Biomedical subjects

R Wallin

Publications and source records attributed to R Wallin.

At least 73 records · Page 4Linked to original sources

PubMed1989

Vitamin K1 reduction in human liver. Location of the coumarin-drug-insensitive enzyme.

The antidotal effect of vitamin K in overcoming poisoning by coumarin anticoagulant drugs is mediated by a vitamin K-reducing enzyme of the endoplasmic reticulum [Wallin & Martin (1987) Biochem. J. 241, 389-396]. With microsomes obtained from human liver biopsies, we have investigated the localization and the transverse orientation of this enzyme in the endoplasmic reticulum and compared its orientation to that of the other enzymes of the vitamin K-dependent carboxylation system. All enzymes were protected by the microsomal membrane and thus appear to have a luminal orientation in the endoplasmic reticulum, consistent with their role in the vitamin K-dependent modification of secretory glycoproteins. Separation of rough and smooth microsomes showed that vitamin K-dependent carboxylase activity was 6-fold higher in rough than in smooth microsomes. Vitamin K1 reduction by the coumarin-drug-sensitive (pathway I) and -insensitive (pathway II) enzymes of the vitamin K-dependent carboxylation system was the same in rough and smooth microsomes. The data suggest a close association between the pathway I and II enzymes in the endoplasmic reticulum. These pathways may be partial reactions of multienzyme complex which carries out the various activities associated with the vitamin K-dependent carboxylation system.

Carbon-Carbon Ligases↗

PubMed1989

Identification of mRNA coding for factor VII protein in human alveolar macrophages--coagulant expression may be limited due to deficient postribosomal processing.

Clotting factors synthesized by monocytes and macrophages may initiate coagulation reactions during inflammation. Functional vitamin K-dependent coagulation factors have been found to be associated with human monocytes/macrophages, but there are no reports identifying mRNA coding for vitamin K-dependent proteins in these cells. In the present studies, factor VII mRNA was found in total RNA extracted from freshly isolated human alveolar macrophages using hybridization with a complementary DNA probe. On the other hand, vitamin K-dependent carboxylase activity which is required for postribosomal modification of the protein, was not detectable in the macrophages before or after culture, and human blood mononuclear leukocytes also lacked this enzyme activity. Control human and rat hepatoma cells exhibited high levels of carboxylase activity within the same experiments. Using sensitive kinetic assays, no increase in factor VII activity was detected during culture of alveolar macrophages under conditions promoting 1.78 +/- .24 (n = 8) fold increases of tissue factor activity. These findings with freshly isolated cells demonstrate that alveolar macrophages synthesize factor VII mRNA in vivo. However, the mRNA was found in the absence of evidence for gamma-carboxylase activity or processing of the factor into a functional clotting enzyme. The results imply that functional expression of any synthesized coagulation factor VII in alveolar macrophages may be limited or prevented due to a cellular deficiency at the level of postribosomal processing.

Carbon-Carbon Ligases↗

PubMed1989

Neutrophil function in ischemic heart disease.

Neutrophils contribute to the healing of and scar formation in myocardium after ischemic injury. Many recent studies indicate that neutrophils may be involved in the genesis and propagation of myocardial ischemia. To characterize neutrophil function in ischemic heart disease, neutrophil chemotaxis, leukotriene B4 (LTB4) generation, and elastase release in plasma were measured in 20 patients with stable angina, 17 patients with unstable angina or acute myocardial infarction (AMI), and 20 age-matched control subjects. Neutrophils from patients with stable angina exhibited markedly increased chemotactic activity and LTB4 generation as compared with the age-matched control subjects (p less than 0.01). Neutrophils of nine of 17 patients with unstable angina or AMI clumped spontaneously ex vivo and exhibited marked pseudopod formation and granule extrusion on electron microscopy. Subsequent chemotactic activity and LTB4 generation by neutrophils from these patients was less than in patients with stable angina, suggesting previous in vivo activation. Plasma levels of peptide B beta, a product of fibrin degradation by human neutrophil elastase, were approximately 15-fold higher (p less than 0.001) in patients with unstable angina or AMI (588 +/- 171 pmol/l, mean +/- SEM) compared with those in patients with stable angina (37 +/- 25 pmol/l) or control subjects (40 +/- 22 pmol/l), confirming intense in vivo neutrophil activation. Our study shows enhanced neutrophil function in patients with ischemic heart disease. The increased neutrophil chemotactic activity and LTB4 generation may be markers of stable angina pectoris. Intense neutrophil activation in unstable angina or AMI, as manifested by morphologic changes in neutrophils and elastase release, may relate to ongoing in vivo cellular activation.

Chemotaxis, Leukocyte↗

PubMed1989

Vitamin K-dependent carboxylation in the developing rat: evidence for a similar mechanism of action of warfarin in fetal and adult livers.

Our report presents data on maturation of the vitamin K-dependent carboxylation system in fetal and neonatal rat livers. This system which converts precursors of clotting factors II, VII, IX, X, protein S, and protein C to gamma-carboxylated proteins exhibited low gamma-carboxylation activity before birth. However, around the time of birth there was a sudden increase in all enzyme activities associated with the vitamin K-dependent carboxylation system. In 2-d neonatal rats these activities dropped to levels that were measured in fetal livers whereupon the activities had risen to adult levels in 7-d neonatal rats. However, the activities of the two pathways that provide the carboxylase with reduced vitamin KH2 cofactor were never as high as that measured in maternal livers. It appeared that the pathway which is insensitive to coumarin anticoagulant drugs matures later than the coumarin drug-sensitive pathway. This conclusion is supported by the finding of a late appearance in development of the vitamin K-reducing enzyme DT-diaphorase. Warfarin, when administered to the mother, affected the fetal livers at all stages of development studied (d 16-21). This was clearly demonstrated by vitamin K-dependent 14C-labeling of a 70-kD liver protein that has been shown previously to be a marker for the effect of this drug on the liver. The data demonstrate a similar mechanism of action of warfarin in fetal and neonatal rat livers and an ongoing maturation process of the vitamin K-dependent carboxylation system in these rats.

Animals↗

PubMed1989

Differences in coagulation and fibrinolysis after traumatic and septic shock in man.

In a prospective investigation of 19 patients with traumatic (n = 11) and septic (n = 8) shock admitted to the Intensive Care Unit of the University Hospital of Uppsala, differences in coagulation and fibrinolysis between the two conditions were evaluated. It was found that plasma coagulation variables such as thrombotest, normotest and antithrombin III were significantly lower in the septic patients, thus indicating a more intense and/or altered intravascular coagulation. Significantly higher levels of the von Willerbrand factor were found in septic patients, indicating a greater release from endothelial cells and platelets. The plasminogen activator inhibitor and the plasminogen-binding form of alpha 2-antiplasmin showed significantly higher values in septic patients, indicating a greater fibrinolysis inhibition in these patients. The greater disturbance in the protease-inhibitor balance in septicaemia was also verified by significantly lower levels of plasminogen and alpha 2-macroglobulin than after traumatic shock. Two out of 11 patients in traumatic shock developed the adult respiratory distress syndrome with a slow onset and benign course, whereas six out of eight septic shock patients developed a similar syndrome with a rapid onset and malignant course. These laboratory and clinical results lend further support to the hypothesis that the differences in coagulation and fibrinolysis systems reflect partly different pathogenic mechanisms underlying septic- and traumatic-induced adult respiratory distress syndrome.

Adolescent↗

PubMed1989

Initiation of the extrinsic pathway of coagulation by human and rabbit alveolar macrophages: a kinetic study.

We examined assembly and expression of the factor X activating complex on human and rabbit alveolar macrophages. Kinetic parameters of the factor X activating reaction were determined by functional titrations of factors VII and X with macrophage tissue factor (TF) added. We found rapid activation of factor X to Xa on alveolar macrophage surfaces. Detection of rapid factor Xa formation on macrophages required addition of exogenous factors VII and X. At plasma concentrations of the purified factors, factor Xa was formed on freshly isolated macrophages at approximately 5.4 pmol/min/10(6) cells. After macrophage maturation in culture for 20 hours with LPS (endotoxin) added, the factor X activation rate was increased two- to sixfold. The km' (apparent km) of TF-factor VII enzymatic complexes assembled on alveolar macrophages for factor X were (258 +/- 55 and 475 +/- 264 nmol/L for human and rabbit cells, respectively). The km' did not change during macrophage maturation in culture, but V'max (apparent Vmax) was consistently increased. The K1/2 of human factor VII (concentrations giving half maximal rates of factor X activation) for the interaction with human and rabbit alveolar macrophage TF were 0.191 +/- 0.096 and 1.7 +/- 0.7 etamol/L, respectively. The K1/2 were not significantly changed after maturation, whereas rates of Xa formation at saturation with factor VII were increased. The fast rates of factor X activation observed at physiologic concentrations of plasma-derived factors VII and X indicate that TF on alveolar macrophages is likely to provide sites for binding of factor VII and activation of factor X in vivo during clotting reactions associated with alveolar edema and inflammation.

Animals↗

PubMed1988

Early processing of prothrombin and factor X by the vitamin K-dependent carboxylase.

Binding interactions between the membrane-associated vitamin K-dependent carboxylase and its prothrombin and factor X substrates have been investigated in liver microsomes. Both substrates are firmly attached to microsomal membrane fragments which also harbor the carboxylase. In vitro 14CO2 gamma-carboxylation of these substrates, triggered by reduced vitamin K1H2, resulted in release of 14C-labeled prothrombin precursors from the membrane fragments, but no release of 14C-labeled factor X precursors could be demonstrated, which suggested a difference in early processing of these substrates by the carboxylase. Warfarin treatment of rats resulted in a 3-fold increase in the membrane concentration of factor X antigens and a 20-fold increase in 14C gamma-carboxylation of the membrane pool of factor X carboxylase substrates. There was a dose-response relationship between the amount of drug administered to the rats and 14C labeling of the membrane pool of factor X carboxylase substrates. On the other hand, the membrane concentration of prothrombin antigens did not increase in response to the drug, and 14CO2 gamma-carboxylation of the membrane pool of prothrombin carboxylase substrates was the same in warfarin and saline-treated rats. The results demonstrate significant differences in the interaction between the carboxylase and its prothrombin and factor X substrates. It appears that the different interactions result from binding of the prothrombin and the factor X precursors to separate microsomal membrane proteins that are involved in the gamma-carboxylation reaction. Warfarin appears to induce the factor X precursor-specific but not the prothrombin precursor-specific binding proteins, which suggests a new mechanism for the action of warfarin. These binding proteins may be under different genetic control. Treatment of the prothrombin and the factor X carboxylase substrates with endonuclease H showed that the rat prothrombin and the human factor X carboxylase substrates are high mannose glycoproteins. The human prothrombin and the rat factor X carboxylase substrates did not, on the other hand, change their migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels after endonuclease H treatment. The data demonstrate differences in the glycoprotein nature of the rat and the human carboxylase substrates.

Animals↗

PubMed1988

No evidence for vitamin K-dependent carboxylation of canine surfactant apoproteins, 28-36 kDa.

Recent research has shown that rat surfactant apoproteins (26-38 kDa) are vitamin K-dependent [Rannels, Gallaher, Wallin & Rannels (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5952-5956]. We have investigated the effect of the vitamin K antagonist warfarin on this family of apoproteins in surfactant from dog lung. Our data suggest that warfarin does not interfere with synthesis and secretion of these proteins into dog lung surfactant. Abnormal surfactant apoproteins, produced in response to warfarin treatment of the dog, were also not found in lung surfactant. 4-Carboxyglutamic acid analysis of purified dog apoproteins also failed to detect the vitamin K-modification. When vitamin K-dependent 14C labelling of precursors of vitamin K-dependent proteins was carried out, fluorography of these precursors, when electrophoresed into SDS/polyacrylamide gels, revealed 14C-labelled proteins of apparent molecular mass 74, 46, 42, 34, 31 and 23 kDa. Antibodies produced against purified dog surfactant apoproteins recognized precursors of the surfactant apoproteins in lung microsomes but did not recognize any 14C-labelled carboxylase substrates. These precursors appeared on immunoblots with apparent molecular mass 29, 32, 33 and 50 kDa. Our data suggest that there are significant differences between this class of surfactant apoproteins in the rat and the dog.

Animals↗

PubMed1988

Identification of vitamin K-dependent carboxylase activity in lung type II cells but not in lung macrophages.

Fluorography of 14C-labelled glutamic acid residues in vitamin K-dependent protein precursors in lung microsomes (microsomal fractions) shows that the lung has several substrates that are not found in the liver. These precursor proteins unique to the lung have apparent molecular masses of 65, 53, 50, 36, 31 and 13 kDa. Type II epithelial cells appear to synthesize most of the vitamin K-dependent proteins in the lung. The 36 and the 31 kDa precursors also found in Type-II-cell microsomes have a similar molecular mass to those of surfactant-associated proteins, and we have previously shown [Rannels, Gallaher, Wallin & Rannels (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5952-5956] that the 36 kDa protein is one of the precursors for these proteins. Immunoblotting of membrane fragments of Type-II-cell microsomes with plasma prothrombin antibodies identified two prothrombin-like antigens of apparent molecular masses 68 and 65 kDa. This raises the question as to whether Type II cells are also a potential site for synthesis of prothrombin and possibly other vitamin K-dependent clotting factors. Pulmonary macrophages appear to be devoid of vitamin K-dependent carboxylase activity. However, Type II epithelial cells have significant activity, and this activity was unaltered when these cells were maintained in primary culture for 3 days, suggesting that carboxylase activity is expressed in lung alveolar epithelium independently of culture-induced changes in cellular differentiation. Carboxylase activity in Type II cells was enhanced 2-fold when cells were cultured for 24 h in the presence of 50 microM-warfarin. Type II cells, therefore, resemble hepatocytes with regard to their response to coumarin anticoagulant drugs.

Animals↗

PubMed1987

3-Methylcholanthrene induction of enzymes in the vitamin K-dependent carboxylation system.

The effect of 3-methylcholanthrene on liver enzymes in the vitamin K-dependent carboxylation system has been investigated in normal rats and rats treated with the anticoagulant warfarin. It was found that 3-methylcholanthrene did not interfere with the anticoagulant function of the drug. Treatment of rats with 3-methylcholanthrene resulted in a 2.7-fold increase in liver cytosolic DT-diaphorase activity and a 1.5-fold increase in liver microsomal vitamin K-dependent carboxylase activity. A pathway for production of reduced vitamin K cofactor for the vitamin K-dependent carboxylase is catalyzed by DT-diaphorase and an as yet unidentified NADH-specific dehydrogenase(s). The data suggest that the unidentified enzyme(s) in the pathway is not induced by 3-methylcholanthrene.

Biomarkers↗

PubMed1987

Warfarin poisoning and vitamin K antagonism in rat and human liver. Design of a system in vitro that mimics the situation in vivo.

The present paper describes a system in vitro that has been developed to mimic vitamin K metabolism and vitamin K function in liver. In this system the two pathways that are known to participate in vitamin K reduction are active and the vitamin K-dependent carboxylase accepts a synthetic pentapeptide as substrate. With this system in vitro the effect of warfarin on both pathways was examined under conditions which simulated a warfarin-poisoned liver. Identical experiments were completed with rat and human liver. All activities currently associated with vitamin K metabolism and vitamin K function were similar in the rat and human systems. Warfarin neutralized the ability of pathway I (the vitamin K epoxide reductase pathway) to produce reduced and active vitamin K cofactor for the carboxylase. In both the rat and the human system, however, when warfarin was present, reduced vitamin K cofactor was produced by pathway II (the dehydrogenase pathway). The data are consistent with observations in vivo on the effect of vitamin K1 when used as an antidote. This suggests that the system in vitro reflects the mechanism in vivo by which vitamin K1 overcomes warfarin poisoning.

Animals↗

PubMed1987

Rat and human liver vitamin K epoxide reductase: inhibition by thiol blockers and vitamin K1.

1. Reduction of vitamin K1 2,3-epoxide by rat and human liver vitamin K epoxide reductase is inhibited by N-ethylmaleimide and iodoacetamide. 2. Both enzymes are protected from inhibition by N-ethylmaleimide by vitamin K1 or vitamin K1 2,3-epoxide. 3. Vitamin K1 inhibits reduction of vitamin K1 2,3-epoxide to vitamin K1 which suggests product inhibition of the enzyme.

Animals↗

PubMed1987

DT-diaphorase in morbidly obese patients.

Dicumarol sensitive DT-diaphorase (EC 1.6.99.2) activity has been measured in human cytosol from liver and various extrahepatic tissues not containing tumors which were removed during elective operations. The specific activity was found to be highest in tissues of the gastrointestinal tract. In contrast to rodent liver, the human liver has extremely low DT-diaphorase activity. Tissue activity was found to be highly variable among the patients studied which suggests that nutritional and/or genetic factors may be involved. The variability ranged from undetectable levels in liver to 223 nmol/mg X min in stomach. The data question whether the current concept concerning the biological function of this enzyme is applicable to man.

Cytosol↗

PubMed1987

Vitamin K-dependent carboxylation of pulmonary surfactant-associated proteins.

Rat type II pneumocytes expressed vitamin K-dependent carboxylase activity that incorporated 14CO2 into microsomal protein precursors of molecular weights similar to those of surfactant-associated proteins (SAP). Compared to carboxylated precursor proteins present in the liver, these molecules appeared to be unique to the lung. Antibodies raised against purified rat surfactant reacted with SAP resolved by NaDodSO4/PAGE and with surfactant-containing lamellar bodies in type II pneumocyte cytoplasm. NaDodSO4/PAGE of microsomal proteins, after carboxylase-catalyzed incorporation of 14CO2, demonstrated radiolabeled, immunoreactive products identical to SAP. The presence of gamma-carboxyglutamic acid in these proteins was confirmed by HPLC analysis of SAP hydrolysates. Furthermore, lung carboxylase activity and SAP matured over similar time courses during fetal lung development. These results show that SAP are carboxylated by type II cells via a vitamin K-dependent pathway analogous to that for hepatic carboxylation of clotting factors. Further analogy to the clotting system suggests that gamma-carboxyglutamic acid residues in SAP polypeptides play a role in Ca2+ binding and thus in the known requirements for both the cation and SAP in the physiological function of pulmonary surfactant.

Animals↗

PubMed1986

Vitamin K antagonism of coumarin anticoagulation. A dehydrogenase pathway in rat liver is responsible for the antagonistic effect.

In the liver, it appears that there are two different pathways for vitamin K reduction. One pathway is irreversibly inhibited by coumarin anticoagulant drugs. The other pathway has been shown in the present study to be composed of enzymes that are not effected by physiological 'in vivo' concentrations of these drugs. This pathway appears to be responsible for the antidotal effect of vitamin K in overcoming coumarin poisoning. In rat liver the pathway has been shown to be composed of DT-diaphorase (EC.1.6.99.2) and a microsomal dehydrogenase(s). The activity of the microsomal dehydrogenase(s) was 3.6-fold higher with NADH than with NADPH present in the test system. It appears that this enzyme is the physiologically important enzyme in the pathway. In contrast with DT-diaphorase, this enzyme(s) is shown to be tightly associated with the mirosomal membrane. The enzyme(s) is not identical with either of the quinone-reducing enzymes cytochrome P-450 reductase or cytochrome-b5 reductase. Our data thus postulate the existence of an as-yet-unidentified microsomal dehydrogenase that appears to have an important function in the pathway.

4-Hydroxycoumarins↗

PubMed1986

Vitamin K antagonism of coumarin intoxication in the rat.

An in vitro system which expresses all enzyme activities related to vitamin K-dependent carboxylation of blood clotting factors was prepared from livers of rats overdosed with warfarin, difenacoum and dicumarol respectively. In this system, the activities of the two pathways that are known to produce active reduced vitamin K1 cofactor for the carboxylation reaction were measured. Also the ability of high concentrations of vitamin K1 to overcome inhibition of clotting factor synthesis was studied. In the systems prepared from livers of warfarin and difenacoum intoxicated rats, pathway I was inactive. Vitamin K epoxide reductase was also inactive which strongly suggests that this enzyme catalyzes the activity of pathway I in vivo. Reduction of vitamin K1 by pathway II bypassed the inactive pathway I and resulted in carboxylation activity. This pathway therefore mediates the antidotic effect of vitamin K1 in the coumarin intoxicated liver. In the in vitro system prepared from dicumarol intoxicated livers the activity of pathway I was not significantly affected. Dicumarol however was a strong inhibitor when added to liver microsomes in vitro.

4-Hydroxycoumarins↗

PubMed1986

Rat liver vitamin K-dependent carboxylase: a study of antibodies raised against partially purified preparations of the enzyme.

Antibodies raised against three preparations of increasing purity of the microsomal vitamin K-dependent carboxylase did not neutralize essential proteins in the enzyme complex. When immobilized on Sepharose the antibodies removed 75% of contaminating proteins in the starting material, including cytochrome P-450. Immunoaffinity chromatography was more efficient when carried out in the presence of the detergent CHAPS than in the presence of Triton X-100. Immunoabsorption stimulated carboxylase activity 2.9-fold and resulted in a 66-fold increase in the specific activity of the complex.

Animals↗

PubMed1986

Adriamycin and DT-diaphorase.

It has been suggested that DT-diaphorase (EC 1.6.99.2) can oppose the cytotoxic effect of the antineoplastic drug adriamycin. In this study, adriamycin was tested as a substrate for purified DT-diaphorase from rat liver. Purified DT-diaphorase was unable to use adriamycin as substrate. Also, the drug did not have any significant inhibitory effect on the enzyme.

Animals↗