In vivo release of anti-Xa clotting activity by a heparin analogue

The parenteral injection of a semi-synthetic heparin analogue (SSHA) releases anti-Xa clotting activity, lipoprotein lipase activity and PF₄ antigen. The increased anti-Xa activity is not neutralized by PF₄ or protamine sulphate. A second injection of the drug after 90 minutes, or an increase in dose, does not increase the level of induced anti-Xa clotting activity. Possible mechanisms of action include the release by SSHA of endogenous glycosaminoglycans with anti-Xa activity, and interference by released lipoprotein lipase of a modulator of anti-Xa activity. It is concluded that a drug with weak anticoagulant activity in vitro may nevertheless have significant antithrombotic potential.     

In vitro and in vivo studies of the anti-Xa activity of heparin

Several heparin preparations have been assayed using the Denson and Bonnar anti-Xa assay. The heat defibrination step involved in this assay has been shown to introduce discrepancies into the results, due mainly to co-precipitation of the heparin with fibrinogen. The amount of heparin lost is dependant on the molecular weight. The anti-Xa activity of ex vivo samples from subjects given heparin was much more resistant to loss during defibrination than in vitro samples, resulting in artificially high potency estimates. A modified assay has been proposed, omitting the defibrination step. The results provide further evidence that the anti-Xa activity observed after subcutaneous injection of heparin differs from that measured when the drug is added to plasma in vitro.     

A new low molecular weight heparin derivative. In vitro and in vivo studies

We present here the in vitro and in vivo evaluation of a new heparin low molecular weight (LMW) derivative that can be prepared on an industrial scale. The in vitro anti activated factor Xa (anti Xa) activity was 67 per cent that of standard heparin while anti-thrombin and anticoagulant activities were respectively limited to 16 and 10 per cent. The same discrepancy between anti Xa and anticoagulant activity was found after intravenous (IV) or subcutaneous (SC) injection to ten healthy volunteers. From the kinetic of the anti Xa activity disappearance it can be assumed that the mechanism of elimination of LMW heparin is not strongly different from that of traditional heparin.     

In vivo antithrombotic synergy of oral heparin and arginine: endothelial thromboresistance without changes in coagulation parameters

On the basis of suggested clinical efficacy in an uncontrolled study in ninety-seven patients with unstable angina, an animal study was conducted to investigate antithrombotic synergy between orally administered heparin and arginine. A rat venous thrombosis model tested the difference in thrombus formation when heparin (7.5 mg/kg) and arginine (113 mg/kg) were administered, alone or in combination, by stomach tube with a minimum of 20 rats/group. Oral heparin, arginine, and heparin plus arginine reduced thrombus formation by 50%, 75%, and 90%, respectively, when compared to saline administration. Heparin was recovered from endothelium, yet there was little or no observable plasma anticoagulant activity. An orally administered low-molecular-weight anticoagulant glycosaminoglycan mixture, sulodexide (7.5 mg/kg), showed an 88% reduction in stable thrombus formation when administered alone but showed no synergy with oral arginine. A 28-day study with oral sulodexide (2.9 mg/kg) and arginine (43.9 mg/kg), 20 rats/group, showed antithrombotic activity with minimal anticoagulant activity indicating suitability for long term treatment. These findings suggest the endothelial localization of heparin and a synergistic antithrombotic effect for orally administered heparin and arginine.     

Structural features of heparin and their effect on heparin cofactor II mediated inhibition of thrombin

Heparins from different species and tissues show similar levels of ATIII and HCII mediated anti-IIa activities. On fractionation, chains containing predominantly ATIII or HCII activities could not be separated. Oligosaccharide mapping demonstrates that the concentration of an oligosaccharide comprising a portion of heparin's ATIII binding site in a particular heparin fraction correlates with ATIII mediated anti-IIa activity, but does not correlate with HCII mediated anti-IIa activity. These results suggest that ATIII and HCII do not share a common binding site. Partial enzymatic depolymerization of heparin resulted in large oligosaccharides which could be purified and partially characterized. Although oligosaccharides of degree of polymerization (dp) 18 and 20 showed significant ATIII and HCII mediated anti-IIa activities no separation of these activities resulted. These data suggest however that a minimum chain length of dp18 was required for HCII mediated anti-IIa activity.     

In vivo studies on the enhancement of serotonin reuptake by tianeptine

The present study investigates the in vivo effects of the serotonin uptake enhancer tianeptine. The serotonin metabolite, 5-hydroxy-indolacetic acid (5-HIAA) was measured by in vivo voltammetry and carbon fiber electrodes chronically implanted in different brain areas of freely moving rats. Tianeptine (10 mg/kg i.p.) increased extracellular 5-HIAA in the hippocampus and hypothalamus. The interaction between tianeptine and drugs known to interfere with the uptake or release of serotonin (sertraline, buspirone, D-norfenfluramine) was then studied and, to ascertain the in vivo pharmacological relevance of tianeptine's effects, its ability to reduce the serotoninergic syndrome was evaluated. Both the biochemical and behavioral data indicate that in vivo tianeptine's effects on the serotoninergic system are likely to be due to serotonin uptake enhancement.     

Effects of heparin and a semi-synthetic heparin analogue on platelet aggregation, lipoprotein lipase and other laboratory tests in surgical patients

Platelet aggregation, lipoprotein lipase activity, coagulation parameters and routine blood chemistry were measured in a randomised study of 21 surgical patients before, immediately after and 3 months after operation. Sodium heparin 5000 IU was given subcutaneously to 11 patients every 12 hours for 7 days, the first injection 2 hours preoperatively; 10 patients received a semi-synthetic heparin analogue (SSHA 75 mg) in the same manner. The groups were sex and age matched. No conclusive changes were found in platelet aggregation. The increase in lipoprotein lipase activity in SSHA patients 2 hours after injection was significantly greater than in heparin patients. Neither of the two drugs induced significant changes in coagulation parameters or routine blood chemistry. The results indicate a difference in the effect on lipoprotein lipase release between heparin and SSHA at the used dosage schedules.     

Conventional heparin and semisynthetic heparin analogue (SSHA) alteration of blood coagulation after embolic occlusion of human renal circulation

Blood coagulation and fibrinolytic variables were measured in a peripheral vein in a study of 21 consecutive patients before and after angio-embolization of renal carcinoma. The first ten patients received conventional heparin, 5000 IU twice daily, and the following eleven a semi-synthetic heparin analogue (SSHA), 50 mg twice daily, for 5 days. The first injection was given 2 hours before embolization and the last injection at least 12 hours before the last blood sampling. Both groups showed increased levels of FPA on day 5-7, indicating that the anticoagulant influence had ceased. F VII levels decreased only in the SSHA group from embolization to day 3, but were increased in both groups on day 5-7. Levels of thrombin-antithrombin complexes (TAT) were significantly increased in the heparin group 2 hours after embolization, indicating that thrombin activity had been formed. The corresponding TAT level in the SSHA group was not significantly increased. The differences could possibly indicate a different mechanism of action on blood coagulation of SSHA as compared with heparin, with involvement of extrinsic pathway and maybe by-passing antithrombin III inhibition.     

In vitro study of the inhibition of coagulation induced by different radiocontrast molecules

The anticoagulant activity of seven intravascular radiocontrast molecules (RCM) was evaluated in different in vitro systems using citrated human plasma. Each RCM was tested in a concentration range of 5 to 50 mM. The thrombin time and the reptilase time showed a dose-dependent lengthening of fibrinoformation, the recording of fibrinoformation exhibited a significant delay of fibrin monomer generation and polymerization although the amplitude of the fibrino-formation was not decreased. The interfering effect with fibrin clot formation impairs also global coagulation tests and monospecific coagulation tests using fibrinoformation as the final step of the assay, but a possible interaction between RCM and some specific coagulation factors cannot be excluded. RCM potentiated the anti-thrombin action of heparin but the inhibition or delay of fibrino-formation is not related to an antithrombinic effect of contrast media. The thrombin amidolytic activity is not modified by RCM but the generation of FpA is delayed and decreased. The ultrastructure of the fibrin clot is not altered at the end of the polymerization.     

In vitro comparison of the effect of heparin,enoxaparin and fondaparinux on tests of coagulation

Low-molecular weight heparin (LMWH) is increasingly used in place of unfractionated heparin (UFH) in patients with unstable angina, and phase II clinical trials using fondaparinux for this indication are underway. Because unstable angina patients often require urgent percutaneous coronary interventions (PCI) or aortocoronary bypass surgery, a point-of-care test is needed to monitor the anticoagulant effect of these agents. The activated clotting time (ACT) and activated partial thromboplastin time (aPTT) are the tests most often used to monitor heparin. The purpose of this in vitro study was to determine whether the ACT or the aPTT can be used to monitor the anticoagulant effect of low-molecular weight heparin and fondaparinux.The ACT and aPTT were measured after heparin, enoxaparin or fondaparinux was added to the blood of healthy volunteers, in doses with equivalent inhibitory activity against activated factor X (factor Xa). To mimic the clinical scenario where an unstable angina patient, who has already received enoxaparin, is urgently taken for PCI or bypass surgery, the ACT was determined after heparin was added to blood containing clinically relevant doses of enoxaparin.We determined that enoxaparin produced significantly less prolongation of both the ACT and the aPTT than heparin, whereas fondaparinux had no effect on either of these tests. Addition of enoxaparin to heparin-containing plasma did not prolong the ACT beyond that produced by heparin alone. The ACT and aPTT therefore cannot be used to monitor low-molecular weight heparin or fondaparinux, highlighting the need for a point-of-care anti-factor Xa assay.     

In vitro and in vivo inhibition of glycolytic enzymes by acrylamide

The effect of acrylamide on glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, and lactate dehydrogenase has been studied both in vitro and in vivo. Acrylamide inhibited crystalline GAPDH and PFK from rabbit muscle as well as the enzyme present in rat brain and sciatic nerve homogenates in vitro. Inhibition of enzyme activity was a function of the concentration and the duration of preincubation with acrylamide. Enzyme inhibition was prevented by dithiothreitol. Acrylamide did not inhibit LDH activity even at high concentrations. Rats intoxicated with acrylamide had approximately 33% less GAPDH in sciatic nerves, but normal levels were found in liver and brain homogenates. The significance of selective GAPDH inhibition is discussed in relation to the pathogenesis of peripheral neuropathy induced by acrylamide.     

Mechanism of potentiation of antithrombin III and heparin cofactor II inhibition by sulfated xylans

Kinetic analyses of antithrombin III (AT-III)-thrombin or heparin cofactor II (HC-II)-thrombin or AT-III-factor Xa interactions were carried out in the absence or in the presence of one of the sulfated xylans or unfractionated heparin or low molecular weight (LMW) heparin utilizing chromogenic substrates. These studies demonstrated that under pseudo first order conditions the inhibitions were proportional to the AT-III or HC-II concentrations used and the apparent second order rate constants determined from the slopes of the pseudo first order plots of log of thrombin or Xa remaining as a function of time were significantly elevated in presence of the sulfated compounds. On a molar basis oat spelts xylan sulfate was the most effective compound in accelerating the rate of thrombin-AT-III interaction followed by commercial heparin while the latter was most effective in accelerating the rate of thrombin-HC-II interaction. Heparin and LMW heparin were more effective in that order in accelerating the rate of Xa-AT-III interaction while oat spelts xylan sulfate, corn cob xylan sulfate, SP-54 were less effective than the heparins in that order. Studies were also conducted on the concentrations of the sulfated compounds required to inhibit by 50% the thrombin activity by AT-III or HC-II or that required to inhibit by 50% the factor Xa activity by AT-III. The results showed an inverse relationship between the increase in the rate of acceleration by the sulfated compound with the decrease in the amount required for 50% inhibition. SDS-polyacrylamide gel study of the reaction mixture containing thrombin, AT-III or HC-II along with heparin or oat spelts xylan sulfate showed that like heparin, oat spelts xylan sulfate potentiated the formation of thrombin-AT-III or thrombin-HC-II complexes which were stable in presence of denaturing or reducing agents. Chemical modification of arginine or lysine of AT-III significantly lowered its potentiation of thrombin or Xa inhibition by oat spelts xylan sulfate.     

Purification, characterization and in vivo studies of salmon heparin

Heparin was purified from gills and intestines from farmed Atlantic salmon (Salmo salar).Heparin activity was determined after size exclusion chromatography in the molecular weight range from above 8,000 to near 1,500. A specific activity of 110.1 antifactor Xa units/mg was measured in the less than 3,500 molecular weight fraction while 136.8 antifactor Xa units/mg was detected in a 8,000-3,500 molecular weight fraction.The presence of high affinity salmon heparin was demonstrated by using chromatography on antithrombin-Sepharose. Heparin with molecular weights lower than 3,500 was found both in high and low affinity fractions. NMR-analysis detected N- and O-sulfated oligosaccharides essential for heparin activity.The amount of salmon heparin with molecular weight lower than 8,000 varied from 12% to almost 100%. The factors determining this variation is not known, but appears to reside in the fish at the time of slaughter.The in vivo effect of salmon heparin was tested in rabbits using dalteparin as control. Salmon heparin activity was recovered in plasma samples expressed as antifactor Xa activity after intravenous administration. Based on a small number of samples and animals, the results indicate that in vivo half-life time of salmon heparin was higher than that of dalteparin.     

Factor IX a inhibition contributes to the heparin effect.

We investigated whether the inactivation of factor IXa contributes to the partial inhibition of thrombin formation that is observed at therapeutic concentrations of heparin. The action of standard unfractionated heparin (0.05 U/ml) on thrombin formation in the intrinsic system was compared to that of a mixture of dermatan sulfate (DS) and a synthetic pentasaccharide (PS). DS enhances the action of heparin cofactor II which inhibits thrombin only. PS specifically enhances the anti-factor Xa activity of antithrombin III (AT III). The concentrations of DS and PS were chosen so as to obtain equal anti-thrombin and anti-factor Xa activities as in 0.05 U/ml heparin. An extra inhibitory effect of heparin over the mixture is observed in situations where free factor IXa, not bound to factor VIIIa and phospholipid, limits the rate of thrombin formation, notably in contact activated plasma. We conclude that the inactivation of free factor IXa by heparin contributes importantly to the inhibition of thrombin formation in the intrinsic system such as e.g. measured in the activated partial thromboplastin time.     

In vivo and in vitro studies on putative interneurones in the rat hippocampus: Possible mediators of feed-forward inhibition

Extracellular and intracellular recordings have been made from non-pyramidal neurones in the rat hippocampus in vivo and in vitro. These cells were situated in the stratum pyramidale but were orthodromically activated with a lower threshold than pyramidal neurones in response to stimulation of the Schaffer collateral/commissural afferents. Cells fired earlier than pyramidal neurones in response to suprathreshold stimulation and, in contrast to pyramidal cells, often fired a burst of action potentials. The non-pyramidal neurones also appeared to be orthodromically activated on stimulating the alveus and fired later than antidromically activated pyramidal cells. A very short action potential duration and the ability to fire at high frequencies in response to long depolarizing current pulses also distinguished these neurons from pyramidal cells. It is suggested that these non-pyramidal cells are interneurones which could mediate an early feed-forward activity onto pyramidal cells such as feed-forward inhibition. They may also be recurrently activated and hence could conceivably mediate a recurrent inhibition.     

In vivo and in vitro NGF studies on developing cerebellar cells.

The biological effect of nerve growth factor (NGF) in the early prenatal cerebellar cell was studied in vivo and in vitro with autoradiographic, tissue culture and immunohistochemical techniques. Iodinated NGF (125I-NGF) injected into the cerebella of 16-day-old rat embryos showed accumulation of this ligand in the Purkinje cell layers. The ability of these cells to accumulate NGF lasted to the 19th day of embryonic life. Cerebellar cells isolated from embryos of the same age, but not older embryos, cultured in vitro for two weeks in the presence of NGF, showed morphological characteristics similar to Purkinje-like cells. These findings suggest that NGF exerts a time-limited trophic effect on immature Purkinje cell precursors.     

In vivo inspection of Wallerian degeneration. MRI studies

11 cases of infarction with Wallerian Degeneration studied by Magnetic Resonance Imaging. It showed that MR Imaging is a very useful method to inspect Wallerian degeneration in vivo after cerebral infarction and provides a new way to study neural pathways. T2-Weighted Images can demonstrate the degenerated pyramid tract and the extent of the degeneration. T1-Weighted Images can demonstrate structural changes clearly such as the atrophy of the cerebral peduncle.     

Anticoagulant properties of heparin fractionated by affinity chromatography on matrix-bound antithrombin III and by gel filtration

Heparin purified by affinity chromatography on anti-thrombin III-Sepharose has been studied by various methods. The specific activities of the materials obtained were in the range 170–230 units/mg as determined by a whole plasma clotting method and 360–780 units/mg as determined by a F.X_a inhibition method. Gel filtration of the material showed that there was a definite molecular size dependency of the specific activities and the activity profiles were markedly different when assay -ed by different methods. These features were also observed (at generally lower activities) with gel filtration fractions of commercial heparin. The possible conclusions regarding the mechanism of heparin anticoagulant action are discussed.