Reversal of Anticoagulation as Assessed by Thrombin Generation Measurement.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 876-876
Author(s):  
Alex Gatt ◽  
Joost van Veen ◽  
Peter Cooper ◽  
Steve Kitchen ◽  
Michael Makris
Keyword(s):  
Thrombin Generation ◽  
Fresh Frozen Plasma ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Protamine Sulphate ◽  
Reversal Agents ◽  
Time To Peak ◽  
High Doses ◽  
Vitro Effect

Abstract Antidotes to the ever-growing number of anticoagulants are always desirable in order to placate bleeding in emergencies. In general, the older anticoagulants like coumarins and unfractionated heparin (UFH) have proven reversal agents, whereas the newer ones do not. We studied the in vitro effect of 5 different heparinoids (UFH, Tinzaparin, Enoxaparin, Fondaparinux and Danaparoid) on the calibrated automated thrombogram. The assay uses a fluorogenic substrate that is cleaved by the thrombin formed after the addition of plasma to 5pM tissue factor, 4μM phospholipids and calcium chloride. We investigated all the parameters generated by dedicated software (Thrombinoscope™) ie the lag time (LT), the time to peak (ttpeak), the endogenous thrombin potential (ETP) and the peak thrombin. All the five anticoagulants tested inhibited thrombin generation (TG) in a concentration dependent manner. We subsequently analysed the in vitro effect of different concentrations of six potential reversal agents on correcting TG parameters of maximally inhibited plasma for each anticoagulant. These were protamine sulphate at 2.5, 5 & 8μg/ml, activated FVIIa (Novoseven®) at 5, 10 & 50μg/ml, FEIBA® at 0.5,1 & 2U/ml, Beriplex® at 0.3, 0.6 & 1.2U/ml, Prothromplex® TIM4 at 0.4, 0.8 & 1.8U/ml and fresh frozen plasma (FFP) at 250, 500 & 750μl/ml. The three concentrations reflect the recommended therapeutic doses for each agent together with lower and higher doses than normally used. As predicted, UFH (final concentration 0.527U/ml) was completely reversed with a standard protamine concentration of 5μg/ml. However, the highest dose of protamine gave slightly lower TG, indicating that higher concentrations of protamine sulphate can have a paradoxical ‘anticoagulant’ effect. High doses of FEIBA (2U/ml) and FVIIa (50μg/ml) restored ~50% of thrombin generation parameters. Tinzaparin (at 1antiXaUnit/ml) was also completely neutralised by protamine. However, a higher concentration of 8μg/ml protamine was required. This effect was not seen with Enoxaparin with this higher concentration of protamine reversing only ~40% of the ETP, 21% of the peak thrombin, 71% of ttpeak and 72% of the LT. There was no positive effect of protamine on Fondaparinux (3μg/ml) and Danaparoid (1antiXa U/ml)-treated plasma. Whereas Danaparoid seemed relatively resistant to all six reversal agents, Fondaparinux effect was completely neutralised by FVIIa at concentrations between 10–50μg/ml. This study highlights the differences in neutralisation of different low molecular weight heparins and UFH. In particular, Tinzaparin was much more readily reversed with protamine sulphate than Enoxaparin. It also indicates that high doses of FVIIa could completely reverse Fondaparinux anticoagulation.

Thrombin generation assays are superior to traditional tests in assessing anticoagulation reversal in vitro

2008 ◽  
Vol 100 (08) ◽  
pp. 350-355 ◽  
Author(s):  
Alex Gatt ◽  
Joost J. van Veen ◽  
Anita M. Woolley ◽  
Steve Kitchen ◽  
Peter Cooper ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
High Dose ◽  
Protamine Sulphate ◽  
Reversal Agents ◽  
New Anticoagulants ◽  
Spiked Plasma ◽  
Coagulation Tests ◽  
Complete Reversal ◽  
High Doses

SummaryEven though new anticoagulants are being devised with the notion that they do not require regular monitoring, when bleeding occurs, it is important to have an antidote and a reliable test to confirm whether the anticoagulant effects are persisting. We examined the effects of five heparinoids, unfractionated heparin (UFH), tinzaparin, enoxaparin, danaparoid and fondaparinux on the traditional APTT and anti-Xa assays as well as on the calibrated automated thrombogram (CAT). We also studied the ability of protamine sulphate (PS), NovoSeven®, FEIBA® and FFP to reverse maximum anticoagulation induced by the different heparinoids. The CAT was the only test to detect the coagulopathy of all the anticoagulants. PS produced complete reversal of UFH, and this could be monitored with all three tests. Tinzaparin can also be completely neutralised in vitro with high doses of PS, but the maximum enoxaparin reversal achieved with PS is only approximately 60%. Fondaparinux does not significantly affect the APTT and PS has no significant effect on its reversal. Only NovoSeven was able to correct the fondaparinux induced CAT abnormalities whilst having no effect on the anti-Xa level. None of the reversal agents was very effective in danaparoid spiked plasma but NovoSeven, at high dose, increased the ETP by 40% and reduced the anti-Xa level from 0.93 to 0.78 IU/ml. We conclude that the CAT is superior to the traditional coagulation tests in that it not only detects the coagulopathy of all the he-parinoids but can be also be used to monitor their reversal.

In Vitro Effect of Danaparoid Sodium (Orgaran®) on Thrombin Generation after Minimal Tissue Factor Pathway Activation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4151-4151
Author(s):  
Ismail Elalamy ◽  
Anna D. Petropoulou ◽  
Mohamed Hatmi ◽  
Meyer M. Samama ◽  
Grigoris T. Gerotziafas
Keyword(s):  
Molecular Weight ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Lag Time ◽  
Dependent Manner ◽  
Pathway Activation ◽  
Coagulation Tests ◽  
Vitro Effect ◽  
Routine Coagulation

Abstract Introduction: Orgaran® (Org 10172) is a low molecular weight heparinoid which consists of natural sulphated glycosaminoglycans (heparan, dermatan, chondroitin sulphate). It has a mean molecular weight of approximately 6 kDa (4–10 kDa), an excellent bioavailability following subcutaneous administration and an anti-Xa/anti-IIa activity ratio superior to 22. It is the anticoagulant of choice in patients developping Heparin-Induced Thrombocytopenia (HIT), whereas its’ use is also proposed for surgical thromboprophylaxis. Orgaran® has no effect on routine coagulation tests (aPTT, PT, TT). Thrombin generation test(TG) is a global clotting assay proven to be sensitive to the anticoagulant effect of LMWHs and specific FXa inhibitors (i.e. fondaparinux and BAY-597939). In this in vitro study, we determined the tissue factor (TF)-induced TG inhibition potency of Orgaran® using the Thrombogram-Thrombinoscope® assay. Materials and Methods: TG was assessed after TF pathway activation in Platelet Rich Plasma (PRP) (1.5x105 platelets/μl) using diluted thromboplastin (Dade Innovin®, 1:1000 final dilution). The clotting process is provoked by a physiologically relevant TF concentration. Orgaran® was added to control plasma from 8 healthy volunteers at five different final concentrations (0.2, 0.4, 0.6, 0.8 and 1IU anti-Xa/ml). TG was initiated by adding the triggering solution containing CaCl2 and the fluorogenic substrate. The analyzed TG parameters are the lag time, the maximal concentration of thrombin (Cmax), the time to reach Cmax (Tmax), the TG velocity and the endogenous thrombin potential (ETP). Results: Orgaran® prolonged significantly the lag time and the Tmax at a concentration over 0.40 IU anti-Xa/ml (p<0.05). At the lowest studied concentration (0.20 IU anti-Xa/ml), lag time and Tmax were only prolonged by 12%, whereas their maximal prolongation (around 50%) was observed at 1IU anti-Xa/ml. Furthermore, Orgaran® inhibited ETP, Cmax and TG velocity in an almost linear dose dependent manner. A significant inhibition of ETP, Cmax and TG velocity was obtained at concentrations superior to 0.20 IU anti-Xa/ml. (p<0.05). At the highest studied concentration (1IU anti-Xa/ml) Orgaran® suppressed all TG parameters by about 80% (Table 1). Conclusion: Orgaran® exhibited a significant inhibitory activity of in vitro TG. At concentrations achieved in clinical practice (prophylactic or therapeutic dose), Orgaran® modified in vitro TG profile while it has no effect on routine coagulation tests. Thus, TG assay is a sensitive method for monitoring Orgaran® and this test requires a clinical prospective evaluation. Table 1. Determination of IC20 and IC50 anti-Xa inhibitory concentrations of Orgaran® on TG parameters Lag Time Tmax ETP Cmax Velocity IC: Inhibitory Concentration * or Concentration increasing 20% and 50% the lag time and the Tmax respectively IC 20 (IU/ml) 0.30 0.30 0.18 0.18 0.15 IC 50 (IU/ml) 0.83 >1 0.30 0.50 0.35 1IU anti-Xa/ml 53% 47% 68% 76% 84%

Functional Characterization of BAX 855, a PEGylated Recombinant FVIII

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4359-4359
Author(s):  
Gerald Schrenk ◽  
Katalin Varadi ◽  
Herbert Gritsch ◽  
Hanspeter Rottensteiner ◽  
Hartmut J. Ehrlich ◽  
...  
Keyword(s):  
Functional Properties ◽  
Thrombin Generation ◽  
Binding Capacity ◽  
Functional Characterization ◽  
Activated Protein C ◽  
Density Lipoprotein ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Recombinant Fviii

Abstract Abstract 4359 Baxter and Nektar have developed BAX 855, a longer-acting PEGylated form of Baxter’s recombinant FVIII (ADVATE process) using stable PEG technology from Nektar. BAX 855 was functionally characterized in vitro and its features were compared with those of the unmodified parent rFVIII. The overall hemostatic potency of BAX 855 was assessed using a thrombin generation assay. Human FVIII-deficient plasma containing less than 1% of FVIII was supplemented with different concentrations of BAX 855 and unmodified rFVIII and coagulation was triggered by adding a small amount of recombinant human tissue factor complexed with phospholipid (PL) micelles to the plasma. Similar to unmodified rFVIII, BAX 855 corrected the impaired thrombin generation of the FVIII deficient plasma in a concentration-dependent manner. The role of FVIII within the tenase complex was determined by measuring the kinetics of FXa generation with a FIXa-cofactor activity assay, using either untreated or thrombin activated BAX 855. Comparison of the kinetic parameters and the maximum FXa generated revealed similar characteristics between BAX 855 and unmodified rFVIII. A similar approach revealed that BAX 855 fully retained its ability to be activated and inactivated by thrombin. The susceptibility of BAX 855 to activated protein C (APC) inactivation was also similar for BAX 855 and unmodified rFVIII. The binding affinities for VWF were similar for unmodified rFVIII (KD 0.6 nM) and BAX 855 (KD 0.8 nM) and the binding capacity of BAX 855 was also only slightly reduced. In contrast, the binding capacity of BAX 855 to the low-density lipoprotein-receptor-related protein (LRP) clearance receptor was 55% less than that of the unmodified rFVIII. In summary, the functional properties of BAX 855 were fully retained, indicating that PEGylation did not have an impact on the functional properties of rFVIII. Disclosures: Schrenk: Baxter Innovations GmbH: Employment. Varadi:Baxter Innovations GmbH: Employment. Gritsch:Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment.

scholarly journals Cytotoxic potential of camel whey and milk on cervix cancer (HeLa) cell line

2019 ◽  
Vol 5 (3) ◽  
pp. 231-236
Author(s):  
Lubna A Abdallah ◽  
Ashraf M Sawafta ◽  
Sanad A Ben Ali ◽  
Hasan A Baradia
Keyword(s):  
Hela Cells ◽  
Culture Technique ◽  
Cervix Cancer ◽  
Camel Milk ◽  
Dependent Manner ◽  
Anticancer Effect ◽  
Concentration Dependent Manner ◽  
Diphenyltetrazolium Bromide ◽  
Vitro Effect

Camel milk is an important nutritional source that historically been used in the treatment of cancer. Therefore, the main aim of the present study is to determine the in vitro anticancer effect of both camel milk and whey against cervix cancer (HeLa) cells. To perform that, skimmed milk as well as whey immunoglobulins concentrate samples were prepared at different concentrations (0, 1, 2.5, 5, 7.5 and 10 mg/ml). Then, the in vitro effect of the prepared concentrations on HeLa cells morphology and growth was investigated by tissue culture technique. Moreover, the anticancer activity of camel milk and whey against HeLa cells was estimated by the 3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. The obtained results displayed that both camel milk and whey reduced the viability of HeLa cells specially at 7.5 and 10 mg/ml. In addition, the viability of treated HeLa cells reduced after addition of both camel milk and whey to approximately 15% in a concentration dependent manner. In conclusion, this study showed the in vitro cytotoxic effect of camel milk and whey as they inhibited the growth of HeLa cells. Asian J. Med. Biol. Res. June 2019, 5(3): 231-236

In-vitro evaluation of anti-factor IXa aptamer on thrombin generation, clotting time, and viscoelastometry

2009 ◽  
Vol 101 (05) ◽  
pp. 827-833 ◽  
Author(s):  
Kenichi Tanaka ◽  
Fania Szlam ◽  
Christopher Rusconi ◽  
Jerrold Levy
Keyword(s):  
Thrombin Generation ◽  
Lag Time ◽  
Clot Formation ◽  
Dependent Manner ◽  
Plasma Samples ◽  
Clotting Time ◽  
Concentration Dependent Manner ◽  
Factor Ixa ◽  
Prolonged Aptt

SummaryThe REG1 system consists of factor IXa inhibitor, RB006, an ap-tamer-based anticoagulant and its antidote, RB007. The optimal use of RB006 can be facilitated by understanding its effect on the formation of thrombin and fibrin, and other standard tests of coagulation. Blood from consented volunteers was drawn into 3.2% citrate (9:1 v/v) and either used immediately or centrifuged to obtain platelet-poor plasma. Increasing concentrations of ap-tamer (6–24 μg/ml) alone or in combination with heparin (0.1 U/ml) or lepirudin (0.2 μg/ml) were added to blood and plasma samples. Activated clotting times (ACT+, low range-ACT), thrombelastometry (ROTEM™) or thrombelastography (TEG®) were performed in recalcified whole blood samples. Thrombin generation, prothrombin time (PT) and activated partial thromboplastin time (aPTT) were performed in plasma samples. To some samples the antidote RB007 was added to neutralise the anticoagulation activity of RB006. In all experiment the ratio of RB006 to RB007 was kept 1:2. RB006 dose-dependently prolonged aPTT and low range-ACT, but, as expected, had no effect on PT. RB006 prolonged the lag time and decreased the peak of Actin-triggered thrombin generation. Thrombin-activated TEG demonstrated that RB006 decreases the rate of clot formation. These effects were potentiated when RB006 was combined with heparin or lepirudin. In all experiments RB007 reversed the effects of RB006 back to baseline. In conclusion, RB006 inhibits thrombin generation and clot formation in a concentration-dependent manner. It is feasible to monitor RB006 and its reversal with RB007 using aPTT, low range-ACT, and thrombin-activated TEG.

The Calcium Modulator Nifedipine Exerts Its Antiaggregatory Property via a Nitric Oxide Mediated Process

1994 ◽  
Vol 72 (02) ◽  
pp. 309-312 ◽  
Author(s):  
R Berkels ◽  
W Klaus ◽  
M Boiler ◽  
R Rösen
Keyword(s):  
Nitric Oxide ◽  
Platelet Aggregation ◽  
Channel Blocker ◽  
Platelet Rich Plasma ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Endogenous Nitric Oxide ◽  
Vitro Effect ◽  
Dose Dependent

SummaryThe in vitro effect of nifedipine, a calcium channel blocker of the dihydropyridine (DHP) type, on platelet aggregation was reinvestigated considering especially the capability of platelets to form endogenous nitric oxide (NO). We studied the dose-dependent antiaggregatory property of nifedipine in porcine platelet rich plasma. Aggregation was stimulated by collagen (7.5 ¼g/ml). Nifedipine inhibited collagen-induced platelet aggregation with an IC50 of 380 nmol/1. The antiaggregatory effect of nifedipine could be significantly diminished by N-nitro-L-arginine (NNA) in a concentration dependent manner, whereas oxy haemoglobin (4 ¼M), a NO scavenger, totally abolished the effect of nifedipine. L-Arginine, the precursor of NO, dose-dependently inhibited the collagen-induced platelet aggregation but did not potentiate the effects of nifedipine. Therefore, we propose that in platelet rich plasma the nifedipine induced inhibition of platelet aggregation is mediated by NO, a potent endogenous inhibitor of aggregation. We could confirm this hypothesis by measuring NO directly with a specific electrode.

scholarly journals In vitro Effect of Aqueous Extract of Fresh Leaves of Abroma augusta L on the Diffusion of Glucose

2013 ◽  
Vol 16 (1) ◽  
pp. 21-26 ◽  
Author(s):  
Md Tariqul Islam ◽  
Md Ajijur Rahman ◽  
Md Anwar-Ul Islam
Keyword(s):  
Dietary Fiber ◽  
Aqueous Extract ◽  
Leaf Extract ◽  
Pharmaceutical Journal ◽  
Glucose Absorption ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Aqueous Leaf Extract ◽  
Vitro Effect

There have been a number of reports concerning the role of dietary fiber in hampering the diffusion of glucose and lowering the postprandial serum glucose. The present study investigates the effect of viscous aqueous leaf extract of Abroma augusta L (Family: Sterculiaceae, Bengali name: Ulatkambal, English name: Devil's cotton, DC) on the diffusion of glucose in vitro. Different mixtures were prepared using varying concentrations of sodium carboxymethylcellulose (Na-CMC) and aqueous extract of A. augusta with a fixed concentration of glucose. The diffusion of glucose from these systems into the outer medium through the ultra-fine membrane was measured. The results showed that both Na-CMC and aqueous extract of ulatkambal significantly (p<0.05) reduced the diffusion of glucose compared to control in a concentration-dependent manner. The result of this study suggested that dietary fiber present in the aqueous leaf extract of A. augusta may be potentially effective in the management of type 2 diabetes mellitus by reducing post-prandial glucose absorption from the gastrointestinal tract. DOI: http://dx.doi.org/10.3329/bpj.v16i1.14486 Bangladesh Pharmaceutical Journal 16(1): 21-26, 2013

Inhibition of Factors Xa and Iia Results in Synergistic Inhibition of Thrombin Generation with Minimal Clot Time Elevation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1877-1877
Author(s):  
Kathleen M. Welch ◽  
Kamlai Saiya-Cork ◽  
Weston R. Gould ◽  
Robert J. Leadley
Keyword(s):  
Thrombin Generation ◽  
Ex Vivo ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Specific Factor ◽  
Synergistic Activity ◽  
Dependent Manner ◽  
Clotting Time ◽  
Concentration Dependent Manner

Abstract Inhibition of either coagulation factor Xa (FXa) or thrombin (FIIa) alters ex vivo biomarkers of coagulation and decreases thrombus size in animal models of experimental thrombosis. The objective of this study was to determine if combining FXa and FIIa inhibitors would synergistically reduce the magnitude of IIa generated without elevating markers of bleeding. A synergistic combination may translate into lower doses, more effective anticoagulation and better safety. To predict the FXa and FIIa inhibitor combinations with the maximum potential for synergy, PD 0313052, a potent, selective FXa inhibitor, and argatroban, a potent FIIa inhibitor, were each tested independently and in combination using an in vitro thrombin generation assay. Individually, PD 0313052 and argatroban reduced total thrombin generation (TG) in a concentration dependent manner with IC50’s of 497±148 and 882±193 nM, respectively. Subsequently, PD 0313052 and argatroban were combined in 96 well plates at concentrations ranging from 0.125x to 8x their respective IC50 concentrations. Analysis using the Bliss Independence Model identified statistically significant synergistic activity, with the greatest increase (33%) over simple additivity at 249 and 441 nM FXa to FIIa, respectively, both below their respective IC50 concentrations. Furthermore, combinations of PD 0313052 and argatroban were evaluated in an assay measuring activated clotting time (ACT). Although both PD 0313052 and argatroban dose-dependently elongated the ACT, the combination of 0.5x the TG IC50 concentrations, which demonstrated the greatest synergy in the TG assay, showed the smallest increase in the ACT, prolonging clotting time by only 15%. These data demonstrate that the combination of a specific factor Xa inhibitor and a specific IIa inhibitor can synergistically reduce thrombin generation without appreciable elevation of ACT, suggesting that dual inhibition of FXa and FIIa, using relatively low doses of each, may provide efficacious and safe treatment for thromboembolic diseases.

Lipoxin A4 methyl ester protects PC12 cells from ketamine-induced neurotoxicity via the miR-22/BAG5 pathway

2021 ◽  
pp. 096032712110516
Author(s):  
Xue-song Wang ◽  
Long-cheng Li ◽  
Xue Zhang ◽  
Jin Gao
Keyword(s):  
Methyl Ester ◽  
Pc12 Cells ◽  
In Vitro Assays ◽  
Protective Mechanism ◽  
Dependent Manner ◽  
Protective Effects ◽  
Concentration Dependent Manner ◽  
Pheochromocytoma Pc12 ◽  
High Doses

Objective Ketamine is an anesthetic that induces neurotoxicity when administered at high doses. In this work, we explored the protective effects of lipoxin A4 methyl ester (LXA4 ME) against ketamine-induced neurotoxicity and the underlying protective mechanism in pheochromocytoma (PC12) cells. Methods PC12 cells were treated with 50 μM of ketamine and different LXA4 ME concentrations of LXA4 ME (5–50 nM) for 24 h, and their viability, apoptosis, and oxidative status were assessed. Results Quantitative real-time polymerase chain reaction experiments showed that ketamine downregulated miR-22 expression and upregulated Bcl-2-associated athanogene 5 (BAG5) in PC12 cells in a concentration-dependent manner. LXA4 ME induced the opposite effects, thus attenuating ketamine-induced neurotoxicity. Further in vitro assays showed that miR-22 directly targeted BAG5, thus promoting cell viability by suppressing cell apoptosis and oxidative stress. Under expression miR-22 or upregulation of BAG5 antagonized the effects of LXA4 ME. Conclusion LXA4 ME can protect PC12 cells from ketamine-induced neurotoxicity by activating the miR-22/BAG5 signaling pathway. Thus, LXA4 ME can be used as a protective drug against ketamine-induced neural damage.

In vitro Effects of Chlorpyrifos on the Acetylcholinesterase Activity of Euryhaline Fish, Oreochromis mossambicus

2010 ◽  
Vol 65 (3-4) ◽  
pp. 303-306 ◽  
Author(s):  
Janapala Venkateswara Rao ◽  
Pathakoti Kavitha
Keyword(s):  
Ache Activity ◽  
Kinetic Studies ◽  
Gill Tissue ◽  
Acetylcholinesterase Activity ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
In Vitro Effects ◽  
Brain Ache Activity ◽  
Vitro Effect

The in vitro effect of a widely used organophosphorus insecticide, chlorpyrifos (CPP), on the acetylcholinesterase (AChE) activity was studied in vitro. The kinetic constants Km and Vmax and the bimolecular constant ki were determined in vitro. The in vitro AChE study indicated that CPP is neurotoxic and that it alters the apparent Km values widely in a concentration-dependent manner, resulting in a competitive type of inhibition. Based on the ki values, the sensitivity of AChE in brain is greater than that in gill tissue, at 7.3 · 10 - 5 M and 11.92 · 10 - 5 M, respectively. The study points to the importance of kinetic studies and the results suggest that in biomonitoring programmes brain AChE activity can be a good diagnostic tool for CPP toxicity.

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