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.

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

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.

Thrombin generation potential and clot-forming capacity of thawed fresh-frozen plasma, plasma frozen within 24 h and solvent/detergent-treated plasma (octaplasLG® ), during 5-day storage at 1-6°C.

PubMed

Heger, A; Neisser-Svae, A; Trawnicek, L; Triulzi, D

2018-04-23

To enable rapid availability of plasma in emergency situations, the shelf-life of thawed fresh-frozen plasma (FFP) has been extended from 24 h to 5 days. The aim of this study was to evaluate the thrombin generation (TG) potential and clot-forming ability during 5 days of refrigerated storage of thawed FFP, plasma frozen within 24 h and solvent/detergent-treated plasma octaplasLG ® . During storage for 5 days, TG capacity decreased significantly over time, and rotational thromboelastometry showed significantly prolonged clotting times. However, the stability studies confirmed comparable in vitro haemostatic potentials of all three thawed plasma products at day 5. © 2018 International Society of Blood Transfusion.

Thrombin generation and fibrin formation under flow on biomimetic tissue factor-rich surfaces.

PubMed

Onasoga-Jarvis, A A; Puls, T J; O'Brien, S K; Kuang, L; Liang, H J; Neeves, K B

2014-01-01

Blood flow regulates coagulation and fibrin assembly by controlling the rate of transport of zymogens, enzymes and plasma proteins to and from the site of an injury. The objective of this work was to define the hemodynamic conditions under which fibrin can form under flow on tissue factor (TF)-rich substrates. TF-coated silica beads (~ 800 nm) were patterned into 18-85-μm spots. Normal pooled plasma and factors VIII, IX and XI deficient plasmas were perfused over the beads coated with 0.08, 0.8 and 8 molecules-TF μm(-2) at shear rates of 50-1000 s(-1) . Fibrin deposition and thrombin generation were measured by fluorescence microscopy in a hydrodynamic focusing microfluidic device. Fibrin deposition was supported on patterned bead spots, but not planar TF substrates at the same surface TF concentration. There was a threshold spot size and a shear rate dependent TF concentration that was necessary to support fibrin polymerization. FVIII and FIX had minor effects on fibrin dynamics at 8 molecules-TF μm(-2) , but were essential at 0.8 molecules-TF μm(-2) . The absence of FXI influenced thrombin generation and fibrin deposition at both 0.8 and 8 molecules-TF μm(-2) . These results show that fibrin deposition requires perturbations in the flow field that protect reactions from dilution by flow under venous and arterial conditions. FVIII and FIX have a modest effect on fibrin deposition at high TF concentrations, but are necessary for fibrin deposition at low TF concentrations. FXI amplifies thrombin generation under flow at both low and high TF concentrations. © 2013 International Society on Thrombosis and Haemostasis.

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.

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.

Effects of recombinant human prothrombin on thrombin generation in plasma from patients with hemophilia A and B.

PubMed

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

2015-07-01

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

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.

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

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.

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.

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

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.

Differential Contributions of Intrinsic and Extrinsic Pathways to Thrombin Generation in Adult, Maternal and Cord Plasma Samples

PubMed Central

Rice, Nicklaus T.; Szlam, Fania; Varner, Jeffrey D.; Bernstein, Peter S.; Szlam, Arthur D.; Tanaka, Kenichi A.

2016-01-01

Background Thrombin generation (TG) is a pivotal process in achieving hemostasis. Coagulation profiles during pregnancy and early neonatal period are different from that of normal (non-pregnant) adults. In this ex vivo study, the differences in TG in maternal and cord plasma relative to normal adult plasma were studied. Methods Twenty consented pregnant women and ten consented healthy adults were included in the study. Maternal and cord blood samples were collected at the time of delivery. Platelet-poor plasma was isolated for the measurement of TG. In some samples, anti-FIXa aptamer, RB006, or a TFPI inhibitor, BAX499 were added to elucidate the contribution of intrinsic and extrinsic pathway to TG. Additionally, procoagulant and inhibitor levels were measured in maternal and cord plasma, and these values were used to mathematically simulate TG. Results Peak TG was increased in maternal plasma (393.6±57.9 nM) compared to adult and cord samples (323.2±38.9 nM and 209.9±29.5 nM, respectively). Inhibitory effects of RB006 on TG were less robust in maternal or cord plasma (52% vs. 12% respectively) than in adult plasma (81%). Likewise the effectiveness of BAX499 as represented by the increase in peak TG was much greater in adult (21%) than in maternal (10%) or cord plasma (12%). Further, BAX499 was more effective in reversing RB006 in adult plasma than in maternal or cord plasma. Ex vivo data were reproducible with the results of the mathematical simulation of TG. Conclusion Normal parturient plasma shows a large intrinsic pathway reserve for TG compared to adult and cord plasma, while TG in cord plasma is sustained by extrinsic pathway, and low levels of TFPI and AT. PMID:27196067

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.

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 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

Augmentation of thrombin generation in neonates undergoing cardiopulmonary bypass.

PubMed

Guzzetta, N A; Szlam, F; Kiser, A S; Fernandez, J D; Szlam, A D; Leong, T; Tanaka, K A

2014-02-01

Factor concentrates are currently available and becoming increasingly used off-label for treatment of bleeding. We compared recombinant activated factor VII (rFVIIa) with three-factor prothrombin complex concentrate (3F-PCC) for the ability to augment thrombin generation (TG) in neonatal plasma after cardiopulmonary bypass (CPB). First, we used a computer-simulated coagulation model to assess the impact of rFVIIa and 3F-PCC, and then performed similar measurements ex vivo using plasma from neonates undergoing CPB. Simulated TG was computed according to the coagulation factor levels from umbilical cord plasma and the therapeutic levels of rFVIIa, 3F-PCC, or both. Subsequently, 11 neonates undergoing cardiac surgery were enrolled. Two blood samples were obtained from each neonate: pre-CPB and post-CPB after platelet and cryoprecipitate transfusion. The post-CPB products sample was divided into control (no treatment), control plus rFVIIa (60 nM), and control plus 3F-PCC (0.3 IU ml(-1)) aliquots. Three parameters of TG were measured ex vivo. The computer-simulated post-CPB model demonstrated that rFVIIa failed to substantially improve lag time, TG rate and peak thrombin without supplementing prothrombin. Ex vivo data showed that addition of rFVIIa post-CPB significantly shortened lag time; however, rate and peak were not statistically significantly improved. Conversely, 3F-PCC improved all TG parameters in parallel with increased prothrombin levels in both simulated and ex vivo post-CPB samples. Our data highlight the importance of prothrombin replacement in restoring TG. Despite a low content of FVII, 3F-PCC exerts potent procoagulant activity compared with rFVIIa ex vivo. Further clinical evaluation regarding the efficacy and safety of 3F-PCC is warranted.

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

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

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

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

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

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.

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

Statistical analysis plan for the WOMAN-ETAPlaT study: Effect of tranexamic acid on platelet function and thrombin generation

PubMed Central

Dallaku, Kastriot; Shakur, Haleema; Edwards, Phil; Beaumont, Danielle; Roberts, Ian; Huque, Sumaya; Delius, Maria; Mansmann, Ulrich

2017-01-01

Background. Postpartum haemorrhage (PPH) is a potentially life-threatening complication for women, and the leading cause of maternal mortality. Tranexamic acid (TXA) is an antifibrinolytic used worldwide to treat uterine haemorrhage and to reduce blood loss in general surgery. TXA may have effects on thrombin generation, platelet function and coagulation factors as a result of its inhibition on the plasmin. Methods. WOMAN ETAPlaT is a sub-study of the World Maternal Antifibrinolitic trial (WOMAN trial). All adult women clinically diagnosed with PPH after a vaginal delivery or caesarean section, are eligible for inclusion in the study. Blood samples will be collected at the baseline and 30 minutes after the first dose of study treatment is given. Platelet function will be evaluated in whole blood immediately after sampling with Multiplate® tests (ADPtest and TRAPtest). Thrombin generation, fibrinogen, D-dimer, and coagulation factors vW, V and VIII will be analysed using platelet poor plasma. Results. Recruitment to WOMAN ETAPlaT started on 04 November 2013 and closed on 13 January 2015, during this time  188 patients were recruited. The final participant follow-up was completed on 04 March 2015. This article introduces the statistical analysis plan for the study, without reference to unblinded data.   Conclusion. The data from this study will provide evidence for the effect of TXA on thrombin generation, platelet function and coagulation factors in women with PPH. Trial registration: ClinicalTrials.gov Identifier: NCT00872469; ISRCTN76912190 PMID:28413832

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.

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.

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.

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.

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.

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.

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

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

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.

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

Red blood cell-derived microparticles isolated from blood units initiate and propagate thrombin generation.

PubMed

Rubin, Olivier; Delobel, Julien; Prudent, Michel; Lion, Niels; Kohl, Kid; Tucker, Erik I; Tissot, Jean-Daniel; Angelillo-Scherrer, Anne

2013-08-01

Red blood cell-derived microparticles (RMPs) are small phospholipid vesicles shed from RBCs in blood units, where they accumulate during storage. Because microparticles are bioactive, it could be suggested that RMPs are mediators of posttransfusion complications or, on the contrary, constitute a potential hemostatic agent. This study was performed to establish the impact on coagulation of RMPs isolated from blood units. Using calibrated automated thrombography, we investigated whether RMPs affect thrombin generation (TG) in plasma. We found that RMPs were not only able to increase TG in plasma in the presence of a low exogenous tissue factor (TF) concentration, but also to initiate TG in plasma in absence of exogenous TF. TG induced by RMPs in the absence of exogenous TF was neither affected by the presence of blocking anti-TF nor by the absence of Factor (F)VII. It was significantly reduced in plasma deficient in FVIII or F IX and abolished in FII-, FV-, FX-, or FXI-deficient plasma. TG was also totally abolished when anti-XI 01A6 was added in the sample. Finally, neither Western blotting, flow cytometry, nor immunogold labeling allowed the detection of traces of TF antigen. In addition, RMPs did not comprise polyphosphate, an important modulator of coagulation. Taken together, our data show that RMPs have FXI-dependent procoagulant properties and are able to initiate and propagate TG. The anionic surface of RMPs might be the site of FXI-mediated TG amplification and intrinsic tenase and prothrombinase complex assembly. © 2012 American Association of Blood Banks.

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.

Unfavorably Altered Fibrin Clot Properties in Patients with Eosinophilic Granulomatosis with Polyangiitis (Churg-Strauss Syndrome): Association with Thrombin Generation and Eosinophilia.

PubMed

Mastalerz, Lucyna; Celińska-Lӧwenhoff, Magdalena; Krawiec, Piotr; Batko, Bogdan; Tłustochowicz, Witold; Undas, Anetta

2015-01-01

Given reports on the increased prevalence of thromboembolic incidents in patients with eosinophilic granulomatosis with polyangiitis (EGPA; Churg-Strauss syndrome), we investigated whether fibrin clot properties are unfavorably altered in EGPA. Ex vivo plasma fibrin clot characteristics, including clot permeability, turbidimetry and efficiency of fibrinolysis using two assays, were investigated in 34 consecutive patients with remission in EGPA according to the Birmingham Vasculitis Activity Score version 3 (23 female, 11 male), aged 48 (range, 21-80) years. The control group comprised 34 age- and sex- matched volunteers. Compared with controls, patients with EGPA were characterized by denser fiber clots (estimated pore size, Ks, 7.30±0.93 vs 10.14±1.07 10-9 cm2), faster fibrin polymerization (lag phase in a turbidimetric curve, 41.8±3.6 vs 47.4±2.9 s), thicker fibrin fibers (maximum absorbance, ΔAbs, 0.87±0.09 vs 0.72±0.07), higher maximum levels of D-dimer released from clots (DDmax 4.10±0.46 vs 3.54±0.35 mg/L), and prolonged clot lysis time (t50%; 9.50±1.45 vs 7.56±0.87 min); all p<0.0001. Scanning electron microscopy images confirmed denser plasma fibrin networks composed of thinner fibers formed in EGPA. Antineutrophil cytoplasmic antibody status and C-reactive protein did not affect clot variables. Multivariate analysis adjusted for fibrinogen showed that Ks was predicted by eosinophil count, peak thrombin generation, factor VIII, and soluble CD40 ligand, whereas eosinophil count, peak thrombin generation and antiplasmin predicted t50%. This study is the first to show that EGPA is associated with prothrombotic plasma fibrin clot phenotype, which may contribute to thromboembolic manifestations reported in this disease.

Unfavorably Altered Fibrin Clot Properties in Patients with Eosinophilic Granulomatosis with Polyangiitis (Churg-Strauss Syndrome): Association with Thrombin Generation and Eosinophilia

PubMed Central

Mastalerz, Lucyna; Celińska-Lӧwenhoff, Magdalena; Krawiec, Piotr; Batko, Bogdan; Tłustochowicz, Witold; Undas, Anetta

2015-01-01

Objectives Given reports on the increased prevalence of thromboembolic incidents in patients with eosinophilic granulomatosis with polyangiitis (EGPA; Churg-Strauss syndrome), we investigated whether fibrin clot properties are unfavorably altered in EGPA. Methods Ex vivo plasma fibrin clot characteristics, including clot permeability, turbidimetry and efficiency of fibrinolysis using two assays, were investigated in 34 consecutive patients with remission in EGPA according to the Birmingham Vasculitis Activity Score version 3 (23 female, 11 male), aged 48 (range, 21–80) years. The control group comprised 34 age- and sex- matched volunteers. Results Compared with controls, patients with EGPA were characterized by denser fiber clots (estimated pore size, Ks, 7.30±0.93 vs 10.14±1.07 10−9 cm2), faster fibrin polymerization (lag phase in a turbidimetric curve, 41.8±3.6 vs 47.4±2.9 s), thicker fibrin fibers (maximum absorbance, ΔAbs, 0.87±0.09 vs 0.72±0.07), higher maximum levels of D-dimer released from clots (DDmax 4.10±0.46 vs 3.54±0.35 mg/L), and prolonged clot lysis time (t50%; 9.50±1.45 vs 7.56±0.87 min); all p<0.0001. Scanning electron microscopy images confirmed denser plasma fibrin networks composed of thinner fibers formed in EGPA. Antineutrophil cytoplasmic antibody status and C-reactive protein did not affect clot variables. Multivariate analysis adjusted for fibrinogen showed that Ks was predicted by eosinophil count, peak thrombin generation, factor VIII, and soluble CD40 ligand, whereas eosinophil count, peak thrombin generation and antiplasmin predicted t50%. Conclusion This study is the first to show that EGPA is associated with prothrombotic plasma fibrin clot phenotype, which may contribute to thromboembolic manifestations reported in this disease. PMID:26540111

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

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.

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.

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.

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

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

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

[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.

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.

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

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.

Hemostatic properties and protein expression profile of therapeutic apheresis plasma treated with amotosalen and ultraviolet A for pathogen inactivation.

PubMed

Ohlmann, Philippe; Hechler, Béatrice; Chafey, Philippe; Ravanat, Catherine; Isola, Hervé; Wiesel, Marie-Louise; Cazenave, Jean-Pierre; Gachet, Christian

2016-09-01

The INTERCEPT Blood System (IBS) using amotosalen-HCl and ultraviolet (UV)A inactivates a large spectrum of microbial pathogens and white blood cells in therapeutic plasma. Our aim was to evaluate to what extent IBS modifies the capacity of plasma to generate thrombin and induces qualitative or quantitative modifications of plasma proteins. Plasma units from four donors were collected by apheresis. Samples were taken before (control [CTRL]) and after IBS treatment and stored at -80°C until use. The activities of plasma coagulation factors and inhibitors and the thrombin generation potential were determined using assays measuring clotting times and the calibrated automated thrombogram (CAT), respectively. The proteomic profile of plasma proteins was examined using a two-dimensional differential in-gel electrophoresis (2D-DIGE) method. Nearly all of the procoagulant and antithrombotic factors tested retained at least 78% of their initial pre-IBS activity. Only FVII and FVIII displayed a lower level of conservation (67%), which nevertheless remained within the reference range for conventional plasma coagulation factors. The thrombin generation profile of plasma was conserved after IBS treatment. Among the 1331 protein spots revealed by 2D-DIGE analysis, only four were differentially expressed in IBS plasma compared to CTRL plasma and two were identified by mass spectrometric analysis as transthyretin and apolipoprotein A1. The IBS technique for plasma moderately decreases the activities of plasma coagulation factors and antithrombotic proteins, with no impact on the thrombin generation potential of plasma and very limited modifications of the proteomic profile. © 2016 AABB.

Tissue factor activity in women with preeclampsia or SGA: a potential explanation for the excessive thrombin generation in these syndromes.

PubMed

Erez, Offer; Romero, Roberto; Vaisbuch, Edi; Than, Nandor Gabor; Kusanovic, Juan Pedro; Mazaki-Tovi, Shali; Gotsch, Francesca; Mittal, Pooja; Dong, Zhong; Chaiworapongsa, Tinnakorn; Kim, Chong Jai; Nhan-Chang, Chia-Ling; Kim, Sun Kwon; Yeo, Lami; Mazor, Moshe; Hassan, Sonia S

2018-06-01

The aim of this study was to determine whether the activity of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in the plasma of women with preeclampsia (PE) and small for gestational age (SGA) neonate differ from that of normal pregnant women and whether they are related to specific placental lesions. This cross-sectional study included the following groups: (1) normal pregnancy (n = 68); (2) PE (n= 128); and (3) SGA (n = 56). Maternal plasma TF and TFPI activity was determined with chromogenic assays. (1) The median maternal plasma TF activity, but not TFPI activity, differed among the study groups (p < .0001 and p = .4, respectively); (2) patients with PE had a higher median maternal plasma TF activity than women with normal pregnancies (p < .0001) and mothers with SGA fetuses (p = .002); (3) among patients with PE, those with distal villous hypoplasia had a higher median maternal TF activity than those without these placental lesions (p = .018); and (4) following adjustment for confounding variables, maternal plasma TF and TFPI activity were not associated with an SGA neonate. Plasma TF activity is higher in women with PE than in those with SGA or normal pregnancies. We propose that these changes may be responsible, at least in part, for the increased in-vivo thrombin generation observed in this obstetrical syndrome.

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

PubMed

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

2010-10-01

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

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.

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

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.

Effect of two oral doses of 17beta-estradiol associated with dydrogesterone on thrombin generation in healthy menopausal women: a randomized double-blind placebo-controlled study.

PubMed

Rousseau, Alexandra; Robert, Annie; Gerotziafas, Grigoris; Torchin, Dahlia; Zannad, Faiez; Lacut, Karine; Libersa, Christian; Dasque, Eric; Démolis, Jean-Louis; Elalamy, Ismail; Simon, Tabassome

2010-04-01

Oral hormone therapy is associated with an increased risk of venous thrombosis. Drug agencies recommend the use of the lowest efficient dose to treat menopausal symptoms for a better risk/ratio profile, although this profile has not been totally investigated yet. The aim of the study was to compare the effect of the standard dose of 17beta-estradiol to a lower one on thrombin generation (TG). In a 2-month study, healthy menopausal women were randomized to receive daily 1mg or 2 mg of 17beta-estradiol (E1, n = 24 and E2, n = 26; respectively) with 10 mg dydrogesterone or placebo (PL, n = 22). Plasma levels factors VII, X, VIII and II were assessed before and after treatment as well as Tissue factor triggered TG, which allows the investigation of the different phases of coagulation process. The peak of thrombin was higher in hormone therapy groups (E1: 42.39 +/- 50.23 nm, E2: 31.08 +/- 85.86 nm vs. 10.52 +/- 40.63 nm in PL, P = 0.002 and P = 0.01). Time to reach the peak was also shortened (PL: 0.26 +/- 0.69 min vs. E1: -0.26 +/- 0.80 min, E2: -0.55 +/- 0.79 min, P <10(-3) for both comparisons) and mean rate index of the propagation phase of TG was significantly increased. Among the studied clotting factors, only the levels of FVII were significantly increased after treatment administration. The two doses of 17beta-estradiol induced in a similar degree an acceleration of the initiation and propagation phase of tissue factor triggered thrombin generation and a significant increase of FVII coagulant activity.

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.

In vitro sealing of iatrogenic fetal membrane defects by a collagen plug imbued with fibrinogen and plasma.

PubMed

Engels, A C; Hoylaerts, M F; Endo, M; Loyen, S; Verbist, G; Manodoro, S; DeKoninck, P; Richter, J; Deprest, J A

2013-02-01

We aimed to demonstrate local thrombin generation by fetal membranes, as well as its ability to generate fibrin from fibrinogen concentrate. Furthermore, we aimed to investigate the efficacy of collagen plugs, soaked with plasma and fibrinogen, to seal iatrogenic fetal membrane defects. Thrombin generation by homogenized fetal membranes was measured by calibrated automated thrombography. To identify the coagulation caused by an iatrogenic membrane defect, we analyzed fibrin formation by optical densitometry, upon various concentrations of fibrinogen. The ability of a collagen plug soaked with fibrinogen and plasma was tested in an ex vivo model for its ability to seal an iatrogenic fetal membrane defect. Fetal membrane homogenates potently induced thrombin generation in amniotic fluid and diluted plasma. Upon the addition of fibrinogen concentrate, potent fibrin formation was triggered. Measured by densiometry, fibrin formation was optimal at 1250 µg/mL fibrinogen in combination with 4% plasma. A collagen plug soaked with fibrinogen and plasma sealed an iatrogenic membrane defect about 35% better than collagen plugs without these additives (P = 0.037). These in vitro experiments suggest that the addition of fibrinogen and plasma may enhance the sealing efficacy of collagen plugs in closing iatrogenic fetal membrane defects. © 2013 John Wiley & Sons, Ltd.

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

Effects of normoxic and hypoxic exercise regimens on monocyte-mediated thrombin generation in sedentary men.

PubMed

Wang, Jong-Shyan; Chang, Ya-Lun; Chen, Yi-Ching; Tsai, Hsing-Hua; Fu, Tieh-Cheng

2015-08-01

Exercise and hypoxia paradoxically modulate vascular thrombotic risks. The shedding of procoagulant-rich microparticles from monocytes may accelerate the pathogenesis of atherothrombosis. The present study explores the manner in which normoxic and hypoxic exercise regimens affect procoagulant monocyte-derived microparticle (MDMP) formation and monocyte-promoted thrombin generation (TG). Forty sedentary healthy males were randomized to perform either normoxic (NET; 21% O2, n=20) or hypoxic (HET; 15% O2, n=20) exercise training (60% VO(2max)) for 30 min/day, 5 days/week for 5 weeks. At rest and immediately after HET (100 W under 12% O2 for 30 min), the MDMP characteristics and dynamic TG were measured by flow cytometry and thrombinography respectively. The results demonstrated that acute 12% O2 exercise (i) increased the release of coagulant factor V (FV)/FVIII-rich, phosphatidylserine (PS)-exposed and tissue factor (TF)-expressed microparticles from monocytes, (ii) enhanced the peak height and rate of TG in monocyte-rich plasma (MRP) and (iii) elevated concentrations of norepinephrine/epinephrine, myeloperoxidase (MPO) and interleukin-6 (IL-6) in plasma. Following the 5-week intervention, HET exhibited higher enhancements of peak work-rate and cardiopulmonary fitness than NET did. Moreover, both NET and HET decreased the FV/FVIII-rich, PS-exposed and TF-expressed MDMP counts and the peak height and rate of TG in MRP following the HET. However, HET elicited more suppression for the HE (hypoxic exercise)-enhanced procoagulant MDMP formation and dynamic TG in MPR and catecholamine/peroxide/pro-inflammatory cytokine levels in plasma than NET. Hence, we conclude that HET is superior to NET for enhancing aerobic capacity. Furthermore, HET effectively suppresses procoagulant MDMP formation and monocyte-mediated TG under severe hypoxic stress, compared with NET.

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.

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

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

Different profile of thrombin generation in children with acute lymphoblastic leukaemia treated with native or pegylated asparaginase: A cohort study.

PubMed

Rozen, Laurence; Noubouossie, Denis; Dedeken, Laurence; Huybrechts, Sophie; Lê, Phu Quoc; Ferster, Alina; Demulder, Anne

2017-02-01

Asparaginase (Asp) and corticosteroid (CS) treatment in patients with acute lymphoblastic leukaemia (ALL) is associated with an increased risk of thrombotic events. Characterization of global haemostatic phenotypes of patients with ALL during Asp therapy. Thrombin generation (TG) was monitored in platelet-poor plasma of 56 children treated for a B lineage ALL (36 with native, 20 with PEG Asp) using 1 pM tissue factor and 4 μM phospholipids, with and without thrombomodulin. Protein C activity (PC), free protein S (PS), antithrombin (AT) and fibrinogen levels were also measured. Elevated endogenous thrombin potential (ETP) and peak of TG were noted at diagnosis, throughout the Induction phase and Late Intensification but was significantly less for PEG than for native Asp (P < 0.001), while age, sex, type of corticosteroid during Induction and molecular response had no significant effect. The reduction of ETP after addition of thrombomodulin was significantly lower in ALL children compared with that in controls, suggesting impairment in PS/PC pathway. Three patients experienced thrombosis: two treated with native and one with PEG Asp. The two patients with native Asp had, at the time of thrombosis, a prothrombotic profile. Treatment with Asp, in combination with CS, enhances TG in children with ALL, more significantly with native than PEG Asp, which is present early at diagnosis, persists during Induction and reappears during Late Intensification. This is consistent with the high incidence of thrombotic events described during these phases of therapy. The less pronounced effect of PEG Asp remains to be elucidated. © 2016 Wiley Periodicals, Inc.

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.

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.

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

In vivo increase in thrombin generation by four-factor prothrombin complex concentrate in apixaban-treated healthy volunteers.

PubMed

Cheung, Y W; Barco, S; Hutten, B A; Meijers, J C M; Middeldorp, S; Coppens, M

2015-10-01

Four-factor prothrombin complex concentrate (PCC) (Cofact; Sanquin Blood Supply) 50 IU kg(-1) increased thrombin generation beyond baseline values in healthy, rivaroxaban-treated subjects. To assess whether infusion with doses of 37.5 IU kg(-1) and 25 IU kg(-1) PCC reverses the anticoagulant effect of high-dose apixaban, another oral direct factor Xa inhibitor. In a randomized, double-blind, placebo-controlled, crossover study, six healthy subjects received twice-daily apixaban 10 mg for 3.5 days followed by a single bolus of 37.5 IU kg(-1) PCC, 25 IU kg(-1) PCC, or placebo. The primary outcome was the effect of PCC 15 min after infusion on thrombin generation (endogenous thrombin potential [ETP]); secondary outcomes were the immediate effect of PCC on prothrombin time (PT) and the effect of PCC as compared with placebo over a period of 24 h on ETP and PT. Fifteen minutes after infusion of 37.5 IU kg(-1) and 25 IU kg(-1) PCC, ETP increased from 41% ± 11% to 56% ± 23% (P = 0.06) and from 44% ± 12% to 51% ± 15% (P = 0.03), respectively. ETP significantly differed over time between 37.5 IU kg(-1) PCC and placebo during 24 h after infusion (P < 0.01). Both PCC doses restored apixaban-induced PT prolongation after 15 min (P < 0.01), and this was sustained over a period of 24 h. Both 37.5 IU kg(-1) PCC and 25 IU/kg PCC improved coagulation parameters in healthy subjects, suggesting partial reversal of the anticoagulant effect of apixaban. This implies that PCC might be considered in patients with apixaban-associated bleeding. However, ETP was not immediately restored to pre-apixaban levels, suggesting that these doses are too low to instantly and fully restore hemostasis at peak apixaban levels. © 2015 International Society on Thrombosis and Haemostasis.

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

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.

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

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.

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.

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.

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

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.

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

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

Factor V Has Anticoagulant Activity in Plasma in the Presence of TFPIα: Difference between FV1 and FV2.

PubMed

van Doorn, Peter; Rosing, Jan; Duckers, Connie; Hackeng, Tilman M; Simioni, Paolo; Castoldi, Elisabetta

2018-06-04

 Activated factor V (FVa) is a potent procoagulant cofactor in the prothrombinase complex, whereas its precursor factor V (FV) stimulates the inhibition of factor Xa (FXa) by tissue factor pathway inhibitor-α (TFPIα), presumably by promoting TFPIα binding to phospholipids. Plasma FV comprises two glycosylation isoforms (FV1 and FV2) with low and high phospholipid-binding affinity, respectively. The FV1/FV2 ratio is increased in carriers of the FV R2 haplotype.  This article demonstrates the TFPIα-cofactor function of FV in plasma and compares FV1 and FV2.  Thrombin generation at low TF concentration was measured in FV-depleted plasma reconstituted with 0 to 100% FV, FV1 or FV2, and in 122 individuals genotyped for the R2 haplotype. The TFPIα-cofactor activities of FV1 and FV2 were also investigated in a model system of TFPIα-mediated FXa inhibition.  In the FV titration, thrombin generation first increased (up to 5% FV) and then progressively decreased at higher FV concentrations. This anticoagulant effect of FV, which was also observed with FV2 but not with FV1, was largely abolished by anti-TFPIα antibodies, suggesting that it reflects TFPIα-cofactor activity of FV. In the model system of TFPIα-mediated FXa inhibition, FV2 was a more potent TFPIα-cofactor than FV1, in line with their respective phospholipid affinities. Accordingly, FV R2 carriers had higher thrombin generation than non-carriers, even after correction for demographics and plasma levels of coagulation factors and inhibitors.  FV (and particularly its FV2 isoform) contributes to the TFPIα-dependent down-regulation of thrombin generation in plasma triggered with low TF. Schattauer GmbH Stuttgart.

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.

Quarantine versus pathogen-reduced plasma-coagulation factor content and rotational thromboelastometry coagulation.

PubMed

Theusinger, Oliver M; Goslings, David; Studt, Jan-Dirk; Brand-Staufer, Brigitte; Seifert, Burkhardt; Spahn, Donat R; Frey, Beat M

2017-03-01

Different types of fresh-frozen plasma (FFP) exist, and the concentrations of plasma proteins vary between individuals and blood groups. Furthermore, processing may also influence the content. Quarantine-stored plasma (qFFP) and plasma that was pathogen-reduced using blood-safety (Intercept) technology (piFFP) were analyzed regarding procoagulant and anticoagulant hemostasis proteins, including endogenous thrombin (thrombin-generation) potential (ETP). Thirty-five samples of each type of FFP were analyzed using only male Blood Group O donors. FFP units were stored frozen for comparable periods of time before plasma protein content was assessed. Once the units were thawed, all tests were completed within 4 hours. The results are presented as means ± standard deviations or as median (minimum; maximum) and were compared using independent-sample t tests (significance, p < 0.01). Significantly higher concentrations of adintegrin-like and metalloprotease with thrombospondin type-13 motifs (ADAMTS13), fibrinogen, Factor (F)V, FVIII, FXIII, protein S, protein S activity, antithrombin, microvesicle (<900 nm), and α2 antiplasmin were observed in qFFP. The variability of factors was significantly lower in piFFP. Tissue factor (TF) at 1 picomolar (pM) exhibited significantly longer lag time, a lower peak, lower ETP, and a lower velocity index in qFFP compared with piFFP. In TF at 5 pM, significant differences in lag time (longer in qFFP), velocity index (lower in qFFP), and peak (lower in qFFP) were observed. Rotational thromboelastometry revealed a significantly longer (p = 0.002) clot-formation time with intrinsic thromboelastometry for piFFP and a significantly shorter clotting time (p = 0.004) with thromboelastometry fibrinogen testing for piFFP. Pathogen reduction reduces procoagulant and anticoagulant coagulation factors as well as variability. A thrombin-generation assay showed no reduced ETP and no supraphysiological thrombin generation. None of the

Aptamer-based detection of plasma proteins by an electrochemical assay coupled to magnetic beads.

PubMed

Centi, Sonia; Tombelli, Sara; Minunni, Maria; Mascini, Marco

2007-02-15

The DNA thrombin aptamer has been extensively investigated, and the coupling of this aptamer to different transduction principles has demonstrated the wide applicability of aptamers as bioreceptors in bioanalytical assays. The goal of this work was to design an aptamer-based sandwich assay with electrochemical detection for thrombin analysis in complex matrixes, using a simple target capturing step by aptamer-functionalized magnetic beads. The conditions for the aptamer immobilization and for the protein binding have been first optimized by surface plasmon resonance, and then transferred to the electrochemical-based assay performed onto screen-printed electrodes. The assay was then applied to the analysis of thrombin in buffer, spiked serum, and plasma and high sensitivity and specificity were found. Moreover, thrombin was generated in situ in plasma by the conversion of its precursor prothrombin, and the formation of thrombin was followed at different times. The concentrations detected by the electrochemical assay were in agreement with a simulation software that mimics the formation of thrombin over time (thrombogram). The proposed work demonstrates that the high specificity of aptamers together with the use of magnetic beads are the key features for aptamer-based analysis in complex matrixes, opening the possibility of a real application to diagnostics or medical investigation.

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.

Aptamer-based SERRS Sensor for Thrombin Detection

PubMed Central

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

2012-01-01

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

Centripetal myosin redistribution in thrombin-stimulated platelets. Relationship to platelet Factor 4 secretion.

PubMed

Painter, R G; Ginsberg, M H

1984-11-01

We have examined the F-actin and myosin distribution in resting and thrombin-activated platelets by double label immunofluorescence microscopy. In resting, discoid platelets, F-actin and myosin staining was distributed in a diffuse pattern throughout the interior of the cell with slight accentuation at the cell periphery. In contrast, platelet factor 4 antigen (PF4) was more centrally localized in a fine punctate distribution which is consistent with its localization in alpha-granules. Within 5 sec after thrombin stimulation both F-actin and myosin staining were increased at the periphery of the now spherical platelets. Subsequently, a myosin-containing spherical structure decreased in diameter closely surrounding a phase-dense central zone. In contrast, F-actin staining continued to be accentuated at the cell periphery and was prominent in filopodia and blebs. As previously shown, PF4 staining was localized after 30 sec within large intracellular masses that corresponded to closed vacuolar structures at the ultrastructural level. Morphometric analysis of electron micrographs showed that formation of these vacuolar structures kinetically paralleled alpha-granule disappearance and preceded PF4 release. These PF4-containing structures translocated to the cell periphery after 1-3 min, where they appeared to fuse with the plasma membrane. Ultrastructural analysis of thin sections showed that the myosin-rich spherical structure spatially and temporally correlated with a band of microfilaments that closely surrounded the organelle-rich central zone of the cell. Morphometric analysis of these micrographs showed that the absolute volume of this central zone decreased with time after thrombin addition, showing a significant change after 15 sec and reaching a maximum value after 3-5 min. Changes in the volume of this compartment kinetically preceded PF4 release. On the basis of these data, we propose that an actomyosin contractile force is generated which centripetally

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

Two distinct forms of Factor VIII coagulant protein in human plasma. Cleavage by thrombin, and differences in coagulant activity and association with von Willebrand factor.

PubMed Central

Weinstein, M J; Chute, L E

1984-01-01

We have characterized Factor VIII coagulant protein, present in normal human plasma, that reacts with a specific human 125I-labeled anti-human VIII:C antigen Fab antibody fragment. Two major Factor VIII coagulant antigen populations were present. The first, approximately 85% of the total antigen, was bound to von Willebrand factor and when tested in a standard one-stage assay had Factor VIII coagulant activity. The second antigenic population, eluting near fibrinogen when plasma was gel filtered, was not bound to von Willebrand protein, did not have Factor VIII coagulant activity unless activated, but did block anti-VIII:C Fab neutralization of clotting activity. The two antigenic populations were separable by cryoprecipitation and agarose gel electrophoresis. Although the two antigenic populations differed in their Factor VIII coagulant activity and in their binding to von Willebrand factor, the principal member of both populations is of mol wt 2.4 X 10(5). Both antigens, when proteolyzed by thrombin, were quickly converted to a 1 X 10(5)-mol wt form in association with the appearance of VIII:C activity. The 1 X 10(5)-mol wt antigen was further slowly degraded to an 8 X 10(4)-mol wt form while Factor VIII coagulant activity declined. These results demonstrate the presence of an inactive Factor VIII coagulant protein in plasma, not associated with von Willebrand factor, that can react with thrombin to yield Factor VIII coagulant activity. Images PMID:6421875

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

PubMed

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

2017-05-01

Essentials It is unknown if hemophilia patients with atrial fibrillation need anticoagulation. Endogenous thrombin potentials (ETP) in hemophilia patients and patients on coumarins were compared. Severe hemophilia patients had comparable ETP to therapeutic international normalized ratio (INR). In non-severe hemophilia, 33% had higher ETP than therapeutic INR and may need anticoagulation. Click to hear Dr Negrier's perspective on global assays for assessing coagulation SUMMARY: Background It is unknown whether patients with hemophilia A with atrial fibrillation require treatment with vitamin K antagonists (VKAs) to the same extent as the normal population. Objective To compare hemostatic potential in hemophilia patients and patients on VKAs using thrombin generation (TG). Methods In this cross-sectional study, TG, initiated with 1pM tissue factor, was measured in 133 patients with severe (FVIII < 1%, n = 15) and non-severe (FVIII 1-50%, n = 118) hemophilia A, 97 patients on a VKA with an international normalized ratio (INR) ≥ 1.5 and healthy controls. Endogenous thrombin potential (ETP) (nm*min) was compared according to FVIII level (< 1%, 1-19% and 20-50%) with healthy controls and patients with sub-therapeutic INR (1.5-1.9) and therapeutic INR (≥ 2.0). Medians and interquartile ranges (IQRs) were calculated. Results Compared with healthy controls (898 [IQR 803-1004]), both hemophilia patients and patients on VKAs had lower median ETPs at 304 (196-449) and 176 (100-250), respectively. ETP was quite similar in severe hemophilia patients (185 [116-307]) and patients with a therapeutic INR (156 [90-225]). Compared with patients with therapeutic INR, ETP in patients with FVIII 1-19% and patients with FVIII 20-50% was higher at 296 (203-430) and 397 (219-632), respectively. All patients with therapeutic INR had an ETP < 400. Considering this threshold, 93% of severe hemophilia patients, 70% of patients with FVIII 1-19% and 52% of patients with FVIII 20-50% had an

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

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.

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.

Aptamer Based Microsphere Biosensor for Thrombin Detection

PubMed Central

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

2006-01-01

We have developed an optical microsphere resonator biosensor using aptamer as receptor for the measurement of the important biomolecule thrombin. The sphere surface is modified with anti-thrombin aptamer, which has excellent binding affinity and selectivity for thrombin. Binding of the thrombin at the sphere surface is monitored by the spectral position of the microsphere's whispering gallery mode resonances. A detection limit on the order of 1 NIH Unit/mL is demonstrated. Control experiments with non-aptamer oligonucleotide and BSA are also carried out to confirm the specific binding between aptamer and thrombin. We expect that this demonstration will lead to the development of highly sensitive biomarker sensors based on aptamer with lower cost and higher throughput than current technology.

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

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

Electrode assemblies, plasma generating apparatuses, and methods for generating plasma

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

Kong, Peter C.; Grandy, Jon D.; Detering, Brent A.

Electrode assemblies for plasma reactors include a structure or device for constraining an arc endpoint to a selected area or region on an electrode. In some embodiments, the structure or device may comprise one or more insulating members covering a portion of an electrode. In additional embodiments, the structure or device may provide a magnetic field configured to control a location of an arc endpoint on the electrode. Plasma generating modules, apparatus, and systems include such electrode assemblies. Methods for generating a plasma include covering at least a portion of a surface of an electrode with an electrically insulating membermore » to constrain a location of an arc endpoint on the electrode. Additional methods for generating a plasma include generating a magnetic field to constrain a location of an arc endpoint on an electrode.« less

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

PLASMA GENERATOR

DOEpatents

Foster, J.S. Jr.

1958-03-11

This patent describes apparatus for producing an electricity neutral ionized gas discharge, termed a plasma, substantially free from contamination with neutral gas particles. The plasma generator of the present invention comprises a plasma chamber wherein gas introduced into the chamber is ionized by a radiofrequency source. A magnetic field is used to focus the plasma in line with an exit. This magnetic field cooperates with a differential pressure created across the exit to draw a uniform and uncontaminated plasma from the plasma chamber.

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.

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.

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 ...

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.

Increased thrombin generation in women with polycystic ovary syndrome: A pilot study on the effect of metformin and oral contraceptives.

PubMed

Glintborg, Dorte; Sidelmann, Johannes J; Altinok, Magda Lambaa; Mumm, Hanne; Andersen, Marianne

2015-10-01

Polycystic ovary syndrome (PCOS) is associated with risk factors for cardiovascular disease (CVD) which may be modified by the use of metformin and oral contraceptives (OC). Thrombin generation (TG) measures are risk markers of CVD and address the composite of multiple factors that influence blood coagulation. This prospective, randomized, intervention study evaluated the potential influence of PCOS on TG measures and the effect of OC and/or metformin on TG measures in women with PCOS. Ninety patients with PCOS and 35 controls were included. Patients were randomized to 12 months of treatment with metformin, metformin+OC or OC alone. C-reactive protein (CRP), fibrinogen, total cholesterol, trunk fat mass, body mass index, estradiol, testosterone, sex hormone binding globulin (SHBG) as well as TG measures, i.e. the lag time for formation of thrombin, the endogenous thrombin potential (ETP), peak thrombin concentration (peak) and time to peak were determined at baseline and after 12 months of treatment. CRP and total testosterone were significantly higher and SHBG significantly lower in PCOS women than in controls (P=0.012, P<0.001 and P=0.008, respectively). The TG measures ETP, peak and lag time were increased in women with PCOS compared to controls (P<0.01). Significant correlations were observed between TG measures and fibrinogen, CRP, SHBG and fat trunk mass (P>0.01). ETP (P=0.006), peak (P=0.003) and lag time (P=0.023) remained increased after adjustment for these potential confounders. Treatment with OC and metformin+OC further increased ETP (P<0.001) and peak (P<0.005) and reduced time to peak (P<0.04). The increase in ETP was significantly lower in the metformin+OC group than in the OC group (P<0.05). Metformin alone did not affect TG significantly. PCOS is associated with increase in TG measures independent of other risk factors of CVD. OC increase TG measures further and may thus add to the increased risk of CVD already present in women with PCOS. Copyright

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.

Solid expellant plasma generator

NASA Technical Reports Server (NTRS)

Stone, Nobie H. (Inventor); Poe, Garrett D. (Inventor); Rood, Robert (Inventor)

2010-01-01

An improved solid expellant plasma generator has been developed. The plasma generator includes a support housing, an electrode rod located in the central portion of the housing, and a mass of solid expellant material that surrounds the electrode rod within the support housing. The electrode rod and the solid expellant material are made of separate materials that are selected so that the electrode and the solid expellant material decompose at the same rate when the plasma generator is ignited. This maintains a point of discharge of the plasma at the interface between the electrode and the solid expellant material.

Study of thrombinic activation indexes, in patients with lower limb critic ischaemia, before and after prostaglandin E1 therapy.

PubMed

Trifiletti, A; Bartolone, S; Scamardi, R; Pizzoleo, M A; Attinà, A; Sottilotta, G; Canobbio, V; Soraci, S; Barbera, N

1999-03-01

In this study the action of a prostaglandin, PGE1, was studied in a group of patients with peripheral arterial occlusive disease (PAOD). In 16 patients (14 men and 2 women, aged 47-70 years, mean 57 +/- 7) with PAOD, Fontaine stage IIb and III in critical ischemia, the effects on two indexes of thrombin generation and action of the endovenous administration (2 hours) of 60 micrograms of Alprostadil-PGE1 for four weeks were evaluated. In all artheriopathic patients, before and after pharmacological treatment, the following haemostatic parameters were evaluated: the prothrombin fragment 1 + 2 (F1 + 2) and the fibrinopeptide A(FPA). The patients showed plasma levels of FPA significantly decreased at the end of the treatment. On the other hand, no significant difference in plasma F1 + 2 levels was observed after treatment. These results seem to indicate that plasma F1 + 2 levels are significantly elevated, as a marker of thrombosis status, in patients with PAOD before and after treatment with PGE1.

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.

A host-guest-recognition-based electrochemical aptasensor for thrombin detection.

PubMed

Fan, Hao; Li, Hui; Wang, Qingjiang; He, Pingang; Fang, Yuzhi

2012-05-15

A sensitive electrochemical aptasensor for thrombin detection is presented based on the host-guest recognition technique. In this sensing protocol, a 15 based thrombin aptamer (ab. TBA) was dually labeled with a thiol at its 3' end and a 4-((4-(dimethylamino)phenyl)azo) benzoic acid (dabcyl) at its 5' end, respectively, which was previously immobilized on one Au electrode surface by AuS bond and used as the thrombin probe during the protein sensing procedure. One special electrochemical marker was prepared by modifying CdS nanoparticle with β-cyclodextrins (ab. CdS-CDs), which employed as electrochemical signal provider and would conjunct with the thrombin probe modified electrode through the host-guest recognition of CDs to dabcyl. In the absence of thrombin, the probe adopted linear structure to conjunct with CdS-CDs. In present of thrombin, the TBA bond with thrombin and transformed into its special G-quarter structure, which forced CdS-CDs into the solution. Therefore, the target-TBA binding event can be sensitively transduced via detecting the electrochemical oxidation current signal of Cd of CdS nanoparticles in the solution. Using this method, as low as 4.6 pM thrombin had been detected. Copyright © 2012 Elsevier B.V. All rights reserved.

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

Whole blood clots are more resistant to lysis than plasma clots--greater efficacy of rivaroxaban.

PubMed

Varin, Rémi; Mirshahi, Shahsultan; Mirshahi, Pezhman; Klein, Christophe; Jamshedov, Jovid; Chidiac, Jean; Perzborn, Elisabeth; Mirshahi, Massoud; Soria, Claudine; Soria, Jeannette

2013-03-01

Defective thrombolysis, a thrombotic risk factor, can be attributed to the formation of a compact clot poorly accessible to fibrinolytic enzymes. Venous thrombi, rich in red blood cells (RBCs), and arterial thrombi containing various amounts of RBCS, plasma and whole blood (WB) clot permeability and degradability were compared. The effect of rivaroxaban, a potent direct factor Xa inhibitor, was also evaluated. Fibrin permeability was determined by flow measurement through the clot. Clot degradability was evaluated by the amount of D-dimer generated by clot perfusion with plasminogen and tissue plasminogen activator. Fibrin clot structure was assessed by confocal microscopy. WB clot permeability (KS) and degradability were 6.7- and 38-fold lower, respectively, compared with plasma clots. This is attributed to 1) occlusion of fibrin pores by RBCs and 2) a consistent increase in thrombin generation due to platelets and RBCs inducing formation of a tighter clot. Rivaroxaban added to plasma or WB before clotting, in reducing thrombin generation, led to the formation of a looser clot that is more degradable by fibrinolytic enzymes. Permeability and degradability of whole blood clots formed in the presence of rivaroxaban were very similar to those of plasma clots. The resistance to fibrinolysis of WB clots was reduced considerably when clots were formed with rivaroxaban. These results may have implications for the development of antithrombotic agents. Copyright © 2012 Elsevier Ltd. All rights reserved.

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.

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.

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-stimulated platelet aggregation is inhibited by kallikrein in a time- and concentration-dependent manner.

PubMed

Veloso, D

2003-01-01

Many in vitro studies have shown that activation of prekallikrein (PK) to kallikrein (KAL) in normal plasma triggers rapid activation of the coagulation cascade. In agreement, the coagulation activation is impaired in PK-deficient plasma. Paradoxically, PK-deficient patients show a tendency to thrombosis. To investigate the discrepancy between the in vitro and in vivo findings, we analyzed the effect of KAL on the rate of platelet aggregation. For this research, physiologic concentrations of washed human platelets were incubated for 5 and/or 10 min with approximately 2.2 to 88 nM human plasma KAL (< 1/100 to approximately 1/3 of PK concentrations in plasma) prior to the addition of high concentrations of alpha-thrombin (54 nM) or fibrinogen plus ADP. KAL concentrations were arbitrarily selected on the assumption that concentrations of free KAL (the enzymatically active species) were minute in normal plasma and higher when KAL production was enhanced, and/or inhibitors were depleted. Full platelet aggregation was that seen in the absence of KAL or PK. Inhibition of platelet aggregation stimulated by thrombin was markedly increased with increased KAL concentrations and incubation times. The degree of inhibition by KAL was smaller when ADP was the agonist. The data suggest that KAL may play a role in the modulation of platelet aggregation in vivo under normal conditions as well as when prolonged, high concentrations of KAL occur in blood. The data may also help to explain the intriguing observation that PK-deficient patients show a tendency to thrombotic episodes and myocardial infarction whereas in vitro assays predict bleeding.

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.

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.

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

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.

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

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

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

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

Development of a microplate coagulation assay for Factor V in human plasma

PubMed Central

2011-01-01

Background 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. Methods 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. Results 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. Conclusions 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

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

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.

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

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.

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

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

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

PubMed Central

Tilley, Derek; Levit, Irina; Samis, John A.

2012-01-01

In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase 1, 2. Manual FV assays have been described 3, 4, but they are time consuming and subjective. Automated FV assays have been reported 5-7, but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput 8, 9. Microplate assays have been reported for clot lysis 10, platelet aggregation 11, and coagulation Factors 12, but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma. This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405nm during fibrin formation in human plasma (Figure 1) 13. The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only μl quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity). Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections 14. DIC is associated with a poor prognosis and increases mortality above the pre

Measurement of factor v activity in human plasma using a microplate coagulation assay.

PubMed

Tilley, Derek; Levit, Irina; Samis, John A

2012-09-09

In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase (1, 2). Manual FV assays have been described (3, 4), but they are time consuming and subjective. Automated FV assays have been reported (5-7), but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput (8, 9). Microplate assays have been reported for clot lysis (10), platelet aggregation (11), and coagulation Factors (12), but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma. This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405 nm during fibrin formation in human plasma (Figure 1) (13). The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only μl quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80 pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity). Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections (14). DIC is associated with a poor prognosis and increases mortality

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

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.

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.

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

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.

A chemiluminescence biosensor for the detection of thrombin based on the aptamer composites

NASA Astrophysics Data System (ADS)

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

2018-03-01

An efficient, rapid, simple and ultrasensitive chemiluminescence (CL) approach was proposed for thrombin detection based on the aptamer-thrombin recognition. The aptamer composites were synthesized in this work using graphene oxide (GO) as the backing material. The thrombin was adsorbed on the aptamer composites based on the aptamer-thrombin recognition. Thus, thrombin could be quantified by the difference value of the CL intensity between supernate of the sample and the mixture which composed of thrombin and coexisted substances. The CL intensity exhibits a stable response to thrombin over a concentration range from 2.5 × 10- 10 to 1 × 10- 9 mol·L- 1 with a detection limit as low as 8.3 × 10- 11 mol·L- 1, the relative standard deviation (RSD) was found to be 4.9% for 11 determinations of 1.25 × 10- 9 mol·L- 1 thrombin. Finally, the applicability of the method was verified by applying to serum samples. The recoveries were in the range of 90.3-101.0% with RSD of 2.6-3.2%.

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

Toehold strand displacement-driven assembly of G-quadruplex DNA for enzyme-free and non-label sensitive fluorescent detection of thrombin.

PubMed

Xu, Yunying; Zhou, Wenjiao; Zhou, Ming; Xiang, Yun; Yuan, Ruo; Chai, Yaqin

2015-02-15

Based on a new signal amplification strategy by the toehold strand displacement-driven cyclic assembly of G-quadruplex DNA, the development of an enzyme-free and non-label aptamer sensing approach for sensitive fluorescent detection of thrombin is described. The target thrombin associates with the corresponding aptamer of the partial dsDNA probes and liberates single stranded initiation sequences, which trigger the toehold strand displacement assembly of two G-quadruplex containing hairpin DNAs. This toehold strand displacement reaction leads to the cyclic reuse of the initiation sequences and the production of DNA assemblies with numerous G-quadruplex structures. The fluorescent dye, N-Methyl mesoporphyrin IX, binds to these G-quadruplex structures and generates significantly amplified fluorescent signals to achieve highly sensitive detection of thrombin down to 5 pM. Besides, this method shows high selectivity towards the target thrombin against other control proteins. The developed thrombin sensing method herein avoids the modification of the probes and the involvement of any enzyme or nanomaterial labels for signal amplification. With the successful demonstration for thrombin detection, our approach can be easily adopted to monitor other target molecules in a simple, low-cost, sensitive and selective way by choosing appropriate aptamer/ligand pairs. Copyright © 2014 Elsevier B.V. All rights reserved.

Hollow-Cathode Source Generates Plasma

NASA Technical Reports Server (NTRS)

Deininger, W. D.; Aston, G.; Pless, L. C.

1989-01-01

Device generates argon, krypton, or xenon plasma via thermionic emission and electrical discharge within hollow cathode and ejects plasma into surrounding vacuum. Goes from cold start up to full operation in less than 5 s after initial application of power. Exposed to moist air between operations without significant degradation of starting and running characteristics. Plasma generated by electrical discharge in cathode barrel sustained and aided by thermionic emission from emitter tube. Emitter tube does not depend on rare-earth oxides, making it vulnerable to contamination by exposure to atmosphere. Device modified for use as source of plasma in laboratory experiments or industrial processes.

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.

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.

The effect of platelets on fibrin gel structure formed in the presence of recombinant factor VIIa in hemophilia plasma and in plasma from a patient with Glanzmann thrombasthenia.

PubMed

He, S; Ekman, G Jacobsson; Hedner, U

2005-02-01

Fibrin gel structure has been shown to be dependent on the thrombin concentration as well as the rate of thrombin generation. Accordingly, factor VIII (FVIII)- and FIX-deficient plasma (hemophilia A and B) form loose fibrin clots with high permeability constants. By adding rFVIIa in vitro to FVIII-deficient plasma containing platelets (frozen and thawed), the fibrin gel permeability constant normalized, indicating that extra rFVIIa (1.2 microg mL(-1) or higher) induced a tight fibrin structure. Thrombin generation is highly dependent on the number of platelets, and in this study it was demonstrated that the addition of rFVIIa (5 microg mL(-1)) normalizes the fibrin gel permeability in samples containing platelets (frozen-thawed) in numbers of at least down to 20 x 10(6) mL(-1). The effect of rFVIIa was not observed when unfrozen platelets instead of frozen-thawed platelets were added. Neither was any effect on the fibrin permeability seen, in the presence of annexin V, known to block the effect of phospholipids on the platelet surface. This indicates an important role of platelet phospholipids for the effect of rFVIIa. A similar effect on the fibrin permeability of rFVIIa was observed when added to platelet-rich plasma from a patient with Glanzmann thrombasthenia. Recombinant FVIIa has been found to induce hemostasis in patients with hemophilia and inhibitors against FVIII/FIX as well as in patients with Glanzmann thrombasthenia, indicating the importance of the formation of a tight fibrin gel structure, more resistant against premature proteolysis, for maintaining hemostasis. In conclusion, the addition of rFVIIa (5 microg mL(-1)) also substantially decreased the permeability constant of fibrin gels formed in FVIII-deficient plasma in the presence of low numbers of frozen-thawed platelets (down to 20 x 10(6) mL(-1)). A similar pattern was obtained in plasma from a Glanzmann patient. No effect was found in the presence of unfrozen instead of frozen-thawed platelets

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

High frequency plasma generator for ion thrusters

NASA Technical Reports Server (NTRS)

Goede, H.; Divergilio, W. F.; Fosnight, V. V.; Komatsu, G.

1984-01-01

The results of a program to experimentally develop two new types of plasma generators for 30 cm electrostatic argon ion thrusters are presented. The two plasma generating methods selected for this study were by radio frequency induction (RFI), operating at an input power frequency of 1 MHz, and by electron cyclotron heating (ECH) at an operating frequency of 5.0 GHz. Both of these generators utilize multiline cusp permanent magnet configurations for plasma confinement and beam profile optimization. The program goals were to develop a plasma generator possessing the characteristics of high electrical efficiency (low eV/ion) and simplicity of operation while maintaining the reliability and durability of the conventional hollow cathode plasma sources. The RFI plasma generator has achieved minimum discharge losses of 120 eV/ion while the ECH generator has obtained 145 eV/ion, assuming a 90% ion optical transparency of the electrostatic acceleration system. Details of experimental tests with a variety of magnet configurations are presented.

Absolute hypoxic exercise training enhances in vitro thrombin generation by increasing procoagulant platelet-derived microparticles under high shear stress in sedentary men.

PubMed

Chen, Yu-Wen; Chen, Yi-Ching; Wang, Jong-Shyan

2013-05-01

HS (high shear) stress associated with artery stenosis facilitates TG (thrombin generation) by increasing the release of procoagulant PDMPs (platelet-derived microparticles). Physical exercise and hypoxia may paradoxically modulate vascular thrombotic risks. The aim of the present study was to investigate how exercise training with/without hypoxia affected TG mediated by PDMPs under physio-pathological shear flows. A total of 75 sedentary males were randomly divided into five groups (n=15 in each group): 21% O2 [NC (normoxic control)] or 15% O2 [HC (hypoxic control)] at rest or were trained at 50% of peak work rate under 21% O2 [NT (normoxic training)] or 15% O2 [HAT (hypoxic-absolute training)], or 50% of HR (heart rate) reserve under 15% O2 [HRT (hypoxic-relative training)] for 30 min/day, 5 days/week for 4 weeks. The PDMP characteristics and dynamic TG were measured by flow cytometry and thrombinography respectively. Before the intervention, strenuous exercise markedly increased the PDMP count (14.8%) and TG rate (19.5%) in PDMP-rich plasma at 100 dynes/cm2 of shear stress (P<0.05). After the interventions, both NT and HRT significantly attenuated the enhancement of HS-induced PDMPs (4.7 and 4.9%) and TG rate (3.8 and 3.0%) (P<0.05) by severe exercise. Conversely, HAT notably promoted the PDMP count (37.3%) and TG rate (38.9%) induced by HS (P<0.05), concurrent with increasing plasma TF (tissue factor) and coagulation factor V levels at rest or following exercise. We conclude that both HRT and NT depress similarly HS-mediated TG during exercise, but HAT accelerates the prothrombotic response to vigorous exercise. These findings provide new insights into how exercise training under a hypoxic condition influences the risk of thrombosis associated with stenotic arteries.

Effect of Chronic Blood Transfusion on Biomarkers of Coagulation Activation and Thrombin Generation in Sickle Cell Patients at Risk for Stroke

PubMed Central

Hyacinth, Hyacinth I.; Adams, Robert J.; Greenberg, Charles S.; Voeks, Jenifer H.; Hill, Allyson; Hibbert, Jacqueline M.; Gee, Beatrice E.

2015-01-01

Hypercoagulability in sickle cell disease (SCD) is associated with multiple SCD phenotypes, association with stroke risk has not been well described. We hypothesized that serum levels of biomarkers of coagulation activation correlate with high transcranial Doppler ultrasound velocity and decreases with blood transfusion therapy in SCD patients. Stored serum samples from subjects in the Stroke Prevention in Sickle Cell Anemia (STOP) trial were analyzed using ELISA and protein multiplexing techniques. 40 subjects from each treatment arm (Standard Care [SC] and Transfusion [Tx]) at three time points—baseline, study exit and one year post-trial and 10 each of age matched children with SCD but normal TCD (SNTCD) and with normal hemoglobin (HbAA) were analyzed. At baseline, median vWF, TAT and D-dimer levels were significantly higher among STOP subjects than either HbAA or SNTCD. At study exit, median hemoglobin level was significantly higher while median TCD velocity was significantly lower in Tx compared to SC subjects. Median vWF (409.6 vs. 542.9 μg/ml), TAT (24.8 vs. 40.0 ng/ml) and D-dimer (9.2 vs. 19.1 μg/ml) levels were also significantly lower in the Tx compared to the SC group at study exit. Blood levels of biomarkers coagulation activation/thrombin generation correlated positively with TCD velocity and negatively with number of blood transfusions. Biomarkers of coagulation activation/thrombin generation were significantly elevated in children with SCD, at high risk for stroke. Reduction in levels of these biomarkers correlated with reduction in stroke risk (lower TCD velocity), indicating a possible role for hypercoagulation in SCD associated stroke. PMID:26305570

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.

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.

Generator of chemically active low-temperature plasma

NASA Astrophysics Data System (ADS)

Tyuftyaev, A. S.; Gadzhiev, M. Kh; Sargsyan, M. A.; Demirov, N. A.; Spector, N. O.

2016-11-01

A new generator of high enthalpy (H 0 > 40 kJ/g), chemically active nitrogen and air plasmas was designed and constructed. Main feature of the generator is an expanding channel of an output electrode; the generator belongs to the class of DC plasma torches with thermionic cathode with an efficiency of 80%. The generator ensures the formation of a slightly divergent plasma jet (2α = 12°) with a diameter of D = 10-12 mm, an electric arc maximum power of 20-50 kW, plasma forming gas flow rate 1.0-2.0 g/s, and the average plasma temperature at an outlet of 8000-11000 K.

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

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

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

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.

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 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

Human plasma platelet-derived exosomes: effects of aspirin.

PubMed

Goetzl, Edward J; Goetzl, Laura; Karliner, Joel S; Tang, Norina; Pulliam, Lynn

2016-05-01

Platelet-derived exosomes mediate platelet atherogenic interactions with endothelial cells and monocytes. A new method for isolation of plasma platelet-derived exosomes is described and used to examine effects of aging and aspirin on exosome cargo proteins. Exosome secretion by purified platelets in vitro did not increase after exposure to thrombin or collagen, as assessed by exosome counts and quantification of the CD81 exosome marker. Thrombin and collagen increased exosome content of α-granule chemokines CXCL4 and CXCL7 and cytoplasmic high-mobility group box 1 (HMGB1) protein, but not membrane platelet glycoprotein VI (GPVI), with dependence on extracellular calcium. Aspirin consumption significantly blocked thrombin- and collagen-induced increases in exosome cargo levels of chemokines and HMGB1, without altering total exosome secretion or GPVI cargo. Plasma platelet-derived exosomes, enriched by absorption with mouse antihuman CD42b [platelet glycoprotein Ib (GPIb)] mAb, had sizes and cargo protein contents similar to those of exosomes from purified platelets. The plasma platelet-derived exosome number is lower and its chemokine and HMGB1 levels higher after age 65 yr. Aspirin consumption significantly suppressed cargo protein levels of plasma platelet-derived exosomes without altering total levels of exosomes. Cargo proteins of human plasma platelet-derived exosomes may biomark platelet abnormalities and in vivo effects of drugs.- Goetzl, E. J., Goetzl, L., Karliner, J. S., Tang, N., Pulliam, L. Human plasma platelet-derived exosomes: effects of aspirin. © FASEB.

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

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

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.

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

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.

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

Electrostatic interaction based approach to thrombin detection by surface-enhanced Raman spectroscopy.

PubMed

Hu, Juan; Zheng, Peng-Cheng; Jiang, Jian-Hui; Shen, Guo-Li; Yu, Ru-Qin; Liu, Guo-Kun

2009-01-01

We have developed an electrostatic interaction based biosensor for thrombin detection using surface-enhanced Raman spectroscopy (SERS). This method utilized the electrostatic interaction between capture (thrombin aptamer) and probe (crystal violet, CV) molecules. The specific interaction between thrombin and aptamer could weaken the electrostatic barrier effect from the negative charged aptamer SAMs to the diffusion process of the positively charged CV from the bulk solution to the Au nanoparticle surface. Therefore, the more the bound thrombin, the more the CV molecules near the Au nanoparticle surface and the stronger the observed Raman signal of CV, provided the Raman detections were set at the same time point for each case. This procedure presented a highly specific selectivity and a linear detection of thrombin in the range from 0.1 nM to 10 nM with a detection limit of about 20 pM and realized the thrombin detection in human blood serum solution directly. The electrostatic interaction based technique provides an easy and fast-responding optical platform for a "signal-on" detection of proteins, which might be applicable for the real time assay of proteins.

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.

PLASMA GENERATOR

DOEpatents

Wilcox, J.M.; Baker, W.R.

1963-09-17

This invention is a magnetohydrodynamic device for generating a highly ionized ion-electron plasma at a region remote from electrodes and structural members, thus avoiding contamination of the plasma. The apparatus utilizes a closed, gas-filled, cylindrical housing in which an axially directed magnetic field is provided. At one end of the housing, a short cylindrical electrode is disposed coaxially around a short axial inner electrode. A radial electrical discharge is caused to occur between the inner and outer electrodes, creating a rotating hydromagnetic ionization wave that propagates aiong the magnetic field lines toward the opposite end of the housing. A shorting switch connected between the electrodes prevents the wave from striking the opposite end of the housing. (AEC)

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

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

γT -S195A thrombin reduces the anticoagulant effects of dabigatran in vitro and in vivo.

PubMed

Sheffield, W P; Lambourne, M D; Eltringham-Smith, L J; Bhakta, V; Arnold, D M; Crowther, M A

2014-07-01

Dabigatran etexilate (DE) is an oral direct thrombin inhibitor used to prevent strokes in patients with atrial fibrillation. No licensed DE antidote is currently available. We hypothesized that active site-mutated S195A thrombin (S195A-IIa) and/or its trypsinized derivative (γT -S195A-IIa) would sequester dabigatran, the active form of DE, and reduce its anticoagulant effects. To assess active site-mutated S195A or γT -S195A-IIa as dabigatran reversal agents in vitro and in vivo. Diluted thrombin time (dTT) assays were performed using human or murine plasma containing dabigatran, combined with S195A-IIa, γT -S195A-IIa or FPR-chloromethyl ketone-treated thrombin (FPR-IIa). Bleeding times were determined in anesthetized DE-treated mice also receiving γT -S195A-IIa or vehicle 15 min prior to tail transection. The time to occlusion of carotid arteries of DE-treated mice also receiving S195A-IIa, γT -S195A-IIa, prothrombin complex concentrate (PCC) or vehicle, 15 min prior to topical FeCl3 , was determined using Doppler ultrasound. γT-S195A-IIa reduced dTT values of dabigatran-containing human and murine plasma more effectively than S195-IIa; FPR-IIa had no effect. A dose of 13 mg kg(-1) DE abrogated occlusive thrombus formation in the carotid arteries of FeCl3 -treated mice; γT -S195A-IIa (6 mg kg(-1) ) or PCC (14.3 IU kg(-1) ), but not saline vehicle or S195A-IIa (6 mg kg(-1) ), was equally effective in restoring thrombus formation. Bleeding times of mice treated with 60 mg kg(-1) DE and γT -S195A-IIa (6 mg kg(-1) ) or saline vehicle did not differ. Our data suggest that γT -S195A-IIa decreases the anticoagulant effects of dabigatran in vitro and is partially effective at restoring hemostasis-related thrombus formation in DE-treated mice in vivo. © 2014 International Society on Thrombosis and Haemostasis.

Plasma generating apparatus for large area plasma processing

DOEpatents

Tsai, C.C.; Gorbatkin, S.M.; Berry, L.A.

1991-07-16

A plasma generating apparatus for plasma processing applications is based on a permanent magnet line-cusp plasma confinement chamber coupled to a compact single-coil microwave waveguide launcher. The device creates an electron cyclotron resonance (ECR) plasma in the launcher and a second ECR plasma is created in the line cusps due to a 0.0875 tesla magnetic field in that region. Additional special magnetic field configuring reduces the magnetic field at the substrate to below 0.001 tesla. The resulting plasma source is capable of producing large-area (20-cm diam), highly uniform (.+-.5%) ion beams with current densities above 5 mA/cm[sup 2]. The source has been used to etch photoresist on 5-inch diam silicon wafers with good uniformity. 3 figures.

Plasma generating apparatus for large area plasma processing

DOEpatents

Tsai, Chin-Chi; Gorbatkin, Steven M.; Berry, Lee A.

1991-01-01

A plasma generating apparatus for plasma processing applications is based on a permanent magnet line-cusp plasma confinement chamber coupled to a compact single-coil microwave waveguide launcher. The device creates an electron cyclotron resonance (ECR) plasma in the launcher and a second ECR plasma is created in the line cusps due to a 0.0875 tesla magnetic field in that region. Additional special magnetic field configuring reduces the magnetic field at the substrate to below 0.001 tesla. The resulting plasma source is capable of producing large-area (20-cm diam), highly uniform (.+-.5%) ion beams with current densities above 5 mA/cm.sup.2. The source has been used to etch photoresist on 5-inch diam silicon wafers with good uniformity.

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

Mechanisms generating kappa distributions in plasmas

NASA Astrophysics Data System (ADS)

Livadiotis, Georgios

2017-10-01

Kappa distributions have become increasingly widespread across plasma physics. Publication records reveal an exponential growth of papers relevant to kappa distributions. However, the vast majority of publications refer to statistical fits and applications of these distributions in plasmas. Up to date, there is no systematic analysis on the origin of kappa distributions, that is, the mechanisms that can generate kappa distributions in plasmas. The general scheme that characterizes these mechanisms is composed of two parts: (1) the generation of local correlations among particles, and (2) the thermalization, that is, the stabilization of the particle system into stationary states described by kappa distributions or combinations thereof. Several mechanisms are known in the literature, each characterized by a specific relationship between the plasma properties. These relationships serve as conditions that need to be fulfilled for the corresponding mechanisms to be applied in the plasma. Using these relationships, we identify three mechanisms that generate kappa distributions in the solar wind plasma: (i) Debye shielding, (ii) magnetic field binding, and (iii) thermal fluctuations, each one prevailing in different scales of the solar wind plasma and magnetic field properties. The work was supported in part by the project NNX17AB74G of NASA's HGI Program.

Endogenous Thrombin Potential Changes during the First Cycle of Oral Contraceptive Use

PubMed Central

Westhoff, Carolyn L.; Pike, Malcolm C.; Cremers, Serge; Eisenberger, Andrew; Thomassen, Stella; Rosing, Jan

2017-01-01

Objectives Venous thromboembolism (VTE) risk increases within months of combination oral contraceptive (COC) initiation. Because elevated endogenous thrombin potential (ETP) has been found in several studies to be a VTE risk factor, we evaluated the extent of ETP changes during the initial cycle of an ethinyl estradiol (EE) and levonorgestrel (LNG) COC. We also assessed the relationship between ETP changes and systemic EE and LNG concentrations. Study Design Participants provided multiple blood samples during a first 21-day cycle of a 30 µg EE/150 µg LNG COC and after a further 7 days without an active COC. Thrombin generation measured with and without addition of activated protein C (APC) yielded ETP+APC and ETP−APC and the normalized APC sensitivity ratio (nAPCsr). EE and LNG pharmacokinetic analyses were conducted over 24 hours after the first COC tablet and again at steady state. Results Thrombin generation was determined in 16 of the 17 women who completed the study. Mean ETP−APC increased steadily to 21% above baseline at 24 hours after the 6th COC tablet (COC624; p < 0.001) and to 28% above baseline at steady state (COC21; p < 0.001). Mean ETP+APC increased considerably more – by 54% at COC624 and by 79% at steady state. Mean nAPCsr increased by 28% at COC624 and by 41% at steady state. Higher concentrations of EE or LNG were not correlated with greater increases in ETP. Conclusions ETP increases during the first COC cycle were substantial. Implications The early increases in ETP may provide biological support for the rapid increase in VTE risk during initial COC use. The lack of association between this clotting system perturbation and the systemic EE concentration is surprising and deserves further study. PMID:28088496

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.

An electrochemical label-free and sensitive thrombin aptasensor based on graphene oxide modified pencil graphite electrode.

PubMed

Ahour, F; Ahsani, M K

2016-12-15

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

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.

The effect of corn trypsin inhibitor and inhibiting antibodies for FXIa and FXIIa on coagulation of plasma and whole blood.

PubMed

Hansson, K M; Nielsen, S; Elg, M; Deinum, J

2014-10-01

Corn trypsin inhibitor (CTI), an inhibitor of FXIIa, is used to prevent plasma coagulation by contact activation, to specifically investigate tissue factor (TF)-initiated coagulation. In the present work the specificity of CTI for factor (F) XIIa is questioned. In the commercial available plasma coagulation assays CTI was found to double activated partial thromboplastin time (APTT) at a plasma concentration of 7.3 ± 1.5 μm CTI (assay concentration 2.4 μm). No effect was found on the prothrombin time (PT) when high TF concentrations were used. Also, with specific antibodies for FXIIa and for FXIa only APTT was found to be extended but not PT. With specific enzyme assays using chromogenic substrates CTI was shown to be a strong inhibitor of FXIIa and a competitive inhibitor of FXIa with Ki  = 8.1 ± 0.3 μm, without effect on the coagulation factors FVIIa, FIXa, FXa and thrombin. In thrombin generation and coagulation (free oscillation rheometry, FOR) assays, initiated with low TF concentrations, no effect of CTI (plasma concentrations of 4.4 and 13.6 μm CTI, 25 resp. 100 mg L(-1) in blood) was found with ≥ 1 pm TF. At ≤ 0.1 pm TF in the FOR whole blood assay the coagulation time (CT) concentration dependently increased while the plasma CT became longer than the observation time. To avoid inhibition of FXIa and the thrombin feedback loop we recommend that for coagulation assays the concentration of CTI in blood should be below 20 mg L(-1) (1.6 μm) and in plasma below 3 μm. © 2014 International Society on Thrombosis and Haemostasis.

Plasma Generator Using Spiral Conductors

NASA Technical Reports Server (NTRS)

Szatkowski, George N. (Inventor); Dudley, Kenneth L. (Inventor); Ticatch, Larry A. (Inventor); Smith, Laura J. (Inventor); Koppen, Sandra V. (Inventor); Nguyen, Truong X. (Inventor); Ely, Jay J. (Inventor)

2016-01-01

A plasma generator includes a pair of identical spiraled electrical conductors separated by dielectric material. Both spiraled conductors have inductance and capacitance wherein, in the presence of a time-varying electromagnetic field, the spiraled conductors resonate to generate a harmonic electromagnetic field response. The spiraled conductors lie in parallel planes and partially overlap one another in a direction perpendicular to the parallel planes. The geometric centers of the spiraled conductors define endpoints of a line that is non-perpendicular with respect to the parallel planes. A voltage source coupled across the spiraled conductors applies a voltage sufficient to generate a plasma in at least a portion of the dielectric material.

The immobilization of a direct thrombin inhibitor to a polyurethane as a nonthrombogenic surface coating for extracorporeal circulation.

PubMed

Yu, Jane; Brisbois, Elizabeth; Handa, Hitesh; Annich, Gail; Meyerhoff, Mark; Bartlett, Robert; Major, Terry

2016-04-07

A biomaterial with both antithrombin and antiplatelet properties is the ideal surface for use in extracorporeal circulation (ECC) as it targets both fibrin generation and platelet adhesion. A hemocompatible surface coating avoids the need for systemic anticoagulation by providing a local anticoagulant effect at the polymer-blood interface. Previous work has demonstrated the potential use of argatroban, a direct thrombin inhibitor, as a nonthrombogenic material for extracorporeal devices. The work reported here focuses on the characterization of argatroban linked to a polyurethane-silicone polymer, CarboSil®. Chemical immobilization, the amount of argatroban, incubation times, and saturation point were evaluated to achieve maximal antithrombin activity at the polymer surface. Cross-linked polymer coatings reacted with 10 and 30 µmole of argatroban were prepared and tested. These coatings resulted in argatroban activity levels of 0.131 µM and 0.446 µM, respectively. After refining the cross-linking process, argatroban activity increased by approximately 3.6 fold. Maintenance of activity and leaching from the polymer surface were also evaluated. Using the refined process for linking argatroban to polymer, the resulting polymer was applied as a surface coating to the inner lumen of poly(vinyl chloride) ECC circuit tubing and its antithrombin effect evaluated using a 4 h rabbit ECC model. Following 4 h of blood exposure, the argatroban circuit demonstrated significantly less thrombus formation compared to the control CarboSil® coating with a 4.1 cm 2 thrombus average area for the control coating compared to 1.2 cm 2 for the argatroban coating (n=4). There was no significant change in thrombin time from baseline in plasma from animals in which the argatroban coated circuit was used, with a thrombin time of 16.2 s at t=0 and 14.5 s after 4 h. These results demonstrate the potential efficacy of immobilized argatroban as a hemocompatible biomaterial for extracorporeal

The immobilization of a direct thrombin inhibitor to a polyurethane as a nonthrombogenic surface coating for extracorporeal circulation

PubMed Central

Yu, Jane; Brisbois, Elizabeth; Handa, Hitesh; Annich, Gail; Meyerhoff, Mark; Bartlett, Robert; Major, Terry

2016-01-01

A biomaterial with both antithrombin and antiplatelet properties is the ideal surface for use in extracorporeal circulation (ECC) as it targets both fibrin generation and platelet adhesion. A hemocompatible surface coating avoids the need for systemic anticoagulation by providing a local anticoagulant effect at the polymer-blood interface. Previous work has demonstrated the potential use of argatroban, a direct thrombin inhibitor, as a nonthrombogenic material for extracorporeal devices. The work reported here focuses on the characterization of argatroban linked to a polyurethane-silicone polymer, CarboSil®. Chemical immobilization, the amount of argatroban, incubation times, and saturation point were evaluated to achieve maximal antithrombin activity at the polymer surface. Cross-linked polymer coatings reacted with 10 and 30 µmole of argatroban were prepared and tested. These coatings resulted in argatroban activity levels of 0.131 µM and 0.446 µM, respectively. After refining the cross-linking process, argatroban activity increased by approximately 3.6 fold. Maintenance of activity and leaching from the polymer surface were also evaluated. Using the refined process for linking argatroban to polymer, the resulting polymer was applied as a surface coating to the inner lumen of poly(vinyl chloride) ECC circuit tubing and its antithrombin effect evaluated using a 4 h rabbit ECC model. Following 4 h of blood exposure, the argatroban circuit demonstrated significantly less thrombus formation compared to the control CarboSil® coating with a 4.1 cm2 thrombus average area for the control coating compared to 1.2 cm2 for the argatroban coating (n=4). There was no significant change in thrombin time from baseline in plasma from animals in which the argatroban coated circuit was used, with a thrombin time of 16.2 s at t=0 and 14.5 s after 4 h. These results demonstrate the potential efficacy of immobilized argatroban as a hemocompatible biomaterial for extracorporeal

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

Pulsed metallic-plasma generators.

NASA Technical Reports Server (NTRS)

Gilmour, A. S., Jr.; Lockwood, D. L.

1972-01-01

A pulsed metallic-plasma generator is described which utilizes a vacuum arc as the plasma source. The arc is initiated on the surface of a consumable cathode which can be any electrically conductive material. Ignition is accomplished by using a current pulse to vaporize a portion of a conductive film on the surface of an insulator separating the cathode from the ignition electrode. The film is regenerated during the ensuing arc. Over 100 million ignition cycles have been accomplished by using four 0.125-in. diameter zinc cathodes operating in parallel and high-density aluminum-oxide insulators. Among the applications being investigated for the generator are metal deposition, vacuum pumping, electric propulsion, and high-power dc arc interruption.

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

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.

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.

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.

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

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.

Plasma motor generator system

NASA Technical Reports Server (NTRS)

Hite, Gerald E.

1987-01-01

The significant potential advantages of a plasma motor generator system over conventional systems for the generation of electrical power and propulsion for spacecraft in low Earth orbits warrants its further investigation. The two main components of such a system are a long insulated wire and the plasma generating hollow cathodes needed to maintain electrical contact with the ionosphere. Results of preliminary theoretical and experimental investigations of this system are presented. The theoretical work involved the equilibrium configurations of the wire and the nature of small oscillation about these equilibrium positions. A particularly interesting result was that two different configurations are allowed when the current is above a critical value. Experimental investigations were made of the optimal starting and running conditions for the proposed, low current hollow cathodes. Although optimal ranges of temperature, argon pressure and discharge voltage were identified, start up became progressively more difficult. This supposed depletion or contamination of the emissive surface could be countered by the addition of new emissive material.

D.C. - ARC plasma generator for nonequilibrium plasmachemical processes

NASA Astrophysics Data System (ADS)

Kvaltin, J.

1990-06-01

The analysis of conditions for generation of nonequilibrium plasma to plasmachemical processes is made and the design of d.c.-arc plasma generator on the base of integral criterion is suggested. The measurement of potentials on the plasma column of that generator is presented.

Increased coagulation and fibrinolytic potential of solvent-detergent plasma: a comparative study between Omniplasma and fresh frozen plasma.

PubMed

van Beers, J J B C; van Egmond, L T; Wetzels, R J H; Verhezen, P W M; Beckers, E A M; van Oerle, R; Spronk, H M H; Straat, R J M H E; Henskens, Y M C

2016-07-01

In this study, differences in levels of proteins involved in coagulation and fibrinolysis were compared between fresh frozen (quarantine plasma) and Omniplasma. Furthermore, thawing conditions and plasma stability after thawing were studied. 10 Omniplasma and 10 quarantine plasma units were used to study different procoagulation, anticoagulation and fibrinolytic parameters. Analysis took place at different time-points during plasma storage at 2-6°C. At baseline, significant reduced levels of factor V, free protein S, α2-antiplasmin and tPA-induced ROTEM lysis time were observed in Omniplasma as compared to quarantine plasma. Moreover, thrombin generation, IXa-AT complex levels and factor XIa were significantly increased in Omniplasma. The majority of the parameters studied remained stable in Omniplasma 48 h after thawing, with the exception of factor VIII (decrease) and IXa-AT (increase). Our results suggest an increased coagulation potential, presumingly as a result of contact activation during the production process and also, an increased fibrinolytic potential in Omniplasma. The stability of Omniplasma, based upon the different parameters studied, is comparable to Q-plasma. A maximum post-thawing time of 48 hfor Omniplasma can be suggested. © 2016 International Society of Blood Transfusion.

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

[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.

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

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.

Plasma plume MHD power generator and method

DOEpatents

Hammer, James H.

1993-01-01

Highly-conducting plasma plumes are ejected across the interplanetary magnetic field from a situs that is moving relative to the solar wind, such as a spacecraft or an astral body, such as the moon, having no magnetosphere that excludes the solar wind. Discrete plasma plumes are generated by plasma guns at the situs extending in opposite directions to one another and at an angle, preferably orthogonal, to the magnetic field direction of the solar wind plasma. The opposed plumes are separately electrically connected to their source by a low impedance connection. The relative movement between the plasma plumes and the solar wind plasma creates a voltage drop across the plumes which is tapped by placing the desired electrical load between the electrical connections of the plumes to their sources. A portion of the energy produced may be used in generating the plasma plumes for sustained operation.

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.

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.

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

An amplitude modulated radio frequency plasma generator

NASA Astrophysics Data System (ADS)

Lei, Fan; Li, Xiaoping; Liu, Yanming; Liu, Donglin; Yang, Min; Xie, Kai; Yao, Bo

2017-04-01

A glow discharge plasma generator and diagnostic system has been developed to study the effects of rapidly variable plasmas on electromagnetic wave propagation, mimicking the plasma sheath conditions encountered in space vehicle reentry. The plasma chamber is 400 mm in diameter and 240 mm in length, with a 300-mm-diameter unobstructed clear aperture. Electron densities produced are in the mid 1010 electrons/cm3. An 800 W radio frequency (RF) generator is capacitively coupled through an RF matcher to an internally cooled stainless steel electrode to form the plasma. The RF power is amplitude modulated by a waveform generator that operates at different frequencies. The resulting plasma contains electron density modulations caused by the varying power levels. A 10 GHz microwave horn antenna pair situated on opposite sides of the chamber serves as the source and detector of probe radiation. The microwave power feed to the source horn is split and one portion is sent directly to a high-speed recording oscilloscope. On mixing this with the signal from the pickup horn antenna, the plasma-induced phase shift between the two signals gives the path-integrated electron density with its complete time dependent variation. Care is taken to avoid microwave reflections and extensive shielding is in place to minimize electronic pickup. Data clearly show the low frequency modulation of the electron density as well as higher harmonics and plasma fluctuations.

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

Hollow laser plasma self-confined microjet generation

NASA Astrophysics Data System (ADS)

Sizyuk, Valeryi; Hassanein, Ahmed; CenterMaterials under Extreme Environment Team

2017-10-01

Hollow laser beam produced plasma (LPP) devices are being used for the generation of the self-confined cumulative microjet. Most important place by this LPP device construction is achieving of an annular distribution of the laser beam intensity by spot. An integrated model is being developed to detailed simulation of the plasma generation and evolution inside the laser beam channel. The model describes in two temperature approximation hydrodynamic processes in plasma, laser absorption processes, heat conduction, and radiation energy transport. The total variation diminishing scheme in the Lax-Friedrich formulation for the description of plasma hydrodynamic is used. Laser absorption and radiation transport models on the base of Monte Carlo method are being developed. Heat conduction part on the implicit scheme with sparse matrixes using is realized. The developed models are being integrated into HEIGHTS-LPP computer simulation package. The integrated modeling of the hollow beam laser plasma generation showed the self-confinement and acceleration of the plasma microjet inside the laser channel. It was found dependence of the microjet parameters including radiation emission on the hole and beam radiuses ratio. This work is supported by the National Science Foundation, PIRE project.

Analysis of plasmas generated by fission fragments. [nuclear pumped lasers and helium plasma

NASA Technical Reports Server (NTRS)

Deese, J. E.; Hassan, H. A.

1977-01-01

A kinetic model is developed for a plasma generated by fission fragments and the results are employed to study helium plasma generated in a tube coated with fissionable material. Because both the heavy particles and electrons play important roles in creating the plasma, their effects are considered simultaneously. The calculations are carried out for a range of neutron fluxes and pressures. In general, the predictions of the theory are in good agreement with available intensity measurements. Moreover, the theory predicts the experimentally measured inversions. However, the calculated gain coefficients are such that lasing is not expected to take place in a helium plasma generated by fission fragments. The effects of an externally applied electric field are also considered.

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.

Back-diffusion plasma generator for ionosphere study

NASA Astrophysics Data System (ADS)

Fang, H. K.; Oyama, K.-I.; Chen, A. B.

2017-11-01

To produce ionospheric plasma environments at ground level is essential to get information not only for the development of CubeSat-class spacecraft but also for the design of ionospheric plasma instruments and to confirm their performance. In this paper, we describe the principle of plasma generation and characteristics of the back-diffusion plasma source, which can produce in-lab plasma similar to the Earth’s ionosphere, E and F regions, conditions of electron and ion temperature and density. The ion and electron energy distributions of the plasma generated by a back-diffusion source are measured by means of a cleaned Langmuir probe and gridded particle energy analyzers. The ion motion in front of the source is investigated by a hard-sphere collision model in SIMION software and the simulation results are comparable with the findings of our experiment. Furthermore, plasma densities and ion temperatures at different positions in front of the source are also demonstrated. The back-diffusion source has been accommodated for ionospheric plasma productions in several Asian institutes. The plasma characteristics of the source shown in this paper will benefit space research groups in the development of space plasma instruments.

Functional assembly of intrinsic coagulation proteases on monocytes and platelets. Comparison between cofactor activities induced by thrombin and factor Xa

PubMed Central

1992-01-01

Generation of coagulation factor Xa by the intrinsic pathway protease complex is essential for normal activation of the coagulation cascade in vivo. Monocytes and platelets provide membrane sites for assembly of components of this protease complex, factors IXa and VIII. Under biologically relevant conditions, expression of functional activity by this complex is associated with activation of factor VIII to VIIIa. In the present studies, autocatalytic regulatory pathways operating on monocyte and platelet membranes were investigated by comparing the cofactor function of thrombin-activated factor VIII to that of factor Xa-activated factor VIII. Reciprocal functional titrations with purified human factor VIII and factor IXa were performed at fixed concentrations of human monocytes, CaCl2, factor X, and either factor IXa or factor VIII. Factor VIII was preactivated with either thrombin or factor Xa, and reactions were initiated by addition of factor X. Rates of factor X activation were measured using chromogenic substrate specific for factor Xa. The K1/2 values, i.e., concentration of factor VIIIa at which rates were half maximal, were 0.96 nM with thrombin- activated factor VIII and 1.1 nM with factor Xa-activated factor VIII. These values are close to factor VIII concentration in plasma. The Vsat, i.e., rates at saturating concentrations of factor VIII, were 33.3 and 13.6 nM factor Xa/min, respectively. The K1/2 and Vsat values obtained in titrations with factor IXa were not significantly different from those obtained with factor VIII. In titrations with factor X, the values of Michaelis-Menten coefficients (Km) were 31.7 nM with thrombin- activated factor VIII, and 14.2 nM with factor Xa-activated factor VIII. Maximal rates were 23.4 and 4.9 nM factor Xa/min, respectively. The apparent catalytic efficiency was similar with either form of factor VIIIa. Kinetic profiles obtained with platelets as a source of membrane were comparable to those obtained with monocytes

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.

Plasma cell treatment device Plasma-on-Chip: Monitoring plasma-generated reactive species in microwells

PubMed Central

Oh, Jun-Seok; Kojima, Shinya; Sasaki, Minoru; Hatta, Akimitsu; Kumagai, Shinya

2017-01-01

We have developed a plasma cell treatment device called Plasma-on-Chip that enables the real-time monitoring of a single cell culture during plasma treatment. The device consists of three parts: 1) microwells for cell culture, 2) a microplasma device for generating reactive oxygen and nitrogen species (RONS) for use in cell treatment, and 3) through-holes (microchannels) that connect each microwell with the microplasma region for RONS delivery. Here, we analysed the delivery of the RONS to the liquid culture medium stored in the microwells. We developed a simple experimental set-up using a microdevice and applied in situ ultraviolet absorption spectroscopy with high sensitivity for detecting RONS in liquid. The plasma-generated RONS were delivered into the liquid culture medium via the through-holes fabricated into the microdevice. The RONS concentrations were on the order of 10–100 μM depending on the size of the through-holes. In contrast, we found that the amount of dissolved oxygen was almost constant. To investigate the process of RONS generation, we numerically analysed the gas flow in the through-holes. We suggest that the circulating gas flow in the through-holes promotes the interaction between the plasma (ionised gas) and the liquid, resulting in enhanced RONS concentrations. PMID:28176800

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

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.

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

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

Role of plasma electrons in the generation of a gas discharge plasma

NASA Astrophysics Data System (ADS)

Gruzdev, V. A.; Zalesski, V. G.; Rusetski, I. S.

2012-12-01

The role of different ionization mechanisms in penning-type gas discharges used to generate an emitting plasma in plasma electron sources is considered. It is shown that, under certain conditions, a substantial contribution to the process of gas ionization is provided by plasma electrons.

High Frequency Plasma Generators for Ion Thrusters

NASA Technical Reports Server (NTRS)

Divergilio, W. F.; Goede, H.; Fosnight, V. V.

1981-01-01

The results of a one year program to experimentally adapt two new types of high frequency plasma generators to Argon ion thrusters and to analytically study a third high frequency source concept are presented. Conventional 30 cm two grid ion extraction was utilized or proposed for all three sources. The two plasma generating methods selected for experimental study were a radio frequency induction (RFI) source, operating at about 1 MHz, and an electron cyclotron heated (ECH) plasma source operating at about 5 GHz. Both sources utilize multi-linecusp permanent magnet configurations for plasma confinement. The plasma characteristics, plasma loading of the rf antenna, and the rf frequency dependence of source efficiency and antenna circuit efficiency are described for the RFI Multi-cusp source. In a series of tests of this source at Lewis Research Center, minimum discharge losses of 220+/-10 eV/ion were obtained with propellant utilization of .45 at a beam current of 3 amperes. Possible improvement modifications are discussed.

Atmospheric plasma generation for LCD panel cleaning

NASA Astrophysics Data System (ADS)

Kim, Gyu-Sik; Won, Chung-Yuen; Choi, Ju-Yeop; Yim, C. H.

2007-12-01

UV lamp systems have been used for cleaning of display panels of TFT LCD or Plasma Display Panel (PDP). However, the needs for high efficient cleaning and low cost made high voltage plasma cleaning techniques to be developed and to be improved. Dielectric-barrier discharges (DBDs), also referred to as barrier discharges or silent discharges have for a long time been exclusively related to ozone generation. In this paper, a 6kW high voltage plasma power supply system was developed for LCD cleaning. The -phase input voltage is rectified and then inverter system is used to make a high frequency pulse train, which is rectified after passing through a high-power transformer. Finally, bi-directional high voltage pulse switching circuits are used to generate the high voltage plasma. Some experimental results showed the usefulness of atmospheric plasma for LCD panel cleaning.

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

Cascade generation in Al laser induced plasma

NASA Astrophysics Data System (ADS)

Nagli, Lev; Gaft, Michael; Raichlin, Yosef; Gornushkin, Igor

2018-05-01

We found cascade IR generation in Al laser induced plasma. This generation includes doublet transitions 3s 25s 2S1/2 → 3s24p 2P1/2,3/2 → 3s24s 2S1/2; corresponding to strong lines at 2110 and 2117 nm, and much weaker lines at 1312-1315 nm. The 3s25s2S 1/2 starting IR generation level is directly pumped from the 3s23p 2P3/2 ground level. The starting level for UV generation at 396.2 nm (transitions 3s24s 2S1/2 → 4p 2P3/2) is populated due to the fast collisional processes in the plasma plume. These differences led to different time and special dependences on the lasing in the IR and UV spectral range within the aluminum laser induced plasma.

Slot-Antenna/Permanent-Magnet Device for Generating Plasma

NASA Technical Reports Server (NTRS)

Foster, John E.

2007-01-01

A device that includes a rectangular-waveguide/slot-antenna structure and permanent magnets has been devised as a means of generating a substantially uniform plasma over a relatively large area, using relatively low input power and a low gas flow rate. The device utilizes electron cyclotron resonance (ECR) excited by microwave power to efficiently generate plasma in a manner that is completely electrodeless in the sense that, in principle, there is no electrical contact between the plasma and the antenna. Plasmas generated by devices like this one are suitable for use as sources of ions and/or electrons for diverse material-processing applications (e.g., etching or deposition) and for ion thrusters. The absence of plasma/electrode contact essentially prevents plasma-induced erosion of the antenna, thereby also helping to minimize contamination of the plasma and of objects exposed to the plasma. Consequently, the operational lifetime of the rectangular-waveguide/ slot-antenna structure is long and the lifetime of the plasma source is limited by the lifetime of the associated charged-particle-extraction grid (if used) or the lifetime of the microwave power source. The device includes a series of matched radiating slot pairs that are distributed along the length of a plasma-source discharge chamber (see figure). This arrangement enables the production of plasma in a distributed fashion, thereby giving rise to a uniform plasma profile. A uniform plasma profile is necessary for uniformity in any electron- or ion-extraction electrostatic optics. The slotted configuration of the waveguide/ antenna structure makes the device scalable to larger areas and higher powers. All that is needed for scaling up is the attachment of additional matched radiating slots along the length of the discharge chamber. If it is desired to make the power per slot remain constant in scaling up, then the input microwave power must be increased accordingly. Unlike in prior ECR microwave plasma-generating

A multifunctional label-free electrochemical impedance biosensor for Hg(2+), adenosine triphosphate and thrombin.

PubMed

Chen, Lifen; Chen, Zhong-Ning

2015-01-01

A multifunctional label-free biosensor for the detection of Hg(2+), adenosine triphosphate and thrombin has been developed based on the changing of the electrochemical impedance spectroscopy (EIS) from the modified electrodes when nucleic acid subunits interacting with different targets. The modified electrode consists of three interaction sections, including DNA with T-T mismatch recognizing Hg(2+) to form T-Hg(2+)-T complex, split DNA chip against ATP, and DNA domin against thrombin to form G-quadruplex. Upon DNA interaction with thrombin or ATP, an increased charge transfer resistance (Rct) had been detected. However, a decreased Rct against Hg(2+) was obtained. The Rct difference (ΔRct) has relationship with the concentration of the different targets, Hg(2+), ATP and thrombin can be selectively detected with the detection limit of 0.03, 0.25, and 0.20 nmol L(-1), respectively. To separately detect the three analytes existing in the same sample, ATP aptamer, G-rich DNA strands and EDTA were applied to mask ATP, Hg(2+) or thrombin separately. Copyright © 2014 Elsevier B.V. All rights reserved.

On-site SiH4 generator using hydrogen plasma generated in slit-type narrow gap

NASA Astrophysics Data System (ADS)

Takei, Norihisa; Shinoda, Fumiya; Kakiuchi, Hiroaki; Yasutake, Kiyoshi; Ohmi, Hiromasa

2018-06-01

We have been developing an on-site silane (SiH4) generator based on use of the chemical etching reaction between solid silicon (Si) and the high-density H atoms that are generated in high-pressure H2 plasma. In this study, we have developed a slit-type plasma source for high-efficiency SiH4 generation. High-density H2 plasma was generated in a narrow slit-type discharge gap using a 2.45 GHz microwave power supply. The plasma’s optical emission intensity distribution along the slit was measured and the resulting distribution was reflected by both the electric power distribution and the hydrogen gas flow. Because the Si etching rate strongly affects the SiH4 generation rate, the Si etching behavior was investigated with respect to variations in the experimental parameters. The weight etch rate increased monotonically with increasing input microwave power. However, the weight etch rate decreased with increasing H2 pressure and an increasing plasma gap. This reduction in the etch rate appears to be related to shrinkage of the plasma generation area because increased input power is required to maintain a constant plasma area with increasing H2 pressure and the increasing plasma gap. Additionally, the weight etch rate also increases with increasing H2 flow rate. The SiH4 generation rate of the slit-type plasma source was also evaluated using gas-phase Fourier transform infrared absorption spectroscopy and the material utilization efficiencies of both Si and the H2 gas for SiH4 gas formation were discussed. The main etch product was determined to be SiH4 and the developed plasma source achieved a SiH4 generation rate of 10 sccm (standard cubic centimeters per minute) at an input power of 900 W. In addition, the Si utilization efficiency exceeded 60%.

Electrode assemblies, plasma apparatuses and systems including electrode assemblies, and methods for generating plasma

DOEpatents

Kong, Peter C; Grandy, Jon D; Detering, Brent A; Zuck, Larry D

2013-09-17

Electrode assemblies for plasma reactors include a structure or device for constraining an arc endpoint to a selected area or region on an electrode. In some embodiments, the structure or device may comprise one or more insulating members covering a portion of an electrode. In additional embodiments, the structure or device may provide a magnetic field configured to control a location of an arc endpoint on the electrode. Plasma generating modules, apparatus, and systems include such electrode assemblies. Methods for generating a plasma include covering at least a portion of a surface of an electrode with an electrically insulating member to constrain a location of an arc endpoint on the electrode. Additional methods for generating a plasma include generating a magnetic field to constrain a location of an arc endpoint on an electrode.

Pharmacodynamics of recombinant activated factor VII and plasma-derived factor VII in a cohort of severe FVII deficient patients.

PubMed

van Geffen, Mark; Mathijssen, Natascha C J; Holme, Pål A; Laros-van Gorkom, Britta A P; van Kraaij, Marian G J; Masereeuw, Roselinde; Peyvandi, Flora; van Heerde, Waander L

2013-07-01

Recombinant activated factor VII (rFVIIa) and plasma-derived factor VII (pdFVII) are used to prevent bleedings in severe FVII deficient patients, despite their short half-lifes. It is suggested that FVII levels of 15-20 IU/dL are sufficient to maintain hemostasis. We analyzed the pharmacodynamic effects of FVII substitution therapy in the Nijmegen Hemostasis Assay (NHA) that simultaneously measures thrombin and plasmin generation. Ten severe FVII deficient patients were treated with 20 μg/kg rFVIIa or 25 IU/kg pdFVII in a cross-over design. Thrombin generation lag-time (TG-LT) was identified as an effect-response parameter. Pharmacodynamic analysis using a maximum effect model showed 50% reduction of the TG-LT effect at ~2 IU/dL FVII activity for both rFVIIa and pdFVII. The FVII activity to obtain TG-LT comparable to the upper limit of normal range in healthy controls (4 min) was given by the effective concentration (ECnormal), showing sufficient hemostasis at 3-4 IU/dL FVII activity. No association was seen between FVII activity and other thrombin or plasmin generation parameters as measured by NHA. In conclusion, 3-4 IU/dL FVII activity seems sufficient to maintain hemostasis in patients with severe FVII deficiency during prophylaxis. These data may suggest a potential value for measurement of TG-LT in the monitoring of FVII(a) therapy. Copyright © 2013 Elsevier Ltd. All rights reserved.

Plasma driven neutron/gamma generator

DOEpatents

Leung, Ka-Ngo; Antolak, Arlyn

2015-03-03

An apparatus for the generation of neutron/gamma rays is described including a chamber which defines an ion source, said apparatus including an RF antenna positioned outside of or within the chamber. Positioned within the chamber is a target material. One or more sets of confining magnets are also provided to create a cross B magnetic field directly above the target. To generate neutrons/gamma rays, the appropriate source gas is first introduced into the chamber, the RF antenna energized and a plasma formed. A series of high voltage pulses are then applied to the target. A plasma sheath, which serves as an accelerating gap, is formed upon application of the high voltage pulse to the target. Depending upon the selected combination of source gas and target material, either neutrons or gamma rays are generated, which may be used for cargo inspection, and the like.

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.

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.

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.

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.

Generation conditions of CW Diode Laser Sustained Plasma

NASA Astrophysics Data System (ADS)

Nishimoto, Koji; Matsui, Makoto; Ono, Takahiro

2016-09-01

Laser sustained plasma was generated using 1 kW class continuous wave diode laser. The laser beam was focused on the seed plasma generated by arc discharge in 1 MPa xenon lamp. The diode laser has advantages of high energy conversion efficiency of 80%, ease of maintenance, compact size and availability of conventional quartz based optics. Therefore, it has a prospect of further development compared with conventional CO2 laser. In this study, variation of the plasma shape caused by laser power is observed and also temperature distribution in the direction of plasma radius is measured by optical emission spectroscopy.

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

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.

Impact of Pre-Plasma on Electron Generation and Transport in Laser Plasma Interactions

NASA Astrophysics Data System (ADS)

Peebles, Jonathan Lee

Relativistic laser plasma interactions in conjunction with an underdense pre-plasma have been shown to generate a two temperature component electron spectrum. The lower temperature component described by "ponderomotive scaling'" is relatively well known and understood and is useful for applications such as the fast ignition inertial confinement fusion scheme. The higher energy electrons generated due to pre-plasma are denoted as "super-ponderomotive" electrons and facilitate interesting and useful applications. These include but are not limited to table top particle acceleration and generating high energy protons, x-rays and neutrons from secondary interactions. This dissertation describes experimental and particle-in-cell computational studies of the electron spectra produced from interactions between short pulse high intensity lasers and controlled pre-plasma conditions. Experiments were conducted at 3 laser labs: Texas Petawatt (University of Texas at Austin), Titan (Lawrence Livermore National Laboratory) and OMEGA-EP (University of Rochester). These lasers have different capabilities, and multiple experiments were carried out in order to fully understand super-ponderomotive electron generation and transport in the high intensity laser regime (I > 1018 W/cm2). In these experiments, an additional secondary long pulse beam was used to generate different scale lengths of "injected" pre-plasma while the pulse length and intensity of the short pulse beam were varied. The temperature and quantity of super-ponderomotive electrons were monitored with magnetic spectrometers and inferred via bremsstrahlung spectrometers while trajectory was estimated via Cu-Kalpha imaging. The experimental and simulation data show that super-ponderomotive electrons require pulse lengths of at least 450 fs to be accelerated and that higher intensity interactions generate large magnetic fields which cause severe deflection of the super-ponderomotive electrons. Laser incidence angle is

Enhanced pair plasma generation in the relativistic transparency regime

NASA Astrophysics Data System (ADS)

Liu, W. Y.; Luo, W.; Yuan, T.; Yu, J. Y.; Chen, M.; Sheng, Z. M.

2017-10-01

Electron-positron ( e - e + ) pair plasma generation in the relativistic transparency regime in a thin foil with a fixed thickness irradiated by two counter-propagating laser pulses is investigated through multi-dimensional particle-in-cell simulations. It is shown that target transparency can significantly enhance the pair generation due to the formation of a stable standing wave. An optimum foil density of 200-280 n c (with nc being the critical plasma density of the incident laser at the wavelength of 1 μm) is found for enhanced e - e + pair generation for laser intensity around 10 PW. With such foil density, laser energy transformed to pair plasma formation is approximately four times higher than that with a foil density of 710 nc, while the laser energy transformed to γ-photons remains almost the same. Dense e - e + plasma with a density as high as 10 22 cm - 3 ( ≃ 10 n c ) can be produced accordingly. Comparison of pair plasma generation between cases with fundamental and double frequency driver lasers further demonstrates such an enhancement effect. It shows that when the duration of the laser pulse is relatively short, the double frequency driver can generate more pair plasmas due to the earlier excitation of relativistic transparency.

Ultra-High Intensity Magnetic Field Generation in Dense Plasma

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

Fisch, Nathaniel J.

2014-01-08

The main objective of this grant proposal was to explore the efficient generation of intense currents. Whereas the efficient generation of electric current in low-­energy-­density plasma has occupied the attention of the magnetic fusion community for several decades, scant attention has been paid to carrying over to high-­energy-­density plasma the ideas for steady-­state current drive developed for low-­energy-­density plasma, or, for that matter, to inventing new methodologies for generating electric current in high-­energy-­density plasma. What we proposed to do was to identify new mechanisms to accomplish current generation, and to assess the operation, physics, and engineering basis of new formsmore » of current drive in regimes appropriate for new fusion concepts.« less

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

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.

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.

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.

Does Lipid Profile Affect Thrombin Generation During Ramadan Fasting in Patients With Cardiovascular Risks?

PubMed

Sassi, Mouna; Chakroun, Taher; Chouchène, Saoussen; Hellara, Ilhem; Boubaker, Hamdi; Grissa, Mohamed Habib; Khochtali, Ines; Hassine, Mohsen; Addad, Faouzi; Elalamy, Ismail; Nouira, Semir

2017-11-01

There is evidence that diet and variation in lipid metabolism can influence blood coagulation, but little is known about the effect of Ramadan fasting on plasmatic coagulation pattern. We investigated the effect of Ramadan fasting on thrombin generation (TG) in patients with cardiovascular disease (CVD) risks, and we aimed to assess the effect of lipid profile on TG parameters. The study was conducted in 36 adults having at least 2 CVD risks and in 30 healthy controls. Coagulation pattern was assessed by both classical clotting times and TG test. A complete lipid profile was performed simultaneously. Patients were invited 2 times: 1 week before Ramadan and during the last week of the Ramadan. The TG parameters were not different in patients with CVD risks compared to healthy controls. Fasting had no effect on plasmatic coagulation parameters and on TG profile. Individual analysis of the mean rate index (MRI) of TG revealed 3 groups: group 1 with no modification of MRI, group 2 with a significant increase in MRI (81.64 nM/min vs 136.07 nM/min; P < .001), and group 3 with a significant decrease in MRI (125.27 nM/min vs 73.18 nM/min; P = .001). Only in group 2, a significant increase was observed in total cholesterol and low-density lipoprotein cholesterol. Changes in lipid profile during Ramadan fasting did not influence the global coagulation pattern in patients with CVD risks. Whereas, a significant increase in the propagation phase of TG was associated with a significant increase in cholesterol levels, which was not found with the other TG parameters.

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.

Arterial thrombosis associated with immune thrombocytopenia: presence of a platelet aggregating IgG synergistic with thrombin and adrenalin.

PubMed

Jackson, S P; Jane, S M; Mitchell, C A; Fernando Cortizo, W; Hau, L; Pfueller, S L; Salem, H H

1989-11-24

We report the case of a 50-year-old lady who presented with arterial thrombosis in the setting of thrombocytopenia. Investigations confirmed the diagnosis of idiopathic thrombocytopenic purpura. A spontaneous platelet aggregating factor (SPAF) was isolated from the immunoglobulin fraction of the patient's plasma. The isolated IgG irreversibly aggregated platelet-rich plasma and washed platelets, an effect abolished by pretreating the platelets with aspirin. The activity of the IgG was greatly enhanced by subaggregatory concentrations of thrombin and adrenalin and was localized to the F(ab')2 of the molecule. Plasmapheresis in combination with anti-platelet therapy resulted in an increase in the patient's platelet count, reduced platelet aggregating activity of plasma and significant clinical improvement. We suggest that the presence of this platelet aggregating IgG contributed to the development of thrombosis in our patient and postulate that a similar factor may explain the paradox of thrombosis observed in a select group of thrombocytopenic patients.

Edge plasma boundary layer generated by kink modes in tokamaks

NASA Astrophysics Data System (ADS)

Zakharov, Leonid E.

2011-06-01

This paper describes the structure of the electric current generated by external wall touching and free boundary kink modes at the plasma edge using the ideally conducting plasma model. Both kinds of modes generate δ-functional surface current at the plasma edge. Free boundary kink modes also perturb the core plasma current, which in the plasma edge compensates the difference between the δ-functional surface currents of free boundary and wall touching kink modes. In addition, the resolution of an apparent paradox with the pressure balance across the plasma boundary in the presence of the surface currents is provided.

Conceptual Design of Electron-Beam Generated Plasma Tools

NASA Astrophysics Data System (ADS)

Agarwal, Ankur; Rauf, Shahid; Dorf, Leonid; Collins, Ken; Boris, David; Walton, Scott

2015-09-01

Realization of the next generation of high-density nanostructured devices is predicated on etching features with atomic layer resolution, no damage and high selectivity. High energy electron beams generate plasmas with unique features that make them attractive for applications requiring monolayer precision. In these plasmas, high energy beam electrons ionize the background gas and the resultant daughter electrons cool to low temperatures via collisions with gas molecules and lack of any accelerating fields. For example, an electron temperature of <0.6 eV with densities comparable to conventional plasma sources can be obtained in molecular gases. The chemistry in such plasmas can significantly differ from RF plasmas as the ions/radicals are produced primarily by beam electrons rather than those in the tail of a low energy distribution. In this work, we will discuss the conceptual design of an electron beam based plasma processing system. Plasma properties will be discussed for Ar, Ar/N2, and O2 plasmas using a computational plasma model, and comparisons made to experiments. The fluid plasma model is coupled to a Monte Carlo kinetic model for beam electrons which considers gas phase collisions and the effect of electric and magnetic fields on electron motion. The impact of critical operating parameters such as magnetic field, beam energy, and gas pressure on plasma characteristics in electron-beam plasma processing systems will be discussed. Partially supported by the NRL base program.

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.

Detection of Thrombin Based on Fluorescence Energy Transfer between Semiconducting Polymer Dots and BHQ-Labelled Aptamers.

PubMed

Liu, Yizhang; Jiang, Xuekai; Cao, Wenfeng; Sun, Junyong; Gao, Feng

2018-02-14

Carboxyl-functionalized semiconducting polymer dots (Pdots) were synthesized as an energy donor by the nanoprecipitation method. A black hole quenching dye (BHQ-labelled thrombin aptamers) was used as the energy acceptor, and fluorescence resonance energy transfer between the aptamers and Pdots was used for fluorescence quenching of the Pdots. The addition of thrombin restored the fluorescence intensity. Under the optimized experimental conditions, the fluorescence of the system was restored to the maximum when the concentration of thrombin reached 130 nM, with a linear range of 0-50 nM (R² = 0.990) and a detection limit of 0.33 nM. This sensor was less disturbed by impurities, showing good specificity and signal response to thrombin, with good application in actual samples. The detection of human serum showed good linearity in the range of 0-30 nM (R² = 0.997), with a detection limit of 0.56 nM and a recovery rate of 96.2-104.1%, indicating that this fluorescence sensor can be used for the detection of thrombin content in human serum.

Plasma generators, reactor systems and related methods

DOEpatents

Kong, Peter C [Idaho Falls, ID; Pink, Robert J [Pocatello, ID; Lee, James E [Idaho Falls, ID

2007-06-19

A plasma generator, reactor and associated systems and methods are provided in accordance with the present invention. A plasma reactor may include multiple sections or modules which are removably coupled together to form a chamber. Associated with each section is an electrode set including three electrodes with each electrode being coupled to a single phase of a three-phase alternating current (AC) power supply. The electrodes are disposed about a longitudinal centerline of the chamber and are arranged to provide and extended arc and generate an extended body of plasma. The electrodes are displaceable relative to the longitudinal centerline of the chamber. A control system may be utilized so as to automatically displace the electrodes and define an electrode gap responsive to measure voltage or current levels of the associated power supply.

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

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

Arc plasma generator of atomic driver for steady-state negative ion source.

PubMed

Ivanov, A A; Belchenko, Yu I; Davydenko, V I; Ivanov, I A; Kolmogorov, V V; Listopad, A A; Mishagin, V V; Putvinsky, S V; Shulzhenko, G I; Smirnov, A

2014-02-01

The paper reviews the results of development of steady-state arc-discharge plasma generator with directly heated LaB6 cathode. This arc-discharge plasma generator produces a plasma jet which is to be converted into an atomic one after recombination on a metallic plate. The plate is electrically biased relative to the plasma in order to control the atom energies. Such an intensive jet of hydrogen atoms can be used in negative ion sources for effective production of negative ions on a cesiated surface of plasma grid. All elements of the plasma generator have an augmented water cooling to operate in long pulse mode or in steady state. The thermo-mechanical stresses and deformations of the most critical elements of the plasma generator were determined by simulations. Magnetic field inside the discharge chamber was optimized to reduce the local power loads. The first tests of the steady-state arc plasma generator prototype have performed in long-pulse mode.

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

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.

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

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

Apparatus for generating quasi-free-space microwave-driven plasmas

NASA Astrophysics Data System (ADS)

Hoff, Brad W.; French, David M.; Reid, Remington R.; Lawrance, Julie E.; Lepell, P. David; Maestas, Sabrina S.

2016-03-01

An apparatus for generating quasi-free-space microwave-driven plasmas has been designed, constructed, and tested. The plasma is driven by a multi-kW, ˜5 GHz microwave beam focused at the center of a vacuum chamber using a Koch-type metal plate lens. Sustained plasma discharges have been generated in argon at pressures ranging from 150 to 200 mTorr, at beam power levels ranging from 5 to 10 kW, and at gas flow rates of approximately 200 SCCM.

Apparatus for generating quasi-free-space microwave-driven plasmas

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

Hoff, Brad W.; French, David M.; Reid, Remington R.

An apparatus for generating quasi-free-space microwave-driven plasmas has been designed, constructed, and tested. The plasma is driven by a multi-kW, ∼5 GHz microwave beam focused at the center of a vacuum chamber using a Koch-type metal plate lens. Sustained plasma discharges have been generated in argon at pressures ranging from 150 to 200 mTorr, at beam power levels ranging from 5 to 10 kW, and at gas flow rates of approximately 200 SCCM.

Apparatus for generating quasi-free-space microwave-driven plasmas.

PubMed

Hoff, Brad W; French, David M; Reid, Remington R; Lawrance, Julie E; Lepell, P David; Maestas, Sabrina S

2016-03-01

An apparatus for generating quasi-free-space microwave-driven plasmas has been designed, constructed, and tested. The plasma is driven by a multi-kW, ∼5 GHz microwave beam focused at the center of a vacuum chamber using a Koch-type metal plate lens. Sustained plasma discharges have been generated in argon at pressures ranging from 150 to 200 mTorr, at beam power levels ranging from 5 to 10 kW, and at gas flow rates of approximately 200 SCCM.

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.

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

PubMed Central

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

2012-01-01

Here, we report the application of a label-free electrochemical aptasensor based on a graphite-epoxy composite electrode for the detection of thrombin; in this work, aptamers were immobilized onto the electrodes surface using wet physical adsorption. The detection principle is based on the changes of the interfacial properties of the electrode; these were probed in the presence of the reversible redox couple [Fe(CN)6]3−/[Fe(CN)6]4− using impedance measurements. The electrode surface was partially blocked due to formation of aptamer-thrombin complex, resulting in an increase of the interfacial electron-transfer resistance detected by Electrochemical Impedance Spectroscopy (EIS). The aptasensor showed a linear response for thrombin in the range of 7.5 pM to 75 pM and a detection limit of 4.5 pM. The aptasensor was regenerated by breaking the complex formed between the aptamer and thrombin using 2.0 M NaCl solution at 42 °C, showing its operation for different cycles. The interference response caused by main proteins in serum has been characterized. PMID:22736991

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

Aptamer based label free thrombin assay based on the use of silver nanoparticles incorporated into self-polymerized dopamine.

PubMed

Xu, Qingjun; Wang, Guixiang; Zhang, Mingming; Xu, Guiyun; Lin, Jiehua; Luo, Xiliang

2018-04-13

The authors describe an electrochemical aptasensor for thrombin that is based on the use of a glassy carbon electrode (GCE) modified with polydopamine that is loaded with silver nanoparticles (PDA/AgNPs). The use of AgNPs improves the conductivity of the film and increases the surface area of the GCE. PDA was deposited on the GCE via self-polymerization, and the thrombin binding aptamer was grafted onto the PDA-modified GCE by a single step reaction. Residual electrode surface was blocked with 6-mercapto-1-hexanol. On exposure to thrombin, the electrochemical impedance of the modified electrode increases gradually. Response is linear in the 0.1 pM to 5.0 nM thrombin concentration range, and the limit of detection is as low as 36 fM. The method is selective and capable of detecting thrombin in diluted human serum. In our perception, such a GCE modified with AgNP in a PDA matrix may be applied to many other analytes for which appropriate aptamers are available. Graphical abstract Schematic of an electrochemical aptasensor for sensitive and selective thrombin detection based on the use of a self-polymerized polydopamine film loaded with silver nanoparticles.

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

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.

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.

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

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

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.

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.

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.

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

Laser-pulse shape effects on magnetic field generation in underdense plasmas

NASA Astrophysics Data System (ADS)

Gopal, Krishna; Raja, Md. Ali; Gupta, Devki Nandan; Avinash, K.; Sharma, Suresh C.

2018-07-01

Laser pulse shape effect has been considered to estimate the self-generated magnetic field in laser-plasma interaction. A ponderomotive force based physical mechanism has been proposed to investigate the self-generated magnetic field for different spatial profiles of the laser pulse in inhomogeneous plasmas. The spatially inhomogeneous electric field of a laser pulse imparts a stronger ponderomotive force on plasma electrons. Thus, the stronger ponderomotive force associated with the asymmetric laser pulse generates a stronger magnetic field in comparison to the case of a symmetric laser pulse. Scaling laws for magnetic field strength with the laser and plasma parameters for different shape of the pulse have been suggested. Present study might be helpful to understand the plasma dynamics relevant to the particle trapping and injection in laser-plasma accelerators.

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

Generation of Platelet Microparticles after Cryopreservation of Apheresis Platelet Concentrates Contributes to Hemostatic Activity.

PubMed

Eker, İbrahim; Yılmaz, Soner; Çetinkaya, Rıza Aytaç; Pekel, Aysel; Ünlü, Aytekin; Gürsel, Orhan; Yılmaz, Sebahattin; Avcu, Ferit; Muşabak, Uğur; Pekoğlu, Ahmet; Ertaş, Zerrin; Açıkel, Cengizhan; Zeybek, Nazif; Kürekçi, Ahmet Emin; Avcı, İsmail Yaşar

2017-03-01

In the last decade, substantial evidence has accumulated about the use of cryopreserved platelet concentrates, especially in trauma. However, little reference has been made in these studies to the morphological and functional changes of platelets. Recently platelets have been shown to be activated by cryopreservation processes and to undergo procoagulant membrane changes resulting in the generation of platelet-derived microparticles (PMPs), platelet degranulation, and release of platelet-derived growth factors (PDGFs). We assessed the viabilities and the PMP and PDGF levels of cryopreserved platelets, and their relation with thrombin generation. Apheresis platelet concentrates (APCs) from 20 donors were stored for 1 day and cryopreserved with 6% dimethyl sulfoxide. Cryopreserved APCs were kept at -80 °C for 1 day. Thawed APCs (100 mL) were diluted with 20 mL of autologous plasma and specimens were analyzed for viabilities and PMPs by flow cytometry, for thrombin generation by calibrated automated thrombogram, and for PDGFs by enzyme-linked immunosorbent assay testing. The mean PMP and PDGF levels in freeze-thawed APCs were significantly higher (2763±399.4/µL vs. 319.9±80.5/µL, p<0.001 and 550.9±73.6 pg/mL vs. 96.5±49 pg/mL, p<0.001, respectively), but the viability rates were significantly lower (68.2±13.7% vs. 94±7.5%, p<.001) than those of fresh APCs. The mean endogenous thrombin potential (ETP) of freeze-thawed APCs was significantly higher than that of the fresh APCs (3406.1±430.4 nM.min vs. 2757.6±485.7 nM.min, p<0.001). Moreover, there was a significant positive poor correlation between ETP levels and PMP levels (r=0.192, p=0.014). Our results showed that, after cryopreservation, while levels of PMPs were increasing, significantly higher and earlier thrombin formation was occurring in the samples analyzed despite the significant decrease in viability. Considering the damage caused by the freezing process and the scarcity of evidence for their in

Turn-on fluorescence sensor based on single-walled-carbon-nanohorn-peptide complex for the detection of thrombin.

PubMed

Zhu, Shuyun; Liu, Zhongyuan; Hu, Lianzhe; Yuan, Yali; Xu, Guobao

2012-12-14

Proteases play a central role in several widespread diseases. Thus, there is a great need for the fast and sensitive detection of various proteolytic enzymes. Herein, we have developed a carbon nanotube (CNT)-based protease biosensing platform that uses peptides as a fluorescence probe for the first time. Single-walled carbon nanohorns (SWCNHs) and thrombin were used to demonstrate this detection strategy. SWCNHs can adsorb a fluorescein-based dye (FAM)-labeled peptide (FAM-pep) and quench the fluorescence of FAM. In contrast, thrombin can cleave FAM-pep on SWCNHs and recover the fluorescence of FAM, which allows the sensitive detection of thrombin. This biosensor has a high sensitivity and selectivity toward thrombin, with a detection limit of 100 pM. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Use of Plasma Actuators as a Moving-Wake Generator

NASA Technical Reports Server (NTRS)

Corke, Thomas C.; Thomas, Flint O.; Klapetzky Michael J.

2007-01-01

The work documented in this report tests the concept of using plasma actuators as a simple and easy way to generate a simulated moving-wake and the disturbances associated with it in turbines. This wake is caused by the blades of the upstream stages of the turbine. Two types of devices, one constructed of arrays of NACA 0018 airfoils, and the one constructed of flat plates were studied. The airfoils or plates were equipped with surface mounted dielectric barrier discharge (DBD) plasma actuators, which were used to generate flow disturbances resembling moving-wakes. CTA hot-wire anemometry and flow visualization using a smoke-wire were used to investigate the wake independence at various spacings and downstream locations. The flat plates were found to produce better results than the airfoils in creating large velocity fluctuations in the free-stream flow. Different dielectric materials, plasma actuator locations, leading edge contours, angles of attack and plate spacings were investigated, some with positive results. The magnitudes of the velocity fluctuations were found to be comparable to existing mechanical moving-wake generators, thus proving the feasibility of using plasma actuators as a moving-wake generator.

Studying the Generation Stage of a Plasma Jet in a Plasma Focus Discharge

NASA Astrophysics Data System (ADS)

Polukhin, S. N.; Gurei, A. E.; Nikulin, V. Ya.; Peregudova, E. N.; Silin, P. V.; Kharrasov, A. M.

2017-12-01

A dense compact plasmoid generated at the pinch collapse stage is revealed in a plasma focus discharge by laser optical methods. The initial size of the plasmoid is 1 mm, its electron density is more than 2 × 1019 cm-3, and the plasmoid propagates along the axis from the anode at an average velocity of more than 107 cm/s. A shock wave is generated in the residual argon plasma during the motion of the bunch, its density decreases to 1018 cm-3 at a distance of 3 cm from its place of generation, and the plasmoid expands by 3-5 times and almost merges together with the leading edge of the shock wave.

Surface plasma source with saddle antenna radio frequency plasma generator.

PubMed

Dudnikov, V; Johnson, R P; Murray, S; Pennisi, T; Piller, C; Santana, M; Stockli, M; Welton, R

2012-02-01

A prototype RF H(-) surface plasma source (SPS) with saddle (SA) RF antenna is developed which will provide better power efficiency for high pulsed and average current, higher brightness with longer lifetime and higher reliability. Several versions of new plasma generators with small AlN discharge chambers and different antennas and magnetic field configurations were tested in the plasma source test stand. A prototype SA SPS was installed in the Spallation Neutron Source (SNS) ion source test stand with a larger, normal-sized SNS AlN chamber that achieved unanalyzed peak currents of up to 67 mA with an apparent efficiency up to 1.6 mA∕kW. Control experiments with H(-) beam produced by SNS SPS with internal and external antennas were conducted. A new version of the RF triggering plasma gun has been designed. A saddle antenna SPS with water cooling is fabricated for high duty factor testing.

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

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.

Explosively Generated Plasmas: Measurement and Models of Shock Generation and Material Interactions

NASA Astrophysics Data System (ADS)

Emery, Samuel; Elert, Mark; Giannuzzi, Paul; Le, Ryan; McCarthy, Daniel; Schweigert, Igor

2017-06-01

Explosively generated plasmas (EGPs) are created by the focusing of a shock produced from an explosive driver via a conical waveguide. In the waveguide, the gases from the explosive along with the trapped air are accelerated and compressed (via Mach stemming) to such extent that plasma is produced. These EGPs have been measured in controlled experiments to achieve temperatures on the order of 1 eV and velocities as high as 25 km/s. We have conducted a combined modeling and measurement effort to increase the understanding for design purposes of the shock generation of EGPs and the interaction of EGP with explosive materials. Such efforts have led to improved measures of pressure and temperature, spatial structure of the plasma, and the decomposition/deflagration behavior of RDX upon exposure to an EGP. Funding provided by the Environmental Security Technology Certification Program (ESTCP) Munitions Response program area.

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.

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.

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.

Non-Equilibrium Plasma MHD Electrical Power Generation at Tokyo Tech

NASA Astrophysics Data System (ADS)

Murakami, T.; Okuno, Y.; Yamasaki, H.

2008-02-01

This paper reviews the recent activities on radio-frequency (rf) electromagnetic-field-assisted magnetohydrodynamic (MHD) power generation experiments at the Tokyo Institute of Technology. An inductively coupled rf field (13.56 MHz) is continuously supplied to the disk-shaped Hall-type MHD generator. The first part of this paper describes a method of obtaining increased power output from a pure Argon plasma MHD power generator by incorporating an rf power source to preionize and heat the plasma. The rf heating enhances ionization of the Argon and raises the temperature of the free electron population above the nominally low 4500 K temperatures obtained without rf heating. This in turn enhances the plasma conductivity making MHD power generation feasible. We demonstrate an enhanced power output when rf heating is on approximately 5 times larger than the input power of the rf generator. The second part of this paper is a demonstration of a physical phenomenon of the rf-stabilization of the ionization instability, that had been conjectured for some time, but had not been seen experimentally. The rf heating suppresses the ionization instability in the plasma behavior and homogenizes the nonuniformity of the plasma structures. The power-generating performance is significantly improved with the aid of the rf power under wide seeding conditions. The increment of the enthalpy extraction ratio of around 2% is significantly greater than the fraction of the net rf power, that is, 0.16%, to the thermal input.

Aptamer-based turn-on fluorescent four-branched quaternary ammonium pyrazine probe for selective thrombin detection.

PubMed

Yan, Shengyong; Huang, Rong; Zhou, Yangyang; Zhang, Ming; Deng, Minggang; Wang, Xiaolin; Weng, Xiaocheng; Zhou, Xiang

2011-01-28

In this thrombin detection system, the bright fluorescence of TASPI is almost eliminated by the DNA aptamer TBA (turn-off); however, in the presence of thrombin, it specifically binds to TBA by folding unrestricted TBA into an anti-parallel G-quadruplex structure and then releasing TASPI molecules, resulting in vivid and facile fluorescence recovery (turn-on).

Stability of pro-gastrin-releasing peptide in serum versus plasma.

PubMed

Yoshimura, Toru; Fujita, Kenju; Kawakami, Satoshi; Takeda, Katsumichi; Chan, Sabrina; Beligere, Gangamani; Dowell, Barry

2008-01-01

Although serum assays for pro-gastrin-releasing peptide (ProGRP) assays have been commercially available in Japan for several years, the stability of ProGRP in serum and plasma has not been well documented. We investigated the stability of ProGRP in serum and plasma with fresh and stored (frozen) specimens, as well as the cause of the observed instability in serum. ProGRP concentrations in fresh serum were decreased by 6-28% after room temperature storage for 2 h and by 8-32% after 2-8 degrees C storage for 24 h. The average change in ProGRP concentrations in fresh plasma was within +/-10% of baseline for more than 4 h at room temperature and for more than 24 h at 2-8 degrees C. The incubation of a serine protease, thrombin (activated blood coagulation factor II), in a buffer solution containing ProGRP caused decreases in ProGRP concentrations. Following the addition of phenylmethylsulfonyl fluoride, a serine protease inhibitor, to serum, the serum stability for ProGRP was similar to that in plasma. ProGRP is significantly more stable in plasma than in serum. We speculate that thrombin in serum is one of the factors that inactivate ProGRP in serum by proteolysis of the ProGRP antigen. The use of plasma samples for ProGRP may improve the clinical reliability of this marker by minimizing preanalytical changes in ProGRP concentrations. (c) 2008 S. Karger AG, Basel.

Peculiarities of Efficient Plasma Generation in Air and Water by Short Duration Laser Pulses

NASA Technical Reports Server (NTRS)

Adamovsky, Grigory; Floyd, Bertram M.

2017-01-01

We have conducted experiments to demonstrate an efficient generation of plasma discharges by focused nanosecond pulsed laser beams in air and provided recommendations on the design of optical systems to implement such plasma generation. We have also demonstrated generation of the secondary plasma discharge using the unused energy from the primary one. Focused nanosecond pulsed laser beams have also been utilized to generate plasma in water where we observed self-focusing and filamentation. Furthermore, we applied the laser generated plasma to the decomposition of methylene blue dye diluted in water.

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.

Generation of low-temperature air plasma for food processing

NASA Astrophysics Data System (ADS)

Stepanova, Olga; Demidova, Maria; Astafiev, Alexander; Pinchuk, Mikhail; Balkir, Pinar; Turantas, Fulya

2015-11-01

The project is aimed at developing a physical and technical foundation of generating plasma with low gas temperature at atmospheric pressure for food industry needs. As known, plasma has an antimicrobial effect on the numerous types of microorganisms, including those that cause food spoilage. In this work an original experimental setup has been developed for the treatment of different foods. It is based on initiating corona or dielectric-barrier discharge in a chamber filled with ambient air in combination with a certain helium admixture. The experimental setup provides various conditions of discharge generation (including discharge gap geometry, supply voltage, velocity of gas flow, content of helium admixture in air and working pressure) and allows for the measurement of the electrical discharge parameters. Some recommendations on choosing optimal conditions of discharge generation for experiments on plasma food processing are developed.

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.

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.

Characterization of pulsed atmospheric-pressure plasma streams (PAPS) generated by a plasma gun

NASA Astrophysics Data System (ADS)

Robert, E.; Sarron, V.; Riès, D.; Dozias, S.; Vandamme, M.; Pouvesle, J.-M.

2012-06-01

An experimental study of atmospheric-pressure rare gas plasma propagation in a high-aspect-ratio capillary is reported. The plasma is generated with a plasma gun device based on a dielectric barrier discharge (DBD) reactor powered by either nanosecond or microsecond rise-time high-voltage pulses at single-shot to multi-kHz frequencies. The influence of the voltage waveform, pulse polarity, pulse repetition rate and capillary material have been studied using nanosecond intensified charge-coupled device imaging and plasma-front velocity measurements. The evolution of the plasma appearance during its propagation and the study of the role of the different experimental parameters lead us to suggest a new denomination of pulsed atmospheric-pressure plasma streams to describe all the plasma features, including the previously so-called plasma bullet. The unique properties of such non-thermal plasma launching in capillaries, far from the primary DBD plasma, are associated with a fast ionization wave travelling with velocity in the 107-108 cm s-1 range. Voltage pulse tailoring is shown to allow for a significant improvement of such plasma delivery. Thus, the plasma gun device affords unique opportunities in biomedical endoscopic applications.

Determination of Plasma Screening Effects for Thermonuclear Reactions in Laser-generated Plasmas

NASA Astrophysics Data System (ADS)

Wu, Yuanbin; Pálffy, Adriana

2017-03-01

Due to screening effects, nuclear reactions in astrophysical plasmas may behave differently than in the laboratory. The possibility to determine the magnitude of these screening effects in colliding laser-generated plasmas is investigated theoretically, having as a starting point a proposed experimental setup with two laser beams at the Extreme Light Infrastructure facility. A laser pulse interacting with a solid target produces a plasma through the Target Normal Sheath Acceleration scheme, and this rapidly streaming plasma (ion flow) impacts a secondary plasma created by the interaction of a second laser pulse on a gas jet target. We model this scenario here and calculate the reaction events for the astrophysically relevant reaction 13C(4He, n)16O. We find that it should be experimentally possible to determine the plasma screening enhancement factor for fusion reactions by detecting the difference in reaction events between two scenarios of ion flow interacting with the plasma target and a simple gas target. This provides a way to evaluate nuclear reaction cross-sections in stellar environments and can significantly advance the field of nuclear astrophysics.

Generator of the low-temperature heterogeneous plasma flow

NASA Astrophysics Data System (ADS)

Yusupov, D. I.; Gadzhiev, M. Kh; Tyuftyaev, A. S.; Chinnov, V. F.; Sargsyan, M. A.

2018-01-01

A generator of low-temperature dc plasma with an expanding channel of an output electrode for gas-thermal spraying was designed and constructed. The delivery of the sprayed powder into the cathode and anode arc-binding zones or into the plasma jet below the anode binding was realized. The electrophysical characteristics of both the plasma torch and the heterogeneous plasma flow with Al2O3 powder are studied. It is shown that the current-voltage characteristic (CVC) of a plasma torch depends on the gas flow rate. If the flow rate varies from 1 to 3 g/s, the falling CVC becomes gradually increasing. The speed and temperature of the sprayed powder are determined.

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.

Electron beam generation in the turbulent plasma of Z-pinch discharges

NASA Astrophysics Data System (ADS)

Vikhrev, Victor V.; Baronova, Elena O.

1997-05-01

Numerical modeling of the process of electron beam generation in z-pinch discharges are presented. The proposed model represents the electron beam generation under turbulent plasma conditions. Strong current distribution inhomogeneity in the plasma column has been accounted for the adequate generation process investigation. Electron beam is generated near the maximum of compression due to run away mechanism and it is not related with the current break effect.

Pooled thrombin-activated platelet-rich plasma: a substitute for fetal bovine serum in the engineering of osteogenic/vasculogenic grafts.

PubMed

Tchang, Laurent A; Pippenger, Benjamin E; Todorov, Atanas; Wolf, Francine; Burger, Maximilian G; Jaquiery, Claude; Bieback, Karen; Martin, Ivan; Schaefer, Dirk J; Scherberich, Arnaud

2017-05-01

The use of fetal bovine serum (FBS) as a culture medium supplement in cell therapy and clinical tissue engineering is challenged by immunological concerns and the risk of disease transmission. Here we tested whether human, thrombin-activated, pooled, platelet-rich plasma (tPRP) can be substituted for FBS in the engineering of osteogenic and vasculogenic grafts, using cells from the stromal vascular fraction (SVF) of human adipose tissue. SVF cells were cultured under perfusion flow into porous hydroxyapatite scaffolds for 5 days, with the medium supplemented with either 10% tPRP or 10% FBS and implanted in an ectopic mouse model. Following in vitro culture, as compared to FBS, the use of tPRP did not modify the fraction of clonogenic cells or the different cell phenotypes, but increased by 1.9-fold the total number of cells. After 8 weeks in vivo, bone tissue was formed more reproducibly and in higher amounts (3.7-fold increase) in constructs cultured with tPRP. Staining for human-specific ALU sequences and for the human isoforms of CD31/CD34 revealed the human origin of the bone, the formation of blood vessels by human vascular progenitors and a higher density of human cells in implants cultured with tPRP. In summary, tPRP supports higher efficiency of bone formation by SVF cells than FBS, likely by enhancing cell expansion in vitro while maintaining vasculogenic properties. The use of tPRP may facilitate the clinical translation of osteogenic grafts with intrinsic capacity for vascularization, based on the use of adipose-derived cells. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

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

Generation of filamentary structures by beam-plasma interaction

NASA Astrophysics Data System (ADS)

Wang, X. Y.; Lin, Y.

2006-05-01

The previous simulations by Wang and Lin [Phys. Plasmas. 10, 3528, (2003)] showed that filaments, frequently observed in space plasmas, can form via the interaction between an ion beam and a background plasma. In this study, the physical mechanism for the generation of the filaments is investigated by a two-dimensional hybrid simulation, in which a field-aligned ion beam with relative beam density nb=0.1 and beam velocity Vb=10VA is initiated in a uniform plasma. Right-hand nonresonant ion beam modes, consistent with the linear theory, are found to be dominant in the linear stage of the beam-plasma interaction. In the later nonlinear stage, the nonresonant modes decay and the resonant modes grow through a nonlinear wave coupling. The interaction among the resonant modes leads to the formation of filamentary structures, which are the field-aligned structures (k⊥B) of magnetic field B, density, and temperature in the final stage. The filaments are nonlinearly generated in a prey-predator fashion by the parallel and oblique resonant ion beam modes, which meanwhile evolve into two types of shear Alfvén modes, with one mainly propagating along the background field B0 and the other obliquely propagating. The filamentary structures are found to be phase standing in the plasma frame, but their amplitude oscillates with time. In the dominant filament mode, fluctuations in the background ion density, background ion temperature, and beam density are in phase with the fluctuations in B, whereas the significantly enhanced beam temperature is antiphase with B. It is found that the filaments are produced by the interaction of at least two ion beam modes with comparable amplitudes, not by only one single mode, thus their generation mechanism is different from other mechanisms such as the stimulated excitation by the decay of an Alfvén wave.

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.

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

THE DYNAMICAL GENERATION OF CURRENT SHEETS IN ASTROPHYSICAL PLASMA TURBULENCE

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

Howes, Gregory G.

2016-08-20

Turbulence profoundly affects particle transport and plasma heating in many astrophysical plasma environments, from galaxy clusters to the solar corona and solar wind to Earth's magnetosphere. Both fluid and kinetic simulations of plasma turbulence ubiquitously generate coherent structures, in the form of current sheets, at small scales, and the locations of these current sheets appear to be associated with enhanced rates of dissipation of the turbulent energy. Therefore, illuminating the origin and nature of these current sheets is critical to identifying the dominant physical mechanisms of dissipation, a primary aim at the forefront of plasma turbulence research. Here, we presentmore » evidence from nonlinear gyrokinetic simulations that strong nonlinear interactions between counterpropagating Alfvén waves, or strong Alfvén wave collisions, are a natural mechanism for the generation of current sheets in plasma turbulence. Furthermore, we conceptually explain this current sheet development in terms of the nonlinear dynamics of Alfvén wave collisions, showing that these current sheets arise through constructive interference among the initial Alfvén waves and nonlinearly generated modes. The properties of current sheets generated by strong Alfvén wave collisions are compared to published observations of current sheets in the Earth's magnetosheath and the solar wind, and the nature of these current sheets leads to the expectation that Landau damping of the constituent Alfvén waves plays a dominant role in the damping of turbulently generated current sheets.« less

Waves generated in the plasma plume of helicon magnetic nozzle

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

Singh, Nagendra; Rao, Sathyanarayan; Ranganath, Praveen

2013-03-15

Experimental measurements have shown that the plasma plume created in a helicon plasma device contains a conical structure in the plasma density and a U-shaped double layer (US-DL) tightly confined near the throat where plasma begins to expand from the source. Recently reported two-dimensional particle-in-cell simulations verified these density and US-DL features of the plasma plume. Simulations also showed that the plasma in the plume develops non-thermal feature consisting of radial ion beams with large densities near the conical surface of the density structure. The plasma waves that are generated by the radial ion beams affecting the structure of themore » plasma plume are studied here. We find that most intense waves persist in the high-density regions of the conical density structure, where the transversely accelerated ions in the radial electric fields in the plume are reflected setting up counter-streaming. The waves generated are primarily ion Bernstein modes. The nonlinear evolution of the waves leads to magnetic field-aligned striations in the fields and the plasma near the conical surface of the density structure.« less

Magnetic Field Generation During the Collision of Narrow Plasma Clouds

NASA Astrophysics Data System (ADS)

Sakai, Jun-ichi; Kazimura, Yoshihiro; Haruki, Takayuki

1999-06-01

We investigate the dynamics of the collision of narrow plasma clouds,whose transverse dimension is on the order of the electron skin depth.A 2D3V (two dimensions in space and three dimensions in velocity space)particle-in-cell (PIC) collisionless relativistic code is used toshow the generation of a quasi-staticmagnetic field during the collision of narrow plasma clouds both inelectron-ion and electron-positron (pair) plasmas. The localizedstrong magnetic fluxes result in the generation of the charge separationwith complicated structures, which may be sources of electromagneticas well as Langmuir waves. We also present one applicationof this process, which occurs during coalescence of magnetic islandsin a current sheet of pair plasmas.

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.

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.

Exploration to generate atmospheric pressure glow discharge plasma in air

NASA Astrophysics Data System (ADS)

Wenzheng, LIU; Chuanlong, MA; Shuai, ZHAO; Xiaozhong, CHEN; Tahan, WANG; Luxiang, ZHAO; Zhiyi, LI; Jiangqi, NIU; Liying, ZHU; Maolin, CHAI

2018-03-01

Atmospheric pressure glow discharge (APGD) plasma in air has high application value. In this paper, the methods of generating APGD plasma in air are discussed, and the characteristics of dielectric barrier discharge (DBD) in non-uniform electric field are studied. It makes sure that APGD in air is formed by DBD in alternating current electric field with using the absorbing electron capacity of electret materials to provide initial electrons and to end the discharge progress. Through designing electric field to form two-dimensional space varying electric field and three-dimensional space varying electric field, the development of electron avalanches in air-gap is suppressed effectively and a large space of APGD plasma in air is generated. Further, through combining electrode structures, a large area of APGD plasma in air is generated. On the other hand, by using the method of increasing the density of initial electrons, millimeter-gap glow discharge in atmospheric pressure air is formed, and a maximum gap distance between electrodes is 8 mm. By using the APGD plasma surface treatment device composed of contact electrodes, the surface modification of high polymer materials such as aramid fiber and polyester are studied and good effect of modifications is obtained. The present paper provides references for the researchers of industrial applications of plasma.

S-Duct Engine Inlet Flow Control Using SDBD Plasma Streamwise Vortex Generators

NASA Astrophysics Data System (ADS)

Kelley, Christopher; He, Chuan; Corke, Thomas

2009-11-01

The results of a numerical simulation and experiment characterizing the performance of plasma streamwise vortex generators in controlling separation and secondary flow within a serpentine, diffusing duct are presented. A no flow control case is first run to check agreement of location of separation, development of secondary flow, and total pressure recovery between the experiment and numerical results. Upon validation, passive vane-type vortex generators and plasma streamwise vortex generators are implemented to increase total pressure recovery and reduce flow distortion at the aerodynamic interface plane: the exit of the S-duct. Total pressure recovery is found experimentally with a pitot probe rake assembly at the aerodynamic interface plane. Stagnation pressure distortion descriptors are also presented to show the performance increase with plasma streamwise vortex generators in comparison to the baseline no flow control case. These performance parameters show that streamwise plasma vortex generators are an effective alternative to vane-type vortex generators in total pressure recovery and total pressure distortion reduction in S-duct inlets.

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

A study of single and binary ion plasma expansion into laboratory-generated plasma wakes

NASA Technical Reports Server (NTRS)

Wright, Kenneth Herbert, Jr.

1988-01-01

Plasma expansion into the wake of a large rectangular plate immersed in a collisionless, supersonic plasma was investigated in laboratory experiments. The experimental conditions address both single ion and binary ion plasma flows for the case of a body whose size is large in comparison with the Debye length, when the potential difference between the body and the plasma is relatively small. A new plasma source was developed to generate equi-velocity, binary ion plasma flows, which allows access to new parameter space that have previously been unavailable for laboratory studies. Specifically, the new parameters are the ionic mass ratio and the ionic component density ratio. In a series of experiments, a krypton-neon plasma is employed where the ambient density ratio of neon to krypton is varied more than an order of magnitude. The expansion in both the single ion and binary ion plasma cases is limited to early times, i.e., a few ion plasma periods, by the combination of plasma density, plasma drift speed, and vacuum chamber size, which prevented detailed comparison with self-similar theory.

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

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

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.

About plasma points' generation in Z-pinch

NASA Astrophysics Data System (ADS)

Afonin, V. I.; Potapov, A. V.; Lazarchuk, V. P.; Murugov, V. M.; Senik, A. V.

1997-05-01

The streak tube study results (at visible and x-ray ranges) of dynamics of fast Z-pinch formed at explosion of metal wire in diode of high current generator are presented. Amplitude of current in the load reached ˜180 kA at increase time ˜50 ns. The results' analysis points to capability of controlling hot plasma points generation process in Z-pinch.

Dual-colored graphene quantum dots-labeled nanoprobes/graphene oxide: functional carbon materials for respective and simultaneous detection of DNA and thrombin

NASA Astrophysics Data System (ADS)

Qian, Zhao Sheng; Shan, Xiao Yue; Chai, Lu Jing; Chen, Jian Rong; Feng, Hui

2014-10-01

Convenient and simultaneous detection of multiple biomarkers such as DNA and proteins with biocompatible materials and good analytical performance still remains a challenge. Herein, we report the respective and simultaneous detection of DNA and bovine α-thrombin (thrombin) entirely based on biocompatible carbon materials through a specially designed fluorescence on-off-on process. Colorful fluorescence, high emission efficiency, good photostability and excellent compatibility enables graphene quantum dots (GQDs) as the best choice for fluorophores in bioprobes, and thus two-colored GQDs as labeling fluorophores were chemically bonded with specific oligonucleotide sequence and aptamer to prepare two probes targeting the DNA and thrombin, respectively. Each probe can be assembled on the graphene oxide (GO) platform spontaneously by π-π stacking and electrostatic attraction; as a result, fast electron transfer in the assembly efficiently quenches the fluorescence of probe. The presence of DNA or thrombin can trigger the self-recognition between capturing a nucleotide sequence and its target DNA or between thrombin and its aptamer due to their specific hybridization and duplex DNA structures or the formation of apatamer-substrate complex, which is taken advantage of in order to achieve a separate quantitative analysis of DNA and thrombin. A dual-functional biosensor for simultaneous detection of DNA and thrombin was also constructed by self-assembly of two probes with distinct colors and GO platform, and was further evaluated with the presence of various concentrations of DNA and thrombin. Both biosensors serving as a general detection model for multiple species exhibit outstanding analytical performance, and are expected to be applied in vivo because of the excellent biocompatibility of their used materials.

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.

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.

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

Chemically reactive species in liquids generated by atmospheric-pressure plasmas and their roles in plasma medicine

NASA Astrophysics Data System (ADS)

Hamaguchi, Satoshi

2013-07-01

Plasmas whose gas temperatures are close to room temperature may be generated in ambient air or a gas at atmospheric pressure with the use of low-frequency high voltage or low-power radio-frequency (RF) or microwave power applied to electrodes. Such plasmas can serve as a powerful source of free radicals and/or chemically reactive species that arise from atoms and molecules of the ambient gas. Recently use of such plasmas for medical purposes has attracted much attention as they can be implemented in possible medical devices that can cause blood coagulation, heal wounds, facilitate angiogenesis, sterilize surgical devices as well as living tissues without harming healthy cells, and selectively inactivate cancer cells. Especially of interest among reactive species generated by atmospheric-pressure plasmas (APP) are reactive oxygen species (ROS) and reactive nitrogen species (RNS) that are generated in liquid phase. Since most living tissues and cells are immersed in liquids (such as blood or culture media), reactive species generated by APPs in the gas phase are transported to the liquid phase and possibly converted to different types of reactive species therein before causing some influence on the tissues or cells. In this study, the rate equations are solved to evaluate concentrations of various reactive species in pure water that are originated by plasma reactions in atmosphere and possible effects of such species (including ROS/RNS) on living tissues and cells are discussed.

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.

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.

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

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.

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.

Defect generation in electronic devices under plasma exposure: Plasma-induced damage

NASA Astrophysics Data System (ADS)

Eriguchi, Koji

2017-06-01

The increasing demand for higher performance of ULSI circuits requires aggressive shrinkage of device feature sizes in accordance with Moore’s law. Plasma processing plays an important role in achieving fine patterns with anisotropic features in metal-oxide-semiconductor field-effect transistors (MOSFETs). This article comprehensively addresses the negative aspect of plasma processing — plasma-induced damage (PID). PID naturally not only modifies the surface morphology of materials but also degrades the performance and reliability of MOSFETs as a result of defect generation in the materials. Three key mechanisms of PID, i.e., physical, electrical, and photon-irradiation interactions, are overviewed in terms of modeling, characterization techniques, and experimental evidence reported so far. In addition, some of the emerging topics — control of parameter variability in ULSI circuits caused by PID and recovery of PID — are discussed as future perspectives.

Study of Plasma Motor Generator (PMG) tether system for orbit reboost

NASA Technical Reports Server (NTRS)

1987-01-01

Detailed designs were produced for a 2 kW plasma motor generator tether system based largely on existing hardware and hardware designs. Specifically, the hollow cathode design and electronics are derived from ion propulsion equipment. A prototype tether was constructed and will be tested for deployment, strength, resistance to breakage and abrasion and electrical properties. In addition, laboratory development models of the electronics will be used to operate two plasma motor generator hollow cathode assemblies with this tether to verify electrical performance parameters for the complete system. Results show that a low cost demonstration of a plasma motor generator tether system appears to be feasible by the middle of the 1990s.

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.

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.

Shock Generation and Control Using DBD Plasma Actuators

NASA Technical Reports Server (NTRS)

Patel, Mehul P.; Cain, Alan B.; Nelson, Christopher C.; Corke, Thomas C.; Matlis, Eric H.

2012-01-01

This report is the final report of a NASA Phase I SBIR contract, with some revisions to remove company proprietary data. The Shock Boundary Layer Interaction (SBLI) phenomena in a supersonic inlet involve mutual interaction of oblique shocks with boundary layers, forcing the boundary layer to separate from the inlet wall. To improve the inlet efficiency, it is desired to prevent or delay shock-induced boundary layer separation. In this effort, Innovative Technology Applications Company (ITAC), LLC and the University of Notre Dame (UND) jointly investigated the use of dielectric-barrier-discharge (DBD) plasma actuators for control of SBLI in a supersonic inlet. The research investigated the potential for DBD plasma actuators to suppress flow separation caused by a shock in a turbulent boundary layer. The research involved both numerical and experimental investigations of plasma flow control for a few different SBLI configurations: (a) a 12 wedge flow test case at Mach 1.5 (numerical and experimental), (b) an impinging shock test case at Mach 1.5 using an airfoil as a shock generator (numerical and experimental), and (c) a Mach 2.0 nozzle flow case in a simulated 15 X 15 cm wind tunnel with a shock generator (numerical). Numerical studies were performed for all three test cases to examine the feasibility of plasma flow control concepts. These results were used to guide the wind tunnel experiments conducted on the Mach 1.5 12 degree wedge flow (case a) and the Mach 1.5 impinging shock test case (case b) which were at similar flow conditions as the corresponding numerical studies to obtain experimental evidence of plasma control effects for SBLI control. The experiments also generated data that were used in validating the numerical studies for the baseline cases (without plasma actuators). The experiments were conducted in a Mach 1.5 test section in the University of Notre Dame Hessert Laboratory. The simulation results from cases a and b indicated that multiple

A research program in magnetogasdynamics utilizing hypervelocity coaxial plasma generators

NASA Technical Reports Server (NTRS)

Spight, C.

1976-01-01

A broadly-gauged research program in magnetogasdynamics utilizing hypervelocity coaxial plasma generators is presented. A complete hypervelocity coaxial plasma generator facility was assembled and tested. Significant progress was made in the direction of understanding the important processes in the interaction of hypervelocity MGD flow with transverse applied fields. It is now proposed to utilize the accumulated experimental capability and theoretical analysis in application to the analysis and design parameterization of pulsed magnetogasdynamic direct energy convertor configurations.

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

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.

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.

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

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.

Reactive species in non-equilibrium atmospheric-pressure plasmas: Generation, transport, and biological effects

NASA Astrophysics Data System (ADS)

Lu, X.; Naidis, G. V.; Laroussi, M.; Reuter, S.; Graves, D. B.; Ostrikov, K.

2016-05-01

Non-equilibrium atmospheric-pressure plasmas have recently become a topical area of research owing to their diverse applications in health care and medicine, environmental remediation and pollution control, materials processing, electrochemistry, nanotechnology and other fields. This review focuses on the reactive electrons and ionic, atomic, molecular, and radical species that are produced in these plasmas and then transported from the point of generation to the point of interaction with the material, medium, living cells or tissues being processed. The most important mechanisms of generation and transport of the key species in the plasmas of atmospheric-pressure plasma jets and other non-equilibrium atmospheric-pressure plasmas are introduced and examined from the viewpoint of their applications in plasma hygiene and medicine and other relevant fields. Sophisticated high-precision, time-resolved plasma diagnostics approaches and techniques are presented and their applications to monitor the reactive species and plasma dynamics in the plasma jets and other discharges, both in the gas phase and during the plasma interaction with liquid media, are critically reviewed. The large amount of experimental data is supported by the theoretical models of reactive species generation and transport in the plasmas, surrounding gaseous environments, and plasma interaction with liquid media. These models are presented and their limitations are discussed. Special attention is paid to biological effects of the plasma-generated reactive oxygen and nitrogen (and some other) species in basic biological processes such as cell metabolism, proliferation, survival, etc. as well as plasma applications in bacterial inactivation, wound healing, cancer treatment and some others. Challenges and opportunities for theoretical and experimental research are discussed and the authors' vision for the emerging convergence trends across several disciplines and application domains is presented to

Material for electrodes of low temperature plasma generators

DOEpatents

Caplan, Malcolm; Vinogradov, Sergel Evge'evich; Ribin, Valeri Vasil'evich; Shekalov, Valentin Ivanovich; Rutberg, Philip Grigor'evich; Safronov, Alexi Anatol'evich

2008-12-09

Material for electrodes of low temperature plasma generators. The material contains a porous metal matrix impregnated with a material emitting electrons. The material uses a mixture of copper and iron powders as a porous metal matrix and a Group IIIB metal component such as Y.sub.2O.sub.3 is used as a material emitting electrons at, for example, the proportion of the components, mass %: iron: 3-30; Y.sub.2O.sub.3:0.05-1; copper: the remainder. Copper provides a high level of heat conduction and electric conductance, iron decreases intensity of copper evaporation in the process of plasma creation providing increased strength and lifetime, Y.sub.2O.sub.3 provides decreasing of electronic work function and stability of arc burning. The material can be used for producing the electrodes of low temperature AC plasma generators used for destruction of liquid organic wastes, medical wastes, and municipal wastes as well as for decontamination of low level radioactive waste, the destruction of chemical weapons, warfare toxic agents, etc.

Material for electrodes of low temperature plasma generators

DOEpatents

Caplan, Malcolm; Vinogradov, Sergel Evge'evich; Ribin, Valeri Vasil'evich; Shekalov, Valentin Ivanovich; Rutberg, Philip Grigor'evich; Safronov, Alexi Anatol'evich; Shiryaev, Vasili Nikolaevich

2010-03-02

Material for electrodes of low temperature plasma generators. The material contains a porous metal matrix impregnated with a material emitting electrons. The material uses a mixture of copper and iron powders as a porous metal matrix and a Group IIIB metal component such as Y.sub.2O.sub.3 is used as a material emitting electrons at, for example, the proportion of the components, mass %: iron:3-30; Y.sub.2O.sub.3:0.05-1; copper: the remainder. Copper provides a high level of heat conduction and electric conductance, iron decreases intensity of copper evaporation in the process of plasma creation providing increased strength and lifetime, Y.sub.2O.sub.3 provides decreasing of electronic work function and stability of arc burning. The material can be used for producing the electrodes of low temperature AC plasma generators used for destruction of liquid organic wastes, medical wastes, municipal wastes as well as for decontamination of low level radioactive waste, the destruction of chemical weapons, warfare toxic agents, etc.

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.

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.

Current Interruption and Particle Beam Generation by a Plasma Focus.

DTIC Science & Technology

1982-11-30

Through collaboration with Dr. K. H. Schoenbach of Texas Tech University the plasma focus opening switch (PFOS) was revised to answer basic questions...results are consistent with the snowplow model. The final analysis of the plasma focus particle beam generation experiments was completed and a...strong correlation was found for the beam-target model as the mechanism for neutron production in the Illinois plasma focus device. (Author)

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

PubMed

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

2016-11-15

A sensitive aptasensor based on a robust nanocomposite of titanium dioxide nanoparticles, multiwalled carbon nanotubes (MWCNT), chitosan and a novel synthesized Schiff base (SB) (TiO2/MWCNT/CHIT/SB) on the surface of a glassy carbon electrode (GCE) was developed for thrombin detection. The resultant nanocomposite can provide a large surface area, excellent electrocatalytic activity, and high stability, which would improve immobilization sites for biological molecules, allow remarkable amplification of the electrochemical signal and contribute to improved sensitivity. Thrombin aptamers were simply immobilized onto the TiO2-MWCNT/CHIT-SB nanocomposite matrix through simple π - π stacking and electrostatic interactions between CHIT/SB and aptamer strands. The electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to analyze the surface characterization of unmodified GCE and TiO2-MWCNT/CHIT-SB modified GCE, and also the interaction between aptamer and thrombin. In the presence of thrombin, the aptamer on the adsorbent layer captures the target on the electrode interface, which makes a barrier for electrons and inhibits electron transfer, thereby resulting in decreased DPV and increased impedance signals of the TiO2-MWCNT/CHIT-SB modified GCE. Furthermore, the proposed aptasensor has a very low LOD of 1.0fmolL(-1) thrombin within the detection range of 0.00005-10nmolL(-1). The aptasensor also presents high specificity and reproducibility for thrombin, which is unaffected by the coexistence of other proteins. Clinical application was performed with analysis of the thrombin levels in blood and CSF samples obtained from patients with MS, Parkinson, Epilepsy and Polyneuropathy using both the aptasensor and commercial ELISA kit. The results revealed the proposed system to be a promising candidate for clinical analysis of thrombin. Copyright © 2016 Elsevier B.V. All rights reserved.

Nonlinear currents generated in plasma by a radiation pulse with a frequency exceeding the electron plasma frequency

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

Grishkov, V. E.; Uryupin, S. A., E-mail: uryupin@sci.lebedev.ru

2016-09-15

It is shown that the nonlinear currents generated in plasma by a radiation pulse with a frequency exceeding the electron plasma frequency change substantially due to a reduction in the effective electron–ion collision frequency.

Preliminary characterization of a laser-generated plasma sheet

DOE PAGES

Keiter, P. A.; Malamud, G.; Trantham, M.; ...

2014-12-10

We present the results from recent experiments to create a flowing plasma sheet. Two groups of three laser beams with nominally 1.5 kJ of energy per group were focused to separate pointing locations, driving a shock into a wedge target. As the shock breaks out of the wedge, the plasma is focused on center, creating a sheet of plasma. Measurements at 60 ns indicate the plasma sheet has propagated 2825 microns with an average velocity of 49 microns/ns. These experiments follow previous experiments, which are aimed at studying similar physics as that found in the hot spot region of cataclysmicmore » variables. Krauland et al created a flowing plasma, which represents the flowing plasma from the secondary star. This flow interacted with a stationary object, which represented the disk around the white dwarf. A reverse shock is a shock formed when a freely expanding plasma encounters an obstacle. Reverse shocks can be generated by a blast wave propagating through a medium. As a result, they can also be found in binary star systems where the flowing gas from a companion star interacts with the accretion disk of the primary star.« less

Generation of nano roughness on fibrous materials by atmospheric plasma

NASA Astrophysics Data System (ADS)

Kulyk, I.; Scapinello, M.; Stefan, M.

2012-12-01

Atmospheric plasma technology finds novel applications in textile industry. It eliminates the usage of water and of hazard liquid chemicals, making production much more eco-friendly and economically convenient. Due to chemical effects of atmospheric plasma, it permits to optimize dyeing and laminating affinity of fabrics, as well as anti-microbial treatments. Other important applications such as increase of mechanical resistance of fiber sleeves and of yarns, anti-pilling properties of fabrics and anti-shrinking property of wool fabrics were studied in this work. These results could be attributed to the generation of nano roughness on fibers surface by atmospheric plasma. Nano roughness generation is extensively studied at different conditions. Alternative explanations for the important practical results on textile materials and discussed.

Coagulation indices in very preterm infants from cord blood and postnatal samples.

PubMed

Neary, E; McCallion, N; Kevane, B; Cotter, M; Egan, K; Regan, I; Kirkham, C; Mooney, C; Coulter-Smith, S; Ní Áinle, F

2015-11-01

Very premature infants are at high risk of bleeding complications; however, few data exist on ranges for standard coagulation tests. The primary objective of this study was to measure standard plasma coagulation tests and thrombin generation in very premature infants compared with term infants. The secondary objective was to evaluate whether an association existed between coagulation indices and intraventricular hemorrhage (IVH). Cord and peripheral blood of neonates < 30 weeks gestational age (GA) was drawn at birth, on days 1 and 3 and fortnightly until 30 weeks corrected gestational age. Prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen and coagulation factor levels were measured and tissue factor-stimulated thrombin generation was characterized. Control plasma was obtained from cord blood of term neonates. One hundred and sixteen infants were recruited. Median (range) GA was 27.7 (23.7-29.9) weeks and mean (SD) birth weight was 1020 (255) g. Median (5th-95th percentile) day 1 PT, APTT and fibrinogen were 17.5 (12.7-26.6) s, 78.7 (48.7-134.3) s and 1.4 (0.72-3.8) g L(-1) , respectively. No difference in endogenous thrombin potential between preterm and term plasma was observed, where samples were available. Levels of coagulation factors II, VII, IX and X, protein C, protein S and antithrombin were reduced in preterm compared with term plasma. Day 1 APTT and PT were not associated with IVH. In the largest cross-sectional study to date of very preterm infants, typical ranges for standard coagulation tests were determined. Despite long clotting times, thrombin generation was observed to be similar in very preterm and term infants. © 2015 International Society on Thrombosis and Haemostasis.

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.

Pulsed Energy Systems for Generating Plasmas

NASA Technical Reports Server (NTRS)

Rose, M. Franklin; Shotts, Z.

2005-01-01

This paper will describe the techniques needed to electrically generate highly ionized dense plasmas for a variety of applications. The components needed in pulsed circuits are described in terms of general performance parameters currently available from commercial vendors. Examples of pulsed systems using these components are described and technical data from laboratory experiments presented. Experimental data are given for point designs, capable of multi-megawatt power levels.

Control of ultra-intense single attosecond pulse generation in laser-driven overdense plasmas.

PubMed

Liu, Qingcao; Xu, Yanxia; Qi, Xin; Zhao, Xiaoying; Ji, Liangliang; Yu, Tongpu; Wei, Luo; Yang, Lei; Hu, Bitao

2013-12-30

Ultra-intense single attosecond pulse (AP) can be obtained from circularly polarized (CP) laser interacting with overdense plasma. High harmonics are naturally generated in the reflected laser pulses due to the laser-induced one-time drastic oscillation of the plasma boundary. Using two-dimensional (2D) planar particle-in-cell (PIC) simulations and analytical model, we show that multi-dimensional effects have great influence on the generation of AP. Self-focusing and defocusing phenomena occur in front of the compressed plasma boundary, which lead to the dispersion of the generated AP in the far field. We propose to control the reflected high harmonics by employing a density-modulated foil target (DMFT). When the target density distribution fits the laser intensity profile, the intensity of the attosecond pulse generated from the center part of the plasma has a flatten profile within the center range in the transverse direction. It is shown that a single 300 attosecond (1 as = 10(-18)s) pulse with the intensity of 1.4 × 10(21) W cm(-2) can be naturally generated. Further simulations reveal that the reflected high harmonics properties are highly related to the modulated density distribution and the phase offset between laser field and the carrier envelope. The emission direction of the AP generated from the plasma boundary can be controlled in a very wide range in front of the plasma surface by combining the DMFT and a suitable driving laser.

Short-range wakefields generated in the blowout regime of plasma-wakefield acceleration

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

Stupakov, G.

In the past, calculation of wakefields generated by an electron bunch propagating in a plasma has been carried out in linear approximation, where the plasma perturbation can be assumed small and plasma equations of motion linearized. This approximation breaks down in the blowout regime where a high-density electron driver expels plasma electrons from its path and creates a cavity void of electrons in its wake. Here in this paper, we develop a technique that allows us to calculate short-range longitudinal and transverse wakes generated by a witness bunch being accelerated inside the cavity. Our results can be used for studiesmore » of the beam loading and the hosing instability of the witness bunch in plasma-wakefield and laser-wakefield acceleration.« less

Short-range wakefields generated in the blowout regime of plasma-wakefield acceleration

NASA Astrophysics Data System (ADS)

Stupakov, G.

2018-04-01

In the past, calculation of wakefields generated by an electron bunch propagating in a plasma has been carried out in linear approximation, where the plasma perturbation can be assumed small and plasma equations of motion linearized. This approximation breaks down in the blowout regime where a high-density electron driver expels plasma electrons from its path and creates a cavity void of electrons in its wake. In this paper, we develop a technique that allows us to calculate short-range longitudinal and transverse wakes generated by a witness bunch being accelerated inside the cavity. Our results can be used for studies of the beam loading and the hosing instability of the witness bunch in plasma-wakefield and laser-wakefield acceleration.

Short-range wakefields generated in the blowout regime of plasma-wakefield acceleration

DOE PAGES

Stupakov, G.

2018-04-02

In the past, calculation of wakefields generated by an electron bunch propagating in a plasma has been carried out in linear approximation, where the plasma perturbation can be assumed small and plasma equations of motion linearized. This approximation breaks down in the blowout regime where a high-density electron driver expels plasma electrons from its path and creates a cavity void of electrons in its wake. Here in this paper, we develop a technique that allows us to calculate short-range longitudinal and transverse wakes generated by a witness bunch being accelerated inside the cavity. Our results can be used for studiesmore » of the beam loading and the hosing instability of the witness bunch in plasma-wakefield and laser-wakefield acceleration.« less

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.

Contribution of photocurrent mechanism and influence of plasma length in THz generation by two-color laser induced plasma

NASA Astrophysics Data System (ADS)

Alirezaee, Hajar; Sharifian, Mehdi

2018-04-01

The contribution of the photocurrent mechanism has been investigated in terahertz (THz) generation in laser-induced gas plasma. This study was done through comparing the THz amplitude for two phase differences (" separators="| φ = 0 , π / 2 ) between the fundamental pulse and its second harmonic. Our particle-in-cell simulation result showed that this mechanism played the most important role in the two-color regime with a low-frequency range. We also investigated the effect of the plasma channel length on generated THz amplitude, and it was found that THz amplitude would depend significantly on the plasma channel length.

Charge-exchange plasma generated by an ion thruster

NASA Technical Reports Server (NTRS)

Kaufman, H. R.

1977-01-01

The charge exchange plasma generated by an ion thruster was investigated experimentally using both 5 cm and 15 cm thrusters. Results are shown for wide ranges of radial distance from the thruster and angle from the beam direction. Considerations of test environment, as well as distance from the thruster, indicate that a valid simulation of a thruster on a spacecraft was obtained. A calculation procedure and a sample calculation of charge exchange plasma density and saturation electron current density are included.

Investigation Of Plasma Critical Surface Rippling By Harmonics Generation In Laser Plasmas

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

Racz, E.; Foeldes, I. B.; Szatmari, S.

2006-01-15

Experiments were carried out by a tightly focused, prepulse-free hybrid KrF excimer-dye laser system (700fs pulse duration, 248nm wavelength, 15mJ pulse energy). Intense 2{omega}, 3{omega} and near threshold 4{omega} were generated in laser plasmas on solid surfaces for p- and s-polarized 1.5{center_dot}1017 W/cm2 radiation intensity. Directionality and polarization properties were investigated depending on the laser intensity and polarization. The observations showed diffuse propagation of harmonics for intensities above 1016 W/cm2 and the polarization of harmonics was mixed for the highest intensities. The explanation of these results is surface rippling of the plasma critical surface because of the Rayleigh-Taylor instability, whichmore » is an intrinsic consequence of the unstable balance between light pressure and plasma expansion.« less

Generation and acceleration of neutral atoms in intense laser plasma experiments

NASA Astrophysics Data System (ADS)

Tata, Sheroy; Mondal, Angana; Sarkar, Shobhik; Ved, Yash; Lad, Amit D.; Pasley, John; Colgan, James; Krishnamurthy, M.

2017-10-01

The interaction of a high intensity (>=1018 W/cm2), high contrast (>=109), ultra-short (30fs) laser with solid targets generates a highly dense hot plasma. The quasi-static electric fields in such plasmas are well known for ion acceleration via the target normal sheath acceleration process. Under such conditions charge reduction to generate fast neutral atoms is almost inhibited. Improvised Thomson parabola spectrometry with improved signal to noise ratio has enabled us to measure the signals of fast neutral atoms and negative ions having energies in excess of tens of keV. A study on the neutralization of accelerated protons in plasma shows that the neutral atom to all particle ratio rises sharply from a few percent at the highest detectable energy to 50 % at 15 keV. Using usual charge transfer reactions the generation of neutral atoms can not be explained, thus we conjecture that the neutralization of the accelerated ions is not from the hot dense region of the plasma but neutral atom formation takes place by co-propagating ions with low energy electrons enhancing the effective neutral ratio.

Impact of plasma jet vacuum ultraviolet radiation on reactive oxygen species generation in bio-relevant liquids

NASA Astrophysics Data System (ADS)

Jablonowski, H.; Bussiahn, R.; Hammer, M. U.; Weltmann, K.-D.; von Woedtke, Th.; Reuter, S.

2015-12-01

Plasma medicine utilizes the combined interaction of plasma produced reactive components. These are reactive atoms, molecules, ions, metastable species, and radiation. Here, ultraviolet (UV, 100-400 nm) and, in particular, vacuum ultraviolet (VUV, 10-200 nm) radiation generated by an atmospheric pressure argon plasma jet were investigated regarding plasma emission, absorption in a humidified atmosphere and in solutions relevant for plasma medicine. The energy absorption was obtained for simple solutions like distilled water (dH2O) or ultrapure water and sodium chloride (NaCl) solution as well as for more complex ones, for example, Rosewell Park Memorial Institute (RPMI 1640) cell culture media. As moderate stable reactive oxygen species, hydrogen peroxide (H2O2) was studied. Highly reactive oxygen radicals, namely, superoxide anion (O2•-) and hydroxyl radicals (•OH), were investigated by the use of electron paramagnetic resonance spectroscopy. All species amounts were detected for three different treatment cases: Plasma jet generated VUV and UV radiation, plasma jet generated UV radiation without VUV part, and complete plasma jet including all reactive components additionally to VUV and UV radiation. It was found that a considerable amount of radicals are generated by the plasma generated photoemission. From the experiments, estimation on the low hazard potential of plasma generated VUV radiation is discussed.

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.

Increased plasma fibrinopeptide A levels during attacks induced by hyperventilation in patients with coronary vasospastic angina.

PubMed

Oshima, S; Ogawa, H; Yasue, H; Okumura, K; Matsuyama, K; Miyagi, H

1989-07-01

Plasma fibrinopeptide A levels, beta-thromboglobulin levels and platelet factor 4 levels were estimated by enzyme-linked immunosorbent assay before and after hyperventilation in 12 patients with coronary vasospastic angina and in 12 control subjects matched for age and gender. In all 12 study patients, anginal attacks accompanied by electrocardiographic (ECG) changes (ST elevation in 11 patients and ST depression in 1 patient) were induced by hyperventilation. Coronary angiography was performed on 11 of the 12 patients, and coronary artery spasm with the same ECG changes was induced by intracoronary injection of acetylcholine in all 11. The plasma fibrinopeptide A levels increased significantly from 2.0 +/- 0.4 to 10.0 +/- 2.4 ng/ml during the attack (p less than 0.001) in the study patients, but remained unchanged before and after hyperventilation in the control subjects. The plasma levels of beta-thromboglobulin and platelet factor 4 remained unchanged after hyperventilation in both groups. Our data indicate that coronary artery spasm may induce thrombin generation and trigger thrombus formation in the coronary artery.

Minimally invasive therapy of pseudoaneurysms of the trunk: application of thrombin.

PubMed

Schellhammer, Frank; Steinhaus, Daniele; Cohnen, Mathias; Hoppe, Jonas; Mödder, Ulrich; Fürst, Günter

2008-01-01

Thrombin injection has been proven to be successful in postcatheterization pseudoaneurysms. However, there are only a few reports on the treatment of pseudoaneurysms of the trunk. We report our first experiences using a percutaneous as well as an endovascular access. Eight iatrogenic pseudoaneurysms of the trunk (aorta, n = 4; pulmonary artery, n = 1; gastroduodenal artery, n = 1; left gastric artery, n = 1, renal artery, n = 1) were treated either percutaneously using CT guidance (n = 3) or via an endovascular access (n = 5). Noninvasive control angiograms were performed at day 1 and weeks 1 and 3 by either CT or MR angiography. The total volume of the pseudoaneurysms was 31.2 +/- 23.1 ml on average, with a mean volume of the perfused aneurysmal lumen of 12.9 +/- 7.2 ml. The maximum diameter was 4.1 +/- 1.39 cm on average. In each case, the aneurysmal neck was not wider than 2 mm. One pseudoaneurysm occluded spontaneously following selective catheterization. The remaining pseudoaneurysms were successfully treated by injection of 765 +/- 438.1 IU thrombin. In one individual, a nontarget embolization occurred, as well as an intervention-associated rupture of a pseudoaneurysm. High-grade stenoses of the donor artery were found in a different case. Only once was the endoluminal access converted to a percutaneous one. Thrombin injection might be a future first-line treatment of vascular lesions such as pseudoaneurysms of the trunk. In our experience both percutanous and endoluminal access are technically feasible and safe. However, further experiences are mandatory, especially concerning the question of dosage and long-term results.

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.

Terahertz generation by beating two Langmuir waves in a warm and collisional plasma