Deep venous thrombosis (DVT) poses a major challenge to public health worldwide. Endothelial cell injury evokes inflammatory and oxidative responses that contribute to thrombus formation. Tea polyphenol (TP) in the form of epigallocatechin-3-gallate (EGCG) has anti-inflammatory and oxidative effect that may ameliorate DVT. However, the precise mechanism remains incompletely understood. The current study was designed to investigate the anti-DVT mechanism of EGCG in combination with warfarin (an oral anticoagulant). Rabbits were randomly divided into five groups. A DVT model of rats was established through ligation of the inferior vena cava (IVC) and left common iliac vein, and the animals were orally administered with EGCG, warfarin, or vehicle for seven days. In vitro studies included pretreatment of human umbilical vein endothelial cells (HUVECs) with different concentrations of EGCG for 2 h before exposure to hydrogen peroxide. Thrombus weight and length were examined. Histopathological changes were observed by hematoxylin-eosin staining. Blood samples were collected for detecting coagulation function, including thrombin and prothrombin times, activated partial thromboplastin time, and fibrinogen levels. Protein expression in thrombosed IVCs and HUVECs was evaluated by Western blot, immunohistochemical analysis, and/or immunofluorescence staining. RT-qPCR was used to determine the levels of AGTR-1 and VEGF mRNA in IVCs and HUVECs. The viability of HUVECs was examined by CCK-8 assay. Flow cytometry was performed to detect cell apoptosis and ROS generation was assessed by 2',7'-dichlorofluorescein diacetate reagent. In vitro and invivo studies showed that EGCG combined with warfarin significantly reduced thrombus weight and length, and apoptosis in HUVECs. Our findings indicated that the combination of EGCG and warfarin protects HUVECs from oxidative stress and prevents apoptosis. However, HIF-1α silencing weakened these effects, which indicated that HIF-1α may participate in DVT. Furthermore, HIF-1α silencing significantly up-regulated cell apoptosis and ROS generation, and enhanced VEGF expression and the activation of the PI3K/AKT and ERK1/2 signaling pathways. In conclusion, our results indicate that EGCG combined with warfarin modifies HIF-1α and VEGF to prevent DVT in rabbits through anti-inflammation via the PI3K/AKT and ERK1/2 signaling pathways.
Animals ; Anticoagulants/pharmacology* ; Catechin/analogs & derivatives* ; Eosine Yellowish-(YS)/pharmacology* ; Fibrinogen/pharmacology* ; Hematoxylin/pharmacology* ; Human Umbilical Vein Endothelial Cells ; Humans ; Hydrogen Peroxide/pharmacology* ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; MAP Kinase Signaling System ; Phosphatidylinositol 3-Kinases/metabolism* ; Polyphenols/pharmacology* ; Proto-Oncogene Proteins c-akt/metabolism* ; RNA, Messenger ; Rabbits ; Rats ; Reactive Oxygen Species/metabolism* ; Signal Transduction ; Sincalide/pharmacology* ; Tea ; Thrombin/pharmacology* ; Vascular Endothelial Growth Factor A/metabolism* ; Venous Thrombosis/pathology* ; Warfarin/pharmacology*

The study aimed to establish a population pharmacokinetic/pharmacodynamic (PPK/PD) model of warfarin. PCR-RFLP technique was used to genotype the CYP2C9 and VKORC1 polymorphisms of 73 patients. RP-HPLC-UV method was used to determine the 190 plasma concentrations of warfarin. Application of NONMEM, the clinical information and 263 international normalized ratio (INR) monitoring data were used to investigate the effect of genetic, physiological, pathological factors, other medication on clearance and anticoagulant response. The final model of warfarin PPK/PD was described as follows: CL = θCL · (WT/60)θWT · θCYP · eηCL (if CYP2C9*1/*1, θCYP = 1; if *1/*3, θCYP = 0.708); EC50 = θEC50 · θVKOR · eηEC50 (if VKORC1- 1639AA, θVKOR = 1; if GA, θVKOR = 2.01; V = θV; K(E0) = θK(E0); Emax = θEmax; E0 = θE0 · eηE0. Among them, the body weight (WT), CYP2C9 and VKORC1 genotype had conspicuous effect on warfarin PK/PD parameters. The goodness diagnosis, Bootstrap, NPDE verification showed that the final model was stable, effective and predictable. It may provide a reference for opitimizing the dose regimen of warfarin.
Anticoagulants ; pharmacology ; Body Weight ; Cytochrome P-450 CYP2C9 ; genetics ; Genotype ; Humans ; International Normalized Ratio ; Nonlinear Dynamics ; Polymorphism, Genetic ; Vitamin K Epoxide Reductases ; genetics ; Warfarin ; pharmacokinetics

OBJECTIVETo 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.

METHODSOne 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.

RESULTSRats 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.

CONCLUSIONAnticoagulation 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.

Animals ; Anticoagulants ; pharmacology ; Disease Models, Animal ; Endothelin-1 ; metabolism ; Heparin, Low-Molecular-Weight ; pharmacology ; Hypertension, Pulmonary ; drug therapy ; metabolism ; Lung ; pathology ; Morpholines ; pharmacology ; Pulmonary Embolism ; drug therapy ; metabolism ; Rats ; Rivaroxaban ; Thiophenes ; pharmacology ; Warfarin ; pharmacology

The present study was designed to determine the effects of puerarin pre-treatment on the pharmacokinetics of the oral anticoagulant agent warfarin and the antiplatelet agent clopidogrel in rats. In the treatment group, rats was gavaged with warfarin or clopidogrel after repeated treatment with puerarin at intraperitoneal doses of 20, 60, or 200 mg·kg(-1) for 7 days, while rats in the control group were administrated only with the same dose warfarin or clopidogrel. Plasma samples were obtained at the prescribed times and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The results showed that rats treated with puerarin at all the test doses of 20, 60 and 200 mg·kg(-1) were found to affect the pharmacokinetics of warfarin, but not clopidogrel, suggesting a potential herb-drug interaction between puerarin and warfarin.
Animals ; Anticoagulants ; pharmacokinetics ; Chromatography, Liquid ; Clopidogrel ; Drug Administration Schedule ; Herb-Drug Interactions ; Injections, Intraperitoneal ; Isoflavones ; administration & dosage ; pharmacology ; Male ; Platelet Aggregation Inhibitors ; pharmacokinetics ; Rats ; Rats, Wistar ; Tandem Mass Spectrometry ; Ticlopidine ; analogs & derivatives ; pharmacokinetics ; Vasodilator Agents ; administration & dosage ; pharmacology ; Warfarin ; pharmacokinetics

Anticoagulants ; adverse effects ; pharmacology ; Blood Coagulation Disorders ; chemically induced ; Drug Interactions ; Humans ; Warfarin ; adverse effects ; pharmacology

OBJECTIVETo study the effects of Ginkgo biloba extract (GBE) on the pharmacokinetics and pharmacodynamics of warfarin and observe the anticoagulant activity of GBE.

METHODA randomized, double-blinded, placebo-controlled, two-way cross-over trial was conducted. Twelve healthy volunteers (sex ratio was 1: 1) were randomized into two groups and received GBE (three pill, tid) or placebo (three pill, tid) for 5 weeks respectively. the subjects received a single dose of warfarin (5 mg) on the day 29. Blood samples for pharmacokinetics and pharmacodynamics assessment were collectd.

RESULTCompared with placebo, BE had no significant pharmacodynamics effects on warfarin and had no effects on prothrombin time (PT) and activated partial thromboplastin time (APTT). GBE extract increased C(max), AUC(0-144 min), AUC(0-infinity), t1/2, of warfarin significantly and decreased CL(F) of warfarin significantly (P < 0.05), and there were no singnificant difference of V(d) (F).

CONCLUSIONGBE has limited effects on the pharmacokinetics but no effects on the pharmacodynamics of single dose warfain in health subjects. GBE has no effects on clotting process alone.

Adult ; Anticoagulants ; pharmacology ; Double-Blind Method ; Female ; Ginkgo biloba ; Herb-Drug Interactions ; Humans ; Male ; Plant Extracts ; pharmacology ; Warfarin ; blood ; pharmacology ; Young Adult

OBJECTIVETo evaluate the effects of epicutaneous application of anticoagulant warfarin, by examining the presence of tissue injury and immune/inflammatory activity in exposed skin.

METHODSRats were exposed to warfarin by applying 10 μg of warfarin-sodium to 10-12 cm(2) skin (range 0.8-1 μg per 1 cm(2)) for 3 consecutive days. Tissue injury was evaluated by lipid peroxidation, histomorphological changes and signs of reparative activity in skin. T cell infiltration and selected aspects of epidermal cell activity were examined as indicators of immune/inflammatory skin response to warfarin application.

RESULTSRepeated warfarin application exerted no effect on skin metabolic viability, but resulted in tissue injury (increased malondialdehyde, MDA, production, evident histo-morphological changes in epidermis and dermis depicting cell injury and death). Increased numbers of proliferating cell nuclear antigen (PCNA(+)) cells indicated reparative processes in injured skin. Infiltration of CD3(+) cells (T lymphocytes) along with the increased production of tumor necrosis factor-a (TNF-a) by epidermal cells from warfarin-treated skin and their co-stimulatory effect in an in vitro T-cell activation assay demonstrated immunomodulatory effects of epicutaneous warfarin.

CONCLUSIONPresented data have documented tissue damage associated with immune/inflammatory activity in skin exposed to warfarin. Observed effects are relevant to immunotoxic potential of this anticoagulant in settings of external exposure.

Animals ; CD3 Complex ; genetics ; metabolism ; Dermatitis, Contact ; pathology ; Epidermis ; cytology ; Gene Expression Regulation ; physiology ; Inflammation ; metabolism ; Lipid Peroxidation ; Male ; Proliferating Cell Nuclear Antigen ; genetics ; metabolism ; Rats ; Rodenticides ; pharmacology ; Skin ; cytology ; drug effects ; metabolism ; T-Lymphocytes ; physiology ; Warfarin ; pharmacology

OBJECTIVETo investigate the association of the polymorphisms of cytochrome P450 2C9 (CYP2C9) exon 4 608T/G, 561A/C, 537A/C and 527A/C, and -65G/C with warfarin sensitivity.

METHODSA total of 102 patients under warfarin anticoagulant therapy were selected. During follow-up, warfarin dosage and associated Prothrombin Time-International Normalized Ratio (P-INR) values were recorded. Simultaneous monitoring of incidence of bleeding and thrombosis adverse effect was recommended. Genetic polymorphisms of the above mentioned loci were identified by polymerase chain reaction and DNA sequencing.

RESULTSThe average age of the 102 patients was (62.1+/-10.5) years. The body mass index (BMI) was (24.7+/-3.8) kg/m2. Mean daily warfarin requirement was from 1.250 to 5.077 mg/day when therapeutic PT-INR (1.5-2.5) was maintained. DNA sequencing showed no polymorphisms of 608T/G, 561A/C, 537A/C, 527A/C in CYP2C9 exon 4. Warfarin daily dosage in CYP2C9 exon 4 -65C carriers was 3.106+/-0.619 mg/d, while it was (2.555+/-0.708) mg/d in individuals with wild-type -65G (P=0.020). Receiver operating characteristic (ROC) analysis showed that warfarin daily dosage of more than 2.5 mg/d can be used to predict the CYP2C9 exon 4 -65GC genotype (AUC: 0.770, P=0.005, 95%CI:0.626-0.915). Logistic regression indicated that BMI was an independent factor of bleeding during anti-coagulation therapy (OR=0.794, 95%CI: 0.651-0.970, P=0.024).

CONCLUSIONThe Chinese population are, generally, warfarin-sensitive. Exon 4 of the CYP2C9 gene is highly conserved in this population. The warfarin maintenance dosage in CYP2C9 exon 4 -65CG carriers was significantly higher than those with wild-type -65GG. The clinical significance needs further investigation with more large-scale, multi-center trials.

Adult ; Alleles ; Anticoagulants ; pharmacology ; therapeutic use ; Aryl Hydrocarbon Hydroxylases ; genetics ; Cytochrome P-450 CYP2C9 ; Exons ; genetics ; Female ; Genetic Predisposition to Disease ; Genotype ; Humans ; Male ; Middle Aged ; Point Mutation ; Polymorphism, Genetic ; Thrombosis ; drug therapy ; Warfarin ; pharmacology ; therapeutic use

The effect of atorvastatin on warfarin-induced aortic medial calcification and systolic blood pressure (SBP) of rats induced by warfarin was studied. Thirty healthy and adult rats were randomly divided into Warfarin group (n=10), Atorvastatin group (n=10) and normal control group (n=10). Caudal arterial pressure of rats was measured once a week, and 4 weeks later, aorta was obtained. Elastic fiber, collagen fiber and calcium accumulation in tunica media of cells were measured by Von Kossa staining. The results showed that warfarin treatment led to elevation of systolic blood pressure and aortic medial calcification. The chronic treatment also increased collagen, but decreased elastin in the aorta. However, the atorvastatin treatment had adverse effects. It was concluded that treatment with atorvastatin presented evidence of blood pressure lowing and calcification reducing. These data demonstrate that atorvastatin protected aortic media from warfarin-induced calcification and elevation of systolic blood pressure.
Aortic Diseases/chemically induced ; Aortic Diseases/drug therapy ; Aortic Diseases/*pathology ; Blood Pressure/*drug effects ; Calcinosis/chemically induced ; Calcinosis/*drug therapy ; Calcinosis/pathology ; Heptanoic Acids/*pharmacology ; Heptanoic Acids/therapeutic use ; Hypertension/chemically induced ; Hypertension/*drug therapy ; Pyrroles/*pharmacology ; Pyrroles/therapeutic use ; Random Allocation ; Rats, Wistar ; Warfarin

We report a case of intolerance to warfarin dosing due to impaired drug metabolism in a patient with CYP2C9*3/*4. A 73-yr-old woman with atrial fibrilation was taking warfarin. She attained a high prothrombin time international normalized ratio (INR) at the standard doses during the induction of anticoagulation and extremely low dose of warfarin (6.5 mg/week) was finally chosen to reach the target INR. Genotyping for CYP2C9 revealed that this patient had a genotype CYP2C9*3/*4. This is the first Korean compound heterozygote for CYP2C9*3 and *4. This case suggests the clinical usefulness of pharmacogenetic testing for individualized dosage adjustments of warfarin.
Aged ; Anticoagulants/pharmacology ; Aryl Hydrocarbon Hydroxylases/*genetics ; Atrial Fibrillation/*drug therapy/*genetics ; Female ; *Genotype ; Heterozygote ; Humans ; International Normalized Ratio ; Pharmacogenetics ; Polymorphism, Genetic ; Prothrombin Time ; Warfarin/*pharmacology