Antithrombin III inhibition of the reaction to thrombin excitation of the anticoagulation system

It has been established that intravenous administration of alpha-thrombin-antithrombin III preparations (1 mkM) has practically no effect on anticoagulation parameters (thrombin time, additive fibrinolytic activity, nonenzymatic fibrinolysis and nonenzymatic fibrinolytic activity). Administration of 1 mkM of alpha-thrombin caused a statistically significant increase of all the parameters. The experiments on perfusion of the humorally isolated sinocarotid area of the rabbit with alpha-thrombin-antithrombin III preparations (1.25 mkM) showed no changes peculiar to the induction of anticoagulation response with thrombin. It is concluded that antithrombin III blocks the ability of thrombin to activate anticoagulation system function.     

Mast cell population in rats with experimental atherosclerosis

Mast cell population was studied in rats with experimental atherosclerosis. It has been established that animals kept for 8 months on atherogenic diet revealed marked changes in mast cell population. Predominance of light cells and cell defects were noted. Heparin saturation index was reduced (0.35), as compared to the control (3.9). Stimulation of anticoagulation system by DIP-alpha-thrombin in such animals revealed no heparin in the blood. Mast cell subpopulation was characterized by light cell predominance and low heparin saturation index. The nature of cell degradation remained unchanged. The data obtained indicate the defects in mast cell pool in animals with experimental atherosclerosis.     

Prevention of intravascular blood coagulation in rats by DIP-alpha-thrombin administration

The possibility of prevention of intravascular blood coagulation in rats by DIP-alpha-thrombin devoid of proteolytic activity and capable of stimulating the reaction of anticoagulation system was studied. The injection of lethal thromboplastin dose was shown to produce a sharp increase in soluble fibrin blood content, total disappearance of fibrinolytic activity and intravascular blood coagulation. The animals died of thrombosis in 90% of cases. It was established that the injection of lethal thromboplastin dose 5 min after DIP-alpha-thrombin injection caused a 13% lethality from thrombosis. No reliable changes in fibrinolytic activity and soluble fibrin content were observed. A significant increase in thrombin and recalcification time was recorded. It is suggested that DIP-alpha-thrombin prevents intravascular blood coagulation induced by lethal thromboplastin dose due to mobilization of the reserve capacities of neuro-humoral anticoagulation system.     

Heparin secretion by mast cells as an index of the status of the anticoagulant system

The status of the mast cell population was studied and compared after administration of trypsin or alpha-thrombin in similar molar concentrations. Morphometry disclosed a substantial shift of the mast cell population towards light, heparin-free cells within one minute after alpha-thrombin administration. The index of mast cell saturation with heparin dropped below 1. The maximal heparin secretion was observed at the 5th minute of experiment. The morphometric criteria of the mast cell population returned to basal level in 120 minutes. These data along with a significant increase in the level of complex heparin compounds and plasma thrombin time indicate heparin release as a result of the effector action of the anticoagulation system. No changes were observed in the activity of complex heparin compounds and in thrombin time after intravenous injection of trypsin. It is suggested that high heparin secretion by mast cells may serve as criterion of the active status of the anticoagulation system.     

Degradation of mono-oleoylglycerol, trioleoylglycerol and phosphatidylcholine in emulsions and lipoproteins by rat hepatic acylglycerol lipase.

The purpose of this study was to characterize the lipolytic activities released by heparin from rat livers. Heparin perfusates of rat livers degraded monooleoylglycerol, trioleoylglycerol and phosphatidylcholine in emulsions as well as in chylomicrons, chylomicron remnants, low-density lipoprotein/high-density lipoprotein-1 (LDL/HDL-1) and high-density lipoprotein-2 (HDL-2). The preferred substrate was mono-oleoylglycerol. Heparin perfusates were separated by chromatography on either heparin-Sepharose or N-desulphated, N-acetylated heparin-Sepharose into at least two related lipases which differed in their ability to hydrolyse HDL-2 phosphatidylcholine, but not in their ability to degrade mono-oleoylglycerol, trioleoylglycerol and phosphatidylcholine in emulsions. The sodium dodecyl sulphate (SDS)/polyacrylamide-gel-electrophoretic patterns of heparin perfusates purified on either normal or N-desulphated N-acetylated heparin-Sepharose were the same, despite differences in their ability to degrade HDL-2 phosphatidylcholine. There was a single band of Mr 56000 without 2-mercaptoethanol in the SDS disruption buffer and three major bands, of Mr 62000, 59000 and 56000, with 2-mercaptoethanol present. When mono-oleoylglycerol lipase was purified 161-fold, there was a concomitant enrichment of the Mr-56000 protein.     

Perfusion of the septum of the rabbit with vasopressin antiserum enhances endotoxin fever

The septal region of the brains of conscious, adult, male New Zealand White rabbits were perfused by means of a push-pull system before and after an intravenous administration of bacterial pyrogen extracted from Salmonella abortus equi. Perfusion of the septal area with sucrose solution (260 mM) had no significant effect on the resulting fever (1.13 +/- 0.09 degrees C) when compared to a control fever without the push-pull perfusion (1.06 +/- 0.12 degrees C). Arginine vasopressin (AVP) added to the perfusing solution (20 micrograms/ml) caused a significant attenuation of the fever (0.81 +/- 0.20 degrees C). An antiserum specific to AVP when added to the perfusing solution resulted in a fever which was significantly greater (2.38 +/- 0.13 degrees C) than the control. Radioimmunoassay of perfusates collected from the control perfusions before and during fever showed that, as the body temperature rose in response to the pyrogen, the level of AVP in the perfusate collected from the septal area decreased. These results provide further evidence that AVP may act in the septal area of the brain to modulate the febrile response.     

Modulation of the platelet aggregation capacity by modified forms of thrombin

It was found that human platelets possess a high sensitivity towards alpha-thrombin (Km = 2 nM). Modified thrombin forms (beta/gamma-thrombin) with an impaired recognition site of high molecular weight substrates and DIP-alpha-thrombin and trypsin are incapable of inducing platelet aggregation when taken at concentrations corresponding to effective concentrations of alpha-thrombin. Beta/gamma-Thrombin and trypsin, unlike DIP-alpha-thrombin, cause platelet aggregation at concentrations of 100-200 nM. Studies on the modulating effects of modified thrombin forms, alpha-thrombin and trypsin, on platelet aggregation induced by alpha-thrombin revealed that beta/gamma-thrombin, alpha-thrombin and trypsin at concentrations causing no cell aggregation potentiate the platelet response after 2 min incubation and inhibit platelet aggregation upon prolonged (15 min) incubation. However, DIP-alpha-thrombin, irrespective of the incubation time (up to 30 min) increased the sensitivity of platelets to alpha-thrombin-induced aggregation. The activating effect of DIP-alpha-thrombin is characterized by an equilibrium constant (KA) of 17 nM. The experimental data confirm the hypothesis that the necessary prerequisite for an adequate physiological response of platelets to alpha-thrombin is the maintenance in the thrombin molecule of an intact active center and a recognition site for high molecular weight substrates. The specificity of thrombin as a potent platelet aggregation inducer is determined by the recognition site for high molecular weight substrates.     

Complex Compound of Heparin and Glutamic Acid: Synthesis and Effect on Hemostatic Indices in vitro and in vivo

A complex compound of high-molecular heparin and glutamic acid has been synthesized. This compound has anticoagulant, fibrinolytic, and antithrombotic properties in vitro. Single intravenous or chronic peroral introduction of the complex in normal animals imitates the activation of anticoagulation system. When the anticoagulation system in depressed, the complex restores or even activates the anticoagulant–fibrinolytic background of the serum it. The obtained data are discussed in physiological terms.     

Metabolism of two forms of apolipoprotein B of VLDL by rat liver

Apolipoprotein B (apoB) is composed of metabolically distinct fractions of higher molecular weight (apoBh) and lower molecular weight (apoBl). When 125I-labeled very low density lipoprotein (VLDL) prepared from recirculating liver perfusates was injected into rats, labeled apoBl was preferentially removed from the plasma and apoBh entered low density lipoprotein (LDL). The time-related movement of labeled apoBh into higher density fractions was independent of that of labeled apoBl. When 125I-labeled triglyceride-rich lipoprotein (TRL) prepared from sucrose-fed rats was incubated with plasma from rats injected with heparin and then studied in a recirculating liver perfusion, apoBl was preferentially removed compared to apoBh. Thus, the loss of apoBl of hepatic VLDL in vivo was similar to the loss of apoBl of lipase-treated TRL in vitro. In control perfusions where TRL was incubated with heat-treated postheparin plasma, not only was there less initial hepatic clearance of apoB but the early phase of preferential apoBl removal during 30 min of perfusion was not observed. ApoE removal from perfusates was the same whether or not the TRL had been treated with heparin-releasable lipases. Apoprotein degradation, as indicated by the appearance in the perfusate of labeled degradation products, occurred 30 min after the preferential phase of apoBl removal. These results suggest that hepatic clearance of VLDL and TRL remnants is favored by lipolysis and by the presence of apoBl on the particle that enhances their hepatic binding and degradation.     

Antidiabetic and Anticoagulant Properties of Heparin–Glutamic Acid Complex

Natural heparin complexes proved to activate the anticoagulation system. The obtained experimental data convincingly confirm that glutamic acid alone, and particularly in a complex with heparin, has a considerable preventive potential and efficiently protects experimental animals with induced diabetes mellitus.     

Alpha-thrombin stimulation of heparin secretion by the peritoneal mast cells in rats

The interaction of alpha-thrombin with connective tissue-type mast cells (CTMC) purified by Ficoll density gradient centrifugation has been examined. It was demonstrated that exposure of CTMC to polymixin (widely used histamine liberator) (3 mg/ml) induced the release of heparin and histamine. Exposure of CTMC to 10(-11) M alpha-thrombin resulted in increase of heparin secretion by 75.5% in relation to basal level. CTMC which were stimulated by very low concentrations of alpha-thrombin (10(-11)-10(-8) M) can release high level of heparin, but not histamine. We have a suggestion that the thrombin specificity is connected with the additional recognition binding site for high molecular substrates (HMS) distinct from the active centre. Unlike alpha-thrombin which has both the active centre and the recognition site for HMS, beta/gamma-thrombin with catalytic activity but with disrupted recognition site induced the heparin release from mast cells only at higher concentrations than alpha-thrombin. It was revealed that DIP-alpha-thrombin without proteolytic activity was unable to activate mast cells in contrast to alpha-thrombin. We consider that alpha-thrombin induced release of heparin by CTMC account for proteolytic and hormone-like activity enzyme by means of both the active centre and the additional recognition site for HMS.     

Decreased sensitivity to the hypoglycemic action of insulin in animals with a lowered blood heparin concentration

It has been shown that in animals with reduced heparin blood concentration the hypoglycemic insulin action was considerably low. This was established for rats with alloxan diabetes and ageing rats with depressed anticoagulation system and atherosclerosis enhanced by prolonged atherogenic diet. With insulin injection (0.2-0.3 U/200 g), blood sugar concentration in such animals was 2-2.5 times lower than in normal ones. The compensation of endogenous heparin deficiency by an intravenous injection of heparin normalizes the reaction of animals to exogenous insulin.     

Structure of the endothelium of the thoracic and abdominal aorta of intact rats studied by various methods of preparation and handling of the specimens

By means of scanning and transmissive electron microscopy structural peculiarities of endothelium of the thoracic and abdominal parts of the intact rat aorta have been studied at various regimens of preparation and making specimens . The greatest changes endotheliocytes (EC) undergo at using immersion fixation after dissection of the aortal segments. These changes are less pronounced at immersion fixation in situ. A decreased perfusion pressure results in appearance of intimal folds and microfolds on the surface of EC. Increasing time for washing more than 1 min results in appearance of inflations and craters on the surfice of EC. For analysis by means of transmissive electron microscopy it is not necessary to remove blood completely out of the vascular bed. The most essential factor is to maintain perfusion pressure at the average systolic level in the given area of the vessel. However, to make the analysis by means of scanning electron microscopy this method is not suitable. The most optimal condition for initial stages of preparing vessels for morphological investigation is their washing for 1 min in the medium 199 with addition of heparin (10 units/ml) during no more than 1 min with a subsequent perfusive fixation in 2.5% solution of glutaraldehyde in the medium 199 no less than 5 min under the average arterial pressure in the given area of the vessel.     

ON THE ELIMINATION OF CENTRALLY FORMED 5-HYDROXYINDOLEACETIC ACID BY CEREBROSPINAL FLUID AND URINE

Abstract— In rats, the release of centrally formed 5-hydroxyindoleacetic acid (5-HIAA) into brain and spinal cord perfusates and urine was measured. Data from spinal cord perfusion of anaesthetized rats indicate that more than about 36% of the spinal production (122ng/h) of 5-HIAA is eliminated via the cerebrospinal fluid (CSF). More than 30% of cerebrally formed 5-HIAA (265.0 ng/h) was calculated to be released into ventricular-cisternal perfusates. Of the total amount of 5-HIAA found in the urine we estimated that about 8% originates in the central nervous system (CNS).
In probenecid treated animals there was a substantial increase in the outflow of 5-HIAA in both perfusion systems. In the combined perfusion experiments no proportional increase of the cerebral contribution to the cisternal outflow was found after probenecid. Our data indicate that a significant proportion of centrally formed 5-HIAA is eliminated by the CSF. No evidence was found for an increased contribution of cerebral 5-HIAA to lumbar CSF after application of the probenecid test. Urinary levels of 5-HIAA do not reflect quantitatively central 5-hydroxytryptamine metabolism.     

Hormonal mediation of rat heart lipoprotein lipase activity after fat feeding

The effect of acute fat feeding on the response of two fractions of lipoprotein lipase in heart was explored. In rats, previously fasted, lipoprotein lipase activity released into the perfusate by heparin increased approximately 50% 4 h after fat feeding. The lipase activity remaining in the heart tissue after heparin perfusion showed no significant difference. When rats maintained ad libitum were intubated with glucose 2 h before the fat dose, a relatively larger increase (5-10-fold) in the heparin-releasable lipase activity was observed. The capacity of these hearts to hydrolyze 14C-labeled chylomicrons was also increased 4-5-fold over the controls. Fat ingestion has been reported to elevated plasma corticosteroid levels in rats. When adrenalectomized rats were fed fat, no significant changes in the heparin-releasable lipase activity were observed Hydrocortisone and corticotropin treatment increased the heparin-releasable lipase activity to the same degree as observed with fat feeding. These data suggest that the increase in heart lipoprotein lipase activity following fat feeding is mediated via corticosteroids.     

I. Determination of the endogenous and evoked release of catecholamines from the hypothalamus and caudate nucleus of the conscious and unrestrained rat

The action of cathecholamines within the CNS is important for the expression of numerous vegetative and behavioral functions. To understand the role these amines play, it is necessary to measure changes in the levels of these transmitter substances by utilizing new developments and methodology in the behaving animal. Utilizing new developments in methodology, it is possible to measure the release of amines into perfusates obtained from specific sites in the brain of the rat under basal and evoked conditions without prior purification or concentration.Using the push-pull perfusion technique, perfusates were obtained from the hypothalamus and caudate nucleus and analyzed by liquid chromatography with electrochemical detection. It is possible to readily determine basal release of dopamine from the caudate nucleus. Detection of both dopamine and noradrenaline is possible under ephedrine stimulated conditions from both the caudate nucleus and the hypothalamus. Although levels of serotonin (5-HT) were detected in brain perfusates, it may not be of neuronal origin. It may be possible to use these techniques to delineate the roles these amines play in various physiological functions.     

An in vitro evaluation of inflammation response of titanium functionalized with heparin/fibronectin complex

Immobilization of biomolecules with a variety of biological functions has been a promising method to improve the biocompatibility of biomaterials. However, little is known about their inflammatory property and cytotoxicity, which are both key aspects to most biomaterials designed for tissue engineering applications and in vivo implantation. In this in vitro study, heparin/fibronectin complex (Hep/Fn) was coimmobilized onto titanium surface (HF-Ti), which had been proven to have the properties of both anticoagulation and endothelialization in our previous study. Fourier transform infrared (FTIR) spectroscopy and water contact angle measurement were utilized to determine the surface chemical compositions and physical properties. Toluidine Blue O (TBO) and immunochemistry methods were performed to quantify the surface-immobilized heparin and fibronectin. The early inflammatory responses elicited by pristine Ti and HF-Ti were investigated by proinflammatory cytokine secretion of tumor necrosis factor-alpha (TNF-α) released by attached peritoneal macrophages, monocyte chemoattractant protein-1 (MCP-1) and interleukin-1β (IL-1β) released by attached human umbilical vein endothelial cells (ECs), respectively. Scanning electronic microscopy (SEM) and immunofluorescence were employed to investigate the changes in macrophages and ECs morphologies. The incubation period for both cells was 24 h and the results showed that HF-Ti revealed a weaker inflammatory response than pristine Ti, which provoked a stronger inflammatory response and higher activation of macrophages. Our data suggest that Hep/Fn coimmobilized biomaterials surface may develop to be a new generation of biomaterials with both biocompatibility and anti-inflammatory properties, especially for used as cardiovascular implants and in tissue engineering applications.     

Neurochemical changes associated with schedule-controlled behavior.

Studies of monoamine metabolites within the cerebrospinal fluid compartment have indicated that this approach may be useful in examining central metabolic changes in vivo. By combining the technologies of radioisotope chemistry, operant behavior control and modification, and brain perfusion with push-pull cannulas, we have been able to examine minute to minute changes in the disposition of radiolabeled monoamine transmitter candidates and their metabolites. These substances appear to co-vary with changes in complex behavior maintained by operant schedules of reinforcement and affected by changes in schedules or administration of psychotropic drugs. In agreement with other perfusion studies, we have observed changes in fractional distribution of radiolabeled urea, a so-called extracellular marker, along with shifts in monoamines; but the former appear more transient. These observations nevertheless support the concept of dynamic changes within the extracellular environment of the CNS that may be part of a hormone-like communicating system with functional significance. Furthermore, the presence of peaks and/or troughs, in perfusates of [14C]urea of similar substances should not be taken as a priori evidence for nonspecificity of the technic, since selective release or inhibition of release of monoamines can be shown with appropriate drugs that are thought to act through these aminergic systems. Destruction of catecholamine nerve terminals with 6-hydroxydopamine likewise attenuates the signal-locked release of radiolabeled norepinephrine by a conditioned stimulus after conditioning occurs. No such release is seen on presentation of the to-be-conditioned neutral stimulus in control or 6-hydroxydopamine treated rats. These initial studies indicate the availability of a powerful tool for the study of drug-neurochemical-behavioral interactions using subjects as their own controls for extended periods of time so that phenomena of plasticity, tolerance and dependence may likewise be examined.     

Cryoprotective concentrations of polyethyleneoxide exhibit low toxicity toward the isolated perfused rat liver

Isolated perfused rat livers were exposed for 30 min at 35 °C to 10 and 15% (v/v) solutions of polyethyleneoxide with a mean molecular weight of 400. A dual-circuit perfusion system was employed to ensure efficient removal of the polyethyleneoxide from the liver.Bile production and urea synthesis by the livers was depressed during exposure to polyethyleneoxide but resumed within 30 min after its removal. The ability of the livers to maintain a constant concentration of glucose in perfusates and their retention of both potassium and aspartate aminotransferase were also altered after exposure to the cryoprotectant.Polyethyleneoxide at 10% (v/v) was considered to be relatively nontoxic toward the isolated rat liver and it therefore shows promise as a cryoprotective compound which may allow long-term storage of the liver at subzero temperatures.     

Anticoagulation in large-volume leukapheresis: comparison between citrate- versus heparin-based anticoagulation on safety and CD34 (+) cell collection efficiency

Background aimsLittle is known of the effect of anticoagulation on peripheral blood progenitor cell (PBPC) harvest during large-volume leukapheresis (LVL). Because of the interaction of heparin with stromal cell-derived factor (SDF)-1α, it has been proposed that a heparin-based anticoagulation may result in an increased PBPC collection efficiency compared with standard citrate-based anticoagulation.MethodsWe conducted a prospective randomized trial to address the effect of both anticoagulation regimes on safety, subjective comfort and CD34 ⁺ collection efficiency in 90 adult patients undergoing standardized LVL. Anticoagulation consisted of either citrate (group C) or a combination of heparin and low-dose citrate (group H).ResultsThe overall incidence of adverse reactions (AR) during LVL was 17%. AR consisted only of citrate-related AR; no bleeding complications were observed. Determination of parameters of the acid–base balance revealed a higher frequency of metabolic alkalosis in group C. Analysis of serum SDF-1α revealed no differences in SDF-1α plasma levels. There were no differences in the CD34 ⁺ cell collection efficiency, resulting in the harvest of equal CD34 ⁺ cell yields independent of the anticoagulation used.ConclusionsOur data show no clinical relevant effect of a heparin containing anticoagulation in terms of an increased overall CD34 ⁺ cell collection during LVL, although this regime shows some benefits in terms of the incidence and subjective tolerance towards AR. Based on our results the decision between a citrate- and heparin-substituted anticoagulation for LVL should be driven by patient-related factors, and should concern potential contraindications of both methods.