Activation of human platelets by 2-arachidonoylglycerol: role of PKC in NO/cGMP pathway modulation

We demonstrated that the endocannabinoid 2-arachidonoylglycerol (2-AG) activated dose-dependently washed human platelets and increased intracellular calcium levels. Moreover 2-AG activated protein kinase C measured as p47pleckstrin phosphorylation. These parameters were prevented by the tromboxane A2 receptor antagonist SQ29548, by phospholipase C pathway (U73122) and protein kinase C (GF109203X) inhibitors. No effect on 2-AG-induced platelet activation and calcium elevation in the presence of inhibitors of fatty acid amide hydrolase or monoacylglycerol lipase was observed. In addition we have shown that 2-AG dose-dependently increased NO and cGMP levels. These effects were abolished by U73122, GF109203X, EGTA and the intracellular calcium chelator BAPTA/AM. Moreover, 2-AG enhanced eNOS activity through the phosphorylation of its positive regulatory residue ser1177 and by dephosphorylation of the negative one thr495. The eNOS ser1177 phosphorylation was inhibited by U73122 and GF109203X but it was unaffected by the PI3K/AKT pathway inhibitors LY294002 and MK2206. The dephosphorylation of thr495 was reversed by low concentrations of calyculin A. Taken together these data suggest that 2-AG behaves as a true platelet agonist stimulating PKC activation and calcium elevation. Likely 2-AG can modulate platelet activation by increasing NO levels through eNOS activation.     

Microbubble-induced serotonin secretion in human platelets

The effect of nitrogen (N2) microbubbles on platelets resembles that of common platelet agonists with respect to aggregation (Thorsen T et al., Undersea Biomed Res 1986; 13: 289-303). In the present study we examined the effect of microbubbles on platelet secretion of preloaded 14C-serotonin. We demonstrate that stirring of platelet-rich plasma with N2-microbubbles causes a loss of single platelets that is associated with secretion. However, secretion did not increase above baseline values until after 20 min of microbubble exposure, when platelet aggregation had reached 40%. After that time the secretion rate increased. There was no correlation between secreted serotonin and the degree of platelet aggregation. Although no 14C-serotonin secretion occurred in presence of acetylsalicyclic acid (ASA), microbubble-induced platelet aggregation was only marginally reduced. Epinephrine alone caused significant platelet aggregation but no 14C-serotonin secretion and it enhanced N2-microbubble-induced platelet aggregation and secretion; ASA completely prevented secretion under these circumstances but failed to abolish the enhancement of aggregation compared with microbubbles alone. Earlier studies have shown that platelets adhere to the bubble surfaces (Thorsen T et al., Undersea Biomed Res 1987; 14: 45-59). The results in the present study indicate that non-adhering platelets in the bulk phase are not activated by means of autocrine stimulation through dense granule material.     

Microsomal CA2+ uptake in human platelets is stimulated by calmodulin

Calcium accumulating vesicles were prepared from washed human platelets. Ca2+ uptake could be stimulated with calmodulin. The stimulation of Ca2+ uptake was not abolished by the adenylate cyclase inhibitor 2',5'-dideoxyadenosine, thus excluding enhanced cAMP formation as a possible cause for the calmodulin effect. Our findings suggest that, in addition to cAMP, calmodulin is also involved in the regulation of platelet free Ca2+ concentrations, i.e. Ca2+ homeostasis in platelets is under dual control.     

Activation of human microglia by fibrillar prion protein-related peptides is enhanced by amyloid-associated factors SAP and C1q

Complement activation products C1q and C3d, serum amyloid P component (SAP) and activated glial cells accumulate in amyloid deposits of conformationally changed prion protein (PrP(Sc)) in Creutzfeldt-Jakob disease, Gerstmann-Str?ussler-Scheinker disease and scrapie-infected mouse brain. Biological properties, including the potential to activate microglia, relate to prion (PrP) peptide fibrillogenic abilities. We investigated if SAP and C1q influence the fibrillogenic properties of human and mouse PrP peptide and concomitantly their stimulatory effects on human microglia in vitro. PrP-peptide induced microglial IL-6 and TNF-alpha release significantly increased in the presence of SAP and C1q. Also, SAP and C1q enhanced PrP-peptide fibril formation as revealed by electron microscopy and thioflavin S-based quantitative assays. This suggests that SAP and C1q contribute to fibrillar state-dependent cellular effects of PrP. Combined, ultrastructural and thioflavin assays, together with microglial cytokine release measurements, provide a test system to screen potential, fibrillarity impeding therapeutics for prion disease.     

Heterologous supersensitization between serotonin2 and alpha2-adrenergic receptor-mediated intracellular calcium mobilization in human platelets

Summary Recent reports suggest that serotonin (5-HT)₂ receptor-mediated second messenger systems are enhanced in platelets of affective disorders. To make the mechanism of the enhanced response clear, we investigated 5-HT₂ and alpha ()₂-adrenergic receptor-induced intracellular calcium (Ca²⁺) mobilization in platelets of healthy volunteers, using fura-2. 5-HT₂ and ₂-adrenergic receptor-mediated Ca²⁺ mobilization was enhanced by prior exposure to the other type of agonist, so called heterologous supersensitization. The supersensitization was due to the enhancement of maximal response without change in agonist affinity. Chelating extracellular Ca²⁺ did not diminish the supersensitization. This enhancement of Ca²⁺ mobilization was not inhibited by H-7, an inhibitor of protein kinase C. However, this supersensitization was inhibited by pretreatment with sodium fluoride which directly activates guanine nucleotide binding regulatory proteins (G proteins). These results suggest that the supersensitization was caused from intracellular Ca²⁺ storage sites through a G protein-coupled pathway.Abbreviations fura-2/AM 1-(2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy)-2-(2-amino-5-methylphenoxy)-ethane-N,N,N, N-tetraacetic acid, pentaacetoxymethyl ester - H-7 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride - EGTA ethylenedioxybis(ethylamine)-N,N,N,N-tetraacetic acid - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - NaF sodium fluoride - Fmax maximal fluorescence intensity - Fmin minimal fluorescence intensity     

Effects of various anions on serotonin transport in human platelets.

A survey is given of some results from the author's studies on serotonin (5HT) uptake in human blood platelets in vitro. These findings are relevant also for serotonin reuptake at serotonergic synapses, since it has been shown that the serotonin transporter (5HTT) in platelets is identical to that in the brain. The main findings: 5HT uptake is dependent on the presence of Cl-, which shows simple saturation kinetics in relation to 5HT uptake rate, probably by acting on a hypothetical "anion site". Chloride can, however, be partly replaced by other small anions like bromide, iodide and nitrite. Unexpectedly, increasing concentrations of nitrite gives a sigmoidal 5HT uptake curve, indicating a double effect: being able to replace chloride in the "anion site", and exerting an allosteric effect by being bound to a separate "activating site". It was later found that the monocarboxylic acids acetate, lactate and pyruvate, which can not replace chloride, have an allosteric stimulatory effect on 5HT uptake in the presence of chloride, presumably by acting at the same activating site as shown for nitrite. The chloride concentration, as well as lactate and puruvate, may exert a regulatory effect on serotonin reuptake in the brain.     

Collagen-induced calcium influx is enhanced in platelets from hypercholesterolemic rabbits

We investigated the mechanism of hyperreactivity to collagen of the platelets from hypercholesterolemic rabbits, as compared with cholesterol-enriched platelets prepared in vitro by incubation of normal platelets with hypercholesterolemic plasma or cholesterol-lecithin dispersion. The platelets from rabbits fed high cholesterol diet for only 1 to 2 weeks, had an enhanced reactivity in aggregation and secretion, as well as an increase in cholesterol content and a decrease in membrane lipid fluidity. The cholesterol-enriched platelets prepared in vitro also had the same properties. In all these cholesterol-enriched platelets, two membrane functions, Ca2+-influx and arachidonate liberation from membrane phospholipids, were markedly enhanced in response to collagen, probably due to the influence of cholesterol upon physical properties of the membranes. These data suggest that in platelets from hypercholesterolemic rabbits, the decrease in membrane fluidity, accompanying the increase in membrane cholesterol content, brings about the enhancement of Ca2+-influx and liberation of arachidonate, thus resulting in hypersensitivity to collagen.     

Activation of permeabilized platelets by inositol-1,4,5-trisphosphate

The effect of inositol 1,4,5-trisphosphate (IP3) was studied using human platelets permeabilized with saponin and suspended in a high potassium, Ca2+-free buffer containing 40 microM EGTA and 1.2 mM magnesium. Under these conditions IP3 stimulated aggregation at a concentration of 0.5 microM with maximum aggregation at 5.0 microM. Aggregation was associated with phosphorylation of myosin light chain and a 47,000 dalton protein, and with a change in platelet shape including granule centralization and pseudopod formation similar to changes seen when cytoplasmic calcium is raised by other means. IP3 stimulated [14C]-serotonin release from platelet dense granules, [14C]-arachidonic acid release from platelet phospholipids and production of thromboxane B2. Preincubation of platelets with aspirin which blocked thromboxane formation also inhibited protein phosphorylation, serotonin secretion and partially inhibited aggregation. These results support the concept that IP3 is a major intracellular messenger in platelets and suggests that its effects are mediated both through Ca2+ flux and thromboxane formation.     

Activation of human and rabbit blood platelets by synthetic structural analogs of platelet activating factor

The proaggregatory potencies of synthetic analogs of platelet activating factor (PAF-acether: 1-0-alkyl 2-0-acetyl sn-glycero-3-phosphorylcholine) were tested in rabbit and human platelet rich plasma (PRP). For activation of human compared to rabbit platelets about 30 to 100 times higher concentrations of the compounds were needed, but the relative potencies of the analogs tested were quite similar in both species. The absolute stereospecificity required for platelet activation could be confirmed and extended also to the pair of enantiomers with a cis-9 double bond in the 0-alkyl chain and to the enantiomers with the 0-alkyl chain at C-2 of glycerol. The propionyl ester at C-2 of glycerol proved to be as potent as the acetyl ester but replacement by the formyl as well as by the butyryl ester reduced the activity to less than three percent. Variations at the 3-phosphorylcholine substituent all abolished the activity. It appears that the correct stereochemistry and a phosphorylcholine group in a strictly defined position towards the asymmetric C-2 of the glycerol backbone are absolute requirements for activity whereas variations in the alkyl and the short chain acyl groups have not as dramatic consequences.     

Effects of enhanced P2X1 receptor Ca2+ influx on functional responses in human platelets

G-protein-coupled P2Y1 and P2Y12 receptors play key roles in platelet activation, however the importance of ionotropic P2X1 receptors remains unclear. Platelet P2X1 responses are highly labile in vitro, but were greatly enhanced by increasing [Ca2+]o in the range 1-10 mM. The P2X1 agonist alpha,beta-MeATP stimulated a shape change which saturated at peak [Ca2+]i of > or = 400 nM, without evidence for aggregation. The maximal P2X1-evoked transmission decrease was 82% of that obtained via P2Y1 receptors. alpha,beta-MeATP caused a disc to sphere transformation in virtually all platelets, but lacked the long processes produced by ADP. Following block of P2Y1 receptors with A3P5PS, co-stimulation with alpha,beta,-MeATP and ADP failed to induce aggregation despite the generation of peak [Ca2+]i responses similar to those stimulated via P2Y1 receptors. Therefore early, transient Ca2+ influx via P2X1 receptors can contribute to platelet activation by stimulating a significant morphological change, but does not readily synergise with P2Y12 receptors to support aggregation.     

Activation of guinea pig platelets by a monoclonal antibody

We herein describe the characterization of a monoclonal anti-guinea pig platelet antibody, termed GT2-12, which causes aggregation, ATP and [³h]serotonin release, and platelet protein phosphorylation. GT212 can bind to platelets and megakaryocytes in guinea pig bone marrow cells. A protein of 34,000 daltons was detected by immunoprecipitation of platelet lysates with GT2-12. Incubation of ³²P-labeled guinea pig platelets with GT2-12 resulted in rapid phosphorylation of proteins of 40,000 and 20,000 daltons. GT2-12-induced aggregation and protein phosphorylation of platelets were inhibited by diltiazem, TMB-8 and dibutyryl cAMP and partially inhibited by indometacin. The F(ab′)₂ fragment of GT2-12 IgG also induced platelet aggregation, indicating that activation is not mediated by Fc receptors. This type of antibody should be useful for studying platelet function in the guinea pig model.     

Protein phosphorylation and diglyceride production during serotonin release induced by epinephrine plus ADP in human platelets

Epinephrine (1 microM) plus ADP (5 microM) induced serotonin release from human platelets although neither epinephrine nor ADP alone brought about such a release. During this release reaction, the phosphorylation of 40K-dalton protein was induced to an extent similar to that induced by 0.5 unit/ml thrombin. The amount of diglyceride (DG) produced by epinephrine plus ADP, however, was much smaller than that produced by thrombin. The reaction velocities of these reactions induced by epinephrine plus ADP were slower than those induced by thrombin. Epinephrine or ADP alone hardly produced any DG and induced 40K-dalton protein phosphorylation only slightly. Indomethacin (1 microgram/ml), a cyclooxygenase inhibitor, remarkably inhibited epinephrine plus ADP induced serotonin release, 40K-dalton protein phosphorylation and DG production although this agent had little effect on the same reactions induced by thrombin. These results suggest that prostaglandin endoperoxides or thromboxane A2 may be involved in serotonin release, 40K-dalton protein phosphorylation and DG production induced by epinephrine plus ADP.     

Dual antagonism by L-636,499 of serotonin and thromboxane A2 induced aggregation of canine platelets

We evaluated the ability of L-636,499 (3-carboxyl-dibenzo-[b,f] thiepen-5,5-dioxide), a compound structurally similar to cyproheptadine, to antagonize U46619 (a TXA2/PGH2 mimetic) and serotonin (5-hydroxytryptamine; 5HT)-induced aggregation of canine platelets in vitro. L-636,499 antagonized competitively and dose-dependently aggregation induced by both 5HT and U46619, with pA2 values of 5.8 +/- 0.6 and 4.8 +/- 0.2, respectively. L-670,596, a potent TXA2/PGH2 receptor antagonist, and ketanser in, a potent 5HT2 receptor antagonist, yielded pA2 values of 7.0 +/- 0.3 and 9.0 +/- 0.2 vs. their respective agonists. These results show that despite its low potency vs. TXA2- and 5HT2-induced aggregation, L-636,499 antagonizes both physiologic mediators comparably.     

Phencyclidine-induced locomotor hyperactivity is enhanced in mice after stereotaxic brain serotonin depletion

The aim of this study was to investigate the role of forebrain serotonin projections in behavioural models with relevance to schizophrenia. Mice received stereotaxic micro-injections of the serotonin neurotoxin 5,7-dihydroxytryptamine into the median raphe nucleus (MRN). Two weeks later, MRN-lesioned mice were hyperactive at baseline and showed enhanced locomotor hyperactivity induced by phencyclidine. In contrast, no lesion effect was observed on the locomotor hyperactivity induced by amphetamine treatment or on prepulse inhibition. Lesioned mice showed a 68% depletion of serotonin in the hippocampus and 31% depletion in the striatum. These data confirm previous studies in rats that selective serotonin depletion in the brain enhances the effect of phencyclidine, but not amphetamine, on locomotor activity. This enhanced action of phencyclidine is likely to be mediated by the absence of serotonin-mediated behavioural inhibition in the hippocampus, leaving the psychostimulant effects of phencyclidine unopposed. Taken together with previous studies in rats, these studies in mice suggest that serotonin release in the dorsal hippocampus constitutes a behavioural inhibitory pathway normally involved in dampening excessive behavioural stimulation. Dysfunction of this pathway could be involved in psychosis and its stimulation could be a potential mechanism of action of antipsychotic drugs.     

Activation of dorsal raphe serotonin neurons is necessary for waiting for delayed rewards

The forebrain serotonergic system is a crucial component in the control of impulsive behaviors. We previously reported that the activity of serotonin neurons in the midbrain dorsal raphe nucleus increased when rats performed a task that required them to wait for delayed rewards. However, the causal relationship between serotonin neural activity and the tolerance for the delayed reward remained unclear. Here, we test whether the inhibition of serotonin neural activity by the local application of the 5-HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin in the dorsal raphe nucleus impairs rats' tolerance for delayed rewards. Rats performed a sequential food-water navigation task that required them to visit food and water sites alternately via a tone site to get rewards at both sites after delays. During the short (2 s) delayed reward condition, the inhibition of serotonin neural activity did not significantly influence the numbers of reward choice errors (nosepoke at an incorrect reward site following a conditioned reinforcer tone), reward wait errors (failure to wait for the delayed rewards), or total trials (sum of reward choice errors, reward wait errors, and acquired rewards). By contrast, during the long (7-11 s) delayed reward condition, the number of wait errors significantly increased while the numbers of total trials and choice errors did not significantly change. These results indicate that the activation of dorsal raphe serotonin neurons is necessary for waiting for long delayed rewards and suggest that elevated serotonin activity facilitates waiting behavior when there is the prospect of forthcoming rewards.     

Brain-derived neurotrophic factor is stored in human platelets and released by agonist stimulation

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, plays critical roles in the survival, growth, and maintenance of brain and peripheral neurons. We report the presence of BDNF protein in human platelets and its release upon agonist stimulation. The BDNF content of washed platelets varied widely, from 3.5 to 67 ng/4 x 10(8) platelets, averaging 25.2 +/- 21.2 ng/4 x 10(8) platelets (mean+/-SD). The BDNF concentration in platelet-poor plasma was low (1.7+/-1.7 ng/ml, n = 11). Thrombin, collagen, the Ca++ ionophore A23187, and shear stress each induced a rapid release of BDNF from platelets. Up to only half of platelet BDNF was secreted upon agonist stimulation, suggesting that platelets may have a non-releasable pool of BDNF, or that the released BDNF binds to a recognition site on the platelet surface and is internalized, as occurs with serotonin. However, the cognate BDNF receptor, TrkB, was not detected in platelets. Nevertheless, the ability of BDNF to bind washed platelets was shown by FACS analysis confocal microscopy and by the binding and apparent internalization of [125I]-BDNF by platelets. A very high affinity site (Kd = 130 x 10(-15) M, approximately 80 sites/platelet) and a moderately high affinity site (Kd = 20 nM, approximately 3750 sites/platelet) were identified. The BDNF content in two megakaryocytic cell lines, DAMI and Meg-01, was only 0.1% of the content measured in platelets. No BDNF mRNA was detected by Northern blotting in these cell lines or in platelets. The pituitary gland was also ruled out as a source for platelet BDNF, since the BDNF content of rat platelets did not decrease 2 weeks after hypophysectomy. Thus, platelet BDNF is not acquired from the megakaryocyte or pituitary gland, but is probably acquired from other sources via the blood circulation. Platelets appear to bind, store and release BDNF upon activation at the site of traumatic injury to facilitate the repair of peripheral nerves or other tissues that contain TrkB.