Administration Of Dicumarol Research Articles

ATP-releasing SWELL1 channel in spinal microglia contributes to neuropathic pain.

Following peripheral nerve injury, extracellular adenosine 5'-triphosphate (ATP)-mediated purinergic signaling is crucial for spinal cord microglia activation and neuropathic pain. However, the mechanisms of ATP release remain poorly understood. Here, we show that volume-regulated anion channel (VRAC) is an ATP-releasing channel and is activated by inflammatory mediator sphingosine-1-phosphate (S1P) in microglia. Mice with microglia-specific deletion of Swell1 (also known as Lrrc8a), a VRAC essential subunit, had reduced peripheral nerve injury-induced increase in extracellular ATP in spinal cord. The mutant mice also exhibited decreased spinal microgliosis, dorsal horn neuronal hyperactivity, and both evoked and spontaneous neuropathic pain-like behaviors. We further performed high-throughput screens and identified an FDA-approved drug dicumarol as a novel and potent VRAC inhibitor. Intrathecal administration of dicumarol alleviated nerve injury-induced mechanical allodynia in mice. Our findings suggest that ATP-releasing VRAC in microglia is a key spinal cord determinant of neuropathic pain and a potential therapeutic target for this debilitating disease.

The mitochondrial uncoupler dicumarol disrupts the MTT assay

Dicumarol is routinely added to the 3-[4,4-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay to study the role of NAD(P)H:quinone oxido-reductase in drug activation and detoxification. We assessed the direct impact of dicumarol (a mitochondrial uncoupler) on the MTT assay. Mouse mammary tumor (EMT6) and Chinese hamster ovary (CHO) cells were treated with media containing either 10 or 1% fetal bovine serum and dicumarol (0–1000 μM) mimicking standard assay conditions. MTT, clonogenic, total reactive oxygen species (ROS), and oxygen consumption assays were performed. Significant increases in the apparent viability of EMT6 and CHO cells were observed with MTT assays after short time periods with maximum effects at 2 hr. Reduced serum concentrations intensified this effect. Conversely, significant decreases in viability for both cell lines occurred after longer incubations and serum withdrawal enhanced this effect in both cell lines. Clonogenic assays provided contrasting results where viability increased significantly only in EMT6 cells (not CHO) and was smaller than that reported by MTT. Furthermore, greater dicumarol toxicity was observed in clonogenic assays. Significant toxicity compared to control occurred after 4-hr treatment (vs. 12 hr MTT) and serum withdrawal also increased the toxicity of dicumarol with extended culture. ROS production in EMT6 and CHO cells increased in a concentration-dependent manner with 20-min dicumarol administration and thereafter declined. The ec 50 for dicumarol-induced oxygen consumption was 0.84 μM in CHO compared to 1.18 μM in EMT6 cells. Cell lines are differentially sensitive to the toxicity of dicumarol and cell survival data may be skewed by its inclusion, probably due to ROS production and mitochondrial uncoupling. Dicumarol is not recommended for inclusion in the MTT assay.

Mode of action of menaquinone-4 on blood coagulation.

The prothrombin activity in plasma was highly correlated with that in liver in normal rats, bishydroxycoumarin (dicumarol)-treated rats and CCl4-treated rats. Menaquinone-4(K2(20)) significantly increased the prothrombin activities in plasma and in liver of rats which had hypoprothrombinemia induced by administration of dicumarol or CCl4. K2(20) significantly promoted the prothrombin synthesis in liver homogenate in the rats treated with dicumarol or CCl4. Furthermore, it was observed that K2(20) exerted a promoting effect on prothrombin synthesis in the microsomal fraction of liver. However, when plasma was incubated with K2(20), the prothrombin time of the plasma was not shortened. The effects of K2(20) on the prothrombin activity and on the prothrombin synthesis of liver were more rapid and stronger than those of vitamin K1. It is concluded that K2(20) promotes prothrombin synthesis in liver parenchymal cells and improves hypoprothrombinemia caused by dicumarol or CCl4 in rats.

Alterations in Absorption of Dicumarol by Various Excipient Materials

A plasma level study was conducted in dogs to determine the effects of various excipient materials on the absorption of dicumarol. The drug was combined with the excipients by an equilibration process and administered orally. Plasma concentrations of dicumarol after administration with excipients were compared to control levels produced by the drug given alone. Significant differences in the plasma levels of dicumarol were observed with six of the 10 excipients used in the study. Significantly higher plasma levels (up to 180% of control values) were observed when dicumarol was administered with magnesium oxide or hydroxide. This effect may be due to chelate formation because the magnesium chelate of dicumarol produced higher plasma levels of dicumarol than the drug administered alone. Dicumarol administration with talc, colloidal magnesium aluminum silicate, aluminum hydroxide, or starch resulted in significantly lower plasma levels of the drug. It is suggested that these types of interactions may be an explanation for differences in the bioavailability of dicumarol from different dosage formulations.

Effect of dicumarol upon protein synthesis in the rat

The effect of dicumarol upon protein synthesis at three levels of cellular organization in the rat has been studied. It has been demonstrated that dicumarol administration to rats at such concentrations as to extend the one-stage Quick prothrombin time to greater than 240 sec and to reduce the concentrations of factors II, VII and X levels of 8, 14 and 16 per cent of normal, respectively, did not impair the biosynthesis of serum albumin when studied over a 10- to 20-hr period. Furthermore, dicumarol did not impair the biosynthesis of nascent peptide in liver as revealed by incorporation of radioactivity from amino acids into hepatic polysemes. Finally, no effect of dicumarol at concentrations between 10 −5 and 2 × 10 −4 could be detected upon protein biosynthesis in vitro by normal polysomes plus soluble supernatant extract from rat liver. These data do not support the hypothesis that coumarin anticoagulant drugs act by uncoupling oxidative phosphorylation and depressing protein synthesis in vivo. Likewise, no effect of dicumarol upon the intrinsic reactions of protein synthesis in the presence of an ATP generating system in vitro could be detected.

Genetic control of dicumarol levels in man.

The mean half-life of dicumarol in the plasma of seven sets of identical and seven sets of fraternal twins after a single oral dose of 4 mg/kg was 43.6+/-SD 17.9 hr. Half-lives ranged from 7 to 74 hr in these 28 normal adults not receiving other drugs for 2 wk preceding dicumarol administration. Large differences among unrelated individuals in dicumarol half-life disappeared almost completely in identical twins, but persisted to some extent in most sets of fraternal twins. These results indicate that marked differences among subjects in dicumarol half-life are under genetic rather than environmental control. Reproducibility of values for dicumarol half-life was demonstrated. A direct relationship between the dose and the half-life of dicumarol occurred in unrelated volunteers administered progressively larger doses at 10-day intervals. Dose dependence of the half-life of a drug results in increased variability of half-life and hence in greater risks of toxicity on long-term therapy. Risks of toxicity on the one hand and of failure to anticoagulate adequately on the other can be reduced by determining dicumarol half-life before starting long-term therapy. Half-lives for dicumarol and phenylbutzone tended to be correlated in the 28 twins, but no correlation occurred between dicumarol and antipyrine half-lives.

Dicumarol therapy. Some effects on platelets and their relationship to clotting tests.

One of the problems of Dicumarol administration has been determination of the adequacy of therapy. A fundamental stage in thrombus formation in the circulation is platelet clumping. Since platelets are involved in both hemostasis and thrombosis, a study of changes in platelet function may prove useful. The effects of Dicumarol on platelet clumping and adhesiveness have, therefore, been studied and related to changes in other clotting mechanisms. Comparison of platelet function in control periods and during Dicumarol administration shows that this drug lowers the adhesive index and prolongs the platelet clumping time to a highly significant degree. The accuracy of estimates of these 2 platelet functions given by 3 other tests, prothrombin time, whole blood clotting time and plasma activity in the thromboplastin generation tests have been compared. By any criterion, degree of correlation, consistency of regression slopes, intercepts or scatter about regression lines, clotting time was the most efficient of the 3 tests for evaluting platelet function. The prothrombin time was the least efficient. Furthermore, hemorrhage from excessive dosage was closely related to the platelet clumping time and clotting time. It should be pointed out, however, that in about one-third of the patients the prothrombin time was as good an index of platelet clumping time as clotting time. While prothrombin times done with 2 different sources of thromboplastin may give congruous results in untreated patients, considerable discrepancies may appear during Dicumarol therapy. If prothrombin time is to be used as a guide to Dicumarol therapy, it is important to establish in each patient separately its reliability as a measure of the behavior of the clotting mechanism as a whole. For those in whom it seems reliable, an individual "therapeutic range" should be determined. For other cases, some other suitable test should be employed. It is emphasized that regulation of Dicumarol dosage is an individual problem and that only those tests shown to be reliable indices of safe effective modifications of clotting mechanism in the individual patient should be used.

THROMBOCYTOPENIA IN THE EXPERIMENTAL PRODUCTION OF HEMORRHAGIC DEATH BY MULTIPLE FACTORS

Three experimental conditions were established in rats either singly or in combination: (a) thrombocytopenia by the intraperitoneal administration of a single dose of radioactive phosphorus, 4 μc/g of body weight, (b) prothrombopenia by the oral administration of dicumarol, 2 mg/100 g of body weight per day for 7 days, (c) stress by the intraperitoneal administration of a single dose of 10% NaCl, 2.5 ml/100 g of body weight. The response to the treatment was measured by the level of circulating platelets and the prothrombin time. The cause of death was assessed by gross and, occasionally, histological examination of the organs of the animals that had died within 15 days of the injection of the radioactive phosphorus.The percentage mortality from spontaneous hemorrhage when thrombocytopenia was combined with either hypocoagulability or stress was very much greater than the mortality resulting from a single treatment, or from a simple additive effect (P «0.01). Mortality due to hemorrhage was thrombocytopenia, 20%; P32 without thrombocytopenia, 0% (also together with dicumarol and with stress); dicumarol, 16%; stress, 0%; thrombocytopenia and dicumarol, 81%; thrombocytopenia and stress, 67%; controls, 0%. These results provide additional support for the hypothesis of spontaneous hemorrhage being caused by multiple factors acting simultaneously.

THROMBOCYTOPENIA IN THE EXPERIMENTAL PRODUCTION OF HEMORRHAGIC DEATH BY MULTIPLE FACTORS

Three experimental conditions were established in rats either singly or in combination: (a) thrombocytopenia by the intraperitoneal administration of a single dose of radioactive phosphorus, 4 μc/g of body weight, (b) prothrombopenia by the oral administration of dicumarol, 2 mg/100 g of body weight per day for 7 days, (c) stress by the intraperitoneal administration of a single dose of 10% NaCl, 2.5 ml/100 g of body weight. The response to the treatment was measured by the level of circulating platelets and the prothrombin time. The cause of death was assessed by gross and, occasionally, histological examination of the organs of the animals that had died within 15 days of the injection of the radioactive phosphorus.The percentage mortality from spontaneous hemorrhage when thrombocytopenia was combined with either hypocoagulability or stress was very much greater than the mortality resulting from a single treatment, or from a simple additive effect (P «0.01). Mortality due to hemorrhage was thrombocytopenia, 20%; P32 without thrombocytopenia, 0% (also together with dicumarol and with stress); dicumarol, 16%; stress, 0%; thrombocytopenia and dicumarol, 81%; thrombocytopenia and stress, 67%; controls, 0%. These results provide additional support for the hypothesis of spontaneous hemorrhage being caused by multiple factors acting simultaneously.

Hageman trait (Hageman factor deficiency)

The seventh case of HT is reported, of a twenty-six year old woman of German-Irish-French descent in whom abnormal clinical bleeding was absent despite the presence of a severe coagulation defect discovered at the age of five. Coagulation studies of the patient's parents were normal. Fourteen family relatives gave no history of abnormal bleeding. The coagulation defect consisted of a prolonged coagulation time in glass, but only slightly prolonged time in siliconized tubes. The patient's barium sulfate-treated plasma and serum combined produced an abnormal thromboplastin generation test. The patient's prolonged plasma recalcification time was corrected by normal plasma in extremely minute amounts by barium sulfate-treated plasma, by serum and plasmas deficient in AHG, PTC, PTA and PTF-D, but not by plasma deficient in HF. The patient's plasma corrected the prolonged plasma recalcification times of AHG, PTC and PTA-deficient plasmas. HF present in plasma and serum was found to be non-dialyzable, poorly absorbed by barium sulfate, resistant to heating at 60 °C. for fifteen minutes, present in the 25 to 33 and 33 to 50 per cent ammonium sulfate fractionated normal plasma precipitates, present in Cohn's plasma fractions III, IV-1 and IV-4, to migrate electrophoretically between the beta and gamma globulins and not to be decreased by dicumarol administration or in the newborn.

Long-term dicumarol administration as a therapeutic trial in sicklemia; report of a case.

THE management of sicklemia to the present has been restricted to the transfusion of whole blood during the frequent crises of hemolytic anemia. No remedy has been found that will beneficially infl...

The reduction of autoprothrombin. II. Activity with dicumarol.

It is generally considered that the administration of dicumarol is followed by a decrease in prothrombin concentration, and it has been suggested that another plasma factor may also decrease. We have found that there are two derivatives of prothrombin called autoprothrombin and autoprothrombin II. In this paper we present data which show that the concentration of autoprothrombin II can be reduced practically to zero with the use of Dicumarol without observing a bleeding tendency. The decrease in concentration coincides with a decrease in prothrombin concentration but is relatively more rapid.

On the nature of the blood coagulation mechanisms in certain clinical states.

Current concepts of deficiency of serum prothrombin conversion accelerator (SPCA) metabolism are all related to prothrombin and its activation. SPCA deficiency is a hemorrhagic diathesis first described by Alexander and associates and subsequently by Owren and others. Patients with hemorrhagic tendencies were helped following administration of serum but this was of short duration, and the use of whole blood was of doubtful value. Plasma of Dicumarol-treated patients as well as plasma of normal persons only partly corrected the function of the coagulation mechanisms. The concentrations of Ac-globulin and prothrombin in the plasma were only slightly below normal. I t has been postulated that the patient with SPCA deficiency is deficient in a precursor of a factor found in serum. This precursor is supposed to be in plasma and to be different from any previously recognized coagulation factor. Owren is especially emphatic concerning its reality, and states that was discovered as a contaminant of prothrombin preparations. .In 1949 proconvertin was separated from prothrombin. .The discovery of patients with a hemorrhagic disease due to a lack of proconvertin has provided a final proof of the existence of this factor. . We have not found it possible to correlate the results of our extensive work with those assertions or with similar points of view. Our data, based on experiments with purified prothrombin, lead us to believe with even more assurance than formerly that the factor found in serum, and variously called factor VII, convertin, or SPCA, is a derivative of prothrombin itself and not of something distinctly different from prothrombin. Even apart from our work on purified prothrombin, it is interesting to note that no one has been able to prepare the hypothetical precursor of the accelerator found in serum and to establish that it is distinctly different from all other clotting factors. It is generally stated that the administration of Dicumarol is followed by a decrease in concentration of prothrombin. That view has been supported for many years and we also uphold it. The hypothetical precursor of SPCA (proSPCA, proconvertin, precursor of factor VII) has recently been reported as reduced in concentration, parallel with reduction of prothrombin. Indeed Biggs and Macfarlane found so great a divergence with Tromexan that they suggested that prothrombin concentration remains at the normal level while factor VII decreases. On the other hand, Owren and Aas, and to a lesser extent, Koller, Loeligpr and Duckert, using a method that is supposed to measure the concentration of the hypothetical precursor (proconvertin, pro-SPCA) in plasma, found

The Clotting Action of Russell Viper Venom

Abstract 1. It has been shown that Russell viper venom in the presence of lipoid cofactor can clot hemophilia A plasma, hemophilia B plasma and proconvertin deficient plasma within 5 seconds. It is unable to clot proaccelerin deficient plasma rapidly. Thus the clotting behavior of venom-lipoid is similar to convertin. 2. A rapid presumptive test for hypoproconvertinemia is simply to do a Quick clotting time with brain thromboplastin and then with viper venom. Proconvertin deficient plasma will give a prolonged brain thromboplastin time but a normal venom time. Prothrombin or proaccelerin deficient plasma will give long clotting times with both agents. 3. The observation that Russell viper venom acts independently of the proconvertin content of plasma explains the differences between the venom and brain thromboplastins. It also explains the failure of venom to measure the full effect of dicumarol administration.

Prevention of systemic arterial embolism in chronic rheumatic heart disease by means of protracted anticoagulant therapy.

Seven clinic outpatients with mitral stenosis and auricular fibrillation complicated by systemic embolism have been maintained on Dicumarol for periods of 20 to 48 months. None has had a recognized embolism while on therapy. Dicumarol administration has been interrupted in each case for periods of six days to four months. Three additional systemic embolic episodes in two individuals occurred during this interval. Hemorrhagic manifestations have occurred on three occasions. The study suggests that: (a) protracted Dicumarol therapy is an effective means of preventing recurrent embolism in individuals with chronic rheumatic heart disease; (b) it is applicable to the average clinic outpatient.

Dicumarol and plasma antithrombin activity.

SummaryWith the administration of dicumarol to dogs there are no significant changes in the activity of antithrombin-II, and -III. Antithrombin-IV activity remained but no accurate quantitative measurements were made. In human subjects antithrombin-IV activity also remains and is probably not decreased. The thrombin-titer method of analysis shows that there are very marked changes in the activation rate of prothrombin soon after dicumarol is administered to human subjects. The addition of purfied prothrombin does not restore the activation rate to normal. In contrast to human subjects the activation rate of prothrombin is not reduced in dog plasma when dicumarol is administered.

White Thrombo-embolism and Vascular Fragility in the Hamster Cheek Pouch after Anticoagulants

Abstract 1. Heparin, dicumarol, Tromexan and phenylindanedione did not prevent, but actually enhanced, the formation of platelet plugs at the site of hemorrhages produced at the tip of a stimulating microelectrode in contact with the walls of small blood vessels in the hamster cheek pouch. Furthermore, these anticoagulants per se produced an increased adhesiveness of platelets and leukocytes to the endothelium of small venules. In particular, heparin produced platelet embolism immediately after administration. The circulating emboli varied in size from that of leukocytes to cylindrical masses which filled the lumina of vessels measuring up to 75 µ in diameter. Platelet embolism was not found during the first five days of administration of dicumarol, but appeared subsequently. 2. The significance of increased platelet agglutinability during the hypocoagulable state induced by anticoagulants and the need for a critical distinction between agglutinability of the formed elements and coagulation involving fibrin are discussed. 3. An increase in the fragility of the walls of venules followed the administration of heparin, dicumarol, Tromexan and phenylindanedione. A brief series of relatively weak faradic shocks from a micro-electrode produced a hemorrhage. Shocks of much greater strength applied to venules of normal untreated hamsters produced no visible effect. This method is proposed as a new semiquantitative procedure for investigation of vascular fragility in accessible membrane preparations. It may possibly distinguish between fragility in the sense of rhexis or breaking of the wall and petechial formation such as that produced by the application of negative pressure and perhaps erroneously referred to in the literature as "fragility." 4. Petechial hemorrhages appeared at the points of junction of the postcapillary venules after anticoagulant administration, but not on the capillaries. Spontaneous ruptures were not found in capillaries. No evidence was found for an increase in capillary fragility. Consequently, the term "vascular fragility" is proposed to replace "capillary" fragility.

Severe hemorrhage due to tromexan; report of three cases.

TROMEXAN, a new anticoagulant, has recently been accepted for general use. Chemically, it is described as ethyl biscoumacetate (3,3′ carboxymethylenebis- [4-hydroxycoumarin] ethyl ester). It is most widely recognized as Tromexan but is also known as Pelentan, B.O.E.A. and D.E.A. Dicumarol1 and Tromexan2 are related members of the coumarin series of drugs. Each is presumed to act in similar fashion, by interfering with the normal production of prothrombin by the liver. The exact mode of action is as yet unknown.3 , 4 The possibility of hemorrhage after Dicumarol administration is well known, and precautions for its use have been repeatedly stressed.5 6 7 Utilizing an . . .

The Effect of Dicumarol on Co-Thromboplastin, a Factor in Blood Concerned with the Conversion of Prothrombin to Thrombin

The deficiency in the conversion of prothrombin to thrombin which develops after administration of dicumarol appears to be due largely to decreased cothromboplastin activity. Both the development of this deficiency and the recovery from it tend to precede the accompanying changes in prothrombin itself, as measured by the two-stage method. In terms of deviation from normal, the deficiency in conversion often exceeds in magnitude the deficiency of prothrombin itself.

Assay of plasma antihemophilic activity in normal heterozygous (hemophilia) and prothrombinopenic dogs.

Summary1. A method is described for the assay of antihemophilic factor; results may be expressed in per cent of a control plasma or in units. Hemophilic blood, canine or human, is used as substrate. 2. In normal dogs, regardless of sex, and in females heterozygous for hemophilia, a steady state of plasma AHF appears to exist. The antihemophilic activity of heterozygotes is approximately the same as that of normal dogs. 3. The antihemophilic activity of plasma is maintained at normal levels following acute liver injury, as well as after dicumarol administration.