The effect of heparin adsorbed on Sepharose-lysine on the inactivation of thrombin by anti-thrombin III [proceedings].
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Antithrombins↗
Biomedical subjects
E Regoeczi
Publications and source records attributed to E Regoeczi.
The molecular components of human transferrin type C.
Immunologically pure human transferrin type C (TfC) was isolated from the plasmas of 11 individual healthy donors. After conversion into the 2Fe-form, the preparations were analysed by polyacrylamide gel electrophoresis and chromatography on DEAE-cellulose. In all samples studied by either method the presence of three components, designated A, B and C, was observed. Calculations from eight chromatograms yielded the following relative proportions for the components: A:6%, B:62% and C:32%. The quantity of iron bound played no role in this chromatographic resolution. The components were immunologically identical but their sialic acid content increased inthe order of A less than B less than C. The presence of galactose as an ultimate residue of the oligosaccharide chains in TfC component A was confirmed by a biological test. This observation together with the results of earlier analyses for hexose, hexosamine and galactose in the subfractions from Behringwerke human transferrin, suggests that sialic acid is probably the only variable among TfC components A, B and C. Loss of sialic acid from component C during the isolation of TfC was excluded as an explanation for the presence of the other two components. The electrophoretic appearance of TfC samples from five patients with liver disease (chronic active hepatitis, cirrhosis or alcoholic liver) did not noticeably differ from that of TfC FROM HEALTHY PERSONS. Baboon transferrin resembles TfC with respect to sialic acid heterogeneity. This species was therefore studied to decide whether sialic acid is gradually lost from transferrin in the circulation or whether transferrin is not fully sialylated before discharge from the hepatocyte. Using DEAE-cellulose chromatography no difference was found between baboon transferrin molecules which were less than 6h old and those which had a mean age of 8.9 days. By inference it is suggested that the reason for the multiplicity of TfC is also likely to be biosynthetic.
Adult↗
Some observations on the carbohydrate composition of purified transferrin.
Five subfractions were isolated on DEAE-cellulose from samples of a commercially available human transferrin preparation and their carbohydrate composition was analysed. Hexosamine, galactose and total hexose were determined in four subfractions and sialic acid in all five. The data obtained indicate that the excess number of electrophoretic bands observed in transferrin from this source is due to the loss of carbohydrates which only affects sialic acid and none of the other sugar types. The availability of the penultimate galactose residues as the terminal residues in the subfractions deficient in sialic acid was also confirmed by a biological test utilizing the rat liver. The reason for the gradual loss of sialic acid from transferrin is unknown. Freezing-thawing and lyophilization did not detectably affect the sialic acid content of purified transferrin. However, free sialic acid did appear in some preparations on storage. It is concluded that similar changes in the carbohydrate composition of other plasma glycoproteins before, during, or after purification can be expected to exert an adverse effect on their usefulness as metabolic tracers.
Animals↗
Relationship between the anticoagulant and antithrombotic effects of heparin in experimental venous thrombosis.
The relationship between the antithrombotic and anticoagulant effects of heparin was assessed using venous thrombi in rabbits. Accretion of 125I-fibrinogen onto jugular vein thrombi was used to assess the antithrombotic effect of heparin, and the protamine sulfate titration test (heparin activity) and the activated partial thromboplastin time (APTT) were used to measure its anticoagulant effect. The effect of heparin on jugular vein bleeding times was also measured in a separate group of animals. Fibrinogen accretion was significantly lower with continuous infusion than with intermittent injection. Heparin, given by continuous infusion, produced marked inhibition of fibrinogen accretion (to less than 10% of control accretion) at an APTT value of between 75 and 80 sec (control 34 sec) and at a level of heparin activity of 0.4-0.5 U/ml. Infusion of cryoprecipitate reduced the effect of heparin on the APTT relative to its effect on heparin activity. In these cryoprecipitate-treated animals, marked inhibition of fibrinogen accretion occurred at a similar level of heparin activity (0.4-0.6 U/ml) but at a significantly lower APTT (35-50 sec) than in normal animals. On the other hand, there was a progressive increase in jugular vein bleeding time with both increasing APTT values and heparin levels in both groups of animals.
Animals↗
Increased heparin clearance in experimental pulmonary embolism.
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Animals↗
The physicochemical and chemical properties of alpha 1-acid glycoproteins from mammalian and avian plasmas.
We have isolated from the plasma of man, rabbit, pig, rat and chicken alpha 1-acid glycoproteins that show a single band on polyacrylamide gel electrophoresis and a single arc in immunodiffusion. Amino acid and carbohydrate compositions are presented. The human protein is very similar to preparations described by earlier workers and, although significant differences in amino acid composition exist among them, the proteins from the other species are assumed to be analogous to it. Ultra centrifugal studies, despite showing single, fairly symmetrical peaks at high speed, produced evidence for the physical heterogeneity of each protein in agreement with some earlier reports. Although many models consisting of mixtures of components were tested, none was found that fitted adequately all observations on any one of the glycoproteins.
Amino Acids↗
The proteolytic nature of commercial samples of galactose oxidase. Purification of the enzyme by a simple affinity method.
Several commercially available samples of galactose oxidase (D-galactose: oxygen 6-oxidoreductase, EC 1.1.3.9) were found to contain high proteolytic activity on proteins such as fibrinogen, transferrin, albumin and casein. A simple, efficient method was devised for the purification of galactose oxidase which relies on the affinity of the enzyme for agarose (Sepharose 6B). The purified galactose oxidase was recovered in high yield free from proteolytic activity. The enzymic affinity for Sepharose and Sephadex was investigated to clarify the absorption mechanism.
Alcohol Oxidoreductases↗
The affinity of human, rabbit and bovine thrombins for sepharose-lysine and other conjugates.
Human, rabbit and bovine thrombins are shown to possess marked affinities for Sepharose-lysine. Using either Xa-activated crude prothrombins (human, rabbit) or a commercial thrombin sample (bovine), the enzyme was isolated in a single chromatographic step by the affinity medium and preparations of high specific activity were obtained. The relevance of bound-lysine for the affinity of the thrombins was studied using other Sepharose conjugates with structures related to Sepharose-lysine. Using freshly activated prothrombins it was found that human and rabbit thrombin uptake required a conjugate with a spacer chain containing a minimum of four carbon atoms in length which supported a terminal amino group. As the thrombin activity aged, affinity for the terminal amino group decreased but the hydrophobic spacer chain became essential for enzyme binding. The active centre of thrombin was not involved in binding to Sepharose-lysine.
Animals↗
Studies of the metabolism of asialotransferrins: nonspecific changes in the metabolic behaviour of human asialotransferrin in avians.
In a previous study (Regoeczi, E. & Hatton, M.W.C. (1976) Can J. Physiol. Pharmacol. 54, 27-34) it was shown that the chicken (Gallus domesticus) does not possess a hepatic asialoglycoprotein receptor. In the present study the same conclusion is drawn for the duck (Anas platyrhynchos). For this reason these avian species were used to assess those changes in the distribution and catabolism of human asialotransferrin which takes place in the absence of the asialoglycoprotein receptor of the liver.
Animals↗
Asialoglycoprotein clearance in the chicken: evidence for the lack of a hepatic asialoglycoprotein receptor.
The behaviour of desialylated human and chicken acid alpha1-glycoproteins is reported in chickens. Although desialylation resulted in accelerated disappearance rates from the plasma of both proteins, nevertheless the asialoproteins were eliminated much more slowly than expected on the basis of earlier observations in mammals. Analysis of tissue radioactivities, including kidney, liver, lung and spleen, failed to demonstrate any accumulation of the labeled asialoproteins in the liver, which is contrary to the situation in mammals. The main pathway for the elimination of desialylated human acid alpha1-glycoprotein in the chicken is via the kidney (tubular catabolism and/or urinary excretion). The clearance of desialylated chicken acid alpha1-glycoprotein is more complex as it involves the kidney as well as the reticuloendothelial system. These findings indicate that, unlike mammals, chickens do not possess a hepatic plasma membrane receptor for asialoglycoproteins. This raises the possibility that the presence or absence of this specific receptor may constitute a fundamental biological difference between mammals and birds.
Animals↗
The effect of experimental hematoma on serum levels of fibrinogen-related antigen.
The effect of experimental hematomas on serum levels of fibrinogen-related antigen (FRA) was studied in rabbits. Homologous blood used for production of hematomas was mixed with 131I-fibrinogen, and the measurement of FRA was made using a radioactive technique (representing FRA from hematoma) and immunologically (representing total FRA). Total body radiation of animals bearing hematomas containing 131I-fibrinogen decayed slowly (mean t 1/2 = 189 hr) over the first 2-3 days and then rapidly (mean t 1/2 = 43 hr) during the subsequent days. Serum FRA levels measured immunologically rose transiently from 4 to 11 mug/ml during the slow phase of hematoma resolution, and returned to baseline levels during the rapid phase of resolution. The appearance of a small amount of protein-bound radioactivity in the blood of animals with hematomas paralleled the time course of the changes in immunologically determined FRA, but, even at its peak, circulating levels only averaged 0.36% of the administered tracer. From these observations, it seemed unlikely that the dissolution of fibrin which may form as the result of bleeding into tissues would significatnly contribute to levels of circulating FRA.
Animals↗
Labelled polyvinylpyrrolidone as an in vivo indicator of reticuloendothelial activity.
It is shown that after a single i.v. dose of [131I]-polyvinylpyrrolidone ([131I]-PVP) the total body and plasma radioactivities of rabbits decrease at distinctly different rates. The difference between these two rates is utilized to calculate the phagocytic rate of [131I]-PVP by reticuloendothelial cells.A number of experimental conditions are reported in which enhanced reticuloendothelial uptake of [131I]-PVP is readily demonstrable. They include the injection of small quantities of heterologous plasma, certain proteolytic fragments of the fibrinogen molecule, the clearance of antigen-antibody complexes, and the acute phase reaction (inflammatory response) as brought about by serum sickness, sterile abscess and vaccination. Based on these observations it is suggested that [131I]-PVP may provide a convenient technique for the long-term monitoring of the activity of reticuloendothelial cells, presumably mainly that of the histiocytes. The pronounced polydispersity of commercially available [131I]-PVP is a serious problem in this respect which can be largely overcome, but not completely abolished, by the screening techniques described herein. Post-mortem analyses of rabbit tissues showed most of the [131I]-PVP to be present in the skin (20%), followed by the liver (14%), bone marrow (10%), muscle (7%) and kidney (5%). Gel filtration studies with [131]-PVP in the presence and in the absence of plasma proteins failed to demonstrate any association between PVP and the proteins. [131I]-PVP kept at physiological pH and 37 degrees C lost less than 5% of its radioactivity over one month due to spontaneous deiodination.
Ancrod↗
The effect of heparin on the affinity chromatography of plasminogen. Demonstration of heparin-plasminogen interaction.
Evidence is presented that heparin binds rabbit plasminogen types I and II under affinity chromatographic conditions using the single stage technique earlier described (Hatton, M.W.C. and Regoeczi, E. (1974) Biochim. Biophys. Acta 359, 55-65). Thus, the affinity of types I and II for Sepharose-lysine is markedly increased in the presence of heparin and elution by epsilon-aminohexanoic acid requires a steeper gradient to recover the plasminogen types. Furthermore by adding sufficient epsilon-aminohexanoic acid to non-heparinised plasma to suppress plasminogen affinity, the presence of heparin is shown to encourage binding of plasminogen (type II more so than type I) to the gel. However, the heparin effect is quickly reversed by washing the column with 0.5 M NaCl prior to elution by epsilon-aminohexanoic acid. No evidence of a stable plasminogen-heparin complex has been found from gel filtration studies and any interaction between plasminogen and heparin probably only takes place when heparin is bound to an affinity site. Studies with 35-S-labelled heparin have shown the mucopolysaccharide to bind to the free amino group of Sepharose-lysine and Sepharose-cadaverine and to be displaced by 0.5 M NaCl elution but not by 0.1 M epsilon-aminohexanoic acid. The plasminogen types produced from heparinised plasma are free from heparin and closely resemble preparations from non-heparinised plasma when compared by polyacrylamide gel electrophoresis, Sephadex gel filtration and arginine esterase activity after urokinase activation.
Aminocaproates↗
Carbohydrate-mediated elimination of avian plasma glycoprotein in mammals.
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Animals↗
The relevance of the structure of lysine bound to Sepharose for the affinity of rabbit plasminogen;.
The features of the structure of lysine, linked to Sepharose by the alpha-amino group, which are important for affinity chromatography of rabbit plasminogem were studied. Nine lysine and lysine-like conjugates, including epsilon-aminohexanoic acid DL-norleucine, DL-alpha-aminoadipic acid, DL-alpha-epsilon-diaminopimelic acid, cadaverine L-ornithine, L-arginine and D-lysine, were prepared; Using labelled rabbit plasminogen added to plasma, the ability of each conjugate to absorb plasminogen and separate the allomeric forms, type I and type II, during epsilon-aminohexanoic acid gradient elution was compared to Sepharose-L-lysine. Plasminogen had no affinity for Sepharose-epsilon-aminohexanoic acid, and was only weakly attracted by Sepharose-norleucine, Sepharose-cadaverine and others. Sepharose-ornithine held a greater attraction to the protein but the strongest binding was obtained with Sepharose-arginine. The affinity of plasminogen type I was always less than type II for the Sepharose-lysine analogues and the recovery of type II greater than type I from Sepharose-ornithine and Sepharose-arginine. Plasminogen affinity was in the order of Sepharose-arginine greater than Sepharose-lysine greater than Sepharose-ornithine. However, because of the present difficulty in recovering plasminogen from Sepharose-arginine the use of Sepharose-lysine in the affinity chromatography of rabbit plasminogen remains unchallenged. It is concluded that binding of rabbit plasminogen to conjugates of lysine and its analogues is determined by the presence of both a free carboxyl and a free amino group and that the distance between these groups is critical.
Animals↗
Studies of the metabolism of asialotransferrins: the metabolic heterogeneity of human asialotransferrin.
Catabolism of human transferrin and human asialotransferrin was simultaneously studied in guinea pigs by means of total body radioactivity measurements. Total body activity representing transferrin decreased at a constant rate with an average half-life of 88 h. Decrease of the total body activity representing asialotransferrin exhibited at least two rates; the half-life of the fast initial component averaged at 25 h, whereas the half-life of the slower component averaged at 55 h. Transition occurred between the 50th and 80th hours of the experiments. The complex character of the elimination curves could not be explained by differences in the iron content of asialotransferrin, by the presence of transferrin variants or of denatured protein in the injected material, by residual sialic acid in the preparations, by accumulation of radioactive terminal catabolic products in the body, by an association of asialotransferrin with any other macromolecular plasma constituent, by changing conditions for mass action, or by a continuing return of labeled protein from the extravascular space. Injection of bovine asialotransferrin into guinea pigs did not result in complex total body curves. Analyses of guinea pig tissues demonstrated that human asialotransferrin had marked affinity for the liver and none for the kidney, lung, or spleen. These observations are consistent with the hypothesis that the glycopeptides in human transferrin are heterogeneous in that removal of the sialyl residues exposes structures with different affinities for the hepatic asialoglycoprotein receptor. The precise chemical basis for the metabolic heterogeneity is unknown.
Animals↗
Studies of the metabolism of asialotrasferrins: the in vivo behavior of baboon and rhesus asialotransferrins.
The catabolism and distribution of rhesus and baboon asialotransferrins relative to the corresponding parent proteins were studied in rabbits using a dual isotope tracer technique. Also a similar study with the baboon proteins in a baboon is reported. The metabolic data obtained in rabbits with both rhesus and baboon transferrins was close to the values established in a previous study for rabbit transferrin. Desialylation resulted in an average increase in the fractional catabolic rate of rhesus trasferrin by 22.7%. This change is similar to that found earlier with asialotransferrins from several nonprimate mammals which are thought not to interact with the hepatic asialoglyco-protein receptor. Two kinetically distinct fractions were identified in baboon asialotransferrin. One of these, amounting to approximately one-third of the protein, was eliminated from the circulation very rapidly. The remaining two-thirds constituted a slowly catabolized fraction which behaved in vivo similarly to rhesus asialotransferrin. Unlike the rapidly cleared fraction, elimination of the slowly catabolized fraction in baboon asialotransferrin is probably not mediated by the hepatic asialoglycoprotein receptor. An amount comparable to the rapidly eliminated fraction in baboon asialotransferrin was recovered with the liver of rats in short-term experiments. In rats which were preinjected with chicken acid alpha1-glycoprotein the hepatic uptake of baboon asialotransferrin was markedly reduced. Data obtained in the baboon agreed with the findings in rabbits, although transferrin turnover was slower in the baboon. From its behavior in vivo as an asialoglycoprotein, baboon transferrin shows greater resemblance to human transferrin than rhesus transferrin. The conclusion is supported by carbohydrate analyses which show an intermediate position for baboon transferrin between man and a nonprimate mammal (rabbit), and a similarity between rhesus and rabbit transferrins.
Animals↗
Mechanisms for elevated fibrin/fibrinogen degradation products in acute experimental pulmonary embolism.
The mechanism and significance of elevated levels of serum fibrin degradation products (FDP) in pulmonary embolism were investigated experimentally. Dogs were embolized with autologous blood clot-incorporating canine 125I-fibrin and were infused with either saline, heparin, or streptokinase. Serial measurements were made of total FDP by hemagglutination inhibition assay and of radioactive FDP. After saline, the peak level of total FDP was 323 mug/ml, but radioactive FDP was only 8 mug/ml. After heparin, these values were 44 and 11 mug/ml, respectively, and after streptokinase, 415 and 20 mug/ml. The results suggest that under these experimental conditions the elevated levels of FDP in pulmonary embolism are derived mainly from lysis of fibrin deposited after embolization rather than from lysis of the original embolus. Heparin inhibits both fibrin deposition and elevation of FDP levels after embolism.
Animals↗