warfarin and Disease-Models--Animal

Atherosclerosis is a pathological process underpinning many cardiovascular diseases; it is the main cause of global mortality. Atherosclerosis is characterized by an invasion of inflammatory cells, accumulation of lipids and the formation of fatty streaks (plaques) which subsequently allow accumulation of calcium and other minerals leading to a disturbance in the vascular endothelium and its regulatory role in arterial function. Vascular calcification is a different process, stringently regulated mainly by local factors, in which osteoblast-like cells accumulate in the muscular layer of arteries ultimately taking on the physiological appearance of bone. The elevated stiffness of the arteries leads to severe vascular complications in brain, heart and kidneys. Recently, evidence from animal experiments as well as clinical and epidemiological results suggests that long-term treatment with warfarin, but not with the novel direct anticoagulants, can increase the risk or even induce vascular calcification in some individuals. Gamma-carboxylation is an enzymatic process not only needed for activation of vitamin K but also other proteins which participate in bone formation and vascular calcification. Thus, reduced expression of the vitamin K-dependent proteins which physiologically inhibit calcification of cellular matrix could be postulated to lead to vascular calcification. Published clinical data, describing at present a few thousand patients, need to be supplemented with controlled studies to confirm this interesting hypothesis.

Topics: Animals; Anticoagulants; Arteries; Atherosclerosis; Dietary Supplements; Disease Models, Animal; Humans; Time Factors; Vascular Calcification; Vascular Stiffness; Vitamin K; Warfarin

Anticoagulant-related nephropathy (ARN) is a newly recognized form of AKI in which overanticoagulation causes profuse glomerular hemorrhage, which manifests on renal biopsy as numerous renal tubules filled with red cells and red cell casts. The glomeruli show changes, but they are not sufficient to account for the glomerular hemorrhage. We were the first to study ARN, and since then, our work has been confirmed by numerous other investigators. Oral anticoagulants have been in widespread use since the 1950s; today, >2 million patients with atrial fibrillation take an oral anticoagulant. Despite this history of widespread and prolonged exposure to oral anticoagulants, ARN was discovered only recently, suggesting that the condition may be a rare occurrence. This review chronicles the discovery of ARN, its confirmation by others, and our animal model of ARN. We also provide new data on analysis of "renal events" described in the

Topics: Acute Kidney Injury; Administration, Oral; Animals; Anticoagulants; Creatinine; Disease Models, Animal; Humans; International Normalized Ratio; Kidney; Practice Guidelines as Topic; Renal Insufficiency, Chronic; Risk Factors; Warfarin

The discovery of bioactive molecules from botanical sources is an expanding field, preferentially oriented to plants having a tradition of use in medicine and providing high yields and availability. Temperate forage legumes are Fabaceae species that include worldwide-important crops. These plants possess therapeutic virtues that have not only been used in veterinary and folk medicine, but have also attracted the interest of official medicine. We have examined here Medicago sativa (alfalfa), Trifolium pratense and T. repens (clovers), Melilotus albus and M. officinalis (sweet clovers), Lotus corniculatus (birdsfoot trefoil), Onobrychis viciifolia (sainfoin), Lespedeza capitata (roundhead lespedeza), and Galega officinalis (goat's rue). The phytochemical complexes of these species contain secondary metabolites whose pharmacological potentials deserve investigation. Major classes of compounds include alkaloids and amines, cyanogenic glycosides, flavonoids, coumarins, condensed tannins, and saponins. Some of these phytochemicals have been related to antihypercholesterolemia, antidiabetic, antimenopause, anti-inflammatory, antiedema, anthelmintic, and kidney protective effects. Two widely prescribed drugs have been developed starting from temperate forage legumes, namely, the antithrombotic warfarin, inspired from sweet clover's coumarin, and the antidiabetic metformin, a derivative of sainfoin's guanidine. Available evidence suggests that temperate forage legumes are a potentially important resource for the extraction of active principles to be used as nutraceuticals and pharmaceuticals.

Topics: Animals; Anti-Inflammatory Agents; Clinical Trials as Topic; Coumarins; Disease Models, Animal; Fabaceae; Fibrinolytic Agents; Flavonoids; Galega; Humans; Hypoglycemic Agents; Lespedeza; Lotus; Medicago; Medicago sativa; Melilotus; Phytochemicals; Plants, Medicinal; Proanthocyanidins; Saponins; Trifolium; Warfarin

Venous thromboembolism (VTE) can be life-threatening and requires anticoagulant treatment; for many years, vitamin K antagonists, e.g. warfarin, were the only oral anticoagulants available for long-term treatment. Although highly effective, they have many limitations including a slow onset, a multitude of drug-drug and drug-food interactions, and a narrow therapeutic range. These limitations spurred the search for non-vitamin K antagonist oral anticoagulants (NOACs), such as dabigatran etexilate.. The authors illustrate the progression of preclinical and clinical studies leading to the development of dabigatran, the only approved NOAC to act by direct thrombin inhibition. They focus on molecule discovery, animal models of thrombosis, clinical trials and post-launch activities in VTE treatment.. Dabigatran demonstrated comparable efficacy to the highly effective warfarin, and a more favourable safety profile in trials of VTE treatment. A favourable anticoagulant safety profile in addition to efficacy is essential for VTE treatment. Availability of the dabigatran-specific reversal agent, idarucizumab, provides a means of rapidly reversing the anticoagulant effect if required. Future investigations into the optimal duration of VTE treatment and an evaluation of the impact of idarucizumab, in real-world studies, could provide valuable information to help optimise treatment for selected patients.

Topics: Administration, Oral; Animals; Antibodies, Monoclonal, Humanized; Anticoagulants; Antidotes; Dabigatran; Disease Models, Animal; Drug Interactions; Humans; Venous Thromboembolism; Warfarin

Although the current therapeutic armamentarium of venous thrombosis encompasses the use of vitamin K antagonists, heparins, and direct oral anticoagulants, these drugs have several important drawbacks. Antisense oligonucleotides are relatively short single-stranded nucleic acid sequences, which hybridize with a target messenger RNA (mRNA) and suppress protein synthesis. Coagulation factor XI is a key player in blood coagulation, and thus represents a potential target for antisense therapy. The available evidence reviewed in this article suggests that factor XI antisense oligonucleotides may be more effective than conventional anticoagulants in preventing the onset and propagation of thrombosis, do not require factor measurement since the reduction of mRNA synthesis appears dose-dependently, robustly, and stably decreased for 3 to 5 weeks after the end of administration, with an incidence of major bleeding that is at least not greater than that associated with warfarin or low-molecular-weight heparin therapy. Despite conceptual simplicity, rational design, and relatively inexpensive cost, the preliminary findings in animal models and in patients undergoing knee surgery need to be validated in other prospective trials and cost-effective analyses before this attractive treatment option can be advocated as a new paradigm in prevention and treatment of venous thrombosis.

Topics: Animals; Clinical Trials as Topic; Disease Models, Animal; Factor XI; Fibrinolytic Agents; Heparin, Low-Molecular-Weight; Humans; Oligodeoxyribonucleotides, Antisense; RNA, Messenger; Venous Thrombosis; Warfarin

Anticoagulant-related nephropathy, a recently recognized entity, manifests as unexplained acute kidney injury in the setting of excessive anticoagulation with oral agents. Histologic findings in warfarin-related nephropathy include glomerular hemorrhage and renal tubular obstruction by red blood cells. Affected patients are at increased risk of mortality as well as irreversible kidney injury. Patients with chronic kidney disease are particularly vulnerable to this complication. Similar case reports of anticoagulant-related nephropathy have been linked to the more novel oral anticoagulant, dabigatran. Anticoagulant-related nephropathy has been successfully reproduced in rat models. These animal models shed light on the pathogenesis of the disease including the potential role of direct thrombin and protease-activated receptor-1 inhibition. Warfarin and dabigatran also cause an increase in systolic blood pressure in rats, a risk factor for developing nephropathy. This article reviews the current evidence for anticoagulant-related nephropathy and provides data for the suggested possible mechanisms underlying this adverse effect.

Topics: Acute Kidney Injury; Administration, Oral; Animals; Anticoagulants; Dabigatran; Disease Models, Animal; Humans; Rats; Renal Insufficiency, Chronic; Risk Factors; Warfarin

Venous thromboembolism (VTE), which includes deep vein thrombosis and pulmonary embolism, is a common and potentially preventable cause of morbidity and mortality. Unfractionated heparin, low-molecular-weight heparin, and warfarin have been the cornerstone of VTE prevention and treatment but are being replaced by recently approved non-vitamin K antagonist oral anticoagulants (NOACs): dabigatran, rivaroxaban, apixaban, and edoxaban. The NOACs are at least as effective and as safe as heparins and warfarin for VTE prevention and treatment and are more convenient because they have a low propensity for food and drug interactions and are given in fixed doses without routine coagulation monitoring. The remaining limitations of currently available NOACs include their dependence on renal and hepatic function for clearance, and the lack of an approved antidote. Betrixaban is a new NOAC with distinct pharmacological characteristics: minimal renal clearance, minimal hepatic metabolism, and long half-life. It has undergone successful Phase II studies in orthopedic thromboprophylaxis, and in stroke prevention in atrial fibrillation. Currently, it is being evaluated in a Phase III trial of extended thromboprophylaxis in medical patients (APEX study). In this article, we describe the development of betrixaban, review its pharmacological profile, discuss the results of clinical trials, and examine its potential for VTE prevention and treatment.

Topics: Administration, Oral; Animals; Benzamides; Clinical Trials, Phase II as Topic; Clinical Trials, Phase III as Topic; Disease Models, Animal; Factor Xa Inhibitors; Heparin; Humans; Pyridines; Venous Thromboembolism; Vitamin K; Warfarin

Topics: Animals; Anticoagulants; Atrial Fibrillation; Clinical Trials as Topic; Disease Models, Animal; Factor Xa Inhibitors; Humans; Platelet Aggregation; Pyrazoles; Pyridones; Stroke; Thrombin; Thrombosis; Warfarin

Coagulation factor (F)XI was first described as a member of the contact pathway of coagulation. However, the 'classic' theory of the extrinsic and intrinsic pathway has been revised and FXI was found to be activated by thrombin and to play a role in sustained thrombin generation and fibrinolysis inhibition. Recent studies have pointed to a disproportionate role of FXI in thrombosis and hemostasis. The observations that human congenital FXI deficiency is generally accompanied by mild and injury-related bleeding, and that experimental, provoked bleeding in animals is unaffected by FXI deficiency or FXI inhibition, suggest that the FXI amplification pathway is less important for normal hemostasis in vivo. In contrast, elevated plasma levels of FXI may contribute to human thromboembolic disease and the antithrombotic efficacy of FXI inhibition has been demonstrated in numerous animal models of arterial, venous and cerebral thrombosis. Whether severe FXI deficiency in humans protects against thromboembolic events remains unclear, although some evidence exists that the occurrence of ischemic stroke or venous thrombosis is low in severely FXI-deficient patients. Because of its distinctive function in thrombosis and hemostasis, FXI is an attractive target for the treatment and prevention of thromboembolism. A novel strategy for FXI inhibition is the use of antisense technology which has been studied in various thrombosis and bleeding animal models. The results are promising and support the concept that targeting FXI might serve as a new, effective and potentially safer alternative for the treatment of thromboembolic disease in humans.

Topics: Animals; Blood Coagulation; Disease Models, Animal; Dose-Response Relationship, Drug; Factor XI; Factor XI Deficiency; Fibrinolytic Agents; Hemostasis; Humans; Mice; Models, Biological; Oligonucleotides, Antisense; Reperfusion Injury; Thromboembolism; Thrombosis; Warfarin

There is in vitro and in vivo evidence that anticoagulants impair normal bone metabolism, and it is widely believed that this may impair fracture healing. However, there are only a few heterogeneous in vivo animal studies confirming this and the mechanisms are not fully understood.. To review the literature concerning the effects of anticoagulants on fracture healing, and to present current understanding of the mechanisms involved by reviewing in vivo studies of bone biology and in vitro studies of bone cells.. A systematic search of Medline and other databases was combined with manual searching of bibliographies of key papers to identify relevant studies in the English and German languages.. There is strong evidence that warfarin, heparin and aspirin retard fracture healing. The preferential use of low molecular weight heparins is advocated to minimise this. Fondaparinux has not shown any impairment in vitro. Further studies of fondaparinux, the timing of anticoagulation therapy and the mechanisms of action of these agents are of paramount importance.

Topics: Animals; Anticoagulants; Aspirin; Bone and Bones; Bone Remodeling; Disease Models, Animal; Fondaparinux; Fracture Healing; Heparin; Humans; Polysaccharides; Warfarin

We describe that high-dose methylprednisolone (20 mg/kg) can induce multifocal osteonecrosis (ON) in conjunction with thrombocytopenia, hypofibrinogenemia, and hyperlipemia. Detailed clinical and laboratory evaluations of coagulation system are recommended in those patients who develop manifestations of an abnormal lipid metabolism shortly after high-dose corticosteroid therapy. Moreover, we investigated the effects of the combination treatment with an anticoagulant (warfarin) plus a lipid-lowering agent (probucol) on prevention of steroid-induced osteonecrosis (ON) in this animal model. The incidence of ON in warfarin plus probucol (5%) was significantly lower than that observed in the control group (70%) (p <0.0001). Our results experimentally showed that the combined use of an anticoagulant and a lipid-lowering agent helps prevent steroid-induced ON in rabbits.

Topics: Animals; Anticholesteremic Agents; Anticoagulants; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Therapy, Combination; Glucocorticoids; Humans; Hyperlipidemias; Methylprednisolone; Osteonecrosis; Probucol; Rabbits; Thrombocytopenia; Warfarin

Topics: Animals; Anticoagulants; Biopsy; Bronchoscopy; Disease Models, Animal; Hemorrhage; Humans; Iatrogenic Disease; Lung Diseases; Swine; Warfarin

Evidence indicates that progression of the Lewis lung carcinoma in mice and small cell carcinoma of the lung in humans is retarded by warfarin administration. This suggests that vitamin K-dependent pathways are of importance in the pathogenesis of these tumors. Available data were reviewed for these tumor types in an attempt to explore mechanisms and to gain insights that might guide the selection of other coagulation-reactive drugs for testing in future controlled clinical trials in small cell carcinoma of the lung. While many differences exist between the Lewis lung tumor and small cell carcinoma of the lung, both are rapidly growing malignancies of pulmonary origin that metastasize early to kill the host after a short time. Both are favorably influenced by combination chemotherapy and radiation therapy as well as anticoagulant treatment. Peripheral blood changes indicative of disseminated intravascular coagulation occur in each of these tumor types, and tumor cells from both are capable of interacting with the coagulation mechanism. While many details concerning the host-tumor interaction remain to be elucidated, the considerable and diverse information available for these tumor types provides a secure base for future investigation. It is postulated that certain drugs in addition to warfarin might reasonably be studied in controlled clinical trials of small cell carcinoma of the lung and that drugs other than warfarin might be effective for tumor types that are not responsive to this agent.

Topics: Animals; Anticoagulants; Antineoplastic Agents; Carcinoma, Small Cell; Clinical Trials as Topic; Disease Models, Animal; Humans; Lung Neoplasms; Mice; Warfarin

Topics: Animals; Blood Coagulation; Blood Platelets; Coumarins; Disease Models, Animal; Fibrin; Fibrinolysis; Hemostasis; Humans; Neoplasm Metastasis; Neoplasms; Neoplasms, Experimental; Platelet Aggregation; Warfarin

Topics: Animals; Anticoagulants; Aspirin; Blood Platelet Disorders; Blood Platelets; Clinical Trials as Topic; Cold Temperature; Dextrans; Dipyridamole; Disease Models, Animal; Dogs; Heparin; Humans; Placebos; Platelet Adhesiveness; Postoperative Care; Rabbits; Rats; Thromboembolism; Thrombosis; Warfarin

Evidence indicates that progression of the Lewis lung carcinoma in mice and small cell carcinoma of the lung in humans is retarded by warfarin administration. This suggests that vitamin K-dependent pathways are of importance in the pathogenesis of these tumors. Available data were reviewed for these tumor types in an attempt to explore mechanisms and to gain insights that might guide the selection of other coagulation-reactive drugs for testing in future controlled clinical trials in small cell carcinoma of the lung. While many differences exist between the Lewis lung tumor and small cell carcinoma of the lung, both are rapidly growing malignancies of pulmonary origin that metastasize early to kill the host after a short time. Both are favorably influenced by combination chemotherapy and radiation therapy as well as anticoagulant treatment. Peripheral blood changes indicative of disseminated intravascular coagulation occur in each of these tumor types, and tumor cells from both are capable of interacting with the coagulation mechanism. While many details concerning the host-tumor interaction remain to be elucidated, the considerable and diverse information available for these tumor types provides a secure base for future investigation. It is postulated that certain drugs in addition to warfarin might reasonably be studied in controlled clinical trials of small cell carcinoma of the lung and that drugs other than warfarin might be effective for tumor types that are not responsive to this agent.

Topics: Animals; Anticoagulants; Antineoplastic Agents; Carcinoma, Small Cell; Clinical Trials as Topic; Disease Models, Animal; Humans; Lung Neoplasms; Mice; Warfarin

Topics: Animals; Anticoagulants; Aspirin; Blood Platelet Disorders; Blood Platelets; Clinical Trials as Topic; Cold Temperature; Dextrans; Dipyridamole; Disease Models, Animal; Dogs; Heparin; Humans; Placebos; Platelet Adhesiveness; Postoperative Care; Rabbits; Rats; Thromboembolism; Thrombosis; Warfarin

Recent studies have revealed that atrial fibrillation (AF) patients have a high risk of developing cognitive impairment, vascular dementia, and Alzheimer's disease (AD). Some reports suggest that the application of oral anticoagulant with an appropriate dose may have a preventive effect on AD. However, which oral anticoagulant drug is more appropriate for preventing AD and the underlying mechanism(s) is still unknown.. The aim of the present study was to assess the treatment effect of rivaroxaban administration as well as investigate the roles of PAR-1 and PAR-2 in the AD + CAA mice model.. In the present study, we compared a traditional oral anticoagulant, warfarin, and a direct oral anticoagulant (DOAC), rivaroxaban, via long-term administration to an AD with cerebral amyloid angiopathy (CAA) mice model.. Rivaroxaban treatment attenuated neuroinflammation, blood-brain barrier dysfunction, memory deficits, and amyloid-β deposition through PAR-1/PAR-2 inhibition in the AD + CAA mice model compared with warfarin and no-treatment groups.. The present study demonstrates that rivaroxaban can attenuate AD progress and can be a potential choice to prevent AD.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Anticoagulants; Cerebral Amyloid Angiopathy; Disease Models, Animal; Humans; Mice; Neuroinflammatory Diseases; Rivaroxaban; Warfarin

 Intracranial hemorrhage (ICH) is one of the major devastating complications of anticoagulation. Matrix metalloproteinase (MMP) inhibition has been proposed as a novel pharmacological approach for ICH treatment..  We evaluated the effects of CM-352 (MMP-fibrinolysis inhibitor) in an experimental ICH model associated with oral anticoagulants as compared with clinically used prothrombin complex concentrate (PCC)..  ICH was induced by collagenase injection into the striatum of wild type (C57BL/6J) anticoagulated mice (warfarin or rivaroxaban) and.  Only PCC reduced hemorrhage volume and improved functional outcome in warfarin-ICH, but both PCC and CM-352 treatments diminished hemorrhage volume (46%,.  CM-352 treatment, by diminishing MMPs and rivaroxaban-associated fibrinolytic effects, might be a novel antihemorrhagic strategy for rivaroxaban-associated ICH.

Topics: Animals; Anticoagulants; Benzamides; Blood Coagulation Factors; Cerebral Hemorrhage; Disease Models, Animal; Hydroxamic Acids; Interleukin-6; Intracranial Hemorrhages; Matrix Metalloproteinase 10; Mice; Mice, Inbred C57BL; Plasminogen Activator Inhibitor 1; Rivaroxaban; Warfarin

Vascular calcification is common among aging populations and mediated by vascular smooth muscle cells (VSMCs). The endoplasmic reticulum (ER) is involved in protein folding and ER stress has been implicated in bone mineralization. The role of ER stress in VSMC-mediated calcification is less clear. Approach and Results: mRNA expression of the ER stress markers PERK (PKR (protein kinase RNA)-like ER kinase), ATF (activating transcription factor) 4, ATF6, and Grp78 (glucose-regulated protein, 78 kDa) was detectable in human vessels with levels of PERK decreased in calcified plaques compared to healthy vessels. Protein deposition of Grp78/Grp94 was increased in the matrix of calcified arteries. Induction of ER stress accelerated human primary VSMC-mediated calcification, elevated expression of some osteogenic markers (Runx2 [RUNX family transcription factor 2], OSX [Osterix], ALP [alkaline phosphatse], BSP [bone sialoprotein], and OPG [osteoprotegerin]), and decreased expression of SMC markers. ER stress potentiated extracellular vesicle (EV) release via SMPD3 (sphingomyelin phosphodiesterase 3). EVs from ER stress-treated VSMCs showed increased Grp78 levels and calcification. Electron microscopy confirmed the presence of Grp78/Grp94 in EVs. siRNA (short interfering RNA) knock-down of Grp78 decreased calcification. Warfarin-induced Grp78 and ATF4 expression in rat aortas and VSMCs and increased calcification in an ER stress-dependent manner via increased EV release.. ER stress induces vascular calcification by increasing release of Grp78-loaded EVs. Our results reveal a novel mechanism of action of warfarin, involving increased EV release via the PERK-ATF4 pathway, contributing to calcification. This study is the first to show that warfarin induces ER stress and to link ER stress to cargo loading of EVs.

Topics: Activating Transcription Factor 4; Adolescent; Adult; Aged; Animals; Cells, Cultured; Disease Models, Animal; eIF-2 Kinase; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Extracellular Vesicles; Female; Gene Expression Regulation; Heat-Shock Proteins; Humans; Male; Middle Aged; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Rats, Sprague-Dawley; Signal Transduction; Vascular Calcification; Warfarin; Young Adult

Calcific aortic valve disease is a common heart disease that contributes to increased cardiovascular morbidity and mortality. There is a lack of effective pharmaceutical therapy because its mechanisms are not yet fully known. Ginkgo biloba extract (EGB761) is reported to alleviate vascular calcification. However, whether EGB761 protects against aortic valve calcification, a disease whose pathogenesis shares many similarities with vascular calcification, and potential molecular mechanisms remain unknown. In this study, porcine aortic valve interstitial cell (pAVIC) calcification was induced by warfarin with or without the presence of EGB761. Immunostaining was performed to establish and characterize the pAVIC phenotype. Calcium deposition and calcium content were examined by Alizarin Red S staining and an intracellular calcium content assay. Alkaline phosphatase activity was detected by the p-nitrophenyl phosphate method. The expression levels of bone morphogenetic protein-2 (BMP2), Runt-related transcription factor 2 (Runx2), homeobox protein MSX-2, and phosphorylated (p)-Smad1/5 were detected by reverse transcription-quantitative polymerase chain reaction (PCR) and Western blot analysis. Consistent with these in vitro data, we also confirmed the suppression of in vivo calcification by EGB761 in the warfarin-induced C57/Bl6 mice. The results indicated that both pAVICs and aortic valves tissue of mice stimulated with warfarin showed increased calcium deposition and expression of osteogenic markers (alkaline phosphatase, BMP2, homeobox protein MSX-2, and Runx2) and promoted p-Smad1/5 translocation from the cytoplasm to the nucleus. The addition of EGB761 significantly inhibited p-Smad1/5 translocation from the cytoplasm to the nucleus, thus suppressing calcification. In conclusion, EGB761 could ameliorate warfarin-induced aortic valve calcification through the inhibition of the BMP2-medicated Smad1/5/Runx2 signaling pathway.

Topics: Active Transport, Cell Nucleus; Animals; Aortic Valve; Bone Morphogenetic Protein 2; Calcinosis; Calcium; Cells, Cultured; Core Binding Factor Alpha 1 Subunit; Disease Models, Animal; Ginkgo biloba; Heart Valve Diseases; Homeodomain Proteins; Male; Mice, Inbred C57BL; Osteogenesis; Phosphorylation; Plant Extracts; Signal Transduction; Smad1 Protein; Smad5 Protein; Sus scrofa; Warfarin

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

Oral anticoagulation prevents thromboembolism in atrial fibrillation. Factor Xa inhibitors, like edoxaban, are known to reduce inflammation and proliferation of smooth muscle cells, while vitamin K antagonism can cause vascular calcific damage. The influence of edoxaban compared to warfarin on vascular remodeling, atherosclerosis and arteriogenesis is unknown.. Apolipoprotein E knockout (ApoE. There was no difference in hind-limb perfusion restoration between the three groups after 14 days (Co 0.36 ± 0.05 vs. Warf 0.39 ± 0.09 (p = .39), Co vs. Edo 0.51 ± 0.06 (p = .089), Warf vs. Edo (p = .83)) after ligation. Immuno-histologically, there was no difference in smooth muscle cell count in both hindlimbs between the three groups or in the amount of perivascular macrophages in collateral-bearing hindlimb tissue. Edoxaban showed the lowest amount of plaque tissue in the aortic sinus tissue (Co 74 ± 11% vs. Edo 62 ± 12% (p = .024), Co vs. Warf 69 ± 14% (p = .30), Edo vs. Warf (p = .14)) as well as the least amount of fibrosis (Co 3.1 ± 0.9% vs. Edo 1.7 ± 0.6% (p = .027), Co vs. Warf 4.1 ± 0.7% (p = .081), Edo vs. Warf (p < .001)). No difference in mRNA content of inflammatory cytokines in muscle tissue or spleen was detected between the three groups.. These data suggest that treatment with edoxaban unlike warfarin prevents vascular maladaptive remodeling, which may be clinically important.

Topics: Animals; Anticoagulants; Atherosclerosis; Collateral Circulation; Disease Models, Animal; Factor Xa Inhibitors; Fibrosis; Hindlimb; Ischemia; Mice, Inbred C57BL; Mice, Knockout, ApoE; Muscle, Skeletal; Neovascularization, Physiologic; Plaque, Atherosclerotic; Pyridines; Thiazoles; Vascular Remodeling; Warfarin

Vitamin K (VK) is a co-factor in the post-translational gamma glutamic carboxylation of Gla-proteins. VK-dependent coagulation factors are carboxylated in the liver by VK1. Osteocalcin and Matrix-Gla protein (MGP) are carboxylated in extrahepatic tissues by VK2. A model of VK deficiency would be suitable for studying extrahepatic Gla-proteins provided that severe bleeding is prevented.. The aim of this work was to adapt an established protocol of vascular calcification by warfarin-induced inactivation of MGP as a calcification inhibitor, in an attempt to create a broader state of subclinical VK deficiency and to verify its safety.. Two consecutive experiments, each lasting 4 weeks, were required to modify the dosing schedule of warfa-rin and VK1 and to adapt it to the Wistar rats used. The original high doses of warfarin used initially had to be halved and the protective dose of VK1 to be doubled, in order to avoid treatment-induced hemorrhagic deaths. The second experiment aimed to confirm the efficacy and safety of the modified doses. To verify the VK deficiency, blood vessels were examined histologically for calcium deposits and serum osteocalcin levels were mea-sured.. The original dosing schedule induced VK deficiency, manifested by arterial calcifications and dramatic changes in carboxyl-ated and uncarboxylated osteocalcin. The modified dosing regimen caused similar vascular calcification and no bleeding.. The modified protocol of carefully balanced warfarin and VK1 doses is an effective and safe way to induce subclinical VK deficiency that can be implemented to investigate VK-dependent proteins like osteocalcin.

Topics: Animals; Anticoagulants; Antifibrinolytic Agents; Arteries; Asymptomatic Diseases; Calcium-Binding Proteins; Carbon-Carbon Ligases; Disease Models, Animal; Extracellular Matrix Proteins; Matrix Gla Protein; Osteocalcin; Rats; Vascular Calcification; Vitamin K 1; Vitamin K 2; Vitamin K Deficiency; Warfarin

We aimed to investigate the role of the miR-29b and its effect on TGF-β3 pathway in vascular and valvular calcification in a rat model of calcific aortic valve diseases (CAVD). A rat model of CAVD was established by administration of warfarin plus vitamin K. The expression levels of miR-29b, osteogenic markers and other genes were determined by qRT-PCR, Western blot and/or immunofluorescence and immunohistochemistry. The calcium content and alkaline phosphatase (ALP) activity were measured. The calcium content, ALP activity and osteogenic markers levels in calcified aorta and aortic valve were augmented compared to controls. The expression of miR-29b, p-Smad3, and Wnt3 and β-catenin was significantly up-regulated, whereas TGF-β3 was markedly down-regulated. However, compared with the CAVD model group, the calcium content and ALP activity in rats treated with antagomiR-29b were significantly decreased, and antagomiR-29b administration reversed the effects of CAVD model on the expression of miR-29b and osteogenic markers. Inhibition of miR-29b in CAVD rats prevented from vascular and valvular calcification and induced TGF-β3 expression, suggesting that the miR-29b/TGF-β3 axis may play a regulatory role in the pathogenesis of vascular and valvular calcification and could play a significant role in the treatment of CAVD and other cardiovascular diseases.

Topics: Animals; Antagomirs; Aortic Valve; Aortic Valve Stenosis; Calcification, Physiologic; Calcinosis; Disease Models, Animal; Heart; Male; MicroRNAs; Osteogenesis; Osteopontin; Rats, Sprague-Dawley; Smad3 Protein; Transforming Growth Factor beta3; Up-Regulation; Vascular Calcification; Warfarin; Wnt Signaling Pathway

Acute pancreatitis is associated with activation of coagulation and there is a close relationship between coagulation and the severity of this disease. Administration of anticoagulants such as heparin or acenocoumarol has shown to reduce the severity of acute pancreatitis and accelerate the recovery. The aim of the current study was to determine the impact of warfarin administration on the course of ischemia/reperfusion-induced acute pancreatitis. Acute pancreatitis was induced in rats by pancreatic ischemia followed by reperfusion. Vehicle (1 ml/dose) or warfarin (45, 90 or 180 μg/kg/dose in 1 ml of vehicle) were administered intragastrically once a day. The first dose of warfarin was given 24 h after the start of pancreatic reperfusion. The severity of acute pancreatitis was assessed 2, 5, 9 and 14 days after the beginning of pancreatic reperfusion. Treatment with warfarin reduces pancreatic damage and accelerates recovery in histological examination and this effect is accompanied by a faster reduction in serum activity of pancreatic digestive enzymes, lipase and amylase. In addition, warfarin led to an earlier decrease in serum concentration of pro-inflammatory interleukin-1β and plasma level of D-dimer. These effects were associated with an improvement of pancreatic blood flow. We conclude that warfarin exhibits a therapeutic effect in acute pancreatitis evoked by pancreatic ischemia followed by reperfusion.

Topics: Amylases; Animals; Anticoagulants; Biomarkers; Disease Models, Animal; Fibrin Fibrinogen Degradation Products; Interleukin-1beta; Lipase; Male; Pancreas; Pancreatitis; Rats, Wistar; Reperfusion Injury; Time Factors; Warfarin

Vitamin K is present in the testes though its actual function in male reproduction is poorly understood. This study investigated the harmful effect of extrahepatic vitamin K insufficiency on the testicular structure. Sprague-Dawley rats were fed with a diet containing warfarin for 2, 4 and 8 weeks; control animals received a standard diet without warfarin. It was found that extrahepatic vitamin K deficiency that is induced by warfarin results in histopathological features that range from delayed spermiation, presence of multinucleated giant cells in the seminiferous tubules, germ cells degeneration, asthenozoospermia, oligozoospermia and increase in the percentage of abnormal sperm morphology when compared to the controls. Data obtained from the two groups were analysed using the Student t test. It is concluded that warfarin-induced vitamin K deficiency has a negative impact on spermatogenesis.

Topics: Administration, Oral; Animals; Disease Models, Animal; Humans; Infertility, Male; Male; Rats; Rats, Sprague-Dawley; Seminiferous Tubules; Spermatogenesis; Spermatozoa; Vitamin K; Vitamin K Deficiency; Warfarin

BACKGROUND Worldwide, the treatment of patients with chronic kidney disease (CKD) remains a challenge as warfarin treatment can be associated with severe adverse events related to bleeding. Alternative anticoagulants that can be used in CKD remain to be identified. This study aimed to compare the effects of indobufen, a new antiplatelet agent, with warfarin in a rat model of adenine-induced CKD. MATERIAL AND METHODS Forty-eight male Wistar rats were treated with intragastric adenine to create the rat model of CKD and were divided into four groups: an untreated control group (N=12), a group treated with dimethyl sulfoxide (DMSO) (N=12), a group treated with indobufen, (N=12) and a group treated with warfarin (N-12). Treatment was given for 4 weeks and 8 weeks. Kidney histology was performed, and the degree of fibrosis was quantified using Masson trichrome staining. RESULTS In the rat model of adenine-induced CKD, Masson trichrome staining showed that the degree of kidney fibrosis in the indobufen group (26%) was significantly reduced (p<0.05) when compared the DMSO group (58%) and the warfarin group (49%). Kidney fibrosis was associated with upregulation of 6-keto-PGI2/TXB2 in the rat kidney tissue. CONCLUSIONS In a rat model of adenine-induced CKD, preliminary findings showed that indobufen was associated with reduced kidney fibrosis when compared with warfarin.

Topics: Adenine; Animals; Anticoagulants; China; Disease Models, Animal; Fibrosis; Isoindoles; Kidney; Kidney Failure, Chronic; Male; Phenylbutyrates; Platelet Aggregation Inhibitors; Rats; Rats, Wistar; Renal Insufficiency, Chronic; Warfarin

Warfarin is the most worldwide used anticoagulant drug and rodenticide. Since it crosses placental barrier it can induce warfarin embryopathy (WE), a fetal mortality in neonates characterized by skeletal deformities in addition to brain hemorrhages. Although the effects of warfarin exposure in aquatic off target species were already described, the particular molecular toxicological mechanisms during early development are still unclear. Here, we used zebrafish (Danio rerio) to describe and compare the developmental effects of warfarin exposure (0, 15.13, 75.68 and 378.43 mM) on two distinct early developmental phases (embryos and eleuthero-embryos). Although exposure to both developmental phases induced fish mortality, only embryos exposed to the highest warfarin level exhibited features mimicking mammalian WE, e.g. high mortality, higher incidence of hemorrhages and altered skeletal development, among other effects. To gain insights into the toxic mechanisms underlying warfarin exposure, the transcriptome of embryos exposed to warfarin was explored through RNA-Seq and compared to that of control embryos. 766 differentially expressed (564 up- and 202 down-regulated) genes were identified. Gene Ontology analysis revealed particular cellular components (cytoplasm, extracellular matrix, lysosome and vacuole), biological processes (mainly amino acid and lipid metabolism and response to stimulus) and pathways (oxidative stress response and apoptosis signaling pathways) being significantly overrepresented in zebrafish embryos upon warfarin exposure. Protein-protein interaction further evidenced an altered redox system, blood coagulation and vasculogenesis, visual phototransduction and collagen formation upon warfarin exposure. The present study not only describes for the first time the WE in zebrafish, it provides new insights for a better risk assessment, and highlights the need for programming the rat eradication actions outside the fish spawning season to avoid an impact on off target fish community. The urge for the development of more species-specific anticoagulants for rodent pest control is also highlighted.

Topics: Abnormalities, Drug-Induced; Animals; Anticoagulants; Disease Models, Animal; Embryo, Nonmammalian; Humans; Nasal Bone; Oxidative Stress; Rodenticides; Transcriptome; Warfarin; Water Pollutants, Chemical; Zebrafish

The most frequently used oral anti-coagulant warfarin has been implicated in inducing calcification of aortic valve interstitial cells (AVICs), whereas the mechanism is not fully understood. The low-level activation of p53 is found to be involved in osteogenic transdifferentiation and calcification of AVICs. Whether p53 participates in warfarin-induced AVIC calcification remains unknown. In this study, we investigated the role of low-level p53 overexpression in warfarin-induced porcine AVIC (pAVIC) calcification. Immunostaining, quantitative PCR, and Western blotting revealed that p53 was expressed in human and pAVICs and that p53 expression was slightly increased in calcific human aortic valves compared with non-calcific valves. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining indicated that apoptosis slightly increased in calcific aortic valves than in non-calcific valves. Warfarin treatment led to a low-level increase of p53 mRNA and protein in both pAVICs and mouse aortic valves. Low-level overexpression of p53 in pAVICs via an adenovirus vector did not affect pAVIC apoptosis but promoted warfarin-induced calcium deposition and expression of osteogenic markers. shRNA-mediated p53 knockdown attenuated the pAVIC calcium deposition and osteogenic marker expression. Moreover, ChIP and luciferase assays showed that p53 was recruited to the

Topics: Animals; Anticoagulants; Antifibrinolytic Agents; Aortic Valve; Atrial Fibrillation; Calcinosis; Cells, Cultured; Disease Models, Animal; Epigenesis, Genetic; Gene Expression Regulation; Genes, Reporter; Heart Valve Diseases; Humans; Male; Mice, Inbred C57BL; Promoter Regions, Genetic; Recombinant Proteins; Rheumatic Heart Disease; RNA Interference; Snail Family Transcription Factors; Sus scrofa; Tumor Suppressor Protein p53; Vitamin K 1; Warfarin

Chronic kidney disease (CKD) causes dysregulation of mineral metabolism, vascular calcification and renal osteodystrophy, an entity called 'CKD-Mineral and Bone Disorder' (CKD-MBD). Here we determine whether metformin, an anti-diabetic drug, exerts favorable effects on progressive, severe CKD and concomitant mineral metabolism disturbances. Rats with CKD-MBD, induced by a 0.25% adenine diet for eight weeks, were treated with 200 mg/kg/day metformin or vehicle from one week after CKD induction onward. Severe, stable CKD along with marked hyperphosphatemia and hypocalcemia developed in these rats which led to arterial calcification and high bone turnover disease. Metformin protected from development toward severe CKD. Metformin-treated rats did not develop hyperphosphatemia or hypocalcemia and this prevented the development of vascular calcification and inhibited the progression toward high bone turnover disease. Kidneys of the metformin group showed significantly less cellular infiltration, fibrosis and inflammation. To study a possible direct effect of metformin on the development of vascular calcification, independent of its effect on renal function, metformin (200 mg/kg/day) or vehicle was dosed for ten weeks to rats with warfarin-induced vascular calcification. The drug did not reduce aorta or small vessel calcification in this animal model. Thus, metformin protected against the development of severe CKD and preserved calcium phosphorus homeostasis. As a result of its beneficial impact on renal function, associated comorbidities such as vascular calcification and high bone turnover disease were also prevented.

Topics: Adenine; Animals; Chronic Kidney Disease-Mineral and Bone Disorder; Disease Models, Animal; Humans; Hypoglycemic Agents; Male; Metformin; Rats; Rats, Wistar; Renal Insufficiency, Chronic; Severity of Illness Index; Treatment Outcome; Vascular Calcification; Warfarin

Vitamin K antagonists, such as warfarin, are known to promote arterial calcification through blockade of gamma-carboxylation of Matrix-Gla-Protein. It is currently unknown whether other oral anticoagulants such as direct inhibitors of Factor Xa can have protective effects on the progression of aortic valve calcification.. To compare the effect of warfarin and rivaroxaban on the progression of aortic valve calcification in atherosclerotic mice.. 42 ApoE-/- mice fed with Western-type Diet (WTD) were randomized to treatment with warfarin (n = 14), rivaroxaban (n = 14) or control (n = 14) for 8 weeks. Histological analyses were performed to quantify the calcification of aortic valve leaflets and the development of atherosclerosis. The analyses showed a significant increase in valve calcification in mice treated with warfarin as compared to WTD alone (P = .025) or rivaroxaban (P = .005), whereas no significant differences were found between rivaroxaban and WTD (P = .35). Quantification of atherosclerosis and intimal calcification was performed on the innominate artery of the mice and no differences were found between the 3 treatments as far as atherogenesis and calcium deposition is concerned. In vitro experiments performed using bovine interstitial valve cells (VIC) showed that treatment with rivaroxaban did not prevent the osteogenic conversion of the cells but reduce the over-expression of COX-2 induced by inflammatory mediators.. We showed that warfarin, but not rivaroxaban, could induce calcific valve degeneration in a mouse model of atherosclerosis. Both the treatments did not significantly affect the progression of atherosclerosis. Overall, these data suggest a safer profile of rivaroxaban on the risk of cardiovascular disease progression.

Topics: Animals; Anticoagulants; Aortic Valve; Aortic Valve Stenosis; Atherosclerosis; Calcinosis; Cattle; Cells, Cultured; Cyclooxygenase 2; Disease Models, Animal; Disease Progression; Factor Xa Inhibitors; Female; Male; Mice, Knockout, ApoE; Risk Assessment; Rivaroxaban; Time Factors; Vascular Calcification; Warfarin

Thrombin has been demonstrated to be involved in several viral diseases including human metapneumovirus (hMPV) infections. We previously showed that immediate administration of thrombin inhibitor argatroban post-infection protected mice against hMPV disease. This current work aims at determining whether warfarin and heparin, two other anticoagulants inhibiting thrombin formation and activities, may also be used for treatment against hMPV in vivo. We found that immediate injections of argatroban, warfarin or heparin after virus challenge protected mice against hMPV infection, as evidenced by decreased or no mortality, less weight loss, reduced viral load and attenuated inflammation. However, delayed treatments starting 1 day post-infection with argatroban or warfarin almost did not impact the survival whereas delayed treatment with heparin induced an increased mortality during infection. Moreover, these treatments also did not reduce weight loss, viral replication and inflammation. In agreement with these results, thrombin generation was decreased upon immediate anticoagulant treatments but was unaltered upon delayed treatments. Thus, thrombin generation occurs at the onset of hMPV infection and thrombin inhibition may be only useful for the treatment of this disease when initiated in the early stage. In this case, heparin is not recommended because of its reduced efficacy on mortality in infected mice whereas argatroban and warfarin appear as safe and effective drugs for the treatment of hMPV disease. The antiviral and anti-inflammatory effects of argatroban occur via thrombin-dependent pathways whereas the mechanisms by which warfarin exerts its beneficial effects against hMPV infection were not elucidated and need to be further studied.

Topics: Animals; Anticoagulants; Arginine; Disease Models, Animal; Heparin; Metapneumovirus; Mice; Paramyxoviridae Infections; Pipecolic Acids; Sulfonamides; Survival Analysis; Treatment Outcome; Viral Load; Virus Replication; Warfarin

Recent global deregulation of ginseng as the table food raises our concern about the possible ginseng-warfarin interaction that could be life-threatening to patients who take warfarin for preventing fatal strokes and thromboembolism while using ginseng products for bioenergy recovery. Here we show that quality-control ginsenosides, extracted from ginseng and containing its major active ingredients, produce dose- and time-dependent antagonism in rats against warfarin's anti-coagulation assessed by INR and rat thrombosis model. The interactions between ginsenosides and warfarin on thrombosis, pharmacokinetics, activities of coagulation factors and liver cytochrome P450 isomers are determined by using thrombosis analyzer, UPLC/MS/MS, ELISA and real-time PCR, respectively. The antagonism correlates well with the related pharmacokinetic interaction showing that the blood plateaus of warfarin reached by one-week warfarin administration are significantly reduced after three-week co-administration of warfarin with ginsenosides while 7-hydroxywarfarin is increased. The one-week warfarin and three-week warfarin-ginsenosides regimen result in restoring the suppressed levels by warfarin of the coagulating factors II, VII and protein Z, and significantly enhance activities of P450 3A4 and 2C9 that metabolize warfarin. The present study, for the first time, provides the solid evidence to demonstrate the warfarin-ginsenoside interaction, and warns the warfarin users and regulation authorities of the dangerous interaction.

Topics: Animals; Anticoagulants; Blood Coagulation Factors; Carrageenan; Disease Models, Animal; Dose-Response Relationship, Drug; Ginsenosides; Herb-Drug Interactions; International Normalized Ratio; Internationality; Panax; Quality Control; Rats; Rats, Sprague-Dawley; Social Control, Formal; Thrombosis; Time Factors; Warfarin

In multiple sclerosis, coagulation factors have been shown to modulate inflammation. In this translational study, we investigated whether long-term anticoagulation with warfarin or rivaroxaban has beneficial effects on the course of autoimmune experimental encephalomyelitis (EAE).. Female SJL/J mice treated with anticoagulants namely warfarin or rivaroxaban were immunized with PLP. In preventive settings, both treatment with warfarin and rivaroxaban reduced the maximum EAE score as compared to the control group and led to a reduction of inflammatory lesions in the spinal cord. In contrast, therapeutic treatment with warfarin had no beneficial effects on the clinical course of EAE. Signs of intraparenchymal hemorrhage at the site of the inflammatory lesions were not observed.. We developed long-term anticoagulation models that allowed exploring the course of EAE under warfarin and rivaroxaban treatment. We found a mild preventive effect of both warfarin and rivaroxaban on neurological deficits and local inflammation, indicating a modulation of the disease induction by anticoagulation.

Topics: Animals; Anticoagulants; Brain; Disease Models, Animal; Electric Impedance; Encephalomyelitis, Autoimmune, Experimental; Endothelial Cells; Freund's Adjuvant; Mice; Myelin Proteolipid Protein; Peptide Fragments; Random Allocation; Rivaroxaban; Swine; Thrombin; Time Factors; Warfarin

Intracerebral hemorrhage (ICH) during oral anticoagulation therapy with an oral vitamin K epoxidase reductase such as warfarin is a life-threatening complication. However, whether direct oral anticoagulants (DOACs) are associated with larger hematoma volume and higher mortality rates remains controversial. We evaluated the hematoma volume and pathophysiology of ICH during anticoagulation with warfarin or rivaroxaban, an orally active direct factor Xa inhibitor.. Mice were orally pretreated with rivaroxaban (10 or 30 mg/kg), warfarin (4 mg/kg), or vehicle. ICH was induced by intrastriatal collagenase-injection. Hematoma volume and neurological deficits 24 h after ICH induction were significantly decreased in the rivaroxaban-pretreated group in comparison with the warfarin-pretreated group. Rivaroxaban did not increase the hematoma volume relative to that observed for vehicle, and improved survival rate 7 days after ICH induction compared with warfarin.. We evaluated blood-brain barrier (BBB) permeability 6 h after ICH induction using Evans blue spectrophotometry. Evans blue extravasation was significantly reduced in the rivaroxaban group compared with the warfarin group. To investigate the mechanism underlying hematoma expansion and BBB permeability, we focused on thrombin, a clot-derived factor and one of the major contributors to ICH-induced brain injury. To investigate the effects of anticoagulant agents on thrombin-induced injuries, human brain endothelial cells were used in membrane permeability assays. Rivaroxaban, but not warfarin, significantly mitigated the thrombin-induced increase in membrane permeability.. These findings indicate that rivaroxaban decreases BBB disruption after ICH, and limits early hematoma expansion in these experimental models compared with warfarin. Our study suggests that rivaroxaban has advantages over warfarin with respect to ICH, an important complication during long-term anticoagulation therapy.

Topics: Animals; Anticoagulants; Blood-Brain Barrier; Cells, Cultured; Cerebral Hemorrhage; Disease Models, Animal; Electric Impedance; Endothelial Cells; Factor Xa Inhibitors; Hematoma; Isothiocyanates; Mice; Neurologic Examination; Permeability; Rivaroxaban; Treatment Outcome; Warfarin

Resveratrol is a popular ingredient in dietary supplements. Some patients concomitantly use dietary supplements and medicines in Japan. In the present study, we determined whether trans-resveratrol and melinjo (Gnetum gnemon L.) seed extract (MSE), which contains resveratrol dimers, interacted with drugs using a mouse model.. Male C57BL/6J mice were fed experimental diets containing 0.005%, 0.05%, or 0.5% (w/w) trans-resveratrol or MSE for 1 or 12 weeks. The expression of liver cytochrome P-450 (CYP) mRNA and activity of liver microsomal CYP were measured. To determine the influence of resveratrol or MSE on drug efficacy, the anticoagulant activity of warfarin was examined in mice that were fed diets containing trans-resveratrol or MSE for 12 weeks.. When the mice were fed experimental diets for 1 week, none of the doses of trans-resveratrol and MSE affected body weight, liver weight, or plasma AST and ALT levels. Trans-resveratrol also did not affect CYP1A1, CYP1A2, CYP2C, or CYP3A activities. In contrast, 0.5% MSE slightly increased CYP1A1 activity. When the mice were fed experimental diets for 12 weeks, 0.05% trans-resveratrol increased CYP1A1, CYP2C, and CYP3A activities, whereas 0.5% MSE suppressed CYP3A activity. Under these conditions, 0.5% trans-resveratrol enhanced the anticoagulant activity of warfarin, although CYP2C activity increased. However, MSE did not affect the anticoagulant activity of warfarin.. The 0.05% trans-resveratrol did not interact with warfarin in a mouse model, whereas 0.5% trans-resveratrol may have enhanced the anticoagulant activity of warfarin.

Topics: Animals; Anticoagulants; Antioxidants; Blood Coagulation; Cytochrome P-450 Enzyme System; Disease Models, Animal; Drug Combinations; Drug Synergism; Gene Expression Regulation, Enzymologic; Gnetum; Male; Mice; Mice, Inbred C57BL; Microsomes, Liver; Plant Extracts; Real-Time Polymerase Chain Reaction; Resveratrol; Seeds; Stilbenes; Warfarin

Warfarin and rivaroxaban are highly effective in reducing stroke risk in patients with atrial fibrillation (AF). However, their effects on anticoagulation and neurovascular unit (NVU) change remain elusive. In this study, we assessed the risks and benefits of pre-treatment with warfarin or rivaroxaban after tissue-type plasminogen activator (tPA) thrombolysis in ischemic rat brain.. Pre-treatment with warfarin (.2 mg/kg/day), low dose rivaroxaban (60 mg/kg/day), high dose rivaroxaban (120 mg/kg/day) or vehicle was performed for 2 weeks, transient middle cerebral artery occlusion (tMCAO) was induced for 90 min, then followed by reperfusion with tPA. At 24 hours (h) after reperfusion, we observed the changes of matrix metalloproteinase-9 (MMP-9), tissue factor, caspase 3 and NVU dissociation.. Prothrombin time (PT) was significantly prolonged in the warfarin and rivaroxaban pretreated groups. MMP-9 expression greatly increased in the warfarin group, and this was reduced in the rivaroxaban groups compared with the vehicle group. Tissue factor expression remarkably decreased in the warfarin and rivaroxaban groups. The number of caspase 3-positive cells had no difference among all the groups. Marked dissociations between astrocyte foot processes and the basal lamina or pericytes were observed in the warfarin pretreated group, but such dissociations were improved in the rivaroxaban groups.. Our present study shows that pre-treatment with rivaroxaban was noninferior to warfarin in the anticoagulation, but a lower risk of NVU dysfunction and dissociation after tPA treatment in rivaroxaban. This finding could partly explain the mechanism of reducing hemorrhagic complications by rivaroxaban in clinical studies.

Topics: Animals; Anticoagulants; Body Weight; Brain; Caspase 3; Collagen; Disease Models, Animal; Drug Administration Schedule; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Infarction, Middle Cerebral Artery; Male; Matrix Metalloproteinase 9; Plant Lectins; Rats; Rats, Wistar; Receptors, Platelet-Derived Growth Factor; Rivaroxaban; Warfarin

Thrombin is a potent regulator of brain function in health and disease, modulating glial activation and brain inflammation. Thrombin inhibitors, several of which are in clinical use as anti-coagulants, can reduce thrombin-dependent neuroinflammation in pathological conditions. However, their effects in a healthy CNS are largely unknown. In adult healthy mice, we compared the effects of treatment by the direct thrombin inhibitor dabigatran etexilate (DE), to those of warfarin, which acts by preventing vitamin K recycling essential for coagulation. After 4weeks, warfarin increased both astrocyte GFAP and microglia Iba-1 staining throughout the CNS; whereas DE reduced expression of both markers. Warfarin, but not DE, reduced sulfatide levels; and warfarin showed longer lasting changes in cerebellar gene expression. DE also reduced glial activation in a mouse model of Alzheimer's disease, although no changes in amyloid plaque burden were observed. These results suggest that treatment with direct thrombin inhibitors may be preferable to those agents which reduce vitamin K levels and have the potential to increase glial activation.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Anticoagulants; Brain; Calcium-Binding Proteins; Dabigatran; Disease Models, Animal; Female; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Humans; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microfilament Proteins; Mutation; Neuroglia; Peptide Fragments; Presenilin-1; Warfarin

As the number of patients with cardioembolic ischemic stroke is predicted to be double by 2030, increased burden of warfarin-associated hemorrhagic transformation (HT) after cerebral ischemia is an expected consequence. However, thus far, no effective treatment strategy is available for HT prevention in routine clinical practice. While the glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 (Ex-4) is known to protect against oxidative stress and neuronal cell death caused by ischemic brain damage, its effect on preventing warfarin-associated HT after cerebral ischemia is yet unknown. Therefore, we hypothesized that Ex-4 would stabilize the blood-brain barrier (BBB) and suppress neuroinflammation through PI3K-Akt-induced inhibition of glycogen synthase kinase-3β (GSK-3β) after warfarin-associated HT post-cerebral ischemia.. We used male C57BL/6 mice for all experiments. A 5-mg warfarin sodium tablet was dissolved in animals' drinking water (effective warfarin uptake 0.04 mg (2 mg/kg) per mouse). The mice were fed for 0, 6, 12, and 24 h with ad libitum access to the treated water. To study the effects of Ex-4, temporary middle cerebral artery occlusion (MCAO) was performed. Then, either Ex-4 (10 mg/kg) or saline was injected through the tail vein, and in the Ex-4 + wortmannin group, PI3K inhibitor wortmannin was intravenously injected, after reperfusion. The infarct volume, neurological deficits, and integrity of the BBB were assessed 72 h post MCAO. One- or two-way ANOVA was used to test the difference between means followed by Newman-Keuls post hoc testing for pair-wise comparison.. We observed that Ex-4 ameliorated warfarin-associated HT and preserved the integrity of the BBB after cerebral ischemia through the PI3K/Akt/GSK-3β pathway. Furthermore, Ex-4 suppressed oxidative DNA damage and lipid peroxidation, attenuated pro-inflammatory cytokine expression levels, and suppressed microglial activation and neutrophil infiltration in warfarin-associated HT post-cerebral ischemia. However, these effects were totally abolished in the mice treated with Ex-4 + the PI3K inhibitor-wortmannin. The PI3K/Akt-GSK-3β signaling pathway appeared to contribute to the protection afforded by Ex-4 in the warfarin-associated HT model.. GLP-1 administration could reduce warfarin-associated HT in mice. This beneficial effect of GLP-1 is associated with attenuating neuroinflammation and BBB disruption by inactivating GSK-3β through the PI3K/Akt pathway.

Topics: Animals; Anticoagulants; Blood-Brain Barrier; Brain; Brain Infarction; Brain Ischemia; Cytokines; Disease Models, Animal; Exenatide; Gene Expression Regulation; Glucagon-Like Peptide-1 Receptor; Hemorrhage; Hypoglycemic Agents; Infarction, Middle Cerebral Artery; Male; Mice; Mice, Inbred C57BL; Microglia; Nervous System Diseases; Peptides; Signal Transduction; Venoms; Warfarin

Hypertension is a common comorbidity in patients with chronic kidney disease (CKD). We reported earlier that oral anticoagulants, including warfarin and dabigatran, may induce acute kidney injury. No effects of oral anticoagulants on blood pressure (BP) have been previously reported. The aim of this study was to examine in detail the relationship of anticoagulant therapy and BP in rats.. Sham-operated and 5/6 nephrectomy rats were treated with different doses of oral anticoagulants (warfarin and dabigatran), superoxide scavenger N-acetylcysteine (NAC), vitamin K, and protease activated receptor 1 (PAR-1) inhibitor SCH79797. BP was measured by a tail cuff daily.. Warfarin and dabigatran both increased systolic BP in sham-operated and 5/6 nephrectomy rats in a dose-dependent manner. SCH79797 also increased systolic BP in a dose-dependent manner. Vitamin K prevented warfarin-induced increase in BP but did not affect BP when administered alone. NAC delayed the warfarin-associated increase in BP. Warfarin effects on BP were similar in 5/6 nephrectomy rats with different CKD stages.. Both warfarin and dabigatran increase systolic BP in rats. The mechanism of this effect is not clear, but our data suggest that it is related to decreased thrombin activity associated with anticoagulant treatment. The superoxide scavenger NAC delayed, but did not prevent, warfarin-induced hypertension.

Topics: Acetylcysteine; Animals; Anticoagulants; Antifibrinolytic Agents; Antithrombins; Benzimidazoles; beta-Alanine; Blood Pressure; Dabigatran; Disease Models, Animal; Dose-Response Relationship, Drug; Free Radical Scavengers; Nephrectomy; Pyrroles; Quinazolines; Rats; Receptor, PAR-1; Renal Insufficiency, Chronic; Vitamin K; Warfarin

Chronic kidney disease (CKD) affects the nonrenal clearance of drugs by modulating the functional expression of hepatic drug-metabolizing enzymes and transporters. The impact of CKD on oxidative and conjugative metabolism has been extensively studied. However, its effect on hepatic drug reduction, an important phase I drug-metabolism pathway, has not been investigated. We aimed to assess the effect of experimental CKD on hepatic reduction using warfarin as a pharmacological probe substrate. Cytosolic and microsomal cellular fractions were isolated from liver tissue harvested from five-sixths-nephrectomized and control rats (n = 10 per group). The enzyme kinetics for warfarin reduction were evaluated in both fractions, and formation of warfarin alcohols was used as an indicator of hepatic reductase activity. Selective inhibitors were employed to identify reductases involved in warfarin reduction. Gene and protein expression of reductases were determined using quantitative real-time polymerase chain reaction and Western blotting, respectively. Formation of RS/SR-warfarin alcohol was decreased by 39% (P < 0.001) and 43% (P < 0.01) in cytosol and microsomes, respectively, in CKD rats versus controls. However, RR/SS-warfarin alcohol formation was unchanged in the cytosol, and a trend toward its decreased production was observed in microsomes. Gene and protein expression of cytosolic carbonyl reductase 1 and aldo-keto reductase 1C3/18, and microsomal 11β-hydroxysteroid dehydrogenase type 1 were significantly reduced by >30% (P < 0.05) in CKD rats compared with controls. Collectively, these results suggest that the functional expression of hepatic reductases is selectively decreased in kidney disease. Our findings may explain one mechanism for altered nonrenal clearance, exposure, and response of drugs in CKD patients.

Topics: 11-beta-Hydroxysteroid Dehydrogenase Type 1; Alcohol Oxidoreductases; Aldehyde Reductase; Aldo-Keto Reductases; Animals; Cytosol; Disease Models, Animal; Kidney Diseases; Liver; Male; Microsomes, Liver; Oxidoreductases; Rats; Rats, Sprague-Dawley; Warfarin

Little is known about the pathophysiology of oral anticoagulation-associated intracerebral hemorrhage (OAC-ICH). We compared hematoma volume, number of terminal deoxynucleotidyl dUTP nick-end labeling (TUNEL)-positive cells (indicating cell death), MMP-9 levels, and perilesional edema formation between warfarin-treated mice and controls. Intracerebral hemorrhage was induced by an injection of collagenase into the right striatum. Twenty-four hours later, hematoma volume was measured using a photometric hemoglobin assay. Cell death was quantified using TUNEL staining. MMP-9 levels were determined by zymography, and edema formation was assessed via the wet-dry method. Warfarin increased hematoma volume by 2.6-fold. The absolute number of TUNEL-positive cells in the perihematomal zone was lower in warfarin-treated animals (300.5 ± 39.8 cells/mm2) than in controls (430.5 ± 38.9 cells/mm2; p = 0.034), despite the larger bleeding volume. MMP-9 levels were reduced in anticoagulated mice as compared to controls (p = 0.018). Perilesional edema formation was absent in warfarin mice and modestly present in controls. Our results suggest differences in the pathophysiology of OAC-ICH compared to intracerebral hemorrhage occurring under normal coagulation. A likely explanation is that thrombin, a strong inductor of apoptotic cell death and blood-brain barrier disruption, is produced to a lesser extent in OAC-ICH. In humans, however, we assume that the detrimental effects of a larger hematoma volume in OAC-ICH by far outweigh potential protective effects of thrombin deficiency.

Topics: Animals; Anticoagulants; Brain Edema; Cell Death; Cerebral Hemorrhage; Disease Models, Animal; Drug Administration Schedule; Hematoma; In Situ Nick-End Labeling; Male; Matrix Metalloproteinase 8; Matrix Metalloproteinase 9; Mice; Neurologic Examination; Statistics, Nonparametric; Warfarin

Lack of specific antidotes is a major concern in intracerebral hemorrhage (ICH) related to direct anticoagulants including dabigatran (OAC-ICH). We examined the efficacy of idarucizumab, an antibody fragment binding to dabigatran, in a mouse model of OAC-ICH. Dabigatran etexilate (DE) dose-dependently prolonged diluted thrombin time and tail-vein bleeding time, which were reversed by idarucizumab. Pretreatment with DE increased intracerebral hematoma volume and cerebral hemoglobin content. Idarucizumab in equimolar dose prevented excess hematoma expansion for both DE doses. In more extensive ICH, idarucizumab significantly reduced mortality. Thus, idarucizumab prevents excess intracerebral hematoma formation in mice anticoagulated with dabigatran and reduces mortality.

Topics: Animals; Antibodies, Monoclonal, Humanized; Anticoagulants; Antithrombins; Benzimidazoles; beta-Alanine; Bleeding Time; Blood Coagulation; Brain; Cerebral Hemorrhage; Dabigatran; Disease Models, Animal; Hematoma; Mice; Thrombin Time; Warfarin

To establish the rat model of acute pulmonary embolism, and study the changes of vascular active substances in pulmonary embolism rats, and investigate the interventive effect of anticoagulant drugs on vascular active substances.. One hundred and twenty-eight rats were randomly divided into four groups: control group, model group, low-molecular-weight heparin and warfarin treated group and rivaroxaban-treated group (n = 32 in each group). The method of autologous thrombosis was used to establish the animal model of acute pulmonary embolism. The animals were treated with saline or different anticoagulant drugs. The physiological and biochemical parameters were detected at different time points after embolization. The rats were killed after embolism of 24 h, 3 d, 5 d or 1 week respectively and the pathologic samples of lung tissues were collected to analyze the pulmonary pathological changes in different groups.. Rats in embolization group after blood clots injection showed shortness of breath, oral cyanosis; quicken heart rates and other symptoms. All embolization groups had pulmonary hypertension, the levels of type B natriuretic peptide (BNP) were increased significantly. The ratio of endothelin-1 (ET-1)/NO and thromboxane (TXB2) and prostacyclin (6-k-PGFla) were abnormal. After treated with effective anticoagulant drugs, the levels of BNP, ET-1, NO, TXB2 and 6-k-PGF1a were tended to the normal levels in the control group. The pulmonary hypertensions were gradually decreased. The efficacy of rivaroxaban on pulmonary embolism was the same as that of the low molecular weight heparin or warfarin.. Anticoagulation therapy can effectively improve endothelial function after pulmonary embolism, reduce pulmonary hypertension, and revise the increased BNP levels to normal levels. The efficacy of rivaroxaban is not inferior to that of low molecular weight heparin and warfarin.

Topics: Animals; Anticoagulants; Disease Models, Animal; Endothelin-1; Heparin, Low-Molecular-Weight; Hypertension, Pulmonary; Lung; Morpholines; Pulmonary Embolism; Rats; Rivaroxaban; Thiophenes; Warfarin

The aim of this study is to evaluate the effect of these new generation hemostatic agents on early-stage soft tissue healing of warfarin-treated rats by measuring the tissue factor (TF) activities. Rats in the warfarin group were treated intraperitonally with 0.1 mg/kg warfarin, and rats in the control group were treated with 1 mL/kg saline. All rats had 3 incisions on dorsal dermal tissue applied Celox, Ankaferd Blood Stopper (ABS), or no hemostatic agent. Six rats from each group were killed on day 4, and the other 6 were killed on day 8. Prothrombin time (PT) and TF activities were evaluated, respectively. Both the hemostatic agents positively affected the hemostasis. Warfarin treatment increased the PT levels as expected. Celox-treated dermal tissues had higher TF activity when compared to ABS-treated ones. The ABS affected the early-stage healing positively in clinical aspect, whereas Celox was more effective on hemostasis by means of increasing TF activities.

Topics: Animals; Biopolymers; Disease Models, Animal; Drug Interactions; Hemostasis; Male; Plant Extracts; Prothrombin Time; Rats; Rats, Wistar; Skin; Thromboplastin; Warfarin

Excessive anticoagulation with warfarin can result in acute kidney injury (AKI) by causing glomerular hemorrhage and renal tubular obstruction by red blood cell (RBC) casts in some patients, especially in those with chronic kidney disease (CKD). This condition was described as warfarin-related nephropathy (WRN). Recent evidence suggests that WRN-like syndromes are not confined to anticoagulation with warfarin, but may be seen with other anticoagulants, such as dabigatran. The aim of this study was to investigate dabigatran effects on kidney function in an animal model of CKD and possible pathogenic mechanisms of AKI.. Control and 5/6 nephrectomy rats were treated with different doses of dabigatran and protease-activated receptor 1 (PAR-1) inhibitor SCH79797.. Dabigatran resulted in changes in coagulation in rats similar to those in humans at 50 mg/kg/day. Dabigatran resulted in a dose-dependent increase in serum creatinine (Scr) and hematuria in both control and 5/6 nephrectomy rats. SCH79797 also increased Scr and hematuria, more prominent in animals with CKD. Morphologically, numerous RBC tubular casts were seen in 5/6 nephrectomy rats treated with either dabigatran or SCH79797 and only occasional RBC casts in control rats.. Our data indicate that WRN represents part of a broader syndrome, anticoagulant-related nephropathy (ARN). ARN, at least partially, is mediated via PAR-1. Our findings suggest that not only CKD patients, but other patients as well, are at high risk of developing AKI if the therapeutic range of anticoagulation with dabigatran is exceeded. Close monitoring of kidney function in patients on dabigatran therapy is warranted.

Topics: Acute Kidney Injury; Animals; Anticoagulants; Antithrombins; Benzimidazoles; beta-Alanine; Dabigatran; Disease Models, Animal; Hemorrhage; Kidney; Kidney Glomerulus; Male; Rats; Rats, Sprague-Dawley; Warfarin

This study assesses the risks and benefits of tissue plasminogen activator (tPA) treatment under oral anticoagulation with dabigatran compared with warfarin or vehicle control in transient middle cerebral artery occlusion (tMCAO). After pretreatment with warfarin (0.2 mg/kg/day), dabigatran (20 mg/kg/day), or vehicle (0.5% carboxymethyl cellulose sodium salt) for 7 days, tMCAO was induced for 120 min, followed by reperfusion and tPA (10 mg/kg/10 ml). Clinical parameters, including cerebral infarction volume, hemorrhagic volume, and blood coagulation, were examined. At 24 hr after reperfusion, markers for the neurovascular unit at the peri-ischemic lesion were immunohistochemically examined in brain sections, and MMP-9 activity was measured by zymography. Paraparesis and intracerebral hemorrhage volume were significantly improved in the dabigatran-pretreated group compared with the warfarin-pretreated group. A marked dissociation between astrocyte foot processes and the basal lamina or pericyte was observed in the warfarin-pretreated group, which was greatly improved in the dabigatran-pretreated group. Furthermore, a remarkable activation of MMP-9 in the ipsilateral warfarin-pretreated rat brain was greatly reduced in dabigatran-pretreated rats. The present study reveals that the mechanism of intracerebral hemorrhage with warfarin-pretreatment plus tPA in ischemic stroke rats is the dissociation of the neurovascular unit, including the pericyte. Neurovascular protection by dabigatran, which was first shown in this study, could partially explain the reduction in hemorrhagic complication by dabigatran reported from clinical study.

Topics: Animals; Antithrombins; Benzimidazoles; beta-Alanine; Brain Ischemia; Dabigatran; Disease Models, Animal; Fibrinolytic Agents; Intracranial Hemorrhages; Male; Rats; Rats, Wistar; Stroke; Tissue Plasminogen Activator; Warfarin

Assessment of the bleeding risk of antithrombotic agents is usually performed in healthy animals with some form of vascular injury to peripheral organs to induce bleeding. However, bleeding observed in patients with currently marketed antithrombotic drugs is typically spontaneous in nature such as intracranial haemorrhage (ICH) and gastrointestinal (GI) bleeding, which happens most frequently on top of preexisting pathologies such as GI ulcerations and polyps. Apc(min/+) mice are reported to develop multiple adenomas through the entire intestinal tract and display progressive anaemia.In this study, we evaluated the potential utility of Apc(min/+) mice as a model for assessing spontaneous GI bleeding with antithrombotic agents. Apc(min/+) mice exhibited progressive blood loss starting at the age of nine weeks. Despite the increase in bleeding, Apc(min/+) mice were in a hypercoagulable state and displayed an age-dependent increase in thrombin generation and circulating fibrinogen as well as a significant decrease in clotting times. We evaluated the effect of warfarin, dabigatran etexilate, apixaban and clopidogrel in this model by administering them in diet or in the drinking water to mice for 1-4 weeks. All of these marketed drugs significantly increased GI bleeding in Apc(min/+) mice, but not in wild-type mice. Although different exposure profiles of these antithrombotic agents make it challenging to compare the bleeding risk of compounds, our results indicate that the Apc(min/+) mouse may be a sensitive preclinical model for assessing the spontaneous GI bleeding risk of novel antithrombotic agents.

Topics: Age Factors; Animals; Benzimidazoles; Clopidogrel; Dabigatran; Disease Models, Animal; Drug Evaluation, Preclinical; Fibrinolytic Agents; Gastrointestinal Hemorrhage; Genes, APC; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Pyrazoles; Pyridines; Pyridones; Risk Factors; Ticlopidine; Warfarin

Bleeding is a major problem in warfarin pretreated patients who need emergency surgical procedures. APH is a hemostatic agent with ultra-hydrophilic and particulate properties. This study aimed to evaluate the in vivo hemostatic effect of APH in rats pretreated with warfarin.. Forty-eight Wistar rats were divided into two main groups: one group was pretreated with warfarin and the other group was not. These two groups were further divided into three subgroups according to the administration of APH, wheat meal, or saline, for a total of six subgroups. Standard full thickness tissue defects were performed on the backs of the rats. Saline, wheat meal, or APH were administered to the bleeding defect site in both main groups. Hemostasis time and amount of bleeding were calculated.. The bleeding time in rats administered APH was significantly shorter than those administered wheat meal and saline. Consequently, the amount of bleeding was significantly less in the APH groups than in the control groups.. APH has an effective hemostatic property in rats pretreated or non-pretreated with anticoagulants. Hemostatic agents can be useful for incidences of external bleedings, which are increasing because of anticoagulation.

Topics: Administration, Cutaneous; Animals; Anticoagulants; Disease Models, Animal; Hemorrhage; Hemostasis; Hemostatics; Male; Phytotherapy; Plant Extracts; Random Allocation; Rats; Rats, Wistar; Warfarin

Immune responses to therapeutic factor VIII remain a major problem, affecting 30% of patients with severe hemophilia A. The primary factors that drive immune responses in these patients remain elusive. There have been conflicting reports on a role of coagulation (or thrombin) in anti-FVIII immune responses.. To assess the importance of coagulation-associated processes for the onset of the anti-FVIII immune response.. Using FVIII-deficient mice, we compared the immunogenicity of recombinant FVIII or the inactive FVIII(V) (634M) mutant. In parallel, the involvement of tissue factor (TF) activity in the anti-FVIII immune response was investigated upon injection of a neutralizing anti-TF antibody or by the use of chimeric mice that lack TF expression in myeloid cells. The development of the anti-FVIII immune response was also monitored after treatment with warfarin.. The kinetics of the development of antibody responses to FVIII(V) (634M) were indistinguishable from those of wild-type FVIII. Inhibition of TF activity did not modulate immune responses to exogenous FVIII. Additionally, global inhibition of coagulation with warfarin failed to reduce the anti-FVIII immune response.. Thrombin generation or coagulation-associated processes do not modulate the anti-FVIII antibody response in mouse model of severe hemophilia A.

Topics: Animals; Antibodies, Neutralizing; Blood Coagulation; Disease Models, Animal; Factor VIII; Hemophilia A; Immunity, Humoral; Inflammation; Mice; Mutation; Plasmids; Protein Structure, Tertiary; Recombinant Proteins; Thrombin; Thromboplastin; Warfarin

Pseudoxanthoma elasticum (PXE) is a multisystem ectopic mineralization disorder caused by mutations in the ABCC6 gene. Warfarin, a commonly used anticoagulant, is associated with increased mineralization of the arterial blood vessels and cardiac valves. We hypothesized that warfarin may accelerate ectopic tissue mineralization in PXE, with clinical consequences. To test this hypothesis, we developed a model in which Abcc6(-/-) mice, which recapitulate features of PXE, were fed a diet supplemented with warfarin and vitamin K1. Warfarin action was confirmed by significantly increased serum levels of oxidized vitamin K. For mice placed on a warfarin-containing diet, quantitative chemical and morphometric analyses revealed massive accumulation of mineral deposits in a number of tissues. Mice fed a warfarin-containing diet were also shown to have abundant uncarboxylated form of matrix Gla protein, which allowed progressive tissue mineralization to ensue. To explore the clinical relevance of these findings, 1747 patients with PXE from the approximately 4000 patients in the PXE International database were surveyed about the use of warfarin. Of the 539 respondents, 2.6% reported past or present use of warfarin. Based on the prevalence of PXE (approximately 1:50,000), thousands of patients with PXE worldwide may be at risk for worsening of PXE as a result of warfarin therapy.

Topics: Animals; ATP-Binding Cassette Transporters; Calcification, Physiologic; Calcium; Calcium-Binding Proteins; Dermis; Diet; Dietary Supplements; Disease Models, Animal; Extracellular Matrix Proteins; Humans; Magnesium; Matrix Gla Protein; Mice; Minerals; Multidrug Resistance-Associated Proteins; Organ Specificity; Phosphates; Phosphorus; Pseudoxanthoma Elasticum; Vitamin K; Warfarin; X-Ray Microtomography

Here, we investigated the anticoagulant effects of darexaban in mice and human plasma in vitro, effects of darexaban in thrombosis and bleeding models in mice, and reversal effects of anti-inhibitor coagulant complex (ACC) and recombinant factor VIIa (rFVIIa) on anticoagulant effects of darexaban. In mice, darexaban inhibited FXa activity in plasma with an ED50 value of 24.8 mg/kg. Both darexaban and warfarin prolonged prothrombin time (PT) at 3 mg/kg and 0.3 mg/kg/day, respectively. PT and activated partial thromboplastin time (aPTT) prolonged by darexaban were dose-dependently reversed by intravenously-administered rFVIIa, significantly so at 1 mg/kg. In a pulmonary thromboembolism (PE) mouse model, both darexaban and warfarin dose-dependently reduced the mortality rate, significantly so at 10 mg/kg and 3 mg/kg/day, respectively. In a FeCl3-induced venous thrombosis (VT) mouse model, darexaban (0.3-10 mg/kg) dose-dependently decreased the thrombus protein content, significantly so at doses of 3 mg/kg or higher. In a tail-transection mouse model, darexaban had no significant effect on the amount of blood loss at doses up to 10 mg/kg, while warfarin showed a dose-dependent increase in blood loss, significantly so from 1 mg/kg/day. Darexaban and its metabolite darexaban glucuronide significantly prolonged PT and aPTT in human plasma in vitro, and while rFVIIa concentration-dependently reversed the prolonged PT in this plasma, ACC dose-dependently reversed both PT and aPTT changes prolonged by darexaban. Taken together, these results suggest that darexaban has a potential to be an oral anticoagulant with a better safety profile than warfarin, and that rFVIIa and ACC may be useful as antidotes to darexaban in cases of overdose.

Topics: Animals; Anticoagulants; Azepines; Benzamides; Disease Models, Animal; Factor VIIa; Hemorrhage; Humans; Male; Mice; Mice, Inbred ICR; Partial Thromboplastin Time; Prothrombin Time; Recombinant Proteins; Thrombosis; Warfarin

Dabigatran etexilate (DE) has recently been introduced for stroke prevention in atrial fibrillation, but management of acute ischaemic stroke during therapy with DE is a challenge. Thrombolysis is contraindicated because of a presumed increased risk of intracerebral haemorrhagic complications. We studied in different ischaemia models whether DE increases secondary haemorrhage after thrombolysis. C57BL/6 mice were anticoagulated with high-dose DE or warfarin. After 2 hour (h) or 3 h transient filament MCAO, rt-PA was injected. At 24 h after MCAO, secondary haemorrhage was quantified using a macroscopic haemorrhage score and haemoglobin spectrophotometry. Post-ischaemic blood-brain-barrier (BBB) damage was assessed using Evans blue. To increase the validity of findings, the duration of anticoagulation was prolonged in mice (5 x DE over 2 days), and the effect of DE after thrombolysis was also examined in thromboembolic MCAO in rats.Pretreatment with warfarin resulted in significantly more secondary haemorrhage (mean haemorrhage score 2.6 ± 0.2) compared to non-anticoagulated animals (1.7 ± 0.3) and DE (9 mg/kg, 1.6 ± 0.3) in 2 h ischaemia. Also after a 3 h period of ischaemia, haemorrhage was more severe in animals anticoagulated with warfarin compared to 9 mg/kg DE and non-anticoagulated control. Prolonged or enteral dabigatran pretreatment led to identical results. Also, thrombolysis after thromboembolic MCAO in rats did not induce more severe bleeding in DE-treated animals. Mice pretreated with warfarin had higher BBB permeability and increased activation of matrix-metalloproteinase 9. In conclusion, DE does not increase the risk of secondary haemorrhage after thrombolysis in various rodent models of ischaemia and reperfusion. The implications of this finding for stroke patients have to be determined in the clinical setting.

Topics: Animals; Anticoagulants; Antithrombins; Benzimidazoles; beta-Alanine; Blood-Brain Barrier; Cerebral Arteries; Cerebral Hemorrhage; Dabigatran; Disease Models, Animal; Humans; Ischemia; Mice; Mice, Inbred C57BL; Rats; Stroke; Thrombolytic Therapy; Warfarin

Edoxaban is a novel, potent and orally active direct Factor Xa (FXa) inhibitor under development for prophylaxis and treatment of thromboembolic diseases. Properties of dose response and margin of safety of anticoagulants are the key factors for a positive risk/benefit of novel oral anticoagulants.. To compare the dose response of antithrombotic effect and margin of safety between antithrombotic and hemorrhagic effects of edoxaban with conventional anticoagulants, unfractionated heparin (UFH), dalteparin (low molecular weight heparin), lepirudin, and warfarin in rat models of thrombosis and hemorrhage.. Rats were treated with edoxaban, UFH, dalteparin, and lepirudin by continuous intravenous (iv) infusion, or with oral warfarin for 4 days before inducing thrombosis or bleeding. Thrombosis was induced by inserting a platinum wire into the inferior vena cava for 60 minutes. Tail template bleeding time was measured after making an incision on the tail.. In rats, iv infusion of edoxaban inhibited venous thrombosis in a dose-dependent manner. The other anticoagulants also exerted dose-dependent antithrombotic effects. The slopes of the dose-response curves of edoxaban were significantly shallower than the slopes of UFH, dalteparin, and warfarin. At supratherapeutic doses, edoxaban prolonged bleeding time in a rat tail bleeding model. To determine bleeding risk, the margins between antithrombotic and bleeding-time prolongation were compared. The margins of safety of edoxaban were wider than those of UFH, dalteparin, lepirudin, and warfarin.. These results suggest that edoxaban may be more easily controlled and has the potential for a more positive risk/benefit ratio compared to conventional anticoagulants.

Topics: Animals; Anticoagulants; Disease Models, Animal; Dose-Response Relationship, Drug; Factor Xa Inhibitors; Heparin; Hirudins; Male; Pyridines; Rats; Rats, Wistar; Recombinant Proteins; Thiazoles; Thromboembolism; Warfarin

Vascular calcification is an independent risk factor for cardiovascular disease. Once thought to be a passive process, vascular calcification is now known to be actively prevented by proteins acting systemically (fetuin-A) or locally (matrix Gla protein). Warfarin is a vitamin K antagonist, widely prescribed to reduce coagulation by inhibiting vitamin K-dependent coagulation factors. Recently, it became clear that vitamin K antagonists also affect vascular calcification by inactivation of matrix Gla protein. Here, we investigated functional cardiovascular characteristics in a mouse model with warfarin-induced media calcification.. DBA/2 mice received diets with variable concentrations of warfarin (0.03, 0.3, and 3 mg/g) with vitamin K1 at variable time intervals (1, 4, and 7 weeks). Von Kossa staining revealed that warfarin treatment induced calcified areas in both medial layer of aorta and heart in a dose- and time-dependent fashion, which could be inhibited by simultaneous vitamin K2 treatment. With ongoing calcification, matrix Gla protein mRNA expression decreased, and inactive matrix Gla protein expression increased. TdT-mediated dUTP-biotin nick end labeling-positive apoptosis increased, and vascular smooth muscle cell number was concomitantly reduced by warfarin treatment. On a functional level, warfarin treatment augmented aortic peak velocity, aortic valve-peak gradient, and carotid pulse-wave velocity.. Warfarin induced significant calcification with resulting functional cardiovascular damage in DBA/2 wild-type mice. The model would enable future researchers to decipher mechanisms of vascular calcification and may guide them in the development of new therapeutic strategies.

Topics: Animals; Anticoagulants; Antifibrinolytic Agents; Aorta; Apoptosis; Calcium; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Muscle, Smooth, Vascular; Pulsatile Flow; Risk Factors; Vascular Calcification; Vitamin K 1; Vitamin K 2; Warfarin

In vitro, transglutaminase-2 (TG2)-mediated activation of the β-catenin signaling pathway is central in warfarin-induced calcification, warranting inquiry into the importance of this signaling axis as a target for preventive therapy of vascular calcification in vivo.. The adverse effects of warfarin-induced elastocalcinosis in a rat model include calcification of the aortic media, loss of the cellular component in the vessel wall, and isolated systolic hypertension, associated with accumulation and activation of TG2 and activation of β-catenin signaling. These effects of warfarin can be completely reversed by intraperitoneal administration of the TG2-specific inhibitor KCC-009 or dietary supplementation with the bioflavonoid quercetin, known to inhibit β-catenin signaling. Our study also uncovers a previously uncharacterized ability of quercetin to inhibit TG2. Quercetin reversed the warfarin-induced increase in systolic pressure, underlying the functional consequence of this treatment. Molecular analysis shows that quercetin diet stabilizes the phenotype of smooth muscle and prevents its transformation into osteoblastic cells.. Inhibition of the TG2/β-catenin signaling axis seems to prevent warfarin-induced elastocalcinosis and to control isolated systolic hypertension.

Topics: Animals; Aorta; Aortic Diseases; beta Catenin; Blood Pressure; Cell Line; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; GTP-Binding Proteins; Isoxazoles; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Smooth, Vascular; Osteogenesis; Phosphorylation; Protein Glutamine gamma Glutamyltransferase 2; Quercetin; Rats; Rats, Wistar; Signal Transduction; Transglutaminases; Vascular Calcification; Warfarin

The leading cause of death in patients with chronic kidney disease (CKD) is cardiovascular disease, with vascular calcification being a key modifier of disease progression. A local regulator of vascular calcification is vitamin K. This γ-glutamyl carboxylase substrate is an essential cofactor in the activation of several extracellular matrix proteins that inhibit calcification. Warfarin, a common therapy in dialysis patients, inhibits the recycling of vitamin K and thereby decreases the inhibitory activity of these proteins. In this study, we sought to determine whether modifying vitamin K status, either by increasing dietary vitamin K intake or by antagonism with therapeutic doses of warfarin, could alter the development of vascular calcification in male Sprague-Dawley rats with adenine-induced CKD. Treatment of CKD rats with warfarin markedly increased pulse pressure and pulse wave velocity, as well as significantly increased calcium concentrations in the thoracic aorta (3-fold), abdominal aorta (8-fold), renal artery (4-fold), and carotid artery (20-fold). In contrast, treatment with high dietary vitamin K1 increased vitamin K tissue concentrations (10-300-fold) and blunted the development of vascular calcification. Thus, vitamin K has an important role in modifying mechanisms linked to the susceptibility of arteries to calcify in an experimental model of CKD.

Topics: Adenine; Animals; Anticoagulants; Arteries; Biomarkers; Blood Pressure; Dietary Supplements; Disease Models, Animal; Disease Progression; Male; Osteocalcin; Pulse Wave Analysis; Rats; Rats, Sprague-Dawley; Renal Insufficiency, Chronic; Time Factors; Vascular Calcification; Vitamin K 1; Vitamin K 2; Warfarin

The number of patients who are on long-term anticoagulation therapy while experiencing traumatic brain injury (TBI) is rising. This experimental study evaluated whether warfarin pre-treatment increases brain hemorrhage and worsens functional outcome after TBI, and whether the rapid reversal of anticoagulation after TBI prevents warfarin-exacerbated brain damage. Normal CD-1 mice (C) and mice pre-treated with warfarin (W) to an International Normalized Ratio of 3.5±0.9 underwent TBI using a controlled cortical impact model. Mean hemorrhage volume 24 h after TBI was 1.2±0.4 μL in C mice and 10.9±6.9 μL in W mice (p=0.029, n=4 per group). In a second study, anticoagulated mice received either saline (W-S) or prothrombin complex concentrate (W-PCC, 100 U/kg) intravenously 60 min after TBI. Anticoagulation reversal using PCC (W-PCC mice) reduced hemorrhage volumes as compared to W-S animals (7.3±6.0 versus 19.8±14.0 μL, p=0.045, n=8 per group). In a third study, we examined motor deficits and lesion volume in C, W-S, and W-PCC mice until 33 days after injury. Functional outcome and lesion volume were no different between groups (n=10 per group). In conclusion, we characterized an experimental model of TBI occurring during warfarin anticoagulation. Anticoagulation led to higher intracerebral blood volumes, but did not significantly worsen functional outcome. The rapid reversal of anticoagulation may be effective in preventing excess bleeding.

Topics: Animals; Anticoagulants; Brain Injuries; Disease Models, Animal; Hemorrhage; Intracranial Hemorrhages; Male; Mice; Recovery of Function; Warfarin

Anticoagulation with dabigatran etexilate (DE) has a favorable risk-to-benefit profile for the prevention of ischemic events in patients with atrial fibrillation compared to warfarin. Whereas warfarin constitutes a strong contraindication for thrombolysis, it is unclear whether patients anticoagulated with DE can be thrombolysed. We compared the risk of thrombolysis-associated hemorrhagic transformation (HT) after pretreatment with DE or warfarin in a mouse model of ischemic stroke.. Thirty-nine C57BL/6 mice were pretreated orally with 75 mg/kg DE, 112.5mg/kg DE, 2mg/kg warfarin, or saline. We performed right middle cerebral artery occlusion for 3 hours, administered recombinant tissue plasminogen activator (rt-PA) directly before reperfusion, and assessed neurological deficit and HT blood volume after 24 hours.. Warfarin anticoagulation increased HT secondary to rt-PA treatment as compared to nonanticoagulated controls (6.9 ± 5.5 μl vs 0.8 ± 0.6 μl, p < 0.05). In contrast, the rate of HT after pretreatment with 75 mg/kg DE, which led to plasma levels comparable to the highest plasma levels observed in participants of the RE-LY trial, did not differ significantly from controls (1.6 ± 0.8; p > 0.05 vs control). However, a high-dose group receiving 112.5mg/kg DE showed a considerable extent of HT (9.2 ± 5.6 μl, p < 0.01).. Our experimental data suggest that the risk of thrombolysis-associated HT may not be increased under DE pretreatment with standard doses leading to plasma levels of up to 400 ng/ml, a concentration that was not exceeded in the majority of DE trial patients. At higher DE plasma levels, however, the risk of severe HT rises considerably, emphasizing the need for a readily available assay of DE anticoagulant activity.

Topics: Animals; Anticoagulants; Benzimidazoles; beta-Alanine; Cerebral Hemorrhage; Dabigatran; Disease Models, Animal; Drug Interactions; Fibrinolytic Agents; Mice; Mice, Inbred C57BL; Stroke; Tissue Plasminogen Activator; Warfarin

intracerebral hemorrhage associated with oral anticoagulants has a poor prognosis. Current treatment guidelines are based on case series and plausibility only, and a common consensus on effective hemostatic therapy is missing. We compared the effectiveness of diverse hemostatic approaches in a mouse model of warfarin-associated intracerebral hemorrhage.. male C57BL/6 mice received anticoagulant treatment with warfarin (0.4 mg/kg for 3 days). Intracerebral hemorrhage was induced by striatal injection of collagenase, and 30 minutes later, mice received an intravenous injection of saline (200 μL n=15), prothrombin complex concentrate (100 U/kg, n=10), fresh-frozen plasma (200 μL, n=13), recombinant human Factor VII activated (3.5 mg/kg, n=8 and 10 mg/kg, n=8), or tranhexamic acid (400 mg/kg, n=12). Intracerebral hemorrhage volume was quantified on T2-weighted images after 24 hours.. mean hematoma volumes were 7.4 ± 1.8 mm(3) in the nonwarfarin controls and 21.9 ± 5.0 mm(3) in the warfarin group receiving saline. Prothrombin complex concentrate (7.5 ± 2.3 mm(3)) and fresh-frozen plasma (8.7 ± 2.1) treatment resulted in significantly smaller hematoma volume compared with saline. Recombinant human Factor VII activated (10 mg/kg: 14.7 ± 3.4; 3.5 mg/kg: 15.0 ± 6.8 mm(3)) and tranexamic acid (16.2 ± 4.1 mm(3)) were less effective. Water content in the hemorrhagic hemisphere was similar in all groups except for tranexamic acid in which it was significantly increased.. prothrombin complex concentrate and fresh-frozen plasma effectively prevent hematoma growth in murine warfarin-associated intracerebral hemorrhage, whereas Factor VIIa was less effective. Tranexamic acid exacerbates perihematoma edema in this mouse warfarin-associated intracerebral hemorrhage model.

Topics: Animals; Anticoagulants; Blood Coagulation Factors; Cerebral Hemorrhage; Collagenases; Disease Models, Animal; Factor VIIa; Hematoma; Hemostasis; Humans; Male; Mice; Plasma; Recombinant Proteins; Tranexamic Acid; Warfarin

The prevalence of long-term oral anticoagulant therapy is rising. Treatment options for patients who have an ischemic stroke under oral anticoagulant therapy are limited, and clinical data on the risk of hemorrhagic transformation (HT) in this condition are scarce. We therefore aimed to establish a mouse model of ischemic stroke occurring during oral anticoagulant therapy to assess the frequency and characteristics of HT.. C57BL/6 mice (n=59) were pretreated with warfarin. Untreated mice (n=32) served as controls. We performed a 3-hour transient filament occlusion of the right middle cerebral artery. In a first set of animals, ischemic lesion size and HT were evaluated macroscopically at 24 hours after middle cerebral artery occlusion. In a second set of mice, quantitative analysis of HT was performed at different time points after middle cerebral artery occlusion and in animals with different international normalized ratio levels using a photometric hemoglobin assay.. Oral anticoagulant therapy at the onset of ischemia led to HT in all anticoagulated mice, whereas only 14% of the control mice showed HT. Mean HT blood volume 24 hours after middle cerebral artery occlusion was 0.3±0.4 μL in controls, 4.2±1.7 μL in mice anticoagulated to a mean international normalized ratio of 1.9±0.5 (P<0.05 versus controls), and 5.2±2.7 μL in mice with an international normalized ratio of 2.9±0.9 (P<0.001 versus controls). Anticoagulated mice euthanized at the time point of reperfusion had less HT than mice euthanized after 21 hours of reperfusion (1.6±0.5 μL versus 5.9±3.6 μL, P<0.05).. We present a mouse model of ischemic stroke occurring during oral anticoagulant therapy. Warfarin pretreatment dramatically increases the risk of HT 24 hours after middle cerebral artery occlusion. Reperfusion injury seems to be a critical component in this condition.

Topics: Animals; Anticoagulants; Brain Ischemia; Cerebral Hemorrhage; Cerebrovascular Circulation; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Infarction, Middle Cerebral Artery; Male; Mice; Mice, Inbred C57BL; Reperfusion Injury; Risk Factors; Stroke; Warfarin

In this study, the hemostatic efficacy of Celox® in rats under hypothermia or warfarin treatment was investigated.. A total of forty-eight Sprague-Dawley female rats weighing 200-350 g were used in the study. Six experimental study groups were designed, as follows: Group 1: Normothermia + compression; Group 2: normothermia + Celox®; Group 3: hypothermia + compression; Group 4: hypothermia + Celox®; Group 5: normothermia + warfarin + compression; and Group 6: normothermia + warfarin + Celox®.. Celox® provided effective hemorrhage control in all three tested groups. There was a statistically significant difference between compression and Celox® implementation in all groups in terms of hemostasis (p-values for the normothermia, hypothermia and warfarin groups were p<0.05, p<0.01 and p<0.01, respectively). Furthermore, the compression numbers were significantly lower in all of the groups that received Celox ® than in those in which compression alone was applied (p-values for the normothermia, hypothermia and warfarin groups were p<0.01, p<0.01 and p<0.001, respectively).. Celox® provides effective hemorrhage control under conditions of normothermia, hypothermia and use of the oral anticoagulant agent warfarin.

Topics: Animals; Anticoagulants; Chitosan; Disease Models, Animal; Female; Femoral Artery; Hemorrhage; Hemostatics; Hypothermia, Induced; Rats; Rats, Sprague-Dawley; Warfarin

Darexaban (YM150) is an oral factor Xa inhibitor developed for the prophylaxis of venous and arterial thromboembolic disease. This study was conducted to investigate the biochemical and pharmacological profiles of darexaban and its active metabolite darexaban glucuronide (YM-222714), which predominantly determines the antithrombotic effect after oral administration of darexaban. In vitro activity was evaluated by enzyme and coagulation assays, and a prothrombin activation assay using reconstituted prothrombinase or whole blood clot. In vivo effects were examined in venous thrombosis, arterio-venous (A-V) shunt thrombosis, and bleeding models in rats. Both darexaban and darexaban glucuronide competitively and selectively inhibited human factor Xa with Ki values of 0.031 and 0.020 μM, respectively. They showed anticoagulant activity in human plasma, with doubling concentrations of darexaban and darexaban glucuronide for prothrombin time of 1.2 and 0.95 μM, respectively. Anticoagulant activity was independent of antithrombin. Darexaban and darexaban glucuronide inhibited the prothrombin activation induced by prothrombinase complex or whole blood clot with similar potency to free factor Xa. In contrast, prothrombinase- and clot-induced prothrombin activation were resistant to inhibition by enoxaparin. In venous and A-V shunt thrombosis models in rats, darexaban strongly suppressed thrombus formation without affecting bleeding time, with ID₅₀ values of 0.97 and 16.7 mg/kg, respectively. Warfarin also suppressed thrombus formation in these models, but caused a marked prolongation of bleeding time at antithrombotic dose. In conclusion, darexaban is a selective and direct factor Xa inhibitor and a promising oral anticoagulant for the prophylaxis and treatment of thromboembolic diseases.

Topics: Administration, Oral; Animals; Anticoagulants; Azepines; Benzamides; Bleeding Time; Disease Models, Animal; Dogs; Dose-Response Relationship, Drug; Enoxaparin; Factor Xa Inhibitors; Glucuronides; Humans; Macaca fascicularis; Male; Mice; Mice, Inbred ICR; Prothrombin Time; Rabbits; Rats; Rats, Sprague-Dawley; Thrombosis; Venous Thrombosis; Warfarin

The study aimed to predict effective human jejunal permeability (P(eff)) using a biophysical model based on parametrized paracellular, aqueous boundary layer, and transcellular permeabilities, and the villus-fold surface area expansion factor (k(VF)). Published human jejunal data (119 P(eff), 53 compounds) were analyzed by a regression procedure incorporating a dual-pore size paracellular model. Transcellular permeability, scaled by k(VF), was equated to that of Caco-2 at pH 6.5. The biophysical model predicted human jejunal permeability data within the experimental uncertainty. This investigation revealed several surprising predictions: (i) many molecules permeate predominantly (but not exclusively) by the paracellular route, (ii) the aqueous boundary layer thickness in the intestinal perfusion experiments is larger than expected, (iii) the mucosal surface area in awake humans is apparently nearly entirely accessible to drug absorption, and (iv) the relative "leakiness" of the human jejunum is not so different from that observed in a number of published Caco-2 studies.

Topics: Animals; Disease Models, Animal; Dogs; Humans; Jejunal Diseases; Kidney Diseases; Models, Biological; Permeability; Porosity; Regression Analysis

The burden of intracerebral hemorrhage associated with oral anticoagulants (OAC-ICH) is growing. However, little is known about the pathophysiology of W-ICH. Herein, we refine a mouse model of OAC-ICH using repetitive T2* MRI to describe kinetics of hematoma enlargement, and establish a benchside point of care INR assay (PoC) for assessment of anticoagulation.. C57/BL6 mice drank warfarin (0.4mg/kg/24h) in their water. ICH was induced by stereotactic injection of collagenase type VII (0.045U) into the left striatum. Hemorrhagic blood volume was quantified by MRI T2* images and on cryosections 48h after ICH induction. Kinetics of hematoma expansion were compared in strongly, moderately, and non-anticoagulated mice using repeated MRI T2* imaging. The PoC INR technique was validated against standard laboratory INR, and tail vein bleeding time (TVBT).. PoC INR correlated with central laboratory measurements (r=0.989; p<0.0001) and with TVBT (r=0.982; p<0.0001). Hematoma volume was 21.2+/-6.7mm(3) in heavily (PoC INR 4-5), 12.3+/-4.8 in moderately (INR 2-3), and 8.6+/-3.3 in non-anticoagulated mice (INR<1.2). Hematoma volume determined from cryosections and T2* MRI correlated well (r=0.922). Strength of anticoagulation was associated with neurologic outcome. Hematoma enlargement occurred mainly during the first 3h in anticoagulated mice.. PoC allows repeated benchside INR measurements in individual mice which reflect the level of anticoagulation. Stronger anticoagulation results in larger hematoma volumes. As hematoma enlargement occurs mainly during the first hours, potential hemostatic therapies should be tested early in this OAC-ICH model.

Topics: Animals; Bleeding Time; Brain; Calibration; Cerebral Hemorrhage; Collagenases; Disease Models, Animal; Disease Progression; International Normalized Ratio; Kinetics; Magnetic Resonance Imaging; Male; Mice; Mice, Inbred C57BL; Severity of Illness Index; Tail; Veins; Warfarin

The antithrombotic activity of tecarfarin, a novel orally active vitamin K epoxide reductase inhibitor, was assessed in canine and rabbit thrombosis models. In dogs, once-daily oral doses of 0.5mg/kg tecarfarin selectively reduced the levels of the vitamin K-dependent coagulation factors (factors II, VII, IX, and X) and prolonged the prothrombin time (PT). A 4 to 7day course of oral tecarfarin (0.05 - 0.5mg/kg) prolonged the PT by 3 to 5-fold and reduced thrombus formation in arterial and venous segments subjected to a combination of electrical injury and flow-limiting constriction. To compare the effects of tecarfarin with those of warfarin, rabbits were given once-daily oral doses of 1mg/kg tecarfarin, 1.5mg/kg warfarin, or saline for 2days. After verifying increases in the PT with tecarfarin and warfarin, blood loss from standardized ear incisions was measured to assess hemorrhagic potential. Then, after intravenous injection of (125)I-labeled rabbit fibrinogen, thrombosis was induced in an isolated jugular vein segment by a combination of balloon catheter-induced endothelial denudation and venous occlusion. Compared with the saline control, both tecarfarin and warfarin prolonged the PT, increased blood loss from the ear incisions, and attenuated thrombus formation. Thus, like warfarin, tecarfarin attenuates venous and arterial thrombus formation in animal models by reducing the levels of the vitamin K-dependent coagulation factors.

Topics: Animals; Anticoagulants; Benzoates; Coumarins; Disease Models, Animal; Dogs; Female; Humans; Male; Prothrombin Time; Rabbits; Thrombosis; Warfarin

Warfarin-associated intracerebral hemorrhage (W-ICH) is a severe type of stroke. There is no consensus on the optimal treatment for W-ICH. Using a mouse model, we tested whether the rapid reversal of anticoagulation using human prothrombin complex concentrate (PCC) can reduce hemorrhagic blood volume. Male CD-1 mice were treated with warfarin (2 mg/kg over 24 h), resulting in a mean (+/-s.d.) International Normalized Ratio of 3.5+/-0.9. First, we showed that an intravenous administration of human PCC rapidly reversed anticoagulation in mice. Second, a stereotactic injection of collagenase was administered to induce hemorrhage in the right striatum. Forty-five minutes later, the animals were randomly treated with PCC (100 U/kg) or saline i.v. (n=12 per group). Twenty-four hours after hemorrhage induction, hemorrhagic blood volume was quantified using a photometric hemoglobin assay. The mean hemorrhagic blood volume was reduced in PCC-treated animals (6.5+/-3.1 microL) compared with saline controls (15.3+/-11.2 microL, P=0.015). In the saline group, 45% of the mice developed large hematomas (i.e., >15 microL). In contrast, such extensive lesions were never found in the PCC group. We provide experimental data suggesting PCC to be an effective acute treatment for W-ICH in terms of reducing hemorrhagic blood volume. Future studies are needed to assess the therapeutic potential emerging from our finding for human W-ICH.

Topics: Animals; Blood Coagulation; Blood Coagulation Factors; Cerebral Hemorrhage; Coagulants; Disease Models, Animal; Drug Administration Schedule; Humans; Injections, Intravenous; Male; Mice; Mice, Inbred Strains; Time Factors; Treatment Outcome; Warfarin

Matrix-Gla Protein (MGP) is a vitamin K-dependent protein acting as a local inhibitor of vascular calcification. Vitamin K-antagonists (oral anticoagulant; OAC) inhibit the activation of MGP by blocking vitamin K-metabolism. The aim of this study was to investigate the effect of long-term OAC treatment on circulating MGP levels in humans and on MGP expression in mice. Additionally, we tested the association between circulating inactive MGP (ucMGP) levels and the presence and severity of AVC in patients with aortic valve disease (AVD). We analysed circulating ucMGP levels in 191 consecutive patients with echocardiographically proven calcific AVD and 35 control subjects. The extent of AVC in the patients was assessed by multislice spiral computed tomography. Circulating ucMGP levels were significantly lower in patients with AVD (348.6 +/- 123.1 nM) compared to the control group (571.6 +/- 153.9 nM, p < 0.001). Testing the effect of coumarin in mice revealed that also the mRNA expression of MGP in the aorta was downregulated. Multifactorial analysis revealed a significant effect of glomerular filtration rate and long-term OAC therapy on circulating ucMGP levels in the patient group. Subsequently, patients on long-term OAC had significantly increased AVC scores. In conclusion, patients with calcific AVD had significantly lower levels of circulating ucMGP as compared to a reference population, free of coronary and valvular calcifications. In addition, our data suggest that OAC treatment may decrease local expression of MGP, resulting in decreased circulating MGP levels and subsequently increased aortic valve calcifications as an adverse side effect.

Topics: Administration, Oral; Adult; Aged; Aged, 80 and over; Animals; Anticoagulants; Aorta; Aortic Valve; Biomarkers; Calcinosis; Calcium-Binding Proteins; Case-Control Studies; Cross-Sectional Studies; Disease Models, Animal; Down-Regulation; Echocardiography; Extracellular Matrix Proteins; Female; Glomerular Filtration Rate; Heart Valve Diseases; Humans; Male; Matrix Gla Protein; Mice; Mice, Inbred DBA; Middle Aged; Osteopontin; Prognosis; Risk Assessment; Risk Factors; RNA, Messenger; Severity of Illness Index; Time Factors; Tomography, Spiral Computed; Vitamin K; Warfarin

Future demographic changes predict an increase in the number of patients with atrial fibrillation. As long-term anticoagulation for the prevention of ischemic strokes becomes more prevalent, the burden of warfarin-associated intracerebral hemorrhage (W-ICH) is likely to grow. However, little is known about the clinical aspects and pathophysiologic mechanisms of W-ICH. This study describes the development of a mouse model of W-ICH in which hematoma growth and outcomes can be correlated with anticoagulation parameters.. CD-1 mice were treated with warfarin (2 mg/kg per 24 hours) added to drinking water. ICH was induced by stereotactic injection of collagenase type VII (0.075 U) into the right striatum. Hemorrhagic blood volume was quantified by means of a photometric hemoglobin assay 2 and 24 hours after hemorrhage induction. Neurologic outcomes were assessed on a 5-point scale.. The international normalized ratio in nonanticoagulated mice was 0.8+/-0.1. After 24 (W-24) and 30 (W-30) hours of warfarin pretreatment, international normalized ratio values increased to 3.5+/-0.9 and 7.2+/-3.4, respectively. Compared with nonanticoagulated mice, mean hemorrhagic blood volume determined 24 hours after hemorrhage induction was found to be 2.5-fold larger in W-24 mice (P=0.019) and 3.1-fold larger in W-30 mice (P<0.001, n=10 per group). Mortality at 24 hours after hemorrhage induction was 0% in nonanticoagulated mice, 10% in W-24 mice, and 30% in W-30 mice. Hematoma enlargement between 2 and 24 hours after hemorrhage induction was -1.4% for nonanticoagulated mice, 22.9% for W-24 mice, and 62.2% for W-30 mice.. This study characterizes the first experimental model of W-ICH. It may be helpful in gaining further insights into the pathophysiology of W-ICH and may be used for testing the efficacy of treatment strategies, such as hemostatic therapy, in this severe subtype of stroke.

Topics: Administration, Oral; Animals; Anticoagulants; Brain Damage, Chronic; Cerebral Hemorrhage; Collagenases; Corpus Striatum; Disease Models, Animal; Disease Progression; Hematoma; Injections; International Normalized Ratio; Male; Mice; Microbial Collagenase; Movement Disorders; Warfarin

Both abnormal lipid metabolisms and coagulopathy have been suggested to be associated with the development of steroid-induced osteonecrosis (ON). We examined plasma risk factors for development of steroid-induced ON in rabbits after prophylaxis with a lipid-lowering agent and/or an anticoagulant.. Seventy adult male rabbits were injected intramuscularly once with 20 mg/kg methylprednisolone acetate. Fifty-five rabbits received prophylaxis with probucol (a lipid-lowering agent; n = 20) or warfarin (an anticoagulant; n = 14) or both (n = 21). Probucol and warfarin were administered beginning 1 to 2 weeks prior to steroid injection. Two weeks after steroid injection, the bilateral femora and humeri were examined histopathologically for the presence of ON. Based on a logistic regression model, laboratory variables before steroid injection were assessed to determine whether they demonstrated any association with the risk of ON.. Twenty-one rabbits developed ON. In the univariate analyses, significant positive associations were observed between plasma concentrations of triglyceride and low-density lipoprotein and the risk of development of ON. In the multivariate model, only the plasma triglyceride level suggested a positive association. Even after adjusting for probucol and warfarin use, the plasma triglyceride level was still suggested to be a predictor for development of ON. Rabbits with higher baseline triglyceride levels had a more pronounced triglyceride increase in their response to steroids.. Our study suggests that, after prophylaxis with probucol and/or warfarin, plasma triglyceride level is associated with the development of steroid-induced ON in rabbits.

Topics: Adrenal Cortex Hormones; Animals; Anticholesteremic Agents; Anticoagulants; Biomarkers; Chemoprevention; Disease Models, Animal; Fatty Acids, Nonesterified; Lipoproteins, LDL; Male; Methylprednisolone; Methylprednisolone Acetate; Odds Ratio; Osteonecrosis; Probucol; Rabbits; Risk Factors; Triglycerides; Warfarin

Isolated systolic hypertension is associated with increased elastase activity, vascular calcification, and vascular stiffness. We sought to determine the importance of elastase activity and matrix degradation in the development of elastocalcinosis.. Elastocalcinosis was induced in vivo and ex vivo using warfarin. Hemodynamic parameters, calcium deposition, elastin degradation, transforming growth factor (TGF)-beta signaling, and elastase activity were evaluated at different time points in the in vivo model. Metalloproteinases, serine proteases, and cysteine proteases were blocked to measure their relative implication in elastin degradation. Gradual elastocalcinosis was obtained, and paralleled the elastin degradation pattern. Matrix metalloproteinase (MMP)-9 activity was increased at 5 days of warfarin treatment, whereas TGF-beta signaling was increased at 7 days. Calcification was significantly elevated after 21 days. Blocking metalloproteinases activation with doxycycline and TGF-beta signaling with SB-431542 were able to prevent calcification.. Early MMP-9 activation precedes the increase of TGF-beta signaling, and overt vascular elastocalcinosis and stiffness. Modulation of matrix degradation could represent a novel therapeutic avenue to prevent the gradual age-related stiffening of large arteries, leading to isolated systolic hypertension.

Topics: Animals; Aorta, Abdominal; Calcinosis; Collagen; Disease Models, Animal; Durapatite; Elastin; Femoral Artery; Male; Matrix Metalloproteinase 9; Osteopontin; Rats; Rats, Wistar; Signal Transduction; Transforming Growth Factor beta; Vascular Diseases; Warfarin

Emerging findings have demonstrated the critical role of blood clotting factors in the formation and stabilization of embryonic blood vessels. Whether a similar role is true during post-natal angiogenesis remains to be determined. Here we sought to determine whether the suppression of thrombin generation with anticoagulant drugs at doses routinely used for therapeutic purposes would affect the angiogenesis pattern following hindlimb ischemia in rats. Animals were treated with r-hirudin or enoxaparin within six hours post induction of hindlimb ischemia, whereas two other groups received oral anticoagulation warfarin beginning at day 3 post-ischemia or saline (as control). The revascularization anatomical and functional responses were evaluated 30 days following tissue ischemia. Chronic administration of the drugs resulted in stable anticoagulation in all animals throughout the experiment. Animals that received drugs with fast anticoagulation effects (i.e. r-hirudin and enoxaparin) presented a significant decrease in capillary density and capillary-to-myocyte ratio compared to control animals. These effects were not associated with changes in relative perfusion of the hindlimb at steady state. These anti-angiogenic effects occur in a time-dependent manner, since delayed inhibition of coagulation (>72 hours) presents no adverse effect on the angiogenic response. We conclude that the use of anticoagulant drugs immediately after tissue ischemia induction hampers in vivo angiogenic response in a rodent hindlimb ischemia model.

Topics: Animals; Anticoagulants; Blood Coagulation Factors; Disease Models, Animal; Enoxaparin; Hindlimb; Hirudins; Ischemia; Kinetics; Neovascularization, Physiologic; Rats; Rats, Inbred Lew; Time Factors; Warfarin

A significant need exists for new chronic oral anticoagulation therapies to replace warfarin. Previous studies have shown that beta-D-xylosides, which prime glycosaminoglycan (GAG) synthesis, have antithrombin and antithrombotic activity. In the following report, a new orally active beta-D-xyloside (odiparcil) has been characterized in a rat model of venous thrombosis and its efficacy and bleeding liability compared to warfarin. Additionally, studies were conducted to investigate odiparcil's ex vivo antithrombin and antiplatelet activity, and also to explore the potential utility of protamine sulfate as a neutralizing agent.. In vivo thrombosis studies were conducted in a rat inferior vena cava model, and bleeding studies in a rat tail transection model. Following oral dosing, warfarin and odiparcil produced dose-related suppression of thrombus formation. A therapeutically relevant dose of warfarin in this model (international normalized ratio; INR 3.0) achieved approximately 65% inhibition of thrombus formation. Warfarin caused dose-related significant increases in bleeding indices. Odiparcil antithrombotic activity was limited by its mechanism to a maximum suppression of thrombus formation of 65-70%, and did not prolong bleeding indices. Additionally, odiparcil-induced heparin cofactor II (HCII)-dependent antithrombin activity was shown to be a function of dermatan sulfate-like GAG production. Other than thrombin-related effects, no odiparcil effects on platelet function were observed. In antidote studies, it was demonstrated that odiparcil-induced antithrombotic activity could be partially neutralized by protamine sulfate.. These experiments suggest that an antithrombotic approach based upon xyloside induction of circulating GAGs may have the potential to approximate the efficacy of warfarin and yet with a reduced risk to hemostasis.

Topics: Animals; Anticoagulants; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Glycosaminoglycans; Glycosides; Hemorrhage; Heparin Cofactor II; Protamines; Rats; Vena Cava, Inferior; Venous Thrombosis; Warfarin

The antithrombotic and bleeding properties of the oral direct thrombin inhibitor ximelagatran and of warfarin were investigated in an experimental venous thrombosis and bleeding model in anaesthetized rats. Rats were randomized to receive ximelagatran (1-20 micromol/kg), warfarin (0.20-0.82 micromol/kg), or vehicle (tap water) once daily orally for 4 days before surgery. Thrombosis was induced by partial stenosis and application of ferric chloride to the wall of the abdominal vena cava under anaesthesia. Sixty minutes after thrombus induction, rats were sacrificed, thrombi harvested, and their fresh weight determined. Bleeding was determined as haemoglobin in fluid collected from the abdominal cavity. Blood samples were taken before thrombus induction and sacrifice for determination of coagulation parameters and plasma concentrations of melagatran, the active form of ximelagatran. Ximelagatran and warfarin dose-dependently reduced thrombus formation. The highest doses of ximelagatran and warfarin almost completely prevented thrombus formation; however, the increase in bleeding (versus vehicle) was significantly lower with the highest dose of ximelagatran than with the highest dose of warfarin. The oral direct thrombin inhibitor ximelagatran is thus as at least as effective as warfarin in the prevention of thrombus formation in this animal model, but with a wider separation between antithrombotic effects and bleeding.

Topics: Animals; Azetidines; Benzylamines; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Glycine; Hemorrhage; Male; Rats; Rats, Sprague-Dawley; Venae Cavae; Venous Thrombosis; Warfarin

The antiphospholipid syndrome is an antibody-mediated hypercoagulable state characterized by recurrent venous and arterial thromboembolic events. Several studies have determined that the frequency of antiphospholipid syndrome in patients presenting with a venous thromboembolic event is between 4% and 14%. Because of the high risk for recurrent thromboembolism in these patients, current recommendations suggest a longer, potentially lifelong, course of antithrombotic therapy following an initial event. Although most authorities agree on an extended course of therapy, considerable controversy surrounds the optimal target therapeutic INR for patients with antiphospholipid syndrome. For an initial venous thromboembolic event, a target INR of 2.0 to 3.0 is supported by two prospective, randomized clinical trials. In contrast, relatively limited data exist for an initial arterial thromboembolic event in patients who have the antiphospholipid syndrome, and therapeutic recommendations range from aspirin to warfarin with a high target INR. Recurrent thromboembolic events can be extremely difficult to treat, and some patients may benefit from the addition of immunosuppressive therapies. Importantly, as many as 50% of the initial thromboembolic events sustained by patients with antiphospholipid antibodies occur in the setting of additional, coincident prothrombotic risk factors, indicating the importance of addressing any additional risk factors, such as hypercholesterolemia, in these patients. Prospective studies are needed to address the role of thromboprophylactic strategies in asymptomatic individuals with antiphospholipid antibodies in the absence of additional risk factors.

Topics: Animals; Anticoagulants; Antiphospholipid Syndrome; Autoantibodies; Cardiolipins; Disease Models, Animal; Humans; Incidence; International Normalized Ratio; Kidney Failure, Chronic; Lupus Erythematosus, Systemic; Renal Dialysis; Venous Thromboembolism; Warfarin

We have previously shown that an endothelin receptor antagonist can regress medial arterial calcification in a rat model. The aim of this study was to characterize the phenotypic changes of vascular smooth muscle cells during calcification and mineral loss, in order to understand better the underlying mechanisms. Control Wistar rats were compared with rats treated only with warfarin/ vitamin K1 (15 mg/kg per day) for 8 weeks, or in combination with darusentan (30 mg/kg per day) for the final 4 weeks. Vascular smooth muscle cell, bone cell and macrophage phenotypes were evaluated by the local expression of alpha-actin, tartrate-resistant acid phosphatase and ED-1, respectively. Proteins involved in the modulation of bone resorption like osteopontin and osteoprotegerin were also evaluated by immunohistochemistry. The warfarin/vitamin K1 treatment increased medial arterial calcification ninefold (P < 0.05). At sites of calcification, there was a decrease in alpha-actin localization, and an appearance of osteopontin immunostaining. Histochemical and immunostaining for osteoclast and macrophage markers, as well as for osteoprotegerin, were negative. Although the extent of calcification foci was reduced by darusentan, protein localization in the calcified areas was not modified. Thus, the development of medial arterial calcification produces a phenotypic change in vascular smooth muscle cells that does not appear to be normalized in regions remaining calcified during mineral loss.

Topics: Animals; Aorta, Thoracic; Aortic Diseases; Calcinosis; Disease Models, Animal; Endothelin Receptor Antagonists; Endothelin-1; Macrophages; Male; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Osteoclasts; Osteopontin; Osteoprotegerin; Phenotype; Phenylpropionates; Pyrimidines; Rats; Rats, Wistar; Receptors, Endothelin; Vitamin K 1; Warfarin

In this study, we investigated the effects of warfarin potassium on the incidence of the femoral head osteonecrosis in spontaneously hypertensive rats (SHR). Twenty-four SHRs were divided into two equal groups, one given normal water (water group) and another provided with water containing warfarin (warfarin group). We compared the two groups histologically and observed the incidence of osteonecrosis. We also studied 17 Wistar Kyoto rats (WKY) to compare with SHR. Coagulation time, platelet count, and protein C activity were measured. Immunohistochemistry was also performed using endothelial nitric oxide synthetase (eNOS) antibody to investigate the function of endothelial cells. The incidence of osteonecrosis was significantly less in the warfarin group (10.5%) than in the water group (52.6%). Coagulation time was significantly longer in the warfarin group than the water group. Platelet count and protein C activity were not statistically different between the warfarin group and the water group. Results of immunohistochemistry revealed that endothelial cells in the femoral head were positive for eNOS in WKY but not in SHR. Our results indicated that warfarin reduced the incidence of femoral head necrosis in SHR.

Topics: Animals; Anticoagulants; Disease Models, Animal; Femur Head Necrosis; Hemostasis; Male; Platelet Count; Protein C; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Warfarin; Whole Blood Coagulation Time

Isolated systolic hypertension is the predominant form of hypertension in the elderly population. Reduction of arterial compliance appears to contribute to the elevation of pulse pressure (PP) and among potential mechanisms, gradual vascular calcification, fragmentation of elastic lamellae, and augmentation of rigid component like collagen could contribute to increase aortic stiffening. Few experimental models of the disease are currently available.. To induce large artery calcification, rats were treated with warfarin and vitamin K(1) (WK) for 4 and 8 weeks, to inhibit the maturation of matrix Gla protein. The impact of chronic PP elevation was determined on large artery and cardiac remodeling and on aortic endothelial function.. The WK treatment led to aortic medial calcification and a proportional elevation of PP, attributable mainly to a selective elevation of systolic blood pressure. The chronic treatment also increased collagen, whereas elastin decreased in the aorta. Pulse wave velocity, an index of aortic stiffening, increased in rats treated with WK. However, indices of left ventricular and aortic hypertrophy and remodeling remained normal. In addition, the WK treatment did not modify the vasoconstriction to norepinephrine and endothelin-1, and the vasodilatory response to acetylcholine and sodium nitroprusside.. Chronic treatment with WK represents a new model of isolated systolic hypertension with several characteristics of the human disease. The relative ease to induce calcification in this model may help to foster more fundamental research, which is lacking in this type of hypertension.

Topics: Animals; Anticoagulants; Antifibrinolytic Agents; Aorta; Calcinosis; Calcium; Collagen; Disease Models, Animal; Drug Administration Schedule; Elastin; Hemodynamics; Hypertension; Male; Rats; Rats, Wistar; Renin; Systole; Vascular Diseases; Vasomotor System; Vitamin K 1; Warfarin

Thrombin has been proposed to play a key role in the development of atherosclerosis, both by promoting fibrin deposition into the atherosclerotic vessel wall and also by signalling through thrombin receptors. Unfortunately, mice homozygous for a deletion of the prothrombin gene (FII) die in utero, making a direct assessment of the role of thrombin during atherogenesis difficult. We have assessed the contribution of thrombin-dependent processes to vascular lipid lesion formation in the atherosclerosis-prone apolipoprotein E (ApoE)-deficient mice by inhibiting thrombin generation with warfarin. ApoE-/- mice were treated with warfarin at a dose that increased the prothrombin time (PT) more than 10-fold (250-375 microg/kg body weight/day) for 12 weeks from the age of 12 weeks onwards. The extent and composition of the vascular lipid lesions that developed were assessed using oil red O to measure neutral lipid in the vessel wall and quantitative immunofluoresence to measure fibrin(ogen) levels as well as macrophage and smooth muscle cell numbers. Mice treated with warfarin developed lesions both in the aortic sinus and the descending aorta to the same degree as mice receiving no treatment (28,351+/-350 microm2/mouse treated with warfarin versus 27,952+/-750 micro2/control mouse; P = .86). However, the amount of fibrin(ogen) deposited in the vessel wall was decreased by more than 60% (34+/-11 arbitrary units in warfarin treated mice versus 92+/-11 arbitrary units in control mice; P < .01). Staining of macrophage and for smooth muscle cell markers was unaltered by treatment with warfarin. We conclude that suppressing thrombin generation does not alter the development of vascular lipid lesions in mice with a severe disorder of lipid metabolism, despite a marked reduction in fibrin(ogen) deposition.

Topics: Animals; Anticoagulants; Aorta; Apolipoproteins E; Arteriosclerosis; Depression, Chemical; Disease Models, Animal; Fibrin; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Smooth, Vascular; Prothrombin Time; Thrombin; Warfarin

The thrombomodulin (TM) gene was ablated in mice in a cell type-restricted manner from vascular endothelium by Cre-recombinase-mediated excision controlled by the endothelial cell lineage-specific Tie2 promoter. Forty percent of mutant (TMLox-) mice display a distinct lethal embryonic phenotype not observed in completely TM-deficient embryos. The remaining 60% of TMLox mice survive beyond birth, but invariably succumb to a severe hypercoagulable state and massive thrombosis after 3 weeks, terminating in a lethal consumptive coagulopathy. The progression of thrombosis was age- and sex-dependent. Disruption of the TM/protein C pathway was not associated with a latent proinflammatory state. Disease onset and progression could be prevented by warfarin anticoagulation. These results show that in mice, loss of endothelial cell TM function causes spontaneous and fatal thrombosis in the arterial and venous circulation, resulting from unfettered activation of the coagulation system. The combination of complete disease penetrance, uniform disease onset at young age, large vessel thrombosis of the extremities and multiple organ systems, and consumptive coagulopathy as the disease end-point provides a unique mouse model of human thrombotic disease.

Topics: Age Factors; Animals; Anticoagulants; Blood Coagulation; Cardiomegaly; Disease Models, Animal; Disease Progression; Disseminated Intravascular Coagulation; Endothelium, Vascular; Female; Gene Expression Regulation; Gene Targeting; Genes, Lethal; Genes, Synthetic; Humans; Integrases; Male; Mice; Mice, Transgenic; Myocardium; Organ Specificity; Protein C; Receptor Protein-Tyrosine Kinases; Receptor, TIE-2; Recombinant Fusion Proteins; Recombination, Genetic; Sexual Maturation; Thrombomodulin; Thrombophilia; Thrombosis; Viral Proteins; Warfarin

To investigate the relationship among lipids, coagulation and thrombosis in the absence of atherosclerosis, spontaneous or dietary-induced hyperlipidemic (FHL) rats were studied. FHL showed higher levels of coagulation factors VII, IX, X, VIII and XII and a shortening of the occlusion time (OT) of an artificial arterial prosthesis as compared with normolipidemic (FNL) animals. Damage of abdominal aorta of FHL was followed by increased fibrin deposition in the vascular intima as compared to FNL. After 5 months of cholesterol-rich diet FNL showed increased cholesterol, triglycerides and factor II, VII, IX, X, XII levels. A significant shortening of the OT and increased fibrin deposition was also observed. Two-month diet withdrawal restored the initial condition. Warfarin treatment, at a dose decreasing vitamin K-dependent factor to levels found in FNL, prolonged the OT and reduced fibrin deposition, without modifying F XII or changing lipid profile. An increase in the activated form of F VII was observed. In contrast, no difference was found in F VII clearance. High lipid levels favour the process of thrombus formation by increasing the activation of vitamin K-dependent coagulation factors. Low-dose warfarin treatment reverts the prothrombotic effect of hyperlipidemia.

Topics: Administration, Oral; Animals; Anticoagulants; Aorta, Abdominal; Aortic Diseases; Blood Coagulation Factors; Blood Vessel Prosthesis; Cholesterol, Dietary; Diet, Atherogenic; Disease Models, Animal; Enzyme Activation; Factor VII; Hypercholesterolemia; Hyperlipidemias; Hypertriglyceridemia; Postoperative Complications; Rats; Rats, Inbred Strains; Rats, Sprague-Dawley; Thrombophilia; Thrombosis; Vitamin K; Warfarin

Automated human plasma, continuous monitoring of the formation and inactivation of thrombin during the coagulation process provides an adequate way to detect hypo- and hypercoagulant conditions. Here, we describe an analogous procedure to determine the endogenous thrombin potential (ETP), i. e. the free thrombin concentration-time integral, of coagulating rat plasma. When activated with tissue factor, the ETP of plasma from Wistar rats was comparable to the ETP of human plasma, in spite of a relatively short half-life time of free thrombin in rat plasma. The ETP was highly sensitive to heparin as well as to administration of vitamin K antagonist or feeding of the animals with a vitamin K-deficient diet. In plasma that was activated under sub-optimal conditions (reduced levels of tissue factor or vitamin K-dependent coagulation factors), the ETP increased with the rate of thrombin formation in the first minutes of the coagulation process. Since both parameters are dependent of the prothrombin concentration, it appears that this level plays an important role in determining both the initial and total activity of the coagulation system. Thus, automated measurement of free thrombin during the coagulation process of rat plasma allows a detailed analysis of hypocoagulability in this animal model.

Topics: Animals; Blood Coagulation Disorders; Blood Coagulation Tests; Disease Models, Animal; Electronic Data Processing; Humans; Kinetics; Male; Rats; Rats, Wistar; Sensitivity and Specificity; Thrombin; Thromboplastin; Warfarin

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

Topics: Animals; Anticoagulants; Arterial Occlusive Diseases; Bleeding Time; Blood Coagulation Tests; Disease Models, Animal; Dose-Response Relationship, Drug; Humans; Male; Rats; Rats, Sprague-Dawley; Thrombin; Thrombosis; Warfarin

Topics: Animals; Aspirin; Blood Cell Count; Carotid Artery Thrombosis; Catheterization; Disease Models, Animal; Fibrinolytic Agents; Heparin; Male; Rats; Time Factors; Warfarin

The anticoagulant and antithrombotic effects of YM-75466 (N-[4-[(1-acetimidoyl-4-piperidyl)oxy]phenyl]-N-[(7-amidino-2-naph thyl)methyl]sulfamoyl acetic acid monomethanesulfonate), a novel orally-active factor Xa (FXa) inhibitor, and warfarin were compared in mice. Both agents were orally administered in all studies. In ex vivo studies, the peak effects of YM-75466 occurred 1 hr after administration while the peak of warfarin activity occurred 18 hr after administration. At each peak, both YM-75466 and warfarin prolonged coagulation time dose-dependently. The dose response curve of warfarin for prothrombin time was steeper than that of YM-75466. In a thromboplastin-induced thromboembolism model, administration of 30 mg/kg YM-75466 or 3 mg/kg warfarin significantly improved the lethality ratio. In blood loss studies, YM-75466 did not increase blood loss from the tail even at 30 mg/kg, while warfarin markedly increased blood loss at 3 mg/kg. Agents that interfere with warfarin action did not interfere with YM-75466 action. In conclusion, this study shows that YM-75466 has advantages over warfarin: i) rapid onset of anticoagulant activity, ii) wide therapeutic range, iii) little effect on bleeding and iv) lack of drug interaction with agents that interfere with warfarin. These results suggest that YM-75466 may be promising as a novel oral anticoagulant agent.

Topics: Administration, Oral; Analgesics, Non-Narcotic; Animals; Anti-Bacterial Agents; Anticoagulants; Anticonvulsants; Antifibrinolytic Agents; Blood Coagulation; Carbamazepine; Cimetidine; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Erythromycin; Factor Xa Inhibitors; Fibrinolytic Agents; Hemorrhage; Male; Mice; Mice, Inbred ICR; Partial Thromboplastin Time; Phenytoin; Piperidines; Prothrombin Time; Rifampin; Sulfonamides; Thromboembolism; Thromboplastin; Vitamin K 1; Warfarin

The physiologic role of osteocalcin (OC), a vitamin K-dependent protein specific to bone, remains elusive. It has been shown that rats maintained on chronic treatment with vitamin K1 and its antagonist warfarin exhibit a marked decrease in bone osteocalcin because noncarboxylated osteocalcin does not bind to bone hydroxyapatite. To assess the role of OC in bone remodeling, we applied the warfarin model to growing lambs. We analyzed the bone changes after 3 months of concurrent warfarin and vitamin K1 treatment. Four groups of four lambs were constituted at birth and received daily a saline solution (control group, CT), 4 mg/kd/day of vitamin K1 (vitamin K group), 4 mg/kg/day of vitamin K1 + 75 or 150 mg/kg/day of warfarin (W75 and W150 group, respectively). In warfarin-treated animals, bone osteocalcin levels were decreased, both in the metaphysis (9% compared to controls) and the diaphysis (30% compared to controls) of the metacarpals. The fraction of noncarboxylated osteocalcin measured every month in the serum was significantly higher in warfarin-treated lambs than in controls at each timing point (37.6 +/- 2.6% in W75 and 48.7 +/- 5.2% in W150 versus 14.4 +/- 3.8% in controls at 3 months). Compared to non-warfarin-treated animals (NW), the main histomorphometric parameters measured on the iliac crest after tetracycline double labeling were significantly reduced in the warfarin-treated lambs: 12.2 +/- 5.2 versus 18.6 +/- 4.7% in NW (p < 0.03) for the cancellous bone area, which reflects the trabecular bone density; 14.7 +/- 6.1 versus 21.0 +/- 3.6% in NW (p < 0.03) for the eroded perimeter, and 0.315 +/- 0.064 versus 0.561 +/- 0.23 microns 3/microns 2/day in NW (p < 0.02) for the tetracycline-based bone formation rate. In conclusion, the depletion of osteocalcin in the bone of lambs induced within 3 months a marked osteopenia that resulted from a decrease in resorption and a more pronounced decrease in bone formation. Our data suggest that the presence of osteocalcin, the major gla-containing protein of bone, may be important for the maintenance of a normal bone mass and remodeling of trabecular bone.

Topics: Animals; Animals, Newborn; Body Weight; Bone and Bones; Bone Diseases, Metabolic; Bone Remodeling; Calcium; Creatinine; Disease Models, Animal; Male; Osteocalcin; Parathyroid Hormone; Radioimmunoassay; Random Allocation; Sheep; Vitamin K 1; Warfarin

1. Thrombus formation induced by electrical stimulation of the carotid artery was investigated in anesthetized rabbits and rats. Occlusive Grade III thrombi were produced consistently in 34 normal New Zealand rabbits and 58 untreated albino Wistar rats. Thrombus formation was monitored continuously in some of the animals with a magnetic flowmeter or a thermistor probe applied on the carotid. 2. The usefulness of the model for the screening of drugs was tested by treating the animals with warfarin, heparin, prostacyclin (PGI2), dihydroprostacyclin (DiHPGI2), prostaglandin E1 (PGE1), and prostaglandin D2 (PGD2). 3. All of the drugs except warfarin were infused continuously into the venous circulation during the entire experimental period at a rate of 0.2 ml/min. 4. Warfarin (10 mg/kg), administered by gavage 24 h before the experiment, prevented thrombus formation, as did heparin iv (greater than 34 U/kg). 5. Of the four platelet antiaggregatory prostaglandins tested, PGI2 was the most potent inhibitor of thrombus formation and DiHPGI2 the least active, as evaluated by visual inspection of stimulated arterial segments which were excised 30 min (rabbits) or 15 min (rats) after the stimulation was stopped. PGI2 was less active in rats than in rabbits (Threshold Protective Dose ratio ca. 4:1). PGE1 and PGD2 showed intermediate activity in both animal models.

Topics: Animals; Carotid Artery Thrombosis; Disease Models, Animal; Electric Stimulation; Epoprostenol; Female; Heparin; Male; Prostaglandins; Prostaglandins D; Prostaglandins E; Rabbits; Rats; Rats, Inbred Strains; Warfarin

Topics: Animals; Disease Models, Animal; Hypoprothrombinemias; Male; Prothrombin Time; Rabbits; Warfarin

Anticoagulant drugs are known to have an effect on tumour growth. However, the mechanisms by which they act are poorly understood, and have therefore been investigated in this study. Wistar rats were given eight weekly subcutaneous injections of azoxymethane, at a dose of 10 mg kg-1 week-1. Following this they were randomized into two groups: a control group, which received no further treatment, and a warfarin treated group, which received warfarin at 'non-therapeutic' doses in their drinking water, for a further 8 weeks. Pairs of rats from each group were killed at 5-weekly intervals from 10 to 35 weeks after the first azoxymethane injection. At 40 weeks all remaining rats were killed. Samples of colonic mucosa from the descending colon and rectum were taken for scanning electron microscopy. The number of microadenomas per low power field was determined in both groups at each time interval. Tumour incidence and distribution were noted in animals killed at 40 weeks. The median number of microadenomas was significantly lower in warfarin treated animals than in controls at all time intervals. Tumour number was also significantly decreased by warfarin treatment (27 in azoxymethane treated animals, 10 in animals receiving azoxymethane and warfarin, P less than 0.05). The distribution of tumours along the colon was similar to that seen previously, following 12 weeks of azoxymethane. These effects occurred despite the non-concurrent administration of azoxymethane and warfarin.

Topics: Adenoma; Animals; Azoxymethane; Colon; Colonic Neoplasms; Disease Models, Animal; Intestinal Mucosa; Male; Microscopy, Electron, Scanning; Precancerous Conditions; Random Allocation; Rats; Rats, Inbred Strains; Rectal Neoplasms; Rectum; Warfarin

Topics: Animals; Antithrombin III; Blood Coagulation; Disease Models, Animal; Factor X; Factor Xa; Female; Fibrinolysis; Heparin; Humans; Pregnancy; Prothrombin Time; Rabbits; Thrombin; Thromboembolism; Vitamin K; Warfarin

The present study was designed for long-term evaluation of the recently developed Hall-Kaster heart valve prosthesis. Mitral valve replacement was performed in 33 dogs. A detailed description of the operative procedure used is given. Extracorporeal circulation was instituted using a nonhemic priming volume resulting in an extreme hemodilution with hematocrit of about 15%. 19 of the animals died within 24 h, while 7 survived the observation period of 4--6 weeks. Thrombus on the valve was the major cause of early and late deaths. Anticoagulants were therefore given, and the therapy was made more extensive as the study progressed. The anticoagulation regime finally comprised warfarin administration preoperatively and throughout the experiment, and heparin infusion during the first 24 h postoperatively. Neomycin was used for preoperative sterilization of the gut. Later, cephalotin and penicillin were given. The use of these anticoagulants and antibiotics was found to be of major significance in providing us with a satisfactory canine model for long-term in vivo evaluation of the prosthesis.

Topics: Animals; Disease Models, Animal; Dogs; Heart Valve Prosthesis; Mitral Valve; Postoperative Complications; Thrombosis; Warfarin

1 The decreased binding of drugs and dyes to plasma proteins from male and female rats with acute renal failure has been investigated using equilibrium dialysis at 37 degrees C. 2 Acute renal failure induced by bilateral ligation of the ureters produced a greater than threefold increase in the unbound fraction of o-methyl red relative to normal rat plasma. Unbound dye concentration in plasma from sham-operated control rats was also significantly increased but to a lesser extent. 3 Glycerol-induced acute renal failure produced a significant increase in the unbound fractions of o-methyl red, methyl orange, bromocresol green (BCG), 2-(4'-hydroxybenzeneazo) benzoic acid (HABA), phenytoin and salicylic acid. A marginally significant increase in unbound warfarin concentration was observed. 4 Glycerol-induced renal failure had no effect on total plasma protein concentration and experiments with o-methyl red and salicylic acid indicated that a direct effect of glycerol was not responsible for the diminution of binding. 5 Glycerol-induced acute renal failure, which produced decreases in drug and dye binding similar to those reported for human uraemic plasma, provides a convenient non-surgical animal model for the investigation of this phenomenon.

Topics: Acute Kidney Injury; Animals; Blood Proteins; Coloring Agents; Creatinine; Disease Models, Animal; Female; Glycerol; Male; Phenytoin; Rats; Salicylates; Sex Factors; Urea; Ureter; Warfarin

Topics: Animals; Aspirin; Dipyridamole; Disease Models, Animal; Rats; Thrombosis; Warfarin

Topics: Acetates; Adenosine Diphosphate; Animals; Anti-Inflammatory Agents; Arteriosclerosis; Arthritis, Rheumatoid; Blood Coagulation; Collagen; Coumarins; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Ethyl Biscoumacetate; Fibrinolysis; Humans; In Vitro Techniques; Methods; Phenylbutazone; Platelet Adhesiveness; Pyrazoles; Rats; Thrombophlebitis; Warfarin