Discordance between the anti-Xa activity and the antithrombotic activity of an ultra-low molecular weight heparin fraction

The relationship between the in vivo antithrombotic effect of heparin and ex vivo anti-Xa activity has been investigated using an animal thrombosis model. Three low molecular weight heparins were compared with the standard heparin from which they were fractionated. All four heparins showed a dose-dependent antithrombotic effect enabling the relative antithrombotic and anti-Xa activities to be compared over a dosage range. A correlation between ex vivo anti-Xa heparin levels and antithrombotic effect was demonstrated for the standard (MW 16,000), intermediate (MW 7,600) and low (MW 4,600) molecular weight heparins but not for the ultra-low molecular weight (MW 3,000) fraction. The lack of relationship between anti-Xa activity and inhibition of thrombosis for the very low molecular weight fraction indicates that a very high anti-Xa activity (measured in vitro or ex vivo) is not always predictive of in vivo antithrombotic efficacy. These findings suggest that other properties of low molecular weight heparins contribute to their antithrombotic effectiveness.     

Comparison of the in vivo hemorrhagic and antithrombotic effects of a low antithrombin-iii affinity heparin fraction

Antithrombin III (ATIII) affinity chromatography of commercial grade heparin yields fractions of high and low affinity for ATIII. In vitro, the high affinity fraction accounts for most of the anticoagulant effect while there is evidence that the low affinity material interferes with platelet function. We have studied the relative antithrombotic and hemorrhagic effects of low affinity heparin. The low affinity heparin fraction, specific activity 43 USP units/mg, was compared with standard heparin (150 USP units/mg) in rabbit experimental models. A residual 12.5% by weight of this low affinity heparin showed high affinity for ATIII. Inhibition of thrombosis in a stasis-hypercoagulability model was directly related to the weight (mg) of high affinity material in each of the heparins. In the bleeding model, when similar weights (mg) of high affinity material were infused, significantly more bleeding was demonstrated with the low affinity fraction which contained a 5-fold excess by weight of low affinity material. We have demonstrated that a low affinity heparin depleted of in vitro anticoagulant and in vivo antithrombotic activity significantly contributes to hemorrhage.     

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.     

Structural studies and "in vivo" and "in vitro" pharmacological activities of heparin fractions and fragments prepared by chemical and enzymic depolimerization

Two heparin families (SM- and FM- heparins) have been fractionated with barium by selective precipitation at different temperatures. Fragments were obtained from these two fractions by enzymic and chemical degradation. Some structural features and pharmacological activities of FM- and SM-heparins as well as the fragments are now reported. The fragments, prepared by ascorbate/H2O2 oxidation (M.W. 4,500) or heparitinase II (M.W. 5,500), are enriched with trisulfated disaccharide units whereas heparin (M.W.12,500) and FM-heparin (M.W. 10,200) contain also N-sulfated and N-acetylated disaccharides. The chemical and enzymic fragments show 1/3 to 1/5 of the anticoagulant activity of heparin by the USP and APTT methods. The LPL releasing activity is also low in the fragments as well as the AXa activity measured by the chromogenic method. On the other hand the AXa activity measured by the Yin and Wessler procedure is two times higher in the fragments when compared to heparin. The fragments resulting from heparin after heparitinase II degradation and ascorbate/H2O2 oxidation were rich in trisulfated disaccharide units whereas heparin and FM-heparin contained also measurable amounts of monosulfated and N-acetylated disaccharide units. The mechanism of ascorbate oxidation and the structural requirements for the pharmacological activities of heparin is discussed in view of these and other findings.     

Comparative study of antithrombotic effect of a low molecular weight heparin and unfractionated heparin in an ex vivo model of deep arterial injury

Thrombosis after plaque rupture triggers the onset of acute coronary events. The treatment of choice for patients with acute coronary syndromes is conventional unfractionated heparin. Low molecular weight heparin has recently been reported to be as effective and even safer than unfractionated heparin. In this study, the effects of the low molecular weight heparin reviparin and unfractionated heparin on thrombus formation were examined under dynamic conditions using an extracorporeal perfusion chamber in a porcine model. Thrombus formation was assessed by the deposition of porcine ¹²³I-fibrin(ogen) and autologous ¹¹¹In-platelets on porcine tunica media at high and low shear rates. Reviparin reduced the fibrinogen molecules deposited on injured vessels at high shear rates (252±80 molecules×10¹²/cm² for reviparine (200 U/kg/hour) vs. 624±70×10¹²/cm² for unfractionated heparin (200 U/kg/hour) (p<0.05). At low shear rates, fibrinogen deposition was also significantly reduced by reviparin (130±15 molecules×10¹²/cm²) compared to unfractionated heparin (192±40×10¹²/cm² at 200 U/kg/hour; p<0.05). No change in platelet deposition was detected after heparin administration in either treatment group. In conclusion, the low molecular weight heparin reviparin has a higher antithrombotic potential than unfractionated heparin. Reviparin may have advantages over unfractionated heparin in treatment and prevention of acute coronary syndromes.     

In vitro and in vivo effects of a peptide mimetic (SC-47643) of RGD as an antiplatelet and antithrombotic agent

Platelet aggregation requires binding of fibrinogen (fgn) to activated platelets and inhibition of this binding blocks platelet aggregation. Synthetic peptides modeled after the platelet binding sequence on fgn block the platelet glycoprotein IIb/IIIa receptor and effectively inhibit aggregation. SC-47643 (SC) is a mimetic of the RGD-containing peptide sequence that is recognized by the platelet IIb/IIIa receptor. SC inhibited fgn binding to activated platelets (IC50: 1.0 x 10(-5) M) and prevented platelet aggregation in response to a variety of platelet agonists in both washed human platelets and platelet rich plasma (IC50's ranging from 4 x 10(-6) to 1 x 10(-5) M, respectively). SC inhibited collagen induced thrombocytopenia in the rat (ED50 0.07 mg/kg and t1/2 36 min). In dogs ex vivo collagen induced platelet aggregation was inhibited 50% after a bolus injection of 1.7 mg/kg. After a steady state infusion (2 hr), the ED50 was 0.03 mg/kg/min, with no effects on blood pressure, heart rate or platelet count. These data demonstrate that SC, a peptide mimetic of the natural fgn binding sequence, is capable of blocking platelet-fgn interactions and platelet aggregation.     

Lack of correlation between cholinergic-induced changes in chemosensory activity and dopamine release from the cat carotid body in vitro

We studied the effects of nicotine, acetylcholine (ACh) and dopamine (DA) on the frequency of chemosensory discharges (f_x) and catecholamine (CA) efflux in the cat carotid body superfused in vitro. CA efflux was measured by changes in CA concentration (ΔCA) determined by chronoamperometry with nafionated carbon-fiber microelectrodes inserted in the carotid body, while f_x was recorded simultaneously from the carotid (sinus) nerve. Nicotine (10–20 μg) and ACh (>100 μg) increased f_x in all carotid bodies (n=16), but produced a delayed ΔCA (0.65 μM) in only half of them. Eserine potentiated ACh-evoked increases in f_x and CA effluxes. Nicotine and ACh-induced ΔCA were rapidly reduced upon repeated administration. While f_x increases evoked by low doses of nicotine or ACh were reduced or abolished by prior administration of exogenous DA (>100 μg), CA effluxes were enhanced and hastened. Thus, cholinergic-induced changes in f_x are dissociated from CA efflux.     

Acute and repeated administration of fluoxetine, citalopram, and paroxetine significantly alters the activity of midbrain dopamine neurons in rats: an in vivo electrophysiological study

We examined the effect of the administration of the selective serotonin reuptake inhibitors (SSRIs) fluoxetine, citalopram, and paroxetine on the activity of spontaneously active dopamine (DA) neurons in the substantia nigra pars compacta (SNC) and ventral tegmental area (VTA) in anesthetized adult male Sprague-Dawley rats. This was accomplished using the technique of in vivo extracellular recording. A single injection of 2.5 mg/kg (i.p.) of fluoxetine significantly increased the number of spontaneously active SNC and VTA DA neurons. In contrast, a single injection of either 1 mg/kg (i.p.) of paroxetine or 5 mg/kg of fluoxetine significantly increased the number of spontaneously active VTA DA neurons. The repeated administration (one injection per day for 21 days) of all of the SSRIs produced a significant increase in the number of spontaneously active VTA DA neurons. Overall, our results indicate that the systemic administration of SSRI alters the activity of midbrain DA neurons with differential effects on VTA compared with SNC DA neurons.     

Behaviour of [11C]R(-)- and [11C]S(+)-rolipram in vitro and in vivo, and their use as PET radiotracers for the quantificative assay of PDE4

Cyclic AMP (cAMP) is a continually produced nucleotide which is inactivated by hydrolysis to 5'AMP via phosphodiesterase 4 (PDE4) enzymes. Rolipram is a selective PDE4 inhibitor which exists in two enantiomeric forms, R(-) and S(+). Both of these enantiomers have previously been labelled with carbon-11 and used as positron emission tomography (PET) ligands for measuring PDE4 expression and function, and indirectly to explore the function of the cAMP second messenger, in vivo, using PET. The aim of these studies was to relate the in vitro affinities of the two rolipram enantiomers using standard pharmacological assays with the in vivo behaviour of the two enantiomers using PET. In vitro competition assays were performed using rat cortical membranes and [(3)H]R(-)- and [(3)H]S(+)-rolipram with increasing concentrations of either unlabelled R(-)- or S(+)-rolipram. In vivo, a series of PET studies were performed in the porcine brain using [(11)C]R(-)-rolipram with co-administration of increasing doses of either unlabelled R(-)- or S(+)-rolipram. Additional in vivo PET studies were performed using [(11)C]S(+)-rolipram with saturating doses of rolipram. In all studies, R(-)-rolipram exhibited a higher affinity for the PDE4 enzyme than S(+)-rolipram. The calculated affinity ratios were 7.97 from the in vitro studies; 12.5 from the in vivo studies using [(11)C]R(-)-rolipram; and 14.7 from the in vivo studies using [(11)C]S(+)-rolipram. To conclude, the in vitro affinities of R(-)- and S(+)-rolipram predict their apparent in vivo behaviour in the porcine brain, as measured by PET.     

Acute and chronic administration of the selective 5-HT1A receptor antagonist WAY-405 significantly alters the activity of midbrain dopamine neurons in rats: an in vivo electrophysiological study

In this study, we examined the effect of the acute and chronic administration of WAY-405, a selective 5-HT(1A) receptor antagonist, on the number and firing pattern of spontaneously active substantia nigra pars compacta (A9) and ventral tegmental area (A10) dopamine (DA) neurons in anesthetized rats. This was accomplished using in vivo, extracellular single unit recording. The i.v. administration of WAY-405 (5-640 microg/kg) did not significantly alter the basal firing rate or pattern of spontaneously active A9 and A10 DA neurons. A single injection of 10 microg/kg of WAY-405 decreased the number of spontaneously active A10 DA neurons and the 100 microg/kg dose significantly decreased the number of spontaneously active A9 and A10 DA neurons compared to vehicle-treated animals. A single injection of 1, 10, or 100 microg/kg of WAY-405 significantly decreased the degree of bursting of A10 DA neurons. In contrast, 1 microg/kg i.p. of WAY-405 significantly increased the percent of A9 DA neurons exhibiting a bursting pattern. The repeated administration of 10 or 100 microg/kg i.p. of WAY-405 (21 days) significantly decreased the number of spontaneously active DA neurons in both the A9 and A10 compared to vehicle-treated animals and this decrease was not reversed by i.v. (+)-apomorphine. The repeated administration of WAY-405 significantly altered the firing pattern of DA neurons, particularly those in the A10 area. Overall, these results indicate that the antagonism of the 5-HT(1A) receptor significantly alters the activity of midbrain DA neurons in anesthetized rats.     

Catecholamine metabolism in the rat locus coeruleus as studied by in vivo differential pulse voltammetry. I. Nature and origin of contributors to the oxidation current at +0.1v

Differential pulse voltammetry was used together with treated carbon fiber microelectrodes to study the in vivo catecholamine (CA) metabolism in the locus coeruleus (LC), a brain region densely packed with noradrenergic neurons. In chronically implanted rats, an in vivo oxidation current that peaks at +0.1 V has been detected inside the LC complex. This current whose potential is characteristic of the oxidation of the catechols, had the same anatomical localization as the noradrenergic cells. Pharmacological experiments have been made to ascertain which catechols contribute to this in vivo current. Monoamine oxidase inhibition by pargyline was followed by a total and rapid suppression of the in vivo signal. Blockade of dopamine-beta-hydroxylase by FLA-63 induced a significant increase in the electrochemical signal. Post-mortem analysis of LC catechol levels after administration of this drug revealed a considerable decrease in NA and its major catechol metabolite, 3,4-dihydroxyphenylglycol (DOPEG) although DA and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were significantly increased. Comparison of these results led us to conclude that DOPAC is probably the most important contributor to the in vivo oxidation current. This assertion is corroborated by results obtained after tyrosine hydroxylase inhibition with alpha-methyl-p-tyrosine: the in vivo catechol current was rapidly suppressed and post-mortem levels of DOPAC were significantly reduced while DOPEG remained almost normal. An attempt was made to selectively destroy the LC cell bodies by a unilateral injection of ibotenic acid (10 micrograms). Eight to 15 days after injection, no current was detectable in the injected side although it was still present in the contralateral intact side. Post-mortem levels of DOPAC and DOPEG levels of the lesioned side were 29% and 17%, respectively, of those in the intact side. Thus, we assumed that the in vivo catechol current in the LC comes from the oxidation of DOPAC most probably synthesized by the noradrenergic cell bodies.     

Effect of the acute and chronic administration of the selective 5-HT6 receptor antagonist SB-271046 on the activity of midbrain dopamine neurons in rats: an in vivo electrophysiological study

This study examined the effect of the acute and repeated per os (p.o.) administration of the selective 5-HT(6) receptor antagonist SB-271046, on the number, as well as the firing pattern of spontaneously active dopamine (DA) neurons in the rat substantia nigra pars compacta (SNC) and ventral tegmental area (VTA) in anesthetized male Sprague-Dawley rats. This was accomplished using the technique of extracellular in vivo electrophysiology. A single p.o. administration of either 1, 3, or 10 mg/kg of SB-271046 did not significantly alter the number of spontaneously active SNC DA neurons per stereotaxic electrode tract compared to vehicle-treated animals. The acute administration of either 1 or 3 mg/kg of SB-271046 did not significantly alter the number of spontaneously active VTA DA neurons. In contrast, a significant decrease in the number of spontaneously active VTA DA neurons was observed after a single administration of 10 mg/kg of SB-271046 compared to vehicle-treated animals. The acute p.o. administration of SB-271046 significantly altered the firing pattern parameters of all (bursting + nonbursting DA neurons) DA neurons, particularly those in the VTA, compared to vehicle-treated animals. The repeated p.o. administration (once per day for 21 days) of 1, 3, or 10 mg/kg of SB-271046 did not significantly alter the number of spontaneously active VTA DA neurons compared to vehicle-treated animals. The repeated administration of 3 or 10 mg/kg of SB-271046 significantly increased the number of spontaneously active SNC DA neurons compared to vehicle controls. Overall, the repeated administration of SB-271046 had relatively little effect on the firing pattern of midbrain DA neurons. The results obtained following the chronic administration of SB-271046 show that this compound has a profile different from that of typical or atypical antipsychotic drugs in this model. Clinical studies are required to understand what role 5-HT(6) receptor blockade might eventually play in the treatment of schizophrenia.     

Effects of d- and l-amphetamine on dopamine metabolism and ascorbic acid levels in nucleus accumbens and olfactory tubercle as studied by in vivo differential pulse voltammetry

Differential pulse voltammetry used together with electrochemically pretreated carbon fibre microelectrodes allowed us to detect in vivo two well-separated peaks in nucleus accumbens and olfactory tubercle. The two peaks situated at −50 mV (peak 1) and +100 mV (peak 2) correspond, respectively, to the oxidation current of the ascorbic acid and to the oxidation current of the 3,4-dihydroxyphenylacetic acid (DOPAC). The experiments were carried out on anesthetized rats. Voltammograms were recorded in nucleus accumbens and olfactory tubercle every minute alternately in each structure. In control conditions, peak 1 height was greater in olfactory tubercle than in nucleus accumbens and peak 2 height was greater in nucleus accumbens than in olfactory tubercle. Both isomers of amphetamine induced a decrease of the peak 2 height in the two structures. The decrease was greater in olfactory tubercle. Higher doses of l-amphetamine were required to induce peak 2 height decrease of the same extent. Both isomers induced a marked increase of the peak 1 height in nucleus accumbens whereas peak 1 height in olfactory tubercle was slightly augmented. d-amphetamine was more effective than l-amphetamine in increasing peak 1 height.     

The acute and chronic administration of (+/-)-8-hydroxy-2-(Di-n-propylamino)tetralin significantly alters the activity of spontaneously active midbrain dopamine neurons in rats: an in vivo electrophysiological study

This study examined the effect of the acute and chronic systemic administration of (+/-)-8-Hydroxy-2-(Di-n-propylamino)Tetralin(8-OH-DPAT) on the number and firing pattern of spontaneously active dopamine (DA) neurons in the ventral tegmental area (VTA or A10) and substantia nigra pars compacta (SNC or A9) in anesthetized male rats. These parameters were measured using extracellular in vivo electrophysiology. A single s.c. injection of 0.01, 0.1, or 1 mg/kg of 8-OH-DPAT did not significantly alter the number of spontaneously active SNC DA neurons compared to vehicle-treated animals (controls). The acute administration of 0.01 or 0.1 mg/kg of 8-OH-DPAT did not significantly alter, whereas the 1 mg/kg dose significantly decreased the number of spontaneously active VTA DA neurons compared to controls. The acute administration of 8-OH-DPAT significantly increased the percentage of VTA DA neurons firing in a bursting pattern. In contrast, there was a significant decrease in the percentage of SNC DA neurons firing in a bursting pattern following the acute administration of 8-OH-DPAT. The number of spontaneously active SNC DA neurons was not significantly altered by the chronic s.c. administration of 8-OH-DPAT (0.01, 0.1, or 1 mg/kg s.c.) as compared to controls. However, the chronic s.c. administration of all doses of 8-OH-DPAT significantly decreased the number of spontaneously active VTA DA neurons compared to controls. The i.v. administration of (+)-apomorphine (50 microg/kg) did not reverse the 8-OH-DPAT-induced decrease in the number of spontaneously active VTA DA neurons, suggesting that this effect is unlikely due to depolarization blockade. The percentage of VTA DA neurons exhibiting burst firing was significantly increased by 0.01 and 0.1 mg/kg, but significantly decreased by 1 mg/kg of 8-OH-DPAT. Overall, the systemic administration of 8-OH-DPAT preferentially affects the activity of spontaneously active A10 DA neurons in rats.