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1

Topical thrombin preparations and their use in cardiac surgery  

Directory of Open Access Journals (Sweden)

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

Brianne L Dunn

2009-10-01

2

Topical thrombin preparations and their use in cardiac surgery  

OpenAIRE

Brianne L Dunn1, Walter E Uber1, John S Ikonomidis21Department of Pharmacy Services and 2Division of Cardiothoracic Surgery, Medical University of South Carolina, Charleston, South Carolina, USAAbstract: Coagulopathic bleeding may lead to increased morbidity and mortality after cardiac surgery. Topical bovine thrombin has been used to promote hemostasis after surgical procedures for over 60 years and is used frequently as a topical hemostatic agent in cardiac surgery. Recently, use o...

Dunn, Brianne L.; Uber, Walter Amp Nbsp E.; Ikonomidis, John S.

2009-01-01

3

Optimization of expression of untagged and histidine-tagged human recombinant thrombin precursors in Escherichia coli.  

Science.gov (United States)

The present study is focused on preparation of proper Escherichia coli expression system to ensure high yields of various modified precursors of human recombinant thrombin, a potential biopharmaceutical reagent. Two thrombin precursors, the smallest single-chain ?-thrombin precursor prethrombin-2 and its shortened form prethrombin-2?13, and their His-tagged forms were used. In order to determine the effect of the different lengths and amino acid compositions of affinity His-tag on the target protein expression level, a variety of the His-tag sequences were used. We found out that the protein expression efficiency was closely related to the codons used for encoding of amino acids of fusion histidine tag. Optimization of culture medium composition is another way to increase yield of the target protein. Suitable medium composition can ensure cell growth to high densities which is related to total yield of expressed protein. In this study, a new optimized complex medium for batch fermentation was developed. Addition of nutrients like a yeast extract and enzymatic casein hydrolysate to the defined medium components had a positive impact on protein expression, where relatively high expression level of the target protein from total amount of cellular proteins was achieved. Further, we have focused on trace element solution composition, and the optimized nickel and selenium concentrations were determined. Our results show that the composition of essential trace metal solution has a major impact not only on expression level, but it can also affect cell growth rate. PMID:24878753

Osadská, Michaela; Bo?ková, Hana; Krahulec, Ján; Stuchlík, Stanislav; Tur?a, Ján

2014-11-01

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

International Nuclear Information System (INIS)

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

5

Thrombin Time  

Science.gov (United States)

... by thrombin into insoluble threads called fibrin that crosslink together to form a fibrin net that adheres ... Reference 11th Edition: Mosby, Inc., Saint Louis, MO. Pp 445-446. (© 2014). Thrombic Risk Reflexive Panel. ARUP ...

6

The use of thrombin in the radiology department.  

LENUS (Irish Health Repository)

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

Ward, E

2009-03-01

7

Thrombin and platelet activation.  

Science.gov (United States)

The accumulation of thrombin at sites of vascular injury provides one of the chief means for recruiting platelets into a growing hemostatic plug. Studies completed over the past 10 years show that platelet responses to thrombin are mediated by a subset of G protein-coupled receptors known as protease-activated receptors. These receptors are activated on cleavage by thrombin, initiating the intracellular signaling events needed to transform mobile, nonadhesive platelets into cells that can participate in the growth of an immobile hemostatic plug. How this is accomplished is the subject of this review. PMID:12970120

Brass, Lawrence F

2003-09-01

8

Molecular mapping of the heparin-binding exosite of thrombin.  

OpenAIRE

Thrombin contains electropositive patches at opposite poles of the molecule which represent potential exosites for the binding of macromolecular ligands. The function of anion-binding exosite I, the fibrin(ogen) recognition site, has been well described. Anion-binding exosite II, located near the carboxyl terminus of the molecule, has been proposed to bind heparin on the basis of chemical modification studies. To define the functional heparin-binding site on thrombin, purified recombinant alp...

Sheehan, J. P.; Sadler, J. E.

1994-01-01

9

Recombiner  

International Nuclear Information System (INIS)

Purpose: To shorten the pre-heating time for a recombiner and obtain a uniform temperature distribution for the charged catalyst layer in a BWR type reactor. Constitution: A pre-heating heater is disposed to the outer periphery of a vessel for a recombiner packed with catalysts for recombining hydrogen and oxygen in gases flowing through a radioactive gaseous wastes processing system. Heat pipes for transmitting the heat applied to said container to the catalyst are disposed vertically and horizontally within the container. Different length of the heat pipes are combined. In this way, pre-heating time for the recombiner before the operation start and before the system switching can be shortened and the uniform pre-heating for the inside of the recombiner is also made possible. Further, heater control in the pre-heating can be carried out effectively and with ease. (Moriyama, K.)

10

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

OpenAIRE

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

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

1992-01-01

11

Nanocomplexation of thrombin with cationic amylose derivative for improved stability and hemostatic efficacy  

Science.gov (United States)

As a topical hemostatic agent, thrombin has wide application for many surgical treatments. However, native thrombin always suffers from its physical and chemical instabilities. In this work, a nanocomplexation strategy was developed for modifying the stability and hemostatic efficacy of thrombin, in which a water-soluble cationic amylose derivative containing poly(l-lysine) dendrons was prepared by a click reaction and then used to complex thrombin in an aqueous system. For resultant thrombin nanocomplexes, their morphology and particle size distribution were investigated. Their stabilities were studied in terms of activity retention percentages under different storage time, pH values, and illumination time. In addition, their ability to achieve in vitro fibrinogen and blood coagulation were evaluated. Via a rat hepatic hemorrhage model and a rat iliac artery hemorrhage model, these thrombin nanocomplexes were confirmed to have good tissue biocompatibility and in vivo hemostatic effectiveness. PMID:25673989

Zhuang, Baoxiong; Li, Zhihua; Pang, Jiadong; Li, Wenbin; Huang, Pinbo; Wang, Jie; Zhou, Yu; Lin, Qing; Zhou, Quanbo; Ye, Xiao; Ye, Huilin; Liu, Yimin; Zhang, Li-Ming; Chen, Rufu

2015-01-01

12

Recombineering  

Science.gov (United States)

Recombineering, a recently developed technique for efficient genetic manipulation of bacteria, is facilitated by phage-derived recombination proteins and has the advantage of using DNA substrates with short regions of homology. This system was first developed in E. coli but has since been adapted for use in other bacteria. It is now widely used in a number of different systems for a variety of purposes, and the construction of chromosomal gene knockouts, deletions, insertions, point mutations, as well as in vivo cloning, mutagenesis of bacterial artificial chromosomes and phasmids, and the construction of genomic libraries has been reported. However, these methods also can be effectively applied to the genetic modification of bacteriophage genomes, in both their prophage and lytically growing states. The ever-growing collection of fully sequenced bacteriophages raises more questions than they answer, including the unknown functions of vast numbers of genes with no known homologs and of unknown function. Recombineering of phage genomes is central to addressing these questions, enabling the simple construction of mutants, determination of gene essentiality, and elucidation of gene function. In turn, advances in our understanding of phage genomics should present similar recombineering tools for dissecting a multitude of other genetically naïve bacterial systems. PMID:22666652

Marinelli, Laura J.; Hatfull, Graham F.; Piuri, Mariana

2012-01-01

13

Topical Application of Recombinant Type VII Collagen Incorporates Into the Dermal–Epidermal Junction and Promotes Wound Closure  

OpenAIRE

Patients with recessive dystrophic epidermolysis bullosa (RDEB) have incurable skin fragility, blistering, and skin wounds due to mutations in the gene that codes for type VII collagen (C7) that mediates dermal–epidermal adherence in human skin. In this study, we evaluated if topically applied human recombinant C7 (rC7) could restore C7 at the dermal–epidermal junction (DEJ) and enhance wound healing. We found that rC7 applied topically onto murine skin wounds stably incorp...

Wang, Xinyi; Ghasri, Pedram; Amir, Mahsa; Hwang, Brian; Hou, Yingpin; Khilili, Michael; Lin, Andrew; Keene, Douglas; Uitto, Jouni; Woodley, David T.; Chen, Mei

2013-01-01

14

Factor XI contributes to thrombin generation in the absence of factor XII.  

Science.gov (United States)

During surface-initiated blood coagulation in vitro, activated factor XII (fXIIa) converts factor XI (fXI) to fXIa. Whereas fXI deficiency is associated with a hemorrhagic disorder, factor XII deficiency is not, suggesting that fXI can be activated by other mechanisms in vivo. Thrombin activates fXI, and several studies suggest that fXI promotes coagulation independent of fXII. However, a recent study failed to find evidence for fXII-independent activation of fXI in plasma. Using plasma in which fXII is either inhibited or absent, we show that fXI contributes to plasma thrombin generation when coagulation is initiated with low concentrations of tissue factor, factor Xa, or alpha-thrombin. The results could not be accounted for by fXIa contamination of the plasma systems. Replacing fXI with recombinant fXI that activates factor IX poorly, or fXI that is activated poorly by thrombin, reduced thrombin generation. An antibody that blocks fXIa activation of factor IX reduced thrombin generation; however, an antibody that specifically interferes with fXI activation by fXIIa did not. The results support a model in which fXI is activated by thrombin or another protease generated early in coagulation, with the resulting fXIa contributing to sustained thrombin generation through activation of factor IX. PMID:19351955

Kravtsov, Dmitri V; Matafonov, Anton; Tucker, Erik I; Sun, Mao-Fu; Walsh, Peter N; Gruber, Andras; Gailani, David

2009-07-01

15

Topical Application of Recombinant Type VII Collagen Incorporates Into the Dermal–Epidermal Junction and Promotes Wound Closure  

Science.gov (United States)

Patients with recessive dystrophic epidermolysis bullosa (RDEB) have incurable skin fragility, blistering, and skin wounds due to mutations in the gene that codes for type VII collagen (C7) that mediates dermal–epidermal adherence in human skin. In this study, we evaluated if topically applied human recombinant C7 (rC7) could restore C7 at the dermal–epidermal junction (DEJ) and enhance wound healing. We found that rC7 applied topically onto murine skin wounds stably incorporated into the newly formed DEJ of healed wounds and accelerated wound closure by increasing re-epithelialization. Topical rC7 decreased the expression of fibrogenic transforming growth factor-?2 (TGF-?2) and increased the expression of anti-fibrogenic TGF-?3. These were accompanied by the reduced expression of connective tissue growth factor, fewer ? smooth muscle actin (?-SMA)–positive myofibroblasts, and less deposition of collagen in the healed neodermis, consistent with less scar formation. In addition, using a mouse model in which skin from C7 knock out mice was grafted onto immunodeficient mice, we showed that applying rC7 onto RDEB grafts with wounds restored C7 and anchoring fibrils (AFs) at the DEJ of the grafts and corrected the dermal–epidermal separation. The topical application of rC7 may be useful for treating patients with RDEB and patients who have chronic skin wounds. PMID:23670575

Wang, Xinyi; Ghasri, Pedram; Amir, Mahsa; Hwang, Brian; Hou, Yingpin; Khilili, Michael; Lin, Andrew; Keene, Douglas; Uitto, Jouni; Woodley, David T; Chen, Mei

2013-01-01

16

Topical application of recombinant type VII collagen incorporates into the dermal-epidermal junction and promotes wound closure.  

Science.gov (United States)

Patients with recessive dystrophic epidermolysis bullosa (RDEB) have incurable skin fragility, blistering, and skin wounds due to mutations in the gene that codes for type VII collagen (C7) that mediates dermal-epidermal adherence in human skin. In this study, we evaluated if topically applied human recombinant C7 (rC7) could restore C7 at the dermal-epidermal junction (DEJ) and enhance wound healing. We found that rC7 applied topically onto murine skin wounds stably incorporated into the newly formed DEJ of healed wounds and accelerated wound closure by increasing re-epithelialization. Topical rC7 decreased the expression of fibrogenic transforming growth factor-?2 (TGF-?2) and increased the expression of anti-fibrogenic TGF-?3. These were accompanied by the reduced expression of connective tissue growth factor, fewer ? smooth muscle actin (?-SMA)-positive myofibroblasts, and less deposition of collagen in the healed neodermis, consistent with less scar formation. In addition, using a mouse model in which skin from C7 knock out mice was grafted onto immunodeficient mice, we showed that applying rC7 onto RDEB grafts with wounds restored C7 and anchoring fibrils (AFs) at the DEJ of the grafts and corrected the dermal-epidermal separation. The topical application of rC7 may be useful for treating patients with RDEB and patients who have chronic skin wounds. PMID:23670575

Wang, Xinyi; Ghasri, Pedram; Amir, Mahsa; Hwang, Brian; Hou, Yingpin; Khalili, Michael; Khilili, Michael; Lin, Andrew; Keene, Douglas; Uitto, Jouni; Woodley, David T; Chen, Mei

2013-07-01

17

Refined structure of the hirudin-thrombin complex.  

Science.gov (United States)

The structure of a recombinant hirudin (variant 2, Lys47) human alpha-thrombin complex has been refined using restrained least-squares methods to a crystallographic R-factor of 0.173. The hirudin structure consists of an N-terminal domain folded into a globular unit and a long 17-peptide C-terminal in an extended chain conformation. The N-terminal domain binds at the active-site of thrombin where Ile1' to Tyr3' penetrates to the catalytic triad. The alpha-amino group of Ile1' of hirudin makes a hydrogen bond with OG of Ser195 of thrombin, the side-chains of Ile1' and Tyr3' occupy the apolar site, Thr2' is at the entrance to, but does not enter, the S1 specificity site and Ile1' to Tyr3' form a parallel beta-strand with Ser214 to Gly219. The latter interaction is antiparallel in all other serine proteinase-protein inhibitor complexes. The extended C-terminal segment of hirudin, which is abundant in acidic residues, makes many electrostatic interactions with the fibrinogen binding exosite while the last five residues are in a 3(10) helical turn residing in a hydrophobic patch on the thrombin surface. The precision of the complementarity displayed by these two molecules produces numerous interactions, which although independently generally weak, together are responsible for the high degree of affinity and specificity. Although hirudin-thrombin and D-Phe-Pro-Arg-chloromethyl ketone-thrombin differ in conformation in the autolysis loop (Lys145 to Gly150), this is most likely due to different crystal packing interactions and changes in circular dichroism between the two are probably due to the inherent flexibility of the loop. An RGD sequence, which is generally known to be involved in cell surface receptor interactions, occurs in thrombin and is associated with a long solvent channel filled with water molecules leading to the surface from the end of the S1 site. However, the RGD triplet does not appear to be able to interact in concert in a surface binding mode. PMID:1920434

Rydel, T J; Tulinsky, A; Bode, W; Huber, R

1991-09-20

18

Monoclonal antibody to the thrombin receptor stimulates DNA synthesis in combination with gamma-thrombin or phorbol myristate acetate  

OpenAIRE

Studies with various thrombin derivatives have shown that initiation of cell proliferation by thrombin requires two separate types of signals: one, generated by high affinity interaction of thrombin or DIP-thrombin (alpha-thrombin inactivated at ser 205 of the B chain by diisopropylphosphofluoridate) with receptors and the other, by thrombin's enzymic activity. To further study the role of high affinity thrombin receptors in initiation, we immunized mice with whole human fibroblasts and selec...

1987-01-01

19

Zinc modulates thrombin adsorption to fibrin  

International Nuclear Information System (INIS)

Human thrombin with high affinity to Sepharose insolubilized fibrin monomers (high-affinity thrombin) was used to investigate the effect of Zn(II) on the thrombin adsorption to fibrin. Results showed that at Zn(II) concentrations exceeding 100 mumols/l, thrombin binding to fibrin was decreased concomitant with the Zn(II) concentration and time; at lower Zn(II) concentrations, thrombin adsorption was enhanced. Experimental results were identical by using 125I-labelled high-affinity alpha-thrombin or by measuring the thrombin activity either by chromogenic substrate or by a clotting time method. In contrast, Ca(II) alone (final conc. 3 mmol/l) or in combination with Zn(II) was not effective. However, at higher Ca(II) concentrations (7.5-15 mmol/l), thrombin adsorption was apparently decreased. Control experiments revealed that Zn(II) had no impact on the clottability of fibrinogen, and that the results of the experiments with Ca(II) were not altered by possible cross-linking of fibrin. We conclude that unlike Ca(II), Zn(II) is highly effective in modulating thrombin adsorption to fibrin

20

The unresolved safety concerns of bovine thrombin  

Directory of Open Access Journals (Sweden)

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

Javidroozi Mazyar

2008-09-01

21

The unresolved safety concerns of bovine thrombin.  

Science.gov (United States)

A recent review has suggested that bovine thrombin is not associated with an increased risk of bleeding in surgical populations. In spite of extremely limited evidence available, many valuable resources (e.g. safety surveillance and post-marketing programs, case reports) were excluded in reaching this conclusion. While waiting for the adequately powered, controlled clinical trials to address the effects of bovine thrombin on bleeding and thrombotic events, the potential risk cannot be simply ignored. Rather, continued vigilance in the post-surgical setting for bleeding events that may be associated with the development of acquired coagulation factor inhibitors following bovine thrombin administration is warranted. PMID:18808708

Shander, Aryeh; Javidroozi, Mazyar

2008-01-01

22

Limited efficacy of topical recombinant feline interferon-omega for treatment of cats with acute upper respiratory viral disease.  

Science.gov (United States)

Despite a lack of controlled studies confirming its efficacy, recombinant feline interferon-omega (rfeIFN-?) is used in the treatment of feline upper respiratory tract disease (FURTD), which is usually caused by feline calicivirus (FCV) or feline herpesvirus-1 (FHV-1). The aims of the present study were to investigate whether administration of rfeIFN-? improves clinical signs in cats with acute FURTD and whether this treatment reduces shedding of FCV. Thirty-seven cats affected with acute FURTD were recruited into a prospective, randomised, placebo-controlled, double-blinded clinical trial. The presence of FCV and/or FHV-1 was determined by performing quantitative polymerase chain reaction (qPCR) on oropharyngeal and conjunctival swabs. Cats were randomly assigned to treatment groups, receiving either placebo or rfeIFN-? (2.5?MU/kg) subcutaneously, followed by 0.5?MU topically at 8-h intervals via the conjunctiva, intranasally, and orally for 21 days. All cats received additional treatment with antibiotics, expectorants, and inhalation of nebulised physiological saline with camomile. Clinical signs and FCV shedding were evaluated over 42 days. All cats demonstrated improvement in clinical signs during the course of the study, with no significant difference in any of the assessed variables when comparing the two groups. FCV copy numbers decreased more rapidly in cats receiving rfeIFN-?. Treatment with rfeIFN-? was not effective in ameliorating clinical signs of acute viral FURTD compared to placebo, but might accelerate a reduction in FCV load in infected cats. PMID:25457261

Ballin, Anne C; Schulz, Bianka; Helps, Christopher; Sauter-Louis, Carola; Mueller, Ralf S; Hartmann, Katrin

2014-12-01

23

Thrombin has a bimodal effect on glioma cell growth.  

OpenAIRE

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

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

1997-01-01

24

Antiplatelet therapy: thrombin receptor antagonists.  

Science.gov (United States)

Activated platelets stimulate thrombus formation in response to rupture of an atherosclerotic plaque or endothelial cell erosion, promoting atherothrombotic disease. Multiple pathways contribute to platelet activation. Aspirin, an irreversible inhibitor of thromboxane A2 synthesis, in combination with clopidogrel, an inhibitor of P2Y(12) adenosine diphosphate platelet receptors, represent the current standard-of-care of antiplatelet therapy for patients with acute coronary syndrome and for those undergoing percutaneous coronary intervention. Although these agents have demonstrated significant clinical benefit, the increased risk of bleeding and the recurrence of thrombotic events represent substantial limitations. Thrombin is one of the most important platelet activators. The inhibition of protease-activated receptor 1 showed a good safety profile in preclinical studies. In fact, phase II studies with vorapaxar (SCH530348) and atopaxar (E5555) showed no increase of bleeding events in addition to the current standard-of-care of antiplatelet therapy. Although the results of phase III trials for both drugs are awaited, this family is a promising new addition to the current clinical practice for patients with atherothrombotic disease, not only as an alternative, but also as additional therapy. PMID:21906120

Tello-Montoliu, Antonio; Tomasello, Salvatore D; Ueno, Masafumi; Angiolillo, Dominick J

2011-10-01

25

Correlated motions and residual frustration in thrombin.  

Science.gov (United States)

Thrombin is the central protease in the cascade of blood coagulation proteases. The structure of thrombin consists of a double ?-barrel core surrounded by connecting loops and helices. Compared to chymotrypsin, thrombin has more extended loops that are thought to have arisen from insertions in the serine protease that evolved to impart greater specificity. Previous experiments showed thermodynamic coupling between ligand binding at the active site and distal exosites. We present a combined approach of molecular dynamics (MD), accelerated molecular dynamics (AMD), and analysis of the residual local frustration of apo-thrombin and active-site-bound (PPACK-thrombin). Community analysis of the MD ensembles identified changes upon active site occupation in groups of residues linked through correlated motions and physical contacts. AMD simulations, calibrated on measured residual dipolar couplings, reveal that upon active site ligation, correlated loop motions are quenched, but new ones connecting the active site with distal sites where allosteric regulators bind emerge. Residual local frustration analysis reveals a striking correlation between frustrated contacts and regions undergoing slow time scale dynamics. The results elucidate a motional network that probably evolved through retention of frustrated contacts to provide facile conversion between ensembles of states. PMID:23621631

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

2013-10-24

26

Thrombin receptor expression in normal and atherosclerotic human arteries.  

OpenAIRE

Thrombin is a multifunctional serine protease generated at sites of vascular injury. A host of thrombin actions on vascular endothelial cells, smooth muscle cells, and macrophages has been defined in cell culture systems, but the in vivo significance of these activities is unknown. We have defined the expression of the recently identified receptor for thrombin in human arteries by both in situ hybridization and immunohistochemistry. In normal-appearing arteries, thrombin receptor was expresse...

Nelken, N. A.; Soifer, S. J.; O Keefe, J.; Vu, T. K.; Charo, I. F.; Coughlin, S. R.

1992-01-01

27

Allosteric networks in thrombin distinguish procoagulant vs. anticoagulant activities.  

Science.gov (United States)

The serine protease ?-thrombin is a dual-action protein that mediates the blood-clotting cascade. Thrombin alone is a procoagulant, cleaving fibrinogen to make the fibrin clot, but the thrombin-thrombomodulin (TM) complex initiates the anticoagulant pathway by cleaving protein C. A TM fragment consisting of only the fifth and sixth EGF-like domains (TM56) is sufficient to bind thrombin, but the presence of the fourth EGF-like domain (TM456) is critical to induce the anticoagulant activity of thrombin. Crystallography of the thrombin-TM456 complex revealed no significant structural changes in thrombin, suggesting that TM4 may only provide a scaffold for optimal alignment of protein C for its cleavage by thrombin. However, a variety of experimental data have suggested that the presence of TM4 may affect the dynamic properties of the active site loops. In the present work, we have used both conventional and accelerated molecular dynamics simulation to study the structural dynamic properties of thrombin, thrombin:TM56, and thrombin:TM456 across a broad range of time scales. Two distinct yet interrelated allosteric pathways are identified that mediate both the pro- and anticoagulant activities of thrombin. One allosteric pathway, which is present in both thrombin:TM56 and thrombin:TM456, directly links the TM5 domain to the thrombin active site. The other allosteric pathway, which is only present on slow time scales in the presence of the TM4 domain, involves an extended network of correlated motions linking the TM4 and TM5 domains and the active site loops of thrombin. PMID:23197839

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

2012-12-26

28

Fibrinogen and thrombin concentrations are critical for fibrin glue adherence in rat high-risk colon anastomoses  

Scientific Electronic Library Online (English)

Full Text Available OBJECTIVE: Fibrin glues have not been consistently successful in preventing the dehiscence of high-risk colonic anastomoses. Fibrinogen and thrombin concentrations in glues determine their ability to function as sealants, healers, and/or adhesives. The objective of the current study was to compare [...] the effects of different concentrations of fibrinogen and thrombin on bursting pressure, leaks, dehiscence, and morphology of high-risk ischemic colonic anastomoses using fibrin glue in rats. METHODS: Colonic anastomoses in adult female Sprague-Dawley rats (weight, 250-350 g) treated with fibrin glue containing different concentrations of fibrinogen and thrombin were evaluated at post-operative day 5. The interventions were low-risk (normal) or high-risk (ischemic) end-to-end colonic anastomoses using polypropylene sutures and topical application of fibrinogen at high (120 mg/mL) or low (40 mg/mL) concentrations and thrombin at high (1000 IU/mL) or low (500 IU/mL) concentrations. RESULTS: Ischemia alone, anastomosis alone, or both together reduced the bursting pressure. Glues containing a low fibrinogen concentration improved this parameter in all cases. High thrombin in combination with low fibrinogen also improved adherence exclusively in low-risk anastomoses. No differences were detected with respect to macroscopic parameters, histopathology, or hydroxyproline content at 5 days post-anastomosis. CONCLUSIONS: Fibrin glue with a low fibrinogen content normalizes the bursting pressure of high-risk ischemic left-colon anastomoses in rats at day 5 after surgery.

Eliseo Portilla-de, Buen; Abel, Orozco-Mosqueda; Caridad, Leal-Cortés; Gonzalo, Vázquez-Camacho; Clotilde, Fuentes-Orozco; Andrea Socorro, Alvarez-Villaseñor; Michel Dassaejv, Macías-Amezcua; Alejandro, González-Ojeda.

2014-04-01

29

Thrombin regulates the function of human blood dendritic cells  

International Nuclear Information System (INIS)

Thrombin is the key enzyme in the coagulation cascade and activates endothelial cells, neutrophils and monocytes via protease-activated receptors (PARs). At the inflammatory site, immune cells have an opportunity to encounter thrombin. However little is known about the effect of thrombin for dendritic cells (DC), which are efficient antigen-presenting cells and play important roles in initiating and regulating immune responses. The present study revealed that thrombin has the ability to stimulate blood DC. Plasmacytoid DC (PDC) and myeloid DC (MDC) isolated from PBMC expressed PAR-1 and released MCP-1, IL-10, and IL-12 after thrombin stimulation. Unlike blood DC, monocyte-derived DC (MoDC), differentiated in vitro did not express PAR-1 and were unresponsive to thrombin. Effects of thrombin on blood DC were significantly diminished by the addition of anti-PAR-1 Ab or hirudin, serine protease inhibitor. Moreover, thrombin induced HLA-DR and CD86 expression on DC and the thrombin-treated DC induced allogenic T cell proliferation. These findings indicate that thrombin plays a role in the regulation of blood DC functions

30

Thrombin-induced increase in albumin permeability across the endothelium  

International Nuclear Information System (INIS)

We studied the effect of thrombin on albumin permeability across the endothelial monolayer in vitro. Bovine pulmonary artery endothelial cells were grown on micropore membranes. Morphologic analysis confirmed the presence of a confluent monolayer with interendothelial junctions. Albumin permeability was measured by the clearance of 125I-albumin across the endothelial monolayer. The control 125I-albumin clearance was 0.273 +/- 0.02 microliter/min. The native enzyme, alpha-thrombin (10(-6) to 10(-10) M), added to the luminal side of the endothelium produced concentration-dependent increases in albumin clearance (maximum clearance of 0.586 +/- 0.08 microliter/min at 10(-6) M). Gamma (gamma) thrombin (10(-6) M and 10(-8) M), which lacks the fibrinogen recognition site, also produced a concentration-dependent increase in albumin clearance similar to that observed with alpha-thrombin. Moreover, the two proteolytically inactive forms of the native enzyme, i-Pr2 P-alpha-thrombin and D-Phe-Pro-Arg-CH2-alpha-thrombin, increased the 125I-albumin clearance (0.610 +/- 0.09 microliter/min and 0.609 +/- 0.02 microliter/min for i-Pr2 P-alpha-thrombin and D-Phe-Pro-Arg-CH2-alpha-thrombin at 10(-6) M, respectively). Since the modified forms of thrombin lack the fibrinogen recognition and active serine protease sites, the results indicate that neither site is required for increased albumin permeability. The increase in albumin clearance with alpha-thrombin was not secondary to endothelial cell lysis because lactate dehydrogenase concentration in the medium following thrombin was not significantly different from baseline values. There was also no morphological evidence of cell lysis. Moreover, the increase in 125I-albumin clearance induced by alpha-thrombin was reversible by washing thrombin from the endothelium

31

Thrombin Receptor Antagonism in Antiplatelet Therapy  

OpenAIRE

Activated platelets play a crucial role in the pathogenesis of atherothrombotic disease and its complications. Even under treatment of antiplatelet drugs, such as acetylsalicylic acid and P2Y12 antagonists, morbidity and mortality rates of thromboembolic complications remain high. Hence, the therapeutic inhibition of protease-activated receptor (PAR)-1, which is activated by thrombin, is a novel promising approach in antiplatelet therapy. Recent data suggest that PAR-1 is mainly involved in p...

Olivier, C.; Diehl, P.; Bode, C.; Moser, M.

2013-01-01

32

Thrombin inhibition by cyclic peptides from thrombomodulin.  

Science.gov (United States)

Peptides corresponding to the loop regions of the fourth, fifth, and sixth epidermal growth factor (EGF)-like domains of thrombomodulin (TM) have been synthesized and assayed for thrombin inhibition, as indicated by both inhibition of thrombin-mediated fibrinogen clotting and inhibition of the association of thrombin with TM that results in protein C activation. Peptides from the fifth EGF-like domain showed significant inhibition of fibrinogen clotting and protein C activation, whereas peptides from the fourth and sixth EGF-like domains were weak inhibitors in both assays. Two structural features were important for inhibitory potency of the peptides from the fifth EGF-like domain: cyclization by a disulfide bond and attachment of the "tail" amino acids C-terminal to the disulfide loop. Linear control peptides did not significantly inhibit clotting or protein C activation. The C-terminal loop alone, the "tail" peptide, or a mixture of the two were at least 10-fold less potent inhibitors of clotting or protein C activation. A more constrained peptide analog was designed by deletion of an isoleucine within the C5-C6 disulfide loop, TM52-1 + 5C. This analog was a better inhibitor in both assay systems, having a Ki for protein C activation of 26 microM. PMID:7613475

Lougheed, J. C.; Bowman, C. L.; Meininger, D. P.; Komives, E. A.

1995-01-01

33

Prophylactic thrombolysis by thrombin-activated latent prourokinase targeted to PECAM-1 in the pulmonary vasculature  

OpenAIRE

A recombinant prodrug, single-chain urokinase-type plasminogen activator (scuPA) fused to an anti–PECAM-1 antibody single-chain variable fragment (anti–PECAM scFv/scuPA) targets endothelium and augments thrombolysis in the pulmonary vasculature.1 To avoid premature activation and inactivation and to limit systemic toxicity, we replaced the native plasmin activation site in scFv/low-molecular-weight (lmw)–scuPA with a thrombin activation site, generating anti–PECAM scFv/uPA-T that (1) ...

Ding, Bi-sen; Hong, Nankang; Murciano, Juan-carlos; Ganguly, Kumkum; Gottstein, Claudia; Christofidou-solomidou, Melpo; Albelda, Steven M.; Fisher, Aron B.; Cines, Douglas B.; Muzykantov, Vladimir R.

2008-01-01

34

Subconjunctival and topical application of recombinant tissue plasminogen activator in rabbits / Uso tópico e subconjuntival de ativador de plasminogênio tecidual recombinante em coelhos  

Scientific Electronic Library Online (English)

Full Text Available Objetivo: Quantificar produtos de degradação de fibrina (PDF) após uso tópico e subconjunctival de ativador de plasminogênio tecidual recombinante (r-TPA) em coelhos. Métodos: Formação de fibrina foi induzida na câmara anterior em 25 coelhos. Cinco coelhos foram submetidos a injeção intracameral de [...] r-TPA (controle positivo). Dez coelhos foram submetidos a injeção subconjuntival de r-TPA e dez coelhos foram submetidos a instilação tópica de r-TPA. Amostras de humor aquoso foram coletados e uma análise quantitativa dos produtos de degradação de fibrina foi realizada. Resultados: Não foi observado diferença estatisticamente significativa na degradação de fibrina em nenhum dos momentos estudados quando comparados com o controle. Porém foi observado diferença estatisticamente significante na quantificação do produtos de degradação de fibrina no grupo controle e no grupo subconjuntival. Conclusão: Produtos de degradação de fibrina foi observado nas amostras do grupo subconjunctival, porém, provavelmente não foi suficiente para degradar a fibrin presente. r-TPA tópico não foi efetivo em absorver fibrina na câmara anterior. Abstract in english Purpose: To quantify fibrin degradation products after topical and subconjunctival administration of recombinant tissue plasminogen activator in rabbits. Methods: Fibrin formation was induced in the anterior chamber in 25 rabbits. Subsequently, five rabbits received an injection of r-TPA (positive [...] control) in the anterior chamber, another 10 received a subconjunctival injection of r-TPA, and the remaining 10 received instillations of topical r-TPA. Afterwards, samples of aqueous humor were collected and semi-quantitative analysis of fibrin degradation products (FDP) was performed. Results: No statistical differences were noted between the treatment and control groups at any time point. Fibrin degradation products semi-quantification showed statistical improvement in the control group and the subconjunctival group. Conclusion: Fibrin degradation products were observed in the anterior chamber after subconjunctival administration of r-TPA. However, it was probably not sufficient to cause fibrin degradation. Topical r-TPA did not effectively absorb anterior chamber fibrin.

José Ricardo de Abreu, Reggi; Richard Yudi, Hida; Milton Massato, Hida; Maria Cristina, Nishiwaki-Dantas; Hisashi, Suzuki.

2015-02-01

35

Thrombin induces endothelial arginase through AP-1 activation  

OpenAIRE

Arterial thrombosis is a common disease leading to severe ischemia beyond the obstructing thrombus. Additionally, endothelial dysfunction at the site of thrombosis can be rescued by l-arginine supplementation or arginase blockade in several animal models. Exposure of rat aortic endothelial cells (RAECs) to thrombin upregulates arginase I mRNA and protein levels. In this study, we further investigated the molecular mechanism of thrombin-induced arginase changes in endothelial cells. Thrombin s...

Zhu, Weifei; Chandrasekharan, Unni M.; Bandyopadhyay, Smarajit; Morris, Sidney M.; Dicorleto, Paul E.; Kashyap, Vikram S.

2009-01-01

36

Influence of Aromatic and Aliphatic Moieties on Thrombin Inhibitors Potency  

OpenAIRE

Thrombin is a plasma serine protease that plays a key role in coagulation and hemostasis but also in thromboembolic diseases. Direct thrombin inhibitors could be beneficial for future anticoagulant therapy in the prophylaxis of venous and arterial thrombosis as well as myocardial infarction. To design the efficient thrombin inhibitors we have synthesized and studied peptide-based inhibitors resistant to enzymatic degradation. Compounds with general formula X-DArg-D-Phe-OMe, where X = residue ...

Poyarkov, Alexey; Rocabayera, Xavier; Poyarkova, Svetlana; Kukhar, Valery

2008-01-01

37

Investigation of a thrombin-complexing protein associated with platelets  

International Nuclear Information System (INIS)

A fraction of the 125I-thrombin that binds to human platelets is taken into a sodium dodecyl sulfate-resistant 77k Da complex with a platelet factor. This platelet factor is in several respects similar to protease nexin I (PNI), a fibroblasts thrombin inhibitor. The complexes are of the right size, bind to agarose that has been derivatized with either anti-PNI antibody or heparin, do not form when the thrombin active site has been blocked with diisopropylphosphofluoridate, and do not appear on platelets when heparin is present. The interaction with the platelet surface may modulate the conformation and function of this platelet form of protease nexin I (PNIp) because: (i) an antibody against protease nexi I inhibited released PNIp, but not platelet-bound PNIp from complexing 125I-thrombin, and (ii) whereas PNIp extracted from platelets bound both thrombin and urokinase, platelet-bound PNIp bound only thrombin. In experiments employing several different platelet isolation methods, PNIp accounted for a large fraction of the rapid high affinity binding of 125I-thrombin to platelets. However, platelets isolated and maintained in the presence of metabolic inhibitors failed to take added thrombin into 125I-thrombine-PNIp complexes

38

Interactions of some commonly used drugs with human ?-thrombin.  

Science.gov (United States)

Adverse side effects of drugs are often caused by the interaction of drug molecules to targets other than the intended ones. In this study, we investigated the off-target interactions of some commercially available drugs with human ?-thrombin. The drugs used in the study were selected from Super Drug Database based on the structural similarity to a known thrombin inhibitor argatroban. Interactions of these drugs with thrombin were initially checked by in silico docking studies and then confirmed by thrombin inhibition assay using a fluorescence microplate-based method. Results show that the three commonly used drugs piperacillin (anti-bacterial), azlocillin (anti-bacterial), and metolazone (anti-hypertensive and diuretic) have thrombin inhibitory activity almost similar to that of argatroban. The Ki values of piperacillin, azlocillin, and metolazone with thrombin are .55, .95, and .62?nM, respectively. The IC50 values of piperacillin, azlocillin, and metolazone with thrombin are 1.7, 2.9, and 1.92?nM, respectively. This thrombin inhibitory activity might be a reason for the observed side effects of these drugs related to blood coagulation and other thrombin activities. Furthermore, these compounds (drugs) may be used as anti-coagulants as such or with structural modifications. PMID:24819365

Nair, Divya Gopalakrishnan; Narayanan, Sunilkumar Puthenpurackal; Chittalakkottu, Sadasivan

2015-05-01

39

A thrombin receptor in resident rat peritoneal macrophages  

International Nuclear Information System (INIS)

Resident rat peritoneal macrophages possess 6 x 10(2) high-affinity binding sites per cell for bovine thrombin with a Kd of 11 pM, and 7.5 x 10(4) low-affinity sites with a Kd of 5.8 nM. These binding sites are highly specific for thrombin. Half-maximal binding of 125I-labeled bovine thrombin is achieved after 1 min at 37 degrees C, and after 12 min at 4 degrees C. The reversibly bound fraction of the ligand dissociates according to a biexponential time course with the rate constants 0.27 and 0.06 min-1 at 4 degrees C. Part of the tracer remains cell-associated even after prolonged incubation, but all cell-associated radio-activity migrates as intact thrombin upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bound thrombin is minimally endocytosed as judged by the resistance to pH 3 treatment, and the receptor does not mediate a quantitatively important degradation of the ligand. The binding is not dependent on the catalytic site of thrombin, since irreversibly inactivated thrombin also binds to the receptor. 125I-labeled thrombin covalently cross-linked to its receptor migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr 160,000, corresponding to an approximate receptor size of Mr 120,000

40

Platelet tyrosine-specific protein phosphorylation is regulated by thrombin.  

OpenAIRE

Intact human platelets, terminally differentiated cells with no growth potential, were found to possess unusually high levels of tyrosine-specific protein phosphorylation. The physiological platelet activator thrombin transiently elevated platelet phosphotyrosine content, apparently through stimulation of one or more tyrosine-specific protein kinases. Immunoblotting with antiphosphotyrosine antiserum showed that thrombin caused dramatic changes in the tyrosine phosphorylation of a number of i...

Ferrell, J. E.; Martin, G. S.

1988-01-01

41

Characterization of the thrombin receptor and its involvement in stimulating cell proliferation  

International Nuclear Information System (INIS)

To study the role of high-affinity receptors in thrombin mitogenesis, experiments have been carried out in three main areas: (1) development and selection of a monoclonal antibody to the thrombin receptor; (2) use of synthetic peptides as receptor probes; and (3) purification and characterization of the thrombin receptor. The monoclonal antibody that was developed and selected, TR-9 inhibits from 80 to 100% of 125I-alpha-thrombin binding, exhibits an immunofluorescent pattern indistinguishable from that of thrombin bound to receptors on these cells, and selectively binds solubized thrombin receptors. By itself, TR-9 neither initiated DNA synthesis nor blocked thrombin initiation, but TR-9 in combination with alpha-thrombin, gamma-thrombin or phorbol myristate acetate (PMA) stimulated thymidine incorporation up to 3-fold over controls. These results demonstrate that the binding of TR-9 to the thrombin receptor can mimic the effects of high-affinity interaction of thrombin with this receptor

42

Injury to cultured endothelial cells by thrombin-stimulated platelets  

International Nuclear Information System (INIS)

In vivo, stimulated platelets may injure the endothelium. We have used cultured endothelial cells to assess endothelial cell damage caused by platelet stimulation with thrombin. Endothelial cells were cultured from umbilical veins and semiconfluent cultures were labeled with Na251CrO4. Twenty four hours later washed human platelets (final concentration 200,000 platelets/microliters) and thrombin (final concentration 4 units/ml) were added to the medium and the culture dish was shaken for 15 minutes. The percentage of cells detached from the culture dish and the percentage of 51Cr lost from the endothelial cells into the ambient fluid during the shaking were determined and used as indicators of cell injury. Increased percentages of loosened cells and 51Cr in the ambient fluid were observed with platelet suspension and thrombin compared to controls with neither platelet suspension nor thrombin and controls with either platelet suspension or thrombin. The platelet-free supernatant obtained after reaction of the platelets with thrombin also increased the percentage of loosened cells, but it did not increase the percentage of 51Cr in the ambient fluid to a significant degree. Thrombin alone caused a moderate loss of 51Cr, but no increased loosening of cells. Treatment of the platelets with acetylsalicylic acid prior to the experiment depressed the detachment effect of thrombin-stimulated plateletsnt effect of thrombin-stimulated platelets, but did not alter the effect on the release of 51Cr into the ambient fluid. Scanning and transmission electron microscopy of cultured endothelial cells exposed to thrombin-stimulated platelets confirmed the presence of loosening and injury to the endothelial cells

43

Planar Hall magnetoresistive aptasensor for thrombin detection.  

Science.gov (United States)

The use of aptamer-based assays is an emerging and attractive approach in disease research and clinical diagnostics. A sensitive aptamer-based sandwich-type sensor is presented to detect human thrombin using a planar Hall magnetoresistive (PHR) sensor in cooperation with superparamagnetic labels. A PHR sensor has the great advantages of a high signal-to-noise ratio, a small offset voltage and linear response in the low-field region, allowing it to act as a high-resolution biosensor. In the system presented here, the sensor has an active area of 50 µm × 50 µm with a 10-nm gold layer deposited onto the sensor surface prior to the binding of thiolated DNA primary aptamer. A polydimethylsiloxane well of 600-µm radius and 1-mm height was prepared around the sensor surface to maintain the same specific area and volume for each sensor. The sensor response was traced in real time upon the addition of streptavidin-functionalized magnetic labels on the sensor. A linear response to the thrombin concentration in the range of 86 pM-8.6 µM and a lower detection limit down to 86 pM was achieved by the proposed present method with a sample volume consumption of 2 µl. The proposed aptasensor has a strong potential for application in clinical diagnosis. PMID:24727201

Sinha, B; Ramulu, T S; Kim, K W; Venu, R; Lee, J J; Kim, C G

2014-09-15

44

Reduced surface expression and binding of fibronectin by thrombin-stimulated thrombasthenic platelets.  

OpenAIRE

Thrombin stimulation results in increased surface expression of endogeneous fibronectin and binding of plasma fibronectin to human platelets. Platelets of patients with Glanzmann's thrombasthenia, a bleeding disorder, exhibit reduced thrombin-induced platelet aggregation, little or no clot retraction, and abnormal platelet spreading on glass surfaces. Thrombin stimulation of patient platelets from four thrombasthenic kindreds resulted in little fibronectin binding. Nevertheless, thrombin did ...

Ginsberg, M. H.; Forsyth, J.; Lightsey, A.; Chediak, J.; Plow, E. F.

1983-01-01

45

APTAMER-BASED SERRS SENSOR FOR THROMBIN DETECTION  

Energy Technology Data Exchange (ETDEWEB)

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

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

2008-07-02

46

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

International Nuclear Information System (INIS)

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

47

The interaction of thrombin with platelet protease nexin  

International Nuclear Information System (INIS)

Thrombin interacts with a platelet protein which is immunologically related to fibroblast protease nexin and has been termed platelet protease nexin I (PNI). Conflicting hypotheses about the relationship of the thrombin-PNI complex formation to platelet activation have been proposed. The studies presented here demonstrate that the platelet-associated and supernatant complexes with added 125I-thrombin are formed only under conditions which produce platelet activation in normal and chymotrypsin-modified platelets. The platelet-associated complex is formed prior to the appearance of complexes in supernatants. Appearance of the supernatant complex coincides with the appearance of thrombospondin in the reaction supernatants. Excess native thrombin, dansylarginine N-(3-ethyl-1,5-pentanediyl) amide or hirudin can prevent radiolabeled platelet-associated complex formation if added before 125I-thrombin. DAPA or hirudin can prevent or dissociate complex formation if added up to one minute after thrombin but not at later time points. The surface associated complex is accessible to trypsin although a portion remains with the cytoskeletal proteins when thrombin-activated platelets are solubilized with Triton X 100. The surface-associated complex formation parallels many aspects of the specific measurable thrombin binding, yet it does not appear to involve other identified surface glycoprotein thrombin receptors or substrates. Although the time course of appearance of the complexestime course of appearance of the complexes in supernatants is consistent with other data which suggest that PNI may be released from platelet granules during platelet activation, other explanations for the appearance of PNI on the platelet surface and in supernatants during platelet activation are possible

48

Injury to cultured endothelial cells by thrombin-stimulated platelets  

Energy Technology Data Exchange (ETDEWEB)

In vivo, stimulated platelets may injure the endothelium. We have used cultured endothelial cells to assess endothelial cell damage caused by platelet stimulation with thrombin. Endothelial cells were cultured from umbilical veins and semiconfluent cultures were labeled with Na/sub 2/ /sup 51/CrO/sub 4/. Twenty four hours later washed human platelets (final concentration 200,000 platelets/microliters) and thrombin (final concentration 4 units/ml) were added to the medium and the culture dish was shaken for 15 minutes. The percentage of cells detached from the culture dish and the percentage of /sup 51/Cr lost from the endothelial cells into the ambient fluid during the shaking were determined and used as indicators of cell injury. Increased percentages of loosened cells and /sup 51/Cr in the ambient fluid were observed with platelet suspension and thrombin compared to controls with neither platelet suspension nor thrombin and controls with either platelet suspension or thrombin. The platelet-free supernatant obtained after reaction of the platelets with thrombin also increased the percentage of loosened cells, but it did not increase the percentage of /sup 51/Cr in the ambient fluid to a significant degree. Thrombin alone caused a moderate loss of /sup 51/Cr, but no increased loosening of cells. Treatment of the platelets with acetylsalicylic acid prior to the experiment depressed the detachment effect of thrombin-stimulated platelets, but did not alter the effect on the release of /sup 51/Cr into the ambient fluid. Scanning and transmission electron microscopy of cultured endothelial cells exposed to thrombin-stimulated platelets confirmed the presence of loosening and injury to the endothelial cells.

Jorgensen, L.; Grothe, A.G.; Larsen, T.; Kinlough-Rathbone, R.L.; Mustard, J.F.

1986-04-01

49

Regulation of mesangial cell adhesion and shape by thrombin.  

Science.gov (United States)

Adenosine 3',5'-cyclic monophosphate (cAMP) elevation in cultured rat mesangial cells causes urokinase-dependent adhesion loss, stress-fiber fragmentation, and shape change. Thrombin cleaves single-chain urokinase (scu-PA), causing its inactivation, but not two-chain u-PA [tcu-plasminogen activator (PA)] or tissue-type PA. We tested the ability of thrombin to inhibit the effects of cAMP elevation in mesangial cells and inactivate cell-associated scu-PA. In an assay of trypsin-sensitive adhesion, 65.9% of control cells and 5.5% of cells treated with isoproterenol + methylisobutylxanthine (IM) remained adherent. In the presence of 0.01, 0.1, 1.0, and 10.0 unit/ml thrombin, 20.9, 46.6, 50.4, and 53.3%, respectively, of IM-treated cells remained attached. Thrombin also inhibited stress-fiber fragmentation and shape change. The effects of thrombin were blocked by hirudin or antithrombin III plus heparin. Direct zymography in gels containing gelatin and plasminogen revealed loss of a closely spaced pair of PA bands with thrombin treatment (1.0 unit/ml). Hirudin blocked the loss. alpha-Thrombin inactivated by diisopropyl fluorophosphate neither inhibited shape change nor caused loss of the PA bands; however, gamma-thrombin was nearly as active as native alpha-thrombin in both regards. Pretreatment of the cells with as little as 1.0 unit/ml thrombin for 1.0 min caused marked inhibition of shape change and near total loss of the slower migrating u-PA band (of the doublet). The faster migrating band was inhibited less. The results indicate that the slower migrating band represents scu-PA; the nature of the faster migrating band is less certain. Thrombin reversed the adhesion loss and shape change caused by 8-(4-chlorophenylthio)-cAMP and MIX. Thus physiological concentrations of thrombin rapidly inactivate mesangial cell scu-PA and inhibit and reverse cAMP-stimulated adhesion loss and shape change.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1652208

Glass, W F; Rampt, E; Garoni, J A; Fenton, J W; Kreisberg, J I

1991-08-01

50

Cytochalasins inhibit arachidonic acid metabolism in thrombin-stimulated platelets.  

OpenAIRE

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

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

1982-01-01

51

Thrombin binding to human brain and spinal cord  

International Nuclear Information System (INIS)

Thrombin, a serine protease that regulates hemostasis, has been shown to stimulate the formation of cGMP in murine neuroblastoma cells. The nervous system in vivo thus may be postulated to respond to this blood-borne factor after it breaches the blood-brain barrier, as in trauma. Human alpha-thrombin was radiolabeled with 125I and shown to bind rapidly, reversibly, and with high affinity to human brain and spinal cord. These findings indicate the presence of specific thrombin-binding sites in nervous tissue and may have important clinical implications

52

Thrombin induces broad spectrum proteolysis in human serum samples  

OpenAIRE

Background: During clotting, ? thrombin cleaves fibrinogen releasing fibrinopeptide A (FPA). FPA is easily identified in serum using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Using MALDI-TOF MS, we observed multiple, progressively shorter fragments of serum FPA. Following ambient incubation of serum, variations in the content of FPA fragments occur over time. Denaturation of ? thrombin by heating the serum sample appears to minimize this ...

O Mullan, Patrick; Craft, David; Yi, Jizu; Gelfand, Craig A.

2008-01-01

53

Direct thrombin inhibitors, but not the direct factor Xa inhibitor rivaroxaban, increase tissue factor-induced hypercoagulability in vitro and in vivo  

Science.gov (United States)

Background Increased hypercoagulability has been reported with low doses of direct thrombin inhibitors but not with direct factor Xa inhibitors. Objectives To compare the effects of rivaroxaban with those of melagatran and dabigatran on thrombin generation (TG) and tissue factor-induced hypercoagulability and to explore the possible involvement of the thrombin–thrombomodulin/activated protein C system. Methods In normal human plasma and in protein C-deficient plasma, TG was investigated in vitro in the presence and absence of recombinant human soluble thrombomodulin (rhs-TM). TG was determined by calibrated automated thrombography and an ELISA for prothrombin fragments 1+2 (F1+2). In an in vivo rat model, hypercoagulability was induced by tissue factor; levels of thrombin–antithrombin (TAT) and fibrinogen and the platelet count were determined. Results Rivaroxaban inhibited TG in a concentration-dependent manner. In the absence of rhs-TM, melagatran and dabigatran also inhibited TG concentration dependently. However, in the presence of rhs-TM, lower concentrations of melagatran (119–474 nmol L–1) and dabigatran (68–545 nmol L?1) enhanced endogenous thrombin potential, peak TG, and F1+2 formation in normal plasma but not in protein C-deficient plasma. In vivo, rivaroxaban dose-dependently inhibited TAT generation, whereas melagatran showed a paradoxical effect, with an increase in TAT and a small decrease in fibrinogen and platelet count at lower doses. Conclusion Low concentrations of the direct thrombin inhibitors melagatran and dabigatran enhanced TG and hypercoagulability, possibly via inhibition of the protein C system. In contrast, rivaroxaban reduced TG and hypercoagulability under all conditions studied, suggesting that it does not suppress this negative-feedback system. PMID:24766850

Perzborn, E; Heitmeier, S; Buetehorn, U; Laux, V

2014-01-01

54

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

Directory of Open Access Journals (Sweden)

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

Kristensen Torsten

2009-05-01

55

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

DEFF Research Database (Denmark)

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

Valnickova, Zuzana; Thaysen-Andersen, Morten

2009-01-01

56

High density fermentation and activity of a recombinant lumbrokinase (PI239) from Pichia pastoris.  

Science.gov (United States)

A system for the expression of recombinant lumbrokinase (rPI239) was developed in the yeast Pichia pastoris. A total supernatant protein content of 0.174 g/L of high density fermentation broth was obtained. The rPI239 exhibited in vitro fibrinolytic activity. The in vivo activity of rPI239 was measured by prothrombin time, kaolin part thrombin time, thrombin time, and fibrinolytic activity. This work presents the high-density fermentation of rPI239 from P. pastoris and shows that the recombinant protein has similar fibrinolytic activity both in vivo and in vitro. PMID:17118673

Ge, Tao; Fu, Shi-Hong; Xu, Li-Hong; Tang, Qing; Wang, Huan-Yu; Guan, Kun-Ping; Liang, Guo-Dong

2007-03-01

57

Antithrombotic effects and bleeding time of thrombin inhibitors and warfarin in the rat.  

Science.gov (United States)

Warfarin limits the synthesis of y-glutamyl carboxylated forms of coagulation factors, factor II, factor VII, factor IX, and factor X, protein C, and protein S and as a result impairs the function of these proteins. In contrast, direct inhibitors of thrombin only affect one enzyme in the coagulation cascade. The aim of this study was to investigate the antithrombotic effect and the slope of the dose-response curves of the multifactorial coagulation inhibitor warfarin in comparison with the single factor low-molecular-weight thrombin inhibitors melagatran and inogatran. An arterial thrombosis model in rats was used, and vessel damage was induced by topical application of ferric chloride to the carotid artery. The slopes of the dose-response curves were 3.6, 1.8, 1.1, and 1.2, for warfarin, heparin, inogatran, and melagatran, respectively. For warfarin the antithrombotic effect increased from 23% to 81% when the dose was doubled. In contrast, 10-fold increases in the doses of inogatran and melagatran were necessary to obtain a similar increase in antithrombotic effect. The doses needed to obtain 80% antithrombotic effect for heparin, warfarin, and melagatran were investigated in a tail transection bleeding model. For heparin, this dose significantly prolonged the bleeding time and the blood loss; for warfarin, only the total bleeding time was increased while for melagatran there was no increase in bleeding. We conclude that, thrombin inhibitors affecting only one enzyme in the coagulation cascade seem preferable to inhibitors affecting multiple enzymes, such as warfarin, due to shallower dose-response curves and a wider therapeutic interval. PMID:10326765

Elg, M; Gustafsson, D; Carlsson, S

1999-05-01

58

A plasmin-activatable thrombin inhibitor reduces experimental thrombosis and assists experimental thrombolysis in murine models.  

Science.gov (United States)

The leech protein hirudin is a potent natural thrombin inhibitor. Its potential as an antithrombotic agent is limited by its promotion of bleeding. We attempted to modify this profile by positioning albumin and a plasmin cleavage site on its N-terminus, in recombinant protein HSACHV3 [comprising hirudin variant 3 (HV3) fused to the C-terminus of human serum albumin (HSA) via a plasmin cleavage site (C)], Previously we showed that HSACHV3 inhibited thrombin in a plasmin-dependent manner, and that, unlike HV3, it did not increase bleeding in vivo when administered to mice. Here we tested HSACHV3 for the ability to reduce thrombosis and assist enzymatic thrombolysis in animal models. Intravenous administration of HSACHV3, but not a control protein lacking the plasmin cleavage site (HSAHV3), reduced thrombus weight by 2.1-fold in the ferric chloride-injured mouse vena cava. Similarly, thrombi formed in a rabbit jugular vein stasis model were 1.7-fold lighter in animals treated with HSACHV3 compared to those receiving HSAHV3. Administration of 60 mg/kg body weight HSACHV3 prolonged the time to occlusion in the ferric chloride-injured mouse carotid artery by threefold compared to vehicle controls, while equimolar HSAHV3 had no effect. HSACHV3 had no ability to restore flow to the murine carotid arteries occluded by ferric chloride treatment, but combining HSACHV3 (60 mg/kg) with recombinant mutant tissue plasminogen activator (TNKase) significantly reduced the time to restore patency to the artery compared to TNKase alone. Unlike unfused HV3, HSACHV3 did not increase bleeding in a mouse liver laceration model. Our results show that HSACHV3 acts as an antithrombotic agent that does not promote bleeding and which speeds the time to flow restoration when used as an adjunct to pharmacological thrombolysis in animal models. PMID:25481811

Sheffield, W P; Eltringham-Smith, L J; Gataiance, S; Bhakta, V

2015-05-01

59

Thrombin Increases Expression of Fibronectin Antigen on the Platelet Surface  

Science.gov (United States)

Fibronectins (fn) are adhesive glycoproteins which bind to collagen and to fibrin and appear to be important in cellular adhesion to other cells or surfaces. Fn-related antigen is present in human platelets, suggesting a possible role for fn in the adhesive properties of platelets. We have studied the localization of fn in resting and thrombin-stimulated platelets by immunofluorescence and quantitative binding of radiolabeled antibody. In resting fixed platelets, variable light surface staining for fn was observed. When these cells were made permeable to antibody with detergent, staining for fn was markedly enhanced and was present in a punctate distribution, suggesting intracellular localization. Stimulation with thrombin, which is associated with increased platelet adhesiveness, resulted in increased staining for fn antigen on intact platelets. These stimulated cells did not leak 51Cr nor did they stain for F-actin, thus documenting that the increased fn staining was not due to loss of plasma membrane integrity. The thrombin-induced increase in accessible platelet fn antigen was confirmed by quantitative antibody binding studies in which thrombin-stimulated platelets specifically bound 15 times as much radiolabeled F(ab')2 anti-fn as did resting cells. Thus, thrombin stimulation results in increased expression of fn antigen on the platelet surface. Here it may participate in interactions with fibrin, connective tissue, or other cells.

Ginsberg, Mark H.; Painter, Richard G.; Forsyth, Jane; Birdwell, Charles; Plow, Edward F.

1980-02-01

60

Scanning electrochemical microscopy for study of aptamer-thrombin interfacial interactions on gold disk microelectrodes.  

Science.gov (United States)

A feasibility for the determination of thrombin on gold disk microelectrodes (GDMs) using scanning electrochemical microscopy (SECM) is reported. The assembly process step-by-step of thrombin aptasensor on GDMs is monitored by SECM. SECM analysis reveals the immobilization of thrombin aptamers on GDMs. The interaction between thrombin aptamers and thrombin on GDMs is imaged by SECM with feedback mode using ferrocenemethanol as an electrochemical mediator. The formation of thrombin/thrombin aptamer complex on GDMs results in a decrease in the tip peak current on spatial SECM images. This method is able to linearly and selectively detect thrombin over a linear range from 10(-12) to 10(-5)M with a detection limit of 6.07 fM. PMID:24407695

Bai, Huei-Yu; del Campo, F Javier; Tsai, Yu-Chen

2014-03-01

61

Thrombin receptor antagonism in antiplatelet therapy.  

Science.gov (United States)

Activated platelets play a crucial role in the pathogenesis of atherothrombotic disease and its complications. Even under treatment of antiplatelet drugs, such as acetylsalicylic acid and P2Y12 antagonists, morbidity and mortality rates of thromboembolic complications remain high. Hence, the therapeutic inhibition of protease-activated receptor (PAR)-1, which is activated by thrombin, is a novel promising approach in antiplatelet therapy. Recent data suggest that PAR-1 is mainly involved in pathological thrombus formation, but not in physiological hemostasis. Therefore, PAR-1 inhibition offers the possibility to reduce atherothrombotic events without increasing bleeding risk. So far, two emerging PAR-1 antagonists have been tested in clinical trials: vorapaxar (SCH530349; Merck & Co., Whitehouse Station, NJ, USA) and atopaxar (E5555; Eisai, Tokyo, Japan). Although in TRA-CER vorapaxar showed an unfavorable profile for patients with acute coronary syndrome in addition to standard therapy, it revealed promising results for patients with prior myocardial infarction in TRA 2P-TIMI50. Depending on the status of clinical approval, vorapaxar might be an option for patients with peripheral arterial disease to reduce limb ischemia. The second PAR-I antagonist, atopaxar, tended towards reducing major cardiovascular adverse events in acute coronary syndrome patients in a phase II trial. However, although statistically not significant, bleeding events were numerically increased in atopaxar-treated patients compared with placebo. Furthermore, liver enzymes were elevated and the relative corrected QT interval was prolonged in atopaxar-treated patients. Currently, the development of atopaxar by Eisai is discontinued. The future of this novel class of antithrombotic drugs will depend on the identification of patient groups in which the risk-benefit ratio is favorable. PMID:25135289

Olivier, C; Diehl, P; Bode, C; Moser, M

2013-06-01

62

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

OpenAIRE

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 o...

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

2011-01-01

63

Identification of the thrombin receptor on human platelets by chemical crosslinking.  

OpenAIRE

To identify the molecular site of thrombin binding to the platelet membrane, we covalently linked 125I-thrombin to platelets by using the bifunctional chemical cross-linking agents disuccinimidyl suberate and dithiobis(succinimidyl propionate). The proteins cross-linked to 125I-thrombin by this method were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and followed by autoradiography. Two radiolabeled thrombin complexes were identified, a major species of Mr approximate...

Takamatsu, J.; Horne, M. K.; Gralnick, H. R.

1986-01-01

64

Fractal gold modified electrode for ultrasensitive thrombin detection  

Science.gov (United States)

We report a label-free and ultrasensitive aptasensor based on a fractal gold modified (FracAu) electrode for thrombin detection with a femtomolar detection limit. The FracAu electrode was prepared by electrodeposition of hydrogen tetrachloroaurate (HAuCl4) onto a bare indium tin oxide (ITO) electrode surface. After this process the electrode was characterized by SEM. A thiol-modified aptamer against thrombin was immobilized on the FracAu electrode through a self-assembling process. Upon thrombin binding, the interfacial electron transfer of the FracAu electrode was perturbed by the formation of an aptamer-thrombin complex. The concentration of thrombin in the sample solution was determined by measuring the change in the oxidation peak current of hydroxymethyl ferrocene (C11H12FeO) with differential pulse voltammetry (DPV). The current response (reduced peak current) had a linear relationship with the logarithm of thrombin concentrations in the range of 10-15 to 10-10 M with a detection limit of 5.7 fM. Furthermore, the as-prepared FracAu electrode exhibited high selectivity. The application of FracAu electrodes may be extended to prepare other types of biosensors, such as immunosensors, enzyme biosensors and DNA biosensors. These results show that FracAu electrodes have great promise for clinical diagnosis of disease-related biomarkers.We report a label-free and ultrasensitive aptasensor based on a fractal gold modified (FracAu) electrode for thrombin detection with a femtomolar detection limit. The FracAu electrode was prepared by electrodeposition of hydrogen tetrachloroaurate (HAuCl4) onto a bare indium tin oxide (ITO) electrode surface. After this process the electrode was characterized by SEM. A thiol-modified aptamer against thrombin was immobilized on the FracAu electrode through a self-assembling process. Upon thrombin binding, the interfacial electron transfer of the FracAu electrode was perturbed by the formation of an aptamer-thrombin complex. The concentration of thrombin in the sample solution was determined by measuring the change in the oxidation peak current of hydroxymethyl ferrocene (C11H12FeO) with differential pulse voltammetry (DPV). The current response (reduced peak current) had a linear relationship with the logarithm of thrombin concentrations in the range of 10-15 to 10-10 M with a detection limit of 5.7 fM. Furthermore, the as-prepared FracAu electrode exhibited high selectivity. The application of FracAu electrodes may be extended to prepare other types of biosensors, such as immunosensors, enzyme biosensors and DNA biosensors. These results show that FracAu electrodes have great promise for clinical diagnosis of disease-related biomarkers. Electronic supplementary information (ESI) available: Fig. S1 showing current reductions of FracAu and bulk Au biosensors, Fig. S2 showing cyclic voltammogram of FracAu and bulk Au electrode in 0.5 M H2SO4 aqueous solution. See DOI: 10.1039/c2nr30826f

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

2012-05-01

65

Multiple active forms of thrombin. IV. Relative activities of meizothrombins  

International Nuclear Information System (INIS)

The prothrombin activation intermediates meizothrombin and meizothrombin(desF1) (meizothrombin that has been autoproteolyzed to remove fragment 1) have been obtained in a relatively pure, active form with minimal autolysis, making them suitable for enzymatic characterization. When compared at equimolar concentrations, alpha-thrombin, fragment 1.2+ alpha-thrombin, meizothrombin(desF1), and meizothrombin have approximately 100, 100, 10, and 1% activity, respectively, toward the macromolecular substrates factor V, fibrinogen, and platelets. The difference in activity of these four enzymes cannot be attributed to alterations in the catalytic triad, as all four enzymes have nearly identical catalytic efficiency toward the chromogenic substrate S2238. Further, the ability of meizothrombin and meizothrombin(desF1) to activate protein C was 75% of the activity exhibited by alpha-thrombin or fragment 1.2+ alpha-thrombin. All four enzymes bind to thrombomodulin, as judged by the enhanced rate of protein C activation upon preincubation of the enzymes with thrombomodulin. The extent of rate enhancement varied, with meizothrombin/thrombomodulin exhibiting only 50% of the alpha-thrombin/thrombomodulin rate. This difference in rate is not due to a decreased affinity of the meizothrombin for thrombomodulin since the apparent dissociation constants for the alpha-thrombin-thrombomodulin complex and the meizothrombin-thrombomodulin complex are virtually identical. The difference in the irtually identical. The difference in the observed rate is due in part to the higher Km for protein C exhibited by the meizothrombin-thrombomodulin complex. Incubation of the thrombomodulin-enzyme complex with phospholipid vesicles caused an increase in the protein C activation rates. The kinetic constants for protein C activation in the presence of phospholipid are virtually identical for these enzyme-thrombomodulin complexes

66

Radioimmunoassay for fibrinopeptide A using a tripeptide thrombin inhibitor  

International Nuclear Information System (INIS)

In a radioimmunoassay method for determining the concentration of fibrinopeptide A in plasma, a sample of blood is collected, a thrombin inhibitor is added and plasma is separated. The plasma is contacted under radioimmunoassay competitive binding conditions with a sufficient amount of an antibody to fibrinopeptide A and radioactively labelled fibrinoepetide A. The antibody bound fibrinopeptide A is separated from the unbound fibrinopeptide A and the radioactivity measured. An improvement comprises using D-Phe-Pro-ArgCh2Cl or its hydrocloric, hydroflouric, acetic, or citric acid addition salts as the thrombin inhibitor. The reagent may also include a chelating agent and an antiproteolytic agent

67

Preparation and characterization of human thrombin for use in a fibrin glue.  

Science.gov (United States)

Cryoprecipitate is frequently combined with thrombin to produce a fibrin sealant to enhance haemostasis during surgical procedures. We evaluated the thrombin produced from plasma using the Thrombin Processing Device (TPD)trade mark (Thermogenesis, Rancho Cordova, CA, USA). Plasma (250 mL) was processed in the CryoSeal FS System using the CP-3 disposable to produce cryoprecipitate by automated freezing and thawing. Simultaneously, thrombin was generated using the attached TPD. The cryoprecipitate and thrombin were harvested after approximately 50 min and then frozen and stored at -80 degrees C until analysis of total protein, fibrinogen, factor VIII (FVIII) activity, von Willebrand factor (vWF) and thrombin activity. Sodium dodecyl sulphate (SDS) gel electrophoresis was used to compare thrombin. After combining the thrombin with cryoprecipitate, the rate of clot initiation and strength was measured using a Thromboelastograph (TEG) (Haemoscope Corp, Skokie, IL, USA). Cryoprecipitate was produced, with a fibrinogen concentration of 22 +/- 7.7 g L(-1) (20 +/- 2% recovery), FVIII activity of 14.2 +/- 4.0 IU mL(-1) and vWF of 19.9 +/- 5.2 IU mL(-1). The separate thrombin product had a concentration of 64.3 +/- 16.7 IU mL(-1) of thrombin and a total protein of 0.39 +/- 0.1 g, with SDS gel electrophoresis showing a major band at 37 kD, as did the commercial human thrombin. The TEG curves of cryoprecipitate and TPD-produced or commercial thrombin were compared. The R values (time to clot initiation) were somewhat slower with the TPD-produced thrombin, but the maximum strength (MA) of the clots was similar. In conclusion, human thrombin can be produced during automated cryoprecipitate production. This thrombin is in sufficient concentration to initiate clotting and cross-linking of fibrin from cryoprecipitate to produce an entirely autologous fibrin glue. PMID:17561860

Rock, G; Neurath, D; Semple, E; Harvey, M; Freedman, M

2007-06-01

68

Thrombin regulates components of the fibrinolytic system in human mesangial cells  

Energy Technology Data Exchange (ETDEWEB)

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

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

1990-11-01

69

Catalytically competent human and bovine zeta-thrombin and chimeras generated from unfolded polypeptide chains.  

OpenAIRE

Human and bovine alpha-thrombin cleaved at the B-chain by chymotrypsin generates catalytically competent zeta-thrombins, which are comprised of two noncovalently linked fragments: a 36-(human) or 49-(bovine) residue A-chain linked by a disulfide to B-chain residues B1-148 (zeta 1-thrombin) and B-chain residues B149-259 (zeta 2-thrombin). Human and bovine D-Phe-Pro-Arg-CH2-zeta- and PhMeSO2-zeta-thrombins were prepared by reaction of the active-site histidine (H-B43) and serine (S-B205) with P...

Lewis, S. D.; Brezniak, D. V.; Fenton, J. W.; Shafer, J. A.

1992-01-01

70

Thrombin induces rapid PAR1-mediated non-classical FGF1 release  

International Nuclear Information System (INIS)

Thrombin induces cell proliferation and migration during vascular injury. We report that thrombin rapidly stimulated expression and release of the pro-angiogenic polypeptide fibroblast growth factor 1 (FGF1). Thrombin failed to induce FGF1 release from protease-activated receptor 1 (PAR1) null fibroblasts, indicating that this effect was dependent on PAR1. Similarly to thrombin, FGF1 expression and release were induced by TRAP, a specific oligopeptide agonist of PAR1. These results identify a novel aspect of the crosstalk between FGF and thrombin signaling pathways which both play important roles in tissue repair and angiogenesis

71

Expression and function of thrombin receptors (PAR1, PAR3 and PAR4) in human umbilical vein endothelial cells: thrombin-mediated CX3CL1 expression  

OpenAIRE

Upregulation of the inflammatory pathways is a major event in pathological condition. Thrombin, the key protease involved in coagulation process has been desribed as a potent activator of endothelial cells. This study shows that response to thrombin might be potentiated in endothelial cells by infection via a common virus, the human cytomegalovirus (HCMV). Indeed, HCMV induces expression of thrombin receptors PAR1 and PAR3 but not of PAR4. This effect of HCMV may be relevant for various funct...

Popovic, Milan

2008-01-01

72

A Guided Mode Resonance Aptasensor for Thrombin Detection  

Directory of Open Access Journals (Sweden)

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

Wen-Yih Chen

2011-09-01

73

Effects of thrombin on the integrity of monolayers of cultured human endothelial cells  

International Nuclear Information System (INIS)

51Cr-prelabelled endothelial cells (EC) in confluent monolayers were incubated in RPMI 1640 + foetal calf serum 20% (v/v) to which purified thrombin was added. Thrombin (greater than or equal to 0.1 NIH U/ml) significantly accelerated 51Cr-release and caused extensive but reversible cell contraction. Thrombin-exposed EC reacted to a new dose of thrombin with no appreciable shape change, but 51Cr-efflux was again accelerated. EC exposed to thrombin pretreated with N-bromosuccinimide (modifying the macromolecular site) or phenylmethylsulfonyl fluoride (blocking the serine site) retained normal morphology and did not leak excess amounts of 51Cr. Antithrombin III also inhibited the effect of thrombin. Pretreatment of EC with either indomethacin, aspirin, sulfinpyrazone, pronase or neuraminidase did not influence the effect of subsequent thrombin exposure

74

Regulation of thrombin generation by TFPI in plasma without and with heparin.  

Science.gov (United States)

The purpose of this study was to investigate the impact of recombinant glycosylated TFPI (rg-TFPI) from BHK cells, nonglycosylated TFPI (r-TFPI) from Escherichia coli, and truncated TFPI (1-161) on thrombin generation (TG) in plasma treated with and without heparin in vitro and ex vivo. Fasting plasma samples were collected from 6 healthy persons. TG was assessed by the calibrated automated thrombography (CAT) method. The addition of increasing concentrations (0-200 ng/mL) of different TFPI caused a 5% to 30% prolongation of lag time for TF (3.0 pM) induced TG, with the most pronounced effect for rg-TFPI and the least pronounced effect for truncated TFPI, but without affecting endogenous thrombin potential (ETP) in TF-induced coagulation. Removal of native TFPI from plasma by anti-TFPI IgG treatment shortened lag time by 35 +/- 4% without affecting ETP. Increasing concentrations (0-200 ng/mL) of various TFPI in the presence of low heparin concentrations (0.1 IU/mL) prolonged lag time and decreased ETP by 25% to 75% with the most prominent effect promoted by glycosylated full-length TFPI. The effect of neutralizing antibodies against TFPI and antithrombin (AT) was studied in plasma in the presence of heparin administered in vitro or ex vivo. The results revealed that TFPI and AT acted in synergy as inhibitors of coagulation in terms of the effect on both initiation (lag time) and propagation (ETP). Our data demonstrated that the CAT assay appropriately assessed the impact of TFPI on initiation and propagation of TG in a physiological plasma milieu with and without heparin. TFPI contributed significantly to regulation of coagulation initiation (lag time). The C-terminal region and, to a lesser extent, glycosylation of the TFPI molecule were essential for its anticoagulant function in the absence and presence of heparin. PMID:19218095

Brodin, Ellen; Appelbom, Hege; Osterud, Bjarne; Hilden, Ida; Petersen, Lars C; Hansen, John-Bjarne

2009-03-01

75

Nonradiative recombination in semiconductors  

CERN Document Server

In recent years, great progress has been made in the understandingof recombination processes controlling the number of excessfree carriers in semiconductors under nonequilibrium conditions. As a result, it is now possible to give a comprehensivetheoretical description of these processes. The authors haveselected a number of experimental results which elucidate theunderlying physical problems and enable a test of theoreticalmodels. The following topics are dealt with: phenomenological theory ofrecombination, theoretical models of shallow and deep localizedstates, cascade model of carrier captu

Abakumov, VN; Yassievich, IN

1991-01-01

76

Thrombin increases inflammatory cytokine and angiogenic growth factor secretion in human adipose cells in vitro  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Abdominal obesity is associated with pro-thrombotic and inflammatory states. Therefore, the purpose of this study was to examine the expression of thrombin receptors (PAR1 and PAR4 human adipose tissue and whether thrombin stimulates an inflammatory cytokine and growth factor profile in human adipose tissue. Methods Human adipose tissue, isolated preadipocytes and differentiated adipocytes were used in this study. PAR1 and PAR4 mRNA and protein were detected by RT-PCR and immunoblot analysis in both adipose tissue and adipose microvessels. In separate studies, IL-1?, IL-6, MCP-1, TNF-?, IL-10, FGF-2, VEGF, and PDGF production were measured from adipose tissue (n = 5, adipocytes (n = 5, and preadipocytes (n = 3 supernatants with and without thrombin (1 or 10 U/ml; 24 hrs treatment. Results Thrombin increased cytokine secretion of IL-1?, IL-6, MCP-1 and TNF-? and growth factor secretion of VEGF from adipocytes along with MCP-1 and VEGF from preadipocytes. The direct thrombin inhibitor lepirudin given in conjunction with thrombin prevented the thrombin-mediated increase in cytokine and growth factor secretion. Conclusion Here we show that thrombin PAR1 and PAR4 receptors are present and that thrombin stimulates inflammatory cytokine generation and growth factor release in human adipose tissue and cells in vitro. These data suggest that thrombin may represent a molecular link between obesity and associated inflammation.

Phillips Shane A

2009-03-01

77

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

International Nuclear Information System (INIS)

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

78

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

Energy Technology Data Exchange (ETDEWEB)

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

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

2003-01-01

79

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

Science.gov (United States)

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

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

2010-08-01

80

Randomized, multicenter, double-blind, and placebo-controlled trial using topical recombinant human acidic fibroblast growth factor for deep partial-thickness burns and skin graft donor site.  

Science.gov (United States)

Wound healing is a dynamic and complex biologic process that could be accelerated by growth factors. To investigate the efficacy of topical recombinant human acidic fibroblast growth factor (rh-aFGF) treatment in deep partial-thickness burn or skin graft donor sites, we designed a randomized, multicenter, double-blind, and placebo-controlled clinical trial. The healing rate, fully healed rate, and healing time were evaluated to assess the efficacy of rh-aFGF application. Laboratory examinations and abnormal signs were used to assess the side and toxic effects. The results showed that the healing rate of burn wounds and skin graft donor sites treated by rh-aFGF was significantly higher than that by placebo, and the mean healed time of burn wounds and skin graft donor sites in the rh-aFGF group was significantly the shorter than that in the placebo group. In conclusion, topical administration of rh-aFGF can accelerate the wound healing process and shorten the healed time. It is a potential therapeutic application for promoting healing of deep partial-thickness burns or skin graft donor sites. PMID:18028126

Ma, Bing; Cheng, Da-Sheng; Xia, Zhao-Fan; Ben, Dao-Feng; Lu, Wei; Cao, Zhi-Fang; Wang, Qiang; He, Jia; Chai, Jia-Ke; Shen, Chuan-An; Sun, Yong-Hua; Zhang, Guo-An; Hu, Xiao-Hua

2007-01-01

81

Endothelial cells stimulated with tumor necrosis factor-alpha express varying amounts of tissue factor resulting in inhomogenous fibrin deposition in a native blood flow system. Effects of thrombin inhibitors.  

OpenAIRE

TNF-alpha induces changes in endothelial cell functions, such as upregulation of tissue factor, resulting in endothelial procoagulant activity which may play a role in disseminated intravascular coagulation. The procoagulant activity of TNF-alpha-stimulated endothelial cell monolayers was studied in a human ex vivo native (nonanticoagulated) blood flow system using the three thrombin inhibitors recombinant hirudin, Ro 46-6240, and heparin. Under venous blood flow conditions (shear rate 65 s-1...

Kirchhofer, D.; Tschopp, T. B.; Hadva?ry, P.; Baumgartner, H. R.

1994-01-01

82

Human coagluation factor V purification and thrombin-catalyzed activation.  

OpenAIRE

Factor V was isolated from human plasma by barium citrate adsorption, polyethylene glycol fractionation, DEAE-Sepharose CL-6B chromatography, ammonium sulfate fractionation, and gel chromatography on Ultrogel 22. Degradation of Factor V during purification was largely prevented by ample use of inhibitors of proteolytic enzyme. The purified Factor V was a stable, single-chain molecule with an apparent molecular weight of 330,000. Activation of human Factor V by thrombin resulted in a 10- to 15...

Dahlba?ck, B.

1980-01-01

83

Thrombin increases expression of fibronectin antigen on the platelet surface.  

OpenAIRE

Fibronectins (fn) are adhesive glycoproteins which bind to collagen and to fibrin and appear to be important in cellular adhesion to other cells or surfaces. Fn-related antigen is present in human platelets, suggesting a possible role for fn in the adhesive properties of platelets. We have studied the localization of fn in resting and thrombin-stimulated platelets by immunofluorescence and quantitative binding of radiolabeled antibody. In resting fixed platelets, variable light surface staini...

Ginsberg, M. H.; Painter, R. G.; Forsyth, J.; Birdwell, C.; Plow, E. F.

1980-01-01

84

Thrombin-receptor antagonist vorapaxar in acute coronary syndromes  

OpenAIRE

Background: Vorapaxar is a new oral protease-activatedreceptor 1 (PAR-1) antagonist that inhibits thrombin-induced platelet activation. Methods: In this multinational, double-blind, randomized trial, we compared vorapaxar with placebo in 12,944 patients who had acute coronary syndromes without ST-segment elevation. The primary end point was a composite of death from cardiovascular causes, myocardial infarction, stroke, recurrent ischemia with rehospitalization, or urgent coronary revasculariz...

Tricoci, Pierluigi; Huang, Zhen; Held, Claes; et al.

2012-01-01

85

Recombination instability  

DEFF Research Database (Denmark)

A recombination instability is considered which may arise in a plasma if the temperature dependence of the volume recombination coefficient, alpha, is sufficiently strong. Two cases are analyzed: (a) a steady-state plasma produced in a neutral gas by X-rays or high energy electrons; and (b) an afterglow plasma.

D'Angelo, N.

1967-01-01

86

Metabolic plasticity in resting and thrombin activated platelets.  

Science.gov (United States)

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

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

87

Atorvastatin neutralises the thrombin-induced tissue factor expresion in endothelial cells via geranylgeranyl pyrophosphate  

OpenAIRE

Statins may have beneficial effects in atherogenesis given their antithrombotic properties involving non-lipid mechanisms that modify endothelial function of tissue factor induction by thrombin. In this study, we investigate the effect of atorvastatin on tissue factor (TF) activity in thrombin-stimulated endothelial cells and its regulation through mevalonate or its derivatives. First subculture of human umbilical endothelial cells was used for this study. Cells were treated with thrombin and...

Marti?nez-sales, Vicenta; Vila, Virtudes; Ferrando, Marcos; Reganon, Edelmiro

2010-01-01

88

Thrombin stimulates proliferation of cultured rat aortic smooth muscle cells by a proteolytically activated receptor.  

OpenAIRE

Thrombin has been implicated in the stimulation of smooth muscle cell (SMC) proliferation that contributes to post angioplasty restenosis. The present studies demonstrated that human alpha-thrombin was a potent and efficacious mitogen for cultured rat aortic SMC, stimulating an increase in 3H-thymidine incorporation, as well as an increase in cell number at 1 to 10 nM concentration. gamma-Thrombin, which is enzymatically active but lacks fibrinogen clotting activity, stimulated SMC mitogenesi...

Mcnamara, C. A.; Sarembock, I. J.; Gimple, L. W.; Fenton, J. W.; Coughlin, S. R.; Owens, G. K.

1993-01-01

89

Thrombin stimulates the adherence of neutrophils to human endothelial cells in vitro.  

OpenAIRE

Highly purified human thrombin stimulates the adherence of polymorphonuclear leukocytes (PMNs) to vascular endothelial cells (EC). When Indium-labeled PMNs were incubated with primary monolayers of cultured human umbilical vein EC, the basal adherence was 10 +/- 1% of the PMNs at 5 min. Addition of thrombin (2 U/ml) increased the mean adherence to 42 +/- 15%. Enhanced neutrophil adherence in response to thrombin was confirmed by experiments with unlabeled leukocytes, examined by phase contras...

Zimmerman, G. A.; Mcintyre, T. M.; Prescott, S. M.

1985-01-01

90

Aptamer-Crosslinked Microbubbles: Smart Contrast Agents for Thrombin-Activated Ultrasound Imaging  

OpenAIRE

Thrombosis, or malignant blood clotting, is associated with numerous cardiovascular diseases and cancers. This report describes a microbubble contrast agent that produces ultrasound harmonic signal only when exposed to elevated thrombin levels. Silenced initially, microbubbles 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.

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

2012-01-01

91

Inhibition of thrombin activity by selected natural products in comparison to neutrophil elastase.  

Science.gov (United States)

Enzymatic thrombin activity is significantly inhibited only by a few selected natural phenolic compounds (myricetin, rosmarinic acid, caffeic acid phenethyl ester) but more strongly by unsaturated fatty acids like erucic acid and oleic acids. Compared to the inhibitory potential against neutrophil elastase, thrombin activity is rather weakly inhibited by phenolic compounds and fatty acids. Because of the importance of thrombin as a ligand for protease-activated receptor 1 (PAR-1), which is involved in inflammation, the inhibition of thrombin activity by natural compounds might enhance the anti-inflammatory effects of neutrophil elastase inhibition. PMID:16142650

Melzig, Matthias F; Henke, Kristin

2005-08-01

92

Low anticoagulant heparin blocks thrombin-induced endothelial permeability in a PAR-dependent manner.  

Science.gov (United States)

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

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

2014-08-01

93

Thrombomodulin tightens the thrombin active site loops to promote protein C activation.  

Science.gov (United States)

Thrombomodulin (TM) forms a 1:1 complex with thrombin. Whereas thrombin alone cleaves fibrinogen to make the fibrin clot, the thrombin-TM complex cleaves protein C to initiate the anticoagulant pathway. Crystallographic investigations of the complex between thrombin and TMEGF456 did not show any changes in the thrombin active site. Therefore, research has focused recently on how TM may provide a docking site for the protein C substrate. Previous work, however, showed that when the thrombin active site was occupied with substrate analogues labeled with fluorophores, the fluorophores responded differently to active (TMEGF1-6) versus inactive (TMEGF56) fragments of TM. To investigate this further, we have carried out amide H/(2)H exchange experiments on thrombin in the presence of active (TMEGF45) and inactive (TMEGF56) fragments of TM. Both on-exchange and off-exchange experiments show changes in the thrombin active site loops, some of which are observed only when the active TM fragment is bound. These results are consistent with the previously observed fluorescence changes and point to a mechanism by which TM changes the thrombin substrate specificity in favor of protein C cleavage. PMID:16274226

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

2005-11-15

94

Thrombin generation and platelet activation induced by rFVIIa (NovoSeven) and NN1731 in a reconstituted cell-based model mimicking haemophilia conditions.  

Science.gov (United States)

Replacement therapy with factor VIII (FVIII) and factor IX (FIX) is routinely used in haemophilia patients with haemophilia A and B, respectively, while recombinant activated FVII (rFVIIa) has proven to induce haemostasis in haemophilia patients with inhibitors. To evaluate the effect of therapeutic intervention in patients with residual factor activities, the effects of increasing concentrations of rFVIIa or NN1731 on thrombin generation and platelet activation were measured in a cell-based model system mimicking severe, moderate and mild haemophilia A or B. Purified monocytes stimulated to express tissue factor and non-activated platelets from peripheral blood of healthy donors were incubated with a mixture of purified human coagulation factors in the absence or presence of increasing concentrations of FVIII or FIX. Sub-samples were analysed for thrombin activity and platelet activation measured as exposure of P-selectin by flow cytometry. Dose-dependent increases in thrombin generation and platelet activation were observed following increasing concentrations of rFVIIa or NN1731 in both haemophilia A- and B-like conditions. At 25 nm rFVIIa, which nears the peak levels in patient plasma after 90 microg kg(-1) intravenous dosing, the effects on maximum thrombin generation rate (maxTG) at 1-10% FVIII were comparable to those at 100% and 200% FVIII in the absence of rFVIIa. Normalization of maxTG required 500 nm rFVIIa and 25 nm NN1731 or 25-100 nm rFVIIa and 5 nm NN1731 in severe or moderate/mild haemophilia A and haemophilia B, respectively. This suggests that NN1731 holds its promise as a future bypassing agent for haemophilia patients with and without inhibitors. PMID:19659796

Aljamali, M N; Kjalke, M; Hedner, U; Ezban, M; Tranholm, M

2009-11-01

95

Thrombin regulates components of the fibrinolytic system in human mesangial cells  

International Nuclear Information System (INIS)

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

96

Pulmonary epithelial clearance of 99mTc-DTPA after thrombin-induced pulmonary microembolism  

International Nuclear Information System (INIS)

We investigated the effect of thrombin-induced pulmonary microembolism on the pulmonary clearance rate of aerosolized 99mTc diethylenetriamine pentaacetic acid (99mTc-DTPA) in awake, chronically prepared sheep. Chest activity was recorded after administration of a 0.44 micron aerosol of 99mTc-DTPA. Decay-corrected data were fit to an exponential and expressed as percent decrease per min (%/min). Sheep were given alpha-thrombin intravenously (80 U/kg for 10 min) 60 min after the aerosol administration. The clearance rate prior to alpha-thrombin was 0.35 +/- 0.05 %/min (mean +/- SEM). During alpha-thrombin administration, the clearance rate increased to 5.84 +/- 0.70 %/min (p less than 0.001 from baseline), but returned to 0.41 +/- 0.06 %/min within 30 min after the end of the thrombin infusion. The increased clearance rate during alpha-thrombin administration was not due to increased lung volume since alpha-thrombin did not change functional residual capacity. Moreover, the clearance rate was unchanged during gamma-thrombin administration, which does not induce coagulation, or during alpha-thrombin challenge in defibrinogenated animals. alpha-thrombin administration in neutrophil-depleted sheep caused a transient increase in DTPA clearance similar to that in control sheep, suggesting that the increase occurred independently of neutrophils. The results indicate that alpha-thrombin causes a large, transient increase in 99mTc-DTPA clearance, which may be the result of increased epithelial permeability. This response is dependent on the activation of intravascular coagulation

97

Amide H/2H exchange reveals a mechanism of thrombin activation.  

Science.gov (United States)

Thrombin is a dual action serine protease in the blood clotting cascade. Similar to other clotting factors, thrombin is mainly present in the blood in a zymogen form, prothrombin. Although the two cleavage events required to activate thrombin are well-known, little is known about why the thrombin precursors are inactive proteases. Although prothrombin is much larger than thrombin, prethrombin-2, which contains all of the same amino acids as thrombin, but has not yet been cleaved between Arg320 and Ile321, remains inactive. Crystal structures of both prethrombin-2 and thrombin are available and show almost no differences in the active site conformations. Slight differences were, however, seen in the loops surrounding the active site, which are larger in thrombin than in most other trypsin-like proteases, and have been shown to be important for substrate specificity. To explore whether the dynamics of the active site loops were different in the various zymogen forms of thrombin, we employed amide H/(2)H exchange experiments to compare the exchange rates of regions of thrombin with the same regions of prothrombin, prethrombin-2, and meizothrombin. Many of the surface loops showed less exchange in the zymogen forms, including the large loop corresponding to anion binding exosite 1. Conversely, the autolysis loop and sodium-binding site exchanged more readily in the zymogen forms. Prothrombin and prethrombin-2 gave nearly identical results while meizothrombin in some regions more closely resembled active thrombin. Thus, cleavage of the Arg320-Ile321 peptide bond is the key to formation of the active enzyme, which involves increased dynamics of the substrate-binding loops and decreased dynamics of the catalytic site. PMID:16784223

Koeppe, Julia R; Komives, Elizabeth A

2006-06-27

98

Thrombin drives tumorigenesis in colitis-associated colon cancer.  

Science.gov (United States)

The established association between inflammatory bowel disease and colorectal cancer underscores the importance of inflammation in colon cancer development. On the basis of evidence that hemostatic proteases are powerful modifiers of both inflammatory pathologies and tumor biology, gene-targeted mice carrying low levels of prothrombin were used to directly test the hypothesis that prothrombin contributes to tumor development in colitis-associated colon cancer (CAC). Remarkably, imposing a modest 50% reduction in circulating prothrombin in fII+/- mice, a level that carries no significant bleeding risk, dramatically decreased adenoma formation following an azoxymethane/dextran sodium sulfate challenge. Similar results were obtained with pharmacologic inhibition of prothrombin expression or inhibition of thrombin proteolytic activity. Detailed longitudinal analyses showed that the role of thrombin in tumor development in CAC was temporally associated with the antecedent inflammatory colitis. However, direct studies of the antecedent colitis showed that mice carrying half-normal prothrombin levels were comparable to control mice in mucosal damage, inflammatory cell infiltration, and associated local cytokine levels. These results suggest that thrombin supports early events coupled to inflammation-mediated tumorigenesis in CAC that are distinct from overall inflammation-induced tissue damage and inflammatory cell trafficking. That prothrombin is linked to early events in CAC was strongly inferred by the observation that prothrombin deficiency dramatically reduced the formation of very early, precancerous aberrant crypt foci. Given the importance of inflammation in the development of colon cancer, these studies suggest that therapeutic interventions at the level of hemostatic factors may be an effective means to prevent and/or impede colitis-associated colon cancer progression. PMID:24710407

Turpin, Brian; Miller, Whitney; Rosenfeldt, Leah; Kombrinck, Keith; Flick, Matthew J; Steinbrecher, Kris A; Harmel-Laws, Eleana; Mullins, Eric S; Shaw, Maureen; Witte, David P; Revenko, Alexey; Monia, Brett; Palumbo, Joseph S

2014-06-01

99

Wound healing and the immune response in swine treated with a hemostatic bandage composed of salmon thrombin and fibrinogen  

OpenAIRE

We investigated the inflammatory response in pigs exposed to salmon fibrinogen/thrombin dressings. Animals were exposed to the material in 3 ways: (a) thrombin and fibrinogen were injected intravenously, (b) dual full-thickness skin lesions were surgically created on the dorsal aspect of the swine and treated with the fibrinogen/thrombin bandage and a commercial bandage or (c) a fibrinogen/thrombin bandage was inserted through an abdominal incision into the peritoneal cavity. Blood was collec...

Rothwell, Stephen W.; Sawyer, Evelyn; Dorsey, Jennifer; Flournoy, William S.; Settle, Timothy; Simpson, David; Cadd, Gary; Janmey, Paul; White, Charles; Szabo, Kathleen A.

2009-01-01

100

Phospholipase A2-Catalyzed Hydrolysis of Plasmalogen Phospholipids in Thrombin-Stimulated Human Platelets  

OpenAIRE

In the present study, phospholipase A2 (PLA2)-catalyzed hydrolysis of platelet membrane phospholipids was investigated by measuring PLA2 activity, phospholipid hydrolysis, arachidonic acid release and choline lysophospholipid production in thrombin-stimulated human platelets. Thrombin-stimulated platelets demonstrated selective hydrolysis of arachidonylated plasmenylcholine and plasmenylethanolamine, with little change in diacyl phospholipids. Accelerated plasmalogen hydrolysis was accompanie...

Beckett, Caroline S.; Kell, Pamela J.; Creer, Michael H.; Mchowat, Jane

2006-01-01

101

A Novel allosteric pathway of thrombin inhibition: Exosite II mediated potent inhibition of thrombin by chemo-enzymatic, sulfated dehydropolymers of 4-hydroxycinnamic acids*  

OpenAIRE

Thrombin and factor Xa, two important pro-coagulant proteinases, can be regulated through direct and indirect inhibition mechanisms. Recently, we designed sulfated dehydropolymers (DHPs) of 4-hydroxycinnamic acids that displayed interesting anticoagulant properties (Monien, B. H., Henry, B. L., Raghuraman, A., Hindle, M., and Desai, U. R. (2006) Bioorg. Med. Chem. 14, 7988–7998). To better understand their mechanism of action, we studied the direct inhibition of thrombin, factor Xa, factor ...

Henry, Brian L.; Monien, Bernhard H.; Bock, Paul E.; Desai, Umesh R.

2007-01-01

102

THE EVALUATION OF CLOTTING TIME IN BOVINE THROMBIN, REPTILASE ® , AND THROMBIN-LIKE FRACTION OF Crotalus durissus terrificus VENOM USING BOVINE, EQUINE, OVINE, BUBALINE AND HUMAN CRYOPRECIPITATES  

OpenAIRE

The objective of this study was to evaluate the effects of the thrombin-like fraction of Crotalus durissus terrificus venom, Reptilase , and bovine thrombin of fibrinogen pools on bovine, equine, ovine, bubaline and human cryoprecipitates. The authors also made a comparative study between animal and human cryoprecipitates to see if any there was any possibility of future use in medicine. Fibrinogen levels in cryoprecipitate were studied using 48 blood samples obtained as follows:12 samples fr...

Thomazini-santos, I. A.; Giannini, M. J. S. M.; Toscano, E.; Machado, P. E. A.; Lima, C. R. G.; Barraviera, B.

1998-01-01

103

Screening of direct thrombin inhibitors from Radix Salviae Miltiorrhizae by a peak fractionation approach.  

Science.gov (United States)

Thrombin plays a significant role in thromboembolic disease. In this work, a peak fractionation approach combined with an activity assay method was used to screen direct thrombin inhibitors from Radix Salviae Miltiorrhizae (RSM), a famous herbal remedy for the treatment of cardiovascular diseases in China. A total of 91 fractions were collected from the RSM extract, and 19 fractions out of them showed thrombin inhibitory effects with dose-effect relationship. Among them, three compounds were unambiguously identified as 15, 16-dihydrotanshinone I, cryptotanshinone and tanshinone IIA with IC50 values of 29.39, 81.11 and 66.60?M, respectively. The three compounds were reported with direct thrombin inhibition activities for the first time and their ligand-thrombin interactions were explored by a molecular docking research. These results may contribute to explain the medical benefit of RSM for the prevention and treatment of cardiovascular diseases. PMID:25819728

Lu, Jun; Song, Hui-Peng; Li, Ping; Zhou, Ping; Dong, Xin; Chen, Jun

2015-05-10

104

Thrombin-free fibrin coating on small caliber vascular prostheses has high antithrombogenicity in rabbit model.  

Science.gov (United States)

Fibrin coatings on prosthetic vascular graft, which are conventionally produced by fibrinogen and thrombin, are expected to improve antithrombogenicity and healing characteristics. Thrombin is one of the factors of blood coagulation cascade; however, it has a possibility to play a negative role in the graft antithrombogenicity. The purpose of this study was to evaluate the performance of our new grafts, thrombin-free fibrin-coated small caliber vascular prostheses. Knitted polyester fabric vascular prostheses 2 mm in internal diameter were coated with fibrin coating with thrombin (Graft I) or without thrombin (Graft II). Both grafts were implanted in bilateral common carotid arteries of 35 Japanese white rabbits, with Graft I in one side and Graft II in the contralateral side. Graft patency, histology, thrombin activity, and platelet deposition were compared between both grafts on postoperative days (PODs) 1, 3, 7, 10, 14, 30, and 60. Both grafts were patent without thrombus or stenosis at each end point (maximal period, POD 60). Macro- and microscopic findings revealed that no obvious difference was observed between both grafts. Before graft implantation, thrombin activities in Grafts I and II were 0.711 +/- 0.086 and 0.009 +/- 0.007 optical density at 405 nm, respectively. Thrombin activity of Graft II was significantly less than that of Graft I in every period after graft implantation, and platelet deposition of Graft II was significantly less than that of Graft I until POD 30. Thrombin-free fibrin-coated vascular prostheses have superior performance of antithrombogenicity to conventional fibrin-coated vascular prostheses with thrombin. PMID:16266301

Hasegawa, Tomomi; Okada, Kenji; Takano, Yoshihito; Hiraishi, Yoshiaki; Okita, Yutaka

2005-11-01

105

Thrombin-specific inactivation of endothelial cell derived plasminogen activator  

International Nuclear Information System (INIS)

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

106

Novel anti-platelet agents: focus on thrombin receptor antagonists.  

Science.gov (United States)

Platelets are the key in the pathogenesis of atherothrombotic disease such as acute coronary syndromes, stroke, and peripheral arterial disease. Current anti-platelet treatments are mainly based on inhibition of two important pathways of platelet activation: thromboxane A2 (TXA2) mediated (aspirin) and adenosine diphosphate (ADP)-P2Y12 receptor mediated (clopidogrel, prasugrel, and ticagrelor). Despite the dual anti-platelet therapy with aspirin and P2Y12 inhibitors have reduced ischemic events in patients with acute coronary syndromes (ACS), the rate of recurrent ischemic complication after ACS remains high. Combination of multiple anti-platelet agents is also associated with increased risk of bleeding. Thrombin is a potent platelet agonist and the increase of its activity has been reported in patients with ACS. Platelet effects of thrombin are mediated by protease-activated receptors (PAR), and PAR-1 is the most important receptor in human platelets. Two PAR-1 antagonists, vorapaxar and atopaxar, have undergone clinical investigation. In this review, we will describe the pharmacology of PAR-1 antagonists and will review and discuss results of randomized clinical trials with PAR-1 antagonists. PMID:23435863

de Souza Brito, Flavio; Tricoci, Pierluigi

2013-06-01

107

Recombining WMAP: Beyond standard recombination  

CERN Document Server

We place new constraints on sources of ionizing and resonance radiation at the epoch of the recombination process using the recent CMB temperature and polarization spectra coming from WMAP. We find that non-standard recombination scenarios are still consistent with the current data. In light of this we study the impact that such models can have on the determination of several cosmological parameters. In particular, the constraints on curvature and baryon density appear to be weakly affected by a modified recombination scheme. However, it may affect the current WMAP constraints on inflationary parameters like the spectral index and its running. Physically motivated models, like those based on primordial black hole or super heavy dark matter decay, are able to provide a good fit to the current data. Future observations in both temperature and polarization will be needed to more stringently test these models.

Bean, R; Silk, J; Bean, Rachel; Melchiorri, Alessandro; Silk, Joe

2003-01-01

108

Mycobacterial recombineering.  

Science.gov (United States)

The precise knockout or modification of Mycobacterium tuberculosis genes has been critical for the identification of functions important for the growth and pathogenicity of this important bacterium. Schemes have been previously described, using both non-replicating vectors and transducing particles, for the introduction of gene knockout substrates into M. tuberculosis, where the endogenous recombination systems of the host (both homologous and illegitimate) compete for transfer of the modified allele to the chromosome. Recombineering technologies, first introduced in laboratory and pathogenic strains of Escherichia coli over the last 16 years, have been developed for use in M. tuberculosis. Described in this chapter is the use of the mycobacterial Che9c phage RecET recombination system, which has been used to make gene knockouts, reporter fusions, promoter replacements, and single base pair modifications within the M. tuberculosis and M. smegmatis chromosomes at very high frequency. Higher success rates, in a shorter period of time, are routinely observed when recombineering is compared to previously described M. tuberculosis gene knockout protocols. PMID:25779316

Murphy, Kenan C; Papavinasasundaram, Kadamba; Sassetti, Christopher M

2015-01-01

109

Substitution of valine for glycine-558 in the congenital dysthrombin thrombin Quick II alters primary substrate specificity  

International Nuclear Information System (INIS)

Thrombin Quick II is one of two dysfunctional forms of thrombin derived from the previously described congenital dysprothrombin prothrombin Quick. Thrombin Quick II does not clot fibrinogen, hydrolyze p-nitroanilide substrates of thrombin, or bind N2-[5-(dimethylamino)naphthalene-1-sulfonyl]arginine N,N-(3-ethyl-1,5-pentanediyl)amide, a high-affinity competitive inhibitor of thrombin. To determine the structural alteration in thrombin Quick II, the reduced, carboxymethylated protein was hydrolyzed by a lysyl endopeptidase. A peptide not present in a parallel thrombin hydrolysate was identified by reverse-phase chromatography. This Gly residue, which is highly conserved in the chymotrypsin family of serine proteases, forms part of the substrate binding pocket for bulky aromatic and basic side chains in chymotrypsin and trypsin, respectively. However, in porcine elastase 1, the corresponding residue is threonine. Consistent with the identified structural alteration, thrombin Quick II incorporates [3H]diisopropyl fluorophosphate stoichiometrically and hydrolyzes the elastase substrate succinyl-Ala-Ala-Pro-Leu-p-nitroanilide with a relative kcat/KM of 0.14 when compared to thrombin. This results from a 3-fold increase in KM and a 2.5-fold decrease in kcat for thrombin Quick II when compared to thrombin acting on the same substrate. These results and those of other investigators studying mutant trypsins suppnvestigators studying mutant trypsins support the conclusion that the catalytic activity of serine proteases is very sensitive to structural alterations in the primary substrate binding pocket

110

Platelet activation and aggregation : the importance of thrombin activity--a laboratory model  

DEFF Research Database (Denmark)

This study introduces a new laboratory model of whole blood platelet aggregation stimulated by endogenously generated thrombin, and explores this aspect in haemophilia A in which impaired thrombin generation is a major hallmark. The method was established to measure platelet aggregation initiated by tissue factor evaluated by means of impedance aggregometry. Citrated whole blood from healthy volunteers and haemophilia A patients with the addition of inhibitors of the contact pathway and fibrin polymerization was evaluated. In healthy persons, a second wave of platelet aggregation was found to coincide with the thrombin burst and to be abolished by thrombin inhibitors. In this system, platelet aggregation in severe haemophilia A (n = 10) was found to be significantly decreased as compared with healthy individuals (912 ± 294 vs. 1917 ± 793 AU × min, P = 0.003), most probably due to the weak level of thrombin generation. For the first time, analysis of platelet aggregation as induced by endogenously generated thrombin was demonstrated. The new method makes it possible to explore the influence of the coagulation system on platelet function. In contrast to the general understanding, the data suggest that the impaired thrombin generation in haemophilia may affect platelet activation. Future studies will address whether our results may contribute to understanding differences in bleeding phenotypes and response to haemostatic substitution observed among patients.

Jensen, Maria Sander; Larsen, O H

2013-01-01

111

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

LENUS (Irish Health Repository)

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

Ni Ainle, Fionnuala

2009-08-20

112

Isoproterenol attenuates the thrombin-induced increase in pulmonary endothelial permeability  

International Nuclear Information System (INIS)

The proposed permeability-decreasing effect of the beta-agonist, isoproterenol, was tested by measurement of the clearance rate of 125I-albumin across bovine pulmonary artery endothelial cell monolayers. These cells were seeded on gelatinized polycarbonate filters and mounted in a modified Boyden chamber with Dulbecco's Modified Eagle Medium (DMEM) and 1% BSA in the luminal and abluminal chambers. Baseline clearance rates were measured for an initial 30 min with the cells incubated at 370C in DMEM. At 30 min, 70 ul of either DMEM, ?-thrombin (10-6M), ?-thrombin and isoproterenol (10-6M) or isoproterenol alone was added to the luminal chamber. Clearance rates were determined at 30-60 min in each group and normalized to the baseline clearance rates of each (n = 8) cell monolayer. The experimental/baseline values were 0.98 +/- 0.08 (mean +/- SEM) for DMEM, 1.90 +/- 0.10 for thrombin, 1.52 +/- 0.08 for thrombin and isoproterenol, and 0.83 +/- 0.10 for isoproterenol alone. Thrombin increased (p < 0.05) the clearance rate from the control DMEM value and isoproterenol reduced (p < 0.05) the response to thrombin. Therefore, isoproterenol attenuates the thrombin-induced increase in pulmonary endothelial permeability

113

Hyperbranched rolling circle amplification based electrochemiluminescence aptasensor for ultrasensitive detection of thrombin.  

Science.gov (United States)

An ultrasensitive electrochemiluminescence (ECL) aptamer sensor for protein (thrombin as an example) detection based on hyperbranched rolling circle amplification (HRCA) had been developed. A complementary single-strand DNA (CDNA) of the thrombin aptamer had been modified on the gold electrode firstly, and then hybridized with thrombin aptamer to make the aptamer immobilized on the electrode surface, in the presence of thrombin, aptamer-thrombin bioaffinity complexes formed and made thrombin aptamer leave the electrode surface. Thus, the linear padlock probe hybridized with the free CDNA on the electrode surface and circularized by Escherichia coli DNA ligase. Subsequently, the linear padlock probe was served as a template for the initiation of HRCA reaction, and a lot of dsDNA modified on the electrode surface. Then Ru(phen)?²? (acted as the ECL indicator) intercalates specifically into double-stranded DNA (dsDNA) grooves to generate ECL signal. The ECL intensity of the system has a linear relationship with thrombin concentration in the range of 3.0-300 aM with a detection limit of 1.2 aM (S/N=3). The proposed method combines the high sensitivity of ECL, exponential ampli?cation of HRCA for signal enhancement and high selectivity of aptamer. PMID:25086328

Jin, Guixiao; Wang, Chunmei; Yang, Linlin; Li, Xiaojuan; Guo, Longhua; Qiu, Bin; Lin, Zhenyu; Chen, Guonan

2015-01-15

114

Thrombin selectively induces transcription of genes in human monocytes involved in inflammation and wound healing.  

Science.gov (United States)

Thrombin is essential for blood coagulation but functions also as a mediator of cellular signalling. Gene expression microarray experiments in human monocytes revealed thrombin-induced upregulation of a limited subset of genes, which are almost exclusively involved in inflammation and wound healing. Among these, the expression of F3 gene encoding for tissue factor (TF) was enhanced indicating that this physiological initiator of coagulation cascade may create a feed-forward loop to enhance blood coagulation. Activation of protease-activated receptor type 1 (PAR1) was shown to play a main role in promoting TF expression. Moreover, thrombin induced phosphorylation of ERK1/2, an event that is required for expression of thrombin-regulated genes. Thrombin also increased the expression of TF at the protein level in monocytes as evidenced by Western blot and immunostaining. Furthermore, FXa generation induced by thrombin-stimulated monocytes was abolished by a TF blocking antibody and therefore it is entirely attributable to the expression of tissue factor. This cellular activity of thrombin provides a new molecular link between coagulation, inflammation and wound healing. PMID:25057055

López, Mercedes L; Bruges, Gustavo; Crespo, Gustavo; Salazar, Victor; Deglesne, Pierre-Antoine; Schneider, Heike; Cabrera-Fuentes, Hector; Schmitz, M Lienhard; Preissner, Klaus T

2014-11-01

115

A convenient sandwich assay of thrombin in biological media using nanoparticle-enhanced fluorescence polarization.  

Science.gov (United States)

A new aptamer biosensor was presented for the detection of thrombin in this work, which was based on fluorescence polarization (FP) using silica nanoparticles as enhancement probe. The silica nanoparticles covered by streptavidin were tagged with a thrombin aptamer (5'-biotin-GGTTGGTGTGGTTGG-3'), which was bound to the surface of silica nanoparticle through the specific interaction between streptavidin and biotin. In the presence of thrombin, it induced the aptamer to form quadruplex structure. When the other thrombin aptamer labeled with fluorescein (5'-FAM-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3') was added to the above system, a sandwich structure can form at the surface of silica nanoparticles. The fluorescence polarization was therefore enhanced and quantification between fluorescence polarization signal and concentration of thrombin was built. The sensor provided a linear range from 0.6 to 100 nM for thrombin with a detection limit of 0.20 nM (3.29 SB/m, according to the recent recommendation of IUPAC) in a homogeneous media. The same linear range was obtained in spiked human serum samples with a slightly higher detection limit (0.26 nM), demonstrating high anti-interference of the sensor in a complex biological sample matrix. And the sensor can be used to monitor spiked concentration of thrombin level in real human plasma with satisfactory results obtained. PMID:24508546

Yue, Qiaoli; Shen, Tongfei; Wang, Lei; Xu, Shuling; Li, Haibo; Xue, Qingwang; Zhang, Yuanfu; Gu, Xiaohong; Zhang, Shuqiu; Liu, Jifeng

2014-06-15

116

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

Energy Technology Data Exchange (ETDEWEB)

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

Nieman, M.T.; Burke, F.; Warnock, M.; Zhou, Y.; Sweigart, J.; Chen, A.; Ricketts, D.; Lucchesi, B.R.; Chen, Z.; Cera, E.Di; Hilfinger, J.; Kim, J.S.; Mosberg, H.I.; Schmaier, A.H. (Case Western); (Michigan); (TSRL); (WU-MED)

2008-04-29

117

Thrombin-inhibiting nanoparticles rapidly constitute versatile and detectable anticlotting surfaces  

Science.gov (United States)

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.

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

2014-09-01

118

Fluorescent reporters of thrombin, heparin cofactor II, and heparin binding in a ternary complex  

OpenAIRE

Thrombin inactivation by heparin cofactor II (HCII) is accelerated by ternary complex formation with heparin. The novel active-site-labeled thrombins, [4?F]FPR-T and [6F]FFR-T, and the exosite I probe, Hir-(54–65)( SO3?), characterized thrombin exosite I and II interactions with HCII and heparin in the complex. HCII binding to exosite I of heparin-bound [4?F]FPR-T caused a saturable fluorescence increase, absent with antithrombin. Heparin binding to exosite II and a second weaker s...

Verhamme, Ingrid M.

2011-01-01

119

Lysosomotropic agents selectively potentiate thrombin-induced acid hydrolase secretion from platelets.  

OpenAIRE

Thrombin induces partial secretion (up to 60%) of beta-N-acetyl-D-hexosaminidase (EC 3.2.1.52) from untreated platelets. Preincubation of platelets with 10 mM NH4Cl for up to 2 hr resulted in a time-dependent and marked stimulation of thrombin-induced secretion of both this enzyme and other acid glycosidases from platelets. The enhancement of the thrombin-induced secretion was not due to cell lysis, and NH4Cl alone did not cause leakage of lysosomal enzymes into the medium. The effect could b...

Oost, B. A.; Smith, J. B.; Holmsen, H.; Vladutiu, G. D.

1985-01-01

120

The thrombin receptor is a critical extracellular switch controlling myelination.  

Science.gov (United States)

Hemorrhagic white matter injuries in the perinatal period are a growing cause of cerebral palsy yet no neuroprotective strategies exist to prevent the devastating motor and cognitive deficits that ensue. We demonstrate that the thrombin receptor (protease-activated receptor 1, PAR1) exhibits peak expression levels in the spinal cord at term and is a critical regulator of the myelination continuum from initiation to the final levels achieved. Specifically, PAR1 gene deletion resulted in earlier onset of spinal cord myelination, including substantially more Olig2-positive oligodendrocytes, more myelinated axons, and higher proteolipid protein (PLP) levels at birth. In vitro, the highest levels of PAR1 were observed in oligodendrocyte progenitor cells (OPCs), being reduced with differentiation. In parallel, the expression of PLP and myelin basic protein (MBP), in addition to Olig2, were all significantly higher in cultures of PAR1-/- oligodendroglia. Moreover, application of a small molecule inhibitor of PAR1 (SCH79797) to OPCs in vitro increased PLP and MBP expression. Enhancements in myelination associated with PAR1 genetic deletion were also observed in adulthood as evidenced by higher amounts of MBP and thickened myelin sheaths across large, medium, and small diameter axons. Enriched spinal cord myelination in PAR1-/- mice was coupled to increases in extracellular-signal-regulated kinase 1/2 and AKT signaling developmentally. Nocturnal ambulation and rearing activity were also elevated in PAR1-/- mice. These studies identify the thrombin receptor as a powerful extracellular regulatory switch that could be readily targeted to improve myelin production in the face of white matter injury and disease. GLIA 2015;63:846-859. PMID:25628003

Yoon, Hyesook; Radulovic, Maja; Drucker, Kristen L; Wu, Jianmin; Scarisbrick, Isobel A

2015-05-01

121

P3-P3' residues flanking scissile bonds in factor VIII modulate rates of substrate cleavage and procofactor activation by thrombin.  

Science.gov (United States)

Thrombin-catalyzed activation of factor VIII (FVIII) occurs through proteolysis at three P1 Arg residues: Arg(372) and Arg(740) in the FVIII heavy chain and Arg(1689) in the FVIII light chain. Cleavage at the latter two sites is relatively fast compared with cleavage at Arg(372), which appears to be rate-limiting. Examination of the P3-P3' residues flanking each P1 site revealed that those sequences at Arg(740) and Arg(1689) are more optimal for thrombin cleavage than at Arg(372), suggesting these sequences may impact reaction rates. Recombinant FVIII variants were prepared with mutations swapping scissile bond flanking sequences in the heavy chain individually and in combination with a second swap or with a P1 point mutation. Rates of generation of A1 and A3-C1-C2 subunits were determined by Western blotting and correlated with rates of cleavage at Arg(372) and Arg(1689), respectively. Rates of thrombin cleavage at Arg(372) were increased ~10- and ~3-fold compared with that of wild-type FVIII when it was replaced with P3-P3' residues flanking Arg(740) and Arg(1689), respectively, and these values paralleled increased rates of A2 subunit generation and procofactor activation. Positioning of more optimal residues flanking Arg(372) abrogated the need for initial cleavage at Arg(740) to facilitate this step. These results show marked changes in cleavage rates correlate with the extent of cleavage-optimal residues flanking the scissile bond and modulate the mechanism for procofactor activation. PMID:22455313

Newell-Caito, Jennifer L; Griffiths, Amy E; Fay, Philip J

2012-04-24

122

The direct thrombin inhibitors (argatroban, bivalirudin and lepirudin) and the indirect Xa-inhibitor (danaparoid) increase fibrin network porosity and thus facilitate fibrinolysis.  

Science.gov (United States)

The present study aimed to assess whether the fibrin network structure is modified by the direct thrombin-inhibitors lepirudin, argatroban or bivalirudin and by the indirect Xa-inhibitor danaparoid. Using an in vitro assay that imitates the physiological process of coagulation from thrombin generation to fibrin formation, we examined a normal plasma pool spiked with one of the inhibitors. At concentrations considered to be the plasma levels observed during therapy, almost no influence was detected for lepirudin despite clear-cut effects on "clotting time". However, argatroban, bivalirudin and danaparoid increased the fibrin gel permeability (Ks) to a similar extent. At concentrations higher than the "therapeutic" levels, the dose-response curve in the Ks assay became very steep for lepirudin while those were shallow for the others. In parallel with the drug-induced increases of Ks, larger network pores in 3D-microscopic images and significant shortenings in "clot lysis time" induced by addition of rtPA were observed. Recombinant factor VIII (rFVIII) added to danaparoid-treated samples profoundly counteracted the increase of Ks but had only a slight or no effect on the other drugs. Thus, in vitro, argatroban, bivalirudin and danaparoid have comparable anticoagulating effects, rendering the fibrin network more permeable and less resistant to fibrinolysis. For lepirudin, the steep dose-response curve supports previous clinical findings, i.e. this thrombin inhibitor has a narrow therapeutic window. Furthermore, our data suggest that the haemostatic agent, rFVIII, might be effective in treatment of bleeding complications induced by danaparoid. PMID:20216982

He, Shu; Blombäck, Margareta; Bark, Niklas; Johnsson, Hans; Wallén, N Hakan

2010-05-01

123

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

International Nuclear Information System (INIS)

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

124

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

Directory of Open Access Journals (Sweden)

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

Alice Sosic

2011-10-01

125

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

DEFF Research Database (Denmark)

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

Rangnekar, Abhijit; Zhang, Alex M.

2012-01-01

126

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

International Nuclear Information System (INIS)

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

127

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

International Nuclear Information System (INIS)

Hirudin, isolated from the European leech Hirudo medicinalis, is a potent inhibitor of thrombin, forming an almost irreversible thrombin-hirudin complex. Previously, the authors have shown that the carboxyl terminus of hirudin (residues 45-65) inhibits clotting activity and without binding to the catalytic site of thrombin. In the present study, a series of peptides corresponding to this carboxyl-terminal region of hirudin have been synthesized, and their anticoagulant activity and binding properties to thrombin were examined. Binding was assessed by their ability to displace 125I-hirudin 45-65 from Sepharose-immobilized thrombin and by isolation of peptide-thrombin complexes. They show that the carboxyl-terminal 10 amino acid residues 56-65 (Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln) are minimally required for binding to thrombin and inhibition of clotting. Phe-56 was critical for maintaining anticoagulant activity as demonstrated by the loss of activity when Phe-56 was substituted with D-Phe, Glu, or Leu. In addition, they found that the binding of the carboxyl-terminal peptide of hirudin with thrombin was associated with a significant conformational change of thrombin as judged by circular dichroism. This conformational change might be responsible for the loss of clotting activity of thrombin

128

Highly specific detection of thrombin using an aptamer-based suspension array and the interaction analysis via microscale thermophoresis.  

Science.gov (United States)

A novel aptamer-based suspension array detection platform was designed for the sensitive, specific and rapid detection of human ?-thrombin as a model. Thrombin was first recognized by a 29-mer biotinylated thrombin-binding aptamer (TBA) in solution. Then 15-mer TBA modified magnetic beads (MBs) captured the former TBA-thrombin to form an aptamer-thrombin-aptamer sandwich complex. The median fluorescence intensity obtained via suspension array technology was positively correlated with the thrombin concentration. The interactions between TBAs and thrombin were analyzed using microscale thermophoresis (MST). The dissociation constants could be respectively achieved to be 44.2 ± 1.36 nM (TBA1-thrombin) and 15.5 ± 0.637 nM (TBA2-thrombin), which demonstrated the high affinities of TBA-thrombin and greatly coincided with previous reports. Interaction conditions such as temperature, reaction time, and coupling protocol were optimized. The dynamic quantitative working range of the aptamer-based suspension array was 18.37-554.31 nM, and the coefficients of determination R(2) were greater than 0.9975. The lowest detection limit of thrombin was 5.4 nM. This method was highly specific for thrombin without being affected by other analogs and interfering proteins. The recoveries of thrombin spiked in diluted human serum were in the range 82.6-114.2%. This innovative aptamer-based suspension array detection platform not only exhibits good sensitivity based on MBs facilitating highly efficient separation and amplification, but also suggests high specificity by the selective aptamer binding, thereby suggesting the expansive application prospects in research and clinical fields. PMID:25710359

Liu, Yanan; Liu, Nan; Ma, Xinhua; Li, Xiaoli; Ma, Jia; Li, Ya; Zhou, Zhijiang; Gao, Zhixian

2015-04-21

129

Proton bridging in the interactions of thrombin with hirudin and its mimics.  

Science.gov (United States)

Thrombin is the pivotal serine protease enzyme in the blood cascade system and thus a target of drug design for control of its activity. The most efficient nonphysiologic inhibitor of thrombin is hirudin, a naturally occurring small protein. Hirudin and its synthetic mimics employ a range of hydrogen bonding, salt bridging, and hydrophobic interactions with thrombin to achieve tight binding with K(i) values in the nano- to femtomolar range. The one-dimensional (1)H nuclear magnetic resonance spectrum recorded at 600 MHz reveals a resonance 15.33 ppm downfield from silanes in complexes between human ?-thrombin and r-hirudin in pH 5.6-8.8 buffers and between 5 and 35 °C. There is also a resonance between 15.17 and 15.54 ppm seen in complexes of human ?-thrombin with hirunorm IV, hirunorm V, an N?(Me)Arg peptide, RGD-hirudin, and N?-2-naphthylsulfonyl-glycyl-DL-4-amidinophenylalanyl-piperidide acetate salt (NAPAP), while there is no such low-field resonance observed in a complex of porcine trypsin and NAPAP. The chemical shifts suggest that these resonances represent H-bonded environments. H-Donor-acceptor distances in the corresponding H-bonds are estimated to be 2.7 Å. Addition of Phe-Pro-Arg-chloromethylketone (PPACK) to a complex of human ?-thrombin with r-hirudin results in an additional signal at 18.03 ppm, which is 0.10 ppm upfield from the observed signal [Kovach, I. M., et al. (2009) Biochemistry 48, 7296-7304] for thrombin covalently modified with PPACK. In contrast, the peak at 15.33 ppm remains unchanged. The fractionation factors for the thrombin-hirudin complexes are near 1.0 within 20% error. The most likely site of the short H-bond in complexes of thrombin with the hirudin family of inhibitors is in the hydrophobic patch of the C-terminus of hirudin where Glu(57') and Glu(58') are embedded and interact with Arg(75) and Arg(77) and their solvate water (on thrombin). Glu(57') and Glu(58') present in the hirudin family of inhibitors make up a key binding epitope of fibrinogen, thrombin's prime substrate, which lends substantial interest to the short hydrogen bond as a binding element at the fibrinogen recognition site. PMID:23517305

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

2013-04-01

130

Percutaneous Repair of Radial Artery Pseudoaneurysm in a Hemodialysis Patient Using Sonographically Guided Thrombin Injection  

International Nuclear Information System (INIS)

We report a case of a radial artery pseudoaneurysm complicating an incorrect puncture of a Brescia-Cimino hemodialysis fistula that was treated with percutaneous ultrasound-guided thrombin injection. The pseudoaneurysm recurred after the initial successful thrombin injection. With a second injection we obtained permanent pseudoaneurysm occlusion. Our case illustrates that this procedure is an effective treatment in this type of arteriovenous fistula complication. We compare this case with the only similar one we could find in the literature

131

Pericellular Ca2+ recycling potentiates thrombin-evoked Ca2+ signals in human platelets  

OpenAIRE

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 ...

Sage, Stewart O.; Pugh, Nicholas; Farndale, Richard W.; Harper, Alan G. S.

2013-01-01

132

Impact of Moderate Blast Exposures on Thrombin Biomarkers Assessed by Calibrated Automated Thrombography in Rats  

OpenAIRE

Severe blast exposures are frequently complicated with fatal intracranial hemorrhages. However, many more sustain low level blasts without tissue damage detectable by brain imaging. To investigate effects of nonlethal blast on thrombin-related biomarkers, rats were subjected to two different types of head-directed blast: 1) moderate “composite” blast with strong head acceleration or 2) moderate primary blast, without head acceleration. Thrombin generation (TG) ex vivo after blast was stud...

Prima, Victor; Serebruany, Victor L.; Svetlov, Artem; Hayes, Ronald L.; Svetlov, Stanislav I.

2013-01-01

133

Characterization of Enhanced Monovalent and Bivalent Thrombin DNA Aptamer Binding Using Single Molecule Force Spectroscopy  

OpenAIRE

Thrombin aptamer binding strength and stability is dependent on sterical parameters when used for atomic force microscopy sensing applications. Sterical improvements on the linker chemistry were developed for high-affinity binding. For this we applied single molecule force spectroscopy using two enhanced biotinylated thrombin aptamers, BFF and BFA immobilized on the atomic force microscopy tip via streptavidin. BFF is a dimer composed of two single-stranded aptamers (aptabody) connected to ea...

Neundlinger, Isabel; Poturnayova, Alexandra; Karpisova, Ivana; Rankl, Christian; Hinterdorfer, Peter; Snejdarkova, Maja; Hianik, Tibor; Ebner, Andreas

2011-01-01

134

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

Energy Technology Data Exchange (ETDEWEB)

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

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

1991-01-31

135

Systems Biology of Coagulation Initiation: Kinetics of Thrombin Generation in Resting and Activated Human Blood  

OpenAIRE

Blood function defines bleeding and clotting risks and dictates approaches for clinical intervention. Independent of adding exogenous tissue factor (TF), human blood treated in vitro with corn trypsin inhibitor (CTI, to block Factor XIIa) will generate thrombin after an initiation time (Ti) of 1 to 2 hours (depending on donor), while activation of platelets with the GPVI-activator convulxin reduces Ti to ?20 minutes. Since current kinetic models fail to generate thrombin in the absence of a...

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

2010-01-01

136

Expression of monocyte chemotactic protein-1 by monocytes and endothelial cells exposed to thrombin.  

OpenAIRE

Thrombin, in addition to being a key enzyme in hemostasis, affects a series of endothelial and leukocyte functions and thus may be involved in the regulation of inflammatory reactions. Because leukocyte recruitment and activation are important events in inflammatory and thrombotic processes, in this study we have examined the possibility that thrombin induces the production of a cytokine chemotactic for mononuclear phagocytes. Human peripheral blood mononuclear cells (PBMC) exposed in vitro t...

Colotta, F.; Sciacca, F. L.; Sironi, M.; Luini, W.; Rabiet, M. J.; Mantovani, A.

1994-01-01

137

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

International Nuclear Information System (INIS)

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

138

Nanoparticles That Sense Thrombin Activity As Synthetic Urinary Biomarkers of Thrombosis  

OpenAIRE

Thrombin is a serine protease and regulator of hemostasis that plays a critical role in the formation of obstructive blood clots, or thrombosis, that is a life-threatening condition associated with numerous diseases such as atherosclerosis and stroke. To detect thrombi in living animals, we design and conjugate thrombin-sensitive peptide substrates to the surface of nanoparticles. Following intravenous infusion, these “synthetic biomarkers” survey the host vasculature for coagulation and,...

Lin, Kevin Y.; Kwong, Gabriel A.; Warren, Andrew D.; Wood, David K.; Bhatia, Sangeeta N.

2013-01-01

139

Electrostatic interactions in the association of proteins: an analysis of the thrombin-hirudin complex.  

OpenAIRE

The role of electrostatic interactions in stabilization of the thrombin-hirudin complex has been investigated by means of two macroscopic approaches: the modified Tanford-Kirkwood model and the finite-difference method for numerical solution of the Poisson-Boltzmann equations. The electrostatic potentials around the thrombin and hirudin molecules were asymmetric and complementary, and it is suggested that these fields influence the initial orientation in the process of the complex formation. ...

Karshikov, A.; Bode, W.; Tulinsky, A.; Stone, S. R.

1992-01-01

140

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

International Nuclear Information System (INIS)

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

141

Targeting the GPIb? binding site of thrombin to simultaneously induce dual anticoagulant and antiplatelet effects.  

Science.gov (United States)

Exosite 2 of human thrombin contributes to two opposing pathways, the anticoagulant pathway and the platelet aggregation pathway. We reasoned that an exosite 2 directed allosteric thrombin inhibitor should simultaneously induce anticoagulant and antiplatelet effects. To assess this, we synthesized SbO4L based on the sulfated tyrosine-containing sequence of GPIb?. SbO4L was synthesized in three simple steps in high yield and found to be a highly selective, direct inhibitor of thrombin. Michelis-Menten kinetic studies indicated a noncompetitive mechanism of inhibition. Competitive inhibition studies suggested ideal competition with heparin and glycoprotein Ib?, as predicted. Studies with site-directed mutants of thrombin indicated that SbO4L binds to Arg233, Lys235, and Lys236 of exosite 2. SbO4L prevented thrombin-mediated platelet activation and aggregation as expected on the basis of competition with GPIb?. SbO4L presents a novel paradigm of simultaneous dual anticoagulant and antiplatelet effects achieved through the GPIb? binding site of thrombin. PMID:24635452

Mehta, Akul Y; Thakkar, Jay N; Mohammed, Bassem M; Martin, Erika J; Brophy, Donald F; Kishimoto, Takao; Desai, Umesh R

2014-04-10

142

A label-free electrochemical aptasensor for sensitive thrombin detection in whole blood  

International Nuclear Information System (INIS)

In this paper, we reported a novel label-free electrochemical aptasensors for thrombin detection in whole blood using self-assembled multilayers with carboxymethyl-PEG-carboxymethyl (CM-PEG-CM) and thrombin-binding aptamer (TBA). In the sensing strategy, CM-PEG-CM and TBA were assembled on the electrode surface via covalent binding. In the presence of target, the TBA on the outermost layer of the self-assembled multilayer would catch the target on the electrode interface, which makes a barrier for electrons and inhibits the electro-transfer, resulting in the decreased DPV signals. Using this strategy, a wide detection range (1 pM–160 nM) for target thrombin was obtained, with a low detection limit of 1.56 × 10?14 M. The control experiments were also carried out by using bull serum albumin (BSA) and lysozyme in the absence of thrombin. The results showed that the aptasensors had good specificity, stability and reproducibility to thrombin. Moreover, the aptasensors could be used for detection of thrombin in whole blood which could provide a promising platform for fabrication of aptamer based biosensors in clinical application

143

Presence of plasma proteins facilitates the uptake of 125I-thrombin by the rabbit thoracic aorta endothelium in vitro  

International Nuclear Information System (INIS)

Various purified proteins, protein derivatives and two polysaccharides were added individually to a physiological medium in order to effect uptake of 125I-thrombin by the rabbit aorta endothelium. Over a wide range of concentration (0.004-40 mg/ml), the presence of either purified rabbit or bovine albumin during thrombin uptake encouraged an increase (70-110%) in 125I-thrombin binding by the endothelium and subendothelium compared to uptake by aorta segments in the absence of added protein. Pretreatment of aorta segments with albumin before incubation with 125I-thrombin in the absence of albumin did not encourage thrombin uptake to the same extent as having 125I-thrombin and albumin together. Purified human transferrin, rabbit IgG, chicken ovalbumin or denatured bovine casein could replace albumin to produce a similar enhancement of thrombin uptake. Replacing active concentrations of albumin by either reduced-carboxymethylated albumin, defatted albumin, plasmin-treated or thermolysin-treated albumin also caused an increase (50-130%) in thrombin binding, whereas replacement by acid-hydrolysed albumin or with polyglutamic acid was either ineffective or even inhibitory. Lysine-modified or arginine-modified albumins caused a small enhancement (14-32%) and no enhancement of thrombin uptake, respectively. Dextran, at low concentration (0.04-0.4 mg/ml) did not influence thrombin uptake, and at higher concentration (4-40 mg/ml) caused a decrease in uptake by both the endothelium and subendothelial layers. Low concentration of dextran sulphate inhibited thrombin uptake to 20-30% of control values. These data express the importance of accompanying protein in the response of the vascular endothelium during binding of thrombin. The possibility that other protein-cell interactions may be similarly influenced by macromolecular solutes is also discussed

144

Evidence for the presence of a protease-activated receptor distinct from the thrombin receptor in human keratinocytes.  

OpenAIRE

Thrombin receptor activation was explored in human epidermal keratinocytes and human dermal fibroblasts, cells that are actively involved in skin tissue repair. The effects of thrombin, trypsin, and the receptor agonist peptides SFLLRN and TFRIFD were assessed in inositolphospholipid hydrolysis and calcium mobilization studies. Thrombin and SFLLRN stimulated fibroblasts in both assays to a similar extent, whereas TFRIFD was less potent. Trypsin demonstrated weak efficacy in these assays in co...

Santulli, R. J.; Derian, C. K.; Darrow, A. L.; Tomko, K. A.; Eckardt, A. J.; Seiberg, M.; Scarborough, R. M.; Andrade-gordon, P.

1995-01-01

145

Thrombin regulates intracellular cyclic AMP concentration in human platelets through phosphorylation/activation of phosphodiesterase 3A  

OpenAIRE

Thrombin-induced cyclic AMP (cAMP) reduction potentates several steps in platelet activation, including Ca++ mobilization, cytoskeletal reorganization, and fibrinogen receptor conformation. We now reinvestigate the signaling pathways by which intracellular cAMP content is controlled after platelet activation by thrombin. When washed human platelets were stimulated with thrombin, cAMP-dependent phosphodiesterase (PDE3A) activity was significantly increased. A nonselective PDE inhibitor, 3-isob...

Zhang, Wei; Colman, Robert W.

2007-01-01

146

Thrombin generation as a predictor of radiotherapy induced skin erythema  

International Nuclear Information System (INIS)

Background and purpose: Biological mechanisms underlying radiation induced erythema remain largely unknown, with no simple way to accurately predict or prevent extreme cases. Based on the recent findings in patients suffering from chronic urticaria, we sought to determine if similar mechanisms of hypercoagulation contributed to comparable skin reactions during radiotherapy. Materials and methods: Plasma levels of prothrombin factor 1+2 (F1+2), D-dimers and plasminogen activator inhibitor-1 (Pai-1) were tested in 32 women undergoing irradiation following breast conserving surgery for early breast cancer. Reflectance spectrophotometry was used to objectively assess erythema throughout the treatment by measuring the amount of light reflected from the skin surface as a function of wavelength. Correlations between peak levels of erythema and plasma biomarkers were then assessed. Results: Individual peak reflectance readings generally occurred between day 29 of treatment and 2 weeks post radiotherapy, and represented a median increase of 66% (range: 11-146%; p < 0.001) from baseline. Peak reflectance correlated with F1+2 and Pai-1 levels measured both at baseline and day 29 of treatment, and multivariate analysis indicated that these two baseline measurements were the best predictors of peak reflectance, accounting for 59% of the variability in erythema (p = 0.000004). Conclusions: Patients with signs of intravascular thrombin generation are at higher risk of radiotherapy-iation are at higher risk of radiotherapy-induced skin reactions, providing a new therapeutic avenue for possibly predicting and preventing this side effect of cancer treatment

147

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

DEFF Research Database (Denmark)

Thrombin Activatable Fibrinolysis inhibitor (TAFI) is a basic carboxypeptidase, circulating in plasma as an enzymatic inactive precursor. TAFI shares ~40% overall sequence identity with pancreas Carboxypeptidase B (PCPB) with the activation peptide being less conserved. Following activation of TAFI we observed a change in the isoelectric point (IP) of TAFIa. The IP of TAFIa was significantly more basic than the IP of TAFI. This was not observed in PCPB. Due to the change in IP, we have investigated the structural basis for this observation by mapping the N- and O-linked glycans. TAFI has 5 potential N-linked glycosylation sites, four of them located on the activation peptide. Only one potential O-linked glycosylation site is seen. In addition, TAFIa is unstable and loose enzymatic activity quickly. This is in contrast to the homologous PCPB. The structural basis for this observation is not known but could be caused by differences in thermodynamic stability. Disulfides are a major contributor to the structuralintegrity of proteins and disulfide permutations could explain the difference in enzymatic stability. To test this hypothesis we have identified the disulfide pattern of the eight cysteines in TAFI. In this study we have purified and characterized TAFI from human plasma.

Christensen, Trine; Skottrup, Peter Durand

148

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

International Nuclear Information System (INIS)

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 ?-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)4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Tyr-Leu] inhibits the thrombin-catalyzed hydrolysis of a tripeptide p-nitroanilide substrate with Ki = 2.3 nM. In contrast, the synthetic C-terminal hirudin peptide S-Hir53-64, which binds to the thrombin ABE, blocked the fibrinogen clotting activity of the enzyme with Ki = 144 nM but failed to inhibit the hydrolysis of p-nitroanilide substrates at concentrations as high as 1 mM. Hirulog-1, but not S-Hir53-64, was found to inhibit the incorporation of [14C]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 nt activities of hirulog-1, hirudin, and S-Hir53-64 showed that the synthetic hirulog-1 is 2-fold more potent than hirudin and 100-fold more active than S-Hir53-64 in increasing the activated partial thromboplastin time of normal human plasma

149

Ultrasensitive electrochemical detection for thrombin using hybridization chain reaction with enzyme-amplification.  

Science.gov (United States)

In this work, a new electrochemical aptasensor using hybridization chain reaction (HCR) for signal amplification was developed for highly sensitive detection of thrombin. The sandwich system of aptamer/thrombin/aptamer-primer complex was fabricated as the sensing platform. As the initiator strands, aptamer-primer complex could propagate a chain reaction of hybridization events between the two hairpin probes, and whether long nicked DNA polymers could be formed on the modified electrode. Then the biotin-labeled dsDNA polymers could introduce numerous avidin-labeled horseradish peroxidase (HRP), resulting in significantly amplified electrochemical signal through the electrocatalysis of HRP. On the basis of the enzymatic oxidization of Fe(2+) by H2O2 to yield Fe(3+), the imaging of thrombin was detected by the reduction current of Fe(3+) with the scanning electrochemical microscopic tip. The electrochemical signals had a good linear with logarithm of thrombin concentration in the range from 1.0fM to 100fM, reaching a detection limit of thrombin as low as 0.04fM. In addition, the proposed strategy exhibited excellent specificity and was successfully applied in real sample assay which demonstrated the potential application in clinical diagnostics. PMID:25682250

Song, Weiling; Xie, Xuxu; Sun, Wenbo; Zhang, Ningbo; Li, Chunxiang

2015-02-20

150

Modeling of factor XIII activation peptide (28-41) V34L mutant bound to thrombin.  

Science.gov (United States)

Factor XIII (FXIII) is a transglutaminase involved in blood coagulation. The enzyme is activated by thrombin cleaving the peptide bond R37-G38. A common mutation V34L found in FXIII has been correlated with protection from myocardial infarction. Also FXIII V34L is activated more quickly than the wild type. In the present study, FXIII (28-41) V34L mutant peptide bound to thrombin has been modeled and molecular dynamics simulations were carried out using Insight II. An average structure was calculated after simulation. The structure showed significant difference from the crystal structure of the wild type FXIII (28-37) peptide bound to thrombin. In the crystal structure the peptide adopts a folded conformation in such a way that the hydrophobic side chains of V29 and V34 occupy the apolar binding site of thrombin. The modeled V34L peptide adopts a significantly different conformation and only the bulkier L34 occupies the apolar binding site while V29 side chain is exposed to the bulk solvent. Hence, this may speed up the release of FXIII from thrombin after its activation. PMID:18808204

Nair, Divya G; Sunilkumar, P N; Sadasivan, C

2008-12-01

151

A sensitive nanoporous gold-based electrochemical aptasensor for thrombin detection.  

Science.gov (United States)

An attempt was made in the present paper to develop a nanoporous gold (NPG)-based electrochemical aptasensor for thrombin detection. The substrate electrode NPG was in situ fabricated by a facile one-step square wave potential pulse (SWPP) treatment. The treatment involved repeated gold oxidation-reduction and intensive H(2) bubbles evolution. After 100min treatment, the active surface area of Au increased greatly (34 times). The electrochemical aptasensor was fabricated using a layer-by-layer assembling strategy. A "sandwich" structure was formed via thrombin connecting the aptamer-modified NPG and the aptamer-modified Au nanoparticles (AuNPs). The AuNPs was modified with two kinds of single strand DNA (ssDNA). One was aptamer of thrombin, but the other was not, reducing the cross-reaction between thrombin and its aptamer on the same AuNP. The electrochemical signal produced by the [Ru(NH(3))(6)](3+) bound to ssDNA via electrostatic interaction was measured by chronocoulometry. Due to the amplification effects of both NPG and AuNPs, this novel NPG-based aptasensor could detect thrombin quantitatively in the range of 0.01-22nM with a detection limit as low as 30fM. The present aptasensor also exhibited excellent selectivity, stability and reusability. PMID:20452755

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

2010-08-01

152

Label-free impedimetric thrombin sensor based on poly(pyrrole-nitrilotriacetic acid)-aptamer film.  

Science.gov (United States)

A label-free and highly sensitive impedimetric aptasensor was developed based on electropolymerized film for the determination of thrombin. The first step is the electrogeneration of a poly(pyrrole-nitrilotriacetic acid) (poly(pyrrole-NTA)) film onto the surface of electrodes followed by complexation of Cu(2+) ions. Then, the histidine labeled thrombin aptamer was immobilized onto the electrode through coordination of the histidine groups on the NTA-Cu(2+) complex. The aptamer sensor was applied for the detection and quantification of thrombin via impedimetric detection without a labeling step. A linear quantification of thrombin was obtained in the range 4.7×10(-12)-5.0×10(-10) mol L(-1) with a sensitivity of 2838 ?/log unit (R(2)=0.9984). The impedance modulus at 0.3 Hz as a function of thrombin concentration was used to elaborate a similar linear relationship from 4.7×10(-12) to 5×10(-10) mol L(-1). In addition, aptamer-poly(pyrrole-NTA) electrodes incubated for 40 min in aqueous solutions of bovine serum albumin (BSA), lysozyme and IgG (5×10(-7) mol L(-1)) did not exhibit non-specific adsorption of proteins. Moreover, it has been demonstrated that the selective sensor can be regenerated several times with a good reproducibility. PMID:22959014

Xu, Hui; Gorgy, Karine; Gondran, Chantal; Le Goff, Alan; Spinelli, Nicolas; Lopez, Christian; Defrancq, Eric; Cosnier, Serge

2013-03-15

153

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

Science.gov (United States)

Thrombocytosis is a commonly encountered clinical scenario and can be either a secondary process (reactive thrombocytosis), or due to clonal disorder (i.e., essential thrombocythemia). This distinction is important as it carries implications for evaluation, prognosis and treatment. In this study we compared procoagulant potential in essential thrombocythemia and reactive thrombocytosis by measuring the thrombin generation and the level of circulating procoagulant phospholipids with functional tests. Twenty nine patients with essential thrombocythemia and 24 with reactive thrombocytosis were studied. Thrombin generation was determined by calibrated automated thrombography. Procoagulant phospholipids were detected by a chronometric standardised method (STA-Procoag-PPL). Patients with reactive thrombocytosis had a longer lag time, higher endogenous thrombin potential, peak of thrombin generation and velocity index than patients with essential thrombocythemia. The level of circulating procoagulant phospholipids was increased in patients with essential thrombocythemia as observed with the procoagulant phospholipids assay. Each parameter was analysed using ROC curves. Highest areas under the curve (AUC) were found for lag time and procoagulant phospholipids ratio (0.817 and 0.853, respectively), associated with high negative predictive value for ET (92.3% and 80%, respectively). In conclusion, patients with essential thrombocythemia and reactive thrombocytosis displayed significant differences in terms of thrombin generation and levels of procoagulant phospholipids. Among these parameters, lag time and procoagulant phospholipids ratio could help to differentiate between reactive thrombocytosis and essential thrombocythemia patients. PMID:23873831

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

2013-12-01

154

Allosteric changes in solvent accessibility observed in thrombin upon active site occupation.  

Science.gov (United States)

The solvent accessibility of thrombin in its substrate-free and substrate-bound forms has been compared by amide hydrogen/deuterium (H/(2)H) exchange. The optimized inhibitor peptide dPhe-Pro-Arg chloromethyl ketone (PPACK) was used to simulate the substrate-bound form of thrombin. These studies were motivated by the lack of observed changes in the active site of thrombin in the crystal structure of the thrombin-thrombomodulin complex. This result appeared to contradict amide exchange studies on the thrombin-thrombomodulin complex that suggested subtle changes occur in the active site loops upon thrombomodulin binding. Our results show that two active site loops, residues 214-222 and residues 126-132, undergo decreases in solvent accessibility due to steric contacts with PPACK substrate. However, we also observe two regions outside the active site undergoing solvent protection upon substrate binding. The first region corresponds to anion binding exosite 1, and the second is a beta-strand-containing loop which runs through the core of the molecule and contains Trp141 which makes critical contacts with anion binding exosite 1. These results indicate two pathways of allosteric change that connect the active site to the distal anion binding exosite 1. PMID:15122890

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

2004-05-11

155

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

OpenAIRE

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 mig...

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; Kim, Sung-soo

2011-01-01

156

The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex.  

Science.gov (United States)

Protein C activation on the surface of the endothelium is critical to the negative regulation of blood coagulation. We now demonstrate that monoclonal antibodies that block protein C binding to the endothelial cell protein C receptor (EPCR) reduce protein C activation rates by the thrombin-thrombomodulin complex on endothelium, but that antibodies that bind to EPCR without blocking protein C binding have no effect. The kinetic result of blocking the EPCR-protein C interaction is an increased apparent Km for the activation without altering the affinity of thrombin for thrombomodulin. Activation rates of the protein C derivative lacking the gamma-carboxyglutamic acid domain, which is required for binding to EPCR, are not altered by the anti-EPCR antibodies. These data indicate that the protein C activation complex involves protein C, thrombin, thrombomodulin, and EPCR. These observations open new questions about the control of coagulation reactions on vascular endothelium. Images Fig. 6 PMID:8816778

Stearns-Kurosawa, D J; Kurosawa, S; Mollica, J S; Ferrell, G L; Esmon, C T

1996-01-01

157

Characterisation of Rac activation in thrombin- and collagen-stimulated human blood platelets.  

Science.gov (United States)

In this study, we characterised the mechanisms of Rac GTPase activation in human platelets stimulated by two physiological agonists, either thrombin, acting through membrane receptors coupled to heterotrimeric G-proteins, or collagen which is known to mobilise a tyrosine kinase-dependent pathway. Both agonists induced a rapid activation of Rac that was not significantly affected by the inhibition of integrin alpha(IIb)beta(3) engagement. Using pharmacological inhibitors, we found that phospholipase C activation and calcium mobilisation were essential for platelet Rac activation by either thrombin or collagen whereas protein kinase C inhibition was without effect. In contrast to Rac, Cdc42 activation was independent of phospholipase C activation, indicating that the two GTPases are differently regulated. We also found that phosphoinositide 3-kinase was not required for Rac activation in response to thrombin but was involved in its activation by collagen. PMID:11696351

Soulet, C; Gendreau, S; Missy, K; Benard, V; Plantavid, M; Payrastre, B

2001-11-01

158

Thrombin facilitates invasion of ovarian cancer along peritoneum by inducing monocyte differentiation toward tumor-associated macrophage-like cells.  

Science.gov (United States)

Peritoneal metastasis is a distinct pathologic characteristic of advanced epithelial ovarian cancer (EOC), which is the most deadly disease of the female reproductive tract. The inflammatory environment of the peritoneum in EOC contains abundant macrophages, activated thrombin, and thrombin-associated receptors. However, little is known about the mechanism by which the thrombin-macrophages interaction contributes to tumor invasion and metastasis. We investigated the phenotype and cytokine/chemokine expression of thrombin-treated peripheral blood monocytes (MOs)/macrophages, it was found that the phenotype of MOs was altered toward a TAM-like macrophage CD163(high)IL-10(high)CCL18(high)IL-8(high) after thrombin stimulation. By Matrigel invasion assay, the conditioned medium of thrombin-stimulated MOs accelerated remarkable invasion of ES-2, SKOV3, and HO-8910, which was similar to invasive cell numbers of ascites stimuli (P neutralizing antibody attenuated EOC cell invasion in a concentration-dependent manner. Increased transcriptional activation of NF-kappaB p50/p65 was identified in thrombin-treated MOs. This study provided insight the role of thrombin in the regulation of EOC peritoneal invasion via "educating" MOs. PMID:20352429

Zhang, Ting; Ma, Zhengwen; Wang, Ruili; Wang, Ying; Wang, Shujun; Cheng, Zhongping; Xu, Hong; Jin, Xinjuan; Li, Weiping; Wang, Xipeng

2010-07-01

159

The protective role of (-)-epigallocatechin-3-gallate in thrombin-induced neuronal cell apoptosis and JNK-MAPK activation.  

Science.gov (United States)

(-)-Epigallocatechin-3-gallate (EGCG), the major polyphenolic component of green tea, has anti-inflammatory and antioxidant properties and provides neuroprotection against central nervous system diseases. Yet, it is not known whether EGCG may be neuroprotective against intracerebral hemorrhage. In this study, we used a simplified in-vitro model of thrombin neurotoxicity to test whether EGCG provides neuroprotection against thrombin-associated toxicity. Exposure of primary cortical neurons to thrombin (100?U/ml) caused dose-dependent and time-dependent cytotoxicity. Cell Counting Kit 8 and lactate dehydrogenase were used to monitor cell viability after exposure of neurons to thrombin or EGCG and after EGCG pretreatment. Flow cytometric analysis and western blotting demonstrated that thrombin-induced neuron degeneration occurs through apoptosis. A concentration of 25??M EGCG significantly abolished thrombin-induced toxicity and prevented apoptosis by suppressing c-Jun-N-terminal kinase (JNK) phosphorylation, and the JNK inhibitor SP600125 reduced thrombin-induced caspase 3 activation and apoptosis. These data suggest that EGCG may have protective effects against thrombin-induced neuroapoptosis by inhibiting the activation of JNK, leading to caspase 3 cleavage. EGCG is a novel candidate neuroprotective agent against intracerebral hemorrhage-induced neurotoxicity. PMID:25839175

He, Qianqian; Bao, Lei; Zimering, Jeffrey; Zan, Kun; Zhang, Zuohui; Shi, Hongjuan; Zu, Jie; Yang, Xinxin; Hua, Fang; Ye, Xinchun; Cui, Guiyun

2015-05-01

160

The protective role of (?)-epigallocatechin-3-gallate in thrombin-induced neuronal cell apoptosis and JNK-MAPK activation  

Science.gov (United States)

(?)-Epigallocatechin-3-gallate (EGCG), the major polyphenolic component of green tea, has anti-inflammatory and antioxidant properties and provides neuroprotection against central nervous system diseases. Yet, it is not known whether EGCG may be neuroprotective against intracerebral hemorrhage. In this study, we used a simplified in-vitro model of thrombin neurotoxicity to test whether EGCG provides neuroprotection against thrombin-associated toxicity. Exposure of primary cortical neurons to thrombin (100?U/ml) caused dose-dependent and time-dependent cytotoxicity. Cell Counting Kit 8 and lactate dehydrogenase were used to monitor cell viability after exposure of neurons to thrombin or EGCG and after EGCG pretreatment. Flow cytometric analysis and western blotting demonstrated that thrombin-induced neuron degeneration occurs through apoptosis. A concentration of 25??M EGCG significantly abolished thrombin-induced toxicity and prevented apoptosis by suppressing c-Jun-N-terminal kinase (JNK) phosphorylation, and the JNK inhibitor SP600125 reduced thrombin-induced caspase 3 activation and apoptosis. These data suggest that EGCG may have protective effects against thrombin-induced neuroapoptosis by inhibiting the activation of JNK, leading to caspase 3 cleavage. EGCG is a novel candidate neuroprotective agent against intracerebral hemorrhage-induced neurotoxicity. PMID:25839175

He, Qianqian; Bao, Lei; Zimering, Jeffrey; Zan, Kun; Zhang, Zuohui; Shi, Hongjuan; Zu, Jie; Yang, Xinxin; Hua, Fang; Ye, Xinchun

2015-01-01

161

Mutations in the fourth EGF-like domain affect thrombomodulin-induced changes in the active site of thrombin.  

Science.gov (United States)

A number of alanine and more conservative mutants of residues in the fourth domain of thrombomodulin (TM) were prepared and assayed for protein C activation and for thrombin binding. Several of the alanine mutations appeared to cause misfolding or structural defects as assessed by poor expression and/or NMR HSQC experiments, while more conservative mutations at the same site appeared to allow correct folding and preserved activity. Several of the conservative mutants bound more weakly to thrombin despite the fact that the fourth domain does not directly contact thrombin in the crystal structure of the thrombin-TM complex. A few of the mutant TM fragments bound thrombin with an affinity similar to that of the wild type but exhibited decreases in k cat for protein C activation. These mutants were also less able to cause a change in the steady state fluorescence of fluorescein-EGR-chloromethylketone bound to the active site of thrombin. These results suggest that some residues within the fourth domain of TM may primarily interact with protein C but others are functionally important for altering the way TM interacts with thrombin. Residues in the fourth domain that primarily affect k cat for protein C activation may do this by changing the active site of thrombin. PMID:18803401

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

2008-10-14

162

Recombinant Technology and Probiotics  

OpenAIRE

Recombinant technology has led the way to monumental advances in the development of useful molecules, including the development of safe probiotics. The development of novel approaches using recombinant technology and probiotics that allow accurate targeting of therapeutics to the mucosa is an interesting area of research. The creation and use of recombinant probiotics expressing recombinantovalbumin, recombinant ovalbumin mutants and yet-to-be-designed recombinant hypo/non-allergenic molecule...

Icy D’Silva

2011-01-01

163

Atorvastatin limits the pro-inflammatory response of rat aortic smooth muscle cells to thrombin.  

Science.gov (United States)

Thrombin, a serine protease, plays an important role in the progression of atherosclerosis. How atorvastatin could limit the pro-inflammatory response to thrombin was studied in cultured rat aortic smooth muscle cells. The variations in expression of interleukin-6, heme oxygenase-1, p(22phox) and Mox-1 mRNAs were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). Interleukin-6 release was determined using the B9 cell assay. Nuclear factor-kappa B (NF-kappaB) translocation was analysed by electrophoretic mobility shift assay (EMSA) and RhoA protein translocation by Western blot. Thrombin activated interleukin-6 secretion and mRNA expression in smooth muscle cells in a dose-dependent manner. The greatest effect on mRNA expression was obtained after 1 h of stimulation. Preincubation (72 h) of the cells with various concentrations of atorvastatin prevented this effect. Simultaneous addition of mevalonate overcame this statin effect. Thrombin was without effect on p(22phox) and heme oxygenase-1 mRNA expression but, after 3 h of stimulation, induced a two-fold increase in that of Mox-1. Preincubation with atorvastatin dose-dependently downregulated this Mox-1 mRNA expression. In addition, thrombin induced NF-kappaB translocation and membrane translocation of RhoA in smooth muscle cells which were both prevented by pre-treatment of the cells by atorvastatin. These data demonstrate the ability of atorvastatin to prevent the induction by thrombin of a pro-inflammatory phenotype in smooth muscle cells. PMID:12921859

Haloui, Mounsif; Meilhac, Olivier; Jandrot-Perrus, Martine; Michel, Jean Baptiste

2003-08-01

164

Thrombin activity is unaltered by N-terminal truncation of factor XIII activation peptides.  

Science.gov (United States)

In blood coagulation, thrombin helps to activate factor XIII by cleaving the activation peptide at the R37-G38 peptide bond. The residues N-terminal to the scissile bond are important in determining rates of hydrolysis. Solution studies of wild-type and mutant peptides of factor XIII AP (28-37) suggest residues P(4)-P(1) are most critical in substrate recognition. By contrast, the X-ray crystal structure of FXIII AP (28-37) displays all of the residues, P(10)-P(1), interacting with the thrombin active site in a conformation similar to that of fibrinogen Aalpha (7-16) [Sadasivan, C., and Yee, V. C. (2000) J. Biol. Chem. 275, 36942-36948]. Peptides were therefore synthesized with the N-terminal P(10)-P(6) residues removed to further characterize interactions of thrombin with factor XIII activation peptides. The truncations have no adverse effects on thrombin's ability to bind and to hydrolyze the shortened peptides. The wild-type FXIII AP (33-41) V34 sequence actually exhibits a decrease in K(m) relative to the longer (28-41) sequence whereas the cardioprotective FXIII AP (33-41) V34L exhibits a further increase in k(cat) relative to its longer parent sequence. One-dimensional proton line broadening NMR and 2D transferred-NOESY studies indicate that the shortened peptides maintain similar bound conformations as their FXIII AP (28-37) counterparts. Furthermore, the distinctive NOE between the L34 and P36 side chains is preserved. Kinetic and NMR studies thus reveal that the N-terminal portions of FXIII AP (28-37) (V34 and V34L) are not necessary for effective interaction with the thrombin active site surface. FXIII activation peptides bind to thrombin in a manner more like PAR1 than fibrinogen Aalpha. PMID:15065858

Isetti, Giulia; Maurer, Muriel C

2004-04-13

165

Headache Topics  

Science.gov (United States)

Home » My Headache » Headache Topic Sheets Headache Topic Sheets Consumer Topics Consumer Medications Spanish Abdominal Migraine Acupuncture Alcohol and Headaches Allergy and Headaches Analgesic Rebound Anemia Arteriovenous Malformation Arthritis Aura Basilar ...

166

Glu-192----Gln substitution in thrombin mimics the catalytic switch induced by thrombomodulin.  

OpenAIRE

In serine proteases, residue 192, three residues prior to the active site Ser-195, plays an important role in determining substrate specificity. In trypsin (EC 3.4.21.4) and most trypsin-like enzymes with relatively broad specificity, this position is occupied by Gln. In thrombin (EC 3.4.21.5), an enzyme with restricted specificity, position 192 is occupied by Glu. The potential importance of Glu-192 in restricting the specificity of thrombin was investigated by isosterically replacing Glu-19...

Le Bonniec, B. F.; Esmon, C. T.

1991-01-01

167

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

OpenAIRE

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

Hill-eubanks, D. C.; Parker, C. G.; Lollar, P.

1989-01-01

168

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

LENUS (Irish Health Repository)

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

Tobin, W O

2013-02-01

169

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

Energy Technology Data Exchange (ETDEWEB)

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

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

2010-01-01

170

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

International Nuclear Information System (INIS)

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

171

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

DEFF Research Database (Denmark)

A 15-mer DNA aptamer (named TBA) adopts a G-quadruplex structure that strongly inhibits fibrin-clot formation by binding to thrombin. We have performed thermodynamic analysis, binding affinity and biological activity studies of TBA variants modified by unlocked nucleic acid (UNA) monomers. UNA-U placed in position U3, U7 or U12 increases the thermodynamic stability of TBA by 0.15-0.50?kcal/mol. In contrast, modification of any position within the two G-quartet structural elements is unfavorable for quadruplex formation. The intramolecular folding of the quadruplexes is confirmed by T(m) versus ln c analysis. Moreover, circular dichroism and thermal difference spectra of the modified TBAs displaying high thermodynamic stability show bands that are characteristic for antiparallel quadruplex formation. Surface plasmon resonance studies of the binding of the UNA-modified TBAs to thrombin show that a UNA monomer is allowed in many positions of the aptamer without significantly changing the thrombin-binding properties. The biological effect of a selection of the modified aptamers was tested by a thrombin time assay and showed that most of the UNA-modified TBAs possess anticoagulant properties, and that the construct with a UNA-U monomer in position 7 is a highly potent inhibitor of fibrin-clot formation.

Pasternak, Anna; Hernandez, Frank J

2011-01-01

172

Fibrin formation is more impaired than thrombin generation and platelets immediately following cardiac surgery  

DEFF Research Database (Denmark)

Cardiac surgery performed on cardio-pulmonary bypass (CPB) may be complicated by coagulopathy and bleeding. This prospective observational study investigated the CPB-induced changes in thrombin generation, fibrin formation, and in the platelet component of the whole blood clot elasticity. The effects of haemostatic therapy with fresh frozen plasma (FFP) and platelet concentrate on these parameters were also evaluated.

Solomon, Cristina; Rahe-Meyer, Niels

2011-01-01

173

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

International Nuclear Information System (INIS)

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

174

A non-aggregation colorimetric assay for thrombin based on catalytic properties of silver nanoparticles.  

Science.gov (United States)

In this paper, we developed a simple and rapid colorimetric assay for protein detection based on the reduction of dye molecules catalyzed by silver nanoparticles (AgNPs). Aptamer-modified magnetic particles and aptamer-functionalized AgNPs were employed as capture and detection probes, respectively. Introduction of thrombin as target protein could form a sandwich-type complex involving catalytically active AgNPs, whose catalytic activity was monitored on the catalytic reduction of rhodamine B (RhB) by sodium borohydride (NaBH4). The amount of immobilized AgNPs on the complex increased along with the increase of the thrombin concentration, thus the detection of thrombin was achieved via recording the decrease in absorbance corresponding to RhB. This method has adopted several advantages from the key factors involved, i.e., the sandwich binding of affinity aptamers contributed to the increased specificity; magnetic particles could result in rapid capture and separation processes; the conjugation of AgNPs would lead to a clear visual detection. It allows for the detection limit of thrombin down to picomolar level by the naked eye, with remarkable selectivity over other proteins. Moreover, it is possible to apply this method to the other targets with two binding sites as well. PMID:24356228

Li, Jie; Li, Wei; Qiang, Weibing; Wang, Xi; Li, Hui; Xu, Danke

2014-01-01

175

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

International Nuclear Information System (INIS)

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

176

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

International Nuclear Information System (INIS)

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

177

Two different proteins that compete for binding to thrombin have opposite kinetic and thermodynamic profiles.  

Science.gov (United States)

Thrombin binds thrombomodulin (TM) at anion binding exosite 1, an allosteric site far from the thrombin active site. A monoclonal antibody (mAb) has been isolated that competes with TM for binding to thrombin. Complete binding kinetic and thermodynamic profiles for these two protein-protein interactions have been generated. Binding kinetics were measured by Biacore. Although both interactions have similar K(D)s, TM binding is rapid and reversible while binding of the mAb is slow and nearly irreversible. The enthalpic contribution to the DeltaG(bind) was measured by isothermal titration calorimetry and van't Hoff analysis. The contribution to the DeltaG(bind) from electrostatic steering was assessed from the dependence of the k(a) on ionic strength. Release of solvent H(2)O molecules from the interface was assessed by monitoring the decrease in amide solvent accessibility at the interface upon protein-protein binding. The mAb binding is enthalpy driven and has a slow k(d). TM binding appears to be entropy driven and has a fast k(a). The favorable entropy of the thrombin-TM interaction seems to be derived from electrostatic steering and a contribution from solvent release. The two interactions have remarkably different thermodynamic driving forces for competing reactions. The possibility that optimization of binding kinetics for a particular function may be reflected in different thermodynamic driving forces is discussed. PMID:14691232

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

2004-01-01

178

Structural resiliency of an EGF-like subdomain bound to its target protein, thrombin.  

Science.gov (United States)

The thrombin-bound structures of native peptide fragments from the fifth EGF-like domain of thrombomodulin were determined by use of NMR and transferred NOE spectroscopy. The bound peptides assume an EGF-like structure of an antiparallel beta-sheet, a novel structural motif observed for a bound peptide in protein-peptide complexes. There is a remarkable structural resiliency of this structure motif manifested in its ability to accommodate a different number of residues within the disulfide loop. Docking experiments revealed that the key contacts with thrombin are hydrophobic interactions between the side chains of residues Ile 414 and Ile 424 of thrombomodulin and a hydrophobic pocket on the thrombin surface. Residues Leu 415, Phe 419, and Ile 420, which would have been buried in intact EGF-like domains, are unfavorably exposed in the complex of thrombin with the EGF-like thrombomodulin fragment, thus providing a rationale for the enhancement of binding affinity upon the deletion of Ile 420. The unique beta-sheet structures of the bound peptides are specified by the presence of disulfide bridges in the peptides because a corresponding linear thrombomodulin fragment folds into a sheet structure with a different backbone topology. The different bound conformations for the linear and the cyclized peptides indicate that side-chain interactions within a specific environment may dictate the folding of bound peptides in protein-peptide complexes. PMID:8745396

Hrabal, R.; Komives, E. A.; Ni, F.

1996-01-01

179

Thrombin inhibition with dabigatran protects against high-fat diet-induced fatty liver disease in mice.  

Science.gov (United States)

Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of obesity and metabolic syndrome. Robust coagulation cascade activation is common in obese patients with NAFLD. We identified a critical temporal relationship between thrombin generation and the manifestation of hepatic steatosis, inflammation, and injury in C57BL/6J mice fed a high-fat diet (HFD) for 1, 2, and 3 months. Mice fed a HFD exhibited dramatic increases in hepatocellular injury and inflammation over time. Hepatic fibrin deposition preceded an increase in serum alanine aminotransferase, and the most dramatic changes in liver histopathology occurred in conjunction with a detectable increase in plasma thrombin-antithrombin levels at 3 months. To directly determine whether thrombin activity promotes NAFLD pathogenesis, mice were fed a HFD and simultaneously treated with the direct thrombin inhibitor dabigatran etexilate for 3 months. Notably, dabigatran treatment significantly reduced hepatic fibrin deposition, hepatic inflammation, hepatocellular injury, and steatosis in mice fed a HFD. Of interest, dabigatran treatment also significantly attenuated HFD-induced body weight gain. Gene expression analysis suggested that thrombin potentially drives NAFLD pathogenesis by altering the expression of genes associated with lipid metabolism and bile acid synthesis. Collectively, the results suggest that thrombin activity is central to HFD-induced body weight gain, liver injury, and inflammation and provide the proof-of-principle evidence that pharmacological thrombin inhibition could be effective in limiting NAFLD and associated pathologies. PMID:25138021

Kopec, Anna K; Joshi, Nikita; Towery, Keara L; Kassel, Karen M; Sullivan, Bradley P; Flick, Matthew J; Luyendyk, James P

2014-11-01

180

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

Energy Technology Data Exchange (ETDEWEB)

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

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

1989-12-01

181

An antibody that binds to primary specific pocket-associated structure in the active site of bovine thrombin.  

Science.gov (United States)

We attempted to produce a monoclonal antibody (MAb) against the active site of native thrombin. Bovine thrombin was treated with diisopropyl fluorophosphate, and prepared diisopropylphosphoryl-thrombin was used for the immunization to BALB/c mice. Spleen cells of immunized mice were hybridized with mouse myeloma cells P3U1, and a hybridoma clone CC2, which produced a MAb against bovine thrombin was established. The MAb produced by hybridoma clone CC2 (MAb(CC2)), consisting of IgG(1) and kappa light chain, was purified using protein A affinity chromatography. Purified MAb(CC2) prolonged the fibrin forming time of bovine thrombin and inhibited the release of fibrinopeptide A from rabbit fibrinogen. In addition, it was found that argatroban partially, but competitively, interfere the binding between MAb(CC2) and bovine thrombin. It was then considered that MAb(CC2) would bind to the molecular structure associating primary specific pocket in the active site of bovine thrombin. PMID:11991818

Kinjoh, Kiyohiko; Nakamura, Mariko; Zeng, Gang; Kosugi, Tadayoshi

2002-02-01

182

PAR1 antagonists inhibit thrombin-induced platelet activation whilst leaving the PAR4-mediated response intact.  

Science.gov (United States)

Abstract Thrombin-induced platelet activation is initiated by PAR1 and PAR4 receptors. Vorapaxar, a PAR1 antagonist, has been assessed in patients with acute coronary syndromes (ACS) and stable atherosclerotic disease in addition to standard-of-care treatment. In clinical trials, vorapaxar has been observed to reduce the frequency of ischaemic events in some subgroups though in others has increased the frequency of bleeding events. Among patients undergoing CABG surgery, which is associated with excess thrombin generation, bleeding was not increased. The aim of these studies was to investigate the effects of selective PAR1 antagonism on thrombin-induced platelet activation in patients receiving vorapaxar or placebo in the TRACER trial and to explore the roles of PAR1 and PAR4 in thrombin-induced platelet activation in healthy volunteers. ACS patients receiving vorapaxar or placebo in the TRACER trial were studied at baseline and 4 hours, 1 and 4 months during drug administration. Thrombin-induced calcium mobilisation in platelet-rich plasma was assessed by flow cytometry. In vitro studies were performed in healthy volunteers using the PAR1 antagonist SCH79797 or PAR4 receptor desensitisation. Vorapaxar treatment significantly inhibited thrombin-induced calcium mobilisation, leaving a residual, delayed response. These findings were consistent with calcium mobilisation mediated via the PAR4 receptor and were reproduced in vitro using SCH79797. PAR4 receptor desensitization, in combination with SCH79797, completely inhibited thrombin-induced calcium mobilisation confirming that the residual calcium mobilisation was mediated via PAR4. In conclusion vorapaxar selectively antagonises the PAR1-mediated component of thrombin-induced platelet activation, leaving the PAR4-mediated response intact, which may explain why vorapaxar is well tolerated in patients undergoing CABG surgery since higher thrombin levels in this setting may override the effects of PAR1 antagonism through PAR4 activation, thus preserving haemostasis. Further assessment may be warranted. PMID:24750101

Judge, Heather M; Jennings, Lisa K; Moliterno, David J; Hord, Edward; Ecob, Rosemary; Tricoci, Pierluigi; Rorick, Tyrus; Kotha, Jayaprakash; Storey, Robert F

2014-04-21

183

Pericellular Ca(2+) recycling potentiates thrombin-evoked Ca(2+) signals in human platelets.  

Science.gov (United States)

We have previously demonstrated that Na(+)/Ca(2+) exchangers (NCXs) potentiate Ca(2+) 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 Ca(2+) 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 Ca(2+) in a pericellular region around the platelets. To test whether this pericellular Ca(2+) accumulation might be responsible for the influence of NCXs over platelet function, platelets were exposed to fast Ca(2+) chelators or had their glycocalyx removed. Both manipulations of the pericellular Ca(2+) rise reduced thrombin-evoked Ca(2+) signals and dense granule secretion. Blocking Ca(2+)-permeable ion channels had a similar effect, suggesting that Ca(2+) 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 [Ca(2+)] 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 [Ca(2+)] 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 [Ca(2+)]. PMID:24303163

Sage, Stewart O; Pugh, Nicholas; Farndale, Richard W; Harper, Alan G S

2013-10-01

184

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

OpenAIRE

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

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

1986-01-01

185

Synergistic inhibition of thrombin-induced platelet aggregation by the novel nitric oxide-donor GEA 3175 and adenosine.  

OpenAIRE

1. The influence of the novel nitric oxide-donor GEA 3175 on thrombin- and ionomycin-stimulated human platelets was investigated. The effect of GEA 3175 was compared with that of adenosine, an activator of platelet adenylyl cyclase. 2. GEA 3175 inhibited thrombin-induced secretion of ATP but did not affect aggregation; similar results were obtained with adenosine. 3. Thrombin-stimulated rises in the cytosolic free Ca2+ concentration, [Ca2+]i, were dose-dependently inhibited by GEA 3175 and ad...

Grenega?rd, M.; Gustafsson, M. C.; Andersson, R. G.; Bengtsson, T.

1996-01-01

186

Phorbol myristate acetate inhibits thrombin-stimulated Ca2+ mobilization and phosphatidylinositol 4,5-bisphosphate hydrolysis in human platelets.  

OpenAIRE

The tumor-promoting phorbol diester 4 beta-phorbol 12-myristate 13-acetate (PMA) inhibited mobilization of intracellular Ca2+ in platelets by thrombin (also trypsin and 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine). PMA was effective over the same concentration range that activates protein kinase C in intact platelets; IC50 vs. thrombin = 2 ng/ml, 3.4 nM: greater than 90% inhibition at 10-20 ng/ml. Suppression of thrombin-induced Ca2+ mobilization was evident within 30 sec of pretreatment ...

Zavoico, G. B.; Halenda, S. P.; Sha Afi, R. I.; Feinstein, M. B.

1985-01-01

187

Induction of MMP-9 release from human dermal fibroblasts by thrombin: involvement of JAK/STAT3 signaling pathway in MMP-9 release  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background It has been recognized that dermal fibroblasts and matrix metalloproteases (MMP play crucial roles in wound healing process in skin. Thrombin was found to stimulate IL-8 release from human dermal fibroblasts (HDFs. However, little is known of the effect of thrombin on secretion of MMPs from dermal fibroblasts. In the present study, the influence of thrombin on proMMP-2 and proMMP-9 activity release from primary cultured HDFs, and its potential signaling pathways were investigated. Results The results showed that thrombin induced proMMP-9, but not proMMP-2 release from HDFs in a dose dependent manner at 6 h following incubation. Thrombin also upregulated expression of proMMP-9 mRNA in HDFs. Hirudin completely abolished the action of thrombin on HDFs. An agonist peptide of protease-activated receptor-1, SFLLR-NH2 stimulated an enhanced release of proMMP-9 from HDFs. AG490, an inhibitor of STAT3 inhibited basal and thrombin-provoked proMMP-9 release and phosphorylation of STAT3. PD98059, an inhibitor of MAPK and LY294002, an inhibitor PI3K failed to significantly inhibit thrombin induced proMMP-9 release. Conclusion Thrombin is a potent stimulus of proMMP-9 release from HDFs. Thrombin induced proMMP-9 release is most likely through activation of PAR-1. JAK/STAT3 signaling pathway is involved in proMMP-9 release from HDFs.

He Shaoheng

2007-05-01

188

Fundamental study of recombination and recombineering in Escherichia coli  

OpenAIRE

Recombination and recombineering systems have been used in Escherichia coli to recombinant DNA sequences. With endonuclease and DNA lipase the bacterial plasmid and target DNA fragment can bind together and recombinant for a new DNA sequences. Red Proteins have been used in recombineering system to perform the function as the enzymes in recombination system, and faster and easier than the other way of recombinant new DNA sequences in E.coli. In this report we get to know the pr...

Sun, Xiaohang; Huang, Yang

2008-01-01

189

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

International Nuclear Information System (INIS)

An ultrasound-guided direct injection of thrombin is currently the first choice of treatment for the postcatheterization pseudoaneurysm, mainly in the femoral artery. A pseudoaneurysm in the posterior tibial artery is very rare, so there are not enough reports about proper treatment yet. We report a case of a balloon-assisted injection of thrombin under ultrasonography-guidance to manage a pseudoaneurysm in the posterior tibial artery and concurrently to prevent a distal artery embolization.

190

Expression of Allosteric Linkage between the Sodium Ion Binding Site and Exosite I of Thrombin during Prothrombin Activation*  

OpenAIRE

The specificity of thrombin for procoagulant and anticoagulant substrates is regulated allosterically by Na+. Ordered cleavage of prothrombin (ProT) at Arg320 by the prothrombinase complex generates proteolytically active, meizothrombin (MzT), followed by cleavage at Arg271 to produce thrombin and fragment 1.2. The alternative pathway of initial cleavage at Arg271 produces the inactive zymogen form, the prethrombin 2 (Pre 2)·fragment 1.2 complex, which is cleaved subsequently at Arg320. Clea...

Kroh, Heather K.; Tans, Guido; Nicolaes, Gerry A. F.; Rosing, Jan; Bock, Paul E.

2007-01-01

191

Fucosylated chondroitin sulfate inhibits plasma thrombin generation via targeting of the factor IXa heparin-binding exosite  

OpenAIRE

Depolymerized holothurian glycosaminoglycan (DHG) is a fucosylated chondroitin sulfate with antithrombin-independent antithrombotic properties. Heparin cofactor II (HCII)-dependent and -independent mechanisms for DHG inhibition of plasma thrombin generation were evaluated. When thrombin generation was initiated with 0.2 pM tissue factor (TF), the half maximal effective concentration (EC50) for DHG inhibition was identical in mock- or HCII-depleted plasma, suggesting a serpin-independent mecha...

Buyue, Yang; Sheehan, John P.

2009-01-01

192

Plasmid-Mediated Resistance to Thrombin-Induced Platelet Microbicidal Protein in Staphylococci: Role of the qacA Locus  

OpenAIRE

Thrombin-induced platelet microbicidal protein 1 (tPMP-1) is a small, cationic peptide released from rabbit platelets following thrombin stimulation. In vitro resistance to this peptide among strains of Staphylococcus aureus correlates with the survival advantage of such strains at sites of endothelial damage in humans as well as in experimental endovascular infections. The mechanisms involved in the phenotypic resistance of S. aureus to tPMP-1 are not fully delineated. The plasmid-encoded st...

Kupferwasser, Leon Iri; Skurray, Ronald A.; Brown, Melissa H.; Firth, Neville; Yeaman, Michael R.; Bayer, Arnold S.

1999-01-01

193

The impact of elective knee/hip replacement surgery and thromboprophylaxis with Rivaroxaban or Dalteparin on Thrombin Generation  

OpenAIRE

Abstract Total hip/knee replacement surgeries are associated with an increased risk of venous thromboembolism and post-operative thromboprophylaxis has become standard treatment. The aims of this study were to: (1) assess the impact of hip/knee replacement surgery on ex vivo thrombin generation (TG), prothrombin-fragments 1+2 (F1+2), thrombin-antithrombin complexes (TAT) and D-dimer; (2) compare the anticoagulant effects of dalteparin and rivaroxaban on TG 24 hours after surgery. H...

Green, Laura; Lawrie, Andrew S.; Patel, Shelain; Hossain, Fahad; Chitolie, Andrew; Mackie, Ian; Haddad, Fares S.; Machin, Sam

2010-01-01

194

Calcium/calmodulin transduces thrombin-stimulated secretion: studies in intact and minimally permeabilized human umbilical vein endothelial cells  

OpenAIRE

Thrombin stimulates cultured endothelial cells (EC) to secrete stored von Willebrand factor (vWF), but the signal transduction pathways are poorly defined. Thrombin is known to elevate the concentration of intracellular calcium ([Ca2+]i) and to activate protein kinase C (PKC) in EC. Since both calcium ionophores and phorbol esters release vWF, both second messenger pathways have been postulated to participate in vWF secretion in response to naturally occurring agonists. We find that in intact...

1992-01-01

195

Regulation of the Actin Cytoskeleton by Thrombin in Human Endothelial Cells: Role of Rho Proteins in Endothelial Barrier Function  

OpenAIRE

Endothelial barrier function is regulated at the cellular level by cytoskeletal-dependent anchoring and retracting forces. In the present study we have examined the signal transduction pathways underlying agonist-stimulated reorganization of the actin cytoskeleton in human umbilical vein endothelial cells. Receptor activation by thrombin, or the thrombin receptor (proteinase-activated receptor 1) agonist peptide, leads to an early increase in stress fiber formation followed by cortical actin ...

Vouret-craviari, Vale?rie; Boquet, Patrice; Pouysse?gur, Jacques; Obberghen-schilling, Ellen

1998-01-01

196

Stimulation of neutrophil adherence to vascular endothelial cells by histamine and thrombin and its inhibition by PAF antagonists and dexamethasone.  

OpenAIRE

1. In order to clarify the roles of platelet-activating factor (PAF) in histamine- and thrombin-induced neutrophil adhesion to vascular endothelial cells, the effects of several PAF antagonists were examined. The effects of the glucocorticoid dexamethasone were also examined in order to gain further insight into the anti-inflammatory actions of glucocorticoids. 2. In culture, histamine and thrombin stimulated the adherence of rat peritoneal neutrophils to human endothelial cells from the umbi...

Watanabe, M.; Yagi, M.; Omata, M.; Hirasawa, N.; Mue, S.; Tsurufuji, S.; Ohuchi, K.

1991-01-01

197

THROMBIN REGULATES METASTATIC POTENTIAL OF HUMAN RHABDOMYOSARCOMA CELLS – DISTINCT ROLE OF PAR1 AND PAR3 SIGNALING  

OpenAIRE

We observed that human rhabdomyosarcoma (RMS) cells highly express a tissue factor (TF) that promotes thrombin formation, which indirectly and directly affects RMS progression. First, we found that thrombin activates platelets to generate microvesicles (PMVs), which transfer to RMS cells’ ?2?3 integrin and increase their adhesiveness to endothelial cells. Accordingly, RMS cells covered with PMVs showed higher metastatic potential after intravenous injection into immunodeficient mice. Furt...

Wysoczynski, Marcin; Rui, Liu; Kucia, Magda; Drukala, Justyna; Ratajczak, Mariusz Z.

2010-01-01

198

[Promoting effect of thrombin on proliferation of bone marrow-derived mesenchymal stem cells and its mechanisms].  

Science.gov (United States)

This study was aimed to investigate the growth-promoting activity of thrombin on mesenchymal stem cells (MSC) and its mechanisms. Human bone marrow MSC were cultured in serum-free medium supplemented with graded concentrations of thrombin, and the proliferation status of MSC was detected by MTT test. The expression levels of protease-activated receptors (PAR) and c-MYC gene were detected by PCR. Activated Akt signaling pathway was revealed by Western blot, and specific inhibitors of the signaling pathways were used to confirm the effects. The results showed that thrombin stimulated MSC proliferation in a dose-dependent manner; the minimal concentration of thrombin for stimulating MSC growth was 0.5 U/ml, and the promoting effect reached its maximum when thrombin at a dose of 8 U/ml was employed. PCR results showed that MSC expressed the two types of PAR1 and PAR2. After PAR1 was blocked with a specific inhibitor SCH79797, the growth-promoting effect of thrombin was inhibited, while this phenomenon was not observed when MSC were exposed to FSLLRY-NH2, a specific inhibitor for PAR2. Further experiments showed that after exposure to thrombin, the AKT signaling pathway in MSC was promptly activated, and c-MYC expression was greatly up-regulated. Meanwhile, when LY294002, a specific AKT inhibitor, was added into the culture medium, the up-regulation of c-MYC expression was reduced, accompanied by the low rate of MSC growth. It is concluded that thrombin can stimulate MSC proliferation by eliciting PAR1-mediated AKT activation and subsequent up-regulation of c-MYC expression. PMID:24763028

Chen, Jin; Ma, Yu-Jie; Wang, Zi; Lin, Shan-Shan; Xiao, Feng-Jun; Wang, Hua; Wang, Li-Sheng; Guo, Zi-Kuan

2014-04-01

199

Thrombin and interleukin-1? decrease HOX gene expression in human first trimester decidual cells: implications for pregnancy loss  

OpenAIRE

Bleeding or inflammation in early pregnancy may result in pregnancy loss or defective implantation. Their effect on HOX gene expression in first trimester decidua is unknown. Bleeding results in thrombin generation, although infection or inflammation results in production of cytokines typified by Interleukin-1? (IL-1?). First trimester decidual cells were pretreated with 17? estradiol (E2), medroxyprogesterone acetate (MPA) or both and subsequently treated with thrombin or IL-1?. Affymetr...

Sarno, Jennifer; Schatz, Frederick; Huang, S. Joseph; Lockwood, Charles; Taylor, Hugh S.

2009-01-01

200

Evaluation of the Profile of Thrombin Generation during the Process of Whole Blood Clotting as Assessed by Thromboelastography  

OpenAIRE

Thromboelastography is useful for assessment of whole blood coagulation. The objective of this study was to evaluate the possibility of linking the tracing of whole blood clotted in a thromboelastograph TEG with the generation of thrombin assessed by thrombin/antithrombin complex (TAT). Citrated whole blood containing corn trypsin inhibitor from volunteers was clotted in the presence of CaCl2 and tissue factor. Clotting was monitored with 8 channels of a TEG system. At different time points t...

Rivard, Georges E.; Brummel, Kathleen; Mann, Kenneth G.; Fan, Li; Hofer, Ange?lique; Cohen, Eli

2005-01-01

201

Spectroscopic and Electrochemical Detection of Thrombin/5'-SH or 3'-SH Aptamer Immobilized on (porous) Gold Substrates  

International Nuclear Information System (INIS)

Thrombin is a serine protease that catalyzes the conversion of soluble fibrinogen to insoluble fibrin, and thus induces physiological and pathological blood coagulation. Therefore, it is important to detect thrombin in blood serum for purposes of diagnosis. To achieve this goal, it has been suggested that a 15-mer aptamer strongly binds with thrombin to form a G-quartet structure of the aptamer. Generally, 5'-end thiol-functionalized aptamer has been used as an anti-thrombin binder. Herein, we evaluate the possibility of utilizing a 3'-SH aptasensor for thrombin detection using SPR spectroscopy, and compare the enhancement of the electrochemical signal of the thrombin-aptamer bound on a porous gold substrate. Although the two aptamers have similar configurations, in SPR analysis, the 3'-SH aptamer was a effective aptasensor as well as 5'-SH aptamer. Results from electrochemical analysis showed that the porous gold substrate acted as a good substrate for an aptasensor and demonstrated 5-fold enhancement of current change, as compared to gold thin film

202

Thrombin-activated platelets promote leukotriene B4 synthesis in polymorphonuclear leucocytes stimulated by physiological agonists.  

OpenAIRE

1. The addition of 2 x 10(8) human platelets to 8 x 10(6) polymorphonuclear leucocytes (PMNL) incubated in presence of 2.5 u ml-1 thrombin and 0.1 microM N-formyl-Met-Leu-Phe (FMLP) (or C5a or PAF) led to enhancement of leukotriene B4 (LTB4) synthesis by the PMNL (measured by h.p.l.c. as 20-hydroxy- and 20-carboxy-LTB4) from 4 +/- 1 pmol (in absence of platelets) to 26 +/- 4 pmol (mean +/- s.e.mean, n = 9). Platelets and thrombin were both essential for the enhancement of LTB4 synthesis. 2. P...

Palmantier, R.; Borgeat, P.

1991-01-01

203

First Steps in the Direction of Synthetic, Allosteric, Direct Inhibitors of Thrombin and Factor Xa  

OpenAIRE

Designing non-saccharide functional mimics of heparin is a major challenge. In this work, a library of small, aromatic molecules based on the sulfated DHP scaffold was synthesized and screened against thrombin and factor Xa. The results reveal that i) selected monomeric benzofuran derivatives inhibit the two enzymes, albeit weakly; ii) the two enzymes recognize different structural features in the benzofurans studied suggesting significant selectivity of recognition; and iii) the mechanism of...

Verghese, Jenson; Liang, Aiye; Sidhu, Preet Pal Singh; Hindle, Michael; Zhou, Qibing; Desai, Umesh R.

2009-01-01

204

The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex.  

OpenAIRE

Protein C activation on the surface of the endothelium is critical to the negative regulation of blood coagulation. We now demonstrate that monoclonal antibodies that block protein C binding to the endothelial cell protein C receptor (EPCR) reduce protein C activation rates by the thrombin-thrombomodulin complex on endothelium, but that antibodies that bind to EPCR without blocking protein C binding have no effect. The kinetic result of blocking the EPCR-protein C interaction is an increased ...

Stearns-kurosawa, D. J.; Kurosawa, S.; Mollica, J. S.; Ferrell, G. L.; Esmon, C. T.

1996-01-01

205

Quantum and Molecular Dynamics Study for Binding of Macrocyclic Inhibitors to Human ?-Thrombin  

OpenAIRE

Molecular dynamics simulations followed by quantum mechanical calculation and Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) analysis have been carried out to study binding of proline- and pyrazinone-based macrocyclic inhibitors (L86 and T76) to human ?-thrombin. Detailed binding interaction energies between these inhibitors and individual protein fragments are calculated using DFT method based on a new quantum mechanical approach for computing protein-ligand interaction energy...

Wu, Emilia L.; Mei, Ye; Han, Keli; Zhang, John Z. H.

2007-01-01

206

Gold nanoparticle enhanced electrochemiluminescence of CdS thin films for ultrasensitive thrombin detection.  

Science.gov (United States)

Interactions between surface plasmons (SP) of metallic surfaces and photoluminescence (PL) of semiconductor nanocrystal (S-NC) surfaces have been extensively investigated, and SP-induced PL enhancement has been used as a sensitive analytical technique. However, this SP induced electrochemiluminescence (ECL) enhancement is rarely studied. In this work, we report greatly enhanced ECL of CdS thin films by gold nanoparticles (Au NPs) for ultrasensitive detection of thrombin. The system was composed of a CdS NC film on glassy carbon electrode (GCE) as ECL emitter attached an aptamer of thrombin. Then, ssDNA-AuNP conjugates hybridized with the aptamer to form a separation length of ca. 12 nm between CdS NCs and Au NPs. The system showed 5-fold enhancement of ECL intensity as compared to that without Au NPs, which might be attributed to the long-distance interaction between the S-NCs and SPR field of noble metal nanoparticles (MNPs).We also found that the enhanced ECL could be influenced by the involving factors such as the separation distance, spectral overlap, and magnetic field. Such enhancement in combination with smart recognition of aptamer and target protein allowed us to construct an ultrasensitive aptasensor for attomolar detection of thrombin. The presence of target protein was reflected by the ECL signal decrease caused by the target-induced removal of ssDNA-AuNP conjugates. The decrease of ECL signal was logarithmically linear with the concentration of thrombin in a wide range from 100 aM to 100 fM. The principle described in this work could be also applied to many other bioassays. PMID:21517100

Wang, Jing; Shan, Yun; Zhao, Wei-Wei; Xu, Jing-Juan; Chen, Hong-Yuan

2011-06-01

207

Gelatin-thrombin hemostatic matrix injection to salvage refractory post-renal graft biopsy bleed  

OpenAIRE

Post-renal biopsy bleeding refractory to angioembolization usually requires graft nephrectomy as a life-saving measure. Gelatin-thrombin hemostatic matrix injection in the needle tract is a novel attempt to control bleeding in such cases and to salvage the allograft. We hereby describe two cases of post-graft biopsy bleed. Both these patients continued to bleed even after angioembolization. They were shifted to the operating room upon developing hypotension, having received multiple blood tra...

Jain, V.; Gupta, A.; Gulia, A.; Singhal, M.; Gulati, S.; Tiwari, S. C.; Kumar, A.

2013-01-01

208

Cleavage-based hybridization chain reaction for electrochemical detection of thrombin.  

Science.gov (United States)

In the present work, we constructed a new label-free "inter-sandwich" electrochemical aptasensor for thrombin (TB) detection by employing a cleavage-based hybridization chain reaction (HCR). The designed single-stranded DNA (defined as binding DNA), which contained the thrombin aptamer binding sequence, a DNAzyme cleavage site and a signal reporter sequence, was first immobilized on the electrode. In the absence of a target TB, the designed DNAzymes could combine with the thrombin aptamer binding sequence via complementary base pairing, and then Cu(2+) could cleave the binding DNA. In the presence of a target TB, TB could combine with the thrombin aptamer binding sequence to predominantly form an aptamer-protein complex, which blocked the DNAzyme cleavage site and prevented the binding DNA from being cleaved by Cu(2+)-dependent DNAzyme. As a result, the signal reporter sequence could leave the electrode surface to trigger HCR with the help of two auxiliary DNA single-strands, A1 and A2. Then, the electron mediator hexaammineruthenium (III) chloride ([Ru(NH3)6](3+)) was embedded into the double-stranded DNA (dsDNA) to produce a strong electrochemical signal for the quantitative measurement of TB. For further amplification of the electrochemical signal, graphene reduced by dopamine (PDA-rGO) was introduced as a platform in this work. With this strategy, the aptasensor displayed a wide linearity in the range of 0.0001 nM to 50 nM with a low detection limit of 0.05 pM. Moreover, the resulting aptasensor exhibited good specificity and acceptable reproducibility and stability. Because of these factors, the fabrication protocol proposed in this work may be extended to clinical application. PMID:24971937

Chang, Yuanyuan; Chai, Yaqin; Xie, Shunbi; Yuan, Yali; Zhang, Juan; Yuan, Ruo

2014-09-01

209

Anti-thrombin III, Protein C, and Protein S deficiency in acute coronary syndrome  

OpenAIRE

The final most common pathway for the majority of coronary artery disease is occlusion of a coronary vessel. Under normal conditions, antithrombin III (AT III), protein C, and protein S as an active protein C cofactor, are natural anticoagulants (hemostatic control) that balances procoagulant activity (thrombin antithrombin complex balance) to prevent thrombosis. If the condition becomes unbalanced, natural anticoagulants and the procoagulants can lead to thrombosis. Thirty subjects with a...

Dasnan Ismail; Harun, S.; Idrus Alwi; Tambunan, Karmel L.; Shufrie Effendy

2002-01-01

210

Successful endovascular treatment of a hemodialysis graft pseudoaneurysm by covered stent and direct percutaneous thrombin injection.  

LENUS (Irish Health Repository)

Vascular access for hemodialysis remains a challenge for nephrologists, vascular surgeons, and interventional radiologists alike. Arteriovenous fistula and synthetic grafts remain the access of choice for long-term hemodialysis; however, they are subject to complications from infection and repeated needle cannulation. Pseudoaneurysms are an increasingly recognized adverse event. At present, there are many minimally invasive methods to repair these wall defects. We present a graft pseudoaneurysm, which required a combination of endovascular stent graft placement and percutaneous thrombin injection for successful occlusion.

Keeling, Aoife N

2011-07-25

211

Intraovarian Thrombin and Activated Protein C Signaling System Regulates Steroidogenesis during the Periovulatory Period  

OpenAIRE

In addition to its role in blood coagulation, thrombin directly stimulates protease-activated receptors (PAR) or interacts with thrombomodulin (THBD) to activate membrane-bound protein C which stimulates PAR1 and PAR4 receptors to promote downstream pleiotropic effects. Our DNA microarray, RT-PCR, and immunostaining analyses demonstrated ovarian expression of THBD, activated protein C (APC) receptor [endothelial protein C receptor (EPCR)], as well as PAR1 and PAR4 receptors in mice. After tre...

Cheng, Yuan; Kawamura, Kazuhiro; Deguchi, Masashi; Takae, Seido; Mulders, Sabine M.; Hsueh, Aaron J. W.

2011-01-01

212

Thrombin Stimulates Human Endothelial Arginase Enzymatic Activity via RhoA/ROCK Pathway  

OpenAIRE

Background— Arginase competes with endothelial nitric oxide synthase (eNOS) for the substrate L-arginine and decreases NO production. This study investigated regulatory mechanisms of arginase activity in endothelial cells and its role in atherosclerosis. Methods and Results— In human endothelial cells isolated from umbilical veins, thrombin concentration- and time-dependently stimulated arginase enzymatic activity, reaching a 1.9-fold increase (P

Ming, Xiu-fen; Barandier, Christine; Viswambharan, Hema; Kwak, Brenda R.; Mach, Franc?ois; Mazzolai, Lucia; Hayoz, Daniel; Ruffieux, Jean; Rusconi, Sandro; Montani, Jean-pierre; Yang, Zhihong

2005-01-01

213

Thrombin-stimulated immunoprecipitation of phosphatidylinositol 3-kinase from human platelets.  

OpenAIRE

Growth factors and transforming proteins that activate tyrosine phosphorylation have been shown to cause an increased labeling of 3-phosphate-containing phosphatidylinositols. Turnover correlates with the formation of a complex between phosphatidylinositol 3-kinase, the activated protein-tyrosine kinase, and other proteins thought to participate in transmembrane signaling. When human platelets are treated with thrombin, labeling of 3-phosphate-containing phosphatidylinositols is stimulated wi...

Mitchell, C. A.; Jefferson, A. B.; Bejeck, B. E.; Brugge, J. S.; Deuel, T. F.; Majerus, P. W.

1990-01-01

214

Protease-activated receptor 1 is the primary mediator of thrombin-stimulated platelet procoagulant activity  

OpenAIRE

The activation of human platelets by thrombin is mediated primarily by protease-activated receptors (PARs). PAR1 and PAR4 are present on human platelets and are activated by the hexapeptides SFLLRN and GYPGQV, respectively. To further characterize the involvement of PAR1 and PAR4 in platelet activation, the ability of SFLLRN or GYPGQV to generate annexin V binding to newly exposed phospholipids on the platelet surface and generate procoagulant activity has been examined. Exposure of phosphati...

Andersen, Henrik; Greenberg, Daniel L.; Fujikawa, Kazuo; Xu, Wenfeng; Chung, Dominic W.; Davie, Earl W.

1999-01-01

215

Thrombin Production and Human Neutrophil Elastase Sequestration by Modified Cellulosic Dressings and Their Electrokinetic Analysis  

OpenAIRE

Wound healing is a complex series of biochemical and cellular events. Optimally, functional material design addresses the overlapping acute and inflammatory stages of wound healing based on molecular, cellular, and bio-compatibility issues. In this paper the issues addressed are uncontrolled hemostasis and inflammation which can interfere with the orderly flow of wound healing. In this regard, we review the serine proteases thrombin and elastase relative to dressing functionality that improve...

Nicolette Prevost; Judson Vincent Edwards

2011-01-01

216

Effect of thrombin inhibition on synovial inflammation in antigen induced arthritis  

OpenAIRE

OBJECTIVE—To determine the effect of the thrombin inhibitor, hirudin, on the pathogenesis of murine antigen induced arthritis (AIA).?METHODS—AIA was induced by intra-articular injection of methylated bovine serum albumin in the knee joints of previously immunised mice. Hirudin (injected subcutaneously 3 × 200 µg/mouse/day) was given over 13 days, starting three days before arthritis onset, and its anticoagulant effect monitored by clotting times. Arthritis severity was evaluated b...

Varisco, P. A.; Peclat, V.; Ness, K.; Bischof-delaloye, A.; So, A.; Busso, N.

2000-01-01

217

Effect of thrombin fragment (TP508) on myocardial ischemia-reperfusion injury in hypercholesterolemic pigs  

OpenAIRE

Myocardial ischemia-reperfusion (IR) injury occurs frequently in the setting of hypercholesterolemia. We investigated the potential efficacy of a novel thrombin fragment (TP508) on IR injury in a hypercholesterolemic porcine model. Twenty-one hypercholesterolemic male Yucatan pigs underwent 60 min of mid-left anterior descending coronary artery occlusion followed by 120 min of reperfusion. Pigs received either placebo (control, n = 7) or TP508 in two doses (TP508 low dose, n = 7, as bolus of ...

Osipov, Robert M.; Robich, Michael P.; Feng, Jun; Clements, Richard T.; Liu, Yuhong; Glazer, Hilary P.; Wagstaff, John; Bianchi, Cesario; Sellke, Frank W.

2009-01-01

218

Permeability of Three-Dimensional Fibrin Constructs Corresponds to Fibrinogen and Thrombin Concentrations  

OpenAIRE

Research in the last few years have focused on the use of three-dimensional (3D) fibrin construct to deliver growth factors and cells. Three-dimensional construct permeability and porosity are important aspects for proper nutrient uptake, gas exchange, and waste removal—factors that are critical for cell growth and survival. We have previously reported that the mechanical strength (stiffness) of 3D fibrin constructs is dependent on the fibrinogen and thrombin concentration. In this study, w...

Chiu, Cecilia L.; Hecht, Vivian; Duong, Haison; Wu, Benjamin; Tawil, Bill

2012-01-01

219

Reduced thrombin generation increases host susceptibility to group A streptococcal infection  

OpenAIRE

Bacterial plasminogen activators are commonplace among microbial pathogens, implying a central role of host plasmin in supporting bacterial virulence. Group A streptococci (GAS) secrete streptokinase, a specific activator of human plasminogen (PLG). The critical contribution of the streptokinase-PLG interaction to GAS pathogenicity was recently demonstrated using mice expressing human PLG. To examine the importance of thrombin generation in antimicrobial host defense, we challenged mice with ...

Sun, Hongmin; Wang, Xixi; Degen, Jay L.; Ginsburg, David

2009-01-01

220

Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin.  

Science.gov (United States)

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 ?H 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. PMID:21526769

Treuheit, Nicholas A; Beach, Muneera A; Komives, Elizabeth A

2011-05-31

221

Recombination and peak jumping  

OpenAIRE

We find an advantage of recombination for a category of complex fitness landscapes. Recent studies of empirical fitness landscapes reveal complex gene interactions and multiple peaks, and recombination can be a powerful mechanism for escaping suboptimal peaks. However classical work on recombination largely ignores the effect of complex gene interactions. The advantage we find has no correspondence for 2-locus systems or for smooth landscapes. The effect is sometimes extreme...

Crona, Kristina

2014-01-01

222

Heparin cofactor II-thrombin complex: a biomarker of MPS disease.  

Science.gov (United States)

The mucopolysaccharidoses are a group of lysosomal storage disorders caused by defects in the degradation of glycosaminoglycans. Each disorder is characterized by progressive multi-system disease with considerable clinical heterogeneity. The clinical heterogeneity of these disorders is thought to be related to the degree of the metabolic block in glycosaminoglycan degradation which in turn is related to the underlying mutation at the respective locus. There are currently no objective means other than longitudinal clinical observation, or the detection of a recurrent genetic mutation to accurately predict the clinical course for an individual patient, particularly when diagnosed early. In addition, there are no specific disease biomarkers that reflect the total body burden of disease. The lack of specific biomarkers has made monitoring treatment responses and predicting disease course difficult in these disorders. The recent introduction of enzyme replacement therapy for MPS I, II, and VI highlights the need for objective measures of disease burden and disease responsiveness. We show that serum levels of heparin cofactor II-thrombin complex is a reliable biomarker of the mucopolysaccharidoses. Untreated patients have serum levels that range from 3- to 112-fold above control values. In a series of patients with varying severity of mucopolysaccharidosis I, the serum complex concentration was reflective of disease severity. In addition, serum heparin cofactor II-thrombin levels showed responsiveness to various treatment regimens. We propose that serum levels of heparin cofactor II-thrombin complex may provide an important assessment and monitoring tool for patients with mucopolysaccharidosis. PMID:18511319

Randall, Derrick R; Colobong, Karen E; Hemmelgarn, Harmony; Sinclair, Graham B; Hetty, Elly; Thomas, Anita; Bodamer, Olaf A; Volkmar, Barbara; Fernhoff, Paul M; Casey, Robin; Chan, Alicia K; Mitchell, Grant; Stockler, Silvia; Melancon, Serge; Rupar, Tony; Clarke, Lorne A

2008-08-01

223

N-terminal cysteinyl proteins can be prepared using thrombin cleavage.  

Science.gov (United States)

Expressed protein ligation--which allows native proteins to be selectively linked together by a normal peptide bond in an aqueous environment--has emerged as a powerful technique. The technique requires the formation of a C-terminal alpha-thioester and an N-terminal Cys. An N-terminal Cys can be formed by enzymatic cleavage, commonly using the Factor Xa and TEV proteases. We show that thrombin can be used for the formation of N-terminal Cys, providing another choice of reagents for expressed protein ligation. Proteins with N-terminal Cys can be obtained by the convenient modification of vectors with the putative thrombin cleavage site LVPRG to LVPRC. Two example protein domains (Csk and Abl tyrosine kinase domain) with N-terminal Cys are demonstrated using this method. The use of thrombin protease to generate N-terminal Cys overcomes some of the limitations of existing methods, making it generally useful for expressed protein ligation and other biotechnological applications. PMID:18331839

Liu, Dongsheng; Xu, Rong; Dutta, Kaushik; Cowburn, David

2008-04-01

224

Electrochemiluminescence biosensor based on CdSe quantum dots for the detection of thrombin  

International Nuclear Information System (INIS)

A novel QDs electrochemiluminescence (ECL) biosensor for the determination of thrombin was described. The CdSe QDs solution was dripped onto the clear surface of the ITO and then immersed in PBS which contained EDC and NHS as a coupling agent to activate the carboxyl-terminated surface of the CdSe QDs. The ITO electrode was immersed in the PBS containing 0.4 ?M aptamer, followed by rinsing with PBS and dried with N2 again, then dipped in the BSA solution for 30 min to decrease the non-specific binding. After that, the aptamer modified ITO was soaked in PBS to remove unbound aptamer. Under optimal conditions, the linear range was obtained from 0 to 64 ?g mL?1 with a correlation coefficient of 0.9986 (n = 16). The control experiment was also carried out by using BSA, lysozyme and IgG in the absence of thrombin. The results showed that the aptasensor had good specificity, stability and reproducibility to the thrombin. Moreover, the aptasensor could be used for detection of real sample with consistent results in comparison with those obtained by electrochemical method which could provide a promising platform for fabrication of aptamer based biosensors.

225

Percutaneous Ablation of Peripheral Pseudoaneurysms Using Thrombin: A Simple and Effective Solution  

International Nuclear Information System (INIS)

Purpose: To assess the effectiveness of tissue adhesive and thrombin solution in the percutaneous ablation of peripheral artery pseudoaneurysms.Methods: Twenty-five pseudoaneurysms were treated over a 33-month period; all had failed ultrasound-guided compression. Tissue adhesive or thrombin solution was injected percutaneously, with needle tip position and changes within the aneurysm confirmed with color Doppler ultrasound. In 19 cases we utilized a protective balloon inflated across the aneurysm neck prior to the injection of tissue adhesive and in six cases used thrombin injection alone. Seven patients were anticoagulated. Patients were followed up after the procedure.Results: All 25 aneurysms were treated successfully; two patients required a return visit and there were no immediate complications or peripheral emboli detected. One patient developed a contralateral pseudoaneurysm.Conclusions: The percutaneous injection of pseudoaneurysms is a safe, a traumatic, and effective treatment for femoral artery pseudoaneurysms in the peripheral circulation. There are significant advantages over ultrasound-guided compression or surgical repair

226

Direct thrombin inhibitor-bivalirudin functionalized plasma polymerized allylamine coating for improved biocompatibility of vascular devices.  

Science.gov (United States)

The direct thrombin inhibitor of bivalirudin (BVLD), a short peptide derived from hirudin, has drawn an increasing attention in clinical application because it is safer and more effective than heparin for diabetic patients with moderate- or high-risk for acute coronary syndromes (ACS). In this study, BVLD was covalently conjugated on plasma polymerized allylamine (PPAam) coated 316L stainless steel (SS) to develop an anticoagulant surface. QCM-D real time monitoring result shows that 565±20 ng/cm2 of BVLD was bound to the PPAam surface. Infrared spectroscopy (IR) and X-ray photoelectron spectroscopy (XPS) confirmed the immobilization of BVLD. The conjugation of BVLD onto the PPAam coating led to enhanced binding of thrombin, and the activity of the thrombin adsorbed on its surface was effectively inhibited. As a result, the BVLD immobilized PPAam (BVLD-PPAam) substrate prolonged the clotting times, and exhibited inhibition in adhesion and activation of platelets and fibrinogen. We also found that the BVLD-PPAam coating significantly enhanced endothelial cell adhesion, proliferation, migration and release of nitric oxide (NO) and secretion of prostaglandin I2 (PGI2). In vivo results indicate that the BVLD-PPAam surface restrained thrombus formation by rapidly growing a homogeneous and intact endothelium on its surface. These data suggest the potential of this multifunctional BVLD-PPAam coating for the application not only in general vascular devices such as catheters, tubes, oxygenator, hemodialysis membranes but also vascular grafts and stents. PMID:22877639

Yang, Zhilu; Tu, Qiufen; Maitz, Manfred F; Zhou, Shuo; Wang, Jin; Huang, Nan

2012-11-01

227

Alginate-calcium microsphere loaded with thrombin: A new composite biomaterial for hemostatic embolization.  

Science.gov (United States)

To date, transcatheter arterial embolization (TAE) has become a standard treatment to control intracavitary bleeding as an alternative to surgery. Due to excellent biocompatibility and no residual in vivo, biodegradable materials are preferred in TAE. However, gelfoam is the only commercially available biodegradable embolic material used to treat blunt trauma of solid abdominal viscera until now, and controversial on its stability and reliability never stopped in the past five decades. In this study, a new biodegradable macromolecule material (thrombin-loaded alginate-calcium microspheres, TACMs) was prepared using electrostatic droplet techniques and a special method was developed for hemostatic embolization. Thrombin was successfully loaded into microspheres with high encapsulation efficiency and drug loading capacity. A burst release of TACMs was observed at early stage and sustained release later on, with the activity of thrombin preserved well. The strength of TACMs mixed thrombus, which was used as embolic agent, increased in a dose-dependent manner after TACMs were added. In addition, the TACMs were verified to be of no cytotoxicity and systemic toxicity, and biodegradable in vivo. Finally, the results of preliminary applications revealed that the TACMs could serve as an effective and promising embolic material for blunt trauma and hemorrhage of solid abdominal viscera. PMID:25583022

Rong, Jing-Jing; Liang, Ming; Xuan, Feng-Qi; Sun, Jing-Yang; Zhao, Li-Jun; Zhen, Hui-Zhen; Tian, Xiao-Xiang; Liu, Dan; Zhang, Quan-Yu; Peng, Cheng-Fei; Yao, Tian-Ming; Li, Fei; Wang, Xiao-Zeng; Han, Ya-Ling; Yu, Wei-Ting

2015-04-01

228

The membrane potential modulates thrombin-stimulated Ca²? mobilization and platelet aggregation.  

Science.gov (United States)

G protein-coupled receptors can be directly modulated by changes in transmembrane voltage in a variety of cell types. Here we show that, while changes in the membrane voltage itself do not induce detectable modifications in the cytosolic Ca(2+) concentration, platelet stimulation with thrombin or the PAR-1 and PAR-4 agonist peptides SFLLRN and AYPGKF, respectively, results in Ca(2+) release from intracellular stores that is sensitive to the membrane depolarisation. Direct activation of G proteins or phospholipase C by AlF4(-) and m-3M3FBS, respectively, leads to Ca(2+) release that is insensitive to changes in the membrane potential. Thapsigargin-, as well as OAG-induced Ca(2+) entry are affected by the membrane voltage, probably as a result of the modification in the driving force for Ca(2+) influx; however, hyperpolarisation does not enhance thrombin- or OAG-evoked Ca(2+) entry probably revealing the presence of a voltage-sensitive regulatory mechanism. Transmembrane voltage also modulates the activity of the plasma membrane Ca(2+)-ATPase (PMCA) most likely due to a decrease in the phosphotyrosine content of the pump. Thrombin-stimulated platelet aggregation is modulated by membrane depolarisation by a mechanism that is, at least partially, independent of Ca(2+). These observations indicate that PAR-1 and PAR-4 receptors are modulated by the membrane voltage in human platelets. PMID:23988350

Albarrán, Letizia; Dionisio, Natalia; López, Esther; Salido, Ginés M; Rosado, Juan A

2013-10-15

229

Induction of MMP-9 release from human dermal fibroblasts by thrombin: involvement of JAK/STAT3 signaling pathway in MMP-9 release  

OpenAIRE

Abstract Background It has been recognized that dermal fibroblasts and matrix metalloproteases (MMP) play crucial roles in wound healing process in skin. Thrombin was found to stimulate IL-8 release from human dermal fibroblasts (HDFs). However, little is known of the effect of thrombin on secretion of MMPs from dermal fibroblasts. In the present study, the influence of thrombin on proMMP-2 and proMMP-9 activity release from primary cultured HDFs, and its potential signaling pathways were inv...

He Shaoheng; Luo Jianmin; Wang Li

2007-01-01

230

Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors.  

OpenAIRE

Propagation of venous thrombi or rethrombosis after coronary thrombolytic therapy can occur despite heparin administration. To explore potential mechanisms, we set out to determine whether clot-bound thrombin is relatively protected from inhibition by heparin-antithrombin III but susceptible to inactivation by antithrombin III-independent inhibitors. Using plasma fibrinopeptide A (FPA) levels as an index of thrombin activity, we compared the ability of thrombin inhibitors to block FPA release...

Weitz, J. I.; Hudoba, M.; Massel, D.; Maraganore, J.; Hirsh, J.

1990-01-01

231

?2-Glycoprotein I binds factor XI and inhibits its activation by thrombin and factor XIIa: Loss of inhibition by clipped ?2-glycoprotein I  

OpenAIRE

Activation of factor XI (FXI) by thrombin in vivo plays a role in coagulation by providing an important positive feedback mechanism for additional thrombin generation. FXI is activated in vitro by thrombin, or FXIIa in the presence of dextran sulfate. In this report, we investigated the effect of ?2-glycoprotein I (?2GPI) on the activation of FXI. ?2GPI bound FXI in vitro and inhibited its activation to FXIa by thrombin and FXIIa. The affinity of the interaction between ?2GPI and FXI was ...

Shi, Tong; Iverson, G. Michael; Qi, Jian C.; Cockerill, Keith A.; Linnik, Matthew D.; Konecny, Pamela; Krilis, Steven A.

2004-01-01

232

Protein C inhibits endocytosis of thrombin-thrombomodulin complexes in A549 lung cancer cells and human umbilical vein endothelial cells  

Energy Technology Data Exchange (ETDEWEB)

We investigated the effect of protein C on the endocytosis of thrombin-thrombomodulin complexes. We previously showed that exposure of umbilical vein endothelial cells to thrombin stimulated the internalization and degradation of thrombin. A similar internalization was stimulated by a monoclonal antithrombomodulin antibody. We have repeated these studies in the presence of protein C and found that endocytosis of /sup 125/I-thrombin-thrombomodulin complexes, but not /sup 125/I-antithrombomodulin-thrombomodulin complexes, is inhibited. Activated protein C did not inhibit endocytosis of thrombin-thrombomodulin complexes. Protein C inhibited both internalization and degradation of /sup 125/I-thrombin and diisopropylphosphoryl (DIP) /sup 125/I-thrombin in human lung cancer cells (A549). These effects were observed at protein C concentrations found in human plasma. Protein S had no effect on the inhibition of endocytosis of thrombin-thrombomodulin complexes by protein C. We propose that protein C may regulate the rate of endocytosis of thrombin-thrombomodulin complexes in vivo and thereby control the capacity for endothelium to activate protein C.

Maruyama, I.; Majerus, P.W.

1987-05-01

233

Protein C inhibits endocytosis of thrombin-thrombomodulin complexes in A549 lung cancer cells and human umbilical vein endothelial cells  

International Nuclear Information System (INIS)

We investigated the effect of protein C on the endocytosis of thrombin-thrombomodulin complexes. We previously showed that exposure of umbilical vein endothelial cells to thrombin stimulated the internalization and degradation of thrombin. A similar internalization was stimulated by a monoclonal antithrombomodulin antibody. We have repeated these studies in the presence of protein C and found that endocytosis of 125I-thrombin-thrombomodulin complexes, but not 125I-antithrombomodulin-thrombomodulin complexes, is inhibited. Activated protein C did not inhibit endocytosis of thrombin-thrombomodulin complexes. Protein C inhibited both internalization and degradation of 125I-thrombin and diisopropylphosphoryl (DIP) 125I-thrombin in human lung cancer cells (A549). These effects were observed at protein C concentrations found in human plasma. Protein S had no effect on the inhibition of endocytosis of thrombin-thrombomodulin complexes by protein C. We propose that protein C may regulate the rate of endocytosis of thrombin-thrombomodulin complexes in vivo and thereby control the capacity for endothelium to activate protein C

234

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

DEFF Research Database (Denmark)

BACKGROUND AND OBJECTIVES: Transfusion based on the Thrombelastograph (TEG) results reduces transfusion requirements in cardiac surgery and in liver transplantation. Taking the pivotal role of thrombin generation in the coagulation process into consideration, the clinical utility of the TEG may, in part, depend on its reflection of the dynamics of thrombin generation. MATERIAL AND METHODS: The kinetics of thrombin generation of platelets stored for 2 and 7 days, respectively, was assessed by calibrated automated thrombogram (CAT) and the lag time (min), time to peak (ttPeak; min), peak (nm thrombin) and endogenous thrombin potential (ETP; nm thrombin*min) were registered. Clot formation was evaluated by TEG and the R time (min), maxial amplitude (MA; mm), time to maximum thrombus generation (TMG; min) and maximum thrombus generation (MTG; dynes cm(-2) s(-1)) and total thrombus generation (TTG; dyne cm(-2)) were registered. RESULTS: Platelets become more procoagulant, evaluated both by TEG and CAT during storage. The reduction in CAT lag time and the ttPeak correlated with a decrease in the TEG R time and TMG (P < 0.0001) as did the CAT peak thrombin generation and the TEG MTG (P = 0.0035). No correlation between ETP and TTG was found (P = 0.65). CONCLUSION: The kinetics of thrombin generation, as evaluated by CAT, correlates with the thrombus generation, as evaluated by thrombelastography and this may in part explain the clinical utility of the TEG in identifying clinically relevant coagulopathies, secondary to impaired thrombin generation Udgivelsesdato: 2008/2

Johansson, Per Ingemar; Svendsen, M.S.

2008-01-01

235

Genetic recombination in Micromonospora.  

Science.gov (United States)

Biochemical mutants were obtained from Micromonospora chalcea, M. purpurea, and M. echinospora by using ultraviolet radiation or nitrosoguanidine. Crosses carried out between complementary nutritional mutants of the same species showed positive genetic interaction. Data are reported which indicate that the interaction between the crossed strains is due to genetic recombination. No evidence for interspecific genetic recombination was found. PMID:5113596

Beretta, M; Betti, M; Polsinelli, M

1971-08-01

236

Oxygen-hydrogen recombiner  

International Nuclear Information System (INIS)

Purpose: To improve the oxygen-hydrogen removing performance, as well as enable to maintain the high performance in a range of increasing the processing gas flow rate within a recombiner with the reduced pressure and reduced amount of charged catalyst. Constitution: A plate-like metal catalyst comprising alumina added as a binder to the surface of a sponge-like metal support made of nickel-chromium alloy and particles of platinum type novel metal such as platinum or palladium having a catalytic activity supported on alumina is contained in a cartridge and filled within an oxygen-hydrogen recombiner. The recombiner is adapted so that the exhaust gas flow rate therein is within a range from 1 nm/sec to 4 nm/sec. It is possible with such a constitution to improve the recombining performance, reduce the pressure loss, decrease the size of the recombiner and facilitate the maintenance and check thereof. (Kawakami, Y.)

237

AI Topics  

OpenAIRE

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

Buchanan, Bruce G.; Glick, Jonathan

2002-01-01

238

Signal amplification aptamer biosensor for thrombin based on a glassy carbon electrode modified with graphene, quantum dots and gold nanoparticles  

Science.gov (United States)

A novel electrogenerated chemiluminescence (ECL) assay for sensitive determination of thrombin is designed employing CdSe/ZnS quantum dots served as an ECL label. This ECL sensor is fabricated on graphene modified glassy carbon electrode which is then covered with a low surface coverage of gold nanoparticles (AuNPs). An aptamer is used to selectively recognize the target. The thiol-terminated aptamer is first immobilized on AuNPs/graphene modified electrode, and then thrombin is imported to form the aptamer-thrombin complexes. After blocking the nonspecifically bound oligonucleotides with MCH solution, another CdSe/ZnS quantum dots modified aptamer is hybridized with the free thiol-terminated aptamer to form a DNA complexe. A decreased ECL signal is observed upon recognition of the target thrombin. The integrated ECL intensity versus the concentration of thrombin is linear in the range from 0.01 to 50 nM. The detection limit is 10 fM. The present aptasensor also exhibits excellent selectivity, stability and reusability. This sensing system can provide a promising label-free model for aptamer-based compounds sensitive detection.

Xie, Lingling; You, Liqin; Cao, Xiaoyu

2013-05-01

239

Thrombin stimulation of synthesis and secretion of fibronectin by human A549 epithelial cells and mouse LB fibroblasts  

International Nuclear Information System (INIS)

Thrombin, a serine protease generated at wound sites, takes part in multiple biological functions, including wound healing. The present report elucidates the effect of thrombin on fibronectin (FN) synthesis and secretion in fibroblasts and epithelial cells. Subconfluent cultures of mouse LB fibroblasts and human A549 epithelial cells were exposed to various concentrations of bovine plasma thrombin at 37 degrees C for 16 hr. After exposure, cells were processed for determination of cell-associated and secreted FN by metabolic labeling, immunoprecipitation, immunofluorescence, and peroxidase immunocytochemistry. The correlation of FN production with cell growth was studied by a combined procedure of peroxidase immunocytochemistry and light microscopic autoradiography. The amounts of cell-associated and secreted FN were significantly increased with dose increments of thrombin. The increases were most evident in secreted FN. The increase of cell-associated FN was also evidenced by results from immunofluorescence and immunocytochemical studies. Ultrastructurally, the intracellular FN was localized in rough endoplasmic reticulum, Golgi complexes, and secretory granules, whereas non-released extracellular FN was localized in the plasma membrane of cell-to-cell contacts and in the extracellular fibrils. More intense cytoplasmic FN staining was observed in cells that were not labeled with [3H]-thymidine, indicating that FN production may vary with different phases of cell groway vary with different phases of cell growth. The results imply that thrombin may play an important role in the early phases of wound healing

240

Thrombin and interleukin-1beta decrease HOX gene expression in human first trimester decidual cells: implications for pregnancy loss.  

Science.gov (United States)

Bleeding or inflammation in early pregnancy may result in pregnancy loss or defective implantation. Their effect on HOX gene expression in first trimester decidua is unknown. Bleeding results in thrombin generation, although infection or inflammation results in production of cytokines typified by Interleukin-1beta (IL-1beta). First trimester decidual cells were pretreated with 17beta estradiol (E(2)), medroxyprogesterone acetate (MPA) or both and subsequently treated with thrombin or IL-1beta. Affymetrix microarray analysis was used to assess the expression of all HOX genes and confirmed using real-time RT-PCR. E(2) or MPA treatment resulted in significant increases in HOXA10 and HOXA11. Subsequent treatment with thrombin resulted in diminished expression of HOXA10 and HOXA9. Treatment with IL-1beta resulted in decreased expression of HOXA1, 3, 9, 10 and 11. HOXA10 expression was reduced by 70% after thrombin treatment (P = 0.018) and by 90% after IL-1beta treatment (P = 0.004). HOXA11 mRNA expression was decreased by 88% after IL-1beta treatment (P HOXA10 and HOXA11 RNA and protein expression in the decidua of spontaneous pregnancy loss compared with that of viable pregnancies. In conclusion, multiple HOX genes are expressed in decidual cells and inhibited by thrombin and IL-1beta. Since HOXA10 and HOXA11 are known to be necessary for successful pregnancy, these findings suggest a molecular mechanism by which bleeding or inflammation may affect pregnancy outcome. PMID:19389728

Sarno, Jennifer; Schatz, Frederick; Huang, S Joseph; Lockwood, Charles; Taylor, Hugh S

2009-07-01

241

Magnetic relaxation switch and colorimetric detection of thrombin using aptamer-functionalized gold-coated iron oxide nanoparticles  

Energy Technology Data Exchange (ETDEWEB)

We describe a sensitive biosensing system combining magnetic relaxation switch diagnosis and colorimetric detection of human {alpha}-thrombin, based on the aptamer-protein interaction induced aggregation of Fe{sub 3}O{sub 4}-Au nanoparticles. To demonstrate the concept, gold-coated iron oxide nanoparticle was synthesized by iterative reduction of HAuCl{sub 4} onto the dextran-coated Fe{sub 3}O{sub 4} nanoparticles. The resulting core-shell structure had a flowerlike shape with pretty narrow size distribution (referred to as 'nanorose'). The two aptamers corresponding to human {alpha}-thrombin were conjugated separately to two distinct nanorose populations. Once a solution containing human {alpha}-thrombin was introduced, the nanoroses switched from a well dispersed state to an aggregated one, leading to a change in the spin-spin relaxation time (T{sub 2}) as well as the UV-Vis absorption spectra of the solution. Thus the qualitative and quantitative detection method for human {alpha}-thrombin was established. The dual-mode detection is clearly advantageous in obtaining a more reliable result; the detection range is widened as well. By using the dual-mode detection method, a detectable T{sub 2} change is observed with 1.0 nM human {alpha}-thrombin, and the detection range is from 1.6 nM to 30.4 nM.

Liang Guohai; Cai Shaoyu; Zhang Peng [Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433 (China); Peng Youyuan [Department of Chemistry, Quanzhou Normal University, Quanzhou 362000 (China); Chen Hui; Zhang Song [Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433 (China); Kong Jilie, E-mail: jlkong@fudan.edu.cn [Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433 (China)

2011-03-18

242

Magnetic relaxation switch and colorimetric detection of thrombin using aptamer-functionalized gold-coated iron oxide nanoparticles  

International Nuclear Information System (INIS)

We describe a sensitive biosensing system combining magnetic relaxation switch diagnosis and colorimetric detection of human ?-thrombin, based on the aptamer-protein interaction induced aggregation of Fe3O4-Au nanoparticles. To demonstrate the concept, gold-coated iron oxide nanoparticle was synthesized by iterative reduction of HAuCl4 onto the dextran-coated Fe3O4 nanoparticles. The resulting core-shell structure had a flowerlike shape with pretty narrow size distribution (referred to as 'nanorose'). The two aptamers corresponding to human ?-thrombin were conjugated separately to two distinct nanorose populations. Once a solution containing human ?-thrombin was introduced, the nanoroses switched from a well dispersed state to an aggregated one, leading to a change in the spin-spin relaxation time (T2) as well as the UV-Vis absorption spectra of the solution. Thus the qualitative and quantitative detection method for human ?-thrombin was established. The dual-mode detection is clearly advantageous in obtaining a more reliable result; the detection range is widened as well. By using the dual-mode detection method, a detectable T2 change is observed with 1.0 nM human ?-thrombin, and the detection range is from 1.6 nM to 30.4 nM.

243

Thrombin inhibits HMGB1-mediated proinflammatory signaling responses when endothelial protein C receptor is occupied by its natural ligand  

Directory of Open Access Journals (Sweden)

Full Text Available High mobility group box 1 (HMGB1 is involved in thepathogenesis of vascular diseases. Unlike activated protein C(APC, the activation of PAR-1 by thrombin is known to elicitproinflammatory responses. To determine whether the occupancyof EPCR by the Gla-domain of APC is responsible for thePAR-1-dependent antiinflammatory activity of the protease, wepretreated HUVECs with the PC zymogen and then activatedPAR-1 with thrombin. It was found that thrombin downregulatesthe HMGB1-mediated induction of both TNF-? andIL-6 and inhibits the activation of both p38 MAPK and NF-?B inHUVECs pretreated with PC. Furthermore, thrombin inhibitedHMGB1-mediated hyperpermeability and leukocyte adhesion/migration by inhibiting the expression of cell adhesion moleculesin HUVECs if EPCR was occupied. Collectively, theseresults suggest the concept that thrombin can initiate proinflammatoryresponses in vascular endothelial cells through theactivation of PAR-1 may not hold true for normal vesselsexpressing EPCR under in vivo conditions. [BMB Reports 2013;46(11: 544-549

Jong-Sup Bae

2013-11-01

244

Dissociative recombination in aeronomy  

Science.gov (United States)

The importance of dissociative recombination in planetary aeronomy is summarized, and two examples are discussed. The first is the role of dissociative recombination of N2(+) in the escape of nitrogen from Mars. A previous model is updated to reflect new experimental data on the electronic states of N produced in this process. Second, the intensity of the atomic oxygen green line on the nightside of Venus is modeled. Use is made of theoretical rate coefficients for production of O (1S) in dissociative recombination from different vibrational levels of O2(+).

Fox, J. L.

1989-01-01

245

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

International Nuclear Information System (INIS)

Blood coagulation factor VIII (fVIII) is a plasma protein that is decreased or absent in hemophilia A. It is isolated as a mixture of heterodimers that contain a variably sized heavy chain and a common light chain. Thrombin catalyzes the activation of fVIII in a reaction that is associated with cleavages in both types of chain. The authors isolated a serine protease from Bothrops jararacussu snake venom that catalyzes thrombin-like heavy-chain cleavage but not light-chain cleavage in porcine fVIII as judged by NaDodSO4/PAGE and N-terminal sequence analysis. Using a plasma-free assay of the ability of activated 125I-fVIII to function as a cofactor in the activation of factor X by factor IXa, they found that fVIII is activated by the venom enzyme. The venom enzyme-activated fVIII was isolated in stable form by cation-exchange HPLC. von Willebrand factor inhibited venom enzyme-activated fVIII but not thrombin-activated fVIII. These results suggest that the binding of fVIII to von Willebrand factor depends on the presence of an intact light chain and that activated fVIII must dissociate from von Willebrand factor to exert its cofactor effect. Thus, proteolytic activation of fVIII-von Willebrand factor complex appears to be differentially regulated by light-chain cleavage to dissociate the complex and heavy-chain cleavage to activate the cofactor function

246

Thrombin related peptide TP508 promoted fracture repair in a mouse high energy fracture model  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Thrombin related peptide (TP508 is a 23 amino-acid synthetic peptide that represents a portion of the receptor-binding domain of thrombin molecule. Previous studies have shown that TP508 can accelerate musculoskeletal tissue repair including fracture healing. Objectives The aim of this study was to investigate the effect of TP508 on fracture healing in a murine fracture model representing high energy fracture situation. Methods Eighty CD 1 mice underwent controlled quadriceps muscle crush and open transverse mid diaphyseal femoral fracture that was then fixed with an external fixator. Animals were randomised into four groups to receive an intra-operative dose of either 100 ?g TP508 into the fracture gap; 100 ?g TP508 into the surrounding damaged muscle tissues; 10 ?g TP508 into the fracture gap, or control equal amount of saline into the fracture gap. Radiographic assessment was performed weekly for 5 weeks; histological analysis was at 3 and 5 weeks post fracture and biomechanical testing of the fractured bone was performed at 5 weeks post fracture. Results Mechanical testing data showed that the fracture stiffness was significantly higher in the group receiving 100 ?g TP508 into the fracture gap than other groups. Histological and radiographic analysis revealed a trend of increase in bone formation in the 100 ?g TP508 injected into the fracture gap group compared to the saline control group. It was noted that the scar tissues was significantly less in Group II comparing with the saline control group and there was increased blood vessel formation in the crushed muscles and fracture gap areas in the groups receiving TP508 comparing to the saline control group. Conclusion The results from this study demonstrated the use of thrombin related peptide TP508 in the situation of a high energy fracture can promote fracture healing and reduce the potential complications such as muscle fibrosis and fracture delayed or non-union.

Pan Xiao-Hua

2009-01-01

247

Monitoring of dabigatran therapy using Hemoclot(®) Thrombin Inhibitor assay in patients with atrial fibrillation.  

Science.gov (United States)

Dabigatran, a new direct thrombin inhibitor, achieves strong anticoagulation that is more predictable than warfarin. Nevertheless, a patient on dabigatran therapy (DT) may suffer from thrombotic or bleeding events. The routine monitoring of DT is not recommended, and standard coagulation tests are not sensitive enough for the assessment of DT activity. The aim of this study was to examine the clinical usefulness of the Hemoclot(®) Thrombin Inhibitor (HTI) assay in the assessment of dabigatran plasma levels in patients with non-valvular AF. Nineteen patients (12 men, 7 women) on DT were included in this preliminary prospective observational study. Dabigatran was administrated twice daily in a two dose regimens: 150 mg (5 patients) and 110 mg (14 patients). Blood samples were taken for the assessment of trough and peak levels of dabigatran. Dabigatran concentrations were measured with the HTI assay. The average dabigatran trough level was 69.3 ± 55.5 ng/ml and the average dabigatran peak level was 112.7 ± 66.6 ng/ml. The dabigatran trough plasma concentration was in the established reference range in 15 patients and the dabigatran peak plasma concentration was in the established reference range in 9 patients, respectively. Despite the fact that the activated partial thromboplastin and thrombin times were generally changed (prolonged), these tests failed to identify the patients with too low or too high dabigatran concentrations. The study confirmed the high sensitivity of the HTI assay for the assessment of dabigatran plasma levels. When compared to standard coagulation tests, the HTI is a more suitable assay for the monitoring of patients treated with dabigatran. Monitoring of DT may be beneficial in selected patients; however, further studies will be needed for the final clarification of this issue. PMID:25103614

Samoš, Matej; Stan?iaková, Lucia; Ivanková, Jela; Staško, Ján; Ková?, František; Dobrotová, Miroslava; Galajda, Peter; Kubisz, Peter; Moká?, Marián

2015-01-01

248

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

Energy Technology Data Exchange (ETDEWEB)

Blood coagulation factor VIII (fVIII) is a plasma protein that is decreased or absent in hemophilia A. It is isolated as a mixture of heterodimers that contain a variably sized heavy chain and a common light chain. Thrombin catalyzes the activation of fVIII in a reaction that is associated with cleavages in both types of chain. The authors isolated a serine protease from Bothrops jararacussu snake venom that catalyzes thrombin-like heavy-chain cleavage but not light-chain cleavage in porcine fVIII as judged by NaDodSO{sub 4}/PAGE and N-terminal sequence analysis. Using a plasma-free assay of the ability of activated {sup 125}I-fVIII to function as a cofactor in the activation of factor X by factor IXa, they found that fVIII is activated by the venom enzyme. The venom enzyme-activated fVIII was isolated in stable form by cation-exchange HPLC. von Willebrand factor inhibited venom enzyme-activated fVIII but not thrombin-activated fVIII. These results suggest that the binding of fVIII to von Willebrand factor depends on the presence of an intact light chain and that activated fVIII must dissociate from von Willebrand factor to exert its cofactor effect. Thus, proteolytic activation of fVIII-von Willebrand factor complex appears to be differentially regulated by light-chain cleavage to dissociate the complex and heavy-chain cleavage to activate the cofactor function.

Hill-Eubanks, D.C.; Parker, C.G.; Lollar, P. (Univ. of Vermont, Burlington (USA))

1989-09-01

249

Use of a Thrombin-gelatin Hemostatic Matrix (Surgiflo) in Spinal Surgery.  

Science.gov (United States)

A variety of techniques have been used to stop venous bleeding from the spinal epidural space. These generally consist of packing with Surgicel®, fibrillar collagen or Gelfoam®. Bipolar coagulation may also be used to control bleeding from spinal venous plexus, but it may bear the risk of healthy nervous tissue injury: dissipation of heat from the tips of the bipolar forceps may induce thermal injury to adjacent neural structures. In the case of intraspinal bleeding, quick and safe hemostasis is mandatory to ensure adequate visualization and safe preparation so as to avoid damaging nerves and spinal medulla. In addition, quick and safe hemostasis reduces the duration of surgery. Efficient control of bleeding can thereby reduce perioperative morbidity. During 6 months, the authors performed more than 170 major spinal surgeries, and in 67 procedures they used injection of thrombin-gelatin hemostatic matrix (Surgiflo, Johnson & Johnson Wound Management, Somerville, NJ) into spinal epidural space to assist in hemostasis. When the venous bleeding continued from the epidural space after packing with hemostatic agents as Surgicel and fibrillar collagen, gelatin matrix was used to stop venous bleeding. In all cases, the results were judged to be excellent, with immediate stoppage of epidural bleeding, or good. No complications related to the thrombin-gelatin hemostatic matrix were encountered. The thrombin-gelatin matrix could represent a valuable tool when other hemostatic strategies are ineffective or suboptimal. It is safe and biocompatible when compared with hemostatic agents currently in use. This is the first study reporting the use of Surgiflo hemostatic matrix in spinal surgery. PMID:25419955

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

2014-11-01

250

The selection of DNA aptamers for two different epitopes of thrombin was not due to different partitioning methods.  

Science.gov (United States)

Nearly all aptamers identified so far for any given target molecule have been specific for the same binding site (epitope). The most notable exception to the 1 aptamer per target molecule rule is the pair of DNA aptamers that bind to different epitopes of thrombin. This communication refutes the suggestion that these aptamers exist because different partitioning methods were used when they were selected. The possibility that selection of these aptamers was biased by conflicting secondary structures was also investigated and found not to contribute. The preparation of protein-coated magnetic beads for systematic evolution of ligands by exponential enrichment (SELEX) and the different specificities of the thrombin aptamers for the ? and ? forms of thrombin are also reported. PMID:23216233

Wilson, Robert; Cossins, Andrew; Nicolau, Dan V; Missailidis, Sotiris

2013-02-01

251

Effects of suramin on human platelet aggregation and Ca2+ mobilization induced by thrombin and other agonists.  

Science.gov (United States)

The purpose of this study was to investigate the effect of suramin, a polyanionic napthalene sulfonic acid, on human platelet aggregation and Ca2+ mobilization induced by various agonists. Our results show that suramin completely inhibited aggregation by thrombin, platelet activating factor (PAF), alkyllysophosphatidic acid (ALPA), or arachidonic acid in a concentration-dependent manner. The IC50 values of suramin for inhibition of aggregation by PAF, arachidonic acid, and thrombin were 76.7, 239, and 1.49 microg/ml, respectively. Ca2+ mobilization induced by thrombin was inhibited by suramin with an approximate IC50 value of 20 microg/ml. This concentration of suramin had no effect on PAF or oleic acid-induced Ca2+ mobilization. The mechanism by which suramin inhibits aggregation is not clear, but our results suggest that suramin inhibits the ligand-receptor interaction. PMID:9820121

Siafaka-Kapadai, A; Svetlov, S; Hanahan, D J; Javors, M A

1998-01-01

252

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

OpenAIRE

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 unfav...

Pasternak, Anna; Hernandez, Frank J.; Rasmussen, Lars M.; Vester, Birte; Wengel, Jesper

2010-01-01

253

First Steps in the Direction of Synthetic, Allosteric, Direct Inhibitors of Thrombin and Factor Xa  

Science.gov (United States)

Designing non-saccharide functional mimics of heparin is a major challenge. In this work, a library of small, aromatic molecules based on the sulfated DHP scaffold was synthesized and screened against thrombin and factor Xa. The results reveal that i) selected monomeric benzofuran derivatives inhibit the two enzymes, albeit weakly; ii) the two enzymes recognize different structural features in the benzofurans studied suggesting significant selectivity of recognition; and iii) the mechanism of inhibition is allosteric. The molecules represent the first allosteric small molecule inhibitors of the two enzymes. PMID:19540113

Verghese, Jenson; Liang, Aiye; Sidhu, Preet Pal Singh; Hindle, Michael; Zhou, Qibing; Desai, Umesh R.

2009-01-01

254

N-terminal cysteinyl proteins can be prepared using thrombin cleavage  

OpenAIRE

Expressed protein ligation – which allows native proteins to be selectively linked together by a normal peptide bond in an aqueous environment – has emerged as a powerful technique. The technique requires the formation of a C-terminal ?-thioester and an N-terminal Cys. An N-terminal Cys can be formed by enzymatic cleavage, commonly using the Factor Xa and TEV proteases. We show that thrombin can be used for the formation of N-terminal Cys, providing another choice of reagents for express...

Liu, Dongsheng; Xu, Rong; Dutta, Kaushik; Cowburn, David

2008-01-01

255

Factor XI contributes to thrombin generation in the absence of factor XII  

OpenAIRE

During surface-initiated blood coagulation in vitro, activated factor XII (fXIIa) converts factor XI (fXI) to fXIa. Whereas fXI deficiency is associated with a hemorrhagic disorder, factor XII deficiency is not, suggesting that fXI can be activated by other mechanisms in vivo. Thrombin activates fXI, and several studies suggest that fXI promotes coagulation independent of fXII. However, a recent study failed to find evidence for fXII-independent activation of fXI in plasma. Using plasma in wh...

Kravtsov, Dmitri V.; Matafonov, Anton; Tucker, Erik I.; Sun, Mao-fu; Walsh, Peter N.; Gruber, Andras; Gailani, David

2009-01-01

256

Oxygen-hydrogen recombiner  

International Nuclear Information System (INIS)

Purpose: To reduce unreacted hydrogen flowing through a space between a catalyst and a recombiner to thereby reduce the hydrogen density at the exit of the recombiner. Constitution: A catalyst support plate is disposed on a recombiner, on which is placed catalysts comprising noble metals such as platinum and palladium supported on plate-like sponge metals. 10 or more buffle plates each with about 1 mm gap to the wall surface of the recombiner are inserted in perpendicular to the flowing direction in the catalysts. The catalysts are closely contacted with the buffle plates by their own weight and the pressure resulted from the gas stream, by which the gas tending to flow through the gap are changed in the flowing direction and flow in the catalyst layer to perform recombining reaction. While on the other hand, since the gap is retained to about 1 mm and 10 or more buffle plates are disposed, the amount of the unreacted hydrogen can be decreased. In this way, hydrogen density at the exit of the recombiner can be reduced and packing and exchange of the catalysts can be facilitated. (Yoshihara, H.)

257

Thrombin stimulates the release of arachidonate but not 8,11,14-eicosatrienoate from endothelial cell glycerolipids  

International Nuclear Information System (INIS)

Previous studies in their laboratory have shown that thrombin stimulates the release of arachidonate (20:4(n-6)) but not 22:4(n-6) from endothelial cell glycerolipids. The authors now report that thrombin also does not significantly stimulate the release of 8,11,14-[14C]eicosatrienoate per se. Human umbilical vein endothelial cells were radiolabeled for 24 hr with 1.25 ?M [14C]20:3(n-6) or [14C]20:4(n-6). When incubated for 10 min in buffered saline with 50 ?M fat-free albumin and 1 U/ml thrombin, these cells released 4.1% and 7.6%, respectively, of the previously incorporated radioactivity. Analysis of released 14C-fatty acids by radio-gas chromatography indicated that the thrombin-stimulated release from cells prelabelled with [14C]20:3(n-6) was essentially due to release of [14C]arachidonate synthesized endogenously by desaturation of the [14C]20:3(n-6). Expressed as a percentage of each 14C-fatty acyl present moiety in cellular glycerolipids of cells prelabelled with [14C]20:3(n-6), release was 8.2% for arachidonate but only 0.63% for 20:3(n-6). Studies with other 14C-fatty acids indicate that 5,8,11-20:3 is released in response to thrombin (5-9%); 8,11,14,17-20:4 is not (<1%). These results suggest that a ?5 double bond in the fatty acid is necessary for thrombin-stimulated release from endothelial cell glycerolipids

258

Thrombin stimulates the release of arachidonate but not 8,11,14-eicosatrienoate from endothelial cell glycerolipids  

Energy Technology Data Exchange (ETDEWEB)

Previous studies in their laboratory have shown that thrombin stimulates the release of arachidonate (20:4(n-6)) but not 22:4(n-6) from endothelial cell glycerolipids. The authors now report that thrombin also does not significantly stimulate the release of 8,11,14-(/sup 14/C)eicosatrienoate per se. Human umbilical vein endothelial cells were radiolabeled for 24 hr with 1.25 ..mu..M (/sup 14/C)20:3(n-6) or (/sup 14/C)20:4(n-6). When incubated for 10 min in buffered saline with 50 ..mu..M fat-free albumin and 1 U/ml thrombin, these cells released 4.1% and 7.6%, respectively, of the previously incorporated radioactivity. Analysis of released /sup 14/C-fatty acids by radio-gas chromatography indicated that the thrombin-stimulated release from cells prelabelled with (/sup 14/C)20:3(n-6) was essentially due to release of (/sup 14/C)arachidonate synthesized endogenously by desaturation of the (/sup 14/C)20:3(n-6). Expressed as a percentage of each /sup 14/C-fatty acyl present moiety in cellular glycerolipids of cells prelabelled with (/sup 14/C)20:3(n-6), release was 8.2% for arachidonate but only 0.63% for 20:3(n-6). Studies with other /sup 14/C-fatty acids indicate that 5,8,11-20:3 is released in response to thrombin (5-9%); 8,11,14,17-20:4 is not (<1%). These results suggest that a ..delta..5 double bond in the fatty acid is necessary for thrombin-stimulated release from endothelial cell glycerolipids.

Rosenthal, M.D.

1986-05-01

259

[Use of thrombin generation assay for the evaluation of coagulation and anticoagulant activity of hemostasis system in patients with abdominal sepsis].  

Science.gov (United States)

Generally recognized factor, which complicates the course of sepsis, is the development of hypercoagulation syndrome. The increase of thrombin coagulation indicates on the elevation of risk of thrombus formation in microcirculation vessels, which could cause the formation of multiple organ failure. The thrombin generation assay is a new method of the evaluation of homeostasis system status. The test reflects the fermentation activity of thrombin and shows the functional condition, which arises in the interaction of procoagulant and anticoagulant. The diagnosis of generalized peritonitis had 30 patients (18 men and 12 women, aged 61+/-18,3 years) and they were included in the research. It was shown, that the use of thrombin generation assay in patients with the abdominal sepsis could give the well-timed analysis of hypercoagulation changes and the assessment of protein C system investment in the thrombin generation. PMID:24640752

Gamzatov, Kh A; Gurzhi?, D V; Lazarev, S M; Namestnikov, Iu A; Lerner, A A; Papaian, L P; Golovina, O G; Khait, E A; Smirnova, O A; Matvienko, O Iu

2013-01-01

260

Halcinonide Topical  

Science.gov (United States)

... you are taking, especially cancer chemotherapy agents, other topical medications, and vitamins.tell your doctor if you have an infection or have ever had diabetes, glaucoma, cataracts, a circulation disorder, or an immune disorder. ...

261

Flurandrenolide Topical  

Science.gov (United States)

... you are taking, especially cancer chemotherapy agents, other topical medications, and vitamins.tell your doctor if you have an infection or have ever had diabetes, glaucoma, cataracts, a circulation disorder, or an immune disorder. ...

262

Fluocinonide Topical  

Science.gov (United States)

... you are taking, especially cancer chemotherapy agents, other topical medications, and vitamins.tell your doctor if you have an infection or have ever had diabetes, glaucoma, cataracts, a circulation disorder, or an immune disorder. ...

263

Fluocinolone Topical  

Science.gov (United States)

... you are taking, especially cancer chemotherapy agents, other topical medications, and vitamins.tell your doctor if you have an infection or have ever had diabetes, glaucoma, cataracts, a circulation disorder, or an immune disorder. ...

264

Betamethasone Topical  

Science.gov (United States)

... you are taking, especially cancer chemotherapy agents, other topical medications, and vitamins.tell your doctor if you have an infection or have ever had diabetes, glaucoma, cataracts, a circulation disorder, or an immune disorder. ...

265

Estradiol Topical  

Science.gov (United States)

... symptoms are vaginal burning, itching, and dryness may benefit more from a medication that is applied topically ... your doctor about eating grapefruit and drinking grapefruit juice while taking this medicine.

266

Tacrolimus Topical  

Science.gov (United States)

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

267

Cyclic nucleotides attenuate thrombin-evoked alterations in parameters of platelet Na/H antiport. The role of cytosolic Ca.  

OpenAIRE

In this work, we explored the role of cyclic nucleotides in modulating parameters of the Na/H antiport in human platelets. Sodium nitroprusside and iloprost, as well as cyclic nucleotide analogues, were used to raise cellular levels of cAMP and cGMP. Cyclic nucleotides reversed the thrombin-evoked alkaline shift in cytosolic pH set point and the activity of the Na/H antiport, concurrently with attenuation of thrombin-induced rise in cytosolic free Ca. No effect of cyclic nucleotides was obser...

Kimura, M.; Lasker, N.; Aviv, A.

1992-01-01

268

Pertussis toxin inhibits thrombin-induced activation of phosphoinositide hydrolysis and Na+/H+ exchange in hamster fibroblasts.  

OpenAIRE

Prior treatment with pertussis toxin of G0-arrested hamster fibroblasts (CCL39) results in a dose-dependent inhibition of two early events of the mitogenic response elicited by alpha-thrombin: accumulation of inositol phosphates in Li+-treated cells, and activation of the Na+/H+ antiport, measured either by the amiloride-sensitive 22Na+ influx or by the increase in intracellular pH. At 10(-1) U/ml of alpha-thrombin, the maximal inhibition was approximately 50% for these two early cellular res...

Paris, S.; Pouysse?gur, J.

1986-01-01

269

Phospholipid-esterified Eicosanoids Are Generated in Agonist-activated Human Platelets and Enhance Tissue Factor-dependent Thrombin Generation*  

OpenAIRE

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 × 107 cells (n = 5 separate donors, mean ±...

Thomas, Christopher P.; Morgan, Lloyd T.; Maskrey, Benjamin H.; Murphy, Robert C.; Ku?hn, Hartmut; Hazen, Stanley L.; Goodall, Alison H.; Hamali, Hassan A.; Collins, Peter W.; O'Donnell, Valerie B

2010-01-01

270

Modulation of human uterine smooth muscle cell collagen contractility by thrombin, Y-27632, TNF alpha and indomethacin  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Preterm labour occurs in approximately 10% of pregnancies and is a major cause of infant morbidity and mortality. However, the pathways involved in regulating contractility in normal and preterm labour are not fully elucidated. Our aim was to utilise a human myometrial contractility model to investigate the effect of a number of uterine specific contractility agents in this system. Therefore, we investigated the contractile response of human primary uterine smooth muscle cells or immortalised myometrial smooth muscle cells cultured within collagen lattices, to known mediators of uterine contractility, which included thrombin, the ROCK-1 inhibitor Y-27632, tumour necrosis factor alpha (TNF alpha and the non-steroidal anti-inflammatory indomethacin. Methods Cell contractility was calculated over time, with the collagen gel contraction assay, utilising human primary uterine smooth muscle cells (hUtSMCs and immortalised myometrial smooth muscle cells (hTERT-HM: a decrease in collagen gel area equated to an increase in contractility. RNA was isolated from collagen embedded cells and gene expression changes were analysed by real time fluorescence reverse transcription polymerase chain reaction. Scanning electron and fluorescence microscopy were employed to observe cell morphology and cell collagen gel interactions. Statistical analysis was performed using ANOVA followed by Tukey's post hoc tests. Results TNF alpha increased collagen contractility in comparison to the un-stimulated collagen embedded hUtSMC cells, which was inhibited by indomethacin, while indomethacin alone significantly inhibited contraction. Thrombin augmented the contractility of uterine smooth muscle cell and hTERT-HM collagen gels, this effect was inhibited by the thrombin specific inhibitor, hirudin. Y-27632 decreased both basal and thrombin-induced collagen contractility in the hTERT-HM embedded gels. mRNA expression of the thrombin receptor, F2R was up-regulated in hUtSMCs isolated from collagen gel lattices, following thrombin-stimulated contractility. Conclusion TNF alpha and thrombin increased uterine smooth muscle cell collagen contractility while indomethacin had the opposite effect. Thrombin-induced collagen contractility resulted in F2R activation which may in part be mediated by the ROCK-1 pathway. This study established the in vitro human myometrial model as a viable method to assess the effects of a range of uterotonic or uterorelaxant agents on contractility, and also permits investigation of the complex regulatory pathways involved in mediating myometrial contractility at labour.

Smith Terry J

2009-01-01

271

Role of Na+/H+ exchange in thrombin-induced platelet-activating factor production by human endothelial cells.  

OpenAIRE

Thrombin-stimulated endothelial cells produce platelet-activating factor (PAF) in a dose-dependent manner: the activation of a Ca2+-dependent lyso-PAF acetyltransferase is the rate-limiting step in this process. The present study shows that acetyltransferase activation and consequent PAF production induced by thrombin in human endothelial cells are markedly inhibited in Na+-free media or after addition of the amiloride analog 5-(N-ethyl-N-isopropyl)amiloride, suggesting that a Na+/H+ antiport...

Bosia, Amalia; Pescarmona, Gianpiero; Turrini, Francesco Michelangelo; Garbarino, Giovanni; Ghigo, Dario Antonio; Bussolino, Federico

1988-01-01

272

The Use of Direct Thrombin Injection to Treat a Type II Endoleak Following Endovascular Repair of Abdominal Aortic Aneurysm  

International Nuclear Information System (INIS)

This report describes the use of thrombin to treat a type II endoleak which was causing continued abdominal aortic aneurysm expansion in a patient who had undergone endovascular repair. A small quantity of thrombin was injected into the leak by a percutaneous approach directly into the aneurysm sac using color doppler ultrasound. The procedure was successful and required only a few minutes to perform. We believe this procedure is an alternative to some of the more complex and technically challenging means of treating this lesion

273

Thrombin generation during collection of blood from donors taking oral contraceptives.  

Science.gov (United States)

Thrombin generation, as evidenced by plasma fibrinopeptide A (FPA) concentrations, was studied during blood collection from donors taking oral contraceptives (OC). 450 ml blood were drawn into Fenwal PVC bags from 26 OC users and 28 nonusers. Blood samples for determination of FPA, beta-thromboglobulin (BTG), thrombotest (TT), prekallikrein (PKK), antithrombin-III (AT-III) and factor VIII procoagulant activity (FVIII:C) were drawn from the bags immediately after ending blood donation and following storage for 24 h at 4 degrees C. The FPA concentrations following donation were significantly higher in the OC than in the control group (p less than 0.05). The levels of PKK were also higher in blood obtained from OC users (p less than 0.001), as was the FVIII:C level, the latter difference, however, was not significant (p = 0.06). No cold-promoted activation of factor VII, as evidenced from TT, was detected following storage at 4 degrees C, neither was any change observed in the FPA, PKK and AT-III levels. The BTG concentrations increased significantly during storage, most pronounced in the control group (p less than 0.05). The decay of FVIII:C was similar in the two groups, averaging 24.7%. No correlation was observed between the FPA levels and the other parameters determined. We conclude that thrombin generation is more pronounced during routine blood collection from donors taking OC. PMID:2955572

Skjønsberg, O H; Kierulf, P; Engebretsen, L F; Gjønnes, G; Godal, H C

1987-01-01

274

Cysteine proteases from the Asclepiadaceae plants latex exhibited thrombin and plasmin like activities.  

Science.gov (United States)

In the present study we evaluated the presence of cysteine protease from the latex of four plants Asclepias curassavica L., Calotropis gigantea R.Br., Pergularia extensa R.Br. and Cynanchum puciflorum R.Br. belongs to the family Asclepiadaceae. Cysteine proteases from these plants latex exhibited both thrombin and plasmin like activities. Latex enzyme fraction in a concentration dependent manner induced the formation of clot in citrated blood plasma. Direct incubation of fibrinogen with latex enzyme fraction resulted in the formation of fibrin clot similar to thrombin enzyme. However prolonged incubation resulted in degradation of the formed fibrin clot suggesting plasmin like activity. Latex enzyme fraction preferentially hydrolyzed Aalpha and Bbeta chains of fibrinogen to form fibrin clot. Latex enzyme fraction also hydrolyzed the subunits of fully cross linked fibrin efficiently, the order of hydrolysis was alpha-polymer > alpha-chains > beta-chain and gamma-gamma dimer. Cysteine proteases from all the four Asclepiadaceae plants latex exhibited similar action on fibrinogen and fibrin. This study scientifically validate the use of plant latex in stop bleeding and wound healing by traditional healers all over the world. PMID:18979066

Shivaprasad, H V; Riyaz, M; Venkatesh Kumar, R; Dharmappa, K K; Tarannum, Shaista; Siddesha, J M; Rajesh, R; Vishwanath, B S

2009-10-01

275

Antithrombotic effects due to pharmacological modulation of thrombin-activatable fibrinolysis inhibitor in rats.  

Science.gov (United States)

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a basic carboxypeptidase zymogen that can be activated by thrombin. Activated TAFI (TAFIa) cleaves carboxyl-terminal lysine residues from partially degraded fibrin, rendering it resistant to fibrinolysis by endogenous tissue plasminogen activator (tPA). Carboxypeptidase inhibitor (CPI) isolated from potato inhibits TAFIa and reduces clot lysis time in rabbit and mouse plasma. In the present study, we report the effect of CPI on tPA-mediated clot lysis using rat plasma in vitro. CPI at 400, 600 and 800 ng/ml caused a dose-dependent enhancement of tPA-induced clot lysis. In vivo effect of CPI was also investigated using ferric chloride-induced arterial thrombosis model in rat. The results showed that i.v. administration of CPI significantly prolonged the 'time to occlusion' at the dose of 2 and 4 mg/kg. At 2 mg/kg i.v. dose in rat, CPI showed no effect on prothrombin time and activated partial thromboplastin time, indicating noninterference of CPI with other clotting factors in mediating its thrombolytic effect through TAFI inhibition. Furthermore, 2 mg/kg i.v. dose of CPI did not produce significant increase in bleeding time when tested in rat tail-transection bleeding model. These results provide evidence for a role of TAFI in arterial thrombosis in rats and suggest that TAFI inhibition could be explored as an attractive target for the development of new antithrombotic drugs. PMID:18936552

Soni, Hitesh; Sharma, Ajay; Bhatt, Shvetank; Jain, Mukul R; Patel, Pankaj R

2008-01-01

276

A sensitive HIV-1 envelope induced fusion assay identifies fusion enhancement of thrombin  

International Nuclear Information System (INIS)

To evaluate the interaction between HIV-1 envelope glycoprotein (Env) and target cell receptors, various cell-cell-fusion assays have been developed. In the present study, we established a novel fusion system. In this system, the expression of the sensitive reporter gene, firefly luciferase (FL) gene, in the target cells was used to evaluate cell fusion event. Simultaneously, constitutively expressed Renilla luciferase (RL) gene was used to monitor effector cell number and viability. FL gave a wider dynamic range than other known reporters and the introduction of RL made the assay accurate and reproducible. This system is especially beneficial for investigation of potential entry-influencing agents, for its power of ruling out the false inhibition or enhancement caused by the artificial cell-number variation. As a case study, we applied this fusion system to observe the effect of a serine protease, thrombin, on HIV Env-mediated cell-cell fusion and have found the fusion enhancement activity of thrombin over two R5-tropic HIV strains.

277

A sensitive HIV-1 envelope induced fusion assay identifies fusion enhancement of thrombin  

Energy Technology Data Exchange (ETDEWEB)

To evaluate the interaction between HIV-1 envelope glycoprotein (Env) and target cell receptors, various cell-cell-fusion assays have been developed. In the present study, we established a novel fusion system. In this system, the expression of the sensitive reporter gene, firefly luciferase (FL) gene, in the target cells was used to evaluate cell fusion event. Simultaneously, constitutively expressed Renilla luciferase (RL) gene was used to monitor effector cell number and viability. FL gave a wider dynamic range than other known reporters and the introduction of RL made the assay accurate and reproducible. This system is especially beneficial for investigation of potential entry-influencing agents, for its power of ruling out the false inhibition or enhancement caused by the artificial cell-number variation. As a case study, we applied this fusion system to observe the effect of a serine protease, thrombin, on HIV Env-mediated cell-cell fusion and have found the fusion enhancement activity of thrombin over two R5-tropic HIV strains.

Cheng, De-Chun; Zhong, Guo-Cai; Su, Ju-Xiang [Department of Microbiology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081 (China); Liu, Yan-Hong [Second Affiliated Hospital of Harbin Medical University, 246 Xuefu Road, Harbin, Heilongjiang 150081 (China); Li, Yan; Wang, Jia-Ye [Department of Microbiology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081 (China); Hattori, Toshio [Department of Emerging Infectious Diseases, Division of Internal Medicine, Graduate School of Medicine, Tohoku University, Sendai 9808574 (Japan); Ling, Hong, E-mail: lingh@ems.hrbmu.edu.cn [Department of Microbiology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081 (China); Department of Parasitology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081 (China); Key Lab of Heilongjiang Province for Infection and Immunity, Key Lab of Heilongjiang Province Education Bureau for Etiology, Harbin, Heilongjiang 150081 (China); Zhang, Feng-Min, E-mail: fengminzhang@yahoo.com.cn [Department of Microbiology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081 (China); Key Lab of Heilongjiang Province for Infection and Immunity, Key Lab of Heilongjiang Province Education Bureau for Etiology, Harbin, Heilongjiang 150081 (China)

2010-01-22

278

Regulation of Meiotic Recombination  

Energy Technology Data Exchange (ETDEWEB)

Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system for assaying recombination using tetrad analysis in a higher eukaryotic system (6). This system enabled the measurement of the frequency and distribution of recombination events at a genome wide level in wild type Arabidopsis (7), construction of genetic linkage maps which include positions for each centromere (8), and modeling of the strength and pattern of interference (9). This proposal extends the use of tetrad analysis in Arabidopsis by using it as the basis for assessing the phenotypes of mutants in genes important for recombination and the regulation of crossover interference and performing a novel genetic screen. In addition to broadening our knowledge of a classic genetic problem - the regulation of recombination by crossover interference - this proposal also provides broader impact by: generating pedagogical tools for use in hands-on classroom experience with genetics, building interdisciplinary collegial partnerships, and creating a platform for participation by junior scientists from underrepresented groups. There are three specific aims: (1) Isolate mutants in Arabidopsis MUS81 homologs using T-DNA and TILLING (2) Characterize recombination levels and interference in mus81 mutants (3) Execute a novel genetic screen, based on tetrad analysis, for genes that regulate meiotic recombination

Gregory p. Copenhaver

2011-11-09

279

The antithrombotic efficacy of AT-1459, a novel, direct thrombin inhibitor, in rat models of venous and arterial thrombosis.  

Science.gov (United States)

The antithrombotic efficacy of AT-1459, a novel, direct thrombin inhibitor (Ki = 4.9 nM) was evaluated in rat models of venous thrombosis combined with a bleeding time test and arterial thrombosis. After drugs were given by i. v. bolus injection plus a continuous infusion, the ID50, (a dose that exhibits 50% inhibition of thrombus formation over each vehicle group) values of AT-1459, argatroban, and dalteparin were 0.04 mg/kg plus 0.04 mg/kg/h, 0.1 mg/kg plus 0.4 mg/ kg/h, and 13.0 IU/kg plus 26.0 IU/kg/h, respectively, in the venous thrombosis study. The BT2 (a dose that causes 2-fold prolongation of bleeding time over each vehicle group) values of AT-1459, argatroban, and dalteparin were 0.9 mg/kg plus 0.9 mg/kg/h, 1.0 mg/kg plus 0.6 mg/kg/h, and 345.5 IU/kg plus 691.0 IU/kg/h in the rat tail transection model. The ratios of BT2/ID50 of AT-1459, argatroban, and dalteparin were 22.5, 10.0, and 26.6, respectively. In a rat model of arterial thrombosis induced by topical FeCl2 application, intravenous administration of AT-1459, argatroban, and dalteparin improved the vessel patency significantly (P < 0.01) at 0.6 mg/kg plus 0.6 mg/kg/h, 0.6 mg/kg plus 2.4 mg/kg/h, and 300 IU/kg plus 600 IU/kg/h, respectively. The oral antithrombotic effect of AT-1459 lasted for 6 after administering 30 mg/kg and improved the vessel patency significantly 1 h after administering the same dose in venous and arterial thrombosis models, respectively, with a rapid onset of action. Warfarin also inhibited thrombus weight and improved the vessel patency significantly after oral administration of 0.3 mg/kg for three consecutive days in the same study. The antithrombotic and hemorrhagic effects of all drugs studied were correlated with plasma concentration or clotting times. These results suggest that AT-1459 may be clinically useful as an orally available antithrombotic agent for the prevention of venous and arterial thrombosis. PMID:11776321

Cho, J H; Yun, C H; Seo, H S; Koga, T; Dan, T; Koo, B A; Kim, H Y

2001-12-01

280

Rolling-circle amplification detection of thrombin using surface-enhanced Raman spectroscopy with core-shell nanoparticle probe.  

Science.gov (United States)

An ultrasensitive surface-enhanced Raman spectroscopy (SERS) sensor based on rolling-circle amplification (RCA)-increased "hot-spot" was developed for the detection of thrombin. The sensor contains a SERS gold nanoparticle@Raman label@SiO2 core-shell nanoparticle probe in which the Raman reporter molecules are sandwiched between a gold nanoparticle core and a thin silica shell by a layer-by-layer method. Thrombin aptamer sequences were immobilized onto the magnetic beads (MBs) through hybridization with their complementary strand. In the presence of thrombin, the aptamer sequence was released; this allowed the remaining single-stranded DNA (ssDNA) to act as primer and initiate in situ RCA reaction to produce long ssDNAs. Then, a large number of SERS probes were attached on the long ssDNA templates, causing thousands of SERS probes to be involved in each biomolecular recognition event. This SERS method achieved the detection of thrombin in the range from 1.0×10(-12) to 1.0×10(-8) ?M and a detection limit of 4.2×10(-13) ?M, and showed good performance in real serum samples. PMID:25766032

Li, Xuemei; Wang, Linlin; Li, Chunxiang

2015-04-27

281

Endogenous thrombin potential as a novel method for the characterization of procoagulant snake venoms and the efficacy of antivenom.  

Science.gov (United States)

Venom-induced consumption coagulopathy occurs in snake envenoming worldwide but the interaction between procoagulant snake venoms and human coagulation remains poorly understood. We aimed to evaluate an assay using endogenous thrombin potential (ETP) to investigate the procoagulant properties of a range of Australian whole venoms in human plasma and compared this to traditional clotting and prothrombinase activity studies. We developed a novel modification of ETP using procoagulant snake venoms to trigger thrombin production. This was used to characterise the relative potency, calcium and clotting factor requirements of five important Australian snake venoms and efficacy of commercial antivenom, and compared this to prothrombinase activity and clotting assays. All five venoms initiated thrombin generation in the absence and presence of calcium. Pseudonaja textilis (Brown snake; psnake; psnake; p=0.0073) all had statistically significant increases in ETP with calcium. Venom potency varied between assays, with ETP ranging from least potent with Oxyuranus scutellatus (Taipan) venom to intermediate with N. scutatus and H. stephensii venoms to most potent with P. textilis and Tropidechis carinatus (Rough-scale snake) venoms. ETPs for N. scutatus, T. carinatus and H. stephensii venoms were severely reduced with factor V deficient plasma. Antivenom neutralized the thrombin generating capacity but not prothrombin substrate cleaving ability of the venoms. Contrary to previous studies using clotting tests and factor Xa substrates, these venoms differ in calcium requirement. ETP is a useful assay to investigate mechanisms of other procoagulant venoms and is a robust method of assessing antivenom efficacy. PMID:20338189

Isbister, Geoffrey K; Woods, David; Alley, Steven; O'Leary, Margaret A; Seldon, Michael; Lincz, Lisa F

2010-08-01

282

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

International Nuclear Information System (INIS)

Guanosine 5'-O-thiotriphosphate (GTP gamma S) and thrombin stimulate the activity of phospholipase C in platelets that have been permeabilized with saponin and whose inositol phospholipids have been prelabeled with [3H]inositol. Ca2+ has opposite effects on the formation of [3H]inositol phosphates induced by thrombin or GTP gamma S. While the action of GTP gamma S on the formation of [3H]inositol phosphates is inhibited by Ca2+, action of thrombin is stimulated by Ca2+. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which inhibits the function of GTP-binding proteins, also inhibits the effect of GTP gamma S on phospholipase C stimulation but, surprisingly, increases the effect of thrombin. Ca2+ increases the inhibitory effect of GDP beta S on GTP gamma S activation of phospholipase C, but Ca2+ further enhances the stimulatory effect of GDP beta S on the thrombin activation of phospholipase C. This indicates that two mechanisms are responsible for the activation of phospholipase C in platelets. A GTP-binding protein is responsible for regulation of phospholipase C induced by GTP gamma S, while the effect of thrombin on the stimulation of phospholipase C is independent of GTP-binding proteins. However, the effect of thrombin may be modulated by the action of an inhibitory GTP-binding protein

283

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

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Full Text Available Here we tested the ability to augment the biological activity of the thrombin aptamer, d(GGTTGGTGTGGTTGG, by using locked nucleic acid (LNA to influence its G-quadruplex structure. Compared to un-substituted control aptamer, LNA-containing aptamers displayed varying degrees of thrombin inhibition. Aptamers with LNA substituted in either positions G5, T7, or G8 showed decreased thrombin inhibition, whereas LNA at position G2 displayed activity comparable to un-substituted control aptamer. Interestingly, the thermal stability of the substituted aptamers does not correlate to activity – the more stable aptamers with LNA in position G5, T7, or G8 showed the least thrombin inhibition, while a less stable aptamer with LNA at G2 was as active as the un-substituted aptamer. These results suggest that LNA substitution at sites G5, T7, and G8 directly perturbs aptamer-thrombin affinity. This further implies that for the thrombin aptamer, activity is not dictated solely by the stability of the G-quadruplex structure, but by specific interactions between the central TGT loop and thrombin and that LNA can be tolerated in a biologically active nucleic acid structure albeit in a position dependent fashion.

Michael B. Jarstfer

2008-03-01

284

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

Energy Technology Data Exchange (ETDEWEB)

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

Oberdisse, E.; Lapetina, E.G.

1987-05-14

285

Thrombin Enhances NGF-Mediated Neurite Extension via Increased and Sustained Activation of p44/42 MAPK and p38 MAPK  

Science.gov (United States)

Rapid neurite remodeling is fundamental to nervous system development and plasticity. It involves neurite extension that is regulated by NGF through PI3K/AKT, p44/42 MAPK and p38 MAPK. It also involves neurite retraction that is regulated by the serine protease, thrombin. However, the intracellular signaling pathway by which thrombin causes neurite retraction is unknown. Using the PC12 neuronal cell model, we demonstrate that thrombin utilizes the PI3K/AKT pathway for neurite retraction in NGF-differentiated cells. Interestingly, however, we found that thrombin enhances NGF-induced neurite extension in differentiating cells. This is achieved through increased and sustained activation of p44/42 MAPK and p38 MAPK. Thus, thrombin elicits opposing effects in differentiated and differentiating cells through activation of distinct signaling pathways: neurite retraction in differentiated cells via PI3K/AKT, and neurite extension in differentiating cells via p44/42 MAPK and p38 MAPK. These findings, which also point to a novel cooperative role between thrombin and NGF, have significant implications in the development of the nervous system and the disease processes that afflicts it as well as in the potential of combined thrombin and NGF therapy for impaired learning and memory, and spinal cord injury which all require neurite extension and remodeling. PMID:25061982

Mufti, Rania E.; Sarker, Krishna; Jin, Yan; Fu, Songbin; Rosales, Jesusa L.; Lee, Ki-Young

2014-01-01

286

cAMP controls the restoration of endothelial barrier function after thrombin-induced hyperpermeability via Rac1 activation.  

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Inflammatory mediators like thrombin disrupt endothelial adherens junctions (AJs) and barrier integrity leading to oedema formation followed by resealing of AJs and a slow recovery of the barrier function. The molecular mechanisms of this process have not yet been fully delineated. The aim of the present study was to analyse the molecular mechanism of endothelial barrier recovery and thrombin was used as model inflammatory mediator. Thrombin caused a strong increase in endothelial permeability within 10 min accompanied by loss of Rac1 but not cdc42 activity, drop in cellular cAMP contents, and a strong activation of the endothelial contractile machinery mainly via RhoA/Rock signalling. Activation of RhoA/Rock signalling precedes and is dependent upon a rise in the cytosolic Ca(2+) concentration. Inhibition of cytosolic Ca(2+) rise but not MLCK or Rock enhances the recovery of endothelial barrier function. The cellular cAMP contents increased gradually during the barrier recovery phase (30-60 min after thrombin challenge) accompanied by an increase in Rac1 activity. Inhibition of Rac1 activity using a specific pharmacological inhibitor (NSC23766) abrogated the endothelial barrier recovery process, suggesting a Rac1-dependent phenomenon. Likewise, inhibition of either adenylyl cyclase or the cAMP-effectors PKA and Epac (with PKI and ESI-09, respectively) caused an abrogation of Rac1 activation, resealing of endothelial AJs and recovery of endothelial barrier function. The data demonstrate that endothelial barrier recovery after thrombin challenge is regulated by Rac1 GTPase activation. This Rac1 activation is due to increased levels of cellular cAMP and activation of downstream signalling during the barrier recovery phase. PMID:25344477

Aslam, Muhammad; Tanislav, Christian; Troidl, Christian; Schulz, Rainer; Hamm, Christian; Gündüz, Dursun

2014-10-01

287

Adiabatic cooling and recombination heating in expanding recombination plasma  

International Nuclear Information System (INIS)

Objective region of gain in Ne/Te plane for 3d5/2-2p3/2 transitions in H-like ion in recombination C plasma is given. The characteristics of plasma at the juncture of ionization and recombination are obtained. Adiabatic cooling Wp and recombination heating Ws are studied and their approximate expressions are given

288

Recombineering Pseudomonas syringae  

Science.gov (United States)

Here we report the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecE...

289

Recombinant hormones in osteoporosis  

DEFF Research Database (Denmark)

For the last 10 years, bone anabolic therapy with the recombinant human parathyroid hormone (rhPTH) analogue, teriparatide (rhPTH[1 - 34]), or full-length rhPTH(1 - 84) has been an option in the treatment of osteoporosis. Both drugs are given as a daily subcutaneous injection. In the USA, only teriparatide is marketed.

Rejnmark, Lars; Rejnmark, Lars

2013-01-01

290

Introduction to dissociative recombination  

Science.gov (United States)

Dissociative recombination (DR) of molecular ions with electrons has important consequences in many areas of physical science. Ab-initio calculations coupled with resonant scattering theory and multichannel quantum defect studies have produced detailed results illuminating the role of ion vibrational excitation, the quantum yields of the DR products, and the role of Rydberg states. The theoretical and experimental results are discussed.

Guberman, Steven L.; Mitchell, J. Brian A.

1989-01-01

291

Incorporation of noncoded amino acids into the N-terminal domain 1-47 of hirudin yields a highly potent and selective thrombin inhibitor.  

OpenAIRE

Hirudin is an anticoagulant polypeptide isolated from a medicinal leech that inhibits thrombin with extraordinary potency (Kd = 0.2-1.0 pM) and selectivity. Hirudin is composed of a compact N-terminal region (residues 1-47, cross-linked by three disulfide bridges) that binds to the active site of thrombin, and a flexible C-terminal tail (residues 48-64) that interacts with the exosite I of the enzyme. To minimize the sequence of hirudin able to bind thrombin and also to improve its therapeuti...

Filippis, V.; Russo, I.; Vindigni, A.; Di Cera, E.; Salmaso, S.; Fontana, A.

1999-01-01

292

Thrombin induces Egr-1 expression in fibroblasts involving elevation of the intracellular Ca2+ concentration, phosphorylation of ERK and activation of ternary complex factor  

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Full Text Available Abstract Background The serine protease thrombin catalyzes fibrin clot formation by converting fibrinogen into fibrin. Additionally, thrombin stimulation leads to an activation of stimulus-responsive transcription factors in different cell types, indicating that the gene expression pattern is changed in thrombin-stimulated cells. The objective of this study was to analyze the signaling cascade leading to the expression of the zinc finger transcription factor Egr-1 in thrombin-stimulated lung fibroblasts. Results Stimulation of 39M1-81 fibroblasts with thrombin induced a robust and transient biosynthesis of Egr-1. Reporter gene analysis revealed that the newly synthesized Egr-1 was biologically active. The signaling cascade connecting thrombin stimulation with Egr-1 gene expression required elevated levels of cytosolic Ca2+, the activation of diacylgycerol-dependent protein kinase C isoenzymes, and the activation of extracellular signal-regulated protein kinase (ERK. Stimulation of the cells with thrombin triggered the phosphorylation of the transcription factor Elk-1. Expression of a dominant-negative mutant of Elk-1 completely prevented Egr-1 expression in stimulated 39M1-81 cells, indicating that Elk-1 or related ternary complex factors connect the intracellular signaling cascade elicited by activation of protease-activated receptors with transcription of the Egr-1 gene. Lentiviral-mediated expression of MAP kinase phosphatase-1, a dual-specific phosphatase that dephosphorylates and inactivates ERK in the nucleus, prevented Elk-1 phosphorylation and Egr-1 biosynthesis in thrombin stimulated 39M1-81 cells, confirming the importance of nuclear ERK and Elk-1 for the upregulation of Egr-1 expression in thrombin-stimulated lung fibroblasts. 39M1-81 cells additionally express M1 muscarinic acetylcholine receptors. A comparison between the signaling cascades induced by thrombin or carbachol showed no differences, except that signal transduction via M1 muscarinic acetylcholine receptors required the transactivation of the EGF receptor, while thrombin signaling did not. Conclusion This study shows that stimulus-transcription coupling in thrombin-treated lung fibroblasts relies on the elevation of the intracellular Ca2+-concentration and the activation of PKC and ERK. In the nucleus, ternary complex factors function as key proteins linking the intracellular signaling cascade with enhanced transcription of the Egr-1 gene. This study further shows that the dominant-negative Elk-1 mutant is a valuable tool to study Elk-1-mediated gene transcription.

Thiel Gerald

2009-05-01

293

Evidence supporting the use of recombinant activated factor VII in congenital bleeding disorders  

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Full Text Available Pär I Johansson, Sisse R OstrowskiCapital Region Blood Bank, Section for Transfusion Medicine, Rigshospitalet, University of Copenhagen, Copenhagen, DenmarkBackground: Recombinant activated factor VII (rFVIIa, NovoSeven® was introduced in 1996 for the treatment of hemophilic patients with antibodies against coagulation factor VIII or IX.Objective: To review the evidence supporting the use of rFVIIa for the treatment of patients with congenital bleeding disorders.Patients and methods: English-language databases were searched in September 2009 for reports of randomized controlled trials (RCTs evaluating the ability of rFVIIa to restore hemostasis in patients with congenital bleeding disorders.Results: Eight RCTs involving 256 hemophilic patients with antibodies against coagulation factors, also known as inhibitors, were identified. The evidence supporting the use of rFVIIa in these patients was weak with regard to dose, clinical setting, mode of administration, efficacy, and adverse events, given the limited sample size of each RCT and the heterogeneity of the studies.Conclusion: The authors suggest that rFVIIa therapy in hemophilic patients with inhibitors should be based on the individual’s ability to generate thrombin and form a clot, and not on the patient’s weight alone. Therefore, assays for thrombin generation, such as whole-blood thromboelastography, have the potential to significantly improve the treatment of these patients.Keywords: hemophilia, inhibitors, coagulation factor VIII, coagulation factor IX, rFVIIa, NovoSeven, FEIBA, hemostasis, RCT

Pär I Johansson

2010-06-01

294

Constructing minimal ancestral recombination graphs.  

OpenAIRE

By viewing the ancestral recombination graph as defining a sequence of trees, we show how possible evolutionary histories consistent with given data can be constructed using the minimum number of recombination events. In contrast to previously known methods, which yield only estimated lower bounds, our method of detecting recombination always gives the minimum number of recombination events if the right kind of rooted trees are used in our algorithm. A new lower bound can be defined if rooted...

Song, Ys; Hein, J.

2005-01-01

295

Evidence of recombination within human alpha-papillomavirus  

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Full Text Available Abstract Background Human papillomavirus (HPV has a causal role in cervical cancer with almost half a million new cases occurring each year. Presence of the carcinogenic HPV is necessary for the development of the invasive carcinoma of the genital tract. Therefore, persistent infection with carcinogenic HPV causes virtually all cervical cancers. Some aspects of the molecular evolution of this virus, as the putative importance of recombination in its evolutionary history, are an opened current question. In addition, recombination could also be a significant issue nowadays since the frequency of co-infection with more than one HPV type is not a rare event and, thus, new recombinant types could be currently being generated. Results We have used human alpha-PV sequences from the public database at Los Alamos National Laboratory to report evidence that recombination may exist in this virus. A model-based population genetic approach was used to infer the recombination signal from the HPV DNA sequences grouped attending to phylogenetic and epidemiological information, as well as to clinical manifestations. Our results agree with recently published ones that use a different methodology to detect recombination associated to the gene L2. In addition, we have detected significant recombination signal in the genes E6, E7, L2 and L1 at different groups, and importantly within the high-risk type HPV16. The method used has recently been shown to be one of the most powerful and reliable procedures to detect the recombination signal. Conclusion We provide new support to the recent evidence of recombination in HPV. Additionally, we performed the recombination estimation assuming the best-fit model of nucleotide substitution and rate variation among sites, of the HPV DNA sequence sets. We found that the gene with recombination in most of the groups is L2 but the highest values were detected in L1 and E6. Gene E7 was recombinant only within the HPV16 type. The topic deserves further study because recombination is an important evolutionary mechanism that could have high impact both in pharmacogenomics (i.e. on the influence of genetic variation on the response to drugs and for vaccine development.

Carvajal-Rodríguez Antonio

2007-03-01

296

Aptamer-directed lanthanide chelate self-assembly for rapid thrombin detection.  

Science.gov (United States)

We report a sensitive assay method for homogeneous thrombin detection. The method is based on lanthanide chelate complementation, where the luminescent complex is split into two separate label moieties, which are intrinsically non-luminescent. A luminescent mixed chelate complex is formed only when the label moieties are brought into close proximity directed by two separate binding events of aptamers to the analyte. This results in high specificity in signal generation while time-resolved fluorescence detection eliminates the short lifetime autofluorescence, which is inherent to many homogeneous assays and limits their applicability. The developed method is also very rapid as the maximum signal is obtained in just five minutes. Lanthanide chelate complementation can be applied for the detection of other proteins when two binders recognizing separate epitopes of the analyte are available. PMID:23807946

Päkkilä, Henna; Blom, Sami; Kopra, Kari; Soukka, Tero

2013-09-01

297

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

Science.gov (United States)

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. The objective of the present study was to evaluate these two proteins in plasma from patients with aortic stenosis and establish their relationship with the severity of the disease. Fifty-four consecutive patients with aortic stenosis, including 38 (70.4%) severe aortic stenosis patients, were studied. Plasma FXIa and TF activity were determined in clotting assays by measuring the response to inhibitory monoclonal antibodies. TF activity was detectable in plasma from 14 of 54 patients (25.9%), including 13 of 38 with severe aortic stenosis (34.2%) and one of 16 (6.25%) with moderate aortic stenosis (P=0.052). FXIa activity was found in 12 (22.2%) patients, mostly in individuals with severe aortic stenosis (11 of 38, 28.9%, P=0.067). All 12 patients with circulating FXIa had active TF in their plasma as well. Severe aortic stenosis patients with detectable TF had higher maximal (111±20 vs. 97±16 mmHg, P=0.02) and mean (61±12 vs. 53±8 mmHg, P=0.02) transvalvular gradient, compared with those without such activity in plasma. In severe aortic stenosis patients with detectable active TF, prothrombin fragment 1.2, a thrombin generation marker, was higher than that in patients without TF (375±122 vs. 207±64 pM, PFXIa and TF activity was observed for the first time in aortic stenosis patients, primarily in severe ones. This activity correlates with thrombin generation in those patients. PMID:21519234

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

2011-09-01

298

Resolution of radiolabeled molecular species of phospholipid in human platelets: effect of thrombin  

Energy Technology Data Exchange (ETDEWEB)

Resolution of individual molecular species of human platelet 1,2-diradyl-sn-glycero-3-phosphocholines and 1,2-diradyl-sn-glycero-3-phosphoethanolamines by reverse phase high pressure liquid chromatography (HPLC) allowed a thorough analysis of those phospholipids labeled with (/sup 3/H)arachidonic acid. Approximately 54% and 16% of the total incorporated radiolabel was found in choline glycerophospholipids and ethanolamine glycerophospholipids, respectively, with ca. 90% of this being found in the 1,2-diacyl molecular species. Eighty percent of (/sup 3/H)-arachidonic acid incorporated into 1-acyl-2-arachidonoyl-sn-glycero-3-phosphocholine in resting platelets was equally distributed between 1-palmitoyl-2-arachidonoyl and 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, while 70% of the radiolabel in 1-acyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine was found in 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine. Thrombin stimulation (5 U/ml for 5 min) resulted in deacylation of all 1-acyl-2-(/sup 3/H)arachidonoyl molecular species of 1-acyl-2-arachidonoyl-sn-glycero-3-phosphocholine and 1-acyl-2-arachidonoyl-sn-glycero-3-ethanolamine. There was also a slight increase in 1-O-alkyl-2-(/sup 3/H)arachidonoyl-sn-glycero-3-phosphocholine and a significant increase in 1-O-alk-1'-enyl-2-(/sup 3/H)arachidonoyl-sn-glycero-3-phosphoethanolamine molecular species of over 300%. Thus, HPLC methodology indicates that arachidonoyl-containing molecular species of phosphatidylcholine and phosphatidylethanolamine are the major source of arachidonic acid in thrombin-stimulated human platelets, while certain ether phospholipid molecular species become enriched in arachidonate.

Purdon, A.D.; Patelunas, D.; Smith, J.B.

1987-02-01

299

Characterization of Ixophilin, a thrombin inhibitor from the gut of Ixodes scapularis.  

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Ixodes scapularis, the black-legged tick, vectors several human pathogens including Borrelia burgdorferi, the agent of Lyme disease in North America. Pathogen transmission to the vertebrate host occurs when infected ticks feed on the mammalian host to obtain a blood meal. Efforts to understand how the tick confronts host hemostatic mechanisms and imbibes a fluid blood meal have largely focused on the anticoagulation strategies of tick saliva. The blood meal that enters the tick gut remains in a fluid state for several days during the process of feeding, and the role of the tick gut in maintaining the blood-meal fluid is not understood. We now demonstrate that the tick gut produces a potent inhibitor of thrombin, a key enzyme in the mammalian coagulation cascade. Chromatographic fractionation of engorged tick gut proteins identified one predominant thrombin inhibitory activity associated with an approximately 18 kDa protein, henceforth referred to as Ixophilin. The ixophilin gene was preferentially transcribed in the guts of feeding nymphs. Expression began after 24 hours of feeding, coincident with the flow of host blood into the tick gut. Immunity against Ixophilin delayed tick feeding, and decreased feeding efficiency significantly. Surprisingly, immunity against Ixophilin resulted in increased Borrelia burgdorferi transmission to the host, possibly due to delayed feeding and increased transmission opportunity. These observations illuminate the potential drawbacks of targeting individual tick proteins in a functional suite. They also underscore the need to identify the "anticoagulome" of the tick gut, and to prioritize a critical subset of anticoagulants that could be targeted to efficiently thwart tick feeding, and block pathogen transmission to the vertebrate host. PMID:23874485

Narasimhan, Sukanya; Perez, Oriana; Mootien, Sara; DePonte, Kathleen; Koski, Raymond A; Fikrig, Erol; Ledizet, Michel

2013-01-01

300

Cyclodextrin functionalized graphene-gold nanoparticle hybrids with strong supramolecular capability for electrochemical thrombin aptasensor.  

Science.gov (United States)

We demonstrate a facile one-pot synthetic strategy for controlled synthesis of thio-?-cyclodextrin functionalized graphene/gold nanoparticles (SH-?-CD-Gr/AuNPs) composites using SH-?-CD as both the dispersant and linker. The obtained SH-?-CD-Gr/AuNPs integrate the excellent electrical properties and large surface area of graphene and AuNPs with supramolecular recognition ability of CD, which show more effective electron transfer and higher enriched ability for the ferrocene probe via the host-guest interaction between CD and ferrocene than SH-?-CD-Gr. In the presence of target, the stronger interaction between aptamer and target makes the ferrocene move closer to the electrode surface, thus facilitating the electron transfer. Based on this sensing mechanism, a new and highly sensitive biosensing concept by the use of SH-?-CD-Gr/AuNPs as enhancing materials is demonstrated for "signal-on" detection of targets (thrombin as a model target). This biosensor exhibits a wide linear range for thrombin from 1.6×10(-17) M to 8.0×10(-15) M and a very low limit of detection 5.2×10(-18) M, which is two-order magnitude better than those of SH-?-CD-Gr (the detection linear range from 1.6×10(-15) M to 8.0×10(-13) M and detection limit of 1.0×10(-15) M). Our proposed electrochemical aptasensor based on SH-?-CD-Gr/AuNPs shows good selectivity against other proteins such as human serum albumin, lysozyme and insulin. To the best of our knowledge, the present SH-?-CD-Gr/AuNPs hybrids are the most efficient graphene-based electrochemical active probes ever reported for biosensors. PMID:25618374

Xue, Qiong; Liu, Zhiguang; Guo, Yujing; Guo, Shaojun

2015-06-15

301

Oxygen-hydrogen recombiner  

International Nuclear Information System (INIS)

Purpose: To reduce the amount of a catalyst and improve the hydrogen removing performance of a catalyst type oxygen-hydrogen recombiner in a radioactive gaseous wastes processing facility of BWR type nuclear power plants. Constitution: The catalyst (of palladium or platinum group) has a low activity at a low temperature. In view of the above, hydrogen gases injected from the outside for improving the catalyst activity even under a low temperature state such as reactor start-up and shut-down. When the hydrogen gas concentration increases, chemical combination between oxygen and hydrogen in the recombiner is increased, by which the amount of heat generation is increased to rise the temperature of the catalyst. In this way, the catalyst activity can be improved. Corresponding to the injection of the hydrogen gas, oxygen is also injected so as to attain 1 : 2 ratio. (Ikeda, J.)

302

AECL passive autocatalytic recombiners  

International Nuclear Information System (INIS)

Atomic Energy of Canada Limited's (AECL) Passive Autocatalytic Recombiner (PAR) is a passive device used for hydrogen mitigation under post-accident conditions in nuclear reactor containment. The PAR employs a proprietary AECL catalyst which promotes the exothermal reaction between hydrogen and oxygen to form water vapour. The heat of reaction combined with the PAR geometry establishes a convective flow through the recombiner, where ambient hydrogen-rich gas enters the PAR inlet and hot, humid, hydrogen-depleted gas exits the outlet. AECL's PAR has been extensively qualified for CANDU and light water reactors (LWRs), and has been supplied to France, Finland, Ukraine, South Korea and is currently being deployed in Canadian nuclear power plants. (author)

303

Intrachromosomal recombination in plants.  

OpenAIRE

Molecular evidence for intrachromosomal recombination between closely linked DNA repeats within the plant genome is presented. The non-overlapping complementary deletion derivatives of the selectable neomycin phosphotransferase gene (nptII), when intact conferring kanamycin resistance, were inserted into the genome of Nicotiana tabacum. The functional marker gene was restored with frequencies between 10(-4) and 10(-6) per proliferating cell clone. Prolonged tissue culture prior to kanamycin s...

Peterhans, A.; Schlu?pmann, H.; Basse, C.; Paszkowski, J.

1990-01-01

304

Thrombin induces ICAM-1 expression in human lung epithelial cells via c-Src/PDGFR/PI3K/Akt-dependent NF-?B/p300 activation.  

Science.gov (United States)

Up-regulation of ICAM-1 (intercellular adhesion molecule-1) is frequently implicated in lung inflammation and lung diseases, such as IPF (idiopathic pulmonary fibrosis). Thrombin has been shown to play a key role in inflammation via the induction of adhesion molecules, which then causes lung injury. However, the mechanisms underlying thrombin-induced ICAM-1 expression in HPAEpiCs (human pulmonary alveolar epithelial cells) remain unclear. In the present study, we have shown that thrombin induced ICAM-1 expression in HPAEpiCs. Pre-treatment with the inhibitor of thrombin [PPACK (D-Phe-Pro-Arg-chloromethyl ketone)], c-Src (PP1), PDGFR (platelet-derived growth factor receptor) (AG1296), PI3K (phosohinositide 3-kinase) (LY294002), NF-?B (nuclear factor ?B) (Bay11-7082) or p300 (GR343) and transfection with siRNAs of c-Src, PDGFR, Akt, p65 and p300 markedly reduced thrombin-induced ICAM-1 expression and monocyte adherence to HPAEpiCs challenged with thrombin. In addition, we established that thrombin stimulated the phosphorylation of c-Src, PDGFR, Akt and p65, which were inhibited by pre-treatment with their respective inhibitors PP1, AG1296, LY294002 or Bay11-7082. In addition, thrombin also enhanced Akt and NF-?B translocation from the cytosol to the nucleus, which was reduced by PP1, AG1296 or LY294002. Thrombin induced NF-?B promoter activity and the formation of the p65-Akt-p300 complex, which were inhibited by AG1296, LY294002 or PP1. Finally, we have shown that thrombin stimulated in vivo binding of p300, Akt and p65 to the ICAM-1 promoter, which was reduced by AG1296, LY294002, SH-5 or PP1. These results show that thrombin induced ICAM-1 expression and monocyte adherence via a c-Src/PDGFR/PI3K/Akt/NF-?B-dependent pathway in HPAEpiCs. Increased understanding of the signalling mechanisms underlying ICAM-1 gene regulation will create opportunities for the development of anti-inflammatory therapeutic strategies. PMID:24506791

Cheng, Shin-Ei; Lee, I-Ta; Lin, Chih-Chung; Hsiao, Li-Der; Yang, Chuen-Mao

2014-08-01

305

Dielectronic recombination theory  

International Nuclear Information System (INIS)

A theory now in wide use for the calculation of dielectronic recombination cross sections (?DR) and rate coefficients (?DR) was one introduced originally by Feshbach for nuclear physics applications, and then later adapted for atomic scattering problems by Hahn. In the following, we briefly review this theory in a very general form, which allows one to account for the effects of overlapping and interacting resonances, as well as continuum-continuum coupling. An extension of our notation will then also allow for the inclusion of the effects of direct radiative recombination, along with a treatment of the interference between radiative and dielectronic recombination. Other approaches to the calculation of ?DR have been described by Fano and by Seaton. We will not consider those theories here. Calculations of ?DR have progressed considerably over the last 25 years, since the early work of Burgess. Advances in the reliability of theoretical predictions have also been promoted recently b a variety of direct laboratory measurements of ?DR. While the measurements of ?DR for ?n ? 0 excitations have tended to agree very well with calculations, the case of ?n = 0 has been much problematic. However, by invoking a mechanism originally proposed by Jacobs, which takes into account the effect of stray electric fields on high Rydberg states (HRS) participating in the DR process, new calculations have improved the agreement between theory and experiment for these cases. Nevertheless, certain discrepancies still remain

306

Poly(methyl methacrylate) microchip affinity capillary gel electrophoresis of aptamer-protein complexes for the analysis of thrombin in plasma.  

Science.gov (United States)

Thrombin generation in blood serves as an important marker for various hemostasis-related diseases and conditions. Analytical techniques currently utilized for determining the thrombin potential of patients rely primarily on the enzymatic activity of thrombin. Microfluidic-based ACE using fluorescently labeled aptamers as affinity probes could provide a simple and efficient technique for the real-time analysis of thrombin levels in plasma. In this study, aptamers were used for the analysis of thrombin by affinity microchip CGE. The CGE used a poly(methyl methacrylate) (PMMA) microfluidic device for the sorting of the affinity complexes with a linear polyacrylamide (LPA) serving as the sieving matrix. Due to the fact that the assay was run under nonequilibrium electrophoresis conditions, the presence of the sieving gel was found to stabilize the affinity complex, providing improved electrophoretic performance compared to free-solution electrophoresis. Two fluorescently labeled aptamer affinity probes, HD1 and HD22, which bind to exosites I and II, respectively, of thrombin were investigated. With an electric field strength of 300 V/cm, two well-resolved peaks corresponding to free aptamer and the thrombin-aptamer complex were obtained in less than 1 min of separation time with a run-to-run and chip-to-chip reproducibility (RSD) of migration times plasma. Assays were performed directly on plasma that was diluted to 10% v/v. Thrombin was successfully analyzed by microchip CGE at a concentration level of 543.5 nM for the human plasma sample. PMID:18702051

Obubuafo, Anne; Balamurugan, Subramanian; Shadpour, Hamed; Spivak, David; McCarley, Robin L; Soper, Steven A

2008-08-01

307

Stimulation of activin A expression in rat aortic smooth muscle cells by thrombin and angiotensin II correlates with neointimal formation in vivo.  

OpenAIRE

Vasoactive GTP-binding protein-coupled receptor agonists (e.g., angiotensin II [AII] and alpha-thrombin) stimulate the production of mitogenic factors from vascular smooth muscle cells. In experiments to identify mitogens secreted from AII- or alpha-thrombin-stimulated rat aortic smooth muscle (RASM) cells, neutralizing antibodies directed against several growth factors (e.g., PDGF and basic fibroblast growth factor [basic FGF]) failed to inhibit the mitogenic activity of conditioned media sa...

Pawlowski, J. E.; Taylor, D. S.; Valentine, M.; Hail, M. E.; Ferrer, P.; Kowala, M. C.; Molloy, C. J.

1997-01-01

308

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

Science.gov (United States)

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 ?-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 ?–? 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.

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

2014-10-01

309

Experiência inicial com o uso de adesivo tissular contendo trombina para tratamento do pseudo-aneurisma femoral Treatment of femoral pseudoaneurysm with thrombin tissue adhesive: initial experience  

Directory of Open Access Journals (Sweden)

Full Text Available O pseudo-aneurisma (PSA após cateterização femoral tem sido diagnosticado com regularidade em serviços com grande movimento de intervenções percutâneas, com incidência variando de 0,05 a 6%. PSA femorais pequenos podem ser acompanhados até a resolução espontânea. As opções de tratamento são: compressão guiada por ultra-som, injeção de trombina para trombose do PSA e tratamento cirúrgico. A injeção percutânea de trombina tem a vantagem de ser um procedimento indolor e rápido. Podem ser utilizados trombina isolada ou preparados contendo trombina associada a fibrinogênio e fatores de coagulação. A experiência inicial dos autores de cinco casos tratados com injeção de adesivo tissular contendo trombina mostrou resultado satisfatório em quatro; um caso necessitou tratamento cirúrgico. Não houve sucesso com uso isolado de trombina humana, porém, ocorreu trombose imediata após injeção de preparado de trombina associada a fibrinogênio/fator XIII. Neste artigo, são discutidas as opções de tratamento dos PSA femorais e a técnica do uso de trombina percutânea.Pseudoaneurysms caused by femoral artery catheterization have been regularly diagnosed in medical units with a great number of percutaneous interventions, with a documented incidence between 0.05 and 6%. Small femoral pseudoaneurysms undergo spontaneous resolution. Treatment options are: ultrasound-guided compression, thrombin injection to induce pseudoaneurysm thrombosis and surgical treatment. Percutaneous thrombin injection has the advantage of being a fast and painless procedure. Both isolated thrombin and thrombin preparations with fibrinogen and coagulation factors can be used. The authors' initial experience with five cases treated with thrombin tissue adhesive showed successful results in four; one case required surgery. There was no success with isolated human thrombin, but immediate thrombosis was achieved after injection of thrombin associated to fibrinogen and factor XIII. In this article, the treatment options for femoral pseudoaneurysms and the technique of percutaneous thrombin are discussed.

Daniel Mendes Pinto

2006-03-01

310

Thrombin Activates AMP-Activated Protein Kinase in Endothelial Cells via a Pathway Involving Ca2+/Calmodulin-Dependent Protein Kinase Kinase ?  

OpenAIRE

AMP-activated protein kinase (AMPK) is a sensor of cellular energy state in response to metabolic stress and other regulatory signals. AMPK is controlled by upstream kinases which have recently been identified as LKB1 or Ca2+/calmodulin-dependent protein kinase kinase ? (CaMKK?). Our study of human endothelial cells shows that AMPK is activated by thrombin through a Ca2+-dependent mechanism involving the thrombin receptor protease-activated receptor 1 and Gq-protein-mediated phospholipase C...

Stahmann, Nadine; Woods, Angela; Carling, David; Heller, Regine

2006-01-01

311

Inhibition by atrial and brain natriuretic peptides of endothelin-1 secretion after stimulation with angiotensin II and thrombin of cultured human endothelial cells.  

OpenAIRE

We examined the inhibition by atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) of endothelin-1 secretion stimulated by angiotensin II (ANGII) and thrombin using cultured human umbilical-vein endothelial cells. ANGII and thrombin dose-dependently stimulated immunoreactive (ir) endothelin-1 secretion. Human ANP(1-28) and human BNP-32 both inhibited such secretion in a dose-dependent way. Inhibition of this secretion by ANP and BNP was paralleled by an increase in the level o...

Kohno, M.; Yasunari, K.; Yokokawa, K.; Murakawa, K.; Horio, T.; Takeda, T.

1991-01-01

312

Platelet protease-activated receptor (PAR)4, but not PAR1, associated with neutral sphingomyelinase responsible for thrombin-stimulated ceramide-NF-?B signaling in human platelets  

OpenAIRE

Thrombin activates platelets mainly through protease-activated receptor (PAR)1 and PAR4. However, downstream platelet signaling between PAR1 and PAR4 is not yet well understood. This study investigated the relationship between nSMase/ceramide and the NF-?B signaling pathway in PARs-mediated human platelet activation. The LC-MS/MS, aggregometry, flow cytometry, immunoprecipitation, and mesenteric microvessels of mice were used in this study. Human platelets stimulated by thrombin, 3-OMS (a ne...

Chen, Wei-fan; Lee, Jie-jen; Chang, Chao-chien; Lin, Kuan-hong; Wang, Shwu-huey; Sheu, Joen-rong

2013-01-01

313

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

Science.gov (United States)

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. PMID:25240130

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

2015-02-15

314

Secretory products from thrombin-stimulated human platelets exert an inhibitory effect on NK-cytotoxic activity.  

DEFF Research Database (Denmark)

We have investigated the interaction between human platelets and the NK-system, with special emphasis on the action of secretory products from platelets in an NK assay with 51Cr-labelled K562 as target cells. Supernatants from thrombin-stimulated platelets added to the NK assay consistently decreased the NK-cytotoxicity by 40% +/- 4.3%, indicating the existence of secreted products from platelets as a source of NK-inhibiting substances. In contrast, no direct cytotoxic effect of these secretory products on the target cells (K562) was seen. Thus, normal human platelets, when stimulated with thrombin, are capable of secreting different, yet undefined factors, which significantly inhibit NK activity in vitro. The results also suggest that the role of products from contaminating in vitro activated platelets should be borne in mind when performing conventional NK assays. Udgivelsesdato: 1986-Oct

Skov Madsen, P; Hokland, P

1987-01-01

315

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

Science.gov (United States)

In this work, we reported a new label-free electrochemical aptasensor for highly sensitive detection of thrombin using direct electron transfer of glucose oxidase (GOD) as a redox probe and a gold nanoparticle-polyaniline-graphene (Au-PANI-Gra) hybrid for amplification. The Au-PANI-Gra hybrid with large surface area provided a biocompatible sensing platform for the immobilization of GOD. GOD was encapsulated into the three-dimensional netlike (3-mercaptopropyl)trimethoxysilane (MPTS) to form the MPTS-GOD biocomposite, which not only retained the native functions and properties, but also exhibited tunable porosity, high thermal stability, and chemical inertness. With abundant thiol tail groups on MPTS, MPTS-GOD was able to chemisorb onto the surface of the Au-PANI-Gra modified electrode through the strong affinity of the Au-S bond. The electrochemical signal originated from GOD, avoiding the addition or labeling of other redox mediators. After immobilizing the thiolated thrombin binding aptamer through gold nanoparticles (AuNPs), GOD as a blocking reagent was employed to block the remaining active sites of the AuNPs and avoid the nonspecific adsorption. The proposed method avoided the labeling process of redox probes and increased the amount of electroactive GOD. The concentration of thrombin was monitored based on the decrease of current response through cyclic voltammetry (CV) in 0.1 M PBS (pH 7.4). With the excellent direct electron transfer of double layer GOD membranes, the resulting aptasensor exhibited high sensitivity for detection of thrombin with a wide linear range from 1.0 × 10(-12) to 3.0 × 10(-8) M. The proposed aptasensor also showed good stability, satisfactory reproducibility and high specificity, which provided a promising strategy for electrochemical aptamer-based detection of other biomolecules. PMID:24003439

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

2013-11-01

316

High-sensitive determination of human alpha-thrombin by its 29-mer aptamer in affinity probe capillary electrophoresis.  

Science.gov (United States)

ACE technique provides an effective tool for the separation and identification of disease-related biomarkers in clinical analysis. In recent years, a couple of synthetic DNA or RNA oligonucleotides, known as aptamers, rival the specificity and affinity for targets to antibodies and are employed as one kind of powerful affinity probe in ACE. In this work, based on high affinity between antithrombin aptamer and thrombin (their dissociation constant is 0.5 nM), a carboxyfluorescein-labeled 29-nucleotide (nt) aptamer (F29-mer) was used and an aptamer-based affinity probe CE (aptamer-based APCE) method was successfully established for high-sensitive detection and quantitative analysis of thrombin. Experimental conditions including incubation temperature and time, buffer composition, and concentration of cations were investigated and optimized. Under the optimized condition, the linear range was from 0 to 400 nM and the LOD was 2 nM (74 ng/mL, S/N = 3), i.e., 40 amol, both in running buffer and in 5% v/v human serum. This LOD is the lowest one than those achieved by the previous APCE methods but based on a 15-mer aptamer. This approach offers a promising method for the rapid, selective, and sensitive detection of thrombin in practical utility. Further binding experiments using one carboxyfluorescein-labeled aptamer and the other nonlabeled aptamer or vice versa were carried out to deduce the formation of ternary complex when these two aptamers coexisted in the free solution with thrombin. PMID:18481835

Li, Yilin; Guo, Lei; Zhang, Fucheng; Zhang, Zhaoyang; Tang, Jijun; Xie, Jianwei

2008-06-01

317

Association of fibrinogen with human platelets pretreated with chymotrypsin or aggregated with ADP or thrombin: an immunocytochemical study.  

OpenAIRE

Although platelets can be induced to aggregate in the absence of external fibrinogen, the response is greatly potentiated by fibrinogen and fibrinogen becomes associated with the surface of stimulated platelets. We compared the aggregation response and association of fibrinogen with the surface of platelets aggregated by ADP or thrombin, and of chymotrypsin-treated platelets aggregated by fibrinogen. The association of fibrinogen with the surface of the platelets was visualized using an elect...

Suzuki, H.; Kinlough-rathbone, R. L.; Packham, M. A.; Yamazaki, H.; Mustard, J. F.

1989-01-01

318

Membrane Changes Associated with Platelet Activation: EXPOSURE OF ACTIN ON THE PLATELET SURFACE AFTER THROMBIN-INDUCED SECRETION  

OpenAIRE

The effect of aggregation and secretion on membrane proteins was studied in washed human platelets. Reversible aggregation without secretion was stimulated by ADP and secretion without aggregation was stimulated by thrombin in the presence of EDTA. No loss of platelet surface glycoproteins occurred during reversible ADP-induced platelet aggregation, as measured by quantitative polyacrylamide gel electrophoresis analysis of platelets that were labeled with 125I-diazotized diiodosulfanilic acid...

George, James N.; Lyons, Roger M.; Morgan, Rebecca K.

1980-01-01

319

Thrombin Receptors and Protease-Activated Receptor-2 in Human Placentation: Receptor Activation Mediates Extravillous Trophoblast Invasion in Vitro  

OpenAIRE

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-...

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

320

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

OpenAIRE

Plasma progesterone levels remain elevated throughout human pregnancy, suggesting that reduced reproductive-tract progesterone receptor (PR) initiates labor. Placental abruption and excess thrombin generation elicit preterm delivery (PTD). PR, glucocorticoid receptor (GR), and total and p-ERK1/2 in decidual cells (DCs) and interstitial trophoblasts (IT) were assessed via immunohistochemical staining in abruption-associated PTD versus gestational-age matched control placentas, and in cultured ...

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

2012-01-01

321

Calibration of Fura-2 signals introduces errors into measurement of thrombin-stimulated calcium mobilisation in human platelets.  

Science.gov (United States)

The intracellular calcium indicator dye Fura-2 has been widely used for the study of human platelet thrombin receptor-mediated calcium mobilisation in disease states. In general, authors (a) use a Fura-2/AM concentration of 2-3 mumol/l and (b) calibrate fluorescence signals on the basis of maximum and minimum ratios of fluorescence (Rmax and Rmin) and the ratio of the calcium-free and calcium-bound fluorescence at an excitation wavelength of 380 nm ("C2/B2"). In the present study, it is found (a) that a greater peak response to thrombin is seen when 1 mumol/l Fura-2/AM rather than 2 or 3 mumol/l is used; and (b) that calibration leads to a poorer test-retest reliability and in general a greater variability of the obtained calcium signal than when the simple measurement of the 340 nm/380 nm fluorescence ratio is used. It is suggested that this poor variability is due to the presence of an extracellular factor that can quench the Fura-2 signal once the platelets have been permeabilised by detergent treatment. Consistent with this, addition of bovine serum albumin to the assay medium has no significant effect on the fluorescence ratio response to thrombin, but greatly increases the observed calibrated calcium signal. PMID:9385466

Fowler, C J; Tiger, G

1997-09-30

322

Anti-thrombin III, Protein C, and Protein S deficiency in acute coronary syndrome  

Directory of Open Access Journals (Sweden)

Full Text Available The final most common pathway for the majority of coronary artery disease is occlusion of a coronary vessel. Under normal conditions, antithrombin III (AT III, protein C, and protein S as an active protein C cofactor, are natural anticoagulants (hemostatic control that balances procoagulant activity (thrombin antithrombin complex balance to prevent thrombosis. If the condition becomes unbalanced, natural anticoagulants and the procoagulants can lead to thrombosis. Thirty subjects with acute coronary syndrome (ACS were studied for the incidence of antithrombin III (AT III, protein C, and protein S deficiencies, and the result were compare to the control group. Among patients with ACS, the frequency of distribution of AT-III with activity < 75% were 23,3% (7 of 30, and only 6,7% ( 2 of 30 in control subject. No one of the 30 control subject have protein C activity deficient, in ACS with activity < 70% were 13,3% (4 of 30. Fifteen out of the 30 (50% control subjects had protein S activity deficiency, while protein S deficiency activity < 70% was found 73.3.% (22 out of 30. On linear regression, the deterministic coefficient of AT-III activity deficiency to the development ACS was 13,25 %, and the deterministic coefficient of protein C activity deficient to the development of ACS was 9,06 %. The cut-off point for AT-III without protein S deficiency expected to contribute to the development of vessel disease was 45%. On discriminant analysis, protein C activity deficiency posed a risk for ACS of 4,5 greater than non deficient subjects, and AT-III activity deficiency posed a risk for ACS of 3,5 times greater than non deficient subjects. On binary logistic regression, protein S activity acted only as a reinforcing factor of AT-III activity deficiency in the development of ACS. Protein C and AT III deficiency can trigger ACS, with determinant coefficients of 9,06% and 13,25% respectively. Low levels of protein C posed a greater risk of ACS than low levels of AT III. Protein S deficiency was a reinforcing factor on AT-III deficient to development of ACS. The cut-off point of AT-III without protein S deficiency expected to give single vessel disease was 45%, and 9,5% for the development of triple vessel disease. (Med J Indones 2002; 11: 87-92Keywords: acute coronary syndrome, Anti-thrombin III, Protein C, Protein S

Dasnan Ismail

2002-05-01

323

Recombinant Collagenlike Proteins  

Science.gov (United States)

A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

Fertala, Andzej

2007-01-01

324

Proteomic analysis of the porcine platelet proteome and alterations induced by thrombin activation.  

Science.gov (United States)

Platelets are enucleated cells derived from bone marrow megakaryocytes and defects in platelet functions could be involved in many cardiovascular diseases. Proteomics can be used to provide a new insight in the study of these platelet functions and can help to identify the biochemical events underlying platelet activation. In this study, we have obtained a reference 2-DE map of porcine platelet proteins. A large number of cytoskeletal and metabolic proteins were found as well as some proteins related to cell mobility and immunological functions. Other proteins implicated in the cell signalling process, transport or apoptosis were also identified. Moreover, we have analysed, by 2D-DIGE methodology, quantitative modifications of platelet proteins following their activation by thrombin. 26 spots exhibited statistically significant differences, and a total of 16 spots corresponding to 13 different proteins were successfully identified. Using Ingenuity Pathway Analysis, the association of the deregulated proteins with canonical pathways highlighted two major pathways; coagulation system and integrin signalling. These results confirm that this proteomic approach (based on 2D-DIGE, mass spectrometry and bioinformatic and pathway databases) has proved to be a powerful tool when applied to studying signalling pathways that could play a relevant role in the activation of platelets. PMID:18824148

Esteso, Gloria; Mora, María Isabel; Garrido, Juan José; Corrales, Fernando; Moreno, Angela

2008-12-01

325

Thrombin and plasmin-like activities in the latices of Cryptostegia grandiflora and Plumeria rubra.  

Science.gov (United States)

Latex proteins have drawn attention because they have shown several pharmacological activities. Herein, the fibrin(ogen)olytic activity of Cryptostegia grandiflora (CgLP) and Plumeria rubra (PrLP) latices were evaluated and characterized. Ion-exchange chromatography separated CgLP in proteolytic (CgLP PI) and nonproteolytic proteins (CgLP PII). CgLP and CgLP PI hydrolyzed azocasein in a dose-dependent manner, whereas CgLP PII and PrLP showed negligible activities. CgLP and CgLP PI accelerated plasmatic clot formation and digested all fibrinogen chains in a time/dose-dependent manner, though in a nonspecific way. CgLP and CgLP PI did not fully hydrolyze the subunits of the fibrin clot since fibrin ?-chain showed resistance to proteolysis. No fibrinogenolytic activity was noticed after incubation of CgLP and CgLP PI with E-64. These results suggested that fibrinogenolytic and procoagulant activities of C. grandiflora were performed by cysteine proteases and confirm the activity of latex cysteine proteases as thrombin and plasmin-like proteins. PMID:23314383

Viana, Carolina A; Oliveira, Jefferson S; Freitas, Cleverson D T; Alencar, Nylane M N; Carvalho, Cristina P S; Nishi, Beatriz C; Ramos, Márcio V

2013-06-01

326

Sub-nanomolar detection of thrombin activity on a microfluidic chip.  

Science.gov (United States)

Bioluminescence resonance energy transfer (BRET) is a form of Förster resonance energy transfer. BRET has been shown to support lower limits of detection than fluorescence resonance energy transfer (FRET) but, unlike FRET, has not been widely implemented on microfluidic devices for bioanalytical sensing. We recently reported a microscope-based microfluidic system for BRET-based biosensing, using a hybrid, high quantum-efficiency, form of BRET chemistry. This paper reports the first optical fiber-based system for BRET detection on a microfluidic chip, capable of quantifying photon emissions from the low quantum-efficiency BRET(2) system. We investigated the effects of varying core diameter and numerical aperture of optical fibers, as well as varying microfluidic channel design and measurement conditions. We optimized the set-up in order to maximize photon counts and minimize the response time. The optimized conditions supported measurement of thrombin activity, with a limit of detection of 20 pM, which is lower than the microscope-based system and more than 20 times lower than concentrations reported to occur in plasma clots. PMID:25553187

Le, Nam Cao Hoai; Gel, Murat; Zhu, Yonggang; Wang, Jian; Dacres, Helen; Anderson, Alisha; Trowell, Stephen C

2014-11-01

327

Thrombin stimulates the production of a novel polyphosphoinositide in human platelets  

Energy Technology Data Exchange (ETDEWEB)

The sequential actions of phosphoinositide 4-kinase and 5-kinase and hydrolysis of phosphatidylinositol (PtdIns) 4,5-P2 are stimulated during platelet activation. Recently, a phosphoinositide 3-kinase has been implicated in signal transduction in several cell types. Stimulation of PtdIns(3,4)P2 synthesis has been shown in polyoma middle T-transformed and platelet-derived growth factor-stimulated cells, and this novel lipid has been implicated in signal transduction and regulation of cell proliferation. We demonstrate the formation of PtdIns(3,4)P2 in human platelets and show that the synthesis of this lipid (and of PtdIns(4,5)P2) is stimulated during activation of platelets by thrombin. This indicates the presence of phosphoinositide 3-kinase activity in platelets. We postulate that PtdIns(3,4)P2 is involved in signal transduction in platelets and discuss the possibility that this novel lipid is a substrate for phospholipase C.

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

1990-02-15

328

Thrombin stimulates the production of a novel polyphosphoinositide in human platelets  

International Nuclear Information System (INIS)

The sequential actions of phosphoinositide 4-kinase and 5-kinase and hydrolysis of phosphatidylinositol (PtdIns) 4,5-P2 are stimulated during platelet activation. Recently, a phosphoinositide 3-kinase has been implicated in signal transduction in several cell types. Stimulation of PtdIns(3,4)P2 synthesis has been shown in polyoma middle T-transformed and platelet-derived growth factor-stimulated cells, and this novel lipid has been implicated in signal transduction and regulation of cell proliferation. We demonstrate the formation of PtdIns(3,4)P2 in human platelets and show that the synthesis of this lipid (and of PtdIns(4,5)P2) is stimulated during activation of platelets by thrombin. This indicates the presence of phosphoinositide 3-kinase activity in platelets. We postulate that PtdIns(3,4)P2 is involved in signal transduction in platelets and discuss the possibility that this novel lipid is a substrate for phospholipase C

329

Selective deacylation of 1-acyl-2-arachidonoyl PC and PE in thrombin-stimulated human platelets  

International Nuclear Information System (INIS)

Previously the authors have shown that uptake and stimulated release of 3H-arachidonate (AA) in human platelets involves mainly 1-acyl-2-arachidonoyl phospholipids. To determine deacylation of molecular species of 1-acyl-2-arachidonoyl phosphatidylcholine (PC) and phosphatidylethanolamine (PE), phospholipid was extracted by the method of Bligh and Dyer from cells (109/ml) stimulated or not by thrombin (THR, 5 U/ml .370C, 5 min). Total PC and PE were isolated by thin-layer chromatography (TLC) and converted to 1-radyl-2-acyl glycerobenzoates. The 1,2 diacyl glycerobenzoates were separated from other subclasses by TLC. Individual molecular species of 1,2 diacyl glycerobenzoates were resolved by reverse phase HPLC. Mass (O.D.230) and 3H-AA radioactivity (i.e. specific activity) were determined on-line, and changes in individual molecular species could be deduced. Significant deacylation of all 1-acyl-2-arachidonoyl PC and PE molecular species occurred with no apparent deacylation in any non-AA-containing molecular species of 1,2 diacyl PC/PE. At 5 minutes the net deacylation of 1-acyl-2-arachidonoyl PC and PE was approximately 15 and 5 nanomoles, respectively/109 cells. These results indicate that selective deacylation of arachidonoyl-containing molecular species compared to non-arachidonoyl-containing molecular species of PC/PE occurs in THR-stimulated cells. This suggests certain AA-containing phospholipids are compartmentalized with and susceptible to, the action of phospholipase A2

330

Thrombin antithrombin complex and IL-18 serum levels in stroke patients  

Directory of Open Access Journals (Sweden)

Full Text Available The complex picture of inflammation and coagulation alterations comes to life in acute stroke phases. Increasing evidence points to a strong interaction and extensive crosstalk between the inflammation and coagulation systems: the interest towards this relationship has increased since recent experimental research showed that the early administration of antithrombin III (ATIII decreases the volume of ischemia in mice and might be neuroprotective, playing an antiinflammatory role. We aimed to establish the extent of the relationship among markers of inflammation (S100B and IL-18 and procoagulant and fibrinolytic markers (ATIII, thrombin-antithrombin III complex (TAT, Fibrin Degradation Products (FDP, D-dimer in 13 comatose patients affected by focal cerebral ischemia. Plasma levels of TAT, D-dimer and FDP, IL18 and S100B were increased. IL-18 and S100B high serum levels in ischemic patients suggest an early activation of the inflammatory cascade in acute ischemic injury. The basic principles of the interaction between inflammatory and coagulation systems are revised, from the perspective that simultaneous modulation of both coagulation and inflammation, rather than specific therapies aimed at one of these systems could be more successful in stroke therapy.

Rosalba Tufano

2010-06-01

331

Effect of thrombin fragment (TP508) on myocardial ischemia-reperfusion injury in hypercholesterolemic pigs.  

Science.gov (United States)

Myocardial ischemia-reperfusion (IR) injury occurs frequently in the setting of hypercholesterolemia. We investigated the potential efficacy of a novel thrombin fragment (TP508) on IR injury in a hypercholesterolemic porcine model. Twenty-one hypercholesterolemic male Yucatan pigs underwent 60 min of mid-left anterior descending coronary artery occlusion followed by 120 min of reperfusion. Pigs received either placebo (control, n = 7) or TP508 in two doses (TP508 low dose, n = 7, as bolus of 0.5 mg/kg 50 min into ischemia and an infusion of 1.25 mg.kg(-1).h(-1) during reperfusion period or TP508 high dose, n = 7, a double dose of TP508 low-dose group). Myocardial function was monitored throughout the experiment. The area at risk and myocardial necrosis were determined by Monastryl blue/triphenyl tetrazolium chloride staining. Apoptosis in the ischemic territory was assessed. Coronary microvascular reactivity to endothelium-dependent and -independent factors was measured. Myocardial necrosis was lower in both TP508-treated groups vs. control (P pigs, TP508 decreases myocardial necrosis and apoptosis after IR. Thus TP508 may offer a novel approach in protecting the myocardium from IR injury. PMID:19372304

Osipov, Robert M; Robich, Michael P; Feng, Jun; Clements, Richard T; Liu, Yuhong; Glazer, Hilary P; Wagstaff, John; Bianchi, Cesario; Sellke, Frank W

2009-06-01

332

Peptide affinity labels for thrombin and other trypsin-like proteases  

Science.gov (United States)

A peptide affinity label of the formula (I): ##STR1## wherein X is a radical capable of acting as a leaving group in a nucleophilic substitution reaction; A is an aromatic amino acid residue; B is H, or a C.sub.1 -C.sub.4 alkyl group, or aryl; Y is selected from the group consisting of hydrogen, aroyl, C.sub.1 -C.sub.6 acyl, and Q--(A)--.sub.n, wherein Q=hydrogen, aroyl, or C.sub.1 -C.sub.6 acyl, n=1-10, A is an amino acid residue selected from the aliphatic, hydroxy-containing, carboxylic acid group, and amide-thereof-containing, aromatic, sulfur-containing and imino-containing amino acids; and wherein J is selected from the group consisting of --CH.sub.2 --, --CH.sub.2 --CH.sub.2 --,--CH.sub.2 --CH.sub.2 --CH.sub.2 --, --CH.dbd.CH-- and --CH(OH)--CH.sub.2. The affinity label is useful for irreversibly inactivating thrombin and trypsin-like enzymes and may be used as a potential anticlotting agent.

Shaw, Elliott N. (Shoreham, NY); Kettner, Charles A. (Yaphank, NY)

1982-03-09

333

Vorapaxar expands antiplatelet options. Which patients may benefit from thrombin receptor antagonism?  

Science.gov (United States)

Vorapaxar is the first substance of a new class of antiplatelet drugs that has been tested in large clinical trials. The protease-activated receptor 1 (PAR-1) antagonist inhibits thrombin-induced platelet activation to prevent atherothrombosis. In the phase 3 trials TRACER (acute coronary syndrome) and TRA 2P-TIMI 50 (stable atherosclerosis) reducing ischemic events with vorapaxar came at the cost of bleeding. TRACER compared vorapaxar to placebo in 12,944 patients who had non-ST-segment elevation acute coronary syndromes on top of contemporary treatment including dual antiplatelet therapy (aspirin and clopidogrel). Vorapaxar reduced ischemic events non-significantly, but increased bleeding significantly, therefore not justifying triple antiplatelet therapy in this setting. Follow-up was stopped early because of bleeding. TRA 2P-TIMI 50 examined 26,449 patients who had a history of myocardial infarction, ischemic stroke, or peripheral arterial disease. Vorapaxar reduced ischemic events and increased bleeding both significantly. Recruitment of patients with prior stroke was stopped early. Net clinical outcome and subgroup analyses suggested that vorapaxar could be beneficial for patients with prior myocardial infarction - but no history of stroke. PMID:22777302

Duerschmied, D; Bode, C

2012-01-01

334

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

Science.gov (United States)

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 ?M) 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. PMID:21306887

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

2011-03-15

335

An electrochemical aptasensor for thrombin detection based on the recycling of exonuclease III and double-stranded DNA-templated copper nanoparticles assisted signal amplification.  

Science.gov (United States)

In this paper, we report an improved electrochemical aptasensor based on exonuclease III and double-stranded DNA (dsDNA)-templated copper nanoparticles (CuNPs) assisted signal amplification. In this sensor, duplex DNA from the hybridization of ligated thrombin-binding aptamer (TBA) subunits and probe DNA can act as an effective template for the formation of CuNPs on the electrode surface, so copper ions released from acid-dissolution of CuNPs may catalyze the oxidation of ?-phenylenediamine to produce an amplified electrochemical response. In the presence of thrombin, a short duplex domain with four complementary base pairs can be stabilized by the binding of TBA subunits with thrombin, in which TBA subunit 2 can be partially digested from 3' terminal with the cycle of exonuclease III, so the ligation of TBA subunits and the subsequent formation of CuNPs can be inhibited. By electrochemical characterization of dsDNA-templated CuNPs on the electrode surface, our aptasensor can display excellent performances for the detection of thrombin in a broad linear range from 100fM to 1nM with a low detection limit of 20.3fM, which can also specially distinguish thrombin in both PBS and serum samples. Therefore, our aptasensor might have great potential for clinical diagnosis of biomarkers in the future. PMID:25682243

Zhao, Jing; Xin, Meiling; Cao, Ya; Yin, Yongmei; Shu, Yongqian; Ma, Wenli

2015-02-20

336

Thermodynamic and biological evaluation of a thrombin binding aptamer modified with several unlocked nucleic acid (UNA) monomers and a 2'-C-piperazino-UNA monomer  

DEFF Research Database (Denmark)

Thrombin binding aptamer is a DNA 15-mer which forms a G-quadruplex structure and possess promising anticoagulant properties due to specific interactions with thrombin. Herein we present the influence of a single 2'-C-piperazino-UNA residue and UNA residues incorporated in several positions on thermodynamics, kinetics and biological properties of the aptamer. 2'-C-Piperazino-UNA is characterized by more efficient stabilization of quadruplex structure in comparison to regular UNA and increases thermodynamic stability of TBA by 0.28-0.44kcal/mol in a position depending manner with retained quadruplex topology and molecularity. The presence of UNA-U in positions U3, U7, and U12 results in the highest stabilization of G-quadruplex structure (??G(37)(°)=-1.03kcal/mol). On the contrary, the largest destabilization mounting to 1.79kcal/mol was observed when UNA residues were placed in positions U7, G8, and U9. Kinetic studies indicate no strict correlation between thermodynamic stability of modified variants and their binding affinity to thrombin. Most of the studied variants bind thrombin, albeit with decreased affinity in reference to unmodified TBA. Thrombin time assay studies indicate three variants as being as potent as TBA in fibrin clotting inhibition.

Jensen, Troels B; Henriksen, Jonas R

2011-01-01

337

A 2D-DIGE-based proteomic analysis reveals differences in the platelet releasate composition when comparing thrombin and collagen stimulations.  

Science.gov (United States)

Upon stimulation, platelets release a high number of proteins (the releasate). There are clear indications that these proteins are involved in the pathogenesis of several diseases, such as atherosclerosis. In the present study we compared the platelet releasate following platelet activation with two major endogenous agonists: thrombin and collagen. Proteome analysis was based on 2D-DIGE and LC-MS/MS. Firstly, we showed the primary role of thrombin and collagen receptors in platelet secretion by these agonists; moreover, we demonstrated that GPVI is the primary responsible for collagen-induced platelet activation/aggregation. Proteomic analysis allowed the detection of 122 protein spots differentially regulated between both conditions. After excluding fibrinogen spots, down-regulated in the releasate of thrombin-activated platelets, 84 differences remained. From those, we successfully identified 42, corresponding to 37 open-reading frames. Many of the differences identified correspond to post-translational modifications, primarily, proteolysis induced by thrombin. Among others, we show vitamin K-dependent protein S, an anticoagulant plasma protein, is up-regulated in thrombin samples. Our results could have pathological implications given that platelets might be playing a differential role in various diseases and biological processes through the secretion of different subsets of granule proteins and microvesicles following a predominant activation of certain receptors. PMID:25645904

Vélez, Paula; Izquierdo, Irene; Rosa, Isaac; García, Ángel

2015-01-01

338

Antithrombotic properties of SSR182289A, a new, orally active thrombin inhibitor.  

Science.gov (United States)

N-[3-[[[(1S)-4-(5-Amino-2-pyridinyl)-1-[[4-difluoromethylene)-1-piperidinyl]carbonyl]butyl]amino]sulfonyl][1,1'-biphenyl]-2-yl]acetamide hydrochloride (SSR182289A) is a novel, potent, and selective thrombin inhibitor. We have examined the antithrombotic properties of SSR182289A administered by i.v. and p.o. routes in several different animal thrombosis models in comparison with reference antithrombotic agents. Oral administration of SSR182289A produced dose-related antithrombotic effects in the following models; rat venous thrombosis (ED(50) 0.9 mg/kg p.o.), rat silk thread arterio-venous (AV) shunt (ED(50) 3.8 mg/kg p.o.), rat thromboplastin-induced AV shunt (ED(50) 3.1 mg/kg p.o.), rat carotid artery thrombosis (ED(200) 5.9 mg/kg p.o.), and rabbit venous thrombosis (ED(50) 7.5 mg/kg p.o.). Administered as an i.v. bolus, SSR182289A showed antithrombotic activity in the above models with ED(50)/ED(200) values in the range of 0.2 to 1.9 mg/kg i.v. SSR182289A increased rat tail transection bleeding time at doses > or =10 mg/kg p.o. In the rat thromboplastin-induced AV shunt model, SSR182289A 10 mg/kg p.o. produced marked antithrombotic effects at 30, 60, 120, and 240 min after administration. Hence, SSR182289A demonstrates potent oral antithrombotic properties in animal venous, AV-shunt, and arterial thrombosis models. PMID:12538808

Lorrain, J; Millet, L; Lechaire, I; Lochot, S; Ferrari, P; Visconte, C; Sainte-Marie, M; Lunven, C; Berry, C N; Schaeffer, P; Herbert, J-M; O'Connor, S E

2003-02-01

339

Thrombin-dependent Incorporation of von Willebrand Factor into a Fibrin Network.  

Science.gov (United States)

Attachment of platelets from the circulation onto a growing thrombus is a process involving multiple platelet receptors, endothelial matrix components, and coagulation factors. It has been indicated previously that during a transglutaminase reaction activated factor XIII (FXIIIa) covalently cross-links von Willebrand factor (VWF) to polymerizing fibrin. Bound VWF further recruits and activates platelets via interactions with the platelet receptor complex glycoprotein Ib (GPIb). In the present study we found proof for binding of VWF to a fibrin monomer layer during the process of fibrinogen-to-fibrin conversion in the presence of thrombin, arvin, or a snake venom from Crotalus atrox. Using a domain deletion mutant we demonstrated the involvement of the C domains of VWF in this binding. Substantial binding of VWF to fibrin monomers persisted in the presence of the FXIIIa inhibitor K9-DON, illustrating that cross-linking via factor XIII is not essential for this phenomenon and suggesting the identification of a second mechanism through which VWF multimers incorporate into a fibrin network. Under high shear conditions, platelets were shown to adhere to fibrin only if VWF had been incorporated. In conclusion, our experiments show that the C domains of VWF and the E domain of fibrin monomers are involved in the incorporation of VWF during the polymerization of fibrin and that this incorporation fosters binding and activation of platelets. Fibrin thus is not an inert end product but partakes in further thrombus growth. Our findings help to elucidate the mechanism of thrombus growth and platelet adhesion under conditions of arterial shear rate. PMID:25381443

Miszta, Adam; Pelkmans, Leonie; Lindhout, Theo; Krishnamoorthy, Ganeshram; de Groot, Philip G; Hemker, Coenraad H; Heemskerk, Johan W M; Kelchtermans, Hilde; de Laat, Bas

2014-12-26

340

Selective deacylation of 1-acyl-2-arachidonoyl PC and PE in thrombin-stimulated human platelets  

Energy Technology Data Exchange (ETDEWEB)

Previously the authors have shown that uptake and stimulated release of /sup 3/H-arachidonate (AA) in human platelets involves mainly 1-acyl-2-arachidonoyl phospholipids. To determine deacylation of molecular species of 1-acyl-2-arachidonoyl phosphatidylcholine (PC) and phosphatidylethanolamine (PE), phospholipid was extracted by the method of Bligh and Dyer from cells (10/sup 9//ml) stimulated or not by thrombin (THR, 5 U/ml .37/sup 0/C, 5 min). Total PC and PE were isolated by thin-layer chromatography (TLC) and converted to 1-radyl-2-acyl glycerobenzoates. The 1,2 diacyl glycerobenzoates were separated from other subclasses by TLC. Individual molecular species of 1,2 diacyl glycerobenzoates were resolved by reverse phase HPLC. Mass (O.D./sub 230/) and /sup 3/H-AA radioactivity (i.e. specific activity) were determined on-line, and changes in individual molecular species could be deduced. Significant deacylation of all 1-acyl-2-arachidonoyl PC and PE molecular species occurred with no apparent deacylation in any non-AA-containing molecular species of 1,2 diacyl PC/PE. At 5 minutes the net deacylation of 1-acyl-2-arachidonoyl PC and PE was approximately 15 and 5 nanomoles, respectively/10/sup 9/ cells. These results indicate that selective deacylation of arachidonoyl-containing molecular species compared to non-arachidonoyl-containing molecular species of PC/PE occurs in THR-stimulated cells. This suggests certain AA-containing phospholipids are compartmentalized with and susceptible to, the action of phospholipase A/sub 2/.

Purdon, A.D.; Patelunas, D.; Smith, J.B.

1986-03-01

341

Novel magnetic fibrin hydrogel scaffolds containing thrombin and growth factors conjugated iron oxide nanoparticles for tissue engineering  

Directory of Open Access Journals (Sweden)

Full Text Available Ofra Ziv-Polat1, Hadas Skaat1, Abraham Shahar2, Shlomo Margel11Department of Chemistry, Bar-Ilan Institute of Nanotechnology and Advanced Materials, Ramat-Gan 52900, Israel; 2NVR Research Ltd, Nes-Ziona 74031, IsraelAbstract: Novel tissue-engineered magnetic fibrin hydrogel scaffolds were prepared by the interaction of thrombin-conjugated iron oxide magnetic nanoparticles with fibrinogen. In addition, stabilization of basal fibroblast growth factor (bFGF was achieved by the covalent and physical conjugation of the growth factor to the magnetic nanoparticles. Adult nasal olfactory mucosa (NOM cells were seeded in the transparent fibrin scaffolds in the absence or presence of the free or conjugated bFGF-iron oxide nanoparticles. The conjugated bFGF enhanced significantly the growth and differentiation of the NOM cells in the fibrin scaffolds, compared to the same or even five times higher concentration of the free bFGF. In the presence of the bFGF-conjugated magnetic nanoparticles, the cultured NOM cells proliferated and formed a three-dimensional interconnected network composed mainly of tapered bipolar cells. The magnetic properties of these matrices are due to the integration of the thrombin- and bFGF-conjugated magnetic nanoparticles within the scaffolds. The magnetic properties of these scaffolds may be used in future work for various applications, such as magnetic resonance visualization of the scaffolds after implantation and reloading the scaffolds via magnetic forces with bioactive agents, eg, growth factors bound to the iron oxide magnetic nanoparticles.Keywords: thrombin, fibroblast growth factor, fibrin scaffold, iron oxide nanoparticles, tissue engineering, magnetism, bioactive nanoparticle

Ziv-Polat O

2012-03-01

342

Anticoagulant profile of iopamidol and meglumine amidotrizoate and their lack of thrombin generation: an in vitro study  

International Nuclear Information System (INIS)

The aim of this in vitro study was to sketch the subtle anticoagulant profile of iopamidol 300 mg l/ml (low osmolality non ionic contrast medium) and meglumine amidotrizoate 370 mg l/ml (high osmolality ionic contrast medium) in situations where variable amounts of clotting factors are observed and to check whether thrombin-generation significantly occurred in non anti-coagulated blood-contrast materials mixtures. In the first experiment, mixtures of deficient plasmas with a routine plasma pool provided different ranges with a variable amounts of clotting factor II, V, VIII, X, XI and XII. For each clotting factor level studied within these ranges, an activated partial thromboplastin time was determined with either contrast material loaded thromboplastin (5% v/v) used as a control. In the second experiment fibrino-peptide A (FpA) or modified anti-thrombin III (ATM) assays were performed in either (9:1) non anti-coagulated blood contrast materials mixtures or blood-glucose mixtures (control). Differing aPTT prolongation profiles were observed when clotting factors V, VIII, XI and XII were lowered in the plasma. However, neither iopamidol not amidotrizoate induced an aPTT prolongation with decreasing clotting factor II. In the second experiment no significant thrombin generation was observed as both blood - contrast materials mixtures showed significantly lower FpA and ATM levels (p < 0.001) than glucose control after 5 minutes and 10 minutes incubation at room temperatand 10 minutes incubation at room temperature. These findings provide evidence that the use of iopamidol in angiographic procedures does not increase risk of clotting or hemorrhage. (author)

343

Thrombin-binding affinities of different disulfide-bonded isomers of the fifth EGF-like domain of thrombomodulin.  

Science.gov (United States)

The fifth EGF-like domain of thrombomodulin (TM), both with and without the amino acids that connect the fifth domain to the sixth domain, has been synthesized and refolded to form several different disulfide-bonded isomers. The domain without the connecting region formed three disulfide-bonded isomers upon refolding under redox conditions. Of these three isomers, the (1-2,3-4,5-6) bonded isomer was the best inhibitor of fibrinogen clotting and also of the thrombin-TM interaction that results in protein C activation, but all the isomers were inhibitors in both assays. The isomer containing an EGF-like disulfide-bonding pattern (1-3,2-4,5-6) was not found among the oxidation products. The domain with the connecting region amino acids (DIDE) at the C-terminus formed two isolable products upon refolding in redox buffer. These products had the same two disulfide-bonding patterns as the earliest and latest eluting isomers of the domain without the DIDE. In order to compare the thrombin-binding affinities of these isomers to the isomer with the EGF-like disulfide bonds, acetamidomethyl protection of the second and fourth cysteines was used to force the disulfide bonds into the EGF-like pattern. Thrombin-binding affinity, measured as inhibition of fibrinogen clotting and as inhibition of protein C activation correlated inversely with the number of crossed disulfide bonds. As was found for the domain without the connecting region, the isomer that was the best inhibitor of fibrinogen clotting and of protein C activation was the isomer with no crossing disulfide bonds (1-2,3-4,5-6).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8535250

Hunter, M. J.; Komives, E. A.

1995-01-01

344

'Pergularain e I'--a plant cysteine protease with thrombin-like activity from Pergularia extensa latex.  

Science.gov (United States)

Pergularain e I, a cysteine protease with thrombin-like activity, was purified by ion exchange chromatography from the latex of Pergularia extensa. Its homogeneity was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), native PAGE and reverse-phase high-performance liquid chromatography (RP-HPLC). The molecular mass of pergularain e I by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) was found to be 23.356 kDa and the N-terminal sequence is L-P-H-D-V-E. Pergularain e I is a glycoprotein containing approximately 20% of carbohydrate. Pergularain e I constituted 6.7% of the total protein with a specific activity of 9.5 units/mg/min with a 2.11-fold increased purity. Proteolytic activity of the pergularain e I was completely inhibited by iodoacetic acid (IAA). Pergularain e I exhibited procoagulant activity with citrated plasma and fibrinogen similar to thrombin. Pergularain e I increases the absorbance of fibrinogen solution in concentration-dependent and time-dependent manner. At 10 microg concentration, an absorbance of 0.48 was reached within 10 min of incubation time. Similar absorbance was observed when 0.2 NIH units of thrombin were used. Thrombin-like activity of pergularain e I is because of the selective hydrolysis of A alpha and B beta chains of fibrinogen and gamma-chain was observed to be insusceptible to hydrolysis. Molecular masses of the two peptide fragments released from fibrinogen due to the hydrolysis by pergularain e I at 5-min incubation time were found to be 1537.21 and 1553.29 and were in close agreement with the molecular masses of 16 amino acid sequence of fibrinopeptide A and 14 amino acid sequence of fibrinopeptide B, respectively. Prolonged fibrinogen-pergularain e I incubation releases additional peptides and their sequence comparison of molecular masses of the released peptides suggested that pergularain e I hydrolyzes specifically after arginine residues. PMID:19853890

Shivaprasad, Holenarasipura V; Rajaiah, Rajesh; Frey, Brigitte M; Frey, Felix J; Vishwanath, Bannikuppe S

2010-03-01

345

The heparin-binding exosite of factor IXa is a critical regulator of plasma thrombin generation and venous thrombosis  

OpenAIRE

The role of the factor IXa heparin-binding exosite in coagulation was assessed with mutations that enhance (R170A) or reduce (R233A) stability of the protease-factor VIIIa A2 domain interaction. After tissue factor (TF) addition to reconstituted factor IX-deficient plasma, factor IX R170A supported a 2-fold increase in velocity index (slope) and peak thrombin concentration, whereas factor IX R233A had a 4- to 10-fold reduction relative to factor IX wild-type. In the absence of TF, 5 to 100 pM...

Buyue, Yang; Whinna, Herbert C.; Sheehan, John P.

2008-01-01

346

The interaction between fibrinogen and 3H-arginine-labelled proteins derived from fibrosarcoma in the presence of thrombin  

International Nuclear Information System (INIS)

With the use of 3H-arginine-labelled cationic proteins derived from fibrosarcoma induced by methylcholanthrene it has been shown in studies in vitro that these proteins interact with fibrinogen under the influence of thrombin. The effect of this reaction depends on the concentration of cationic proteins. It was calculated that 1 mg of fibrinogen can be interacted with 2.5 ?g of 3H-arginine-labelled cationic proteins. The clinical role of cationic proteins appearing in circulation in malignancy have been discussed briefly. (author)

347

Differential effects of formoterol on thrombin- and PDGF-induced proliferation of human pulmonary arterial vascular smooth muscle cells  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Increased pulmonary arterial vascular smooth muscle (PAVSM cell proliferation is a key pathophysiological component of pulmonary vascular remodeling in pulmonary arterial hypertension (PH. The long-acting ?2-adrenergic receptor (?2AR agonist formoterol, a racemate comprised of (R,R- and (S,S-enantiomers, is commonly used as a vasodilator in chronic obstructive pulmonary disease (COPD. PH, a common complication of COPD, increases patients’ morbidity and reduces survival. Recent studies demonstrate that formoterol has anti-proliferative effects on airway smooth muscle cells and bronchial fibroblasts. The effects of formoterol and its enantiomers on PAVSM cell proliferation are not determined. The goals of this study were to examine effects of racemic formoterol and its enantiomers on PAVSM cell proliferation as it relates to COPD-associated PH. Methods Basal, thrombin-, PDGF- and chronic hypoxia-induced proliferation of primary human PAVSM cells was examined by DNA synthesis analysis using BrdU incorporation assay. ERK1/2, mTORC1 and mTORC2 activation were determined by phosphorylation levels of ERK1/2, ribosomal protein S6 and S473-Akt using immunoblot analysis. Results We found that (R,R and racemic formoterol inhibited basal, thrombin- and chronic hypoxia-induced proliferation of human PAVSM cells while (S,S formoterol had lesser inhibitory effect. The ?2AR blocker propranolol abrogated the growth inhibitory effect of formoterol. (R,R, but not (S,S formoterol attenuated basal, thrombin- and chronic hypoxia-induced ERK1/2 phosphorylation, but had little effect on Akt and S6 phosphorylation levels. Formoterol and its enantiomers did not significantly affect PDGF-induced DNA synthesis and PDGF-dependent ERK1/2, S473-Akt and S6 phosphorylation in human PAVSM cells. Conclusions Formoterol inhibits basal, thrombin-, and chronic hypoxia-, but not PDGF-induced human PAVSM cell proliferation and ERK1/2, but has little effect on mTORC1 and mTORC2 signaling. Anti-proliferative effects of formoterol depend predominantly on its (R,R enantiomer and require the binding with ?2AR. These data suggest that (R,R formoterol may be considered as potential adjuvant therapy to inhibit PAVSM cell proliferation in COPD-associated PH.

Goncharova Elena A

2012-11-01

348

The crystal structure of human alpha-thrombin complexed with LY178550, a nonpeptidyl, active site-directed inhibitor.  

OpenAIRE

The crystal structure of human alpha-thrombin in complex with LY178550, a nonpeptidyl, active site-directed inhibitor, has been solved to 2.07 A resolution by the method of X-ray crystallography. The final model of the complex has a crystallographic R-value of 21.5% (Rfree = 23.1%) with 0.014 A and 2.4 degrees standard deviation from ideal bond lengths and angles, respectively. Well-defined electron density was observed for the inhibitor in the active site. The inhibitor binds to the active s...

Chirgadze, N. Y.; Sall, D. J.; Klimkowski, V. J.; Clawson, D. K.; Briggs, S. L.; Hermann, R.; Smith, G. F.; Gifford-moore, D. S.; Wery, J. P.

1997-01-01

349

Topic: A Literature Review  

Directory of Open Access Journals (Sweden)

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

Dandan Zhang

2009-08-01

350

Recombining WMAP: constraints on ionizing and resonance radiation at recombination  

OpenAIRE

We place new constraints on sources of ionizing and resonance radiation at the epoch of the recombination process using the recent CMB temperature and polarization spectra coming from WMAP. We find that non-standard recombination scenarios are still consistent with the current data. In light of this we study the impact that such models can have on the determination of several cosmological parameters. In particular, the constraints on curvature and baryon density appear to be...

Bean, Rachel; Melchiorri, Alessandro; Silk, Joe

2003-01-01

351

Optimization of the purification methods for recovery of recombinant growth hormone from Paralichthys olivaceus  

Science.gov (United States)

This study aimed to optimize the purification of recombinant growth hormone from Paralichthys olivaceus. Recombinant flounder growth hormone (r-fGH) was expressed by Escherichia coli in form of inclusion body or as soluble protein under different inducing conditions. The inclusion body was renatured using two recovery methods, i.e., dilution and dialysis. Thereafter, the refolded protein was purified by Glutathione Sepharase 4B affinity chromatography and r-fGH was obtained by cleavage of thrombin. For soluble products, r-fGH was directly purified from the lysates by Glutathione Sepharase 4B affinity chromatography. ELISA-receptor assay demonstrated that despite its low receptor binding activity, the r-fGH purified from refolded inclusion body had a higher yield (2.605 mg L-1) than that from soluble protein (1.964 mg L-1). Of the tested recovery methods, addition of renaturing buffer (pH 8.5) into denatured inclusion body yielded the best recovery rate (17.9%). This work provided an optimized purification method for high recovery of r-fGH, thus contributing to the application of r-fGH to aquaculture.

Zang, Xiaonan; Zhang, Xuecheng; Mu, Xiaosheng; Liu, Bin

2013-03-01

352

Cloning and expression of a new recombinant thrombolytic and anthithrombotic agent - a staphylokinase variant.  

Science.gov (United States)

To develop a more potent antithrombin agent with thrombolytic and antiplatelet properties, a new staphylokinase (SAK) variant was constructed. The kringle 2 domain (K2) of tissue type-plasminogen activator (t-PA) containing a fibrin-specific binding site (i), the RGD sequence (Arg-Gly-Asp) for the prevention of platelet aggregation (ii) and the antithrombotic agent - hirulog (iii) was assembled to the C-terminal part of recombinant staphylokinase (r-SAK). cDNA for the hybrid protein SAK-RGD-K2-Hirul was cloned into Pichia pastoris pPIC9K yeast expression vector. The introduction of K2 t-PA, the RGD sequence and hirulog into the C-terminus of r-SAK did not alter the staphylokinase activity. We observed a higher clot lysis potency of SAK-RGD-K2-Hirul as evidenced by a faster and more profound lysis of (125)I-labeled human fibrin clots. The potency of thrombin inhibition by the hirulog C-terminal part of the recombinant fusion protein was almost identical to that of r-Hir alone. These results suggest that the SAK-RGD-K2-Hirul construct can be a more potent and faster-acting thrombolytic agent with better antithrombin and antiplatelet properties compared to r-SAK and SAK-RGD-K2-Hir. PMID:19018330

Kowalski, Micha?; Brown, George; Bieniasz, Magdalena; Oszajca, Katarzyna; Chabielska, Ewa; Pietras, Tadeusz; Szemraj, Zofia; Makandjou-Ola, Eusebio; Bartkowiak, Jacek; Szemraj, Janusz

2009-01-01

353

High-resolution NMR studies of fibrinogen-like peptides in solution: Interaction of thrombin with residues 1-23 of the A? chain of human fibrinogen  

International Nuclear Information System (INIS)

The interaction of the following human fibrinogen-like peptides with bovine thrombin was studied by use of one- and two-dimensional NMR techniques in aqueous solution: Ala(1)-Asp-Ser-Gly-Glu-Gly-Asp-Phe(8)-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg(16)-Gly(17)-Pro-Arg(19)-Val(20)-Val-Glu-Arg (F10), residues 1-16 of F10 (fibrinopeptide A), residues 17-23 of F10 (F12), residues 1-20 of F10 (F13), residues 6-20 of F10 with Arg(16) replaces by a Gly residue (F14), and residues 6-19 of F10 with Arg(16) replaced by a Leu residue (F15). At pH 5.3 and 25 degree C, the Arg(16)-Gly(17) peptide bonds of both peptides F10 and F13 were cleaved instantaneously in the presence of 0.6 mM thrombin, whereas the cleavage of the Arg(19)-Val(20) peptide bonds in peptides F12, F13, and F14 took over 1 h for completion. On the basis of observations of line broadening, fibrinopeptide A was found to bind to thrombin. While resonances from residues Ala(1)-Glu(5) were little affected, binding of fibrinopeptide A to thrombin caused significant line broadening of NH and side-chain proton resonances within residues Asp(7)-Arg(16). Peptides with Arg(16) replaced by Gly and Leu, respectively, i.e.; F14 and F15, were also found to bind to thrombin but with a different conformation, as indicated by the absence of the long-range NOEs observed with fibrinopeptide A. Residues Asp(7)-Arg(16) constitute an essential structural element in the interaction of thrombin with fibrinogennogen

354

Plasma membrane associated phospholipase C from human platelets: Synergistic stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis by thrombin and guanosine 5'-O-(3-thiotriphosphate)  

International Nuclear Information System (INIS)

The effects of thrombin and GTP?S on the hydrolysis of phosphoinositides by membrane-associated phospholipase C (PLC) from human platelets were examined with endogenous [3H]inositol-labeled membranes or with lipid vesicles containing either [3H]phosphatidylinositol or [3H]phosphatidylinositol 4,5-bisphosphate. GTP?S (1 ?M) or thrombin (1 unit/mL) did not stimulate release of inositol trisphosphate (IP3), inositol bisphosphate (IP2), or inositol phosphate (IP) from [3H]inositol-labeled membranes. IP2 and IP3, but not IP, from [3H]inositol-labeled membranes were, however, stimulated 3-fold by GTP?S (1 ?M) plus thrombin (1 unit/mL). A higher concentration of GTP?S (100 ?M) alone also stimulated IP2 and IP3, but not IP, release. In the presence of 1 mM calcium, release of IP2 and IP3 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 (PIP2) by platelet membrane associated PLC was also markedly enhanced by GTP?S (100 ?M) or GTP?S (1 ?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 hydrolysiium. Thrombin-induced hydrolysis of PIP2 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?S (100 ?M) or calcium (1 mM) dependent PIP2 breakdown, while TPA inhibited GTP?S-dependent but not calcium-dependent phospholipase C activity

355

Topical report review status  

International Nuclear Information System (INIS)

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

356

Topical report review status  

Energy Technology Data Exchange (ETDEWEB)

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

NONE

1997-08-01

357

Human endothelial cells in culture produce platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) when stimulated with thrombin.  

OpenAIRE

Cultured human endothelial cells produce platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) when stimulated with human thrombin. The response to thrombin is dose dependent, with a half-maximal effect at 0.17 unit/ml. The product is identified as PAF by the incorporation of radiolabeled precursors, its behavior in chromatographic systems, the recovery of biological activity, and the effect of treatment with phospholipase A2. Incorporation of [3H]acetate into PAF is ...

Prescott, S. M.; Zimmerman, G. A.; Mcintyre, T. M.

1984-01-01

358

P3-P3' Residues Flanking Scissile Bonds in Factor VIII Modulate Rates of Substrate Cleavage and Procofactor Activation by Thrombin  

OpenAIRE

Thrombin-catalyzed activation of factor VIII (FVIII) occurs through proteolysis at three P1 Arg residues: Arg372 and Arg740 in the FVIII heavy chain and Arg1689 in the FVIII light chain. Cleavage at the latter two sites is relatively fast compared with cleavage at Arg372, which appears rate limiting. Examination of the P3-P3? residues flanking each P1 site revealed that those sequences at Arg740 and Arg1689 are more optimal for thrombin cleavage than at Arg372, suggesting these sequences ma...

Newell-caito, Jennifer L.; Griffiths, Amy E.; Fay, Philip J.

2012-01-01

359

Hydrogen recombiner development at AECL  

International Nuclear Information System (INIS)

Catalytic recombiners have been developed at AECL for the purpose of hydrogen removal in post-accident nuclear containment buildings. The recombiners are based on a particular catalyst designed by AECL which has extraordinary resistance to fouling from water and water vapour and a large thermodynamic range of operation. The catalysts were developed, originally, for the purpose of heavy water manufacturing by way of a catalytic exchange process. Application of these catalyst materials in recombiners for containment applications began in the late 1980's. The first application was a passive recombiner, qualified for use in control of radiolytic hydrogen in the headspace of a pool-type experimental reactor of AECL design in 1988. The passive, or natural convection recombiner concept has continued development to commercial stage for application in power reactor containments. This paper reviews the AECL recombiner development, describes the current model and shows results from tests of full-scale recombiners in the Large Scale Vented Combustion Test Facility at AECL-WL. The AECL recombiner is designed for compactness and ease of engineering into containment. The design is a simple, open-ended rectangular enclosure with catalyst elements arranged inside to promote optimum convective flow driven by heat of recombination at the catalyst surface. Self start, as evidenced by catalyst heating and initiation of flow, is achieved in less than 1% hydrogen, with available oxygen, at room temperature and 100% relative humidity. This low temperature start-up in condensing atmospheres is viewed as the most challenging condition for wet-proofing effectiveness. Cold start-up is a vital performance requirement in containments, such as CANDU, where engineered air-cooling systems are operating and where long-term hydrogen control is required, after containment atmospheres have cooled. Once started, the removal capacity scales linearly with the inlet cross-section area and the partial pressure of hydrogen. The recombiner also reacts carbon monoxide, in the presence of hydrogen, at approximately the same rate as the hydrogen. The catalyst materials and wet-proofing are unaffected by radiation or high temperatures. Large scale tests confirm self-start behavior and demonstrate strong mixing, irrespective of recombiner placement. (author)

360

Progenitors of Recombining Supernova Remnants  

OpenAIRE

Usual supernova remnants have either ionizing plasma or plasma in collisional ionization equilibrium, i.e., the ionization temperature is lower than or equal to the electron temperature. However, the existence of recombining supernova remnants, i.e., supernova remnants with the ionization temperature higher than the electron temperature, is recently confirmed. One suggested way to have recombining plasma in a supernova remnant is to have a dense circumstellar medium at the t...

Moriya, Takashi J.

2012-01-01

361

Two-center dielectronic recombination  

CERN Document Server

In the presence of a neighboring atom, electron-ion recombination can proceed resonantly via excitation of an electron in the atom, with subsequent relaxation through radiative decay. It is shown that this two-center dielectronic process can largely dominate over single-center radiative recombination at internuclear distances as large as several nanometers. The relevance of the predicted process is demonstrated by using examples of water-dissolved alkali cations and warm dense matter.

Müller, C; López-Urrutia, J R Crespo; Harman, Z

2010-01-01

362

Biochemistry of eukaryotic homologous recombination  

OpenAIRE

The biochemistry of eukaryotic homologous recombination caught fire with the discovery that Rad51 is the eukaryotic homolog of the bacterial RecA and T4 UvsX proteins; and this field is still hot. The core reaction of homologous recombination, homology search and DNA strand invasion, along with the proteins catalyzing it, are conserved throughout evolution in principle. However, the increased complexity of eukaryotic genomes and the diversity of eukaryotic cell biology pose additional challen...

Heyer, Wolf-dietrich

2007-01-01

363

Intermolecular homologous recombination in plants.  

OpenAIRE

To study DNA topological requirements for homologous recombination in plants, we have constructed pairs of plasmids that contain nonoverlapping deletions in the neomycin phosphotransferase gene [APH(3')II], which, when intact, confers kanamycin resistance to plant cells. Protoplasts isolated from Nicotiana tabacum were cotransformed with complementary pairs of plasmids containing these truncated gene constructs. Homologous recombination or gene conversion within the homologous sequences (6 to...

Baur, M.; Potrykus, I.; Paszkowski, J.

1990-01-01

364

Do mitochondria recombine in humans?  

OpenAIRE

Until very recently, mitochondria were thought to be clonally inherited through the maternal line in most higher animals. However, three papers published in 2000 claimed population-genetic evidence of recombination in human mitochondrial DNA. Here I review the current state of the debate. I review the evidence for the two main pathways by which recombination might occur: through paternal leakage and via a mitochondrial DNA sequence in the nuclear genome. There is no strong evidence for either...

Eyre-walker, A.

2000-01-01

365

Thrombin hydrolysis of V29F and V34L mutants of factor XIII (28-41) reveals roles of the P(9) and P(4) positions in factor XIII activation.  

Science.gov (United States)

In blood coagulation, thrombin helps to activate factor XIII by cleaving the activation peptide at the R37-G38 peptide bond. The more easily activated factor XIII V34L has been correlated with protection from myocardial infarction. V34L and V29F factor XIII mutant peptides were designed to further characterize substrate binding to thrombin. HPLC kinetic studies have been carried out on thrombin hydrolysis of FXIII activation peptide (28-41), FXIII (28-41) V34L, FXIII (28-41) V29F, and FXIII (28-41) V29F V34L. The V34L mutations lead to improvements in both K(m) and k(cat) whereas the V29F mutation primarily affects K(m). Interactions of the peptides with thrombin have been monitored by 1D proton line broadening NMR and 2D transferred NOESY studies. The results were compared with previously published X-ray crystal structures of thrombin-bound fibrinogen Aalpha (7-16), thrombin receptor PAR1 (38-60), and factor XIII (28-37). In solution, the (34)VVPR(37) and (34)LVPR(37) segments of the factor XIII activation peptide serve as the major anchor points onto thrombin. The N-terminal segments are proposed to interact transiently with the enzyme surface. Long-range NOEs from FXIII V29 or F29 toward (34)V/LVPR(37) have not been observed by NMR studies. Overall, the kinetic and NMR results suggest that the factor XIII activation peptide binds to thrombin in a manner more similar to the thrombin receptor PAR1 than to fibrinogen Aalpha. The V29 and V34 positions affect, in different ways, the ability of thrombin to effectively hydrolyze the activation peptide. Mutations at these sites may prove useful in controlling factor XIII activation. PMID:11851434

Trumbo, Toni A; Maurer, Muriel C

2002-02-26

366

Ethanol production by recombinant hosts  

Science.gov (United States)

Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

Fowler, David E. (Gainesville, FL); Horton, Philip G. (Gainesville, FL); Ben-Bassat, Arie (Gainesville, FL)

1996-01-01

367

Ethanol production by recombinant hosts  

Science.gov (United States)

Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

Ingram, Lonnie O. (Gainesville, FL); Beall, David S. (Gainesville, FL); Burchhardt, Gerhard F. H. (Gainesville, FL); Guimaraes, Walter V. (Vicosa, BR); Ohta, Kazuyoshi (Miyazaki, JP); Wood, Brent E. (Gainesville, FL); Shanmugam, Keelnatham T. (Gainesville, FL)

1995-01-01

368

Recombination of H and He in Yang-Mills Gravity  

CERN Document Server

We investigate some aspects of the thermal history of the early universe according to Yang-Mills Gravity (YMG); a gauge theory of gravity set in flat spacetime. Specifically, equations for the ionization fractions of hydrogen and singly ionized helium during the recombination epoch are deduced analytically and then solved numerically. By considering several approximations we find that the presence of primordial helium and its interaction with Lyman series photons has a much stronger effect on the overall free electron density in YMG than it does in the standard, General Relativity (GR) based, model. Compared to the standard model recombination happens over a much larger range of temperatures, although there is still a very sharp temperature of last scattering around 2000 K. Since the ionization history of the universe is not directly observable we discuss how one may use it to predict the CMB power spectrum and thus test YMG. This topic will be explored in detail in an upcoming paper.

Katz, Daniel

2014-01-01

369

REGULATION OF HUMAN PLATELET AGGREGATION BY GENETICALLY MODIFIED PIG ENDOTHELIAL CELLS AND THROMBIN INHIBITION  

Science.gov (United States)

Background Coagulation disorders remain barriers to successful pig-to-primate organ xenotransplantation. In vitro, we investigated the impact of pig genetic modifications on human platelet aggregation in response to pig aortic endothelial cells (pAEC). Methods In comparison to human (h)AEC and wild-type (WT) pAEC, the expression of human complement- (CD46, CD55) or coagulation (thrombomodulin [TBM], endothelial protein C receptor [EPCR]) -regulatory proteins on pAEC from WT or ?1,3-galactosyltransferase gene-knockout (GTKO) pigs was studied by flow cytometry. Using platelet-aggregometry, human whole blood platelet aggregation was evaluated after co-incubation with various AEC. Further, the inhibitory effect on aggregation of heparin, low molecular weight heparin, and hirudin was assessed. Results Heparin, low molecular weight heparin and hirudin almost completely prevented platelet aggregation induced by WT pAEC. The level of expression of human CD46, CD55, TBM and EPCR on pAEC was comparable to that on hAEC. Platelet aggregation induced by all genetically-modified pAEC was significantly less (p<0.05) than that by WT pAEC (which was 54%). GTKO/CD46/TBM pAEC induced the least platelet aggregation (27%) – a reduction of almost 50% - but this remained significantly greater (p<0.01) than aggregation induced by hAEC (4%). There was significant positive correlation between reduction of aggregation and TBM or EPCR expression on pAEC (r2=0.89 and r2=0.86, respectively; p<0.01). Platelet aggregation induced by GTKO/CD46/TBM pAEC in the presence of hirudin (1IU/ml) was comparable to platelet aggregation induced by hAEC. Conclusions Genetic-modification of pAEC is associated with significant reduction of human platelet aggregation in vitro. With concomitant thrombin inhibition, platelet aggregation was comparable to that stimulated by hAEC. PMID:24188473

Iwase, Hayato; Ekser, Burcin; Hara, Hidetaka; Phelps, Carol; Ayares, David; Cooper, David K.C.; Ezzelarab, Mohamed B.

2013-01-01

370

3D photonic crystal-based biosensor functionalized with quantum dot-based aptamer for thrombine detection  

Science.gov (United States)

In this paper, we propose a new technique for protein detection by using the enhancement of intensity in quantum dots (Qdot) whose emission is guided by 3D photonic crystal (PC) structures. For easy to use, we design the emitted light from the sensor can be recovered, when the chemical antibody (aptamer) conjugated with guard DNA (g-DNA) labeled with a quencher (Black FQ) hybridizes with the target proteins. In detail, we synthesis a Qdot-aptamer complex and then immobilize these complex on the PC surfaces. Next, we perform the hybridization of the Qdot-aptamer complex with g-DNA labeled with the quencher. It induces the quenching effect of fluoresce intensity in the Qdot-aptamer. In presence of target protein (thrombin), the Qdot-aptamer complex prefers to form the thrombin-aptamer complex: this results in the release of Black FQ-g-DNA and the quenched light intensity recovers into the original high intensity with Qdot. The intensity recovery varies quantitatively according to the level of the target protein concentration. This proposed sensor shows much higher detection sensitivity than the general fluorescent detection mechanism, which is functionalized on the flat surfaces because of the light guiding effect from 3D photonic crystal structures.

Lim, Chae Young; Choi, Eunpyo; Park, Youngkyu; Park, Jungyul

2013-05-01

371

Rapid stiffening of integrin receptor-actin linkages in endothelial cells stimulated with thrombin: a magnetic bead microrheology study.  

Science.gov (United States)

By using magnetic bead microrheology we study the effect of inflammatory agents and toxins on the viscoelastic moduli of endothelial cell plasma membranes in real time. Viscoelastic response curves were acquired by applying short force pulses of ~500 pN to fibronectin-coated magnetic beads attached to the surface membrane of endothelial cells. Upon addition of thrombin, a rapid stiffening of the membrane was observed within 5 s, followed by recovery of the initial deformability within 2 min. By using specific inhibitors, two known pathways by which thrombin induces actin reorganization in endothelial cells, namely activation of Ca2+-calmodulin-dependent myosin light chain kinase and stimulation of Rho/Rho-kinase, were excluded as possible causes of the stiffening effect. Interestingly, the cytotoxic necrotizing factor of Escherichia coli, a toxin which, in addition to Rho, activates the GTPases Rac and CDC42Hs, also induced a dramatic stiffening effect, suggesting that the stiffening may be mediated through a Rac- or Cdc42Hs-dependent pathway. This work demonstrates that magnetic bead microrheometry is not only a powerful tool to determine the absolute viscoelastic moduli of the composite cell plasma membrane, but also a valuable tool to study in real time the effect of drugs or toxins on the viscoelastic parameters of the plasma membrane. PMID:11371441

Bausch, A R; Hellerer, U; Essler, M; Aepfelbacher, M; Sackmann, E

2001-06-01

372

A label-free electrochemical aptasensor based on the catalysis of manganese porphyrins for detection of thrombin.  

Science.gov (United States)

A novel manganese porphyrin (MnPP)-catalyzed aerobic oxidation of l-cysteine to disulfides (RSSR) was firstly found and applied into electrochemical aptasensor with a label-free technique for signal amplification. The possible catalytic mechanism of the catalytic reaction where MnPP catalyzed l-cysteine with thiol (RSH) structure to RSSR was discussed in detail. For fabrication of the aptasensor, thionine (Thi), which served as an electron mediator, was mixed with MnPP and immobilized on the nafion coated carbon electrode through ion exchange adsorption. Gold nanoparticle (nano-Au) was assembled on the Thi for immobilizing thrombin binding aptamer (TBA). In the presence of thrombin (TB), TBA will capture TB and form TBA-TB composite thus perturbed electron transfer, leading to decrease of the current for quantitatively detecting TB. Under optimal condition, the electrochemical aptasensor exhibited a linear range of 0.1-25nM with a detection limit of 0.02nM. This work opens a novel way for signal amplification study about porphyrins that served as mimetic enzyme to thiol in electrochemical aptasensor. PMID:25530538

Zheng, Yingning; Yuan, Yali; Chai, Yaqin; Yuan, Ruo

2015-04-15

373

Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper  

Directory of Open Access Journals (Sweden)

Full Text Available A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13% of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 µg and fibrinogen (minimum coagulant dose = 4.2 µg in vitro, and promotes defibrin(ogenation in vivo (minimum defibrin(ogenating dose = 1.0 µg. In addition, when injected intravenously in mice at doses of 5 and 10 µg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the `gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.

A.V Pérez

2008-01-01

374

Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for a [...] pproximately 0.13% of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 µg) and fibrinogen (minimum coagulant dose = 4.2 µg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 µg). In addition, when injected intravenously in mice at doses of 5 and 10 µg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the `gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.

A.V, Pérez; A, Rucavado; L, Sanz; J.J, Calvete; J.M, Gutiérrez.

2008-01-01

375

The crystal structure of human alpha-thrombin complexed with LY178550, a nonpeptidyl, active site-directed inhibitor.  

Science.gov (United States)

The crystal structure of human alpha-thrombin in complex with LY178550, a nonpeptidyl, active site-directed inhibitor, has been solved to 2.07 A resolution by the method of X-ray crystallography. The final model of the complex has a crystallographic R-value of 21.5% (Rfree = 23.1%) with 0.014 A and 2.4 degrees standard deviation from ideal bond lengths and angles, respectively. Well-defined electron density was observed for the inhibitor in the active site. The inhibitor binds to the active site in an L-shaped manner, mimicking the bound conformation of the tripeptide arginal series of thrombin inhibitors (Chirgadze NY et al., 1992, American Crystallographic Association Meeting 20: 116 [Abstr. PB311]). The basic amidine of LY178550 forms a salt bridge with Asp 189 within the specificity pocket, while the 4-benzylpiperidine side chain engages in a number of hydrophobic interactions at the S2 and S3 binding sites. The inhibitor does not interact in any fashion with the active site sequence Ser 214-Gly 216, as occurs with many of the inhibitors studied previously. The indole N-H of the inhibitor forms a hydrogen bond to the gamma-oxygen of the catalytic serine (Ser 195). PMID:9232642

Chirgadze, N Y; Sall, D J; Klimkowski, V J; Clawson, D K; Briggs, S L; Hermann, R; Smith, G F; Gifford-Moore, D S; Wery, J P

1997-07-01

376

Recombinant protein scaffolds for tissue engineering  

International Nuclear Information System (INIS)

New biological materials for tissue engineering are now being developed using common genetic engineering capabilities to clone and express a variety of genetic elements that allow cost-effective purification and scaffold fabrication from these recombinant proteins, peptides or from chimeric combinations of these. The field is limitless as long as the gene sequences are known. The utility is dependent on the ease, product yield and adaptability of these protein products to the biomedical field. The development of recombinant proteins as scaffolds, while still an emerging technology with respect to commercial products, is scientifically superior to current use of natural materials or synthetic polymer scaffolds, in terms of designing specific structures with desired degrees of biological complexities and motifs. In the field of tissue engineering, next generation scaffolds will be the key to directing appropriate tissue regeneration. The initial period of biodegradable synthetic scaffolds that provided shape and mechanical integrity, but no biological information, is phasing out. The era of protein scaffolds offers distinct advantages, particularly with the combination of powerful tools of molecular biology. These include, for example, the production of human proteins of uniform quality that are free of infectious agents and the ability to make suitable quantities of proteins that are found in low quantity or are hard to isolate from tissue. For the particular needs of from tissue. For the particular needs of tissue engineering scaffolds, fibrous proteins like collagens, elastin, silks and combinations of these offer further advantages of natural well-defined structural scaffolds as well as endless possibilities of controlling functionality by genetic manipulation. (topical review)

377

Investigations for designing catalytic recombiners  

International Nuclear Information System (INIS)

In case of a severe accident in pressurised water reactors (PWR) a high amount of hydrogen up to about 20,000 m3 might be generated and released into the containments. The mixture consisting of hydrogen and oxygen may either burn or detonate, if ignited. In case of detonation the generated shock wave may endanger the components of the plant or the plant itself. Consequently, effective removal of hydrogen is required. The fact that hydrogen and oxygen react exo-thermally on catalytically acting surfaces already at low temperatures generating steam and heat is made use of in catalytic recombiners. They consist of substrates coated with catalyst (mainly platinum or palladium) which are arranged inside a casing. Being passively acting measures, recombiners do not need any additional energy supply. Experimental investigations on catalytic hydrogen recombination are conducted at FZJ (Forschungszentrum Juelich) using three test facilities. The results yield insight in the development potential of contemporary recombiner systems as well as of innovative systems. Detailed investigations on a recombiner section show strong temperature gradients over the surface of a catalytically coated sample. Dependent on the flow velocity, ignition temperature may be reached at the leading edge already at an inlet hydrogen concentration of about 5 vol.-%. The thermal strain of the substrate leads to considerable detachment of catalyst particles probably causing unintended ignition of the flammable mixture. Temperature peaks can be prevented effectively by leaving the first part of the plate uncoated. In order to avoid overheating of the catalyst elements of a recombiner even at high hydrogen concentrations a modular system of porous substrates is proposed. The metallic substrates are coated with platinum at low catalyst densities thus limiting the activity of the single specimen. A modular arrangement of these elements provides high recombination rates over a large hydrogen concentration range without igniting the mixture

378

Recombining WMAP: Constraints on ionizing and resonance radiation at recombination  

International Nuclear Information System (INIS)

We place new constraints on sources of ionizing and resonance radiation at the epoch of the recombination process using the recent cosmic microwave background temperature and polarization spectra coming from the Wilkinson Microwave Anisotropy Probe (WMAP). We find that non-standard recombination scenarios are still consistent with the current data. In light of this we study the impact that such models can have on the determination of several cosmological parameters. In particular, the constraints on curvature and baryon density appear to be weakly affected by a modified recombination scheme. However, it may affect the current WMAP constraints on inflationary parameters such as the spectral index ns and its running. Physically motivated models, such as those based on primordial black holes or super heavy dark matter decay, are able to provide a good fit to the current data. Future observations in both temperature and polarization will be needed to more stringently test these models

379

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

Science.gov (United States)

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

Henry, Brian L; Desai, Umesh R

2014-11-01

380

Epitope mapping of a monoclonal antibody against human thrombin by H/D-exchange mass spectrometry reveals selection of a diverse sequence in a highly conserved protein  

Science.gov (United States)

The epitope of a monoclonal antibody raised against human thrombin has been determined by hydrogen/deuterium exchange coupled to MALDI mass spectrometry. The antibody epitope was identified as the surface of thrombin that retained deuterium in the presence of the monoclonal antibody compared to control experiments in its absence. Covalent attachment of the antibody to protein G beads and efficient elution of the antigen after deuterium exchange afforded the analysis of all possible epitopes in a single MALDI mass spectrum. The epitope, which was discontinuous, consisting of two peptides close to anion-binding exosite I, was readily identified. The epitope overlapped with, but was not identical to, the thrombomodulin binding site, consistent with inhibition studies. The antibody bound specifically to human thrombin and not to murine or bovine thrombin, although these proteins share 86% identity with the human protein. Interestingly, the epitope turned out to be the more structured of two surface regions in which higher sequence variation between the three species is seen. PMID:12021429

Baerga-Ortiz, Abel; Hughes, Carrie A.; Mandell, Jeffrey G.; Komives, Elizabeth A.

2002-01-01

381

The Anopheles gambiae cE5, a tight- and fast-binding thrombin inhibitor with post-transcriptionally regulated salivary-restricted expression.  

Czech Academy of Sciences Publication Activity Database

Ro?. 42, ?. 9 (2012), s. 610-620. ISSN 0965-1748 R&D Projects: GA ?R GAP502/12/2409 Institutional research plan: CEZ:AV0Z60220518 Keywords : Anopheles * Salivary protein * Anti-thrombin * Anophelin * Hematophagy * Post-transcriptional regulation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.234, year: 2012

Ronca, R.; Kotsyfakis, Michalis; Lombardo, F.; Rizzo, C.; Currà, C.; Ponzi, M.; Fiorentino, G.; Ribeiro, J.M.C.; Arcà, B.

2012-01-01

382

Diclofenac Topical (osteoarthritis pain)  

Science.gov (United States)

Diclofenac topical gel (Voltaren) is used to relieve pain from osteoarthritis (arthritis caused by a breakdown of ... the knees, ankles, feet, elbows, wrists, and hands. Diclofenac topical liquid (Pennsaid) is used to relieve osteoarthritis ...

383

Current Drive in Recombining Plasma  

Energy Technology Data Exchange (ETDEWEB)

The Langevin equations describing the average collisional dynamics of suprathermal particles in nonstationary plasma remarkably admit an exact analytical solution in the case of recombining plasma. The current density produced by arbitrary particle fluxes is derived including the effect of charge recombination. Since recombination has the effect of lowering the charge density of the plasma, thus reducing the charged particle collisional frequencies, the evolution of the current density can be modified substantially compared to plasma with fixed charge density. The current drive efficiency is derived and optimized for discrete and continuous pulses of current, leading to the discovery of a nonzero "residual" current density that persists indefinitely under certain conditions, a feature not present in stationary plasmas.

P.F. Schmit and N.J. Fisch

2012-05-15

384

Inhomogeneous recombinations during cosmic reionization  

CERN Document Server

By depleting the ionizing photon budget available to expand cosmic HII regions, recombining systems (or Lyman limit systems) can have a large impact during (and following) cosmic reionization. Unfortunately, directly resolving such structures in large-scale reionization simulations is computationally impractical. Instead, here we implement a sub-grid prescription for tracking inhomogeneous recombinations in the intergalactic medium. Building on previous work parameterizing photo-heating feedback on star-formation, we present large-scale, semi-numeric reionization simulations which self-consistently track the local (sub-grid) evolution of both sources and sinks of ionizing photons. Our simple, single-parameter model naturally results in both an extended reionization and a modest, slowly-evolving emissivity, consistent with observations. Recombinations are instrumental in slowing the growth of large HII regions, and damping the rapid rise of the ionizing background in the late stages of (and following) reioniza...

Sobacchi, Emanuele

2014-01-01

385

The Dissociative Recombination of OH(+)  

Science.gov (United States)

Theoretical quantum chemical calculations of the cross sections and rates for the dissociative recombination of the upsilon = 0 level of the ground state of OH(+) show that recombination occurs primarily along the 2 (2)Pi diabatic route. The products are 0((1)D) and a hot H atom with 6.1 eV kinetic energy. The coupling to the resonances is very small and the indirect recombination mechanism plays only a minor role. The recommended value for the rate coefficient is (6.3 +/- 0.7) x 10(exp -9)x (T(e)/1300)(exp -0.48) cu.cm/s for 10 less than T(e) less than 1000 K.

Guberman, Steven L.

1995-01-01

386

Freshman Health Topics  

Science.gov (United States)

This article examines a cluster of health topics that are frequently selected by students in lower division classes. Topics address issues relating to addictive substances, including alcohol and tobacco, eating disorders, obesity, and dieting. Analysis of the topics examines their interrelationships and organization in the reference literature.…

Hovde, Karen

2011-01-01

387

Melagatran, a direct thrombin inhibitor, but not edoxaban, a direct factor Xa inhibitor, nor heparin aggravates tissue factor-induced hypercoagulation in rats.  

Science.gov (United States)

There are concerns that some anticoagulants can paradoxically increase thrombogenesis under certain circumstances. We have shown that low-dose administration of a direct thrombin inhibitor, melagatran, significantly worsens the coagulation status induced by tissue factor injection in rats. We compared the effect of inhibition of thrombin and factor Xa for their potential to aggravate tissue factor-induced coagulation in rats. Hypercoagulation was induced by the injection of 2.8 U/kg tissue factor after administration of melagatran, heparin and edoxaban in rats. Blood samples were collected 10min after tissue factor injection. Platelet numbers, thrombin-antithrombin complex concentrations and plasma compound concentrations were measured. Though a high dose of melagatran (1mg/kg, i.v.) suppressed platelet consumption and thrombin-antithrombin complex generation induced by tissue factor, lower doses of melagatran (0.01, 0.03 and 0.1mg/kg, i.v.) significantly enhanced platelet consumption and thrombin-antithrombin complex generation. In addition, although melagatran (3mg/kg, i.v.) improved coagulation status when tissue factor was given 5min after the drug administration, and 2, 4 and 8h after melagatran dosing, it deteriorated coagulation status. These results were well explained by the plasma melagatran concentration. Low concentrations (15-234ng/ml) of melagatran aggravated coagulation status whereas it was mended by high concentrations (1190ng/ml or more) of the compound. In contrast, edoxaban and heparin did not show any exacerbation under these examination conditions. These results show that subtherapeutic concentrations of melagatran are associated with coagulation pathway activation, whereas factor Xa inhibition with edoxaban has a low risk of paradoxical hypercoagulation. PMID:22546231

Furugohri, Taketoshi; Fukuda, Toshio; Tsuji, Naoki; Kita, Akemi; Morishima, Yoshiyuki; Shibano, Toshiro

2012-07-01

388

Dissociative recombination of protonated methanol  

OpenAIRE

The branching ratios of the different reaction pathways and the overall rate coefficients of the dissociative recombination reactions of CH3OH2+ and CD3OD2+ have been measured at the CRYRING storage ring located in Stockholm, Sweden. Analysis of the data yielded the result that formation of methanol or deuterated methanol accounted for only 3 and 6% of the total rate in CH3OH2+ and CD3OD2+, respectively. Dissociative recombination of both isotopomeres mainly involves fragmentation of the C–...

Geppert, Wolf; Hamberg, Mathias; Thomas, Richard D.; O?sterdahl, Fabian; Hellberg, Fredrik; Zhaunerchyk, Vitali; Ehlerding, Anneli; Millar, Tom; Roberts, Helen; Semaniak, Jacek; Af Ugglas, Magnus; Ka?llberg, Anders; Simonsson, Ansgar; Kaminska, Magdalena; Larsson, Mats

2006-01-01

389

Recombination in the evolution of human bocavirus.  

Science.gov (United States)

Whole genome sequencing of Novosibirsk human bocavirus (HBoV) isolates has detected an isolate that emerged via recombination between HBoV3 and HBoV4 genotypes. The recombination site is located between regions with abnormally low and abnormally high GC contents in the genome. This site is a bocavirus recombination hotspot and coincides with one of two parvovirus recombination hotspots. The Novosibirsk recombinant isolate, which is similar to a previously studied isolate from Thailand, utilizes the strategy of borrowing ORF3, which encodes structural proteins, of a rare genotype HBoV4. The role of recombination in HBoV evolution is discussed. PMID:25193564

Tyumentsev, Alexander I; Tikunova, Nina V; Tikunov, Artem Yu; Babkin, Igor V

2014-12-01

390

Health Topic XML File Description  

Science.gov (United States)

... Topics Drugs & Supplements Videos & Cool Tools MedlinePlus Health Topic XML File Description To use the sharing features ... information categories assigned. Example of a Full Health Topic Record - topic title =" Abdominal Pain " url =" http:// ...

391

Radioiodination and biodistribution study of a thrombin-like enzyme/gyroxin from Lachesis muta muta venom  

International Nuclear Information System (INIS)

Recently, our group isolated and sequenced a thrombin-like enzyme (TLE) of 40kDa from the snake Lachesis muta muta. This protein hydrolyses synthetic substrates with specificity similar to that of trypsin and may be involved in the haemorrhagic, proteolytic and blood-clotting activities of the Lachesis venom. When injected into the tail veins of mice at levels of 0.015-0.130?g/g mouse, the TLE induce temporary episodes of opisthotonus and rapid rolling around the long axis of the animals and that is the reason why it is also called gyroxin. If this gyroxin activity is caused by direct interaction with the brain or by indirect effect remains to be investigated. We report in this work the radioiodination of TLE and the biodistribution of I-TLE in order to investigate its pharmacokinetics and verify if this enzyme are able to penetrate the blood brain barrier to evoke directly the gyroxin effects. (author)

392

Thrombin activity, fibrinogen level and prostacyclin release in mice treated with WR-2721 before whole body x-irradiation  

International Nuclear Information System (INIS)

SAS mice, treated intraperitoneally with 400 mg/kg of WR-2721 survived 9.5 Gy dose of X-radiation. Prostacyclin release from lung tissue decreased in mice treated with WR-2721 and remained unchanged in mice treated with this drug and exposed to the lethal dose of X-rays. Thrombin activity in blood plasma of mice not affected either by WR-2721 or X-radiation. Also in mice radio-protected and then irradiated this parameter remained unchanged. Fibrinogen level in blood plasma temporarily increased in mice treated with WR-2721 and significantly increased about a week after exposure of mice to the lethal dose of X-rays. This latter increase did not subside in mice which were treated with WR-2721 before irradiation. (author). 35 refs, 5 tabs

393

Thrombin activity, fibrinogen level and prostacyclin release in mice treated with WR-2721 before whole body x-irradiation  

Energy Technology Data Exchange (ETDEWEB)

SAS mice, treated intraperitoneally with 400 mg/kg of WR-2721 survived 9.5 Gy dose of X-radiation. Prostacyclin release from lung tissue decreased in mice treated with WR-2721 and remained unchanged in mice treated with this drug and exposed to the lethal dose of X-rays. Thrombin activity in blood plasma of mice not affected either by WR-2721 or X-radiation. Also in mice radio-protected and then irradiated this parameter remained unchanged. Fibrinogen level in blood plasma temporarily increased in mice treated with WR-2721 and significantly increased about a week after exposure of mice to the lethal dose of X-rays. This latter increase did not subside in mice which were treated with WR-2721 before irradiation. (author). 35 refs, 5 tabs.

Sochanowicz, B.; Dancewicz, A.M. [Institute of Nuclear Chemistry and Technology, Warsaw (Poland)

1993-12-31

394

Three months of strictly controlled daily endurance exercise reduces thrombin generation and fibrinolytic risk markers in younger moderately overweight men  

DEFF Research Database (Denmark)

PURPOSE: Physical activity is associated with a decreased risk of cardiovascular disease, but dose dependency of long-term physical exercise on biomarkers within coagulation and fibrinolysis is unknown. We aimed to investigate effects of two doses of daily endurance exercise on biomarkers of the haemostatic balance in overweight men. METHODS: Haemostatic variables were investigated in 53 healthy, younger (20-40 years), moderately overweight (BMI 25-30 kg/m(2)) men randomly assigned to 3 months of strictly controlled endurance exercise at two different doses corresponding to an energy expenditure of 600 kcal/day (HIGH), 300 kcal/day (MOD), or to maintain their habitual lifestyle (CON). Fasting blood samples were collected before and after the intervention and analysed for thrombin generation (endogenous thrombin potential, ETP) and concentrations of tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor type 1 (PAI-1), and von Willebrand factor (vWF). RESULTS: We observed significant within-group decreases in ETP (MOD 7 %; HIGH 6 %) and in t-PA (MOD 22 %; HIGH 21 %) and PAI-1 (MOD 16 %; HIGH 32 %) in both training groups, and no changes in the CON group. At 3 months, between-group differences were observed for ETP (p = 0.016) and t-PA (p = 0.012) due to significantly lower values in MOD and HIGH compared with CON. Borderline significant between-group differences were observed for PAI-1 (p = 0.082). A significant increase was observed in vWF in HIGH, but with no between-group differences. CONCLUSIONS: Our results demonstrate an effect of 3 months of daily endurance exercise on biomarkers of the haemostatic balance in the direction of reduced cardiovascular risk, independent of exercise dose.

Gram, Anne Sofie; Bladbjerg, Else-Marie

2015-01-01

395

Effect of low molecular weight heparins and fondaparinux upon thrombin generation triggered by human pancreatic cancer cells BXPC3.  

Science.gov (United States)

Low molecular weight heparins (LMWHs) and fondaparinux are widely used for prophylaxis and treatment of venous thromboembolic disease in cancer patients. However, the optimization of the antithrombotic treatment especially in patients with adenocarcinoma of the pancreas is a challenging issue. The understanding of the mechanism of action of the LMWHs and fondaparinux in cancer-induced hypercoagulability might help to optimize antithrombotic treatment. To this aim, we investigated the influence of BXPC3 pancreas adenocarcinoma cells on the antithrombotic activity of LMWHs and fondaparinux. Thrombin generation (TG) in normal platelet poor (PPP) and platelet rich plasma (PRP) spiked with clinically relevant concentrations of dalteparin, enoxaparin, nadroparin tinzaparin and fondaparinux was assessed with the Calibrated Automated Thrombogram assay. BXPC3 (5 cells/?l) were added to plasma. The mean rate index (MRI) of the propagation phase of TG and the endogenous thrombin potential (ETP) were analyzed. The IC50 of the studied compounds were determined and compared on the basis of anti-Xa and anti-IIa equivalent units. We demonstrate that the specific antithrombin (AT)-dependent anti-Xa activity of LMWHs and fondaparinux almost selectively inhibits the propagation phase of TG. The synergy between the anti-Xa and anti-IIa activities of LMWHs rather than the selective inhibition of FXa warrants abrogation of TG. The mean molecular weight and anti-Xa/anti-IIa ratio of the AT-dependent agents cannot predict the alteration of their capacity to inhibit TG. Tinzaparin was the most potent inhibitor of TG than the other LMWHs. Enoxaparin was more potent than nadroparin and dalteparin. PMID:22724467

Gerotziafas, Grigoris T; Galea, Vassiliki; Mbemba, Elisabeth; Sassi, Mouna; Roman, Marie-Paule; Khaterchi, Amir; van Dreden, Patrick; Japcowitz, Max; Lotz, Jean Pierre; Bernaudin, Jean Francois; Fareed, Jawed; Hatmi, Mohamed; Elalamy, Ismail

2014-01-01

396

Low-density lipoprotein, collagen, and thrombin models reveal that Rosemarinus officinalis L. exhibits potent antiglycative effects.  

Science.gov (United States)

Using the low-density lipoprotein (LDL), collagen, and thrombin models, we report here that the rosemary extracts (REs), either the aqueous (REw) or the acetonic (REA), all possessed many antiglycation-related features, and the effective concentrations required were as follows: 0.1 mg/mL for suppressing the relative electrophoretic mobility, 1.3 microg/mL for anticonjugated diene induction, 0.5 mg/mL for inhibition of thiobarbituric acid reactive substances production, 0.1 mg/mL for AGEs (advanced glycation end products) formation, 0.1 mg/mL to block glucose incorporation, and 0.05 mg/mL as an effective anti-antithrombin III. Using high-performance liquid chromatography/mass spectrometry, we identified five major constituents among eight major peaks, including rosmarinic acid, carnosol, 12-methoxycarnosic acid, carnosic acid, and methyl carnosate. In the LDL model, REA was proven to be more efficient than REw; yet, the reverse is true for the collagen and the thrombin III models, the reason of which was ascribed to the higher lipid-soluble antioxidant content (such as rosmarinic acid, carnosol, carnosic acid, 12-methoxycarnosic acid and methyl carnosate) in REA than in REw and the different surface lipid characteristics between LDL and collagen; although to act as anti-AGEs, both extracts were comparable. To assist the evidence, a larger 2,2-diphenyl-1-picrylhydrazyl radical scavenging capability with less total polyphenolic content was found in REA. We conclude that rosemary is an excellent multifunctional therapeutic herb; by looking at its potential potent antiglycative bioactivity, it may become a good adjuvant medicine for the prevention and treatment of diabetic, cardiovascular, and other neurodegenerative diseases. PMID:17385882

Hsieh, Chiu-Lan; Peng, Chiung-Huei; Chyau, Charng-Cherng; Lin, Yuh-Charn; Wang, Hui-Er; Peng, Robert Y

2007-04-18

397

Increased expression of protease nexin-1 in fibroblasts during idiopathic pulmonary fibrosis regulates thrombin activity and fibronectin expression.  

Science.gov (United States)

Idiopathic pulmonary fibrosis (IPF) is a chronic diffuse lung disease characterized by an accumulation of excess fibrous material in the lung. Protease nexin-1 (PN-1) is a tissue serpin produced by many cell types, including lung fibroblasts. PN-1 is capable of regulating proteases of both coagulation and fibrinolysis systems, by inhibiting, respectively, thrombin and plasminergic enzymes. PN-1 is thus a good candidate for regulating tissue remodeling occurring during IPF. We demonstrated a significant increase of PN-1 expression in lung tissue extracts, lung fibroblasts and bronchoalveolar lavage fluids of patients with IPF. The increase of PN-1 expression was reproduced after stimulation of control lung fibroblasts by transforming growth factor-?, a major pro-fibrotic cytokine involved in IPF. Another serpin, plasminogen activator inhibitor-1 (PAI-1) is also overexpressed in fibrotic fibroblasts. Unlike PAI-1, cell-bound PN-1 as well as secreted PN-1 from IPF and stimulated fibroblasts were shown to inhibit efficiently thrombin activity, indicating that both serpins should exhibit complementary roles in IPF pathogenesis, via their different preferential antiprotease activities. Moreover, we observed that overexpression of PN-1 induced by transfection of control fibroblasts led to increased fibronectin expression, whereas PN-1 silencing induced in fibrotic fibroblasts led to decreased fibronectin expression. Overexpression of PN-1 lacking either its antiprotease activity or its binding capacity to glycosaminoglycans had no effect on fibronectin expression. These novel findings suggest that modulation of PN-1 expression in lung fibroblasts may also have a role in the development of IPF by directly influencing the expression of extracellular matrix proteins. Our data provide new insights into the role of PN-1 in the poorly understood pathological processes involved in IPF and could therefore give rise to new therapeutic approaches. PMID:25199049

François, Déborah; Venisse, Laurence; Marchal-Somme, Joëlle; Jandrot-Perrus, Martine; Crestani, Bruno; Arocas, Véronique; Bouton, Marie-Christine

2014-11-01

398

Radiative recombination of complex ions  

Science.gov (United States)

The rates of radiative capture by atomic systems is computed for the first four ionization stages of the abundant elements C, N, O, Ne, Mg, Si, S, and Ar. A simple prescription is given for calculating the rates for systems in higher ionization stages. Results for capture to atomic helium are also given. In the general case, recent calculations of photoionization cross sections have been used to compute capture rates to levels of the ground-state configuration of the recombined species. Captures to other levels of the valence shell and to the higher shells are evaluated using hydrogenic formulae but with a correction factor which takes into account the nonhydrogenic nature of these states. This correction factor is due to the incomplete shielding by the inner electrons and represents essentially an effective charge for the recombining ion; it is determined semiempirically from an appropriate weighting of values derived from the observed level structure for each species. In some cases, for the capture to neutral atomic systems, its application increases the resulting computed recombination rate by a significant amount (about 50%). Computed recombination rates are tabulated for the four ionization stages at an electron temperature of 10,000 K and for the first stage at 100 K. A convenient procedure is outlined for evaluating the rates at other temperatures in the general neighborhood of these values.

Gould, R. J.

1978-01-01

399

Recombination luminescence in rigid media  

International Nuclear Information System (INIS)

When separated ion-pairs result from the ?-irradiation of pure or doped matrices or from solute photoionization, some of the photoejected electrons undergo spontaneous recombination, giving rise to the so-called isothermal luminescence (ITL). Cation-anion recombination takes place when molecular diffusion is possible; that is, it appears mostly in thermoluminescence (TL), upon warming irradiated samples. Electrons are photoextracted from matrix traps or from anions, and the neutralization luminescence is designated as stimulated luminescence (SL). ITL, TL, and SL all constitute very sensitive test for the presence of charged species. ITL decay kinetics throw some light on the recombination mechanism. In TL studies, the maxima of the glow curves correlate with phase transition temperatures (crystal ? crystal or glass ? crystal), providing insight into matrix molecular dynamics. From SL spectra, positive and negative charges can be discriminated, and various characteristics of the negative ions - electrons or anions - can be attained. The global SL irrespective of its spectrum composition, SL emission spectra, and SL excitation spectra or stimulation spectra are reviewed. The SL spectra, being specific of negative charged species, complement optical absorption spectroscopy when cations and anions have undistinguishable spectra. Even though radiative charge recombination constitutes the final step of ion-pair existence, it may serve to track back the successive stagrve to track back the successive stages of photoelectron life: electron ejection, matrix trapping or the attachments of electrons to molecules or radicals, and the release of trapped electrons. (Yamashita, S.)

400

Improving recombinant protein purification yield  

Science.gov (United States)

Production of adequate amounts of recombinant proteins is essential for antibody production, biochemical activity study, and structural determination during the post-genomic era. It’s technologically challenging and a limiting factor for tung oil research because analytical reagents such as high qua...

401

Genetics Home Reference: Recombinant 8 syndrome  

Science.gov (United States)

... this population have been found. What are the genetic changes related to recombinant 8 syndrome? Recombinant 8 ... Center . Where can I find general information about genetic conditions? The Handbook provides basic information about genetics ...

402

The ?0 polarization and the recombination mechanism  

International Nuclear Information System (INIS)

We use the recombination and the Thomas Precession Model to obtain a prediction for the ?0 polarization in the p+p??0+X reaction. We study the effect of the recombination function on the ?0 polarization

403

The ?0 polarization and the recombination mechanism  

International Nuclear Information System (INIS)

We use the recombination and the Thomas Precession Model to obtain a prediction for the ?0 polarization in the p+p ? ?0 + X reaction. We study the effect of the recombination function on the ?0 polarization. (author)

404

The effect of a single recombination event  

DEFF Research Database (Denmark)

We investigate the variance in how visible a single recombination event is in a SNP data set as a function of the type of recombination event and its age. Data is simulated under the coalescent with recombination and inference is by the popular composite likelihood methods. The major determinant of the effect of a recombination event is the genealogical type of the event and whether SNP variation is present that can reveal the genealogical consequences of the recombination event. Recombination events that only change some branch lengths in the genealogy have a very small, but detectable, effect. The more lineages left when the recombination event occurs, the larger effect it has, implying that it is mainly young recombination events that we detect when estimating the rate. If the population is growing, though, more lineages are present back in time and relatively more ancient recombination events may leave a stronger effect on data. We also investigate the amount of recombination events expected to be shared by two populations as a function of their separation time and explicitly model the European and African population in at attempt to survey how large an effect recombination events shared by these two populations are expected to contribute compared to the effect of private recombination events

Schierup, Mikkel Heide; Jensen, Thomas Mailund

405

Topical report review status  

International Nuclear Information System (INIS)

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

406

Extended recombinant bacterial ghost system.  

Science.gov (United States)

Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts from a variety of bacteria are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extends the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying foreign epitopes further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts have inherent adjuvant properties, they can be used as adjuvants in combination with subunit vaccines. Subunits or other ligands can also be coupled to matrixes like dextran which are used to fill the internal lumen of ghosts. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in this production. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. The endotoxic component of the outer membrane does not limit the use of ghosts as vaccine candidates but triggers the release of several potent immunoregulatory cytokines. As carriers, there is no limitation in the size of foreign antigens that can be inserted in the membrane and the capacity of all spaces including the membranes, peri-plasma and internal lumen of the ghosts can be fully utilized. This extended recombinant ghost system represents a new strategy for adjuvant free combination vaccines. PMID:10486935

Lubitz, W; Witte, A; Eko, F O; Kamal, M; Jechlinger, W; Brand, E; Marchart, J; Haidinger, W; Huter, V; Felnerova, D; Stralis-Alves, N; Lechleitner, S; Melzer, H; Szostak, M P; Resch, S; Mader, H; Kuen, B; Mayr, B; Mayrhofer, P; Geretschläger, R; Haslberger, A; Hensel, A

1999-08-20

407

Detection of Quantitative Trait Loci Influencing Recombination Using Recombinant Inbred Lines  

OpenAIRE

The genetic basis of variation in recombination in higher plants is polygenic and poorly understood, despite its theoretical and practical importance. Here a method of detecting quantitative trait loci (QTL) influencing recombination in recombinant inbred lines (RILs) is proposed that relies upon the fact that genotype data within RILs carry the signature of past recombination. Behavior of the segregational genetic variance in numbers of chromosomal crossovers (recombination) over generations...

Dole, Jefferey; Weber, David F.

2007-01-01

408

Sparse Topical Coding  

OpenAIRE

We present sparse topical coding (STC), a non-probabilistic formulation of topic models for discovering latent representations of large collections of data. Unlike probabilistic topic models, STC relaxes the normalization constraint of admixture proportions and the constraint of defining a normalized likelihood function. Such relaxations make STC amenable to: 1) directly control the sparsity of inferred representations by using sparsity-inducing regularizers; 2) be seamlessl...

Zhu, Jun; Xing, Eric P.

2012-01-01

409

TOPICAL TREATMENT OF MELASMA  

OpenAIRE

Melasma is a common hypermelanotic disorder affecting the face that is associated with considerable psychological impacts. The management of melasma is challenging and requires a long-term treatment plan. In addition to avoidance of aggravating factors like oral pills and ultraviolet exposure, topical therapy has remained the mainstay of treatment. Multiple options for topical treatment are available, of which hydroquinone (HQ) is the most commonly prescribed agent. Besides HQ, other topical ...

Bandyopadhyay Debabrata

2009-01-01

410

Low-Level Resistance of Staphylococcus aureus to Thrombin-Induced Platelet Microbicidal Protein 1 In Vitro Associated with qacA Gene Carriage Is Independent of Multidrug Efflux Pump Activity  

OpenAIRE

Thrombin-induced platelet microbial protein 1 (tPMP-1), a cationic antimicrobial polypeptide released from thrombin-stimulated rabbit platelets, targets the Staphylococcus aureus cytoplasmic membrane to initiate its microbicidal effects. In vitro resistance to tPMP-1 correlates with survival advantages in vivo. In S. aureus, the plasmid-carried qacA gene encodes a multidrug transporter, conferring resistance to organic cations (e.g., ethidium [Et]) via proton motive force (PMF)-energized expo...

Bayer, A. S.; Kupferwasser, L. I.; Brown, M. H.; Skurray, R. A.; Grkovic, S.; Jones, T.; Mukhopadhay, K.; Yeaman, M. R.

2006-01-01

411

Recombination properties of P1 dlac.  

OpenAIRE

The P1 dlac prophage plasmid of Escherichia coli K-12 has been utilized as the recipient DNA substrate in experiments with lambda plac5 transduction and with Hfr and F' conjugation. The P1 dlac plasmid does not recombine with lambda plac5 at the elevated levels seen for the F42lac plasmid. Recombination between lambda plac5 and P1 dlac is essentially indistinguishable from recombination between lambda plac5 and a chromosomal lac gene in tems of both level of recombination and recombination pa...

Porter, R. D.

1982-01-01

412

Recombinant protein production in bacterial hosts.  

Science.gov (United States)

The production of recombinant proteins is crucial for both the development of new protein drugs and the structural determination of drug targets. As such, recombinant protein production has a major role in drug development. Bacterial hosts are commonly used for the production of recombinant proteins, accounting for approximately 30% of current biopharmaceuticals on the market. In this review, I introduce fundamental concepts in recombinant protein production in bacteria, from drug development to production scales. Recombinant protein production processes can often fail, but how can this failure be minimised to rapidly deliver maximum yields of high-quality protein and so accelerate drug discovery? PMID:24246684

Overton, Tim W

2014-05-01

413

Homology-associated nonhomologous recombination in mammalian gene targeting.  

OpenAIRE

Nonhomologous (illegitimate) recombination of DNA underlies many changes in the genome. It involves no or little homology between recombining DNAs and has been considered unrelated with homologous recombination, which requires long homology. In mouse cells, however, we found recombination products whose sequences suggest that homologous interaction between DNAs caused nonhomologous recombination with another DNA. The intermediates of homologous recombination were apparently trapped at various...

Sakagami, K.; Tokinaga, Y.; Yoshikura, H.; Kobayashi, I.

1994-01-01

414

CSA Hot Topics Series  

Science.gov (United States)

The Hot Topics series provides a free sampling of the resources in Cambridge Scientific Abstracts (CSA) and the Internet Database Service (IDS). The 30 topics span subjects in the humanities, engineering, environmental policy, and medicine. Each Hot Topic gives an overview of the subject, key citations with abstracts, a list of Websites, a glossary, a comment form so that users can correspond with the editors, and a "source" section, which explains from which of CSA's paid services the resources were culled. The latest hot topic is MicroElectroMechanical systems (MEMS), "small integrated devices or systems that combine electrical and mechanical components."

415

Sexual recombination in Aspergillus tubingensis.  

Science.gov (United States)

Aspergillus tubingensis from section Nigri (black Aspergilli) is closely related to A. niger and is used extensively in the industrial production of enzymes and organic acids. We recently discovered sexual reproduction in A. tubingensis, and in this study we demonstrate that the progeny are products of meiosis. Progeny were obtained from six crosses involving five MAT1-1 strains and two MAT1-2 strains. We examined three loci, including mating type (MAT), RNA polymerase II (RPB2) and ?-tubulin (BT2), and found that 84% (58/69) of progeny were recombinants. Recombination associated with sexual reproduction in A. tubingensis provides a new option for the genetic improvement of industrial strains for enzyme and organic acid production. PMID:25572097

Olarte, Rodrigo A; Horn, Bruce W; Singh, Rakhi; Carbone, Ignazio

2015-01-01

416

Synthesis of modified homo-N-nucleosides from the reactions of mesityl nitrile oxide with 9-allylpurines and their influence on lipid peroxidation and thrombin inhibition.  

Science.gov (United States)

9-(3-Mesityl-4,5-dihydroisoxazol-5-yl) homo-N-nucleosides were prepared from the 1,3-dipolar cycloaddition reactions of mesityl nitrile oxide with 9-allyl derivatives of 6-chloropurine, 6-piperidinylpurine, 6-morpholinylpurine, 6-pyrrolidinylpurine, and 6-N,N-dibenzoyladenine. The new compounds were tested in vitro for their ability: (i) to interact with 1,1-diphenyl-2-picryl-hydrazyl (DPPH) stable free radical, (ii) to inhibit lipid peroxidation, (iii) to scavenge the superoxide anion, (iv) to inhibit the activity of soybean lipoxygenase, and (v) to inhibit in vitro thrombin. Most of them found to be potent thrombin inhibitors and to inhibit in vitro lipid peroxidation. The majority of the compounds showed significant lipoxygenase inhibitory activity. PMID:19811914

Thalassitis, Andreas; Hadjipavlou-Litina, Dimitra J; Litinas, Konstantinos E; Miltiadou, Panagiotis

2009-11-15

417

The pCri System: a vector collection for recombinant protein expression and purification.  

Science.gov (United States)

A major bottleneck in structural, biochemical and biophysical studies of proteins is the need for large amounts of pure homogenous material, which is generally obtained by recombinant overexpression. Here we introduce a vector collection, the pCri System, for cytoplasmic and periplasmic/extracellular expression of heterologous proteins that allows the simultaneous assessment of prokaryotic and eukaryotic host cells (Escherichia coli, Bacillus subtilis, and Pichia pastoris). By using a single polymerase chain reaction product, genes of interest can be directionally cloned in all vectors within four different rare restriction sites at the 5'end and multiple cloning sites at the 3'end. In this way, a number of different fusion tags but also signal peptides can be incorporated at the N- and C-terminus of proteins, facilitating their expression, solubility and subsequent detection and purification. Fusion tags can be efficiently removed by treatment with site-specific peptidases, such as tobacco etch virus proteinase, thrombin, or sentrin specific peptidase 1, which leave only a few extra residues at the N-terminus of the protein. The combination of different expression systems in concert with the cloning approach in vectors that can fuse various tags makes the pCri System a valuable tool for high throughput studies. PMID:25386923

Goulas, Theodoros; Cuppari, Anna; Garcia-Castellanos, Raquel; Snipas, Scott; Glockshuber, Rudi; Arolas, Joan L; Gomis-Rüth, F Xavier

2014-01-01

418

IP Internal Movement and Topicalization  

Science.gov (United States)

In this dissertation, I investigate the phenomenon of internal topicalization cross-linguistically, using Chinese as a starting point. Internal topicalization refers to constructions in which a topic phrase is placed between the subject and the verb (in contrast to external topicalization, which involves a topic in the CP domain). I argue that…

Kuo, Pei-Jung

2009-01-01

419

An evaluation of platelet-rich plasma without thrombin activation with or without anorganic bone mineral in the treatment of human periodontal intrabony defects.  

Science.gov (United States)

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. PMID:21381872

Rodrigues, Silvia V; Acharya, Anirudh B; Thakur, Srinath L

2011-01-01

420

Molecular basis of thrombin recognition by protein C inhibitor revealed by the 1.6-? structure of the heparin-bridged complex  

OpenAIRE

Protein C inhibitor (PCI) is a serpin with many roles in biology, including a dual role as pro- and anticoagulant in blood. The protease specificity and local function of PCI depend on its interaction with cofactors such as heparin-like glycosaminoglycans (GAGs) and thrombomodulin (TM). Both cofactors significantly increase the rate of thrombin inhibition, but GAGs serve to promote the anticoagulant activity of PCI, and TM promotes its procoagulant function. To gain insight into how PCI recog...

Li, Wei; Adams, Ty E.; Nangalia, Jyoti; Esmon, Charles T.; Huntington, James A.

2008-01-01

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