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

Science.gov (United States)

Coleman, Lewis S

2007-01-01

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

Endogenous fibrinolysis facilitates clot retraction in vivo.

Science.gov (United States)

Samson, Andre L; Alwis, Imala; Maclean, Jessica A A; Priyananda, Pramith; Hawkett, Brian; Schoenwaelder, Simone M; Jackson, Shaun P

2017-12-07

Clot retraction refers to the process whereby activated platelets transduce contractile forces onto the fibrin network of a thrombus, which over time increases clot density and decreases clot size. This process is considered important for promoting clot stability and maintaining blood vessel patency. Insights into the mechanisms regulating clot retraction at sites of vascular injury have been hampered by a paucity of in vivo experimental models. By pairing localized vascular injury with thrombin microinjection in the mesenteric circulation of mice, we have demonstrated that the fibrin network of thrombi progressively compacts over a 2-hour period. This was a genuine retraction process, as treating thrombi with blebbistatin to inhibit myosin IIa-mediated platelet contractility prevented shrinkage of the fibrin network. Real-time confocal analysis of fibrinolysis after recombinant tissue-type plasminogen activator (tPA) administration revealed that incomplete proteolysis of fibrin polymers markedly facilitated clot retraction. Similarly, inhibiting endogenous fibrinolysis with tranexamic acid reduced retraction of fibrin polymers in vivo. In vitro clot retraction experiments indicated that subthreshold doses of tPA facilitated clot retraction through a plasmin-dependent mechanism. These effects correlated with changes in the elastic modulus of fibrin clots. These findings define the endogenous fibrinolytic system as an important regulator of clot retraction, and show that promoting clot retraction is a novel and complementary means by which fibrinolytic enzymes can reduce thrombus size. © 2017 by The American Society of Hematology.

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

DEFF Research Database (Denmark)

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

2012-01-01

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...

Systems biology of coagulation initiation: kinetics of thrombin generation in resting and activated human blood.

Directory of Open Access Journals (Sweden)

Manash S Chatterjee

2010-09-01

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

Clotting mechanism in beagles irradiated by 4.5 Gy γ-rays

International Nuclear Information System (INIS)

Zhao Zhenhu; Wang Ning; Li Ming; Xing Shuang; Huang Haixiao; Ou Hongling; Xiong Guolin; Zhao Yanfang; Xie Ling; Wang Jinxiang; Miao Jingcheng; Zhu Nankang; Zhang Xueguang; Luo Qingliang; Cong Yuwen

2010-01-01

Objective: To explore the clotting mechanism in beagles irradiated by 4.5 Gy γ-rays after treatment with supportive care, or supportive care and combined cytokines. Methods: Sixteen beagles were divided into irradiation control group, Supportive care group and combined cytokines treatment group. Platelet aggregation test, thrombelastography (TEG) and the time measurement were analyzed in vitro. Results: In irradiation group and supportive care group, the platelet aggregation rates in beagles were decreased markedly and the k value of TEG was increased 7 d post-irradiation, while those indexes in combined cytokines treatment group changed little. At 14 d post-irradiation, each parameter of TEG in irradiated group changed obviously. The values of r, k, r + k and M were elevated significantly, clotting time and the maximum coagulation time of thrombus delayed, the Ma value was decreased markedly, and the maximum elasticity amplitude of thrombus was diminished. All parameters in combined cytokines treatment group were better than those in supportive care group. The thrombin time was prolonged obviously in irradiated group 14 d post-irradiation, while the thrombin time was the longest at 2-3 weeks post irradiation in supportive care group and combined cytokines treatment group (P>0.05). Conclusions: Cytokines could improve the platelet aggregation and the blood clotting functions of beagles suffering from acute radiation sickness. (authors)

Thrombin-Activatable Microbubbles as Potential Ultrasound Contrast Agents for the Detection of Acute Thrombosis.

Science.gov (United States)

Lux, Jacques; Vezeridis, Alexander M; Hoyt, Kenneth; Adams, Stephen R; Armstrong, Amanda M; Sirsi, Shashank R; Mattrey, Robert F

2017-11-01

Acute deep vein thrombosis (DVT) is the formation of a blood clot in the deep veins of the body that can lead to fatal pulmonary embolism. Acute DVT is difficult to distinguish from chronic DVT by ultrasound (US), the imaging modality of choice, and is therefore treated aggressively with anticoagulants, which can lead to internal bleeding. Here we demonstrate that conjugating perfluorobutane-filled (PFB-filled) microbubbles (MBs) with thrombin-sensitive activatable cell-penetrating peptides (ACPPs) could lead to the development of contrast agents that detect acute thrombosis with US imaging. Successful conjugation of ACPP to PFB-filled MBs was confirmed by fluorescence microscopy and flow cytometry. Fluorescein-labeled ACPP was used to evaluate the efficiency of thrombin-triggered cleavage by measuring the mean fluorescence intensity of ACPP-labeled MBs (ACPP-MBs) before and after incubation at 37 °C with thrombin. Lastly, control MBs and ACPP-MBs were infused through a tube containing a clot, and US contrast enhancement was measured with or without the presence of a thrombin inhibitor after washing the clot with saline. With thrombin activity, 91.7 ± 14.2% of the signal was retained after ACPP-MB infusion and washing, whereas only 16.7 ± 4% of the signal was retained when infusing ACPP-MBs in the presence of hirudin, a potent thrombin inhibitor.

Thrombin-Accelerated Quick Clotting Serum Tubes: An Evaluation with 22 Common Biochemical Analytes

Directory of Open Access Journals (Sweden)

Wai-Yoong Ng

2013-01-01

Full Text Available Clot activator serum tubes have significantly improved turnaround times for result reporting compared to plain tubes. With increasing workload and service performance expectations confronting clinical laboratories with high-volume testing and with particular emphasis on critical analytes, attention has focussed on preanalytical variables that can be improved. We carried out a field study on the test performance of BD vacutainer rapid serum tubes (RSTs compared to current institutional issued BD vacutainer serum separator tubes (SSTs in its test result comparability, clotting time, and stability on serum storage. Data from the study population (n=160 of patients attending outpatient clinics and healthy subjects showed that results for renal, liver, lipids, cardiac, thyroid, and prostate biochemical markers were comparable between RSTs and SSTs. Clotting times of the RSTs were verified to be quick with a median time of 2.05 min. Analyte stability on serum storage at 4°C showed no statistically significant deterioration except for bicarbonate, electrolytes, and albumin over a period of 4 days. In conclusion, RSTs offered savings in the time required for the clotting process of serum specimens. This should translate to further trimming of the whole process from blood collection to result reporting without too much sacrifice on test accuracy and performance compared to the current widely used SSTs in most clinical laboratories.

Discovery of thrombin activatable fibrinolysis inhibitor (TAFI)

NARCIS (Netherlands)

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

2006-01-01

CAS: blood clotting factor 11, 9013-55-2; thrombin, 9002-04-4; tissue plasminogen activator, 105913-11-9; protein C, 60202-16-6; Carboxypeptidase U, 3.4.17.20; Protein C; Tissue Plasminogen Activator, 3.4.21.68

21 CFR 864.7875 - Thrombin time test.

Science.gov (United States)

2010-04-01

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

Biological variation in tPA-induced plasma clot lysis time.

Science.gov (United States)

Talens, Simone; Malfliet, Joyce J M C; Rudež, Goran; Spronk, Henri M H; Janssen, Nicole A H; Meijer, Piet; Kluft, Cornelis; de Maat, Moniek P M; Rijken, Dingeman C

2012-10-01

Hypofibrinolysis is a risk factor for venous and arterial thrombosis, and can be assessed by using a turbidimetric tPA-induced clot lysis time (CLT) assay. Biological variation in clot lysis time may affect the interpretation and usefulness of CLT as a risk factor for thrombosis. Sufficient information about assay variation and biological variation in CLT is not yet available. Thus, this study aimed to determine the analytical, within-subject and between-subject variation in CLT. We collected blood samples from 40 healthy individuals throughout a period of one year (average 11.8 visits) and determined the CLT of each plasma sample in duplicate. The mean (± SD) CLT was 83.8 (± 11.1) minutes. The coefficients of variation for total variation, analytical variation, within-subject variation and between-subject variation were 13.4%, 2.6%, 8.2% and 10.2%, respectively. One measurement can estimate the CLT that does not deviate more than 20% from its true value. The contribution of analytical variation to the within-subject variation was 5.0%, the index of individuality was 0.84 and the reference change value was 23.8%. The CLT was longer in the morning compared to the afternoon and was slightly longer in older individuals (> 40 years) compared to younger (≤40 years) individuals. There was no seasonal variation in CLT and no association with air pollution. CLT correlated weakly with fibrinogen, C-reactive protein, prothrombin time and thrombin generation. This study provides insight into the biological variation of CLT, which can be used in future studies testing CLT as a potential risk factor for thrombosis.

Elevated NT-proBNP is associated with unfavorably altered plasma fibrin clot properties in atrial fibrillation.

Science.gov (United States)

Matusik, Paweł T; Matusik, Patrycja S; Kornacewicz-Jach, Zdzisława; Małecka, Barbara; Ząbek, Andrzej; Undas, Anetta

2017-09-15

Dense fibrin clot formation and hypofibrinolysis have been reported in atrial fibrillation (AF). It is unclear which factors affect fibrin clot properties in AF. We investigated plasma fibrin clot permeability (K s ), clot lysis time (CLT), endogenous thrombin potential (ETP) as well as other coagulation and fibrinolysis parameters along with N-terminal pro-B-type natriuretic peptide (NT-proBNP) in 160 AF patients (median age, 70.5years). Previous stroke (n=15; 9.4%) was associated with decreased K s (P=0.04) and longer CLT (P=0.005), together with higher antiplasmin (P=0.03) and lower tissue-type plasminogen activator (P=0.01). Lower K s (P=0.04) and tendency towards longer CLT (P=0.10) were observed in patients with a left atrium diameter>40mm. Patients with a CHA 2 DS 2 -VASc score of 3 or more (82.5%) were characterized by higher thrombin-activatable fibrinolysis inhibitor antigen (P=0.009). K s was inversely correlated with log NT-proBNP (r=-0.34, PCLT was positively correlated with log NT-proBNP (R=0.61, PCLT (the top quartile,≥109min). In AF patients prothrombotic fibrin clot properties assessed ex vivo are determined by PAI-1 and NT-proBNP and this phenotype is associated with prior ischemic stroke. Copyright © 2017 Elsevier B.V. All rights reserved.

Clot retraction is mediated by factor XIII-dependent fibrin-αIIbβ3-myosin axis in platelet sphingomyelin-rich membrane rafts.

Science.gov (United States)

Kasahara, Kohji; Kaneda, Mizuho; Miki, Toshiaki; Iida, Kazuko; Sekino-Suzuki, Naoko; Kawashima, Ikuo; Suzuki, Hidenori; Shimonaka, Motoyuki; Arai, Morio; Ohno-Iwashita, Yoshiko; Kojima, Soichi; Abe, Mitsuhiro; Kobayashi, Toshihide; Okazaki, Toshiro; Souri, Masayoshi; Ichinose, Akitada; Yamamoto, Naomasa

2013-11-07

Membrane rafts are spatially and functionally heterogenous in the cell membrane. We observed that lysenin-positive sphingomyelin (SM)-rich rafts are identified histochemically in the central region of adhered platelets where fibrin and myosin are colocalized on activation by thrombin. The clot retraction of SM-depleted platelets from SM synthase knockout mouse was delayed significantly, suggesting that platelet SM-rich rafts are involved in clot retraction. We found that fibrin converted by thrombin translocated immediately in platelet detergent-resistant membrane (DRM) rafts but that from Glanzmann's thrombasthenic platelets failed. The fibrinogen γ-chain C-terminal (residues 144-411) fusion protein translocated to platelet DRM rafts on thrombin activation, but its mutant that was replaced by A398A399 at factor XIII crosslinking sites (Q398Q399) was inhibited. Furthermore, fibrin translocation to DRM rafts was impaired in factor XIII A subunit-deficient mouse platelets, which show impaired clot retraction. In the cytoplasm, myosin translocated concomitantly with fibrin translocation into the DRM raft of thrombin-stimulated platelets. Furthermore, the disruption of SM-rich rafts by methyl-β-cyclodextrin impaired myosin activation and clot retraction. Thus, we propose that clot retraction takes place in SM-rich rafts where a fibrin-αIIbβ3-myosin complex is formed as a primary axis to promote platelet contraction.

Clotting of mammalian fibrinogens by papain: A re-examination

OpenAIRE

Doolittle, RF

2014-01-01

© 2014 American Chemical Society. Papain has long been known to cause the gelation of mammalian fibrinogens. It has also been reported that papain-fibrin is insoluble in dispersing solvents like strong urea or sodium bromide solutions, similar to what is observed with thrombin-generated clots in the presence of factor XIIIa and calcium. In those old studies, both the gelation and subsequent clot stabilization were attributed to papain, although the possibility that the second step might be du...

Platelet glycoprotein VI binds to polymerized fibrin and promotes thrombin generation.

Science.gov (United States)

Mammadova-Bach, Elmina; Ollivier, Véronique; Loyau, Stéphane; Schaff, Mathieu; Dumont, Bénédicte; Favier, Rémi; Freyburger, Geneviève; Latger-Cannard, Véronique; Nieswandt, Bernhard; Gachet, Christian; Mangin, Pierre H; Jandrot-Perrus, Martine

2015-07-30

Fibrin, the coagulation end product, consolidates the platelet plug at sites of vascular injury and supports the recruitment of circulating platelets. In addition to integrin αIIbβ3, another as-yet-unidentified receptor is thought to mediate platelet interaction with fibrin. Platelet glycoprotein VI (GPVI) interacts with collagen and several other adhesive macromolecules. We evaluated the hypothesis that GPVI could be a functional platelet receptor for fibrin. Calibrated thrombin assays using platelet-rich plasma (PRP) showed that tissue factor-triggered thrombin generation was impaired in GPVI-deficient patients and reduced by the anti-GPVI Fab 9O12. Assays on reconstituted PRP and PRP from fibrinogen-deficient patients revealed a fibrinogen-dependent enhancement of thrombin generation, which relied on functional GPVI. The effect of GPVI was found to depend on fibrin polymerization. A binding assay showed a specific interaction between GPVI-Fc and fibrin, inhibited by the Fab 9O12. This Fab also reduced platelet adhesion to fibrin at low (300 s(-1)) and high (1500 s(-1)) wall shear rates. Platelets adherent to fibrin displayed shape change, exposure of procoagulant phospholipids, and the formation of small clots. When hirudinated blood was perfused at 1500 s(-1) over preformed fibrin-rich clots, the Fab 9O12 decreased the recruitment of platelets by up to 85%. This study identifies GPVI as a platelet receptor for polymerized fibrin with 2 major functions: (1) amplification of thrombin generation and (2) recruitment of circulating platelets to clots. These so-far-unrecognized properties of GPVI confer on it a key role in thrombus growth and stabilization. © 2015 by The American Society of Hematology.

Platelet factor XIII increases the fibrinolytic resistance of platelet-rich clots by accelerating the crosslinking of alpha 2-antiplasmin to fibrin

Science.gov (United States)

Reed, G. L.; Matsueda, G. R.; Haber, E.

1992-01-01

Platelet clots resist fibrinolysis by plasminogen activators. We hypothesized that platelet factor XIII may enhance the fibrinolytic resistance of platelet-rich clots by catalyzing the crosslinking of alpha 2-antiplasmin (alpha 2AP) to fibrin. Analysis of plasma clot structure by polyacrylamide gel electrophoresis and immunoblotting revealed accelerated alpha 2AP-fibrin crosslinking in platelet-rich compared with platelet-depleted plasma clots. A similar study of clots formed with purified fibrinogen (depleted of factor XIII activity), isolated platelets, and specific factor XIII inhibitors indicated that this accelerated crosslinking was due to the catalytic activity of platelet factor XIII. Moreover, when washed platelets were aggregated by thrombin, there was evidence of platelet factor XIII-mediated crosslinking between platelet alpha 2AP and platelet fibrin(ogen). Specific inhibition (by a monoclonal antibody) of the alpha 2AP associated with washed platelet aggregates accelerated the fibrinolysis of the platelet aggregate. Thus in platelet-rich plasma clots, and in thrombin-induced platelet aggregates, platelet factor XIII actively formed alpha 2AP-fibrin crosslinks, which appeared to enhance the resistance of platelet-rich clots to fibrinolysis.

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

Science.gov (United States)

Mitrophanov, Alexander Y; Reifman, Jaques

2011-10-01

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

THROMBIN GENERATION AND BLEEDING IN HEMOPHILIA A

Science.gov (United States)

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

2012-01-01

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

Clot lysis time in platelet-rich plasma: method assessment, comparison with assays in platelet-free and platelet-poor plasmas, and response to tranexamic acid.

Science.gov (United States)

Panes, Olga; Padilla, Oslando; Matus, Valeria; Sáez, Claudia G; Berkovits, Alejandro; Pereira, Jaime; Mezzano, Diego

2012-01-01

Fibrinolysis dysfunctions cause bleeding or predisposition to thrombosis. Platelets contain several factors of the fibrinolytic system, which could up or down regulate this process. However, the temporal relationship and relative contributions of plasma and platelet components in clot lysis are mostly unknown. We developed a clot lysis time (CLT) assay in platelet-rich plasma (PRP-CLT, with and without stimulation) and compared it to a similar one in platelet-free plasma (PFP) and to another previously reported test in platelet-poor plasma (PPP). We also studied the differential effects of a single dose of tranexamic acid (TXA) on these tests in healthy subjects. PFP- and PPP-CLT were significantly shorter than PRP-CLT, and the three assays were highly correlated (p plasma PAI-1, von Willebrand factor, fibrinogen, LDL-cholesterol, and triglycerides (p platelet aggregation/secretion, platelet counts, and pro-coagulant tests to explore factor X activation by platelets, PRP clotting time, and thrombin generation in PRP. Among all the studied variables, PFP-CLT was independently associated with plasma PAI-1, LDL-cholesterol, and triglycerides and, additionally, stimulated PRP-CLT was also independently associated with plasma fibrinogen. A single 1 g dose of TXA strikingly prolonged all three CLTs, but in contrast to the results without the drug, the lysis times were substantially shorter in non-stimulated or stimulated PRP than in PFP and PPP. This standardized PRP-CLT may become a useful tool to study the role of platelets in clot resistance and lysis. Our results suggest that initially, the platelets enmeshed in the clot slow down the fibrinolysis process. However, the increased clot resistance to lysis induced by TXA is overcome earlier in platelet-rich clots than in PFP or PPP clots. This is likely explained by the display of platelet pro-fibrinolytic effects. Focused research is needed to disclose the mechanisms for the relationship between CLT and plasma

Adenosine diphosphate-decorated chitosan nanoparticles shorten blood clotting times, influencing the structures and varying the mechanical properties of the clots

Directory of Open Access Journals (Sweden)

Chung TW

2014-03-01

Full Text Available Tze-Wen Chung,1,3 Pei-Yi Lin,2 Shoei-Shen Wang,2 Yen-Fung Chen31Department of Biomedical Engineering, National Yang-Ming University, 2Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan, Republic of China; 3Department of Chemical and Materials Engineering, National Yunlin University of Science and Technology, Yunlin, Taiwan, Republic of ChinaAbstract: Chitosan nanoparticles (NPs decorated with adenosine diphosphate (ADP (ANPs or fibrinogen (FNPs were used to fabricate hemostatic NPs that can shorten blood clotting time and prevent severe local hemorrhage. The structure and mechanical properties of the blood clot induced with ANP (clot/ANP or FNP (clot/FNP were also investigated. The NPs, ANPs, and FNPs, which had particle sizes of 245.1±14.0, 251.0±9.8, and 326.5±14.5 nm and zeta potentials of 24.1±0.5, 20.6±1.9, and 15.3±1.5 mV (n=4, respectively, were fabricated by ionic gelation and then decorated with ADP and fibrinogen. The zeta potentials and Fourier transform infrared (FTIR spectroscopy of the NPs confirmed that their surfaces were successfully coated with ADP and fibrinogen. The scanning electron microscope (SEM micrographs of the structure of the clot induced with "undecorated" chitosan NPs (clot/NP, clot/ANP, and clot/FNP (at 0.05 wt% were different, after citrated bloods had been recalcified by a calcium chloride solution containing NPs, ANPs, or FNPs. This indicated that many NPs adhered on the membrane surfaces of red blood cells, that ANPs induced many platelet aggregates, and that FNPs were incorporated into the fibrin network in the clots. Measurements of the blood clotting times (Tc of blood clot/NPs, clot/ANPs, and clot/FNPs, based on 90% of ultimate frequency shifts measured on a quartz crystal microbalance (QCM, were significantly (P<0.05 (n=4 shorter than that of a clot induced by a phosphate-buffered solution (PBS (clot/PBS (63.6%±3.1%, 48.3%±6.2%, and 63.2%±4.7%, respectively. The ∆F2

Inactivation of single-chain urokinase-type plasminogen activator by thrombin in human subjects

NARCIS (Netherlands)

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

1999-01-01

Thrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) into a virtually inactive two-chain form (tcu-PA/T), a process that may protect a blood clot from early fibrinolysis. It is not known under what circumstances tcu-PA/T can be generated in vivo. We have studied the occurrence

Ex vivo reversal of effects of rivaroxaban evaluated using thromboelastometry and thrombin generation assay

Science.gov (United States)

Schenk, B.; Würtinger, P.; Streif, W.; Sturm, W.; Fries, D.; Bachler, M.

2016-01-01

Background In major bleeding events, the new direct oral anticoagulants pose a great challenge for physicians. The aim of the study was to test for ex vivo reversal of the direct oral anticoagulant rivaroxaban with various non-specific reversal agents: prothrombin complex concentrate (PCC), activated prothrombin complex concentrate (aPCC), recombinant activated factor VII (rFVIIa), and fibrinogen concentrate (FI). Methods Blood was obtained from healthy volunteers and from patients treated with rivaroxaban. Blood samples from healthy volunteers were spiked with rivaroxaban to test the correlation between rivaroxaban concentration and coagulation tests. Patient blood samples were spiked with various concentrations of the above-mentioned agents and analysed using thromboelastometry and thrombin generation. Results When added in vitro, rivaroxaban was significantly (P<0.05) correlated with ROTEM® thromboelastometry EXTEM (extrinsic coagulation pathway) clotting time (CT), time to maximal velocity (MaxV−t), and with all measured thrombin generation parameters. In vivo, CT, MaxV−t, lag time, and peak thrombin generation (Cmax) were significantly correlated with rivaroxaban concentrations. Regarding reversal of rivaroxaban, all tested agents significantly (P<0.05) reduced EXTEM CT, but to different extents: rFVIIa by 68%, aPCC by 47%, PCC by 17%, and FI by 9%. Only rFVIIa reversed EXTEM CT to baseline values. Both PCC (+102%) and aPCC (+232%) altered overall thrombin generation (area under the curve) and increased Cmax (+461% for PCC, +87.5% for aPCC). Conclusions Thromboelastometry and thrombin generation assays do not favour the same reversal agents for rivaroxaban anticoagulation. Controlled clinical trials are urgently needed to establish doses and clinical efficacy of potential reversal agents. Clinical trial registration EudracCT trial no. 213-00474-30. PMID:27623677

Rigidification of the autolysis loop enhances Na+ binding to thrombin

Science.gov (United States)

Pozzi, Nicola; Chen, Raymond; Chen, Zhiwei; Bah, Alaji; Di Cera, Enrico

2011-01-01

Binding of Na+ to thrombin ensures high activity toward physiological substrates and optimizes the procoagulant and prothrombotic roles of the enzyme in vivo. Under physiological conditions of pH and temperature, the binding affinity of Na+ is weak due to large heat capacity and enthalpy changes associated with binding, and the Kd=80 mM ensures only 64% saturation of the site at the concentration of Na+ in the blood (140 mM). Residues controlling Na+ binding and activation have been identified. Yet, attempts to improve the interaction of Na+ with thrombin and possibly increase catalytic activity under physiological conditions have so far been unsuccessful. Here we report how replacement of the flexible autolysis loop of human thrombin with the homologous rigid domain of the murine enzyme results in a drastic (up to 10-fold) increase in Na+ affinity and a significant improvement in the catalytic activity of the enzyme. Rigidification of the autolysis loop abolishes the heat capacity change associated with Na+ binding observed in the wild-type and also increases the stability of thrombin. These findings have general relevance to protein engineering studies of clotting proteases and trypsin-like enzymes. PMID:21536369

Investigation of Interactions between Thrombin and Ten Phenolic Compounds by Affinity Capillary Electrophoresis and Molecular Docking

Directory of Open Access Journals (Sweden)

Qiao-Qiao Li

2018-01-01

Full Text Available Thrombin plays a vital role in blood coagulation, which is a key process involved in thrombosis by promoting platelet aggregation and converting fibrinogen to form the fibrin clot. In the receptor concept, drugs produce their therapeutic effects via interactions with the targets. Therefore, investigation of interaction between thrombin and small molecules is important to find out the potential thrombin inhibitor. In this study, affinity capillary electrophoresis (ACE and in silico molecular docking methods were developed to study the interaction between thrombin and ten phenolic compounds (p-hydroxybenzoic acid, protocatechuic acid, vanillic acid, gallic acid, catechin, epicatechin, dihydroquercetin, naringenin, apigenin, and baicalein. The ACE results showed that gallic acids and six flavonoid compounds had relative strong interactions with thrombin. In addition, the docking results indicated that all of optimal conformations of the six flavonoid compounds were positioned into the thrombin activity centre and had interaction with the HIS57 or SER195 which was the key residue to bind thrombin inhibitors such as argatroban. Herein, these six flavonoid compounds might have the potential of thrombin inhibition activity. In addition, the developed method in this study can be further applied to study the interactions of other molecules with thrombin.

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

Energy Technology Data Exchange (ETDEWEB)

Maraganore, J.M.; Bourdon, P.; Jablonski, J.; Ramachandran, K.L. (Biogen, Inc., Cambridge, MA (USA)); Fenton, J.W. II (New York State Department of Health, Albany (USA))

1990-07-31

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

Rigidification of the autolysis loop enhances Na(+) binding to thrombin.

Science.gov (United States)

Pozzi, Nicola; Chen, Raymond; Chen, Zhiwei; Bah, Alaji; Di Cera, Enrico

2011-11-01

Binding of Na(+) to thrombin ensures high activity toward physiological substrates and optimizes the procoagulant and prothrombotic roles of the enzyme in vivo. Under physiological conditions of pH and temperature, the binding affinity of Na(+) is weak due to large heat capacity and enthalpy changes associated with binding, and the K(d)=80 mM ensures only 64% saturation of the site at the concentration of Na(+) in the blood (140 mM). Residues controlling Na(+) binding and activation have been identified. Yet, attempts to improve the interaction of Na(+) with thrombin and possibly increase catalytic activity under physiological conditions have so far been unsuccessful. Here we report how replacement of the flexible autolysis loop of human thrombin with the homologous rigid domain of the murine enzyme results in a drastic (up to 10-fold) increase in Na(+) affinity and a significant improvement in the catalytic activity of the enzyme. Rigidification of the autolysis loop abolishes the heat capacity change associated with Na(+) binding observed in the wild-type and also increases the stability of thrombin. These findings have general relevance to protein engineering studies of clotting proteases and trypsin-like enzymes. Copyright © 2011 Elsevier B.V. All rights reserved.

Effect of chitosan and coagulation factors on the wound repair phenotype of bioengineered blood clots.

Science.gov (United States)

Hoemann, Caroline D; Marchand, Catherine; Rivard, Georges-Etienne; El-Gabalawy, Hani; Poubelle, Patrice E

2017-11-01

Controlling the blood clot phenotype in a surgically prepared wound is an evolving concept in scaffold-guided tissue engineering. Here, we investigated the effect of added chitosan (80% or 95% Degree of Deacetylation, DDA) or coagulation factors (recombinant human Factor VIIa, Tissue Factor, thrombin) on inflammatory factors released by blood clots. We tested the hypothesis that 80% DDA chitosan specifically enhances leukotriene B 4 (LTB 4 ) production. Human or rabbit whole blood was combined with isotonic chitosan solutions, coagulation factors, or lipopolysaccharide, cultured in vitro at 37°C, and after 4hours the serum was assayed for LTB 4 or inflammatory factors. Only 80% DDA chitosan clots produced around 15-fold more LTB 4 over other clots including 95% DDA chitosan clots. All serum contained high levels of PDGF-BB and CXCL8. Normal clots produced very low type I cytokines compared to lipopolysaccharide clots, with even lower IL-6 and IL-12 and more CCL3/CCL4 produced by chitosan clots. Coagulation factors had no detectable effect on clot phenotype. Conclusion In blood clots from healthy individuals, 80% DDA chitosan has a unique influence of inducing more LTB 4 , a potent neutrophil chemoattractant, with similar production of PDGF-BB and CXCL8, and lower type I cytokines, compared to whole blood clots. Copyright © 2017 Elsevier B.V. All rights reserved.

Factor XI dependent and independent activation of thrombin activatable fibrinolysis inhibitor (TAFI) in plasma associated with clot formation

NARCIS (Netherlands)

Bouma, B. N.; Mosnier, L. O.; Meijers, J. C.; Griffin, J. H.

1999-01-01

Thrombin Activatable Fibrinolysis Inhibitor (TAFI) also known as plasma procarboxypeptidase B is activated by relatively high concentrations of thrombin in a reaction stimulated by thrombomodulin. In plasma an intact factor XI-dependent feed back loop via the intrinsic pathway is necessary to

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

Science.gov (United States)

Yu, Johnson Chung Sing

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

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

Energy Technology Data Exchange (ETDEWEB)

Henriksen, R.A.; Mann, K.G. (Univ. of Vermont, Burlington (USA))

1989-03-07

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 N{sup 2}-(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 ({sup 3}H)diisopropyl fluorophosphate stoichiometrically and hydrolyzes the elastase substrate succinyl-Ala-Ala-Pro-Leu-p-nitroanilide with a relative k{sub cat}/K{sub M} of 0.14 when compared to thrombin. This results from a 3-fold increase in K{sub M} and a 2.5-fold decrease in k{sub cat} for thrombin Quick II when compared to thrombin acting on the same substrate. These results and those of other investigators 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.

Indium-111 platelet scintigraphy and two-dimensional echocardiography for detection of left ventricular thrombus: influence of clot size and age

International Nuclear Information System (INIS)

Seabold, J.E.; Schroeder, E.C.; Conrad, G.R.

1987-01-01

Two-dimensional echocardiography and indium-111 platelet scintigraphy were performed on 50 dogs to determine the influence of clot age and size on the detection of experimentally induced left ventricular mural thrombus. Thrombus was induced by apical infarction and injection of a sclerosing agent and thrombin. The animals were classified into four groups according to the time of indium-111 platelet injection after thrombus induction: Group I (17 dogs, 1/2 hour after induction; 3 dogs, before induction), Group II (12 dogs, 24 hours after induction) and Group III (12 dogs, 1 week after induction). In Group IV (six control dogs) apical infarction was produced, but thrombin was not injected; indium-111 platelets were injected 1/2 to 1 hour after infarction. The dogs were studied by indium-111 platelet scintigraphy and by two-dimensional echocardiography 1/2 to 5 hours (Group I) and 1 to 5 and up to 72 hours (Groups II to IV) after platelet administration and before death was induced. Two-dimensional echocardiography showed the best overall sensitivity for detection of acute thrombus (97%; 29 of 30). The sensitivity of indium-111 platelet scintigraphy was 86% (18 of 21) for clots greater than or equal to 0.08 ml in size, and 67% (20 of 30) for detection of all clots. Thrombus did not form in 14 dogs of Groups I to III and in 6 of 6 control dogs. The specificity of scintigraphy was 100% (20 of 20) compared with 80% (16 of 20) for echocardiography. Echocardiography was more sensitive than scintigraphy for detecting very small clots in this experimental model

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

Science.gov (United States)

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

2009-01-01

Topical thrombins are active hemostatic agents that can be used to minimize blood loss during surgery. Before 2007, the only topical thrombins available were derived from bovine plasma. Antibody formation to bovine thrombin and/or factor V, with subsequent risk of cross-reactivity with human factor V, and hemorrhagic complications associated with human factor-V deficiencies have been described in case reports of surgeries in which bovine thrombins were used. This risk is now included in the boxed warning section of the bovine thrombin prescribing information. In 2007 and 2008, 2 new topical thrombins from nonbovine sources received approval for use from the US Food and Drug Administration. The 3 active topical thrombins that are currently marketed are bovine plasma-derived thrombin, human plasma-derived thrombin, and human recombinant thrombin. The purpose of this review was to evaluate the literature on the efficacy and safety of topical thrombins and discuss the pharmacoeconomic considerations associated with their use. PubMed, EMBASE, and International Pharmaceutical Abstracts were searched for relevant papers published in English through October 10,2008, using the terms thrombin, human recombinant thrombin, bovine thrombin, plasma derived thrombin, and topical thrombin. Manufacturer-provided materials were also reviewed. Abstracts and unpublished data, as well as evaluations of sealants, adhesives, glues, and other hemostats that contain thrombin mixed with fibrinogen and other clotting factors, were excluded. Four randomized, double-blind studies involving the active, stand-alone topical thrombins were found. The bovine thrombin involved in these studies was the predecessor to the currently marketed, highly purified bovine formulation. No studies comparing the human products, studies involving the highly purified bovine preparation, or placebo-controlled studies involving bovine thrombin were found. In a Phase III comparison of human recombinant thrombin and

Bufadienolides from Kalanchoe daigremontiana as thrombin inhibitors-In vitro and in silico study.

Science.gov (United States)

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

2017-06-01

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

Clot formation and lysis in platelet rich plasma of healthy donors and patients with resistant hypertension

Directory of Open Access Journals (Sweden)

I. I. Patalakh

2018-04-01

Full Text Available Hemostatic balance in blood is affected by numerous factors, including coagulation and fibrinolytic proteins, the wide spectrum of their inhibitors, and blood cells. Since platelets can participate in contradictory processes, they significantly complicate the whole picture. Therefore, nowadays the development of global assays of hemostasis, which can reflect the physiological process of hemostasis and can be used for point-of-care diagnosis of thrombosis, is crucial. This paper outlines a new approach we used to analyze the capabilities of clot waveform analysis tools to distinguish the response of platelet-rich plasma from healthy donors and patients with arterial hypertension caused by stimulation of coagulation and lysis (with exogenous thrombin and recombinant tissue-type plasminogen activator, respectively. In donor plasma, when the clot degradation was accompanied by 40 IU/ml of recombinant tissue-type plasminogen activator, platelets potentiated fibrinolysis more than coagulation, which ultimately shifts the overall balance to a profibrinolytic state. At the same time, for patients with hypertension, platelets, embedded in clot obtained from platelet-rich plasma, showed a weaker ability to stimulate fibrinolysis. The obtained data gives the evidence that platelets can act not only as procoagulants but also as profibrinolytics. By simultaneously amplifying coagulation and fibrinolysis, making their rates comparable, platelets would control plasma procoagulant activity, thereby regulating local hemostatic balance, the size and lifetime of the clot. Moreover, clot waveform analysis may be used to distinguish the effects of platelet-rich plasma on clotting or lysis of fibrin clots in healthy donors and patients with essential hypertension.

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

Energy Technology Data Exchange (ETDEWEB)

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

2011-09-20

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

Percutaneous treatment of femoral pseudoaneurysms: comparison of fibrin sealant against thrombin

Directory of Open Access Journals (Sweden)

Daniel Mendes Pinto

2013-12-01

Full Text Available INTRODUCTION: Femoral pseudoaneurysms are a complication that occurs in connection with up to 8% of percutaneous procedures. Of the available treatments, ultrasound guided thrombin injection has a high success rate and is well-tolerated by patients. The combination of thrombin and fibrinogen known as fibrin sealant forms a stable clot and can be used to treat pseudoaneurysms, particularly those with complex anatomy and larger size. OBJECTIVE: To compare the results of treating femoral pseudoaneurysm in two ways: Group T was treated with thrombin alone and Group T+F was treated with fibrin sealant (thrombin+fibrinogen. METHODS: A retrospective analysis was conducted of femoral pseudoaneurysm cases treated between January 2005 and December 2012. RESULTS: Twenty-eight patients were treated, 21 with thrombin alone and seven with fibrin sealant. All patients in group T were treated successfully, but only four patients in group T+F were treated successfully (57.1% success rate in Group T+F, p<0.01. The three cases of failure in group T+F needed surgery and in one of these cases the complication was embolization to the femoral bifurcation. The pseudoaneurysms that were treated with fibrin sealant were larger (25 cm3 in Group T and 57.7 cm3 in Group T+F, p=0.02 and required larger volumes of thrombin (0.5 mL in Group T and 1.0 mL in Group T+F, p<0.01. There was one complication in Group T and two complications in Group T+F (p<0.01. CONCLUSIONS: Irrespective of the small number of cases reviewed, treatment with thrombin alone was superior to treating with fibrin sealant, since it caused few complications and was more effective at correcting pseudoaneurysms.

Plasma fibrin clot properties in the G20210A prothrombin mutation carriers following venous thromboembolism: the effect of rivaroxaban.

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Janion-Sadowska, Agnieszka; Natorska, Joanna; Siudut, Jakub; Ząbczyk, Michal; Stanisz, Andrzej; Undas, Anetta

2017-08-30

We sought to investigate whether the G20210A prothrombin mutation modifies plasma fibrin clot properties in patients after venous thromboembolism (VTE) and how rivaroxaban treatment affects these alterations. We studied 34 prothrombin mutation heterozygous carriers and sex- and age-matched 34 non-carriers, all at least three months since the first VTE episode, before and during treatment with rivaroxaban. Clot permeability (K s ) and clot lysis time (CLT) with or without elimination of thrombin activatable fibrinolysis inhibitor (TAFI) were assessed at baseline, 2-6 hours (h) after and 20-25 h after intake of rivaroxaban (20 mg/day). At baseline, the prothrombin mutation group formed denser clots (K s -12 %, p=0.0006) and had impaired fibrinolysis (CLT +14 %, p=0.004, and CLT-TAFI +13 %, p=0.03) compared with the no mutation group and were similar to those observed in 15 healthy unrelated prothrombin mutation carriers. The G20210A prothrombin mutation was the independent predictor for K s and CLT before rivaroxaban intake. At 2-6 h after rivaroxaban intake, clot properties improved in both G20210A carriers and non-carriers (K s +38 %, and +37 %, CLT -25 % and -25 %, CLT-TAFI -20 % and -24 %, respectively, all pCLT +17 %, CLT-TAFI +13 %, all p<0.001). Rivaroxaban concentration correlated with fibrin clot properties. After 20-25 h since rivaroxaban intake most clot properties returned to baseline. Rivaroxaban-related differences in clot structure were confirmed by scanning electron microscopy images. In conclusion, rivaroxaban treatment, though improves fibrin clot properties, cannot abolish more prothrombotic fibrin clot phenotype observed in prothrombin mutation carriers following VTE.

BjussuSP-I: a new thrombin-like enzyme isolated from Bothrops jararacussu snake venom.

Science.gov (United States)

Sant' Ana, Carolina D; Ticli, Fabio K; Oliveira, Leandro L; Giglio, Jose R; Rechia, Carem G V; Fuly, André L; Selistre de Araújo, Heloisa S; Franco, João J; Stabeli, Rodrigo G; Soares, Andreimar M; Sampaio, Suely V

2008-11-01

A thrombin-like enzyme named BjussuSP-I, isolated from B. jararacussu snake venom, is an acidic single chain glycoprotein with approximately 6% sugar, Mr=61,000 under reducing conditions and pI approximately 3.8, representing 1.09% of the chromatographic A(280) recovery. BjussuSP-I is a glycosylated serine protease containing both N-linked carbohydrates and sialic acid in its structure. BjussuSP-I showed a high clotting activity upon human plasma, which was inhibited by PMSF, leupeptin, heparin and 1,10-phenantroline. This enzyme showed high stability regarding coagulant activity when analyzed at different temperatures (-70 to 37 degrees C), pHs (4.5 to 8.0), and presence of two divalent metal ions (Ca(2+) and Mg(2+)). It also displayed TAME esterase and proteolytic activities toward natural (fibrinogen and fibrin) and synthetic (BAPNA) substrates, respectively, being also inhibited by PMSF and leupeptin. BjussuSP-I can induce production of polyclonal antibodies able to inhibit its clotting activity, but unable to inhibit its proteolytic activity on fibrinogen. The enzyme also showed crossed immunoreactivity against 11 venom samples of Bothrops, 1 of Crotalus, and 1 of Calloselasma snakes, in addition of LAAO isolated from B. moojeni venom. It displayed neither hemorrhagic, myotoxic, edema-inducing profiles nor proteolytic activity on casein. BjussuSP-I showed an N-terminal sequence (VLGGDECDINEHPFLA FLYS) similar to other thrombin-like enzymes from snake venoms. Based on its biochemical, enzymatic and pharmacological characteristics, BjussuSP-I was identified as a new thrombin-like enzyme isoform from Bothrops jararacussu snake venom.

Effects of temperature on bleeding time and clotting time in normal male and female volunteers.

Science.gov (United States)

Valeri, C R; MacGregor, H; Cassidy, G; Tinney, R; Pompei, F

1995-04-01

This study was done to assess the effects of temperature on bleeding time and clotting time in normal male and female volunteers. Open study utilizing normal volunteers. University research laboratory. Fifty-four healthy male and female volunteers, ranging in age from 19 to 35 yrs, who were not receiving medications. The study was done and the samples of venous blood and shed blood collected at the template bleeding time site were obtained at a convenient time for each volunteer. Skin temperature was changed from +20 degrees to +38 degrees C and blood samples were obtained from the antecubital vein of each volunteer. The effect of local skin temperature ranging from +20 degrees to +38 degrees C on bleeding time was evaluated in 38 normal volunteers (19 male and 19 female). Skin temperature was maintained at +20 degrees to +38 degrees C by cooling or warming the forearm. At each temperature, measurements were made of complete blood count, bleeding time, and thromboxane B2 concentrations in shed blood collected at the template bleeding time site and in serum and plasma isolated from blood collected from the antecubital vein. Clotting time studies were measured in 16 normal volunteers (eight male and eight female) at temperatures ranging from +22 degrees to +37 degrees C. At +32 degrees C, the bleeding time was longer and hematocrit was lower in female than in male volunteers. However, at local skin temperatures of < +32 degrees C, both the males and females exhibited significantly increased bleeding times, which were associated with a reduction in shed blood thromboxane B2. Each 1 degree C decrease in temperature was associated with a 15% decrease in the shed blood thromboxane B2 concentration. Clotting times were three times longer at +22 degrees C than at +37 degrees C. Each 1 degree C reduction in the temperature of the clotted blood was associated with a 15% reduction in the serum thromboxane B2 concentration. Our data indicate that during surgical procedures, it

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

Directory of Open Access Journals (Sweden)

Nicolette Prevost

2011-12-01

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

The effects of Aloe vera [gel] on clotting time, prothrombin time and ...

African Journals Online (AJOL)

Background: Published reports on the effects of Aloe vera gel on blood coagulation in experimental animals are relatively scanty. Aim: To determine the effect of Aloe vera gel on clotting time, prothrombin time and plasma fibrinogen concentration in albino Wistar rats. Methods: A total of 28 adult albino Wistar rats divided ...

Clotting of mammalian fibrinogens by papain: a re-examination.

Science.gov (United States)

Doolittle, Russell F

2014-10-28

Papain has long been known to cause the gelation of mammalian fibrinogens. It has also been reported that papain-fibrin is insoluble in dispersing solvents like strong urea or sodium bromide solutions, similar to what is observed with thrombin-generated clots in the presence of factor XIIIa and calcium. In those old studies, both the gelation and subsequent clot stabilization were attributed to papain, although the possibility that the second step might be due to contaminating factor XIII in fibrinogen preparations was considered. I have revisited this problem in light of knowledge acquired over the past half-century about thiol proteases like papain, which mostly cleave peptide bonds, and transglutaminases like factor XIIIa that catalyze the formation of ε-lysyl-γ-glutamyl cross-links. Recombinant fibrinogen, inherently free of factor XIII and other plasma proteins, formed a stable gel when treated with papain alone. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the intermolecular cross-linking in papain-fibrin leads to γ-chain dimers, trimers, and tetramers, just as is the case with thrombin-factor XIIIa-stabilized fibrin. Mass spectrometry of bands excised from gels showed that the cross-linked material is quite different from what occurs with factor XIIIa, however. With papain, the cross-linking occurs between γ chains in neighboring protofibrils becoming covalently linked in a "head-to-tail" fashion by a transpeptidation reaction involving the α-amino group of γ-Tyr1 and a papain cleavage site at γ-Gly403 near the carboxy terminus, rather than by the (reciprocal) "tail-to-tail" manner that occurs with factor XIIIa and that depends on cross-links between γ-Lys406 and γ-Gln398.

Four-factor prothrombin complex concentrate improves thrombin generation and prothrombin time in patients with bleeding complications related to rivaroxaban: a single-center pilot trial.

Science.gov (United States)

Schenk, Bettina; Goerke, Stephanie; Beer, Ronny; Helbok, Raimund; Fries, Dietmar; Bachler, Mirjam

2018-01-01

Direct oral anticoagulants (DOACs) pose a great challenge for physicians in life-threatening bleeding events. The aim of this study was to test the efficacy of reversing the DOAC rivaroxaban using four-factor PCC (prothrombin complex concentrate), a non-specific reversing agent. Patients with life-threatening bleeding events during rivaroxaban treatment were included and administered 25 U kg -1 of PCC. Blood samples were collected immediately prior to as well as after PCC treatment at predefined time intervals. The primary endpoint was defined as the difference in thrombin generation (TG) parameters ETP (endogenous thrombin potential) and C max (peak thrombin generation) prior to and ten minutes subsequent to PCC treatment. Thirteen patients, of whom the majority suffered from intra-cranial haemorrhage (ICH) or subdural haemorrhage (SDH), were included and administered PCC. The results show that the ETP (TG) significantly ( p  = 0.001) improved by 68% and C max (TG) by 54% (p = 0.001) during PCC treatment. In addition, the Quick value (prothrombin time: Quick PT ) significantly improved by 28% and the activated partial thromboplastin time (aPTT) was decreased by 7% ten minutes after PCC administration. C max was reduced at baseline, but not ETP, aPTT or Quick PT . Lag time until initiation (TG, t lag ), thromboelastometry clotting time (CT EXTEM ) and time to peak (TG, t max ) correlated best with measured rivaroxaban levels and were out of normal ranges at baseline, but did not improve after PCC administration. In 77% of the patients bleeding (ICH/SDH-progression) ceased following PCC administration. During the study three participants passed away due to other complications not related to PCC treatment. The possibility of thrombosis formation was also evaluated seven days after administering PCC and no thromboses were found. This study shows that use of PCC improved ETP, C max, Quick PT and aPTT. However, of these parameters, only C max was reduced at the

Comparison of standard fibrinogen measurement methods with fibrin clot firmness assessed by thromboelastometry in patients with cirrhosis.

Science.gov (United States)

Vucelic, Dragica; Jesic, Rada; Jovicic, Snezana; Zivotic, Maja; Grubor, Nikica; Trajkovic, Goran; Canic, Ivana; Elezovic, Ivo; Antovic, Aleksandra

2015-06-01

The Clauss fibrinogen method and thrombin clotting time (TCT) are still routinely used in patients with cirrhosis to define fibrinogen concentration and clotting potential. The thromboelastometric functional fibrinogen FIBTEM assay evaluates the strength of fibrin-based clots in whole blood, providing information on both quantitative deficit and fibrin polymerization disorders. To compare these three methods of assessing fibrinogen in patients with cirrhosis of different aetiologies, characterized by impairment in fibrinogen concentration as well as functional aberrance. Sixty patients with alcoholic and 24 patients with cholestatic cirrhosis were included (Child-Pugh score (CPs)A, n=24; B, n=32; C, n=28). All parameters were compared with those from a control group. Maximum clot firmness (MCF) in the FIBTEM test was assessed in regard to its relevance in detection of qualitative fibrinogen disorders in comparison with results obtained by standard measurement methods, i.e. the Clauss fibrinogen method and TCT. With increased cirrhosis severity, fibrinogen and FIBTEM-MCF levels significantly declined (p=0.002), while TCT was significantly prolonged (p=0.002). In all CPs groups, fibrinogen strongly correlated with FIBTEM-MCF (r=0.77, r=0.72, r=0.74; pmeasurement in cirrhotic patients, especially in evaluating fibrin polymerization disorders in these patients. Further studies are needed to evaluate the usefulness of this assay in predicting bleeding complications in cirrhotic patients as well as monitoring replacement treatment. Copyright © 2015 Elsevier Ltd. All rights reserved.

Honey Bee Venom (Apis mellifera) Contains Anticoagulation Factors and Increases the Blood-clotting Time.

Science.gov (United States)

Zolfagharian, Hossein; Mohajeri, Mohammad; Babaie, Mahdi

2015-12-01

Bee venom (BV) is a complex mixture of proteins and contains proteins such as phospholipase and melittin, which have an effect on blood clotting and blood clots. The mechanism of action of honey bee venom (HBV, Apis mellifera) on human plasma proteins and its anti-thrombotic effect were studied. The purpose of this study was to investigate the anti-coagulation effect of BV and its effects on blood coagulation and purification. Crude venom obtained from Apis mellifera was selected. The anti-coagulation factor of the crude venom from this species was purified by using gel filtration chromatography (sephadex G-50), and the molecular weights of the anti-coagulants in this venom estimated by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Blood samples were obtained from 10 rabbits, and the prothrombin time (PT) and the partial thromboplastin time (PTT) tests were conducted. The approximate lethal dose (LD) values of BV were determined. Crude BV increased the blood clotting time. For BV concentrations from 1 to 4 mg/mL, clotting was not observed even at more than 300 seconds, standard deviations (SDs) = ± 0.71; however, clotting was observed in the control group 13.8 s, SDs = ± 0.52. Thus, BV can be considered as containing anti-coagulation factors. Crude BV is composed 4 protein bands with molecular weights of 3, 15, 20 and 41 kilodalton (kDa), respectively. The LD50 of the crude BV was found to be 177.8 μg/mouse. BV contains anti-coagulation factors. The fraction extracted from the Iranian bees contains proteins that are similar to anti-coagulation proteins, such as phospholipase A2 (PLA2) and melittin, and that can increase the blood clotting times in vitro.

Honey Bee Venom (Apis mellifera Contains Anticoagulation Factors and Increases the Blood-clotting Time

Directory of Open Access Journals (Sweden)

Hossein Zolfagharian

2015-12-01

Full Text Available Objectives: Bee venom (BV is a complex mixture of proteins and contains proteins such as phospholipase and melittin, which have an effect on blood clotting and blood clots. The mechanism of action of honey bee venom (HBV, Apis mellifera on human plasma proteins and its anti-thrombotic effect were studied. The purpose of this study was to investigate the anti-coagulation effect of BV and its effects on blood coagulation and purification. Methods: Crude venom obtained from Apis mellifera was selected. The anti-coagulation factor of the crude venom from this species was purified by using gel filtration chromatography (sephadex G-50, and the molecular weights of the anti-coagulants in this venom estimated by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE. Blood samples were obtained from 10 rabbits, and the prothrombin time (PT and the partial thromboplastin time (PTT tests were conducted. The approximate lethal dose (LD values of BV were determined. Results: Crude BV increased the blood clotting time. For BV concentrations from 1 to 4 mg/mL, clotting was not observed even at more than 300 seconds, standard deviations (SDs = ± 0.71; however, clotting was observed in the control group 13.8 s, SDs = ± 0.52. Thus, BV can be considered as containing anti-coagulation factors. Crude BV is composed 4 protein bands with molecular weights of 3, 15, 20 and 41 kilodalton (kDa, respectively. The LD50 of the crude BV was found to be 177.8 μg/mouse. Conclusion: BV contains anti-coagulation factors. The fraction extracted from the Iranian bees contains proteins that are similar to anti-coagulation proteins, such as phospholipase A2 (PLA2 and melittin, and that can increase the blood clotting times in vitro.

Salmon and human thrombin differentially regulate radicular pain, glial-induced inflammation and spinal neuronal excitability through protease-activated receptor-1.

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Jenell R Smith

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

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

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Jérémy Lagrange

2017-11-01

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

Rational and timely haemostatic interventions following cardiac surgery - coagulation factor concentrates or blood bank products.

Science.gov (United States)

Tang, Mariann; Fenger-Eriksen, Christian; Wierup, Per; Greisen, Jacob; Ingerslev, Jørgen; Hjortdal, Vibeke; Sørensen, Benny

2017-06-01

Cardiac surgery may cause a serious coagulopathy leading to increased risk of bleeding and transfusion demands. Blood bank products are commonly first line haemostatic intervention, but has been associated with hazardous side effect. Coagulation factor concentrates may be a more efficient, predictable, and potentially a safer treatment, although prospective clinical trials are needed to further explore these hypotheses. This study investigated the haemostatic potential of ex vivo supplementation of coagulation factor concentrates versus blood bank products on blood samples drawn from patients undergoing cardiac surgery. 30 adults were prospectively enrolled (mean age=63.9, females=27%). Ex vivo haemostatic interventions (monotherapy or combinations) were performed in whole blood taken immediately after surgery and two hours postoperatively. Fresh-frozen plasma, platelets, cryoprecipitate, fibrinogen concentrate, prothrombin complex concentrate (PCC), and recombinant FVIIa (rFVIIa) were investigated. The haemostatic effect was evaluated using whole blood thromboelastometry parameters, as well as by thrombin generation. Immediately after surgery the compromised maximum clot firmness was corrected by monotherapy with fibrinogen or platelets or combination therapy with fibrinogen. At two hours postoperatively the coagulation profile was further deranged as illustrated by a prolonged clotting time, a reduced maximum velocity and further diminished maximum clot firmness. The thrombin lagtime was progressively prolonged and both peak thrombin and endogenous thrombin potential were compromised. No monotherapy effectively corrected all haemostatic abnormalities. The most effective combinations were: fibrinogen+rFVIIa or fibrinogen+PCC. Blood bank products were not as effective in the correction of the coagulopathy. Coagulation factor concentrates appear to provide a more optimal haemostasis profile following cardiac surgery compared to blood bank products. Copyright © 2017

21 CFR 864.7140 - Activated whole blood clotting time tests.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Activated whole blood clotting time tests. 864.7140 Section 864.7140 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864...

Low-grade endotoxemia and clotting activation in the early phase of pneumonia.

Science.gov (United States)

Cangemi, Roberto; Della Valle, Patrizia; Calvieri, Camilla; Taliani, Gloria; Ferroni, Patrizia; Falcone, Marco; Carnevale, Roberto; Bartimoccia, Simona; D'Angelo, Armando; Violi, Francesco

2016-11-01

Community-acquired pneumonia (CAP) is associated with an increased risk of arterial and venous thrombosis but the underlying pathophysiological mechanisms are still unclear. We investigated if, in patients with CAP, a pro-thrombotic state does exist and its relationship with serum levels of endotoxins. A total of 104 consecutive patients with CAP were prospectively recruited and followed up until discharge. At admission and at discharge, serum endotoxins, systemic markers of clotting activation and zonulin, a marker of gut permeability, were analysed. Hospitalized patients matched for gender, age and comorbidities but without infections were used as control. At admission, CAP patients showed higher plasma levels of F 1 +2 , a marker of thrombin generation (P = 0.023), and lower levels of protein C (PC; P zonulin were higher in CAP patients than controls (P zonulin was detected (R = 0.575; P < 0.001) CONCLUSION: This study provides the first evidence that CAP patients disclose an ongoing pro-thrombotic state and suggests a role for endotoxemia in determining enhanced thrombin generation. © 2016 Asian Pacific Society of Respirology.

The interaction of thrombin with platelet protease nexin

International Nuclear Information System (INIS)

Knupp, C.L.

1989-01-01

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 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

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

Science.gov (United States)

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

2012-10-01

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

Determining Human Clot Lysis Time (in vitro with Plasminogen/Plasmin from Four Species (Human, Bovine, Goat, and Swine

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Omaira Cañas Bermúdez

2015-05-01

Full Text Available Cardiovascular disease is the leading cause of death worldwide, including failures in the plasminogen/plasmin system which is an important factor in poor lysis of blood clots. This article studies the fibrinolytic system in four species of mammals, and it identifies human plasminogen with highest thrombolysis efficiency. It examines plasminogen from four species (human, bovine, goat, and swine and identifies the most efficient one in human clot lysis in vitro. All plasminogens were identically purified by affinity chromatography. Human fibrinogen was purified by fractionation with ethanol. The purification of both plasminogen and fibrinogen was characterized by one-dimensional SDS-PAGE (10%. Human clot formation in vitro and its dissolution by plasminogen/plasmin consisted of determining lysis time from clot formation to its dilution. Purification of proteins showed greater than 95% purity, human plasminogen showed greater ability to lyse clot than animal plasminogen. The article concludes that human plasminogen/plasmin has the greatest catalysis and efficiency, as it dissolves human clot up to three times faster than that of irrational species.

Graft Product for Autologous Peripheral Blood Stem Cell Transplantation Enhances Thrombin Generation and Expresses Procoagulant Microparticles and Tissue Factor.

Science.gov (United States)

Sidibe, Fatoumata; Spanoudaki, Anastasia; Vanneaux, Valerie; Mbemba, Elisabeth; Larghero, Jerome; Van Dreden, Patrick; Lotz, Jean-Pierre; Elalamy, Ismail; Larsen, Annette K; Gerotziafas, Grigoris T

2018-05-01

The beneficial effect of autologous peripheral blood stem cell transplantation (APBSCT) may be compromised by acute vascular complications related to hypercoagulability. We studied the impact of graft product on thrombin generation of normal plasma and the expression of tissue factor (TF) and procoagulant platelet-derived procoagulant microparticles (Pd-MPs) in samples of graft products. Graft products from 10 patients eligible for APBSCT were mixed with platelet-poor plasma (PPP) or platelet-rich plasma (PRP) from healthy volunteers and assessed for in vitro thrombin generation. In control experiments, thrombin generation was assessed in (1) PPP and PRP without any exogenous TF and/or procoagulant phospholipids, (2) PPP with the addition of TF (5 pM) and procoagulant phospholipids (4 μM), (3) in PRP with the addition of TF (5 pM). Graft products were assessed with Western blot assay for TF expression, with a specific clotting assay for TF activity and with flow cytometry assay for Pd-MPs. The graft product enhanced thrombin generation and its procoagulant activity was related to the presence of Pd-MPs and TF. The concentration of Pd-MPs in the graft product was characterized by a significant interindividual variability. The present study reveals the need for a thorough quality control of the graft products regarding their procoagulant potential.

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

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Herrera Maria

2006-04-01

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

Renal function and plasma dabigatran level measured at trough by diluted thrombin time assay

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Marta E. Martinuzzo

2017-02-01

Full Text Available Dabigatran etexilate (direct thrombin inhibitor is effective in preventing embolic stroke in patients with atrial fibrillation. It does not require laboratory control, but given the high renal elimination, its measurement in plasma is important in renal failure. The objectives of the study were to verify the analytical quality of the diluted thrombin time assay for measurement of dabigatran plasma concentration (cc, correlate cc with classic coagulation assays, prothrombin time (PT and activated partial thromboplastin time (APTT, and evaluate them according to the creatinine clearance (CLCr. Forty plasma samples of patients (34 consecutive and 6 suspected of drug accumulation receiving dabigatran at 150 (n = 19 or 110 (n = 21 mg/12 hours were collected. Blood samples were drawn at 10-14 hours of the last intake. Dabigatran concentration was determined by diluted thrombin time (HemosIl DTI, Instrumentation Laboratory (IL. PT and APTT (IL were performed on two fotooptical coagulometers, ACL TOP 300 and 500 (IL. DTI presented intra-assay coefficient of variation < 5.4% and inter-assay < 6%, linearity range 0-493 ng/ml. Patients' cc: median 83 (4-945 ng/ml. Individuals with CLCr in the lowest tertile (22.6-46.1 ml/min showed significantly higher median cc: 308 (49-945, compared to the average 72 (12-190 and highest tertile, 60 (4-118 ng/ml. Correlation between cc and APTT or PT were moderate, r2 = 0.59 and -0.66, p < 0.0001, respectively. DTI test allowed us to quantify plasma dabigatran levels, both in patients with normal or altered renal function, representing a useful tool in clinical situations such as renal failure, pre surgery or emergencies

Genome scan of clot lysis time and its association with thrombosis in a protein C deficient kindred

Science.gov (United States)

Meltzer, M.E.; Hasstedt, S.J.; Vossen, C.Y.; Callas, P.W.; de Groot, Ph.G.; Rosendaal, F.R.; Lisman, T.; Bovill, E.G.

2011-01-01

Summary Background Previously we found increased clot lysis time (CLT), as measured with a plasma-based assay, to increase the risk of venous thrombosis in two population-based case-control studies. Genes influencing CLT are yet unknown. Objectives and Patients/Methods We tested CLT as risk factor for venous thrombosis in Kindred Vermont II (n=346), a pedigree suffering from a high thrombosis risk, partially attributable to a type I protein C deficiency. Furthermore we tested for quantitative trait loci (QTL) for CLT using variance component linkage analysis. Results Protein C deficient family members had shorter CLT than non-deficient members (median CLT 67 versus 75 minutes). One standard deviation increase in CLT increased risk of venous thrombosis 2.4-fold in non-deficient family members. Protein C deficiency without elevated CLT increased risk 6.9-fold. Combining both risk factors yielded a 27.8-fold increased risk. Heritability of CLT was 42-52%. We found suggestive evidence of linkage on chromosome 11 (62 cM), partly explained by the prothrombin 20210A mutation, and on chromosome 13 (52 cM). Thrombin Activatable Fibrinolysis Inhibitor genotypes did not explain the variation in CLT. Conclusion Hypofibrinolysis appears to increase thrombosis risk in this family especially in combination with protein C deficiency. Protein C deficiency is associated with short CLT. CLT is partly genetically regulated. Suggestive QTL were found on chromosome 11 and 13. PMID:21575129

Real-time monitoring of human blood clotting using a lateral excited film bulk acoustic resonator

Science.gov (United States)

Chen, Da; Wang, Jingjng; Wang, Peng; Guo, Qiuquan; Zhang, Zhen; Ma, Jilong

2017-04-01

Frequent assay of hemostatic status is an essential issue for the millions of patients using anticoagulant drugs. In this paper, we presented a micro-fabricated film bulk acoustic sensor for the real-time monitoring of blood clotting and the measurement of hemostatic parameters. The device was made of an Au/ZnO/Si3N4 film stack and excited by a lateral electric field. It operated under a shear mode resonance with the frequency of 1.42 GHz and had a quality factor of 342 in human blood. During the clotting process of blood, the resonant frequency decreased along with the change of blood viscosity and showed an apparent step-ladder curve, revealing the sequential clotting stages. An important hemostatic parameter, prothrombin time, was quantitatively determined from the frequency response for different dilutions of the blood samples. The effect of a typical anticoagulant drug (heparin) on the prothrombin time was exemplarily shown. The proposed sensor displayed a good consistency and clinical comparability with the standard coagulometric methods. Thanks to the availability of direct digital signals, excellent potentials of miniaturization and integration, the proposed sensor has promising application for point-of-care coagulation technologies.

Blood Clotting and Pregnancy

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Full Text Available ... Blood Basics Blood Disorders Anemia Bleeding Disorders Blood Cancers Blood Clots Blood Clotting and Pregnancy Clots and ... Increased maternal age Other medical illness (e.g., cancer, infection) back to top How are Blood Clots ...

Blood Clotting and Pregnancy

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Full Text Available ... For Patients Blood Disorders Blood Clots Blood Clotting & Pregnancy If you are pregnant, or you have just ... The risk of developing a blood clot during pregnancy is increased by the following: Previous blood clots ...

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

LENUS (Irish Health Repository)

Ni Ainle, Fionnuala

2009-08-20

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.

Genome scan of clot lysis time and its association with thrombosis in a protein C-deficient kindred.

Science.gov (United States)

Meltzer, M E; Hasstedt, S J; Vossen, C Y; Callas, P W; DE Groot, Ph G; Rosendaal, F R; Lisman, T; Bovill, E G

2011-07-01

 Previously, we found increased clot-lysis time (CLT), as measured with a plasma-based assay, to increase the risk of venous thrombosis in two population-based case-control studies. The genes influencing CLT are as yet unknown.  We tested CLT as risk factor for venous thrombosis in Kindred Vermont II (n = 346), a pedigree suffering from a high thrombosis risk, partially attributable to a type I protein C deficiency. Furthermore, we tested for quantitative trait loci (QTLs) for CLT, using variance component linkage analysis.  Protein C-deficient family members had shorter CLTs than non-deficient members (median CLT 67 min vs. 75 min). One standard deviation increase in CLT increased the risk of venous thrombosis 2.4-fold in non-deficient family members. Protein C deficiency without elevated CLT increased the risk 6.9-fold. Combining both risk factors yielded a 27.8-fold increased risk. The heritability of CLT was 42-52%. We found suggestive evidence of linkage on chromosome 11 (62 cM), partly explained by the prothrombin 20210A mutation, and on chromosome 13 (52 cM). Thrombin-activatable fibrinolysis inhibitor genotypes did not explain the variation in CLT. Hypofibrinolysis appears to increase thrombosis risk in this family, especially in combination with protein C deficiency. Protein C deficiency is associated with short CLT. CLT is partly genetically regulated. Suggestive QTLs were found on chromosomes 11 and 13. © 2011 International Society on Thrombosis and Haemostasis.

Antithrombotic/anticoagulant and anticancer activities of selected ...

African Journals Online (AJOL)

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2008-02-05

Feb 5, 2008 ... ... plant parts. The thrombin assay and clotting time assays (thrombin-induced and CaCl2- ... twigs, stem, bark and underground parts of plants are .... Cells were seeded in 96-well plates (Nunc, Denmark) at a density of.

Blood Clotting and Pregnancy

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Full Text Available ... with Your Doctor Patient Group Links Advocacy Toolkit Home For Patients Blood Disorders Blood Clots Blood Clotting & Pregnancy If you are pregnant, or you have just had a baby, you are at greater risk of developing a blood clot. Blood clots in pregnant women tend to form in the deep veins of ...

Blood Clotting and Pregnancy

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Full Text Available ... all publications For Patients Blood Basics Blood Disorders Anemia Bleeding Disorders Blood Cancers Blood Clots Blood Clotting and Pregnancy Clots and Travel DVT Myths vs. Facts Blood ...

Blood Clotting and Pregnancy

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

Science.gov (United States)

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

2002-11-04

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

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

Science.gov (United States)

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

2017-01-01

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

Blood Clotting and Pregnancy

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Full Text Available ... blood clots are treated with an anticoagulant, a medicine that prevents the blood from clotting. Certain anticoagulants are safe to use during pregnancy. back to top Are Blood Clots Preventable? There ...

Thrombin regulates components of the fibrinolytic system in human mesangial cells

International Nuclear Information System (INIS)

Villamediana, L.M.; Rondeau, E.; He, C.J.; Medcalf, R.L.; Peraldi, M.N.; Lacave, R.; Delarue, F.; Sraer, J.D.

1990-01-01

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

Electrokinetic and Hemostatic Profiles of Nonwoven Cellulosic/Synthetic Fiber Blends with Unbleached Cotton

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J. Vincent Edwards

2014-11-01

Full Text Available Greige cotton contains waxes and pectin on the outer surface of the fiber that are removed when bleached, but these components present potential wound dressing functionality. Cotton nonwovens blended with hydrophobic and hydrophilic fibers including viscose, polyester, and polypropylene were assessed for clotting activity with thromboelastography (TEG and thrombin production. Clotting was evaluated based on TEG measurements: R (time to initiation of clot formation, K (time from end of R to a 20 mm clot, α (rate of clot formation according to the angle tangent to the curve as K is reached, and MA (clot strength. TEG values correlate to material surface polarity as measured with electrokinetic parameters (ζplateau, Δζ and swell ratio. The material surface polarity (ζplateau varied from −22 to −61 mV. K values and thrombin concentrations were found to be inversely proportional to  ζplateau with an increase in material hydrophobicity. An increase in the swell ratios of the materials correlated with decreased K values suggesting that clotting rates following fibrin formation increase with increasing material surface area due to swelling. Clot strength (MA also increased with material hydrophobicity. Structure/function implications from the observed clotting physiology induced by the materials are discussed.

Blood Clotting and Pregnancy

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Full Text Available ... of articles from the 2017 ASH Annual Meeting Education Program Blood: How I Treat A compendium of Blood articles updated ... Clots Blood Clotting and Pregnancy Clots and Travel DVT Myths vs. Facts Blood ...

Blood Clotting and Pregnancy

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Full Text Available ... Blood Clots Blood Clotting and Pregnancy Clots and Travel DVT Myths vs. Facts Blood Disorder Fact Sheets ... that occurs when a DVT breaks off and travels to the blood vessels of the lungs. DVT ...

Blood Clotting and Pregnancy

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Full Text Available ... Section Action Alerts Advocacy Toolkit Policy News Sickle Cell Disease Initiative Policy Statements Congressional Fellowship Testimony and ... Blood Basics Blood Disorders Anemia Bleeding Disorders Blood Cancers Blood Clots Blood Clotting and Pregnancy Clots and ...

Blood Clotting and Pregnancy

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Full Text Available ... back to top How are Blood Clots in Pregnant Women Treated? Typically, blood clots are treated with ... you think you have one. If you are pregnant and have concerns about blood clots, talk with ...

Blood Clotting and Pregnancy

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Full Text Available ... clot. Blood clots in pregnant women tend to form in the deep veins of the legs or ... potentially dangerous to your baby. Blood clots can form inside the placenta, cutting off blood flow and ...

Modeling thrombin generation: plasma composition based approach.

Science.gov (United States)

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

2014-01-01

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

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

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Romy M W Kremers

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

Plasma Clot Lysis Time and Its Association with Cardiovascular Risk Factors in Black Africans

NARCIS (Netherlands)

Z. de Lange (Zelda); M. Pieters (Marlien); J.C. Jerling (Johann); A. Kruger (Annamarie); D.C. Rijken (Dingeman)

2012-01-01

textabstractStudies in populations of European descent show longer plasma clot lysis times (CLT) in patients with cardiovascular disease (CVD) than in controls. No data are available on the association between CVD risk factors and fibrinolytic potential in black Africans, a group undergoing rapid

APTAMER-BASED SERRS SENSOR FOR THROMBIN DETECTION

Energy Technology Data Exchange (ETDEWEB)

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

2008-07-02

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.

Determination of enoxaparin with rotational thrombelastometry using the prothrombinase-induced clotting time reagent.

Science.gov (United States)

Schaden, Eva; Schober, Andreas; Hacker, Stefan; Spiss, Christian; Chiari, Astrid; Kozek-Langenecker, Sibylle

2010-04-01

Drug monitoring of low molecular weight heparin is generally not recommended, but could be reasonable in critically ill patients, whose risk for bleeding or thrombosis shows a high interpatient variability. Anti-Xa assays are not available around the clock even in central hospitals, whereas rotational thrombelastometry (ROTEM) becomes increasingly used at the bedside. Prothrombinase-induced clotting time (PiCT) reagent allows determination of factor Xa-inhibition in plasma. The aim of our study was to evaluate enoxaparin determination in whole blood with the ROTEM using specific test modifications, including PiCT. After ethics committee's approval, citrated whole blood obtained from overall 16 healthy volunteers was incubated with enoxaparin at 16 different anti-Xa concentrations. Main endpoint was the clotting time (CT) in ROTEM representing initial activation of clot formation. CT was determined in the new PiCT-ROTEM test, in a low-tissue factor-activated modification (LowTF-ROTEM) as well as in the commercially available heparin-sensitive ROTEM assays (HEPTEM and INTEM). In the absence of enoxaparin, CT values were 168.6 +/- 6.1 s (PiCT-ROTEM), 247.3 +/- 18.6 s (LowTF-ROTEM), and -6.2 +/- 7.9 s (INTEM-HEPTEM). A linear dependency (P anti-Xa concentration and CT was found for PiCT-ROTEM, LowTF-ROTEM, and for INTEM-HEPTEM with correlation coefficients of 0.93 for PiCT-ROTEM, 0.94 for LowTF-ROTEM, and 0.81 for INTEM-HEPTEM. This in-vitro experiment demonstrates a strong correlation between enoxaparin anti-Xa concentrations and specific ROTEM tests. These promising assays should be further evaluated for monitoring anticoagulation in high-risk patients in clinical studies.

Host defense peptides of thrombin modulate inflammation and coagulation in endotoxin-mediated shock and Pseudomonas aeruginosa sepsis.

Science.gov (United States)

Kalle, Martina; Papareddy, Praveen; Kasetty, Gopinath; Mörgelin, Matthias; van der Plas, Mariena J A; Rydengård, Victoria; Malmsten, Martin; Albiger, Barbara; Schmidtchen, Artur

2012-01-01

Gram-negative sepsis is accompanied by a disproportionate innate immune response and excessive coagulation mainly induced by endotoxins released from bacteria. Due to rising antibiotic resistance and current lack of other effective treatments there is an urgent need for new therapies. We here present a new treatment concept for sepsis and endotoxin-mediated shock, based on host defense peptides from the C-terminal part of human thrombin, found to have a broad and inhibitory effect on multiple sepsis pathologies. Thus, the peptides abrogate pro-inflammatory cytokine responses to endotoxin in vitro and in vivo. Furthermore, they interfere with coagulation by modulating contact activation and tissue factor-mediated clotting in vitro, leading to normalization of coagulation responses in vivo, a previously unknown function of host defense peptides. In a mouse model of Pseudomonas aeruginosa sepsis, the peptide GKY25, while mediating a modest antimicrobial effect, significantly inhibited the pro-inflammatory response, decreased fibrin deposition and leakage in the lungs, as well as reduced mortality. Taken together, the capacity of such thrombin-derived peptides to simultaneously modulate bacterial levels, pro-inflammatory responses, and coagulation, renders them attractive therapeutic candidates for the treatment of invasive infections and sepsis.

Host defense peptides of thrombin modulate inflammation and coagulation in endotoxin-mediated shock and Pseudomonas aeruginosa sepsis.

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Martina Kalle

Full Text Available Gram-negative sepsis is accompanied by a disproportionate innate immune response and excessive coagulation mainly induced by endotoxins released from bacteria. Due to rising antibiotic resistance and current lack of other effective treatments there is an urgent need for new therapies. We here present a new treatment concept for sepsis and endotoxin-mediated shock, based on host defense peptides from the C-terminal part of human thrombin, found to have a broad and inhibitory effect on multiple sepsis pathologies. Thus, the peptides abrogate pro-inflammatory cytokine responses to endotoxin in vitro and in vivo. Furthermore, they interfere with coagulation by modulating contact activation and tissue factor-mediated clotting in vitro, leading to normalization of coagulation responses in vivo, a previously unknown function of host defense peptides. In a mouse model of Pseudomonas aeruginosa sepsis, the peptide GKY25, while mediating a modest antimicrobial effect, significantly inhibited the pro-inflammatory response, decreased fibrin deposition and leakage in the lungs, as well as reduced mortality. Taken together, the capacity of such thrombin-derived peptides to simultaneously modulate bacterial levels, pro-inflammatory responses, and coagulation, renders them attractive therapeutic candidates for the treatment of invasive infections and sepsis.

Development and comparison of a minimally-invasive model of autologous clot pulmonary embolism in Sprague-Dawley and Copenhagen rats

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Sanapareddy Nina

2010-02-01

Full Text Available Abstract Background Experimental models of pulmonary embolism (PE that produce pulmonary hypertension (PH employ many different methods of inducing acute pulmonary occlusion. Many of these models induce PE with intravenous injection of exogenous impervious objects that may not completely reproduce the physiological properties of autologous thromboembolism. Current literature lacks a simple, well-described rat model of autlogous PE. Objective: Test if moderate-severity autologous PE in Sprague-Dawley (SD and Copenhagen (Cop rats can produce persistent PH. Methods blood was withdrawn from the jugular vein, treated with thrombin-Ca++ and re-injected following pretreatment with tranexamic acid. Hemodynamic values, clot weights and biochemical measurements were performed at 1 and 5 days. Results Infusion of clot significantly increased the right ventricular peak systolic pressure to 45-55 mm Hg, followed by normalization within 24 hours in SD rats, and within 5 days in COP rats. Clot lysis was 95% (24 hours and 97% (5 days in SD rats and was significantly lower in COP rats (70%, 24 hours; 87% 5 days. Plasma D-dimer was elevated in surgical sham animals and was further increased 8 hours after pulmonary embolism. Neither strain showed a significant increase in bronchoalveolar chemotactic activity, myeloperoxidase activity, leukocyte infiltration, or chemokine accumulation, indicating that there was no significant pulmonary inflammation. Conclusions Both SD and COP rats exhibited near complete fibrinolysis of autologous clot PE within 5 days. Neither strain developed persistent PH. Experimental models of PE designed to induce sustained PH and a robust inflammatory response appear to require significant, persistent pulmonary vascular occlusion.

Protein Z efficiently depletes thrombin generation in disseminated intravascular coagulation with poor prognosis.

Science.gov (United States)

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

2016-01-01

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

Comparison of Hemostasis Times With a Kaolin-Based Hemostatic Pad (QuikClot Radial) vs Mechanical Compression (TR Band) Following Transradial Access: A Pilot Prospective Study.

Science.gov (United States)

Roberts, Jonathan S; Niu, Jianli; Pastor-Cervantes, Juan A

2017-10-01

Hemostasis following transradial access (TRA) is usually achieved by mechanical compression. We investigated use of the QuikClot Radial hemostasis pad (Z-Medica) compared with the TR Band (Terumo Medical) to shorten hemostasis after TRA. Thirty patients undergoing TRA coronary angiography and/or percutaneous coronary intervention were randomized into three cohorts post TRA: 10 patients received mechanical compression with the TR Band, 10 patients received 30 min of compression with the QuikClot Radial pad, and 10 patients received 60 min of compression with the QuikClot Radial pad. Times to hemostasis and access-site complications were recorded. Radial artery patency was evaluated 1 hour after hemostasis by the reverse Barbeau's test. There were no differences in patient characteristics, mean dose of heparin (7117 ± 1054 IU), or mean activated clotting time value (210 ± 50 sec) at the end of procedure among the three groups. Successful hemostasis was achieved in 100% of patients with both the 30-min and 60-min compression groups using the QuikClot pad. Hemostasis failure occurred in 50% of patients when the TR Band was initially weaned at the protocol-driven time (40 min after sheath removal). Mean compression time for hemostasis with the TR Band was 149.4 min compared with 30.7 min and 60.9 min for the 30-min and 60-min QuikClot groups, respectively. No radial artery occlusion occurred in any subject at the end of the study. Use of the QuikClot Radial pad following TRA in this pilot trial significantly shortened hemostasis times when compared with the TR Band, with no increased complications noted.

Prediction of recurrent venous thromboembolism by clot lysis time: a prospective cohort study.

Science.gov (United States)

Traby, Ludwig; Kollars, Marietta; Eischer, Lisbeth; Eichinger, Sabine; Kyrle, Paul A

2012-01-01

Venous thromboembolism (VTE) is a chronic disease, which tends to recur. Whether an abnormal fibrinolytic system is associated with an increased risk of VTE is unclear. We assessed the relationship between fibrinolytic capacity (reflected by clot lysis time [CLT]) and risk of recurrent VTE. We followed 704 patients (378 women; mean age 48 yrs) with a first unprovoked VTE for an average of 46 months after anticoagulation withdrawal. Patients with natural coagulation inhibitor deficiency, lupus anticoagulant, cancer, homozygosity for factor V Leiden or prothrombin mutation, or requirement for indefinite anticoagulation were excluded. Study endpoint was symptomatic recurrent VTE. For measurement of CLT, a tissue factor-induced clot was lysed by adding tissue-type plasminogen activator. Time between clot formation and lysis was determined by measuring the turbidity. 135 (19%) patients had recurrent VTE. For each increase in CLT of 10 minutes, the crude relative risk (RR) of recurrence was 1.13 (95% CI 1.02-1.25; p = 0.02) and was 1.08 (95% CI 0.98-1.20; p = 0.13) after adjustment for age and sex. For women only, the adjusted RR was 1.14 (95% CI, 0.91-1.42, p = 0.22) for each increase in CLT of 10 minutes. CLT values in the 4(th) quartile of the female patient population, as compared to values in the 1(st) quartile, conferred a risk of recurrence of 3.28 (95% CI, 1.07-10.05; p = 0.04). No association between CLT and recurrence risk was found in men. Hypofibrinolysis as assessed by CLT confers a moderate increase in the risk of recurrent VTE. A weak association between CLT and risk of recurrence was found in women only.

Prediction of recurrent venous thromboembolism by clot lysis time: a prospective cohort study.

Directory of Open Access Journals (Sweden)

Ludwig Traby

Full Text Available Venous thromboembolism (VTE is a chronic disease, which tends to recur. Whether an abnormal fibrinolytic system is associated with an increased risk of VTE is unclear. We assessed the relationship between fibrinolytic capacity (reflected by clot lysis time [CLT] and risk of recurrent VTE. We followed 704 patients (378 women; mean age 48 yrs with a first unprovoked VTE for an average of 46 months after anticoagulation withdrawal. Patients with natural coagulation inhibitor deficiency, lupus anticoagulant, cancer, homozygosity for factor V Leiden or prothrombin mutation, or requirement for indefinite anticoagulation were excluded. Study endpoint was symptomatic recurrent VTE. For measurement of CLT, a tissue factor-induced clot was lysed by adding tissue-type plasminogen activator. Time between clot formation and lysis was determined by measuring the turbidity. 135 (19% patients had recurrent VTE. For each increase in CLT of 10 minutes, the crude relative risk (RR of recurrence was 1.13 (95% CI 1.02-1.25; p = 0.02 and was 1.08 (95% CI 0.98-1.20; p = 0.13 after adjustment for age and sex. For women only, the adjusted RR was 1.14 (95% CI, 0.91-1.42, p = 0.22 for each increase in CLT of 10 minutes. CLT values in the 4(th quartile of the female patient population, as compared to values in the 1(st quartile, conferred a risk of recurrence of 3.28 (95% CI, 1.07-10.05; p = 0.04. No association between CLT and recurrence risk was found in men. Hypofibrinolysis as assessed by CLT confers a moderate increase in the risk of recurrent VTE. A weak association between CLT and risk of recurrence was found in women only.

Aptamer Based Microsphere Biosensor for Thrombin Detection

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Xudong Fan

2006-08-01

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

Limitations of using synthetic blood clots for measuring in vitro clot capture efficiency of inferior vena cava filters

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Robinson RA

2013-05-01

Full Text Available Ronald A Robinson, Luke H Herbertson, Srilekha Sarkar Das, Richard A Malinauskas, William F Pritchard, Laurence W GrossmanOffice of Science and Engineering Laboratories, Center for Devices and Radiological Health, US Food and Drug Administration, Silver Spring, MD, USAAbstract: The purpose of this study was first to evaluate the clot capture efficiency and capture location of six currently-marketed vena cava filters in a physiological venous flow loop, using synthetic polyacrylamide hydrogel clots, which were intended to simulate actual blood clots. After observing a measured anomaly for one of the test filters, we redirected the focus of the study to identify the cause of poor clot capture performance for large synthetic hydrogel clots. We hypothesized that the uncharacteristic low clot capture efficiency observed when testing the outlying filter can be attributed to the inadvertent use of dense, stiff synthetic hydrogel clots, and not as a result of the filter design or filter orientation. To study this issue, sheep blood clots and polyacrylamide (PA synthetic clots were injected into a mock venous flow loop containing a clinical inferior vena cava (IVC filter, and their captures were observed. Testing was performed with clots of various diameters (3.2, 4.8, and 6.4 mm, length-to-diameter ratios (1:1, 3:1, 10:1, and stiffness. By adjusting the chemical formulation, PA clots were fabricated to be soft, moderately stiff, or stiff with elastic moduli of 805 ± 2, 1696 ± 10 and 3295 ± 37 Pa, respectively. In comparison, the elastic moduli for freshly prepared sheep blood clots were 1690 ± 360 Pa. The outlying filter had a design that was characterized by peripheral gaps (up to 14 mm between its wire struts. While a low clot capture rate was observed using large, stiff synthetic clots, the filter effectively captured similarly sized sheep blood clots and soft PA clots. Because the stiffer synthetic clots remained straight when approaching the

Thermodynamic, Anticoagulant, and Antiproliferative Properties of Thrombin Binding Aptamer Containing Novel UNA Derivative

DEFF Research Database (Denmark)

Kotkowiak, Weronika; Lisowiec-Wachnicka, Jolanta; Grynda, Jakub

2018-01-01

Thrombin is a serine protease that plays a crucial role in hemostasis, fibrinolysis, cell proliferation, and migration. Thrombin binding aptamer (TBA) is able to inhibit the activity of thrombin molecule via binding to its exosite I. This 15-nt DNA oligonucleotide forms an intramolecular, antipar......Thrombin is a serine protease that plays a crucial role in hemostasis, fibrinolysis, cell proliferation, and migration. Thrombin binding aptamer (TBA) is able to inhibit the activity of thrombin molecule via binding to its exosite I. This 15-nt DNA oligonucleotide forms an intramolecular......, antiparallel G-quadruplex structure with a chair-like conformation. In this paper, we report on our investigations on the influence of certain modified nucleotide residues on thermodynamic stability, folding topology, and biological properties of TBA variants. In particular, the effect of single incorporation......-quadruplex thermodynamic and biological stability, and that the effect is strongly position dependent. Interestingly, TBA variants containing the modified nucleotide residues are characterized by unchanged folding topology. Thrombin time assay revealed that incorporation of certain UNA residues may improve G...

The use of thrombin in the radiology department.

LENUS (Irish Health Repository)

Ward, E

2009-03-01

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.

The use of thrombin in the radiology department

Energy Technology Data Exchange (ETDEWEB)

Ward, E.; Buckley, O.; Browne, R.F. [Adelaide and Meath Hospitals incorporating the National Children' s Hospital (AMNCH), Department of Radiology, Dublin 24 (Ireland); Collins, A. [Royal Victoria Hospital, Department of Radiology, Belfast (United Kingdom); Torreggiani, W.C. [Adelaide and Meath Hospitals incorporating the National Children' s Hospital (AMNCH), Department of Radiology, Dublin 24 (Ireland)]|[Adelaide and Meath Hospital, Department of Radiology, Dublin 24 (Ireland)

2009-03-15

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. (orig.)

Topical thrombin preparations and their use in cardiac surgery

Directory of Open Access Journals (Sweden)

Brianne L Dunn

2009-10-01

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 

Blood Clotting and Pregnancy

Science.gov (United States)

... blood clots A genetic predisposition to blood clots Obesity Prolonged immobility (e.g., bedrest, long distance travel) Multiple births Increased maternal age Other medical illness (e.g., cancer, infection) ...

Thrombin-induced increase in albumin permeability across the endothelium

International Nuclear Information System (INIS)

Garcia, J.G.; Siflinger-Birnboim, A.; Bizios, R.; Del Vecchio, P.J.; Fenton, J.W. II; Malik, A.B.

1986-01-01

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

Hypersensitivity to thrombin of platelets from hypercholesterolemic rats

International Nuclear Information System (INIS)

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

1986-01-01

Hypersensitivity of platelets to thrombin has been associated with hypercholesterolemia. The authors have examined the mechanisms involved in this hypersensitivity. Rats were given diets rich in milk fat and containing added cholesterol and taurocholate to produce hypercholesterolemia (HC) (262 +/- 25 mg%) or added sitosterol as a normocholesterolemic control (NC) (89 +/- 6 mg%). Washed platelets were prelabelled with 14 C-serotonin. In the presence of acetylsalicyclic acid (ASA) (to inhibit thromboxane A 2 (TXA 2 ) formation) and creatine phosphate/creatine phosphokinase (CP/CPK) (to remove released ADP), HC platelets aggregated more (26 +/- 1%) and released more 14 C (9.1 +/- 2.0%) than NC platelets (aggregation: 0%, p 14 C release: 1.5 +/- 0.5%, p 2 formation is involved in the hypersensitivity of HC platelets to thrombin. Total binding of 125 I-thrombin to HC platelets was less than that to NC platelets but HC platelets were smaller and had less protein than NC platelets; the thrombin binding per mg platelet protein was the same for HC and NC platelets, indicating that hypersensitivity to thrombin of HC platelets does not result from increased thrombin binding. Thus, hypersensitivity of HC platelets to thrombin is not due to TXA 2 formation, the action of released ADP or increased thrombin binding

Blood Clotting and Pregnancy

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

Energy Technology Data Exchange (ETDEWEB)

Ocaña, Cristina; Valle, Manel del, E-mail: manel.delvalle@uab.cat

2016-03-17

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

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

International Nuclear Information System (INIS)

Ocaña, Cristina; Valle, Manel del

2016-01-01

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

Blood Clotting and Pregnancy

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Synergy in thrombin-graphene sponge for improved hemostatic efficacy and facile utilization.

Science.gov (United States)

Li, Guofeng; Quan, Kecheng; Xu, CongCong; Deng, Bo; Wang, Xing

2018-01-01

Composites are attractive for its potential synergistic effects that can result in high-performance, but the synergy depends on subtle design. In this study, a hemostatic composite, a thrombin/cross-linked graphene sponge (TCGS), was developed through a facile gradient composite strategy. The porous structure of the CGS assures that the thrombin is stably embedded in the TCGS, avoiding a burst release but maintaining its bioactivity. In the synergy between proper thrombin stimulation and the fast absorption of the sponge, TCGS exhibits outstanding hemostatic performance, ultrafast bleeding cessation, within 100s, which is superior to both CGS and equal amounts of native thrombin. Lower or excessive thrombin dosages prolong the bleeding time. The study revealed that the balance between plasma absorption and thrombin stimulation at the interface is critical for improving hemostatic efficacy. TCGS is also highlighted for its biosafety and stability, even after 6 months of storage in environment. This potentially ultra-long shelf life is conducive to its practical applications. Therefore, TCGS not only provides a new strategy for developing a hemostatic composite but also provides a new method and understanding for the design of hemostatic materials. Copyright © 2017 Elsevier B.V. All rights reserved.

Human Thrombin Injection for the Percutaneous Treatment of Iatrogenic Pseudoaneurysms

International Nuclear Information System (INIS)

Elford, Julian; Burrell, Christopher; Freeman, Simon; Roobottom, Carl

2002-01-01

Purpose: Thrombin injection is becoming well established for the percutaneous management of iatrogenic pseudoaneurysms. All the published series to date use bovine thrombin,and there have been reports of adverse immunologic effects following its use. Our study aimed to assess the efficacy of human thrombin injection for pseudoaneurysm occlusion. Methods:Fourteen patients with iatrogenic pseudoaneurysms underwent a color Doppler ultrasound examination to assess their suitability for percutaneous human thrombin injection. Human thrombin 1000 IU was then injected into the pseudoaneurysm sac under sterile conditions and with ultrasound guidance. A further color Doppler ultrasound examination was performed 24 hr later to confirm occlusion. Results: All 14 pseudoaneurysms were successfully occluded by human thrombin injection. In two cases a second injection of thrombin was required,but there were no other complications, and all pseudoaneurysms remained occluded at 24 hr. Conclusion: Ultrasound-guided human thrombin injection is simple to perform, effective and safe. We recommend that human thrombin becomes the agent of choice for percutaneous injection into iatrogenic pseudoaneurysms

Blood Clotting and Pregnancy

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

Science.gov (United States)

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

2016-01-01

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

Blood Clotting and Pregnancy

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Intrinsic clotting factors in dependency of age, sex, body mass index, and oral contraceptives: definition and risk of elevated clotting factor levels.

Science.gov (United States)

Luxembourg, Beate; Schmitt, Joern; Humpich, Marek; Glowatzki, Matthias; Seifried, Erhard; Lindhoff-Last, Edelgard

2009-10-01

Elevated clotting factors have been demonstrated to be a risk factor for venous thromboembolism (VTE). The aim of our study was to investigate the impact of age, sex, body mass index, and oral contraceptives on the clotting factor activities of factors VIII, IX, XI, and XII and their impact on the cutoff definition and risk of VTE associated with elevated clotting factors. Factor VIII, IX, XI, and XII activities were measured in 499 blood donors and 286 patients with VTE. Age and body mass index predicted significantly and independently the clotting factor activities of factors VIII, IX, and XI, whereas use of oral contraceptives predicted factor IX, XI, and XII levels. Percentiles of clotting factor activities, which are often used for the cutoff definition of elevated clotting factors, varied due to the effect of age, body mass index, and oral contraceptives. The adjusted odds ratios for VTE were 10.3 [95% confidence interval (CI) 5.1-20.7], 6.1 (95% CI 3.1-12.0), and 3.3 (95% CI 1.9-5.8) for elevated factors VIII, IX, and XI, respectively. Furthermore, our study demonstrates for the first time that elevated factor XII is associated with an increased risk of VTE (adjusted odds ratio 2.9, 95% CI 1.6-5.3).

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

LENUS (Irish Health Repository)

Tobin, W O

2013-02-01

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.

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

Science.gov (United States)

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

2017-01-01

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

Activation of human factor V by factor Xa and thrombin

International Nuclear Information System (INIS)

Monkovic, D.D.; Tracy, P.B.

1990-01-01

The activation of human factor V by factor Xa and thrombin was studied by functional assessment of cofactor activity and sodium dodecyl sulfate-polycarylamide gel electrophoresis followed by either autoradiography of 125 I-labeled factor V activation products or Western blot analyses of unlabeled factor V activation products. Cofactor activity was measured by the ability of the factor V/Va peptides to support the activation of prothrombin. The factor Xa catalyzed cleavage of factor V was observed to be time, phospholipid, and calcium ion dependent, yielding a cofactor with activity equal to that of thrombin-activated factor V (factor Va). The cleavage pattern differed markedly from the one observed in the bovine system. The factor Xa activated factor V subunits expressing cofactor activity were isolated and found to consist of peptides of M r 220,000 and 105,000. Although thrombin cleaved the M r 220,000 peptide to yield peptides previously shown to be products of thrombin activation, cofactor activity did not increase. N-Terminal sequence analysis confirmed that both factor Xa and thrombin cleave factor V at the same bond to generate the M r 220,000 peptide. The factor Xa dependent functional assessment of 125 I-labeled factor V coupled with densitometric analyses of the cleavage products indicated that the cofactor activity of factor Xa activated factor V closely paralleled the appearance of the M r 220,000 peptide. The data indicate that factor Xa is as efficient an enzyme toward factor V as thrombin

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

Science.gov (United States)

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

2010-04-01

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

Fluid Mechanics of Blood Clot Formation.

Science.gov (United States)

Fogelson, Aaron L; Neeves, Keith B

2015-01-01

Intravascular blood clots form in an environment in which hydrodynamic forces dominate and in which fluid-mediated transport is the primary means of moving material. The clotting system has evolved to exploit fluid dynamic mechanisms and to overcome fluid dynamic challenges to ensure that clots that preserve vascular integrity can form over the wide range of flow conditions found in the circulation. Fluid-mediated interactions between the many large deformable red blood cells and the few small rigid platelets lead to high platelet concentrations near vessel walls where platelets contribute to clotting. Receptor-ligand pairs with diverse kinetic and mechanical characteristics work synergistically to arrest rapidly flowing cells on an injured vessel. Variations in hydrodynamic stresses switch on and off the function of key clotting polymers. Protein transport to, from, and within a developing clot determines whether and how fast it grows. We review ongoing experimental and modeling research to understand these and related phenomena.

Pro blood clotting activity of Scoparia dulcis in rats.

Science.gov (United States)

Ediriweera, E R H S S; Jayakody, J R A C; Ratnasooriya, W D

2011-04-01

Scoparia dulcis Linn (Family: Scrophulariaceae, Sinhala: WalKoththamalli) is a perennial herb growing in many tropical countries including Sri Lanka. Traditional Physicians in rural down south areas apply crushed S. dulcis plant on cuts and bruises to stop bleeding. S. dulcis may also have Rakta Sthambhana property. The study on effect of decoction (water extract) of S. dulcis on blood clotting time in rats was carried out to investigate this. Two groups of rats, 12 males and 42 females were used in this experimental study. Forty-two female rats were assigned into seven equal groups (n = 6/gp). Different doses of DE (25, 50, 100, 1000, 1500 mg/kg) (group 1-5) or 2 ml of distilled water (DW) (group 6) were orally administered. 0.1 ml of vitamin K was injected intramuscularly (group 7) as reference drug to seventh the group. Twelve male rats were assigned into two equal groups (n = 6/gp), 2 ml of distilled water (DW) and doses of DE (1500 mg/kg) were orally administered. Clotting time was determined on the Days 1, 2, and 7 using Lee and White method. In the DE treated groups with all doses, there was no reduction in clotting time on the Day 1 but a significant reduction of clotting time (P dulcis has proclotting activity (rakthasthambhana property) and this was faster than vitamin K.

Case series: Bladder clot evacuation using a prostate morcellation device.

Science.gov (United States)

Doersch, Karen M; Navetta, Andrew F; Bird, Erin T; El Tayeb, Marawan M

2017-07-01

We sought to provide a technical update on the use of a prostate morcellator device (PMD) to manage organized blood clots of the bladder following laser prostatectomy. Herein, we describe our experience in using the Wolf Piranha morcellator in managing organized bladder blood clots supplemented with a retrospective chart review of the patients in whom this procedure was performed. Six patients, all male with a mean age of 75 ± 8.9 years, had organized bladder clots following either holmium laser enucleation or photoselective vaporization of the prostate managed with a PMD. Clots were recognized based on hematuria or urinary retention a median of 3.5 days following the aforementioned procedures. Initial management was attempted with more conservative measures, including a three-way Foley catheter, followed by cystoscopy with an Ellik evacuator, or a glass Tommey syringe. Morcellation times were a mean of 10.2 ± 6.15 minutes (range 2-18). This technique was able to manage clots that were an average of 173.3 ± 115.9 cc in size. The procedure was well-tolerated. No patients experienced intraoperative or morcellator-related complications. Benign prostatic hypertrophy frequently requires surgical endoscopic management and can be complicated by hematuria and bladder blood clot formation. When these clots become organized, this can lead to urinary retention and the required management, evacuation, may be difficult. The use of a Wolf Piranha PMD is a safe, well-tolerated, and effective in evacuating organized blood clots of the bladder.

RADIOLOGICAL TIPS Percutaneous thrombin injection for ...

African Journals Online (AJOL)

pseudoaneurysm sac has a typical 'yin-yang' sign. The neck is normally seen posteriorly and is usually thin and longitudinal. A large neck diameter (e.g. >10 mm) is a relative contra-indication for thrombin injection because of a slightly higher risk of distal embolisation. There are several thrombin preparations available.

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

DEFF Research Database (Denmark)

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

2016-01-01

surgery including cardiopulmonary bypass. Thrombin generation was analysed both in platelet-poor plasma and platelet-rich plasma. Analysis of the thrombin generation showed a significantly prolonged lag time (Pplatelet-poorandplatelet-richplasma ... thrombin generation significantly (all P rich plasma...

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

Science.gov (United States)

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

2017-11-01

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

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

Science.gov (United States)

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

2017-06-01

Progestin-only contraceptives induce abnormal uterine bleeding, accompanied by prothrombin leakage from dilated endometrial microvessels and increased thrombin generation by human endometrial stromal cell (HESC)-expressed tissue factor. Initial studies of the thrombin-treated HESC secretome identified elevated levels of cleaved chondroitin sulfate proteoglycan 4 (CSPG4), impairing pericyte-endothelial interactions. Thus, we investigated direct and CSPG4-mediated effects of thrombin in eliciting abnormal uterine bleeding by disrupting endometrial angiogenesis. Liquid chromatography/tandem mass spectrometry, enzyme-linked immunosorbent assay (ELISA) and quantitative real-time-polymerase chain reaction (PCR) evaluated conditioned medium supernatant and cell lysates from control versus thrombin-treated HESCs. Pre- and post-Depo medroxyprogesterone acetate (DMPA)-administered endometria were immunostained for CSPG4. Proliferation, apoptosis and tube formation were assessed in human endometrial endothelial cells (HEECs) incubated with recombinant human (rh)-CSPG4 or thrombin or both. Thrombin induced CSPG4 protein expression in cultured HESCs as detected by mass spectrometry and ELISA (pabnormal uterine bleeding in DMPA users. Mass spectrometry analysis identified several HESC-secreted proteins regulated by thrombin. Therapeutic agents blocking angiogenic effects of thrombin in HESCs can prevent or minimize progestin-only contraceptive-induced abnormal uterine bleeding. Copyright © 2017. Published by Elsevier Inc.

Topical thrombin-related corneal calcification.

Science.gov (United States)

Kiratli, Hayyam; Irkeç, Murat; Alaçal, Sibel; Söylemezoğlu, Figen

2006-09-01

To report a highly unusual case of corneal calcification after brief intraoperative use of topical thrombin. A 44-year-old man underwent sclerouvectomy for ciliochoroidal leiomyoma, during which 35 UNIH/mL lyophilized bovine thrombin mixed with 9 mL of diluent containing 1500 mmol/mL calcium chloride was used. From the first postoperative day, corneal and anterior lenticular capsule calcifications developed, and corneal involvement slightly enlarged thereafter. A year later, 2 corneal punch biopsies confirmed calcification mainly in the Bowman layer. Topical treatment with 1.5% ethylenediaminetetraacetic acid significantly restored corneal clarity. Six months later, a standard extracapsular cataract extraction with intraocular lens placement improved visual acuity to 20/60. This case suggests that topical thrombin drops with elevated calcium concentrations may cause acute corneal calcification in Bowman layer and on the anterior lens capsule.

Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper

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A.V Pérez

2008-01-01

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.

Kaempferol inhibits thrombosis and platelet activation.

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Choi, Jun-Hui; Park, Se-Eun; Kim, Sung-Jun; Kim, Seung

2015-08-01

The objectives of the present study were to investigate whether kaempferol affects pro-coagulant proteinase activity, fibrin clot formation, blood clot and thrombin (or collagen/epinephrine)-stimulated platelet activation, thrombosis, and coagulation in ICR (Imprinting Control Region) mice and SD (Sprague-Dawley) rats. Kaempferol significantly inhibited the enzymatic activities of thrombin and FXa by 68 ± 1.6% and 52 ± 2.4%, respectively. Kaempferol also inhibited fibrin polymer formation in turbidity. Microscopic analysis was performed using a fluorescent conjugate. Kaempferol completely attenuated phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38, c-Jun N-terminal kinase (JNK) 1/2, and phosphoinositide 3-kinase (PI3K)/PKB (AKT) in thrombin-stimulated platelets and delayed aggregation time (clotting) by 34.6% in an assay of collagen/epinephrine-stimulated platelet activation. Moreover, kaempferol protected against thrombosis development in 3 animal models, including collagen/epinephrine- and thrombin-induced acute thromboembolism models and an FeCl3-induced carotid arterial thrombus model. The ex vivo anticoagulant effect of kaempferol was further confirmed in ICR mice. This study demonstrated that kaempferol may be clinically useful due to its ability to reduce or prevent thrombotic challenge. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Label-free aptamer biosensor for selective detection of thrombin

Energy Technology Data Exchange (ETDEWEB)

Na, Weidan; Liu, Xiaotong; Wang, Lei; Su, Xingguang, E-mail: suxg@jlu.edu.cn

2015-10-29

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

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

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

2012-05-15

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

Thrombin stimulates albumin transcytosis in lung microvascular endothelial cells via activation of acid sphingomyelinase.

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Kuebler, Wolfgang M; Wittenberg, Claudia; Lee, Warren L; Reppien, Eike; Goldenberg, Neil M; Lindner, Karsten; Gao, Yizhuo; Winoto-Morbach, Supandi; Drab, Marek; Mühlfeld, Christian; Dombrowsky, Heike; Ochs, Matthias; Schütze, Stefan; Uhlig, Stefan

2016-04-15

Transcellular albumin transport occurs via caveolae that are abundant in lung microvascular endothelial cells. Stimulation of albumin transcytosis by proinflammatory mediators may contribute to alveolar protein leak in lung injury, yet the regulation of albumin transport and its underlying molecular mechanisms are so far incompletely understood. Here we tested the hypothesis that thrombin may stimulate transcellular albumin transport across lung microvascular endothelial cells in an acid-sphingomyelinase dependent manner. Thrombin increased the transport of fluorescently labeled albumin across confluent human lung microvascular endothelial cell (HMVEC-L) monolayers to an extent that markedly exceeds the rate of passive diffusion. Thrombin activated acid sphingomyelinase (ASM) and increased ceramide production in HMVEC-L, but not in bovine pulmonary artery cells, which showed little albumin transport in response to thrombin. Thrombin increased total caveolin-1 (cav-1) content in both whole cell lysates and lipid rafts from HMVEC-L, and this effect was blocked by inhibition of ASM or de novo protein biosynthesis. Thrombin-induced uptake of albumin into lung microvascular endothelial cells was confirmed in isolated-perfused lungs by real-time fluorescence imaging and electron microscopy of gold-labeled albumin. Inhibition of ASM attenuated thrombin-induced albumin transport both in confluent HMVEC-L and in intact lungs, whereas HMVEC-L treatment with exogenous ASM increased albumin transport and enriched lipid rafts in cav-1. Our findings indicate that thrombin stimulates transcellular albumin transport in an acid sphingomyelinase-dependent manner by inducing de novo synthesis of cav-1 and its recruitment to membrane lipid rafts. Copyright © 2016 the American Physiological Society.

THE FLOCCULATION MAXIMUM (pH) OF FIBRINOGEN AND SOME OTHER BLOOD-CLOTTING REAGENTS. (RELATIVE TURBIDIMETRY WITH THE EVELYN PHOTOELECTRIC COLORIMETER).

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Ferguson, J H

1942-03-20

By means of a novel adaptation of the Evelyn photoelectric colorimeter to the measurement of relative turbidities, the question of the flocculation maximum (F.M.) in acetate buffer solutions of varying pH and salt content has been studied on (a) an exceptionally stable prothrombin-free fibrinogen and its solutions after incipient thermal denaturation and incomplete tryptic proteolysis, (b) plasma, similarly treated, (c) prothrombin, thrombin, and (brain) thromboplastin solutions. All the fibrinogens show a remarkable uniformity of the precipitation pattern, viz. F.M. =4.7 (+/-0.2) pH in salt-containing buffer solutions and pH = 5.3 (+/-0.2) in salt-poor buffer (N/100 acetate). The latter approximates the isoelectric point (5.4) obtained by cataphoresis (14). There is no evidence that denaturation or digestion can produce any "second maximum." The data support the view that fibrin formation (under the specific influence of thrombin) is intrinsically unrelated to denaturation and digestion phenomena, although all three can proceed simultaneously in crude materials. A criticism is offered, therefore, of Wöhlisch's blood clotting theory. Further applications of the photoelectric colorimeter to coagulation problems are suggested, including kinetic study of fibrin formation and the assay of fibrinogen, with a possible sensitivity of 7.5 mg. protein in 100 cc. solution.

Thrombin-receptor antagonist vorapaxar in acute coronary syndromes

DEFF Research Database (Denmark)

Tricoci, Pierluigi; Huang, Zhen; Held, Claes

2012-01-01

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

Thrombin induces rapid PAR1-mediated non-classical FGF1 release

International Nuclear Information System (INIS)

Duarte, Maria; Kolev, Vihren; Soldi, Raffaella; Kirov, Alexander; Graziani, Irene; Oliveira, Silvia Marta; Kacer, Doreen; Friesel, Robert; Maciag, Thomas; Prudovsky, Igor

2006-01-01

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

Thrombin binding to human brain and spinal cord

International Nuclear Information System (INIS)

McKinney, M.; Snider, R.M.; Richelson, E.

1983-01-01

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

Blood Clots

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... sitting for long periods. If you travel by airplane, walk the aisle periodically. For long car trips, ... Your-Risk-for-Excessive-Blood-Clotting_UCM_448771_Article.jsp. Accessed April 18, 2016. What causes excessive ...

Low level of procoagulant platelet microparticles is associated with impaired coagulation and transfusion requirements in trauma patients

DEFF Research Database (Denmark)

Windeløv, Nis Agerlin; Johansson, Pär Ingemar; Sørensen, Anne Marie

2014-01-01

BACKGROUND: Following activation, platelets release small vesicles called platelet-derived microparticles (PMPs). PMPs accelerate thrombin generation and thus clot formation at sites of injury by exposing the procoagulant membrane phospholipid phosphatidylserine (PS). The role of PMPs in coagulop......BACKGROUND: Following activation, platelets release small vesicles called platelet-derived microparticles (PMPs). PMPs accelerate thrombin generation and thus clot formation at sites of injury by exposing the procoagulant membrane phospholipid phosphatidylserine (PS). The role of PMPs...

Progestin and thrombin regulate tissue factor expression in human term decidual cells.

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Lockwood, C J; Murk, W; Kayisli, U A; Buchwalder, L F; Huang, S-T; Funai, E F; Krikun, G; Schatz, F

2009-06-01

Perivascular cell membrane-bound tissue factor (TF) initiates hemostasis via thrombin generation. The identity and potential regulation of TF-expressing cells at the human maternal-fetal interface that confers hemostatic protection during normal and preterm delivery is unclear. The objective of the study were to identify TF-expressing cells at the maternal-fetal interface in term and preterm decidual sections by immunohistochemistry and evaluate progestin, thrombin, TNF-alpha, and IL-1beta effects on TF expression by cultured human term decidual cells (DCs). Serial placental sections were immunostained for TF. Leukocyte-free term DC monolayers were incubated with 10(-8) M estradiol (E2) or E2 plus 10(-7) M medroxyprogestrone acetate (MPA) +/- thrombin or TNF-alpha or IL-1beta. ELISA and Western blotting assessed TF in cell lysates. Quantitative real-time RT-PCR measured TF mRNA levels. Immunolocalized TF in DC membranes in preterm and term placental sections displayed higher Histologic Scores than villous mesenchymal cells (P term placental sections, DC-expressed TF exceeds that of other cell types at the maternal-fetal interface and is localized at the cell membranes in which it can bind to factor VII and meet the hemostatic demands of labor and delivery via thrombin formation. Unlike the general concept that TF is constitutive in cells that highly express it, MPA and thrombin significantly enhanced TF expression in term DC monolayers.

Synergistic action between inhibition of P2Y12/P2Y1 and P2Y12/thrombin in ADP- and thrombin-induced human platelet activation

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Nylander, Sven; Mattsson, Christer; Ramström, Sofia; Lindahl, Tomas L

2004-01-01

The objective of this study was to investigate if there is a synergistic effect of a combination of P2Y12 and P2Y1 inhibition and P2Y12 and thrombin inhibition, on ADP- and thrombin-induced platelet activation, respectively. The rationale being that these combinations will cause a concurrent inhibition of both Gαq and Gαi signalling.Blood from healthy volunteers was preincubated with AR-C69931MX, a reversible P2Y12 antagonist; MRS2179, a reversible P2Y1 antagonist; or melagatran, a direct reversible thrombin inhibitor; alone or in various combinations prior to activation with ADP or thrombin. Platelet function in whole blood was assessed by flow cytometry using the antibody PAC-1 to estimate the expression of active αIIbβ3 (the fibrinogen receptor GPIIb/IIIa). A synergistic effect was evaluated by comparing the concentrations in the different combinations with those of corresponding equipotent concentrations of each single inhibitor alone. The equipotent single concentrations were experimentally obtained from concentration response curves performed in parallel.A synergistic effect regarding inhibition of ADP-induced platelet activation (10 μM) was obtained with different combinations of AR-C69931MX and MRS2179.Inhibition of thrombin-induced platelet activation (2 nM) with combinations of AR-C69931MX and the thrombin inhibitor melagatran did also result in a strong synergistic effect.To our knowledge, this is the first time that data supporting a synergistic effect has been published for the inhibitor combinations described.Whether this synergistic effect in vitro also results in an improved antithrombotic effect in vivo with or without an increased risk of bleeding remains to be studied in well-conducted clinical studies. PMID:15265806

Ultrastructure of clots during isometric contraction

OpenAIRE

1982-01-01

We explored the retraction or contraction of platelet-fibrin clots under isometric conditions. In the presence of micromolar calcium clots of normal platelet-rich plasma developed tension at an initial rate of 0.1 to 0.2 g/min per cm2 (initial cross-sectional area). Electron microscopy of clots fixed after attaining a force of 1.6 g/cm2 revealed platelets with elongated bodies and pseudopods in close apposition to fibrin strands which were oriented in cablelike fashion in the direction of ten...

Blood Clots

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... Pregnancy Immobility (including prolonged inactivity, long trips by plane or car ) Smoking Oral contraceptives Certain cancers Trauma Certain surgeries Age (increased risk for people over age 60) A family history of blood clots Chronic inflammatory diseases Diabetes High ...

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

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

1992-01-01

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

Comparison of coagulation activity tests in vitro for selected biomaterials

NARCIS (Netherlands)

van Oeveren, W; Haan, J; Lagerman, P; Schoen, T

Testing of coagulation induced by external communicating medical devices is an International Standardisation Organization (ISO) requirement for products exposed to human blood. Four categories of tests are indicated by ISO 10993/4: a clotting test (partial thromboplastin time; PTT), thrombin

Formation of blood clot on biomaterial implants influences bone healing.

Science.gov (United States)

Shiu, Hoi Ting; Goss, Ben; Lutton, Cameron; Crawford, Ross; Xiao, Yin

2014-12-01

The first step in bone healing is forming a blood clot at injured bones. During bone implantation, biomaterials unavoidably come into direct contact with blood, leading to a blood clot formation on its surface prior to bone regeneration. Despite both situations being similar in forming a blood clot at the defect site, most research in bone tissue engineering virtually ignores the important role of a blood clot in supporting healing. Dental implantology has long demonstrated that the fibrin structure and cellular content of a peri-implant clot can greatly affect osteoconduction and de novo bone formation on implant surfaces. This article reviews the formation of a blood clot during bone healing in relation to the use of platelet-rich plasma (PRP) gels. It is implicated that PRP gels are dramatically altered from a normal clot in healing, resulting in conflicting effect on bone regeneration. These results indicate that the effect of clots on bone regeneration depends on how the clots are formed. Factors that influence blood clot structure and properties in relation to bone healing are also highlighted. Such knowledge is essential for developing strategies to optimally control blood clot formation, which ultimately alter the healing microenvironment of bone. Of particular interest are modification of surface chemistry of biomaterials, which displays functional groups at varied composition for the purpose of tailoring blood coagulation activation, resultant clot fibrin architecture, rigidity, susceptibility to lysis, and growth factor release. This opens new scope of in situ blood clot modification as a promising approach in accelerating and controlling bone regeneration.

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

Science.gov (United States)

Kamisato, Chikako; Furugohri, Taketoshi; Morishima, Yoshiyuki

2016-05-01

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

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

International Nuclear Information System (INIS)

Cooper, J.A.; Feustel, P.J.; Line, B.R.; Malik, A.B.

1986-01-01

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

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

Directory of Open Access Journals (Sweden)

Kathleen E Brummel-Ziedins

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

New synthetic thrombin inhibitors: molecular design and experimental verification.

Science.gov (United States)

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

2011-01-01

The development of new anticoagulants is an important goal for the improvement of thromboses treatments. The design, synthesis and experimental testing of new safe and effective small molecule direct thrombin inhibitors for intravenous administration. Computer-aided molecular design of new thrombin inhibitors was performed using our original docking program SOL, which is based on the genetic algorithm of global energy minimization in the framework of a Merck Molecular Force Field. This program takes into account the effects of solvent. The designed molecules with the best scoring functions (calculated binding energies) were synthesized and their thrombin inhibitory activity evaluated experimentally in vitro using a chromogenic substrate in a buffer system and using a thrombin generation test in isolated plasma and in vivo using the newly developed model of hemodilution-induced hypercoagulation in rats. The acute toxicities of the most promising new thrombin inhibitors were evaluated in mice, and their stabilities in aqueous solutions were measured. New compounds that are both effective direct thrombin inhibitors (the best K(I) was 50) in the thrombin generation assay of approximately 100 nM) were discovered. These compounds contain one of the following new residues as the basic fragment: isothiuronium, 4-aminopyridinium, or 2-aminothiazolinium. LD(50) values for the best new inhibitors ranged from 166.7 to >1111.1 mg/kg. A plasma-substituting solution supplemented with one of the new inhibitors prevented hypercoagulation in the rat model of hemodilution-induced hypercoagulation. Activities of the best new inhibitors in physiological saline (1 µM solutions) were stable after sterilization by autoclaving, and the inhibitors remained stable at long-term storage over more than 1.5 years at room temperature and at 4°C. The high efficacy, stability and low acute toxicity reveal that the inhibitors that were developed may be promising for potential medical applications.

Changes to the structure of blood clots formed in the presence of fine particulate matter

International Nuclear Information System (INIS)

Metassan, Sofian; Routledge, Michael N; Ariens, Robert A S; Scott, D Julian

2009-01-01

Both long-term and short-term exposure (one to two hours) to particulate matter are associated with morbidity and mortality caused by cardiovascular diseases. The underlying mechanisms leading to cardiovascular events are unclear, however, changes to blood coagulability upon exposure to ultrafine particulate matter (UFPM, the smallest of which can enter the circulation) is a plausible mechanism. Objectives: This study aims to investigate the direct effects of particulate matter on fibrin polymerization, lateral aggregation and the formation of fibrin network structure. Methods: Standard Urban Particulate Matter (PM) was suspended in Tris buffer centrifuged and filtered with <200nm filter to obtain ultrafine PM or their water-soluble components. Purified normal fibrinogen was made to clot by adding thrombin and calcium chloride in the presence of varying concentrations of PM. Permeation properties (Darcy constant [Ks]) and turbidity of clots were measured to investigate the effects on flow-rate, pore size, and fibrin polymerization. In addition, confocal microscopy was performed to study detailed clot structure. Results: Total PM increased the Ks of clots in a dose dependant manner (Ks = 4.4, 6.9 and 13.2 x 10-9 cm2 for 0, 50 and 100 |ag/ml total PM concentrations, respectively). Filtered PM also produced a significant increase in Ks at PM concentration of 17 |ag/ml. Final turbidity measurements at 20min were obtained for varying concentrations of PM. Maximum optical density (OD) for 1 mg/ml fibrinogen at 0, 50, 100 and 200 |ag/ml total PM concentrations were 0.39, 0.42, 0.45 and 0.46, respectively. The maximum OD for 0, 17, 34 and 68 |ag/ml filtered PM concentrations were 0.39, 0.42 0.47 and 0.51, respectively, suggesting an increase in fibre diameter with increasing particulate concentration. The lag phase was significantly shorter and the rate of polymerisation was significantly faster in the presence of 68 |ag/ml filtered PM. Confocal microscopy results showed

Thermodynamic, Anticoagulant, and Antiproliferative Properties of Thrombin Binding Aptamer Containing Novel UNA Derivative

Directory of Open Access Journals (Sweden)

Weronika Kotkowiak

2018-03-01

Full Text Available Thrombin is a serine protease that plays a crucial role in hemostasis, fibrinolysis, cell proliferation, and migration. Thrombin binding aptamer (TBA is able to inhibit the activity of thrombin molecule via binding to its exosite I. This 15-nt DNA oligonucleotide forms an intramolecular, antiparallel G-quadruplex structure with a chair-like conformation. In this paper, we report on our investigations on the influence of certain modified nucleotide residues on thermodynamic stability, folding topology, and biological properties of TBA variants. In particular, the effect of single incorporation of a novel 4-thiouracil derivative of unlocked nucleic acid (UNA, as well as single incorporation of 4-thiouridine and all four canonical UNAs, was evaluated. The studies presented herein have shown that 4-thiouridine in RNA and UNA series, as well as all four canonical UNAs, can efficiently modulate G-quadruplex thermodynamic and biological stability, and that the effect is strongly position dependent. Interestingly, TBA variants containing the modified nucleotide residues are characterized by unchanged folding topology. Thrombin time assay revealed that incorporation of certain UNA residues may improve G-quadruplex anticoagulant properties. Noteworthy, some TBA variants, characterized by decreased ability to inhibit thrombin activity, possess significant antiproliferative properties reducing the viability of the HeLa cell line even by 95% at 10 μM concentration.

Percutaneous Ultrasound-Guided Thrombin Injection in Iatrogenic Arterial Pseudoaneurysms: Effectiveness and Complications

International Nuclear Information System (INIS)

Koh, Young Hwan; Kim, Hak Soo; Kim, Hyung Sik; Min, Seung Kee

2005-01-01

To evaluate and describe the efficacy and side effects of a percutaneous thrombin injection under ultrasonography guidance for the treatment of iatrogenic pseudo aneurysms Eighteen consecutive iatrogenic pseudo aneurysm cases were treated with a thrombin injection. The thrombin was injected into the pseudo aneurysm cavity using a 22-gauge needle under ultrasonographic guidance. The causes of the pseudo aneurysms are as follows: post coronary angiography (9 cases), percutaneous coronary balloon angioplasty (5 cases), cerebral angiography (1 case), transhepatic chemo embolization (1 case), percutaneous trans femoral arterial stent insertion (1 case) and bone marrow aspiration for a marrow transplant (1 case). Only one case required a secondary thrombin injection due to recurrent flow in the pseudo aneurysm lumen, which was detected at the follow up Doppler ultrasound. Other seventeen cases were successfully treated on the first trial. There were no technical failures or complication related to the procedure. The average amount of thrombin injected was 733 IU. Nine out of 18 treated patients (50%) showed mild reactions to the thrombin including mild fever (4 cases), chilling sensation (3 cases), a chilling sensation with mild dyspnea (1 case), mild chest discomfort (1 case) after the thrombin injection. All these side effects were transient and improved several hours later. All the iatrogenic pseudo aneurysms were treated successfully with an ultrasound-guided percutaneous thrombin injection. There was a high rate of hypersensitivity to the bovine thrombin, which precaution should be taken to prevent more serious side effects

Percutaneous Ultrasound-Guided Thrombin Injection in Iatrogenic Arterial Pseudoaneurysms: Effectiveness and Complications

Energy Technology Data Exchange (ETDEWEB)

Koh, Young Hwan [Boramae Hospital, Seoul (Korea, Republic of); Kim, Hak Soo; Kim, Hyung Sik; Min, Seung Kee [Gachon Medical School, Incheon (Korea, Republic of)

2005-09-15

To evaluate and describe the efficacy and side effects of a percutaneous thrombin injection under ultrasonography guidance for the treatment of iatrogenic pseudo aneurysms Eighteen consecutive iatrogenic pseudo aneurysm cases were treated with a thrombin injection. The thrombin was injected into the pseudo aneurysm cavity using a 22-gauge needle under ultrasonographic guidance. The causes of the pseudo aneurysms are as follows: post coronary angiography (9 cases), percutaneous coronary balloon angioplasty (5 cases), cerebral angiography (1 case), transhepatic chemo embolization (1 case), percutaneous trans femoral arterial stent insertion (1 case) and bone marrow aspiration for a marrow transplant (1 case). Only one case required a secondary thrombin injection due to recurrent flow in the pseudo aneurysm lumen, which was detected at the follow up Doppler ultrasound. Other seventeen cases were successfully treated on the first trial. There were no technical failures or complication related to the procedure. The average amount of thrombin injected was 733 IU. Nine out of 18 treated patients (50%) showed mild reactions to the thrombin including mild fever (4 cases), chilling sensation (3 cases), a chilling sensation with mild dyspnea (1 case), mild chest discomfort (1 case) after the thrombin injection. All these side effects were transient and improved several hours later. All the iatrogenic pseudo aneurysms were treated successfully with an ultrasound-guided percutaneous thrombin injection. There was a high rate of hypersensitivity to the bovine thrombin, which precaution should be taken to prevent more serious side effects

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

Science.gov (United States)

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

1995-02-10

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

Thrombin Generating Capacity and Phenotypic Association in ABO Blood Groups.

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

2015-01-01

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

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

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

2015-06-01

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

Effects of botropase on clotting factors in healthy human volunteers

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Ashok K Shenoy

2014-01-01

Full Text Available Objective: To evaluate the effects of botropase on various clotting factors in human volunteers. Materials and Methods: It was a prospective open label study conducted on human healthy volunteers. After the baseline screening, subjects fulfilling inclusion criteria were enrolled. On the study day, 1 ml of botropase was administered intravenously and after an hour same dose of botropase (1 ml was given by intramuscular (IM route. The efficacy and safety parameters were monitored up to 72 h from the time of intravenous (IV administration. Results: A total of 15 volunteers, belonging to 24-35 years of age were included in the study. Botropase significantly reduced the plasma level of fibrinogen and fibrin degradation products after 5 min of IV administration (P < 0.05. In addition, factor X was observed to reduce constantly by botropase administration suggesting enhanced turnover between 5 and 20 min of IV administration. Although botropase reduced clotting and bleeding time in all the volunteers, the data remains to be statistically insignificant. Conclusion: Present study demonstrated the safety and efficacy of botropase in human healthy volunteers. The study has shown that it is a factor X activator and reduces effectively clotting and bleeding time.

Massive clot formation after tooth extraction

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Santosh Hunasgi

2015-01-01

Full Text Available Oral surgical procedures mainly tooth extraction can be related with an extended hemorrhage owed to the nature of the process resulting in an "open wound." The attempt of this paper is to present a case of massive postoperative clot formation after tooth extraction and highlight on the oral complications of surgical procedures. A 32-year-old male patient reported to the Dental Clinic for evaluation and extraction of grossly decayed 46. Clinical evaluation of 46 revealed root stumps. Extraction of the root stumps was performed, and it was uneventful. Hemostasis was achieved and postsurgical instructions were specified to the patient. The patient reported to the clinic, the very subsequent morning with a criticism of bleeding at the extraction site. On clinical examination, bleeding was noted from the socket in relation to 46. To control bleeding, oral hemostatic drugs Revici - E (Ethamsylate 500 mg was prescribed and bleeding was stopped in 2 h. However, a massive clot was formed at the extraction site. Further, this clot resolved on its own in 1-week time. Despite the fact that dental extraction is considered to be a minor surgical procedure, some cases may present with life-threatening complications including hemorrhage. Vigilant and significant history taking, physical and dental examinations prior to dental procedures are a must to avoid intraoperative and postoperative complications.

Investigations on blood coagulation in the green iguana (Iguana iguana).

Science.gov (United States)

Kubalek, S; Mischke, R; Fehr, M

2002-05-01

The prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time, kaolin clotting time (KCT), dilute Russell's viper venom time (DRVVT) and reptilase time, as well as five different plasma fibrinogen assays [gravimetry, Jacobsson method (extinction at 280 nm), Millar method (heat precipitation), kinetic turbidometry, Clauss method] and resonance thrombography were performed in 26 clinically healthy green iguanas. All assays were carried out in comparison with pooled normal canine plasma. In iguana plasma, the PT [median (x0.50) = 453-831 s, dependent on the reagent], APTT (x0.50 = 170-242 s, dependent on the reagent), thrombin time (x0.50 = 118 - > 1000 s, dependent on thrombin activity), KCT (x0.50 = 274 s), DRVVT (x0.50 = 349 s) and reptilase time (all samples > 1000 s) were widely scattered at the limit of measurability. Only fibrinogen concentrations measured using the Jacobsson method (x0.50 = 4.40 g/l) correlated well (r = 0.91) with gravimetry (x0.50 = 4.22 g/l). The results of this study indicate a limited suitability and a confined diagnostic significance of the selected methods in the green iguana. This may be caused by the species specificity of certain components of the reagents used, as well as a less optimal test system, i.e. relationship of test reagent to clotting factor concentrations in iguana plasma.

The euglobulin clot lysis time to assess the impact of nanoparticles on fibrinolysis

Energy Technology Data Exchange (ETDEWEB)

Minet, Valentine, E-mail: valentine.minet@unamur.be; Alpan, Lutfiye; Mullier, François [University of Namur – UNamur, Department of Pharmacy, Namur Thrombosis and Hemostasis Center (NTHC), Namur Nanosafety Center (NNC), NAmur Research Institute for Life Sciences NARILIS (Belgium); Toussaint, Olivier [Laboratory of Cellular Biochemistry and Biology (URBC) (Belgium); Lucas, Stéphane [University of Namur (UNamur), Research Centre for the Physics of Matter and Radiation (PMR-LARN), Namur Nanosafety Center NNC, NAmur Research Institute for Life Sciences NARILIS (Belgium); Dogné, Jean-Michel; Laloy, Julie, E-mail: julie.laloy@unamur.be [University of Namur – UNamur, Department of Pharmacy, Namur Thrombosis and Hemostasis Center (NTHC), Namur Nanosafety Center (NNC), NAmur Research Institute for Life Sciences NARILIS (Belgium)

2015-07-15

Nanoparticles (NPs) are developed for many applications in various fields, including nanomedicine. The NPs used in nanomedicine may disturb homeostasis in blood. Secondary hemostasis (blood coagulation) and fibrinolysis are complex physiological processes regulated by activators and inhibitors. An imbalance of this system can either lead to the development of hemorrhages or thrombosis. No data are currently available on the impact of NPs on fibrinolysis. The objectives of this study are (1) to select a screening test to study ex vivo the impact of NPs on fibrinolysis and (2) to test NPs with different physicochemical properties. Euglobulin clot lysis time test was selected to screen the impact of some NPs on fibrinolysis using normal pooled plasma. A dose-dependent decrease in the lysis time was observed with silicon dioxide and silver NPs without disturbing the fibrin network. Carbon black, silicon carbide, and copper oxide did not affect the lysis time at the tested concentrations.

In Vitro Effect of Activated Recombinant Factor VII (rFVIIa) on Coagulation Properties of Human Blood at Hypothermic Temperatures

Science.gov (United States)

2007-11-01

is co- agulopathy, particularly when it is associated with metabolic acidosis and hypothermia, often referred to as the “lethal triad”.3–5 Substantial...that rFVIIa may be able to improve early survival of mas- sively bleeding trauma patients.27 It is also suggested that the correction of acidosis and...ratio and clotting time was measured. Fibrinogen in plasma was also determined based on a clotting assay in which a large excess of bovine thrombin (50

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

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

2016-01-01

We have previously shown that intracerebral hemorrhage-induced brain injury is less in rats bred for high aerobic capacity (high capacity runners; HCR) compared with those bred for low aerobic capacity (low capacity runners; LCRs). Thrombin, an essential component in the coagulation cascade, is produced after cerebral hemorrhage. Intraventricular injection of thrombin causes significant hydrocephalus and white matter damage. In the present study, we examined the effect of exercise capacity on thrombin-induced hydrocephalus and white matter damage. Mid-aged (13-month-old) female LCRs (n = 13) and HCRs (n = 12) rats were used in this study. Rats received an intraventricular injection of thrombin (3 U, 50 μl). All rats underwent magnetic resonance imaging (MRI) at 24 h and were then euthanized for brain histology and Western blot. The mortalities were 20 % in LCRs and 33 % in HCRs after thrombin injection (p > 0.05). No rats died after saline injection. Intraventricular thrombin injection resulted in hydrocephalus and periventricular white matter damage as determined on MRI. In LCR rats, thrombin induced significant ventricle enlargement (23.0 ± 2.3 vs12.8 ± 1.9 mm(3) in LCR saline group; p hydrocephalus in rats with low aerobic capacity. A differential effect of thrombin may contribute to differences in the effects of cerebral hemorrhage with aerobic capacity.

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

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

2012-12-14

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

The Characteristics of Thrombin in Osteoarthritic Pathogenesis and Treatment

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Pei-Yu Chou

2014-01-01

Full Text Available Osteoarthritis (OA is a mechanical abnormality associated with degradation of joints. It is characterized by chronic, progressive degeneration of articular cartilage, abnormalities of bone, and synovial change. The most common symptom of OA is local inflammation resulting from exogenous stress or endogenous abnormal cytokines. Additionally, OA is associated with local and/or systemic activation of coagulation and anticoagulation pathways. Thrombin plays an important role in the stimulation of fibrin deposition and the proinflammatory processes in OA. Thrombin mediates hemostatic and inflammatory responses and guides the immune response to tissue damage. Thrombin activates intracellular signaling pathways by interacting with transmembrane domain G protein coupled receptors (GPCRs, known as protease-activated receptors (PARs. In pathogenic mechanisms, PARs have been implicated in the development of acute and chronic inflammatory responses in OA. Therefore, discovery of thrombin signaling pathways would help us to understand the mechanism of OA pathogenesis and lead us to develop therapeutic drugs in the future.

Developing Mesoscale Model of Fibrin-Platelet Network Representing Blood Clotting =

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Sun, Yueyi; Nikolov, Svetoslav; Bowie, Sam; Alexeev, Alexander; Lam, Wilbur; Myers, David

Blood clotting disorders which prevent the body's natural ability to achieve hemostasis can lead to a variety of life threatening conditions such as, excessive bleeding, stroke, or heart attack. Treatment of these disorders is highly dependent on understanding the underlying physics behind the clotting process. Since clotting is a highly complex multi scale mechanism developing a fully atomistic model is currently not possible. We develop a mesoscale model based on dissipative particle dynamics (DPD) to gain fundamental understanding of the underlying principles controlling the clotting process. In our study, we examine experimental data on clot contraction using stacks of confocal microscopy images to estimate the crosslink density in the fibrin networks and platelet location. Using this data we reconstruct the platelet rich fibrin network and study how platelet-fibrin interactions affect clotting. Furthermore, we probe how different system parameters affect clot contraction. ANSF CAREER Award DMR-1255288.

Prolonged Activated Clotting Time after Protamine Administration Does Not Indicate Residual Heparinization after Cardiopulmonary Bypass in Pediatric Open Heart Surgery.

Science.gov (United States)

Yamamoto, Tomohiro; Wolf, Hans-Gerd; Sinzobahamvya, Nicodème; Asfour, Boulos; Hraska, Victor; Schindler, Ehrenfried

2015-08-01

In open heart surgery, heparinization is commonly neutralized using an empirical heparin:protamine ratio ranging between 1:1 and 1:1.5. However, these ratios may result in protamine overdose that should be avoided for its negative side effects on the coagulation system. This study aimed to indicate the appropriate treatment for prolonged activated clotting time (ACT) after protamine administration following cardiopulmonary bypass (CPB) in pediatric open heart surgery by investigating the underlying reasons for it. Twenty-seven children (open heart surgery were included. Heparin was administered only before CPB (400 IU/kg) and in the pump priming volume for CPB (2,000 IU) and was neutralized by 1:1 protamine after CPB. The blood heparin concentration was measured using anti-Xa assay. ACT and blood concentrations of heparin, coagulation factors, thrombin-antithrombin complex, and prothrombin fragment 1 + 2 were assessed. A rotational thromboelastometry (ROTEM; Tem International GmbH, München, Bayern, Germany) was used to confirm the coagulation status and residual heparin after protamine administration. Anti-Xa assay showed that there is no residual heparin in the blood after 1:1 protamine administration. Nevertheless, ACT (128.89 ± 3.09 seconds before heparin administration) remained prolonged (177.14 ± 5.43 seconds at 10 minutes after protamine, 182.00 ± 5.90 seconds at 30 minutes after protamine). The blood concentrations of coagulation factors were significantly lower than those before heparin administration (p < 0.01). The low FIBTEM MCF of ROTEM (4.43 ± 0.32 mm) at 10 minutes after protamine indicated low fibrinogen concentration. Prolonged ACT after heparin neutralization by 1:1 protamine administration does not necessarily indicate residual heparin, but low blood concentrations of coagulation factors should be considered as a reason as well. Accordingly, supply of coagulation factors instead of additional protamine should be

Clot friction variation with fibrin content; implications for resistance to thrombectomy.

Science.gov (United States)

Gunning, Gillian M; McArdle, Kevin; Mirza, Mahmood; Duffy, Sharon; Gilvarry, Michael; Brouwer, Patrick A

2018-01-01

Despite significant advancements in the procedural efficacy of mechanical thrombectomy in patients with ischemic stroke in recent years, there still remains a portion of the population that does not achieve good recanalization. The reasons for this may be varied. We hypothesized that static friction between the clot and the vessel, or catheter wall might contribute to the difficulty in removing the clot. To determine if there is a relationship between clot composition and the resistance to sliding (friction) which might contribute to resistance to clot removal. As clot composition can vary significantly, we investigated five different types of clot in order to measure their respective frictional properties. To do this, a custom-made testing apparatus was created, consisting of various replaceable low-friction surfaces on which the clots could be placed. The surface was then gradually tilted until the clots began to slide; the angle at which this occurred is related to the coefficient of friction of the clots. The experiment was repeated on a bovine aortic surface in order to confirm the results. We found that fibrin-rich clots (friction than clots with a red blood cell content >20%. This result was confirmed by repeating the experiment on a bovine aortic surface as a representation of the interaction between clots and the arterial wall. The friction properties of clots were found to be related to the content ratio of fibrin to red blood cells. Future imaging techniques that could show fibrin and red blood cell content might help us to predict the 'stickiness' of a clot. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Capture of lipopolysaccharide (endotoxin) by the blood clot: a comparative study.

Science.gov (United States)

Armstrong, Margaret T; Rickles, Frederick R; Armstrong, Peter B

2013-01-01

In vertebrates and arthropods, blood clotting involves the establishment of a plug of aggregated thrombocytes (the cellular clot) and an extracellular fibrillar clot formed by the polymerization of the structural protein of the clot, which is fibrin in mammals, plasma lipoprotein in crustaceans, and coagulin in the horseshoe crab, Limulus polyphemus. Both elements of the clot function to staunch bleeding. Additionally, the extracellular clot functions as an agent of the innate immune system by providing a passive anti-microbial barrier and microbial entrapment device, which functions directly at the site of wounds to the integument. Here we show that, in addition to these passive functions in immunity, the plasma lipoprotein clot of lobster, the coagulin clot of Limulus, and both the platelet thrombus and the fibrin clot of mammals (human, mouse) operate to capture lipopolysaccharide (LPS, endotoxin). The lipid A core of LPS is the principal agent of gram-negative septicemia, which is responsible for more than 100,000 human deaths annually in the United States and is similarly toxic to arthropods. Quantification using the Limulus Amebocyte Lysate (LAL) test shows that clots capture significant quantities of LPS and fluorescent-labeled LPS can be seen by microscopy to decorate the clot fibrils. Thrombi generated in the living mouse accumulate LPS in vivo. It is suggested that capture of LPS released from gram-negative bacteria entrapped by the blood clot operates to protect against the disease that might be caused by its systemic dispersal.

Capture of lipopolysaccharide (endotoxin by the blood clot: a comparative study.

Directory of Open Access Journals (Sweden)

Margaret T Armstrong

Full Text Available In vertebrates and arthropods, blood clotting involves the establishment of a plug of aggregated thrombocytes (the cellular clot and an extracellular fibrillar clot formed by the polymerization of the structural protein of the clot, which is fibrin in mammals, plasma lipoprotein in crustaceans, and coagulin in the horseshoe crab, Limulus polyphemus. Both elements of the clot function to staunch bleeding. Additionally, the extracellular clot functions as an agent of the innate immune system by providing a passive anti-microbial barrier and microbial entrapment device, which functions directly at the site of wounds to the integument. Here we show that, in addition to these passive functions in immunity, the plasma lipoprotein clot of lobster, the coagulin clot of Limulus, and both the platelet thrombus and the fibrin clot of mammals (human, mouse operate to capture lipopolysaccharide (LPS, endotoxin. The lipid A core of LPS is the principal agent of gram-negative septicemia, which is responsible for more than 100,000 human deaths annually in the United States and is similarly toxic to arthropods. Quantification using the Limulus Amebocyte Lysate (LAL test shows that clots capture significant quantities of LPS and fluorescent-labeled LPS can be seen by microscopy to decorate the clot fibrils. Thrombi generated in the living mouse accumulate LPS in vivo. It is suggested that capture of LPS released from gram-negative bacteria entrapped by the blood clot operates to protect against the disease that might be caused by its systemic dispersal.

Postpartum Blood Clots

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... Video) Fetal Ultrasound Scanning Additional Content Medical News Postpartum Blood Clots By Julie S. Moldenhauer, MD, Associate Professor ... Professional Version Postdelivery Period Overview of the Postdelivery (Postpartum) Period Postpartum Infections Postpartum Infections of the Uterus ...

New synthetic thrombin inhibitors: molecular design and experimental verification.

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Elena I Sinauridze

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

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

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

2018-06-01

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

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

International Nuclear Information System (INIS)

Nygaard, Gyrid; Herfindal, Lars; Kopperud, Reidun; Aragay, Anna M.; Holmsen, Holm; Døskeland, Stein Ove; Kleppe, Rune; Selheim, Frode

2014-01-01

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

Variable Resistance to Plasminogen Activator Initiated Fibrinolysis for Intermediate-Risk Pulmonary Embolism.

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Stubblefield, William B; Alves, Nathan J; Rondina, Matthew T; Kline, Jeffrey A

2016-01-01

We examine the clinical significance and biomarkers of tissue plasminogen activator (tPA)-catalyzed clot lysis time (CLT) in patients with intermediate-risk pulmonary embolism (PE). Platelet-poor, citrated plasma was obtained from patients with PE. Healthy age- and sex-matched patients served as disease-negative controls. Fibrinogen, α2-antiplasmin, plasminogen, thrombin activatable fibrinolysis inhibitor (TAFI), plasminogen activator Inhibitor 1 (PAI-1), thrombin time and D-dimer were quantified. Clotting was induced using CaCl2, tissue factor, and phospholipid. Lysis was induced using 60 ng/mL tPA. Time to 50% clot lysis (CLT) was assessed by both thromboelastography (TEG) and turbidimetry (A405). Compared with disease-negative controls, patients with PE exhibited significantly longer mean CLT on TEG (+2,580 seconds, 95% CI 1,380 to 3,720 sec). Patients with PE and a short CLT who were treated with tenecteplase had increased risk of bleeding, whereas those with long CLT had significantly worse exercise tolerance and psychometric testing for quality of life at 3 months. A multivariate stepwise removal regression model selected PAI-1 and TAFI as predictive biomarkers of CLT. The CLT from TEG predicted increased risk of bleeding and clinical failure with tenecteplase treatment for intermediate-risk PE. Plasmatic PAI-1 and TAFI were independent predictors of CLT.

A Novel Photoelectrochemical Biosensor for Tyrosinase and Thrombin Detection

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Jiexia Chen

2016-01-01

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

A Novel Photoelectrochemical Biosensor for Tyrosinase and Thrombin Detection

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

2016-01-01

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

Anticoagulant Effect of Sugammadex: Just an In Vitro Artifact.

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Dirkmann, Daniel; Britten, Martin W; Pauling, Henning; Weidle, Juliane; Volbracht, Lothar; Görlinger, Klaus; Peters, Jürgen

2016-06-01

Sugammadex prolongs activated partial thromboplastin time (aPTT) and prothrombin time (PT) suggestive of anticoagulant effects. To pinpoint its presumed anticoagulant site of action, the authors assessed Sugammadex's impact on a panel of coagulation assays. Sugammadex, Rocuronium, Sugammadex and Rocuronium combined, or saline were added to blood samples from healthy volunteers and analyzed using plasmatic (i.e., aPTT, thrombin time, and fibrinogen concentration) (n = 8 each), PT (quick), activities of plasmatic coagulation factors, and whole blood (extrinsically and intrinsically activated thromboelastometry) assays (n = 18 each). Furthermore, dose-dependent effects of Sugammadex were also assessed (n = 18 each) in diluted Russel viper venom time (DRVVT) assays with low (DRVVT1) and high (DRVVT2) phospholipid concentrations and in a highly phospholipid-sensitive aPTT assay. Sugammadex increased PT (+9.1%; P Sugammadex dose-dependently prolonged both DRVVT1 and the highly phospholipid-sensitive aPTT assays, but additional phospholipids in the DRVVT2 assay almost abolished these prolongations. Thrombin time, a thromboelastometric thrombin generation assay, clot firmness, clot lysis, fibrinogen concentration, and activities of other coagulation factors were unaltered. Rocuronium, Sugammadex and Rocuronium combined, and saline exerted no effects. Sugammadex significantly affects various coagulation assays, but this is explainable by an apparent phospholipid-binding effect, suggesting that Sugammadex`s anticoagulant effects are likely an in vitro artifact.

A constitutive model for developing blood clots with various compositions and their nonlinear viscoelastic behavior.

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van Kempen, Thomas H S; Donders, Wouter P; van de Vosse, Frans N; Peters, Gerrit W M

2016-04-01

The mechanical properties determine to a large extent the functioning of a blood clot. These properties depend on the composition of the clot and have been related to many diseases. However, the various involved components and their complex interactions make it difficult at this stage to fully understand and predict properties as a function of the components. Therefore, in this study, a constitutive model is developed that describes the viscoelastic behavior of blood clots with various compositions. Hereto, clots are formed from whole blood, platelet-rich plasma and platelet-poor plasma to study the influence of red blood cells, platelets and fibrin, respectively. Rheological experiments are performed to probe the mechanical behavior of the clots during their formation. The nonlinear viscoelastic behavior of the mature clots is characterized using a large amplitude oscillatory shear deformation. The model is based on a generalized Maxwell model that accurately describes the results for the different rheological experiments by making the moduli and viscosities a function of time and the past and current deformation. Using the same model with different parameter values enables a description of clots with different compositions. A sensitivity analysis is applied to study the influence of parameter variations on the model output. The relative simplicity and flexibility make the model suitable for numerical simulations of blood clots and other materials showing similar behavior.

Hemostatic interference of Indian king cobra (Ophiophagus hannah) Venom. Comparison with three other snake venoms of the subcontinent.

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Gowtham, Yashonandana J; Kumar, M S; Girish, K S; Kemparaju, K

2012-06-01

Unlike Naja naja, Bungarus caeruleus, Echis carinatus, and Daboia/Vipera russellii venoms, Ophiophagus hannah venom is medically ignored in the Indian subcontinent. Being the biggest poisonous snake, O. hannah has been presumed to inject several lethal doses of venom in a single bite. Lack of therapeutic antivenom to O. hannah bite in India makes any attempt to save the victim a difficult exercise. This study was initiated to compare O. hannah venom with the above said venoms for possible interference in hemostasis. Ophiophagus hannah venom was found to actively interfere in hemostatic stages such as fibrin clot formation, platelet activation/aggregation, and fibrin clot dissolution. It decreased partial thromboplastin time (aPTT), prothrombin time (PT), and thrombin clotting time (TCT). These activities are similar to that shown by E. carinatus and D. russellii venoms, and thus O. hannah venom was found to exert procoagulant activity through the common pathway of blood coagulation, while N. naja venom increased aPTT and TCT but not PT, and hence it was found to exert anticoagulant activity through the intrinsic pathway. Venoms of O. hannah, E. carinatus, and D. russellii lack plasminogen activation property as they do not hydrolyze azocasein, while they all show plasmin-like activity by degrading the fibrin clot. Although N. naja venom did not degrade azocasein, unlike other venoms, it showed feeble plasmin-like activity on fibrin clot. Venom of E. carinatus induced clotting of human platelet rich plasma (PRP), while the other three venoms interfered in agonist-induced platelet aggregation in PRP. Venom of O. hannah least inhibited the ADP induced platelet aggregation as compared to D. russellii and N. naja venoms. All these three venoms showed complete inhibition of epinephrine-induced aggregation at varied doses. However, O. hannah venom was unique in inhibiting thrombin induced aggregation.

Dynamic affinity chromatography in the separation of sulfated lignins binding to thrombin

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Liang, Aiye; Thakkar, Jay N.; Hindle, Michael; Desai, Umesh R.

2013-01-01

Sulfated low molecular weight lignins (LMWLs), a mixture of chemo-enzymatically prepared oligomers, have been found to be potent antagonists of coagulation. However, structures that induce anticoagulation remain unidentified. The highly polar sulfate groups on these molecules and the thousands of different structures present in these mixtures make traditional chromatographic resolution of sulfated LMWLs difficult. We performed dynamic thrombin affinity chromatography monitored using chromogenic substrate hydrolysis assay to isolate sulfated LMWL fractions that differed significantly in their biophysical and biochemical properties. Three fractions, I35, I55 and Peak II, were isolated from the starting complex mixture. Independent plasma clotting assays suggested that I35 possessed good anticoagulation potential (APTT = 4.2 μM; PT = 6.8 μM), while I55 and Peak II were approximately 10- and 100-fold less potent. The ESI-MS spectrum of this oligomeric fraction showed multiple peaks at 684.8, 610.6, 557.4, 541.4, 536.5, and 519.4 m/z, which most probably arise from variably functionalized (β-O4—β-β-linked trimers and/or a β-O4—β-O4-linked dimers. The first direct observation of these structures in sulfated LMWLs will greatly assist in the discovery of more potent sulfated LMWL-based anticoagulants. PMID:23122400

Diastolic timed Vibro-Percussion at 50 Hz delivered across a chest wall sized meat barrier enhances clot dissolution and remotely administered Streptokinase effectiveness in an in-vitro model of acute coronary thrombosis

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Hoffmann Andrew

2012-11-01

Full Text Available Abstract Background Low Frequency Vibro-Percussion (LFVP assists clearance of thrombi in catheter systems and when applied to the heart and timed to diastole is known to enhance coronary flow. However LFVP on a clotted coronary like vessel given engagement over a chest wall sized barrier (to resemble non-invasive heart attack therapy requires study. Methods One hour old clots (n=16 were dispensed within a flexible segment of Soft-Flo catheter (4 mm lumen, weighted, interfaced with Heparinized Saline (HS, secured atop a curved dampening base, and photographed. A ~4 cm meat slab was placed over the segment and randomized to receive intermittent LFVP (engaged, - disengaged at 1 second intervals, or no LFVP for 20 minutes. HS was pulsed (~120/80 mmHg, with the diastolic phase coordinated to match LFVP delivery. The segment was then re-photographed and aspirated of fluid to determine post clot weight. The trial was then repeated with 0.5 mls of Streptokinase (15,000 IU/100 microlitre delivered ~ 2 cm upstream from the clot. Results LFVP - HS only samples (vs. controls showed; a development of clot length fluid channels absent in the control group (p Conclusion Diastolic timed LFVP (50 Hz engaged across a chest wall sized barrier enhances clot disruptive effects to an underlying coronary like system.

Tyrosine sulfation modulates activity of tick-derived thrombin inhibitors

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

2017-09-01

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

In vitro study of the role of thrombin in platelet rich plasma (PRP) preparation: utility for gel formation and impact in growth factors release.

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Huber, Stephany Cares; Cunha Júnior, José Luiz Rosenberis; Montalvão, Silmara; da Silva, Letícia Queiroz; Paffaro, Aline Urban; da Silva, Francesca Aparecida Ramos; Rodrigues, Bruno Lima; Lana, José Fabio Santos Duarte; Annichino-Bizzacchi, Joyce Maria

2016-01-01

The use of PRP has been studied for different fields, with promising results in regenerative medicine. Until now, there is no study in the literature evaluating thrombin levels in serum, used as autologous thrombin preparation. Therefore, in the present study we evaluated the role played by different thrombin concentrations in PRP and the impact in the release of growth factors. Also, different activators for PRP gel formation were evaluated. Thrombin levels were measured in different autologous preparations: serum, L-PRP (PRP rich in leukocytes) and T-PRP (thrombin produced through PRP added calcium gluconate). L-PRP was prepared according to the literature, with platelets and leukocytes being quantified. The effect of autologous thrombin associated or not with calcium in PRP gel was determined by measuring the time of gel formation. The relationship between thrombin concentration and release of growth factors was determined by growth factors (PDGF-AA, VEGF and EGF) multiplex analysis. A similar concentration of thrombin was observed in serum, L-PRP and T-PRP (8.13 nM, 8.63 nM and 7.56 nM, respectively) with a high variation between individuals (CV%: 35.07, 43 and 58.42, respectively). T-PRP and serum with calcium chloride showed similar results in time to promote gel formation. The increase of thrombin concentrations (2.66, 8 and 24 nM) did not promote an increase in growth factor release. The technique of using serum as a thrombin source proved to be the most efficient and reproducible for promoting PRP gel formation, with some advantages when compared to other activation methods, as this technique is easier and quicker with no need of consuming part of PRP. Noteworthy, PRP activation using different thrombin concentrations did not promote a higher release of growth factors, appearing not to be necessary when PRP is used as a suspension.

The Effect of Radioactive Lantern Mantle Powder and Bentonite-Zeoloite Minerals on the Volume of Blood Loss, Bleeding and Clotting Time

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M Atefi

2009-04-01

Full Text Available ABSTRACT Introduction & Objective: Over the past decade the US army has widely studied new technologies for stopping sever hemorrhages and has introduced an effective Zeolite based hemostatic agent. On the other hand, Mortazavi and his colleagues previously reported the bio-stimulatory effects of the topical application of radioactive lantern mantle powder on wound healing. Their subsequent studies showed significant changes in some histological parameters concerning healing. In this light, here the bio-stimulatory effect of burned radioactive lantern mantles powder as well as two minerals bentonite and zeolite are presented. Materials & Methods: This experimental study was conducted in the center for radiological studies, Shiraz University of Medical Sciences in 2008. Fifty male Wistar rats were divided randomly into 5 groups of 10 animals each. Following anesthesia, animals’ tails were cut at a thickness of 5 mm by using a surgical scissor. No intervention was made on the animals of the 1st group. The 2nd to 4th group received topical non-radioactive lantern mantle powder, radioactive lantern mantle powder, Bentonite mineral or a mixture of Bentonite-Zeoliteat minerals respectively. After treatment with above mentioned agents, the volume of blood loss was measured using a scaled test-tube. The bleeding time and clotting time were also measured using a chronometer. SPSS software was used for statistical analysis. ANOVA was used for comparing the means of each parameter in the 5 groups. Results: The the volume of blood loss, bleeding and clotting times in control animals were 4.39±1.92 cc, 112.10±39.60 sec and 94.9±54.26 sec, respectively. In the 5th group in which the animals were treated with a mixture of Bentonite-Zeoliteat minerals, the volume of blood loss, bleeding and clotting times were 1.31±0.60 cc, 34.50±4.65 sec and 24.2±4.61 sec, respectively. Conclusion: This is the 1st investigation that studied the alterations of bleeding

Thrombin induces epithelial-mesenchymal transition and collagen production by retinal pigment epithelial cells via autocrine PDGF-receptor signaling.

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Bastiaans, Jeroen; van Meurs, Jan C; van Holten-Neelen, Conny; Nagtzaam, Nicole M A; van Hagen, P Martin; Chambers, Rachel C; Hooijkaas, Herbert; Dik, Willem A

2013-12-19

De-differentiation of RPE cells into mesenchymal cells (epithelial-mesenchymal transition; EMT) and associated collagen production contributes to development of proliferative vitreoretinopathy (PVR). In patients with PVR, intraocular coagulation cascade activation occurs and may play an important initiating role. Therefore, we examined the effect of the coagulation proteins factor Xa and thrombin on EMT and collagen production by RPE cells. Retinal pigment epithelial cells were stimulated with factor Xa or thrombin and the effect on zonula occludens (ZO)-1, α-smooth muscle actin (α-SMA), collagen, and platelet-derived growth factor (PDGF)-B were determined by real-time quantitative-polymerase chain reaction (RQ-PCR), immunofluorescence microscopy, and HPLC and ELISA for collagen and PDGF-BB in culture supernatants, respectively. PDGF-receptor activation was determined by phosphorylation analysis and inhibition studies using the PDGF-receptor tyrosine kinase inhibitor AG1296. Thrombin reduced ZO-1 gene expression (P production of α-SMA and collagen increased. In contrast to thrombin, factor Xa hardly stimulated EMT by RPE. Thrombin clearly induced PDGF-BB production and PDGF-Rβ chain phosphorylation in RPE. Moreover, AG1296 significantly blocked the effect of thrombin on EMT and collagen production. Our findings demonstrate that thrombin is a potent inducer of EMT by RPE via autocrine activation of PDGF-receptor signaling. Coagulation cascade-induced EMT of RPE may thus contribute to the formation of fibrotic retinal membranes in PVR and should be considered as treatment target in PVR.

A targeted ferritin-microplasmin based thrombolytic nanocage selectively dissolves blood clots.

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Seo, Junyoung; Al-Hilal, Taslim A; Jee, Jun-Goo; Kim, Yong-Lim; Kim, Ha-Jeong; Lee, Byung-Heon; Kim, Soyoun; Kim, In-San

2018-04-01

The use of thrombolytic therapies is limited by an increased risk of systemic hemorrhage due to lysis of hemostatic clots. We sought to develop a plasmin-based thrombolytic nanocage that efficiently dissolves the clot without causing systemic fibrinolysis or disrupting hemostatic clots. Here, we generated a double chambered short-length ferritin (sFt) construct that has an N-terminal region fused to multivalent clot targeting peptides (CLT: CNAGESSKNC) and a C-terminal end fused to a microplasmin (μPn); CLT recognizes fibrin-fibronectin complexes in clots, μPn efficiently dissolves clots, and the assembly of double chambered sFt (CLT-sFt-μPn) into nanocage structure protects the activated-μPn from its circulating inhibitors. Importantly, activated CLT-sFt-μPn thrombolytic nanocage showed a prolonged circulatory life over activated-μPn and efficiently lysed the preexisting clots in both arterial and venous thromboses models. Thus, CLT-sFt-μPn thrombolytic nanocage platform represents the prototype of a targeted clot-busting agent with high efficacy and safety over existing thrombolytic therapies. Copyright © 2018 Elsevier Inc. All rights reserved.

Characterisation of clotting factors, anticoagulant protein activities and viscoelastic analysis in healthy donkeys.

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Perez-Ecija, A; Mendoza, F J

2017-11-01

Studies have demonstrated differences in commonly measured haemostatic parameters between donkeys and horses. Whether clotting factors, anticoagulant protein activities and thromboelastography parameters also differ between species is still unknown. To characterise haemostatic parameters in healthy donkeys and to compare these with those in horses. Cross-sectional study. Clotting factors (V, VII, VIII, IX, X, XI and XII), and antithrombin III, Protein C and Protein S activities were measured in 80 healthy Andalusian and crossbred donkeys and 40 healthy Andalusian crossbred horses with assays based on human deficient plasmas. Thromboelastography was performed in 34 donkeys using a coagulation and platelet function analyser. Donkeys had shorter activated partial thromboplastin time (mean ± s.d. 33.4 ± 5.2 s vs. 38.8 ± 4.2 s; P0.05) and XII (96 ± 21 vs. 108 ± 15; Pdonkeys. Activated clot time (175 [159-189]), time to peak (6.5 [5.8-7.8]) and clot formation rate (26.9 [16.9-36.4]) in donkeys were shorter than reported values in horses. Haemostatic pathways could not be fully evaluated in donkeys because some tests are unavailable. Certain fibrinolytic parameters (plasmin, plasminogen, etc.) have not been characterised in donkeys and this may have affected our results. The haemostatic system in donkeys differs from that in horses and extrapolation of reference values between these species is not appropriate. © 2017 EVJ Ltd.

Mechanical Stability and Fibrinolytic Resistance of Clots Containing Fibrin, DNA, and Histones*

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Longstaff, Colin; Varjú, Imre; Sótonyi, Péter; Szabó, László; Krumrey, Michael; Hoell, Armin; Bóta, Attila; Varga, Zoltán; Komorowicz, Erzsébet; Kolev, Krasimir

2013-01-01

Neutrophil extracellular traps are networks of DNA and associated proteins produced by nucleosome release from activated neutrophils in response to infection stimuli and have recently been identified as key mediators between innate immunity, inflammation, and hemostasis. The interaction of DNA and histones with a number of hemostatic factors has been shown to promote clotting and is associated with increased thrombosis, but little is known about the effects of DNA and histones on the regulation of fibrin stability and fibrinolysis. Here we demonstrate that the addition of histone-DNA complexes to fibrin results in thicker fibers (increase in median diameter from 84 to 123 nm according to scanning electron microscopy data) accompanied by improved stability and rigidity (the critical shear stress causing loss of fibrin viscosity increases from 150 to 376 Pa whereas the storage modulus of the gel increases from 62 to 82 pascals according to oscillation rheometric data). The effects of DNA and histones alone are subtle and suggest that histones affect clot structure whereas DNA changes the way clots are lysed. The combination of histones + DNA significantly prolongs clot lysis. Isothermal titration and confocal microscopy studies suggest that histones and DNA bind large fibrin degradation products with 191 and 136 nm dissociation constants, respectively, interactions that inhibit clot lysis. Heparin, which is known to interfere with the formation of neutrophil extracellular traps, appears to prolong lysis time at a concentration favoring ternary histone-DNA-heparin complex formation, and DNase effectively promotes clot lysis in combination with tissue plasminogen activator. PMID:23293023

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

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Doyle, M.F.; Mann, K.G. (Univ. of Vermont College of Medicine, Burlington (USA))

1990-06-25

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 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.

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

International Nuclear Information System (INIS)

Doyle, M.F.; Mann, K.G.

1990-01-01

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 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

Sucralfate protects blood clots from peptic digestion by gastric juice in vitro.

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Nysaeter, Gunnar; Berstad, Arnold

2006-01-01

To test in vitro the ability of sucralfate to protect a blood clot from peptic digestion by gastric juice. Blood clots adhering to the bottom of plastic tubes were exposed to native acidic gastric juice or gastric juice to which Al-Mg antacids, sucralfate or alkali had been added. The tubes were tilted regularly at room temperature and clot digestion monitored by measuring the diameters of the clots. After 15 h, the liquids, but not the adherent clots, were poured out and the tubes refilled with native acidic gastric juice. Further clot digestion was measured, as before. Native gastric juice digested the clots completely during approximately 7 h, while in neutralized gastric juice or in gastric juice containing antacids or sucralfate no digestion was seen. In the second experiment, native gastric juice completely digested all remaining clots, except those previously exposed to sucralfate. A dose-response study indicated that gastric juice containing 3% or more of sucralfate had this long-lasting, clot-protective effect. In vitro, sucralfate adheres to and protects blood clots from digestion by gastric juice pepsin. This unique effect of sucralfate may be of clinical relevance in the treatment of bleeding peptic ulcers. Copyright 2006 S. Karger AG, Basel.

What Is Deep Vein Thrombosis?

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... the blood to trigger the activity of the enzyme thrombin. Active thrombin then forms long protein strands that clump together with platelets and red blood cells to form clots. Read less Risk Factors Risk factors for VTE include a history of a previous VTE event; surgery; medical conditions ...

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

Energy Technology Data Exchange (ETDEWEB)

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

2011-03-18

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.

Chitosan inhibits platelet-mediated clot retraction, increases platelet-derived growth factor release, and increases residence time and bioactivity of platelet-rich plasma in vivo.

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Deprés-Tremblay, Gabrielle; Chevrier, Anik; Tran-Khanh, Nicolas; Nelea, Monica; Buschmann, Michael D

2017-11-10

Platelet-rich plasma (PRP) has been used to treat different orthopedic conditions, however, the clinical benefits of using PRP remain uncertain. Chitosan (CS)-PRP implants have been shown to improve meniscus, rotator cuff and cartilage repair in pre-clinical models. The purpose of this current study was to investigate in vitro and in vivo mechanisms of action of CS-PRP implants. Freeze-dried formulations containing 1% (w/v) CS (80% degree of deacetylation and number average molar mass 38 kDa), 1% (w/v) trehalose as a lyoprotectant and 42.2 mM calcium chloride as a clot activator were solubilized in PRP. Gravimetric measurements and molecular/cellular imaging studies revealed that clot retraction is inhibited in CS-PRP hybrid clots through physical coating of platelets, blood cells and fibrin strands by chitosan, which interferes with platelet aggregation and platelet-mediated clot retraction. Flow cytometry and ELISA assays revealed that platelets are activated and granules secreted in CS-PRP hybrid clots and that cumulative release of platelet-derived growth factor (PDGF-AB) and epidermal growth factor is higher from CS-PRP hybrid clots compared to PRP clots in vitro. Finally, CS-PRP implants resided for up to 6 weeks in a subcutaneous implantation model and induced cell recruitment and granulation tissue synthesis, confirming greater residency and bioactivity compared to PRP in vivo.

Blood Clotting and Pregnancy

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Full Text Available ... of articles from the 2017 ASH Annual Meeting Education Program Blood: How I Treat A compendium of Blood articles ... Pregnancy Clots and Travel DVT Myths vs. Facts Blood Disorder Fact Sheets ...

Blood Clotting and Pregnancy

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Full Text Available ... predisposition to blood clots Obesity Prolonged immobility (e.g., bedrest, long distance travel) Multiple births Increased maternal age Other medical illness (e.g., cancer, infection) back to top How are Blood ...

Blood Clotting and Pregnancy

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Full Text Available ... genetic predisposition to blood clots Obesity Prolonged immobility (e.g., bedrest, long distance travel) Multiple births Increased maternal age Other medical illness (e.g., cancer, infection) back to top How are ...

In vivo behavior of detergent-solubilized purified rabbit thrombomodulin on intravenous injection into rabbits

International Nuclear Information System (INIS)

Ehrlich, H.J.; Esmon, N.L.; Bang, N.U.

1990-01-01

Thrombomodulin is a thrombin endothelial cell membrane receptor. The thrombomodulin-thrombin complex rapidly activates protein C resulting in anticoagulant activity. We investigated the anticoagulant effects and pharmacokinetic behavior of detergent-solubilized purified rabbit thrombomodulin labeled with iodine 125 when intravenously injected into rabbits. Thrombomodulin half-life (t1/2) was determined by tracking the 125I-radiolabeled protein and the biologic activity as determined by the prolongation of the activated partial thromboplastin time (APTT) and thrombin clotting time (TCT). When 200 micrograms/kg 125I-thrombomodulin was injected into rabbits, the APTT and TCT were immediately prolonged, whereas no effect on the prothrombin time was seen. In vitro calibration curves enabled us to convert the prolongations of the clotting times into micrograms per milliliter thrombomodulin equivalents. The best fit (r greater than 0.99) for the disappearance curves was provided by a two-compartment model with mean t1/2 alpha (distribution phase) of 18 minutes for 125I, 12 minutes for APTT, and 20 minutes for TCT, and mean t1/2 beta (elimination phase) of 385 minutes for 125I, 460 for APTT, and 179 for TCT. The administration of two doses of endotoxin (50 micrograms/kg) 24 hours apart did not accelerate the turnover rate of 125I-thrombomodulin as measured by the disappearance of 125I from the circulation. Thus, detergent-solubilized purified thrombomodulin administered intravenously circulates in a biologically active form for appreciable time periods

Blood Clotting and Pregnancy

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Synthesis of New DNA G-Quadruplex Constructs with Anthraquinone Insertions and Their Anticoagulant Activity

DEFF Research Database (Denmark)

Gouda, Alaa S.; Amine, Mahasen S.; Pedersen, Erik Bjerregaard

2016-01-01

1,4-Dihydroxyanthraquinone and 1,8-dihydroxyanthraquinone were alkylated with 3-bromopropan-1-ol and subsequently transformed into the corresponding DMT protected phosphoramidite building blocks for insertion into loops of the Gquadruplex of the thrombin binding aptamer (TBA). The 1,4-disubstituted...... potassium buffer conditions as previously observed for TBA. Although the majority of the anthraquinone modified TBA analogues showed a decrease in clotting times in a fibrinogen clotting assay when compared to TBA, modified aptamers containing a 1,8-disubstituted anthraquinone linker replacing G8 or T9...

Variable Resistance to Plasminogen Activator Initiated Fibrinolysis for Intermediate-Risk Pulmonary Embolism.

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William B Stubblefield

Full Text Available We examine the clinical significance and biomarkers of tissue plasminogen activator (tPA-catalyzed clot lysis time (CLT in patients with intermediate-risk pulmonary embolism (PE.Platelet-poor, citrated plasma was obtained from patients with PE. Healthy age- and sex-matched patients served as disease-negative controls. Fibrinogen, α2-antiplasmin, plasminogen, thrombin activatable fibrinolysis inhibitor (TAFI, plasminogen activator Inhibitor 1 (PAI-1, thrombin time and D-dimer were quantified. Clotting was induced using CaCl2, tissue factor, and phospholipid. Lysis was induced using 60 ng/mL tPA. Time to 50% clot lysis (CLT was assessed by both thromboelastography (TEG and turbidimetry (A405.Compared with disease-negative controls, patients with PE exhibited significantly longer mean CLT on TEG (+2,580 seconds, 95% CI 1,380 to 3,720 sec. Patients with PE and a short CLT who were treated with tenecteplase had increased risk of bleeding, whereas those with long CLT had significantly worse exercise tolerance and psychometric testing for quality of life at 3 months. A multivariate stepwise removal regression model selected PAI-1 and TAFI as predictive biomarkers of CLT.The CLT from TEG predicted increased risk of bleeding and clinical failure with tenecteplase treatment for intermediate-risk PE. Plasmatic PAI-1 and TAFI were independent predictors of CLT.

Thrombin activatable fibrinolysis inhibitor (TAFI) - A possible link between coagulation and complement activation in the antiphospholipid syndrome (APS).

Science.gov (United States)

Grosso, Giorgia; Vikerfors, Anna; Woodhams, Barry; Adam, Mariette; Bremme, Katarina; Holmström, Margareta; Ågren, Anna; Eelde, Anna; Bruzelius, Maria; Svenungsson, Elisabet; Antovic, Aleksandra

2017-10-01

Thrombosis and complement activation are pathogenic features of antiphospholipid syndrome (APS). Their molecular link is Plasma carboxypeptidase-B, also known as thrombin activatable fibrinolysis inhibitor (TAFIa), which plays a dual role: anti-fibrinolytic, by cleaving carboxyl-terminal lysine residues from partially degraded fibrin, and anti-inflammatory, by downregulating complement anaphylatoxins C3a and C5a. To investigate the levels of TAFI (proenzyme) and TAFIa (active enzyme) in relation to complement activation, fibrin clot permeability and fibrinolytic function in clinical and immunological subsets of 52 APS patients and 15 controls. TAFI (pAPS patients compared to controls. Furthermore, TAFIa was increased (pAPS patients affected by arterial thrombosis compared to other APS-phenotypes. Positive associations were found between TAFI and age, fibrinogen and C5a, and between TAFIa and age, fibrinogen and thrombomodulin. TAFI and TAFIa levels were increased in patients with APS as a potential response to complement activation. Interestingly, TAFI activation was associated with arterial thrombotic APS manifestations. Thus, TAFIa may be considered a novel biomarker for arterial thrombosis in APS. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Science.gov (United States)

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

2018-07-01

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

Blood Clotting and Pregnancy

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Blood Clotting and Pregnancy

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Full Text Available ... A genetic predisposition to blood clots Obesity Prolonged immobility (e.g., bedrest, long distance travel) Multiple births ... treating blood conditions. back to top Antiphospholipid Antibody Syndrome: A Patient's Journey back to top Where Can ...

Blood Clotting and Pregnancy

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Full Text Available ... harming your baby. Jump To: Am I at Risk? The risk of developing a blood clot during ... Advances The Hematologist ASH Clinical News ASH Self-Assessment Program Hematology , ASH Education Program About Awards Membership ...

Blood Clotting and Pregnancy

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

Science.gov (United States)

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

2017-09-01

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

A simple clot based assay for detection of procoagulant cell-derived microparticles.

Science.gov (United States)

Patil, Rucha; Ghosh, Kanjaksha; Shetty, Shrimati

2016-05-01

Cell-derived microparticles (MPs) are important biomarkers in many facets of medicine. However, the MP detection methods used till date are costly and time consuming. The main aim of this study was to standardize an in-house clot based screening method for MP detection which would not only be specific and sensitive, but also inexpensive. Four different methods of MP assessment were performed and the results correlated. Using the flow cytometry technique as the gold standard, 25 samples with normal phosphatidylserine (PS) expressing MP levels and 25 samples with elevated levels were selected, which was cross checked by the commercial STA Procoag PPL clotting time (CT) assay. A simple recalcification time and an in-house clot assay were the remaining two tests. The in-house test measures the CT after the addition of calcium chloride to MP rich plasma, following incubation with Russell viper venom and phospholipid free plasma. The CT obtained by the in-house assay significantly correlated with the results obtained by flow cytometry (R2=0.87, p<0.01). Though preliminary, the in-house assay seems to be efficient, inexpensive and promising. It could definitely be utilized routinely for procoagulant MP assessment in various clinical settings.

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

Science.gov (United States)

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

1988-01-01

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

Blood Clotting and Pregnancy

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The application of large amplitude oscillatory stress in a study of fully formed fibrin clots

Science.gov (United States)

Lamer, T. F.; Thomas, B. R.; Curtis, D. J.; Badiei, N.; Williams, P. R.; Hawkins, K.

2017-12-01

The suitability of controlled stress large amplitude oscillatory shear (LAOStress) for the characterisation of the nonlinear viscoelastic properties of fully formed fibrin clots is investigated. Capturing the rich nonlinear viscoelastic behaviour of the fibrin network is important for understanding the structural behaviour of clots formed in blood vessels which are exposed to a wide range of shear stresses. We report, for the first time, that artefacts due to ringing exist in both the sample stress and strain waveforms of a LAOStress measurement which will lead to errors in the calculation of nonlinear viscoelastic properties. The process of smoothing the waveforms eliminates these artefacts whilst retaining essential rheological information. Furthermore, we demonstrate the potential of LAOStress for characterising the nonlinear viscoelastic properties of fibrin clots in response to incremental increases of applied stress up to the point of fracture. Alternating LAOStress and small amplitude oscillatory shear measurements provide detailed information of reversible and irreversible structural changes of the fibrin clot as a consequence of elevated levels of stress. We relate these findings to previous studies involving large scale deformations of fibrin clots. The LAOStress technique may provide useful information to help understand why some blood clots formed in vessels are stable (such as in deep vein thrombosis) and others break off (leading to a life threatening pulmonary embolism).

Fiber-based optic sensor for detecting human blood clot: present and future revival

Science.gov (United States)

Elshikeri, Nada; Bakhtiar, Hazri

2018-05-01

Sustaining human’s life-frame away from being impeded by the clot - ghost term, we attempt to approach a mobile fiber-based optical sensor (f-s) for detecting blood clot in a blood vessel (intra-arteries/veins). Blood vessels are the part of the circulatory system that transport blood throughout the human body, thus their significance of being protected arise to the monograph focus. MRI (magnetic resonance imaging), X-rays and other medical instruments are diagnostic immobility techniques with a slackest interval. The corer causation of fiber-based optical sensor is to detect a clump of blood in the bloodstream by providing a prompt mobile diagnostic intervals preserving last-minutes-breath of human’s life. The detector (f-s) has been etched by diluting sulphuric acid ~10% at certain zone to sensate its function. The in-vitro monograph peaks its maximal monitoring when the sensor is attached to Raman Spectroscopy (RS) setup. RS quantifies the relative intensities of fibrinogen bond, which is the first type of blood coagulation elements of blood plasma. Blood coagulation parameters are the major concern of the monograph investigation, such as total haemoglobin (tHb), clotting reaction time (t), clot progression time (t2), maximum clot amplitude (ma) and mean refractive index (r). A blood sample will be drawn from the patient and after centrifugation to separate blood plasma from its constituents, then an immediate sloshing of blood plasma in the (f-s) packet which has its plug-in to RS. Estimating the quantitative analysis of blood sample concentration, RS will determine the presence of coagulation in terms of intensity and medical procedures will dominate the treatment process. Thus, the suggestive monograph provides a definite instrument for investigating blood coagulation intra-arteries/veins promptly.

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

Science.gov (United States)

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

2018-05-08

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

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

Science.gov (United States)

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

2011-03-15

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

Cation Coordination Alters the Conformation of a Thrombin-Binding G-Quadruplex DNA Aptamer That Affects Inhibition of Thrombin.

Science.gov (United States)

Zavyalova, Elena; Tagiltsev, Grigory; Reshetnikov, Roman; Arutyunyan, Alexander; Kopylov, Alexey

2016-10-01

Thrombin-binding aptamers are promising anticoagulants. HD1 is a monomolecular antiparallel G-quadruplex with two G-quartets linked by three loops. Aptamer-thrombin interactions are mediated with two TT-loops that bind thrombin exosite I. Several cations were shown to be coordinated inside the G-quadruplex, including K + , Na + , NH 4 + , Ba 2+ , and Sr 2+ ; on the contrary, Mn 2+ was coordinated in the grooves, outside the G-quadruplex. K + or Na + coordination provides aptamer functional activity. The effect of other cations on aptamer functional activity has not yet been described, because of a lack of relevant tests. Interactions between aptamer HD1 and a series of cations were studied. A previously developed enzymatic method was applied to evaluate aptamer inhibitory activity. The structure-function correlation was studied using the characterization of G-quadruplex conformation by circular dichroism spectroscopy. K + coordination provided the well-known high inhibitory activity of the aptamer, whereas Na + coordination supported low activity. Although NH 4 + coordination yielded a typical antiparallel G-quadruplex, no inhibitory activity was shown; a similar effect was observed for Ba 2+ and Sr 2+ coordination. Mn 2+ coordination destabilized the G-quadruplex that drastically diminished aptamer inhibitory activity. Therefore, G-quadruplex existence per se is insufficient for aptamer inhibitory activity. To elicit the nature of these effects, we thoroughly analyzed nuclear magnetic resonance (NMR) and X-ray data on the structure of the HD1 G-quadruplex with various cations. The most reasonable explanation is that cation coordination changes the conformation of TT-loops, affecting thrombin binding and inhibition. HD1 counterparts, aptamers 31-TBA and NU172, behaved similarly with some distinctions. In 31-TBA, an additional duplex module stabilized antiparallel G-quadruplex conformation at high concentrations of divalent cations; whereas in NU172, a different

Thrombin Avtivable Fibrinolysis Inhibitor in Venous and Arterial Thrombosis

NARCIS (Netherlands)

E.L.E. de Bruijne

2011-01-01

textabstractVenous and arterial thromboses are major causes of morbidity and mortality. Venous thrombosis is the result of pathological occlusive clot formation in the veins. It occurs mainly in the deep veins of the leg (deep vein thrombosis), from which parts of the clot frequently embolize to the

Blood Clotting and Pregnancy

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Full Text Available ... are pregnant and have concerns about blood clots, talk with your doctor about your risks and prevention. Depending on your condition, your OB-GYN may refer you to a hematologist, a doctor who specializes in treating blood conditions. back to top ...

Thrombin contributes to protective immunity in pneumonia-derived sepsis via fibrin polymerization and platelet-neutrophil interactions

NARCIS (Netherlands)

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

2017-01-01

Essentials Immunity and coagulation are linked during sepsis but the role of thrombin is not fully elucidated. We investigated the effect of thrombin inhibition on murine Klebsiella pneumosepsis outcome. Thrombin is crucial for survival and limiting bacterial growth in pneumonia derived sepsis.

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

Science.gov (United States)

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

2018-02-01

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

Long-term cytokine and growth factor release from equine platelet-rich fibrin clots obtained with two different centrifugation protocols.

Science.gov (United States)

Jiménez-Aristizabal, Román F; López, Catalina; Álvarez, María E; Giraldo, Carlos; Prades, Marta; Carmona, Jorge U

2017-09-01

To compare the temporal release (over three weeks) of tumor necrosis factor alpha (TNF-α), interleukin 4 (IL-4), IL-1 receptor antagonist (IL-1ra), platelet-derived growth factor BB (PDGF-BB) and transforming growth factor beta-1 (TGF-β 1 ) from two platelet-rich fibrin (PRF) preparations from equine blood obtained at either 240g/8min or 416g/10min. Whole blood from 10 horses was used to obtain PRF clots by two different centrifugation protocols. After 1h of rest, PRF clots were deposited in wells with culture medium, which was changed at 6h, 24h and then every 48h to 21days. Cytokines and GFs were measured by ELISA at 1h (serum supernatants from PRF clots) and all time points of culture medium change. A negative control (plasma) and a positive control (blood lysate) were also included. There were no relevant differences between the two protocols for the temporal release of proteins. However, a significant (p=0.01) effect of time was noted. All cytokines were detected after 6h of PRF clot culture until day 21. GF were detected at 1h until day 21. The concentrations for these proteins diminished gradually over time. A highly significant (p=0.01) correlation was noticed between all the proteins evaluated. Leukocytes enmeshed in PRF clots were able to produce cytokines, TGF-β 1 and PDGF-BB. These findings demonstrate a paramount role of leukocytes in wound healing induced or modified by PRF clots in mammals. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Science.gov (United States)

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

2015-02-15

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

Identification of quantitative trait loci for fibrin clot phenotypes: the EuroCLOT study

DEFF Research Database (Denmark)

Williams, Frances M K; Carter, Angela M; Kato, Bernet

2009-01-01

OBJECTIVE: Fibrin makes up the structural basis of an occlusive arterial thrombus, and variability in fibrin phenotype relates to cardiovascular risk. The aims of the current study from the EU consortium EuroCLOT were to (1) determine the heritability of fibrin phenotypes and (2) identify QTLs as...

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

International Nuclear Information System (INIS)

Setter, R.

1977-01-01

The behaviour of radioactively labelled fibrinogen after infusion of thrombin or ancrod is investigated. Common factors and differences in the behaviour of fibrinogen after infusion of these two enzymes, which act proteolytically on the fibrinogen, are dealt with. Rabbits received an i.v. injection of homologous 125 I-fibrinogen 3 days before ancrod or thrombin infusion. On the day of the experiments, one group of animals received an ancrod infusion (1.5 U/kg body weight for 30 minutes), the other a thrombin infusion (600 U/kg body weight for 60 minutes). Intravenous ancrod and thrombin infusions lowered the fibrinogen level to 30% or 50% of the initial value due to intravascular coagulation. About 50% of the 125 I fibrinogen was transformed after ancrod exposure into a non-coagulating fraction of fibrinogen derivatives which produces no fibrinolytic decomposition products. (orig./AJ) [de

Effect of low frequency ultrasound on combined rt-PA and eptifibatide thrombolysis in human clots.

Science.gov (United States)

Meunier, Jason M; Holland, Christy K; Pancioli, Arthur M; Lindsell, Christopher J; Shaw, George J

2009-01-01

Fibrinolytics such as recombinant tissue plasminogen activator (rt-PA) are used to treat thrombotic disease such as acute myocardial infarction (AMI) and ischemic stroke. Interest in increasing efficacy and reducing side effects has led to the study of adjuncts such as GP IIb-IIIa inhibitors and ultrasound (US) enhanced thrombolysis. Currently, GP IIb-IIIa inhibitor and fibrinolytic treatment are often used in AMI, and are under investigation for stroke treatment. However, little is known of the efficacy of combined GP IIb-IIIa inhibitor, fibrinolytic and ultrasound treatment. We measure the lytic efficacy of rt-PA, eptifibatide (Epf) and 120 kHz ultrasound treatment in an in-vitro human clot model. Blood was drawn from 15 subjects after IRB approval. Clots were made in 20 microL pipettes, and placed in a water tank for microscopic visualization during lytic treatment. Clots were exposed to control, rt-PA (rt-PA), eptifibatide (Epf), or rt-PA+eptifibatide (rt-PA + Epf), with (+US) or without (-US) ultrasound for 30 minutes at 37 degrees C in human plasma. Clot lysis was measured over time, using a microscopic imaging technique. The fractional clot loss (FCL) and initial lytic rate (LR) were used to quantify lytic efficacy. LR values for (- US) treated clots were 0.8+/-0.1(control), 1.8+/-0.3 (Epf), 1.5+/-0.2 (rt-PA), and 1.3+/-0.4 (rt-PA + Epf) (% clot width/minute) respectively. In comparison, the (+ US) group exhibited LR values of 1.6+/-0.2 (control), 4.3+/-0.4 (Epf), 6.3+/-0.4 (rt-PA), and 4.6+/-0.6 (rt-PA + Epf). For (- US) treated clots, FCL was 6.0+/-0.8 (control), 9.2+/-2.5 (Epf), 15.6+/-1.7 (rt-PA), and 28.0+/-2.2% (rt-PA + Epf) respectively. FCL for (+ US) clots was 13.5+/-2.4 (control), 20.7+/-6.4 (Epf), 44.4+/-3.6 (rt-PA) and 30.3+/-3.6% (rt-PA + Epf) respectively. Although the addition of eptifibatide enhances the in-vitro lytic efficacy of rt-PA in the absence of ultrasound, the efficacy of ultrasound and rt-PA is greater than that of combined

Thrombin-specific inactivation of endothelial cell derived plasminogen activator

International Nuclear Information System (INIS)

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

1986-01-01

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 125 I-fibrinogenolytic or caseinolytic assay. T (5 nM), but not plasmin or trypsin at equivalent concentrations, resulted in a time-dependent inhibition of the PA activity in CM. T had no effect on the PA activity of urokinase, streptokinase or preformed plasmin. The ability of T to inactivate the EC-derived PA was abolished by prior treatment of T with active site-directed reagents. SDS-PAGE and zymography with copolymerized fibrinogen and plasminogen revealed further specificity in that only one of the multiple-molecular weight forms of PA present in EC-CM was inactivated by T. The authors conclude that in a highly specific fashion, T inactivates the predominant PA present in EC-CM by limited proteolysis. Thus, another potentially important function of T is suggested which may have particular significance in the temporal regulation of coagulation and fibrinolysis at the blood-endothelium interface

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

International Nuclear Information System (INIS)

Maruyama, I.; Majerus, P.W.

1987-01-01

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 125 I-thrombin-thrombomodulin complexes, but not 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 125 I-thrombin and diisopropylphosphoryl (DIP) 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

14-Day thawed plasma retains clot enhancing properties and inhibits tPA-induced fibrinolysis.

Science.gov (United States)

Huebner, Benjamin R; Moore, Ernest E; Moore, Hunter B; Shepherd-Singh, Raymond; Sauaia, Angela; Stettler, Gregory R; Nunns, Geoffrey R; Silliman, Christopher C

2017-11-01

Plasma-first resuscitation attenuates trauma-induced coagulopathy (TIC); however, the logistics of plasma-first resuscitation require thawed plasma (TP) be readily available due to the obligatory thawing time of fresh frozen plasma (FFP). The current standard is storage of TP for up to 5 days at 4°C, based on factor levels at outdate, for use in patients at risk for TIC, but there remains a 2.2% outdated wastage rate. However, the multitude of plasma proteins in attenuating TIC remains unknown. We hypothesize that TP retains the ability to enhance clotting and reduce tPA-induced fibrinolysis at 14-day storage. FFP was thawed and stored at 4°C at the following intervals: 14, 10, 7, 5, 3, and 1-day prior to the experiment. Healthy volunteers underwent blood draws followed by 50% dilution with TP stored at previously mentioned intervals as well as FFP, normal saline (NS), albumin, and whole blood (WB) control. Samples underwent tPA-modified (75 ng/mL) thrombelastography (TEG) with analysis of R-time, angle, maximum amplitude (MA), and LY30. TEG properties did not change significantly over the thawed storage. 14-day TP retained the ability to inhibit tPA-induced hyperfibrinolysis (median LY30% 9.6%) similar to FFP (5.6%), WB (14.6%), and superior to albumin (59.3%) and NS (58.1%). 14-day TP also retained faster clot formation (median angle, 66.2°) and superior clot strength (MA, 61.5 mm) to albumin (34.8°, 21.6 mm) and NS (41.6°, 32.2 mm). TP plasma stored for 14 days retains clot-enhancing ability and resistance to clot degradation similar to FFP. A clinical trial is needed to validate these in vitro results. Copyright © 2017 Elsevier Inc. All rights reserved.

The hydraulic permeability of blood clots as a function of fibrin and platelet density.

Science.gov (United States)

Wufsus, A R; Macera, N E; Neeves, K B

2013-04-16

Interstitial fluid flow within blood clots is a biophysical mechanism that regulates clot growth and dissolution. Assuming that a clot can be modeled as a porous medium, the physical property that dictates interstitial fluid flow is the hydraulic permeability. The objective of this study was to bound the possible values of the hydraulic permeability in clots formed in vivo and present relationships that can be used to estimate clot permeability as a function of composition. A series of clots with known densities of fibrin and platelets, the two major components of a clot, were formed under static conditions. The permeability was calculated by measuring the interstitial fluid velocity through the clots at a constant pressure gradient. Fibrin gels formed with a fiber volume fraction of 0.02-0.54 had permeabilities of 1.2 × 10(-1)-1.5 × 10(-4)μm(2). Platelet-rich clots with a platelet volume fraction of 0.01-0.61 and a fibrin volume fraction of 0.03 had permeabilities over a range of 1.1 × 10(-2)-1.5 × 10(-5)μm(2). The permeability of fibrin gels and of clots with platelet volume fraction of platelet volume fraction of >0.2 were modeled as a Brinkman medium of coarse solids (platelets) embedded in a mesh of fine fibers (fibrin). Our data suggest that the permeability of clots formed in vivo can vary by up to five orders of magnitude, with pore sizes that range from 4 to 350 nm. These findings have important implications for the transport of coagulation zymogens/enzymes in the interstitial spaces during clot formation, as well as the design of fibrinolytic drug delivery strategies. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Googling Service Boundaries for Endovascular Clot Retrieval Hub Hospitals in a Metropolitan Setting: Proof-of-Concept Study.

Science.gov (United States)

Phan, Thanh G; Beare, Richard; Chen, Jian; Clissold, Benjamin; Ly, John; Singhal, Shaloo; Ma, Henry; Srikanth, Velandai

2017-05-01

There is great interest in how endovascular clot retrieval hubs provide services to a population. We applied a computational method to objectively generate service boundaries for such endovascular clot retrieval hubs, defined by traveling time to hub. Stroke incidence data merged with population census to estimate numbers of stroke in metropolitan Melbourne, Australia. Traveling time from randomly generated addresses to 4 endovascular clot retrieval-capable hubs (Royal Melbourne Hospital [RMH], Monash Medical Center [MMC], Alfred Hospital [ALF], and Austin Hospital [AUS]) estimated using Google Map application program interface. Boundary maps generated based on traveling time at various times of day for combinations of hubs. In a 2-hub model, catchment was best distributed when RMH was paired with MMC (model 1a, RMH 1765 km 2 and MMC 1164 km 2 ) or with AUS (model 1c, RMH 1244 km 2 and AUS 1685 km 2 ), with no statistical difference between models ( P =0.20). Catchment was poorly distributed when RMH was paired with ALF (model 1b, RMH 2252 km 2 and ALF 676 km 2 ), significantly different from both models 1a and 1c (both P AUS was superior to that of RMH, MMC, and ALF in catchment distribution and travel time. The method was also successfully applied to the city of Adelaide demonstrating wider applicability. We provide proof of concept for a novel computational method to objectively designate service boundaries for endovascular clot retrieval hubs. © 2017 American Heart Association, Inc.

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

DEFF Research Database (Denmark)

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

2017-01-01

Dabigatran is an oral anticoagulant and a reversible inhibitor of thrombin. Further, dabigatran might affect platelet function through a direct effect on platelet thrombin receptors. The aim was to investigate the effect of dabigatran on platelet activation and platelet aggregation. Healthy donor...

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

Science.gov (United States)

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

2000-11-17

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

Label-free aptamer biosensor for thrombin detection based on functionalized graphene nanocomposites.

Science.gov (United States)

Wang, Qingqing; Zhou, Zhixue; Zhai, Yanling; Zhang, Lingling; Hong, Wei; Zhang, Zhiquan; Dong, Shaojun

2015-08-15

A label-free and amplified electrochemical impedimetric aptasensor based on functionalized graphene nanocomposites (rGO-AuNPs) was developed for the detection of thrombin, which played a vital role in thrombosis and hemostasis. The thiolated aptamer and dithiothreitol (TBA15-DTT) were firstly immobilized on the gold electrode to capture the thrombin molecules, and then aptamer functionalized graphene nanocomposites (rGO-TBA29) were used to fabricate a sandwich sensing platform for amplifying the impedimetric signals. As numerous negative charges of TBA29 on the electrode repelled to the [Fe(CN)6](4-/3-) anions, resulting in an obvious amplified charge-transfer resistance (Rct) signal. The Rct increase was linearly proportional to the thrombin concentration from 0.3 to 50nM and a detection limit of 0.01nM thrombin was achieved. In addition, graphene could also be labeled with other probes via electrostatic or π-π stacking interactions to produce signals, therefore different detection methods expanding wide application could be used in this model. Copyright © 2015. Published by Elsevier B.V.

Diastolic timed Vibro-Percussion at 50 Hz delivered across a chest wall sized meat barrier enhances clot dissolution and remotely administered Streptokinase effectiveness in an in-vitro model of acute coronary thrombosis

OpenAIRE

Hoffmann, Andrew; Gill, Harjit

2012-01-01

Abstract Background Low Frequency Vibro-Percussion (LFVP) assists clearance of thrombi in catheter systems and when applied to the heart and timed to diastole is known to enhance coronary flow. However LFVP on a clotted coronary like vessel given engagement over a chest wall sized barrier (to resemble non-invasive heart attack therapy) requires study. Methods One hour old clots (n=16) were dispensed within a flexible segment of Soft-Flo catheter (4 mm lumen), weighted, interfaced with Heparin...

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

International Nuclear Information System (INIS)

Hatton, M.W.; Moar, S.L.

1986-01-01

Various purified proteins, protein derivatives and two polysaccharides were added individually to a physiological medium in order to effect uptake of 125 I-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 125 I-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 125 I-thrombin in the absence of albumin did not encourage thrombin uptake to the same extent as having 125 I-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

Deletion of the thrombin cleavage domain of osteopontin mediates breast cancer cell adhesion, proteolytic activity, tumorgenicity, and metastasis

International Nuclear Information System (INIS)

Beausoleil, Michel S; Schulze, Erika B; Goodale, David; Postenka, Carl O; Allan, Alison L

2011-01-01

Osteopontin (OPN) is a secreted phosphoprotein often overexpressed at high levels in the blood and primary tumors of breast cancer patients. OPN contains two integrin-binding sites and a thrombin cleavage domain located in close proximity to each other. To study the role of the thrombin cleavage site of OPN, MDA-MB-468 human breast cancer cells were stably transfected with either wildtype OPN (468-OPN), mutant OPN lacking the thrombin cleavage domain (468-ΔTC) or an empty vector (468-CON) and assessed for in vitro and in vivo functional differences in malignant/metastatic behavior. All three cell lines were found to equivalently express thrombin, tissue factor, CD44, αvβ5 integrin and β1 integrin. Relative to 468-OPN and 468-CON cells, 468-ΔTC cells expressing OPN with a deleted thrombin cleavage domain demonstrated decreased cell adhesion (p < 0.001), decreased mRNA expression of MCAM, maspin and TRAIL (p < 0.01), and increased uPA expression and activity (p < 0.01) in vitro. Furthermore, injection of 468-ΔTC cells into the mammary fat pad of nude mice resulted in decreased primary tumor latency time (p < 0.01) and increased primary tumor growth and lymph node metastatic burden (p < 0.001) compared to 468-OPN and 468-CON cells. The results presented here suggest that expression of thrombin-uncleavable OPN imparts an early tumor formation advantage as well as a metastatic advantage for breast cancer cells, possibly due to increased proteolytic activity and decreased adhesion and apoptosis. Clarification of the mechanisms responsible for these observations and the translation of this knowledge into the clinic could ultimately provide new therapeutic opportunities for combating breast cancer

Sphingosine 1-Phosphate as a Link between Blood Coagulation and Inflammation

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Bernhard Hermann Rauch

2014-06-01

Full Text Available Sphingosine 1-phosphate (S1P is a multifunctional signaling lipid generated from sphingosine by sphingosine kinases. S1P formation has been shown in numerous cells in the circulation, including platelets, vascular endothelial and smooth muscle cells and monocytes. S1P also exerts multiple effects on these cells, i.e. cell proliferation and migration, activation of proinflammatory signaling pathways and release of additional inflammatory mediators. Similar activities and targets have also been identified for activated clotting factors such as thrombin or the activated factor-X (FXa, suggesting a possible involvement of S1P in thrombus-associated cellular signaling and thrombin-induced inflammatory reactions. Several levels of S1P-mediated, thrombin /FXa-induced signaling have already been identified: regulation of sphingosine kinase expression and activity, stimulation of S1P release from platelets and other cells and, possibly regulation of S1P-receptors on target cells. This review summarizes the current state of knowledge about S1P as a clotting factor-regulated molecular link between blood coagulation and inflammation. It is concluded that S1P might represent an until now underestimated lipid mediator of inflammatory reactions following activation of the clotting system and, in this context, also involved in the development and progression of atherosclerosis.

DNA, histones and neutrophil extracellular traps exert anti-fibrinolytic effects in a plasma environment.

Science.gov (United States)

Varjú, Imre; Longstaff, Colin; Szabó, László; Farkas, Ádám Zoltán; Varga-Szabó, Veronika Judit; Tanka-Salamon, Anna; Machovich, Raymund; Kolev, Krasimir

2015-06-01

In response to various inflammatory stimuli, neutrophils secrete neutrophil extracellular traps (NETs), web-like meshworks of DNA, histones and granular components forming supplementary scaffolds in venous and arterial thrombi. Isolated DNA and histones are known to promote thrombus formation and render fibrin clots more resistant to mechanical forces and tissue-type plasminogen activator (tPA)-induced enzymatic digestion. The present study extends our earlier observations to a physiologically more relevant environment including plasma clots and NET-forming neutrophils. A range of techniques was employed including imaging (scanning electron microscopy (SEM), confocal laser microscopy, and photoscanning of macroscopic lysis fronts), clot permeability measurements, turbidimetric lysis and enzyme inactivation assays. Addition of DNA and histones increased the median fibre diameter of plasma clots formed with 16 nM thrombin from 108 to 121 and 119 nm, respectively, and decreased their permeability constant from 6.4 to 3.1 and 3.7×10(-9) cm(2). Histones effectively protected thrombin from antithrombin-induced inactivation, while DNA inhibited plasminogen activation on the surface of plasma clots and their plasmin-induced resolution by 20 and 40 %, respectively. DNA and histones, as well as NETs secreted by phorbol-myristate-acetate-activated neutrophils, slowed down the tPA-driven lysis of plasma clots and the latter effect could be reversed by the addition of DNase (streptodornase). SEM images taken after complete digestion of fibrin in NET-containing plasma clots evidenced retained NET scaffold that was absent in DNase-treated clots. Our results show that DNA and histones alter the fibrin architecture in plasma clots, while NETs contribute to a decreased lytic susceptibility that can be overcome by DNase.

Addition of a sequence from α2-antiplasmin transforms human serum albumin into a blood clot component that speeds clot lysis

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Gataiance Sharon

2009-03-01

Full Text Available Abstract Background The plasma protein α2-antiplasmin (α2AP is cross-linked to fibrin in blood clots by the transglutaminase factor XIIIa, and in that location retards clot lysis. Competition for this effect could be clinically useful in patients with thrombosis. We hypothesized that fusion of N-terminal portions of α2-antiplasmin to human serum albumin (HSA and production of the chimeric proteins in Pichia pastoris yeast would produce a stable and effective competitor protein. Results Fusion protein α2AP(13-42-HSA was efficiently secreted from transformed yeast and purified preparations contained within a mixed population the full-length intact form, while fusions with longer α2AP moieties were inefficiently secreted and/or degraded. The α2AP(13-42-HSA protein, but not recombinant HSA, was cross-linked to both chemical lysine donors and fibrin or fibrinogen by factor XIIIa, although with less rapid kinetics than native α2AP. Excess α2AP(13-42-HSA competed with α2AP for cross-linking to chemical lysine donors more effectively than a synthetic α2AP(13-42 peptide, and reduced the α2AP-dependent resistance to fibrinolysis of plasma clots equally effectively as the peptide. Native α2AP was found in in vivo clots in rabbits to a greater extent than α2AP(13-42, however. Conclusion In this first report of transfer of transglutamination substrate status from one plasma protein to another, fusion protein α2AP(13-42-HSA was shown to satisfy initial requirements for a long-lasting, well-tolerated competitive inhibitor of α2-antiplasmin predicted to act in a clot-localized manner.

Thrombin-specific inactivation of endothelial cell derived plasminogen activator

Energy Technology Data Exchange (ETDEWEB)

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

1986-03-05

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

Endogenous thrombin potential in polycystic ovary syndrome

DEFF Research Database (Denmark)

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

2015-01-01

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

1: 1 Transfusion strategies are right for the wrong reasons.

Science.gov (United States)

Savage, Stephanie A; Zarzaur, Ben L; Brewer, Brian L; Lim, Garrett H; Martin, Ali C; Magnotti, Louis J; Croce, Martin A; Pohlman, Timothy H

2017-05-01

Early assessment of clot function identifies coagulopathies after injury. Abnormalities include a hypercoagulable state from excess thrombin generation, as well as an acquired coagulopathy. Efforts to address coagulopathy have resulted in earlier, aggressive use of plasma emphasizing 1:1 resuscitation. The purpose of this study was to describe coagulopathies in varying hemorrhagic profiles from a cohort of injured patients. All injured patients who received at least one unit of packed red blood cells (PRBC) in the first 24 hours of admission from September 2013 to May 2015 were eligible for inclusion. Group-Based Trajectory Modeling, using volume of transfusion over time, was used to identify specific hemorrhagic phenotypes. The thromboelastography profile of each subgroup was characterized and group features were compared. Four hemorrhagic profiles were identified among 330 patients-minimal (MIN, group 1); patients with large PRBC requirements later in the hospital course (LH, group 2); massive PRBC usage (MH, group 3), and PRBC transfusion limited to shortly after injury (EH, group 4). All groups had an R-time shorter than the normal range (3.2-3.5, p = NS). Patients in group 3 had longer K-times (1.8 vs. 1.2-1.3, p < 0.05), significantly flatter α-angles (66.7 vs. 70.4-72.8, p < 0.05), and significantly weaker clot strength (MA 54.6 vs. 62.3-63.6, p < 0.05). Group 3 had greater physiologic derangements at admission and worse overall outcomes. Hemorrhagic profiles suggest a rapid onset of clot formation in all subgroups but significantly suppressed thrombin burst and diminished clot strength in the most injured. Patients are both hypercoagulable, with early and precipitous clot formation, and also have a demonstrable hypocoagulability. The exact cause of traumatic hypocoagulability is likely multifactorial. Goal-directed resuscitation, as early as institution of the massive transfusion protocol, may be more effective in resuscitating the most coagulopathic

Binding of α2-macroglobulin-thrombin complexes and methylamine-treated α2-macroglobulin to human blood monocytes

International Nuclear Information System (INIS)

Straight, D.L.; Jakoi, L.; McKee, P.A.; Snyderman, R.

1988-01-01

The binding of α 2 -macroglobulin (α 2 M) to human peripheral blood monocytes was investigated. Monocytes, the precursors of tissue macrophages, were isolated from fresh blood by centrifugal elutriation or density gradient centrifugation. Binding studies were performed using 125 I-labeled α 2 M. Cells and bound ligand were separated from free ligand by rapid vacuum filtration. Nonlinear least-squares analysis of data obtained in direct binding studies at 0 0 C showed that monocytes bound the α 2 M-thrombin complex with a K/sub d/ 3.0 +- .09 nM and the monocyte had 1545 +- 153 sitescell. Thrombin alone did not compete for the site. Binding was divalent cation dependent. Direct binding studies also demonstrated that monocytes bound methylamine-treated α 2 M in a manner similar to α 2 M-thrombin. Competitive binding studies showed that α 2 M-thrombin and methylamine-treated α 2 M bound to the same sites on the monocyte. In contrast, native α 2 M did not compete with α 2 M-thrombin for the site. Studies done at 37 0 C suggested that after binding, the monocyte internalized and degraded α 2 M-thrombin and excreted the degradation products. Receptor turnover and degradation of α 2 M-thrombin complexes were blocked in monocytes treated with chloroquine, an inhibitor of lysosomal function. The results indicate that human monocytes have a divalent cation dependent, high-affinity binding site for α 2 M-thrombin and methylamine-treated α 2 M which may function to clear α 2 M-proteinase complexes from the circulation

EFFICACY OF THROMBIN FIBRIN GLUE AND SCLE ROSANT IN THE MANAGEMENT OF BLEEDI NG GASTRIC VARICES

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Sanjay Gupta

2015-01-01

Full Text Available Gastric varices are noted in up to 20 % of patents with portal hypertension , and are more common in those with non - cirrhotic etiology 1 . They bleed at lower portal pressures , bleed more severely and are associated with higher rates of rebleed , encephalopathy and mortality 1,2,3 . Variceal obliteration using tissue adhesives such as N - butyl cyanoacrylate leading to plugging and thrombosis of the gastric varices is currently the first line management option for obliteration of the gastric varices 3 . Although various options have been proposed , gold standard for management of gastric variceal bleeds is yet to be defined. We theorized that injection of the gastric varices using thrombin based glue followed by injection of a sclerosant shall be effective in optimum sclerotherapy and eradication of gastric varices. MATERIAL AND METHODS : All patients presenting with gastric variceal bleed were offered sclerotherapy with Thrombin fibrin based glue and sclerosant (TFG/S . During the study period 18 patients were enrolled in the TGF/S group. 21 patients underwent variceal plugging with n - butyl cyanoacrylate (NBC . There was no significant difference in age/ sex , duration of bleed or time interval between onset of bleed and endotherapy. RESULTS: Patients undergoing endotherapy with TGF/S had less episodes of bleed , and greater eradication of varices. CONCLUSION: The results with thrombin / fibrin glue and sclerotherapy are highly encouraging. Well - designed trials need to be performed KEYWORDS:Gastric varices; Thrombin Sclerotherapy

Alkali treatment of microrough titanium surfaces affects macrophage/monocyte adhesion, platelet activation and architecture of blood clot formation

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V Milleret

2011-05-01

Full Text Available Titanium implants are most commonly used for bone augmentation and replacement due to their favorable osseointegration properties. Here, hyperhydrophilic sand-blasted and acid-etched (SBA titanium surfaces were produced by alkali treatment and their responses to partially heparinized whole human blood were analyzed. Blood clot formation, platelet activation and activation of the complement system was analyzed revealing that exposure time between blood and the material surface is crucial as increasing exposure time results in higher amount of activated platelets, more blood clots formed and stronger complement activation. In contrast, the number of macrophages/monocytes found on alkali-treated surfaces was significantly reduced as compared to untreated SBA Ti surfaces. Interestingly, when comparing untreated to modified SBA Ti surfaces very different blood clots formed on their surfaces. On untreated Ti surfaces blood clots remain thin (below 15 mm, patchy and non-structured lacking large fibrin fiber networks whereas blood clots on differentiated surfaces assemble in an organized and layered architecture of more than 30 mm thickness. Close to the material surface most nucleated cells adhere, above large amounts of non-nucleated platelets remain entrapped within a dense fibrin fiber network providing a continuous cover of the entire surface. These findings might indicate that, combined with findings of previous in vivo studies demonstrating that alkali-treated SBA Ti surfaces perform better in terms of osseointegration, a continuous and structured layer of blood components on the blood-facing surface supports later tissue integration of an endosseous implant.

Iodine based radiopacity of experimental blood clots for testing of mechanical thrombectomy devices

International Nuclear Information System (INIS)

Luo, Zhong Hua; Chung, Alex; Choi, Gibok; Lin, Yih Huie; Pang, Huajin; Uchida, Barry T.; Pavcnik, Dusan; Jeromel, Miran; Keller, Frederick S.; Rösch, Josef

2013-01-01

Barium sulfate powder used for radiopacity of experimental blood clots (EBCs) for testing mechanical thrombectomy devices (MTD) has negative effects on EBCs mechanical properties. In vitro and in vivo exploration was performed to determine if the iodine based contrast medium will have less negative effects on the EBCs than barium. Fresh blood from 2 swine was used to create fibrinogen enhanced and thrombin initiated EBC in tubes. Iodine radiopacity was achieved by mixing the blood with 65% Iohexol or by soaking the EBCs for 2 or 24 hours in Iohexol. The EBCs opacified with barium served as controls. In vitro study: The EBCs were subjected to four tests, manual elongation, catheter injection, radiopacity and contrast wash out tests. In vivo study: The common carotid arteries of 2 swine were embolized by either barium EBC or EBC soaked for 24 hours in Iohexol. The duration of radiopacity of the different EBCs was compared. The EBCs opacified with Iohexol initially had higher radiopacity than the barium opacified EBCs. However, their opacity rapidly decreased with saline soaking and, particularly, after they were embolized in live animals. The mechanical properties of Iohexol opacified EBCs were inferior to barium opacified EBCs. The Iohexol mixed EBCs were less firm and elastic and half of them fragmented during catheter injection. The Iohexol soaked EBCs exhibited decreased tensile strength and elasticity compared to the barium EBCs. Compared to barium, iodine based contrast medium does not offer any advantage for opacifying EBCs

Large-scale preparation of active caspase-3 in E. coli by designing its thrombin-activatable precursors

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Park Sung

2008-12-01

Full Text Available Abstract Background Caspase-3, a principal apoptotic effector that cleaves the majority of cellular substrates, is an important medicinal target for the treatment of cancers and neurodegenerative diseases. Large amounts of the protein are required for drug discovery research. However, previous efforts to express the full-length caspase-3 gene in E. coli have been unsuccessful. Results Overproducers of thrombin-activatable full-length caspase-3 precursors were prepared by engineering the auto-activation sites of caspase-3 precursor into a sequence susceptible to thrombin hydrolysis. The engineered precursors were highly expressed as soluble proteins in E. coli and easily purified by affinity chromatography, to levels of 10–15 mg from 1 L of E. coli culture, and readily activated by thrombin digestion. Kinetic evaluation disclosed that thrombin digestion enhanced catalytic activity (kcat/KM of the precursor proteins by two orders of magnitude. Conclusion A novel method for a large-scale preparation of active caspase-3 was developed by a strategic engineering to lack auto-activation during expression with amino acid sequences susceptible to thrombin, facilitating high-level expression in E. coli. The precursor protein was easily purified and activated through specific cleavage at the engineered sites by thrombin, generating active caspase-3 in high yields.

Thermal Blood Clot Formation and use in Microfluidic Device Valving Applications

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Tai, Yu-Chong (Inventor); Shi, Wendian (Inventor); Guo, Luke (Inventor)

2014-01-01

The present invention provides a method of forming a blood-clot microvalve by heating blood in a capillary tube of a microfluidic device. Also described are methods of modulating liquid flow in a capillary tube by forming and removing a blood-clot microvalve.

A French National Survey on Clotting Disorders in Mastocytosis.

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Carvalhosa, Ana B; Aouba, Achille; Damaj, Gandhi; Canioni, Danielle; Brouzes, Chantal; Gyan, Emmanuel; Durupt, Stéphane; Durieu, Isabelle; Cathebras, Pascal; Costédoat-Chalumeau, Nathalie; Launay, David; Pilmis, Benoit; Barete, Stephane; Frenzel, Laurent; Lortholary, Olivier; Hermine, Olivier; Hermans, Cedric; Chandesris, Marie-Olivia

2015-10-01

Mastocytosis is characterized by a clonal mast cell proliferation with organ infiltration and uncontrolled degranulation. Although not characteristic and poorly explained, some patients develop clotting abnormalities. We retrospectively identified patients with established diagnosis of mastocytosis and related clotting abnormalities (clinical and/or biological) using the national French Reference Centre for Mastocytosis database. From our cohort of 14 adult patients with clotting abnormalities (median age 46 years [range 26-75]), 4 had a presentation suggestive of a primary hemostasis disorder alone (by their symptoms and/or abnormal clotting tests [PFA, von Willebrand's disease [vWD] screening]) and 10 had a laboratory impairment of secondary hemostasis. Among these, 7 had bleeds characteristic of a coagulation cascade disorder (severe/life-threatening in 5 and mild in 2 patients). Clotting abnormalities were of variable severity, typically related to intense crisis of degranulation, such as anaphylactic reactions, and/or to severe organ infiltration by mast cells. Importantly, classical hemostatic management with platelet transfusion, fresh frozen plasma, or vitamin K infusions was unsuccessful, as opposed to the use of agents inhibiting mast cell activity, particularly steroids. This illustrates the crucial role of mast cell mediators such as tryptase and heparin, which interfere both with primary (mainly via inhibition of von Willebrand factor) and secondary hemostasis. There was interestingly an unusually high number of aggressive mastocytosis (particularly mast cell leukemia) and increased mortality in the group with secondary hemostasis disorders (n = 5, 36% of the whole cohort). Mast cell degranulation and/or high tumoral burden induce both specific biologic antiaggregant and anticoagulant states with a wide clinical spectrum ranging from asymptomatic to life-threatening bleeds. Hemostatic control is achieved by mast cell inhibitors such as steroids.

Thrombin-induced, TNFR-dependent miR-181c downregulation promotes MLL1 and NF-κB target gene expression in human microglia.

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Yin, Min; Chen, Zhiying; Ouyang, Yetong; Zhang, Huiyan; Wan, Zhigang; Wang, Han; Wu, Wei; Yin, Xiaoping

2017-06-29

Controlling thrombin-driven microglial activation may serve as a therapeutic target for intracerebral hemorrhage (ICH). Here, we investigated microRNA (miRNA)-based regulation of thrombin-driven microglial activation using an in vitro thrombin toxicity model applied to primary human microglia. A miRNA array identified 22 differential miRNA candidates. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) identified miR-181c as the most significantly downregulated miRNA. TargetScan analysis identified mixed lineage leukemia-1 (MLL1) as a putative gene target for miR-181c. qRT-PCR was applied to assess tumor necrosis factor-alpha (TNF-α), miR-181c, and MLL1 levels following thrombin or proteinase-activated receptor-4-specific activating peptide (PAR4AP) exposure. Anti-TNF-α antibodies and tumor necrosis factor receptor (TNFR) silencing were employed to test TNF-α/TNFR dependence. A dual-luciferase reporter system and miR-181c mimic transfection assessed whether mir-181c directly binds to and negatively regulates MLL1. Nuclear factor kappa-B (NF-κB)-dependent luciferase reporter assays and NF-κB target gene expression were assessed in wild-type (MLL1+) and MLL1-silenced cells. Thrombin or PAR4AP-induced miR-181c downregulation (p < 0.05) and MLL1 upregulation (p < 0.05) that were dependent upon TNF-α/TNFR. miR-181c decreased wild-type MLL1 3'-UTR luciferase reporter activity (p < 0.05), and a miR-181c mimic suppressed MLL1 expression (p < 0.05). Thrombin treatment increased, while miR-181c reduced, NF-κB activity and NF-κB target gene expression in both wild-type (MLL1+) and MLL1-silenced cells (p < 0.05). Thrombin-induced, TNF-α/TNFR-dependent miR-181c downregulation promotes MLL1 expression, increases NF-κB activity, and upregulates NF-κB target gene expression. As miR-181c opposes thrombin's stimulation of pro-inflammatory NF-κB activity, miR-181c mimic therapy may show promise in controlling thrombin

Factors Affecting the Growth and Production of Milk-Clotting Enzyme by Amylomyces rouxii in Rice Liquid Medium

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Pei-Jing Yu

2005-01-01

Full Text Available Amylomyces rouxii is one of the main fungi usually coexisting with yeasts in Chinese yeast ball, the starter of chiu-niang, a traditional Chinese fermented product from rice. In the present study, growth and production of milk-clotting enzyme (MCE in gelatinous rice liquid culture of A. rouxii as influenced by waxy (gelatinous rice content in the medium (5–20 %, temperature (25–40 °C, cultivation time (1–6 days, shaking speeds (0–150 rpm and metal ions (Na+, K+, Zn2+, Mg2+, Mn2+, Cu2+, Ca2+, Fe3+ and Al3+ were investigated. Results revealed that rice content in the medium, shaking speed, temperature and cultivation time all affected the mycelial propagation and the production of milk-clotting enzyme by A. rouxii in the rice liquid culture. The maximum milk-clotting enzyme activity of ca. 1.22 unit/mL of medium was observed in the 3-day static culture of test organism grown at 30 °C in the medium containing 20 % of gelatinous rice, while mycelial propagation increased with the increase of cultivation time and shaking speed. Furthermore, a significant increase (p<0.05 in the milk-clotting enzyme activity of ca. 1.90 unit/mL of medium, which was about 1.55-fold of the control, was observed when Al3+ was added to the rice liquid medium.

A label-free and high sensitive aptamer biosensor based on hyperbranched polyester microspheres for thrombin detection

International Nuclear Information System (INIS)

Sun, Chong; Han, Qiaorong; Wang, Daoying; Xu, Weimin; Wang, Weijuan; Zhao, Wenbo; Zhou, Min

2014-01-01

Highlights: • A label-free thrombin aptamer biosensor applied in whole blood has been developed. • The aptamer biosensor showed a wide detection range and a low detection limit. • The antibiofouling idea utilized for biosensor is significant for diagnostics. - Abstract: In this paper, we have synthesized hyperbranched polyester microspheres with carboxylic acid functional groups (HBPE-CA) and developed a label-free electrochemical aptamer biosensor using thrombin-binding aptamer (TBA) as receptor for the measurement of thrombin in whole blood. The indium tin oxide (ITO) electrode surface modified with HBPE-CA microspheres was grafted with TBA, which has excellent binding affinity and selectivity for thrombin. Binding of the thrombin at the modified ITO electrode surface greatly restrained access of electrons for a redox probe of [Fe(CN) 6 ] 3−/4− . Moreover, the aptamer biosensor could be used for detection of thrombin in whole blood, a wide detection range (10 fM–100 nM) and a detection limit on the order of 0.90 fM were demonstrated. Control experiments were also carried out by using bull serum albumin (BSA) and lysozyme in the absence of thrombin. The good stability and repeatability of this aptamer biosensor were also proved. We expect that this demonstration will lead to the development of highly sensitive label-free sensors based on aptamer with lower cost than current technology. The integration of the technologies, which include anticoagulant, sensor and nanoscience, will bring significant input to high-performance biosensors relevant to diagnostics and therapy of interest for human health

A label-free and high sensitive aptamer biosensor based on hyperbranched polyester microspheres for thrombin detection

Energy Technology Data Exchange (ETDEWEB)

Sun, Chong [Jiangsu Key Laboratory of Biofunctional Materials, Biomedical Functional Materials Collaborative Innovation Center, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023 (China); Institute of Agricultural Products Processing, Jiangsu Academy of Agricultural Sciences, Nanjing 210014 (China); Han, Qiaorong [Jiangsu Key Laboratory of Biofunctional Materials, Biomedical Functional Materials Collaborative Innovation Center, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023 (China); Wang, Daoying; Xu, Weimin [Institute of Agricultural Products Processing, Jiangsu Academy of Agricultural Sciences, Nanjing 210014 (China); Wang, Weijuan [Jiangsu Key Laboratory of Biofunctional Materials, Biomedical Functional Materials Collaborative Innovation Center, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023 (China); Zhao, Wenbo, E-mail: zhaowenbo@njnu.edu.cn [Jiangsu Key Laboratory of Biofunctional Materials, Biomedical Functional Materials Collaborative Innovation Center, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023 (China); Zhou, Min, E-mail: zhouminnju@126.com [Department of Vascular Surgery, the Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008 (China)

2014-11-19

Highlights: • A label-free thrombin aptamer biosensor applied in whole blood has been developed. • The aptamer biosensor showed a wide detection range and a low detection limit. • The antibiofouling idea utilized for biosensor is significant for diagnostics. - Abstract: In this paper, we have synthesized hyperbranched polyester microspheres with carboxylic acid functional groups (HBPE-CA) and developed a label-free electrochemical aptamer biosensor using thrombin-binding aptamer (TBA) as receptor for the measurement of thrombin in whole blood. The indium tin oxide (ITO) electrode surface modified with HBPE-CA microspheres was grafted with TBA, which has excellent binding affinity and selectivity for thrombin. Binding of the thrombin at the modified ITO electrode surface greatly restrained access of electrons for a redox probe of [Fe(CN){sub 6}]{sup 3−/4−}. Moreover, the aptamer biosensor could be used for detection of thrombin in whole blood, a wide detection range (10 fM–100 nM) and a detection limit on the order of 0.90 fM were demonstrated. Control experiments were also carried out by using bull serum albumin (BSA) and lysozyme in the absence of thrombin. The good stability and repeatability of this aptamer biosensor were also proved. We expect that this demonstration will lead to the development of highly sensitive label-free sensors based on aptamer with lower cost than current technology. The integration of the technologies, which include anticoagulant, sensor and nanoscience, will bring significant input to high-performance biosensors relevant to diagnostics and therapy of interest for human health.

Some Important Milestones in the Field of Blood Clotting.

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Doolittle, Russell F

2016-01-01

Several different kinds of 'milestone' in the field of blood coagulation are described from the middle decades of the 20th century. Although viewed from the standpoint of clotting per se, attention is also given to implications for innate immunity. The first milestone considered is the protracted saga of clotting dependence on vitamin K, an adventure that spanned more than five decades beginning in the 1920s. The second has to do with the discovery of a half-dozen 'new' clotting factors during the period immediately following World War II. A third pursues a narrower focus and examines the once mysterious transformation of fibrinogen into fibrin. Finally, the clinical treatment of classical hemophilia had a remarkable turning point in the 1960s as the result of simple but sensible measures. © 2015 S. Karger AG, Basel.

Development of a sandwich ELISA for the thrombin light chain identified by serum proteome analysis

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Kazuyuki Sogawa

2017-08-01

Full Text Available We previously identified novel biomarker candidates in biliary tract cancer (BTC using serum proteome analysis. Among several candidates, we focused on thrombin light chain which is a 4204 Da peptide as the most promising biomarker for BTC. To move thrombin light chain toward potential diagnostic use, we developed an enzyme immunoassay that enables to measure serum thrombin light chain levels.Both one monoclonal antibody specific to the N-termini and one polyclonal antibody were used to develop a sandwich ELISA for thrombin light chain. The assay was evaluated by comparing the results with those obtained by the ClinProt™ system. Serum samples were obtained from 20 patients with BTC, 20 patients with BBTDs and 20 HVs using the ClinProt™ system and ELISA.The results of the established ELISA showed a positive correlation with the findings by ClinProt™ system (slope=0.3386, intercept=34.901, r2=0.9641. The performance of the ELISA was satisfactory in terms of recovery (97.9–102.5% and within-run (1.5–4.8% and between-day (1.9–6.7% reproducibility. Serum thrombin light chain levels were significantly greater in BTC (176.5±47.2 ng/mL than in BBTDs (128.6±17.4 ng/mL and HVs (127.6±16.0 ng/mL (p<0.001.The sandwich ELISA developed in this study will be useful for validation of the diagnostic significance of serum thrombin light chain levels in various cancers. Keywords: Thrombin light chain, Biliary tract cancer, Sandwich ELISA, Serum biomarker

Influence of Acute Normobaric Hypoxia on Hemostasis in Volunteers with and without Acute Mountain Sickness

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Marc Schaber

2015-01-01

Full Text Available Introduction. The aim of the present study was to investigate whether a 12-hour exposure in a normobaric hypoxic chamber would induce changes in the hemostatic system and a procoagulant state in volunteers suffering from acute mountain sickness (AMS and healthy controls. Materials and Methods. 37 healthy participants were passively exposed to 12.6% FiO2 (simulated altitude hypoxia of 4,500 m. AMS development was investigated by the Lake Louise Score (LLS. Prothrombin time, activated partial thromboplastin time, fibrinogen, and platelet count were measured and specific methods (i.e., thromboelastometry and a thrombin generation test were used. Results. AMS prevalence was 62.2% (LLS cut off of 3. For the whole group, paired sample t-tests showed significant increase in the maximal concentration of generated thrombin. ROTEM measurements revealed a significant shortening of coagulation time and an increase of maximal clot firmness (InTEM test. A significant increase in maximum clot firmness could be shown (FibTEM test. Conclusions. All significant changes in coagulation parameters after exposure remained within normal reference ranges. No differences with regard to measured parameters of the hemostatic system between AMS-positive and -negative subjects were observed. Therefore, the hypothesis of the acute activation of coagulation by hypoxia can be rejected.

What is this chocolate milk in my circuit? A cause of acute clotting of a continuous renal replacement circuit: Questions.

Science.gov (United States)

Kakajiwala, Aadil; Chiotos, Kathleen; Brothers, Julie; Lederman, April; Amaral, Sandra

2016-12-01

One of the greatest problems associated with continuous renal replacement therapy (CRRT) is the early clotting of filters. A literature search revealed three case reports of lipemic blood causing recurrent clotting and reduced CRRT circuit survival time in adult patients, but no reports of cases in children. A 23-month-old male infant with Martinez-Frias syndrome and multivisceral transplant was admitted to the hospital with severe sepsis and hemolytic anemia. He developed acute kidney injury, fluid overload and electrolyte imbalances requiring CRRT and was also administered total parenteral nutrition (TPN) and fat emulsion. The first circuit lasted 60 h before routine change was required. The second circuit showed acute clotting after only 18 h, and brownish-milky fluid was found in the circuit tubing layered between the clotted blood. The patient's serum triglyceride levels were elevated at 988 mg/dL. The lipid infusion was stopped and CRRT restarted. Serum triglyceride levels improved to 363 mg/dL. The new circuit lasted 63 h before routine change was required. Clotting of CRRT circuits due to elevated triglyceride levels is rare and has not been reported in the pediatric population. Physicians should be mindful of this risk in patients receiving TPN who have unexpected clotting of CRRT circuits.

Reduced clot debris size using standing waves formed via high intensity focused ultrasound

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Guo, Shifang; Du, Xuan; Wang, Xin; Lu, Shukuan; Shi, Aiwei; Xu, Shanshan; Bouakaz, Ayache; Wan, Mingxi

2017-09-01

The feasibility of utilizing high intensity focused ultrasound (HIFU) to induce thrombolysis has been demonstrated previously. However, clinical concerns still remain related to the clot debris produced via fragmentation of the original clot potentially being too large and hence occluding downstream vessels, causing hazardous emboli. This study investigates the use of standing wave fields formed via HIFU to disintegrate the thrombus while achieving a reduced clot debris size in vitro. The results showed that the average diameter of the clot debris calculated by volume percentage was smaller in the standing wave mode than in the travelling wave mode at identical ultrasound thrombolysis settings. Furthermore, the inertial cavitation dose was shown to be lower in the standing wave mode, while the estimated cavitation bubble size distribution was similar in both modes. These results show that a reduction of the clot debris size with standing waves may be attributed to the particle trapping of the acoustic potential well which contributed to particle fragmentation.

Stabilization of the E* Form Turns Thrombin into an Anticoagulant

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

2009-07-31

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

Pilot study of Alteplase (tissue plasminogen activator) for treatment of urinary clot retention in an in vitro model.

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Ritch, Chad R; Ordonez, Maria A; Okhunov, Zhamshid; Araujo, Juan; Walsh, Rhonda; Baudin, Vania; Lee, Daniel; Badani, Ketan K; Gupta, Mantu; Landman, Jaime

2009-08-01

The management of urinary clot retention and hematuria involves manual irrigation with sterile water or normal saline via a Foley catheter followed by continuous bladder irrigation. Irrigation may become difficult because of the formation of dense blood clots. Tissue plasminogen activator (t-PA/Alteplase) may be a useful pharmacological agent to improve the efficacy of manual irrigation of large, dense clots. The goal of the current study was to compare t-PA to sterile water for clot irrigation in an in vitro model. In vitro models of clot retention were created using 500-cc urinary leg bags each filled with 80 cc of unpreserved whole blood from a healthy volunteer. Each model was incubated at 25 degrees C for 24 hours to allow clot formation. Four models each with 25 mL solution of t-PA at concentrations of 2, 1, 0.5, and 0.25 mg/mL were evaluated and compared to a control (25 mL sterile water). Models were instilled with solution (t-PA or control) and incubated for 30 minutes at 37 degrees C, and then irrigated with sterile water via 18F Foley by a blinded investigator. Three separate experiments were conducted, and statistical analysis was performed comparing various irrigation parameters. Clot evacuation with 25 mL of t-PA at a concentration of 2 mg/mL (50 mg) was significantly easier (p = 0.05) and faster (p < 0.05) than the sterile water control. The mean time for clot evacuation in this model was 2.7 minutes for t-PA solution 2 mg/mL versus 7.3 minutes for the control (p < 0.05). Compared to the control, irrigation with t-PA solution 2 mg/mL also required less irrigant (180 mL vs. 500 mL) (p < 0.05) for complete evacuation. There was a similar trend in efficacy for the lower doses of t-PA, but this was not statistically significant. In this in vitro study, a single 25 mL instillation of t-PA solution 2 mg/mL is significantly better than sterile water alone for clot evacuation. In vivo animal studies are pending.

Stabilization versus inhibition of TAFIa by competitive inhibitors in vitro

NARCIS (Netherlands)

Walker, J.B.; Hughes, B.; James, I.; Haddock, P.; Kluft, C.; Bajzar, L.

2003-01-01

Two competitive inhibitors of TAFIa (activated thrombin-activable fibrinolysis inhibitor), 2-guanidinoethyl-mercaptosuccinic acid and potato tuber carboxypeptidase inhibitor, variably affect fibrinolysis of clotted human plasma. Depending on their concentration, the inhibitors shortened, prolonged,

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

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Yizhang Liu

2018-02-01

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

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

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

2015-04-21

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

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

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Rebeca Cristina Costa Pereira

2017-03-01

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

Assessment of Heparin Anticoagulation Measured Using i-STAT and Hemochron Activated Clotting Time.

Science.gov (United States)

Maslow, Andrew; Chambers, Alison; Cheves, Tracey; Sweeney, Joseph

2018-01-31

Adequate anticoagulation, measured using activated clotting time (ACT), is important during vascular and cardiac surgeries. Unfractionated heparin is the most common anticoagulant used. The purpose of this analysis was to compare the i-STAT ACT (iACT) to the Hemochron ACT (hACT), both of which were then compared to anti-factor Xa (anti-Xa) assay, a representation of heparin level and activity. Prospective study. Tertiary care cardiovascular center. Eleven consecutive elective adult cardiac surgical patients. Prior to cardiopulmonary bypass, ACTs were measured using i-STAT and Hemochron technologies and compared to each other and to anti-Xa assay prior to and during a cumulative administration of heparin. Data were compared using bias analyses. Heparin (300 U/kg) was administered in quarterly doses. Coagulation labs were collected prior to and 3 minutes after each quarterly dose of heparin. The baseline ACTs for i-STAT and Hemochron were 147 and 142 seconds, respectively. A significant association was found between iACT and hACT (p = 0.002). The iACT measurements underestimated hACT at ACT levels >180 seconds or anti-Xa levels >0.75 U/mL. No significant difference was found between ACT data at anti-Xa levels 0.75 U/mL. Copyright © 2018 Elsevier Inc. All rights reserved.

Inherited Anti-Thrombin Deficiency in A Malay-Malaysian Family: A Missense Mutation at Nucleotide g.13267C>A aka anti-thrombin Budapest 5 (p.Pro439Thr) of the SERPINC 1 gene.

Science.gov (United States)

Norlelawati, A T; Rusmawati, I; Naznin, M; Nur Nadia, O; Rizqan Aizzani, R; Noraziana, A W

2014-02-01

Inherited anti-thrombin deficiency is an autosomal dominant disorder which is associated with increased risk for venous thromboembolism (VTE). This condition is very rare in Malaysia and there has been no documented report. Thus, the aim of the present study is to investigate the type of an inherited anti-thrombin deficiency mutation in a 25-year-old Malay woman who presented with deep vein thrombosis in her first pregnancy. DNA was extracted from the patient's blood sample and buccal mucosal swabs from family members. Polymerase chain reaction(PCR) assays were designed to cover all seven exons of the serpin peptidase inhibitor, clade C (antithrombin), member 1 (SERPINC1) gene; and the products were subjected to DNA sequencing. Sequences were referred to NCBI Reference Sequence: NG_012462.1. A heterozygous substitution mutation at nucleotide position 13267 (CCT->ACT) was identified in the patient and two other family members, giving a possible change of codon 439 (Pro→Thr) also known as anti-thrombin Budapest 5. The genotype was absent in 90 healthy controls. The study revealed a heterozygous antithrombin Budapest 5 mutation in SERPINC 1 giving rise to a possible anti-thrombin deficiency in a Malay-Malaysian family.

Artificial Stroke Clots: How Wide is the Gap to the Real World?

Science.gov (United States)

Berndt, Maria; Prothmann, Sascha; Maegerlein, Christian; Oberdieck, Paul; Zimmer, Claus; Hegge, Barbara; Pelisek, Jaroslav; Schirmer, Lucas; Poppert, Holger; Boeckh-Behrens, Tobias

2018-02-01

Especially since the establishment of mechanical thrombectomy as part of standard stroke therapy, artificial thrombi have become important in the training of interventionalists as well as for the development and testing of devices. So far, these in vitro clots have lacked direct comparisons with ex vivo thrombi. We therefore compared the histologic appearance of dynamically produced clots with that of stroke thrombi acquired during mechanical recanalization. Thrombi of 145 consecutive patients with stroke with large-vessel occlusions were histologically compared with 10 artificial clots, dynamically created from human blood and pig's blood using a Chandler loop system. Quantified FP/RBC ratios (fibrin/platelets divided by red blood cell fraction) and white blood cell (WBC) fractions were identified and compared between artificial (human and pig) and ex vivo thrombi obtained from patients with various stroke causes. There were no significant differences in the analysis of FP/RBC ratios between artificial thrombi and ex vivo thrombi (P = 0.42) or in the more precise analyses considering etiologic subgroups. Distinct differences were observed for the WBC fraction, with lower values in artificial thrombi (median, 1.34%) than in ex vivo thrombi (median, 5%) (P < 0.001). The main clot components, FP and RBC, are presumably the most influential factors for clot stability and mechanical resistance. Similarities between artificially generated and ex vivo stroke clots (and when considering different stroke subgroups) support the usefulness of these artificial thrombi in the pre-evaluation of thrombus extraction devices and as appropriate training material. Copyright © 2017 Elsevier Inc. All rights reserved.

Changes in clot lysis levels of reteplase and streptokinase following continuous wave ultrasound exposure, at ultrasound intensities following attenuation from the skull bone

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Roijer Anders

2008-08-01

Full Text Available Abstract Background Ultrasound (US has been used to enhance thrombolytic therapy in the treatment of stroke. Considerable attenuation of US intensity is however noted if US is applied over the temporal bone. The aim of this study was therefore to explore possible changes in the effect of thrombolytic drugs during low-intensity, high-frequency continuous-wave ultrasound (CW-US exposure. Methods Clots were made from fresh venous blood drawn from healthy volunteers. Each clot was made from 1.4 ml blood and left to coagulate for 1 hour in a plastic test-tube. The thrombolytic drugs used were, 3600 IU streptokinase (SK or 0.25 U reteplase (r-PA, which were mixed in 160 ml 0.9% NaCl solution. Continuous-wave US exposure was applied at a frequency of 1 MHz and intensities ranging from 0.0125 to 1.2 W/cm2. For each thrombolytic drug (n = 2, SK and r-PA and each intensity (n = 9 interventional clots (US-exposed, n = 6 were submerged in thrombolytic solution and exposed to CW-US while control clots (also submerged in thrombolytic solution, n = 6 were left unexposed to US. To evaluate the effect on clot lysis, the haemoglobin (Hb released from each clot was measured every 20 min for 1 hour (20, 40 and 60 min. The Hb content (mg released was estimated by spectrophotometry at 540 nm. The difference in effect on clot lysis was expressed as the difference in the amount of Hb released between pairs of US-exposed clots and control clots. Statistical analysis was performed using Wilcoxon's signed rank test. Results Continuous-wave ultrasound significantly decreased the effects of SK at intensities of 0.9 and 1.2 W/cm2 at all times (P 2 and at 1.2 W/cm2, following 40 min exposure at 0.3, 0.6, 0.9 and at 1.2 W/cm2, and following 60 min of exposure at 0.05 0.3, 0.6, 0.9 and at 1.2 W/cm2 (all P Conclusion Increasing intensities of CW-US exposure resulted in increased clot lysis of r-PA-treated blood clots, but decreased clot lysis of SK-treated clots.

Evaluation of the efficacy and safety of rivaroxaban using a computer model for blood coagulation.

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Rolf Burghaus

Full Text Available Rivaroxaban is an oral, direct Factor Xa inhibitor approved in the European Union and several other countries for the prevention of venous thromboembolism in adult patients undergoing elective hip or knee replacement surgery and is in advanced clinical development for the treatment of thromboembolic disorders. Its mechanism of action is antithrombin independent and differs from that of other anticoagulants, such as warfarin (a vitamin K antagonist, enoxaparin (an indirect thrombin/Factor Xa inhibitor and dabigatran (a direct thrombin inhibitor. A blood coagulation computer model has been developed, based on several published models and preclinical and clinical data. Unlike previous models, the current model takes into account both the intrinsic and extrinsic pathways of the coagulation cascade, and possesses some unique features, including a blood flow component and a portfolio of drug action mechanisms. This study aimed to use the model to compare the mechanism of action of rivaroxaban with that of warfarin, and to evaluate the efficacy and safety of different rivaroxaban doses with other anticoagulants included in the model. Rather than reproducing known standard clinical measurements, such as the prothrombin time and activated partial thromboplastin time clotting tests, the anticoagulant benchmarking was based on a simulation of physiologically plausible clotting scenarios. Compared with warfarin, rivaroxaban showed a favourable sensitivity for tissue factor concentration inducing clotting, and a steep concentration-effect relationship, rapidly flattening towards higher inhibitor concentrations, both suggesting a broad therapeutic window. The predicted dosing window is highly accordant with the final dose recommendation based upon extensive clinical studies.

A near-infrared fluorescent bioassay for thrombin using aptamer-modified CuInS2 quantum dots

International Nuclear Information System (INIS)

Lin, Zihan; Hu, Tianyu; Liu, Ziping; Su, Xingguang; Pan, Dong

2015-01-01

We describe a near-infrared (NIR) fluorescent thrombin assay using a thrombin-binding aptamer (TBA) and Zn(II)-activated CuInS 2 quantum dots (Q-dots). The fluorescence of Zn(II)-activated Q-dots is quenched by the TBA via photoinduced electron transfer, but if thrombin is added, it will bind to TBA to form G-quadruplexes and the Q-dots are released. As a result, the fluorescence intensity of the system is restored. This effect was exploited to design an assay for thrombin whose calibration plot, under optimum conditions, is linear in the 0.034 to 102 nmol L −1 concentration range, with a 12 pmol L −1 detection limit. The method is fairly simple, fast, and due to its picomolar detection limits holds great potential in the diagnosis of diseases associated with coagulation abnormalities and certain kinds of cancer. (author)

42 CFR 410.63 - Hepatitis B vaccine and blood clotting factors: Conditions.

Science.gov (United States)

2010-10-01

... 42 Public Health 2 2010-10-01 2010-10-01 false Hepatitis B vaccine and blood clotting factors... Other Health Services § 410.63 Hepatitis B vaccine and blood clotting factors: Conditions... under § 410.10, subject to the specified conditions: (a) Hepatitis B vaccine: Conditions. Effective...

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

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

2018-01-01

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

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

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

2012-11-18

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

[Guidelines for certification of Activated clotting time (ACT) according to the EN ISO 22870 standards].

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Lasne, Dominique; Bauters, Anne; Le Querrec, Agnès; Bourdin, Carole; Voisin, Sophie

2015-01-01

Point of care testing (POCT) must comply with regulatory requirements according to standard EN ISO 22870, which identify biologists as responsible for POCT. Activated clotting time (ACT) is mandatory to monitor on whole blood, anticoagulation achieved by unfractionated heparin during cardiopulmonary bypass (CPB) or cardiac catheterization. This test has no equivalent in the laboratory. With the aim to help the multidisciplinary groups for POCT supervision when they have to analyse the wish of medical departments to use ACT and to help the biologists to be in accordance with the standard, we present the guidelines of the GEHT (Groupe d'étude d'hémostase et thrombose) subcommittee "CEC et Biologie délocalisée" for the certification of ACT. These guidelines are based on the SFBC guidelines for the certification of POCT and on the analysis of the literature to ascertain the justification of clinical need and assess the analytical performance of main analyzers used in France, as well as on a survey conducted with French and Belgian biologists.

Identification and characterization of milk-clotting proteases ...

African Journals Online (AJOL)

SAM

2014-03-12

Mar 12, 2014 ... Two strains of fungi were isolated and identified as Aspergillus tamarii and Penicillium pinophilum ... for milk clotting enzymes by fermentation at acid pH on culture medium containing whey ..... Rhizopus oryzae NBRC 4749.

Influence of Interleukin-1 Beta on Platelet-Poor Plasma Clot Formation: A Potential Impact on Early Bone Healing.

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Xin Wang

Full Text Available Hematoma quality (especially the fibrin matrix plays an important role in the bone healing process. Here, we investigated the effect of interleukin-1 beta (IL-1β on fibrin clot formation from platelet-poor plasma (PPP.Five-milliliter of rat whole-blood samples were collected from the hepatic portal vein. All blood samples were firstly standardized via a thrombelastograph (TEG, blood cell count, and the measurement of fibrinogen concentration. PPP was prepared by collecting the top two-fifths of the plasma after centrifugation under 400 × g for 10 min at 20°C. The effects of IL-1β cytokines on artificial fibrin clot formation from PPP solutions were determined by scanning electronic microscopy (SEM, confocal microscopy (CM, turbidity, and clot lysis assays.The lag time for protofibril formation was markedly shortened in the IL-1β treatment groups (243.8 ± 76.85 in the 50 pg/mL of IL-1β and 97.5 ± 19.36 in the 500 pg/mL of IL-1β compared to the control group without IL-1β (543.8 ± 205.8. Maximal turbidity was observed in the control group. IL-1β (500 pg/mL treatment significantly decreased fiber diameters resulting in smaller pore sizes and increased density of the fibrin clot structure formed from PPP (P < 0.05. The clot lysis assay revealed that 500 pg/mL IL-1β induced a lower susceptibility to dissolution due to the formation of thinner and denser fibers.IL-1β can significantly influence PPP fibrin clot structure, which may affect the early bone healing process.

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

DEFF Research Database (Denmark)

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

2016-01-01

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

Binding of poly(amidoamine), carbosilane, phosphorus and hybrid dendrimers to thrombin-Constants and mechanisms.

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Shcharbin, Dzmitry; Pedziwiatr-Werbicka, Elzbieta; Vcherashniaya, Aliaksandra; Janaszewska, Anna; Marcinkowska, Monika; Goska, Piotr; Klajnert-Maculewicz, Barbara; Ionov, Maksim; Abashkin, Viktar; Ihnatsyeu-Kachan, Aliaksei; de la Mata, F Javier; Ortega, Paula; Gomez-Ramirez, Rafael; Majoral, Jean-Pierre; Bryszewska, Maria

2017-07-01

Thrombin is an essential part of the blood coagulation system; it is a serine protease that converts soluble fibrinogen into insoluble strands of fibrin, and catalyzes many other coagulation-related reactions. Absorption at its surface of small nanoparticles can completely change the biological properties of thrombin. We have analyzed the influence on thrombin of 3 different kinds of small nanoparticles: dendrimers (phosphorus-based, carbosilane based and polyamidoamine) and 2 hybrid systems containing carbosilane, viologen and phosphorus dendritic scaffolds in one single molecule, bearing different flexibility, size and surface charge. There was significant alteration in the rigidity of the rigid dendrimers in contrast to flexible dendrimers. These differences in their action are important in understanding interactions taking place at a bio-nanointerface. Copyright © 2017 Elsevier B.V. All rights reserved.

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

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Akkaya, Alper; Pazarlioglu, Nurdan

2013-01-01

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

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

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

1997-12-01

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

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

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

2011-01-28

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

Activation of PAR-1/NADPH Oxidase/ROS Signaling Pathways is Crucial for the Thrombin-Induced sFlt-1 Production in Extravillous Trophoblasts: Possible Involvement in the Pathogenesis of Preeclampsia

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Qi-tao Huang

2015-03-01

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

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

Science.gov (United States)

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

2015-01-01

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

iTRAQ quantitative proteomics-based identification of cell adhesion as a dominant phenotypic modulation in thrombin-stimulated human aortic endothelial cells.

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Wang, Huang-Joe; Chen, Sung-Fang; Lo, Wan-Yu

2015-05-01

The phenotypic changes in thrombin-stimulated endothelial cells include alterations in permeability, cell shape, vasomotor tone, leukocyte trafficking, migration, proliferation, and angiogenesis. Previous studies regarding the pleotropic effects of thrombin on the endothelium used human umbilical vein endothelial cells (HUVECs)-cells derived from fetal tissue that does not exist in adults. Only a few groups have used screening approaches such as microarrays to profile the global effects of thrombin on endothelial cells. Moreover, the proteomic changes of thrombin-stimulated human aortic endothelial cells (HAECs) have not been elucidated. HAECs were stimulated with 2 units/mL thrombin for 5h and their proteome was investigated using isobaric tags for the relative and absolute quantification (iTRAQ) and the MetaCore(TM) software. A total of 627 (experiment A) and 622 proteins (experiment B) were quantified in the duplicated iTRAQ analyses. MetaCore(TM) pathway analysis identified cell adhesion as a dominant phenotype in thrombin-stimulated HAECs. Replicated iTRAQ data revealed that "Cell adhesion_Chemokines and adhesion," "Cell adhesion_Histamine H1 receptor signaling in the interruption of cell barrier integrity," and "Cell adhesion_Integrin-mediated cell adhesion and migration" were among the top 10 statistically significant pathways. The cell adhesion phenotype was verified by increased THP-1 adhesion to thrombin-stimulated HAECs. In addition, the expression of ICAM-1, VCAM-1, and SELE was significantly upregulated in thrombin-stimulated HAECs. Several regulatory pathways are altered in thrombin-stimulated HAECs, with cell adhesion being the dominant altered phenotype. Our findings show the feasibility of the iTRAQ technique for evaluating cellular responses to acute stimulation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Increased Oxidation as an Additional Mechanism Underlying Reduced Clot Permeability and Impaired Fibrinolysis in Type 2 Diabetes

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Anna Lados-Krupa

2015-01-01

Full Text Available Aims. We sought to investigate whether enhanced oxidation contributes to unfavorable fibrin clot properties in patients with diabetes. Methods. We assessed plasma fibrin clot permeation (Ks, a measure of the pore size in fibrin networks and clot lysis time induced by recombinant tissue plasminogen activator (CLT in 163 consecutive type 2 diabetic patients (92 men and 71 women aged 65 ± 8.8 years with a mean glycated hemoglobin (HbA1c of 6.8%. We also measured oxidative stress markers, including nitrotyrosine, the soluble form of receptor for advanced glycation end products (sRAGE, 8-iso-prostaglandin F2α (8-iso-PGF2α, oxidized low-density lipoprotein (oxLDL, and advanced glycation end products (AGE. Results. There were inverse correlations between Ks and nitrotyrosine, sRAGE, 8-iso-PGF2α, and oxLDL. CLT showed a positive correlation with oxLDL and nitrotyrosine but not with other oxidation markers. All these associations remained significant for Ks after adjustment for fibrinogen, disease duration, and HbA1c (all P<0.05, while oxLDL was the only independent predictor of CLT. Conclusions. Our study shows that enhanced oxidative stress adversely affects plasma fibrin clot properties in type 2 diabetic patients, regardless of disease duration and glycemia control.

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

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Marques, Marisa; Brown, Spencer A; Cordeiro, Natália D S; Rodrigues-Pereira, Pedro; Cobrado, M Luís; Morales-Helguera, Aliuska; Lima, Nuno; Luís, André; Mendanha, Mário; Gonçalves-Rodrigues, Acácio; Amarante, José

2011-03-01

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

Regulation of Thrombin-Induced Lung Endothelial Cell Barrier Disruption by Protein Kinase C Delta.

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Lishi Xie

Full Text Available Protein Kinase C (PKC plays a significant role in thrombin-induced loss of endothelial cell (EC barrier integrity; however, the existence of more than 10 isozymes of PKC and tissue-specific isoform expression has limited our understanding of this important second messenger in vascular homeostasis. In this study, we show that PKCδ isoform promotes thrombin-induced loss of human pulmonary artery EC barrier integrity, findings substantiated by PKCδ inhibitory studies (rottlerin, dominant negative PKCδ construct and PKCδ silencing (siRNA. In addition, we identified PKCδ as a signaling mediator upstream of both thrombin-induced MLC phosphorylation and Rho GTPase activation affecting stress fiber formation, cell contraction and loss of EC barrier integrity. Our inhibitor-based studies indicate that thrombin-induced PKCδ activation exerts a positive feedback on Rho GTPase activation and contributes to Rac1 GTPase inhibition. Moreover, PKD (or PKCμ and CPI-17, two known PKCδ targets, were found to be activated by PKCδ in EC and served as modulators of cytoskeleton rearrangement. These studies clarify the role of PKCδ in EC cytoskeleton regulation, and highlight PKCδ as a therapeutic target in inflammatory lung disorders, characterized by the loss of barrier integrity, such as acute lung injury and sepsis.

Increased thrombin generation in women with polycystic ovary syndrome

DEFF Research Database (Denmark)

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

2015-01-01

Objective. Polycystic ovary syndrome (PCOS) is associated with risk factors for cardiovascular disease (CVD) which may be modified by the use of metformin and oral contraceptives (OC). Thrombin generation (TG) measures are risk markers of CVD and address the composite of multiple factors...

Fluorescence enhancement upon G-quadruplex folding: synthesis, structure, and biophysical characterization of a dansyl/cyclodextrin-tagged thrombin binding aptamer.

Science.gov (United States)

De Tito, Stefano; Morvan, François; Meyer, Albert; Vasseur, Jean-Jacques; Cummaro, Annunziata; Petraccone, Luigi; Pagano, Bruno; Novellino, Ettore; Randazzo, Antonio; Giancola, Concetta; Montesarchio, Daniela

2013-11-20

A novel fluorescent thrombin binding aptamer (TBA), conjugated with the environmentally sensitive dansyl probe at the 3'-end and a β-cyclodextrin residue at the 5'-end, has been efficiently synthesized exploiting Cu(I)-catalyzed azide-alkyne cycloaddition procedures. Its conformation and stability in solution have been studied by an integrated approach, combining in-depth NMR, CD, fluorescence, and DSC studies. ITC measurements have allowed us to analyze in detail its interaction with human thrombin. All the collected data show that this bis-conjugated aptamer fully retains its G-quadruplex formation ability and thrombin recognition properties, with the terminal appendages only marginally interfering with the conformational behavior of TBA. Folding of this modified aptamer into the chairlike, antiparallel G-quadruplex structure, promoted by K(+) and/or thrombin binding, typical of TBA, is associated with a net fluorescence enhancement, due to encapsulation of dansyl, attached at the 3'-end, into the apolar cavity of the β-cyclodextrin at the 5'-end. Overall, the structural characterization of this novel, bis-conjugated TBA fully demonstrates its potential as a diagnostic tool for thrombin recognition, also providing a useful basis for the design of suitable aptamer-based devices for theranostic applications, allowing simultaneously both detection and inhibition or modulation of the thrombin activity.

Utility of single-energy and dual-energy computed tomography in clot characterization: An in-vitro study.

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Brinjikji, Waleed; Michalak, Gregory; Kadirvel, Ramanathan; Dai, Daying; Gilvarry, Michael; Duffy, Sharon; Kallmes, David F; McCollough, Cynthia; Leng, Shuai

2017-06-01

Background and purpose Because computed tomography (CT) is the most commonly used imaging modality for the evaluation of acute ischemic stroke patients, developing CT-based techniques for improving clot characterization could prove useful. The purpose of this in-vitro study was to determine which single-energy or dual-energy CT techniques provided optimum discrimination between red blood cell (RBC) and fibrin-rich clots. Materials and methods Seven clot types with varying fibrin and RBC densities were made (90% RBC, 99% RBC, 63% RBC, 36% RBC, 18% RBC and 0% RBC with high and low fibrin density) and their composition was verified histologically. Ten of each clot type were created and scanned with a second generation dual source scanner using three single (80 kV, 100 kV, 120 kV) and two dual-energy protocols (80/Sn 140 kV and 100/Sn 140 kV). A region of interest (ROI) was placed over each clot and mean attenuation was measured. Receiver operating characteristic curves were calculated at each energy level to determine the accuracy at differentiating RBC-rich clots from fibrin-rich clots. Results Clot attenuation increased with RBC content at all energy levels. Single-energy at 80 kV and 120 kV and dual-energy 80/Sn 140 kV protocols allowed for distinguishing between all clot types, with the exception of 36% RBC and 18% RBC. On receiver operating characteristic curve analysis, the 80/Sn 140 kV dual-energy protocol had the highest area under the curve for distinguishing between fibrin-rich and RBC-rich clots (area under the curve 0.99). Conclusions Dual-energy CT with 80/Sn 140 kV had the highest accuracy for differentiating RBC-rich and fibrin-rich in-vitro thrombi. Further studies are needed to study the utility of non-contrast dual-energy CT in thrombus characterization in acute ischemic stroke.

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

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

2018-01-01

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

Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4

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Semeraro, Fabrizio; Ammollo, Concetta T.; Morrissey, James H.; Dale, George L.; Friese, Paul; Esmon, Naomi L.

2011-01-01

The release of histones from dying cells is associated with microvascular thrombosis and, because histones activate platelets, this could represent a possible pathogenic mechanism. In the present study, we assessed the influence of histones on the procoagulant potential of human platelets in platelet-rich plasma (PRP) and in purified systems. Histones dose-dependently enhanced thrombin generation in PRP in the absence of any trigger, as evaluated by calibrated automated thrombinography regardless of whether the contact phase was inhibited. Activation of coagulation required the presence of fully activatable platelets and was not ascribable to platelet tissue factor, whereas targeting polyphosphate with phosphatase reduced thrombin generation even when factor XII (FXII) was blocked or absent. In the presence of histones, purified polyphosphate was able to induce thrombin generation in plasma independently of FXII. In purified systems, histones induced platelet aggregation; P-selectin, phosphatidylserine, and FV/Va expression; and prothrombinase activity. Blocking platelet TLR2 and TLR4 with mAbs reduced the percentage of activated platelets and lowered the amount of thrombin generated in PRP. These data show that histone-activated platelets possess a procoagulant phenotype that drives plasma thrombin generation and suggest that TLR2 and TLR4 mediate the activation process. PMID:21673343

Effect of anticoagulant administration on blood clotting and some hormones related to rat-fertility

International Nuclear Information System (INIS)

Abdel-Khalek, L.G.

2009-01-01

This study was performed using 30 mature male albino rats divided into 3 equal groups; control and two treated groups to assess the effect of anticoagulant (warfarin) administration on the level of some hormones related to fertility. The two treated groups were injected intraperitoneally every other day with 1 ml (0.03 mg)and 2 ml (0.06 mg)warfarin/ 100 g body weight respectively where, two specimens were taken from each group after two and four weeks. Clotting time (CT), prothrombin time (PT), partial prothrombin time (PTT) platelets count, fasting blood sugar (F.B.S), calcium levels in addition to triiodothyronine (T 3 ), thyroxin (T 4 ), insulin, corticosterone, and testosterone hormones were determined. The results showed that the intraperitoneal injection of warfarin caused significant increase in clotting time, prothrombin time , partial prothrombin time, platelets count and glucose level, while serum calcium level showed significant decrease. Intraperitoneal injection of warfarin caused significant decrease of insulin and significant increase of corticosterone, T 3 showed significant decrease in high dose group while T 4 showed significant decrease in small dose group. The high dose was associated with the highest level of testosterone hormone. these results denoted that warfarin anticoagulant had no negative effect on gonadal sex hormone and hence on male fertility

Phase transitions during compression and decompression of clots from platelet-poor plasma, platelet-rich plasma and whole blood.

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Liang, Xiaojun; Chernysh, Irina; Purohit, Prashant K; Weisel, John W

2017-09-15

Blood clots are required to stem bleeding and are subject to a variety of stresses, but they can also block blood vessels and cause heart attacks and ischemic strokes. We measured the compressive response of human platelet-poor plasma (PPP) clots, platelet-rich plasma (PRP) clots and whole blood clots and correlated these measurements with confocal and scanning electron microscopy to track changes in clot structure. Stress-strain curves revealed four characteristic regions, for compression-decompression: (1) linear elastic region; (2) upper plateau or softening region; (3) non-linear elastic region or re-stretching of the network; (4) lower plateau in which dissociation of some newly made connections occurs. Our experiments revealed that compression proceeds by the passage of a phase boundary through the clot separating rarefied and densified phases. This observation motivates a model of fibrin mechanics based on the continuum theory of phase transitions, which accounts for the pre-stress caused by platelets, the adhesion of fibrin fibers in the densified phase, the compression of red blood cells (RBCs), and the pumping of liquids through the clot during compression/decompression. Our experiments and theory provide insights into the mechanical behavior of blood clots that could have implications clinically and in the design of fibrin-based biomaterials. The objective of this paper is to measure and mathematically model the compression behavior of various human blood clots. We show by a combination of confocal and scanning electron microscopy that compression proceeds by the passage of a front through the sample that separates a densified region of the clot from a rarefied region, and that the compression/decompression response is reversible with hysteresis. These observations form the basis of a model for the compression response of clots based on the continuum theory of phase transitions. Our studies may reveal how clot rheology under large compression in vivo due

Thrombin effectuates therapeutic arteriogenesis in the rabbit hindlimb ischemia model: A quantitative analysis by computerized in vivo imaging

International Nuclear Information System (INIS)

Kagadis, George C.; Karnabatidis, Dimitrios; Katsanos, Konstantinos; Diamantopoulos, Athanassios; Samaras, Nikolaos; Maroulis, John; Siablis, Dimitrios; Nikiforidis, George C.

2006-01-01

We report on an experimental mammalian controlled study that documents arteriogenic capacity of thrombin and utilizes computerized algorithms to quantify the newly formed vessels. Hindlimb ischemia was surgically invoked in 10 New Zealand white rabbits. After quiescence of endogenous angiogenesis heterologous bovine thrombin was intramuscularly injected (1500 units) in one hindlimb per rabbit (Group T). Contralateral limbs were infused with normal saline (Group C). Digital subtraction angiography (DSA) of both limbs was performed after thrombin infusion by selective cannulation of the abdominal aorta and digital images were post-processed with computerized algorithms in order to enhance newly formed vessels. Total vessel area and total vessel length were quantified. In vivo functional evaluation included measurements of blood flow volume at the level of the external iliac artery by Doppler ultrasonography both at baseline and at 20 days after thrombin infusion. Total vessel area and length (in pixels) were 14,713+/-1023 and 5466+/-1327 in group T versus 12,015+/-2557 and 4598+/-1269 in group C (p=0.0062 and 0.1526, respectively). Blood flow volumes (ml/min) at baseline and at 20 days after thrombin infusion were 25.87+/-11.09 and 38.06+/-11.72 in group T versus 26.57+/-11.19 and 20.35+/-7.20 in group C (p=0.8898 and 0.0007, respectively). Intramuscular thrombin effectuates an arteriogenic response in the rabbit hindlimb ischemia model. Computerized algorithms may enable accurate quantification of the neovascularization outcome

7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

Science.gov (United States)

2010-01-01

... 7 Agriculture 3 2010-01-01 2010-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be safe...

A cluster of aspartic residues in the extracellular loop II of PAR 4 is important for thrombin interaction and activation of platelets.

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Sánchez Centellas, Daniel; Gudlur, Sushanth; Vicente-Carrillo, Alejandro; Ramström, Sofia; Lindahl, Tomas L

2017-06-01

Thrombin activates platelets via proteolytic cleavage of protease-activated receptors (PARs) 1 and 4. The two PARs have distinct but complementary roles. The mechanisms responsible for PAR1 activation by thrombin have been extensively studied. However, much less is known regarding thrombin activation of PAR4, especially the potential involvement of regions of PAR4 other than the N-terminal, which is bound to the catalytic site of thrombin. We have studied PAR4 in S. cerevisiae strain MMY12, an expression system in which the GPCR receptors are connected to a Lac Z reporter gene resulting in increased β-galactosidase activity. This approach was used to assess PAR4 mutants to evaluate the contribution of different aspartic residues in facilitating PAR4 activation. Furthermore, peptides mimicking parts of the PAR4 N-terminal and the second extracellular loop (ECLII) were tested for their ability to inhibit platelet activation by thrombin. Binding of these peptides to γ-thrombin was studied by monitoring the decrease in tryptophan fluorescence intensity of thrombin. We conclude that not only the N-terminal but also the electronegative aspartic residues D224, D230 and D235 (located in ECLII) are be important for PAR4 binding to thrombin. We further suggest that they play a role for the tethered ligand binding to the receptor, as mutations also affected activation in response to a PAR4-activating peptide mimicking the new N-terminal formed after cleavage. This agrees with previous results on PAR1 and thrombin binding. We suggest that the ECLII of PAR4 could be a potential target for antithrombotic drug development. Copyright © 2017 Elsevier Ltd. All rights reserved.

Placental vascular pathology and increased thrombin generation as mechanisms of disease in obstetrical syndromes

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Salvatore Andrea Mastrolia

2014-11-01

Full Text Available Obstetrical complications including preeclampsia, fetal growth restriction, preterm labor, preterm prelabor rupture of membranes and fetal demise are all the clinical endpoint of several underlying mechanisms (i.e., infection, inflammation, thrombosis, endocrine disorder, immunologic rejection, genetic, and environmental, therefore, they may be regarded as syndromes. Placental vascular pathology and increased thrombin generation were reported in all of these obstetrical syndromes. Moreover, elevated concentrations of thrombin-anti thrombin III complexes and changes in the coagulation as well as anticoagulation factors can be detected in the maternal circulation prior to the clinical development of the disease in some of these syndromes. In this review, we will assess the changes in the hemostatic system during normal and complicated pregnancy in maternal blood, maternal–fetal interface and amniotic fluid, and describe the contribution of thrombosis and vascular pathology to the development of the great obstetrical syndromes.

Thrombin has biphasic effects on the nitric oxide-cGMP pathway in endothelial cells and contributes to experimental pulmonary hypertension.

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Katrin F Nickel

Full Text Available BACKGROUND: A potential role for coagulation factors in pulmonary arterial hypertension has been recently described, but the mechanism of action is currently not known. Here, we investigated the interactions between thrombin and the nitric oxide-cGMP pathway in pulmonary endothelial cells and experimental pulmonary hypertension. PRINCIPAL FINDINGS: Chronic treatment with the selective thrombin inhibitor melagatran (0.9 mg/kg daily via implanted minipumps reduced right ventricular hypertrophy in the rat monocrotaline model of experimental pulmonary hypertension. In vitro, thrombin was found to have biphasic effects on key regulators of the nitric oxide-cGMP pathway in endothelial cells (HUVECs. Acute thrombin stimulation led to increased expression of the cGMP-elevating factors endothelial nitric oxide synthase (eNOS and soluble guanylate cyclase (sGC subunits, leading to increased cGMP levels. By contrast, prolonged exposition of pulmonary endothelial cells to thrombin revealed a characteristic pattern of differential expression of the key regulators of the nitric oxide-cGMP pathway, in which specifically the factors contributing to cGMP elevation (eNOS and sGC were reduced and the cGMP-hydrolyzing PDE5 was elevated (qPCR and Western blot. In line with the differential expression of key regulators of the nitric oxide-cGMP pathway, a reduction of cGMP by prolonged thrombin stimulation was found. The effects of prolonged thrombin exposure were confirmed in endothelial cells of pulmonary origin (HPAECs and HPMECs. Similar effects could be induced by activation of protease-activated receptor-1 (PAR-1. CONCLUSION: These findings suggest a link between thrombin generation and cGMP depletion in lung endothelial cells through negative regulation of the nitric oxide-cGMP pathway, possibly mediated via PAR-1, which could be of relevance in pulmonary arterial hypertension.

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

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Eliseo Portilla-de Buen

2014-04-01

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.

Incorporation of vitronectin into fibrin clots. Evidence for a binding interaction between vitronectin and gamma A/gamma' fibrinogen.

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Podor, Thomas J; Campbell, Stephanie; Chindemi, Paul; Foulon, Denise M; Farrell, David H; Walton, Philip D; Weitz, Jeffrey I; Peterson, Cynthia B

2002-03-01

Vitronectin is an abundant plasma protein that regulates coagulation, fibrinolysis, complement activation, and cell adhesion. Recently, we demonstrated that plasma vitronectin inhibits fibrinolysis by mediating the interaction of type 1 plasminogen activator inhibitor with fibrin (Podor, T. J., Peterson, C. B., Lawrence, D. A., Stefansson, S., Shaughnessy, S. G., Foulon, D. M., Butcher, M., and Weitz, J. I. (2000) J. Biol. Chem. 275, 19788-19794). The current studies were undertaken to further examine the interactions between vitronectin and fibrin(ogen). Comparison of vitronectin levels in plasma with those in serum indicates that approximately 20% of plasma vitronectin is incorporated into the clot. When the time course of biotinylated-vitronectin incorporation into clots formed from (125)I-fibrinogen is monitored, vitronectin incorporation into the clot parallels that of fibrinogen in the absence or presence of activated factor XIII. Vitronectin binds specifically to fibrin matrices with an estimated K(d) of approximately 0.6 microm. Additional vitronectin subunits are assembled on fibrin-bound vitronectin multimers through self-association. Confocal microscopy of fibrin clots reveals the globular vitronectin aggregates anchored at intervals along the fibrin fibrils. This periodicity raised the possibility that vitronectin interacts with the gamma A/gamma' variant of fibrin(ogen) that represents about 10% of total fibrinogen. In support of this concept, the vitronectin which contaminates fibrinogen preparations co-purifies with the gamma A/gamma' fibrinogen fraction, and clots formed from gamma A/gamma' fibrinogen preferentially bind vitronectin. These studies reveal that vitronectin associates with fibrin during coagulation, and may thereby modulate hemostasis and inflammation.

Monitoring low molecular weight heparins at therapeutic levels: dose-responses of, and correlations and differences between aPTT, anti-factor Xa and thrombin generation assays.

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Owain Thomas

Full Text Available Low molecular weight heparins (LMWH's are used to prevent and treat thrombosis. Tests for monitoring LMWH's include anti-factor Xa (anti-FXa, activated partial thromboplastin time (aPTT and thrombin generation. Anti-FXa is the current gold standard despite LMWH's varying affinities for FXa and thrombin.To examine the effects of two different LMWH's on the results of 4 different aPTT-tests, anti-FXa activity and thrombin generation and to assess the tests' concordance.Enoxaparin and tinzaparin were added ex-vivo in concentrations of 0.0, 0.5, 1.0 and 1.5 anti-FXa international units (IU/mL, to blood from 10 volunteers. aPTT was measured using two whole blood methods (Free oscillation rheometry (FOR and Hemochron Jr (HCJ and an optical plasma method using two different reagents (ActinFSL and PTT-Automat. Anti-FXa activity was quantified using a chromogenic assay. Thrombin generation (Endogenous Thrombin Potential, ETP was measured on a Ceveron Alpha instrument using the TGA RB and more tissue-factor rich TGA RC reagents.Methods' mean aPTT at 1.0 IU/mL LMWH varied between 54s (SD 11 and 69s (SD 14 for enoxaparin and between 101s (SD 21 and 140s (SD 28 for tinzaparin. ActinFSL gave significantly shorter aPTT results. aPTT and anti-FXa generally correlated well. ETP as measured with the TGA RC reagent but not the TGA RB reagent showed an inverse exponential relationship to the concentration of LMWH. The HCJ-aPTT results had the weakest correlation to anti-FXa and thrombin generation (Rs0.62-0.87, whereas the other aPTT methods had similar correlation coefficients (Rs0.80-0.92.aPTT displays a linear dose-response to LMWH. There is variation between aPTT assays. Tinzaparin increases aPTT and decreases thrombin generation more than enoxaparin at any given level of anti-FXa activity, casting doubt on anti-FXa's present gold standard status. Thrombin generation with tissue factor-rich activator is a promising method for monitoring LMWH's.

EVALUATION OF THE COMPUTED TOMOGRAPHIC "SENTINEL CLOT SIGN" TO IDENTIFY BLEEDING ABDOMINAL ORGANS IN DOGS WITH HEMOABDOMEN.

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Specchi, Swan; Auriemma, Edoardo; Morabito, Simona; Ferri, Filippo; Zini, Eric; Piola, Valentina; Pey, Pascaline; Rossi, Federica

2017-01-01

The CT "sentinel clot sign" has been defined as the highest attenuation hematoma adjacent to a bleeding organ in humans with hemoabdomen. The aims of this retrospective descriptive multicenter study were to describe CT findings in a sample of dogs with surgically or necropsy confirmed intra-abdominal bleeding and determine prevalence of the "sentinel clot sign" adjacent to the location of bleeding. Medical records between 2012 and 2014 were searched for dogs with hemoabdomen and in which the origin of the bleeding was confirmed either with surgery or necropsy. Retrieved CT images were reviewed for the presence and localization of the "sentinel clot sign," HU measurements of the "sentinel clot sign" and hemoabdomen, and presence of extravasation of contrast media within the abdominal cavity. Nineteen dogs were included. Three dogs were excluded due to the low amount of blood that did not allow the identification of a "sentinel clot sign." A "sentinel clot sign" was detected in the proximity of the confirmed bleeding organ in 14/16 (88%) of the patients. The mean HU of the "sentinel clot sign" was 56 (range: 43-70) while that of the hemoabdomen was 34 (range: 20-45). Active hemorrhage was identified as extravasation of contrast medium within the peritoneal cavity from the bleeding organ in three dogs. In conclusion, the CT "sentinel clot sign" may be helpful for identifying the source of bleeding in dogs with hemoabdomen. © 2016 American College of Veterinary Radiology.

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

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

1999-10-05

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

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

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

2015-02-15

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

More efficient reversal of dabigatran inhibition of coagulation by activated prothrombin complex concentrate or recombinant factor VIIa than by four-factor prothrombin complex concentrate.

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Lindahl, Tomas L; Wallstedt, Maria; Gustafsson, Kerstin M; Persson, Egon; Hillarp, Andreas

2015-03-01

The number of patients on antithrombotic treatment due to atrial fibrillation and venous thromboembolism is increasing fast due to an aging population. A growing proportion will be treated with novel oral anticoagulants, the first in clinical use was the direct oral thrombin inhibitor dabigatran (Pradaxa®). A small percentage of the patients on dabigatran will experience serious bleeding or be in need of urgent surgery. The aim of this study was to test the effects of different hemostatic agents in potentially reversing the anticoagulant effects in vitro in blood or platelet-rich plasma (PRP) spiked with dabigatran. Whole blood or PRP was spiked with the active substance dabigatran, 200 μg/L. We measured clotting time being induced by 1.4 pmol/L tissue factor using the instrument ReoRox2™ and initial clot growth velocity from a tissue factor covered surface using the instrument Thrombodynamics Analyzer T-2™. Dabigatran prolonged clotting time 5-fold but reduced clot growth velocity only slightly. The reversing effects of prothrombin complex concentrates (PCC), activated PCC (APCC) and recombinant activated factor VII (rFVIIa) were then tested. APCC (1.8 U/mL) reduced the prolonged clotting time by 1/3, rFVIIa (2 μg/L) only slightly (n = 10-20). The reduction was not significant using Mann-Whitney test but significant using t-test with Bonferronis' correction for multiple comparisons, whereas PCC (0.56 U/mL) had no effect on clotting time. APCC doubled initial clot growth velocity, although even more in the absence of dabigatran. In conclusion, APCC and rFVIIa, but not PCC, seem to reverse, at least partially, some effects of dabigatran on coagulation parameters. Systematic evaluation of case reports, registries and, ultimately, randomized clinical trials are needed to elucidate potential benefit for patients. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

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

2010-07-01

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

Patients with deep venous thrombosis and thrombophilia risk factors have a specific prolongation of the lag time in a chromogenic thrombin generation assay

NARCIS (Netherlands)

Haas, F.J.L.M.; Kluft, C.; Biesma, D.H.; Schutgens, R.E.G.

2011-01-01

The objective of the present study was to evaluate the influence of thrombophilia risk factors on variables of a chromogenic thrombin generation assay (ETP) in a setting with acute deep venous thrombosis (DVT) and non-DVT patients. In 152 outpatients suspected for DVT, the results of thrombophilia

A Pseudoaneurysm of the Deep Palmar Arch After Penetrating Trauma to the Hand: Successful Exclusion by Ultrasound Guided Percutaneous Thrombin Injection

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A. Bosman

Full Text Available : Introduction: Pseudoaneurysm of the hand is a rare condition; most are treated surgically. Ultrasound guided thrombin injection has not previously been reported as a treatment option for pseudoaneurysms of the deep palmar arch. Report: A man was referred to the emergency department with a swollen, painful hand after penetrating trauma. On physical examination, a pulsating tumor was found on the dorsum of the hand. Imaging revealed a pseudoaneurysm vascularized by the deep palmar arch. Ultrasound guided percutaneous thrombin injection was successfully performed. Conclusion: Thrombin injection might be a safe alternative option in the treatment of pseudoaneurysm of the deep palmar arch. Keywords: Deep palmar arch, Pseudoaneurysm, Thrombin injection

Tritiated bovine fibrinogen labelled in fibrinopeptide A region

International Nuclear Information System (INIS)

Wegrzynowicz, Z.; Kloczewiak, M.; Kopec, M.

1974-01-01

The method is described for labelling of bovine fibrinogen with 3 H-AcOAc. Preparations labelled at pH 7.8 with 10 to 40 molar excess of 3 H-AcOAc were found to contain 8 to 13 moles of acetyl residues per mole of fibrinogen. The content of clottable protein and UV spectra were unchanged as compared with control unlabelled preprations. The rate of clotting with thrombin was only slightly affected. The investigations on distribution of 3 H in products of proteolysis of 3 H-fibrinogen by thrombin and plasmin demonstrated a preferential labelling of fibrinopeptide A, absence of radioactive tracer in fibrinopeptide B, significantly higher specific radioactivity of fragment E than that of fragment D. Incorporation of the label into fibrinopeptide A opens the possibility for application of 3 H-fibrinogen as a convenient substrate for selective investigations on the enzymatic phase of clotting. (author)

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

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

2017-02-01

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

Platelet Counts in Insoluble Platelet-Rich Fibrin Clots: A Direct Method for Accurate Determination

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Yutaka Kitamura

2018-02-01

Full Text Available Platelet-rich fibrin (PRF clots have been used in regenerative dentistry most often, with the assumption that growth factor levels are concentrated in proportion to the platelet concentration. Platelet counts in PRF are generally determined indirectly by platelet counting in other liquid fractions. This study shows a method for direct estimation of platelet counts in PRF. To validate this method by determination of the recovery rate, whole-blood samples were obtained with an anticoagulant from healthy donors, and platelet-rich plasma (PRP fractions were clotted with CaCl2 by centrifugation and digested with tissue-plasminogen activator. Platelet counts were estimated before clotting and after digestion using an automatic hemocytometer. The method was then tested on PRF clots. The quality of platelets was examined by scanning electron microscopy and flow cytometry. In PRP-derived fibrin matrices, the recovery rate of platelets and white blood cells was 91.6 and 74.6%, respectively, after 24 h of digestion. In PRF clots associated with small and large red thrombi, platelet counts were 92.6 and 67.2% of the respective total platelet counts. These findings suggest that our direct method is sufficient for estimating the number of platelets trapped in an insoluble fibrin matrix and for determining that platelets are distributed in PRF clots and red thrombi roughly in proportion to their individual volumes. Therefore, we propose this direct digestion method for more accurate estimation of platelet counts in most types of platelet-enriched fibrin matrix.

Physical determinants of fibrinolysis in single fibrin fibers.

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Igal Bucay

Full Text Available Fibrin fibers form the structural backbone of blood clots; fibrinolysis is the process in which plasmin digests fibrin fibers, effectively regulating the size and duration of a clot. To understand blood clot dissolution, the influence of clot structure and fiber properties must be separated from the effects of enzyme kinetics and perfusion rates into clots. Using an inverted optical microscope and fluorescently-labeled fibers suspended between micropatterned ridges, we have directly measured the lysis of individual fibrin fibers. We found that during lysis 64 ± 6% of fibers were transected at one point, but 29 ± 3% of fibers increase in length rather than dissolving or being transected. Thrombin and plasmin dose-response experiments showed that the elongation behavior was independent of plasmin concentration, but was instead dependent on the concentration of thrombin used during fiber polymerization, which correlated inversely with fiber diameter. Thinner fibers were more likely to lyse, while fibers greater than 200 ± 30 nm in diameter were more likely to elongate. Because lysis rates were greatly reduced in elongated fibers, we hypothesize that plasmin activity depends on fiber strain. Using polymer physics- and continuum mechanics-based mathematical models, we show that fibers polymerize in a strained state and that thicker fibers lose their prestrain more rapidly than thinner fibers during lysis, which may explain why thick fibers elongate and thin fibers lyse. These results highlight how subtle differences in the diameter and prestrain of fibers could lead to dramatically different lytic susceptibilities.

Pre-analytical variation in glucose concentration due to atmospheric temperature and clot in blood specimens

International Nuclear Information System (INIS)

Butt, T.; Masud, K.; Khan, J.A.; Bhatti, M.S.

2016-01-01

Objective: To determine the effect of temperature and contact of clot with serum on laboratory results of glucose concentration in blood. Study Design: Quasi-experimental study. Place and Duration of Study: December 2014 to August 2015 at the laboratory of Shoaib Hospital, Fateh Jang, Attock Pakistan. Material and Methods: Samples were collected for estimation of blood glucose (Random) concentration from patients reporting to the hospital. Blood specimens (n=94) of such volunteers were analyzed for glucose level. Each sample was put up in five tubes. When the blood clotted the serum from tube-1 was analyzed for glucose level within 30 minutes. In tube-2 and tube-3 serum was kept for 24 hours at room temperature and refrigerator temperature respectively before glucose estimation. In tube-4 and tube-5 serum was not separated from clot and kept at room temperature and refrigerator temperature respectively before glucose estimation. The value of tube 1 was taken as reference value for comparison with other parts of the specimen. The equipment used for blood glucose level estimation was semi auto chemistry analyzer (Rayto, China). The kit used for analysis was Glucose - Liquizyme (Germany). Results: The difference between the mean reference value (tube-1) and refrigerated serum without clot (tube-3) was 4.63 mg/100 ml while that of unrefrigerated portion (tube-2) had a difference of 10.68 mg/100 ml. The mean of unrefrigerated (tube-4) and refrigerated (tube-5) portions of serum kept with the clot had difference of 42.05 mg/100 ml and 25.84 mg/100 ml respectively. The fall in the blood glucose level in all (n=94) the samples in the tube number 3 (serum separated and kept at refrigerated temperature) was 4.63 mg/100 ml +- 3.68 (Mean +- SD) and it ranged from 0 to 20 mg/100 ml whereas fall was maximum in the tube number 4 (serum with clotted blood and kept at room temperature) was 42.04 mg/100 ml +- 10.61 (Mean +- SD) and it ranged from 13 to 82 mg/100 ml. The sample in

Fibrin Clots Are Equilibrium Polymers That Can Be Remodeled Without Proteolytic Digestion

OpenAIRE

Chernysh, Irina N.; Nagaswami, Chandrasekaran; Purohit, Prashant K.; Weisel, John W.

2012-01-01

Fibrin polymerization is a necessary part of hemostasis but clots can obstruct blood vessels and cause heart attacks and strokes. The polymerization reactions are specific and controlled, involving strong knob-into-hole interactions to convert soluble fibrinogen into insoluble fibrin. It has long been assumed that clots and thrombi are stable structures until proteolytic digestion. On the contrary, using the technique of fluorescence recovery after photobleaching, we demonstrate here that the...

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

Science.gov (United States)

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

2015-08-15

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

Alkylation of phosphorothioated thrombin binding aptamers improves the selectivity of inhibition of tumor cell proliferation upon anticoagulation.

Science.gov (United States)

Yang, Xiantao; Zhu, Yuejie; Wang, Chao; Guan, Zhu; Zhang, Lihe; Yang, Zhenjun

2017-07-01

Recently, aptamers have been extensively researched for therapy and diagnostic applications. Thrombin-binding aptamer is a 15nt deoxyribonucleic acid screened by SELEX, it can specifically bind to thrombin and inhibit blood coagulation. Since it is also endowed with excellent antitumor activity, the intrinsic anticoagulation advantage converted to a main potential side effect for its further application in antiproliferative therapy. Site-specific alkylation was conducted through nucleophilic reaction of phosphorothioated TBAs using bromide reagents. Circular dichroism (CD) spectroscopy and surface plasmon resonance (SPR) measurements were used to evaluate anticoagulation activity, and a CCK-8 assay was used to determine cell proliferation activity. The CD spectra of the modified TBAs were weakened, and their affinity for thrombin was dramatically reduced, as reflected by the K D values. On the other hand, their inhibition of A549 cells was retained. Incorporation of different alkyls apparently disrupted the binding of TBA to thrombin while maintaining the antitumor activity. A new modification strategy was established for the use of TBA as a more selective antitumor agent. Copyright © 2017 Elsevier B.V. All rights reserved.

A electrochemiluminescence aptasensor for detection of thrombin incorporating the capture aptamer labeled with gold nanoparticles immobilized onto the thio-silanized ITO electrode

International Nuclear Information System (INIS)

Fang Lanyun; Lue Zhaozi; Wei Hui; Wang Erkang

2008-01-01

A novel electrochemiluminescence (ECL) aptasensor was proposed for sensitive and cost-effective detection of the target thrombin adopted an aptamer-based sandwich format. To detect thrombin, capture aptamers labeled with gold nanoparticles (AuNPs) were first immobilized onto the thio-silanized ITO electrode surface through strong Au-S bonds. After catching the target thrombin, signal aptamers tagged with ECL labels were attached to the assembled electrode surface. As a result, an AuNPs-capture-aptamer/thrombin/ECL-tagged-signal-aptamer sandwich type was formed. Treating the resulting electrode surface with tri-n-propylamine (TPA) and applying a swept potential to the electrode, ECL response was generated which realized the detection of target protein. Spectroscopy and electrochemical impedance techniques were used to characterize and confirm the fabrication of the ECL aptasensor. AuNPs amplification and smart sensor fabrication art were implemented for the sensitive and cost-effective detection purpose. Signal-to-dose curve excellently followed a sandwich format equation and could be used to quantify the protein, and the detection limit was estimated to be 10 nM. Other forms of thrombin such as β- and γ-thrombins had negligible response, which indicated a high specificity of α-thrombin detection. The aptasensor opened up new fields of aptamer applications in ECL domain, a highly sensitive technique, and had a promising perspective to be applied in microarray analysis

Identification and characterization of milk-clotting proteases ...

African Journals Online (AJOL)

SAM

2014-03-12

Mar 12, 2014 ... Biochem. Soc. Trans. 10:285. Hashem AM (2000). Purification and properities of a milk-clotting enzyme produced by Penicillium oxalicum. J. Bioresour. Technol. 75:219-222. Laemmli UK (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227: 680-685.

Two acidic, anticoagulant PLA2 isoenzymes purified from the venom of monocled cobra Naja kaouthia exhibit different potency to inhibit thrombin and factor Xa via phospholipids independent, non-enzymatic mechanism.

Directory of Open Access Journals (Sweden)

Ashis K Mukherjee

Full Text Available The monocled cobra (Naja kaouthia is responsible for snakebite fatality in Indian subcontinent and in south-western China. Phospholipase A2 (PLA2; EC 3.1.1.4 is one of the toxic components of snake venom. The present study explores the mechanism and rationale(s for the differences in anticoagulant potency of two acidic PLA2 isoenzymes, Nk-PLA2α (13463.91 Da and Nk-PLA2β (13282.38 Da purified from the venom of N. kaouthia.By LC-MS/MS analysis, these PLA2s showed highest similarity (23.5% sequence coverage with PLA2 III isolated from monocled cobra venom. The catalytic activity of Nk-PLA2β exceeds that of Nk-PLA2α. Heparin differentially regulated the catalytic and anticoagulant activities of these Nk-PLA2 isoenzymes. The anticoagulant potency of Nk-PLA2α was comparable to commercial anticoagulants warfarin, and heparin/antithrombin-III albeit Nk-PLA2β demonstrated highest anticoagulant activity. The anticoagulant action of these PLA2s was partially contributed by a small but specific hydrolysis of plasma phospholipids. The strong anticoagulant effect of Nk-PLA2α and Nk-PLA2β was achieved via preferential, non-enzymatic inhibition of FXa (Ki = 43 nM and thrombin (Ki = 8.3 nM, respectively. Kinetics study suggests that the Nk-PLA2 isoenzymes inhibit their "pharmacological target(s" by uncompetitive mechanism without the requirement of phospholipids/Ca(2+. The anticoagulant potency of Nk-PLA2β which is higher than that of Nk-PLA2α is corroborated by its superior catalytic activity, its higher capacity for binding to phosphatidylcholine, and its greater strength of thrombin inhibition. These PLA2 isoenzymes thus have evolved to affect haemostasis by different mechanisms. The Nk-PLA2β partially inhibited the thrombin-induced aggregation of mammalian platelets suggesting its therapeutic application in the prevention of unwanted clot formation.In order to develop peptide-based superior anticoagulant therapeutics, future application of Nk-PLA2

Plasma clot lysis time and its association with cardiovascular risk factors in black Africans.

Directory of Open Access Journals (Sweden)

Zelda de Lange

Full Text Available Studies in populations of European descent show longer plasma clot lysis times (CLT in patients with cardiovascular disease (CVD than in controls. No data are available on the association between CVD risk factors and fibrinolytic potential in black Africans, a group undergoing rapid urbanisation with increased CVD prevalence. We investigated associations between known CVD risk factors and CLT in black Africans and whether CLTs differ between rural and urban participants in light of differences in CVD risk.Data from 1000 rural and 1000 urban apparently healthy black South Africans (35-60 years were cross-sectionally analysed.Increased PAI-1(act, BMI, HbA1c, triglycerides, the metabolic syndrome, fibrinogen concentration, CRP, female sex and positive HIV status were associated with increased CLTs, while habitual alcohol consumption associated with decreased CLT. No differences in CLT were found between age and smoking categories, contraceptive use or hyper- and normotensive participants. Urban women had longer CLT than rural women while no differences were observed for men.CLT was associated with many known CVD risk factors in black Africans. Differences were however observed, compared to data from populations of European descent available in the literature, suggesting possible ethnic differences. The effect of urbanisation on CLT is influenced by traditional CVD risk factors and their prevalence in urban and rural communities.

Plasma clot lysis time and its association with cardiovascular risk factors in black Africans.

Science.gov (United States)

de Lange, Zelda; Pieters, Marlien; Jerling, Johann C; Kruger, Annamarie; Rijken, Dingeman C

2012-01-01

Studies in populations of European descent show longer plasma clot lysis times (CLT) in patients with cardiovascular disease (CVD) than in controls. No data are available on the association between CVD risk factors and fibrinolytic potential in black Africans, a group undergoing rapid urbanisation with increased CVD prevalence. We investigated associations between known CVD risk factors and CLT in black Africans and whether CLTs differ between rural and urban participants in light of differences in CVD risk.Data from 1000 rural and 1000 urban apparently healthy black South Africans (35-60 years) were cross-sectionally analysed.Increased PAI-1(act), BMI, HbA1c, triglycerides, the metabolic syndrome, fibrinogen concentration, CRP, female sex and positive HIV status were associated with increased CLTs, while habitual alcohol consumption associated with decreased CLT. No differences in CLT were found between age and smoking categories, contraceptive use or hyper- and normotensive participants. Urban women had longer CLT than rural women while no differences were observed for men.CLT was associated with many known CVD risk factors in black Africans. Differences were however observed, compared to data from populations of European descent available in the literature, suggesting possible ethnic differences. The effect of urbanisation on CLT is influenced by traditional CVD risk factors and their prevalence in urban and rural communities.

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

Science.gov (United States)

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

2012-10-01

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

Identification and characterization of milk-clotting proteases ...

African Journals Online (AJOL)

Two strains of fungi were isolated and identified as Aspergillus tamarii and Penicillium pinophilum which showed good enzymatic activity on casein, 1933.33U and 1822, 21U, respectively. The search for milk clotting enzymes by fermentation at acid pH on culture medium containing whey and, after purification by molecular ...

Translation of cell therapy into clinical practice: validation of an application procedure for bone marrow progenitor cells and platelet rich plasma.

Science.gov (United States)

Nowotny, Joerg; Farack, Jana; Vater, Corina; Johnsen, Matthias; Gelinsky, Michael; Tonn, Torsten; Kasten, Philip

2016-04-06

Tissue regeneration can be improved by local application of autologous bone marrow derived progenitor cells (BMSC) and platelet rich plasma (PRP). However, there is a lack of standardized application procedures for clinical use. Therefore, a technique in accordance with the guidelines for advanced therapies medical products of the European Medicine Agency was developed and established. In detail, a process for the isolation and formulation of autologous bone marrow cells (BMC) and PRP in a clinical setting was validated. To investigate the influence of storage time and temperature on gel formation and gel stability, different concentrations of BMC were stored with and without additional platelets, thrombin and fibrinogen and analyzed over a period of 28 days. In addition, cell vitality using a live-dead staining and migration ability of human mesenchymal stem cells (hMSC) in the gel clot was investigated. For an optimized stable gel clot, human BMC and PRP should be combined with 10% to 20% fibrinogen (9 mg/mL to 18 mg/mL) and 5% to 20% thrombin (25 I.E. to 100 I.E.). Both freshly prepared and stored cells for 1 to 7 days had a stable consistence over 28 days at 37°C. Different platelet concentrations did not influence gel clot formation. The ratio of living cells did not decrease significantly over the observation period of 5 days in the live-dead staining. The study identified an optimal gel texture for local application of BMC and PRP. Seeded hMSC could migrate therein and were able to survive to initiate a healing cascade.

Factor VIII S373L: mutation at P1' site confers thrombin cleavage resistance, causing mild haemophilia A.

Science.gov (United States)

Johnson, D J; Pemberton, S; Acquila, M; Mori, P G; Tuddenham, E G; O'Brien, D P

1994-04-01

A novel CRM+ mutation, factor VIII position 373 serine to leucine substitution (FVIII 373-Leu) was identified during a survey of Factor VIII (FVIII) mutations. We have purified the variant protein from the patient's plasma in order to allow further characterisation of the molecule. The CRM+ plasma contained 120% Factor VIII antigen (FVIII:Ag) and 6% Factor VIII coagulant activity (FVIII:C). After purification the mutant FVIII was subjected to thrombin proteolysis, and was thereby activated 5.6-fold compared with 7-fold for wild type molecule. Subsequently, spontaneous inactivation of the mutant was much slower than noted for wild type FVIII. Western blot analysis using monoclonal antibodies demonstrated that thrombin cleavage of FVIII 373-Leu at positions 740 and 1689 were normal but that cleavage at position 372 was completely absent. Crystallographic coordinates of the active site of thrombin complexed to fibrinopeptide A were used to explore possible mechanistic reasons for the failure of thrombin to cleave the mutant FVIII at position 372. Steric hindrance between the mutant side chain and the side chain of the P1 residue was apparent. We conclude that the functional defect of FVIII 373-Leu results from the inability of thrombin to cleave the mutant at position 372-373, and propose that this is due to steric hindrance by the side chain of leucine 373, preventing correct formation of the enzyme substrate complex.

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

Science.gov (United States)

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

2011-05-31

Several lines of experimental evidence including amide exchange and NMR suggest that ligands binding to thrombin cause reduced backbone dynamics. Binding of the covalent inhibitor dPhe-Pro-Arg chloromethyl ketone to the active site serine, as well as noncovalent binding of a fragment of the regulatory protein, thrombomodulin, to exosite 1 on the back side of the thrombin molecule both cause reduced dynamics. However, the reduced dynamics do not appear to be accompanied by significant conformational changes. In addition, binding of ligands to the active site does not change the affinity of thrombomodulin fragments binding to exosite 1; however, the thermodynamic coupling between exosite 1 and the active site has not been fully explored. We present isothermal titration calorimetry experiments that probe changes in enthalpy and entropy upon formation of binary ligand complexes. The approach relies on stringent thrombin preparation methods and on the use of dansyl-l-arginine-(3-methyl-1,5-pantanediyl)amide and a DNA aptamer as ligands with ideal thermodynamic signatures for binding to the active site and to exosite 1. Using this approach, the binding thermodynamic signatures of each ligand alone as well as the binding signatures of each ligand when the other binding site was occupied were measured. Different exosite 1 ligands with widely varied thermodynamic signatures cause a similar reduction in ΔH and a concomitantly lower entropy cost upon DAPA binding at the active site. The results suggest a general phenomenon of enthalpy-entropy compensation consistent with reduction of dynamics/increased folding of thrombin upon ligand binding to either the active site or exosite 1.

Molecular modeling studies of novel retro-binding tripeptide active-site inhibitors of thrombin.

Science.gov (United States)

Lau, W F; Tabernero, L; Sack, J S; Iwanowicz, E J

1995-08-01

A novel series of retro-binding tripeptide thrombin active-site inhibitors was recently developed (Iwanowicz, E. I. et al. J. Med. Chem. 1994, 37, 2111(1)). It was hypothesized that the binding mode for these inhibitors is similar to that of the first three N-terminal residues of hirudin. This binding hypothesis was subsequently verified when the crystal structure of a member of this series, BMS-183,507 (N-[N-[N-[4-(Aminoiminomethyl)amino[-1-oxobutyl]-L- phenylalanyl]-L-allo-threonyl]-L-phenylalanine, methyl ester), was determined (Taberno, L.J. Mol. Biol. 1995, 246, 14). The methodology for developing the binding models of these inhibitors, the structure-activity relationships (SAR) and modeling studies that led to the elucidation of the proposed binding mode is described. The crystal structure of BMS-183,507/human alpha-thrombin is compared with the crystal structure of hirudin/human alpha-thrombin (Rydel, T.J. et al. Science 1990, 249,227; Rydel, T.J. et al. J. Mol Biol. 1991, 221, 583; Grutter, M.G. et al. EMBO J. 1990, 9, 2361) and with the computational binding model of BMS-183,507.

Percutaneous Thrombin Injection to Complete SMA Pseudoaneurysm Exclusion After Failing of Endograft Placement

International Nuclear Information System (INIS)

Szopinski, Piotr; Ciostek, Piotr; Pleban, Eliza; Iwanowski, Jaroslaw; Krol, Malgorzata Serafin-; Marianowska, Agnieszka; Noszczyk, Wojciech

2005-01-01

Visceral aneurysms are potentially life-threatening vascular lesions. Superior mesenteric artery (SMA) pseudoaneurysms are a rare but well-recognized complication of chronic pancreatitis. Open surgical repair of such an aneurysm, especially in patients after previous surgical treatment, might be dangerous and risky. Stent graft implantation makes SMA pseudoaneurysm exclusion possible and therefore avoids a major abdominal operation. Percutaneous direct thrombin injection is also one of the methods of treating aneurysms in this area. We report a first case of percutaneous ultrasound-guided thrombin injection to complete SMA pseudoaneurysm exclusion after an unsuccessful endograft placement. Six-month follow-up did not demonstrate any signs of aneurysm recurrence

Examining platelet-fibrin interactions during traumatic shock in a swine model using platelet contractile force and clot elastic modulus.

Science.gov (United States)

White, Nathan J; Martin, Erika J; Brophy, Donald F; Ward, Kevin R

2011-07-01

A significant proportion of severely injured patients develop early coagulopathy, characterized by abnormal clot formation, which impairs resuscitation and increases mortality. We have previously demonstrated an isolated decrease in clot strength by thrombelastography in a swine model of nonresuscitated traumatic shock. In order to more closely examine platelet-fibrin interactions in this setting, we define the observed decrease in clot strength in terms of platelet-induced clot contraction and clot elastic modulus using the Hemostasis Analysis System (HAS) (Hemodyne Inc., Richmond, Virginia, USA). Whole blood was sampled for HAS measurements, metabolic measurements, cell counts, and fibrinogen concentration at baseline prior to injury and again at a predetermined level of traumatic shock defined by oxygen debt. Male swine (N=17) received femur fracture and controlled arterial hemorrhage to achieve an oxygen debt of 80 ml/kg. Platelet counts were unchanged, but fibrinogen concentration was reduced significantly during shock (167.6 vs. 66.7 mg/dl, P=0.0007). Platelet contractile force generated during clot formation did not change during shock (11.7 vs. 10.4 kdynes, P=0.41), but clot elastic modulus was dynamically altered, resulting in a lower final value (22.9 vs. 17.3 kdynes/cm, Pshock, platelet function was preserved, whereas terminal clot elastic modulus was reduced during shock in a manner most consistent with early changes in the mechanical properties of the developing fibrin fiber network.

Defining Platelet Function During Polytrauma

Science.gov (United States)

2014-04-01

scaffold of the clot after its thrombin-induced polymerization to fibrin fibers. Formation of a stiff fibrin fiber network and its contraction by platelet...In another study (PROTEC T) Refused Participat ion (Yes=Y, No=N, W=Unqua lified , Refused further draws (agreed to keep previous data

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

Science.gov (United States)

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

2016-11-15

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

High-Quality and -Quantity DNA Extraction from Frozen Archival Blood Clots for Genotyping of Single-Nucleotide Polymorphisms

DEFF Research Database (Denmark)

Bank, Steffen; Nexø, Bjørn Andersen; Andersen, Vibeke

2013-01-01

the efficiency of commercial purification kits for extracting DNA from long-term frozen clotted blood. Methods: Serum tubes with clotted blood were stored at −20°C for 1 to 2.5 years before DNA extraction. DNA was extracted from 10 blood clot samples using PureGene (Qiagen) with and without glycogen, the QIAamp...... with a median of 0.65 μg (range 0.5–2.6 μg) pr 300 μL total blood. Conclusion: The yield obtained by the different commercial kits varied considerably. Our work demonstrates that high-quality and -quantity DNA can be extracted with the Maxwell 16 Blood purification kit (Promega) from cryopreserved blood clots...

Makers of clotting medicine not entitled to liability shield.

Science.gov (United States)

1996-02-23

The Indiana Court of Appeals, Second District, reversed a ruling that protected the manufacturer of a hemophilia blood clotting medicine from legal liability. A boy with hemophilia, known in court records as J.K.B. Jr., contracted HIV from the blood clotting medicine, Factor VIII. His parents sued four pharmaceutical companies, Armour Pharmaceutical Company, Cutter Laboratories, Baxter Healthcare Corporation, and Alpha Therapeutics Corporation, alleging that their products caused J.K.B. Jr.'s death. The companies convinced a Superior Court judge that they were protected under the Indiana Blood Shield Statute. The appeals court ruled that pharmaceutical companies are not included in the Blood Shield Statute. The statute was originally established to protect state-approved blood banks, hospitals, and blood storage facilities because they provide a vital public service.

Cloning and expression of clt genes encoding milk-clotting proteases from Myxococcus xanthus 422.

Science.gov (United States)

Poza, M; Prieto-Alcedo, M; Sieiro, C; Villa, T G

2004-10-01

The screening of a gene library of the milk-clotting strain Myxococcus xanthus 422 constructed in Escherichia coli allowed the description of eight positive clones containing 26 open reading frames. Only three of them (cltA, cltB, and cltC) encoded proteins that exhibited intracellular milk-clotting ability in E. coli, Saccharomyces cerevisiae, and Pichia pastoris expression systems.

A constitutive model for developing blood clots with various compositions and their nonlinear viscoelastic behavior

NARCIS (Netherlands)

van Kempen, T. H. S.; Donders, W. P.; van de Vosse, F. N.; Peters, G. W. M.

2016-01-01

The mechanical properties determine to a large extent the functioning of a blood clot. These properties depend on the composition of the clot and have been related to many diseases. However, the various involved components and their complex interactions make it difficult at this stage to fully

Statistically defining optimal conditions of coagulation time of skim milk

International Nuclear Information System (INIS)

Celebi, M.; Ozdemir, Z.O.; Eroglu, E.; Guney, I

2014-01-01

Milk consist huge amount of largely water and different proteins. Kappa-kazein of these milk proteins can be coagulated by Mucor miehei rennet enzyme, is an aspartic protease which cleavege 105 (phenly alanine)-106 (methionine) peptide bond. It is commonly used clotting milk proteins for cheese production in dairy industry. The aim of this study to measure milk clotting times of skim milk by using Mucor Miehei rennet and determination of optimal conditions of milk clotting time by mathematical modelling. In this research, milk clotting times of skim milk were measured at different pHs (3.0, 4.0, 5.0, 6.0, 7.0, 8.0) and temperatures (20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 degree C). It was used statistical approach for defining best pH and temperature for milk clotting time of skim milk. Milk clotting activity was increase at acidic pHs and high temperatures. (author)

Fabrication and physical and biological properties of fibrin gel derived from human plasma

Science.gov (United States)

Zhao, Haiguang; Ma, Lie; Zhou, Jie; Mao, Zhengwei; Gao, Changyou; Shen, Jiacong

2008-03-01

The fast development of tissue engineering and regenerative medicine drives the old biomaterials, for example, fibrin glue, to find new applications in these areas. Aiming at developing a commercially available hydrogel for cell entrapment and delivery, in this study we optimized the fabrication and gelation conditions of fibrin gel. Fibrinogen was isolated from human plasma by a freeze-thaw circle. Gelation of the fibrinogen was accomplished by mixing with thrombin. Absorbance of the fibrinogen/thrombin mixture at 550 nm as a function of reaction time was monitored by UV-VIS spectroscopy. It was found that the clotting time is significantly influenced by the thrombin concentration and the temperature, while less influenced by the fibrinogen concentration. After freeze-drying, the fibrin gel was characterized by scanning electron microscopy (SEM), revealing fibrous microstructure. Thermal gravimetric analysis found that the degradation temperature of the crosslinked fibrin gel starts from 288 °C, which is about 30 °C higher than that of the fibrinogen. The hydrogel has an initial water-uptake ratio of ~50, decreased to 30-40 after incubation in water for 11 h depending on the thrombin concentration. The fibrin gels lost their weights in PBS very rapidly, while slowly in DMEM/fetal bovine serum and DMEM. In vitro cell culture found that human fibroblasts could normally proliferate in the fibrin gel with spreading morphology. In conclusion, the fibrin gel containing higher concentration of fibrinogen (20 mg ml-1) and thrombin (5 U ml-1) has suitable gelation time and handling properties, and thus is applicable as a delivery vehicle for cells such as fibroblasts.

Optimizing electrode-attached redox-peptide systems for kinetic characterization of protease action on immobilized substrates. Observation of dissimilar behavior of trypsin and thrombin enzymes.

Science.gov (United States)

Anne, Agnès; Chovin, Arnaud; Demaille, Christophe

2012-06-12

In this work, we experimentally address the issue of optimizing gold electrode attached ferrocene (Fc)-peptide systems for kinetic measurements of protease action. Considering human α-thrombin and bovine trypsin as proteases of interest, we show that the recurring problem of incomplete cleavage of the peptide layer by these enzymes can be solved by using ultraflat template-stripped gold, instead of polished polycrystalline gold, as the Fc-peptide bearing electrode material. We describe how these fragile surfaces can be mounted in a rotating disk configuration so that enzyme mass transfer no longer limits the overall measured cleavage kinetics. Finally, we demonstrate that, once the system has been optimized, in situ real-time cyclic voltammetry monitoring of the protease action can yield high-quality kinetic data, showing no sign of interfering effects. The cleavage progress curves then closely match the Langmuirian variation expected for a kinetically controlled surface process. Global fit of the progress curves yield accurate values of the peptide cleavage rate for both trypsin and thrombin. It is shown that, whereas trypsin action on the surface-attached peptide closely follows Michaelis-Menten kinetics, thrombin displays a specific and unexpected behavior characterized by a nearly enzyme-concentration-independent cleavage rate in the subnanomolar enzyme concentration range. The reason for this behavior has still to be clarified, but its occurrence may limit the sensitivity of thrombin sensors based on Fc-peptide layers.

Evaluation of thrombelastographic platelet-mapping in healthy cats.

Science.gov (United States)

Blois, Shauna L; Banerjee, Amrita; Wood, R Darren

2012-06-01

Thrombelastography (TEG) permits analysis of clot formation but it is not specific for platelet activity. TEG PlateletMapping (TEG-PM) is a modification of TEG that uses adenosine diphosphate (ADP) and arachidonic acid (AA) as platelet agonists to define the contribution of platelets to clot formation. The objectives of this study were to determine values for TEG-PM in healthy cats and the interassay variation of TEG-PM. TEG-PM analysis was performed on blood specimens collected from 12 healthy cats and was repeated using a second blood specimen collected 2 hours later. Maximum amplitudes generated by thrombin (MA(thrombin)), fibrin (MA(fibrin)), ADP-stimulated platelet activity (MA(ADP)), and AA-stimulated platelet activity (MA(AA)) were recorded. Mean ± SD for MA(thrombin) was 51.1 ± 8.5 mm, for MA(fibrin) was 32.3 ± 17.7 mm, for MA(ADP) was 32.3 ± 15.0 mm, and for MA(AA) was 24.5 ± 12.2 mm. Mean MA(ADP) and MA(fibrin) were not significantly different, whereas mean MA(AA) was significantly lower than mean MA(fibrin). Results from the first and second specimens were not significantly different. Correlation between the first and second specimens was moderate for MA(thrombin), MA(fibrin), and MA(ADP), but was poor for MA(AA). A high degree of variability (coefficient of variation 47.7-60.0%) was observed for MA(fibrin), MA(ADP), and MA(AA). As MA(ADP) and MA(AA) (AA) were the same as or lower than MA(fibrin), a valid baseline to determine platelet-stimulated clot formation could not be established. Considerable interassay variation and wide intervals for MA(fibrin), MA(ADP), and MA(AA) values in this study indicate that TEG-PM should be used cautiously in feline patients. Several preanalytical factors should be examined in further detail. © 2012 American Society for Veterinary Clinical Pathology.

Relation between lung perfusion defects and intravascular clots in acute pulmonary thromboembolism: assessment with breath-hold SPECT-CT pulmonary angiography fusion images.

Science.gov (United States)

Suga, Kazuyoshi; Yasuhiko, Kawakami; Iwanaga, Hideyuki; Tokuda, Osamu; Matsunaga, Naofumi

2008-09-01

The relation between lung perfusion defects and intravascular clots in acute pulmonary thromboembolism (PTE) was comprehensively assessed on deep-inspiratory breath-hold (DIBrH) perfusion SPECT-computed tomographic pulmonary angiography (CTPA) fusion images. Subjects were 34 acute PTE patients, who had successfully performed DIBrH perfusion SPECT using a dual-headed SPECT and a respiratory tracking system. Automated DIBrH SPECT-CTPA fusion images were used to assess the relation between lung perfusion defects and intravascular clots detected by CTPA. DIBrH SPECT visualized 175 lobar/segmental or subsegmental defects in 34 patients, and CTPA visualized 61 intravascular clots at variable locations in 30 (88%) patients, but no clots in four (12%) patients. In 30 patients with clots, the fusion images confirmed that 69 (41%) perfusion defects (20 segmental, 45 subsegmental and 4 lobar defects) of total 166 defects were located in lung territories without clots, although the remaining 97 (58%) defects were located in lung territories with clots. Perfusion defect was absent in lung territories with clots (one lobar branch and three segmental branches) in four (12%) of these patients. In four patients without clots, nine perfusion defects including four segmental ones were present. Because of unexpected dissociation between intravascular clots and lung perfusion defects, the present fusion images will be a useful adjunct to CTPA in the diagnosis of acute PTE.

Investigation of the effect of kaolin and tissue factor-activated citrated whole blood, on clot forming variables, as evaluated by thromboelastograph

DEFF Research Database (Denmark)

Johansson, Per Ingemar; Bochsen, L.; Andersen, S.

2008-01-01

), and maximum clot strength (amplitude [MA]) were evaluated, together with day-to-day variation, the coefficient of variance (CV%), and the effect of citrate storage time. RESULTS: Clot formation variables were equally affected by TF 1:17,000 and kaolin activation, whereas R was significantly longer when TF 1......:42,500 was used. The CV for the different variables varied from 3 to 13 percent with no significant differences between assays. Storage of citrated WB significantly affected the TEG variables in a hypercoagulable direction. Only the R, however, was significantly affected (12%) when samples rested for 0 and 30...... minutes were evaluated with kaolin as the activator. CONCLUSION: The TEG assays evaluated were reproducible and present with an acceptable CV% for routine clinical practice. Kaolin and TF 1:17,000 equally affected the clot formation variables. Storage of WB for up to 30 minutes in citrate did not, except...

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

International Nuclear Information System (INIS)

Puri, S.; Nicholson, A.A.; Breen, D.J.

2003-01-01

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

Protein kinase C is differentially regulated by thrombin, insulin, and epidermal growth factor in human mammary tumor cells

Energy Technology Data Exchange (ETDEWEB)

Gomez, M.L.; Tellez-Inon, M.T. (Instituto de Ingenieria Genetica y Biologia Molecular, Buenos Aires (Argentina)); Medrano, E.E.; Cafferatta, E.G.A. (Instituto de Investigaciones Bioquimicas Fundacion Campomar, Buenos Aires (Argentina))

1988-03-01

The exposure of serum-deprived mammary tumor cells MCF-7 and T-47D to insulin, thrombin, and epidermal growth factor (EGF) resulted in dramatic modifications in the activity and in the translocation capacity of protein kinase C from cytosol to membrane fractions. Insulin induces a 600% activation of the enzyme after 5 h of exposure to the hormone in MCF-7 cells; thrombin either activates (200% in MCF-7) or down-regulates (in T-47D), and EGF exerts only a moderate effect. Thus, the growth factors studied modulate differentially the protein kinase C activity in human mammary tumor cells. The physiological significance of the results obtained are discussed in terms of the growth response elicited by insulin, thrombin, and EGF.

Monitoring of unfractionated heparin with rotational thrombelastometry using the prothrombinase-induced clotting time reagent (PiCT®).

Science.gov (United States)

Schaden, E; Jilch, S; Hacker, S; Schober, A; Kozek-Langenecker, S

2012-12-24

To achieve sufficient and safe anticoagulation with unfractionated heparin (UFH) a close and reliable drug monitoring is necessary. In general, the activated partial thromboplastin time (APTT) is used for this purpose. In acute phase response, however, the APTT test procedure might be unreliable e.g. with false low results in the presence of elevated factor VIII. In this so called heparin resistance, measurement of anti-Xa activity is recommended over APTT to avoid potentially harmful dose escalation. A combination of anti-Xa measurement and global hemostatic testing with ROTEM® employing the anti-Xa sensitive PiCT® reagent showed high correlation with enoxaparin levels. This test modification could also be suitable for monitoring UFH. Aim of the study was to evaluate the correlation between PiCT®-ROTEM® and levels of UFH. In this in-vitro study blood samples from healthy volunteers were spiked with UFH and subjected to different ROTEM® tests. There was a linear correlation between UFH level and clotting time (CT) in the PiCT®-ROTEM® test with an excellent correlation coefficient of 0.92. Additional endpoints showed similar results (PiCT®-ROTEM® MaxVel r = -0.85 and PiCT®-ROTEM® t_MaxVel r = 0.88). As a point-of-care applicable tool ROTEM® is immediately at hand. If further clinical studies confirm sensitivity in heparin resistance, PiCT®-ROTEM® could permit rapid UFH dose adjustments especially required in critical illness with acute phase response. Copyright © 2012 Elsevier B.V. All rights reserved.

Direct Oral Anticoagulants in Emergency Trauma Admissions.

Science.gov (United States)

Maegele, Marc; Grottke, Oliver; Schöchl, Herbert; Sakowitz, Oliver A; Spannagl, Michael; Koscielny, Jürgen

2016-09-05

Direct (non-vitamin-K-dependent) oral anticoagulants (DOAC) are given as an alternative to vitamin K antagonists (VKA) to prevent stroke and embolic disease in patients with atrial fibrillation that is not due to pathology of the heart valves. Fatal hemorrhage is rarer when DOACs are given (nonvalvular atrial fibrillation: odds ratio [OR] 0.68; 95% confidence interval [95% CI: 0.48; 0.96], and venous thromboembolism: OR 0.54; [0.22; 1.32]). 48% of emergency trauma patients need an emergency operation or early surgery. Clotting disturbances elevate the mortality of such patients to 43%, compared to 17% in patients without a clotting disturbance. This underscores the impor tance of the proper, targeted treatment of trauma patients who are aking DOAC. This review is based on articles retrieved by a selective search in PubMed and on a summary of expert opinion and the recommendations of the relevant medical specialty societies. Peak DOAC levels are reached 2-4 hours after the drug is taken. In patients with normal renal and hepatic function, no drug accumulation, and no drug interactions, the plasma level of DOAC 24 hours after administration is generally too low to cause any clinically relevant risk of bleeding. The risk of drug accumulation is higher in patients with renal dysfunction (creatinine clearance [CrCl] of 30 mL/min or less). Dabigatran levels can be estimated from the thrombin time, ecarin clotting time, and diluted thrombin time, while levels of factor Xa inhibitors can be estimated by means of calibrated chromogenic anti-factor Xa activity tests. Routine clotting studies do not reliably reflect the anticoagulant activity of DOAC. Surgery should be postponed, if possible, until at least 24-48 hours after the last dose of DOAC. For patients with mild, non-life threatening hemorrhage, it suffices to discontinue DOAC; for patients with severe hemorrhage, there are special treatment algorithms that should be followed. DOACs in the setting of hemorrhage are a

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

Science.gov (United States)

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

2008-01-01

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

Building and validation of a prognostic model for predicting extracorporeal circuit clotting in patients with continuous renal replacement therapy.

Science.gov (United States)

Fu, Xia; Liang, Xinling; Song, Li; Huang, Huigen; Wang, Jing; Chen, Yuanhan; Zhang, Li; Quan, Zilin; Shi, Wei

2014-04-01

To develop a predictive model for circuit clotting in patients with continuous renal replacement therapy (CRRT). A total of 425 cases were selected. 302 cases were used to develop a predictive model of extracorporeal circuit life span during CRRT without citrate anticoagulation in 24 h, and 123 cases were used to validate the model. The prediction formula was developed using multivariate Cox proportional-hazards regression analysis, from which a risk score was assigned. The mean survival time of the circuit was 15.0 ± 1.3 h, and the rate of circuit clotting was 66.6 % during 24 h of CRRT. Five significant variables were assigned a predicting score according to the regression coefficient: insufficient blood flow, no anticoagulation, hematocrit ≥0.37, lactic acid of arterial blood gas analysis ≤3 mmol/L and APTT R (2) = 0.232; P = 0.301). A risk score that includes the five above-mentioned variables can be used to predict the likelihood of extracorporeal circuit clotting in patients undergoing CRRT.

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

Science.gov (United States)

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

2017-06-01

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

Bitistatin-functionalized fluorescent nanodiamond particles specifically bind to purified human platelet integrin receptor αIIbβ3 and activated platelets.

Science.gov (United States)

Marcinkiewicz, Cezary; Gerstenhaber, Jonathan A; Sternberg, Mark; Lelkes, Peter I; Feuerstein, Giora

2017-01-01

Thromboembolic events (TEE) underwrite key causes of death in developed countries. While advanced imaging technologies such as computed tomography scans serve to diagnose blood clots during acute cardiovascular events, no such technology is available in routine primary care for TEE risk assessment. Here, we describe an imaging platform technology based on bioengineered fluorescent nanodiamond particles (F-NDPs) functionalized with bitistatin (Bit), a disintegrin that specifically binds to the α IIb β 3 integrin, platelet fibrinogen receptor (PFR) on activated platelets. Covalent linkage of purified Bit to F-NDP was concentration-dependent and saturable, as validated by enzyme-linked immunosorbent assay using specific anti-Bit antibodies. F-NDP-Bit interacted with purified PFR, either in immobilized or soluble form. Lotrafiban, a nonpeptide, α IIb β 3 receptor antagonist, specifically blocked F-NDP-Bit-PFR complex formation. Moreover, F-NDP-Bit specifically binds to activated platelets incorporated into a clot generated by thrombin-activated rat platelet-rich plasma (PRP). Our results suggest that engineered F-NDP-Bit particles could serve as noninvasive, "real-time" optical diagnostics for clots present in blood vessels.

Contribution of thrombin-reactive brain pericytes to blood-brain barrier dysfunction in an in vivo mouse model of obesity-associated diabetes and an in vitro rat model.

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Takashi Machida

Full Text Available Diabetic complications are characterized by the dysfunction of pericytes located around microvascular endothelial cells. The blood-brain barrier (BBB exhibits hyperpermeability with progression of diabetes. Therefore, brain pericytes at the BBB may be involved in diabetic complications of the central nervous system (CNS. We hypothesized that brain pericytes respond to increased brain thrombin levels in diabetes, leading to BBB dysfunction and diabetic CNS complications. Mice were fed a high-fat diet (HFD for 2 or 8 weeks to induce obesity. Transport of i.v.-administered sodium fluorescein and 125I-thrombin across the BBB were measured. We evaluated brain endothelial permeability and expression of tight junction proteins in the presence of thrombin-treated brain pericytes using a BBB model of co-cultured rat brain endothelial cells and pericytes. Mice fed a HFD for 8 weeks showed both increased weight gain and impaired glucose tolerance. In parallel, the brain influx rate of sodium fluorescein was significantly greater than that in mice fed a normal diet. HFD feeding inhibited the decline in brain thrombin levels occurring during 6 weeks of feeding. In the HFD fed mice, plasma thrombin levels were significantly increased, by up to 22%. 125I-thrombin was transported across the BBB in normal mice after i.v. injection, with uptake further enhanced by co-injection of unlabeled thrombin. Thrombin-treated brain pericytes increased brain endothelial permeability and caused decreased expression of zona occludens-1 (ZO-1 and occludin and morphological disorganization of ZO-1. Thrombin also increased mRNA expression of interleukin-1β and 6 and tumor necrosis factor-α in brain pericytes. Thrombin can be transported from circulating blood through the BBB, maintaining constant levels in the brain, where it can stimulate pericytes to induce BBB dysfunction. Thus, the brain pericyte-thrombin interaction may play a key role in causing BBB dysfunction in

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

Directory of Open Access Journals (Sweden)

Benjamin M Scott

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

Circulating endothelial cells and procoagulant microparticles in patients with glioblastoma: prognostic value.

Directory of Open Access Journals (Sweden)

Gaspar Reynés

Full Text Available AIM: Circulating endothelial cells and microparticles are prognostic factors in cancer. However, their prognostic and predictive value in patients with glioblastoma is unclear. The objective of this study was to investigate the potential prognostic value of circulating endothelial cells and microparticles in patients with newly diagnosed glioblastoma treated with standard radiotherapy and concomitant temozolomide. In addition, we have analyzed the methylation status of the MGMT promoter. METHODS: Peripheral blood samples were obtained before and at the end of the concomitant treatment. Blood samples from healthy volunteers were also obtained as controls. Endothelial cells were measured by an immunomagnetic technique and immunofluorescence microscopy. Microparticles were quantified by flow cytometry. Microparticle-mediated procoagulant activity was measured by endogen thrombin generation and by phospholipid-dependent clotting time. Methylation status of MGMT promoter was determined by multiplex ligation-dependent probe amplification. RESULTS: Pretreatment levels of circulating endothelial cells and microparticles were higher in patients than in controls (p<0.001. After treatment, levels of microparticles and thrombin generation decreased, and phospholipid-dependent clotting time increased significantly. A high pretreatment endothelial cell count, corresponding to the 99(th percentile in controls, was associated with poor overall survival. MGMT promoter methylation was present in 27% of tumor samples and was associated to a higher overall survival (66 weeks vs 30 weeks, p<0.004. CONCLUSION: Levels of circulating endothelial cells may have prognostic value in patients with glioblastoma.

Qualification of a select one-stage activated partial thromboplastin time-based clotting assay and two chromogenic assays for the post-administration monitoring of nonacog beta pegol.

Science.gov (United States)

Tiefenbacher, S; Bohra, R; Amiral, J; Bowyer, A; Kitchen, S; Lochu, A; Rosén, S; Ezban, M

2017-10-01

Essentials Nonacog beta pegol (N9-GP) is an extended half-life, recombinant human factor IX (FIX). One-stage clotting (OSC) and chromogenic FIX activity assays were assessed for N9-GP recovery. OSC STA ® -Cephascreen ® , ROX FIX and BIOPHEN FIX chromogenic assays were qualified for N9-GP. Other extended half-life factor products should be assessed in a similar way prior to approval. Background Nonacog beta pegol (N9-GP) is an extended half-life, glycoPEGylated recombinant human factor IX that is under development for the prophylaxis and treatment of bleeding episodes in hemophilia B patients. Considerable reagent-dependent variability has been observed when one-stage clotting assays are used to measure the recovery of recombinant FIX products, including N9-GP. Objective To qualify select one-stage clotting and chromogenic FIX activity assays for measuring N9-GP recovery. Methods The accuracy and precision of the one-stage clotting assay (with the STA-Cephascreen activated partial thromboplastin [APTT] reagent) and the ROX Factor IX and BIOPHEN Factor IX chromogenic assays for measuring N9-GP recovery were assessed in N9-GP-spiked hemophilia B plasma samples in a systematic manner at three independent sites, with manufacturer-recommended protocols and/or site-specific assay setups, including different instruments. Results For each of the three FIX activity assays qualified on five different reagent-instrument systems, acceptable intra-assay and interassay accuracy and precision, dilution integrity, reagent robustness and freeze-thaw and short-term sample stabilities were demonstrated. The STA-Cephascreen assay showed a limited reportable range at one of the three qualification sites, and the BIOPHEN Factor IX assay showed suspect low-end sensitivity at one of the three qualification sites. An individual laboratory would account for these limitations by adjusting the assay's reportable range; thus, these findings are not considered to impact the respective

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

International Nuclear Information System (INIS)

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

1989-01-01

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 4 /PAGE and N-terminal sequence analysis. Using a plasma-free assay of the ability of activated 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

Human Protein C produces anticoagulation and increased fibrinolytic activity in the cat

International Nuclear Information System (INIS)

Burdick, M.D.; Schaub, R.G.

1986-01-01

The effect of activated human Protein C (PCa) infusion on the coagulation and fibrinolytic systems of the Nembutal anesthetized cat was assessed. Human Protein C was activated by incubation with thrombin or by passage over a column of thrombin immobilized on CNBr Sepharose 4B. Cats were given bolus i.v. injections of either vehicle or PCa in a dose range of 3-16 μg/mL of calculated whole body volume. Citrated blood samples (9:1) were taken from a femoral vein prior to and at 5, 10, 20, 30, 60, 120, and 180 min. after PCa. Activated partial thromboplastin time (APTT), thrombin time (TT) euglobulin clot lysis (ECLT) and I-125 fibrin release (FR) was measured. Vehicle treated cats had no change in any parameter. PCa produced a dose and time dependent prolongation of APTT while TT was unchanged. Anticoagulation was evident immediately after PCa infusion and began to normalize within 20 min. Fibrinolytic activity measured by ECLT and FR was also stimulated by PCa but was not evident until 40-60 minutes after PCa injection. The results show that human PCa induces anticoagulation effects in the cat similar to other species. However, stimulation of fibrinolysis requires a longer period of time before expression. This delay of fibrinolytic stimulation should be considered when assessing the effects of human Protein C in other species

Enhanced Effector Function of CD8+ T Cells From Healthy Controls and HIV-Infected Patients Occurs Through Thrombin Activation of Protease-Activated Receptor 1

Science.gov (United States)

Hurley, Amanda; Smith, Mindy; Karpova, Tatiana; Hasley, Rebecca B.; Belkina, Natalya; Shaw, Stephen; Balenga, Nariman; Druey, Kirk M.; Nickel, Erin; Packard, Beverly; Imamichi, Hiromi; Hu, Zonghui; Follmann, Dean; McNally, James; Higgins, Jeanette; Sneller, Michael; Lane, H. Clifford; Catalfamo, Marta

2013-01-01

Disruption of vascular integrity by trauma and other tissue insults leads to inflammation and activation of the coagulation cascade. The serine protease thrombin links these 2 processes. The proinflammatory function of thrombin is mediated by activation of protease-activated receptor 1 (PAR-1). We found that peripheral blood effector memory CD4+ and CD8+ T lymphocytes expressed PAR-1 and that expression was increased in CD8+ T cells from human immunodeficiency virus (HIV)–infected patients. Thrombin enhanced cytokine secretion in CD8+ T cells from healthy controls and HIV-infected patients. In addition, thrombin induced chemokinesis, but not chemotaxis, of CD8+ T cells, which led to structural changes, including cell polarization and formation of a structure rich in F-actin and phosphorylated ezrin-radexin-moesin proteins. These findings suggest that thrombin mediates cross-talk between the coagulation system and the adaptive immune system at sites of vascular injury through increased T-cell motility and production of proinflammatory cytokines. PMID:23204166

Enhanced effector function of CD8(+) T cells from healthy controls and HIV-infected patients occurs through thrombin activation of protease-activated receptor 1.

Science.gov (United States)

Hurley, Amanda; Smith, Mindy; Karpova, Tatiana; Hasley, Rebecca B; Belkina, Natalya; Shaw, Stephen; Balenga, Nariman; Druey, Kirk M; Nickel, Erin; Packard, Beverly; Imamichi, Hiromi; Hu, Zonghui; Follmann, Dean; McNally, James; Higgins, Jeanette; Sneller, Michael; Lane, H Clifford; Catalfamo, Marta

2013-02-15

Disruption of vascular integrity by trauma and other tissue insults leads to inflammation and activation of the coagulation cascade. The serine protease thrombin links these 2 processes. The proinflammatory function of thrombin is mediated by activation of protease-activated receptor 1 (PAR-1). We found that peripheral blood effector memory CD4(+) and CD8(+) T lymphocytes expressed PAR-1 and that expression was increased in CD8(+) T cells from human immunodeficiency virus (HIV)-infected patients. Thrombin enhanced cytokine secretion in CD8(+) T cells from healthy controls and HIV-infected patients. In addition, thrombin induced chemokinesis, but not chemotaxis, of CD8(+) T cells, which led to structural changes, including cell polarization and formation of a structure rich in F-actin and phosphorylated ezrin-radexin-moesin proteins. These findings suggest that thrombin mediates cross-talk between the coagulation system and the adaptive immune system at sites of vascular injury through increased T-cell motility and production of proinflammatory cytokines.

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

OpenAIRE

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

1999-01-01

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...

Kinetic Dissection of the Pre-existing Conformational Equilibrium in the Trypsin Fold*

Science.gov (United States)

Vogt, Austin D.; Chakraborty, Pradipta; Di Cera, Enrico

2015-01-01

Structural biology has recently documented the conformational plasticity of the trypsin fold for both the protease and zymogen in terms of a pre-existing equilibrium between closed (E*) and open (E) forms of the active site region. How such plasticity is manifested in solution and affects ligand recognition by the protease and zymogen is poorly understood in quantitative terms. Here we dissect the E*-E equilibrium with stopped-flow kinetics in the presence of excess ligand or macromolecule. Using the clotting protease thrombin and its zymogen precursor prethrombin-2 as relevant models we resolve the relative distribution of the E* and E forms and the underlying kinetic rates for their interconversion. In the case of thrombin, the E* and E forms are distributed in a 1:4 ratio and interconvert on a time scale of 45 ms. In the case of prethrombin-2, the equilibrium is shifted strongly (10:1 ratio) in favor of the closed E* form and unfolds over a faster time scale of 4.5 ms. The distribution of E* and E forms observed for thrombin and prethrombin-2 indicates that zymogen activation is linked to a significant shift in the pre-existing equilibrium between closed and open conformations that facilitates ligand binding to the active site. These findings broaden our mechanistic understanding of how conformational transitions control ligand recognition by thrombin and its zymogen precursor prethrombin-2 and have direct relevance to other members of the trypsin fold. PMID:26216877

Minimally Invasive Therapy of Pseudoaneurysms of the Trunk: Application of Thrombin

International Nuclear Information System (INIS)

Schellhammer, Frank; Steinhaus, Daniele; Cohnen, Mathias; Hoppe, Jonas; Moedder, Ulrich; Fuerst, Guenter

2008-01-01

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

Intracameral air injection during Ahmed glaucoma valve implantation in neovascular glaucoma for the prevention of tube obstruction with blood clot: Case Report.

Science.gov (United States)

Hwang, Sung Ha; Yoo, Chungkwon; Kim, Yong Yeon; Lee, Dae Young; Nam, Dong Heun; Lee, Jong Yeon

2017-12-01

Glaucoma drainage implant surgery is a treatment option for the management of neovascular glaucoma. However, tube obstruction by blood clot after Ahmed glaucoma valve (AGV) implantation is an unpredictable clinically challenging situation. We report 4 cases using intracameral air injection for the prevention of the tube obstruction of AGV by blood clot. The first case was a 57-year-old female suffering from ocular pain because of a tube obstruction with blood clot after AGV implantation in neovascular glaucoma. Surgical blood clot removal was performed. However, intractable bleeding was noted during the removal of the blood clot, and so intracameral air injection was performed to prevent a recurrent tube obstruction. After the procedure, although blood clots formed around the tube, the tube opening where air could touch remained patent. In 3 cases of neovascular glaucoma with preoperative severe intraocular hemorrhages, intracameral air injection and AGV implantation were performed simultaneously. In all 3 cases, tube openings were patent. It appears that air impeded the blood clots formation in front of the tube opening. Intracameral air injection could be a feasible option to prevent tube obstruction of AGV implant with a blood clot in neovascular glaucoma with high risk of tube obstruction. Copyright © 2017 The Authors. Published by Wolters Kluwer Health, Inc. All rights reserved.

Contact system activation and high thrombin generation in hyperthyroidism.

Science.gov (United States)

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

2017-05-01

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

Sensitivity of the activated partial thromboplastin time, the dilute Russell's viper venom time, and the kaolin clotting time for the detection of the lupus anticoagulant: a direct comparison using plasma dilutions.

Science.gov (United States)

Martin, B A; Branch, D W; Rodgers, G M

1996-01-01

Increasing dilutions of lupus anticoagulant (LA) plasmas from twelve patients were used to directly compare the sensitivity of four tests for LA. The tests evaluated were the modified Bell and Alton activated partial thromboplastin time (APTT), an APTT using a commercially prepared partial thromboplastin (Platelin LS APTT), a modified dilute Russell's viper venom time (DRVVT), and a modified kaolin clotting time (KCT). LAs were detected in all twelve plasmas by each of three tests and eleven of twelve plasmas in a fourth test when undiluted patient plasma was used. Repeating the tests after diluting the LA plasmas with normal platelet-free plasma (PFP) showed that the KCT was the most sensitive test for LA, detecting eleven of twelve LAs at a dilution of 10% patient plasma and ten of twelve LAs at a dilution of 5% patient plasma. The modified Bell and Alton APTT and the modified DRVVT had similar sensitivities at a patient plasma concentration of 10%, detecting seven of twelve and eight of twelve LAs, respectively. The Platelin LS APTT detected only four of twelve LAs at a patient plasma concentration of 10%. Our results indicate that the modified KCT is a sensitive method for the detection of LAs. The modified Bell and Alton APTT and the DRVVT were less sensitive.

Effects of the oral, direct thrombin inhibitor dabigatran on five common coagulation assays.

Science.gov (United States)

Lindahl, Tomas L; Baghaei, Fariba; Blixter, Inger Fagerberg; Gustafsson, Kerstin M; Stigendal, Lennart; Sten-Linder, Margareta; Strandberg, Karin; Hillarp, Andreas

2011-02-01

Dabigatran is an oral, reversible thrombin inhibitor that has shown promising results in large clinical trials. Laboratory monitoring is not needed but the effects on common coagulation assays are incompletely known. Dabigatran was added to plasma from healthy subjects in the concentration range 0-1,000 μg/l and analysed using several reagents for activated thromboplastin time (APTT), prothrombin time (PT), fibrinogen, antithrombin, and activated protein C resistance. Typical trough concentrations are about 50 μg/l, peak concentrations 100-300 μg/l. At 100 μg/l all APTT-results were prolonged. The concentration required to double APTT ranged between 227 and 286 μg/l, the responses for all five reagents were similar. PT-reagents were much less affected with almost no samples above INR 1.2 at 100 μg/l. The effect was sample dilution dependent with PT Quick type more sensitive than PT Owren type methods. If a patient on dabigatran has prolonged APTT, >90 seconds, and Quick PT INR>2 or Owren PT INR>1.5 over-dosing or accumulation of dabigatran should be considered. Two of four fibrinogen reagents underestimated the fibrinogen concentration considerably at expected peak concentration. Methods based on inhibition of thrombin over-estimated the antithrombin concentration, but not Xa-based. The APC-resistance methods over-estimated the APC-ratio, which may lead to miss-classification of factor V Leiden patients as being normal. Different coagulation assays, and even different reagents within an assay group, display variable effects at therapeutic concentrations of dabigatran. Some of these assay variations are of clinical importance, thus knowledge is needed for a correct interpretation of results.

Diagnosis of hemoparasitosis trough the spread-smear technique using a drop of blood clot

OpenAIRE

Neusa Saltiél Stobbe; Eunice Leonora Chaplin; Maria das Graças de Souza Paiva; Nilton Rogério Santos Silva; Flávio A. Pacheco Araújo; Elinor Fortes

1992-01-01

The spread-smear technique using a drop of blood clot and stained by diluted and undimitcd Giemsa stain was evaluated for the search of blood cell types and recognition of Babesia bovis, Babesia bigemina and Anaplasma marginale hemoparasites. The blood clot samples were taken from a 6 month-old calf experimentally inoculated with the blood from a Cattle Tick Fever carrier. The Technique was sumitable for the observation of different blood cells and for the recognition of the hemoparasites.

In vitro anti-thrombotic and anti-coagulant properties of blacklip abalone (Haliotis rubra) viscera hydrolysate.

Science.gov (United States)

Suleria, Hafiz Ansar Rasul; Masci, Paul P; Addepalli, Rama; Chen, Wei; Gobe, Glenda C; Osborne, Simone A

2017-07-01

Abalone viscera contain sulphated polysaccharides with anti-thrombotic and anti-coagulant activities. In this study, a hydrolysate was prepared from blacklip abalone (Haliotis rubra) viscera using papain and bromelain and fractionated using ion exchange and size exclusion chromatography. Hydrolysates and fractions were investigated for in vitro thrombin inhibition mediated through heparin cofactor II (HCII) as well as anti-coagulant activity in plasma and whole blood. On the basis of sulphated polysaccharide concentration, the hydrolysate inhibited thrombin through HCII with an inhibitor concentration at 50% (IC50) of 16.5 μg/mL compared with 2.1 μg/mL for standard heparin. Fractionation concentrated HCII-mediated thrombin inhibition down to an IC50 of 1.8 μg/mL and improved anti-coagulant activities by significantly delaying clotting time. This study confirmed the presence of anti-thrombotic and anti-coagulant molecules in blacklip abalone viscera and demonstrated that these activities can be enriched with a simple chromatography regime. Blacklip abalone viscera warrant further investigation as a source of nutraceutical or functional food ingredients. Graphical abstract Schematic showing preparation of bioactive extracts and fractions from blacklip abalone.

Evaluation of the effects of levobupivacaine on clotting and fibrinolysis using thromboelastography.

LENUS (Irish Health Repository)

Leonard, S A

2012-02-03

Amide local anaesthetics inhibit platelet function. We hypothesized that residual anaesthetic in the epidural space could decrease efficacy of an epidural blood patch in preventing postdural puncture headache. Levobupivacaine has recently been approved for epidural anaesthesia. Its effects on coagulation have not previously been studied. The aim of this study was to determine the effects of levobupivacaine on clotting using thromboelastography. Ten ASA Class I volunteers were studied. Venous blood samples were analysed using a Haemoscope 2000D TEG analyser. Whole blood, a 50% saline control and two levobupivacaine solutions (2.5 mg mL(-1) and 2.5 microg mL(-1) in blood) were compared. The former reproduces that produced in the epidural space by blood (20 mL for an epidural blood patch) and levobupivacaine 0.5% (20 mL). The latter approximates plasma concentrations following epidural injection of levobupivacaine 0.5% (20 mL). P < 0.05 was considered significant. Maximum amplitude (MA), a measure of clot strength, is decreased by levobupivacaine 2.5 mg mL(-1). Levobupivacaine 2.5 mg mL(-1) decreases clot strength and may reduce efficacy of a prophylactic epidural blood patch.

The use of alteplase for the resolution of an intravesical clot in a neonate receiving extracorporeal membrane oxygenation.

Science.gov (United States)

Olarte, J L; Glover, M L; Totapally, B R

2001-01-01

We present a case of the use of alteplase for the lysis of a large urinary bladder clot. A neonate presented with respiratory failure, secondary to a left diaphragmatic hernia necessitating the need for extracorporeal membrane oxygenation (ECMO) support. On day 3 of ECMO support, hematuria was noted, and a subsequent urinary bladder ultrasound revealed a significant urinary bladder clot. Alteplase (0.5-1 mg) was instilled into the urinary bladder via a 10 French Foley catheter (Sherwood Medical, St. Louis, MO). The catheter was clamped for 1 hour, followed by irrigation with normal saline. Multiple doses of alteplase were administered, resulting in complete resolution of the bladder clot. No adverse effects were attributed to the use of the intravesical alteplase. Alteplase seems to be safe and effective for the resolution of bladder clots, thereby potentially avoiding more invasive surgical procedures.

The effect of sepsis and septic shock on the viscoelastic properties of clot quality and mass using rotational thromboelastometry: A prospective observational study.

Science.gov (United States)

Davies, Gareth R; Lawrence, Matthew; Pillai, Suresh; Mills, Gavin M; Aubrey, Robert; Thomas, Dafydd; Williams, Rhodri; Morris, Keith; Evans, Phillip Adrian

2018-04-01

The study purpose was to define changes in coagulation across the sepsis spectrum using rotational thromboelastometry (ROTEM). Sepsis patients were recruited on admission to the Emergency Department and Intensive Care Units of a large teaching hospital in Wales. ROTEM markers of clot development and fibrinolysis were determined, as well as standard coagulation markers. A healthy control group matched for age and gender was also recruited (n=44). 100 patients were recruited (50 sepsis, 20 severe sepsis and 30 septic shock). Maximum clot firmness was significantly higher in the sepsis (p<0.001) and severe sepsis (p=0.012) groups than the healthy control (71.6±4.5 and 70.4±4.1 vs 64.4 respectively). In septic shock there was prolonged clot development; however, maximum clot firmness remained normal. Fibrinolytic function was significantly impaired in septic shock, which was also significantly associated with 28-day mortality (p<0.001). ROTEM indicated significantly enhanced clot structural development in sepsis and severe sepsis, which could be indicative of a hypercoagulable phase. In septic shock, despite there being a prolongation of clotting pathways and impaired fibrinolysis, clot mass was comparably normal, suggestive of the development of a clot with healthy characteristics. Copyright © 2017 Elsevier Inc. All rights reserved.

Structure-activity studies and therapeutic potential of host defense peptides of human thrombin.

Science.gov (United States)

Kasetty, Gopinath; Papareddy, Praveen; Kalle, Martina; Rydengård, Victoria; Mörgelin, Matthias; Albiger, Barbara; Malmsten, Martin; Schmidtchen, Artur

2011-06-01

Peptides of the C-terminal region of human thrombin are released upon proteolysis and identified in human wounds. In this study, we wanted to investigate minimal determinants, as well as structural features, governing the antimicrobial and immunomodulating activity of this peptide region. Sequential amino acid deletions of the peptide GKYGFYTHVFRLKKWIQKVIDQFGE (GKY25), as well as substitutions at strategic and structurally relevant positions, were followed by analyses of antimicrobial activity against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram-positive bacterium Staphylococcus aureus, and the fungus Candida albicans. Furthermore, peptide effects on lipopolysaccharide (LPS)-, lipoteichoic acid-, or zymosan-induced macrophage activation were studied. The thrombin-derived peptides displayed length- and sequence-dependent antimicrobial as well as immunomodulating effects. A peptide length of at least 20 amino acids was required for effective anti-inflammatory effects in macrophage models, as well as optimal antimicrobial activity as judged by MIC assays. However, shorter (>12 amino acids) variants also displayed significant antimicrobial effects. A central K14 residue was important for optimal antimicrobial activity. Finally, one peptide variant, GKYGFYTHVFRLKKWIQKVI (GKY20) exhibiting improved selectivity, i.e., low toxicity and a preserved antimicrobial as well as anti-inflammatory effect, showed efficiency in mouse models of LPS shock and P. aeruginosa sepsis. The work defines structure-activity relationships of C-terminal host defense peptides of thrombin and delineates a strategy for selecting peptide epitopes of therapeutic interest.

Diagnosis of hemoparasitosis trough the spread-smear technique using a drop of blood clot

Directory of Open Access Journals (Sweden)

Neusa Saltiél Stobbe

1992-12-01

Full Text Available The spread-smear technique using a drop of blood clot and stained by diluted and undimitcd Giemsa stain was evaluated for the search of blood cell types and recognition of Babesia bovis, Babesia bigemina and Anaplasma marginale hemoparasites. The blood clot samples were taken from a 6 month-old calf experimentally inoculated with the blood from a Cattle Tick Fever carrier. The Technique was sumitable for the observation of different blood cells and for the recognition of the hemoparasites.

Critical role of non-muscle myosin light chain kinase in thrombin-induced endothelial cell inflammation and lung PMN infiltration.

Science.gov (United States)

Fazal, Fabeha; Bijli, Kaiser M; Murrill, Matthew; Leonard, Antony; Minhajuddin, Mohammad; Anwar, Khandaker N; Finkelstein, Jacob N; Watterson, D Martin; Rahman, Arshad

2013-01-01

The pathogenesis of acute lung injury (ALI) involves bidirectional cooperation and close interaction between inflammatory and coagulation pathways. A key molecule linking coagulation and inflammation is the procoagulant thrombin, a serine protease whose concentration is elevated in plasma and lavage fluids of patients with ALI and acute respiratory distress syndrome (ARDS). However, little is known about the mechanism by which thrombin contributes to lung inflammatory response. In this study, we developed a new mouse model that permits investigation of lung inflammation associated with intravascular coagulation. Using this mouse model and in vitro approaches, we addressed the role of non-muscle myosin light chain kinase (nmMLCK) in thrombin-induced endothelial cell (EC) inflammation and lung neutrophil (PMN) infiltration. Our in vitro experiments revealed a key role of nmMLCK in ICAM-1 expression by its ability to control nuclear translocation and transcriptional capacity of RelA/p65 in EC. When subjected to intraperitoneal thrombin challenge, wild type mice showed a marked increase in lung PMN infiltration via expression of ICAM-1. However, these responses were markedly attenuated in mice deficient in nmMLCK. These results provide mechanistic insight into lung inflammatory response associated with intravascular coagulation and identify nmMLCK as a critical target for modulation of lung inflammation.

Histones Differentially Modulate the Anticoagulant and Profibrinolytic Activities of Heparin, Heparin Derivatives, and Dabigatran.

Science.gov (United States)

Ammollo, Concetta Tiziana; Semeraro, Nicola; Carratù, Maria Rosaria; Colucci, Mario; Semeraro, Fabrizio

2016-02-01

The antithrombin activity of unfractionated heparin (UFH) is offset by extracellular histones, which, along with DNA, represent a novel mediator of thrombosis and a structural component of thrombi. Here, we systematically evaluated the effect of histones, DNA, and histone-DNA complexes on the anticoagulant and profibrinolytic activities of UFH, its derivatives enoxaparin and fondaparinux, and the direct thrombin inhibitor dabigatran. Thrombin generation was assessed by calibrated automated thrombinography, inhibition of factor Xa and thrombin by synthetic substrates, tissue plasminogen activator-mediated clot lysis by turbidimetry, and thrombin-activatable fibrinolysis inhibitor (TAFI) activation by a functional assay. Histones alone delayed coagulation and slightly stimulated fibrinolysis. The anticoagulant activity of UFH and enoxaparin was markedly inhibited by histones, whereas that of fondaparinux was enhanced. Histones neutralized both the anti-Xa and anti-IIa activities of UFH and preferentially blocked the anti-IIa activity of enoxaparin. The anti-Xa activity of fondaparinux was not influenced by histones when analyzed by chromogenic substrates, but was potentiated in a plasma prothrombinase assay. Histones inhibited the profibrinolytic activity of UFH and enoxaparin and enhanced that of fondaparinux by acting on the modulation of TAFI activation by anticoagulants. Histone H1 was mainly responsible for these effects. Histone-DNA complexes, as well as intact neutrophil extracellular traps, impaired the activities of UFH, enoxaparin, and fondaparinux. Dabigatran was not noticeably affected by histones and/or DNA, whatever the assay performed. In conclusion, histones and DNA present in the forming clot may variably influence the antithrombotic activities of anticoagulants, suggesting a potential therapeutic advantage of dabigatran and fondaparinux over heparins. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

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

Science.gov (United States)

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

2002-01-07

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

Tumour imaging by the detection of fibrin clots in tumour stroma using an anti-fibrin Fab fragment

Science.gov (United States)

Obonai, Toshifumi; Fuchigami, Hirobumi; Furuya, Fumiaki; Kozuka, Naoyuki; Yasunaga, Masahiro; Matsumura, Yasuhiro

2016-01-01

The diagnosis of early and aggressive types of cancer is important for providing effective cancer therapy. Cancer-induced fibrin clots exist only within lesions. Previously, we developed a monoclonal antibody (clone 102-10) that recognizes insoluble fibrin but not fibrinogen or soluble fibrin and confirmed that fibrin clots form continuously in various cancers. Here, we describe the development of a Fab fragment probe of clone 102-10 for tumour imaging. The distribution of 102-10 Fab was investigated in genetically engineered mice bearing pancreatic ductal adenocarcinoma (PDAC), and its effect on blood coagulation was examined. Immunohistochemical and ex vivo imaging revealed that 102-10 Fab was distributed selectively in fibrin clots in PDAC tumours 3 h after injection and that it disappeared from the body after 24 h. 102-10 Fab had no influence on blood coagulation or fibrinolysis. Tumour imaging using anti-fibrin Fab may provide a safe and effective method for the diagnosis of invasive cancers by detecting fibrin clots in tumour stroma. PMID:27009516

Laboratory heterogeneity of the lupus anticoagulant: a multicentre study using different clotting assays on a panel of 78 samples. Hemostasis Committee of the "Société Française de Biologie Clinique".

Science.gov (United States)

1992-05-15

The laboratory heterogeneity of the lupus anticoagulant (LA) was investigated in a multicentre study using a panel of 78 plasma samples diagnosed as containing a LA. Consecutive samples were collected by 12 participants using various screening tests, and sent to 7 laboratories which performed one or more clotting assays among the following: activated partial thromboplastin time (APTT), dilute Russell viper venom time, kaolin clotting time (KCT), dilute tissue thromboplastin time (dTTI) and a platelet neutralization test. For APTT and dTTI, 10 versions of these tests including standard and mixing procedures were carried out. They varied by reagents, phospholipid concentration or methodology. Cut-off times were determined for each test by comparing the results of the panel to those of a control population. When the data of all clotting assays were pooled, 70 of the 78 selected plasmas were considered to contain LA, 15 of them having a low-titer inhibitor. Sensitivity, defined as the proportion of positive results among LA-containing plasmas, varied from 62 to 100% and was positively related to responsiveness (defined as the mean ratio of clotting time to cut-off time). Laboratory heterogeneity of LA-containing plasma was illustrated by a star symbol plot analysis. Different populations of samples, with LA preferentially recognized by one assay (or group of assays) irrespective of the overall sensitivity of this assay, were identified. Multiple component analysis demonstrated the heterogeneity of low-titer inhibitors, which complicates their recognition in routine laboratory investigation.

Prothrombin time (PT)

Science.gov (United States)

PT; Pro-time; Anticoagulant-prothrombin time; Clotting time: protime; INR; International normalized ratio ... PT is measured in seconds. Most of the time, results are given as what is called INR ( ...

Growth of hydroxyapatite on physiologically clotted fibrin capped gold nanoparticles

International Nuclear Information System (INIS)

Sastry, T P; Sundaraseelan, J; Swarnalatha, K; Sobhana, S S Liji; Makheswari, M Uma; Sekar, S; Mandal, A B

2008-01-01

The growth of hydroxyapatite (HAp) on physiologically clotted fibrin (PCF)-gold nanoparticles is presented for the first time by employing a wet precipitation method. Fourier transform infrared (FTIR) spectroscopy confirmed the characteristic functionalities of PCF and HAp in the PCF-Au-HAp nanocomposite. Scanning electron microscopy (SEM) images have shown cuboidal nanostructures having a size in the range of 70-300 nm of HAp, whereas 2-50 nm sized particles were visualized in high-resolution transmission electron microscopy (TEM). Energy-dispersive x-ray (EDX) and x-ray diffraction (XRD) studies have confirmed the presence of HAp. These results show that gold nanoparticles with PCF acted as a matrix for the growth of HAp, and that PCF-Au-HAp nanocomposite is expected to have better osteoinductive properties

Shc adaptor proteins are key transducers of mitogenic signaling mediated by the G protein-coupled thrombin receptor

DEFF Research Database (Denmark)

Chen, Y; Grall, D; Salcini, A E

1996-01-01

The serine protease thrombin activates G protein signaling systems that lead to Ras activation and, in certain cells, proliferation. Whereas the steps leading to Ras activation by G protein-coupled receptors are not well defined, the mechanisms of Ras activation by receptor tyrosine kinases have......-insensitive proteins appear to mediate this effect, since (i) pertussis toxin pre-treatment of cells does not blunt the action of thrombin and (ii) Shc phosphorylation on tyrosine can be stimulated by the muscarinic m1 receptor. Shc phosphorylation does not appear to involve protein kinase C, since the addition of 4...

RESEARCH ON THE QUALITY INDICATORS OF CURD PRODUCTS BASED ON PROTEIN-HERBAL CLOTS

Directory of Open Access Journals (Sweden)

Olena GREK

2017-12-01

Full Text Available The article presents the research of qualitative indicators of curd products with different nutritional ingredients based on protein-herbal clots. The effect of the number of Rumex juice and the duration of thermoacid processing on the process of precipitation of milk proteins was determined. It was established that the introduction of vegetative coagulant in the amount (9 ± 0.5% at a temperature (93 ... 95 °C and endurance (3 ... 5 min - provides the optimal yield of protein-herbal clot taking into account restrictions according to organoleptic parameters. The effect of white sugar and apple pectin in fiber on the organoleptic, physico-chemical and rheological indicators curd products was investigated. The dietary fibers increase moisture-proof ability and effective viscosity of samples, and white sugar reduces these indexes due to dehydrating properties. The optimal option is to add to the protein-herbal bunch at the same time two components when mixing - white sugar and apple pectin in fiber in quantities of 15% and 2% respectively. Taking into account the influence of individual non-dairy ingredients - Rumex juice of white sugar and apple pectin in fiber on curd products, the performance of the finished product can purposefully be affected.

Binding of thrombin-activated platelets to a fibrin scaffold through α(IIb)β₃ evokes phosphatidylserine exposure on their cell surface.

Science.gov (United States)

Brzoska, Tomasz; Suzuki, Yuko; Mogami, Hideo; Sano, Hideto; Urano, Tetsumei

2013-01-01

Recently, by employing intra-vital confocal microscopy, we demonstrated that platelets expose phosphatidylserine (PS) and fibrin accumulate only in the center of the thrombus but not in its periphery. To address the question how exposure of platelet anionic phospholipids is regulated within the thrombus, an in-vitro experiment using diluted platelet-rich plasma was employed, in which the fibrin network was formed in the presence of platelets, and PS exposure on the platelet surface was analyzed using Confocal Laser Scanning Microscopy. Almost all platelets exposed PS after treatment with tissue factor, thrombin or ionomycin. Argatroban abrogated fibrin network formation in all samples, however, platelet PS exposure was inhibited only in tissue factor- and thrombin-treated samples but not in ionomycin-treated samples. FK633, an α(IIb)β₃ antagonist, and cytochalasin B impaired platelet binding to the fibrin scaffold and significantly reduced PS exposure evoked by thrombin. Gly-Pro-Arg-Pro amide abrogated not only fibrin network formation, but also PS exposure on platelets without suppressing platelet binding to fibrin/fibrinogen. These results suggest that outside-in signals in platelets generated by their binding to the rigid fibrin network are essential for PS exposure after thrombin treatment.

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

NARCIS (Netherlands)

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

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

Enhanced electrochemiluminescence quenching of CdS:Mn nanocrystals by CdTe QDs-doped silica nanoparticles for ultrasensitive detection of thrombin

Science.gov (United States)

Shan, Yun; Xu, Jing-Juan; Chen, Hong-Yuan

2011-07-01

This work reports an aptasensor for ultrasensitive detection of thrombin based on remarkably efficient energy-transfer induced electrochemiluminescence (ECL) quenching from CdS:Mn nanocrystals (NCs) film to CdTe QDs-doped silica nanoparticles (CdTe/SiO2 NPs). CdTe/SiO2 NPs were synthesized via the Stöber method and showed black bodies' strong absorption in a wide spectral range without excitonic emission, which made them excellent ECL quenchers. Within the effective distance of energy scavenging, the ECL quenching efficiency was dependent on the number of CdTe QDs doped into the silica NPs. Using ca. 200 CdTe QDs doped silica NPs on average of 40 nm in diameter as ECL quenching labels, attomolar detection of thrombin was successfully realized. The protein detection involves a competition binding event, based on thrombin replacing CdTe/SiO2 NPs labeled probing DNA which is hybridized with capturing aptamer immobilized on a CdS:Mn NCs film modified glassy carbon electrode surface by specific aptamer-protein affinity interactions. It results in the displacement of ECL quenching labels from CdS:Mn NCs film and concomitant ECL signal recovery. Owing to the high-content CdTe QDs in silica NP, the increment of ECL intensity (ΔIECL) and the concentration of thrombin showed a double logarithmic linear correlation in the range of 5.0 aM~5.0 fM with a detection limit of 1aM. And, the aptasensor hardly responded to antibody, bovine serum albumin (BSA), haemoglobin (Hb) and lysozyme, showing good detection selectivity for thrombin. This long-distance energy scavenging could have a promising application perspective in the detection of biological recognition events on a molecular level.This work reports an aptasensor for ultrasensitive detection of thrombin based on remarkably efficient energy-transfer induced electrochemiluminescence (ECL) quenching from CdS:Mn nanocrystals (NCs) film to CdTe QDs-doped silica nanoparticles (CdTe/SiO2 NPs). CdTe/SiO2 NPs were synthesized via

Thrombin Time

Science.gov (United States)

... information]. Available online at http://www2.massgeneral.org/pathology/coagbook/CO004600.htm through http://www2.massgeneral.org . Accessed July 2010. Dugdale, D. (Updated 2009 March 2). Congenital afibrinogenemia. MedlinePlus Medical Encyclopedia [On-line ...

Reference values for rotational thromboelastometry (ROTEM) in clinically healthy cats.

Science.gov (United States)

Marly-Voquer, Charlotte; Riond, Barbara; Jud Schefer, Rahel; Kutter, Annette P N

2017-03-01

To establish reference intervals for rotational thromboelastometry (ROTEM) using feline blood. Prospective study. University teaching hospital. Twenty-three clinically healthy cats between 1 and 15 years. For each cat, whole blood was collected via jugular or medial saphenous venipuncture, and blood was placed into a serum tube, a tube containing potassium-EDTA, and tubes containing 3.2% sodium citrate. The tubes were maintained at 37°C for a maximum of 30 minutes before coagulation testing. ROTEM tests included the EXTEM, INTEM, FIBTEM, and APTEM assays. In addition, prothrombin time, activated partial thromboplastin time, thrombin time, and fibrinogen concentration (Clauss method) were analyzed for each cat. Reference intervals for ROTEM were calculated using the 2.5-97.5 th percentile for each parameter, and correlation with the standard coagulation profile was performed. Compared to people, clinically healthy cats had similar values for the EXTEM and INTEM assays, but had lower plasma fibrinogen concentrations (0.9-2.2 g/L), resulting in weaker maximum clot firmness (MCF, 3-10 mm) in the FIBTEM test. In 18 cats, maximum lysis (ML) values in the APTEM test were higher than in the EXTEM test, which seems unlikely to have occurred in the presence of aprotinin. It is possible that the observed high maximum lysis values were due to clot retraction rather than true clot lysis. Further studies will be required to test this hypothesis. Cats have a weaker clot in the FIBTEM test, but have a similar clot strength to human blood in the other ROTEM assays, which may be due to a stronger contribution of platelets compared to that found in people. In cats, careful interpretation of the results to diagnose hyperfibrinolysis is advised, especially with the APTEM test, until further data are available. © Veterinary Emergency and Critical Care Society 2017.

Study of the fibrinogen - fibrin transformation kinetics and modifications caused to this reaction by irradiation (X rays) of the fibrinogen solution

International Nuclear Information System (INIS)

Hollard, D.; Suscillon, M.; Marcille, G.; Rambaud, F.; Baloyan, M.

1966-01-01

The authors present a spectrophotometric method for studying the transformation fibrinogen - fibrin. This method has the advantage of drawing immediately in graph form the three, phases of this transformation: proteolysis or monomerization; polymerisation; clot stabilization. It is a simple, faithful and easily reproductive technic. Owing to this method, they studied modifications of this transformation due to irradiation of fibrinogen solution. Low doses (90 000 R/mn) prevent transverse polymerisation. To upper doses (180 000 R and more), the action of thrombin on fibrinogen does not give an organised clot but a lacteous and fragile gel. There is not here a coagulation in the physiological understanding. (author) [fr

Purification and characterization of a milk-clotting protease from ...

African Journals Online (AJOL)

Crude enzymatic extract obtained from five fermentations (300 g of wheat bran) was characterized by a clotting activity of 0.34 ± 0.08 UP/ml with a strength ratio of 1/1: 200. The comparative study of the summaries from 2 purification protocols showed that it is possible to recover 6% of the initial proteins with a 44.54% activity ...

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

NARCIS (Netherlands)

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

2015-01-01

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

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

International Nuclear Information System (INIS)

Cheng, De-Chun; Zhong, Guo-Cai; Su, Ju-Xiang; Liu, Yan-Hong; Li, Yan; Wang, Jia-Ye; Hattori, Toshio; Ling, Hong; Zhang, Feng-Min

2010-01-01

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.

Elevated Cytokines, Thrombin and PAI-1 in Severe HCPS Patients Due to Sin Nombre Virus

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Virginie Bondu

2015-02-01

Full Text Available Sin Nombre Hantavirus (SNV, Bunyaviridae Hantavirus is a Category A pathogen that causes Hantavirus Cardiopulmonary Syndrome (HCPS with case fatality ratios generally ranging from 30% to 50%. HCPS is characterized by vascular leakage due to dysregulation of the endothelial barrier function. The loss of vascular integrity results in non-cardiogenic pulmonary edema, shock, multi-organ failure and death. Using Electric Cell-substrate Impedance Sensing (ECIS measurements, we found that plasma samples drawn from University of New Mexico Hospital patients with serologically-confirmed HCPS, induce loss of cell-cell adhesion in confluent epithelial and endothelial cell monolayers grown in ECIS cultureware. We show that the loss of cell-cell adhesion is sensitive to both thrombin and plasmin inhibitors in mild cases, and to thrombin only inhibition in severe cases, suggesting an increasing prothrombotic state with disease severity. A proteomic profile (2D gel electrophoresis and mass spectrometry of HCPS plasma samples in our cohort revealed robust antifibrinolytic activity among terminal case patients. The prothrombotic activity is highlighted by acute ≥30 to >100 fold increases in active plasminogen activator inhibitor (PAI-1 which, preceded death of the subjects within 48 h. Taken together, this suggests that PAI-1 might be a response to the severe pathology as it is expected to reduce plasmin activity and possibly thrombin activity in the terminal patients.

Roles of platelet STIM1 and Orai1 in glycoprotein VI- and thrombin-dependent procoagulant activity and thrombus formation.

Science.gov (United States)

Gilio, Karen; van Kruchten, Roger; Braun, Attila; Berna-Erro, Alejandro; Feijge, Marion A H; Stegner, David; van der Meijden, Paola E J; Kuijpers, Marijke J E; Varga-Szabo, David; Heemskerk, Johan W M; Nieswandt, Bernhard

2010-07-30

In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca(2+) entry (SOCE) with Orai1 as principal Ca(2+) entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca(2+) entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1(-/-) and Orai1(-/-) platelets had greatly impaired glycoprotein (GP) VI-dependent Ca(2+) signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2(-/-) platelets reacted normally. Upon blood flow in the presence of thrombin generation and coagulation, Ca(2+) signals of Stim1(-/-) and Orai1(-/-) platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1(-/-) and Orai1(-/-) platelets were deficient in GPVI-induced PS exposure and prothrombinase activity, but not when thrombin was present as co-agonist. Markedly, SKF96365, a blocker of (receptor-operated) Ca(2+) entry, inhibited Ca(2+) and procoagulant responses even in Stim1(-/-) and Orai1(-/-) platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca(2+) entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca(2+) entry and PS exposure, only one relying on STIM1-Orai1 interaction.

Thrombin contributes to bronchoalveolar lavage fluid mitogenicity in lung disease of the premature infant

NARCIS (Netherlands)

Dik, Willem A.; Zimmermann, Luc J. I.; Naber, Brigitta A.; Janssen, Daphne J.; van Kaam, Anton H. L. C.; Versnel, Marjan A.

2003-01-01

Chronic lung disease of prematurity (CLD) is a common consequence of neonatal respiratory distress syndrome (RDS) and is characterized by pulmonary fibrosis. Increased thrombin activity in the alveolar compartment is associated with pulmonary fibrosis in adults and animals, and contributes to

Enhanced electrochemiluminescence quenching of CdS:Mn nanocrystals by CdTe QDs-doped silica nanoparticles for ultrasensitive detection of thrombin.

Science.gov (United States)

Shan, Yun; Xu, Jing-Juan; Chen, Hong-Yuan

2011-07-01

This work reports an aptasensor for ultrasensitive detection of thrombin based on remarkably efficient energy-transfer induced electrochemiluminescence (ECL) quenching from CdS:Mn nanocrystals (NCs) film to CdTe QDs-doped silica nanoparticles (CdTe/SiO(2) NPs). CdTe/SiO(2) NPs were synthesized via the Stöber method and showed black bodies' strong absorption in a wide spectral range without excitonic emission, which made them excellent ECL quenchers. Within the effective distance of energy scavenging, the ECL quenching efficiency was dependent on the number of CdTe QDs doped into the silica NPs. Using ca. 200 CdTe QDs doped silica NPs on average of 40 nm in diameter as ECL quenching labels, attomolar detection of thrombin was successfully realized. The protein detection involves a competition binding event, based on thrombin replacing CdTe/SiO(2) NPs labeled probing DNA which is hybridized with capturing aptamer immobilized on a CdS:Mn NCs film modified glassy carbon electrode surface by specific aptamer-protein affinity interactions. It results in the displacement of ECL quenching labels from CdS:Mn NCs film and concomitant ECL signal recovery. Owing to the high-content CdTe QDs in silica NP, the increment of ECL intensity (ΔI(ECL)) and the concentration of thrombin showed a double logarithmic linear correlation in the range of 5.0 aM∼5.0 fM with a detection limit of 1aM. And, the aptasensor hardly responded to antibody, bovine serum albumin (BSA), haemoglobin (Hb) and lysozyme, showing good detection selectivity for thrombin. This long-distance energy scavenging could have a promising application perspective in the detection of biological recognition events on a molecular level.

Rapid Upregulation of Orai1 Abundance in the Plasma Membrane of Platelets Following Activation with Thrombin and Collagen Related Peptide

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Guilai Liu

2015-11-01

Full Text Available Background: Blood platelets accomplish primary hemostasis following vascular injury and contribute to the orchestration of occlusive vascular disease. Platelets are activated by an increase of cytosolic Ca2+-activity ([Ca2+]i, which is accomplished by Ca2+-release from intracellular stores and subsequent store operated Ca2+ entry (SOCE through Ca2+ release activated Ca2+ channel moiety Orai1. Powerful activators of platelets include thrombin and collagen related peptide (CRP, which are in part effective by activation of small G- protein Rac1. The present study explored the influence of thrombin and CRP on Orai1 protein abundance and cytosolic Ca2+-activity ([Ca2+]i in platelets drawn from wild type mice. Methods: Orai1 protein surface abundance was quantified utilizing CF™488A conjugated antibodies, and [Ca2+]i was determined with Fluo3-fluorescence. Results: In resting platelets, Orai1 protein abundance and [Ca2+]i were low. Thrombin (0.02 U/ml and CRP (5ug/ml within 2 min increased [Ca2+]i and Orai1 protein abundance at the platelet surface. [Ca2+]i was further increased by Ca2+ ionophore ionomycin (1 µM and by store depletion with the sarcoendoplasmatic Ca2+ ATPase inhibitor thapsigargin (1 µM. However, Orai1 protein abundance at the platelet surface was not significantly affected by ionomycin and only slightly increased by thapsigargin. The effect of thrombin and CRP on Orai1 abundance and [Ca2+]i was significantly blunted by Rac1 inhibitor NSC23766 (50 µM. Conclusion: The increase of [Ca2+]i following stimulation of platelets with thrombin and collagen related peptide is potentiated by ultrarapid Rac1 sensitive translocation of Orai1 into the cell membrane.

The state of hemostasis in pregnant women with hydramnion

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I. A. Lyoshenko

2016-05-01

Abstract Purpose. To determine the state of various stages of hemostasis in pregnant women with hydramnion as a risk factor for premature detachment of the placenta. Results. There was investigated the state of hemostasis in pregnant women with hydramnion by low piezoelectric thromboelastography (LPTEG and standard coagulogram. According to the LPTEG results this group of women was found to have increased plasma coagulation potential manifested in elevated constant thrombin activity (TAC by 76.8%, the intensity of coagulation drive (ICD by 74.9%, maximum clot density (MA by 16.8%, shortening of coagulation time (T3 by 7.8% and inhibition of fibrinolysis manifested by decrease in retraction intensity and clot lysis (RICL by 67.7% (p<0.05. Conclusions. The state of hemostasis in pregnant women with hydramnion is characterized by increased plasma coagulation and inhibition of fibrinolysis potential that are factors of preparing the woman’s body for the upcoming birth to control and minimize blood loss.   Keywords: pregnancy, hydramnion, hemostasis, thromboelastography.

Three-dimensional architecture and cell composition of a Choukroun's platelet-rich fibrin clot and membrane.

Science.gov (United States)

Dohan Ehrenfest, David M; Del Corso, Marco; Diss, Antoine; Mouhyi, Jaafar; Charrier, Jean-Baptiste

2010-04-01

Platelet-rich fibrin (PRF; Choukroun's technique) is a second-generation platelet concentrate for surgical use. This easy protocol allows the production of leukocyte and platelet-rich fibrin clots and membranes starting from 10-ml blood samples. The purposes of this study were to determine the cell composition and three-dimensional organization of this autologous biomaterial and to evaluate the influence of different collection tubes (dry glass or glass-coated plastic tubes) and compression procedures (forcible or soft) on the final PRF-membrane architecture. After centrifugation, blood analyses were performed on the residual waste plasmatic layers after collecting PRF clots. The PRF clots and membranes were processed for examination by light microscopy and scanning electron microscopy. Approximately 97% of the platelets and >50% of the leukocytes were concentrated in the PRF clot and showed a specific three-dimensional distribution, depending on the centrifugation forces. Platelets and fibrin formed large clusters of coagulation in the first millimeters of the membrane beyond the red blood cell base. The fibrin network was very mature and dense. Moreover, there was no significant difference in the PRF architecture between groups using the different tested collection tubes and compression techniques, even if these two parameters could have influenced the growth factor content and biologic matrix properties. The PRF protocol concentrated most platelets and leukocytes from a blood harvest into a single autologous fibrin biomaterial. This protocol offers reproducible results as long as the main production principles are respected.

Binding of Thrombin-Activated Platelets to a Fibrin Scaffold through αIIbβ3 Evokes Phosphatidylserine Exposure on Their Cell Surface

Science.gov (United States)

Brzoska, Tomasz; Suzuki, Yuko; Mogami, Hideo; Sano, Hideto; Urano, Tetsumei

2013-01-01

Recently, by employing intra-vital confocal microscopy, we demonstrated that platelets expose phosphatidylserine (PS) and fibrin accumulate only in the center of the thrombus but not in its periphery. To address the question how exposure of platelet anionic phospholipids is regulated within the thrombus, an in-vitro experiment using diluted platelet-rich plasma was employed, in which the fibrin network was formed in the presence of platelets, and PS exposure on the platelet surface was analyzed using Confocal Laser Scanning Microscopy. Almost all platelets exposed PS after treatment with tissue factor, thrombin or ionomycin. Argatroban abrogated fibrin network formation in all samples, however, platelet PS exposure was inhibited only in tissue factor- and thrombin-treated samples but not in ionomycin-treated samples. FK633, an αIIbβ3 antagonist, and cytochalasin B impaired platelet binding to the fibrin scaffold and significantly reduced PS exposure evoked by thrombin. Gly-Pro-Arg-Pro amide abrogated not only fibrin network formation, but also PS exposure on platelets without suppressing platelet binding to fibrin/fibrinogen. These results suggest that outside-in signals in platelets generated by their binding to the rigid fibrin network are essential for PS exposure after thrombin treatment. PMID:23383331

Binding of thrombin-activated platelets to a fibrin scaffold through α(IIbβ₃ evokes phosphatidylserine exposure on their cell surface.

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Tomasz Brzoska

Full Text Available Recently, by employing intra-vital confocal microscopy, we demonstrated that platelets expose phosphatidylserine (PS and fibrin accumulate only in the center of the thrombus but not in its periphery. To address the question how exposure of platelet anionic phospholipids is regulated within the thrombus, an in-vitro experiment using diluted platelet-rich plasma was employed, in which the fibrin network was formed in the presence of platelets, and PS exposure on the platelet surface was analyzed using Confocal Laser Scanning Microscopy. Almost all platelets exposed PS after treatment with tissue factor, thrombin or ionomycin. Argatroban abrogated fibrin network formation in all samples, however, platelet PS exposure was inhibited only in tissue factor- and thrombin-treated samples but not in ionomycin-treated samples. FK633, an α(IIbβ₃ antagonist, and cytochalasin B impaired platelet binding to the fibrin scaffold and significantly reduced PS exposure evoked by thrombin. Gly-Pro-Arg-Pro amide abrogated not only fibrin network formation, but also PS exposure on platelets without suppressing platelet binding to fibrin/fibrinogen. These results suggest that outside-in signals in platelets generated by their binding to the rigid fibrin network are essential for PS exposure after thrombin treatment.

Fibrinolytic Activity and Dose-Dependent Effect of Incubating Human Blood Clots in Caffeic Acid Phenethyl Ester: In Vitro Assays

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Abuzar Elnager

2015-01-01

Full Text Available Background. Caffeic acid phenethyl ester (CAPE has been reported to possess time-dependent fibrinolytic activity by in vitro assay. This study is aimed at investigating fibrinolytic dose-dependent activity of CAPE using in vitro assays. Methods. Standardized human whole blood (WB clots were incubated in either blank controls or different concentrations of CAPE (3.75, 7.50, 15.00, 22.50, and 30.00 mM. After 3 hours, D-dimer (DD levels and WB clot weights were measured for each concentration. Thromboelastography (TEG parameters were recorded following CAPE incubation, and fibrin morphology was examined under a confocal microscope. Results. Overall, mean DD (μg/mL levels were significantly different across samples incubated with different CAPE concentrations, and the median pre- and postincubation WB clot weights (grams were significantly decreased for each CAPE concentration. Fibrin removal was observed microscopically and indicated dose-dependent effects. Based on the TEG test, the Ly30 fibrinolytic parameter was significantly different between samples incubated with two different CAPE concentrations (15.0 and 22.50 mM. The 50% effective dose (ED50 of CAPE (based on DD was 1.99 mg/mL. Conclusions. This study suggests that CAPE possesses fibrinolytic activity following in vitro incubation and that it has dose-dependent activities. Therefore, further investigation into CAPE as a potential alternative thrombolytic agent should be conducted.

Study on sup(99m)Tc-colloid accumulation of in vitro clots and thrombi in rats

Energy Technology Data Exchange (ETDEWEB)

Ohguchi, Manabu [Kanazawa Univ. (Japan). School of Medicine

1983-02-01

The accumulation of different sup(99m)Tc-colloids in vitro clot, venous thrombi and intravascular fibrin deposits in rats was studied. Blood clot was produced by incubating fresh blood at room temperature for three hours. The mean value of % uptake of sup(99m)Tc-Sn colloid in clot was 96.2 % and higher than that of any other sup(99m)Tc-colloids. Venous thrombi in rats were made by clamping the left femoral vein segments for 5 minutes. With sup(99m)Tc-Sn colloid, the mean value of the ratio of the radioactivity in the clamped vein segment to the control segment (L/R ratio) was 54.5. L/R ratios of others were 24.2 with sup(99m)Tc-phytate, 12.1 with sup(99m)Tc-sulfur colloid, 3.9 with sup(99m)Tc-rhenium colloid, 2.4 with sup(99m)Tc-millimicrosphere albumin. L/R ratios were higher for the fresh thrombi, significant uptake was also observed up to seven days. Intravascular fibrin deposits were induced by i.p. injection of endotoxin into rats. Three hours after injection of endotoxin, sup(99m)Tc-colloids were i.v. injected. Significant lung uptake was observed in sup(99m)Tc-colloids except in sup(99m)Tc-rhenium colloid. And the most remarkable change of lung uptake was observed in sup(99m)Tc-Sn colloid. Significant kidney uptake was observed in sup(99m)Tc-Sn colloid and sup(99m)Tc-sulfur colloid. And the most remarkable change of kidney uptake was also observed in sup(99m)Tc-Sn colloid. When heparin was injected i.v. at the same time as endotoxin injection, lung uptake of sup(99m)Tc-Sn colloid was significantly decreased. However, the heparin effect on kidney uptake of sup(99m)Tc-Sn colloid was more significant than on lung uptake. The results in this study indicated that sup(99m)Tc-Sn colloid showed the greatest affinity to in vitro clots, venous thrombi and intravascular fibrin deposits.

Enzymatic milk clotting activity in artichoke (Cynara scolymus) leaves and alpine thistle (Carduus defloratus) flowers. Immobilization of alpine thistle aspartic protease.

Science.gov (United States)

Esposito, Marilena; Di Pierro, Prospero; Dejonghe, Winnie; Mariniello, Loredana; Porta, Raffaele

2016-08-01

Two different milk clotting enzymes, belonging to the aspartic protease family, were extracted from both artichoke leaves and alpine thistle flowers, and the latter was covalently immobilized by using a polyacrylic support containing polar epoxy groups. Our findings showed that the alpine thistle aspartic protease was successfully immobilized at pH 7.0 on Immobeads IB-150P beads and that, under these experimental conditions, an immobilization yield of about 68% and a recovery of about 54% were obtained. Since the enzyme showed an optimal pH of 5.0, a value very similar to the one generally used for milk clotting during cheese making, and exhibited a satisfactory stability over time, the use of such immobilized vegetable rennet for the production of novel dairy products is suggested. Copyright © 2016. Published by Elsevier Ltd.

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

DEFF Research Database (Denmark)

Jensen, Troels B.; Henriksen, Jonas Rosager; Rasmussen, Bjarne E.

2011-01-01

Thrombin binding aptamer is a DNA 15-mer which forms a G-quadruplex structure and possess promising anticoagulant properties due to specific interactions with thrombin. Herein we present the influence of a single 2′-C-piperazino-UNA residue and UNA residues incorporated in several positions on th...

RESULTS OF QUALITY COMPREHENSIVE EVALUATION OF DENTAL CARE FOR CHILDREN WITH BLOOD CLOTTING (PATHOLOGY

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M. A. Gavrilenko

2014-04-01

Full Text Available Background. Treatment methods for diseases of teeth hard tissues and periodontium for children with blood clotting pathology are described in modern literature. But in practice we are faced with the problem of providing dental treatment for these children. Work of pediatric dentist is prevented by the risk of bleeding, fear of dental procedures, child’s psychoemotional tension and refusal of treatment because of bleeding. Taking into account the specificity of blood clotting pathology, surgical methods of dental treatment prevail for these children, which is evidenced by the early loss of deciduous and permanent teeth, occlusal surface disturbances, dentoalveolar anomalies, inflammatory diseases of periodontal tissues. Aim. To evaluate the level of dental care for children with the diseases of blood clotting. Materials and methods. 120 children between 2 and 18 years old with blood clotting disorders (hemophilia A, B, thrombocytopenia, thrombocytopathy were examined. Children were divided into groups: I group – 2-5 years old (40 children, II group – 6-10 years old (40 children, III group – 11-18 years old (40 children, according to the periods of tooth development, with an equal number of children in groups according to diagnoses. Clinical examination was carried out according to the standard scheme, including the analysis of complaints, anamnesis of treatment at the dentist, objective data with the use of statistical method of masticatory efficiency (according to N.I. Agapov, the level of dental care (according to P.A. Leus. Results. During clinical dental examination of children with blood clotting pathology it was found that destruction of crowns of maxillary front teeth, the first deciduous molars and the second deciduous molars, as well as their loss, prevails in the temporary occlusion. During the examination of 2-5 years old children with blood clotting pathology, loss of less than 25% of masticatory efficiency was revealed for 25

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

DEFF Research Database (Denmark)

Skov Madsen, P; Hokland, P; Hokland, M

1987-01-01

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...

Combination of FVIII and by-passing agent potentiates in vitro thrombin production in haemophilia A inhibitor plasma.

Science.gov (United States)

Klintman, Jenny; Astermark, Jan; Berntorp, Erik

2010-11-01

The by-passing agents, recombinant activated factor VII (rFVIIa) and activated prothrombin complex concentrate (APCC), are important tools in the treatment of patients with haemophilia A and high-responding inhibitory antibodies. It has been observed clinically that in some patients undergoing immune tolerance induction the bleeding frequency decreases, hypothetically caused by a transient haemostatic effect of infused FVIII not measurable ex vivo. We evaluated how by-passing agents and factor VIII (FVIII) affect thrombin generation (TG) in vitro using plasma from 11 patients with severe haemophilia A and high titre inhibitors. Samples were spiked with combinations of APCC, rFVIIa and five different FVIII products. Combination of APCC and FVIII showed a synergistic effect in eliciting TG (Pproducts. When rFVIIa and FVIII were combined the interaction between the preparations was found to be additive. APCC and rFVIIa were then combined without FVIII, resulting in an additive effect on thrombin production. Each product separately increased TG above baseline. In conclusion, the amount of thrombin formed in vitro by adding a by-passing agent, was higher in the presence of FVIII. Our findings support the use of FVIII in by-passing therapy to optimize the haemostatic effect. © 2010 Blackwell Publishing Ltd.

Roles of Platelet STIM1 and Orai1 in Glycoprotein VI- and Thrombin-dependent Procoagulant Activity and Thrombus Formation*

Science.gov (United States)

Gilio, Karen; van Kruchten, Roger; Braun, Attila; Berna-Erro, Alejandro; Feijge, Marion A. H.; Stegner, David; van der Meijden, Paola E. J.; Kuijpers, Marijke J. E.; Varga-Szabo, David; Heemskerk, Johan W. M.; Nieswandt, Bernhard

2010-01-01

In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca2+ entry (SOCE) with Orai1 as principal Ca2+ entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca2+ entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1−/− and Orai1−/− platelets had greatly impaired glycoprotein (GP) VI-dependent Ca2+ signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2−/− platelets reacted normally. Upon blood flow in the presence of thrombin generation and coagulation, Ca2+ signals of Stim1−/− and Orai1−/− platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1−/− and Orai1−/− platelets were deficient in GPVI-induced PS exposure and prothrombinase activity, but not when thrombin was present as co-agonist. Markedly, SKF96365, a blocker of (receptor-operated) Ca2+ entry, inhibited Ca2+ and procoagulant responses even in Stim1−/− and Orai1−/− platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca2+ entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca2+ entry and PS exposure, only one relying on STIM1-Orai1 interaction. PMID:20519511

Thrombin-activatable fibrinolysis inhibitor is degraded by Salmonella enterica and Yersinia pestis.

Science.gov (United States)

Valls Serón, M; Haiko, J; DE Groot, P G; Korhonen, T K; Meijers, J C M

2010-10-01

 Pathogenic bacteria modulate the host coagulation system to evade immune responses or to facilitate dissemination through extravascular tissues. In particular, the important bacterial pathogens Salmonella enterica and Yersinia pestis intervene with the plasminogen/fibrinolytic system. Thrombin-activatable fibrinolysis inhibitor (TAFI) has anti-fibrinolytic properties as the active enzyme (TAFIa) removes C-terminal lysine residues from fibrin, thereby attenuating accelerated plasmin formation.  Here, we demonstrate inactivation and cleavage of TAFI by homologous surface proteases, the omptins Pla of Y. pestis and PgtE of S. enterica. We show that omptin-expressing bacteria decrease TAFI activatability by thrombin-thrombomodulin and that the anti-fibrinolytic potential of TAFIa was reduced by recombinant Escherichia coli expressing Pla or PgtE. The functional impairment resulted from C-terminal cleavage of TAFI by the omptins.  Our results indicate that TAFI is degraded directly by the omptins PgtE of S. enterica and Pla of Y. pestis. This may contribute to the ability of PgtE and Pla to damage tissue barriers, such as fibrin, and thereby to enhance spread of S. enterica and Y. pestis during infection. © 2010 International Society on Thrombosis and Haemostasis.

Venom Concentrations and Clotting Factor Levels in a Prospective Cohort of Russell's Viper Bites with Coagulopathy.

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Geoffrey K Isbister

Full Text Available Russell's viper envenoming is a major problem in South Asia and causes venom induced consumption coagulopathy. This study aimed to investigate the kinetics and dynamics of venom and clotting function in Russell's viper envenoming.In a prospective cohort of 146 patients with Russell's viper envenoming, we measured venom concentrations, international normalised ratio [INR], prothrombin time (PT, activated partial thromboplastin time (aPTT, coagulation factors I, II, V, VII, VIII, IX and X, and von Willebrand factor antigen. The median age was 39 y (16-82 y and 111 were male. The median peak INR was 6.8 (interquartile range [IQR]: 3.7 to >13, associated with low fibrinogen [median,3 at 6 h post-antivenom but had reduced to <2, by 24 h. The aPTT had also returned to close to normal (<50 sec at 24 h. Factor VII, VIII and IX levels were unusually high pre-antivenom, median peak concentrations of 393%, 307% and 468% respectively. Pre-antivenom venom concentrations and the INR (r = 0.20, p = 0.02 and aPTT (r = 0.19, p = 0.03 were correlated (non-parametric Spearman analysis.Russell's viper coagulopathy results in prolonged aPTT, INR, low fibrinogen, factors V, VIII and X which recover over 48 h. Severity of clotting abnormalities was associated with venom concentrations.

Correlation between Interleukin-6 and Thrombin-Antithrombin III Complex Levels in Retinal Diseases.

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Ehrlich, Rita; Zahavi, Alon; Axer-Siegel, Ruth; Budnik, Ivan; Dreznik, Ayelet; Dahbash, Mor; Nisgav, Yael; Megiddo, Elinor; Kenet, Gili; Weinberger, Dov; Livnat, Tami

2017-09-01

This study aims to evaluate and correlate the levels of interleukin-6 (IL-6) and thrombin-antithrombin III complex (TAT) in the vitreous of patients with different vitreoretinal pathologies. Vitreous samples were collected from 78 patients scheduled for pars plana vitrectomy at a tertiary medical center. Patients were divided by the underlying vitreoretinal pathophysiology, as follows: macular hole (MH)/epiretinal membrane (ERM) (n = 26); rhegmatogenous retinal detachment (RRD) (n = 32); and proliferative diabetic retinopathy (PDR) (n = 20). Levels of IL-6 and TAT were measured by enzyme-linked immunosorbent assay and compared among the groups. A significant difference was found in the vitreal IL-6 and TAT levels between the MH/ERM group and both the PDR and RRD groups (P Diabetes was associated with higher IL-6 levels in the RRD group. Different relationships between the IL-6 and TAT levels were revealed in patients with different ocular pathologies. Our results imply that variations in vitreal TAT level may be attributable not only to an inflammatory reaction or blood-retinal barrier breakdown, but also to intraocular tissue-dependent regulation of thrombin.

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

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

2018-02-01

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

99mTc-NC100668, a new tracer for imaging venous thromboemboli: pre-clinical biodistribution and incorporation into plasma clots in vivo and in vitro

International Nuclear Information System (INIS)

Edwards, David; Lewis, Joanne; Battle, Mark; Lear, Rochelle; Farrar, Gill; Barnett, D.J.; Godden, Vanessa; Oliveira, Alexandra; Coombes, Catherine; Ahlstroem, Haakan

2006-01-01

99m Tc-NC100668 is a new radiotracer being developed to aid the diagnosis of thromboembolism. The structure of NC100668 is similar to a region of human α 2 -antiplasmin, which is a substrate for factor XIIIa (FXIIIa). The purpose of this study was to confirm the uptake of 99m Tc-NC100668 into forming plasma clot and to establish the biodistribution of 99m Tc-NC100668 in Wistar rats. The in vitro plasma clot uptake of 99m Tc-NC100668 and other compounds with known affinities to FXIIIa was measured using a plasma clot assay. The biodistribution and blood clot uptake of radioactivity of 99m Tc-NC100668 in normal Wistar rats and those bearing experimentally induced deep vein thrombi were investigated. The in vitro uptake of 99m Tc-NC100668 was greater than that for [ 14 C]dansyl cadaverine, a known substrate of FXIIIa in the plasma clot assay. The biodistribution of 99m Tc-NC100668 in male and female Wistar rats up to 24 h p.i. showed that radioactivity was rapidly excreted, predominantly into the urine, with very little background tissue retention. In vivo the uptake and retention of 99m Tc-NC100668 into the blood clot was greater than could be accounted for by non-specific accumulation of the radiotracer within the blood clot. 99m Tc-NC100668 was retained by plasma clots in vitro and blood clots in vivo. No significant tissue retention which could interfere with the ability to image thrombi in vivo was observed. This evidence suggests that 99m Tc-NC100668 might be useful in the detection of thromboembolism. (orig.)

Measuring factor IX activity of nonacog beta pegol with commercially available one-stage clotting and chromogenic assay kits: a two-center study.

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Bowyer, A E; Hillarp, A; Ezban, M; Persson, P; Kitchen, S

2016-07-01

Essentials Validated assays are required to precisely measure factor IX (FIX) activity in FIX products. N9-GP and two other FIX products were assessed in various coagulation assay systems at two sites. Large variations in FIX activity measurements were observed for N9-GP using some assays. One-stage and chromogenic assays accurately measuring FIX activity for N9-GP were identified. Background Measurement of factor IX activity (FIX:C) with activated partial thromboplastin time-based one-stage clotting assays is associated with a large degree of interlaboratory variation in samples containing glycoPEGylated recombinant FIX (rFIX), i.e. nonacog beta pegol (N9-GP). Validation and qualification of specific assays and conditions are necessary for the accurate assessment of FIX:C in samples containing N9-GP. Objectives To assess the accuracy of various one-stage clotting and chromogenic assays for measuring FIX:C in samples containing N9-GP as compared with samples containing rFIX or plasma-derived FIX (pdFIX) across two laboratory sites. Methods FIX:C, in severe hemophilia B plasma spiked with a range of concentrations (from very low, i.e. 0.03 IU mL(-1) , to high, i.e. 0.90 IU mL(-1) ) of N9-GP, rFIX (BeneFIX), and pdFIX (Mononine), was determined at two laboratory sites with 10 commercially available one-stage clotting assays and two chromogenic FIX:C assays. Assays were performed with a plasma calibrator and different analyzers. Results A high degree of variation in FIX:C measurement was observed for one-stage clotting assays for N9-GP as compared with rFIX or pdFIX. Acceptable N9-GP recovery was observed in the low-concentration to high-concentration samples tested with one-stage clotting assays using SynthAFax or DG Synth, or with chromogenic FIX:C assays. Similar patterns of FIX:C measurement were observed at both laboratory sites, with minor differences probably being attributable to the use of different analyzers. Conclusions These results suggest that, of the

Cloning, expression and characterization of a gene from earthworm Eisenia fetida encoding a blood-clot dissolving protein.

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GangQiang Li

Full Text Available A lumbrokinase gene encoding a blood-clot dissolving protein was cloned from earthworm (Eisenia fetida by RT-PCR amplification. The gene designated as CST1 (GenBank No. AY840996 was sequence analyzed. The cDNA consists of 888 bp with an open reading frame of 729 bp, which encodes 242 amino acid residues. Multiple sequence alignments revealed that CST1 shares similarities and conserved amino acids with other reported lumbrokinases. The amino acid sequence of CST1 exhibits structural features similar to those found in other serine proteases, including human tissue-type (tPA, urokinase (uPA, and vampire bat (DSPAα1 plasminogen activators. CST1 has a conserved catalytic triad, found in the active sites of protease enzymes, which are important residues involved in polypeptide catalysis. CST1 was expressed as inclusion bodies in Escherichia coli BL21(DE3. The molecular mass of recombinant CST1 (rCST was 25 kDa as estimated by SDS-PAGE, and further confirmed by Western Blot analysis. His-tagged rCST1 was purified and renatured using nickel-chelating resin with a recovery rate of 50% and a purity of 95%. The purified, renatured rCST1 showed fibrinolytic activity evaluated by both a fibrin plate and a blood clot lysis assay. rCST1 degraded fibrin on the fibrin plate. A significant percentage (65.7% of blood clot lysis was observed when blood clot was treated with 80 mg/mL of rCST1 in vitro. The antithrombotic activity of rCST1 was 912 units/mg calculated by comparison with the activity of a lumbrokinase standard. These findings indicate that rCST1 has potential as a potent blood-clot treatment. Therefore, the expression and purification of a single lumbrokinase represents an important improvement in the use of lumbrokinases.

Staphylococcus chromogenes, a Coagulase-Negative Staphylococcus Species That Can Clot Plasma

OpenAIRE

dos Santos, Danielle Cabral; Lange, Carla Christine; Avellar-Costa, Pedro; dos Santos, Katia Regina Netto; Brito, Maria Aparecida Vasconcelos Paiva; Giambiagi-deMarval, Marcia

2016-01-01

Staphylococcus chromogenes is one of the main coagulase-negative staphylococci isolated from mastitis of dairy cows. We describe S. chromogenes isolates that can clot plasma. Since the main pathogen causing mastitis is coagulase-positive Staphylococcus aureus, the coagulase-positive phenotype of S. chromogenes described here can easily lead to misidentification.

Studies on cytotoxic and clot lysis activity of probiotically fermented cocktail juice prepared using Camellia sinensis and Punica grantum

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Biswas, Ananya; Deori, Meenakshi; Nivetha, A.; Mohansrinivasan, V.

2017-11-01

In the current research the effect of probiotic microorganisms viz; Lactococcus lactis and Lactobacillus plantarum on fermentation of Camellia sinensis and Punica grantum was studied. In vitro test were done to analyze the anticancer, antioxidant and atherosclerosis (clot lysis) properties of fermented juice. The juice was fermented for 48 and 96h, during which concentration of phenolic content, total acid content and free radical scavenging activity of the sample was analyzed by DPPH assay (α, α-diphenyl-β-picrylhydrazyl). Dropping of pH was observed after 48 h of fermentation. The clot lysis activity was found to be 80 % in 100μl concentration of fermented cocktail juice. The 96 h fermented sample has shown around 70% inhibition against colon cancer cell lines. Analytical study of HPLC proves the organic acid production such as ascorbic acid in superior amount for 96h of fermented sample, Based on the retention time, the corresponding peaks were detected at 4.919 and 4.831 min.

Thrombin activatable fibrinolysis inhibitor (TAFI in cord blood.

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Krzysztof Góralczyk

2007-03-01

Full Text Available Thrombin activatable fibrinolysis inhibitor (TAFI is a plasma zymogene (procarboxypeptidase B which can decrease fibrinolysis and thus act as a haemostatic factor. TAFI is now extensively studied in many complications as well as in physiological and complicated pregnancy. The question we posed in the present study was whether TAFI antigen is present in cord blood plasma. The study group consisted of 38 parturient women, 26 primiparous and 12 multiparous with normal course of pregnancy and delivery. The cord blood was sampled from the cord vein, and the mother's blood from the antecubital vein. 3.2% sodium citrate was used as an anticoagulant. TAFIa/ai antigen was measured by ELISA method. TAFIa/ai antigen was identified in all samples of cord blood plasma. Its level was 91.50 ng/ml (range: 71.76 - 160.77 ng/ml vs. 55.46 ng/ml (range: 39.77 - 68.54 ng/ml in the mother's blood, which means that the level of TAFIa/ai antigen was significantly higher in fetal blood than in maternal blood (p<0.00001. TAFIa/ai antigen is an integral component of cord blood plasma. The concentration of TAFIa/ai antigen is about two times higher in fetal blood than in maternal blood.

Staphylococcus chromogenes, a Coagulase-Negative Staphylococcus Species That Can Clot Plasma

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dos Santos, Danielle Cabral; Lange, Carla Christine; Avellar-Costa, Pedro; dos Santos, Katia Regina Netto; Brito, Maria Aparecida Vasconcelos Paiva

2016-01-01

Staphylococcus chromogenes is one of the main coagulase-negative staphylococci isolated from mastitis of dairy cows. We describe S. chromogenes isolates that can clot plasma. Since the main pathogen causing mastitis is coagulase-positive Staphylococcus aureus, the coagulase-positive phenotype of S. chromogenes described here can easily lead to misidentification. PMID:26912749

Use of a Fibrinogen/Thrombin-Based Collagen Fleece (TachoComb, TachoSil) With a Stapled Closure to Prevent Pancreatic Fistula Formation Following Distal Pancreatectomy.

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Mita, Kazuhito; Ito, Hideto; Murabayashi, Ryo; Asakawa, Hideki; Nabetani, Masashi; Kamasako, Akira; Koizumi, Kazuya; Hayashi, Takashi

2015-12-01

Postoperative pancreatic fistula formation remains a source of significant morbidity following distal pancreatectomy. The aim of this study was to evaluate the rate of clinically significant fistulas (International Study Group on Pancreatic Fistula grade B and grade C) after distal pancreatectomy using a fibrinogen/thrombin-based collagen fleece (TachoComb, TachoSil) with a stapled closure. Seventy-five patients underwent distal pancreatectomy at our institution between January 2005 and March 2014. A fibrinogen/thrombin-based collagen fleece was applied to the staple line of the pancreas before stapling. Twenty-six patients (34.7%) developed a pancreatic fistula, 8 patients (10.7%) developed a grade B fistula, and no patients developed a grade C fistula. The duration of the drain was significantly different in patients with or without a pancreatic fistula (8.0 ± 4.5 vs. 5.4 ± 1.3 days, P = .0003). Histological analysis showed that there was a tight covering with the fibrinogen/thrombin-based collagen fleece. The fibrinogen/thrombin-based collagen fleece (TachoComb, TachoSil) with a stapled closure has low rates of fistula formation and provides a safe alternative to the conventional stapled technique in distal pancreatectomy. © The Author(s) 2015.

The Discovery of Dabigatran Etexilate

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Joanne evan Ryn

2013-02-01

Full Text Available Thromboembolic disease is a major cause of mortality and morbidity in the developed world and is caused by an excessive stimulation of coagulation. Thrombin is a key serine protease in the coagulation cascade and numerous efforts have been made to develop safe and effective orally active direct thrombin inhibitors (DTIs. Current anticoagulant therapy includes the use of indirect thrombin inhibitors (e.g. heparins, low-molecular-weight-heparins [LMWHs] and vitamin K antagonists (VKA such as warfarin. However there are several caveats in the clinical use of these agents including narrow therapeutic window, parenteral delivery, and food- and drug-drug interactions. Dabigatran is a synthetic, reversible DTI with high affinity and specificity for its target binding both free and clot-bound thrombin, and offers a favorable pharmacokinetic profile. Large randomized clinical trials have demonstrated that dabigatran provides comparable or superior thromboprophylaxis in multiple thromboembolic disease indications compared to standard of care. This minireview will highlight the discovery and development of dabigatran, the first in a class of new oral anticoagulant (NOAC agents to be licensed worldwide for the prevention of thromboembolism in the setting of orthopedic surgery and stroke prevent in atrial fibrillation.

In vivo chicken model for peripheral intravascular human fibrin clot detection

International Nuclear Information System (INIS)

Hui, K.Y.

1988-01-01

A chicken model was prepared that provides a simple and economical method of evaluating the use of fibrin-specific monoclonal antibody 64C5 in the detection of peripheral vascular thrombi. Human fibrin was clotted in segments of a chicken's femoral artery and vein prior to intravenous injection of radioiodinated antibody 64C5. After a 3-hr perfusion time, the thrombosed and contralateral control segments of the vessels were excised and counted for radioactivity. The radiolabeled 64C5 uptake ratio of the thrombosed segment to the control segment was 5.4 +/- 1.2 (p less than 0.007) in the femoral artery, and 3.8 +/- 1.1 (p less than 0.02) in the femoral vein. This in vivo chicken model may also find application in studies of targeting agents for human fibrin

PAMAM Dendrimers as Potential Carriers of Gadolinium Complexes of Iminodiacetic Acid Derivatives for Magnetic Resonance Imaging

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Magdalena Markowicz-Piasecka

2015-01-01

Full Text Available This is the first study describing the utilization of PAMAM dendrimers as delivery vehicles of novel magnetic resonance imaging (MRI contrast agents. The purpose of this paper was to establish the potential of G4 PAMAM dendrimers as carriers of gadolinium complexes of iminodiacetic acid derivatives and determine imaging properties of synthesized compounds in in vivo studies. Furthermore, we examined the influence of four synthesized complexes on the process of clot formation, stabilization, and lysis and on amidolytic activity of thrombin. Biodistribution studies have shown that the compounds composed of PAMAM G4 dendrimers and gadolinium complexes of iminodiacetic acid derivatives increase signal intensity preferably in liver in range of 59–116% in MRI studies which corresponds with the greatest accumulation of gadolinium after administration of the compounds. Synthesized compounds affect kinetic parameters of the proces of clot formation, its stabilization, and lysis. However, only one synthesized compound at concentration 10-fold higher than potential plasma concentrations contributed to the increase of general parameters such as the overall potential of clot formation and lysis (↑CLAUC and total time of the process (↑T. Results of described studies provide additional insight into delivery properties of PAMAM dendrimers but simultaneously underscore the necessity for further research.

{sup 99m}Tc-NC100668, a new tracer for imaging venous thromboemboli: pre-clinical biodistribution and incorporation into plasma clots in vivo and in vitro