Evaluating a Rapid Field Assessment System for Anticoagulant Rodenticide Exposure of Raptors.

Anticoagulant rodenticides (ARs) are commonly used to control rodent pests. However, worldwide, their use is associated with secondary and tertiary poisoning of nontarget species, especially predatory and scavenging birds. No medical device can rapidly test for AR exposure of avian wildlife. Prothrombin time (PT) is a useful biomarker for AR exposure, and multiple commercially available point-of-care (POC) devices measure PT of humans, and domestic and companion mammals. We evaluated the potential of one commercially available POC device, the Coag-Sense® PT/INR Monitoring System, to rapidly detect AR exposure of living birds of prey. The Coag-Sense device delivered repeatable PT measurements on avian blood samples collected from four species of raptors trapped during migration (Intraclass Correlation Coefficient > 0.9; overall intra-sample variation CV: 5.7%). However, PT measurements reported by the Coag-Sense system from 81 ferruginous hawk (Buteo regalis) nestlings were not correlated to those measured by a one-stage laboratory avian PT assay (r = - 0.017, p = 0.88). Although precise, the lack of agreement in PT estimates from the Coag-Sense device and the laboratory assay indicates that this device is not suitable for detecting potential AR exposure of birds of prey. The lack of suitability may be related to the use of a mammalian reagent in the clotting reaction, suggesting that the device may perform better in testing mammalian wildlife.

3D Cell-Culture Models for the Assessment of Anticoagulant and Anti-Inflammatory Properties of Endothelial Cells.

Endothelial cells (EC) play a crucial role in the pathophysiology of cardiovascular diseases, ischemia/reperfusion injury, and graft rejection in (xeno-)transplantation. In such nonphysiological conditions, EC are known to lose their quiescent phenotype and switch into an actively pro-inflammatory, procoagulant, and anti-fibrinolytic state. This case happens essentially because the endothelial glycocalyx-a layer of proteoglycans and glycoproteins covering the luminal surface of the endothelium-is shed. Heparan sulfate, one of the main components of the endothelial glycocalyx, contributes to its negative charge. In addition, many plasma proteins such as antithrombin III, superoxide dismutase, C1 inhibitor, and growth factors and cytokines bind to heparan sulfate and by this scenario contribute to the establishment of an anticoagulant and anti-inflammatory endothelial surface. Shedding of the glycocalyx results in a loss of plasma proteins from the endothelial surface, and this phenomenon causes the switch in phenotype. Particularly in xenotransplantation, both hyperacute and acute vascular rejection are characterized by coagulation dysregulation, a situation in which EC are the main players.Since many years, EC have been used in vitro in 2D flatbed cell culture models, with or without the application of shear stress. Such models have also been used to assess the effect of human transgenes on complement- and coagulation-mediated damage of porcine EC in the context of xenotransplantation. The methods described in this chapter include the analysis of endothelial cell-blood interactions without the necessity of using anticoagulants as the increased EC surface-to-volume ratio allows for natural anticoagulation of blood. Furthermore, this chapter contains the description of a novel microfluidic in vitro model carrying important features of small blood vessels, such as a 3D round-section geometry, shear stress, and pulsatile flow-all this in a closed circuit, recirculating system aiming at reproducing closely the in vivo situation in small vessels.

In Vitro Assessment of Pharmacokinetic Drug-Drug Interactions of Direct Oral Anticoagulants: Type 5-Phosphodiesterase Inhibitors Are Inhibitors of Rivaroxaban and Apixaban Efflux by P-Glycoprotein.

Because of their lower bleeding risk and simplicity of use, direct oral anticoagulants (DOACs) could represent an interesting alternative to conventional anticoagulant treatment with vitamin K antagonists for patients with pulmonary arterial hypertension (PAH). P-glycoprotein (P-gp) plays a key role in DOAC pharmacokinetics. Type 5-phosphodiesterase inhibitors (PDE5is), a drug class commonly used in the treatment of PAH, have been shown to strongly inhibit P-gp. This work aimed to assess potential P-gp-mediated drug-drug interactions between PDE5is and DOACs using in vitro methods. A cellular model of drug transport assay, using P-gp-overexpressing Madin-Darby canine kidney cells (transfected with the human P-gp gene), was used to determine the bidirectional permeabilities of two DOACs (rivaroxaban and apixaban) in the absence and presence of increasing concentrations (0.5-100 µM) of three PDE5is (sildenafil, tadalafil, and vardenafil). Permeabilities and efflux ratios were calculated from DOAC concentrations, were measured with liquid chromatography coupled with mass spectrometry, and were subsequently used to determine the PDE5i percentage of inhibition and half maximal inhibitory concentration (IC50 ). Rivaroxaban efflux was inhibited by 99%, 66%, and 100% with 100 µM sildenafil, tadalafil, and vardenafil, respectively. Similarly, apixaban efflux was inhibited by 97%, 74%, and 100%, respectively. The IC50 values of the three PDE5is were 8, 28, and 5 µM for rivaroxaban and 23, 15, and 3 µM for apixaban, respectively. This study showed strong in vitro inhibition of DOAC efflux by PDE5is. In vivo studies are required to determine the clinical relevance of these interactions.

Assessment of the protein interaction between coagulation factor XII and corn trypsin inhibitor by molecular docking and biochemical validation.

Essentials Corn Trypsin Inhibitor (CTI) is a selective inhibitor of coagulation Factor XII (FXII). Molecular modelling of the CTI-FXIIa complex suggested a canonical inhibitor binding mode. Mutagenesis revealed the CTI inhibitory loop and helices α1 and α2 mediate the interaction. This confirms that CTI inhibits FXII in canonical fashion and validates the molecular model. SUMMARY: Background Corn trypsin inhibitor (CTI) has selectivity for the serine proteases coagulation factor XII and trypsin. CTI is in widespread use as a reagent that specifically inhibits the intrinsic pathway of blood coagulation but not the extrinsic pathway. Objectives To investigate the molecular basis of FXII inhibition by CTI. Methods We performed molecular docking of CTI, using its known crystal structure, with a model of the activated FXII (FXIIa) protease domain. The interaction model was verified by use of a panel of recombinant CTI variants tested for their ability to inhibit FXIIa enzymatic activity in a substrate cleavage assay. Results The docking predicted that: (i) the CTI central inhibitory loop P1 Arg34 side chain forms a salt bridge with the FXIIa S1 pocket Asp189 side chain; (ii) Trp22 from CTI helix α1 interacts with the FXIIa S3 pocket; and (iii) Arg43 from CTI helix α2 forms a salt bridge with FXIIa H1 pocket Asp60A. CTI amino acid substitution R34A negated all inhibitory activity, whereas the G32W, L35A, W22A and R42A/R43A substitutions reduced activity by large degrees of 108-fold, 41-fold, 158-fold, and 100-fold, respectively; the R27A, W37A, W39A and R42A substitutions had no effect. Synthetic peptides spanning CTI residues 20-44 had inhibitory activity that was three-fold to 4000-fold less than that of full-length CTI. Conclusions The data confirm the validity of a canonical model of the FXIIa-CTI interaction, with helix α1 (Trp22), central inhibitory loop (Arg34) and helix α2 (Arg43) of CTI being required for effective binding by contacting the S1, S3 and H1 pockets of FXIIa, respectively.

Assessment of anticoagulant rodenticide exposure in six raptor species from the Canary Islands (Spain).

Anticoagulant rodenticides are highly toxic compounds that are widely used for pest control of rodents, but that also may threaten the wildlife's health. This work aimed to assess the exposure to first- and second-generation anticoagulant rodenticides (ARs) in six birds of prey species from the Canary Islands (Spain). The concentrations of seven widely used ARs were determined by LC-MS/MS in 104 liver samples of six species of birds of prey (Buteo buteo, Accipiter nisus, Falco pelegrinoides, Falco tinnunculus, Asio otus, and Tyto alba). We determined that 61% of the livers had detectable residues of at least one AR. The most frequently detected AR was bromadiolone, which was detected in 60.3% of the positive cases. The detection frequencies of these compounds varied widely, depending on the species. More than 75% of the A. nisus, T. alba, and A. otus individuals had detectable rodenticide residues in the liver. However, F. tinnunculus exhibited the highest concentrations of AR, with median values above 100 ng/g w.w. We did not detect first-generation ARs in any of the samples. When grouped, nocturnal species exhibited higher AR concentrations than diurnal species (P<0.001). The residue levels were higher among small mammal-eaters than bird-eaters (P<0.01). While most animals exhibited no macroscopic signs of coagulation disorders, approximately 35% exceeded the threshold levels of toxicity, which suggests that these compounds could weaken these animals in their natural environment. In conclusion, the control of rodent populations by ARs suggests that these compounds will enter the food chain and thus threaten the vulnerable populations of raptors on the Canary Islands. Our findings require authorities to ban or strictly control the use of these rodenticides in the natural environment for the conservation of raptors and other predatory species.

Biochemical and biological analysis of Philodryas baroni (Baron's green racer; Dipsadidae) venom: relevance to the findings of human risk assessment.

Philodryas baroni--an attractively colored snake--has become readily available through the exotic pet trade. Most people consider this species harmless; however, it has already caused human envenomation. As little is known about the venom from this South American opisthoglyphous "colubrid" snake, herein, we studied its protein composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as well as its effects on the hemostatic system. Both reducing and nonreducing SDS-PAGE analysis demonstrated that the venom exhibits greatest complexity in the range of 50-80 kDa. The venom displayed proteolytic activity toward azocollagen, with a specific activity of 75.5 U mg⁻¹, and rapidly hydrolyzed the Aα-chain of fibrinogen, exhibiting lower activity toward the Bß- and γ-chains. The venom from P. baroni showed no platelet proaggregating activity per se, but it inhibited collagen- and thrombin-induced platelet aggregation. Prominent hemorrhage developed in mouse skin after intradermal injection of the crude venom, and its minimum hemorrhagic dose was 13.9 µg. When injected intramuscularly into the gastrocnemius of mice, the venom induced local effects such as hemorrhage, myonecrosis, edema, and leucocyte infiltration. Due to its venom toxicity shown herein, P. baroni should be considered dangerous to humans and any medically significant bite should be promptly reviewed by a qualified health professional.

Combination of a two-step fluorescence assay and a two-step anti-Factor Xa assay for detection of heparin falsifications and protein in heparins.

There are several methods for sensitive detection of oversulfated chondroitin sulfate (OSCS) in heparin. Although contamination with OSCS is unlikely to be repeated, use of other compounds to counterfeit heparin must be considered. We have previously developed a two-step fluorescence microplate assay (two-step FI assay) for detection of OSCS. First, the heparin sample is incubated with heparinase I, then its increasing effect on the fluorescence intensity (FI) of the sensor molecule Polymer-H is measured (PolyH assay). The high sensitivity of the assay is shown to be based on heparinase I inhibition by OSCS. The objective of this study was to evaluate another assay option - indirect quantification of OSCS after heparinase I incubation by means of the anti-Factor Xa (aXa) activity of the remaining undegraded heparin (two-step aXa assay). We also examined, whether other heparin mimetics (HepM), direct Factor Xa inhibitors (DXI), and protein impurities are detectable by use of these assays. Heparin was spiked with different amounts of HepM including OSCS, pentosan polysulfate, dextran sulfate, curdlan sulfate, the natural contaminant dermatan sulfate, the DXI rivaroxaban, and BSA as a protein. These samples were compared with pure heparin in the two-step FI assay, the two-step aXa assay, and in the PolyH assay and the aXa assay without heparinase I incubation. Both two-step assays sensitively measured contamination with all the HepM (LOD ≤ 0.5%, LOQ ≤ 0.7%). The two-step aXa assay also detected rivaroxaban (LOD 0.3%, LOQ 0.4%), whereas the two-step FI assay was shown to be suited to determination of protein impurities (LOD 0.11%, LOQ 0.13%). Use of two different heparinase I inactivation procedures enabled clear differentiation between protein, HepM, and both contaminants. Finally, with the aXa assay the heparin potency can be determined in the same assay run, whereas the FI increase in the PolyH assay was shown to be useful for identification. In conclusion, both the two-step FI assay and the two-step aXa assay are sensitive, rapid, and simple tests for the detection of counterfeit heparin. Comprehensive information about heparin quality can be obtained by their combined use and the parallel measurement of non-incubated heparin samples.

Benefits of pharmacogenomics in drug development-earlier launch of drugs and less adverse events.

Currently, pharmaceutical companies are reluctant to introduce pharmacogenomics (PGx) in their practice, since cost-benefit of PGx is obscure and methodology to use PGx in drug development has not been fully established yet. The purpose of this study is to investigate advantages obtained by introducing PGx in clinical trials. Particularly, taking Warfarin as an example, we investigate benefits of Enrichment effect that raises response rate of the drug by PGx. When response rate is raised by only 5%, cost of a clinical trial can be reduced to about 40% of a conventional clinical trial. Furthermore, since period necessary for a trial also can be reduced, development period can be shortened by about 750 days. In summary, PGx enables earlier launch of a drug with less cost, representing benefit to pharmaceutical companies, patients and public as a whole.

Will there be a role for genotyping in warfarin therapy?

PURPOSE OF REVIEW: In North America warfarin is the current standard for oral anticoagulation therapy in the treatment and/or prophylaxis of different thrombotic conditions. In daily clinical practice a significant proportion of patients on long-term warfarin therapy fail to stabilize within their target therapeutic range leading to a resultant increased risk of thromboembolism or bleeding. Various authors and agencies advocate the role of genetic testing to guide warfarin dosing. RECENT FINDINGS: Evidence regarding the clinical efficacy and cost-effectiveness of genotype-based warfarin dosing has been conflicting, although some recent studies have suggested a potential benefit in certain subgroups. SUMMARY: More evidence is needed before the wide adoption of genotype-based warfarin dosing. Future studies should be designed to address outcomes such as major bleeding or recurrent thrombosis, and allow economic evaluations.

Should we be applying warfarin pharmacogenetics to clinical practice? No, not now.

The U.S. Food and Drug Administration modified warfarin labeling in 2007 to suggest, but not mandate, pharmacogenetic testing. Genetic analysis is now commercially available. However, results predict only one third of all dosing variation, the value of testing in reducing bleeding and thrombosis rates remains unproved, and cost-effectiveness is not established. Careful consideration of clinical factors that influence dosing, conscientious prothrombin time monitoring, and sage dosage adjustment remain paramount in warfarin management. Further study is required before routine warfarin pharmacogenetic testing can be recommended.

Prevalence of combinatorial CYP2C9 and VKORC1 genotypes in Puerto Ricans: implications for warfarin management in Hispanics.

Polymorphisms in the cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) genes significantly alter the effective warfarin dose. We determined the frequencies of alleles, single carriers, and double carriers of single nucleotide polymorphisms (SNPs) in the CYP2C9 and VKORC1 genes in a Puerto Rican cohort and gauged the impact of these polymorphisms on warfarin dosage using a published algorithm. A total of 92 DNA samples were genotyped using Luminex x-MAP technology. The polymorphism frequencies were 6.52%, 5.43% and 28.8% for CYP2C9 *2, *3 and VKORC1-1639 C>A polymorphisms, respectively. The prevalence of combinatorial genotypes was 16% for carriers of both the CYP2C9 and VKORC1 polymorphisms, 9% for carriers of CYP2C9 polymorphisms, 35% for carriers of the VKORC1 polymorphism, and the remaining 40% were non-carriers for either gene. Based on a published warfarin dosing algorithm, single, double and triple carriers of functionally deficient polymorphisms predict reductions of 1.0-1.6, 2.0-2.9, and 2.9-3.7 mg/day, respectively, in warfarin dose. Overall, 60% of the population carried at least a single polymorphism predicting deficient warfarin metabolism or responsiveness and 13% were double carriers with polymorphisms in both genes studied. Combinatorial genotyping of CYP2C9 and VKORC1 can allow for individualized dosing of warfarin among patients with gene polymorphisms, potentially reducing the risk of stroke or bleeding.

[Pharmacogenetic testing: soon before every prescription?]. / Tests pharmacogénétiques: bientôt avant chaque prescription?

Genetic polymorphisms have currently been described in more than 200 systems affecting pharmacological responses (cytochromes P450, conjugation enzymes, transporters, receptors, effectors of response, protection mechanisms, determinants of immunity). Pharmacogenetic testing, i.e. the profiling of individual patients for such variations, is about to become largely available. Recent progress in the pharmacogenetics of tamoxifen, oral anticoagulants and anti-HIV agents is reviewed to discuss critically their potential impact on prescription and contribution/limits for improving rational and safe use of pharmaceuticals. Prospective controlled trials are required to evaluate large-scale pharmacogenetic testing in therapeutics. Ethical, social and psychological issues deserve particular attention.

Individual genomes instead of race for personalized medicine.

The cost of sequencing and genotyping is aggressively decreasing, enabling pervasive personalized genomic screening for drug reactions. Drug-metabolizing genes have been characterized sufficiently to enable practitioners to go beyond simplistic ethnic characterization and into the precisely targeted world of personal genomics. We examine six drug-metabolizing genes in J. Craig Venter and James Watson, two Caucasian men whose genomes were recently sequenced. Their genetic differences underscore the importance of personalized genomics over a race-based approach to medicine. To attain truly personalized medicine, the scientific community must aim to elucidate the genetic and environmental factors that contribute to drug reactions and not be satisfied with a simple race-based approach.

Assessment of the heparin-binding AN69 ST hemodialysis membrane: II. Clinical studies without heparin administration.

Binding polyanionic unfractionated heparin over the modified AN69 polyacrylonitrile membrane, the surface electronegativity of which has been neutralized by polyethyleneimine (AN69-ST), renders the membrane more hemocompatible. This property was tested in two groups of long-term hemodialysis patients. Results were rated as massive or partial clotting of a dialyzer at the end of the session. Group I patients were included in a prospective, cross-over study comparing standard dialysis with hemodialysis without systemic administration of unfractionated heparin (n = 12, 123 sessions). In all instances, priming was made with 2 I saline containing 5,000 IU/l heparin. Only patchy or partial clotting was observed in 11% and 39% of the sessions with standard and heparin-free administration, respectively. Group II patients were included in an open, observational pilot study testing the effects of the heparin-coated membrane, without systemic administration of heparin, in patients at high risk of bleeding (n = 68, 331 sessions). Massive clotting was observed in six sessions only (less than 2%) and normal or slightly patchy dialyzers were found in 88% of the sessions. It is concluded that the dialysis AN69 ST membrane, after adequate priming at bedside, can be used without systemic administration of heparin for hemodialysis in patients at high risk of bleeding.

In vitro tests for assessing heparin-induced thrombocytopenia in patients after elective hip replacement. A medico-economical evaluation.

OBJECTIVES: Considering the previously published incidences of heparin-induced thrombocytopenia (HIT) in patients receiving a thromboprophylactic therapy, the role of the hemostasis laboratory is essential in making a clinical decision. The purpose of this project was to compare the strategies of diagnosis and associated care of patients with suspected HIT after elective hip replacement using platelet aggregation assay, carbon 14-serotonin release, and "doing nothing." METHODS: The authors used an incremental cost-effectiveness analysis based on data extracted from the literature. The effectiveness of the strategies was represented by the number of deep venous thromboses prevented. Cost data were collected from the observation of biological and medical practice at Edouard Herriot University Hospital, Lyon, France, in 1999. RESULTS: In comparison with the strategies of doing nothing using no biological test for diagnosis, and clinical care of HIT-suspected patients, the strategy using platelet aggregation test was more expensive and less effective. With respect to the strategy using carbon 14-serotonin release assay, the incremental cost-effectiveness ratio, expressed as U.S. dollars per deep venous thrombosis prevented, reached $200,000, with a marginal effectiveness of eight deep venous thromboses prevented for 10,000 HIT-suspected patients. CONCLUSION: This study suggests that clinical hemostasis laboratories might consider replacing the platelet aggregation test with the carbon 14-serotonin release assay or should use another functional assay such as the flow cytometric assay for the diagnosis and care of patients with suspected HIT.

Assessment of an epoxy-fixed pericardial patch with or without ionically bound heparin in a canine model.

In this study, an epoxy-fixed porcine pericardial patch with or without ionically bound heparin was evaluated in a canine model as an alternative to the glutaraldehyde-fixed biological patch for clinical applications. To evaluate the effectiveness of this epoxy-fixed patch, a composite membrane composed of: an epoxy-fixed porcine patch with ionically bound heparin; a glutaraldehyde-fixed porcine patch with ionically bound heparin; an ePTFE polymeric patch; a polyester polymeric patch; an epoxy-fixed porcine patch without ionically bound heparin; and a glutaraldehyde-fixed porcine patch without ionically bound heparin was made. This membrane was assessed orthopically in a canine model. The early results (1 approximately 4 weeks post implant) revealed that the biological patches with ionically bound heparin had the mildest tissue reactions (inflammatory reaction, fibrosis, and adhesion) among all the test samples. However, by 12 weeks postoperatively, all the test samples had mild to severe tissue reactions. The order of tissue reactions with increasing severity was: the biological patches with ionically bound heparin, the biological patches without ionically bound heparin, and the polymeric patches. The results suggest that heparin may be used to reduce adhesion. Additionally, the epoxy-fixed tissue caused a relatively lower degree of inflammatory reaction than the glutaraldehyde-fixed tissue.