang, Chien-Chung; Lin, Chih-Chung; Chien, Peter Tzu-Yu; Hsiao, Li-Der; Yang, Chuen-Mao

2016-11-01

Thrombin has been known to activate inflammatory genes including matrix metalloproteinases (MMPs). The elevated expression of MMP-9 has been observed in patients with neuroinflammatory diseases and may contribute to the pathology of brain diseases. However, the mechanisms underlying thrombin-induced MMP-9 expression in SK-N-SH cells remain unknown. The effects of thrombin on MMP-9 expression were examined in SK-N-SH cells by gelatin zymography, Western blot, real-time PCR, promoter activity assay, and cell migration assay. The detailed mechanisms were analyzed by using pharmacological inhibitors and small intefering RNA (siRNA) transfection. Here, we demonstrated that thrombin induced the expression of proform MMP-9 and migration of SK-N-SH cells, which were attenuated by pretreatment with the inhibitor of thrombin (PPACK), Gq (GPA2A), PC-PLC (D609), PI-PLC (ET-18-OCH 3 ), nonselective protien kinase C (PKC, GF109203X), PKCα/βII (Gö6983), PKCδ (Rottlerin), p38 mitogen-activated protein kinases (MAPK) (SB202190), JNK1/2 (SP600125), or NF-κB (Bay11-7082 or Helenalin) and transfection with siRNA of Gq, PKCα, PKCβ, PKCδ, p38, JNK1/2, IKKα, IKKβ, or p65. Moreover, thrombin-stimulated PKCα/βII, PKCδ, p38 MAPK, JNK1/2, or p65 phosphorylation was abrogated by their respective inhibitor of PPACK, GPA2A, D609, ET-18-OCH 3 , Gö6983, Rottlerin, SB202190, SP600125, Bay11-7082, or Helenalin. Pretreatment with these inhibitors or transfection with MMP-9 siRNA also blocked thrombin-induced SK-N-SH cell migration. Our results show that thrombin stimulates a Gq/PLC/PKCs/p38 MAPK and JNK1/2 cascade, which in turn triggers NF-κB activation and ultimately induces MMP-9 expression and cell migration in SK-N-SH cells.

Antiplatelet Agents Can Promote Two-Peaked Thrombin Generation in Platelet Rich Plasma: Mechanism and Possible Applications

PubMed Central

Tarandovskiy, Ivan D.; Artemenko, Elena O.; Panteleev, Mikhail A.; Sinauridze, Elena I.; Ataullakhanov, Fazoil I.

2013-01-01

Background Thrombin generation assay is a convenient and widely used method for analysis of the blood coagulation system status. Thrombin generation curve (TGC) is usually bell-shaped with a single peak, but there are exceptions. In particular, TGC in platelet-rich plasma (PRP) can sometimes have two peaks. Objective We sought to understand the mechanism underlying the occurrence of two peaks in the PRP thrombin generation curve. Methods Tissue factor-induced thrombin generation in PRP and platelet-poor plasma (PPP) was monitored using continuous measurement of the hydrolysis rate of the thrombin-specific fluorogenic substrate Z-Gly-Gly-Arg-AMC. Expression of phosphatidylserine (PS) and CD62P on the surface of activated platelets was measured by flow cytometry using corresponding fluorescently labeled markers. Results The addition of the P2Y12 receptor antagonist MeS-AMP (160 µM), 83 nM prostaglandin E1 (PGE1), or 1.6% DMSO to PRP caused the appearance of two peaks in the TGC. The PS exposure after thrombin activation on washed platelets in a suspension supplemented with DMSO, PGE1 or MeS-AMP was delayed, which could indicate mechanism of the second peak formation. Supplementation of PRP with 1.6% DMSO plus 830 nM PGE1 mediated the disappearance of the second peak and decreased the amplitude of the first peak. Increasing the platelet concentration in the PRP promoted the consolidation of the two peaks into one. Conclusions Procoagulant tenase and prothrombinase complexes in PRP assemble on phospholipid surfaces containing PS of two types - plasma lipoproteins and the surface of activated platelets. Thrombin generation in the PRP can be two-peaked. The second peak appears in the presence of platelet antagonists as a result of delayed PS expression on platelets, which leads to delayed assembly of the membrane-dependent procoagulant complexes and a second wave of thrombin generation. PMID:23405196

Computational study of some benzamidine-based inhibitors of thrombin-like snake venom proteinases

NASA Astrophysics Data System (ADS)

Henriques, Elsa S.; Nascimento, Marco A. C.; Ramos, Maria João

Pit viper venoms contain a number of serine proteinases that, despite their observed coagulant thrombin-like action in vitro, exhibit a paradoxical benign defibrinogenating (anticoagulant) action in vivo, with clinical applications in preventing thrombi and improved blood circulation. Considering that several benzamidine-based inhibitors, some highly selective to thrombin, also inhibit the enzymatic activity of such venombins, the modeling of their enzyme-inhibitor interactions could provide valuable information on the topological factors that determine the divergences in activity. The first step, and the object of the present study, was to derive the necessary set of parameters, consistent with the CHARMM force field, and to perform molecular dynamics (MD) simulations on a few selected representatives of the inhibitors in question under physiological conditions. Bonding and van der Waals parameters were derived by analogy to similar ones in the existing force field. Net atomic charges were obtained with a restrained fitting to the molecular electrostatic potential generated at B3LYP/6-31G(d) level. The parameters were refined to reproduce the available experimental geometries and crystal data, and the MD simulations of the free inhibitors in aqueous solution at 298 K provided an insightful description of their available conformational space.

Discovery of potent peptide-mimetic antagonists for the human thrombin receptor, protease-activated receptor-1 (PAR-1).

PubMed

Maryanoff, Bruce E; Zhang, Han-Cheng; Andrade-Gordon, Patricia; Derian, Claudia K

2003-03-01

Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G-protein-coupled receptors, which are enzymatically cleaved to expose a new extracellular N-terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease alpha-thrombin, is expressed in various tissues (e.g. platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. By using a de novo design approach, we have discovered a series of potent heterocycle-based peptide-miimetic antagonists of PAR-1, exemplified by advanced leads RWJ-56110 (22) and RWJ-58259 (32). These compounds are potent, selective PAR-1 antagonists, devoid of PAR-1 agonist and thrombin inhibitory activity: they bind to PAR-1, interfere with calcium mobilization and cellular functions associated with PAR-1, and do not affect PAR-2, PAR-3, or PAR-4. RWJ-56110 was determined to be a direct inhibitor of PAR-1 activation and internalization, without affecting PAR-1 N-terminal cleavage. At high concentrations of alpha-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, but not in human platelets; whereas, at high concentrations of TRAP-6, RWJ-56110 blocked activation responses in both cell types. This result is consistent with the presence of another thrombin receptor on human platelets, namely PAR-4. RWJ-56110 and RWJ-58259 clearly interrupt the binding of a tethered ligand to its receptor. RWJ-58259 demonstrated antirestenotic activity in a rat balloon angioplasty model and antithrombotic activity in a cynomolgus monkey arterial injury model. Such PAR-1 antagonists should not only serve as useful tools to delineate the physiological and pathophysiological roles of PAR-1, but also may have therapeutic potential for treating thrombosis and restenosis in humans.

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

PubMed

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

2006-08-01

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

Outcomes of Ultrasound-Guided Thrombin Injection of Nongroin Arterial Pseudoaneurysms.

PubMed

Valesano, Johnathan C; Schmitz, John J; Kurup, A Nicholas; Schmit, Grant D; Moynagh, Michael R; Atwell, Thomas D; Lewis, Bradley D; Lee, Robert A; Callstrom, Matthew R

2017-08-01

To evaluate success and complication rates of percutaneous ultrasound-guided thrombin injection of nongroin pseudoaneurysms (PSAs). Retrospective review of a prospectively maintained institutional database yielded 39 cases of arterial PSAs occurring at nongroin sites that were treated with percutaneous ultrasound-guided thrombin injection between 2000 and 2016 (average patient age 69.2 y ± 14.0). Of PSAs, 74.4% (29/39) arose in the upper extremities, and 92.3% (36/39) were iatrogenic. The brachial artery was the most commonly affected vessel (51.3% [20/39]), and arterial access was the most common cause (56.4% [22/39]). Average overall PSA size was 2.4 cm (range, 0.5-7.2 cm); average amount of thrombin injected was 320 IU (range, 50-2,000 IU). Technical success was defined as absence of flow within the PSA immediately after thrombin injection. Treatment success was defined as sustained thrombosis on follow-up imaging obtained at 1-3 days after treatment. Technical and treatment success rates of thrombin injections were 100% (39/39) and 84.8% (28/33), respectively. Longer term follow-up imaging (average 71 d; range, 12-201 d) was available for 7 of the treatment successes with 100% (7/7) showing sustained thrombosis. Comparing treatment successes and failures, there was no significant difference in average PSA size (2.3 cm vs 2.0 cm, P = .51) or average amount of thrombin injected (360 IU vs 180 IU, P = .14). There were no complications. Ultrasound-guided thrombin injection is a safe, efficacious treatment option for PSAs arising in nongroin locations. Copyright © 2017 SIR. Published by Elsevier Inc. All rights reserved.

Intracellular Ascorbate Prevents Endothelial Barrier Permeabilization by Thrombin.

PubMed

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

2015-08-28

Intracellular ascorbate (vitamin C) has previously been shown to tighten the endothelial barrier and maintain barrier integrity during acute inflammation in vitro. However, the downstream effectors of ascorbate in the regulation of endothelial permeability remain unclear. In this study, we evaluated ascorbate as a mediator of thrombin-induced barrier permeabilization in human umbilical vein endothelial cells and their immortalized hybridoma line, EA.hy926. We found that the vitamin fully prevented increased permeability to the polysaccharide inulin by thrombin in a dose-dependent manner, and it took effect both before and after subjection to thrombin. Thrombin exposure consumed intracellular ascorbate but not the endogenous antioxidant GSH. Likewise, the antioxidants dithiothreitol and tempol did not reverse permeabilization. We identified a novel role for ascorbate in preserving cAMP during thrombin stimulation, resulting in two downstream effects. First, ascorbate maintained the cortical actin cytoskeleton in a Rap1- and Rac1-dependent manner, thus preserving stable adherens junctions between adjacent cells. Second, ascorbate prevented actin polymerization and formation of stress fibers by reducing the activation of RhoA and phosphorylation of myosin light chain. Although ascorbate and thrombin both required calcium for their respective effects, ascorbate did not prevent thrombin permeabilization by obstructing calcium influx. However, preservation of cAMP by ascorbate was found to depend on both the production of nitric oxide by endothelial nitric-oxide synthase, which ascorbate is known to activate, and the subsequent generation cGMP by guanylate cyclase. Together, these data implicate ascorbate in the prevention of inflammatory endothelial barrier permeabilization and explain the underlying signaling mechanism. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Design and characterization of hirulogs: A novel class of bivalent peptide inhibitors of thrombin

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

Maraganore, J.M.; Bourdon, P.; Jablonski, J.

1990-07-31

A novel class of synthetic peptides has been designed that inhibit the thrombin catalytic site and exhibit specificity for the anion-binding exosite (ABE) of {alpha}-thrombin. These peptides, called hirulogs, consist of (i) an active-site specificity sequence with a restricted Arg-Pro scissile bond, (ii) a polymeric linker of glycyl residues from 6 to 18 {angstrom} in length, and (iii) an ABE recognition sequence such as that in the hirudin C-terminus. Hirulog-1 ((D-Phe)-Pro-Arg-Pro-(Gly){sub 4}-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Tyr-Leu) inhibits the thrombin-catalyzed hydrolysis of a tripeptide p-nitroanilide substrate with K{sub i} = 2.3 nM. In contrast, the synthetic C-terminal hirudin peptide S-Hir{sub 53-64}, which binds to themore » thrombin ABE, blocked the fibrinogen clotting activity of the enzyme with K{sub i} = 144 nM but failed to inhibit the hydrolysis of p-nitroanilide substrates at concentrations as high as 1 mM. Hirulog-1, but not S-Hir{sub 53-64}, was found to inhibit the incorporation of ({sup 14}C)diisopropyl fluorophosphate in thrombin. Hirulog-1 appears specific for thrombin as it lacks inhibitory activities toward human factor Xa, human plasmin, and bovine trypsin at inhibitor:enzyme concentrations 3 orders of magnitude higher than those required to inhibit thrombin. The optimal inhibitory activity of hirulog-1 depends upon all three components of its structure. Comparison of anticoagulant activities of hirulog-1, hirudin, and S-Hir{sub 53-64} showed that the synthetic hirulog-1 is 2-fold more potent than hirudin and 100-fold more active than S-Hir{sub 53-64} in increasing the activated partial thromboplastin time of normal human plasma.« less

Thrombin-inhibiting perfluorocarbon nanoparticles provide a novel strategy for treatment and magnetic resonance imaging of acute thrombosis

PubMed Central

Myerson, J.; He, L.; Lanza, G.; Tollefsen, D.; Wickline, S.

2013-01-01

Background As a regulator of the penultimate step in the coagulation cascade, thrombin represents a principal target of direct and specific anticoagulants. Objective A potent thrombin inhibitor complexed with a colloidal nanoparticle was devised as a first-in-class anticoagulant with prolonged and highly localized therapeutic impact conferred by its multivalent thrombin-absorbing particle surface. Methods PPACK (Phe(D)-Pro-Arg-Chloromethylketone) was secured covalently to the surface of perfluorocarbon-core nanoparticle structures. PPACK and PPACK nanoparticle inhibition of thrombin were assessed in vitro via thrombin activity against a chromogenic substrate. In vivo antithrombotic activity of PPACK, heparin, non-functionalized nanoparticles, and PPACK nanoparticles was assessed through IV administration prior to acute photochemical injury of the common carotid artery. Perfluorocarbon particle retention in extracted carotid arteries from injured mice was assessed via 19F magnetic resonance spectroscopy (MRS) and imaging (MRI) at 11.7 T. APTT measurements determined the systemic effects of the PPACK nanoparticles at various times after injection. Results Optical assay verified that PPACK nanoparticles exceeded PPACK’s intrinsic activity against thrombin. Application of the an in vivo acute arterial thrombosis model demonstrated that PPACK nanoparticles outperformed both heparin (p=.001) and uncomplexed PPACK (p=.0006) in inhibiting thrombosis. 19F MRS confirmed that PPACK nanoparticles specifically bound to sites of acute thrombotic injury. APTT normalized within twenty minutes of PPACK nanoparticles injection. Conclusions PPACK nanoparticles present thrombin-inhibiting surfaces at sites of acutely forming thrombi that continue to manifest local clot inhibition even as systemic effects rapidly diminish and thus represent a new platform for localized control of acute thrombosis. PMID:21605330

Platelet-rich plasma stimulated by pulse electric fields: Platelet activation, procoagulant markers, growth factor release and cell proliferation.

PubMed

Frelinger, A L; Torres, A S; Caiafa, A; Morton, C A; Berny-Lang, M A; Gerrits, A J; Carmichael, S L; Neculaes, V B; Michelson, A D

2016-01-01

Therapeutic use of activated platelet-rich plasma (PRP) has been explored for wound healing, hemostasis and antimicrobial wound applications. Pulse electric field (PEF) stimulation may provide more consistent platelet activation and avoid complications associated with the addition of bovine thrombin, the current state of the art ex vivo activator of therapeutic PRP. The aim of this study was to compare the ability of PEF, bovine thrombin and thrombin receptor activating peptide (TRAP) to activate human PRP, release growth factors and induce cell proliferation in vitro. Human PRP was prepared in the Harvest SmartPreP2 System and treated with vehicle, PEF, bovine thrombin, TRAP or Triton X-100. Platelet activation and procoagulant markers and microparticle generation were measured by flow cytometry. Released growth factors were measured by ELISA. The releasates were tested for their ability to stimulate proliferation of human epithelial cells in culture. PEF produced more platelet-derived microparticles, P-selectin-positive particles and procoagulant annexin V-positive particles than bovine thrombin or TRAP. These differences were associated with higher levels of released epidermal growth factor after PEF than after bovine thrombin or TRAP but similar levels of platelet-derived, vascular-endothelial, and basic fibroblast growth factors, and platelet factor 4. Supernatant from PEF-treated platelets significantly increased cell proliferation compared to plasma. In conclusion, PEF treatment of fresh PRP results in generation of microparticles, exposure of prothrombotic platelet surfaces, differential release of growth factors compared to bovine thrombin and TRAP and significant cell proliferation. These results, together with PEF's inherent advantages, suggest that PEF may be a superior alternative to bovine thrombin activation of PRP for therapeutic applications.

Design, synthesis and biological evaluation of non-peptide PAR1 thrombin receptor antagonists based on small bifunctional templates: arginine and phenylalanine side chain groups are keys for receptor activity.

PubMed

Androutsou, Maria-Eleni; Saifeddine, Mahmoud; Hollenberg, Morley D; Matsoukas, John; Agelis, George

2010-04-01

In the present study, we report the synthesis and biological evaluation of a series of new non-peptide PAR(1) mimetic receptor antagonists, based on conformational analysis of the S(42)FLLR(46) tethered ligand (TL) sequence of PAR(1). These compounds incorporate the key pharmacophore groups in the TL sequence, guanidyl, amino and phenyl, which are essential for triggering receptor activity. Compounds 5 and 15 (50-100 microM) inhibited both TFLLR-amide (10 microM) and thrombin-mediated (0.5 and 1 U/ml; 5 and 10 microM) calcium signaling in a cultured human HEK cell assay.

Antithrombin activity of an algal polysaccharide.

PubMed

Trento, F; Cattaneo, F; Pescador, R; Porta, R; Ferro, L

2001-06-01

In an effort to reduce the risks of a possible iatrogenic transmission of bovine spongiform encephalitis (BSE) through the use of bovine-derived medicinal products, we patented in the USA in 1999 a polysaccharide from brown algae, endowed with interesting pharmacological activities: (a) concentration-dependent inhibition of thromboplastin or cephalin-kaolin-induced thrombin generation from platelets, (b) concentration-dependent inhibition of thrombin-induced platelet aggregation, (c) thrombin has hypotensive effect, which was blunted and zeroed by our fucansulfate in a dose-dependent way, (d) when aortae are stimulated with thrombin, they become stickier for polymorphonucleated leukocytes (PMNs); our fucansulfate decreased concentration-dependently, PMNs sticking to autologous rabbit aortae, (e) dose-dependent inhibition of thrombin-induced thrombosis. All the above data suggest that our fucansulfate could be a heparin substitute endowed with antithrombotic and anti-inflammatory activities, devoid or the problems caused to heparin by its animal origin, i.e., possible prion protein contamination.

A Coffee Ring Aptasensor for Rapid Protein Detection

PubMed Central

Wen, Jessica T.; Ho, Chih-Ming; Lillehoj, Peter B.

2013-01-01

We introduce a new biosensing platform for rapid protein detection that combines one of the simplest methods for biomolecular concentration, coffee ring formation, with a sensitive aptamer-based optical detection scheme. In this approach, aptamer beacons are utilized for signal transduction where a fluorescence signal is emitted in the presence of the target molecule. Signal amplification is achieved by concentrating aptamer-target complexes within liquid droplets, resulting in the formation of coffee ring “spots”. Surfaces with various chemical coatings were utilized to investigate the correlation between surface hydrophobicity, concentration efficiency and signal amplification. Based on our results, we found that the increase in coffee ring diameter with larger droplet volumes is independent of surface hydrophobicity. Furthermore, we show that highly hydrophobic surfaces produce enhanced particle concentration, via coffee ring formation, resulting in signal intensities 6-fold greater than those on hydrophilic surfaces. To validate this biosensing platform for the detection of clinical samples, we detected α-thrombin in human serum and 4x diluted whole blood. Based on our results, coffee ring spots produced detection signals 40x larger than samples in liquid droplets. Additionally, this biosensor exhibits a lower limit of detection of 2 ng/mL (54 pM) in serum, and 4 ng/mL (105 pM) in blood. Based on its simplicity and high performance, this platform demonstrates immense potential as an inexpensive diagnostic tool for the detection of disease biomarkers, particularly for use in developing countries that lack the resources and facilities required for conventional biodetection practices. PMID:23540796

Development of an Efficient G-Quadruplex-Stabilised Thrombin-Binding Aptamer Containing a Three-Carbon Spacer Molecule.

PubMed

Aaldering, Lukas J; Poongavanam, Vasanthanathan; Langkjaer, Niels; Murugan, N Arul; Jørgensen, Per Trolle; Wengel, Jesper; Veedu, Rakesh N

2017-04-18

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-C 3 ), unlocked nucleic acid (UNA) and 3'-amino-modified UNA (amino-UNA) on the structural dynamics and stability of TBA. All three modifications were incorporated at three different loop positions (T3, T7, T12) of the TBA G-quadruplex structure to result in a series of TBA variants and their stability was studied by thermal denaturation; folding was studied by circular dichroism spectroscopy and thrombin clotting time. The results showed that spacer-C 3 introduction at the T7 loop position (TBA-SP7) significantly improved stability and thrombin clotting time while maintaining a similar binding affinity as TBA to thrombin. Detailed molecular modelling experiments provided novel insights into the experimental observations, further supporting the efficacy of TBA-SP7. The results of this study could provide valuable information for future designs of TBA analogues with superior thrombin inhibition properties. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Thrombin generation during cardiopulmonary bypass: the possible role of retransfusion of blood aspirated from the surgical field

PubMed Central

Weerwind, Patrick W; Lindhout, Theo; Caberg, Nicole EH; de Jong, Dick S

2003-01-01

Background In spite of using heparin-coated extracorporeal circuits, cardiopulmonary bypass (CPB) is still associated with an extensive thrombin generation, which is only partially suppressed by the use of high dosages of heparin. Recent studies have focused on the origins of this thrombotic stimulus and the possible role of retransfused suctioned blood from the thoracic cavities on the activation of the extrinsic coagulation pathway. The present study was designed to find during CPB an association between retransfusion of suctioned blood from the pericardium and pleural space, containing activated factor VIIa and systemic thrombin generation. Methods Blood samples taken from 12 consenting patients who had elective cardiac surgery were assayed for plasma factor VIIa, prothrombin fragment 1+2 (F1+2), and thrombin-antithrombin (TAT) concentrations. Blood aspirated from the pericardium and pleural space was collected separately, assayed for F1+2, TAT, and factor VIIa and retransfused to the patient after the aorta occlusion. Results After systemic heparinization and during CPB thrombin generation was minimal, as indicated by the lower than base line plasma levels of F1+2, and TAT after correction for hemodilution. In contrast, blood aspirated from the thoracic cavities had significantly higher levels of factor VIIa, F1+2, and TAT compared to the simultaneous samples from the blood circulation (P < 0.05). Furthermore, after retransfusion of the suctioned blood (range, 200–1600 mL) circulating levels of F1+2, and TAT rose significantly from 1.6 to 2.9 nmol/L (P = 0.002) and from 5.1 to 37.5 μg/L (P = 0.01), respectively. The increase in both F1+2, and TAT levels correlated significantly with the amount of retransfused suctioned blood (r = 0.68, P = 0.021 and r = 0.90, P = 0.001, respectively). However, the circulating factor VIIa levels did not correlate with TAT and F1+2 levels. Conclusions These data suggest that blood aspirated from the thoracic cavities during CPB is highly thrombogenic. Retransfusion of this blood may, therefore, promote further systemic thrombin generation during CPB. PMID:12904260

Protease-Resistant Peptide Ligands from a Knottin Scaffold Library

PubMed Central

Getz, Jennifer A.; Rice, Jeffrey J.; Daugherty, Patrick S.

2011-01-01

Peptides within the knottin family have been shown to possess inherent stability, making them attractive scaffolds for the development of therapeutic and diagnostic agents. Given its remarkable stability to proteases, the cyclic peptide kalata B1 was employed as a scaffold to create a large knottin library displayed on the surface of E. coli. A library exceeding 109 variants was constructed by randomizing seven amino acids within a loop of the kalata B1 scaffold and screened using fluorescence-activated cell sorting to identify peptide ligands specific for the active site of human thrombin. Refolded thrombin binders exhibited high nanomolar affinities in solution, slow dissociation rates, and were able to inhibit thrombin’s enzymatic activity. Importantly, 80% of a knottin-based thrombin inhibitor remained intact after a two hour incubation both with trypsin and with chymotrypsin, demonstrating that modifying the kalata B1 sequence did not compromise its stability properties. In addition, the knottin variant mediated 20-fold enhanced affinity for thrombin, when compared to the same seven residue binding epitope constrained by a single disulfide bond. Our results indicate that peptide libraries derived from the kalata B1 scaffold can yield high affinity protein ligands that retain the remarkable protease resistance associated with the parent scaffold. More generally, this strategy may prove useful in the development of stable peptide ligands suitable for in vivo applications. PMID:21615106

Blood clot detection using magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Khurshid, Hafsa; Friedman, Bruce; Berwin, Brent; Shi, Yipeng; Ness, Dylan B.; Weaver, John B.

2017-05-01

Deep vein thrombosis, the development of blood clots in the peripheral veins, is a very serious, life threatening condition that is prevalent in the elderly. To deliver proper treatment that enhances the survival rate, it is very important to detect thrombi early and at the point of care. We explored the ability of magnetic particle spectroscopy (MSB) to detect thrombus via specific binding of aptamer functionalized magnetic nanoparticles with the blood clot. MSB uses the harmonics produced by nanoparticles in an alternating magnetic field to measure the rotational freedom and, therefore, the bound state of the nanoparticles. The nanoparticles' relaxation time for Brownian rotation increases when bound [A.M. Rauwerdink and J. B. Weaver, Appl. Phys. Lett. 96, 1 (2010)]. The relaxation time can therefore be used to characterize the nanoparticle binding to thrombin in the blood clot. For longer relaxation times, the approach to saturation is more gradual reducing the higher harmonics and the harmonic ratio. The harmonic ratios of nanoparticles conjugated with anti-thrombin aptamers (ATP) decrease significantly over time with blood clot present in the sample medium, compared with nanoparticles without ATP. Moreover, the blood clot removed from the sample medium produced a significant MSB signal, indicating the nanoparticles are immobilized on the clot. Our results show that MSB could be a very useful non-invasive, quick tool to detect blood clots at the point of care so proper treatment can be used to reduce the risks inherent in deep vein thrombosis.

Normal pregnancy is associated with an increase in thrombin generation from the very early stages of the first trimester.

PubMed

Bagot, C N; Leishman, E; Onyiaodike, C C; Jordan, F; Freeman, D J

2017-09-01

Pregnancy is a hypercoagulable state associated with an increased risk of venous thrombosis, which begins during the first trimester, but the exact time of onset is unknown. Thrombin generation, a laboratory marker of thrombosis risk, increases during normal pregnancy but it is unclear exactly how early this increase occurs. We assessed thrombin generation by Calibrated Automated Thrombography in women undergoing natural cycle in vitro fertilization, who subsequently gave birth at term following a normal pregnancy (n=22). Blood samples were taken just prior to conception and repeated five times during very early pregnancy, up to Day 59 estimated gestation. Mean Endogenous Thrombin Potential (ETP), peak thrombin generation and Velocity Index (VI) increased significantly from pre-pregnancy to Day 43 gestation (p=0.024-0.0004). This change persisted to Day 59 gestation. The mean of the percentage change from baseline, accounting for inter-individual variation, in ETP, peak thrombin and VI increased significantly from pre-pregnancy to Day 32 gestation (p=0.0351-<0.0001) with the mean increase from baseline persisting to Day 59 gestation. Thrombin generation increases significantly during the very early stages of normal pregnancy when compared to the pre-pregnancy state. The increased risk of venous thrombosis therefore likely begins very early in a woman's pregnancy, suggesting that women considered clinically to be at high thrombotic risk should start thromboprophylaxis as early as possible after a positive pregnancy test. Copyright © 2017 Elsevier Ltd. All rights reserved.

Involvement of human decidual cell-expressed tissue factor in uterine hemostasis and abruption.

PubMed

Lockwood, C J; Paidas, M; Murk, W K; Kayisli, U A; Gopinath, A; Huang, S J; Krikun, G; Schatz, F

2009-11-01

Vascular injury increases access and binding of plasma-derived factor VII to perivascular cell membrane-bound tissue factor (TF). The resulting TF/VIIa complex promotes hemostasis by cleaving pro-thrombin to thrombin leading to the fibrin clot. In human pregnancy, decidual cell-expressed TF prevents decidual hemorrhage (abruption). During placentation, trophoblasts remodel decidual spiral arteries into high conductance vessels. Shallow trophoblast invasion impedes decidual vascular conversion, producing an inadequate uteroplacental blood flow that elicits abruption-related placental ischemia. Thrombin induces several biological effects via cell surface protease activated receptors. In first trimester human DCs thrombin increases synthesis of sFlt-1, which elicits placental ischemia by impeding angiogenesis-related decidual vascular remodeling. During pregnacy, the fibrillar collagen-rich amnion and choriodecidua extracellular matrix (ECM) provides greater than additive tensile strength and structural integrity. Thrombin acts as an autocrine/paracrine mediator that degrades these ECMs by augmenting decidual cell expression of: 1) matrix metalloproteinases and 2) interleukin-8, a key mediator of abruption-associated decidual infiltration of neutrophils, which express several ECM degrading proteases. Among the cell types at the maternal fetal interface at term, TF expression is highest in decidual cells indicating that this TF meets the hemostatic demands of labor and delivery. TF expression in cultured term decidual cells is enhanced by progestin and thrombin suggesting that the maintenance of elevated circulating progesterone provides hemostatic protection and that abruption-generated thrombin acts in an autocrine/paracrine fashion on decidual cells to promote hemostasis via enhanced TF expression.

Evaluation of Optical Coherence Tomography for the Measurement of the Effects of Activators and Anticoagulants on the Blood Coagulation In Vitro

PubMed Central

Geng, Jinhai; Liu, Gangjun; Chen, Zhongping

2013-01-01

Optical properties of human blood during coagulation were studied using optical coherence tomography (OCT) and the parameter of clotting time derived from the 1/e light penetration depth (d1/e) versus time was developed in our previous work. In this study, in order to know if a new OCT test can characterize the blood-coagulation process under different treatments in vitro, the effects of two different activators (calcium ions and thrombin) and anticoagulants, i.e., acetylsalicylic acid (ASA, a well-known drug aspirin) and melagatran (a direct thrombin inhibitor), at various concentrations are evaluated. A swept-source OCT system with a 1300 nm center wavelength is used for detecting the blood-coagulation process in vitro under a static condition. A dynamic study of d1/e reveals a typical behavior due to coagulation induced by both calcium ions and thrombin, and the clotting time is concentration-dependent. Dose-dependent ASA and melagatran prolong the clotting times. ASA and melagatran have different effects on blood coagulation. As expected, melagatran is much more effective than ASA in anticoagulation by the OCT measurements. The OCT assay appears to be a simple method for the measurement of blood coagulation to assess the effects of activators and anticoagulants, which can be used for activator and anticoagulant screening. PMID:23392340

Evaluation of optical coherence tomography for the measurement of the effects of activators and anticoagulants on the blood coagulation in vitro.

PubMed

Xu, Xiangqun; Geng, Jinhai; Liu, Gangjun; Chen, Zhongping

2013-08-01

Optical properties of human blood during coagulation were studied using optical coherence tomography (OCT) and the parameter of clotting time derived from the 1/e light penetration depth (d(1/e)) versus time was developed in our previous work. In this study, in order to know if a new OCT test can characterize the blood-coagulation process under different treatments in vitro, the effects of two different activators (calcium ions and thrombin) and anticoagulants, i.e., acetylsalicylic acid (ASA, a well-known drug aspirin) and melagatran (a direct thrombin inhibitor), at various concentrations are evaluated. A swept-source OCT system with a 1300 nm center wavelength is used for detecting the blood-coagulation process in vitro under a static condition. A dynamic study of d1/e reveals a typical behavior due to coagulation induced by both calcium ions and thrombin, and the clotting time is concentration-dependent. Dose-dependent ASA and melagatran prolong the clotting times. ASA and melagatran have different effects on blood coagulation. As expected, melagatran is much more effective than ASA in anticoagulation by the OCT measurements. The OCT assay appears to be a simple method for the measurement of blood coagulation to assess the effects of activators and anticoagulants, which can be used for activator and anticoagulant screening.

Highly Tunable Aptasensing Microarrays with Graphene Oxide Multilayers

NASA Astrophysics Data System (ADS)

Jung, Yun Kyung; Lee, Taemin; Shin, Eeseul; Kim, Byeong-Su

2013-11-01

A highly tunable layer-by-layer (LbL)-assembled graphene oxide (GO) array has been devised for high-throughput multiplex protein sensing. In this array, the fluorescence of different target-bound aptamers labeled with dye is efficiently quenched by GO through fluorescence resonance energy transfer (FRET), and simultaneous multiplex target detection is performed by recovering the quenched fluorescence caused by specific binding between an aptamer and a protein. Thin GO films consisting of 10 bilayers displayed a high quenching ability, yielding over 85% fluorescence quenching with the addition of a 2 μM dye-labeled aptamer. The limit for human thrombin detection in the 6- and 10-bilayered GO array is estimated to be 0.1 and 0.001 nM, respectively, indicating highly tunable nature of LbL assembled GO multilayers in controlling the sensitivity of graphene-based FRET aptasensor. Furthermore, the GO chip could be reused up to four times simply by cleaning it with distilled water.

Efficacy and safety of closing postcatheterisation pseudoaneurysms with ultrasound-guided thrombin injections using two approaches: bolus versus slow injection. A prospective randomised trial.

PubMed

Lewandowski, Paweł; Maciejewski, Paweł; Wąsek, Wojciech; Pasierski, Tomasz; Budaj, Andrzej

2011-01-01

Thrombin injection is a widely accepted treatment of an iatrogenic arterial pseudoaneurysm. However, the optimal mode of injection and type of pseudoaneurysm amenable to this therapy have yet been established. To compare efficacy and safety of two approaches to ultrasound-guided thrombin injections into a femoral artery pseudoaneurysm with or without long neck that developed as an iatrogenic complication of cardiac catheterisation. Patients were randomised to thrombin administration in a bolus or slow injection. The length and width of aneurysm neck and blood flow velocity in the neck were measured with color Doppler ultrasonography before the closure procedure. Thrombin dose, time to thrombotic occlusion, blood oxygen saturation in a toe of the extremity with the pseudoaneurysm (a marker of silent microembolisation), and clinical signs of distal embolisation were recorded. Between 2006 and 2009, 73 consecutive patients (33 males; mean age 67.8 ± 11.9 years) with femoral pseudoaneurysms complicating cardiac catheterisation were randomised into two groups that were treated with thrombin bolus (n = 40) or slow injection (n = 33). The efficacy of aneurysm closure with either method was similarly high (100% vs 96.8%, NS, respectively) and did not depend on the length and width of the aneurysm neck. Independent risk factors for distal embolisation were: thrombin dose (OR 4.2; 95% CI 0.92-19.3), the length of aneurysm neck (OR 4.66; 95% CI 1.1-19.9), age above 80 years (OR 10.9; 95% CI 1.0-116.8), and bolus treatment (OR 7.6; 95% CI 1.3-44.9). We observed silent microembolisation phenomenon that was common (occurring in 38% of patients in the bolus group vs 33% of patients in the slow injection group) but in most cases asymptomatic. Femoral pseudoaneurysm closure with a low dose of thrombin is a valid and beneficial treatment. Either method (bolus or slow injection) was similarly efficacious and safe even in the subgroup of patients with neckless aneurysms. We observed and confirmed silent microembolisation phenomenon during thrombin injections.

MicroRNA-146 inhibits thrombin-induced NF-κB activation and subsequent inflammatory responses in human retinal endothelial cells.

PubMed

Cowan, Colleen; Muraleedharan, Chithra K; O'Donnell, James J; Singh, Pawan K; Lum, Hazel; Kumar, Ashok; Xu, Shunbin

2014-07-01

Nuclear factor-κB (NF-κB), a key regulator of immune and inflammatory responses, plays important roles in diabetes-induced microvascular complications including diabetic retinopathy (DR). Thrombin activates NF-κB through protease-activated receptor (PAR)-1, a member of the G-protein-coupled receptor (GPCR) superfamily, and contributes to DR. The current study is to uncover the roles of microRNA (miRNA) in thrombin-induced NF-κB activation and retinal endothelial functions. Target prediction was performed using the TargetScan algorithm. Predicted target was experimentally validated by luciferase reporter assays. Human retinal endothelial cells (HRECs) were transfected with miRNA mimics or antimiRs and treated with thrombin. Expression levels of miR-146 and related protein-coding genes were analyzed by quantitative (q)RT-PCR. Functional changes of HRECs were analyzed by leukocyte adhesion assays. We identified that caspase-recruitment domain (CARD)-containing protein 10 (CARD10), an essential scaffold/adaptor protein of GPCR-mediated NF-κB activation pathway, is a direct target of miR-146. Thrombin treatment resulted in NF-κB-dependent upregulation of miR-146 in HRECs; while transfection of miR-146 mimics resulted in significant downregulation of CARD10 and prevented thrombin-induced NF-κB activation, suggest that a negative feedback regulation of miR-146 on thrombin-induced NF-κB through targeting CARD10. Furthermore, overexpression of miR-146 prevented thrombin-induced increased leukocyte adhesion to HRECs. We uncovered a novel negative feedback regulatory mechanism on thrombin-induced GPCR-mediated NF-κB activation by miR-146. In combination with the negative feedback regulation of miR-146 on the IL-1R/toll-like receptor (TLR)-mediated NF-κB activation in RECs that we reported previously, our results underscore a pivotal, negative regulatory role of miR-146 on multiple NF-κB activation pathways and related inflammatory processes in DR. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

Macrocyclic Prodrugs of a Selective Nonpeptidic Direct Thrombin Inhibitor Display High Permeability, Efficient Bioconversion but Low Bioavailability.

PubMed

Andersson, Vincent; Bergström, Fredrik; Brånalt, Jonas; Grönberg, Gunnar; Gustafsson, David; Karlsson, Staffan; Polla, Magnus; Bergman, Joakim; Kihlberg, Jan

2016-07-28

The only oral direct thrombin inhibitors that have reached the market, ximelagatran and dabigatran etexilat, are double prodrugs with low bioavailability in humans. We have evaluated an alternative strategy: the preparation of a nonpeptidic, polar direct thrombin inhibitor as a single, macrocyclic esterase-cleavable (acyloxy)alkoxy prodrug. Two homologous prodrugs were synthesized and displayed high solubilities and Caco-2 cell permeabilities, suggesting high absorption from the intestine. In addition, they were rapidly and completely converted to the active zwitterionic thrombin inhibitor in human hepatocytes. Unexpectedly, the most promising prodrug displayed only moderately higher oral bioavailability in rat than the polar direct thrombin inhibitor, most likely due to rapid metabolism in the intestine or the intestinal wall. To the best of our knowledge, this is the first in vivo ADME study of macrocyclic (acyloxy)alkoxy prodrugs, and it remains to be established if the modest increase in bioavailability is a general feature of this category of prodrugs or not.

Internal Light Source-Driven Photoelectrochemical 3D-rGO/Cellulose Device Based on Cascade DNA Amplification Strategy Integrating Target Analog Chain and DNA Mimic Enzyme.

PubMed

Lan, Feifei; Liang, Linlin; Zhang, Yan; Li, Li; Ren, Na; Yan, Mei; Ge, Shenguang; Yu, Jinghua

2017-11-01

In this work, a chemiluminescence-driven collapsible greeting card-like photoelectrochemical lab-on-paper device (GPECD) with hollow channel was demonstrated, in which target-triggering cascade DNA amplification strategy was ingeniously introduced. The GPECD had the functions of reagents storage and signal collection, and the change of configuration could control fluidic path, reaction time and alterations in electrical connectivity. In addition, three-dimentional reduced graphene oxide affixed Au flower was in situ grown on paper cellulose fiber for achieving excellent conductivity and biocompatibility. The cascade DNA amplification strategy referred to the cyclic formation of target analog chain and its trigger action to hybridization chain reaction (HCR), leading to the formation of numerous hemin/G-quadruplex DNA mimic enzyme with the presence of hemin. Subjected to the catalysis of hemin/G-quadruplex, the strong chemiluminiscence of luminol-H 2 O 2 system was obtained, which then was used as internal light source to excite photoactive materials realizing the simplification of instrument. In this analyzing process, thrombin served as proof-of-concept, and the concentration of target was converted into the DNA signal output by the specific recognition of aptamer-protein and target analog chain recycling. The target analog chain was produced in quantity with the presence of target, which further triggered abundant HCR and introduced hemin/G-quadruplex into the system. The photocurrent signal was obtained after the nitrogen-doped carbon dots sensitized ZnO was stimulated by chemiluminescence. The proposed GPECD exhibited excellent specificity and sensitivity toward thrombin with a detection limit of 16.7 fM. This judiciously engineered GPECD paved a luciferous way for detecting other protein with trace amounts in bioanalysis and clinical biomedicine.

Thrombin promotes diet-induced obesity through fibrin-driven inflammation.

PubMed

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

2017-08-01

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.

The long term immunological response of swine after two exposures to a salmon thrombin and fibrinogen hemostatic bandage

PubMed Central

Rothwell, Stephen W.; Settle, Timothy; Wallace, Shannon; Dorsey, Jennifer; Simpson, David; Bowman, James R.; Janmey, Paul; Sawyer, Evelyn

2014-01-01

Experimental salmon thrombin/fibrinogen dressings have been shown to provide effective hemostasis in severe hemorrhage situations. The hypothesis for this study was that swine would still remain healthy without coagulopathy six months after exposure to salmon thrombin/fibrinogen dressings. Initial exposure was by insertion of the salmon dressing into the peritoneal cavity. Three months after the initial exposure, the same animals were subjected to two full thickness dermal wounds on the dorsal surface. One wound was bandaged with the salmon thrombin/fibrinogen bandage and the other wound was dressed with a standard bandage. The animals were monitored for an additional three months. Blood was drawn every 14 days over the six months for immunological and coagulation function analysis. All of the animals (8 pigs) remained healthy during the six month period and the dermal wounds healed without incidence. Lymph nodes and spleen showed signs of normal immune response and Western blots showed development of antibodies against salmon fibrinogen, but none of the animals made antibodies that recognized any species of thrombin. Coagulation parameters (fibrinogen concentration, thrombin time, PT and aPTT) and hematological parameters remained normal over the course of the study when compared to initial values of the subject swine. PMID:20705479

Characterization of a potent platelet aggregation inducer from Cerastes cerastes (Egyptian sand viper) venom.

PubMed

Basheer, A R; el-Asmar, M F; Soslau, G

1995-07-03

A potent, proteinaceous inducer of platelet aggregation designated as IVa, has been purified to homogeneity from Cerastes cerastes venom by molecular sieve and ion exchange chromatography. It is composed of 2 subunits with total M(r) of 62,000 as shown by native gel chromatography and chemical cross-linking with disuccinimidyl suberate. It is not clear at the present time whether both subunits are identical gene products, however, both have identical N-terminal sequences for the first 15 amino acids. The protein has a pI above 9.6. IVa (0.1 micrograms/ml) could aggregate platelets up to 80% and was inhibited by p-APMSF, leupeptin, iodoacetamide, protein kinase C inhibitor, phosphatase inhibitor, ATP and PGE1, while it was insensitive to acetylsalicylic acid, ADP scavenger system, protein kinase A inhibitor and hirudin. Protein IVa is a serine proteinase with thrombin-like activity as it hydrolysed thrombin chromogenic substrate CBS 34.47, its aggregatory activity was partially inhibited by monoclonal antibodies against GPIb and the thrombin receptor, as was the thrombin, and its ability to induce intracellular Ca2+ release was blocked by pretreating platelets with thrombin. Unlike thrombin, the IVa protein showed very weak coagulant activity as indicated by plasma recalcification time and fibrinogen clotting time although it could hydrolyse fibrinogen alpha-chains.

Thrombin promotes diet-induced obesity through fibrin-driven inflammation

PubMed Central

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

2017-01-01

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. PMID:28737512

A hypothesis: factor VII governs clot formation, tissue repair and apoptosis.

PubMed

Coleman, Lewis S

2007-01-01

A hypothesis: thrombin is a "Universal Enzyme of Energy Transduction" that employs ATP energy in flowing blood to activate biochemical reactions and cell effects in both hemostasis and tissue repair. All cells possess PAR-1 (thrombin) receptors and are affected by thrombin elevations, and thrombin effects on individual cell types are determined by their unique complement of PAR-1 receptors. Disruption of the vascular endothelium (VE) activates a tissue repair mechanism (TRM) consisting of the VE, tissue factor (TF), and circulating Factors VII, IX and X that governs localized thrombin elevations to activate clot formation and cellular effects that repair tissue damage. The culmination of the repair process occurs with the restoration of the VE followed by declines in thrombin production that causes Apoptosis ("programmed cell death") in wound-healing fibroblasts, which functions as a mechanism to draw wound edges together. The location and magnitude of TRM activity governs the location and magnitude of Factor VIII activity and clot formation, but the large size of Factor VIII prevents it from penetrating the clot formed by its activity, so that its effects are self-limiting. Factors VII, IX and X function primarily as tissue repair enzymes, while Factor VIII and Factor XIII are the only serine protease enzymes in the "Coagulation Cascade" that are exclusively associated with hemostasis.

Partial filling affinity capillary electrophoresis as a useful tool for fragment-based drug discovery: A proof of concept on thrombin.

PubMed

Farcaş, E; Bouckaert, C; Servais, A-C; Hanson, J; Pochet, L; Fillet, M

2017-09-01

With the emergence of more challenging targets, a relatively new approach, fragment-based drug discovery (FBDD), proved its efficacy and gained increasing importance in the pharmaceutical industry. FBDD identifies low molecular-weight (MW) ligands (fragments) that bind to biologically important macromolecules, then a structure-guided fragment growing or merging approach is performed, contributing to the quality of the lead. However, to select the appropriate fragment to be evolved, sensitive analytical screening methods must be used to measure the affinity in the μM or even mM range. In this particular context, we developed a robust and selective partial filling affinity CE (ACE) method for the direct binding screening of a small fragment library in order to identify new thrombin inhibitors. To demonstrate the accuracy of our assay, the complex dissociation constants of three known thrombin inhibitors, namely benzamidine, p-aminobenzamidine and nafamostat were determined and found to be in good concordance with the previously reported values. Finally, the screening of a small library was performed and demonstrated the high discriminatory power of our method towards weak binders compared to classical spectrophotometric activity assay, proving the interest of our method in the context of FBDD. Copyright © 2017 Elsevier B.V. All rights reserved.

Antiplatelet, anticoagulant, and profibrinolytic activities of withaferin A.

PubMed

Ku, Sae-Kwang; Bae, Jong-Sup

2014-03-01

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

Involvement of human decidual cell-expressed tissue factor in uterine hemostasis and abruption

PubMed Central

Lockwood, C.J.; Paidas, M.; Murk, W.K.; Kayisli, U.A.; Gopinath, A.; Krikun, G.; Huang, S.J.; Schatz, F.

2009-01-01

Vascular injury increases access and binding of plasma-derived factor VII to perivascular cell membrane-bound tissue factor (TF). The resulting TF/VIIa complex promotes hemostasis by cleaving pro-thrombin to thrombin leading to the fibrin clot. In human pregnancy, decidual cell-expressed TF prevents decidual hemorrhage (abruption). During placentation, trophoblasts remodel decidual spiral arteries into high conductance vessels. Shallow trophoblast invasion impedes decidual vascular conversion, producing an inadequate uteroplacental blood flow that elicits abruption-related placental ischemia. Thrombin induces several biological effects via cell surface protease activated receptors. In first trimester human DCs thrombin increases synthesis of sFlt-1, which elicits placental ischemia by impeding angiogenesis-related decidual vascular remodeling. During pregnacy, the fibrillar collagen-rich amnion and choriodecidua extracellular matrix (ECM) provides greater than additive tensile strength and structural integrity. Thrombin acts as an autocrine/paracrine mediator that degrades these ECMs by augmenting decidual cell expression of: 1) matrix metalloproteinases and 2) interleukin-8, a key mediator of abruption-associated decidual infiltration of neutrophils, which express several ECM degrading proteases. Our recent observations that: 1) among the cell types at the maternal fetal interface at term TF expression is highest in decidual cells indicates that this TF meets the hemostatic demands of labor and delivery; 2) TF expression in cultured term decidual cells is enhanced by progestin and thrombin suggest that maintenance of elevated circulating progesterone at term provides hemostatic protection, whereas abruption-generated thrombin can act in autocrine/paracrine fashion on DCs to promote hemostasis via enhanced TF expression. PMID:19720393

The influence of prophylactic factor VIII in severe hemophilia A

PubMed Central

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

2013-01-01

Introduction Hemophilia A individuals displaying a similar genetic defect have heterogeneous clinical phenotypes. Aim To evaluate the underlying effect of exogenous factor (f)VIII on tissue factor (Tf)-initiated blood coagulation in severe hemophilia utilizing both empirical and computational models. Methods We investigated twenty-five clinically severe hemophilia A patients. All individuals were on fVIII prophylaxis and had not received fVIII from 0.25 to 4 days prior to phlebotomy. Coagulation was initiated by the addition of Tf to contact-pathway inhibited whole blood ± an anti-fVIII antibody. Aliquots were quenched over 20 min and analyzed for thrombin generation and fibrin formation. Coagulation factor levels were obtained and used to computationally predict thrombin generation with fVIII set to either zero or its value at the time of the draw. Results Due to prophylactic fVIII, at the time of the blood draw, the individuals had fVIII levels that ranged from <1% to 22%. Thrombin generation (maximum level and rate) in both empirical and computational systems increased as the level of fVIII increased. FXIII activation rates also increased as the fVIII level increased. Upon suppression of fVIII, thrombin generation became comparable in both systems. Plasma composition analysis showed a negative correlation between bleeding history and computational thrombin generation in the absence of fVIII. Conclusion Residual prophylactic fVIII directly causes an increase in thrombin generation and fibrin cross-linking in individuals with clinically severe hemophilia A. The combination of each individual's coagulation factors (outside of fVIII) determine each individual's baseline thrombin potential and may affect bleeding risk. PMID:21899664

Anti-inflammatory and anti-fibrinolytic effects of thrombomodulin alfa through carboxypeptidase B2 in the presence of thrombin.

PubMed

Tawara, Shunsuke; Sakai, Takumi; Matsuzaki, Osamu

2016-11-01

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.

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

PubMed Central

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

2012-01-01

Objective Preterm labor is associated with excessive maternal thrombin generation as evidenced by increased circulating thrombin–antithrombin (TAT) III complexes concentration. In addition to its hemostatic functions, thrombin has uterotonic properties that may participate in the mechanism leading to preterm birth in cases of intrauterine bleeding. Thrombin also has a proinflammatory role, and inflammation is associated with increased thrombin generation. The aim of this study was to determine whether intra-amniotic infection/inflammation (IAI) is associated with increased amniotic fluid (AF) thrombin generation in women with preterm and term deliveries. Study design This cross-sectional study included the following groups: 1) mid-trimester (n=74); 2) term not in labor (n=39); 3) term in labor (n=25); 4) term in labor with IAI (n=22); 5) spontaneous preterm labor (PTL) who delivered at term (n=62); 6) PTL without IAI who delivered preterm (n=59); 7) PTL with IAI (n=71). The AF TAT III complexes concentration was measured by ELISA. Non-parametric statistics were used for analysis. Results 1) TAT III complexes were identified in all AF samples; 2) patients with PTL who delivered preterm, with and without IAI, had a significantly higher median AF TAT III complexes concentration than those with an episode of PTL who delivered at term (p<0.001, p=0.03, respectively); 3) among patients with preterm labor without IAI, elevated AF TAT III complexes concentration were independently associated with a shorter amniocentesis-to-delivery interval (hazard ratio- 1.5, 95%CI, 1.07–2.1); 4) among patients at term, those with IAI had a higher median AF TAT III complexes concentration than those without IAI, whether in labor or not in labor (p=0.02); 5) there was no significant difference between the median AF TAT III complexes concentration of patients at term with and without labor; and 6) patients who had a mid-trimester amniocentesis had a lower median AF TAT III complexes concentration than that of patients at term not in labor (p<0.001). Conclusions We present herein a distinct difference in the pattern of intra-amniotic thrombin generation between term and preterm parturition. Preterm labor leading to preterm delivery is associated with an increased intra-amniotic thrombin generation, regardless of the presence of IAI. In contrast, term delivery is associated with an increased intra-amniotic thrombin generation only in patients with IAI. PMID:19900035

Thrombin-induced phosphorylation of the regulatory light chain of myosin II in cultured bovine corneal endothelial cells.

PubMed

Satpathy, M; Gallagher, P; Lizotte-Waniewski, M; Srinivas, S P

2004-10-01

Phosphorylation of the regulatory light chain of myosin II (referred to as myosin light chain or MLC) leads to a loss of barrier integrity in cellular monolayers by an increase in the contractility of the cortical actin cytoskeleton. This effect has been examined in corneal endothelial (CE) cells. Experiments were performed using cultured bovine CE cells (BCEC). MLC phosphorylation was induced by a thrombin-mediated activation of the proteinase-activated receptor-1 (PAR-1). Expression of MLC kinase (MLCK), a Ca2+/calmodulin-dependent protein kinase that phosphorylates MLC at its Ser-19 and Thr-18 residues, was determined by RT-PCR and Western blotting. Expression of PAR-1, RhoA, and Rho kinase-1 (effector of RhoA) was ascertained by RT-PCR. MLC phosphorylation was assessed by urea-glycerol gel electrophoresis followed by immunoblotting. The effects of Rho kinase-1 and PKC were characterized by using their selective inhibitors, Y-27632 and chelerythrine, respectively. Reorganization of the cytoskeleton was evaluated by the phalloidin staining of actin. [Ca2+]i was measured using Fura-2. The barrier integrity was assayed as permeability of BCEC monolayers to horseradish peroxidase (HRP; 44 kDa). RT-PCR showed expression of MLCK, PAR-1, Rho kinase-1, and RhoA. Western blotting indicated expression of the non-muscle and smooth muscle isoforms of MLCK. Exposure to thrombin induced an increase in [Ca2+]i with the peak unaffected by an absence of extracellular Ca2+. Pre-exposure to thrombin (2 U ml(-1); 2 min) led to mono- and di-phosphorylation of MLC. Under both basal conditions and in the presence of thrombin, MLC phosphorylation was prevented by chelerythrine (10 microm) and Y-27632 (<25 microm). Thrombin led to inter-endothelial gaps secondary to the disruption of the cortical actin cytoskeleton, which under resting conditions was organized as a perijunctional actomyosin ring (PAMR). These responses were blocked by pre-treatment with Y-27632. Thrombin also increased permeability to HRP, which was abolished by pre-treatment with Y-27632. Thrombin induces MLC phosphorylation in BCEC. The consequent increase in the contractility of the actin cytoskeleton produces a centripetal force resulting in inter-endothelial gaps and a breakdown of barrier integrity. These responses are PKC- and Rho kinase-dependent. [Ca2+]i increase, as well as sensitivity of the thrombin response to PKC and Rho kinase inhibitors, are consistent with the expression of PAR-1 receptors in BCEC. Thrombin-induced hyperpermeability is a model to investigate barrier dysfunction induced by MLC phosphorylation.

Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation, platelet aggregation, and P-selectin expression

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

Nygaard, Gyrid; Department of Biomedicine, University of Bergen, Bergen; Herfindal, Lars

Highlights: • We investigated the impact of cyclic nucleotide analogues on platelet activation. • Different time dependence were found for inhibition of platelet activation. • Additive effect was found using PKA- and PKG-activating analogues. • Our results may explain some of the discrepancies reported for cNMP signalling. - Abstract: In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigatedmore » whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation.« less

Homogeneous assay of target molecules based on chemiluminescence resonance energy transfer (CRET) using DNAzyme-linked aptamers.

PubMed

Mun, Hyoyoung; Jo, Eun-Jung; Li, Taihua; Joung, Hyou-Arm; Hong, Dong-Gu; Shim, Won-Bo; Jung, Cheulhee; Kim, Min-Gon

2014-08-15

We have designed a single-stranded DNAzyme-aptamer sensor for homogeneous target molecular detection based on chemiluminescence resonance energy transfer (CRET). The structure of the engineered single-stranded DNA (ssDNA) includes the horseradish peroxidase (HRP)-like DNAzyme, optimum-length linker (10-mer-length DNA), and target-specific aptamer sequences. A quencher dye was modified at the 3' end of the aptamer sequence. The incorporation of hemin into the G-quadruplex structure of DNAzyme yields an active HRP-like activity that catalyzes luminol to generate a chemiluminescence (CL) signal. In the presence of target molecules, such as ochratoxin A (OTA), adenosine triphosphate (ATP), or thrombin, the aptamer sequence was folded due to the formation of the aptamer/analyte complex, which induced the quencher dye close to the DNAzyme structure. Consequently, the CRET occurred between a DNAzyme-catalyzed chemiluminescence reaction and the quencher dye. Our results showed that CRET-based DNAzyme-aptamer biosensing enabled specific OTA analysis with a limit of detection of 0.27ng/mL. The CRET platform needs no external light source and avoids autofluorescence and photobleaching, and target molecules can be detected specifically and sensitively in a homogeneous manner. Copyright © 2014 Elsevier B.V. All rights reserved.

Apixaban decreases brain thrombin activity in a male mouse model of acute ischemic stroke.

PubMed

Bushi, Doron; Chapman, Joab; Wohl, Anton; Stein, Efrat Shavit; Feingold, Ekaterina; Tanne, David

2018-05-14

Factor Xa (FXa) plays a critical role in the coagulation cascade by generation of thrombin. During focal ischemia thrombin levels increase in the brain tissue and cause neural damage. This study examined the hypothesis that administration of the FXa inhibitor, apixaban, following focal ischemic stroke may have therapeutic potential by decreasing brain thrombin activity and infarct volume. Male mice were divided into a treated groups that received different doses of apixaban (2, 20, 100 mg/kg administered I.P.) or saline (controls) immediately after blocking the middle cerebral artery (MCA). Thrombin activity was measured by a fluorescence assay on fresh coronal slices taken from the mice brains 24 hr following the MCA occlusion. Infarct volume was assessed using triphenyltetrazolium chloride staining. A high dose of apixaban (100 mg/kg) significantly decreased thrombin activity levels in the ipsilateral hemisphere compared to the control group (Slice#5, p = .016; Slice#6, p = .016; Slice#7, p = .016; Slice#8, p = .036; by the nonparametric Mann-Whitney test). In addition, treatment with apixaban doses of both 100 mg/kg (32 ± 8% vs. 76 ± 7% in the treatment vs. control groups respectively; p = .005 by the nonparametric Mann-Whitney test) and 20 mg/kg (43 ± 7% vs. 76 ± 7% in the treatment vs. control groups respectively; p = .019 by the nonparametric Mann-Whitney test) decreased infarct volumes in areas surrounding the ischemic core (Slices #3 and #8). No brain hemorrhages were observed either in the treated or control groups. In summary, I.P. administration of high dose of apixaban immediately after MCA occlusion decreases brain thrombin activity and reduces infarct size. © 2018 Wiley Periodicals, Inc.

Thrombin and factor Xa link the coagulation system with liver fibrosis.

PubMed

Dhar, Ameet; Sadiq, Fouzia; Anstee, Quentin M; Levene, Adam P; Goldin, Robert D; Thursz, Mark R

2018-05-08

Thrombin activates hepatic stellate cells via protease-activated receptor-1. The role of Factor Xa (FXa) in hepatic fibrosis has not been elucidated. We aimed to evaluate the impact of FXa and thrombin in vitro on stellate cells and their respective inhibition in vivo using a rodent model of hepatic fibrosis. HSC-LX2 cells were incubated with FXa and/or thrombin in cell culture, stained for αSMA and relative gene expression and gel contraction calculated. C57BL/6 J mice were administered thioacetamide (TAA) for 8 weeks with Rivaroxaban (n = 15) or Dabigatran (n = 15). Control animals received TAA alone (n = 15). Fibrosis was scored and quantified using digital image analysis and hepatic tissue hydroxyproline estimated. Stellate cells treated with FXa and thrombin demonstrated upregulation of procollagen, TGF-beta, αSMA and significant cell contraction (43.48%+/- 4.12) compared to culturing with FXa or thrombin alone (26.90%+/- 8.90, p = 0.02; 13.1%+/- 9.84, p < 0.001). Mean fibrosis score, percentage area of fibrosis and hepatic hydroxyproline content (2.46 vs 4.08, p = 0.008; 2.02% vs 3.76%, p = 0.012; 276.0 vs 651.3, p = 0.0001) were significantly reduced in mice treated with the FXa inhibitor compared to control mice. FXa inhibition was significantly more effective than thrombin inhibition in reducing percentage area of fibrosis and hepatic hydroxyproline content (2.02% vs 3.70%,p = 0.031; 276.0 vs 413.1,p = 0.001). FXa promotes stellate cell contractility and activation. Early inhibition of coagulation using a FXa inhibitor significantly reduces TAA induced murine liver fibrosis and may be a viable treatment for liver fibrosis in patients.

Activation of platelet-rich plasma using soluble type I collagen.

PubMed

Fufa, Duretti; Shealy, Blake; Jacobson, May; Kevy, Sherwin; Murray, Martha M

2008-04-01

Platelet-rich plasma (PRP) has recently been found to be a useful delivery system for growth factors important to oral tissue healing. But application of PRP in a liquid form to a wound site within the oral cavity can be complicated by significant loss of the PRP into the surrounding oral space unless gelation through the clotting mechanism is accomplished. Gelation is currently accomplished using bovine thrombin; however, rare but serious complications of this method have led to the search for alternative clotting mechanisms, including the use of soluble collagen as a clotting activator. In this work, our hypothesis was that soluble type I collagen would be as effective as bovine thrombin in causing clotting of the PRP and stimulating growth factor release from the platelets and granulocytes. PRP from human donors was clotted using type I collagen or bovine thrombin. Clot retraction was determined by measuring clot diameters over time. The release of platelet-derived growth factor (PDGF)-AB, transforming growth factor (TGF)-beta1, and vascular endothelial growth factor (VEGF) from both types of clots was measured over 10 days using enzyme-linked immunosorbent assasy. Clots formed using type I collagen exhibited far less retraction than those formed with bovine thrombin. Bovine thrombin and type I collagen stimulated similar release of PDGF-AB and VEGF between 1 and 10 days; however, thrombin activation resulted in a greater release of TGF-beta1 during the first 5 days after activation. The use of type I collagen to activate clotting of PRP may be a safe and effective alternative to bovine thrombin. The use of collagen results in less clot retraction and equal release of PDGF-AB and VEGF compared with currently available methods of clot activation.

ACTIVATION OF PLATELET-RICH PLASMA USING SOLUBLE TYPE I COLLAGEN

PubMed Central

Fufa, Duretti; Shealy, Blake; Jacobson, May; Kevy, Sherwin; Murray, Martha M.

2008-01-01

PURPOSE Platelet-rich plasma (PRP) has recently been found to be a useful delivery system for growth factors important in oral tissue healing. However, application of PRP in a liquid form to a wound site within the oral cavity can be complicated by significant loss of the PRP into the surrounding oral space unless gelation via the clotting mechanism is accomplished. Gelation is currently accomplished using bovine thrombin; however, rare but serious complications of this method have led to the search for alternative clotting mechanisms, including the use of soluble collagen as a clotting activator. In this paper, our hypothesis was that soluble Type I collagen would be as effective as bovine thrombin in causing clotting of the PRP and of stimulating growth factor release from the platelets and granulocytes. MATERIALS AND METHODS PRP from human donors was clotted using Type I collagen or bovine thrombin. Clot retraction was determined by measuring clot diameters over time. The release of PDGF-AB, TGF-β1 and VEGF from both types of clots was measured over 10 days using ELISA. RESULTS Clots formed using Type I collagen had far less retraction than those formed with bovine thrombin. Bovine thrombin and Type I collagen stimulated similar release of PDGF-AB and VEGF between 1 and 10 days; however, thrombin activation resulted in a greater release of TGF-β1 during the first five days after activation. CONCLUSIONS The use of Type I collagen to activate clotting of PRP may be a safe and effective alternative to bovine thrombin. The use of collagen results in less clot retraction and equal release of PDGF-AB and VEGF when compared to currently available methods of clot activation. PMID:18355591

Thrombin-activatable fluorescent peptide incorporated gold nanoparticles for dual optical/computed tomography thrombus imaging.

PubMed

Kwon, Sung-Pil; Jeon, Sangmin; Lee, Sung-Hoon; Yoon, Hong Yeol; Ryu, Ju Hee; Choi, Dayil; Kim, Jeong-Yeon; Kim, Jiwon; Park, Jae Hyung; Kim, Dong-Eog; Kwon, Ick Chan; Kim, Kwangmeyung; Ahn, Cheol-Hee

2018-01-01

Thrombosis is an important pathophysiologic phenomenon in various cardiovascular diseases, which can lead to oxygen deprivation and infarction of tissues by generation of a thrombus. Thus, direct thrombus imaging can provide beneficial in diagnosis and therapy of thrombosis. Herein, we developed thrombin-activatable fluorescent peptide (TAP) incorporated silica-coated gold nanoparticles (TAP-SiO 2 @AuNPs) for direct imaging of thrombus by dual near-infrared fluorescence (NIRF) and micro-computed tomography (micro-CT) imaging, wherein TAP molecules were used as targeted thrombin-activatable peptide probes for thrombin-specific NIRF imaging. The freshly prepared TAP-SiO 2 @AuNPs had an average diameter of 39.8 ± 2.55 nm and they showed the quenched NIRF signal in aqueous condition, due to the excellent quenching effect of TAP molecules on the silica-gold nanoparticle surface. However, 30.31-fold higher NIRF intensity was rapidly recovered in the presence of thrombin in vitro, due to the thrombin-specific cleavage of quenched TAP molecules on the gold particle surface. Furthermore, TAP-SiO 2 @AuNPs were successfully accumulated in thrombus by their particle size-dependent capturing property, and they presented a potential X-ray absorption property in a dose-dependent manner. Finally, thrombotic lesion was clearly distinguished from peripheral tissues by dual NIRF/micro-CT imaging after intravenous injection of TAP-SiO 2 @AuNPs in the in situ thrombotic mouse model, simultaneously. This study showed that thrombin-activatable fluorescent peptide incorporated silica-coated gold nanoparticles can be potentially used as a dual imaging probe for direct thrombus imaging and therapy in clinical applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Catheter-directed Endovascular Application of Thrombin: Report of 3 Cases and Review of the Literature

PubMed Central

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

2017-01-01

Purpose To report 3 new cases of catheter-directed endovascular application of thrombin and explore trends by analysis of published case series. Materials and Methods 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. Results 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. Conclusions 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. PMID:27936421

The effect of defibrotide on thromboembolism in the pulmonary vasculature of mice and rabbits and in the cerebral vasculature of rabbits.

PubMed Central

Paul, W.; Gresele, P.; Momi, S.; Bianchi, G.; Page, C. P.

1993-01-01

1. Administration of bovine thrombin (100 u kg-1) into the carotid artery of rabbits induces a sustained accumulation of 111 Indium-labelled platelets within the cranial vasculature over the subsequent 3 h. 2. Intracarotid (i.c.) administration of defibrotide (64 mg kg-1 bolus plus 64 mg kg-1 h-1 for 1 h) prior to i.c. thrombin (100 u kg-1) significantly reduces the ability of thrombin to induce cranial thromboembolism in rabbits. 3. Intravenous (i.v.) administration of thrombin (20 u kg-1) in rabbits induces a reversible accumulation of radiolabelled platelets into the thoracic circulation which is significantly reduced by i.v. administration of defibrotide (64 mg kg-1 bolus plus 64 mg kg-1 h-1 for 1 h) prior to i.v. thrombin. In contrast, platelet accumulation in response to adenosine diphosphate (ADP; 20 micrograms kg-1, i.v.) or platelet activating factor (PAF; 50 ng kg-1, i.v.) is not significantly affected by this treatment. 4. Intravenous administration of the nitric oxide (NO)-synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 10 mg kg-1) potentiates platelet accumulation induced by low dose thrombin (10 u kg-1, i.v.) within the pulmonary vasculature of rabbits. The potentiated response is significantly abrogated following pretreatment with defibrotide (64 mg kg-1 bolus plus 64 mg kg-1 h-1 for 1 h, i.v.). 5. Intravenous injection of human thrombin (1250 u kg-1) to mice induces death within the majority of animals which is significantly reduced by pretreatment with defibrotide (150-175 mg kg-1, i.v.).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8306102

Thrombin like activity of Asclepias curassavica L. latex: action of cysteine proteases.

PubMed

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

2009-05-04

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.

Molecular design and structure--activity relationships leading to the potent, selective, and orally active thrombin active site inhibitor BMS-189664.

PubMed

Das, Jagabandhu; Kimball, S David; Hall, Steven E; Han, Wen Ching; Iwanowicz, Edwin; Lin, James; Moquin, Robert V; Reid, Joyce A; Sack, John S; Malley, Mary F; Chang, Chiehying Y; Chong, Saeho; Wang-Iverson, David B; Roberts, Daniel G M; Seiler, Steven M; Schumacher, William A; Ogletree, Martin L

2002-01-07

A series of structurally novel small molecule inhibitors of human alpha-thrombin was prepared to elucidate their structure-activity relationships (SARs), selectivity and activity in vivo. BMS-189664 (3) is identified as a potent, selective, and orally active reversible inhibitor of human alpha-thrombin which is efficacious in vivo in a mouse lethality model, and at inhibiting both arterial and venous thrombosis in cynomolgus monkey models.

Manipulation of a DNA aptamer-protein binding site through arylation of internal guanine residues.

PubMed

Van Riesen, Abigail J; Fadock, Kaila L; Deore, Prashant S; Desoky, Ahmed; Manderville, Richard A; Sowlati-Hashjin, Shahin; Wetmore, Stacey D

2018-05-23

Chemically modified aptamers have the opportunity to increase aptamer target binding affinity and provide structure-activity relationships to enhance our understanding of molecular target recognition by the aptamer fold. In the current study, 8-aryl-2'-deoxyguanosine nucleobases have been inserted into the G-tetrad and central TGT loop of the thrombin binding aptamer (TBA) to determine their impact on antiparallel G-quadruplex (GQ) folding and thrombin binding affinity. The aryl groups attached to the dG nucleobase vary greatly in aryl ring size and impact on GQ stability (∼20 °C change in GQ thermal melting (Tm) values) and thrombin binding affinity (17-fold variation in dissociation constant (Kd)). At G8 of the central TGT loop that is distal from the aptamer recognition site, the probes producing the most stable GQ structure exhibited the strongest thrombin binding affinity. However, within the G-tetrad, changes to the electron density of the dG component within the modified nucleobase can diminish thrombin binding affinity. Detailed molecular dynamics (MD) simulations on the modified TBA (mTBA) and mTBA-protein complexes demonstrate how the internal 8-aryl-dG modification can manipulate the interactions between the DNA nucleobases and the amino acid residues of thrombin. These results highlight the potential of internal fluorescent nuclobase analogs (FBAs) to broaden design options for aptasensor development.

Preparation of chondroitin sulfate libraries containing disulfated disaccharide units and inhibition of thrombin by these chondroitin sulfates.

PubMed

Numakura, Mario; Kusakabe, Noriko; Ishige, Kazuya; Ohtake-Niimi, Shiori; Habuchi, Hiroko; Habuchi, Osami

2010-07-01

Chondroitin sulfate (CS) containing GlcA-GalNAc(4,6-SO(4)) (E unit) and CS containing GlcA(2SO(4))-GalNAc(6SO(4)) (D unit) have been implicated in various physiological functions. However, it has been poorly understood how the structure and contents of disulfated disaccharide units in CS contribute to these functions. We prepared CS libraries containing E unit or D unit in various proportions by in vitro enzymatic reactions using recombinant GalNAc 4-sulfate 6-O-sulfotransferase and uronosyl 2-O-sulfotransferase, and examined their inhibitory activity toward thrombin. The in vitro sulfated CSs containing disulfated disaccharide units showed concentration-dependent direct inhibition of thrombin when the proportion of E unit or D unit in the CSs was above 15-17%. The CSs containing both E unit and D unit exhibited higher inhibitory activity toward thrombin than the CSs containing either E unit or D unit alone, if the proportion of the total disulfated disaccharide units of these CSs was comparable. The thrombin-catalyzed degradation of fibrinogen, a physiological substrate for thrombin, was also inhibited by the CS containing both E unit and D unit. These observations indicate that the enzymatically prepared CS libraries containing various amounts of disulfated disaccharide units appear to be useful for elucidating the physiological function of disulfated disaccharide units in CS.

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

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

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

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

Electroactivity of Aptamer at Soft Microinterface Arrays.

PubMed

Felisilda, Bren Mark B; Arrigan, Damien W M

2018-06-26

The electrochemical behavior of a synthetic oligonucleotide, thrombin-binding aptamer (TBA, 15-mer), was explored at a liquid-organogel microinterface array. TBA did not display any response when only background electrolytes were present in both phases. On the basis of literature reports that surfactants can influence nucleic acid detection, the response in the presence of cetyltrimethylammonium (CTA + ) was examined. With both TBA and CTA + in the aqueous phase, the transfer current for CTA + was diminished, signifying the interaction of CTA + with TBA. Experiments with CTA + spiked into the organic phase revealed a sharp current peak, consistent with the interfacial formation of a CTA + -TBA complex. However, use of CTA + as the organic phase electrolyte cation, as the salt with tetrakis(4-chlorophenyl)borate, greatly improved the response to TBA. In this case, a distinctive peak response (at ca. -0.25 V) was attributed to the transfer of CTA + across the soft interface to complex with aqueous phase TBA. Employing this process as a detection step enabled a detection limit of 0.11 μM TBA (by cyclic voltammetry). Furthermore, the presence of magnesium cations at physiological concentration resulted in the disappearance of the TBA response because of Mg 2+ -induced folding of TBA. Also, the current response of TBA was decreased by the addition of thrombin, indicating TBA interacted with this binding partner. Finally, the interfacial surfactant-aptamer interaction was explored in a synthetic urine matrix that afforded a detection limit of 0.29 μM TBA. These results suggest that aptamer-binding interactions can be monitored by electrochemistry at aqueous-organic interfaces and open up a new possibility for detection in aptamer-binding assays.

Fibrin hydrogels to deliver dental stem cells of the apical papilla for regenerative medicine.

PubMed

Germain, Loïc; De Berdt, Pauline; Vanacker, Julie; Leprince, Julian; Diogenes, Anibal; Jacobs, Damien; Vandermeulen, Gaëlle; Bouzin, Caroline; Préat, Véronique; Dupont-Gillain, Christine; des Rieux, Anne

2015-01-01

Evaluation of survival, proliferation and neurodifferentiation of dental stem cells from the apical papilla (SCAP) in fibrin hydrogels. We hypothesized that fibrin composition will influence cell behavior. Modulus, pore and fiber size were measured. SCAP in vitro viability, proliferation and neural differentiation, as well as in vivo proliferation and angiogenesis were studied. Hydrogel moduli were influenced by fibrin formulation but not hydrogel morphology, SCAP in vitro viability and proliferation. In total 60% of SCAP expressed PanNeurofilament in vitro without induction in Fibrinogen50-Thrombin10. SCAP proliferated when implanted in vivo and stimulated host endothelial cell infiltration. Fibrinogen30-Thrombin10 or Thrombin50 would be more favorable to in vitro SCAP viability and in vivo proliferation, while Fibrinogen 50-Thrombin50 would be more adapted to neurodifferentiation.

Changes in thrombin-stimulated platelet malondialdehyde production during the menstrual cycle.

PubMed Central

Tindall, H; Zuzel, M; Paton, R C; McNicol, G P

1981-01-01

Forty normal women had thrombin-stimulated platelet malondialdehyde (MDA) production measured during their menstrual cycle. Twenty women in this group were taking the combined oral contraceptive pill (OCP). Platelet MDA production was found to fall by 30% during normal menstruation and the week when the subjects were not taking a combined OCP, but it remained constant throughout the remainder of the cycle. No significant change in initial platelet aggregation response to stimulation by thrombin, change in plasma thrombin clotting time, plasma heparin neutralising activity (HNA), or plasma antithrombin III (AT-III) activity was seen when the platelet MDA production was reduced. The bleeding time results showed some variation throughout the menstrual cycle but these did not appear to be related to the variation in platelet MDA production. PMID:7251901

Improved Stability of Proline-Derived Direct Thrombin Inhibitors through Hydroxyl to Heterocycle Replacement.

PubMed

Chobanian, Harry R; Pio, Barbara; Guo, Yan; Shen, Hong; Huffman, Mark A; Madeira, Maria; Salituro, Gino; Terebetski, Jenna L; Ormes, James; Jochnowitz, Nina; Hoos, Lizbeth; Zhou, Yuchen; Lewis, Dale; Hawes, Brian; Mitnaul, Lyndon; O'Neill, Kim; Ellsworth, Kenneth; Wang, Liangsu; Biftu, Tesfaye; Duffy, Joseph L

2015-05-14

Modification of the previously disclosed (S)-N-(2-(aminomethyl)-5-chlorobenzyl)-1-((R)-2-hydroxy-3,3-dimethylbutanoyl)pyrrolidine-2-carboxamide 2 by optimization of the P3 group afforded novel, low molecular weight thrombin inhibitors. Heterocycle replacement of the hydroxyl functional group helped maintain thrombin in vitro potency while improving the chemical stability and pharmacokinetic profile. These modifications led to the identification of compound 10, which showed excellent selectivity over related serine proteases as well as in vivo efficacy in the rat arteriovenous shunt. Compound 10 exhibited significantly improved chemical stability and pharmacokinetic properties over 2 and may be utilized as a structurally differentiated preclinical tool comparator to dabigatran etexilate (Pro-1) to interrogate the on- and off-target effects of oral direct thrombin inhibitors.

Dynamics Govern Specificity of a Protein-Protein Interface: Substrate Recognition by Thrombin.

PubMed

Fuchs, Julian E; Huber, Roland G; Waldner, Birgit J; Kahler, Ursula; von Grafenstein, Susanne; Kramer, Christian; Liedl, Klaus R

2015-01-01

Biomolecular recognition is crucial in cellular signal transduction. Signaling is mediated through molecular interactions at protein-protein interfaces. Still, specificity and promiscuity of protein-protein interfaces cannot be explained using simplistic static binding models. Our study rationalizes specificity of the prototypic protein-protein interface between thrombin and its peptide substrates relying solely on binding site dynamics derived from molecular dynamics simulations. We find conformational selection and thus dynamic contributions to be a key player in biomolecular recognition. Arising entropic contributions complement chemical intuition primarily reflecting enthalpic interaction patterns. The paradigm "dynamics govern specificity" might provide direct guidance for the identification of specific anchor points in biomolecular recognition processes and structure-based drug design.

Thrombin Time

MedlinePlus

... Testing Leptin Levetiracetam Lipase Lipid Panel Lipoprotein (a) Lithium Liver Panel Lp-PLA2 Lupus Anticoagulant Testing Luteinizing ... thrombin time is just one component of the battery of tests typically required to evaluate a bleeding ...

Favorable 2'-substitution in the loop region of a thrombin-binding DNA aptamer.

PubMed

Awachat, Ragini; Wagh, Atish A; Aher, Manisha; Fernandes, Moneesha; Kumar, Vaijayanti A

2018-06-01

Simple 2'-OMe-chemical modification in the loop region of the 15mer G-rich DNA sequence GGTTGGTGTGGTTGG is reported. The G-quadruplex structure of this thrombin-binding aptamer (TBA), is stabilized by single modifications (T → 2'-OMe-U), depending on the position of the modification. The structural stability also renders significantly increased inhibition of thrombin-induced fibrin polymerization, a process closely associated with blood-clotting. Copyright © 2018 Elsevier Ltd. All rights reserved.

Ratiometric activatable cell-penetrating peptides provide rapid in vivo readout of thrombin activation.

PubMed

Whitney, Michael; Savariar, Elamprakash N; Friedman, Beth; Levin, Rachel A; Crisp, Jessica L; Glasgow, Heather L; Lefkowitz, Roy; Adams, Stephen R; Steinbach, Paul; Nashi, Nadia; Nguyen, Quyen T; Tsien, Roger Y

2013-01-02

In real time: thrombin activation in vivo can be imaged in real time with ratiometric activatable cell penetrating peptides (RACPPs). RACPPs are designed to combine 1) dual-emission ratioing, 2) far red to infrared wavelengths for in vivo mammalian imaging, and 3) cleavage-dependent spatial localization. The most advanced RACPP uses norleucine (Nle)-TPRSFL as a linker that increases sensitivity to thrombin by about 90-fold. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tissue factor-expressing monocytes inhibit fibrinolysis through a TAFI-mediated mechanism, and make clots resistant to heparins

PubMed Central

Semeraro, Fabrizio; Ammollo, Concetta T.; Semeraro, Nicola; Colucci, Mario

2009-01-01

Background Thrombin is the main activator of the fibrinolysis inhibitor TAFI (thrombin activatable fibrinolysis inhibitor) and heightened clotting activation is believed to impair fibrinolysis through the increase of thrombin activatable fibrinolysis inhibitor activation. However, the enhancement of thrombin generation by soluble tissue factor was reported to have no effect on plasma fibrinolysis and it is not known whether the same is true for cell-associated tissue factor. The aim of this study was to evaluate the effect of tissue factor-expressing monocytes on plasma fibrinolysis in vitro. Design and Methods Tissue factor expression by human blood mononuclear cells (MNC) and monocytes was induced by LPS stimulation. Fibrinolysis was spectrophotometrically evaluated by measuring the lysis time of plasma clots containing LPS-stimulated or control cells and a low concentration of exogenous tissue plasminogen activator. Results LPS-stimulated MNC (LPS-MNC) prolonged fibrinolysis time as compared to unstimulated MNC (C-MNC) in contact-inhibited but not in normal citrated plasma. A significantly prolonged lysis time was observed using as few as 30 activated cells/μL. Fibrinolysis was also impaired when clots were generated on adherent LPS-stimulated monocytes. The antifibrinolytic effect of LPS-MNC or LPS-monocytes was abolished by an anti-tissue factor antibody, by an antibody preventing thrombin-mediated thrombin activatable fibrinolysis inhibitor activation, and by a TAFIa inhibitor (PTCI). Assays of thrombin and TAFIa in contact-inhibited plasma confirmed the greater generation of these enzymes in the presence of LPS-MNC. Finally, the profibrinolytic effect of unfractionated heparin and enoxaparin was markedly lower (~50%) in the presence of LPS-MNC than in the presence of a thromboplastin preparation displaying an identical tissue factor activity. Conclusions Our data indicate that LPS-stimulated monocytes inhibit fibrinolysis through a tissue factor-mediated enhancement of thrombin activatable fibrinolysis inhibitor activation and make clots resistant to the profibrinolytic activity of heparins, thus providing an additional mechanism whereby tissue factor-expressing monocytes/macrophages may favor fibrin accumulation and diminish the antithrombotic efficacy of heparins. PMID:19377079

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

PubMed

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

2018-05-01

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

Thrombin products: economic impact of immune-mediated coagulopathies and practical formulary considerations.

PubMed

Voils, Stacy A

2009-07-01

Thrombin has demonstrated utility in aiding surgical hemostasis since its introduction more than 60 years ago. It is used across a wide variety of surgical procedures by virtually every specialty. Only recently have new equally effective and safe products entered the market, causing decision makers to evaluate formulary selection among products with otherwise modest differences. This evaluation includes identifying costs beyond those of acquisition and storage, as well as indirect factors such as monitoring or specialized distribution requirements. One factor to consider specifically in selection of topical thrombin products is the potential for patients to develop an immune-mediated coagulopathy (IMC) after exposure to bovine-derived thrombin. Costs due to adverse drug events fall into the category of indirect costs and, in some instances, can be substantial if bleeding due to IMC occurs.

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

PubMed

Akkaya, Alper; Pazarlioglu, Nurdan

2013-01-01

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.

Bombesin and thrombin affect discrete pools of intracellular calcium through different G-proteins.

PubMed

Wang, J L; Kalyanaraman, S; Vivo, M D; Gautam, N

1996-11-15

In mouse NIH 3T3 cells, the mitogens bombesin and thrombin induced Ca2+ release from intracellular stores. Ca2+ release induced by bombesin was inhibited by the Ca(2+)-ATPase inhibitor thapsigargin, while Ca2+ release induced by thrombin was unaffected by this agent. The Ca(2+)-release response to bombesin was not affected by pertussis toxin, but the response to thrombin was abolished by the toxin. Stable transfectants overexpressing the G-protein subunit type alpha 9 showed an accentuated response to bombesin, indicating that the bombesin receptor was coupled to a Gq-like G-protein. Together, these results show that the two mitogenic receptors are coupled to distinct G-proteins that affect functionally different pools of Ca2+. Organization of signalling pathways in this manner may allow cells to differentially encode information from different signals.

Platelet-derived microparticles regulates thrombin generation via phophatidylserine in abdominal sepsis.

PubMed

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

2018-02-01

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.

Protocol for obtaining platelet-rich plasma (PRP), platelet-poor plasma (PPP), and thrombin for autologous use.

PubMed

Franco, Diogo; Franco, Talita; Schettino, Angélica Maria; Filho, João Medeiros Tavares; Vendramin, Fabiel Spani

2012-10-01

Plasma has been widely studied and used in many different situations to speed up healing with better tissue adherence and hemostasis. Research projects are now attempting to isolate platelet-rich plasma (PRP) and platelet-poor plasma (PPP), making better use of their properties, particularly during operations and for wounds that are slow to heal. In view of the wide diversity of industrial machines and extraction protocols, together with the variety of industrially produced biologic glues, this article suggests an option for obtaining PRP, PPP, and human thrombin for autologous use. A way of obtaining PRP, PPP, and thrombin is reproduced through a protocol defined and established by the authors. Autologous thrombin and plasma were obtained through the collection and successive centrifugation of ten whole blood samples, until the desired hemocomponents were isolated, followed by quantitative and qualitative analyses of the elements obtained. The mean platelet concentration obtained was 6.03 × 10(8) platelets/ml, with a mean thrombin concentration of 33.54 nM, both values compatible with reports in the literature when different protocols are applied. The protocol described is a good option for the preparation and application of PRP, PPP, and autologous thrombin, particularly as they can be obtained simultaneously, eliminating the possibilities of viral contamination and allergic reactions. Moreover, the cost of this procedure is low, it is easy to perform, and replicable. This journal requires that authors assign a level of evidence to each article.

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

PubMed Central

Pereira, Rebeca Cristina Costa; Lourenço, André Luiz; Terra, Luciana; Abreu, Paula Alvarez; Laneuville Teixeira, Valéria; Castro, Helena Carla

2017-01-01

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. PMID:28335516

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

PubMed

Pereira, Rebeca Cristina Costa; Lourenço, André Luiz; Terra, Luciana; Abreu, Paula Alvarez; Laneuville Teixeira, Valéria; Castro, Helena Carla

2017-03-20

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.

Key role of integrin α(IIb)β (3) signaling to Syk kinase in tissue factor-induced thrombin generation.

PubMed

van der Meijden, Paola E J; Feijge, Marion A H; Swieringa, Frauke; Gilio, Karen; Nergiz-Unal, Reyhan; Hamulyák, Karly; Heemskerk, Johan W M

2012-10-01

The fibrin(ogen) receptor, integrin α(IIb)β(3), has a well-established role in platelet spreading, aggregation and clot retraction. How α(IIb)β(3) contributes to platelet-dependent coagulation is less well resolved. Here, we demonstrate that the potent suppressing effect of clinically used α(IIb)β(3) blockers on tissue factor-induced thrombin generation is linked to diminished platelet Ca(2+) responses and phosphatidylserine (PS) exposure. The same blockers suppress these responses in platelets stimulated with collagen and thrombin receptor agonists, whereas added fibrinogen potentiates these responses. In platelets spreading on fibrinogen, outside-in α(IIb)β(3) signaling similarly enhances thrombin-induced Ca(2+) rises and PS exposure. These responses are reduced in α(IIb)β(3)-deficient platelets from patients with Glanzmann's thrombasthenia. Furthermore, the contribution of α(IIb)β(3) to tissue factor-induced platelet Ca(2+) rises, PS exposure and thrombin generation in plasma are fully dependent on Syk kinase activity. Tyrosine phosphorylation analysis confirms a key role of Syk activation, which is largely but not exclusively dependent on α(IIb)β(3) activation. It is concluded that the majority of tissue factor-induced procoagulant activity of platelets relies on Syk activation and ensuing Ca(2+) signal generation, and furthermore that a considerable part of Syk activation relies on α(IIb)β(3) signaling. These results hence point to a novel role of Syk in integrin-dependent thrombin generation.

FcγRIIa ligation induces platelet hypersensitivity to thrombotic stimuli.

PubMed

Berlacher, Mark D; Vieth, Joshua A; Heflin, Brittany C; Gay, Steven R; Antczak, Adam J; Tasma, Brian E; Boardman, Holly J; Singh, Navinderjit; Montel, Angela H; Kahaleh, M Bashar; Worth, Randall G

2013-01-01

Platelets are known for their important role in hemostasis, however their significance in other functions, including inflammation and infection, are becoming more apparent. Patients with systemic lupus erythematosus (SLE) are known to have circulating IgG complexes in their blood and are highly susceptible to thrombotic events. Because platelets express a single receptor for IgG, we tested the hypothesis that ligation of this receptor (FcγRIIa) induces platelet hypersensitivity to thrombotic stimuli. Platelets from SLE patients were considerably more sensitive to thrombin compared to healthy volunteers, and this correlated with elevated levels of surface IgG on SLE platelets. To test whether FcγRIIa ligation stimulated thrombin hypersensitivity, platelets from healthy volunteers were incubated with buffer or heat-aggregated IgG, then stimulated with increasing concentrations of thrombin. Interestingly, heat-aggregated IgG-stimulated platelets, but not buffer-treated platelets, were hypersensitive to thrombin, and hypersensitivity was blocked by an anti-FcγRIIa monoclonal antibody (mAb). Thrombin hypersensitivity was not due to changes in thrombin receptor expression (GPIbα or PAR1) but is dependent on activation of shared signaling molecules. These observations suggest that ligation of platelet FcγRIIa by IgG complexes induces a hypersensitive state whereby small changes in thrombotic stimuli may result in platelet activation and subsequent vascular complications such as transient ischemic attacks or stroke. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Negative feedback regulation of human platelets via autocrine activation of the platelet-derived growth factor alpha-receptor.

PubMed

Vassbotn, F S; Havnen, O K; Heldin, C H; Holmsen, H

1994-05-13

Human platelets contain platelet-derived growth factor (PDGF) in their alpha-granules which is released during platelet exocytosis. We show by immunoprecipitation and 125I-PDGF binding experiments that human platelets have functionally active PDGF alpha-receptors, but not beta-receptors. The PDGF alpha-receptor (PDGFR-alpha) was identified as a 170-kDa glycosylated protein-tyrosine kinase as found in other cell types. Stimulation of platelets with 0.1 unit/ml thrombin resulted in a significant increase (2-5-fold) of the tyrosine phosphorylation of the PDGFR-alpha, as determined by immunoprecipitation with phosphotyrosine antiserum as well as with PDGFR-alpha antiserum. The observed thrombin-induced autophosphorylation of the PDGFR-alpha was inhibited by the addition of a neutralizing monoclonal PDGF antibody. Thus, our results suggest that the platelet PDGFR-alpha is stimulated in an autocrine manner by PDGF secreted during platelet activation. Preincubation of platelets with PDGF inhibited thrombin-induced platelet aggregation and secretion of ATP + ADP and beta-hexosaminidase. Thrombin-induced platelet aggregation was also reversed when PDGF was added 30 s after thrombin stimulation. Inhibition of the autocrine PDGF pathway during platelet activation by the PDGF antibody led to a potentiation of thrombin-induced beta-hexosaminidase secretion. Thus, the PDGFR-alpha takes part in a negative feedback regulation during platelet activation. Our demonstration of PDGF alpha-receptors on human platelets and its inhibitory function during platelet activation identifies a new possible role of PDGF in the regulation of thrombosis.

Correlation to FVIII:C in Two Thrombin Generation Tests: TGA-CAT and INNOVANCE ETP.

PubMed

Ljungkvist, Marcus; Berndtsson, Maria; Holmström, Margareta; Mikovic, Danijela; Elezovic, Ivo; Antovic, Jovan P; Zetterberg, Eva; Berntorp, Erik

2017-01-01

Several thrombin-generation tests are available, but few have been directly compared. Our primary aim was to investigate the correlation of two thrombin generation tests, thrombin generation assay-calibrated automated thrombogram (TGA-CAT) and INNOVANCE ETP, to factor VIII levels (FVIII:C) in a group of patients with hemophilia A. The secondary aim was to investigate inter-laboratory variation for the TGA-CAT method. Blood samples were taken from 45 patients with mild, moderate and severe hemophilia A. The TGA-CAT method was performed at both centers while the INNOVANCE ETP was only performed at the Stockholm center. Correlation between parameters was evaluated using Spearman's rank correlation test. For determination of the TGA-CAT inter-laboratory variability, Bland-Altman plots were used. The correlation for the INNOVANCE ETP and TGA-CAT methods with FVIII:C in persons with hemophilia (PWH) was r=0.701 and r=0.734 respectively.The correlation between the two methods was r=0.546.When dividing the study material into disease severity groups (mild, moderate and severe) based on FVIII levels, both methods fail to discriminate between them.The variability of the TGA-CAT results performed at the two centers was reduced after normalization; before normalization, 29% of values showed less than ±10% difference while after normalization the number increased to 41%. Both methods correlate in an equal manner to FVIII:C in PWH but show a poor correlation with each other. The level of agreement for the TGA-CAT method was poor though slightly improved after normalization of data. Further improvement of standardization of these methods is warranted.

BjussuSP-I: a new thrombin-like enzyme isolated from Bothrops jararacussu snake venom.

PubMed

Sant' Ana, Carolina D; Ticli, Fabio K; Oliveira, Leandro L; Giglio, Jose R; Rechia, Carem G V; Fuly, André L; Selistre de Araújo, Heloisa S; Franco, João J; Stabeli, Rodrigo G; Soares, Andreimar M; Sampaio, Suely V

2008-11-01

A thrombin-like enzyme named BjussuSP-I, isolated from B. jararacussu snake venom, is an acidic single chain glycoprotein with approximately 6% sugar, Mr=61,000 under reducing conditions and pI approximately 3.8, representing 1.09% of the chromatographic A(280) recovery. BjussuSP-I is a glycosylated serine protease containing both N-linked carbohydrates and sialic acid in its structure. BjussuSP-I showed a high clotting activity upon human plasma, which was inhibited by PMSF, leupeptin, heparin and 1,10-phenantroline. This enzyme showed high stability regarding coagulant activity when analyzed at different temperatures (-70 to 37 degrees C), pHs (4.5 to 8.0), and presence of two divalent metal ions (Ca(2+) and Mg(2+)). It also displayed TAME esterase and proteolytic activities toward natural (fibrinogen and fibrin) and synthetic (BAPNA) substrates, respectively, being also inhibited by PMSF and leupeptin. BjussuSP-I can induce production of polyclonal antibodies able to inhibit its clotting activity, but unable to inhibit its proteolytic activity on fibrinogen. The enzyme also showed crossed immunoreactivity against 11 venom samples of Bothrops, 1 of Crotalus, and 1 of Calloselasma snakes, in addition of LAAO isolated from B. moojeni venom. It displayed neither hemorrhagic, myotoxic, edema-inducing profiles nor proteolytic activity on casein. BjussuSP-I showed an N-terminal sequence (VLGGDECDINEHPFLA FLYS) similar to other thrombin-like enzymes from snake venoms. Based on its biochemical, enzymatic and pharmacological characteristics, BjussuSP-I was identified as a new thrombin-like enzyme isoform from Bothrops jararacussu snake venom.

Molecular Machine Powered Surface Programmatic Chain Reaction for Highly Sensitive Electrochemical Detection of Protein.

PubMed

Zhu, Jing; Gan, Haiying; Wu, Jie; Ju, Huangxian

2018-04-17

A bipedal molecular machine powered surface programmatic chain reaction was designed for electrochemical signal amplification and highly sensitive electrochemical detection of protein. The bipedal molecular machine was built through aptamer-target specific recognition for the binding of one target protein with two DNA probes, which hybridized with surface-tethered hairpin DNA 1 (H1) via proximity effect to expose the prelocked toehold domain of H1 for the hybridization of ferrocene-labeled hairpin DNA 2 (H2-Fc). The toehold-mediated strand displacement reaction brought the electrochemical signal molecule Fc close to the electrode and meanwhile released the bipedal molecular machine to traverse the sensing surface by the surface programmatic chain reaction. Eventually, a large number of duplex structures of H1-H2 with ferrocene groups facing to the electrode were formed on the sensor surface to generate an amplified electrochemical signal. Using thrombin as a model target, this method showed a linear detection range from 2 pM to 20 nM with a detection limit of 0.76 pM. The proposed detection strategy was enzyme-free and allowed highly sensitive and selective detection of a variety of protein targets by using corresponding DNA-based affinity probes, showing potential application in bioanalysis.

Neuroprotective Effects of 17β-Estradiol against Thrombin-Induced Apoptosis in Primary Cultured Cortical Neurons.

PubMed

Bao, Lei; Zhou, Su; Zhao, Hui; Zu, Jie; He, Qianqian; Ye, Xinchun; Cui, Guiyun

2015-01-01

17β-estradiol (E2) is a powerful neuroprotective agent in the central nervous system; however, little is known about its effects on intracerebral hemorrhage. This study examined the effects of E2 on thrombin-induced apoptosis in vitro and investigated the potential mechanisms. Primary cultured cortical neurons were treated with E2 or vehicle and then the cells were exposed to thrombin. Neuronal apoptosis was assessed by flow cytometry. The phosphorylated c-Jun-N-terminal kinase (p-JNK), phosphorylated extracellular signal-regulated kinases 1/2 (p-ERK1/2), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) and caspase-3 were assayed by western blot. Consequently, we found that E2 has significantly reduced the apoptosis in thrombin-treated neurons. E2 also exhibited a downregulation in the ratio of Bax/Bcl-2, caspase-3 and p-JNK. However, E2 had little effect on p-ERK1/2 proteins activation. Taken together, E2 has shown neuroprotective effects on thrombin-induced neuronal apoptosis, and the molecular mechanisms may correlate with the inhibition of the JNK signaling pathway. © 2015 S. Karger AG, Basel.

The possible involvement of protein phosphatase 1 in thrombin-induced Ca2+ influx of human platelets.

PubMed

Murata, K; Sakon, M; Kambayashi, J; Yukawa, M; Yano, Y; Fujitani, K; Kawasaki, T; Shiba, E; Mori, T

1993-04-01

Protein phosphatase 1 is considered to be involved in thrombin-induced platelet activation (Murata et al., Biochem Int 26:327-334, 1992). To clarify the mechanism, we examined the effects of protein phosphatase 1 and 2A inhibitors (calyculin A, tautomycin, okadaic acid) on Ca2+ influx. In the presence of 1 mM Ca2+, thrombin- (0.1 U/ml) induced platelet aggregation and ATP release were inhibited by calyculin A, while this inhibitory effect was abolished in the absence of Ca2+ (EGTA 1 mM). Furthermore, thrombin-induced Mn2+ influx but not intracellular Ca2+ mobilization was inhibited by calyculin A in a dose-related manner. Calyculin A also blocked the ongoing Ca2+ influx when added 3 min after thrombin stimulation. Similar inhibitory effects were observed with okadaic acid and tautomycin in the same potency sequence as the reported one for protein phosphatase 1 (calyculin A > tautomycin > okadaic acid). These results suggest that the anti-platelet effects of phosphatase inhibitors are due to the inhibition of Ca2+ influx and that protein phosphatase 1 plays a key role in the regulation of receptor operated Ca2+ channel of human platelets.

Protein Z efficiently depletes thrombin generation in disseminated intravascular coagulation with poor prognosis.

PubMed

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

2016-01-01

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.

Detection Techniques for Biomolecules using Semi-Conductor Nanocrystals and Magnetic Beads as Labels

NASA Astrophysics Data System (ADS)

Chatterjee, Esha

Continued interest in the development of miniaturized and portable analytical platforms necessitates the exploration of sensitive methods for the detection of trace analytes. Nanomaterials, on account of their unique physical and chemical properties, are not only able to overcome many limitations of traditional detection reagents but also enable the exploration of many new signal transduction technologies. This dissertation presents a series of investigations of alternative detection techniques for biomolecules, involving the use of semi-conductor nanocrystals and magnetic beads as labels. Initial research focused on the development of quantum dot-encapsulating liposomes as a novel fluorescent label for immunoassays. This hybrid nanomaterial was anticipated to overcome the drawbacks presented by traditional fluorophores as well as provide significant signal amplification. Quantum dot-encapsulating liposomes were synthesized by the method of thin film hydration and characterized. The utility of these composite nanostructures for bioanalysis was demonstrated. However, the longterm instability of the liposomes hampered quantitative development. A second approach for assay development exploited the ability of gold nanoparticles to quench the optical signals obtained from quantum dots. The goal of this study was to demonstrate the feasibility of using aptamer-linked nanostructures in FRET-based quenching for the detection of proteins. Thrombin was used as the model analyte in this study. Experimental parameters for the assay were optimized. The assay simply required the mixing of the sample with the reagents and could be completed in less than an hour. The limit of detection for thrombin by this method was 5 nM. This homogeneous assay can be easily adapted for the detection of a wide variety of biochemicals. The novel technique of ferromagnetic resonance generated in magnetic bead labels was explored for signal transduction. This inductive detection technique lends itself to miniaturization, is capable of mass production and is inexpensive to fabricate. The device consisted of a microwave circuit in which a slotline and a coplanar waveguide were integrated with a biochemically activated sensor area. The magnetic beads were immobilized at the sensor area by bio-specific reactions. Experiments conducted on this prototype show promising results for using ferromagnetic resonance -based detection of magnetic labels for fabrication of portable and inexpensive sensor devices. The next stage of work addresses the issue of patterning of sensing surfaces with biomolecules. The ability to selectively immobilize biomolecules on surfaces has far-reaching applications, including sensor development. A simple and widely applicable method for the photopatterning of chitosan films with biotin was presented. Chitosan is a biocompatible and biodegradable polymer. The proposed method was capable of forming spatially defined biotin features on the order of tens of microns, together with a significant reduction of non-specific protein binding and increase in hydrophilicity of the sensor surface. The entire patterning process, inclusive of the blocking step, could be completed in under an hour. This straightforward method for the selective patterning of the biocompatible polymer chitosan is expected to be widely useful in the field of bioanalysis.

Host defense peptides of thrombin modulate inflammation and coagulation in endotoxin-mediated shock and Pseudomonas aeruginosa sepsis.

PubMed

Kalle, Martina; Papareddy, Praveen; Kasetty, Gopinath; Mörgelin, Matthias; van der Plas, Mariena J A; Rydengård, Victoria; Malmsten, Martin; Albiger, Barbara; Schmidtchen, Artur

2012-01-01

Gram-negative sepsis is accompanied by a disproportionate innate immune response and excessive coagulation mainly induced by endotoxins released from bacteria. Due to rising antibiotic resistance and current lack of other effective treatments there is an urgent need for new therapies. We here 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 in vitro and in vivo. Furthermore, they interfere with coagulation by modulating contact activation and tissue factor-mediated clotting in vitro, leading to normalization of coagulation responses in vivo, a previously unknown function of host defense peptides. In a mouse model of Pseudomonas aeruginosa sepsis, the peptide GKY25, while mediating a modest antimicrobial effect, significantly inhibited the pro-inflammatory response, decreased fibrin deposition and leakage in the lungs, as well as reduced mortality. Taken together, the capacity of such thrombin-derived peptides to simultaneously modulate bacterial levels, pro-inflammatory responses, and coagulation, renders them attractive therapeutic candidates for the treatment of invasive infections and sepsis.

Isolation, cloning and structural characterisation of boophilin, a multifunctional Kunitz-type proteinase inhibitor from the cattle tick.

PubMed

Macedo-Ribeiro, Sandra; Almeida, Carla; Calisto, Bárbara M; Friedrich, Thomas; Mentele, Reinhard; Stürzebecher, Jörg; Fuentes-Prior, Pablo; Pereira, Pedro José Barbosa

2008-02-20

Inhibitors of coagulation factors from blood-feeding animals display a wide variety of structural motifs and inhibition mechanisms. We have isolated a novel inhibitor from the cattle tick Boophilus microplus, one of the most widespread parasites of farm animals. The inhibitor, which we have termed boophilin, has been cloned and overexpressed in Escherichia coli. Mature boophilin is composed of two canonical Kunitz-type domains, and inhibits not only the major procoagulant enzyme, thrombin, but in addition, and by contrast to all other previously characterised natural thrombin inhibitors, significantly interferes with the proteolytic activity of other serine proteinases such as trypsin and plasmin. The crystal structure of the bovine alpha-thrombin.boophilin complex, refined at 2.35 A resolution reveals a non-canonical binding mode to the proteinase. The N-terminal region of the mature inhibitor, Q16-R17-N18, binds in a parallel manner across the active site of the proteinase, with the guanidinium group of R17 anchored in the S(1) pocket, while the C-terminal Kunitz domain is negatively charged and docks into the basic exosite I of thrombin. This binding mode resembles the previously characterised thrombin inhibitor, ornithodorin which, unlike boophilin, is composed of two distorted Kunitz modules. Unexpectedly, both boophilin domains adopt markedly different orientations when compared to those of ornithodorin, in its complex with thrombin. The N-terminal boophilin domain rotates 9 degrees and is displaced by 6 A, while the C-terminal domain rotates almost 6 degrees accompanied by a 3 A displacement. The reactive-site loop of the N-terminal Kunitz domain of boophilin with its P(1) residue, K31, is fully solvent exposed and could thus bind a second trypsin-like proteinase without sterical restraints. This finding explains the formation of a ternary thrombin.boophilin.trypsin complex, and suggests a mechanism for prothrombinase inhibition in vivo.

Essential Role of Cofilin-1 in Regulating Thrombin-induced RelA/p65 Nuclear Translocation and Intercellular Adhesion Molecule 1 (ICAM-1) Expression in Endothelial Cells*

PubMed Central

Fazal, Fabeha; Bijli, Kaiser M.; Minhajuddin, Mohd; Rein, Theo; Finkelstein, Jacob N.; Rahman, Arshad

2009-01-01

Activation of RhoA/Rho-associated kinase (ROCK) pathway and the associated changes in actin cytoskeleton induced by thrombin are crucial for activation of NF-κB and expression of its target gene ICAM-1 in endothelial cells. However, the events acting downstream of RhoA/ROCK to mediate these responses remain unclear. Here, we show a central role of cofilin-1, an actin-binding protein that promotes actin depolymerization, in linking RhoA/ROCK pathway to dynamic alterations in actin cytoskeleton that are necessary for activation of NF-κB and thereby expression of ICAM-1 in these cells. Stimulation of human umbilical vein endothelial cells with thrombin resulted in Ser3 phosphorylation/inactivation of cofilin and formation of actin stress fibers in a ROCK-dependent manner. RNA interference knockdown of cofilin-1 stabilized the actin filaments and inhibited thrombin- and RhoA-induced NF-κB activity. Similarly, constitutively inactive mutant of cofilin-1 (Cof1-S3D), known to stabilize the actin cytoskeleton, inhibited NF-κB activity by thrombin. Overexpression of wild type cofilin-1 or constitutively active cofilin-1 mutant (Cof1-S3A), known to destabilize the actin cytoskeleton, also impaired thrombin-induced NF-κB activity. Additionally, depletion of cofilin-1 was associated with a marked reduction in ICAM-1 expression induced by thrombin. The effect of cofilin-1 depletion on NF-κB activity and ICAM-1 expression occurred downstream of IκBα degradation and was a result of impaired RelA/p65 nuclear translocation and consequently, RelA/p65 binding to DNA. Together, these data show that cofilin-1 occupies a central position in RhoA-actin pathway mediating nuclear translocation of RelA/p65 and expression of ICAM-1 in endothelial cells. PMID:19483084

Essential role of cofilin-1 in regulating thrombin-induced RelA/p65 nuclear translocation and intercellular adhesion molecule 1 (ICAM-1) expression in endothelial cells.

PubMed

Fazal, Fabeha; Bijli, Kaiser M; Minhajuddin, Mohd; Rein, Theo; Finkelstein, Jacob N; Rahman, Arshad

2009-07-31

Activation of RhoA/Rho-associated kinase (ROCK) pathway and the associated changes in actin cytoskeleton induced by thrombin are crucial for activation of NF-kappaB and expression of its target gene ICAM-1 in endothelial cells. However, the events acting downstream of RhoA/ROCK to mediate these responses remain unclear. Here, we show a central role of cofilin-1, an actin-binding protein that promotes actin depolymerization, in linking RhoA/ROCK pathway to dynamic alterations in actin cytoskeleton that are necessary for activation of NF-kappaB and thereby expression of ICAM-1 in these cells. Stimulation of human umbilical vein endothelial cells with thrombin resulted in Ser(3) phosphorylation/inactivation of cofilin and formation of actin stress fibers in a ROCK-dependent manner. RNA interference knockdown of cofilin-1 stabilized the actin filaments and inhibited thrombin- and RhoA-induced NF-kappaB activity. Similarly, constitutively inactive mutant of cofilin-1 (Cof1-S3D), known to stabilize the actin cytoskeleton, inhibited NF-kappaB activity by thrombin. Overexpression of wild type cofilin-1 or constitutively active cofilin-1 mutant (Cof1-S3A), known to destabilize the actin cytoskeleton, also impaired thrombin-induced NF-kappaB activity. Additionally, depletion of cofilin-1 was associated with a marked reduction in ICAM-1 expression induced by thrombin. The effect of cofilin-1 depletion on NF-kappaB activity and ICAM-1 expression occurred downstream of IkappaBalpha degradation and was a result of impaired RelA/p65 nuclear translocation and consequently, RelA/p65 binding to DNA. Together, these data show that cofilin-1 occupies a central position in RhoA-actin pathway mediating nuclear translocation of RelA/p65 and expression of ICAM-1 in endothelial cells.

Dynamics Govern Specificity of a Protein-Protein Interface: Substrate Recognition by Thrombin

PubMed Central

Fuchs, Julian E.; Huber, Roland G.; Waldner, Birgit J.; Kahler, Ursula; von Grafenstein, Susanne; Kramer, Christian; Liedl, Klaus R.

2015-01-01

Biomolecular recognition is crucial in cellular signal transduction. Signaling is mediated through molecular interactions at protein-protein interfaces. Still, specificity and promiscuity of protein-protein interfaces cannot be explained using simplistic static binding models. Our study rationalizes specificity of the prototypic protein-protein interface between thrombin and its peptide substrates relying solely on binding site dynamics derived from molecular dynamics simulations. We find conformational selection and thus dynamic contributions to be a key player in biomolecular recognition. Arising entropic contributions complement chemical intuition primarily reflecting enthalpic interaction patterns. The paradigm “dynamics govern specificity” might provide direct guidance for the identification of specific anchor points in biomolecular recognition processes and structure-based drug design. PMID:26496636

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

PubMed

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

2017-02-10

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.

Bombesin and thrombin affect discrete pools of intracellular calcium through different G-proteins.

PubMed Central

Wang, J L; Kalyanaraman, S; Vivo, M D; Gautam, N

1996-01-01

In mouse NIH 3T3 cells, the mitogens bombesin and thrombin induced Ca2+ release from intracellular stores. Ca2+ release induced by bombesin was inhibited by the Ca(2+)-ATPase inhibitor thapsigargin, while Ca2+ release induced by thrombin was unaffected by this agent. The Ca(2+)-release response to bombesin was not affected by pertussis toxin, but the response to thrombin was abolished by the toxin. Stable transfectants overexpressing the G-protein subunit type alpha 9 showed an accentuated response to bombesin, indicating that the bombesin receptor was coupled to a Gq-like G-protein. Together, these results show that the two mitogenic receptors are coupled to distinct G-proteins that affect functionally different pools of Ca2+. Organization of signalling pathways in this manner may allow cells to differentially encode information from different signals. PMID:8947471

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

NASA Astrophysics Data System (ADS)

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

2017-02-01

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.

Procoagulant effects of lung cancer chemotherapy: impact on microparticles and cell-free DNA.

PubMed

Lysov, Zakhar; Dwivedi, Dhruva J; Gould, Travis J; Liaw, Patricia C

2017-01-01

Lung cancer is the second leading type of cancer, with venous thromboembolism being the second leading cause of death. Studies have shown increased levels of microparticles and cell-free DNA (CFDNA) in cancer patients, which can activate coagulation through extrinsic and intrinsic pathways, respectively. However, the impact of lung cancer chemotherapy on microparticle and/or CFDNA generation is not completely understood. The aim of the study was to study the effects of platinum-based chemotherapeutic agents on generation of procoagulant microparticles and CFDNA in vitro and in vivo. Microparticles were isolated from chemotherapy-treated monocytes, human umbilical vein endothelial cells, or cancer cells. Tissue factor (TF) and phosphatidylserine levels were characterized and thrombin/factor Xa generation assays were used to determine microparticle procoagulant activity. CFDNA levels were isolated from cell supernatants and plasma. A murine xenograft model of human lung carcinoma was used to study the procoagulant effects of TF microparticles and CFDNA in vivo. In vitro, platinum-based chemotherapy induced TF/phosphatidylserine microparticle shedding from A549 and A427 lung cancers cells, which enhanced thrombin generation in plasma in a FVII-dependent manner. CFDNA levels were increased in supernatants of chemotherapy-treated neutrophils and plasma of chemotherapy-treated mice. TF microparticles were elevated in plasma of chemotherapy-treated tumour-bearing mice. Plasma CFDNA levels are increased in chemotherapy-treated tumour-free mice and correlate with increased thrombin generation. In tumour-bearing mice, chemotherapy increases plasma levels of CFDNA and TF/phosphatidylserine microparticles. Platinum-based chemotherapy induces the shedding of TF/phosphatidylserine microparticles from tumour cells and the release of CFDNA from host neutrophils.

Solid-state probe based electrochemical aptasensor for cocaine: a potentially convenient, sensitive, repeatable, and integrated sensing platform for drugs.

PubMed

Du, Yan; Chen, Chaogui; Yin, Jianyuan; Li, Bingling; Zhou, Ming; Dong, Shaojun; Wang, Erkang

2010-02-15

Aptamers, which are artificial oligonucleotides selected in vitro, have been employed to design novel biosensors (i.e., aptasensors). In this work, we first constructed a label-free electrochemical aptasensor introducing a probe immobilization technique by the use of a layer-by-layer (LBL) self-assembled multilayer with ferrocene-appended poly(ethyleneimine) (Fc-PEI) on an indium tin oxide (ITO) array electrode for detection of cocaine. The Fc-PEI and gold nanoparticles (AuNPs) were LBL assembled on the electrode surface via electrostatic interaction. Then, cocaine aptamer fragments, SH-C2, were covalently labeled onto the outermost AuNP layer. When the target cocaine and cocaine aptamer C1 were present simultaneously, the SH-C2 layer hybridized partly with C1 to bind the cocaine, which led to a decreased differential pulse voltammetry (DPV) signal of Fc-PEI. This DPV signal change could be used to sensitively detect cocaine with the lowest detectable concentration down to 0.1 microM and the detection range up to 38.8 microM, which falls in the the expected range for medical use of detecting drug abuse involving cocaine. Meanwhile, the sensor was specific to cocaine in complex biologic fluids such as human plasma, human saliva, etc. The sensing strategy had general applicability, and the detection of thrombin could also be realized, displayed a low detection limit, and exhibited worthiness to other analytes. The aptasensor based on the array electrode held promising potential for integration of the sensing ability in multianalysis for simultaneous detection.

A TiS2 nanosheet enhanced fluorescence polarization biosensor for ultra-sensitive detection of biomolecules

NASA Astrophysics Data System (ADS)

Li, Xiang; Ding, Xuelian; Li, Yongfang; Wang, Linsong; Fan, Jing

2016-05-01

Development of new strategies for the sensitive and selective detection of ultra-low concentrations of specific cancer markers is of great importance for assessing cancer therapeutics due to its crucial role in early clinical diagnoses and biomedical applications. In this work, we have developed two types of fluorescence polarization (FP) amplification assay strategies for the detection of biomolecules by using TiS2 as a FP enhancer and Zn2+-dependent self-hydrolyzing deoxyribozymes as catalysts to realize enzyme-catalyzed target-recycling signal amplification. One approach is based on the terminal protection of small-molecule-linked DNA, in which biomolecular binding to small molecules in DNA-small-molecule chimeras can protect the conjugated DNA from degradation by exonuclease I (Exo I); the other approach is based on the terminal protection of biomolecular bound aptamer DNA, in which biomolecules directly bound to the single strand aptamer DNA can protect the ssDNA from degradation by Exo I. We select folate receptor (FR) and thrombin (Tb) as model analytes to verify the current concept. It is shown that under optimized conditions, our strategies exhibit high sensitivity and selectivity for the quantification of FR and Tb with low detection limits (0.003 ng mL-1 and 0.01 pM, respectively). Additionally, this strategy is a simple ``mix and detect'' approach, and does not require any separation steps. This biosensor is also utilized in the analysis of real biological samples, the results agree well with those obtained by the enzyme-linked immunosorbent assay (ELISA).Development of new strategies for the sensitive and selective detection of ultra-low concentrations of specific cancer markers is of great importance for assessing cancer therapeutics due to its crucial role in early clinical diagnoses and biomedical applications. In this work, we have developed two types of fluorescence polarization (FP) amplification assay strategies for the detection of biomolecules by using TiS2 as a FP enhancer and Zn2+-dependent self-hydrolyzing deoxyribozymes as catalysts to realize enzyme-catalyzed target-recycling signal amplification. One approach is based on the terminal protection of small-molecule-linked DNA, in which biomolecular binding to small molecules in DNA-small-molecule chimeras can protect the conjugated DNA from degradation by exonuclease I (Exo I); the other approach is based on the terminal protection of biomolecular bound aptamer DNA, in which biomolecules directly bound to the single strand aptamer DNA can protect the ssDNA from degradation by Exo I. We select folate receptor (FR) and thrombin (Tb) as model analytes to verify the current concept. It is shown that under optimized conditions, our strategies exhibit high sensitivity and selectivity for the quantification of FR and Tb with low detection limits (0.003 ng mL-1 and 0.01 pM, respectively). Additionally, this strategy is a simple ``mix and detect'' approach, and does not require any separation steps. This biosensor is also utilized in the analysis of real biological samples, the results agree well with those obtained by the enzyme-linked immunosorbent assay (ELISA). Electronic supplementary information (ESI) available: Tables S1-S4, Scheme S1, Fig. S1-S10. See DOI: 10.1039/c6nr00946h

Pericellular Ca2+ recycling potentiates thrombin-evoked Ca2+ signals in human platelets

PubMed Central

Sage, Stewart O; Pugh, Nicholas; Farndale, Richard W; Harper, Alan G S

2013-01-01

We have previously demonstrated that Na+/Ca2+ exchangers (NCXs) potentiate Ca2+ signaling evoked by thapsigargin in human platelets, via their ability to modulate the secretion of autocoids from dense granules. This link was confirmed in platelets stimulated with the physiological agonist, thrombin, and experiments were performed to examine how Ca2+ removal by the NCX modulates platelet dense granule secretion. In cells loaded with the near-membrane indicator FFP-18, thrombin stimulation was observed to elicit an NCX-dependent accumulation of Ca2+ in a pericellular region around the platelets. To test whether this pericellular Ca2+ accumulation might be responsible for the influence of NCXs over platelet function, platelets were exposed to fast Ca2+ chelators or had their glycocalyx removed. Both manipulations of the pericellular Ca2+ rise reduced thrombin-evoked Ca2+ signals and dense granule secretion. Blocking Ca2+-permeable ion channels had a similar effect, suggesting that Ca2+ exported into the pericellular region is able to recycle back into the platelet cytosol. Single cell imaging with extracellular Fluo-4 indicated that thrombin-evoked rises in extracellular [Ca2+] occurred within the boundary described by the cell surface, suggesting their presence within the open canalicular system (OCS). FFP-18 fluorescence was similarly distributed. These data suggest that upon thrombin stimulation, NCX activity creates a rise in [Ca2+] within the pericellular region of the platelet from where it recycles back into the platelet cytosol, acting to both accelerate dense granule secretion and maintain the initial rise in cytosolic [Ca2+]. PMID:24303163

Impaired thrombin generation and fibrin clot formation in patients with dilutional coagulopathy during major surgery.

PubMed

Schols, S E M; Lancé, M D; Feijge, M A H; Damoiseaux, J; Marcus, M A; Hamulyák, K; Ten Cate, H; Heemskerk, J W M; van Pampus, E C M

2010-02-01

Patients subjected to haemodilution during surgery are at increased risk of bleeding. We hypothesised that, in the acquired dilutional coagulopathy, insufficient haemostasis is due to either insufficient thrombin generation or insufficient fibrin clot formation. In tissue factor-activated plasmas from patients with coagulation deficiency, we measured time curves of thrombin generation and fibrin clot formation (thromboelastography). Investigated were in study A: 10 patients treated with vitamin K antagonist and five healthy subjects; in study B: 30 patients undergoing cardiopulmonary bypass (CPB) surgery and infused with on average 2,000 ml crystalloids and colloids (no major bleeding); in study C: 58 patients undergoing major general surgery, and transfused with >5,000 ml crystalloids, colloids and red cell concentrates, who experienced major bleeding and were post-transfused with fresh frozen plasma. The treatment with vitamin K antagonist led to a progressive reduction in thrombin generation but not fibrin clot formation. In CPB patients, plasma factor levels post-surgery were 53-60% of normal. This was accompanied by moderate reduction in both haemostatic processes. In plasmas from patients undergoing major surgery, factor levels were 38-41% of normal, and these levels increased after plasma transfusion. Taking preset thresholds for normal thrombin generation and fibrin clot formation, at least one of these processes was low in 88-93% of the patients with (persistent) bleeding, but only in 40-53% of the patients without bleeding. In conclusion, the ability of thrombin generation and fibrin clot formation is independently reduced in acquired dilutional coagulopathy, while minimal levels of both are required for adequate haemostasis.

Probing light chain mutation effects on thrombin via molecular dynamics simulations and machine learning.

PubMed

Xiao, Jiajie; Melvin, Ryan L; Salsbury, Freddie R

2018-03-02

Thrombin is a key component for chemotherapeutic and antithrombotic therapy development. As the physiologic and pathologic roles of the light chain still remain vague, here, we continue previous efforts to understand the impacts of the disease-associated single deletion of LYS9 in the light chain. By combining supervised and unsupervised machine learning methodologies and more traditional structural analyses on data from 10 μs molecular dynamics simulations, we show that the conformational ensemble of the ΔK9 mutant is significantly perturbed. Our analyses consistently indicate that LYS9 deletion destabilizes both the catalytic cleft and regulatory functional regions and result in some conformational changes that occur in tens to hundreds of nanosecond scaled motions. We also reveal that the two forms of thrombin each prefer a distinct binding mode of a Na + ion. We expand our understanding of previous experimental observations and shed light on the mechanisms of the LYS9 deletion associated bleeding disorder by providing consistent but more quantitative and detailed structural analyses than early studies in literature. With a novel application of supervised learning, i.e. the decision tree learning on the hydrogen bonding features in the wild-type and ΔK9 mutant forms of thrombin, we predict that seven pairs of critical hydrogen bonding interactions are significant for establishing distinct behaviors of wild-type thrombin and its ΔK9 mutant form. Our calculations indicate the LYS9 in the light chain has both localized and long-range allosteric effects on thrombin, supporting the opinion that light chain has an important role as an allosteric effector.

Modification of tissue-factor mRNA and protein response to thrombin and interleukin 1 by high glucose in cultured human endothelial cells.

PubMed

Boeri, D; Almus, F E; Maiello, M; Cagliero, E; Rao, L V; Lorenzi, M

1989-02-01

Because diabetic vascular disease is accompanied by a state of hypercoagulability, manifested by increased thrombin activity and foci of intravascular coagulation, we investigated whether a specific procoagulant property of the endothelium--production and surface expression of tissue factor--is modified by elevated glucose concentrations. In unperturbed human vascular endothelial cells, tissue factor mRNA and expression of the functional protein were undetectable and were not induced by 10-12 days of exposure to 30 mM glucose. In thrombin-stimulated cultures, tissue-factor expression was related inversely to cellular density, with confluent cultures producing (per 10(5) cells) half the amount of tissue factor measured in sparse cultures. Cells exposed to high glucose and studied when cell number and thymidine incorporation were identical to control cells manifested increased tissue-factor mRNA level and functional protein production in response to thrombin (P = .002). This effect was not attributable to hypertonicity and was not observed after short exposure to high glucose. In contrast, the tissue-factor response to interleukin 1, a modulator of endothelial function in the context of host defense, was decreased in cells cultured in high glucose (P = .04). These findings indicate that exposure to high glucose can alter tissue-factor gene expression in perturbed vascular endothelium. The reciprocal effects of high glucose on the tissue-factor response to thrombin and interleukin 1 points to different pathways of tissue-factor stimulation by the two agents and suggests functional consequences pertinent to the increased thrombin activity and compromised host-defense mechanisms observed in diabetes.

An Investigation of the Characteristics of the Enzyme Thrombin, Suitable for Classwork

ERIC Educational Resources Information Center

Blofield, B. Ann

1972-01-01

Shows how a simple investigation of the enzyme, thrombin, can provide a series of experiments giving information on enzyme characteristics. The results also provide a basis for discussion of the coagulation mechanism and related phenomena. (Author/AL)

[Pancreatic tail pseudoaneurysm: percutaneous treatment by thrombin injection].

PubMed

Pacheco Jiménez, M; Moreno Sánchez, T; Moreno Rodríguez, F; Guillén Rico, M

2014-01-01

Visceral artery pseudoaneurysms secondary to acute and/or chronic pancreatitis are a relatively common and potentially serious complication. Endovascular techniques are the most currently accepted techniques, given the higher morbidity-mortality of surgery. The thrombosis of the pseudoaneurysm using an ultrasound-guided percutaneous thrombin injection is emerging as a useful option in those cases in which endovascular embolisation is not possible. We present the case of a patient with a pseudoaneurysm of the transverse pancreatic artery secondary to chronic pancreatitis, and successfully treated by administering percutaneous thrombin. Copyright © 2011 SERAM. Published by Elsevier Espana. All rights reserved.

Binding of thrombin-activated platelets to a fibrin scaffold through α(IIb)β₃ evokes phosphatidylserine exposure on their cell surface.

PubMed

Brzoska, Tomasz; Suzuki, Yuko; Mogami, Hideo; Sano, Hideto; Urano, Tetsumei

2013-01-01

Recently, by employing intra-vital confocal microscopy, we demonstrated that platelets expose phosphatidylserine (PS) and fibrin accumulate only in the center of the thrombus but not in its periphery. To address the question how exposure of platelet anionic phospholipids is regulated within the thrombus, an in-vitro experiment using diluted platelet-rich plasma was employed, in which the fibrin network was formed in the presence of platelets, and PS exposure on the platelet surface was analyzed using Confocal Laser Scanning Microscopy. Almost all platelets exposed PS after treatment with tissue factor, thrombin or ionomycin. Argatroban abrogated fibrin network formation in all samples, however, platelet PS exposure was inhibited only in tissue factor- and thrombin-treated samples but not in ionomycin-treated samples. FK633, an α(IIb)β₃ antagonist, and cytochalasin B impaired platelet binding to the fibrin scaffold and significantly reduced PS exposure evoked by thrombin. Gly-Pro-Arg-Pro amide abrogated not only fibrin network formation, but also PS exposure on platelets without suppressing platelet binding to fibrin/fibrinogen. These results suggest that outside-in signals in platelets generated by their binding to the rigid fibrin network are essential for PS exposure after thrombin treatment.

Binding of Thrombin-Activated Platelets to a Fibrin Scaffold through αIIbβ3 Evokes Phosphatidylserine Exposure on Their Cell Surface

PubMed Central

Brzoska, Tomasz; Suzuki, Yuko; Mogami, Hideo; Sano, Hideto; Urano, Tetsumei

2013-01-01

Recently, by employing intra-vital confocal microscopy, we demonstrated that platelets expose phosphatidylserine (PS) and fibrin accumulate only in the center of the thrombus but not in its periphery. To address the question how exposure of platelet anionic phospholipids is regulated within the thrombus, an in-vitro experiment using diluted platelet-rich plasma was employed, in which the fibrin network was formed in the presence of platelets, and PS exposure on the platelet surface was analyzed using Confocal Laser Scanning Microscopy. Almost all platelets exposed PS after treatment with tissue factor, thrombin or ionomycin. Argatroban abrogated fibrin network formation in all samples, however, platelet PS exposure was inhibited only in tissue factor- and thrombin-treated samples but not in ionomycin-treated samples. FK633, an αIIbβ3 antagonist, and cytochalasin B impaired platelet binding to the fibrin scaffold and significantly reduced PS exposure evoked by thrombin. Gly-Pro-Arg-Pro amide abrogated not only fibrin network formation, but also PS exposure on platelets without suppressing platelet binding to fibrin/fibrinogen. These results suggest that outside-in signals in platelets generated by their binding to the rigid fibrin network are essential for PS exposure after thrombin treatment. PMID:23383331

Critical role of non-muscle myosin light chain kinase in thrombin-induced endothelial cell inflammation and lung PMN infiltration.

PubMed

Fazal, Fabeha; Bijli, Kaiser M; Murrill, Matthew; Leonard, Antony; Minhajuddin, Mohammad; Anwar, Khandaker N; Finkelstein, Jacob N; Watterson, D Martin; Rahman, Arshad

2013-01-01

The pathogenesis of acute lung injury (ALI) involves bidirectional cooperation and close interaction between inflammatory and coagulation pathways. A key molecule linking coagulation and inflammation is the procoagulant thrombin, a serine protease whose concentration is elevated in plasma and lavage fluids of patients with ALI and acute respiratory distress syndrome (ARDS). However, little is known about the mechanism by which thrombin contributes to lung inflammatory response. In this study, we developed a new mouse model that permits investigation of lung inflammation associated with intravascular coagulation. Using this mouse model and in vitro approaches, we addressed the role of non-muscle myosin light chain kinase (nmMLCK) in thrombin-induced endothelial cell (EC) inflammation and lung neutrophil (PMN) infiltration. Our in vitro experiments revealed a key role of nmMLCK in ICAM-1 expression by its ability to control nuclear translocation and transcriptional capacity of RelA/p65 in EC. When subjected to intraperitoneal thrombin challenge, wild type mice showed a marked increase in lung PMN infiltration via expression of ICAM-1. However, these responses were markedly attenuated in mice deficient in nmMLCK. These results provide mechanistic insight into lung inflammatory response associated with intravascular coagulation and identify nmMLCK as a critical target for modulation of lung inflammation.

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

PubMed Central

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

2011-01-01

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

Mechanisms of pertussis toxin-induced barrier dysfunction in bovine pulmonary artery endothelial cell monolayers.

PubMed

Patterson, C E; Stasek, J E; Schaphorst, K L; Davis, H W; Garcia, J G

1995-06-01

We have previously characterized several G proteins in endothelial cells (EC) as substrates for the ADP-ribosyltransferase activity of both pertussis (PT) and cholera toxin and described the modulation of key EC physiological responses, including gap formation and barrier function, by these toxins. In this study, we investigated the mechanisms involved in PT-mediated regulation of bovine pulmonary artery endothelial cells barrier function. PT caused a dose-dependent increase in albumin transfer, dependent upon action of the holotoxin, since neither the heat-inactivated PT, the isolated oligomer, nor the protomer induced EC permeability. PT-induced gap formation and barrier dysfunction were additive to either thrombin- or thrombin receptor-activating peptide-induced permeability, suggesting that thrombin and PT utilize distinct mechanisms. PT did not result in Ca2+ mobilization or alter either basal or thrombin-induced myosin light chain phosphorylation. However, PT stimulated protein kinase C (PKC) activation, and both PKC downregulation and PKC inhibition attenuated PT-induced permeability, indicating that PKC activity is involved in PT-induced barrier dysfunction. Like thrombin-induced permeability, the PT effect was blocked by prior increases in adenosine 3',5'-cyclic monophosphate. Thus PT-catalyzed ADP-ribosylation of a G protein (possibly other than Gi) may regulate cytoskeletal protein interactions, leading to EC barrier dysfunction.

Critical Role of Non-Muscle Myosin Light Chain Kinase in Thrombin-Induced Endothelial Cell Inflammation and Lung PMN Infiltration

PubMed Central

Fazal, Fabeha; Bijli, Kaiser M.; Murrill, Matthew; Leonard, Antony; Minhajuddin, Mohammad; Anwar, Khandaker N.; Finkelstein, Jacob N.; Watterson, D. Martin; Rahman, Arshad

2013-01-01

The pathogenesis of acute lung injury (ALI) involves bidirectional cooperation and close interaction between inflammatory and coagulation pathways. A key molecule linking coagulation and inflammation is the procoagulant thrombin, a serine protease whose concentration is elevated in plasma and lavage fluids of patients with ALI and acute respiratory distress syndrome (ARDS). However, little is known about the mechanism by which thrombin contributes to lung inflammatory response. In this study, we developed a new mouse model that permits investigation of lung inflammation associated with intravascular coagulation. Using this mouse model and in vitro approaches, we addressed the role of non-muscle myosin light chain kinase (nmMLCK) in thrombin-induced endothelial cell (EC) inflammation and lung neutrophil (PMN) infiltration. Our in vitro experiments revealed a key role of nmMLCK in ICAM-1 expression by its ability to control nuclear translocation and transcriptional capacity of RelA/p65 in EC. When subjected to intraperitoneal thrombin challenge, wild type mice showed a marked increase in lung PMN infiltration via expression of ICAM-1. However, these responses were markedly attenuated in mice deficient in nmMLCK. These results provide mechanistic insight into lung inflammatory response associated with intravascular coagulation and identify nmMLCK as a critical target for modulation of lung inflammation. PMID:23555849

Dabigatran affects thrombin-dependent platelet aggregation after a week-long therapy.

PubMed

Sokol, Juraj; Nehaj, Frantisek; Ivankova, Jela; Mokan, Michal; Mokan, Marian; Stasko, Jan

2018-05-29

Dabigatran is a direct thrombin inhibitor. As the main adverse event is bleeding, it is relevant whether dabigatran has additional effects on platelet function. If so, it could affect the bleeding risk. We aimed to assess in vitro aggregation in patients with atrial fibrillation (AF) receiving dabigatran. We evaluated 32 AF patients treated with dabigatran (study group) and 18 non-anticoagulated non-AF blood donors (control group). We assessed light transmittance platelet aggregation (LTA) with 100 nmol/L γ-thrombin in both groups. The LTA was performed at two time-points in our dabigatran group of patients. The thrombin-induced platelet aggregation was significantly lower two hours after dabigatran was taken compared to baseline measurement (9% ± 6% vs. 29% ± 21%) in our study group. Moreover, we observed that the baseline value of platelet aggregation in patients on dabigatran treatment was significantly lower compared to healthy volunteers (29% ± 21% vs. 89 ± 8). However, one subanalysis showed that this significant reduction in platelet aggregation at baseline was only observed in patients who received dabigatran for over a week. The thrombin-induced platelet aggregation is reduced in non-valvular AF patients receiving dabigatran after a week-long therapy.

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

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

Pozzi, Nicola; Chen, Raymond; Chen, Zhiwei

2011-09-20

Binding of Na{sup +} to thrombin ensures high activity toward physiological substrates and optimizes the procoagulant and prothrombotic roles of the enzyme in vivo. Under physiological conditions of pH and temperature, the binding affinity of Na{sup +} is weak due to large heat capacity and enthalpy changes associated with binding, and the K{sub d} = 80 mM ensures only 64% saturation of the site at the concentration of Na{sup +} in the blood (140 mM). Residues controlling Na{sup +} binding and activation have been identified. Yet, attempts to improve the interaction of Na{sup +} with thrombin and possibly increase catalyticmore » 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.« less

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

PubMed Central

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

2016-01-01

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

Effect of SanOrg123781A, a synthetic hexadecasaccharide, on clot-bound thrombin and factor Xa in vitro and in vivo.

PubMed

Hérault, J-P; Cappelle, M; Bernat, A; Millet, L; Bono, F; Schaeffer, P; Herbert, J-M

2003-09-01

Factor (F)Xa and thrombin bound to the clot during its formation contribute to the propensity of thrombi to activate the coagulation system. The aim of this work was to study the inhibition of clot-bound FXa and clot-bound thrombin by SanOrg123781A, a synthetic hexadecasaccharide that enhances the inhibition of thrombin and FXa by antithrombin (AT). SanOrg123781A, designed to exhibit low non-specific binding to proteins other than AT, was compared with heparin. In buffer, heparin and SanOrg123781A inhibited FXa and thrombin at similar concentrations [concentration inhibiting 50% (IC50) of Xa and IIa activity were, respectively: heparin 120 +/- 7 and 3 +/- 1 ng mL-1; SanOrg123781A 77 +/- 5 and 4 +/- 1 ng mL-1]. In human plasma, the activity of both compounds was reduced, although the activity of heparin was much more affected than that of SanOrg123781A (IC50 values for inhibition of FXa and FIIa activity were, respectively: heparin 100 +/- 5 and 800 +/- 40 ng mL-1; SanOrg123781A 10 +/- 5 and 30 +/- 3 ng mL-1). We demonstrated, in agreement with our previous results, that the procoagulant activity of the clot is essentially due to clot-bound FXa and to some extent to clot-bound thrombin. We showed that heparin and SanOrg123781A were able to inhibit fragment F1+2 generation induced by clot-bound FXa with IC50 values of 2 +/- 0.5 micro g mL-1 and 0.6 +/- 0.2 micro g mL-1, respectively. Both compounds also inhibited clot-bound thrombin activity, the IC50 values of heparin and SanOrg123781A being 1 +/- 0.01 micro g mL-1 and 0.1 +/- 0.1 micro g mL-1, respectively. Moreover, both heparin and SanOrg123781A significantly inhibited fibrinopeptide A generated by the action of clot-bound thrombin on fibrinogen but also by free thrombin generated from prothrombin by clot-bound FXa with IC50 values of 4 +/- 0.6 and 1 +/- 0.1 micro g mL-1, respectively. As with clot-bound enzymatic activities, SanOrg123781A was three times more active than heparin in vivo on fibrinogen accretion onto a pre-existing thrombus and as activators of recombinant tissue-type plasminogen activator-induced thrombolysis. In conclusion, due to the specific activities of SanOrg123781A, this compound is much more active than heparin in the presence of plasma proteins, on clot-bound enzymes and in in vivo models of thrombosis/thrombolysis.

Self-Propelled Dressings Containing Thrombin and Tranexamic Acid Improve Short-Term Survival in a Swine Model of Lethal Junctional Hemorrhage.

PubMed

Baylis, James R; St John, Alexander E; Wang, Xu; Lim, Esther B; Statz, Matthew L; Chien, Diana; Simonson, Eric; Stern, Susan A; Liggins, Richard T; White, Nathan J; Kastrup, Christian J

2016-09-01

Hemorrhage is the leading cause of preventable death in trauma, and hemorrhage from noncompressible junctional anatomic sites is particularly difficult to control. The current standard is QuikClot Combat Gauze packing, which requires 3 min of compression. We have created a novel dressing with calcium carbonate microparticles that can disperse and self-propel upstream against flowing blood. We loaded these microparticles with thrombin and tranexamic acid and tested their efficacy in a swine arterial bleeding model without wound compression. Anesthetized immature female swine received 5 mm femoral arteriotomies to induce severe junctional hemorrhage. Wounds were packed with kaolin-based QuikClot Combat Gauze (KG), propelled thrombin-microparticles with protonated tranexamic acid (PTG), or a non-propelling formulation of the same thrombin-microparticles with non-protonated tranexamic acid (NPTG). Wounds were not compressed after packing. Each animal then received one 15 mL/kg bolus of hydroxyethyl starch solution followed by Lactated Ringer as needed for hypotension (maximum: 100 mL/kg) for up to 3 h. Survival was improved with PTG (3-h survival: 8/8, 100%) compared with KG (3/8, 37.5%) and NPTG (2/8, 25%) (P <0.01). PTG animals maintained lower serum lactate and higher hemoglobin concentrations than NPTG (P <0.05) suggesting PTG decreased severity of subsequent hemorrhagic shock. However, total blood loss, Lactated Ringer infusion volumes, and mean arterial pressures of surviving animals were not different between groups (P >0.05). Thus, in this swine model of junctional arterial hemorrhage, gauze with self-propelled, prothrombotic microparticles improved survival and 2 indicators of hemorrhagic shock when applied without compression, suggesting this capability may enable better treatment of non-compressible junctional wounds.

Effects of MASP-1 of the Complement System on Activation of Coagulation Factors and Plasma Clot Formation

PubMed Central

Hess, Katharina; Ajjan, Ramzi; Phoenix, Fladia; Dobó, József; Gál, Péter; Schroeder, Verena

2012-01-01

Background Numerous interactions between the coagulation and complement systems have been shown. Recently, links between coagulation and mannan-binding lectin-associated serine protease-1 (MASP-1) of the complement lectin pathway have been proposed. Our aim was to investigate MASP-1 activation of factor XIII (FXIII), fibrinogen, prothrombin, and thrombin-activatable fibrinolysis inhibitor (TAFI) in plasma-based systems, and to analyse effects of MASP-1 on plasma clot formation, structure and lysis. Methodology/Principal Findings We used a FXIII incorporation assay and specific assays to measure the activation products prothrombin fragment F1+2, fibrinopeptide A (FPA), and activated TAFI (TAFIa). Clot formation and lysis were assessed by turbidimetric assay. Clot structure was studied by scanning electron microscopy. MASP-1 activated FXIII and, contrary to thrombin, induced FXIII activity faster in the Val34 than the Leu34 variant. MASP-1-dependent generation of F1+2, FPA and TAFIa showed a dose-dependent response in normal citrated plasma (NCP), albeit MASP-1 was much less efficient than FXa or thrombin. MASP-1 activation of prothrombin and TAFI cleavage were confirmed in purified systems. No FPA generation was observed in prothrombin-depleted plasma. MASP-1 induced clot formation in NCP, affected clot structure, and prolonged clot lysis. Conclusions/Significance We show that MASP-1 interacts with plasma clot formation on different levels and influences fibrin structure. Although MASP-1-induced fibrin formation is thrombin-dependent, MASP-1 directly activates prothrombin, FXIII and TAFI. We suggest that MASP-1, in concerted action with other complement and coagulation proteins, may play a role in fibrin clot formation. PMID:22536427

In Vitro Screening of Synthetic Fluorogenic Substrates for Detection of Cancer Procoagulant Activity.

PubMed

Krause, Jason; Frost, Carminita L

2018-04-01

Cancer procoagulant (CP), a direct activator of coagulation factor X, is among one of the tumour cell products or activities which may promote fibrin formation and has been suggested to be selectively associated with the malignant phenotype. At present, the most reliable assay for the quantification of CP activity is the three-stage chromogenic assay which utilises the ability of CP to activate factor X. In this assay, the activation of factor X leads to the formation of activated thrombin from prothrombin and the eventual hydrolyses of a thrombin chromogenic substrate which contains a p-nitroaniline leaving group. The complexity of the three-stage chromogenic assay suggests a need for a direct method of assaying CP activity. This study focuses on the design of a fluorogenic substrate that would enable the direct quantification of CP activity. The results of the study show two promising substrates for the determination of CP activity: Boc-PQVR-AMC and PQVR-AMC. Further analysis showed that Boc-PQVR-AMC could be excluded as a potential substrate for CP since it was also cleaved by collagenase.

Two-Dimensional Fluorescence Difference Spectroscopy of ZnO and Mg Composites in the Detection of Physiological Protein and RNA Interactions

PubMed Central

Hoffman, Amanda; Wu, Xiaotong; Wang, Jianjie; Brodeur, Amanda; Thomas, Rintu; Thakkar, Ravindra; Hadi, Halena; Glaspell, Garry P.; Duszynski, Molly; Wanekaya, Adam; DeLong, Robert K.

2017-01-01

Two-dimensional fluorescence difference spectroscopy (2-D FDS) was used to determine the unique spectral signatures of zinc oxide (ZnO), magnesium oxide (MgO), and 5% magnesium zinc oxide nanocomposite (5% Mg/ZnO) and was then used to demonstrate the change in spectral signature that occurs when physiologically important proteins, such as angiotensin-converting enzyme (ACE) and ribonuclease A (RNase A), interact with ZnO nanoparticles (NPs). When RNase A is bound to 5% Mg/ZnO, the intensity is quenched, while the intensity is magnified and a significant shift is seen when torula yeast RNA (TYRNA) is bound to RNase A and 5% Mg/ZnO. The intensity of 5% Mg/ZnO is quenched also when thrombin and thrombin aptamer are bound to the nanocomposite. These data indicate that RNA–protein interaction can occur unimpeded on the surface of NPs, which was confirmed by gel electrophoresis, and importantly that the change in fluorescence excitation, emission, and intensity shown by 2-D FDS may indicate specificity of biomolecular interactions. PMID:29244716

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

PubMed

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

2005-09-01

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

Evaluation of procoagulant tissue factor expression in canine hemangiosarcoma cell lines.

PubMed

Witter, Lauren E; Gruber, Erika J; Lean, Fabian Z X; Stokol, Tracy

2017-01-01

OBJECTIVE To evaluate expression of procoagulant tissue factor (TF) by canine hemangiosarcoma cells in vitro. SAMPLES 4 canine hemangiosarcoma cell lines (SB-HSA [mouse-passaged cutaneous tumor], Emma [primary metastatic brain tumor], and Frog and Dal-1 [primary splenic tumors]) and 1 nonneoplastic canine endothelial cell line (CnAoEC). PROCEDURES TF mRNA and TF antigen expression were evaluated by quantitative real-time PCR assay and flow cytometry, respectively. Thrombin generation was measured in canine plasma and in coagulation factor-replete or specific coagulation factor-deficient human plasma by calibrated automated thrombography. Corn trypsin inhibitor and annexin V were used to examine contributions of contact activation and membrane-bound phosphatidylserine, respectively, to thrombin generation. RESULTS All cell lines expressed TF mRNA and antigen, with significantly greater expression of both products in SB-HSA and Emma cells than in CnAoEC. A greater percentage of SB-HSA cells expressed TF antigen, compared with other hemangiosarcoma cell lines. All hemangiosarcoma cell lines generated significantly more thrombin than did CnAoEC in canine or factor-replete human plasma. Thrombin generation induced by SB-HSA cells was significantly lower in factor VII-deficient plasma than in factor-replete plasma and was abolished in factor X-deficient plasma; residual thrombin generation in factor VII-deficient plasma was abolished by incubation of cells with annexin V. Thrombin generation by SB-HSA cells was unaffected by the addition of corn trypsin inhibitor. CONCLUSIONS AND CLINICAL RELEVANCE Hemangiosarcoma cell lines expressed procoagulant TF in vitro. Further research is needed to determine whether TF can be used as a biomarker for hemostatic dysfunction in dogs with hemangiosarcoma.

Evaluation of procoagulant tissue factor expression in canine hemangiosarcoma cell lines

PubMed Central

Witter, Lauren E.; Gruber, Erika J.; Lean, Fabian Z. X.; Stokol, Tracy

2017-01-01

OBJECTIVE To evaluate expression of procoagulant tissue factor (TF) by canine hemangiosarcoma cells in vitro. SAMPLES 4 canine hemangiosarcoma cell lines (SB-HSA [mouse-passaged cutaneous tumor], Emma [primary metastatic brain tumor], and Frog and Dal-1 [primary splenic tumors]) and 1 nonneoplastic canine endothelial cell line (CnAoEC). PROCEDURES TF mRNA and TF antigen expression were evaluated by quantitative real-time PCR assay and flow cytometry, respectively. Thrombin generation was measured in canine plasma and in coagulation factor–replete or specific coagulation factor–deficient human plasma by calibrated automated thrombography. Corn trypsin inhibitor and annexin V were used to examine contributions of contact activation and membrane-bound phosphatidylserine, respectively, to thrombin generation. RESULTS All cell lines expressed TF mRNA and antigen, with significantly greater expression of both products in SB-HSA and Emma cells than in CnAoEC. A greater percentage of SB-HSA cells expressed TF antigen, compared with other hemangiosarcoma cell lines. All hemangiosarcoma cell lines generated significantly more thrombin than did CnAoEC in canine or factor-replete human plasma. Thrombin generation induced by SB-HSA cells was significantly lower in factor VII-deficient plasma than in factor-replete plasma and was abolished in factor X–deficient plasma; residual thrombin generation in FVII-deficient plasma was abolished by incubation of cells with annexin V. Thrombin generation by SB-HSA cells was unaffected by the addition of corn trypsin inhibitor. CONCLUSIONS AND CLINICAL RELEVANCE Hemangiosarcoma cell lines expressed procoagulant TF in vitro. Further research is needed to determine whether TF can be used as a biomarker for hemostatic dysfunction in dogs with hemangiosarcoma. PMID:28029283

In vitro efficacy of pro- and anticoagulant strategies in compensated and acutely ill patients with cirrhosis.

PubMed

Lisman, Ton; Kleiss, Simone; Patel, Vishal C; Fisher, Caleb; Adelmeijer, Jelle; Bos, Sarah; Singanayagam, Arjuna; Stoy, Sidsel Hyldgaard; Shawcross, Debbie L; Bernal, William

2018-05-16

A simultaneous decline in pro- and anticoagulant drivers in patients with liver diseases results in a 'rebalanced' hemostatic system, even in acutely ill patients. Nevertheless, both bleeding and thrombotic events are common. Here, we explored efficacy of pro- and antihemostatic strategies in compensated and acutely ill cirrhotics which may be unpredictable given the profound hemostatic changes. We tested the effects in vitro of the addition of clinically relevant doses of commonly used pro- and antihemostatic strategies in plasma from healthy individuals (n=30) and patients with compensated (n=18) and acutely decompensated cirrhosis (n=18), and acute-on-chronic liver failure (n=10). We used thrombin generation tests and fibrin clot permeability assays to assess potency of various approaches. Fresh frozen plasma and recombinant factor VIIa modestly increased thrombin generation (10-20%). Prothrombin complex concentrate increased thrombin generation 2-fold in controls and 2-4-fold in patients. Clot permeability decreased after addition of fibrinogen concentrate by 51% in controls and by 50-60% in patients. Low molecular weight heparin decreased thrombin generation by 18% in controls and by 23-54% in patients. Similarly, dabigatran decreased thrombin generation by 33% in controls and by 47-100% in patients. In contrast, rivaroxaban decreased thrombin generation by 55% in controls, but only by 11-38% in patients. These in vitro data suggest little prohemostatic effect of fresh frozen plasma and recombinant factor VIIa in acutely ill cirrhotics, whereas prothrombin complex concentrate and fibrinogen concentrate clearly improved hemostasis. Furthermore, our data suggest the requirement for dose-adjustments of commonly used anticoagulants in these patients. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Critical Role for CD38-mediated Ca2+ Signaling in Thrombin-induced Procoagulant Activity of Mouse Platelets and Hemostasis*

PubMed Central

Mushtaq, Mazhar; Nam, Tae-Sik; Kim, Uh-Hyun

2011-01-01

CD38, a multifunctional enzyme that catalyzes the synthesis of intracellular Ca2+ messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), is known to be expressed on platelets. However, the role of CD38 in platelets remains unclear. Our present results show that treatment of platelets with thrombin results in a rapid and sustained Ca2+ signal, resulting from a coordinated interplay of Ca2+-mobilizing messengers, inositol 1,4,5-trisphosphate, cADPR, and NAADP. By dissecting the signaling pathway using various agents, we delineated that cADPR and NAADP are sequentially produced through CD38 internalization by protein kinase C via myosin heavy chain IIA following phospholipase C activation in thrombin-induced platelets. An inositol 1,4,5-trisphosphate receptor antagonist blocked the thrombin-induced formation of cADPR and NAADP as well as Ca2+ signals. An indispensable response of platelets relying on cytosolic calcium is the surface exposure of phosphatidylserine (PS), which implicates platelet procoagulant activity. Scrutinizing this parameter reveals that CD38+/+ platelets fully express PS on the surface when stimulated with thrombin, whereas this response was decreased on CD38−/− platelets. Similarly, PS exposure and Ca2+ signals were attenuated when platelets were incubated with 8-bromo-cADPR, bafilomycin A1, and a PKC inhibitor. Furthermore, in vivo, CD38-deficient mice exhibited longer bleeding times and unstable formation of thrombus than wild type mice. These results demonstrate that CD38 plays an essential role in thrombin-induced procoagulant activity of platelets and hemostasis via Ca2+ signaling mediated by its products, cADPR and NAADP. PMID:21339289

What Is Deep Vein Thrombosis?

MedlinePlus

... the blood to trigger the activity of the enzyme thrombin. Active thrombin then forms long protein strands that clump together with platelets and red blood cells to form clots. Read less Risk Factors Risk factors for VTE include a history of a previous VTE event; surgery; medical conditions ...

Structure and Sequence Search on Aptamer-Protein Docking

NASA Astrophysics Data System (ADS)

Xiao, Jiajie; Bonin, Keith; Guthold, Martin; Salsbury, Freddie

2015-03-01

Interactions between proteins and deoxyribonucleic acid (DNA) play a significant role in the living systems, especially through gene regulation. However, short nucleic acids sequences (aptamers) with specific binding affinity to specific proteins exhibit clinical potential as therapeutics. Our capillary and gel electrophoresis selection experiments show that specific sequences of aptamers can be selected that bind specific proteins. Computationally, given the experimentally-determined structure and sequence of a thrombin-binding aptamer, we can successfully dock the aptamer onto thrombin in agreement with experimental structures of the complex. In order to further study the conformational flexibility of this thrombin-binding aptamer and to potentially develop a predictive computational model of aptamer-binding, we use GPU-enabled molecular dynamics simulations to both examine the conformational flexibility of the aptamer in the absence of binding to thrombin, and to determine our ability to fold an aptamer. This study should help further de-novo predictions of aptamer sequences by enabling the study of structural and sequence-dependent effects on aptamer-protein docking specificity.

Serological analysis of patients treated with a new surgical hemostat containing bovine proteins and autologous plasma.

PubMed

Nelson, P A; Powers, J N; Estridge, T D; Elder, E A; Alea, A D; Sidhu, P K; Sehl, L C; DeLustro, F A

2001-01-01

A randomized, controlled clinical study of the management of diffuse bleeding with CoStasis surgical hemostat, a new hemostat containing bovine thrombin and collagen with the patient's own plasma, included patients undergoing cardiac, hepatic, iliac, and general surgery. Sera from 92 patients treated with CoStasis and 84 control patients were collected preoperatively and at a post surgical follow-up of 8 weeks. Among the control group, 57 patients were treated with Instat collagen sponge in noncardiac indications. Results showed that antibody responses in the CoStasis clinical study were similar to the reported literature for all antigens screened and were not associated with any adverse reactions. The bovine thrombin preparations in CoStasis and other commercially available thrombins were compared with the use of SDS-PAGE and Western blot analyses. Within this clinical study, CoStasis was shown to be a safe and effective hemostatic product containing bovine thrombin and bovine collagen and no pooled human blood products. Copyright 2001 John Wiley & Sons, Inc.

Decidualized Human Endometrial Stromal Cells Mediate Hemostasis, Angiogenesis, and Abnormal Uterine Bleeding

PubMed Central

Lockwood, Charles J.; Krikun, Graciela; Hickey, Martha; Huang, S. Joseph; Schatz, Frederick

2011-01-01

Factor VII binds trans-membrane tissue factor to initiate hemostasis by forming thrombin. Tissue factor expression is enhanced in decidualized human endometrial stromal cells during the luteal phase. Long-term progestin only contraceptives elicit: 1) abnormal uterine bleeding from fragile vessels at focal bleeding sites, 2) paradoxically high tissue factor expression at bleeding sites; 3) reduced endometrial blood flow promoting local hypoxia and enhancing reactive oxygen species levels; and 4) aberrant angiogenesis reflecting increased stromal cell-expressed vascular endothelial growth factor, decreased Angiopoietin-1 and increased endothelial cell-expressed Angiopoietin-2. Aberrantly high local vascular permeability enhances circulating factor VII to decidualized stromal cell-expressed tissue factor to generate excess thrombin. Hypoxia-thrombin interactions augment expression of vascular endothelial growth factor and interleukin-8 by stromal cells. Thrombin, vascular endothelial growth factor and interlerukin-8 synergis-tically augment angiogenesis in a milieu of reactive oxygen species-induced endothelial cell activation. The resulting enhanced vessel fragility promotes abnormal uterine bleeding. PMID:19208784

Selective albumin-binding surfaces modified with a thrombin-inhibiting peptide.

PubMed

Freitas, Sidónio C; Maia, Sílvia; Figueiredo, Ana C; Gomes, Paula; Pereira, Pedro J B; Barbosa, Mário A; Martins, M Cristina L

2014-03-01

Blood-contacting medical devices have been associated with severe clinical complications, such as thrombus formation, triggered by the activation of the coagulation cascade due to the adsorption of certain plasma proteins on the surface of biomaterials. Hence, the coating of such surfaces with antithrombotic agents has been used to increase biomaterial haemocompatibility. Biomaterial-induced clotting may also be decreased by albumin adsorption from blood plasma in a selective and reversible way, since this protein is not involved in the coagulation cascade. In this context, this paper reports that the immobilization of the thrombin inhibitor D-Phe-Pro-D-Arg-D-Thr-CONH2 (fPrt) onto nanostructured surfaces induces selective and reversible adsorption of albumin, delaying the clotting time when compared to peptide-free surfaces. fPrt, synthesized with two glycine residues attached to the N-terminus (GGfPrt), was covalently immobilized onto self-assembled monolayers (SAMs) having different ratios of carboxylate-hexa(ethylene glycol)- and tri(ethylene glycol)-terminated thiols (EG6-COOH/EG3) that were specifically designed to control GGfPrt orientation, exposure and density at the molecular level. In solution, GGfPrt was able to inactivate the enzymatic activity of thrombin and to delay plasma clotting time in a concentration-dependent way. After surface immobilization, and independently of its concentration, GGfPrt lost its selectivity to thrombin and its capacity to inhibit thrombin enzymatic activity against the chromogenic substrate n-p-tosyl-Gly-Pro-Arg-p-nitroanilide. Nevertheless, surfaces with low concentrations of GGfPrt could delay the capacity of adsorbed thrombin to cleave fibrinogen. In contrast, GGfPrt immobilized in high concentrations was found to induce the procoagulant activity of the adsorbed thrombin. However, all surfaces containing GGfPrt have a plasma clotting time similar to the negative control (empty polystyrene wells), showing resistance to coagulation, which is explained by its capacity to adsorb albumin in a selective and reversible way. This work opens new perspectives to the improvement of the haemocompatibility of blood-contacting medical devices. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Effects of an acidic environment on coagulation dynamics.

PubMed

Gissel, M; Brummel-Ziedins, K E; Butenas, S; Pusateri, A E; Mann, K G; Orfeo, T

2016-10-01

Essentials Acidosis, an outcome of traumatic injury, has been linked to impaired procoagulant efficiency. In vitro model systems were used to assess coagulation dynamics at pH 7.4 and 7.0. Clot formation dynamics are slightly enhanced at pH 7.0 in blood ex vivo. Acidosis induced decreases in antithrombin efficacy offset impairments in procoagulant activity. Background Disruption of hydrogen ion homeostasis is a consequence of traumatic injury often associated with clinical coagulopathy. Mechanisms by which acidification of the blood leads to aberrant coagulation require further elucidation. Objective To examine the effects of acidified conditions on coagulation dynamics using in vitro models of increasing complexity. Methods Coagulation dynamics were assessed at pH 7.4 and 7.0 as follows: (i) tissue factor (TF)-initiated coagulation proteome mixtures (±factor [F]XI, ±fibrinogen/FXIII), with reaction progress monitored as thrombin generation or fibrin formation; (ii) enzyme/inhibitor reactions; and (iii) TF-dependent or independent clot dynamics in contact pathway-inhibited blood via viscoelastometry. Results Rate constants for antithrombin inhibition of FXa and thrombin were reduced by ~ 25-30% at pH 7.0. At pH 7.0 (+FXI), TF-initiated thrombin generation showed a 20% increase in maximum thrombin levels and diminished thrombin clearance rates. Viscoelastic analyses showed a 25% increase in clot time and a 25% reduction in maximum clot firmness (MCF). A similar MCF reduction was observed at pH 7.0 when fibrinogen/FXIII were reacted with thrombin. In contrast, in contact pathway-inhibited blood (n = 6) at pH 7.0, MCF values were elevated 6% (95% confidence interval [CI]: 1%-11%) in TF-initiated blood and 15% (95% CI: 1%- 29%) in the absence of TF. Clot times at pH 7.0 decreased 32% (95% CI: 15%-49%) in TF-initiated blood and 51% (95% CI: 35%-68%) in the absence of TF. Conclusions Despite reported decreased procoagulant catalysis at pH 7.0, clot formation dynamics are slightly enhanced in blood ex vivo and suppression of thrombin generation is not observed. A decrease in antithrombin reactivity is one potential mechanism contributing to these outcomes. © 2016 International Society on Thrombosis and Haemostasis.

Dimerization of Matrix Metalloproteinase-2 (MMP-2)

PubMed Central

Koo, Bon-Hun; Kim, Yeon Hyang; Han, Jung Ho; Kim, Doo-Sik

2012-01-01

Matrix metalloproteinase-2 (MMP-2) functions in diverse biological processes through the degradation of extracellular and non-extracellular matrix molecules. Because of its potential for tissue damage, there are several ways to regulate MMP-2 activity, including gene expression, compartmentalization, zymogen activation, and enzyme inactivation by extracellular inhibitors. Enzyme regulation through zymogen activation is important for the regulation of MMP-2 activity. In our previous studies, we showed that thrombin directly cleaved the propeptide of MMP-2 at specific sites for enzyme activation. We also demonstrated that heparan sulfate was required for thrombin-mediated activation of pro-MMP-2 by binding to thrombin, presumably through conformational changes at the active site of the enzyme. This suggests a regulatory mechanism for thrombin-mediated activation of pro-MMP-2. In this study, we found that MMP-2 formed a reduction-sensitive homodimer in a controlled manner and that Ca2+ ion was essential for homodimerization of MMP-2. Homodimerization was not associated with protein kinase C-mediated phosphorylation of MMP-2. MMP-2 formed a homodimer through an intermolecular disulfide bond between Cys102 and the neighboring Cys102. Homodimerization of MMP-2 enhanced thrombin-mediated activation of pro-MMP-2. Moreover, the MMP-2 homodimer could cleave a small peptide substrate without removal of the propeptide. Taken together, our experimental data suggest a novel regulatory mechanism for pro-MMP-2 activation that is modulated through homodimerization of MMP-2. PMID:22577146

Critical role of FcR gamma-chain, LAT, PLCgamma2 and thrombin in arteriolar thrombus formation upon mild, laser-induced endothelial injury in vivo.

PubMed

Kalia, Neena; Auger, Jocelyn M; Atkinson, Ben; Watson, Steve P

2008-05-01

The role of collagen receptor complex GPVI-FcR gamma-chain, PLCgamma2 and LAT in laser-induced thrombosis is unclear. Controversy surrounds whether collagen is exposed in this model or whether thrombosis is dependent on thrombin. This study hypothesized that collagen exposure plays a critical role in thrombus formation in this model, which was tested by investigating contributions of FcR gamma-chain, LAT, PLCgamma2 and thrombin. Thrombi were monitored using intravital microscopy in anesthetized wild-type and FcR gamma-chain, LAT and PLCgamma2 knockout mice. Hirudin (thrombin inhibitor) was administered to wild-type and FcR gamma-chain knockout mice. Significantly reduced thrombus formation was observed in FcR gamma-chain and PLCgamma2 knockouts with a greater decrease observed in LAT knockouts. Dramatic reduction was observed in wild-types treated with hirudin, with abolished thrombus formation only observed in FcR gamma-chain knockouts treated with hirudin. GPVI-FcR gamma-chain, LAT and PLCgamma2 are essential for thrombus generation and stability in this laser-induced model of injury. More importantly, a greater role for LAT was identified, which may reflect a role for it downstream of a second matrix protein receptor. However, inhibition of platelet activation by matrix proteins and thrombin generation are both required to maximally prevent thrombus formation.

Graft Product for Autologous Peripheral Blood Stem Cell Transplantation Enhances Thrombin Generation and Expresses Procoagulant Microparticles and Tissue Factor.

PubMed

Sidibe, Fatoumata; Spanoudaki, Anastasia; Vanneaux, Valerie; Mbemba, Elisabeth; Larghero, Jerome; Van Dreden, Patrick; Lotz, Jean-Pierre; Elalamy, Ismail; Larsen, Annette K; Gerotziafas, Grigoris T

2018-05-01

The beneficial effect of autologous peripheral blood stem cell transplantation (APBSCT) may be compromised by acute vascular complications related to hypercoagulability. We studied the impact of graft product on thrombin generation of normal plasma and the expression of tissue factor (TF) and procoagulant platelet-derived procoagulant microparticles (Pd-MPs) in samples of graft products. Graft products from 10 patients eligible for APBSCT were mixed with platelet-poor plasma (PPP) or platelet-rich plasma (PRP) from healthy volunteers and assessed for in vitro thrombin generation. In control experiments, thrombin generation was assessed in (1) PPP and PRP without any exogenous TF and/or procoagulant phospholipids, (2) PPP with the addition of TF (5 pM) and procoagulant phospholipids (4 μM), (3) in PRP with the addition of TF (5 pM). Graft products were assessed with Western blot assay for TF expression, with a specific clotting assay for TF activity and with flow cytometry assay for Pd-MPs. The graft product enhanced thrombin generation and its procoagulant activity was related to the presence of Pd-MPs and TF. The concentration of Pd-MPs in the graft product was characterized by a significant interindividual variability. The present study reveals the need for a thorough quality control of the graft products regarding their procoagulant potential.

Synthesis and Anticoagulant Activity of Polyureas Containing Sulfated Carbohydrates

PubMed Central

2015-01-01

Polyurea-based synthetic glycopolymers containing sulfated glucose, mannose, glucosamine, or lactose as pendant groups have been synthesized by step-growth polymerization of hexamethylene diisocyanate and corresponding secondary diamines. The obtained polymers were characterized by gel permeation chromatography, nuclear magnetic resonance spectroscopy, and Fourier transform infrared spectroscopy. The nonsulfated polymers showed similar results to the commercially available biomaterial polyurethane TECOFLEX in a platelet adhesion assay. The average degree of sulfation after reaction with SO3 was calculated from elemental analysis and found to be between three and four −OSO3 groups per saccharide. The blood-compatibility of the synthetic polymers was measured using activated partial thromboplastin time, prothrombin time, thrombin time, anti-IIa, and anti-Xa assays. Activated partial thromboplastin time, prothrombin time, and thrombin time results indicated that the mannose and lactose based polymers had the highest anticoagulant activities among all the sulfated polymers. The mechanism of action of the polymers appears to be mediated via an anti-IIa pathway rather than an anti-Xa pathway. PMID:25329742

Application of a clot-based assay to measure the procoagulant activity of stored allogeneic red blood cell concentrates

PubMed Central

Wannez, Adeline; Bailly, Nicolas; Alpan, Lutfiye; Gheldof, Damien; Douxfils, Jonathan; Deneys, Véronique; Bihin, Benoît; Chatelain, Bernard; Dogné, Jean-Michel; Chatelain, Christian; Mullier, François

2018-01-01

Background Thrombotic effects are possible complications of red blood cell transfusion. The generation and accumulation of procoagulant red blood cell extracellular vesicles during storage may play an important role in these thrombotic effects. The objective of this study was to assess the value of a simple phospholipid-dependent clot-based assay (STA®-Procoag-PPL) to estimate the procoagulant activity of stored red blood cells and changes in this activity during storage of the blood component. Materials and methods Extracellular vesicles from 12 red blood cell concentrates were isolated at 13 storage time-points and characterised by quantitative and functional methods: the degree of haemolysis (direct spectrophotometry), the quantification and determination of cellular origin (flow cytometry) and the procoagulant activity (thrombin generation and STA®-Procoag-PPL assays) were assessed. Results The mean clotting time of extracellular vesicles isolated from red blood cell concentrates decreased from 117.2±3.6 sec on the day of collection to 33.8±1.3 sec at the end of the storage period. This illustrates the phospholipid-dependent procoagulant activity of these extracellular vesicles, as confirmed by thrombin generation. Results of the peak of thrombin and the STA®-Procoag-PPL were well correlated (partial r=−0.41. p<0.001). In parallel, an exponential increase of the number of red blood cell-derived extracellular vesicles from 1,779/μL to 218,451/μL was observed. Discussion The STA®-Procoag-PPL is a potentially useful technique for assessing the procoagulant activity of a red blood cell concentrate. PMID:28287378

Phorbol ester stimulates calcium sequestration in saponized human platelets

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

Yoshida, K.; Nachmias, V.T.

1987-11-25

When platelets are activated by agonists, calcium (Ca2+) is released from an intracellular storage site. Recent studies using fura-2 show that, after thrombin stimulation, the rise in free calcium is transient and returns to base-line levels in 2-3 min, while the transient following ADP stimulation lasts only 15-20 s. We reported previously that the phorbol ester 12,13-phorbol myristate acetate (PMA), added at nanomolar levels after thrombin, immediately accelerated the rate of return of calcium to the base line severalfold. In the present study, we used both intact and saponized platelets to determine whether this is due to stimulation of calciummore » sequestration. Using fura-2 and intact platelets, we found 1) that PMA stimulated the restoration of free Ca2+ levels after ADP as well as after thrombin, and 2) that H-7, an inhibitor of protein kinase C (Ca2+/phospholipid-dependent enzyme), slowed the return of Ca2+ to baseline levels. Using saponized platelets, we also found 3) that pretreatment of platelets with PMA before saponin treatment increased the ATP-dependent /sup 45/Ca2+ uptake 2-fold, with a half-maximal effect at 5 nm; 4) that most of the Ca2+ released by ionomycin or by myoinositol 1,4,5-trisphosphate; and 5) that a GTP-binding protein inhibitor, guanosine 5'-O-(2-thiodiphosphate), decreased basal or PMA-stimulated /sup 45/Ca2+ uptake in saponin-treated platelets. Our data suggest that activation of protein kinase C stimulates the sequestration of Ca2+ independently of cAMP or myoinositol 1,4,5-trisphosphate.« less

Non-enzymatic glycation reduces heparin cofactor II anti-thrombin activity.

PubMed

Ceriello, A; Marchi, E; Barbanti, M; Milani, M R; Giugliano, D; Quatraro, A; Lefebvre, P

1990-04-01

The effects of non-enzymatic glycation on heparin cofactor II activity, at glucose concentrations which might be expected in physiological or diabetic conditions have been evaluated in this study. Radiolabelled glucose incorporation was associated with a loss of heparin cofactor anti-thrombin activity. The heparin cofactor heparin and dermatan sulfate-dependent inhibition of thrombin was significantly reduced, showing a remarkable decrease of the maximum second order rate constant. This study shows that heparin cofactor can be glycated at glucose concentrations found in the blood, and that this phenomenon produces a loss of heparin cofactor-antithrombin activity. These data suggest, furthermore, a possible link between heparin cofactor glycation and the pathogenesis of thrombosis in diabetes mellitus.

Vitamin K antagonists and direct thrombin inhibitors: present and future.

PubMed

Pineo, Graham F; Hull, Russell D

2005-02-01

Warfarin and related compounds are efficacious and safe in a variety of clinical thrombotic disorders; however, these drugs have a narrow therapeutic window, whereby inadequate therapy is associated with an increased thrombotic risk and overanticoagulation is associated with bleeding. Therefore, attempts have been made to develop alternatives to warfarin. Ximelagatran, an oral direct thrombin inhibitor, has been shown to be as efficacious and safe as warfarin for the prevention and treatment of different thrombotic disorders. This article reviews the pharmacology of the coumarins, the most commonly used vitamin K antagonists, and the practical aspects regarding their use in the management of thrombotic disorders. The future role of the oral direct thrombin inhibitor ximelagatran also is reviewed.

Severe coagulation factor VII deficiency caused by a novel homozygous mutation (p. Trp284Gly) in loop 140s.

PubMed

Hao, Xiuping; Cheng, XiaoLi; Ye, Jiajia; Wang, Yingyu; Yang, LiHong; Wang, Mingshan; Jin, Yanhui

2016-06-01

Congenital coagulation factor VII (FVII) deficiency is a rare disorder caused by mutation in F7 gene. Herein, we reported a patient who had unexplained hematuria and vertigo with consanguineous parents. He has been diagnosed as having FVII deficiency based on the results of reduced FVII activity (2.0%) and antigen (12.8%). The thrombin generation tests verified that the proband has obstacles in producing thrombin. Direct sequencing analysis revealed a novel homozygous missense mutation p.Trp284Gly. Also noteworthy is the fact that the mutational residue belongs to structurally conserved loop 140s, which majorly undergo rearrangement after FVII activation. Model analysis indicated that the substitution disrupts these native hydrophobic interactions, which are of great importance to the conformation in the activation domain of FVIIa.

Graphene- and aptamer-based electrochemical biosensor

NASA Astrophysics Data System (ADS)

Xu, Ke; Meshik, Xenia; Nichols, Barbara M.; Zakar, Eugene; Dutta, Mitra; Stroscio, Michael A.

2014-05-01

This study investigated the effectiveness of a graphene- and aptamer-based field-effect-transistor-like (FET-like) sensor in detecting lead and potassium ions. The sensor consists of a graphene-covered Si/SiO2 wafer with thrombin binding aptamer (TBA) attached to the graphene layer and terminated by a methylene blue (MB) molecule. K+ and Pb2+ both bind to TBA and cause a conformational change, which results in MB moving closer to the graphene surface and donating an electron. Thus, the abundance of K+ and Pb2+ can be determined by monitoring the current across the source and drain channel. Device transfer curves were obtained with ambipolar field effect observed. Current readings were taken for K+ concentrations of 100 μM to 50 mM and Pb2+ concentrations of 10 μM to 10 mM. As expected, I d decreased as ion concentration increased. In addition, there was a negative shift in V Dirac in response to increased ion concentration.

Sequence of Fibrinogen Proteolysis and Platelet Release after Intrauterine Infusion of Hypertonic Saline

PubMed Central

Nossel, H. L.; Wasser, J.; Kaplan, K. L.; Lagamma, K. S.; Yudelman, I.; Canfield, R. E.

1979-01-01

Plasma fibrinopeptide B (Bβ1-14 or FPB) immunoreactivity was studied by radioimmunoassay in patients who received intrauterine infusion of hypertonic saline to terminate pregnancy. FPB immunoreactivity increased with thrombin treatment (TIFPB) suggesting the presence of a larger FPB-containing peptide, since purified FPB is not altered by thrombin, whereas thrombin increases the immunoreactivity of Bβ1-42 (which includes FPB) 10-fold. TIFPB immunoreactivity in plasma, drawn 4 h after hypertonic saline infusion eluted from Sephadex G-50 similarly to isolated Bβ1-42. Streptokinase, incubated with normal plasma progressively generated TIFPB immunoreactivity, which showed a major component which eluted from Sephadex G-50 similarly to Bβ1-42. Streptokinase generated TIFPB much more rapidly in reptilase-treated plasma that contains fibrin I, (which still includes FPB), indicating that fibrin I is preferred over fibrinogen as a substrate for plasmin cleavage of arginine (Bβ42)-alanine (Bβ43). Serial studies were then made in 10 patients receiving intrauterine hypertonic saline. Fibrinopeptide A (FPA) levels rose immediately, reached a peak between 1 and 2 h, were declining at 4 h, and were normal at 24 and 48 h. TIFPB levels rose slightly in the 1st h, reached a peak at 4 h, and had returned to base-line values at 24 h. Serum fibrinogen degradation product levels were unchanged at 1 h, reached their highest level at 4 h, and were still markedly elevated at 24 and 48 h. Fibrinogen levels dropped slightly being lowest at 4 and 24 h. Platelet counts declined in parallel with the fibrinogen levels over the first 4 h, but continued to decrease through 48 h. Beta thromboglobulin (βTG) levels generally paralleled FPA levels whereas platelet factor 4 (PF4) levels showed only slight changes. The data indicate that immediately after intrauterine hypertonic saline infusion thrombin is formed that cleaves FPA from fibrinogen to produce fibrin I and releases βTG and PF4 from platelets. Later plasmin cleaves Bβ1-42 from fibrin I to produce fragment X, which is further degraded to form serum fibrinogen degradation products. This sequence of proteolysis indicates that plasmin action on fibrin I serves as a mechanism that regulates fibrin II formation by removing the Bβ chain cleavage site, which is required for thrombin action in converting fibrin I to fibrin II. PMID:500818

Molecular modeling studies of novel retro-binding tripeptide active-site inhibitors of thrombin.

PubMed

Lau, W F; Tabernero, L; Sack, J S; Iwanowicz, E J

1995-08-01

A novel series of retro-binding tripeptide thrombin active-site inhibitors was recently developed (Iwanowicz, E. I. et al. J. Med. Chem. 1994, 37, 2111(1)). It was hypothesized that the binding mode for these inhibitors is similar to that of the first three N-terminal residues of hirudin. This binding hypothesis was subsequently verified when the crystal structure of a member of this series, BMS-183,507 (N-[N-[N-[4-(Aminoiminomethyl)amino[-1-oxobutyl]-L- phenylalanyl]-L-allo-threonyl]-L-phenylalanine, methyl ester), was determined (Taberno, L.J. Mol. Biol. 1995, 246, 14). The methodology for developing the binding models of these inhibitors, the structure-activity relationships (SAR) and modeling studies that led to the elucidation of the proposed binding mode is described. The crystal structure of BMS-183,507/human alpha-thrombin is compared with the crystal structure of hirudin/human alpha-thrombin (Rydel, T.J. et al. Science 1990, 249,227; Rydel, T.J. et al. J. Mol Biol. 1991, 221, 583; Grutter, M.G. et al. EMBO J. 1990, 9, 2361) and with the computational binding model of BMS-183,507.

Antiplatelet effects of protopine isolated from Corydalis tubers.

PubMed

Ko, F N; Wu, T S; Lu, S T; Wu, Y C; Huang, T F; Teng, C M

1989-10-15

Protopine inhibited the aggregation and ATP release of rabbit platelets induced by ADP, arachidonic acid, PAF, collagen and ionophore A23187. Although the platelet aggregation caused by thrombin was not inhibited by protopine (100 micrograms/ml), the release reaction was partially suppressed. In rabbit platelet-rich plasma, protopine also inhibited the platelet aggregation caused by ADP, arachidonic acid, PAF and collagen. The thromboxane B2 formation of washed platelets caused by arachidonic acid, collagen, ionophore A23187 and thrombin was suppressed by protopine. Protopine inhibited the intracellular calcium increase caused by arachidonic acid in quin-2/AM loaded rabbit platelets. In the presence of indomethacin, the intracellular calcium increase caused by collagen and PAF was completely suppressed by protopine, and the intracellular calcium increase caused by thrombin was partially inhibited. The phosphoinositides breakdown caused by collagen and PAF was inhibited by protopine, but that by thrombin was not affected significantly. Protopine did not cause the elevation of cyclic AMP level of platelets. It is concluded that the antiplatelet effects of protopine is due to inhibition on thromboxane formation and phosphoinositides breakdown and then lead to the decrease of intracellular calcium concentration.

Contact activation: a revision.

PubMed

Schmaier, A H

1997-07-01

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

Composition of fibrin glues significantly influences axial vascularization and degradation in isolation chamber model.

PubMed

Arkudas, Andreas; Pryymachuk, Galyna; Hoereth, Tobias; Beier, Justus P; Polykandriotis, Elias; Bleiziffer, Oliver; Gulle, Heinz; Horch, Raymund E; Kneser, Ulrich

2012-07-01

In this study, different fibrin sealants with varying concentrations of the fibrin components were evaluated in terms of matrix degradation and vascularization in the arteriovenous loop (AVL) model of the rat. An AVL was placed in a Teflon isolation chamber filled with 500 μl fibrin gel. The matrix was composed of commercially available fibrin gels, namely Beriplast (Behring GmbH, Marburg, Germany) (group A), Evicel (Omrix Biopharmaceuticals S.A., Somerville, New Jersey, USA) (group B), Tisseel VH S/D (Baxter, Vienna, Austria) with a thrombin concentration of 4 IU/ml and a fibrinogen concentration of 80 mg/ml [Tisseel S F80 (Baxter), group C] and with an fibrinogen concentration of 20 mg/ml [Tisseel S F20 (Baxter), group D]. After 2 and 4 weeks, five constructs per group and time point were investigated using micro-computed tomography, and histological and morphometrical analysis techniques. The aprotinin, factor XIII and thrombin concentration did not affect the degree of clot degradation. An inverse relationship was found between fibrin matrix degradation and sprouting of blood vessels. By reducing the fibrinogen concentration in group D, a significantly decreased construct weight and an increased generation of vascularized connective tissue were detected. There was an inverse relationship between matrix degradation and vascularization detectable. Fibrinogen as the major matrix component showed a significant impact on the matrix properties. Alteration of fibrin gel properties might optimize formation of blood vessels.

DNA-based Nanoconstructs for the Detection of Ions and Biomolecules with Related Raman/SERS Signature Studies

NASA Astrophysics Data System (ADS)

Brenneman, Kimber L.

The utilization of DNA aptamers and semiconductor quantum dots (QDs) for the detection of ions and biomolecules was investigated. In recent years, there have been many studies based on the use of DNA and RNA aptamers, which are single stranded oligonucleotides capable of binding to biomolecules, other molecules, and ions. In many of these cases, the conformational changes of these DNA and RNA aptamers are suitable to use fluorescence resonant energy transfer (FRET) or nanometal surface energy transfer (NSET) techniques to detect such analytes. Coupled with this growth in such uses of aptamers, there has been an expanded use of semiconductor quantum dots as brighter, longer-lasting alternatives to fluorescent dyes in labeling and detection techniques of interest in biomedicine and environmental monitoring. Thrombin binding aptamer (TBA) and a zinc aptamer were used to detect mercury, lead, zinc, and cadmium. These probes were tested in a liquid assay as well as on a filter paper coupon. Biomolecules were also studied and detected using surface-enhanced Raman spectroscopy (SERS), including DNA aptamers and C-reactive protein (CRP). Raman spectroscopy is a useful tool for sensor development, label-free detection, and has the potential for remote sensing. Raman spectra provide information on the vibrational modes or phonons, between and within molecules. Therefore, unique spectral fingerprints for single molecules can be obtained. SERS is accomplished through the use of substrates with nanometer scale geometries made of metals with many free electrons, such as silver, gold, or copper. In this research silver SERS substrates were used to study the SERS signature of biomolecules that typically produce very weak Raman signals.

Effect of platelet-derived β-thromboglobulins on coagulation.

PubMed

Egan, Karl; van Geffen, Johanna P; Ma, Hui; Kevane, Barry; Lennon, Aine; Allen, Seamus; Neary, Elaine; Parsons, Martin; Maguire, Patricia; Wynne, Kieran; O' Kennedy, Richard; Heemskerk, Johan W M; Áinle, Fionnuala Ní

2017-06-01

β-thromboglobulins are derived from the cleavage of the CXC chemokine platelet basic protein and are released in high concentrations by activated platelets. Platelet-derived β-thromboglobulins (βTG) share 70% homology with platelet factor 4 (PF4), another CXC chemokine released by activated platelets. PF4 modulates coagulation by inhibiting heparin-antithrombin interactions, promoting protein C activation, and attenuating the activity of activated protein C. In contrast, the effect of βTG on coagulation is unknown. Clotting times, thrombin generation, chromogenic clotting factor assays, and surface plasmon resonance (SPR) were used to assess the effect of purified βTG on coagulation. In normal pooled plasma, βTG shortened the lagtime and time to peak thrombin generation of tissue factor (TF)-dependent and TF-independent thrombin generation. In factor VIII and factor IX-deficient plasmas, βTG induced thrombin generation in the absence of a TF stimulus and in the presence of anti-TF and factor VIIa inhibitory antibodies. The procoagulant effect was not observed when thrombin generation was independent of factor X activation (supplementation of factor X-deficient plasma with factor Xa). Cleavage of a factor Xa-specific chromogenic substrate was observed when βTG was incubated with factor X, suggesting a direct interaction between βTG and factor X. Using SPR, βTG were found to bind to immobilised factor X in a dose dependent manner. βTG modulate coagulation in vitro via an interaction with factor X. Copyright © 2017 Elsevier Ltd. All rights reserved.

In vitro/in vivo effect of Citrus limon (L. Burm. f.) juice on blood parameters, coagulation and anticoagulation factors in rabbits.

PubMed

Riaz, Azra; Khan, Rafeeq Alam; Mirza, Talat; Mustansir, Tazeen; Ahmed, Mansoor

2014-07-01

The genus Citrus of the family Rutaceae includes many species e.g. Citrus indica, Citrus aurantifolia and Citrus limon, among which Citrus limon L. Burm. f. has been reported to have highest antimicrobial activity. It is used as antidote against certain venom, due to its platelet inhibitory effect and also reported to have hypocholesterolemic effect. However its anticoagulant and thrombolytic effect were not been investigated, hence a prospective in-vitro/in-vivo study was designed to determine the effect of Citrus limon on blood parameters, coagulation and anticoagulation factors. In-vitro tests revealed highly significant increase in thrombin time and activated partial thromboplastin time by Citrus limon, whereas fibrinogen concentration was significantly reduced in comparison to control, however prothrombin time was not affected significantly. In-vivo testing of Citrus limon was done at three different doses i.e. 0.2ml/kg, 0.4ml/kg and 0.6ml/kg in healthy rabbits. Significant changes were observed in hematological parameters such as erythrocytes, hemoglobin and mean corpuscular hemoglobin concentration. Bleeding time and thrombin time was significantly prolonged and there was increase in protein C and thrombin antithrombin complex levels. These results may be due to inactivation of thrombin because it significantly decreases fibrinogen concentration and inhibit platelet aggregation. Citrus limon showed maximal anticoagulant effect at 0.4ml/kg, which suggest that Citrus limon possesses an anti-thrombin component and could prevent thrombosis playing a cardio protective role.

Elevated plasma factor VIII enhances venous thrombus formation in rabbits: contribution of factor XI, von Willebrand factor and tissue factor.

PubMed

Sugita, Chihiro; Yamashita, Atsushi; Matsuura, Yunosuke; Iwakiri, Takashi; Okuyama, Nozomi; Matsuda, Shuntaro; Matsumoto, Tomoko; Inoue, Osamu; Harada, Aya; Kitazawa, Takehisa; Hattori, Kunihiro; Shima, Midori; Asada, Yujiro

2013-07-01

Elevated plasma levels of factor VIII (FVIII) are associated with increased risk of deep venous thrombosis. The aim of this study is to elucidate how elevated FVIII levels affect venous thrombus formation and propagation in vivo. We examined rabbit plasma FVIII activity, plasma thrombin generation, whole blood coagulation, platelet aggregation and venous wall thrombogenicity before and one hour after an intravenous infusion of recombinant human FVIII (rFVIII). Venous thrombus induced by the endothelial denudation of rabbit jugular veins was histologically assessed. Thrombus propagation was evaluated as indocyanine green fluorescence intensity. Argatroban, a thrombin inhibitor, and neutralised antibodies for tissue factor (TF), factor XI (FXI), and von Willebrand factor (VWF) were infused before or after thrombus induction to investigate their effects on venous thrombus formation or propagation. Recombinant FVIII (100 IU/kg) increased rabbit plasma FVIII activity two-fold and significantly enhanced whole blood coagulation and total plasma thrombin generation, but did not affect initial thrombin generation time, platelet aggregation and venous wall thrombogenicity. The rFVIII infusion also increased the size of venous thrombus 1 hour after thrombus induction. Argatroban and the antibodies for TF, FXI or VWF inhibited such enhanced thrombus formation and all except TF suppressed thrombus propagation. In conclusion, elevated plasma FVIII levels enhance venous thrombus formation and propagation. Excess thrombin generation by FXI and VWF-mediated FVIII recruitment appear to contribute to the growth of FVIII-driven venous thrombus.

Evidence for actin cytoskeleton-dependent and -independent pathways for RelA/p65 nuclear translocation in endothelial cells.

PubMed

Fazal, Fabeha; Minhajuddin, Mohd; Bijli, Kaiser M; McGrath, James L; Rahman, Arshad

2007-02-09

Activation of the transcription factor NF-kappaB involves its release from the inhibitory protein IkappaBalpha in the cytoplasm and subsequently, its translocation to the nucleus. Whereas the events responsible for its release have been elucidated, mechanisms regulating the nuclear transport of NF-kappaB remain elusive. We now provide evidence for actin cytoskeleton-dependent and -independent mechanisms of RelA/p65 nuclear transport using the proinflammatory mediators, thrombin and tumor necrosis factor alpha, respectively. We demonstrate that thrombin alters the actin cytoskeleton in endothelial cells and interfering with these alterations, whether by stabilizing or destabilizing the actin filaments, prevents thrombin-induced NF-kappaB activation and consequently, expression of its target gene, ICAM-1. The blockade of NF-kappaB activation occurs downstream of IkappaBalpha degradation and is associated with impaired RelA/p65 nuclear translocation. Importantly, thrombin induces association of RelA/p65 with actin and this interaction is sensitive to stabilization/destabilization of the actin filaments. In parallel studies, stabilizing or destabilizing the actin filaments fails to inhibit RelA/p65 nuclear accumulation and ICAM-1 expression by tumor necrosis factor alpha, consistent with its inability to induce actin filament formation comparable with thrombin. Thus, these studies reveal the existence of actin cytoskeleton-dependent and -independent pathways that may be engaged in a stimulus-specific manner to facilitate RelA/p65 nuclear import and thereby ICAM-1 expression in endothelial cells.

Self-assembled, bivalent aptamers on graphene oxide as an efficient anticoagulant.

PubMed

Lai, Pei-Xin; Mao, Ju-Yi; Unnikrishnan, Binesh; Chu, Han-Wei; Wu, Chien-Wei; Chang, Huan-Tsung; Huang, Chih-Ching

2018-06-25

Graphene oxide (GO) has unique structural properties, can effectively adsorb single-strand DNA through π-π stacking, hydrogen bonding and hydrophobic interactions, and is useful in many biotechnology applications. In this study, we developed a thrombin-binding-aptamers (15- and 29-mer) conjugated graphene oxide (TBA15/TBA29-GO) composite for the efficient inhibition of thrombin activity towards the formation of fibrin from fibrinogen. The TBA15/TBA29-GO composite was simply obtained by the self-assembly of TBA15/TBA29 hybrids on GO. The high density and appropriate orientation of TBA15/TBA29 on the GO surface enabled TBA15/TBA29-GO to acquire an ultrastrong binding affinity for thrombin (dissociation constant = 2.9 × 10-12 M). Compared to bivalent TBA15h20A20/TBA29h20A20 hybrids, the TBA15/TBA29-GO composite exhibited a superior anticoagulant potency (ca. 10-fold) against thrombin-mediated coagulation as a result of steric blocking effects and a higher binding affinity for thrombin. In addition, the prolonged thrombin clotting time, prothrombin time (PT), and activated partial thromboplastin time (aPTT) of TBA15/TBA29-GO were at least 2 times longer than those of commercially available drugs (heparin, argatroban, hirudin, and warfarin). The in vitro cytotoxicity and hemolysis analyses revealed the high biocompatibility of TBA15/TBA29-GO. The rat-tail bleeding assay of the hemostasis time and ex vivo PT and aPTT further revealed that TBA15/TBA29-GO is superior (>2-fold) to heparin, which is commonly used in the treatment and prevention of thrombotic diseases. Our multivalent, oligonucleotide-modified GO nanocomposites are easy to prepare, cost-effective, and highly biocompatible and they show great potential as effective anticoagulants for the treatment of thrombotic disorders.

A novel protocol for the production of recombinant LL-37 expressed as a thioredoxin fusion protein.

PubMed

Li, Yifeng

2012-02-01

LL-37 is the only cathelicidin-derived antimicrobial peptide found in humans and it has a multifunctional role in host defense. The peptide has been shown to possess immunomodulatory functions in addition to antimicrobial activity. To provide sufficient material for biological and structural characterization of this important peptide, various systems were developed to produce recombinant LL-37 in Escherichia coli. In one previous approach, LL-37 coding sequence was cloned into vector pET-32a, allowing the peptide to be expressed as a thioredoxin fusion. The fusion protein contains two thrombin cleavage sites: a vector-encoded one that is 30-residue upstream of the insert and an engineered one that is immediately adjacent to LL-37. Cleavage at these two sites shall generate three fragments, one of which is the target peptide. However, when the fusion protein was treated with thrombin, cleavage only occurred at the remote upstream site. A plausible explanation is that the thrombin site adjacent to LL-37 is less accessible due to the peptide's aggregation tendency and cleavage at the remote site generates a fragment, which forms a large aggregate that buries the intended site. In this study, I deleted the vector-encoded thrombin site and S tag in pET-32a, and then inserted the coding sequence for LL-37 plus a thrombin site into the modified vector. Although removing the S tag did not change the oligomeric state of the fusion protein, deletion of the vector-encoded thrombin site allowed the fusion to be cleaved at the engineered site to release LL-37. The released peptide was separated from the carrier and cleavage enzyme by size-exclusion chromatography. This new approach enables a quick production of high quality active LL-37 with a decent amount. Copyright © 2011 Elsevier Inc. All rights reserved.

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

PubMed Central

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

2015-01-01

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

Effects of Antimalarial Tafenoquine on Blood Platelet Activity and Survival.

PubMed

Cao, Hang; Bissinger, Rosi; Umbach, Anja T; Al Mamun Bhuyan, A; Lang, Florian; Gawaz, Meinrad

2017-01-01

The 8-aminoquinoline tafenoquine has been shown to be effective against Plasmodia, Leishmania and Trypanosoma. The substance is at least in part effective by triggering apoptosis of the parasites. Moreover, tafenoquine has been shown to trigger eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. The effect of tafenoquine on eryptosis is in part due to stimulation of Ca2+ entry and oxidative stress. Ca2+ entry is a critical event in the activation of blood platelets by thrombin and collagen related peptide (CRP). The present study explored, whether tafenoquine influences Ca2+ entry, activation and apoptosis of blood platelets. Platelets isolated from wild-type mice were exposed for 30 minutes to tafenoquine (2.5 µg/ml) without or with an additional treatment with thrombin (0.01 U/ml) or CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+] i ) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from α IIb β 3 integrin abundance, phosphatidylserine abundance from annexin-V-binding, relative platelet volume from forward scatter, reactive oxygen species (ROS) from DCF fluorescence, caspase 3 activity with an active caspase-3 Staining kit, and aggregation utilizing staining with CD9-APC and CD9-PE. Both, thrombin (0.01 U/ml) and CRP (2 µg/ml or 5 µg/ml), significantly increased [Ca2+] i , P-selectin abundance, active α IIb β 3 integrin, and annexin-V-binding, and both significantly decreased platelet volume, activated caspase 3 and stimulated aggregation. Administration of tafenoquine (2.5 µg/ml, 30 min) significantly decreased [Ca2+] i both, in the absence and presence of thrombin and CRP. Tafenoquine significantly blunted the effect of thrombin and CRP on [Ca2+] i , P-selectin abundance, and active α IIb β 3 integrin, but significantly increased ROS and annexin-V-binding, significantly augmented the effect of thrombin on caspase 3 activity and platelet volume and significantly enhanced platelet aggregation. Tafenoquine counteracts thrombin and CRP induced increase of cytosolic Ca2+ activity and platelet activation, but enhances platelet apoptosis and platelet aggregation. © 2017 The Author(s) Published by S. Karger AG, Basel.

Effects of cognate, non-cognate and synthetic CXCR4 and ACKR3 ligands on human lung endothelial cell barrier function.

PubMed

Cheng, You-Hong; Eby, Jonathan M; LaPorte, Heather M; Volkman, Brian F; Majetschak, Matthias

2017-01-01

Recent evidence suggests that chemokine CXCL12, the cognate agonist of chemokine receptors CXCR4 and ACKR3, reduces thrombin-mediated impairment of endothelial barrier function. A detailed characterization of the effects of CXCL12 on thrombin-mediated human lung endothelial hyperpermeability is lacking and structure-function correlations are not available. Furthermore, effects of other CXCR4/ACKR3 ligands on lung endothelial barrier function are unknown. Thus, we tested the effects of a panel of CXCR4/ACKR3 ligands (CXCL12, CXCL11, ubiquitin, AMD3100, TC14012) and compared the CXCR4/ACKR3 activities of CXCL12 variants (CXCL12α/β, CXCL12(3-68), CXCL121, CXCL122, CXCL12-S-S4V, CXCL12-R47E, CXCL12-K27A/R41A/R47A) with their effects on human lung endothelial barrier function in permeability assays. CXCL12α enhanced human primary pulmonary artery endothelial cell (hPPAEC) barrier function, whereas CXCL11, ubiquitin, AMD3100 and TC14012 were ineffective. Pre-treatment of hPPAEC with CXCL12α and ubiquitin reduced thrombin-mediated hyperpermeability. CXCL12α-treatment of hPPAEC after thrombin exposure reduced barrier function impairment by 70% (EC50 0.05-0.5nM), which could be antagonized with AMD3100; ubiquitin (0.03-3μM) was ineffective. In a human lung microvascular endothelial cell line (HULEC5a), CXCL12α and ubiquitin post-treatment attenuated thrombin-induced hyperpermeability to a similar degree. CXCL12(3-68) was inefficient to activate CXCR4 in Presto-Tango β-arrestin2 recruitment assays; CXCL12-S-S4V, CXCL12-R47E and CXCL12-K27A/R41A/R47A showed significantly reduced potencies to activate CXCR4. While the potencies of all proteins in ACKR3 Presto-Tango assays were comparable, the efficacy of CXCL12(3-68) to activate ACKR3 was significantly reduced. The potencies to attenuate thrombin-mediated hPPAEC barrier function impairment were: CXCL12α/β, CXCL121, CXCL12-K27A/R41A/R47A > CXCL12-S-S4V, CXCL12-R47E > CXCL122 > CXCL12(3-68). Our findings indicate that CXCR4 activation attenuates thrombin-induced lung endothelial barrier function impairment and suggest that protective effects of CXCL12 are dictated by its CXCR4 agonist activity and interactions of distinct protein moieties with heparan sulfate on the endothelial surface. These data may facilitate development of compounds with improved pharmacological properties to attenuate thrombin-induced vascular leakage in the pulmonary circulation.

Engineering self-contained DNA circuit for proximity recognition and localized signal amplification of target biomolecules

PubMed Central

Ang, Yan Shan; Yung, Lin-Yue Lanry

2014-01-01

Biomolecular interactions have important cellular implications, however, a simple method for the sensing of such proximal events is lacking in the current molecular toolbox. We designed a dynamic DNA circuit capable of recognizing targets in close proximity to initiate a pre-programmed signal transduction process resulting in localized signal amplification. The entire circuit was engineered to be self-contained, i.e. it can self-assemble onto individual target molecules autonomously and form localized signal with minimal cross-talk. α-thrombin was used as a model protein to evaluate the performance of the individual modules and the overall circuit for proximity interaction under physiologically relevant buffer condition. The circuit achieved good selectivity in presence of non-specific protein and interfering serum matrix and successfully detected for physiologically relevant α-thrombin concentration (50 nM–5 μM) in a single mixing step without any further washing. The formation of localized signal at the interaction site can be enhanced kinetically through the control of temperature and probe concentration. This work provides a basic general framework from which other circuit modules can be adapted for the sensing of other biomolecular or cellular interaction of interest. PMID:25056307

Fiber optic immunosensor for cross-linked fibrin concentration

NASA Astrophysics Data System (ADS)

Moskowitz, Samuel E.

2000-08-01

Working with calcium ions in the blood, platelets produce thromboplastin which transforms prothrombin into thrombin. Removing peptides, thrombin changes fibrinogen into fibrin. Cross-linked insoluble fibrin polymers are solubilized by enzyme plasmin found in blood plasma. Resulting D-dimers are elevated in patients with intravascular coagulation, deep venous thrombosis, pulmonary embolism, myocardial infarction, multiple trauma, cancer, impaired renal and liver functions, and sepsis. Consisting principally of a NIR 780 nm GaAlAs laser diode and a 800 nm avalanche photodiode (APD), the fiber-optic immunosensor can determined D-dimer concentration to levels <0.1 ng/ml. A capture monoclonal antibody to the antigen soluble cross-linked fibrin is employed. Immobilized at the tip of an optical fiber by avidin-biotin, the captured antigen is detected by a second antibody which is labeled with NN 382 fluorescent dye. An evanescent wave traveling on an excitation optical fiber excites the antibody-antigen fluorophore complex. Concentration of cross-linked fibrin is directly proportional to the APD measured intensity of fluorescence. NIR fluorescence has advantages of low background interference, short fluorescence lifetime, and large difference between excitation and emission peaks. Competitive ELISA test for D-dimer concentration requires trained personnel performing a time consuming operation.

Light-activated protein inhibition through photoinduced electron transfer of a ruthenium(II)–cobalt(III) bimetallic complex

DOE PAGES

Holbrook, Robert J.; Weinberg, David J.; Peterson, Mark D.; ...

2015-02-11

In this paper, we describe a mechanism of light activation that initiates protein inhibitory action of a biologically inert Co(III) Schiff base (Co(III)-sb) complex. Photoinduced electron transfer (PET) occurs from a Ru(II) bipyridal complex to a covalently attached Co(III) complex and is gated by conformational changes that occur in tens of nanoseconds. Reduction of the Co(III)-sb by PET initiates displacement of the inert axial imidazole ligands, promoting coordination to active site histidines of α-thrombin. Upon exposure to 455 nm light, the rate of ligand exchange with 4-methylimidazole, a histidine mimic, increases by approximately 5-fold, as observed by NMR spectroscopy. Similarly,more » the rate of α-thrombin inhibition increases over 5-fold upon irradiation. Finally, these results convey a strategy for light activation of inorganic therapeutic agents through PET utilizing redox-active metal centers.« less

Inhibitory Effects of Cytosolic Ca(2+) Concentration by Ginsenoside Ro Are Dependent on Phosphorylation of IP3RI and Dephosphorylation of ERK in Human Platelets.

PubMed

Kwon, Hyuk-Woo; Shin, Jung-Hae; Lee, Dong-Ha; Park, Hwa-Jin

2015-01-01

Intracellular Ca(2+) ([Ca(2+)] i ) is platelet aggregation-inducing molecule and is involved in activation of aggregation associated molecules. This study was carried out to understand the Ca(2+)-antagonistic effect of ginsenoside Ro (G-Ro), an oleanane-type saponin in Panax ginseng. G-Ro, without affecting leakage of lactate dehydrogenase, dose-dependently inhibited thrombin-induced platelet aggregation, and the half maximal inhibitory concentration was approximately 155 μM. G-Ro inhibited strongly thrombin-elevated [Ca(2+)] i , which was strongly increased by A-kinase inhibitor Rp-8-Br-cAMPS compared to G-kinase inhibitor Rp-8-Br-cGMPS. G-Ro increased the level of cAMP and subsequently elevated the phosphorylation of inositol 1, 4, 5-triphosphate receptor I (IP3RI) (Ser(1756)) to inhibit [Ca(2+)] i mobilization in thrombin-induced platelet aggregation. Phosphorylation of IP3RI (Ser(1756)) by G-Ro was decreased by PKA inhibitor Rp-8-Br-cAMPS. In addition, G-Ro inhibited thrombin-induced phosphorylation of ERK 2 (42 kDa), indicating inhibition of Ca(2+) influx across plasma membrane. We demonstrate that G-Ro upregulates cAMP-dependent IP3RI (Ser(1756)) phosphorylation and downregulates phosphorylation of ERK 2 (42 kDa) to decrease thrombin-elevated [Ca(2+)] i , which contributes to inhibition of ATP and serotonin release, and p-selectin expression. These results indicate that G-Ro in Panax ginseng is a beneficial novel Ca(2+)-antagonistic compound and may prevent platelet aggregation-mediated thrombotic disease.

Fabrication and physical and biological properties of fibrin gel derived from human plasma.

PubMed

Zhao, Haiguang; Ma, Lie; Zhou, Jie; Mao, Zhengwei; Gao, Changyou; Shen, Jiacong

2008-03-01

The fast development of tissue engineering and regenerative medicine drives the old biomaterials, for example, fibrin glue, to find new applications in these areas. Aiming at developing a commercially available hydrogel for cell entrapment and delivery, in this study we optimized the fabrication and gelation conditions of fibrin gel. Fibrinogen was isolated from human plasma by a freeze-thaw circle. Gelation of the fibrinogen was accomplished by mixing with thrombin. Absorbance of the fibrinogen/thrombin mixture at 550 nm as a function of reaction time was monitored by UV-VIS spectroscopy. It was found that the clotting time is significantly influenced by the thrombin concentration and the temperature, while less influenced by the fibrinogen concentration. After freeze-drying, the fibrin gel was characterized by scanning electron microscopy (SEM), revealing fibrous microstructure. Thermal gravimetric analysis found that the degradation temperature of the crosslinked fibrin gel starts from 288 degrees C, which is about 30 degrees C higher than that of the fibrinogen. The hydrogel has an initial water-uptake ratio of approximately 50, decreased to 30-40 after incubation in water for 11 h depending on the thrombin concentration. The fibrin gels lost their weights in PBS very rapidly, while slowly in DMEM/fetal bovine serum and DMEM. In vitro cell culture found that human fibroblasts could normally proliferate in the fibrin gel with spreading morphology. In conclusion, the fibrin gel containing higher concentration of fibrinogen (20 mg ml(-1)) and thrombin (5 U ml(-1)) has suitable gelation time and handling properties, and thus is applicable as a delivery vehicle for cells such as fibroblasts.

Fabrication and physical and biological properties of fibrin gel derived from human plasma

NASA Astrophysics Data System (ADS)

Zhao, Haiguang; Ma, Lie; Zhou, Jie; Mao, Zhengwei; Gao, Changyou; Shen, Jiacong

2008-03-01

The fast development of tissue engineering and regenerative medicine drives the old biomaterials, for example, fibrin glue, to find new applications in these areas. Aiming at developing a commercially available hydrogel for cell entrapment and delivery, in this study we optimized the fabrication and gelation conditions of fibrin gel. Fibrinogen was isolated from human plasma by a freeze-thaw circle. Gelation of the fibrinogen was accomplished by mixing with thrombin. Absorbance of the fibrinogen/thrombin mixture at 550 nm as a function of reaction time was monitored by UV-VIS spectroscopy. It was found that the clotting time is significantly influenced by the thrombin concentration and the temperature, while less influenced by the fibrinogen concentration. After freeze-drying, the fibrin gel was characterized by scanning electron microscopy (SEM), revealing fibrous microstructure. Thermal gravimetric analysis found that the degradation temperature of the crosslinked fibrin gel starts from 288 °C, which is about 30 °C higher than that of the fibrinogen. The hydrogel has an initial water-uptake ratio of ~50, decreased to 30-40 after incubation in water for 11 h depending on the thrombin concentration. The fibrin gels lost their weights in PBS very rapidly, while slowly in DMEM/fetal bovine serum and DMEM. In vitro cell culture found that human fibroblasts could normally proliferate in the fibrin gel with spreading morphology. In conclusion, the fibrin gel containing higher concentration of fibrinogen (20 mg ml-1) and thrombin (5 U ml-1) has suitable gelation time and handling properties, and thus is applicable as a delivery vehicle for cells such as fibroblasts.

Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin.

PubMed

Treuheit, Nicholas A; Beach, Muneera A; Komives, Elizabeth A

2011-05-31

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.

Coincident regulation of PKCδ in human platelets by phosphorylation of Tyr311 and Tyr565 and phospholipase C signalling

PubMed Central

Hall, Kellie J.; Jones, Matthew L.; Poole, Alastair W.

2007-01-01

PKC (protein kinase C)δ plays a complex role in platelets, having effects on both positive and negative signalling functions. It is phosphorylated on tyrosine residues in response to thrombin and collagen, and it has recently been shown that Tyr311 is phosphorylated in response to PAR (protease-activated receptor) 1 and PAR4 receptor activation. In the present study, we show that Tyr311 and Tyr565 are phosphorylated in response to thrombin, and have examined the interplay between phosphorylation and the classical lipid-mediated activation of PKCδ. Phosphorylation of both Tyr311 and Tyr565 is dependent on Src kinase and PLC (phospholipase C) activity in response to thrombin. Importantly, direct allosteric activation of PKCδ with PMA also induced phosphorylation of Tyr311 and Tyr565, and this was dependent on the activity of Src kinases, but not PLC. Membrane recruitment of PKCδ is essential for phosphorylation of this tyrosine residue, but tyrosine phosphorylation is not required for membrane recruitment of PKCδ. Both thrombin and PMA induce recruitment of PKCδ to the membrane, and for thrombin, this recruitment is a PLC-dependent process. In order to address the functional role of tyrosine residue phosphorylation of PKCδ, we demonstrate that phosphorylation can potentiate the activity of the kinase, although phosphorylation does not play a role in membrane recruitment of the kinase. PKCδ is therefore regulated in a coincident fashion, PLC-dependent signals recruiting it to the plasma membrane and by phosphorylation on tyrosine residues, potentiating its activity. PMID:17570831

Microparticle-associated tissue factor activity correlates with plasma levels of bacterial lipopolysaccharides in meningococcal septic shock.

PubMed

Hellum, Marit; Øvstebø, Reidun; Brusletto, Berit S; Berg, Jens P; Brandtzaeg, Petter; Henriksson, Carola E

2014-03-01

The plasma level of bacterial lipopolysaccharides (LPS) is associated with activation of the coagulation system, inhibition of fibrinolysis and the nature of the clinical presentation and outcome in patients with meningococcal disease. Tissue factor (TF)-bearing microparticles (MPs) appear to contribute to the pathogenesis of disseminated intravascular coagulation (DIC). The aim of this study was to investigate the relationship between MP-associated TF activity and the level of bacterial LPS in plasma from patients with meningococcal septic shock and meningitis. MPs isolated from citrated plasmas were assessed for TF-dependent activity with both a plasma-based thrombin generation assay (CAT) and whole blood-based thromboelastometry (ROTEM). The LPS level was measured using a chromogenic Limulus amebocyte lysate assay. MPs obtained from patients with meningococcal septic shock initiated significantly more efficient and TF-dependent thrombin generation in the CAT assay compared to MPs from patients with meningococcal meningitis. Differences in MP-associated TF activity between the septic shock patients and the meningitis patients were also evident when MPs were added to whole blood using ROTEM. The level of plasma LPS in patients with septic shock (range 2-2,100 EU/mL) was correlated with thrombogram parameters in the CAT assay; lagtime (r(s)=-0.84), time to peak (rs=-0.83), peak (r(s)=0.85) and ETP (r(s)=0.83). MPs obtained from patients with meningococcal septic shock displayed more efficient TF-dependent thrombin generation and clot formation compared to MPs from meningitis patients. MP-associated TF activity was closely associated with plasma LPS levels in the septic shock group. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

PubMed Central

Altman, Raul; Scazziota, Alejandra Silvia; Herrera, Maria de Lourdes; Gonzalez, Claudio

2006-01-01

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 < 0.05) and PTG and AUC→35min were significantly increased (p < 0.05) in platelet rich plasma activated with AA, ADP, Col, and rFVIIa compared to non-activated platelet rich plasma from normal subjects (p = 0.01). Furthermore platelet rich plasma activated by the combined effects of rFVIIa plus AA, ADP or Col had significantly reduced LT and TTP and increased AUC→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. PMID:16630353

Activated protein C plays no major roles in the inhibition of coagulation or increased fibrinolysis in acute coagulopathy of trauma-shock: a systematic review.

PubMed

Gando, Satoshi; Mayumi, Toshihiko; Ukai, Tomohiko

2018-01-01

The pathophysiological mechanisms of acute coagulopathy of trauma-shock (ACOTS) are reported to include activated protein C-mediated suppression of thrombin generation via the proteolytic inactivation of activated Factor V (FVa) and FVIIIa; an increased fibrinolysis via neutralization of plasminogen activator inhibitor-1 (PAI-1) by activated protein C. The aims of this study are to review the evidences for the role of activated protein C in thrombin generation and fibrinolysis and to validate the diagnosis of ACOTS based on the activated protein C dynamics. We conducted systematic literature search (2007-2017) using PubMed, the Cochrane Database of Systematic Reviews (CDSR), and the Cochrane Central Register of Controlled Trials (CENTRAL). Clinical studies on trauma that measured activated protein C or the circulating levels of activated protein C-related coagulation and fibrinolysis markers were included in our study. Out of 7613 studies, 17 clinical studies met the inclusion criteria. The levels of activated protein C in ACOTS were inconsistently decreased, showed no change, or were increased in comparison to the control groups. Irrespective of the activated protein C levels, thrombin generation was always preserved or highly elevated. There was no report on the activated protein C-mediated neutralization of PAI-1 with increased fibrinolysis. No included studies used unified diagnostic criteria to diagnose ACOTS and those studies also used different terms to refer to the condition known as ACOTS. None of the studies showed direct cause and effect relationships between activated protein C and the suppression of coagulation and increased fibrinolysis. No definitive diagnostic criteria or unified terminology have been established for ACOTS based on the activated protein C dynamics.

Novel isoguanine derivative of unlocked nucleic acid-Investigations of thermodynamics and biological potential of modified thrombin binding aptamer.

PubMed

Kotkowiak, Weronika; Czapik, Tomasz; Pasternak, Anna

2018-01-01

Thrombin binding aptamer (TBA), is a short DNA 15-mer that forms G-quadruplex structure and possesses anticoagulant properties. Some chemical modifications, including unlocked nucleic acids (UNA), 2'-deoxy-isoguanosine and 2'-deoxy-4-thiouridine were previously found to enhance the biological activity of TBA. In this paper, we present thermodynamic and biological characteristics of TBA variants that have been modified with novel isoguanine derivative of UNA as well as isoguanosine. Additionally, UNA-4-thiouracil and 4-thiouridine were also introduced simultaneously with isoguanine derivatives. Thermodynamic analysis indicates that the presence of isoguanosine in UNA or RNA series significantly decreases the stability of G-quadruplex structure. The highest destabilization is observed for substitution at one of the G-tetrad position. Addition of 4-thiouridine in UNA or RNA series usually decreases the unfavorable energetic cost of the presence of UNA or RNA isoguanine. Circular dichroism and thermal denaturation spectra in connection with thrombin time assay indicate that the introduction of UNA-isoguanine or isoguanosine into TBA negatively affects G-quadruplex folding and TBA anticoagulant properties. These findings demonstrate that the highly-ordered structure of TBA is essential for inhibition of thrombin activity.

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

PubMed

Minix, R; Doctor, V M

1997-09-01

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

[Bed-side management of post-operative bleeding in transurethral resection of the prostate--hemostatic trial with thrombin solution injected into the resected cavity of the prostate].

PubMed

Hasegawa, S; Nakashima, J; Kimura, S

1987-08-01

To control post-operative bleeding from the resected cavity in transurethral resection of the prostate (TUR-P), 20,000 units of thrombin dissolved in 5-20 ml of physiological saline was injected into the cavity in one shot with a new type of 3-way Foley catheter. The catheter has two holes close to the balloon, and the holes provide outlets through which the solution is directly injected into the cavity. Out of 23 patients between 58 and 87 years old, 11 were given thrombin and 12 were control cases. In each case, urine blood volume (ml/hr) was determined before (a) and after (b) injection. The hemostatic effect was assessed with a "hemostatic index" of b/a. There was a statistically significant difference in the hemostatic indices between thrombin group and the control. (p less than 0.02) This new hemostatic method proved to be a very useful easy bed-side technique to control bleeding even in sudden bleeding after TUR-P. No serious side effects were found during the course of this study.

Development of an Efficient G‐Quadruplex‐Stabilised Thrombin‐Binding Aptamer Containing a Three‐Carbon Spacer Molecule

PubMed Central

Aaldering, Lukas J.; Poongavanam, Vasanthanathan; Langkjær, Niels; Murugan, N. Arul; Jørgensen, Per Trolle; Wengel, Jesper

2017-01-01

Abstract 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‐modified UNA (amino‐UNA) on the structural dynamics and stability of TBA. All three modifications were incorporated at three different loop positions (T3, T7, T12) of the TBA G‐quadruplex structure to result in a series of TBA variants and their stability was studied by thermal denaturation; folding was studied by circular dichroism spectroscopy and thrombin clotting time. The results showed that spacer‐C3 introduction at the T7 loop position (TBA‐SP7) significantly improved stability and thrombin clotting time while maintaining a similar binding affinity as TBA to thrombin. Detailed molecular modelling experiments provided novel insights into the experimental observations, further supporting the efficacy of TBA‐SP7. The results of this study could provide valuable information for future designs of TBA analogues with superior thrombin inhibition properties. PMID:28150905

Stabilization of the E* Form Turns Thrombin into an Anticoagulant

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

Bah, Alaji; Carrell, Christopher J.; Chen, Zhiwei

2009-07-31

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

Platelet-derived growth factor inhibits platelet activation in heparinized whole blood.

PubMed

Selheim, F; Holmsen, H; Vassbotn, F S

1999-08-15

We previously have demonstrated that human platelets have functionally active platelet-derived growth factor alpha-receptors. Studies with gel-filtered platelets showed that an autocrine inhibition pathway is transduced through this tyrosine kinase receptor during platelet activation. The physiological significance of this inhibitory effect of platelet-derived growth factor on gel-filtered platelets activation is, however, not known. In the present study, we investigated whether platelet-derived growth factor inhibits platelet activation under more physiological conditions in heparinized whole blood, which represents a more physiological condition than gel-filtered platelets. Using flow cytometric assays, we demonstrate here that platelet-derived growth factor inhibits thrombin-, thrombin receptor agonist peptide SFLLRN-, and collagen-induced platelet aggregation and shedding of platelet-derived microparticles from the platelet plasma membrane during platelet aggregation in stirred heparinized whole blood. The inhibitory effect of platelet-derived growth factor was dose dependent. However, under nonaggregating conditions (no stirring), we could not demonstrate any significant effect of platelet-derived growth factor on thrombin- and thrombin receptor agonist peptide-induced platelet surface expression of P-selectin. Our results demonstrate that platelet-derived growth factor appears to be a true antithrombotic agent only under aggregating conditions in heparinized whole blood.

Expression of plasma membrane receptor genes during megakaryocyte development

PubMed Central

Sun, Sijie; Wang, Wenjing; Latchman, Yvette; Gao, Dayong; Aronow, Bruce

2013-01-01

Megakaryocyte (MK) development is critically informed by plasma membrane-localized receptors that integrate a multiplicity of environmental cues. Given that the current understanding about receptors and ligands involved in megakaryocytopoiesis is based on single targets, we performed a genome-wide search to identify a plasma membrane receptome for developing MKs. We identified 40 transmembrane receptor genes as being upregulated during MK development. Seven of the 40 receptor-associated genes were selected to validate the dataset. These genes included: interleukin-9 receptor (IL9R), transforming growth factor, β receptor II (TGFBR2), interleukin-4 receptor (IL4R), colony stimulating factor-2 receptor-beta (CSFR2B), adiponectin receptor (ADIPOR2), thrombin receptor (F2R), and interleukin-21 receptor (IL21R). RNA and protein analyses confirmed their expression in primary human MKs. Matched ligands to IL9R, TGFBR2, IL4R, CSFR2B, and ADIPOR2 affected megakaryocytopoiesis. IL9 was unique in its ability to increase the number of MKs formed. In contrast, MK colony formation was inhibited by adiponectin, TGF-β, IL4, and GM-CSF. The thrombin-F2R axis affected platelet function, but not MK development, while IL21 had no apparent detectable effects. ADP-induced platelet aggregation was suppressed by IL9, TGF-β, IL4, and adiponectin. Overall, six of seven of the plasma membrane receptors were confirmed to have functional roles in MK and platelet biology. Also, results show for the first time that adiponectin plays a regulatory role in MK development. Together these data support a strong likelihood that the 40 transmembrane genes identified as being upregulated during MK development will be an important resource to the research community for deciphering the complex repertoire of environmental cues regulating megakaryocytopoiesis and/or platelet function. PMID:23321270

A sensitive electrochemical aptasensor based on palladium nanoparticles decorated graphene-molybdenum disulfide flower-like nanocomposites and enzymatic signal amplification.

PubMed

Jing, Pei; Yi, Huayu; Xue, Shuyan; Chai, Yaqin; Yuan, Ruo; Xu, Wenju

2015-01-01

In the present study, with the aggregated advantages of graphene and molybdenum disulfide (MoS2), we prepared poly(diallyldimethylammonium chloride)-graphene/molybdenum disulfide (PDDA-G-MoS2) nanocomposites with flower-like structure, large surface area and excellent conductivity. Furthermore, an advanced sandwich-type electrochemical assay for sensitive detection of thrombin (TB) was fabricated using palladium nanoparticles decorated PDDA-G-MoS2 (PdNPs/PDDA-G-MoS2) as nanocarriers, which were functionalized by hemin/G-quadruplex, glucose oxidase (GOD), and toluidine blue (Tb) as redox probes. The signal amplification strategy was achieved as follows: Firstly, the immobilized GOD could effectively catalyze the oxidation of glucose to gluconolactone, coupling with the reduction of the dissolved oxygen to H2O2. Then, both PdNPs and hemin/G-quadruplex acting as hydrogen peroxide (HRP)-mimicking enzyme could further catalyze the reduction of H2O2, resulting in significant electrochemical signal amplification. So the proposed aptasensor showed high sensitivity with a wide dynamic linear range of 0.0001 to 40 nM and a relatively low detection limit of 0.062 pM for TB determination. The strategy showed huge potential of application in protein detection and disease diagnosis. Copyright © 2014 Elsevier B.V. All rights reserved.

Universal surface-enhanced Raman scattering amplification detector for ultrasensitive detection of multiple target analytes.

PubMed

Zheng, Jing; Hu, Yaping; Bai, Junhui; Ma, Cheng; Li, Jishan; Li, Yinhui; Shi, Muling; Tan, Weihong; Yang, Ronghua

2014-02-18

Up to now, the successful fabrication of efficient hot-spot substrates for surface-enhanced Raman scattering (SERS) remains an unsolved problem. To address this issue, we describe herein a universal aptamer-based SERS biodetection approach that uses a single-stranded DNA as a universal trigger (UT) to induce SERS-active hot-spot formation, allowing, in turn, detection of a broad range of targets. More specifically, interaction between the aptamer probe and its target perturbs a triple-helix aptamer/UT structure in a manner that activates a hybridization chain reaction (HCR) among three short DNA building blocks that self-assemble into a long DNA polymer. The SERS-active hot-spots are formed by conjugating 4-aminobenzenethiol (4-ABT)-encoded gold nanoparticles with the DNA polymer through a specific Au-S bond. As proof-of-principle, we used this approach to quantify multiple target analytes, including thrombin, adenosine, and CEM cancer cells, achieving lowest limit of detection values of 18 pM, 1.5 nM, and 10 cells/mL, respectively. As a universal SERS detector, this prototype can be applied to many other target analytes through the use of suitable DNA-functional partners, thus inspiring new designs and applications of SERS for bioanalysis.

Cation Coordination Alters the Conformation of a Thrombin-Binding G-Quadruplex DNA Aptamer That Affects Inhibition of Thrombin.

PubMed

Zavyalova, Elena; Tagiltsev, Grigory; Reshetnikov, Roman; Arutyunyan, Alexander; Kopylov, Alexey

2016-10-01

Thrombin-binding aptamers are promising anticoagulants. HD1 is a monomolecular antiparallel G-quadruplex with two G-quartets linked by three loops. Aptamer-thrombin interactions are mediated with two TT-loops that bind thrombin exosite I. Several cations were shown to be coordinated inside the G-quadruplex, including K + , Na + , NH 4 + , Ba 2+ , and Sr 2+ ; on the contrary, Mn 2+ was coordinated in the grooves, outside the G-quadruplex. K + or Na + coordination provides aptamer functional activity. The effect of other cations on aptamer functional activity has not yet been described, because of a lack of relevant tests. Interactions between aptamer HD1 and a series of cations were studied. A previously developed enzymatic method was applied to evaluate aptamer inhibitory activity. The structure-function correlation was studied using the characterization of G-quadruplex conformation by circular dichroism spectroscopy. K + coordination provided the well-known high inhibitory activity of the aptamer, whereas Na + coordination supported low activity. Although NH 4 + coordination yielded a typical antiparallel G-quadruplex, no inhibitory activity was shown; a similar effect was observed for Ba 2+ and Sr 2+ coordination. Mn 2+ coordination destabilized the G-quadruplex that drastically diminished aptamer inhibitory activity. Therefore, G-quadruplex existence per se is insufficient for aptamer inhibitory activity. To elicit the nature of these effects, we thoroughly analyzed nuclear magnetic resonance (NMR) and X-ray data on the structure of the HD1 G-quadruplex with various cations. The most reasonable explanation is that cation coordination changes the conformation of TT-loops, affecting thrombin binding and inhibition. HD1 counterparts, aptamers 31-TBA and NU172, behaved similarly with some distinctions. In 31-TBA, an additional duplex module stabilized antiparallel G-quadruplex conformation at high concentrations of divalent cations; whereas in NU172, a different sequence of loops in the G-quadruplex module provided an equilibrium of antiparallel and parallel G-quadruplexes that shifted with cation binding. In conclusion, structures of G-quadruplex aptamers are flexible enough and are fine-tuned with different cation coordination.

Structure-activity analysis of synthetic alpha-thrombin-receptor-activating peptides.

PubMed

Van Obberghen-Schilling, E; Rasmussen, U B; Vouret-Craviari, V; Lentes, K U; Pavirani, A; Pouysségur, J

1993-06-15

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, second or third N-terminal residue leads to a loss of activity, suggesting that a defined spacing of more than one structural component may be important for ligand-receptor interaction. Finally, we did not observe an antagonistic effect of the inactive peptides on phospholipase C activation or DNA synthesis induced by alpha-thrombin (1 nM) or SFLLRNP (3 microM).

Structure-activity analysis of synthetic alpha-thrombin-receptor-activating peptides.

PubMed Central

Van Obberghen-Schilling, E; Rasmussen, U B; Vouret-Craviari, V; Lentes, K U; Pavirani, A; Pouysségur, J

1993-01-01

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, second or third N-terminal residue leads to a loss of activity, suggesting that a defined spacing of more than one structural component may be important for ligand-receptor interaction. Finally, we did not observe an antagonistic effect of the inactive peptides on phospholipase C activation or DNA synthesis induced by alpha-thrombin (1 nM) or SFLLRNP (3 microM). PMID:7686363

Plasma membrane associated phospholipase C from human platelets: Synergistic stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis by thrombin and guanosine 5 prime -O-(3-thiotriphosphate)

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

Baldassare, J.J.; Henderson, P.A.; Fisher, G.J.

1989-01-10

The effects of thrombin and GTP{gamma}S on the hydrolysis of phosphoinositides by membrane-associated phospholipase C (PLC) from human platelets were examined with endogenous ({sup 3}H)inositol-labeled membranes or with lipid vesicles containing either ({sup 3}H)phosphatidylinositol or ({sup 3}H)phosphatidylinositol 4,5-bisphosphate. GTP{gamma}S (1 {mu}M) or thrombin (1 unit/mL) did not stimulate release of inositol trisphosphate (IP{sub 3}), inositol bisphosphate (IP{sub 2}), or inositol phosphate (IP) from ({sup 3}H)inositol-labeled membranes. IP{sub 2} and IP{sub 3}, but not IP, from ({sup 3}H)inositol-labeled membranes were, however, stimulated 3-fold by GTP{gamma}S (1 {mu}M) plus thrombin (1 unit/mL). A higher concentration of GTP{gamma}S (100 {mu}M) alone also stimulatedmore » IP{sub 2} and IP{sub 3}, but not IP, release. In the presence of 1 mM calcium, release of IP{sub 2} and IP{sub 3} was increased 6-fold over basal levels; however, formation of IP was not observed. At submicromolar calcium concentration, hydrolysis of exogenous phosphatidylinositol 4,5-bisphosphate (PIP{sub 2}) by platelet membrane associated PLC was also markedly enhanced by GTP{gamma}S (100 {mu}M) or GTP{gamma}S (1 {mu}M) plus thrombin (1 unit/mL). Under identical conditions, exogenous phosphatidylinositol (PI) was not hydrolyzed. The same substrate specificity was observed when the membrane-associated PLC was activated with 1 mM calcium. Thrombin-induced hydrolysis of PIP{sub 2} was inhibited by treatment of the membranes with pertussis toxin or pretreatment of intact platelets with 12-O-tetradecanoyl-13-acetate (TPA) prior to preparation of membranes. Pertussis toxin did not inhibit GTP{gamma}S (100 {mu}M) or calcium (1 mM) dependent PIP{sub 2} breakdown, while TPA inhibited GTP{gamma}S-dependent but not calcium-dependent phospholipase C activity.« less

Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets

PubMed Central

Möhle, Robert; Green, David; Moore, Malcolm A. S.; Nachman, Ralph L.; Rafii, Shahin

1997-01-01

We have shown that coculture of bone marrow microvascular endothelial cells with hematopoietic progenitor cells results in proliferation and differentiation of megakaryocytes. In these long-term cultures, bone marrow microvascular endothelial cell monolayers maintain their cellular integrity in the absence of exogenous endothelial growth factors. Because this interaction may involve paracrine secretion of cytokines, we evaluated megakaryocytic cells for secretion of vascular endothelial growth factor (VEGF). Megakaryocytes (CD41a+) were generated by ex vivo expansion of hematopoietic progenitor cells with kit-ligand and thrombopoietin for 10 days and further purified with immunomagnetic microbeads. Using reverse transcription–PCR, we showed that megakaryocytic cell lines (Dami, HEL) and purified megakaryocytes expressed mRNA of the three VEGF isoforms (121, 165, and 189 amino acids). Large quantities of VEGF (>1 ng/106 cells/3 days) were detected in the supernatant of Dami cells, ex vivo-generated megakaryocytes, and CD41a+ cells isolated from bone marrow. The constitutive secretion of VEGF by CD41a+ cells was stimulated by growth factors of the megakaryocytic lineage (interleukin 3, thrombopoietin). Western blotting of heparin–Sepharose-enriched supernatant mainly detected the isoform VEGF165. In addition, immunohistochemistry showed intracytoplasmic VEGF in polyploid megakaryocytes. Thrombin stimulation of megakaryocytes and platelets resulted in rapid release of VEGF within 30 min. We conclude that human megakaryocytes produce and secrete VEGF in an inducible manner. Within the bone marrow microenvironment, VEGF secreted by megakaryocytes may contribute to the proliferation of endothelial cells. VEGF delivered to sites of vascular injury by activated platelets may initiate angiogenesis. PMID:9012841

Platelet glycoprotein VI binds to polymerized fibrin and promotes thrombin generation.

PubMed

Mammadova-Bach, Elmina; Ollivier, Véronique; Loyau, Stéphane; Schaff, Mathieu; Dumont, Bénédicte; Favier, Rémi; Freyburger, Geneviève; Latger-Cannard, Véronique; Nieswandt, Bernhard; Gachet, Christian; Mangin, Pierre H; Jandrot-Perrus, Martine

2015-07-30

Fibrin, the coagulation end product, consolidates the platelet plug at sites of vascular injury and supports the recruitment of circulating platelets. In addition to integrin αIIbβ3, another as-yet-unidentified receptor is thought to mediate platelet interaction with fibrin. Platelet glycoprotein VI (GPVI) interacts with collagen and several other adhesive macromolecules. We evaluated the hypothesis that GPVI could be a functional platelet receptor for fibrin. Calibrated thrombin assays using platelet-rich plasma (PRP) showed that tissue factor-triggered thrombin generation was impaired in GPVI-deficient patients and reduced by the anti-GPVI Fab 9O12. Assays on reconstituted PRP and PRP from fibrinogen-deficient patients revealed a fibrinogen-dependent enhancement of thrombin generation, which relied on functional GPVI. The effect of GPVI was found to depend on fibrin polymerization. A binding assay showed a specific interaction between GPVI-Fc and fibrin, inhibited by the Fab 9O12. This Fab also reduced platelet adhesion to fibrin at low (300 s(-1)) and high (1500 s(-1)) wall shear rates. Platelets adherent to fibrin displayed shape change, exposure of procoagulant phospholipids, and the formation of small clots. When hirudinated blood was perfused at 1500 s(-1) over preformed fibrin-rich clots, the Fab 9O12 decreased the recruitment of platelets by up to 85%. This study identifies GPVI as a platelet receptor for polymerized fibrin with 2 major functions: (1) amplification of thrombin generation and (2) recruitment of circulating platelets to clots. These so-far-unrecognized properties of GPVI confer on it a key role in thrombus growth and stabilization. © 2015 by The American Society of Hematology.

Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin

PubMed Central

Treuheit, Nicholas A.; Beach, Muneera A.; Komives, Elizabeth A.

2011-01-01

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 chloromethylketone to the active site serine, as well as non-covalent 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 the same 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 to exosite 1. PMID:21526769

Laboratory assessment of warfarin reversal with global coagulation tests versus international normalized ratio in patients with intracranial bleeding.

PubMed

Voils, Stacy A; Martin, Erika J; Mohammed, Bassem M; Bayrlee, Ahmad; Brophy, Donald F

2015-06-01

We assess the in-vivo relationship between international normalized ratio (INR) and global coagulation tests in patients with life-threatening bleeding who received prothrombin complex concentrate (PCC) for warfarin reversal. This was a prospective pilot study in adult patients with intracranial bleeding related to anticoagulation with warfarin. Thromboelastography (TEG), thrombin generation parameters and INR were assessed at baseline, 30  min, 2 and 24  h after PCC. Changes in laboratory parameters and relationship between INR and global coagulation tests were assessed over time. Eight patients mean [standard deviation (SD)] age 72 (16) were included and received mean (SD) dose of PCC 24 (5) units/kg. Four patients died during the study, all with INR values more than 1.5 thirty minutes after PCC. Mean (SD) INR was 3.0 (1.3) and decreased significantly to 1.8 (0.48) thirty minutes after PCC (P < 0.01). Baseline endogenous thrombin potential and thrombin peak were 890  nmol/min and 123  nmol and increased significantly to 1943  nmol/min (P < 0.01) and 301  nmol (P < 0.01) 30  min after PCC administration. Reaction (R)-time decreased significantly (P = 0.02), and maximum amplitude and overall coagulation index (CI) significantly increased during treatment (P < 0.01, respectively). Thrombin generation and TEG values corrected after PCC administration; however, INR did not fully correct. Patients that died tended to be older with prolonged INR values across the study period. INR and TEG values correlated well with thrombin generation before administration of PCC, but this relationship was lost afterward.

Fibrin, γ’-fibrinogen, and trans-clot pressure gradient control hemostatic clot growth during human blood flow over a collagen/tissue factor wound

PubMed Central

Muthard, Ryan W.; Welsh, John D.; Brass, Lawrence F.; Diamond, Scott L.

2015-01-01

SUMMARY Objective Biological and physical factors interact to modulate blood response in a wounded vessel, resulting in a hemostatic clot or an occlusive thrombus. Flow and pressure differential (ΔP) across the wound from the lumen to the extravascular compartment may impact hemostasis and the observed core/shell architecture. We examined physical and biological factors responsible for regulating thrombin mediated clot growth. Approach and Results Using factor XIIa-inhibited human whole blood perfused in a microfluidic device over collagen/tissue factor at controlled wall shear rate and ΔP, we found thrombin to be highly localized in the P-selectin+ core of hemostatic clots. Increasing ΔP from 9 to 29 mm-Hg (wall shear rate = 400 s−1) reduced P-selectin+ core size and total clot size due to enhanced extravasation of thrombin. Blockade of fibrin polymerization with 5 mM GPRP dysregulated hemostasis by enhancing both P-selectin+ core size and clot size at 400 s−1 (20 mm-Hg). For whole blood flow (no GPRP), the thickness of the P-selectin-negative shell was reduced under arterial conditions (2000 s−1, 20 mm-Hg). Consistent with the antithrombin-1 activity of fibrin implicated with GPRP, anti-γ’-fibrinogen antibody enhanced core-localized thrombin, core size, and overall clot size, especially at venous (100 s−1) but not arterial wall shear rates (2000 s−1). Pathological shear (15,000 s−1) and GPRP synergized to exacerbate clot growth. Conclusions Hemostatic clotting was dependent on core-localized thrombin that (1) triggered platelet P-selectin display and (2) was highly regulated by fibrin and the trans-clot ΔP. Also, γ’-fibrinogen had a role in venous but not arterial conditions. PMID:25614284

Dual Regulation of Glycogen Synthase Kinase 3 (GSK3)α/β by Protein Kinase C (PKC)α and Akt Promotes Thrombin-mediated Integrin αIIbβ3 Activation and Granule Secretion in Platelets*

PubMed Central

Moore, Samantha F.; van den Bosch, Marion T. J.; Hunter, Roger W.; Sakamoto, Kei; Poole, Alastair W.; Hers, Ingeborg

2013-01-01

Glycogen synthase kinase-3 is a Ser/Thr kinase, tonically active in resting cells but inhibited by phosphorylation of an N-terminal Ser residue (Ser21 in GSK3α and Ser9 in GSK3β) in response to varied external stimuli. Recent work suggests that GSK3 functions as a negative regulator of platelet function, but how GSK3 is regulated in platelets has not been examined in detail. Here, we show that early thrombin-mediated GSK3 phosphorylation (0–30 s) was blocked by PKC inhibitors and largely absent in platelets from PKCα knock-out mice. In contrast, late (2–5 min) GSK3 phosphorylation was dependent on the PI3K/Akt pathway. Similarly, early thrombin-mediated inhibition of GSK3 activity was blocked in PKCα knock-out platelets, whereas the Akt inhibitor MK2206 reduced late thrombin-mediated GSK3 inhibition and largely prevented GSK3 inhibition in PKCα knock-out platelets. More importantly, GSK3 phosphorylation contributes to platelet function as knock-in mice where GSK3α Ser21 and GSK3β Ser9 were mutated to Ala showed a significant reduction in PAR4-mediated platelet aggregation, fibrinogen binding, and P-selectin expression, whereas the GSK3 inhibitor CHIR99021 enhanced these responses. Together, these results demonstrate that PKCα and Akt modulate platelet function by phosphorylating and inhibiting GSK3α/β, thereby relieving the negative effect of GSK3α/β on thrombin-mediated platelet activation. PMID:23239877

A polymeric sealant inhibits anastomotic suture hole bleeding more rapidly than gelfoam/thrombin: results of a randomized controlled trial.

PubMed

Glickman, Marc; Gheissari, Ali; Money, Samuel; Martin, John; Ballard, Jeffrey L

2002-03-01

An experimental polymeric sealant (CoSeal [Cohesion Technologies, Palo Alto, Calif]) provides equivalent anastomotic sealing to Gelfoam (Upjohn, Kalamazoo, Mich)/thrombin during surgical placement of prosthetic vascular grafts. Randomized controlled trial. Nine university-affiliated medical centers. One hundred forty-eight patients scheduled for implantation of polytetrafluoroethylene grafts, mainly for infrainguinal revascularization procedures or the creation of dialysis access shunts, who were treated randomly with either an experimental intervention (n = 74) or control (n = 74). Following polytetrafluoroethylene graft placement, anastomotic suture hole bleeding was treated intraoperatively in all control subjects with Gelfoam/thrombin. Subjects in the experimental group had the polymeric sealant applied directly to the suture lines without concomitant manual compression. Primary treatment success was defined as the proportion of subjects in each group that achieved complete anastomotic sealing within 10 minutes. The proportion of subjects that achieved immediate sealing and the time required to fully inhibit suture hole bleeding also were compared between treatment groups. Overall 10-minute sealing success was equivalent (86% vs 80%; P =.29) between experimental and control subjects, respectively. However, subjects treated with CoSeal achieved immediate anastomotic sealing at more than twice the rate of subjects treated with Gelfoam/thrombin (47% vs 20%; P<.001). Consequently, the median time needed to inhibit bleeding in control subjects was more than 10 times longer than for experimental subjects (16.5 seconds vs 189.0 seconds; P =.01). Strikingly similar findings for all comparisons were observed separately for subgroups of subjects having infrainguinal bypass grafting and for those undergoing placement of dialysis access shunts. The experimental sealant offers equivalent anastomotic sealing performance compared with Gelfoam/thrombin, but it provides this desired effect in a significantly more rapid time frame.

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.

PubMed

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

2013-07-01

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.

An evaluation of platelet-rich plasma without thrombin activation with or without anorganic bone mineral in the treatment of human periodontal intrabony defects.

PubMed

Rodrigues, Silvia V; Acharya, Anirudh B; Thakur, Srinath L

2011-01-01

The efficacy of platelet-rich plasma (PRP) in periodontal regeneration is not well understood and the definite clinical viability of blood derived platelets lacks clarity. Also, the use of thrombin for platelet activation is disputed. Hence, the purpose of this study was to evaluate the efficacy of blood derived platelets without thrombin activation, alone or in combination with bovine anorganic bone mineral (ABM), in the treatment of human periodontal intrabony defects. PRP was prepared using a simple tabletop centrifuge and activated using calcium chloride without the addition of thrombin. This PRP was used alone (in Group A) and in combination with bovine ABM (in Group B) in the treatment of human periodontal angular defects. Both the control and the test groups showed definite improvement in clinical parameters. On comparison, however, there was a statistically significant improvement in the probing pocket depths and relative attachment level in Group B over Group A at 3 and 6 months intervals, whereas at the end of 9 months this difference was not statistically significant. There was no statistically significant difference between the groups with respect to the relative defect depth. Within the limitations of this study and the type of PRP used, i.e. without thrombin mediated activation, it can be concluded that both PRP and PRP combined with bovine ABM results in significant clinical improvement. Albeit statistically insignificant, there is a preponderance of better clinical results with the addition of ABM to PRP. Further studies need to be carried out on a larger sample size to confirm the results of the present study.

In vitro comparison of the effect of two factor XI (FXI) concentrates on thrombin generation in major FXI deficiency.

PubMed

Pike, G N; Cumming, A M; Hay, C R M; Sempasa, B; Sutherland, M; Thachil, J; Burthem, J; Bolton-Maggs, P H B

2016-05-01

Bleeding risk in factor XI (FXI) deficiency following surgery may be reduced by treatment with either of two FXI concentrates, but indications for their use are unclear and treatment has been associated with thrombosis. To quantify and compare the effects of two different FXI concentrates on thrombin generation (TG) in major FXI deficiency (FXI:C < 15 IU dL(-1) ). Thrombin generation was measured in controls (n = 50), FXI-deficient individuals pre and post in vitro spiking with FXI concentrates (n = 10), and in ex vivo samples following treatment with FXI concentrate (n = 3). Thrombin generation was significantly impaired in FXI deficiency but improved following FXI replacement in vitro and in vivo. LFB Hemoleven(®) had greater effect on TG than BPL FXI concentrate in vitro (equivalent in vivo doses 10, 20 and 30 U kg(-1) ): higher endogenous thrombin potential (ETP) (P < 0.0001), peak height (P < 0.01) velocity (P < 0.0002) and shorter lag time and time to peak (both P < 0.003). Some measurements with LFB Hemoleven(®) exceeded the reference range. At lower dose (5 U kg(-1) ), BPL FXI concentrate normalized all TG parameters and LFB Hemoleven(®) normalized the ETP but exceeded the reference range with other parameters. Both FXI concentrates improve TG in vitro in major FXI deficiency but differ in dose response, and for both products, doses lower than previously recommended normalized TG in vitro. Comparison of in vitro spiked and ex vivo samples suggest that in vitro results could be used to estimate an expected in vivo response to FXI replacement. © 2015 John Wiley & Sons Ltd.

Extract of Aronia melanocarpa-modified hemostasis: in vitro studies.

PubMed

Sikora, Joanna; Markowicz-Piasecka, Magdalena; Broncel, Marlena; Mikiciuk-Olasik, Elżbieta

2014-10-01

Aronia melanocarpa has an extremely high content of procyanidins and anthocyanins. The multidirectional benefits of consumption of these berries are widely reported. Although numerous studies confirmed the influence of polyphenols on various stages of hemostasis, the exact mechanism of this phenomenon is not understood. The aim of our study was to evaluate the in vitro effect of A. melanocarpa extract on various parameters of hemostasis. Adenosine 5'-diphosphate (ADP)-induced aggregation was measured with turbidimetric method. Spontaneous and ADP-activated platelet adhesion were investigated using a colorimetric method. The global assay of coagulation and fibrinolysis was performed with the use of optical clotting and lysis (CL) test. Thrombin (0.5 IU/mL) and tissue plasminogen activator (60 ng/mL) were used to obtain a CL curve. The activity of thrombin and plasmin was determined by means of chromogenic substrate (S-2238, S-2251) RESULTS: The aronia extract contributed to the reduction in spontaneous and ADP-activated platelet adhesion. A significant increase in overall potential of CL as well as significant changes in key parameters of these processes (T t-thrombin time, F vo-initial plasma clotting velocity, and L max-maximum lysis) was reported. Chokeberry extract significantly inhibited the amidolytic activity of thrombin and plasmin. Our in vitro findings indicate a complex mechanism of influence of chokeberry polyphenols on platelet activity and the overall potential of CL. We confirmed that chokeberry inhibits the amidolytic activity of thrombin. It was demonstrated for the first time that chokeberry polyphenols inhibit the amidolytic activity of another serine protease, i.e., plasmin, which is the main fibrinolytic enzyme. Furthermore, our research points out a significant contribution of other plasma components and fibrinogen in the modulation of hemostasis by polyphenols.

[Point-of-Care Testing in Trauma Patients - Methods and Evidence].

PubMed

Dirkmann, Daniel; Britten, Martin W; Frey, Ulrich H

2018-06-01

In severely injured patients, trauma-induced coagulopathy (TIC) present at hospital admission is associated with increased transfusion requirements, morbidity and mortality. Early and effective treatment contributes to improved survival rates. Laboratory coagulation assays have long turn-around times and evidence for their usefulness, especially in the context of TIC, is weak. Due to the lack of appropriate guidance, transfusion of allogeneic blood products frequently follows a ratio-based concept (e.g., transfusion of erythrocytes and plasma in a 1 : 1 ratio). Point-of-care (PoC) tests enable the assessment of prothrombin time (PT) and activated partial thromboplastin time in few minutes. However, although normal PT in these tests allows to rule out relevant effects of several anticoagulants, they are not able to detect patients with TIC and/or requiring subsequent massive transfusion. Viscoelastic tests (VETs) make it possible to assess defects in thrombin generation, hypofibrinogenaemia, thrombocytopenia, and hyperfibrinolysis, and thus enable targeted therapy. Impairment of platelet function is the common blind spot not detectable using both standard laboratory-based tests and VETs. However, PoC platelet function tests enable to detect platelet defects and patients taking anti-platelet. Furthermore, impaired platelet function has been identified as a strong predictor for coagulopathy and massive transfusion in trauma patients. In other clinical settings, coagulation management based on VETs is associated with decreased transfusion requirements, incidence of acute kidney failure, and mortality, respectively. Data of the first small prospective randomised trial indicate superiority of VET guided coagulation management solely using coagulation factor concentrates, when compared to plasma transfusions in severe trauma. Georg Thieme Verlag KG Stuttgart · New York.

Thrombin-like enzymes from snake venom: Structural characterization and mechanism of action.

PubMed

Ullah, Anwar; Masood, Rehana; Ali, Ijaz; Ullah, Kifayat; Ali, Hamid; Akbar, Haji; Betzel, Christian

2018-07-15

Snake venom thrombin-like enzymes (SVTLEs) constitute the major portion (10-24%) of snake venom and these are the second most abundant enzymes present in the crude venom. During envenomation, these enzymes had shown prominently the various pathological effects, such as disturbance in hemostatic system, fibrinogenolysis, fibrinolysis, platelet aggregation, thrombosis, neurologic disorders, activation of coagulation factors, coagulant, procoagulant etc. These enzymes also been used as a therapeutic agent for the treatment of various diseases such as congestive heart failure, ischemic stroke, thrombotic disorders etc. Although the crystal structures of five SVTLEs are available in the Protein Data Bank (PDB), there is no single article present in the literature that has described all of them. The current work describes the structural aspects, structure-based mechanism of action, processing and inhibition of these enzymes. The sequence analysis indicates that these enzymes show a high sequence identity (57-85%) with each other and low sequence identity with trypsin (36-43%), human alpha-thrombin (29-36%) and other snake venom serine proteinases (57-85%). Three-dimensional structural analysis indicates that the loops surrounding the active site are variable both in amino acids composition and length that may convey variable substrate specificity to these enzymes. The surface charge distributions also vary in these enzymes. Docking analysis with suramin shows that this inhibitor preferably binds to the C-terminal region of these enzymes and causes the destabilization of their three-dimensional structure. Copyright © 2018 Elsevier B.V. All rights reserved.

Dysfibrinogen Kagoshima with the amino acid substitution gammaThr-314 to Ile: analyses of molecular abnormalities and thrombophilic nature of this abnormal molecule.

PubMed

Niwa, Kazuki; Mimuro, Jun; Miyata, Masaaki; Sugo, Teruko; Ohmori, Tsukasa; Madoiwa, Seiji; Tei, Chuwa; Sakata, Yoichi

2008-01-01

Emerging lines of evidence have suggested that certain dysfibrinogens present a significant risk of thrombosis. The thrombophilic nature of a new-type of dysfibrinogen Kagoshima identified in a 36-year-old female with deep vein thrombosis during the postpartum period was studied. Based on the analyses of the patient fibrinogen and the fibrinogen genes, fibrinogen Kagoshima was shown to have the amino acid substitution of gammaThr-314 to Ile that resulted in impaired function and hypofibrinogenemia. Polymerization of fibrin monomers derived from patient fibrinogen was severely impaired with a partial correction in the presence of calcium ions, causing very low clottability and delayed cross-linking of patient fibrin catalyzed by activated factor XIII. Because of the low clottability, a large amount of soluble fibrin was formed upon thrombin treatment, resulting in an increase of thrombin in the soluble fraction. Additionally, tPA-mediated plasmin generation on fibrin was impaired and calcium-ion-dependent integrity of the gamma-chain D domain of Kagoshima fibrinogen was perturbed. The presence of many tapered-fiber ends inside the tangled fibrin networks, observed by scanning electron microscopy, suggested early termination of fibrin polymerization and the structural alteration. These data suggest that fibrinogen Kagoshima is dysfunctional, giving rise to formation of fibrinolysis-resistant soluble fibrin polymers and entrance of soluble fibrin associating with thrombin to the circulation, partly accounting for the thrombophilic nature of the affected fibrinogen and fibrin molecules.

U.S. Army Medical Department Journal, July-September 2004

DTIC Science & Technology

2004-09-01

on the surface of intestinal, lung, and brain cells. The TF protein then converts FVII into an activated form. Activated factor VII then combines...and FIX, which limits generation of thrombin. Platelets are able to increase production of thrombin when increased FVIIa is present. Normally FVII ...hemorrhage in severe neonatal FVII deficiency. Hemophilia. 2000; 6: 50-54. 12. Gilchrist J. Use of recombinant factor VIIa to treat a severe

New Insights in Thrombin Inhibition Structure-Activity Relationships by Characterization of Octadecasaccharides from Low Molecular Weight Heparin.

PubMed

Mourier, Pierre A J; Guichard, Olivier Y; Herman, Fréderic; Sizun, Philippe; Viskov, Christian

2017-03-08

Low Molecular Weight Heparins (LMWH) are complex anticoagulant drugs that mainly inhibit the blood coagulation cascade through indirect interaction with antithrombin. While inhibition of the factor Xa is well described, little is known about the polysaccharide structure inhibiting thrombin. In fact, a minimal chain length of 18 saccharides units, including an antithrombin (AT) binding pentasaccharide, is mandatory to form the active ternary complex for LMWH obtained by alkaline β-elimination (e.g., enoxaparin). However, the relationship between structure of octadecasaccharides and their thrombin inhibition has not been yet assessed on natural compounds due to technical hurdles to isolate sufficiently pure material. We report the preparation of five octadecasaccharides by using orthogonal separation methods including size exclusion, AT affinity, ion pairing and strong anion exchange chromatography. Each of these octadecasaccharides possesses two AT binding pentasaccharide sequences located at various positions. After structural elucidation using enzymatic sequencing and NMR, in vitro aFXa and aFIIa were determined. The biological activities reveal the critical role of each pentasaccharide sequence position within the octadecasaccharides and structural requirements to inhibit thrombin. Significant differences in potency, such as the twenty-fold magnitude difference observed between two regioisomers, further highlights the importance of depolymerisation process conditions on LMWH biological activity.

Thrombin generation in mesalazine refractory ulcerative colitis and the influence of low molecular weight heparin.

PubMed

Vrij, Anton A; Oberndorff-Klein-Woolthuis, Ardi; Dijkstra, Gerard; de Jong, Andrea E; Wagenvoord, Rob; Hemker, Hendrik C; Stockbrügger, Reinhold W

2007-10-01

In ulcerative colitis (UC), a state of hypercoagulation has frequently been observed. Low molecular weight heparin (LMWH) has shown beneficial effects as an adjuvant treatment of steroid refractory UC in open trials. We assessed potential therapeutic effects of the LMWH reviparin in hospitalised patients with mesalazine refractory UC, as well as its influence on haemostasis factors. Twenty-nine patients with mild-to-moderately active UC were included in a double-blind placebo controlled trial. All patients had a flare-up of disease under mesalazine treatment. Reviparin (Clivarin) 3,436 IU anti-Xa/0.6 ml or placebo s.c. was added, and self-administered twice daily for 8 weeks. Patients were monitored for possible adverse events and changes in clinical symptoms. Endoscopical, histological, biochemical and haemostasis parameters were analysed. Tolerability and compliance were excellent and no serious adverse events occurred. No significant differences were observed on the clinical, endoscopical and histological outcome, as compared to placebo. A high intrinsic and extrinsic thrombin potential was found before LMWH therapy. However, the significant reduction in the thrombin generation by LMWH was not related to the reduction in disease activity. The LMWH reviparine reduces thrombin generation in patients with mild-to-moderately active, mesalazine refractory UC, but is not associated with a reduction in disease activity.

Combination of FVIII and by-passing agent potentiates in vitro thrombin production in haemophilia A inhibitor plasma.

PubMed

Klintman, Jenny; Astermark, Jan; Berntorp, Erik

2010-11-01

The by-passing agents, recombinant activated factor VII (rFVIIa) and activated prothrombin complex concentrate (APCC), are important tools in the treatment of patients with haemophilia A and high-responding inhibitory antibodies. It has been observed clinically that in some patients undergoing immune tolerance induction the bleeding frequency decreases, hypothetically caused by a transient haemostatic effect of infused FVIII not measurable ex vivo. We evaluated how by-passing agents and factor VIII (FVIII) affect thrombin generation (TG) in vitro using plasma from 11 patients with severe haemophilia A and high titre inhibitors. Samples were spiked with combinations of APCC, rFVIIa and five different FVIII products. Combination of APCC and FVIII showed a synergistic effect in eliciting TG (P<0·005) for four FVIII products. When rFVIIa and FVIII were combined the interaction between the preparations was found to be additive. APCC and rFVIIa were then combined without FVIII, resulting in an additive effect on thrombin production. Each product separately increased TG above baseline. In conclusion, the amount of thrombin formed in vitro by adding a by-passing agent, was higher in the presence of FVIII. Our findings support the use of FVIII in by-passing therapy to optimize the haemostatic effect. © 2010 Blackwell Publishing Ltd.

Metabolic plasticity in resting and thrombin activated platelets.

PubMed

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

2015-01-01

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

Label-free electrochemical biosensors based on 3,3',5,5'-tetramethylbenzidine responsive isoporous silica-micelle membrane.

PubMed

Sun, Qinqin; Yan, Fei; Su, Bin

2018-05-15

3,3',5,5'-Tetramethylbenzidine (TMB) has been frequently used as an indicator in G-quadruplex/hemin DNAzyme (G4zyme)-based chemical and biochemical analysis, and its oxidation products are usually monitored by electrochemical or optical methods to quantify G4zyme formation-related analytes. Herein we report a simple electrochemical approach based on isoporous silica-micelle membrane (iSMM) to measure TMB, instead of its oxidation products, in G4zyme-based detection of specific analytes. The iSMM was grown on the indium tin oxide (ITO) electrode, which was composed of highly ordered, vertically oriented silica nanochannels and cylindrical micelles of cetyltrimethylammonium. The iSMM-ITO electrode was selectively responsive to neutral TMB but not its oxidation products, thanks to the sieving and pre-concentration capacity of micellar structures in terms of molecular charge and lipophilicity. In other words, only TMB could be extracted and enriched into micelles and subsequently oxidized at the underlying ITO electrode surface (namely the micelle/ITO interface), generating an amplified anodic current. Since the depletion of TMB was catalyzed by G4zymes formed in the presence of specific analyte, the decrease of this anodic current enabled the quantitative detection of this analyte. The current variation relative to its initial value ((j 0 -j)/j 0 ), termed as the current attenuation ratio, showed the obvious dependence on the analyte concentration. As proof-of-concept experiments, four substances, i.e., potassium cation (K + ), adenosine triphosphate, thrombin and nucleic acid, were detected in aqueous media and the analysis of K + in pre-treated human serum was also performed. Copyright © 2018 Elsevier B.V. All rights reserved.

Reversible inhibition of the platelet procoagulant response through manipulation of the Gardos channel.

PubMed

Wolfs, Jef L; Wielders, Simone J; Comfurius, Paul; Lindhout, Theo; Giddings, John C; Zwaal, Robert F; Bevers, Edouard M

2006-10-01

The platelet procoagulant response requires a sustained elevation of the intracellular Ca2+ concentration, [Ca2+]i, causing exposure of phosphatidylserine (PS) at the outer surface of the plasma membrane. An increased [Ca2+]i also activates Ca2+-dependent K+ channels. Here, we investigated the contribution of the efflux of K+ ions on the platelet procoagulant response in collagen-thrombin-activated platelets using selective K+ channel blockers. The Gardos channel blockers clotrimazol, charybdotoxin, and quinine caused a similar decrease in prothrombinase activity as well as in the number of PS-exposing platelets detected by fluorescence-conjugated annexin A5. Apamin and iberiotoxin, inhibitors of other K+ channels, were without effect. Only clotrimazol showed a significant inhibition of the collagen-plus-thrombin-induced intracellular calcium response. Clotrimazol and charybdotoxin did not inhibit aggregation and release under the conditions used. Inhibition by Gardos channel blockers was reversed by valinomycin, a selective K+ ionophore. The impaired procoagulant response of platelets from a patient with Scott syndrome was partially restored by pretreatment with valinomycin, suggesting a possible defect of the Gardos channel in this syndrome. Collectively, these results provide evidence for the involvement of efflux of K+ ions through Ca2+-activated K+ channels in the procoagulant response of platelets, opening potential strategies for therapeutic interventions.

Modification of Pulsed Electric Field Conditions Results in Distinct Activation Profiles of Platelet-Rich Plasma.

PubMed

Frelinger, Andrew L; Gerrits, Anja J; Garner, Allen L; Torres, Andrew S; Caiafa, Antonio; Morton, Christine A; Berny-Lang, Michelle A; Carmichael, Sabrina L; Neculaes, V Bogdan; Michelson, Alan D

2016-01-01

Activated autologous platelet-rich plasma (PRP) used in therapeutic wound healing applications is poorly characterized and standardized. Using pulsed electric fields (PEF) to activate platelets may reduce variability and eliminate complications associated with the use of bovine thrombin. We previously reported that exposing PRP to sub-microsecond duration, high electric field (SMHEF) pulses generates a greater number of platelet-derived microparticles, increased expression of prothrombotic platelet surfaces, and differential release of growth factors compared to thrombin. Moreover, the platelet releasate produced by SMHEF pulses induced greater cell proliferation than plasma. To determine whether sub-microsecond duration, low electric field (SMLEF) bipolar pulses results in differential activation of PRP compared to SMHEF, with respect to profiles of activation markers, growth factor release, and cell proliferation capacity. PRP activation by SMLEF bipolar pulses was compared to SMHEF pulses and bovine thrombin. PRP was prepared using the Harvest SmartPreP2 System from acid citrate dextrose anticoagulated healthy donor blood. PEF activation by either SMHEF or SMLEF pulses was performed using a standard electroporation cuvette preloaded with CaCl2 and a prototype instrument designed to take into account the electrical properties of PRP. Flow cytometry was used to assess platelet surface P-selectin expression, and annexin V binding. Platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), endothelial growth factor (EGF) and platelet factor 4 (PF4), and were measured by ELISA. The ability of supernatants to stimulate proliferation of human epithelial cells in culture was also evaluated. Controls included vehicle-treated, unactivated PRP and PRP with 10 mM CaCl2 activated with 1 U/mL bovine thrombin. PRP activated with SMLEF bipolar pulses or thrombin had similar light scatter profiles, consistent with the presence of platelet-derived microparticles, platelets, and platelet aggregates whereas SMHEF pulses primarily resulted in platelet-derived microparticles. Microparticles and platelets in PRP activated with SMLEF bipolar pulses had significantly lower annexin V-positivity than those following SMHEF activation. In contrast, the % P-selectin positivity and surface P-selectin expression (MFI) for platelets and microparticles in SMLEF bipolar pulse activated PRP was significantly higher than that in SMHEF-activated PRP, but not significantly different from that produced by thrombin activation. Higher levels of EGF were observed following either SMLEF bipolar pulses or SMHEF pulses of PRP than after bovine thrombin activation while VEGF, PDGF, and PF4 levels were similar with all three activating conditions. Cell proliferation was significantly increased by releasates of both SMLEF bipolar pulse and SMHEF pulse activated PRP compared to plasma alone. PEF activation of PRP at bipolar low vs. monopolar high field strength results in differential platelet-derived microparticle production and activation of platelet surface procoagulant markers while inducing similar release of growth factors and similar capacity to induce cell proliferation. Stimulation of PRP with SMLEF bipolar pulses is gentler than SMHEF pulses, resulting in less platelet microparticle generation but with overall activation levels similar to that obtained with thrombin. These results suggest that PEF provides the means to alter, in a controlled fashion, PRP properties thereby enabling evaluation of their effects on wound healing and clinical outcomes.

The effect of hirudin modification of silk fibroin on cell growth and antithrombogenicity.

PubMed

Wang, Qiongyu; Tu, Fangfang; Liu, Yunfei; Zhang, Yujin; Li, Helei; Kang, Zhao; Yin, Yin; Wang, Jiannan

2017-06-01

Thrombus formation remains a particular challenge for small-diameter vascular grafts. In this study, the direct thrombin inhibitor hirudin (Hir) was used to modify silk fibroin films in an attempt to enhance its antithrombogenic properties. Hir was successfully attached to silk fibroin and uniformly distributed in the regenerative material. Hir-modified films showed good cytocompatibility, and supported adhesion and proliferation of fibroblasts (L929), human umbilical vascular endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs). Proliferation of HAVSMCs was inhibited by increasing Hir concentration. Activated partial thrombin time (APTT), prothrombin time (PT) and thrombin time (TT) of Hir-modified silk fibroin tubular scaffolds (SFTSs) were all increased markedly compared with fresh rabbit blood, ethanol-treated SFTS and unmodified SFTS, demonstrating the improved antithrombogenicity of SFTSs following modification with Hir. Copyright © 2017 Elsevier B.V. All rights reserved.

[THROMBIN-MEDIATED EFFECTS OF BLOOD MICROPARTICLES ON FORMATION, STRUCTURE, AND STABILITY OF FIBRIN CLOTS].

PubMed

Nabiullina, R M; Mustafin, I G; Ataullakhanov, F I; Litvinov, R I; Zubairova, L D

2015-07-01

The effects of blood microparticles (MPs) on the dynamics of fibrin polymerization, clot structure and susceptibility to fibrinolysis were studied. Kinetics of fibrin polymerization, fibrinolysis, thrombin generation in platelet-free, microparticle-depleted and microparticle-depleted plasma replenished with cephalin, from healthy donors were analyzed in parallel. MPs have profound effects on all stages of fibrin formation, decrease its turbidity. All parameters obtained in the absence of MPs were recovered after reconstitution of phospholipids. Thrombin generation rates were reduced in the absence of MPs. In the presence of MPs the fibrin networks had less poro us structures with thinner fibers, while clots formed in the absence of MPs had larger pores and were built of thicker fibers. Clots formed in the presence of MPs were significantly more resistant to fibrinolysis. Results show that normally circulating MPs can support the formation of stable clots at the sites of vascular injury.

The use of argatroban for carotid endarterectomy in heparin-induced thrombocytopenia.

PubMed

Hallman, Sarah E; Hebbar, Latha; Robison, Jay; Uber, Walter E

2005-04-01

Heparin-induced thrombocytopenia (HIT) is a major obstacle in cardiovascular surgeries. In this case report, we used argatroban, a direct thrombin inhibitor, to achieve and maintain anticoagulation for carotid endarterectomy. Unlike heparin, the direct thrombin inhibitors bind directly to thrombin, bypassing antithrombin III and the potential to precipitate HIT. A bolus of argatroban 150 microg/kg followed by an infusion of 5 microg . kg(-1) . min(-1) was used, and adequate anticoagulation was demonstrated with multiple laboratory tests (at 28 min, prothrombin time = 29.8 s, partial thromboplastin time = 69.1 s, international normalized ratio = 3.52 s, and activated clotting time = 220 s). The surgery was successful, and the patient was discharged the next day with no postoperative neurologic sequelae or other complications. We conclude that argatroban can be used safely and successfully for carotid endarterectomy in a patient with a history of HIT.

Na[superscript +] binding to meizothrombin desF1

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

Papaconstantinou, M.E.; Gandhi, P.S.; Chen, Z.

2009-06-10

Meizothrombin is the physiologically active intermediate generated by a single cleavage of prothrombin at R320 to separate the A and B chains. Recent evidence has suggested that meizothrombin, like thrombin, is a Na{sup +}-activated enzyme. In this study we present the first X-ray crystal structure of human meizothrombin desF1 solved in the presence of the active site inhibitor PPACK at 2.1 {angstrom} resolution. The structure reveals a Na{sup +} binding site whose architecture is practically identical to that of human thrombin. Stopped-flow measurements of Na{sup +} binding to meizothrombin desF1 document a slow phase of fluorescence change with a kmore » obs decreasing hyperbolically with increasing [Na{sup +}], consistent with the existence of three conformations in equilibrium, E*, E and E:Na{sup +}, as for human thrombin. Evidence that meizothrombin exists in multiple conformations provides valuable new information for studies of the mechanism of prothrombin activation.« less

Increased thrombin generation in a mouse model of cancer cachexia is partially interleukin-6 dependent.

PubMed

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

2017-03-01

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 hypercoagulable state, which is partly attributable to IL-6 release by the tumor. The findings support the importance of the coagulation state in cancer cachexia and the clinical utility of anti-inflammatory interventions. © 2017 International Society on Thrombosis and Haemostasis.

Enhanced fluorescence of tetrasulfonated zinc phthalocyanine by graphene quantum dots and its application in molecular sensing/imaging.

PubMed

Wang, Jian; Zhang, Yanjun; Ye, Jiqing; Jiang, Zhou

2017-06-01

When excited at 435 nm, tetra-sulfonate zinc phthalocyanine (ZnPcS 4 ) emitted dual fluorescence at 495 and 702 nm. The abnormal fluorescence at 495 nm was experimentally studied and analyzed in detail for the first time. The abnormal fluorescence at 495 nm was deduced to originate from triplet-triplet (T-T) energy transfer of excited phthalocyanine ( 3 *ZnPcS 4 ). Furthermore, graphene quantum dots (GQDs) enhanced the 495 nm fluorescence quantum yield (Q) of ZnPcS 4 . The fluorescence properties of ZnPcS 4 -GQDs conjugate were retained in a cellular environment. Based on the fluorescence of ZnPcS 4 -GQDs conjugate, we designed and prepared an Apt29/thrombin/Apt15 sandwich thrombin sensor with high specificity and affinity. This cost-saving, simple operational sensing strategy can be extended to use in sensing/imaging of other biomolecules. Copyright © 2016 John Wiley & Sons, Ltd.

Development of Microbubble Contrast Agents with Biochemical Recognition and Tunable Acoustic Response

NASA Astrophysics Data System (ADS)

Nakatsuka, Matthew Allan Masao

Microbubbles, consisting of gas-filled cores encapsulated within phospholipid or polymer shells, are the most widely used ultrasound contrast agents in the world. Because of their acoustic impedance mismatch with surrounding tissues and compressible gaseous interiors, they have high echogenicities that allow for efficient backscatter of ultrasound. They can also generate unique harmonic frequencies when insonated near their resonance frequency, depending on physical microbubble properties such as the stiffness and thickness of the encapsulating shell. Microbubbles are used to detect a number of cardiovascular diseases, but current methodologies lack the ability to detect and distinguish small, rapidly growing abnormalities that do not produce visible blockage or slowing of blood flow. This work describes the development, formulation, and validation of microbubbles with various polymer shell architectures designed to modulate their acoustic ability. We demonstrate that the addition of a thick disulfide crosslinked, poly(acrylic acid) encapsulating shell increases a bubble's resistance to cavitation and changes its resonance frequency. Modification of this shell architecture to use hybridized DNA strands to form crosslinks between the polymer chains allows for tuning of the bubble acoustic response. When the DNA crosslinks are in place, shell stiffness is increased so the bubbles do not oscillate and acoustic signal is muted. Subsequently, when these DNA strands are displaced, partial acoustic activity is restored. By using aptamer sequences with a specific affinity towards the biomolecule thrombin as the DNA crosslinking strand, this acoustic "ON/OFF" behavior can be specifically tailored towards the presence of a specific biomarker, and produces a change in acoustic signal at concentrations of thrombin consistent with acute deep venous thrombosis. Incorporation of the emulsifying agent poly(ethylene glycol) into the encapsulating shell improves microbubble yield and circulation half-life substantially, allowing for the in vivo detection of a blood clot in a rabbit model. The results presented here show a unique marriage of highly specific molecular imaging and sonography that could be tailored towards a wide variety of cardiovascular abnormalities.

Multimodal noninvasive monitoring of soft tissue wound healing.

PubMed

Bodo, Michael; Settle, Timothy; Royal, Joseph; Lombardini, Eric; Sawyer, Evelyn; Rothwell, Stephen W

2013-12-01

Here we report results of non-invasive measurements of indirect markers of soft tissue healing of traumatic wounds in an observational swine study and describe the quantification of analog physiological signals. The primary purpose of the study was to measure bone healing of fractures with four different wound treatments. A second purpose was to quantify soft tissue wound healing by measuring the following indirect markers: (1) tissue oxygenation, (2) fluid content, and (3) blood flow, which were all measured by non-invasive modalities, measured with available devices. Tissue oxygenation was measured by near infrared spectroscopy; fluid content was measured by bipolar bio-impedance; and blood flow was measured by Doppler ultrasound. Immediately after comminuted femur fractures were produced in the right hind legs of thirty anesthetized female Yorkshire swine, one of four wound treatments was instilled into each wound. The four wound treatments were as follows: salmon fibrinogen/thrombin-n = 8; commercial bone filler matrix-n = 7; bovine collagen-n = 8; porcine fibrinogen/thrombin-n = 7. Fractures were stabilized with an external fixation device. Immediately following wound treatments, measurements were made of tissue oxygenation, fluid content and blood flow; these measurements were repeated weekly for 3 weeks after surgery. Analog signals of each modality were recorded on both the wounded (right) hind leg and the healthy (left) hind leg, for comparison purposes. Data were processed off-line. The mean values of 10-s periods were calculated for right-left leg comparison. ANOVA was applied for statistical analysis. Results of the bone healing studies are published separately (Rothwell et al. in J Spec Oper Med 13:7-18, 2013). For soft tissue wounds, healing did not differ significantly among the four wound treatments; however, regional oxygenation of wounds treated with salmon fibrinogen/thrombin showed slightly different time trends. Further studies are needed to establish standards for healthy wound healing and for detection of pathological alterations such as infection. Non-invasive measurement and quantification of indirect markers of soft tissue wound healing support the goals and principles of evidence-based medicine and show potential as easy to administer tools for clinicians and battlefield medical personnel to apply when procedures such as the PET scan are not available or affordable. The method we developed for storing analog physiological signals could be used for maintaining electronic health records, by incorporating vital signs such as ECG and EEG, etc.

Contact system activation and high thrombin generation in hyperthyroidism.

PubMed

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

2017-05-01

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  < 0.001), peak thrombin (131.9 (102.2-159.4) vs 31.6 (14.8-83.7), P  < 0.001) and endogenous thrombin potential (649 (538-736) vs 367 (197-1147), P  = 0.021) in TGA with 1 pM tissue factor, neutrophil elastase (1.10 (0.39-2.18) vs 0.23 (0.20-0.35), P  < 0.001), factor XIIa (66.9 (52.8-87.0) vs 73.0 (57.1-86.6), P  < 0.001), HMWK (6.11 (4.95-7.98) vs 3.83 (2.60-5.68), P  < 0.001), prekallikrein (2.15 (1.00-6.36) vs 1.41 (0.63-2.22), P  = 0.026) and bradykinin (152.4 (137.6-180.4) vs 118.3 (97.1-137.9), P  < 0.001) than did normal controls. In age- and sex-adjusted logistic regression analysis, fibrinogen, factor VIII, IX and XIIa, D-dimer, peak thrombin, neutrophil elastase, HMWK and bradykinin showed significant odds ratios representing 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.

Computational Analysis of Intersubject Variability and Thrombin Generation in Dilutional Coagulopathy

DTIC Science & Technology

2012-11-01

proteins: Factor (F)II, FV, FVII , FVIII, F IX, and FX, as well as the anticoagulants antithrombin (AT) and TF pathway inhibi- tor (TFPI). The results...coagulation factors FII, FV, FVII , FVIIa, FVIII, F IX and FX, as well as the anticoagulants TFPI and AT and the throm- bin generation inducer TF. The model...scenario and tissue factor concentration. CONCLUSION: Dilutional effects on thrombin genera- tion in a human population can be predicted from trends

Different Recovery Profiles of Coagulation Factors, Thrombin Generation, and Coagulation Function After Hemorrhagic Shock in Pigs

DTIC Science & Technology

2012-06-06

Different recovery profiles of coagulation factors, thrombin generation, and coagulation function after hemorrhagic shock in pigs Wenjun Z. Martini ...Defense. Address for reprints: Wenjun Z. Martini , PhD, The US Army Institute of Surgical Research, 3698 Chambers Pass, Ft. Sam Houston, San Antonio, TX...control number 1. REPORT DATE 01 SEP 2015 2. REPORT TYPE N/A 3. DATES COVERED - 4. TITLE AND SUBTITLE Different recovery profiles of

Exercise-induced myocardial ischemia in patients with coronary artery disease: lack of evidence for platelet activation or fibrin formation in peripheral venous blood.

PubMed

Marcella, J J; Nichols, A B; Johnson, L L; Owen, J; Reison, D S; Kaplan, K L; Cannon, P J

1983-05-01

The hypothesis that exercise-induced myocardial ischemia is associated with abnormal platelet activation and fibrin formation or dissolution was tested in patients with coronary artery disease undergoing upright bicycle stress testing. In vivo platelet activation was assessed by radioimmunoassay of platelet factor 4, beta-thrombo-globulin and thromboxane B2. In vivo fibrin formation was assessed by radioimmunoassay of fibrinopeptide A, and fibrinolysis was assessed by radioimmunoassay of thrombin-increasable fibrinopeptide B which reflects plasmin cleavage of fibrin I. Peripheral venous concentrations of these substances were measured in 10 normal subjects and 13 patients with coronary artery disease at rest and during symptom-limited peak exercise. Platelet factor 4, beta-thromboglobulin and thromboxane B2 concentrations were correlated with rest and exercise catecholamine concentrations to determine if exercise-induced elevation of norepinephrine and epinephrine enhances platelet activation. Left ventricular end-diastolic and end-systolic volumes, ejection fraction and segmental wall motion were measured at rest and during peak exercise by first pass radionuclide angiography. All patients with coronary artery disease had documented exercise-induced myocardial ischemia manifested by angina pectoris, ischemic electrocardiographic changes, left ventricular segmental dyssynergy and a reduction in ejection fraction. Rest and peak exercise plasma concentrations were not significantly different for platelet factor 4, beta-thromboglobulin, thromboxane B2, fibrinopeptide A and thrombin-increasable fibrinopeptide B. Peripheral venous concentrations of norepinephrine and epinephrine increased significantly (p less than 0.001) in both groups of patients. The elevated catecholamine levels did not lead to detectable platelet activation. This study demonstrates that enhanced platelet activation, thromboxane release and fibrin formation or dissolution are not detectable in peripheral venous blood of patients with coronary disease during exercise-induced myocardial ischemia.

More than a simple lipophilic contact: a detailed thermodynamic analysis of nonbasic residues in the s1 pocket of thrombin.

PubMed

Baum, Bernhard; Mohamed, Menshawy; Zayed, Mohamed; Gerlach, Christof; Heine, Andreas; Hangauer, David; Klebe, Gerhard

2009-07-03

The field of medicinal chemistry aims to design and optimize small molecule leads into drug candidates that may positively interfere with pathological disease situations in humans or combat the growth of infective pathogens. From the plethora of crystal structures of protein-inhibitor complexes we have learned how molecules recognize each other geometrically, but we still have rather superficial understanding of why they bind to each other. This contribution surveys a series of 26 thrombin inhibitors with small systematic structural differences to elucidate the rationale for their widely deviating binding affinity from 185 microM to 4 nM as recorded by enzyme kinetic measurements. Five well-resolved (resolution 2.30 - 1.47 A) crystal structures of thrombin-inhibitor complexes and an apo-structure of the uncomplexed enzyme (1.50 A) are correlated with thermodynamic data recorded by isothermal titration calorimetry with 12 selected inhibitors from the series. Taking solubility data into account, the variation in physicochemical properties allows conclusions to be reached about the relative importance of the enthalpic binding features as well as to estimate the importance of the parameters more difficult to capture, such as residual ligand entropy and desolvation properties. The collected data reveal a comprehensive picture of the thermodynamic signature that explains the so far poorly understood attractive force experienced by m-chloro-benzylamides to thrombin.

Clot retraction is mediated by factor XIII-dependent fibrin-αIIbβ3-myosin axis in platelet sphingomyelin-rich membrane rafts.

PubMed

Kasahara, Kohji; Kaneda, Mizuho; Miki, Toshiaki; Iida, Kazuko; Sekino-Suzuki, Naoko; Kawashima, Ikuo; Suzuki, Hidenori; Shimonaka, Motoyuki; Arai, Morio; Ohno-Iwashita, Yoshiko; Kojima, Soichi; Abe, Mitsuhiro; Kobayashi, Toshihide; Okazaki, Toshiro; Souri, Masayoshi; Ichinose, Akitada; Yamamoto, Naomasa

2013-11-07

Membrane rafts are spatially and functionally heterogenous in the cell membrane. We observed that lysenin-positive sphingomyelin (SM)-rich rafts are identified histochemically in the central region of adhered platelets where fibrin and myosin are colocalized on activation by thrombin. The clot retraction of SM-depleted platelets from SM synthase knockout mouse was delayed significantly, suggesting that platelet SM-rich rafts are involved in clot retraction. We found that fibrin converted by thrombin translocated immediately in platelet detergent-resistant membrane (DRM) rafts but that from Glanzmann's thrombasthenic platelets failed. The fibrinogen γ-chain C-terminal (residues 144-411) fusion protein translocated to platelet DRM rafts on thrombin activation, but its mutant that was replaced by A398A399 at factor XIII crosslinking sites (Q398Q399) was inhibited. Furthermore, fibrin translocation to DRM rafts was impaired in factor XIII A subunit-deficient mouse platelets, which show impaired clot retraction. In the cytoplasm, myosin translocated concomitantly with fibrin translocation into the DRM raft of thrombin-stimulated platelets. Furthermore, the disruption of SM-rich rafts by methyl-β-cyclodextrin impaired myosin activation and clot retraction. Thus, we propose that clot retraction takes place in SM-rich rafts where a fibrin-αIIbβ3-myosin complex is formed as a primary axis to promote platelet contraction.

DELIVERY OF WATER-SOLUBLE DRUGS USING ACOUSTICALLY-TRIGGERED, PERFLUOROCARBON DOUBLE EMULSIONS

PubMed Central

Fabiilli, Mario L.; Lee, James A.; Kripfgans, Oliver D.; Carson, Paul L.; Fowlkes, J. Brian

2010-01-01

Purpose Ultrasound can be used to release a therapeutic payload encapsulated within a perfluorocarbon (PFC) emulsion via acoustic droplet vaporization (ADV), a process whereby the PFC phase is vaporized and the agent is released. ADV-generated microbubbles have been previously used to selectively occlude blood vessels in vivo. The coupling of ADV-generated drug delivery and occlusion has therapeutically, synergistic potentials. Methods Micron-sized, water-in-PFC-in-water (W1/PFC/W2) emulsions were prepared in a two-step process using perfluoropentane (PFP) or perfluorohexane (PFH) as the PFC phase. Fluorescein or thrombin was contained in the W1 phase. Results Double emulsions containing fluorescein in the W1 phase displayed a 5.7±1.4 fold and 8.2±1.3 fold increase in fluorescein mass flux, as measured using a Franz diffusion cell, after ADV for the PFP and PFH emulsions, respectively. Thrombin was stably retained in four out of five double emulsions. For three out of five formulations tested, the clotting time of whole blood decreased, in a statistically significant manner (p < 0.01), when incubated with thrombin-loaded emulsions exposed to ultrasound compared to emulsions not exposed to ultrasound. Conclusions ADV can be used to spatially and temporally control the delivery of water-soluble compounds formulated in PFC double emulsions. Thrombin release could extend the duration of ADV-generated, microbubble occlusions. PMID:20872050

The Non-catalytic B Subunit of Coagulation Factor XIII Accelerates Fibrin Cross-linking*

PubMed Central

Souri, Masayoshi; Osaki, Tsukasa; Ichinose, Akitada

2015-01-01

Covalent cross-linking of fibrin chains is required for stable blood clot formation, which is catalyzed by coagulation factor XIII (FXIII), a proenzyme of plasma transglutaminase consisting of catalytic A (FXIII-A) and non-catalytic B subunits (FXIII-B). Herein, we demonstrate that FXIII-B accelerates fibrin cross-linking. Depletion of FXIII-B from normal plasma supplemented with a physiological level of recombinant FXIII-A resulted in delayed fibrin cross-linking, reduced incorporation of FXIII-A into fibrin clots, and impaired activation peptide cleavage by thrombin; the addition of recombinant FXIII-B restored normal fibrin cross-linking, FXIII-A incorporation into fibrin clots, and activation peptide cleavage by thrombin. Immunoprecipitation with an anti-fibrinogen antibody revealed an interaction between the FXIII heterotetramer and fibrinogen mediated by FXIII-B and not FXIII-A. FXIII-B probably binds the γ-chain of fibrinogen with its D-domain, which is near the fibrin polymerization pockets, and dissociates from fibrin during or after cross-linking between γ-chains. Thus, FXIII-B plays important roles in the formation of a ternary complex between proenzyme FXIII, prosubstrate fibrinogen, and activator thrombin. Accordingly, congenital or acquired FXIII-B deficiency may result in increased bleeding tendency through impaired fibrin stabilization due to decreased FXIII-A activation by thrombin and secondary FXIII-A deficiency arising from enhanced circulatory clearance. PMID:25809477

Structure-activity studies and therapeutic potential of host defense peptides of human thrombin.

PubMed

Kasetty, Gopinath; Papareddy, Praveen; Kalle, Martina; Rydengård, Victoria; Mörgelin, Matthias; Albiger, Barbara; Malmsten, Martin; Schmidtchen, Artur

2011-06-01

Peptides of the C-terminal region of human thrombin are released upon proteolysis and identified in human wounds. In this study, we wanted to investigate minimal determinants, as well as structural features, governing the antimicrobial and immunomodulating activity of this peptide region. Sequential amino acid deletions of the peptide GKYGFYTHVFRLKKWIQKVIDQFGE (GKY25), as well as substitutions at strategic and structurally relevant positions, were followed by analyses of antimicrobial activity against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram-positive bacterium Staphylococcus aureus, and the fungus Candida albicans. Furthermore, peptide effects on lipopolysaccharide (LPS)-, lipoteichoic acid-, or zymosan-induced macrophage activation were studied. The thrombin-derived peptides displayed length- and sequence-dependent antimicrobial as well as immunomodulating effects. A peptide length of at least 20 amino acids was required for effective anti-inflammatory effects in macrophage models, as well as optimal antimicrobial activity as judged by MIC assays. However, shorter (>12 amino acids) variants also displayed significant antimicrobial effects. A central K14 residue was important for optimal antimicrobial activity. Finally, one peptide variant, GKYGFYTHVFRLKKWIQKVI (GKY20) exhibiting improved selectivity, i.e., low toxicity and a preserved antimicrobial as well as anti-inflammatory effect, showed efficiency in mouse models of LPS shock and P. aeruginosa sepsis. The work defines structure-activity relationships of C-terminal host defense peptides of thrombin and delineates a strategy for selecting peptide epitopes of therapeutic interest.

Crosstalk between coagulation and inflammation in mastitis and metritis in dairy cows.

PubMed

Bobowiec, Ryszard; Wessely-Szponder, Joanna; Hola, Piotr

2009-06-01

Coagulation and inflammation are closely related as part of the mechanisms of host defence during a severe infection. The aim of this study was to investigate the relation between thrombin as a factor in both the coagulative and inflammatory processes and neutrophil secretory function on the basis of lactoferrin (LF), elastase and myeloperoxidase release in the course of mastitis and metritis in cows. Thrombin generation was measured on the basis of hydrolysis of SAR-PRO-ARG-pNA and lactoferrin concentration was estimated by an ELISA method. The greatest thrombin generation was observed in the metritis group (1.18 +/- 0.62 IU). The level of LF was the highest in the group of cows with mastitis (0.74 +/- 0.55 mg/ml) in the first phase of the disease. In the second phase of the diseases the level of serum LF in cows with mastitis diminished to the value of 0.41 +/- 0.16 mg/ml, whereas in cows with metritis the level of LF increased to 0.51 +/- 0.17 mg/ml. This study reveals that the excessive production of thrombin not only causes hypercoagulatory disorders but also exaggerates neutrophil function by the release of some enzymes which may play a destructive role during disseminated intravascular coagulation (DIC). These enzymes also inhibit anticoagulative systems, thus potentially worsening the course of the disease.

Exogenous modification of platelet membranes with the omega-3 fatty acids EPA and DHA reduces platelet procoagulant activity and thrombus formation.

PubMed

Larson, Mark K; Tormoen, Garth W; Weaver, Lucinda J; Luepke, Kristen J; Patel, Ishan A; Hjelmen, Carl E; Ensz, Nicole M; McComas, Leah S; McCarty, Owen J T

2013-02-01

Several studies have implicated the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in inhibition of normal platelet function, suggesting a role for platelets in EPA- and DHA-mediated cardioprotection. However, it is unclear whether the cardioprotective mechanisms arise from alterations to platelet-platelet, platelet-matrix, or platelet-coagulation factor interactions. Our previous results led us to hypothesize that EPA and DHA alter the ability of platelets to catalyze the generation of thrombin. We tested this hypothesis by exogenously modifying platelet membranes with EPA and DHA, which resulted in compositional changes analogous to increased dietary EPA and DHA intake. Platelets treated with EPA and DHA showed reductions in the rate of thrombin generation and exposure of platelet phosphatidylserine. In addition, treatment of platelets with EPA and DHA decreased thrombus formation and altered the processing of thrombin precursor proteins. Furthermore, treatment of whole blood with EPA and DHA resulted in increased occlusion time and a sharply reduced accumulation of fibrin under flow conditions. These results demonstrate that EPA and DHA inhibit, but do not eliminate, the ability of platelets to catalyze thrombin generation in vitro. The ability of EPA and DHA to reduce the procoagulant function of platelets provides a possible mechanism behind the cardioprotective phenotype in individuals consuming high levels of EPA and DHA.

Mutant N143P Reveals How Na[superscript +] Activates Thrombin

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

Niu, Weiling; Chen, Zhiwei; Bush-Pelc, Leslie A.

2010-01-12

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

Coagulation under flow: the influence of flow-mediated transport on the initiation and inhibition of coagulation.

PubMed

Fogelson, Aaron L; Tania, Nessy

2005-01-01

A mathematical model of intravascular coagulation is presented; it encompasses the biochemistry of the tissue factor pathway, platelet activation and deposition on the subendothelium, and flow- and diffusion-mediated transport of coagulation proteins and platelets. Simulation experiments carried out with the model indicate the predominant role played by the physical processes of platelet deposition and flow-mediated removal of enzymes in inhibiting coagulation in the vicinity of vascular injury. Sufficiently rapid production of factors IXa and Xa by the TF:VIIa complex can overcome this inhibition and lead to formation of significant amounts of the tenase complex on the surface of activated platelets and, as a consequence, to substantial thrombin production. Chemical inhibitors are seen to play almost no (TFPI) or little (AT-III and APC) role in determining whether substantial thrombin production will occur. The role of APC is limited by the necessity for diffusion of thrombin from the site of injury to nearby endothelial cells to form the thrombomodulin-thrombin complex and for diffusion in the reverse direction of the APC made by this complex. TFPI plays an insignificant part in inhibiting the TF:VIIa complex under the conditions studied whether its action involves sequential binding of TFPI to Xa and then TFPI:Xa to TF:VIIa, or direct binding of TFPI to Xa already bound to the TF:VIIa complex. Copyright 2005 S. Karger AG, Basel.

Management of major bleedings during anticoagulant treatment with the oral direct thrombin inhibitor ximelagatran or warfarin.

PubMed

Fernlöf, Gunilla; Sjöström, Britta M; Lindell, Klas M; Wall, Ulrika E

2009-12-01

Several new oral anticoagulants are currently investigated in phase III programmes, mainly with inhibition of factor Xa or thrombin as their pharmacological target. Advantages are expected with these new drugs compared with vitamin K antagonists, but one potential drawback is the lack of specific antidotes. During the clinical studies with ximelagatran, an oral direct thrombin inhibitor withdrawn due to hepatic side effects, investigators were instructed to manage bleedings with routine measures. We have retrospectively tried to assess whether this was sufficient or whether there was a need for reversal strategies. The study population consisted of patients with major bleedings in three long-term studies (104 ximelagatran, 155 warfarin). All individual patient narratives were reviewed with respect to management of the bleeding. Complementary data were retrieved from the data-based case report forms. Approximately, two of three of the patients in both groups were subject to some kind of treatment. One-third (1/3) in both groups had transfusions documented and/or received specific medication. Vitamin K was given more often to warfarin patients. Two ximelagatran patients received prothrombin complex (four-factor concentrate), but one was a patient with a severe hepatopathy suspected to be drug-induced. Overall, the case descriptions did not reveal any apparent differences in the course of events between groups. We found no indications that the lack of an antidote posed a clinical problem in patients treated with ximelagatran as compared with warfarin. The relatively short half-life of melagatran, the active metabolite of ximelagatran, may have contributed to these results.

Rapid Evaluation of Platelet Function With T2 Magnetic Resonance

PubMed Central

Cuker, Adam; Husseinzadeh, Holleh; Lebedeva, Tatiana; Marturano, Joseph E.; Massefski, Walter; Lowery, Thomas J.; Lambert, Michele P.; Abrams, Charles S.; Weisel, John W.

2016-01-01

Objectives: The clinical diagnosis of qualitative platelet disorders (QPDs) based on light transmission aggregometry (LTA) requires significant blood volume, time, and expertise, all of which can be barriers to utilization in some populations and settings. Our objective was to develop a more rapid assay of platelet function by measuring platelet-mediated clot contraction in small volumes (35 µL) of whole blood using T2 magnetic resonance (T2MR). Methods: We established normal ranges for platelet-mediated clot contraction using T2MR, used these ranges to study patients with known platelet dysfunction, and then evaluated agreement between T2MR and LTA with arachidonic acid, adenosine diphosphate, epinephrine, and thrombin receptor activator peptide. Results: Blood from 21 healthy donors was studied. T2MR showed 100% agreement with LTA with each of the four agonists and their cognate inhibitors tested. T2MR successfully detected abnormalities in each of seven patients with known QPDs, with the exception of one patient with a novel mutation leading to Hermansky-Pudlak syndrome. T2MR appeared to detect platelet function at similar or lower platelet counts than LTA. Conclusions: T2MR may provide a clinically useful approach to diagnose QPDs using small volumes of whole blood, while also providing new insight into platelet biology not evident using plasma-based platelet aggregation tests. PMID:28028118

Conventional liquid chromatography/triple quadrupole mass spectrometer-based metabolite identification and semi-quantitative estimation approach in the investigation of dabigatran etexilate in vitro metabolism

PubMed Central

Hu, Zhe-Yi; Parker, Robert B.; Herring, Vanessa L.; Laizure, S. Casey

2012-01-01

Dabigatran etexilate (DABE) is an oral prodrug that is rapidly converted by esterases to dabigatran (DAB), a direct inhibitor of thrombin. To elucidate the esterase-mediated metabolic pathway of DABE, a high-performance liquid chromatography/mass spectrometer (LC-MS/MS)-based metabolite identification and semi-quantitative estimation approach was developed. To overcome the poor full-scan sensitivity of conventional triple quadrupole mass spectrometry, precursor-product ion pairs were predicted, to search for the potential in vitro metabolites. The detected metabolites were confirmed by the product ion scan. A dilution method was introduced to evaluate the matrix effects of tentatively identified metabolites without chemical standards. Quantitative information on detected metabolites was obtained using ‘metabolite standards’ generated from incubation samples that contain a high concentration of metabolite in combination with a correction factor for mass spectrometry response. Two in vitro metabolites of DABE (M1 and M2) were identified, and quantified by the semi-quantitative estimation approach. It is noteworthy that CES1 convert DABE to M1 while CES2 mediates the conversion of DABE to M2. M1 (or M2) was further metabolized to DAB by CES2 (or CES1). The approach presented here provides a solution to a bioanalytical need for fast identification and semi-quantitative estimation of CES metabolites in preclinical samples. PMID:23239178

Platelet reactivity and mean platelet volume as risk markers of thrombogenesis in atrial fibrillation.

PubMed

Makowski, Marcin; Smorag, Ireneusz; Makowska, Joanna; Bissinger, Andrzej; Grycewicz, Tomasz; Paśnik, Jarek; Kidawa, Michal; Lubiński, Andrzej; Zielińska, Marzenna; Baj, Zbigniew

2017-05-15

Atrial fibrillation (AF) is associated with increased risk of thromboembolic complications. One of the markers of the increased risk of hypercoagulable state is platelet hyperreactivity. The aim of the study was to assess impact of arrhythmia on platelet reactivity. The study included 36 (mean age 48,3; range 21-60) male patients with lone atrial fibrillation, with exclusion of concomitant diseases known to trigger hypercoagulable state. The AF patients underwent cardioversion to restore sinus rhythm and were subsequently under observation for 1month. Echocardiography, ECG and blood collection was performed before cardioversion (T0) and 4weeks after successful cardioversion (T1). During the study period patients have been contacted and examined every week and 24h ECG monitoring was performed. Platelet reactivity was assessed based on changes of CD62 and CD42b expression on platelet surface after stimulation with thrombin. Also changes in MPV were assessed. In all patients sinus rhythm was maintained at the end of the study period, however in 14 patients recurrences of AF were observed, confirmed by 24h ECG monitoring (atrial fibrillation recurrence group - AFR) and 22 patients maintained sinus rhythm throughout the whole study period (SR group). Mean fluorescence intensity (MFI) of CD62 on thrombin stimulated platelets decreased significantly 4weeks after electrical cardioversion as compared to T0 (48.04±22.42 vs 41.47±16.03; p<0.01). Also MFI of CD42b on thrombin stimulated platelets decreased significantly 4weeks after electrical cardioversion as compared to T0 (22.16±10.82 vs 12.06±5.99; p<0.0001). Platelets reactivity estimated by CD 62 expression in SR group decreased significantly after 4weeks observation (58.01±15.26 vs 46.57±13.44; p<0.001) opposite to AFR group 35.66±21.87 vs 34.54±16.4; p-ns). Moreover there were significant differences between basal reactivity during AF between SR and AFR groups (58.01±15.26 vs 35.66±21.87; p-0.01). MFI of CD42b on thrombin stimulated platelets decreased significantly both in AFR and SR groups (22.05±11.36 vs 13.8±6.03; p<0.001 and 21.87±14.18 vs 10.04±5.09; p<0005). MPV decreased significantly 4weeks after electrical cardioversion as compared to T0 (8.81±0.19 vs 8.42±0.14; p<0.0001). The changes of platelet reactivity to thrombin observed after restoration of sinus rhythm in patients prove that arrhythmia intrinsically leads to increased reactivity of platelets. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Development of a microplate coagulation assay for Factor V in human plasma.

PubMed

Tilley, Derek; Levit, Irina; Samis, John A

2011-06-28

Factor V (FV) in its activated form, FVa, is a critical regulator of thrombin generation during fibrin clot formation. There is a need of a simple, fast, and inexpensive microplate-based coagulation assay to measure the functional activity of FV in human plasma. The objective of this study was to develop a microplate-based assay that measures FV coagulation activity during clot formation in human plasma, which is currently not available. The FV assay requires a kinetic microplate reader to measure the change in absorbance at 405nm during fibrin formation in human plasma. The FV assay accurately measures the time, initial rate, and extent of fibrin clot formation in human plasma. The FV microplate assay is simple, fast, economical, sensitive to approx 24-80pM, and multiple samples may be analyzed simultaneously. All the required materials are commercially available. Standard curves of time or initial rate of fibrin clot formation vs FV activity in the 1-stage assay (Without activation by thrombin) may be used to measure FV activity in samples of human plasma. The assay was used to demonstrate that in nine patients with disseminated intravascular coagulation (DIC), the FV 1-stage, 2-stage (With activation by thrombin), and total (2-stage activity - 1-stage activity) activities were decreased, on average, by approximately 54%, 44%, and 42%, respectively, from prolonged clot times when compared to normal pooled human reference plasma (NHP). The results indicate that the FV in the DIC patient plasmas supported both a delayed and slower rate of fibrin clot formation compared with NHP; however, the extent of fibrin clot formation in the DIC patients remained largely unchanged from that observed with NHP. The FV microplate assay may be easily adapted to measure the activity of any coagulation factor using the appropriate factor-deficient plasma and clot initiating reagent. The microplate assay will find use in both research and clinical laboratories to provide measurement of the functional coagulation activity of FV in human plasma.

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

PubMed

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

2014-10-01

Compounds showing dual inhibition of thrombin and factor Xa (FXa) are the subject of great interest owing to their broader specificity for effective anticoagulation therapy against cardiovascular disorders. This is the first report on the functional characterization and assessment of therapeutic potential of a 4423.6 Da inhibitory peptide (Ruviprase) purified from Daboia russelii russelii venom. The secondary structure of Ruviprase is composed of α-helices (61.9%) and random coils (38.1%). The partial N-terminal sequence (E(1)-V(2)-X(3)-W(4)-W(5)-W(6)-A(7)-Q(8)-L(9)-S(10)) of Ruviprase demonstrated significant similarity (80.0%) with an internal sequence of apoptosis-stimulating protein reported from the venom of Ophiophagus hannah and Python bivittatus; albeit Ruviprase did not show sequence similarity with existing thrombin/FXa inhibitors, suggesting its uniqueness. Ruviprase demonstrated a potent in vitro anticoagulant property and inhibited both thrombin and FXa following slow binding kinetics. Ruviprase inhibited thrombin by binding to its active site via an uncompetitive mechanism with a Ki value and dissociation constant (KD) of 0.42 μM and 0.46 μM, respectively. Conversely, Ruviprase demonstrated mixed inhibition (Ki = 0.16 μM) of FXa towards its physiological substrate prothrombin. Furthermore, the biological properties of Ruviprase could not be neutralized by commercial polyvalent or monovalent antivenom. Ruviprase at a dose of 2.0 mg/kg was non-toxic and showed potent in vivo anticoagulant activity after 6 h of intraperitoneal treatment in mice. Because of the potent anticoagulant property as well as non-toxic nature of Ruviprase, the possible application of the peptide as an antithrombotic agent for combating thrombosis-associated ailments appears promising. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Expression of Inflammation-related Intercellular Adhesion Molecules in Cardiomyocytes In Vitro and Modulation by Pro-inflammatory Agents.

PubMed

El-Battrawy, Ibrahim; Tülümen, Erol; Lang, Siegfried; Akin, Ibrahim; Behnes, Michael; Zhou, Xiabo; Mavany, Martin; Bugert, Peter; Bieback, Karen; Borggrefe, Martin; Elmas, Elif

2016-01-01

Cell-surface adhesion molecules regulate multiple intercellular and intracellular processes and play important roles in inflammation by facilitating leukocyte endothelial transmigration. Whether cardiomyocytes express surface-adhesion molecules related to inflammation and the effect of pro-inflammatory mediators remain unknown. In the present study, the expression of different cell-adhesion molecules (CD11a, CD11b, CD31, CD62P, CD162, F11 receptor and mucosal vascular addressin cell adhesion molecule 1 (MADCAM1)) and the effect of pro-inflammatory mediators were investigated in an in vitro model of human cardiomyocytes. Cells were supplied as a primary culture of cardiac alpha actin-positive cells from human heart tissue. The cells were incubated for 24 h with 1 U/ml thrombin or 700 ng/ml lipopolysaccharide (LPS) or with a combination of both. The expression of the cell adhesion molecules was measured by flow cytometry. In cultured human cardiomyocytes, 22.8% of cells expressed CD31, 7.1% MADCAM1 and 2.6% F11R. CD11a, CD11b, CD62P and CD162 were expressed by fewer than 2% of the cells at baseline. CD31 expression increased on incubation of cardiomyocytes with thrombin by 26% (p<0.05) and with LPS by 26% (p=0.06). The combination of thrombin and LPS did not result in increased levels of CD31 (p>0.10). The pro-inflammatory agents LPS and thrombin had no effect on the expression of MADCAM1 and F11R. Inflammation-related cell-adhesion molecules CD31, MADCAM1 and F11R were shown to be expressed on the surface of human cardiomyocytes in an in vitro model. Incubation with LPS or thrombin resulted in increased expression of CD31, however, it did not modify the expression of the cell adhesion molecules MADCAM1 and F11R. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

[Factor V congenital deficiency: about a case].

PubMed

Boujrad, Saloua; El Hasbaoui, Brahim; Echahdi, Hanae; Malih, Mohamed; Agadr, Aomar

2017-01-01

Factor V congenital deficiency is a rare coagulation disorder initially described by Owren in 1947 and known as para hemophilia. It is transmitted through autosomal-recessive inheritance and homozygous cases are usually symptomatic. Factor V is an essential cofactor in the conversion of prothrombin to thrombin by activated factor X. In the absence of factor V, thrombin generation is slowed down and fibrin formation is delayed. This results in a bleeding tendency. We report a case of factor V congenital deficiency in an infant with recurrent epistaxis.

Enzyme-Encapsulated Liposome-Linked Immunosorbent Assay Enabling Sensitive Personal Glucose Meter Readout for Portable Detection of Disease Biomarkers.

PubMed

Lin, Bingqian; Liu, Dan; Yan, Jinmao; Qiao, Zhi; Zhong, Yunxin; Yan, Jiawei; Zhu, Zhi; Ji, Tianhai; Yang, Chaoyong James

2016-03-23

There is considerable demand for sensitive, selective, and portable detection of disease-associated proteins, particularly in clinical practice and diagnostic applications. Portable devices are highly desired for detection of disease biomarkers in daily life due to the advantages of being simple, rapid, user-friendly, and low-cost. Herein we report an enzyme-encapsulated liposome-linked immunosorbent assay for sensitive detection of proteins using personal glucose meters (PGM) for portable quantitative readout. Liposomes encapsulating a large amount of amyloglucosidase or invertase are surface-coated with recognition elements such as aptamers or antibodies for target recognition. By translating molecular recognition signal into a large amount of glucose with the encapsulated enzyme, disease biomarkers such as thrombin or C-reactive protein (CRP) can be quantitatively detected by a PGM with a high detection limit of 1.8 or 0.30 nM, respectively. With the advantages of portability, ease of use, and low-cost, the method reported here has potential for portable and quantitative detection of various targets for different POC testing scenarios, such as rapid diagnosis in clinic offices, health monitoring at the bedside, and chemical/biochemical safety control in the field.

Characterization of the thrombin generation profile in systemic lupus erythematosus.

PubMed

Kern, A; Barabás, E; Balog, A; Burcsár, Sz; Kiszelák, M; Vásárhelyi, B

2017-03-01

Systemic lupus erythematosus (SLE) is a multisystemic inflammatory autoimmune disorder. Thrombotic events occur at a higher incidence among SLE patients. The investigation of thrombin generation (TG) with calibrated automated thrombogram (CAT) test as a global hemostasis assay is applicable for the overall functional assessment of the hemostasis. The aim of this study was to characterize the hemostatic alterations observed in SLE by CAT assay. In this study, CAT parameters and basic coagulation parameters of SLE patients (n = 22) and healthy control subjects (n = 34) were compared. CAT area under the curve (i.e., endogenous thrombin potential) was lower than normal in SLE (807 vs. 1,159 nM*min, respectively), whereas other CAT parameters (peak, lag time, time to peak, and velocity index) and the basic coagulation tests were within the normal range. The presence of anti-phospholipid antibodies and the applied therapy was not associated with hemostasis parameters in SLE. We concluded that the reported high risk of thrombosis is not related to TG potential.

Polyphenol fatty acid esters as serine protease inhibitors: a quantum-chemical QSAR analysis.

PubMed

Viskupicova, Jana; Danihelova, Martina; Majekova, Magdalena; Liptaj, Tibor; Sturdik, Ernest

2012-12-01

We investigated the ability of polyphenol fatty acid esters to inhibit the activity of serine proteases trypsin, thrombin, elastase and urokinase. Potent protease inhibition in micromolar range was displayed by rutin and rutin derivatives esterified with medium and long chain, mono- and polyunsaturated fatty acids (1e-m), followed by phloridzin and esculin esters with medium and long fatty acid chain length (2a-d, 3a-d), while unmodified compounds showed only little or no effect. QSAR study of the compounds tested provided the most significant parameters for individual inhibition activities, i.e. number of hydrogen bond donors for urokinase, molecular volume for thrombin, and solvation energy for elastase. According to the statistical analysis, the action of elastase inhibitors is opposed to those of urokinase and thrombin. Cluster analysis showed two groups of compounds: original polyphenols together with rutin esters with short fatty acid chain length and rutin esters with long fatty acid chain length.

Recombinant activated factor VII: 30 years of research and innovation.

PubMed

Hedner, Ulla

2015-06-01

Recombinant activated factor VII (rFVIIa) was initially developed to treat bleeding episodes in patients with congenital haemophilia and inhibitors. The story of its development began in the 1970s, when FVIIa was identified as one of the activated coagulation factors that has minimal potential for inducing thromboembolic side-effects. Extensive research over the last 30 years has greatly increased our knowledge of the characteristics of FVII, its activation, and the mechanisms by which rFVIIa restores haemostasis. In haemophilia, the haemostatic effect of rFVIIa is mediated via binding to thrombin-activated platelets at the site of injury, thereby enhancing thrombin generation also in the absence of factor (F) VIII or FIX. The mechanism of action of rFVIIa has also allowed its successful use in other clinical scenarios characterised by impaired thrombin generation, and its licensed uses have now been extended to acquired haemophilia, congenital FVII deficiency and Glanzmann's thrombasthenia. Copyright © 2015 Elsevier Ltd. All rights reserved.

A sensitive HIV-1 envelope induced fusion assay identifies fusion enhancement of thrombin

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

Cheng, De-Chun; Zhong, Guo-Cai; Su, Ju-Xiang

2010-01-22

To evaluate the interaction between HIV-1 envelope glycoprotein (Env) and target cell receptors, various cell-cell-fusion assays have been developed. In the present study, we established a novel fusion system. In this system, the expression of the sensitive reporter gene, firefly luciferase (FL) gene, in the target cells was used to evaluate cell fusion event. Simultaneously, constitutively expressed Renilla luciferase (RL) gene was used to monitor effector cell number and viability. FL gave a wider dynamic range than other known reporters and the introduction of RL made the assay accurate and reproducible. This system is especially beneficial for investigation of potentialmore » 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.« less

Mapping the affinity landscape of Thrombin-binding aptamers on 2΄F-ANA/DNA chimeric G-Quadruplex microarrays

PubMed Central

Abou Assi, Hala; Gómez-Pinto, Irene; González, Carlos

2017-01-01

Abstract In situ fabricated nucleic acids microarrays are versatile and very high-throughput platforms for aptamer optimization and discovery, but the chemical space that can be probed against a given target has largely been confined to DNA, while RNA and non-natural nucleic acid microarrays are still an essentially uncharted territory. 2΄-Fluoroarabinonucleic acid (2΄F-ANA) is a prime candidate for such use in microarrays. Indeed, 2΄F-ANA chemistry is readily amenable to photolithographic microarray synthesis and its potential in high affinity aptamers has been recently discovered. We thus synthesized the first microarrays containing 2΄F-ANA and 2΄F-ANA/DNA chimeric sequences to fully map the binding affinity landscape of the TBA1 thrombin-binding G-quadruplex aptamer containing all 32 768 possible DNA-to-2΄F-ANA mutations. The resulting microarray was screened against thrombin to identify a series of promising 2΄F-ANA-modified aptamer candidates with Kds significantly lower than that of the unmodified control and which were found to adopt highly stable, antiparallel-folded G-quadruplex structures. The solution structure of the TBA1 aptamer modified with 2΄F-ANA at position T3 shows that fluorine substitution preorganizes the dinucleotide loop into the proper conformation for interaction with thrombin. Overall, our work strengthens the potential of 2΄F-ANA in aptamer research and further expands non-genomic applications of nucleic acids microarrays. PMID:28100695

Factor XI and Contact Activation as Targets for Antithrombotic Therapy

PubMed Central

Gailani, David; Bane, Charles E.; Gruber, Andras

2015-01-01

Summary The most commonly used anticoagulants produce therapeutic antithrombotic effects either by inhibiting thrombin or factor Xa, or by lowering the plasma levels of the precursors of these key enzymes, prothrombin and factor X. These drugs do not distinguish between thrombin generation contributing to thrombosis from thrombin generation required for hemostasis. Thus, anticoagulants increase bleeding risk, and many patients who would benefit from therapy go untreated because of comorbidities that place them at unacceptable risk for hemorrhage. Studies in animals demonstrate that components of the plasma contact activation system contribute to experimentally-induced thrombosis, despite playing little or no role in hemostasis. Attention has focused on factor XII, the zymogen of a protease (factor XIIa) that initiates contact activation when blood is exposed to foreign surfaces; and factor XI, the zymogen of the protease factor XIa, which links contact activation to the thrombin generation mechanism. In the case of factor XI, epidemiologic data indicate this protein contributes to stroke and venous thromboembolism, and perhaps myocardial infarction, in humans. A phase 2 trial showing that reduction of factor XI may be more effective than low-molecular-weight heparin at preventing venous thrombosis during knee replacement surgery provides proof of concept for the premise that an antithrombotic effect can be uncoupled from an anticoagulant effect in humans by targeting components of contact activation. Here we review data on the role of factor XI and factor XII in thrombosis, and results of pre-clinical and human trials for therapies targeting these proteins. PMID:25976012

Kinetic Dissection of the Pre-existing Conformational Equilibrium in the Trypsin Fold*

PubMed Central

Vogt, Austin D.; Chakraborty, Pradipta; Di Cera, Enrico

2015-01-01

Structural biology has recently documented the conformational plasticity of the trypsin fold for both the protease and zymogen in terms of a pre-existing equilibrium between closed (E*) and open (E) forms of the active site region. How such plasticity is manifested in solution and affects ligand recognition by the protease and zymogen is poorly understood in quantitative terms. Here we dissect the E*-E equilibrium with stopped-flow kinetics in the presence of excess ligand or macromolecule. Using the clotting protease thrombin and its zymogen precursor prethrombin-2 as relevant models we resolve the relative distribution of the E* and E forms and the underlying kinetic rates for their interconversion. In the case of thrombin, the E* and E forms are distributed in a 1:4 ratio and interconvert on a time scale of 45 ms. In the case of prethrombin-2, the equilibrium is shifted strongly (10:1 ratio) in favor of the closed E* form and unfolds over a faster time scale of 4.5 ms. The distribution of E* and E forms observed for thrombin and prethrombin-2 indicates that zymogen activation is linked to a significant shift in the pre-existing equilibrium between closed and open conformations that facilitates ligand binding to the active site. These findings broaden our mechanistic understanding of how conformational transitions control ligand recognition by thrombin and its zymogen precursor prethrombin-2 and have direct relevance to other members of the trypsin fold. PMID:26216877

Phospholipid-esterified eicosanoids are generated in agonist-activated human platelets and enhance tissue factor-dependent thrombin generation.

PubMed

Thomas, Christopher P; Morgan, Lloyd T; Maskrey, Benjamin H; Murphy, Robert C; Kühn, Hartmut; Hazen, Stanley L; Goodall, Alison H; Hamali, Hassan A; Collins, Peter W; O'Donnell, Valerie B

2010-03-05

Here, a group of specific lipids, comprising phosphatidylethanolamine (PE)- or phosphatidylcholine (PC)-esterified 12S-hydroxyeicosatetraenoic acid (12S-HETE), generated by 12-lipoxygenase was identified and characterized. 12S-HETE-PE/PCs were formed within 5 min of activation by thrombin, ionophore, or collagen. Esterified HETE levels generated in response to thrombin were 5.85 +/- 1.42 (PE) or 18.35 +/- 4.61 (PC), whereas free was 65.5 +/- 17.6 ng/4 x 10(7) cells (n = 5 separate donors, mean +/- S.E.). Their generation was stimulated by triggering protease-activated receptors-1 and -4 and signaling via Ca(2+) mobilization secretory phospholipase A2, platelet-activating factor-acetylhydrolase, src tyrosine kinases, and protein kinase C. Stable isotope labeling showed that they form predominantly by esterification that occurs on the same time scale as free acid generation. Unlike free 12S-HETE that is secreted, esterified HETEs remain cell-associated, with HETE-PEs migrating to the outside of the plasma membrane. 12-Lipoxygenase inhibition attenuated externalization of native PE and phosphatidylserine and HETE-PEs. Platelets from a patient with the bleeding disorder, Scott syndrome, did not externalize HETE-PEs, and liposomes supplemented with HETE-PC dose-dependently enhanced tissue factor-dependent thrombin generation in vitro. This suggests a role for these novel lipids in promoting coagulation. Thus, oxidized phospholipids form by receptor/agonist mechanisms, not merely as an undesirable consequence of vascular and inflammatory disease.

Crystal structures of thrombin in complex with chemically modified thrombin DNA aptamers reveal the origins of enhanced affinity.

PubMed

Dolot, Rafal; Lam, Curtis H; Sierant, Malgorzata; Zhao, Qiang; Liu, Feng-Wu; Nawrot, Barbara; Egli, Martin; Yang, Xianbin

2018-05-18

Thrombin-binding aptamer (TBA) is a DNA 15-mer of sequence 5'-GGT TGG TGT GGT TGG-3' that folds into a G-quadruplex structure linked by two T-T loops located on one side and a T-G-T loop on the other. These loops are critical for post-SELEX modification to improve TBA target affinity. With this goal in mind we synthesized a T analog, 5-(indolyl-3-acetyl-3-amino-1-propenyl)-2'-deoxyuridine (W) to substitute one T or a pair of Ts. Subsequently, the affinity for each analog was determined by biolayer interferometry. An aptamer with W at position 4 exhibited about 3-fold increased binding affinity, and replacing both T4 and T12 with W afforded an almost 10-fold enhancement compared to native TBA. To better understand the role of the substituent's aromatic moiety, an aptamer with 5-(methyl-3-acetyl-3-amino-1-propenyl)-2'-deoxyuridine (K; W without the indole moiety) in place of T4 was also synthesized. This K4 aptamer was found to improve affinity 7-fold relative to native TBA. Crystal structures of aptamers with T4 replaced by either W or K bound to thrombin provide insight into the origins of the increased affinities. Our work demonstrates that facile chemical modification of a simple DNA aptamer can be used to significantly improve its binding affinity for a well-established pharmacological target protein.

Amplified voltammetric detection of glycoproteins using 4-mercaptophenylboronic acid/biotin-modified multifunctional gold nanoparticles as labels.

PubMed

Liu, Lin; Xing, Yun; Zhang, Hui; Liu, Ruili; Liu, Huijing; Xia, Ning

2014-01-01

Ultrasensitive detection of protein biomarkers is essential for early diagnosis and therapy of many diseases. Glycoproteins, differing from other types of proteins, contain carbohydrate moieties in the oligosaccharide chains. Boronic acid can form boronate ester covalent bonds with diol-containing species. Herein, we present a sensitive and cost-effective electrochemical method for glycoprotein detection using 4-mercaptophenylboronic acid (MBA)/biotin-modified gold nanoparticles (AuNPs) (MBA-biotin-AuNPs) as labels. To demonstrate the feasibility and sensitivity of this method, recombinant human erythropoietin (rHuEPO) was tested as a model analyte. Specifically, rHuEPO was captured by the anti-rHuEPO aptamer-covered electrode and then derivatized with MBA-biotin-AuNPs through the boronic acid-carbohydrate interaction. The MBA-biotin-AuNPs facilitated the attachment of streptavidin-conjugated alkaline phosphatase for the production of electroactive p-aminophenol from p-aminophenyl phosphate substrate. A detection limit of 8 fmol L(-1) for rHuEPO detection was achieved. Other glycosylated and non-glycosylated proteins, such as horseradish peroxidase, prostate specific antigen, metallothionein, streptavidin, and thrombin showed no interference in the detection assay.

Evaluation of a pretest scoring system (4Ts) for the diagnosis of heparin-induced thrombocytopenia in a university hospital setting.

PubMed

Vatanparast, Rodina; Lantz, Sarah; Ward, Kristine; Crilley, Pamela Ann; Styler, Michael

2012-11-01

The initial diagnosis of heparin-induced thrombocytopenia (HIT) is made on clinical grounds because the assays with the highest sensitivity (eg, heparin-platelet factor 4 antibody enzyme-linked immunosorbent assay [ELISA]) and specificity (eg, serotonin release assay) may not be readily available. The clinical utility of the pretest scoring system, the 4Ts, was developed and validated by Lo et al in the Journal of Thrombosis and Haemostasis in 2006. The pretest scoring system looks at the degree and timing of thrombocytopenia, thrombosis, and the possibility of other etiologies. Based on the 4T score, patients can be categorized as having a high, intermediate, or low probability of having HIT. We conducted a retrospective study of 100 consecutive patients who were tested for HIT during their hospitalization at Hahnemann University Hospital (Philadelphia, PA) in 2009. Of the 100 patients analyzed, 72, 23, and 5 patients had 4T pretest probability scores of low, intermediate, and high, respectively. A positive HIT ELISA (optical density > 1.0 unit) was detected in 0 of 72 patients (0%) in the low probability group, in 5 of 23 patients (22%) in the intermediate probability group, and in 2 of 5 patients (40%) in the high probability group. The average turnaround time for the HIT ELISA was 4 to 5 days. Fourteen (19%) of the 72 patients with a low pretest probability of HIT were treated with a direct thrombin inhibitor. Ten (71%) of the 14 patients in the low probability group treated with a direct thrombin inhibitor had a major complication of bleeding requiring blood transfusion support. In this retrospective study, a low 4T score showed 100% correlation with a negative HIT antibody assay. We recommend incorporating the 4T scoring system into institutional core measures when assessing a patient with suspected HIT, selecting only patients with intermediate to high probability for therapeutic intervention, which may translate into reduced morbidity and lower health care costs.

Intracellular origin and ultrastructure of platelet-derived microparticles.

PubMed

Ponomareva, A A; Nevzorova, T A; Mordakhanova, E R; Andrianova, I A; Rauova, L; Litvinov, R I; Weisel, J W

2017-08-01

Essentials Platelet microparticles play a major role in pathologies, including hemostasis and thrombosis. Platelet microparticles have been analyzed and classified based on their ultrastructure. The structure and intracellular origin of microparticles depend on the cell-activating stimulus. Thrombin-treated platelets fall apart and form microparticles that contain cellular organelles. Background Platelet-derived microparticles comprise the major population of circulating blood microparticles that play an important role in hemostasis and thrombosis. Despite numerous studies on the (patho)physiological roles of platelet-derived microparticles, mechanisms of their formation and structural details remain largely unknown. Objectives Here we studied the formation, ultrastructure and composition of platelet-derived microparticles from isolated human platelets, either quiescent or stimulated with one of the following activators: arachidonic acid, ADP, collagen, thrombin or calcium ionophore A23187. Methods Using flow cytometry, transmission and scanning electron microscopy, we analyzed the intracellular origin, structural diversity and size distributions of the subcellular particles released from platelets. Results The structure, dimensions and intracellular origin of microparticles depend on the cell-activating stimulus. The main structural groups include a vesicle surrounded by one thin membrane or multivesicular structures. Thrombin, unlike other stimuli, induced formation of microparticles not only from the platelet plasma membrane and cytoplasm but also from intracellular structures. A fraction of these vesicular particles having an intracellular origin contained organelles, such as mitochondria, glycogen granules and vacuoles. The size of platelet-derived microparticles depended on the nature of the cell-activating stimulus. Conclusion The results obtained provide a structural basis for the qualitative differences of various platelet activators, for specific physiological and pathological effects of microparticles, and for development of advanced assays. © 2017 International Society on Thrombosis and Haemostasis.

In vitro and in vivo characterization of a reversible synthetic heparin analog.

PubMed

Whelihan, Matthew F; Cooley, Brian; Xu, Yongmei; Pawlinski, Rafal; Liu, Jian; Key, Nigel S

2016-02-01

The global supply of unfractionated heparin (UFH) and all commercially available low molecular weight heparins (LMWH) remain dependent on animal sources, such as porcine intestine or bovine lung. Recent experience has shown that contamination of the supply chain (with over-sulfated chondroitin sulfates) can result in lethal toxicity. Fondaparinux is currently the only commercially available synthetic analog of heparin. We recently described a new class of chemoenzymatically synthesized heparin analogs. One of these compounds (S12-mer) is a dodecasaccharide consisting of an antithrombin-binding moiety with repeating units of IdoA2S-GlcNS6S and two 3-O-sulfate groups that confer the ability to bind protamine. We sought to further characterize this new compound in vitro using biochemical and global coagulation assays and in vivo using thrombosis and hemostasis assays. The anticoagulant activities of the Super 12-mer (S12-mer) and Enoxaparin in anti-factor Xa and plasma-based thrombin generation assays were roughly equivalent with a 50% reduction in peak thrombin generation occurring at approximately 325nM. When protamine was titrated against a fixed concentration of S12-mer in plasma or blood, the S12-mer displayed a significant restitution of thrombin generation and clot formation. In vivo, S12-mer inhibited venous thrombosis to a similar extent as Enoxaparin, with similar bleeding profiles. These data show that the S12-mer has almost identical efficacy to Enoxaparin in terms of FXa inhibition, while displaying significant reversibility with protamine. Taken together with the ability to ensure purity and homogeneity from batch to batch, the S12-mer is a promising new synthetic heparin analog with a potentially enhanced safety profile. Copyright © 2015 Elsevier Ltd. All rights reserved.

Localized hypothermia aggravates bleeding in the collagenase model of intracerebral hemorrhage.

PubMed

John, Roseleen F; Williamson, Michael R; Dietrich, Kristen; Colbourne, Frederick

2015-03-01

Animal studies testing whether therapeutic hypothermia is neuroprotective after intracerebral hemorrhage (ICH) have been inconclusive. In rodents, ICH is often produced in the striatum by infusing collagenase, which causes prolonged hemorrhaging from multiple vessels. Our previous data shows that this bleeding (hematoma) is worsened by systemic hypothermia given soon after collagenase infusion. In this study we hypothesized that localized brain hypothermia would also aggravate bleeding in this model (0.2 U of collagenase in 1.2 μL of saline). We also evaluated cooling after intrastriatal thrombin infusion (1 U in 30 μL of saline)-a simplified model of ICH thought to cause bleeding. Focal hypothermia was achieved by flushing cold water through an implanted cooling device attached to the skull underneath the temporalis muscle of adult rats. Previous work and data at this time shows this method cools the striatum to ∼33°C, whereas the body remains normothermic. In comparison to normothermic groups, cooling significantly worsened bleeding when instituted at 6 hours (∼94 vs. 42 μL, p=0.018) and 12 hours (79 vs. 61 μL, p=0.042) post-ICH (24-hour survival), but not after a 24-hour delay (36-hour survival). Rats were cooled until euthanasia when hematoma size was determined by a hemoglobin-based spectrophotometry assay. Cooling did not influence cerebral blood volume after just saline or thrombin infusion. The latter is explained by the fact that thrombin did not cause bleeding beyond that caused by saline infusion. In summary, local hypothermia significantly aggravates bleeding many hours after collagenase infusion suggesting that bleeding may have confounded earlier studies with hypothermia. Furthermore, these findings serve as a cautionary note on using cooling even many hours after cerebral bleeding.

Selection is more intelligent than design: improving the affinity of a bivalent ligand through directed evolution.

PubMed

Ahmad, Kareem M; Xiao, Yi; Soh, H Tom

2012-12-01

Multivalent molecular interactions can be exploited to dramatically enhance the performance of an affinity reagent. The enhancement in affinity and specificity achieved with a multivalent construct depends critically on the effectiveness of the scaffold that joins the ligands, as this determines their positions and orientations with respect to the target molecule. Currently, no generalizable design rules exist for construction of an optimal multivalent ligand for targets with known structures, and the design challenge remains an insurmountable obstacle for the large number of proteins whose structures are not known. As an alternative to such design-based strategies, we report here a directed evolution-based method for generating optimal bivalent aptamers. To demonstrate this approach, we fused two thrombin aptamers with a randomized DNA sequence and used a microfluidic in vitro selection strategy to isolate scaffolds with exceptionally high affinities. Within five rounds of selection, we generated a bivalent aptamer that binds thrombin with an apparent dissociation constant (K(d)) <10 pM, representing a ∼200-fold improvement in binding affinity over the monomeric aptamers and a ∼15-fold improvement over the best designed bivalent construct. The process described here can be used to produce high-affinity multivalent aptamers and could potentially be adapted to other classes of biomolecules.

Microdrilled cartilage defects treated with thrombin-solidified chitosan/blood implant regenerate a more hyaline, stable, and structurally integrated osteochondral unit compared to drilled controls.

PubMed

Marchand, Catherine; Chen, Gaoping; Tran-Khanh, Nicolas; Sun, Jun; Chen, Hongmei; Buschmann, Michael D; Hoemann, Caroline D

2012-03-01

This study analyzed the long-term cartilage and subchondral bone repair of microdrilled defects treated with chitosan glycerol-phosphate/blood implant, using thrombin (Factor IIa) to accelerate in situ solidification. We also evaluated the cartilage repair response to six smaller microdrill holes compared with two larger holes. Bilateral knee trochlear cartilage defects were created in n=8 skeletally mature rabbits, drilled with six proximal 0.5 mm and two distal 0.9 mm holes, then covered with in situ-solidified IIa-implants (treated) or with IIa-alone (control). After 6.5 months of repair, cartilage repair tissues were analyzed by histological scoring and histomorphometry for hyaline matrix characteristics and osseous integration. Subchondral repair bone was analyzed by 3D microcomputed tomography and compared to acute defects (n=6) and intact trochlea (n=8). Implant-treated cartilage repair tissues had higher structural integrity through the entire defect (p=0.02), twofold higher percent staining for glycosaminoglycan (p=0.0004), and ~24% more collagen type II staining over the smaller drill holes (p=0.008) compared with controls. Otherwise, hole diameter had no specific effect on cartilage repair. The subchondral bone plate was partially restored in treated and control defects but less dense than intact trochlea, with evidence of incomplete regeneration of the calcified cartilage layer. More residual drill holes (p=0.054) were detected in control versus treated defects, and control defects with more than 40% residual holes presented abnormally thicker trabeculae compared with treated defects. Low osteoclast numbers after 6.5 months repair suggested that bone was no longer remodeling. The subchondral bone plate surrounding the defects exhibited a significant thickening compared with age-matched intact trochlea. These data suggest that debridement and drilling can lead to long-term subchondral bone changes outside the cartilage defect. Compared with drilled controls, chitosan implants solidified with thrombin elicited a more hyaline and structurally integrated osteochondral unit, features needed for long-term durability.

Endogenous pro-thrombotic biomarkers from the arm and leg may not have the same value.

PubMed

Lattimer, Christopher R; Kalodiki, Evi; Geroulakos, George; Hoppensteadt, Debra; Fareed, Jawed

2016-05-01

Assessments of endogenous pro-thrombotic biomarkers are performed invariably on arm blood. However, the commonest site for thrombosis is in the leg. A leg blood sample may reflect local pro-thrombotic processes more accurately than systemic arm blood. The aim was to determine whether pro-thrombotic biomarkers from standard venous arm samples differed significantly from leg samples. Concurrent blood samples were taken from an ankle/lower calf varicose vein and an ante-cubital vein in 24 patients awaiting laser treatment as well as age approximated and sex matched healthy controls without venous disease. The following assays were performed: thrombin-antithrombin (ng/ml), antithrombin (%) activity, microparticles (nM), fibrinogen (mg/dl), prothrombin fragment 1.2 (F1.2) (pM) and P-selectin (ng/ml). Expressed as median (inter-quartile range). Significant arm/leg differences were observed in thrombin-antithrombin, antithrombin, prothrombin fragment 1.2 and P-selectin. The legs of patients had significantly reduced antithrombin activity and P-selectin concentrations compared to their arms (leg: 101 (90-108) versus arm: 112 (99-126), P = 0.001 and leg: 42 (26-52) versus 45 (27-52), P = 0.044, respectively). Control leg samples had significantly increased thrombin-antithrombin and P-selectin compared to control arm samples (leg: 2.1 (0.9-3.2) versus arm: 0.8 (0.5-1.7), P = 0.015 and leg: 36 (24-50) versus arm: 30 (23-41), P = 0.007, respectively). However, the control legs had significantly reduced F1.2 (leg: 265 (230-333) versus arm: 299 (236-361), P = 0.028). No significant arm/leg differences were detected in the microparticle or fibrinogen levels. These findings indicate that venous arm blood is significantly different from venous leg blood in four out of six biomarkers studied. Recognition of local venous leg sampling as a site for investigation may unravel why the leg has a greater predisposition to thrombosis and lead the way towards an arm/leg differential test. © The Author(s) 2015.

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

PubMed Central

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

2015-01-01

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

Thrombin-Binding Aptamer Quadruplex Formation: AFM and Voltammetric Characterization

PubMed Central

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

2010-01-01

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

A common standard is inappropriate for determining the potency of ultra low molecular weight heparins such as semuloparin and bemiparin.

PubMed

Jeske, Walter P; Hoppensteadt, Debra; Gray, Angel; Walenga, Jeanine M; Cunanan, Josephine; Myers, Lauren; Fareed, Jawed; Bayol, Alain; Rigal, Hélène; Viskov, Christian

2011-10-01

Lower low-molecular-weight heparins are being developed to improve on the safety and efficacy of antithrombotic therapy. Semuloparin and bemiparin are two depolymerized heparins produced by distinct manufacturing processes. The objective of this investigation was to determine whether a common standard could be used to define their potency. Activities were compared using typical clinical coagulation assays and pharmacological assays required for potency assessment. The activity of semuloparin and bemiparin was comparable in FXa-based assays (anti-FXa, Heptest). However, bemiparin produced a stronger effect in the aPTT, ACT and anti-thrombin assays. Assessment of the parallelism of the concentration-response curves indicated that bemiparin and semuloparin are not equivalent in terms of anti-FIIa activity. Bemiparin had a stronger inhibitory effect on thrombin induced platelet aggregation, and a stronger interaction with HIT antibodies. These data demonstrate that depolymerized heparins can exhibit a range of biologic activities making them unique agents. Pharmacopoeial parameters such as anti-IIa and anti-Xa potency and molecular weight are insufficient to characterize such agents. Copyright © 2011 Elsevier Ltd. All rights reserved.

On-Chip Titration of an Anticoagulant Argatroban and Determination of the Clotting Time within Whole Blood or Plasma Using a Plug-Based Microfluidic System

PubMed Central

Song, Helen; Li, Hung-Wing; Munson, Matthew S.; Van Ha, Thuong G.; Ismagilov, Rustem F.

2006-01-01

This paper describes extending plug-based microfluidics to handling complex biological fluids such as blood, solving the problem of injecting additional reagents into plugs, and applying this system to measuring of clotting time in small volumes of whole blood and plasma. Plugs are droplets transported through microchannels by fluorocarbon fluids. A plug-based microfluidic system was developed to titrate an anticoagulant (argatroban) into blood samples and to measure the clotting time using the activated partial thromboplastin time (APTT) test. To carry out these experiments, the following techniques were developed for a plug-based system: (i) using Teflon AF coating on the microchannel wall to enable formation of plugs containing blood and transport of the solid fibrin clots within plugs, (ii) using a hydrophilic glass capillary to enable reliable merging of a reagent from an aqueous stream into plugs, (iii) using bright-field microscopy to detect the formation of a fibrin clot within plugs and using fluorescent microscopy to detect the production of thrombin using a fluorogenic substrate, and (iv) titration of argatroban (0–1.5 μg/mL) into plugs and measurement of the resulting APTTs at room temperature (23 °C) and physiological temperature (37 °C). APTT measurements were conducted with normal pooled plasma (platelet-poor plasma) and with donor’s blood samples (both whole blood and platelet-rich plasma). APTT values and APTT ratios measured by the plug-based microfluidic device were compared to the results from a clinical laboratory at 37 °C. APTT obtained from the on-chip assay were about double those from the clinical laboratory but the APTT ratios from these two methods agreed well with each other. PMID:16841902

New Signal Amplification Strategy Using Semicarbazide as Co-reaction Accelerator for Highly Sensitive Electrochemiluminescent Aptasensor Construction.

PubMed

Ma, Meng-Nan; Zhuo, Ying; Yuan, Ruo; Chai, Ya-Qin

2015-11-17

A highly sensitive electrochemiluminescent (ECL) aptasensor was constructed using semicarbazide (Sem) as co-reaction accelerator to promote the ECL reaction rate of CdTe quantum dots (CdTe QDs) and the co-reactant of peroxydisulfate (S2O8(2-)) for boosting signal amplification. The co-reaction accelerator is a species that when it is introduced into the ECL system containing luminophore and co-reactant, it can interact with co-reactant rather than luminophore to promote the ECL reaction rate of luminophore and co-reactant; thus the ECL signal is significantly amplified in comparison with that in which only luminophore and co-reactant are present. In this work, the ECL signal probes were first fabricated by alternately assembling the Sem and Au nanoparticles (AuNPs) onto the surfaces of hollow Au nanocages (AuNCs) via Au-N bond to obtain the multilayered nanomaterials of (AuNPs-Sem)n-AuNCs for immobilizing amino-terminated detection aptamer of thrombin (TBA2). Notably, the Sem with two -NH2 terminal groups could not only serve as cross-linking reagent to assemble AuNPs and AuNCs but also act as co-reaction accelerator to enhance the ECL reaction rate of CdTe QDs and S2O8(2-) for signal amplification. With the sandwich-type format, TBA2 signal probes could be trapped on the CdTe QD-based sensing interface in the presence of thrombin (TB) to achieve a considerably enhanced ECL signal in S2O8(2-) solution. As a result, the Sem in the TBA2 signal probes could accelerate the reduction of S2O8(2-) to produce the more oxidant mediators of SO4(•-), which further boosted the production of excited states of CdTe QDs to emit light. With the employment of the novel co-reaction accelerator Sem, the proposed ECL biosensor exhibited ultrahigh sensitivity to quantify the concentration of TB from 1 × 10(-7) to 1 nM with a detection limit of 0.03 fM, which demonstrated that the co-reaction accelerator could provide a simple, efficient, and low-cost approach for signal amplification and hold great potential for other ECL biosensors construction.

A fluorescent radioiodinated oligonucleotidic photoaffinity probe for protein labeling: synthesis and photolabeling of thrombin.

PubMed

Berens, C; Courtoy, P J; Sonveaux, E

1999-01-01

To study the interactions between oligonucleotides and proteins, an original photoaffinity radiolabeling probe has been synthesized. Starting with a 5'-pyridyldithio-3'-amino-oligonucleotide, the photophore benzophenone was first coupled to the 3' end, through acylation by an activated ester of benzoylbenzoic acid. A fluorescein molecule was grafted by alkylation of the free 5'-SH. This compound was finally radiolabeled with 125I using IodoBeads. The selective photolabeling of thrombin in a complex protein mixture by the radioiodinated probe validates this strategy to identify oligonucleotide-binding proteins.

Ultrasound-guided thrombin injection of genicular artery pseudoaneurysm.

PubMed

Rachakonda, Aditya; Qato, Khalil; Khaddash, Tamim; Carroccio, Alfio; Pamoukian, Vicken; Giangola, Gary

2015-07-01

Pseudoaneurysm is a rare complication after arthroscopic procedures involving the knee. A 38-year-old man presented 1 month after right-knee arthroscopy with a 2-cm pulsating mass on the medial side of the right knee. Duplex ultrasound evaluation revealed 2.5 × 2.1-cm pseudoaneurysm just distal to the patella with arterialized flow communicating with the inferior medial genicular artery. Ultrasound-guided thrombin injection was performed in an office setting, and the resolution of active flow within the pseudoaneurysm was confirmed with duplex ultrasonography. Copyright © 2015 Elsevier Inc. All rights reserved.