Discovery of Novel Reductive Elimination Pathway for 10-Hydroxywarfarin

Coumadin (R/S-warfarin) anticoagulant therapy is highly efficacious in preventing the formation of blood clots; however, significant inter-individual variations in response risks over or under dosing resulting in adverse bleeding events or ineffective therapy, respectively. Levels of pharmacologically active forms of the drug and metabolites depend on a diversity of metabolic pathways. Cytochromes P450 play a major role in oxidizing R- and S-warfarin to 6-, 7-, 8-, 10-, and 4′-hydroxywarfarin, and warfarin alcohols form through a minor metabolic pathway involving reduction at the C11 position. We hypothesized that due to structural similarities with warfarin, hydroxywarfarins undergo reduction, possibly impacting their pharmacological activity and elimination. We modeled reduction reactions and carried out experimental steady-state reactions with human liver cytosol for conversion of rac-6-, 7-, 8-, 4′-hydroxywarfarin and 10-hydroxywarfarin isomers to the corresponding alcohols. The modeling correctly predicted the more efficient reduction of 10-hydroxywarfarin over warfarin but not the order of the remaining hydroxywarfarins. Experimental studies did not indicate any clear trends in the reduction for rac-hydroxywarfarins or 10-hydroxywarfarin into alcohol 1 and 2. The collective findings indicated the location of the hydroxyl group significantly impacted reduction selectivity among the hydroxywarfarins, as well as the specificity for the resulting metabolites. Based on studies with R- and S-7-hydroxywarfarin, we predicted that all hydroxywarfarin reductions are enantioselective toward R substrates and enantiospecific for S alcohol metabolites. CBR1 and to a lesser extent AKR1C3 reductases are responsible for those reactions. Due to the inefficiency of reactions, only reduction of 10-hydroxywarfarin is likely to be important in clearance of the metabolite. This pathway for 10-hydroxywarfarin may have clinical relevance as well given its anticoagulant activity and capacity to inhibit S-warfarin metabolism.

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IMPAIREMENT OF PRIMARY HAEMOSTASIS BY LMW-HEPARINS

10.1055/s-0038-1643172 ◽

1987 ◽

Author(s):

A Borowska ◽

D Lauri ◽

A Maggi ◽

E Dejana ◽

G de Gaetano ◽

...

Keyword(s):

Platelet Aggregation ◽

Bleeding Time ◽

Ex Vivo ◽

Anticoagulant Activity ◽

Experimental Studies ◽

Male Rats ◽

Bleeding Complications ◽

Low Molecular Weight ◽

Bleeding Events

Low molecular weight (LMW) heparlns have been developed with the aim of reducing anticoagulant activity thereby minimizing the bleeding complications of conventional heparin. Unexpectedly, bleeding events were reported during treatment with some LMW-heparins, in clinical and experimental studies. We studied the effect of four different LMW-heparlns on primary haemostasis In male rats (CD COBS, Charles River) after l.v. administration of 0.75 mg/kg b.w. of the drugs. LMW heparin A was devoid of any activity on an experimental model of “template” bleeding time in rats (110.6±5.9 sec versus 108.7±4.1 control values) whereas LMW-heparins B, C and D prolonged the bleeding time to a different extent (228.7±19.9, 161.5±6.4 and 161.7±8.6 respectively). Pretreatment of animals with aspirin (100 mg/kg b.w. per o.s). resulted In a significant potentiation of the “template” bleeding time. In vitro platelet aggregation Induced by collagen (20 μg/ml) or by collagen in combination with ADP (5-10 μM) was strongly inhibited by LMW-heparln B, while LMW-heparln A showed no effect. LMW-heparins C and D exerted an Intermediate level of Inhibition of platelet aggregation. The same pattern of aggregating response was found when LMW-heparins A and B were given i.v. to rats (0.75 mg/kg b.w.) and platelet aggregation was studied “ex vivo” 15 min after drug administration.These data may help explain the impairment of primary haemostasis associated with some LMW-heparin preparations.

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How Small Molecules Affect the Thermo-Oxidative Aging Mechanism of Polypropylene: A Reactive Molecular Dynamics Study

Polymers ◽

10.3390/polym13081243 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1243

Author(s):

Fan Zhang ◽

Yufei Cao ◽

Xuan Liu ◽

Huan Xu ◽

Diannan Lu ◽

...

Keyword(s):

Molecular Dynamics ◽

Acetic Acid ◽

Small Molecules ◽

Aging Process ◽

Experimental Studies ◽

Hydroxyl Group ◽

Oxidative Aging ◽

Reactive Molecular Dynamics ◽

Aging Mechanism ◽

Effects Of Temperature

Understanding the aging mechanism of polypropylene (PP) is fundamental for the fabrication and application of PP-based materials. In this paper, we present our study in which we first used reactive molecular dynamics (RMD) simulations to explore the thermo-oxidative aging of PP in the presence of acetic acid or acetone. We studied the effects of temperature and oxygen on the aging process and discussed the formation pathways of typical small molecule products (H2, CO, CO2, CH4, C2H4, and C2H6). The effect of two infection agents, acetic acid and acetone, on the aging reaction was analyzed emphatically. The simulation results showed that acetone has a weak impact on accelerating the aging process, while acetic acid has a significant effect, consistent with previous experimental studies. By tracking the simulation trajectories, both acetic acid and acetone produced small active free radicals to further react with other fragment products, thus accelerating the aging process. The first reaction step of acetic acid is often the shedding of the H atom on the hydroxyl group, while the reaction of acetone is often the shedding of the H atom or the methyl. The latter requires higher energy at lower temperatures. This is why the acceleration effect of acetone for the thermo-oxidative aging of PP was not so significant compared to acetic acid in the experimental temperature (383.15 K).

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Evaluation of anticoagulant activity of aqueous extract of Cestrum nocturnum

International Journal of Phytomedicine ◽

10.5138/09750185.1969 ◽

2017 ◽

Vol 8 (4) ◽

pp. 525

Author(s):

Chandra Kishore Tyagi ◽

Deenanath Jhade ◽

Sunil Kumar Shah

Keyword(s):

Aqueous Extract ◽

Anticoagulant Activity ◽

Ethanolic Extract ◽

Dependent Manner ◽

Thrombin Time ◽

Clotting Time ◽

Standard Procedures ◽

Prolonged Aptt ◽

Pharmacologically Active

The study evaluated anticoagulant properties of the aqueous extract of Cestrum nocturnum using aPTT-Activated Partial Thromboplastin Time, PT- Prothrombin Time & TT-Thrombin Time as standard procedures.For in vitro coagulation assays, aqueous extract of plant prolonged APTT, TT, and PT clotting times in a dose-dependent manner (Table 7). It prolonged APTT clotting time from 45 ± 2 (2mg/mL) to 82.2 ± 2.63s (10mg/mL), PT clotting time from 20.4 ± 1.49 (2mg/mL) to 31.4 ± 2.15s (10mg/mL), and TT clotting time from 9.2 ± 1.16 (2mg/mL) to 17.4 ± 1.01s (10mg/mL) at the concentration of 2 to 10mg/mL. Heparin prolonged APTT and PT clotting times more than 111.8s and 40.8s, respectively, at a concentration of 1 IU/mL. Heparin prolonged TT clotting times more than 20.6s at a concentration of 1 IU/mL.The phytochemical screening of the plant confirm the presence of saponin in the water and ethanolic extract, Alkaloid in all the extract except hexane extract, tannin in water, ethanol and methanol extract, amino acid in water and ethanolic extract, carbohydrate in water and methanolic extract and triterpenoids and glycoside were absent in all the extracts. The results demonstrated that the aqueous extract of Cestrum nocturnum possesses pharmacologically active anticoagulant principles that could be isolated and evaluated for clinical or physiological purposes.

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Crosstalk in Electrical Connectors

Elektrichestvo ◽

10.24160/0013-5380-2021-3-54-59 ◽

2021 ◽

Vol 3 (3) ◽

pp. 54-59

Author(s):

Van Tai NGUYEN ◽

◽

Vladimir Y. Kirillov ◽

Keyword(s):

Frequency Band ◽

Electromagnetic Interference ◽

Experimental Studies ◽

Electrical System ◽

Electrical Wire ◽

Electrical Systems ◽

Efficient Reduction ◽

External Electromagnetic Fields ◽

Resonance Nature ◽

Electrical Connectors

Crosstalk electromagnetic interference occurs in on-board electrical wire bundles of aircraft electrical systems under the effect of external electromagnetic fields. Efficient reduction of crosstalk and elimination of their propagation paths in wire bundles can be achieved by shielding the conductors in the bundles and the bundles as a whole without breaks and discontinuities between the shields and housings of electrical connectors. The shielding of conductors in bundles does not eliminate the crosstalk completely, which can propagate through the contacts of electrical connectors. For estimating the influence of electrical connectors on the levels of crosstalk in electrical system circuits, their experimental studies should be carried out. In the course of these experimental studies, the interference voltage induced at the receptor contacts when voltage is applied to the source contacts in the specified frequency band is measured. The results from experimental studies of crosstalk for various types of electrical connectors in a specified frequency band make it possible to evaluate the resonance nature and levels of crosstalk interference levels depending on the relative position of the contacts and frequency.

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The Interplay between Retinal Pathways of Cholesterol Output and Its Effects on Mouse Retina

Biomolecules ◽

10.3390/biom9120867 ◽

2019 ◽

Vol 9 (12) ◽

pp. 867 ◽

Cited By ~ 3

Author(s):

Alexey M. Petrov ◽

Artem A. Astafev ◽

Natalia Mast ◽

Aicha Saadane ◽

Nicole El-Darzi ◽

...

Keyword(s):

Energy Homeostasis ◽

Cytochromes P450 ◽

Cholesterol Biosynthesis ◽

Mouse Retina ◽

Minor Role ◽

Compensatory Mechanisms ◽

Cholesterol Levels ◽

Transmission Electron ◽

Retinal Pathways ◽

A Minor

In mammalian retina, cholesterol excess is mainly metabolized to oxysterols by cytochromes P450 27A1 (CYP27A1) and 46A1 (CYP46A1) or removed on lipoprotein particles containing apolipoprotein E (APOE). In contrast, esterification by sterol-O-acyltransferase 1 (SOAT) plays only a minor role in this process. Accordingly, retinal cholesterol levels are unchanged in Soat1−/− mice but are increased in Cyp27a1−/−Cyp46a1−/− and Apoe−/− mice. Herein, we characterized Cyp27a1−/−Cyp46a1−/−Soat1−/− and Cyp27a1−/−Cyp46a1−/−Apoe−/− mice. In the former, retinal cholesterol levels, anatomical gross structure, and vasculature were normal, yet the electroretinographic responses were impaired. Conversely, in Cyp27a1−/−Cyp46a1−/−Apoe−/− mice, retinal cholesterol levels were increased while anatomical structure and vasculature were unaffected with only male mice showing a decrease in electroretinographic responses. Sterol profiling, qRT-PCR, proteomics, and transmission electron microscopy mapped potential compensatory mechanisms in the Cyp27a1−/−Cyp46a1−/−Soat1−/− and Cyp27a1−/−Cyp46a1−/−Apoe−/− retina. These included decreased cholesterol biosynthesis along with enhanced formation of intra- and extracellular vesicles, possibly a reserve mechanism for lowering retinal cholesterol. In addition, there was altered abundance of proteins in Cyp27a1−/−Cyp46a1−/−Soat1−/− mice that can affect photoreceptor function, survival, and retinal energy homeostasis (glucose and fatty acid metabolism). Therefore, the levels of retinal cholesterol do not seem to predict retinal abnormalities, and it is rather the network of compensatory mechanisms that appears to determine retinal phenotype.

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A Novel Flow Cytometric Annexin A5 Competition Assay for the Diagnosis of Antiphospholipid Syndrome.

Blood ◽

10.1182/blood.v104.11.4042.4042 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4042-4042

Author(s):

Lisa Senzel ◽

Xiao-Xuan Wu ◽

Jacob Rand

Keyword(s):

Anticoagulant Activity ◽

Anticardiolipin Antibody ◽

Progressive Increase ◽

Fluorescent Labeling ◽

Annexin A5 ◽

Antiphospholipid Antibody ◽

Anionic Phospholipids ◽

Flow Cytometric ◽

Anticoagulant Function ◽

A Minor

Abstract Annexin A5 (A5) is a potent anticoagulant protein that crystallizes over phospholipid surfaces, shielding them from availability for coagulation enzyme reactions. The antiphospholipid antibody (aPL) syndrome (APS) is an autoimmune thrombophilia that is marked by the presence of antibodies against phospholipid-binding proteins and the paradoxical lupus anticoagulant phenomenon. aPL antibodies can promote coagulation by interfering with the crystallization of A5, thereby increasing the exposure of blood to thrombogenic anionic phospholipids. Since a flow cytometric assay for aPL measuring A5 binding to frozen thawed platelets was previously reported, we investigated whether phosphatidylserine-bound polystyrene beads could provide a robust platform for the assay. Beads were incubated with sera from patients with the aPL syndrome and from non-aPL controls. Fluorescence-tagged A5 was added and samples were analyzed by flow cytometry. Similarly treated beads were also used in coagulation assays to determine whether A5 anticoagulant function was also inhibited. Control sera permitted fluorescent labeling of nearly all beads. In contrast, sera from aPL syndrome patients produced a major population of unlabeled beads, sometimes accompanied by a minor population of labeled beads. 7 of 13 confirmed aPL syndrome patients, and 7 of 12 anticardiolipin antibody positive patients, demonstrated reduced A5 binding in this assay, while 10 of 10 healthy blood donor controls did not. Dilution of the aPL sera resulted in progressive increase of A5 binding. Reduction of A5 binding appeared to correlate with reduced A5 anticoagulant activity (r=0.42, n=33, p=.01). This assay shows promise for investigating the effects of aPL antibodies on A5 binding and for the clinical diagnosis of the aPL syndrome. Figure Figure Figure Figure

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Cytochrome P450-mediated metabolism of N-(2-methoxyphenyl)-hydroxylamine, a human metabolite of the environmental pollutants and carcinogens o-anisidine and o-nitroanisole

Interdisciplinary Toxicology ◽

10.2478/v10102-010-0045-8 ◽

2008 ◽

Vol 1 (3-4) ◽

pp. 218-224 ◽

Cited By ~ 1

Author(s):

Karel Naiman ◽

Helena Dračínská ◽

Martin Dračínský ◽

Markéta Martínková ◽

Václav Martínek ◽

...

Keyword(s):

Cytochrome P450 ◽

Cytochromes P450 ◽

Environmental Pollutants ◽

Human Metabolite ◽

Hepatic Microsomes ◽

Minor Metabolite ◽

Reactive Compound ◽

A Minor

Cytochrome P450-mediated metabolism ofN-(2-methoxyphenyl)-hydroxylamine, a human metabolite of the environmental pollutants and carcinogenso-anisidine ando-nitroanisoleN-(2-methoxyphenyl)hydroxylamine is a human metabolite of the industrial and environmental pollutants and bladder carcinogens 2-methoxyaniline (o-anisidine) and 2-methoxynitrobenzene (o-nitroanisole). Here, we investigated the ability of hepatic microsomes from rat and rabbit to metabolize this reactive compound. We found thatN-(2-methoxyphenyl)hydroxylamine is metabolized by microsomes of both species mainly too-aminophenol and a parent carcinogen,o-anisidine, whereas 2-methoxynitrosobenzene (o-nitrosoanisole) is formed as a minor metabolite. AnotherN-(2-methoxyphenyl)hydroxylamine metabolite, the exact structure of which has not been identified as yet, was generated by hepatic microsomes of rabbits, but its formation by those of rats was negligible. To evaluate the role of rat hepatic microsomal cytochromes P450 (CYP) inN-(2-methoxyphenyl)hydroxylamine metabolism, we investigated the modulation of its metabolism by specific inducers of these enzymes. The results of this study show that rat hepatic CYPs of a 1A subfamily and, to a lesser extent those of a 2B subfamily, catalyzeN-(2-methoxyphenyl)hydroxylamine conversion to form both its reductive metabolite,o-anisidine, ando-aminophenol. CYP2E1 is the most efficient enzyme catalyzing conversion ofN-(2-methoxyphenyl)hydroxylamine too-aminophenol.

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Structure of the Antithrombin III-Binding site in Heparin

10.1055/s-0039-1684700 ◽

1979 ◽

Author(s):

U. Lindahl ◽

G. Bäckström ◽

N. Höök ◽

J. Riesenfeld ◽

L. Thunberg ◽

...

Keyword(s):

Antithrombin Iii ◽

Anticoagulant Activity ◽

Periodate Oxidation ◽

Variable Structure ◽

Variable Regions ◽

High Affinity ◽

Swedish Medical ◽

A Minor ◽

Binding Sequence ◽

Partial Chemical

Fragments with high affinity for antithrombin III (AT), composed of 12 to 16 monosaccharide units, were isolated from heparin after partial chemical or enzymatic depolymerization of the polysaccharide. Analysis of such fragments based on identification ot deamination products suggested that nonsulfated L-iduronic acid (a minor constituent) is essential for the anticoagulant activity of heparin. The location of this unit in the AT-binding sequence was determined by periodate oxidation. Furthermore, an N-sulfate group essential for activity was located by structural analysis of partially N-desulfated fragments retaining high affinity for AT. It is proposed that the AT-hinding sequence in heparin has a variable structure containing certain nonvanable regions. A tentative structure for this sequence is presented, with indication of identified constant and variable regions.(Supported by grant No. 2309 from the Swedish Medical Research Council).

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MONOCLONAL ANTIBODIES TO HUMAN PROTEIN S AND C4b-BINDING PROTEIN

10.1055/s-0038-1644291 ◽

1987 ◽

Author(s):

Martin Hessing ◽

Joost C M Meijers ◽

Jan A van Mourik ◽

Bonno N Bouma

Keyword(s):

Monoclonal Antibodies ◽

Complex Formation ◽

Binding Protein ◽

Anticoagulant Activity ◽

Activated Protein C ◽

Protein S ◽

Thrombin Cleavage ◽

Prolonged Aptt ◽

Acid Domain ◽

A Minor

Protein S (PS) circulates in plasma both free and in reversible association with the complement component C4b-binding protein (C4bp). Only free PS is functional as a cofactor for activated protein C (APC). Cleavage of PS by thrombin at a site near the r-carboxyglutamic acid domain is associated with a loss of cofactor activity. This may be a control mechanism for the anticoagulant activity of APC. These observations led us to investigate the role of C4bp and thrombin in the regulation of PS. Complex formation between purified PS and C4bp was studied in plasma and in a system with purified components. 125I-labeled PS was first incubated with either C4bp or citrated plasma and then subjected to polyacrylamide gelelectrophoresis in the absence of SDS. The formation of the C4bp-PS complex in plasma and in the purified system was demonstrated by autoradiography. Crossed immuno-electrophoresis using an antiserum against PS was performed in the presence of 8 mM EDTA. Human citrated plasma showed two precipitin peaks. Free PS migrated rapidly in the first dimension, whereas the C4bp-PS complex was just anodal to the application slot. The addition of C4bp to either plasma or purified PS resulted in the disappearance of the free PS peak and an increase of the slower migrating peak. The effect of purified C4bp on the PS-cofactor function of APC was studied in citrated plasma. The prolongation of the APTT induced by the addition of APC could be inhibited by the addition of increasing amounts of C4bp. Monoclonal antibodies to PS and C4bp were prepared and characterized. The monoclonal antibodies to either PS or C4bp did not block the complex formation between and PS, as was demonstrated by dot blotting of C4bp with 125I-PS and agarose gelelectrophoresis followed by Western blotting. Three out of 7 monoclonal antibodies to PS did not detect PS after thrombin cleavage on an immunoblot after non-reduced SDS polyacrylamide gelelectrophoresis. These 3 antibodies gave a significant shortening of the prolonged APTT induced by the addition of APC to normal plasma, indicating that these monoclonals inhibited the cofactor function of PS. The other 4 monoclonals to PS that did detect PS after thrombin cleavage on an immunoblot, gave only a minor inhibition of the PS cofactor function.

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Cannabinoids and the kidney: effects in health and disease

AJP Renal Physiology ◽

10.1152/ajprenal.00290.2017 ◽

2017 ◽

Vol 313 (5) ◽

pp. F1124-F1132 ◽

Cited By ~ 11

Author(s):

Frank Park ◽

Praveen K. Potukuchi ◽

Hamid Moradi ◽

Csaba P. Kovesdy

Keyword(s):

Kidney Diseases ◽

Experimental Studies ◽

Kidney Injury ◽

New Drugs ◽

Beneficial Effects ◽

Regulatory Changes ◽

Beneficial Impact ◽

Distribution Type ◽

Pharmacologically Active ◽

The Impact

Consumption of cannabis and various related products (cannabinoids) for both medicinal and recreational use is gaining popularity. Furthermore, regulatory changes are fostering a cultural shift toward increasing liberalization of cannabis use, thereby increasing the likelihood of even larger numbers of individuals being exposed in the future. The two different types of receptors (CB1 and CB2) that are activated by the pharmacologically active ingredients of cannabis are found in numerous tissues, including the kidneys. Experimental studies suggest that stimulation of these receptors using pharmacologic agents or their naturally occurring ligands could have both deleterious and beneficial effects on the kidneys, depending on receptor distribution, type of renal insult, or the timing of the activation during acute or chronic states of kidney injury. To date, the mechanisms by which the CB1 or CB2 receptors are involved in the pathology of these renal conditions remain to be fully described. Furthermore, a better understanding of the impact of exocannabinoids and endocannabinoids on the renal system may lead to the development of new drugs to treat kidney disease and its complications. Given the increasing public health relevance of cannabis exposure, it is clear that more research is necessary to clarify the various physiological and pathophysiological effects of cannabis and related analogs on the kidney. This will help limit the deleterious effects of these substances while promoting their potential beneficial impact on renal function in various types of kidney diseases.

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