High Concentrations Of Heparin Research Articles

Inhibition of cell proliferation and protease activity by cartilage factors and heparin.

Proliferating rat smooth muscle cells and fibroblasts have membrane-associated protease activity. High concentrations of heparin inhibited membrane-associated protease activity and cell proliferation, while low concentration of heparin promoted smooth muscle cell proliferation. The inhibition of protease activity and proliferation was abolished when heparin was treated with protamine sulfate or when acid treated fetal calf serum was used. Heparin required the presence of an acid labile factor(s) in serum for the inhibition of protease activity and proliferation. Heparin and antithrombin III in the presence of acid-treated fetal calf serum did not inhibit cell proliferation or protease activity. Cartilage factors isolated from bovine nasal cartilage containing trypsin inhibitory activity, but not papain inhibitory activity, inhibited rat smooth muscle and fibroblast proliferation and surface associated protease activity. The cartilage factors did not require acid-labile components in the fetal calf serum for the inhibitory activity. The inhibitory activity due to heparin and cartilage factors was not permanent under our experimental condition. Protein synthesis was not inhibited by heparin or the cartilage factors. In rat smooth muscle cells and fibroblasts, the expression of surface-associated protease activity was related to the proliferative state of the cells. Surface protease activity was only present on proliferating cells. When surface protease activity was inhibited by high concentrations of heparin in the presence of an acid-labile serum component(s) or cartilage factors, cell proliferation was also inhibited.

Reactivity of a Hereditary Abnormal Antithrombin III Fraction in the Inhibition of Thrombin and Factor Xa

Two different AT-III fractions were purified from the plasma of a patient with recurrent superficial thrombophlebitis. The abnormal AT-III fraction (A-AT) was compared to the normal AT-III fraction (N-AT) in the inhibition of thrombin and factor Xa. Without heparin, both inactivate proteases in a similar manner and at the same rate. However, at low heparin concentration the thrombin inhibition proceeds more slowly with A-AT than with N-AT. At high heparin concentration the difference between A-AT and N-AT becomes very small. The inhibition of factor Xa follows a similar pattern. It is suggested that the heparin binding site of A-AT differs from that of N-AT resulting in a decreased heparin cofactor activity.

Heparin-antithrombin III binding. In vitro and in vivo studies.

Heparin antithrombin III binding was studied by crossed immunoelectrophoresis. In plasma and purified antithrombin III standard, multicomponent patterns were obtained with low concentrations of mucosal heparin. There is evidence that antithrombin III may bind more than one heparin molecule. At high heparin concentration (greater than 16 U/ml), single symmetrical peaks were obtained. Serum samples showed two antithrombin III peaks due to a decreased heparin binding of the slower peak (2.1-3.9 times), which was probably antithrombin III-activated procoagulant complexes. Heparin analogue (A 73025) also bound antithrombin III in vitro but the mobility of the peak was slower than with mucosal heparin and only a single peak was obtained in serum samples. Radioimmunoassay showed a decreased binding of antithrombin III antibody to heparin-antithrombin III complex. Venous occlusion to the forearm resulted in a slow second peak in the plasma. Heparin therapy gave rise to a double peak in the plasma antithrombin III profile and with continuous infusion, quantitative decreases were noted in all subjects studied, two of whom rethrombosed at the end of 7 days therapy.

Interactions between Human Plasma Proteins and Heparin-Poly(Methyl Methacrylate) Copolymer

A solid Heparin-PMMA copolymer has been synthetized by a radical polymerization of methyl methacrylate from oxidative reaction initiated by Ce4+ ions in the presence of heparin. Covalently linked heparin was 10% of copolymer weight. The antithrombin activity of the copolymer corresponded to 1% of grafted heparin. PMMA sequence of the copolymer played the leading role in fibrinogen, immunoglobulins, transferrin and albumin adsorption. These proteins adsorbed on the copolymer, showed different competitive desorption pattern in the presence of whole plasma: fibrinogen presented the highest degree of affinity for the copolymer. The heparin part of the copolymer was responsible for antithrombin III adsorption and for decrease of factor V activity. Active antithrombin III was eluted. An inactivation of factor V in plasma was observed using high concentrations of soluble heparin. This result suggested that copolymer heparin chains, even devoid of antithrombin activity were involved in this inactivation. With Heparin-PMMA copolymer, plasma clotting pro-enzymes behaved differently than on heparin-sepharose copolymer:disappearance of factor XI activity, decrease in prekallikrein activity and activation of factor IX were observed. PMMA sequences were responsible for factor IX activation.

Nitroblue tetrazolium test in neonates

THE NITROBLUE TETRAZOLIUM test was introduced by Parks et al 1 in 1968 as an aid in the diagnosis of acute bacterial infection. Its use is based on the observation that neutrophil reduction of NBT to an intracellular formazan precipitate is increased under conditions of bacterial infection. The test has had limited use in the diagnosis of neonatal sepsis because of spuriously high values found in normal infants. ~ Barker and Farnes 3 observed that falsely high and widely varied NBT values were obtained in adults if high concentrations of heparin were used. Using a micromethod, they further showed that limiting heparin concentration to 1 unit /ml gave normal NBT values of less than 5% in adults and older children. ~ We hypothesized that if the heparin concentration in the NBT solution were similarly controlled, normal values may be defined in newborn subjects, making the test available for future clinical testing in such situations as neonatal sepsis.

The effects of various inhibitors on platelet aggregation induced by polylysine

Cationic polymers such as polylysine, aggregate platelets by reducing the negative surface charge and forming bridges between the adjacent platelet membranes. A controversy however, has arisen about the question whether it can elicit the release reaction.In the present study, three types of polylysines were tested in platelet rich plasma (PRP) and in washed platelet suspension (WPS) for the ability to cause aggregation and release reaction. We also studied the effects of various inhibitors including negative charged acid mucopolysaccharide, heparin.All three types of polylysine induced platelet aggregation which showed biphasic aggregation curve in PRP but showed no secondary curve of aggregation in WPS. At the same concentration of polylysine, maximum aggregation was slightly reduced in both system when the lower molecular one was used. Various inhibitors such as NEM, adenosine, aspirin, PGE1, completely inhibited the aggregation in PRP but had no effect on the aggregation in WPS. Heparin inhibited the both aggregation in PRP and WPS and higher concentration of heparin was needed to inhibit aggregation when the larger molecular one of polylysine was used.Polylysine caused release reaction from the 14C-5HT labeled platelets in PRP but could not elicit release reaction in WPS. Although the small amount of radioactivity was detected in surrounding medium in WPS, these were probably due to the increased permeability of membrane because the same amount of radioactivity was also detected when the various inhibitors were added.Electron microscopic observation of aggregated platelets in PRP and WPS showed the evidence supporting the result of the presence or absence of release reaction in two systems assessed by measuring the radioactivity.Polylysine aggregates platelets by reducing the negative surface charge of platelets and can elicit release reaction in the presence of Ca++ or plasma. The aggregation in PRP and WPS was inhibited by heparin probably due to neutralizing the positive charge of polylysine. In contrast to in WPS, inhibitors other than heparin inhibited the first wave of aggregation in PRP but this mechanism remains unexplained.The average distance between plasma membrane of the aggregated platelets in WPS did not vary with the degree of polymerization of these polylysine. In this respect, platelet aggregation differs from aggregation of red blood cells by polylysine in which widening of the intercellular space were observed by others with an increase in molecular size.

Heparin resistance and decreased hepatic triglyceride hydrolase release during long-term estrogen-progestin treatment

These studies were undertaken to define the mechanism for the depression of postheparin triglyceride hydrolase activity in women treated with estrogen-progestin oral contraceptives. Six treated and six control women were studied. Total, protamine-inhibited, and protamine-resistant triglyceride hydrolase activities were measured after six different intravenous doses of heparin in each subject in order to determine the dose-response relationships for lipase release. As has been reported during short-term treatment with estrogens, long-term treatment with oral contraceptive agents is accompanied by selective depression of protamine-resistant (hepatic) lipase activity. This depression can be partly reversed by the administration of large heparin doses, but maximally releasing heparin doses fail to restore postheparin protamine-resistant activity to control values. These data are compatible with the idea that the releasable pool of hepatic triglyceride hydrolase activity is diminished in women who receive oral contraceptive agents and that the pharmacokinetics of its release are altered in such a way that only relatively high concentrations of heparin displace the enzyme from this pool.

Lipoprotein lipase in cultured heart cells: Characteristics and cellular location

Abstract Lipase activity extracted from cultured neonatal rat heart cells was characterized and identified as lipoprotein lipase. Enzyme activity was stimulated by human apoC-II and rat serum; serum stimulation was prevented by human apoC-I and apoC-III. Lipolysis was maximal at pH 8.0 and was inhibited by protamine sulfate, NaCl and high concentrations of heparin. About 50% of heart cell lipase activity applied to heparin-Sepharose bound to the gel and was eluted with a NaCl gradient. A peak of lipase activity was observed at 0.84 M NaCl. Neonatal rat heart cells in culture are a mixture of muscle and non-muscle cells. To determine the cellular location of the lipoprotein lipase, enzyme activity and muscle cell content of the cultures were determined. Myosin ATPase was used as an index of muscle cell content since ATPase specific activity correlated (r = +0.97) with muscle cell content determined immunofluorescently. When muscle cell content of cultures was decreased or increased by differential plating, lipase specific activity was constant. Moreover, lipase specific activity was constant during culture growth despite a decrease in muscle cell content. It was concluded that lipoprotein lipase activity of cultured heart cells is not associated solely with either muscle or non-muscle cells.

Differential effects of heparin on replicative DNA synthesis and unscheduled DNA synthesis

The effect of heparin on DNA synthesis was compared between replicative DNA synthesis and unscheduled DNA synthesis. Replicative DNA synthesis in permeable cells or nuclei prepared from rapidly growing mouse ascites sarcoma cells was inhibited by heparin. Unscheduled DNA synthesis in nuclei isolated from normal rat liver or from mouse ascites sarcoma cells in stationary phase was stimulated by heparin at low concentrations but inhibited by high heparin concentrations. DNA polymerase activity assayed with activated calf thymus DNA and DNA polymerase α purified partially from mouse ascites sarcoma cells was inhibited with either calf thymus histones or heparin. DNA synthesis inhibited with histones was partially reactivated by heparin. Replicative DNA synthesis in permeable cells was inhibited by adding histones to the assay mixture, and the inhibited DNA synthesis was partially reactivated by low concentrations of heparin. These results indicated that the replicated sites (or replication machinery) in permeable cells or nuclei were largely unrestricted by histones and that heparin inhibition of replicative DNA synthesis was due to the direct inhibitory interaction of heparin with some essential component(s), such as DNA polymerase, of replication machinery.

Electron Microscopy Analysis of the Interaction between Escherichia coli DNA-Dependent RNA Polymerase and the Replicative Form of Phage fd DNA. 2. Analysis of the Dissociation Kinetics

The kinetics of dissociation of the fd DNA - RNA-polymerase complex has been analyzed. Heparin was added to a solution of the enzyme - DNA complex in order to trap free polymerases. At different times after, samples were taken and analyzed by electron microscopy to determine the mean number of enzymes bound per DNA molecule. Unexpectedly, the measured dissociation is not a first-order reaction. The apparent rate constant increases with heparin concentration in the range between 0.001 and 2 mg/ml. These results strongly suggest the existence of a direct transfer process of RNA polymerase to heparin, bypassing the rate-limiting step of dissociation of the enzyme - DNA complex to free enzyme. Theoretical analysis of the direct-transfer model shows that the rate constant of dissociation should level off at high heparin concentrations: measurements of the residual transcription activity show that this is the case. From these experiments, the equilibrium constant of the DNA - RNA-polymerase complex can be determined. The value K = 10(12) M-1 which is obtained solves a striking paradox which existed because measurements performed in other laboratories indicated K = 10(14) M-1, which is greater than the equilibrium constant of the lac-repressor - lac-operator complex (=10(13) M-1).

Lipoprotein lipase in cultured heart cells: Characteristics and cellular location

Lipase activity extracted from cultured neonatal rat heart cells was characterized and identified as lipoprotein lipase. Enzyme activity was stimulated by human apoC-II and rat serum; serum stimulation was prevented by human apoC-I and apoC-III. Lipolysis was maximal at pH 8.0 and was inhibited by protamine sulfate, NaCl and high concentrations of heparin. About 50% of heart cell lipase activity applied to heparin-Sepharose bound to the gel and was eluted with a NaCl gradient. A peak of lipase activity was observed at 0.84 M NaCl. Neonatal rat heart cells in culture are a mixture of muscle and non-muscle cells. To determine the cellular location of the lipoprotein lipase, enzyme activity and muscle cell content of the cultures were determined. Myosin ATPase was used as an index of muscle cell content since ATPase specific activity correlated ( r = +0.97) with muscle cell content determined immunofluorescently. When muscle cell content of cultures was decreased or increased by differential plating, lipase specific activity was constant. Moreover, lipase specific activity was constant during culture growth despite a decrease in muscle cell content. It was concluded that lipoprotein lipase activity of cultured heart cells is not associated solely with either muscle or non-muscle cells.

Spontaneous Platelet Aggregation in Arterial Insufficiency: Mechanisms and Implications

To investigate the clinical implications and mechanisms of spontaneous platelet aggregation (SPA) in man, 150 normal subjects, 22 patient controls and 130 patients with vascular insufficiency were studied. SPA was negative in normal subjects and patient controls whereas it was positive in 36 of 66 (54%) patients with transient ischemic attacks, 6 of 32 (19%) patients with stable angina, 7 of 10 (70%) patients with acute myocardial infarction and 11 of 14 (80%) patients with acute peripheral arterial insufficiency. The SPA was inhibited with aspirin in vivo, and inhibited competitively in vitro by low concentrations of aspirin, 2-chloroadenosine, prostaglandin E1 or apyrase but only by high concentrations of heparin or hirudin. Addition of platelet-poor plasma from patients with positive SPA did not cause normal platelets to aggregate. Treatment of patients who had acute peripheral arterial insufficiency with aspirin and dipyridamole prevented SPA with notable clinical improvement of the ischemic changes.

Interaction of purulent material with antibiotics used to treat Pseudomonas infections.

To define factors contributing to the adverse prognosis of patients with gram-negative bacillemia and abscess formation, we studied the interaction between polymyxin B, colistin sulfate, gentamicin, or carbenicillin with purulent material. Carbenicillin activity was not significantly altered by incubation with pus. Equal volumes of antibiotic and purulent sediment decreased the effective concentration of polymyxin B, colistin sulfate, or gentamicin from 100 mug/ml to 3 to 6 mug/ml. One milliliter of purulent sediment bound more than 700 mug of gentamicin and 1,500 mug of polymyxin B or colistin sulfate. This effect occurred rapidly, proceeded at 4 and 37 C, was stable for 24 to 48 h, and was altered, but not abolished, by varying the pH of the solution. Antibiotic activity could be removed from pus by high concentrations of protamine sulfate, heparin, sodium chloride, or potassium chloride, suggesting binding rather than inactivation.

大動脈壁中の脂質分解酵素について

In order to clarify a part of lipid metabolism in aortic wall, the properties of lipolytic enzymes in the human aortic intima and media have been studied. Human intima and media were homogenized in ice-cold 0.01M phosphate buffer, pH7.0, and the supernatant of homogenate was used as the enzyme source.With cholesterol ester hydrolase, phospholipase and lipase in aortic intima and media, two pH optima of acid and alkaline ranges were consistently observed. In the cases of cholesterol ester hydrolase and lipase, the activities of intimal enzymes were significantly higher than those of medial ones at all pH values. On the contrary, the activity of intimal phospholipase was slightly lower than that of medial one.Comparing with the phospholipases of pancreatic juice, snake venom and starfish, the enzyme of aortic wall was unstable for heat.Alkaline lipases of intima and media were activated by sodium deoxycholate and were slightly inhibited by albumin. On the other hand, both acid lipases were not influenced by these compounds. 1M sodium chloride and protamine sulphate significantly inhibited the activity of the intimal alkaline lipase, but had no effect on the medial alkaline lipase and both the acid lipases. Heparin in low concentration slightly increased the activities of both alkaline lipases, but that of the intimal lipase was inhibited by higher concentration of heparin. These observations lead us to speculate that the main part of the intimal lipase might be lipoprotein lipase, and the medial alkaline lipase might be true lipase. The intimal alkaline lipase had the absolute positional specificity for the primary ester bond of triglyceride. On the contrary, the other lipases had the relative specificities for the primary ester bonds.

Studies of snake venoms on blood coagulation I. The thromboserpentin (thrombin-like) enzyme in the venoms

Abstract An experimental survey was made on the thrombin-like activity of the venoms from forty-three species of all four groups of snakes. The term “thromboserpentin” is introduced for this activity to differentiate it from other endopeptidases contained in snake venoms. Thromboserpentin activity was estimated by the “fibrin time”, denoting the first macroscopic appearance of fibrin strands, and by the gelation time, denoting the appearance of a gel. Thromboserpentin activity was found in most of the Crotalidae group of venoms. Vipera ammodytes venom was the only one in the Viperidae group with thromboserpentin activity, and none was found in the venoms of the Elapidae and Hydrophidae groups of snakes. Heparin and peanut inhibitor were used to characterize the mode of action of the thromboserpentin enzyme. Heat treatment was employed to eliminate other venom containing proteases which might interfere with the gelation time of the fibrinogen-thromboserpentin systems. Rigid fibrin gels were observed in these systems in the presence of high concentrations of heparin. The heparin action on thromboserpentin-fibrinogen systems is discussed in relation to a heparin-thrombin complex and a heparin-fibrin complex. The possibility of the role of heparin on the gelation phase, in addition to its role as an anticoagulant, is discussed in connection with the findings that the fibrin gels induced by the venoms of Agkistrodon mokeson and Lachesis mutus do not dissolve, but remain firm. A close relationship is proposed to exist between heparin and fibrin gel structure, induced either by thrombin or by thromboserpentin. The presented findings of the action of peanut inhibitor suggest that it inhibits specifically thromboserpentin. The application of thromboserpentin is considered useful for studies on the polymerization, gelation and cross-linking of fibrin.

Quantitative Measurement of Ovalbumin Messenger Ribonucleic Acid Activity: LOCALIZATION IN POLYSOMES, INDUCTION BY ESTROGEN, AND EFFECT OF ACTINOMYCIN D

Abstract Procedures are described for the quantitative assay of ovalbumin mRNA in a rabbit reticulocyte lysate protein-synthesizing system. A technique for rapid and efficient separation of labeled immunoprecipitated ovalbumin from nonspecific radioactivity is presented. The capacity of the lysate system for total de novo protein synthesis, determined with the use of aurintricarboxylic acid, was found to be correlated with the capacity for ovalbumin mRNA translation. Hemin, a compound that stimulates hemoglobin synthesis in reticulocyte lysates, also stimulates ovalbumin synthesis. The yield of ovalbumin mRNA activity was used as a criterion in choosing optimal conditions for the isolation of undegraded nucleic acid from oviduct. Phenol extraction of total oviduct homogenate yielded only one-third as much activity as isolation by sodium dodecyl sulfate sucrose gradient. When high concentrations of heparin were used to inhibit ribonuclease, the degradation rate of ovalbumin mRNA activity after homogenization could be slowed enough (half-life = 6.4 hours) to permit nearly quantitative recovery of activity in subcellular fractions. No translation of mRNA fragments could be detected in partially degraded nucleic acid preparations. Seventy-one per cent of the total ovalbumin mRNA activity sedimented with polysomes with only 5% in the 27,000 x g pellet. Administration of estrogen to immature chicks induced ovalbumin mRNA activity, and continued hormone treatment was necessary for the maintenance of activity. In vivo administration of actinomycin D to chicks led to a 65% increase in the rate of ovalbumin synthesis in culture with no change in total or polysomal mRNA levels, providing direct evidence that this drug increases the rate of ovalbumin mRNA translation.

Inhibitory effect of heparin on gentamicin concentrations in blood.

In monitoring gentamicin concentrations in the blood of patients with renal insufficiency, the assayed antibiotic concentration was found to be lower when the sample was drawn as heparinized plasma rather than as serum. This lowering of gentamicin concentrations by heparin was studied further by adding increasing doses of heparin and various amounts of gentamicin to human serum. With a range of 2 to 100 units of heparin per ml, gentamicin concentrations in the serum were lowered by 9 to 14%; with higher heparin concentrations, an even greater and increasing inhibition was noticed, reaching 56% for 1,000 units/ml. This inhibitory effect of heparin on gentamicin was reversible by dilution, indicating that it was not due to degradation or to formation of an inactive chemical complex. Underestimation by the laboratory of gentamicin concentrations in blood is likely to be greatest with capillary tubes, with which the concentration of heparin is especially high. With clinical heparinization, the amount of active heparin in the blood does not exceed 10 units/ml and is for the most part under 3 units/ml; thus, therapeutically significant inhibition of the antibiotic is unlikely in patients receiving anticoagulation.

Dissociation of Hela cells ribosomes by heparin

The present communication describes the dissociation of ribosomes of mammalian origin, induced by high concentrations of heparin. Heparin, like polyvinyl sulfate is a well known inhibitor of RNAse ( Flanagan, 1967). It was systematically used by us in experiments designed to study the variation in the ratio: polysomes — free ribosomes in HeLa cells partially synchronized by thymidine. Recent results with dextran sulfate (Miyazawa & al.), another sulfonated polyanion which depolymerizes ribosomes, prompted us to investigate the effects of heparin.

PARALLEL INHIBITION OF SPONTANEOUS AND ANTIGEN-INDUCED PLATELET HISTAMINE RELEASE.

Small amounts of histamine and serotonin are released spontaneously in vitro from blood cells of normal rabbits. Similar to release induced by antigen-antibody reaction, spontaneous release is calcium dependent but occurs even in the presence of a high concentration of heparin. Spontaneous release is inhibited by tosylarginine and tosylarginine methyl ester, agents which also inhibit antigen-induced release. Benzoylarginine, benzoylarginine methyl ester, lysine ethyl ester, and arginine have little effect on either spontaneous or antigen-induced release. A heat-stable factor in plasma can effect platelet amine release. The results suggest that factors involved early in the coagulation mechanism may be responsible for platelet amine release under these conditions.

THE HEPARIN CO-FACTOR ACTIVITY IN PLASMA AND ITS RELATION TO THE ANTICOAGULANT EFFECT OF INTRAVENOUSLY INJECTED HEPARIN.

AbstractBlombäck, B., M. Blombäck, H. Lagergren and P. Olsson. The heparin co‐factor activity in plasma and its relation to the anticoagulant effect of intravenously injected heparin. Acta physiol. stand. 1963. 58. 306—318. — The heparin co‐factor activity in plasma, defined as the antithrombin activity at high heparin concentrations, was measured with two thrombin‐titration methods in dogs and human subjects following single or repeated intravenous injections of heparin. The co‐factor activity was found to decrease continuously during the period of elimination of heparin from the plasma, after which it rose slowly to the initial level. In dogs, the anticoagulant effect of intravenous heparin was studied at different levels of co‐factor activity in the plasma. The co‐factor activity was increased by infusion of bovine plasma fraction IV—1, prepared according to Cohn. The rise in co‐factor activity was always accompanied by an increase in heparin effect (prolongation of the coagulation time). The co‐factor alone exerted no effect on the coagulation time.