Anticoagulant activities of lung and mucous heparins

It is generally assumed that heparins prepared from different tissues have equivalent actions. However, significant differences were found between lung and mucosal heparins when they were examined in an anti-Factor Xa assay, both $\text{in vivo}$ and $\text{in vitro}$. These differences were not seen when the heparins were assayed by an APTT assay. With both types of heparin, anti-Xa activity increased with low molecular weight fractions, while the APTT activity decreased. However, with lung heparin the specific activities by anti-Xa assay were much lower at all molecular weights examined. It is believed that these observations have important implications for the assay and clinical use of heparin.     

Anticoagulant activities of four unfractionated and fractionated heparins

Four commercial heparins, two of bovine lung and two of porcine mucosal origin, have been fractionated by gel filtration (and one of them also by ion-exchange chromatography) to yield different fractions whose mean MW_s lie in the MW range 7,000–26,000. Three plasma anticoagulant assays have been used to measure the specific activities of the heparin fractions, a kaolin-cephalin clotting time method (KCCT), a calcium-thrombin clotting time method (TCT) and a specific anti-factor Xa assay. The two unfractionated mucosal heparins were found to have higher specific activities when measured in the anti-Xa assay compared to the KCCT assay, while the unfractionated beef lung heparins had lower anti-Xa than KCCT activities. An increase with MW of the heparin fractions was found in specific activities as measured by KCCT and TCT assays over most of the MW range. The low MW heparin fractions (less than 10,500) from all of the heparins had specific activity ratios, KCCT:anti-Xa and TCT:anti-Xa, which were always less than unity. These specific activity ratios increased with increasing MW, usually to greater than unity for the highest MW (greater than 22,000) fractions. There was a tendency for these specific activity ratios to be higher for the beef lung than the mucosal heparin fractions at a given MW. The ability of heparin fractions to inhibit the thrombin induced aggregation of platelets in citrated platelet-rich plasma increased with MW over most of the MW range studied and seemed to follow the specific activities of the fractions when measured by TCT or KCCT assays.     

Anticoagulant and antithrombotic effects of heparin and low molecular weight heparin fragments in rabbits

Heparin and heparin fragments of different molecular weight and with different anti-factor X_a/APTT activity ratios were studied with respect to their ability to inhibit thrombus formation in an animal model. It is concluded that: a) Neither anti-factor X_a nor the APTT activity alone is a good reflector of the antithrombotic activity. b) Anti-factor X_a active fragments must have a minimum molecular weight in order to elicit good antithrombotic activity. c) High affinity for antithrombin III is important for good antithrombotic activity. d) A heparin fragment of molecular weight 4 000 has the same antithrombotic activity as heparin but less effect on the clotting time.     

Orally administered heparins prevent arterial thrombosis in a rat model

Our previous studies demonstrated that orally administered heparins prevent thrombosis in a rat jugular vein thrombosis model, where bovine unfractionated heparin (UFH) and the low molecular weight heparin tinzaparin reduced thrombotic incidence by 50% at 7.5 and 0.1 mg/kg, respectively. Our objectives were to determine if similar antithrombotic effects of oral heparin could be observed in an arterial thrombosis model. In this model, filter paper soaked in 30% ferric chloride was applied to the exposed rat carotid artery. A flowmeter recorded blood flow over a 60 min period determining time when the thrombus began forming (TTB) and time till occlusion (TTO). Immediately following, the thrombus was removed, dried and weighed 24 h later. Bovine UFH (7.5 mg/kg), tinzaparin (0.1 mg/kg) or saline was administered by stomach tube at 2, 5 and 25 h prior to thrombus initiation. TTB was significantly increased when UFH was given at 5 and 25 h but not 2 h prior, and when tinzaparin was given at 5 but not 2 or 25 h prior compared to rats given oral saline. TTO was significantly increased for both UFH and tinzaparin when given 5 and 25 h but not 2 h prior (one-way ANOVA). There was no difference in TTO and TTB between UFH and tinzaparin treated groups. A trend in reduction in thrombus weight was observed for UFH at 5 and 25 h prior and tinzaparin at 5 h prior to thrombus initiation (one-way ANOVA). Although no significant changes were observed in activated partial thromboplastin times, Heptest or anti-Xa activity from plasma of heparin treated rats, endothelial heparin concentrations were significantly greater than controls for UFH at 5 h and for tinzaparin at 2, 5, and 24 h. Thus, heparins administered by the oral route are effective antithrombotic agents in arterial as well as venous models.     

Anticoagulant activity of beef and hog mucosal heparins.

Two purified heparin preparations were obtained from hog and beef mucosa using the same manufacturing procedure. Their activities were compared in vitro for anticoagulant activity using APTT and for anti Xa activity using purified human AT III. Hog mucosal heparin was 25 % more active than beef mucosal heparin in overall clotting assay as well as in purified system. The in vivo response to heparin injection was studied in six healthy volunteers. Heparin levels were measured by anti Xa assay following subcutaneous injection, by APTT and three different anti Xa assays following intravenous injection. No significant difference was found between the respective responses to the two heparin preparations. There was an excellent correlation between anti Xa methods and APTT.     

Effect of age of Armagnac extract and duration of treatment on antithrombotic effects in a rat thrombosis model

In a previous study, we had shown that freeze-dried extracts of 12-year-old Armagnac could inhibit ADP-induced platelet aggregation in a dose- and duration-dependent manner, and reduce thrombus weight in an experimental rat arteriovenous shunt thrombosis model after 2-week oral treatment. Polyphenol content could however vary with age and origin of the brandy, and the onset and offset of the effect were not defined. To this end, we studied the effects of extracts of 5-, 10- and 15-year-old Armagnac from two different producers at 1, 5 and 25 mg/kg orally for 15 days, on the same rat arteriovenous shunt thrombosis model. We then studied the effects of 1, 3, 7 and 15 days of oral treatment with 5 mg/kg extracts of a 5-year-old Armagnac, and the effect 1, 3 and 7 days after a 1-week oral treatment of the same extract at the same dose. There was a dose-dependent decrease in thrombus weight, which was similar for both Armagnac origins for all ages. Extracts of 5- and 10-year-old Armagnac were similar, and more potent than extracts from 15-year-old Armagnac. There was a progressive decrease in thrombus weight over duration of treatment to 7 days, to about 50% of initial thrombus weight. The effect disappeared within 3 days after stopping a 7-day treatment.We confirm the dose-, age- and duration-dependent inhibition of arteriovenous shunt thrombosis in vivo by Armagnac extracts in rats.     

Anticoagulant and antithrombotic properties of synthetic sulfated bis-lactobionic acid amides

Sulfated bis-lactobionic acid amides, a new class of polyanions with heparin-like properties, were synthesized and their antithrombotic and anticoagulant activities were determined. Compared to heparin and Fragmin^R, a low molecular weight heparin, the substances exhibited moderate to low anticoagulant activities in aPTT, thrombin clotting time and Heptest assays. In amidolytic assays no anti-Ila activity and only exceedingly low anti-Xa activity was observed. The antithrombotic activity of the bis-lactobionic acid amides was determined using two thrombosis models. In rabbit and rat models thrombi were induced by a combination of endothelial damage and reduction of blood flow or only by endothelial damage. At least one of the bis-lactobionic acid amides (LW 10082) exhibited a considerable antithrombotic activity which was similar to low molecular weight heparin.     

The role of low-molecular-weight heparins in arterial diseases: optimizing antithrombotic therapy

On the basis of current evidence, all patients with acute coronary syndromes should receive optimized medical therapy, whether or not they ultimately undergo an invasive revascularization procedure, to improve both clinical outcomes and cost effectiveness. While standard aspirin and unfractionated heparin (UFH) have improved short-term outcomes, they do not eliminate the risk of recurrent ischemic episodes. The recent introduction of platelet fibrinogen receptor antagonists and low-molecular-weight heparins (LMWHs) has offered an opportunity to develop more aggressive antithrombotic regimens. The LMWHs have been thoroughly evaluated in unstable angina and non-Q wave myocardial infarction (UA/NQMI), and have demonstrated improved efficacy compared to standard UFH, without an increase in major complications caused by bleeding. Experience has also been gathered using LMWHs in other arterial diseases (such as pregnant patients with prosthetic heart valves) and as an adjunctive therapy with thrombolytics for acute myocardial infarction. Lastly, studies are currently underway evaluating LMWHs in patients with atrial fibrillation.     

Seizure-protecting effects of kyotorphin and related peptides in an animal model of epilepsy

Administration of peptides kyotorphin (KT), neokyotorphin (NKT), and d-ser-2-NKT into the lateral brain ventricle (10 nmol increased the latency and attenuated the severity of picrotoxin-induced convulsions in rats. Anticonvulsive effects of the peptides were also observed after their administration into the CA1 hippocampi (2.5, 5, 10 nmol, with the order of potency d-ser-2-NKT → NKT → KT. When injected into the substantia nigra reticulata, the 10 nmol dose of NKT and d-ser-2-NKT displayed equal seizure-protecting effect, which was higher than that for KT. It is concluded that kyotorphin and its analogs provide structure-dependent, dose-dependent, and target site-dependent antiepileptic effect.     

Pain-relieving effects of acupuncture and electroacupuncture in an animal model of arthritic pain

The effects of acupuncture and electroacupuncture on an animal model of arthritic pain were examined. Under halothane anesthesia, arthritic pain was induced by the injection of carrageenan into the knee joint cavity of male Sprague-Dawley rats. Behavioral performance was tested before and after the termination of acupuncture or electroacupuncture. Electrophysiologically, the responses of afferents to a movement cycle were recorded before and after acupuncture or electroacupuncture. After the acupuncture procedure, the weight-bearing force of the rats was significantly improved and the neural responses to noxious movement stimulation were reduced. Electroacupuncture significantly improved weight-bearing behavior and inhibited neural responses of articular afferents to noxious stimulation. These results indicate that acupuncture and electroacupuncture may provide a potent strategy in relieving arthritic pain.     

Antimyoclonic and neuroprotective effects of lamotrigine in an animal model of cardiac arrest

A major consequence of severe cardiac arrest is impairment of neurological functions. Posthypoxic myoclonus and seizures are two of the major neurological problems following ischemic and hypoxic insults. This condition affects motor function to different degrees of severity ranging from mild to serious debilitation. The pathophysiological mechanism(s) associated with these neurological conditions remain elusive. Glutamate-mediated neuronal overexcitation is thought to play a major role in the neuronal damage and in the neurological consequences of the posthypoxic state. Therefore, lamotrigine, a new anticonvulsant that indirectly modulates glutamatergic neurotransmission by interfering with voltage-dependent sodium channels, was tested for its effectiveness in controlling the neurological and histopathological changes in the animal model of cardiac arrest-induced myoclonus. Lamotrigine dose-dependently attenuated the audiogenic seizures and action myoclonus seen in this rat model. Histological analysis using Nissl staining and the novel Fluoro-Jade histochemistry in cardiac-arrested rats showed an extensive neuronal degeneration in the hippocampus and cerebellum. Lamotrigine treatment significantly attenuated the neuronal degeneration in these brain areas. The neuroprotective effect was more pronounced in hippocampal pyramidal and cerebellar Purkinje neurons. The therapeutic window of lamotrigine in this model was 8 hours. These results suggest that lamotrigine can be viewed as a potential antimyoclonic and neuroprotective agent for the treatment of posthypoxic myoclonus and seizures. The study also suggests that neuronal hyperexcitability may play a role in the etiology of posthypoxic myoclonus and seizure.     

Andrographis Paniculata shows anti-nociceptive effects in an animal model of sensory hypersensitivity associated with migraine

Administration of nitroglycerin (NTG) to rats induces a hyperalgesic condition and neuronal activation of central structures involved in migraine pain. In order to identify therapeutic strategies for migraine pain, we evaluated the anti-nociceptive activity of Andrographis Paniculata (AP), a herbaceous plant, in the hyperalgesia induced by NTG administration in the formalin test. We also analyzed mRNA expression of cytokines in specific brain areas after AP treatment. Male Sprague-Dawley rats were pre-treated with AP extract 30 minutes before NTG or vehicle injection. The data show that AP extract significantly reduced NTG-induced hyperalgesia in phase II of the test, 4 hours after NTG injection. In addition, AP extract reduced IL-6 mRNA expression in the medulla and mesencephalon and also mRNA levels of TNF-alpha in the mesencephalic region. These findings suggest that AP extract may be a potential therapeutic approach in the treatment of general pain, and possibly of migraine.     

Antidepressant-like effects of dopamine agonists in an animal model of depression.

Chronic exposure to mild unpredictable stress (CMS) has previously been found to cause an antidepressant-reversible decrease in the consumption of palatable sweet solutions. There is evidence that the effect of antidepressants in this model is mediated by an increase in transmission at dopamine (DA) synpases. The present study investigated whether another treatment known to increase the functional responsiveness of DA systems, intermittent administration of DA agonists, would have antidepressant-like effects. In three experiments in rats, CMS-induced decreases in sucrose consumption were reversed by three to four twice-weekly injections of quinpirole (100-200 micrograms/kg) or bromocriptine (2.5 mg/kg). The effects lasted for several weeks, and when they waned, could be reinstated by a single additional injection of quinpirole. As with tricyclic antidepressants, the effect of quinpirole was reversed by raclopride, administered acutely immediately prior to a sucrose consumption test; there were no changes in sucrose intake in nonstressed control animals. The results suggest that intermittent administration of DA agonists merits investigation as a novel strategy for the treatment of depression.     

Sedative effects of apomorphine in an animal model of Huntington's disease.

The sedative effectiveness of apomorphine in a newly developed animal model of Huntington's disease was examined. The motor responses of rats with kainic acid lesions of the neostriatum to a sedative dose of apomorphine (50 micrograms/kg) was similar to that observed in intact controls. In contrast, compared to controls, a marked potentiation of the motor stimulant effects of dextroamphetamine was confirmed in the kainic acid-lesioned group. We suggest that the pathological changes underlying the symptoms observed in this animal model and in Huntington's disease do not include abnormalities in presynaptic dopamine receptors in the neostriatum.     

Behavioural effects of parafascicular thalamic lesions in an animal model of parkinsonism

We recently reported that the centromedian-parafascicular thalamic complex (CM-Pf) degenerates in Parkinson's disease and progressive supranuclear palsy. The contribution of such thalamic pathology to disease symptoms has not yet been established. The present study therefore investigated the behavioural impact of lesioning the corresponding thalamic region (termed Pf) on a range of behaviours present in rodents. There were four surgical groups: (1) sham medial forebrain bundle (mfb)+sham Pf, (2) 6-OHDA mfb lesion+sham Pf, (3) sham mfb+NMDA Pf lesion, (4) 6-OHDA+NMDA Pf lesions. Posture, sensory functions and apomorphine-induced rotational asymmetry were assessed before and after each surgery. Other assessments performed including a timed motivational task, grooming behaviours and piloerection. 6-OHDA lesions induced postural (ipsilateral curling and head position biases), sensorimotor (increased latency to respond to tactile stimulation of the contralateral side when eating or grooming) and rotational abnormalities (contralateral circling after apomorphine). The main effects of combined 6-OHDA+Pf lesions were improved performance in a motivational task (decreased latency to retrieve reward) but worsened piloerection, relative to animals with either 6-OHDA or Pf lesions alone. The thalamic zone common to all lesioned animals involved the posterior Pf. Our data suggests that the posterior CM-Pf may be involved in motivational responses and autonomic dysfunction in parkinsonian disorders.     

The relative antithrombotic effectiveness of heparin, a low molecular weight heparin, and a pentasaccharide fragment in an animal model

The antithrombotic efficacy of unfractionated heparin (UFH), a low molecular weight heparin (LMWH) and a synthetic pentasaccharide (PENTA) has been compared in an animal model for stasis thrombosis. We have also compared the relative ability of these three agents to impair thrombin generation in vitro and in vivo, and measured their effects on anti-Xa activity and thrombin clotting times. UFH, LMWH and PENTA all had the capacity to impair thrombogenesis, although there were marked differences in their relative effectiveness. Reduction of thrombin generation to 20% of control values was closely correlated with the prevention of thrombosis after 20 minutes' stasis, but this was only achieved with UFH. The same dry weight dose of LMWH or PENTA reduced thrombin generation to about half control values, and neither significantly impaired thrombus formation after 20 minutes' stasis. Impaired thrombin generation correlated better than anti-Xa activity with prevention of stasis thrombosis. In this model, UFH was clearly superior to LMWH and PENTA as an antithrombotic agent.     

Anticoagulant effects of an antidiabetic drug on monocytes in vitro

Introduction

Monocyte- and microparticle (MP)-associated tissue factor (TF) is upregulated in diabetes. Lipopolysaccharide (LPS) induces expression of TF and alternatively spliced TF (asTF) and increases MP release from monocytes. Using LPS-stimulated TF-bearing human monocytes, we examined whether glibenclamide, a sulfonylurea used to treat diabetes type 2, might possess anticoagulant properties.

Methods

We studied the effects of glibenclamide on cell- and supernatant-associated procoagulant activity (Factor Xa-generating assay and clot formation assay), on expression of TF and asTF (flow cytometry, RT-qPCR, western blot) and on cell viability and MP release (flow cytometry).

Results

Glibenclamide dose-dependently decreased procoagulant activity of cells and supernatants. The reduction in cellular procoagulant activity coincided with reduced expression of TF and asTF in cells, whereas cell viability remained almost unchanged. The glibenclamide-induced reduction in procoagulant activity of supernatants appeared to be associated with a decreased number of released MPs.

Conclusions

Reduction of monocyte- and supernatant-associated procoagulant activity by glibenclamide is associated with decreased expression of TF and asTF and possibly with a reduced MP number. Our data indicate that glibenclamide reduces the prothrombotic state in LPS-stimulated monocytes in vitro. Glibenclamide might therefore also have an anticoagulant effect in vivo, but this needs to be further evaluated.     

Cerebral radioprotective effects of high-dose pentobarbital evaluated in an animal radiosurgery model

Abstract

Because pentobarbital has been shown to reduce cerebral toxicity to single-fraction whole brain irradiation in a rat model, we sought to evaluate its cerebral radioprotective effects for stereotactic radiosurgery. We hypothesized that concurrent high-dose pentobarbital anaesthesia (50 mg kg^–1) during irradiation could delay or prevent the onset of radiation necrosis within the radiosurgical volume. Six rats were placed in pentobarbital or control groups, irradiatedand then evaluated at different intervals (60, 100; 750, 365 days; total = 48 animals studied). All rats had 100 Gy radiosurgery to the right frontal brain region (a threshold dose for focal necrosis at 90 days). The radioprotective effects of pentobarbital were compared to ketamine anaesthesia (control) and evaluated for observed focal necrosis, size of necrotic lesion, blood vessel alterations, and to changes in cell nuclei. There was no difference between groups in the numbers of rats with necrosis at 100 days (p = 0.72), at 150 days (p = 0.77), or at 365 days (p = 0.77); no necrosis was observed in either group at 60 days. There was no difference in the size of the necrotic lesion at 100 days (p = 1.0), at 150 days (p = 0.39), or at 365 days (p = 0.07). There was no difference between groups in observed blood vessel changes or nuclear changes at any time interval (p > 0.6).m There was no animal morbidity related to radiosurgery. While pentobarbital may exhibit a radioprotective effect to lower dose, whole brain irradiation on a metabolic or cellular level, we could not identify any protective benefit for the prevention of focal radiation necrosis or other radiation-related histologic changes in this experiment. The model can be used to evaluate other potential radioprotective agents, and to improve assessments of blood-brain barrier integrity and ultrastructure. [Neurol Res 1994; 16: 456-459]