Human Platelet Serotonin Research Articles

Isolation of Bioactive Compounds That Relate to the Anti‐Platelet Activity of Cymbopogon ambiguus

Infusions and decoctions of Cymbopogon ambiguus have been used traditionally in Australia for the treatment of headache, chest infections and muscle cramps. The aim of the present study was to screen and identify bioactive compounds from C. ambiguus that could explain this plant's anti-headache activity. A dichloromethane extract of C. ambiguus was identified as having activity in adenosine-diphosphate-induced human platelet aggregation and serotonin-release inhibition bioassays. Subsequent fractionation of this extract led to the isolation of four phenylpropenoids, eugenol, elemicin, eugenol methylether and trans-isoelemicin. While both eugenol and elemicin exhibited dose-dependent inhibition of ADP-induced human platelet serotonin release, only eugenol displayed potent inhibitory activity with an IC50 value of 46.6 μM, in comparison to aspirin, with an IC50 value of 46.1 μM. These findings provide evidence to support the therapeutic efficacy of C. ambiguus in the non-conventional treatment of headache and inflammatory conditions.

N-methyl-citalopram: A quaternary selective serotonin reuptake inhibitor

We describe the synthesis and the pharmacological characterization of a new quaternary selective serotonin reuptake inhibitor (SSRI) N-methyl-citalopram (NMC) with periphery restricted action due to its inability to cross the blood brain barrier. NMC recognized and blocked the human platelet serotonin transporter (SERT) with similar affinity to that of citalopram as was evident from competition binding studies with [3H]citalopram and uptake studies with [3H]5-HT. In contrast, the affinity of NMC to rat brain SERT was 10-fold lower than its parent compound citalopram. Similarly to citalopram, NMC did not inhibit dopamine and noradrenaline uptake in rat brain synaptosomes at 10−7M as well as [3H]ketanserin binding to rat brain membranes at 10−5M, demonstrating its SSRI profile. A comparison of radioactivity retained in perfused mice brain following in vivo intraperitoneal injections of tritium-labeled NMC or citalopram showed that unlike citalopram, NMC did not penetrate the brain. Taken together, our observations suggest that N-methyl-citalopram is a selective serotonin reuptake inhibitor that does not penetrate the mouse brain. Epidemiological studies have suggested that chronic use of SSRI drugs may confer a protective effect against myocardial infarction (MI) apparently reflecting reduced platelet aggregation secondary to reduced platelet serotonin levels. N-methyl-citalopram may therefore have a potential as a new anti-platelet drug that does not cross the blood brain barrier and is thus devoid of the adverse CNS effects of SSRI drugs.

Smoking-induced long-lasting modifications of human platelet serotonin catabolism through a MAO epigenetic regulation

AbstractPostulating that serotonin, secreted from smoking-activated platelets, could be involved in smoking-induced vascular modifications, we studied 115 men distributed in smokers (S), former smokers (FS) and never smokers (NS). The platelet serotonin content was similar in S and NS but lower in FS. This was unexpected because the monoamine oxidase (MAO) activity, which catabolizes serotonin, was inhibited during smoking. However, the amount of platelet MAO was higher in S and FS than in NS. The persistent elevated MAO amount in FS prompted us to study the methylation of its gene promoter in an additional series of patients: it was markedly lower for S and FS vs. NS due to cigarette smoke-induced increase of nucleic acid demethylase activity. This smoking-induced demethylation of the MAO gene promoter, resulting in high MAO amount persisting long after quitting smoking, has cardiovascular consequences and could impact fields such as behavior, mental health, and cancer.

Sex-Specific Genetic Architecture of Whole Blood Serotonin Levels

Recently, a quantitative-trait locus (QTL) for whole blood serotonin level was identified in a genomewide linkage and association study in a founder population. Because serotonin level is a sexually dimorphic trait, in the present study, we evaluated the sex-specific genetic architecture of whole blood serotonin level in the same population. Here, we use an extended homozygosity-by-descent linkage method that is suitable for large complex pedigrees. Although both males and females have high broad heritability (H2=0.99), females have a higher additive component (h2=0.63 in females; h2=0.27 in males). Furthermore, the serotonin QTL on 17q that was identified previously in this population, integrin beta 3 (ITGB3), and a novel locus on 2q influence serotonin levels only in males, whereas linkage to a region on chromosome 6q is specific to females. Both sexes contribute to linkage signals on 12q and 16p. There were, overall, more associations meeting criteria for suggestive significance in males than in females, including those of ITGB3 and the serotonin transporter gene (5HTT). This analysis is consistent with heritable sexual dimorphism in whole blood serotonin levels resulting from the effects of a combination of sex-specific and sex-independent loci.

The influence of selective serotonin reuptake inhibitors on human platelet serotonin.

Clinical depression has been proposed to be an independent risk factor for cardiovascular disease. While it is suggested that selective serotonin reuptake inhibitors (SSRIs) reduce the risk of acute cardiovascular problems of depressed patients, the effect of SSRIs on platelets, the only blood cells committed to serotonin (5-HT) transport, remains largely unknown. The goal of this pilot study was to measure the 5-HT levels in platelets of untreated and SSRI-treated depressed patients and normal subjects and to determine whether the interaction of SSRIs with platelets can explain their possible cardiovascular benefit in patients with depression. Platelet 5-HT was determined by an immunocytochemical assay and high-pressure liquid chromatography with electrochemical detection (HPLC-ECD). In normal control subjects without cardiovascular disease, 78 +/- 8% of platelets were 5-HT-positive (n = 14). Depression caused a significant reduction in platelet 5-HT to 46 +/- 21% in untreated patients (n = 13) and 22 +/- 13% in SSRI-treated patients (n = 14). As a class, all selective serotonin reuptake inhibitors significantly reduced the 5-HT concentration in patient platelets. An inverse relationship of 5-HT level and dose of medication might be suggested. These results correlated well with 5-HT data from HPLC (r = 0.8509, p < 0.001). SSRIs did not affect platelet aggregation and dense granule release in response to thrombin, but significantly reduced ADP-induced platelet aggregation and dense granule release in both patient and normal control samples. The active inhibition of platelet aggregation by SSRIs might explain their cardiovascular benefit.

The effect of the anorectic agent, d-fenfluramine, and its primary metabolite, d-norfenfluramine, on intact human platelet serotonin uptake and efflux

Dexfenfluramine, a drug formerly prescribed for treatment of obesity, caused heart valve damage and pulmonary hypertension in some people. The cause of the toxicity has not been defined, but 5-HT has been implicated. The objective of this study was to evaluate the effect of the anorectic agent, d-fenfluramine, and its major metabolite, d-norfenfluramine, on intact human platelet serotonin (5-HT) transport in vitro. The effects of d-fenfluramine and d-norfenfluramine on platelet uptake and efflux of 3H-5-HT were measured in buffer at pH 6.7, to optimize serotonin transporter (SERT) function, and at pH 7.4. Uptake of 3H-5-HT at pH 6.7 and 7.4 was inhibited by both agents at micro m concentrations (IC50, d-fenfluramine approximately 3 microM; d-norfenfluramine approximately 10 microM). However, no efflux of 3H-5-HT from labeled platelets at either pH 6.7 or 7.4 occurred at similar concentrations of d-fenfluramine or d-norfenfluramine. With inhibition of platelet dense granule 3H-5-HT uptake by reserpine, efflux of 3H-5-HT was observed at pH 6, but not at pH 7.4. Fluoxetine, a SERT inhibitor, was a more potent inhibitor of uptake (IC50 0.05 microM) than d-fenfluramine, but the anorectic agent, phentermine, had no effect. Therefore, d-fenfluramine and d-norfenfluramine inhibit human platelet uptake of 5-HT in vitro at tissue concentrations attainable in vivo, but they do not stimulate 5-HT efflux due to dense granule sequestration. Inhibition of platelet 5-HT uptake may play a role in the cardiopulmonary toxicity of d-fenfluramine, but other factors probably contribute, since similar toxicity has not been observed with fluoxetine.

Isolation of two phenylethanoid glycosides from Eremophila gilesii

The leaves of Eremophila gilesii have been used traditionally to treat colds, headaches, sores, and chest pains. Our previous screening of Australian native plants showed that the methanol extract of the aerial parts of E. gilesii demonstrated notable inhibition of ADP-induced human platelet aggregation and serotonin release. Subsequent fractionation on the methanol extract led to the isolation of two phenylethanoid glycosides, verbascoside ( 1) and poliumoside ( 2). This is the first study reporting the presence of phenylethanoid glycosides in E. gilesii.

Gingerols and Related Analogues Inhibit Arachidonic Acid-Induced Human Platelet Serotonin Release and Aggregation

Gingerols, the active components of ginger (the rhizome of Zingiber officinale, Roscoe), represent a potential new class of platelet activation inhibitors. In this study, we examined the ability of a series of synthetic gingerols and related phenylalkanol analogues (G1–G7) to inhibit human platelet activation, compared to aspirin, by measuring their effects on arachidonic acid (AA)-induced platelet serotonin release and aggregation in vitro. The IC 50 for inhibition of AA-induced (at EC 50=0.75 mM) serotonin release by aspirin was 23.4±3.6 μM. Gingerols and related analogues (G1–G7) inhibited the AA-induced platelet release reaction in a similar dose range as aspirin, with IC 50 values between 45.3 and 82.6 μM. G1–G7 were also effective inhibitors of AA-induced human platelet aggregation. Maximum inhibitory (IC max) values of 10.5±3.9 and 10.4±3.2 μM for G3 and G4, respectively, were ∼2-fold greater than aspirin (IC max=6.0±1.0 μM). The remaining gingerols and related analogues maximally inhibited AA-induced platelet aggregation at ∼20–25 μM. The mechanism underlying inhibition of the AA-induced platelet release reaction and aggregation by G1–G7 may be via an effect on cyclooxygenase (COX) activity in platelets because representative gingerols and related analogues (G3–G6) potently inhibited COX activity in rat basophilic leukemia (RBL-2H3) cells. These results provide a basis for the design of more potent synthetic gingerol analogues, with similar potencies to aspirin, as platelet activation inhibitors with potential value in cardiovascular disease.

Decreased reuptake of serotonin in human platelets after surgery.

We have documented earlier a decrease in platelet serotonin and a concurrent increase in plasma serotonin, 5-hydroxytryptamin (5-HT) after various forms of stress, suggesting a disturbed platelet 5-HT reuptake mechanism following stress. In order to further elucidate these findings, we have studied platelet 5-HT reuptake kinetics (Vmax and Km) in nine patients before and 4 days after major, uncomplicated abdominal surgery. We found a significant decrease in the maximal 5-HT reuptake velocity (Vmax) after surgery and changes in Km, verifying alterations in the affinity of the platelet 5-HT transport system. The present results thus confirm the hypothesis that 5-HT reuptake kinetics are altered following adrenergic hyperactivity. A decrease in platelet 5-HT reuptake may bear implications for our understanding of poststress adaptive changes in the cardiovascular system as well as in the central nervous system (CNS) serotonergic neurones following stressful stimulation.

Modulation of adenylyl cyclase activity by noradrenaline and serotonin in human platelets

Modulation of adenylyl cyclase activity by noradrenaline and serotonin in human platelets

Rapid Inhibition by Sodium Azide of the Phosphoinositide-Mediated Calcium Response to Serotonin Stimulation in Human Platelets: Preservation in Alzheimer's Disease

The effect of sodium azide (NaN3) upon platelet Ca2+ signalling has been investigated. A 60 s preincubation with 1 mM NaN3 reduced the Ca2+ response to 1 μM serotonin without a corresponding reduction in the responses to 52 mU/ml thrombin or 70 μM β-amyloid(25–35) (Aβ(25–35)). The effect of NaN3 upon the response to serotonin, which was not blocked by either glutathione ethyl ester (GTEE) or dithiothreitol (DTT), was similar in platelets obtained from patients with Alzheimer's disease and from age- and gender-matched controls. After a preincubation time of 5 min was used, the Ca2+ response to thrombin was greatly reduced by 1 mM NaN3, but not by 50 μM 4-hydroxynonenal (HNE, 50 μM). Platelet levels of HNE and malondialdehyde were not significantly affected by up to 30 min of incubation with NaN3 at room temperature. It is concluded that the rapid effect of NaN3 upon the Ca2+ response to serotonin in human platelets is not mediated by an inhibition of cytochrome c oxidase, and is due to an action proximal to phosphoinositide-specific phospholipase C.

Study of molecular heterogeneity of human platelet serotonin transporter protein using monoclonal antibodies to its C-terminal fragment

Study of molecular heterogeneity of human platelet serotonin transporter protein using monoclonal antibodies to its C-terminal fragment

Interaction of 12-lipoxygenase with cellular proteins

Eicosapentaenoic acid (EPA) has been reported to have a potent anti-aggregatory activity and to be efficiently metabolized by 12-lipoxygenase, not by cyclooxygenase in platelets. In vitro effect of 12-lipoxygenase metabolites of EPA on platelet function was studied and compared with those of arachidonic acid (AA). The 12-lipoxygenase metabolites of AA and EPA; 12-hydroperoxyeicosatetraenoic acid (12-HPETE) and 12-hydroperoxyeicosapentaenoic acid (12-HPEPE), and their hydroxy derivatives, 12-hydroxyeicosatetraenoic acid (12-HETE) and 12-hydroxyeicosapentaenoic acid (12-HEPE) were prepared enzymatically using human platelet lysate. These compounds were purified by high performance liquid chromatography and identified by gas chromatography-mass spectrometry. 12-HPETE and 12-HPEPE inhibited dose-dependently washed human platelet aggregation and serotonin (5-HT) release induced by AA and collagen. The potency of 12-HPEPE was almost equal to that of 12-HPETE. Their hydroxy derivatives, 12-HETE and 12-HEPE were less potent. 12-hydroperoxy derivatives of AA and EPA were the most potent in inhibiting platelet aggregation and 5-HT release among 5-, 12- and 15-hydroperoxy isomers of AA and EPA. The inhibitory effects of 12-HPETE and 12-HPEPE on platelet aggregation were additive.

Evidence for separate serotonin and catecholamine compartments in human platelets

Serotonin (5-HT) and the catecholamines (CA), noradrenaline (NA) and adrenaline (Ad), are platelet dense-granule constituents which influence platelet activity and vessel tone. Platelets accumulate 5-HT via an active process whilst CA uptake occurs mainly by passive diffusion. The platelet contents and collagen-stimulated efflux of 5-HT, NA and Ad were examined in normal individuals to establish whether relationships exist between these monoamines. Regression analysis ievealed that platelet 5-HT was not related to platelet NA or Ad levels. Platelet NA, however, correlated positively with Ad ( r = 0.61, P < 0.01). Collagen-induced release of all three monoamines occurred in a dose-dependent manner. The collagen EC 50 values for 5-HT and CA release, however, differed and were greater for 5-HT release: 9.6 ± 0.8 vs 3.8 ± 0.2 μg/ml collagen, 5-HT vs. Na, P < 0.001; 9.6 ± 0.8 vs. 3.9 ± 0.5 μg/ml, 5-HT vs Ad, P < 0.001. These data may reflect differences regarding the triggering mechanisms for 5-HT and CA release and provide evidence for separate compartments of intra-platelet 5-HT and CA and possibly distinct populations of 5-HT and CA containing dense granules and/or platelets.

Endogenous serotonin in human blood platelets: factors that may influence reference values

The endogenous content of serotonin in human platelets has been used in many clinical studies to indicate platelet activation. A decrease in platelet serotonin compared with controls has been regarded as an indicator of platelet activation. However, the results published are difficult to compare, because of huge variations in endogenous serotonin between control groups in different investigations. This is likely to be because of lack of standardization. Several factors that influence the endogenous platelet serotonin content were studied in more than 200 blood donors. The most important factor was the total g force of the centrifugation used to isolate platelets. Also the age and sex of platelet donors, number of platelets in platelet-rich plasma, and mean platelet volume influenced normal serotonin values. Using a standardized centrifugation procedure (2700 g min) the mean endogenous serotonin was 2.80 nmol/10(9) platelets in young women and 2.58 in elderly women, and 2.67 in young men and 2.30 in elderly men. The differences both for age and sex were statistically significant. Endogenous platelet serotonin shows intrapersonal stability over time, since endogenous platelet serotonin did not change on repeated venepuncture for 9 weeks. Factors such as age, sex and isolation procedure must therefore be considered when endogenous platelet serotonin are studied in relation to disease and treatment.

Human blood platelet serotonin studiedin vitro: endogenous serotonin may stimulate thrombin-induced serotonin release in stored platelets

Human platelets with a high content of endogenous serotonin took up more serotonin when incubated with exogenous serotonin, than platelets with a low endogenous content of serotonin. Also, thrombin-stimulated serotonin secretion (%) was high when endogenous serotonin was high. This was not found with fresh platelets, but was found when platelets were stored as platelet concentrates for 5 and 7 days. With platelets stored for 5 or 7 days, both uptake and secretion were increased after preincubation of the platelets with an amount of exogenous serotonin that was completely taken up. Inhibition of the reuptake of serotonin by imipramine during thrombin-induced secretion increased the secretion in stored platelets. Agonists like collagen, ADP, and a prostaglandin analogue (U46619) gave only 3-7% secretion. Imipramine increased the secretion induced by U46619, but not the secretion induced by ADP or collagen. The specific 5-HT(2) receptor inhibitor, ketanserin, had no effect on agonist stimulated secretion, or secretion stimulated by a calcium ionophore (A23187). Both the uptake and the thrombin-induced secretion of serotonin correlated significantly with endogenous serotonin in stored platelets. In fresh platelets the uptake of serotonin correlated positively, although not significantly, with endogenous serotonin. It is speculated that endogenous serotonin may affect secretion through stimulation of the thrombin receptor, at least in stored platelets.

Acute effect of exercise on human blood platelet serotonin uptake and monoamine oxidase activity

Blood platelet serotonin (5-HT) uptake and monoamine oxidase (MAO) activity have been widely used as peripheral markers for presynaptic 5-HT reuptake and central monoamine metabolism (Lingj~erde 1990; Oreland et al 1980). This has led to interest in studying factors that might affect these parameters. Such studies have shown that both 5-HT uptake and MAO activity depend on the size of the blood platelets (Demet et al 1990; Murphy et al 1978; Gawel et al 1981). Since exercise tends to increase the concentration and the mean volume of circulating blood platelets (Jensen et al 1984), we reasoned that physical activity might influence measurements of 5-HT uptake and MAO activity. Moreover, depression and mania are associated with characteristic changes in the level of physical activity, so we reasoned that these changes might be responsible for differences in blood platelet 5-HT uptake and MAO activity observed in relation to various affective disorders (Meltzer and Arora 199l; Oreland et al 1980). The aim of this study was to determine whether light exercise immediately prior to blood sampling influences blood platelet 5-HT uptake and MAO activity.

Dithiothreithol promotes a higher affinity state of the serotonin transporter for the tricyclic antidepressant, imipramine

Neurotransmitter transporters are present in nerve terminal membranes and are responsible for the inactivation of released neurotransmitter by reuptake. One such transporter is the serotonin transporter which also represents the site of action of many clinically used tricyclic antidepressants, such as imipramine. Recently, most of the known Na+-dependent neurotransmitter transporters have been cloned and sequenced (for review see (I)). On the basis of sequence similarities, the transporters can be divided into two superfamilies. Within each family many features are conserved which may serve important structural, functional or regulatory roles. One such feature, conserved throughout the superfamily of which the serotonin transporter is a member, is two cysteines, nine amino acids apart, which are located on a putative large extracellular loop of the protein. It has been proposed that these cysteines may be involved in ligand binding and/or substrate uptake by the transporters, or perhaps function in maintaining the transporters in an active conformation (2). Thus, the effects of the sulphydryl reducing agent, dithiothreithol (DTT), on ligand binding to the human platelet serotonin transporter were studied. When the binding of the tricyclic antidepressant, [3H]imipramine, to human platelet membranes was measured over a range of increasing DTT concentrations, a concentration dependent increase in [3H]imipramine binding was found with maximum binding being achieved at a DTT concentration of 5 mM. Saturation studies were performed, incubating platelet membranes with a range of [3H]imipramine concentrations in the presence of DTT. Analysis of the saturation curves revealed a 2 to 3 fold decrease in the apparent KD value for [3H]imipramine binding to membranes in the presence of DTT, relative to the control value determined in the absence of DTT. This DTT induced decrease in the KD value was B,, values were unaffected by the presence of DTT (Table 1). The kinetic basis of this DTT induced increase in affinity of the platelet membranes for [3H]imipramine was also studied via dilution-induced dissociation experiments, as described previously (3). Human platelet membranes and [3H]imipramine were incubated in the presence of 10 mM DTT and diluted 50-fold into assay buffer containing 10 mM DTT. A slowing of the rate of dissociation of [3H]imipramine from the membranes was observed relative to the control experiment, which was carried out in the absence of DTT. The dissociation curve was biphasic in the absence of DTT while in the presence of DTT the curve was either monophasic (slowly dissociating) or tended towards this condition (Fig.1.). The dissociation rate of [3H]imipramine from platelet membranes was measured over a range of DTT concentrations. As the DTT concentrations increased, there was an apparent concentrationdependent shift in the [3H]imipramine dissociation curve from the

Hirudin - A new anticoagulant in haemodialysis (HD)

Heparin is the most widely used anticoagulant drug for prevention and treatment of thrombosis. However, inhibition of blood coagulation might not fully explain the antithrombic activity of this drug. The present study shows that different heparin preparations (50 nM) completely human platelet aggregation, serotonin release and thromboxane B2 production induced by purified neutrophil-derived cathepsin G (E.C. No. 3.4.21.20). This inhibitory effect was not related to the anticoagulant property of the compounds, since a heparin preparation with an activated active site for antithrombin II was also effective. Heparins inhibited the protease activity of the enzyme over the same range of concentrations. Since the effect of cathepsin G on platelets requires an intact proteolytic active site, the inhibitory effect of the drugs on cathepsin G-induced platelet activation may be explained by a blockade activity. Heparins were also shownto reduce platelet activation induced by cathepsin G released from activated polymorphonuclear leucocytes in mixed cell suspensions. As polymorphonuclear leucocytes might contribute to both arterial and venous thrombosis through platelet activation induced by the release of cathepsin G, this novel property of heparin could be used its antithrombotic efficacy.

Ligand binding to the serotonin transporter: equilibria, kinetics, and ion dependence.

The effects of Na+ and Cl- on the binding of [3H]imipramine and the cocaine analog [125I]-beta-carbomethoxy-3 beta-(4-iodophenyl)tropane([125I]-beta-CIT) to the human platelet serotonin transporter have been measured. The ion dependence of beta-CIT binding is consistent with binding beta-CIT together with one Na+ ion, but not in an ordered sequence. Imipramine affinity, like beta-CIT affinity, is increased by Na+, but imipramine binding involves at least two Na+ ions. This conclusion is based on the observation that both imipramine association rate constants and equilibrium affinity constants show a sigmoidal Na+ dependence. As with beta-CIT, the imipramine and Na+ binding sequence is not strictly ordered. Cl- increases imipramine affinity, apparently by slowing dissociation. beta-CIT binding occurs even in the absence of Na+ and Cl-. This provided a means to measure substrate and inhibitor affinity in both the presence and absence of cotransported ions. Nontransported inhibitors, such as imipramine and citalopram, as well as the transport substrates serotonin and 3,4-(methylenedioxy)methamphetamine all displaced beta-CIT binding in the absence of NaCl. In the absence of Cl-, Na+ increased the affinity of nontransported inhibitors but not of substrates. The results suggest that Na+ and Cl- induce independent changes in the transporter binding site and that binding of substrates and inhibitors is affected differently by these changes.