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Poisoning caused by coumarin-type anticoagulant rodenticides (ARs) stands as the predominant method for controlling rodents globally. ARs, through secondary poisoning, pose a significant threat to predators due to their lethal and sublethal effects. We examined the concentration of accumulated ARs in liver samples of mostly road-killed steppe polecats (Mustela eversmanii) and European polecats (M. putorius) collected throughout Hungary between 2005 and 2021. The steppe polecat samples were found mainly from Eastern Hungary, while European polecats from Western Hungary. We measured the concentration of six residues by HPLC-FLD. Our analysis revealed the presence of one first-generation and four second-generation ARs in 53% of the steppe polecat (36) and 39% of the European polecat (26) samples. In 17 samples we detected the presence of at least two AR compounds. Although we did not find significant variance in AR accumulation between the two species, steppe polecats displayed greater prevalence and maximum concentration of ARs, whereas European polecat samples exhibited a more diverse accumulation of these compounds. Brodifacoum and bromadiolone were the most prevalent ARs; the highest concentrations were 0.57 mg/kg and 0.33 mg/kg, respectively. The accumulation of ARs was positively correlated with human population density and negatively correlated with the extent of the more natural habitats in both species. To the best of our knowledge, this is the first study to demonstrate anticoagulant rodenticide exposure in steppe polecats globally, and for European polecats in Central European region. Although the extent of AR accumulation in European polecat in Hungary appears comparatively lower than in many other European countries, the issue of secondary poisoning remains a serious problem as these ARs intrude into food webs. Reduced and more prudent usage of pesticides would provide several benefits for wildlife, included humans. However, we advocate a prioritization of ecosystem services through the complete prohibition of the toxicants., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jozsef Lanszki reports was provided by HUN-REN Balaton Limnological Research Institute. Zsolt Pirger reports was provided by HUN-REN Balaton Limnological Research Institute. József Lanszki reports a relationship with HUN-REN Balaton Limnological Research Institute that includes: employment. Zsolt Pirger reports a relationship with HUN-REN Balaton Limnological Research Institute that includes: employment. József Lanszki and Zsolt Pirger was supported by the RRF-2.3.1-21-2022-00014 (National Multidisciplinary Laboratory for Climate Change), the RRF 2.3.1-21-2022-00008, BLRI (National Laboratory for Water Science and Water Security) and the NP2022-II3/2022; BLRI (Sustainable Development and Technologies National Programme of the Hungarian Academy of Sciences). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The first purpose of this study was to determine whether a measurement of the level of direct oral anticoagulants (DOACs) was possible with heparin-calibrated chromogenic anti-factor Xa activity (AXA). The second purpose of this study was to evaluate whether the antidote treatment decision level (30 or 50 ng/mL of DOAC) can be determined by unfractionated heparin (UHF)/low molecular weight heparin (LMWH)-calibrated AXA., Methods: AXA was measured by using two reagents and dedicated analyzers (Sysmex CS-5100 analyzer and STA R Max3). Four types of calibrators were used: 1) Stago DOAC (rivaroxaban, edoxaban, and apixaban)-specific calibrator, 2) Stago LMWH calibrator, 3) Sysmex UHF calibrator, and 4) Sysmex LMWH calibrator. Regression analysis was used between assays. Receiver operating characteristic (ROC) curves were performed, and the concordance rate was calculated., Results: The correlation coefficients were in the range of 0.75 - 0.91 for rivaroxaban and 0.81 - 0.94 for apixaban. The correlation coefficient between edoxaban-calibrated AXA and Sysmex LMWH/Sysmex UHF calibrator-calibrated AXA was low (r = 0.47). Overall correlation between DOAC-calibrated AXA and Stago LMWH-calibrated AXA was linear, at only low concentration in all three DOACs. The concordance rate (89.3 - 100%) is good for de-termining the antidote management level by UFH/LMWH-calibrated AXA, compared with those of DOAC-calibrated AXA in rivaroxaban and apixaban. The concordance rate ranged from 63% to 67% between Sysmex UFH/ LMWH-calibrated AXA and edoxaban-calibrated AXA., Conclusions: The findings of our study suggest limitations in calculating accurate concentrations, when using UFH/LMWH-calibrated AXA to measure DOAC. This study demonstrates that UFH/LMWH-calibrated AXA may be useful in determining the presence of DOACs at the cutoff level for the antidote treatment in rivarovaban and apixaban. However, in edoxaban, UFH/LMWH-calibrated AXA could not accurately measure the presence of DOACs at the cutoff for antidote treatment.

Envenoming resulting from snakebites is recognized as a priority neglected tropical disease by The World Health Organization. The Bothrops genus, consisting of different pitviper species, is considered the most medically significant taxa in Central and South America. Further research into Bothrops venom composition is important to aid in the development of safer and more effective snakebite treatments. In addition, the discovery of Bothrops toxins that could potentially be used for medical or diagnostic purposes is of interest to the pharmaceutical industry. This study aimed to employ high-throughput (HT) venomics to qualitatively analyze venom composition while utilizing coagulation bioassays for identifying coagulopathic toxins and characterizing coagulopathic activity in various Bothrops venoms. Using the recently demonstrated HT venomics workflow in combination with post-column coagulopathic bioassaying, focus was placed at anticoagulant toxins. Well-known procoagulant toxins were also investigated, taking into account that using the HT venomics workflow, procoagulant toxins are especially prone to denaturation during the reversed-phase chromatographic separations performed in the workflow. The findings revealed that the venoms of B. atrox and B. jararaca harbored procoagulant toxins, whereas those of B. alternatus and B. neuwiedi contained both procoagulant and anticoagulant toxins. In general, anticoagulation was associated with phospholipases A 2 s, while procoagulation was associated with snake venom metalloproteinases and snake venom serine proteases. These results showed the identification of coagulopathic venom toxins in the Bothrops venoms analyzed using multiple analytical methods that complement each other. Additionally, each venom underwent qualitative characterization of its composition.

Pesticide contamination poses a significant threat to non-target wildlife, including amphibians, many of which are already highly threatened. This study assessed the extent of pesticide exposure in dead frogs collected during a mass mortality event across eastern New South Wales, Australia between July 2021 and March 2022. Liver tissue from 77 individual frogs of six species were analysed for >600 legacy and contemporary pesticides, including rodenticides. More than a third (36 %) of the liver samples contained at least one of the following pesticides: brodifacoum, dieldrin, DDE, heptachlor/heptachlor epoxide, fipronil sulfone, and 2-methyl-4-chlorophenoxyacetic acid (MCPA). Brodifacoum, a second-generation anticoagulant rodenticide, was found in four of the six frog species analysed: the eastern banjo frog (Limnodynastes dumerilii), cane toad (Rhinella marina), green tree frog (Litoria caerulea) and Peron's tree frog (Litoria peronii). This is the first report of anticoagulant rodenticide detected in wild amphibians, raising concerns about potential impacts on frogs and extending the list of taxa shown to accumulate rodenticides. Dieldrin, a banned legacy pesticide, was also detected in two species: striped marsh frog (Limnodynastes peronii) and green tree frog (Litoria caerulea). The toxicological effects of these pesticides on frogs are difficult to infer due to limited comparable studies; however, due to the low frequency of detection the presence of these pesticides was not considered a major contributing factor to the mass mortality event. Additional research is needed to investigate the effects of pesticide exposure on amphibians, particularly regarding the impacts of second-generation anticoagulant rodenticides. There is also need for continued monitoring and improved conservation management strategies for the mitigation of the potential threat of pesticide exposure and accumulation in amphibian populations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Second-generation anticoagulant rodenticides (SGARs) are widely used to control rodent populations, resulting in the serious secondary exposure of predators to these contaminants. In the United Kingdom (UK), professional use and purchase of SGARs were revised in the 2010s. Certain highly toxic SGARs have been authorized since then to be used outdoors around buildings as resistance-breaking chemicals under risk mitigation procedures. However, it is still uncertain whether and how these regulatory changes have influenced the secondary exposure of birds of prey to SGARs. Based on biomonitoring of the UK Common Buzzard ( Buteo buteo ) collected from 2001 to 2019, we assessed the temporal trend of exposure to SGARs and statistically determined potential turning points. The magnitude of difenacoum decreased over time with a seasonal fluctuation, while the magnitude and prevalence of more toxic brodifacoum, authorized to be used outdoors around buildings after the regulatory changes, increased. The summer of 2016 was statistically identified as a turning point for exposure to brodifacoum and summed SGARs that increased after this point. This time point coincided with the aforementioned regulatory changes. Our findings suggest a possible shift in SGAR use to brodifacoum from difenacoum over the decades, which may pose higher risks of impacts on wildlife.

Bromadiolone and brodifacoum, the most frequently used anticoagulant rodenticides, are highly toxic and pose a threat to public health by causing food poisoning incidents. Here, we developed a fluorescence microplate immunoassay for facile and sensitive detection of bromadiolone and brodifacoum by introducing three commercial chemicals (p-phenylenediamine, polyethyleneimine, H 2 O 2 ) as a new substrate of horseradish peroxidase and then generating fluorescence signals based on an in situ fluorogenic reaction (detection time within 75 min). This assay exhibited higher efficiency in generating fluorescence signals, thereby exhibiting a 6-fold improvement in sensitivity compared with colorimetric ELISA. The limit of detection was 0.23-0.28 ng/mL (ng/g) for bromadiolone and 0.34-0.61 ng/mL (ng/g) for brodifacoum in corn and human serum, with recovery ratios higher than 82.3 %. These satisfactory results illustrated our proposed assay was a potential tool for food analysis and poisoning diagnosis caused by bromadiolone and brodifacoum., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The widespread use of anticoagulant rodenticides (ARs) poses a worldwide threat to farmland wildlife. These compounds accumulate in tissues of both target and non-target species, potentially endangering both direct consumers and their predators. However, investigations on ARs in blood of free-ranging predatory birds are rare. Here, the long-eared owl (Asio otus) has been used as a model predator to assess AR exposure in different agricultural landscapes from a Mediterranean semiarid region. A total of 69 owlets from 38 nests were blood-sampled over 2021 and 2022, aiming to detect AR residues and explore factors that determine their exposure, such as land uses. In addition, prothrombin time (PT) test was conducted to assess potential effects of AR contamination. Overall, nearly all the samples (98.6 %) tested positive for at least one compound and multiple ARs were found in most of the individuals (82.6 %). Among the ARs detected, flocoumafen was the most common compound (88.4 % of the samples). AR total concentration (ΣARs) in blood ranged from 0.06 to 34.18 ng mL -1 , detecting the highest levels in the most intensively cultivated area. The analysis of owl pellets from 19 breeding territories showed relevant among-site differences in the contribution of rodents and birds into the diet of long-eared owls, supporting its high dietary plasticity and indicating AR presence at multiple trophic levels. Moreover, a positive and significant correlation was found between ΣARs and PT (Rho = 0.547, p < 0.001), which demonstrates the direct effect of ARs on free-living nestlings. Our results provide a preliminary overview of AR exposure in a little-studied owl species inhabiting agricultural and rural landscapes. Despite the low detected levels, these findings indicate widespread exposure -often to multiple compounds- from early life stages, which raises concern and draws attention to an ongoing and unresolved contamination issue., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The study deals with the environmental residues of anticoagulant rodenticides (ARs) in Slovenia to evaluate the toxicological risk of secondary poisoning of red foxes (Vulpes vulpes) as representatives of non-target wildlife, and in relation to the investigated use patterns of ARs and specific local parameters in Slovenia. From 2019 to 2022, 148 liver tissue samples of adult red foxes were collected from almost all state geographical regions. The samples were extracted with methanol/water (2:1, v/v), cleaned-up using a solid supported liquid-liquid extraction, and measured by liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) with reporting limits of 0.5 to 5.0 ng/g. Residues of at least one rodenticide were detected in 77.7 % of the samples. The second generation ARs of bromadiolone, brodifacoum and difenacoum were the most frequently found, appearing in 75.0, 51.4, and 18.9 % of the samples, respectively. Concentrations of pooled ARs ranged from 1.5 to 2866.5 ng/g with mean and median values of 601.4 and 350.2 ng/g, respectively. We determined bromadiolone and brodifacoum at concentrations of ≥800 ng/g in 10.8 and 10.1 % of the samples, and 1.4 and 0.7 % of the samples contained residues >2000 ng/g, respectively. These concentrations are much higher than those found in comparable studies in Europe and elsewhere in the world. Residues of ARs were detected in all monitored statistical regions of Slovenia, with higher concentrations in the eastern parts of the country. First generation ARs were found in only 9.5 % of samples, and residues were below 10 ng/g with one exception (coumatetralyl with 55 ng/g). The results of the study indicate a serious toxicological risk for red foxes in Slovenia as part of the Western Balkans, and will contribute to the growing body of knowledge about the protection of European ecosystems, as wildlife is not limited by national borders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Apixaban and dabigatran are the two major direct oral anticoagulant drugs to treat thromboembolic disordered patients. Increasing the clinical application for the thromboembolic disorder and monitoring the concentrations of apixaban, dabigatran, and their metabolites are essential in most clinical circumstances. In this work, we developed a rapid analytical methodology comprising of vortex-assisted salt-enhanced liquid-liquid microextraction technique coupled with UHPLC-MS/MS for the extraction and simultaneous determination of two major direct oral anticoagulant drugs (apixaban, dabigatran), and their two major metabolites from plasma, serum, and urine samples of patients. The developed method was optimized with various procedural steps and validated to study the analytical merits. The developed method yielded a good detection limit of 0.01 ∼ 0.37 ng/mL, 0.01 ∼ 0.32 ng/ml, and 0.01 ∼ 0.27 ng/mL for four target analytes in the plasma, serum, and urine matrices. Moreover, extraction recoveries ranged from 85.11 - 113.57% (for plasma), 89.63 - 110.47% (for serum), and 87.44 -106.79% (for urine samples) with 8.78% RSD. In addition, the method exhibited good R 2 values of 0.999 for all four target analytes, and the specificity and carryover study revealed no carryover effect from the UHPLC-MS/MS system for determining the apixaban, dabigatran, and their metabolites. Due to the above advantages, the developed analytical technique was applied to examine 11 real-time clinical patients' samples, and the observed results were satisfactory for all three different sample matrices. Therefore, this analytical method can be applied for biomonitoring apixaban, dabigatran, and their two major metabolites with high sensitivity in a short time for various clinical applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Anticoagulant rodenticides (ARs), particularly second-generation compounds (SGARs), are extensively used in pest management, impacting non-target wildlife. The California kingsnake (Lampropeltis californiae), an invasive species in Gran Canaria, is under a control plan involving capture and euthanasia. This research aimed to detect 10 different ARs in these snakes, explore geographical and biometrical factors influencing AR exposure, and assess their potential as sentinel species for raptors, sharing similar foraging habits. Liver samples from 360 snakes, euthanized between 2021 and 2022, were analysed for ARs using LC-MS/MS. Results showed all detected rodenticides were SGARs, except for one instance of diphacinone. Remarkably, 90 % of the snakes tested positive for ARs, with over half exposed to multiple compounds. Brodifacoum was predominant, found in over 90 % of AR-positive snakes, while bromadiolone and difenacoum were also frequently detected but at lower levels. The study revealed that larger snakes and those in certain geographic areas had higher AR concentrations. Snakes in less central or more peripheral areas showed lower levels of these compounds. This suggests a correlation between the snakes' size and distribution with the concentration of ARs in their bodies. The findings indicate that the types and prevalence of ARs in California kingsnakes on Gran Canaria mirror those in the island's raptors. This similarity suggests that the kingsnake could serve as a potential sentinel species for monitoring ARs in the ecosystem. However, further research is necessary to confirm their effectiveness in this role., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

This study assesses the impact of second-generation anticoagulant rodenticides (SGARs) on the common kestrel (Falco tinnunculus canariensis) in Tenerife, Canary Islands. The analysis of 390 liver samples over 19 years using HPLC-MS/MS showed that 93.1 % of kestrels were exposed to SGARs in this island. A notable shift in SGAR profiles was observed, with bromadiolone and flocoumafen decreasing, while brodifacoum levels increased sharply from 2018 onwards. Comparatively, Tenerife kestrels had a higher detection frequency of SGARs (93.1 %) than those in the rest of the islands of the archipelago (68.2 %), with median concentrations nearly double (∑AR = 180.9 vs 102.4 ng/g liver, P < 0.0001). Furthermore, on average, kestrels from Tenerife were found to have a higher number of different rodenticide compounds per individual. A Generalized Linear Model (GLM) analysis revealed that several factors contribute to the likelihood of SGAR exposure: being an adult kestrel, the enactment of legal restrictions on SGAR bait concentrations in 2018, higher livestock density, and greater human population density. These findings suggest that both bioaccumulation over the birds' lifespans and environmental factors related to human and agricultural activity are influencing the levels of SGARs detected. Alarmingly, 44.7 % of kestrels had SGAR levels above the toxicity threshold established for other raptor species (200 ng/g liver), signaling a high poisoning risk. This is despite EU regulations to protect wildlife, with our findings indicating an increase in both exposure rates and SGAR concentrations since these laws were enacted. The data highlight a critical environmental threat to endemic species on islands like Tenerife. The common kestrel, not considered globally endangered, is nonetheless facing regional threats from SGAR contamination. These results emphasize the urgent need for effective regulations to address the persistent and growing impact of SGARs on island biodiversity., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: Trypsin inhibitors (TIs) have the ability to competitively or non-competitively bind to trypsin and inhibit its action. These inhibitors are commonly found in plants and are used in protease inhibition studies involved in biochemical pathways of pharmacological interest., Objectives: This work aimed to purify a trypsin inhibitor from Bauhinia pulchella seeds ( Bpu TI), describing its kinetic mechanism and anticoagulant effect., Methods: Affinity chromatography, protein assay, and SDS-PAGE were used to purify the inhibitor. Mass spectrometry, inhibition assays, and enzyme kinetics were used to characterize the inhibitor. In vitro assays were performed to verify its ability to prolong blood clotting time., Results: Affinity chromatography on a Trypsin-Sepharose 4B column gave a yield of 43.1. Bpu TI has an apparent molecular mass of 20 kDa with glycosylation (1.15%). Protein identification was determined by MS/MS, and Bpu TI showed similarity to several Kunitz-type trypsin inhibitors. Bpu TI inhibited bovine trypsin as an uncompetitive inhibitor with IC50 (3 x 10 -6 M) and Ki (1.05 x 10 -6 M). Additionally, Bpu TI showed high stability to temperature and pH variations, maintaining its activity up to 100ºC and in extreme pH ranges. However, the inhibitor was susceptible to reducing agents, such as DTT, which completely abolished its activity. Bpu TI showed an anticoagulant effect in vitro at a concentration of 33 μM, prolonging clotting time by 2.6 times., Conclusion: Our results suggest that Bpu TI can be a biological tool to be used in blood clotting studies., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Anticoagulant rodenticides (ARs) influence predator populations and threaten the stability of ecosystems. Understanding the prevalence and impact of rodenticides in predators is crucial to inform conservation planning and policy. We collected dead birds of four nocturnal predatory species across differing landscapes: forests, agricultural, urban. Liver samples were analysed for eight ARs: three First Generation ARs (FGARs) and five SGARs (Second Generation ARs). We investigated interspecific differences in liver concentrations and whether landscape composition influenced this. FGARs were rarely detected, except pindone at low concentrations in powerful owls Ninox strenua. SGARs, however, were detected in every species and 92 % of birds analysed. Concentrations of SGARs were at levels where potential toxicological or lethal impacts would have occurred in 33 % of powerful owls, 68 % of tawny frogmouths Podargus strigoides, 42 % of southern boobooks N. bookbook and 80 % of barn owls Tyto javanica. When multiple SGARs were detected, the likelihood of potentially lethal concentrations of rodenticides increased. There was no association between landscape composition and SGAR exposure, or the presence of multiple SGARs, suggesting rodenticide poisoning is ubiquitous across all landscapes sampled. This widespread human-driven contamination in wildlife is a major threat to wildlife health. Given the high prevalence and concentrations of SGARs in these birds across all landscape types, we support the formal consideration of SGARs as a threatening process. Furthermore, given species that do not primarily eat rodents (tawny frogmouths, powerful owls) have comparable liver rodenticide concentrations to rodent predators (southern boobook, eastern barn owl), it appears there is broader contamination of the food-web than anticipated. We provide evidence that SGARs have the potential to pose a threat to the survival of avian predator populations. Given the functional importance of predators in ecosystems, combined with the animal welfare impacts of these chemicals, we propose governments should regulate the use of SGARs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Anticoagulant rodenticides (ARs) are used to control rodent populations. Poisoning of non-target species can occur by accidental consumption of commercial formulations used for rodent control. A robust method for determining ARs in animal tissues is important for animal postmortem diagnostic and forensic purposes. We evaluated an ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) method to quantify 8 ARs (brodifacoum, bromadiolone, chlorophacinone, coumachlor, dicoumarol, difethialone, diphacinone, warfarin) in a wide range of animal (bovine, canine, chicken, equine, porcine) liver samples, including incurred samples. We further evaluated UPLC-MS in 2 interlaboratory comparison (ILC) studies; one an ILC exercise (ICE), the other a proficiency test (PT). The limits of detection of UPLC-MS were 0.3-3.1 ng/g, and the limits of quantification were 0.8-9.4 ng/g. The recoveries obtained using UPLC-MS were 90-115%, and relative SDs were 1.2-13% for each of the 8 ARs for the 50, 500, and 2,000 ng/g spiked liver samples. The overall accuracy from the laboratories participating in the 2 ILC studies (4 and 11 laboratories for ICE and PT studies, respectively) were 86-118%, with relative repeatability SDs of 3.7-11%, relative reproducibility SDs of 7.8-31.2%, and Horwitz ratio values of 0.5-1.5. Via the ILC studies, we verified the accuracy of UPLC-MS for AR analysis in liver matrices and demonstrated that ILC can be utilized to evaluate performance characteristics of analytical methods.

Blood platelets play a crucial role in hemostasis, the process responsible for keeping blood flowing in the circulatory system. However, unnecessary platelet activation can lead to aggregation at the site of atherosclerotic plaque rapture and the formation of a thrombus, which promotes atherothrombotic diseases. Various dietary components, such as phenolic compounds, are known to demonstrate antiplatelet and anticoagulant properties, and it is possible that these could form an important element in the prophylaxis and therapy of cardiovascular diseases. Our present study examined the biological activity of isorhamnetin ( 1 ) and two isorhamnetin derivatives, ( 2 ): 3- O -beta-glucoside-7- O -alpha-rhamnoside and ( 3 ): 3- O -beta-glucoside-7- O -alpha-(3″'-isovaleryl)-rhamnoside, isolated from the phenolic fraction of sea buckthorn fruit, against human washed blood platelets and human whole blood in vitro. The anti-platelet and anticoagulant potential was determined using (A) flow cytometry, (B) the thrombus-formation analysis system (T-TAS) and (C) colorimetry. The results of the T-TAS test indicate that the AUC 10 (Area Under the Curve) of the tested phenolic compounds (compounds 1 , 2 and 3 ; 50 µg/mL) was markedly reduced compared to the control values. Moreover, flavonol demonstrated anti-platelet potential, including anti-adhesive activity, with these effects being more intense in compound 2 than isorhamnetin. Different actions of flavonol on platelet activation may depend on their binding ability to various receptors on blood platelets. However, the mechanism of their anti-platelet potential requires further additional studies, including in vitro and in vivo experiments.

Since rodenticides represent a substance group relevant in toxicological analyses, the aim of this work was the development of a complex multi-target screening strategy for the identification with liquid chromatography-tandem mass spectrometry. A simple protein precipitation was used as the sample preparation strategy. Further, a Luna 5 μm C18 (2) 100 Å, 150 × 2 mm analytical column was applied for the separation of relevant analytes with a Shimadzu HPLC. Signal detection was performed with a SCIEX API 5500 QTrap MS/MS system. The rodenticides investigated (α-chloralose, brodifacoum, bromadiolone, coumatetralyl, difenacoum, and warfarin) could be incorporated effectively into a multi-target screening strategy covering about 250 substances representing different groups with a limit of detection appropriate for substance identification. The strategy can easily be modified to perform semi-quantitative measurements for this substance group and could be supplemented by quantification based on standard addition., (© 2022 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Background: Ex vivo assays of platelet function critically inform mechanistic and clinical hematology studies, where effects of divergent blood processing methods on platelet composition are apparent, but unspecified., Objective: Here, we evaluate how different blood anticoagulation options and processing times affect platelet function and protein content ex vivo., Methods: Parallel blood samples were collected from healthy human donors into sodium citrate, acid citrate dextrose, EDTA or heparin, and processed over an extended time course for functional and biochemical experiments, including platelet proteome quantification with multiplexed tandem mass tag (TMT) labeling and triple quadrupole mass spectrometry (MS)., Results: Each anticoagulant had time-dependent effects on platelet function in whole blood. For instance, heparin enhanced platelet agonist reactivity, platelet-monocyte aggregate formation and platelet extracellular vesicle release, while EDTA increased platelet α-granule secretion. Following platelet isolation, TMT-MS quantified 3357 proteins amongst all prepared platelet samples. Altogether, >400 proteins were differentially abundant in platelets isolated from blood processed at 24 h versus 1 h post-phlebotomy, including proteins pertinent to membrane trafficking and exocytosis. Anticoagulant-specific effects on platelet proteomes included increased complement system and decreased α-granule proteins in platelets from EDTA-anticoagulated blood. Platelets prepared from heparinized blood had higher levels of histone and neutrophil-associated proteins in a manner related to neutrophil extracellular trap (NET) formation and platelet:NET interactions in whole blood ex vivo., Conclusion: Our results demonstrate that different anticoagulants routinely used for blood collection have varying effects on platelets ex vivo, where methodology-associated alterations in platelet proteome may influence mechanistic, translational and biomarker studies., (© 2022 International Society on Thrombosis and Haemostasis.)

The powerful owl (Ninox strenua) is a threatened apex predator that consumes mainly arboreal marsupial prey. Low density populations reside in urban landscapes where their viability is tenuous. The catalyst for this research was the reported death of eight powerful owls around Melbourne, Australia, in less than one year (2020/2021). Eighteen deceased owls were toxicologically screened. We assessed toxic metals (Mercury Hg, Lead Pb, Cadmium Cd and Arsenic As) and anticoagulant rodenticides (ARs) in liver (n = 18 owls) and an extensive range of agricultural chemicals in muscle (n = 14). Almost all agricultural chemicals were below detection limits except for p,p-DDE, which was detected in 71% of birds at relatively low levels. Toxic metals detected in some individuals were generally at low levels. However, ARs were detected in 83.3% of powerful owls. The most common second-generation anticoagulant rodenticide (SGAR) detected was brodifacoum, which was present in every bird in which a rodenticide was detected. Brodifacoum was often present at toxic levels and in some instances at potentially lethal levels. Presence of brodifacoum was detected across the complete urban-forest/agriculture gradient, suggesting widespread exposure. Powerful owls do not scavenge but prey upon arboreal marsupials, and generally not rodents, suggesting that brodifacoum is entering the powerful owl food web via accidental or deliberate poisoning of non-target species (possums). We highlight a critical need to investigate SGARs in food webs globally, and not just in species directly targeted for poisoning or their predators., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Anticoagulant rodenticides (ARs) are used globally to control rodent pest infestations in both urban and agricultural settings. It is well documented that non-target wildlife, including predatory birds, are at risk for secondary anticoagulant exposure and toxicosis through the prey they consume. However, there have been no large-scale studies of AR exposure in raptors in Ontario, Canada since new Health Canada legislation was implemented in 2013 in an attempt to limit exposure in non-target wildlife. Our objective was to measure levels of ARs in wild raptors in southern Ontario to assess their exposure. We collected liver samples from 133 raptors representing 17 species submitted to the Canadian Wildlife Health Cooperative (CWHC) in Ontario, Canada, between 2017 and 2019. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to quantitatively assess the level of exposure to 14 first- and second-generation ARs. Detectable levels of one or more ARs were found in 82 of 133 (62%) tested raptors, representing 12 species. The most commonly detected ARs were bromadiolone (54/133), difethialone (40/133), and brodifacoum (33/133). Of AR-positive birds, 34/82 (42%) contained residues of multiple (> 1) anticoagulant compounds. Our results indicate that AR exposure is common in raptors living in southern Ontario, Canada. Our finding that brodifacoum, difethialone, and bromadiolone were observed alone or in combination with one another in the majority of our sampled raptors indicates that legislative changes in Canada may not be protecting non-target wildlife as intended., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Wild raptors are widely used to assess exposure to different environmental contaminants, including anticoagulant rodenticides (ARs). ARs are used on a global scale for rodent control, and act by disruption of the vitamin K cycle that results in haemorrhage usually accompanied by death within days. Some ARs are highly persistent and bioaccumulative, which can cause significant exposure of non-target species. We characterized AR exposure in a heterogeneous sample of dead raptors collected over 12 years (2008-2019) in south-eastern France. Residue analysis of 156 liver samples through LC-MS/MS revealed that 50% (78/156) were positive for ARs, with 13.5% (21/156) having summed second-generation AR (SGAR) concentrations >100 ng/g ww. While SGARs were commonly detected (97.4% of positive samples), first-generation ARs were rarely found (7.7% of positive samples). ARs were more frequently detected and at greater concentration in predators (prevalence: 82.5%) than in scavengers (38.8%). Exposure to multiple ARs was common (64.1% of positive samples). While chlorophacinone exposure decreased over time, an increasing exposure trend was observed for the SGAR brodifacoum, suggesting that public policies may not be efficient at mitigating risk of exposure for non-target species. Haemorrhage was observed in 88 birds, but AR toxicosis was suspected in only 2 of these individuals, and no difference in frequency of haemorrhage was apparent in birds displaying summed SGAR levels above or below 100 ng/g ww. As for other contaminants, 17.2% of liver samples (11/64) exhibited Pb levels compatible with sub-clinical poisoning (>6 μg/g dw), with 6.3% (4/64) above the threshold for severe/lethal poisoning (>30 μg/g dw). Nine individuals with Pb levels >6 μg/g dw also had AR residues, demonstrating exposure to multiple contaminants. Broad toxicological screening for other contaminants was positive for 18 of 126 individuals, with carbofuran and mevinphos exposure being the suspected cause of death of 17 birds. Our findings demonstrate lower but still substantial AR exposure of scavenging birds compared to predatory birds, and also illustrate the complexity of diagnosing AR toxicosis through forensic investigations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Antiplatelet and anticoagulant drugs are classified antithrombotic agents with the purpose to reduce blood clot formation. For a successful treatment of many known complex cardiovascular diseases driven by platelet and/or coagulation activity, the need of more than one antithrombotic agent is inevitable. However, combining drugs with different mechanisms of action enhances risk of bleeding. Dual anticoagulant and antiplatelet (APAC), a novel semisynthetic antithrombotic molecule, provides both anticoagulant and antiplatelet properties in preclinical studies. APAC is entering clinical studies with this new exciting approach to manage cardiovascular diseases. For a better understanding of the biological function of APAC, comprehensive knowledge of its structure is essential. In this study, atomic force microscopy (AFM) was used to characterize APAC according to its structure and to investigate the molecular interaction of APAC with von Willebrand factor (VWF), since specific binding of APAC to VWF could reduce platelet accumulation at vascular injury sites. By the optimization of drop-casting experiments, we were able to determine the volume of an individual APAC molecule at around 600 nm 3 , and confirm that APAC forms multimers, especially dimers and trimers under the experimental conditions. By studying the drop-casting behavior of APAC and VWF individually, we depictured their interaction by using an indirect approach. Moreover, in vitro and in vivo conducted experiments in pigs supported the AFM results further. Finally, the successful adsorption of APAC to a flat gold surface was confirmed by using photothermal-induced resonance, whereby attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) served as a reference method., (© 2021. The Author(s).)

Before a new method is used for clinical testing, it is essential that it is evaluated for suitability for its intended purpose. This document gives guidance for the performance, verification and implementation processes required by regulatory and accreditation bodies. It covers the planning and verification of specialist haemostatic tests, including factor assays, D-dimers, direct anticoagulants and thrombophilia testing., (© 2021 John Wiley & Sons Ltd.)

Anticoagulant rodenticides (ARs) used to control mammalian pest populations cause secondary exposure of predatory species throughout much of the world. It is important to understand the drivers of non-target AR exposure patterns as context for assessing long-term effects and developing effective mitigation for these toxicants. In Australia, however, little is known about exposure and effects of ARs on predators. We detected AR residues in 74% of 50 opportunistically collected carcasses of the Tasmanian wedge-tailed eagle (Aquila audax fleayi), an endangered apex predator. In 22% of birds tested, or 31% of those exposed, liver concentrations of second generation ARs (SGARs) were >0.1 mg/kg ww. Eagles were exposed to flocoumafen, a toxicant only available from agricultural suppliers, at an exceptionally high rate (40% of birds tested). Liver SGAR concentrations were positively associated with the proportion of agricultural habitat and human population density in the area around where each eagle died. The high exposure rate in a species not known to regularly prey upon synanthropic rodents supports the hypothesis that apex predators are vulnerable to SGARs. Our results indicate that AR exposure constitutes a previously unrecognized threat to Tasmanian wedge-tailed eagles and highlight the importance of efforts to address non-target AR exposure in Australia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

Carthami flos, commonly known as Honghua in China, is the dried floret of safflower and widely acknowledged as a blood stasis promoting herb. The study aimed at investigating the relationship between thrombin and carthami flos through a high-performance thrombin affinity chromatography combined with a high-performance liquid chromatography-tandem mass spectrometry system. First, thrombin was immobilized on the glutaraldehyde-modified amino silica gel to prepare the thrombin affinity stationary phase, which was packed into a small column (1.0 × 2.0 mm, id) for recognizing the anticoagulant active components of carthami flos. The target component was enriched and analyzed by the high-performance liquid chromatography-tandem mass spectrometry system. Finally, hydroxysafflor yellow A was screened out and identified as the active component. The anticoagulant effects of hydroxysafflor yellow A were analyzed by anticoagulant experiments in vitro, and the interaction of hydroxysafflor yellow A with thrombin was investigated by the molecular docking method. The results proved that hydroxysafflor yellow A (30 μg/mL, 0.05 mM) and carthami flos extract (30 μg/mL) could prolong activated partial thrombin time and thrombin time by 50 and 11%, respectively. Moreover, hydroxysafflor yellow A exhibits a good hydrogen bond field and stereo field matching with thrombin. Overall, it was concluded that hydroxysafflor yellow A might exert an anticoagulation effect by interacting with thrombin and thus could be potential anticoagulant drugs for the prevention and treatment of venous thrombosis., (© 2021 Wiley-VCH GmbH.)

The common vampire bat ( Desmodus rotundus ) is a hematophagous species responsible for paralytic rabies and bite damage that affects livestock, humans and wildlife from Mexico to Argentina. Current measures to control vampires, based upon coumarin-derived poisons, are not used extensively due in part to the high cost of application, risks for bats that share roosts with vampires and residual environmental contamination. Observations that vampire bat bites may induce resistance in livestock against vampire bat salivary anticoagulants encourage research into novel vaccine-based alternatives particularly focused upon increasing livestock resistance to vampire salivary components. We evaluated the action of vampire bat saliva-Freund's incomplete adjuvant administered to sheep with anticoagulant responses induced by repeated vampire bites in a control group and examined characteristics of vampire bat salivary secretion. We observed that injections induced a response against vampire bat salivary anticoagulants stronger than by repeated vampire bat bites. Based upon these preliminary findings, we hypothesize the utility of developing a control technique based on induction of an immunologically mediated resistance against vampire bat anticoagulants and rabies virus via dual delivery of appropriate host and pathogen antigens. Fundamental characteristics of host biology favor alternative strategies than simple culling by poisons for practical, economical, and ecologically relevant management of vampire populations within a One Health context.

CP3000 coagulation analyzer is a high-throughput, fully automated coagulation analyzer. The objective of this study was to evaluate the analytical performance of CP3000 coagulation system for general and special coagulation analyses. Quality control materials and patient samples were used to evaluate the analytical performance of CP3000 coagulation system. Precision, carryover, linearity, comparability with ACL-TOP 700 coagulation system, and verification of reference range were evaluated or performed according to Clinical and Laboratory Standards Institute guidelines. Within-run and between-run precisions were below 5% for both normal and abnormal ranges. There was no detectable carryover. The linearity of antithrombin and fibrinogen were excellent. The comparability between CP3000 and ACL-TOP 700 coagulation systems was acceptable except for activated partial thromboplastin time and thrombin time due to differences in reagent composition. Reference ranges proposed by the manufacturer were verified to be acceptable. CP3000 coagulation system is a reliable system that can be used to perform routine and special coagulation tests rapidly and accurately. Because of its small footprint as an additional advantage, the implementation of CP3000 coagulation system can be efficient in hospital laboratories of various sizes., (Copyright © 2021 by the Association of Clinical Scientists, Inc.)

Background: Continuous renal replacement therapy (CRRT) with regional citrate anticoagulation (RCA) is now commonly used to treat acute kidney injury in critically ill patients. The concentration of citrate is not routinely measured, with citrate accumulation and/or toxicity primarily assessed using surrogate measures., Objectives: The aim of this study was to measure the concentration of citrate in plasma and ultrafiltrate in patients receiving CRRT with RCA using a modified commercial enzymatic assay., Methods: After meeting inclusion criteria, blood was sampled from 20 patients before, during, and after episodes of filtration. Using spectrophotometry, samples were tested for citrate concentration. Demographic and other clinical and biochemical data were also collected. Throughout, a 15 mmol/L solution of trisodium citrate was used as the prefilter anticoagulant. Results were analysed using STATA (v15.0) and presented as mean (SD), median (IQR), or simple proportion. Comparisons were made using either the Student t test or the Wilcoxon rank-sum test. Correlation was assessed using Pearson's r., Results: Twenty patients (17 males) were enrolled in the study. Mean (SD) age was 63.7 years (9.9). Median (IQR) ICU length of stay was 281 h (199, 422) with 85% undergoing intermittent positive pressure ventilation. Median APACHE 3 score was 95 (87, 117) with an overall 30% mortality rate. Median filtration time was 85 h (46, 149). No difference was found between pre- and post-filtration plasma citrate concentrations (79 µmol/L [50] vs. 71 µmol/L [42], p = 0.65). Mean citrate concentration during filtration was 508 µmol/L (221) with a maximum of 1,070 µmol/L. This was significantly higher than the pre/post levels (p < 0.001)., Conclusions: Plasma concentrations of citrate rose significantly during episodes of CRRT using RCA returning back to normal after cessation of treatment., (© 2021 S. Karger AG, Basel.)

Heparins are linear sulfated polysaccharides widely used as anticoagulant drugs. Their nonreducing-end (NRE) has been little investigated due to challenges in their characterization, but is known to be partly generated by enzymatic cleavage with heparanases, resulting in N -sulfated glucosamines at the NRE. Uronic NRE (specifically glucuronic acids) have been isolated from porcine heparin, with GlcA-GlcNS,3S,6S identified as a porcine-specific NRE marker. To further characterize NRE in heparinoids, a building block analysis involving exhaustive heparinase digestion and subsequent reductive amination with sulfanilic acid was performed. This study describes a new method for identifying heparin classical building blocks and novel NRE building blocks using strong anion exchange chromatography on AS11 columns for the assay, and ion-pair liquid chromatography-mass spectrometry for building block identification. Porcine, ovine, and bovine intestine heparins were analyzed. Generally, NRE on these three heparins are highly sulfated moieties, particularly with 3- O sulfates, and the observed composition of the NRE is highly dependent on heparin origin. At the highest level of specificity, the isolated marker was only detected in porcine heparin. However, the proportion of glucosamines in the NRE and the proportion of glucuronic/iduronic configurations in the NRE uronic moieties greatly varied between heparin types., Competing Interests: The author is an employee of Sanofi.

Prior studies (2006-2016) in birds of prey admitted to a wildlife clinic in Massachusetts, USA, revealed widespread exposure to second-generation anticoagulant rodenticides (SGARs) among red-tailed hawks (Buteo jamaicensis, RTHAs). Continued monitoring of species for which historic data are available can reveal trends in exposure that aid in evaluating the effectiveness of risk-mitigation measures. While the majority of exposure-monitoring studies utilize liver tissue collected postmortem, antemortem modalities, such as serum analysis, may be desirable for risk assessments in certain populations. However, the sensitivity of serum for detecting anticoagulant rodenticides (ARs) is not well studied. Paired liver and serum samples from 43 RTHAs were evaluated from 2017 to 2019. In liver tissue, 100% of birds were positive for ARs, with the SGARs brodifacoum, bromadiolone, and difethialone identified most frequently; 91% of birds had liver residues of 2 to 4 ARs. These findings represent the highest exposure both to ARs overall and to multiple ARs in RTHAs compared to previous studies. All birds diagnosed with AR toxicosis (n = 14) were positive for ARs in serum; however, all subclinically exposed birds (n = 29) were negative in serum. These data show that exposure to SGARs remains widespread in RTHAs in this geographic area. In addition, although serum analysis is not sensitive for detecting sublethal exposures in RTHAs, it can potentially support a diagnosis of AR toxicosis in conjunction with other consistent signs. Environ Toxicol Chem 2020;39:2325-2335. © 2020 SETAC., (© 2020 SETAC.)

Low molecular weight heparins (LMWHs) have largely replaced heparin for the treatment and prevention of thrombosis because of their various advantages over unfractionated heparins (UFHs) such as less bleeding, greater bioavailability, and more predictable anticoagulant effects. For special groups of patients, such as pregnant women, children, and patients with renal failure, it is necessary to monitor the anticoagulant activity of LMWHs in the blood. The traditional method used to determine the anticoagulant activity of heparin is the coagulation test. However, the results are various from different laboratories and different reagents. In contrast, the chromogenic substrate method is more accurate, sensitive and is easy to automate. Here, a method for the determination of the anticoagulant activity of LMWHs was developed by using a capillary-electrophoresis-based substrate chromogenic method. In this method, micellar electrokinetic chromatography (MEKC) was used in combination with electrophoretically mediated microanalysis to determine the anti-factor Xa (FXa) activity of LMWHs. The inhibition was measured by employing a chromogenic peptide substrate (CPS) with a p -nitroaniline ( p -NA) moiety as the chromophore. The injection end of the capillary was used as a microreactor in which solutions of LMWHs, antithrombin Ⅲ (ATⅢ), FXa and CPS were successively injected and mixed by using diffusion, the transverse diffusion of laminar flow profiles and applied voltage. The reaction product p -NA was separated from unreacted CPS and sample matrix by using the MEKC mode with discontinuous background electrolyte system. The produced p -NA was baseline separated from the other components and detected at 380 nm to obtain maximum sensitivity. The amount of p -NA was inversely proportional to the activity of LMWHs in the sample. To improve the accuracy of quantification and the method repeatability of methods, nitrofurantoin (NF) was selected as the internal standard, which was added to the solution of CPS. The method was validated and used to measure a set of samples. The method is characterized by automation, good repeatability, high sensitivity, and cost-effectiveness. Additionally, the method does not interfere by the sample matrix, and thus can be used to monitor the anticoagulant activity of LMWHs in plasma.

Heparin is composed of a highly sulfated linear saccharide and is widely used as an anticoagulant. Low molecular weight heparins (LMWHs) are derived from the unfractionated heparin (UFH) by enzymatic or chemical degradation. LMWHs have largely replaced heparin as an anticoagulant for treatment and prevention of thrombosis because of the advantages of less bleeding, greater bioavailability, and more predictable anticoagulant effects in comparison to heparin. Enoxaparin, produced by the alkaline degradation of UFH through β-eliminative cleavage, represents the most commonly used LMWH. The structural characteristics of LMWHs differ from their parent heparin not only in terms of molecular weight but also in the sulfation degree as a result of losing the sulfate ester groups during the manufacturing process. The resulting compositional variation directly leads to a fluctuation in anticoagulant activity. In vitro functional assays showed that there is a wide variation in anticoagulant activity among the various LMWHs from different manufacturers owing to slight differences in the manufacturing process. This will directly affect heparin drug safety. In order to ensure the stability of product quality, it is necessary to develop a method for detecting the degree of heparin sulfation to monitor the stability of UFH and processing conditions. During the last two decades, various analytical methods based on chromatography or NMR have been developed for structural characterization of UFH and LMWHs. However, the reported methods require expensive equipment and professional data processing. These limitations make it difficult to apply the current methods to quality control via sulfation degree determination. Herein, we report a simple and robust method for the detection of the sulfation degree of UFH and LMWHs. The determination is based on the separation of building blocks of heparin obtained by exhaustive digestion of UFH and LMWHs in a mixture of heparinases. A mixed solution of heparinase Ⅰ, Ⅱ, and Ⅲ was prepared to give a final content of 0.13 IU/mL for each enzyme. The digestion of enoxaparin and heparin samples was performed at 25 ℃ for 48 h. By using a capillary electrophoresis (CE) method, a total of 18 oligosaccharides building blocks of heparin, including ten disaccharides, one trisaccharide, three tetrasaccharides, and four 1,6-anhydro derivatives, can be baseline separated. Then, the compositions of enoxaparin and UFH can be precisely determined. Based on the assumption that the molar extinction coefficient of each oligosaccharide at UV 232 nm is the same, the concentration of each oligosaccharide can be conveniently replaced by their peak area, and the accurate number of sulfate ester groups in each disaccharide unit can be determined, hence the average sulfation degree (SD). The developed method allows us to compare the sulfation degree data between the enoxaparin batches from the different manufacturers to evaluate the composition similarity. Herein, eight batches of commercially available enoxaparin from two manufacturers and four batches of UFH source materials were measured. Each sample was measured in triplicate, and the average values as well as the relative standard deviations (RSD) were calculated. The total sulfation degree (T-SD), the individual degree of N -sulfation ( N -SD) and O -sulfation ( O -SD) data were also determined and compared. A significant difference was observed in the SD of the products from the different manufacturers, which indicated that our method can be used as one of the quantitative compositional analysis methods for quality control of LMWHs and UFH. The variation in terms of the sulfation degree of enoxaparin products from different manufacturers can be precisely identified using this method. This allows us to determine the detailed compositional differences between products from the different manufacturers. The obtained satisfactory data show that high fluctuation in the sulfation degree of UFH could transmit to the final enoxaparin products. The consistency of the products can also be evaluated by using these methods. The CE method has several advantages for quantitative compositional analysis of LMWHs, such as high separation efficiency, high sensitivity, automation, short analysis time and low consumption of both sample and reagents. It has a good application potential in the quality control heparin production.

In this study, high-performance liquid chromatography with fluorescence detection (HPLC-FLD) has been used for the first time, for direct determination of warfarin and its major metabolite, 7-hydroxywarfarin, in rat plasma. The simple and sensitive method was developed using Fortis® reversed-phase diphenyl column (150 × 4.6 mm, 3 μm) and a mobile phase composed of phosphate buffer (25 mmol L-1)/methanol/acetonitrile (70:20:10, V/V/V), adjusted to pH 7.4, at a flow rate of 0.8 mL min-1. The diphenyl chemistry of the stationary phase provided a unique selectivity for separating the structurally related aromatic analytes, warfarin and 7-hydroxywarfarin, allowing their successful quantification in the complex plasma matrix. The method was linear over the range 0.01-25 μg mL-1, for warfarin and 7-hydroxywarfarin, and was found to be accurate, precise and selective in accordance with US FDA guidance for bioanalytical method validation. The method was sensitive enough to quantify 0.01 μg mL-1 of warfarin and 7-hydroxywarfarin (LLOQ) using only 100 μL of plasma. The applicability of this method was demonstrated by analyzing samples obtained from rats after oral administration of a single warfarin dose, and studying warfarin and 7-hydroxywarfarin pharmacokinetics.

Heparin (Hep) is a mucopolysaccharide sulfate anticoagulant drug widely used in clinic and thus the quantification of Hep concentration is of great significance. In this work, a simple resonance light scattering (RLS) method for the determination of Hep concentration using plasmonic copper selenide nanoparticles (Cu 2-x Se NPs) was developed. The positively-charged Cu 2-x Se NPs capped by cetyltrimethyl ammonium bromide (CTAB) were aggregated by negatively-charged Hep, consequently inducing a significant enhancement in the RLS signals of plasmonic Cu 2-x Se NPs. Under the optimal reaction conditions, the increased RLS intensity is linearly correlated with the concentrations of Hep in the range of 0.01-0.60 μg mL -1  (R 2  = 0.999), with a low detection limit (LOD) of 4.0 ng mL -1 (3σ). This method offered a simple, sensitive and selective strategy for determining Hep, which can be further successfully applied to quantify Hep in the heparin sodium injection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Garlic seedlings (GS) and blanched garlic seedlings (BGS) are two kinds of common garlic-derived vegetables in China, but little information is available on their bioactive constituents. In this work, chemical profiles and anticoagulant activities of essential oils from GS (EOGS) and BGS (EOBGS) were disclosed and compared for the first time. Sixteen and fourteen volatile compounds were identified in EOGS and EOBGS by GC-MS analysis, and both of them were rich in sulfur-containing compounds, particularly diallyl sulfides accounting for 74.77% and 85.87%, respectively. EOGS and EOBGS exerted anticoagulant activities via intrinsic, extrinsic, and common coagulation pathways as well as by lowering the content of fibrinogen; EOGS exceeded EOBGS in the activation of intrinsic and extrinsic coagulation pathways, while EOBGS outperformed EOGS on the activation of the common coagulation pathway, which was even superior to that of heparin at the same dose. Herein, the results of the present investigation will give a strong clue that EOGS and EOBGS are more likely to lead to a promising way to vegetable-based anticoagulants.

Rivaroxaban, as a direct oral anticoagulant, has been widely used in the treatment and prevention of thrombosis disease (TD). However, even if the same dose of rivaroxaban is taken, different pathophysiological characteristics of TD patients determine the differences in plasma concentrations between individuals, leading to the difficulties of dosage selection and plasma concentration control. Conventional rivaroxaban detection methods, including prothrombin time method, anti-Xa assay and liquid chromatography-tandem mass spectrometry (LC-MS/MS), are not widely used in clinical practice due to the limitations of accuracy, speed and cost. Here, we present a simple quantitative detection method for rivaroxaban by terahertz (THz) spectroscopy. Combining density functional theory (DFT) method and THz spectroscopy, the THz absorption peaks of rivaroxaban and the corresponding low-frequency vibrational modes are studied theoretically and experimentally. We find linear relationships between the amplitudes of these characteristic peaks and the concentrations of rivaroxaban. Based on these linear functions, we can analyse the rivaroxaban concentration with a detection time of 1 minute per test and the lowest detection limit of 2 μmol mL -1 . As compared to Raman spectroscopy method (its detection limit is about 80 μmol mL -1 ), our method has more potential and is practical for the clinical quantitative detection of rivaroxaban as well as other direct oral anticoagulants.

Background: Coagulation panels are ordered for a variety of conditions in the emergency department (ED)., Objective: This narrative review evaluates specific conditions for which a coagulation panel is commonly ordered but has limited utility in medical decision-making., Discussion: Coagulation panels consist of partial thromboplastin time (PTT) or activated partial thromboplastin time (aPTT), prothrombin time (PT), and international normalized ratio (INR). These tests evaluate the coagulation pathway which leads to formation of a fibrin clot. The coagulation panel can monitor warfarin and heparin therapy, evaluate for vitamin K deficiency, evaluate for malnutrition or severe systemic disease, and assess hemostatic function in the setting of bleeding. The utility of coagulation testing in chest pain evaluation, routine perioperative assessment, prior to initiation of anticoagulation, and as screening for admitted patients is low, with little to no change in patient management based on results of these panels. Coagulation testing should be considered in systemically ill patients, those with a prior history of bleeding or family history of bleeding, patients on anticoagulation, or patients with active hemorrhage and signs of bleeding. Thromboelastography and rotational thromboelastometry offer more reliable measures of coagulation function., Conclusions: Little utility for coagulation assessment is present for the evaluation of chest pain, routine perioperative assessment, initiation of anticoagulation, and screening for admitted patients. However, coagulation panel assessment should be considered in patients with hemorrhage, patients on anticoagulation, and personal history or family history of bleeding., Competing Interests: Declaration of competing interest None., (Published by Elsevier Inc.)

Snakebite is a neglected tropical disease that results in a variety of systemic and local pathologies in envenomed victims and is responsible for around 138,000 deaths every year. Many snake venoms cause severe coagulopathy that makes victims vulnerable to suffering life-threating haemorrhage. The mechanisms of action of coagulopathic snake venom toxins are diverse and can result in both anticoagulant and procoagulant effects. However, because snake venoms consist of a mixture of numerous protein and peptide components, high throughput characterizations of specific target bioactives is challenging. In this study, we applied a combination of analytical and pharmacological methods to identify snake venom toxins from a wide diversity of snake species that perturb coagulation. To do so, we used a high-throughput screening approach consisting of a miniaturised plasma coagulation assay in combination with a venom nanofractionation approach. Twenty snake venoms were first separated using reversed-phase liquid chromatography, and a post-column split allowed a small fraction to be analyzed with mass spectrometry, while the larger fraction was collected and dispensed onto 384-well plates. After fraction collection, any solvent present in the wells was removed by means of freeze-drying, after which it was possible to perform a plasma coagulation assay in order to detect coagulopathic activity. Our results demonstrate that many snake venoms simultaneously contain both procoagulant and anticoagulant bioactives that contribute to coagulopathy. In-depth identification analysis from seven medically-important venoms, via mass spectrometry and nanoLC-MS/MS, revealed that phospholipase A2 toxins are frequently identified in anticoagulant venom fractions, while serine protease and metalloproteinase toxins are often associated with procoagulant bioactivities. The nanofractionation and proteomics approach applied herein seems likely to be a valuable tool for the rational development of next-generation snakebite treatments by facilitating the rapid identification and fractionation of coagulopathic toxins, thereby enabling specific targeting of these toxins by new therapeutics such as monoclonal antibodies and small molecule inhibitors., Competing Interests: The authors have declared that no competing interests exist.

Direct oral anticoagulants (DOACs) have been commonly used for the treatment of venous thromboembolism and for the prevention of stroke in patients with atrial fibrillation. Despite not being initially recommended, monitoring DOACs plasma concentrations is now recognized as essential in emergency situations and in special populations. Moreover, the inter-individual variability found in real studies as well as the high reported non-adherence are corroborating the importance of determining the individual relationship between administered doses, plasma concentrations and pharmacological effects. Therefore, accurate but user-friendly bioanalytical techniques are required to monitor DOACs plasma concentrations in routine clinical practice and phase IV clinical trials. Herein, a fast and simple high performance liquid chromatography (HPLC) method coupled to diode array detection (DAD) was developed, validated and applied to quantify the four currently marketed DOACs (apixaban, edoxaban, dabigatran and rivaroxaban). Sample preparation was performed by solid phase extraction followed by evaporation and concentration of the analytes. Chromatographic separation was accomplished within 6 min on a reversed-phase column (octadecyl-silica packing material; 55 mm × 4 mm, 3 μm particle size), applying a mobile phase composed of an aqueous solution of formic acid (0.1 %, v/v) and acetonitrile, pumped with a gradient elution at 30 °C. The proposed method was linear (r 2 ≥ 0.993) within the concentration ranges of 0.017-5.28 μg mL -1 , 0.066-5.28 μg mL -1 , 0.033-5.28 μg mL -1 and 0.017-5.28 μg mL -1 for apixaban, dabigatran, edoxaban and rivaroxaban, respectively, all of them including the expected range of therapeutic concentrations. Overall, intra- and inter-day trueness of quality control samples, including at the lower limit of quantification (LLOQ), varied between -12.98 to 5.79 %, while imprecision was lower than 16.43 %, supporting that the method is accurate and precise in accordance to international guidelines. Recovery and stability were also assessed and allowed the method to be applied in clinical practice, during therapeutic drug monitoring., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Introduction: The aim of the study was to perform analytical verification and comparison of chromogenic assays for determination of dabigatran, rivaroxaban and apixaban concentration on BCSXP and STA Compact Max analysers., Materials and Methods: Precision, linearity, measurement uncertainty estimation and determination of limit of blank, limit of determination and limit of quantification were calculated. Analytical performance specifications were set according to manufacturer specifications and literature data on between laboratory variability. Comparison of the methods was done using Bland-Altman and Passing-Bablok regression analysis., Results: Obtained results have shown acceptable precision on STA Compact Max only for dabigatran (CV = 3.5%) at lower concentration level comparing to manufacturer declaration (CV = 3.6%). On BCSXP, the highest coefficient of variation has been shown for apixaban (6.1%) at lower concentration level. Within laboratory precision was not met on STA Compact Max for all assays. Bland-Altman analysis has shown statistically significant bias for dabigatran (23.2%, 95%CI 11.2 - 35.3; P < 0.001) and apixaban (8.4%, 95%CI 1.2 - 15.6; P = 0.023). Passing-Bablok regression analysis has shown systematic and proportional deviation between methods for rivaroxaban (y = 6.52 (2.94 to 11.83) + 0.84 (0.80 to 0.89) x., Conclusion: Chromogenic assays for dabigatran, rivaroxaban and apixaban on BCSXP and STA Compact Max analysers are shown as methods with satisfactory long-term analytical performance specifications for determination of direct oral anticoagulants in clinical laboratories. However, we cannot recommend interchangeable use because of the significant bias between assays., Competing Interests: Potential conflict of interest: None declared., (Croatian Society of Medical Biochemistry and Laboratory Medicine.)

In this paper a dispersive magnetic-solid phase extraction (MSPE) using a graphene nanocomposite (rG/Fe 3 O 4 ) followed by ultra high performance liquid chromatography with photodiode array detection has been developed for the simultaneous analysis of new class of oral anticoagulants (NOAs) in human plasma. The performance of the nanocomposite graphene@Fe 3 O 4 on the magnetic solid phase extraction of apixaban, rivaroxaban and dabigatran has been optimized using a Box-Behnken design of experiment. The amount of graphene nanocomposite, the sample pH and the adsorption time were the investigated parameters as a function of the extraction recovery. The analytical method was fully validated based on linearity, limit of detection (LOD), limit of detection (LOQ), inter- and intra-day precision and trueness, and extraction yield. Under optimal condition, excellent linearity (R 2 > 0.9987) over the range (0.001-5.0 μg/mL), limit of detection (0.003 μg/mL), precision (0.81-8.97% RSD) and trueness (-5 to 9 % BIAS%) were observed for the target drugs. The average extraction recovery under optimal from plasma samples ranged between 96.6-98.6% for apixaban, rivaroxaban and dabigatran and the internal standard. The proposed method was developed, validated and successfully applied to the measurement of these NOAs in patients. The new approach offers an attractive alternative for the simultaneous analysis of the selected NOAs from plasma samples, providing several advantages including fewer sample preparation steps, ease of performance, and higher recoveries compared to traditional methodologies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Nonvitamin K antagonist oral anticoagulants (NOACs) have emerged as the preferred choice for the treatment of atrial fibrillation (AF). The establishment of a therapeutic range to minimize bleeding and thrombosis is important for personalized treatment of NOACs. The importance of dried blood spots (DBSs) has increased in medical care. An efficient and effective DBS analytical method could facilitate the concentration management of NOACs. The postcolumn infused internal standard (PCI-IS) method was applied to estimate spot volume and quantify dabigatran, rivaroxaban, and apixaban concentrations on DBS cards. The extraction solvent contented 0.1% formic acid and 70% ACN with a successive extraction procedure. Paired DBS and plasma samples from patients undergoing NOAC therapy ( n = 269) were used to calculate conversion factors. [ 13 C 6 ]-Rivaroxaban was selected as the PCI-IS. The quantification accuracy for the three NOACs was within 88.9-104.3%. The RSDs of the repeatability and intermediate precision were below 10%. The obtained conversion factors of DBS to plasma concentrations of dabigatran, apixaban, and rivaroxaban were 1.81, 1.59, and 1.31, respectively. Bland-Altman analysis showed that the % differences between predicted and measured plasma concentrations were within a bias of ±20%. The result showed that PCI-IS was an accurate and efficient LC-MS/MS method to simultaneously estimate blood volume and NOAC concentrations on DBS cards. The stability results revealed that the DBS sampling strategy could improve compound stability. The developed method offers a new strategy for the therapeutic drug monitoring of NOACs and may improve the safe use of these drugs.

A seminal question in ecotoxicology is the extent to which contaminant exposure evokes prolonged effects on physiological function and fitness. A series of studies were undertaken with American kestrels ingesting environmentally realistic concentrations of the second-generation anticoagulant rodenticide (SGAR) brodifacoum. Kestrels fed brodifacoum at 0.3, 1.0, or 3.0 µg/g diet wet weight for 7 d exhibited dose-dependent hemorrhage, histopathological lesions, and coagulopathy (prolonged prothrombin and Russell's viper venom times). Following termination of a 7-d exposure to 0.5 µg brodifacoum/g diet, prolonged blood clotting time returned to baseline values within 1 wk, but brodifacoum residues in liver and kidney persisted during the 28-d recovery period (terminal half-life estimates >50 d). To examine the hazard of sequential anticoagulant rodenticide (AR) exposure, kestrels were exposed to either the first-generation AR chlorophacinone (1.5 µg/g diet) or the SGAR brodifacoum (0.5 µg/g diet) for 7 d and, following a recovery period, challenged with a low dose of chlorophacinone (0.75 µg/g diet) for 7 d. In brodifacoum-exposed kestrels, the challenge exposure clearly prolonged prothrombin time compared to naive controls and kestrels previously exposed to chlorophacinone. These data provide evidence that the SGAR brodifacoum may have prolonged effects that increase the toxicity of subsequent AR exposure. Because free-ranging predatory and scavenging wildlife are often repeatedly exposed to ARs, such protracted toxicological effects need to be considered in hazard and risk assessments. Environ Toxicol Chem 2020;39:468-481. © 2020 SETAC., (© 2019 SETAC.)

During extracorporeal membrane oxygenation (ECMO), a delicate balance is required to titrate systemic anticoagulation to prevent thrombotic complications within the circuit and prevent bleeding in the patient. Despite focused efforts to achieve this balance, the frequency of both thrombotic and bleeding events remains high. Anticoagulation is complicated to manage in this population due to the complexities of the hemostatic system that are compounded by age-related developmental hemostatic changes, variable effects of the etiology of critical illness on hemostasis, and blood-circuit interaction. Lack of high-quality data to guide anticoagulation management in ECMO patients results in marked practice variability among centers. One aspect of anticoagulation therapy that is particularly challenging is the use of antithrombin (AT) supplementation for heparin resistance. This is especially controversial in the neonatal and pediatric population due to the baseline higher risk of bleeding in this cohort. The indication for AT supplementation is further compounded by the potential inaccuracy of the diagnosis of heparin resistance based on the standard laboratory parameters used to assess heparin effect. With concerns regarding the adverse impact of bleeding and thrombosis, clinicians and institutions are faced with making difficult, real-time decisions aimed at optimizing anticoagulation in this setting. In this clinically focused review, the authors discuss the complexities of anticoagulation monitoring and therapeutic intervention for patients on ECMO and examine the challenges surrounding AT supplementation given both the historical and current perspectives summarized in the literature on these topics.

Diphacinone (DPN) is an extensively used anticoagulant rodenticide that is also considered a hazardous chemical, which poses a threat to nontarget species. DPN poisoning cases in humans or other species frequently occur, while rapid and sensitive detection methods are rarely reported. Thus, it is meaningful to develop an immunoassay for DPN detection with high sensitivity and specificity. In this study, a hapten was synthesized and then conjugated with carrier proteins to prepare the immunogens with different conjugation ratios for the preparation of antibody. After evaluation of the antisera using an indirect competitive enzyme-linked immunosorbent assay (icELISA) and statistical analysis, we found that the immunogen prepared using the N,N-dicyclohexylcarbodiimide (DCC) method with a conjugation ratio of 28.5 could elicit mice to generate antibodies with high performance. Using hybridoma technology, we obtained the specific monoclonal antibody (mAb) 4G5 with a half maximal inhibitory concentration (IC 50 ) of 0.82 ng/mL in buffer solution. We initially explored the recognition mechanism of DPN/CLDPN and mAb from both conformational and electronic aspects. Then, mAb 4G5 was applied to develop icELISA for biological samples. The limits of detection (LODs) of icELISA were 0.28 μg/L, 0.32 μg/L, and 0.55 μg/kg for swine plasma, urine, and liver samples, respectively, and the recoveries ranged from 72.3 to 103.3% with a coefficient of variation (CV) of less than 12.3% in spiked samples. In summary, we developed a sensitive, specific, and accurate icELISA for the detection of DPN in biological samples, which showed potential in food safety analysis and clinical diagnosis. Graphical abstract.

Heparin is a carbohydrate polymer, which is clinically used as an anticoagulant for the treatment of thrombotic disorders. The anticoagulant process is mainly mediated by the interaction of heparin with antithrombin followed by inhibition of clotting factors IIa (FIIa) and Xa (FXa). The influence of polymer disaccharide structure, average molecular weight and impurity profiling (e.g., chloride and water content) was investigated by NMR spectrometry and principal component analysis (PCA) for a representative dataset of porcine heparin samples (n = 509). A significant linear dependence was found between anticoagulant activity and scores on the third principal component (PC3) based on the non-targeted analysis of 1 H NMR fingerprints. The correlation between average molecular values and anticoagulant activity for the 24 porcine heparin samples from two manufacturers was linear (R = 0.85) over typical values for porcine heparin preparations. Chloride and water contents were identified as negatively influencing factors for the actual activity values as their presence decrease the "pharmaceutically active" organic part of heparin preparations. Some suggestions regarding manufacturing process are made according to the results., (Copyright © 2019 Elsevier B.V. All rights reserved.)

NMR spectroscopy has become an essential diagnostic tool for pharmaceutical preparations worldwide, with many runs performed annually on a routine basis. In this study multivariate analysis was performed on a big dataset of 990 NMR heparin spectra recorded over six years (2012-2017) in our laboratory. Several steps of statistical analysis of accumulated data were used to differentiate samples according to animal origin (bovine, porcine and ovine heparin), purity grade (crude and purified heparin), distributing company as well as to estimate their closeness to the heparin reference sample (SST) provided by US Pharmacopeia. The projection of new samples on these models can automatically forecast of all mentioned qualitative heparin properties within one minute. NMR data of heparin pharmaceutical preparations provided a good opportunity to demonstrate the potential of exploratory data analysis considering the same sample preparation and acquisition parameters were used., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Antistasin , which was originally discovered in the salivary glands of the Mexican leech Haementeria officinalis , was newly isolated from Helobdella austinensis . To confirm the temporal expression of antistasin during embryogenesis, we carried out semi-quantitative RT-PCR. Hau-antistasin1 was uniquely expressed at stage 4 of the cleavage and was strongly expressed in the late stages of organogenesis, as were other antistasin members. In order to confirm the spatial expression of antistasin , we performed fluorescence in situ hybridization in the late stages of organogenesis. The expression of each antistasin in the proboscis showed a similar pattern and varied in expression in the body. In addition, the spatial expression of antistasin orthologs in different leeches showed the possibility of different function across leech species. Hau-antistasin1 was expressed in the same region as hedgehog , which is a known mediator of signal transduction pathway. Hau-antistasin1 is probably a downstream target of Hedgehog signaling, involved in segment polarity signal pathway.

Heparin is a glycosaminoglycan with the highest negative charge density of any known biological molecule. Herein, this highly negative charge structure of heparin and the charge effect from positively-charged AuNPs for luminol chemiluminescence (CL) reaction were combined to build a facile and sensitive CL strategy for detection of heparin. The highly negative charge structure of heparin molecules (four negatively-charged side groups per repeat unit) and the effective signal amplification of charge effect from positively-charged AuNPs make this analysis to display high sensitivity for heparin detection, and the detection limit is as low as 0.06 ng/mL. It is about two orders of magnitude lower than the previously reported colorimetric assay and far lower than the current analysis methods. The established CL strategy is to use the electrostatic interaction between heparin and signal probe (positively-charged AuNPs). Since polyanionic heparin has the highest negative charge in biological system, this CL sensing shows high selectivity for the detection of heparin, and hyaluronic acid (HA), an analogue of heparin, cannot cause interference. This CL sensing succeeded in detecting heparin in human serum samples. Besides, polycationic protamine, heparin antidote, can respond to the system's CL signals through its strong interactions with heparin, thus indirectly detecting protamine. For protamine in serum samples, the detection result was basically consistent with Coomassie brilliant blue assay., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: To date, the quantification of the anticoagulant (ACD-A) in plasma units has been based on theoretical calculations. An accurate quantification could help minimize the risks associated with plasmapheresis, given that the total ACD-A used during the procedure is distributed between the donor and the plasma unit. Our aim was to experimentally quantify the volume of ACD-A in units collected by plasmapheresis., Study Design and Methods: We used proton nuclear magnetic resonance spectroscopy to measure the ACD-A volume in 295 plasma units collected by the Azienda USL-IRCCS of Reggio Emilia, Italy. We analyzed the determinants of the differences between estimated and measured ACD-A through multivariate regression models., Results: The experimentally measured ACD-A in plasma units was variable, with 45% of the samples showing a discrepancy of more than 15 mL compared to the manufacturer's estimate. ACD-A was underestimated for higher density of the units (p < 0.0005); a weak association was also observed with triglycerides (underestimated for higher levels, p = 0.015) and sex (overestimated in females, p = 0.008), but our model explained only 35% of the individual variability., Conclusion: The manufacturer's algorithms do not accurately estimate the ACD-A in units collected by plasmapheresis. Donor-related characteristics may affect ACD-A distribution between donor and plasma unit, thereby explaining the discrepancies between estimate and measurement. Errors in the estimate of the ACD-A actually received by donors could hamper studies on dose-response relationship between anticoagulant and adverse reactions. Our work should stimulate research on tailored procedures aimed at minimizing the anticoagulant received by donors and increasing plasmapheresis safety., (© 2019 AABB.)