Endogenous platelet serotonin release monitored during aggregation by means of a new electrochemical technique

Differential pulse voltammetry combined with electrochemically treated carbon fibre microelectrodes was used to monitor endogenous serotonin release occurring during platelet aggreagtion. After platelet stimulation by thrombin, an oxidation peak was recorded at +280 mV. HPLC analyses performed with fluorimetric detection have shwon that this released electroactive compound was essentially serotonin. Moreover, serotonin measurements in the same samples by the technique reported here and by fluorimetry were found to be very similar (1.15 ± 0.30 μM and 1.17 ± 0.15 μM (mean ± dS.D., n = 6), respectively). Extracellular serotonin concentrations could be estimated either directly during aggregation or in supernatants obtained from stimulated or lysed platelets. Maximal serotonin concentrations have been found to be 6.93 ± 0.37 and 3.28 ± 0.39 nmol/10⁹ platelets from rat and human, respectively. Using the reported procedure, we have observed that no serotonin was released from thrombin-stimulated platelets prepared from rats treated with reserpine. Our new technique represents a selective and performant tool for rapid determination of endogenous serotonin platelet secretion.     

Effects of propranolol and pindolol on platelet aggregation and serotonin release

The aggregation of human platelets by adrenaline and adenosine di-phosphate (ADP) and its inhibition by β-blockers was studied by measuring the light transmission of plateletrich plasma (PRP) and suspensions of washed platelets exposed to these agents. Inhibition of aggregation of PRP and washed platelets was dose related in the two β-blockers tested: propranolol and pindolol. The potent β-blockers pindolol was less inhibitory than propranolol when adrenaline and ADP were used to induce platelet aggregation. The aggregation of platelets by adrenaline has two phases. With low doses of the blockers only the second phase was inhibited whereas higher doses blocked both phases. Preincubation of human platelets (PRP and washed platelets) with both blockers per se resulted in release of ¹⁴C-labelled serotonin. Propranolol released more serotonin than pindolol. There was no concomitant release of lactic dehydrogenase. It is concluded that the effects of propranolol and pindolol on platelets do not correlate with the β-blocking activity of these agents. Rather, the more lypophilic agent, propranolol, is more active both in inhibition of aggregation and in releasing platelet serotonin. It is suggested that these actions of the drugs are related to their non-specific membrane effects.     

Quantitation of paf-acether by release of endogenous platelet serotonin assessed by liquid chromatography with electrochemical detection

A new method to quantitate paf-acether (paf) was developed. It is based on the measurement of serotonin released from washed rabbit platelets challenged with paf. Platelets (1 X 10(8)/ml) were exposed with or without stirring to various concentrations of paf (26-130 pM) at 37 degrees C or at room temperature. Supernatants were submitted to a 4-min liquid chromatography run and serotonin was measured by electrochemical detection. We quantitated paf from three different biological sources, human neutrophils, mouse peritoneal macrophages, and cultured mast cells, comparing a classical method, i.e., platelet aggregation with the electrochemical detection of endogenous serotonin. We found similar results since, when compared with the aggregation method, the results differed by 12 to 47%. The sensitivity of both methods was 26 pM. The between-day variation coefficient was 23 and 14% (n = 12) for the aggregation method and the serotonin release, respectively, whereas the within-day variation coefficient for serotonin quantitation was less than 5% (n = 12). The superiority of the new method lies in its simplicity, the economy of platelets, and its possibility of automation. It can be applied to any agonist or any mechanism capable of releasing serotonin from platelets and more generally when a simple and fast method for measuring serotonin is desirable.     

KRDS, a new peptide derived from human lactotransferrin, inhibits platelet aggregation and release reaction

KRDS (Lys-Arg-Asp-Ser), a tetrapeptide from human lactotransferrin, was tested in vitro on human platelet function, and its effects were compared to those of RGDS, a tetrapeptide from human fibrinogen. Both peptides had a high probability of initiating a beta-turn and were highly hydrophilic. KRDS inhibited ADP-induced platelet aggregation [median inhibitory concentration (IC50) 350 microM] and fibrinogen binding (IC50 360 microM) to a lesser extent than RGDS (IC50 75 microM and 20 microM, respectively). Different from RGDS, thrombin-induced serotonin release was inhibited by KRDS (750 microM) on normal platelets (55 +/- 10%) and type I Glanzmann's thrombasthenia platelets (43% +/- 1). However, KRDS had no effect on cytoplasmic Ca2+ mobilization, inositol phospholipid metabolism or protein phosphorylation (myosin light chain P20 and P43). In contrast to RGDS, KRDS does not inhibit the binding of monoclonal antibody PAC-1 to activated platelets. KRDS and RGDS inhibited 4 beta-phorbol-12-myristate-13-acetate (PMA)-induced aggregation and fibrinogen binding, while proteins were normally phosphorylated. Thus, the tetrapeptide KRDS is (a) an inhibitor of serotonin release by a mechanism independent of protein phosphorylation and (b) an inhibitor of fibrinogen binding and, hence, aggregation by a mechanism that may not necessarily involve its direct binding to the glycoprotein IIb-IIIa-complex.     

Thiopental enhances human platelet aggregation by increasing arachidonic acid release.

Conflicting results have been reported regarding the effect of thiopental on aggregation and cytosolic calcium levels in platelets. The present study attempted to clarify these phenomena. Using platelet-rich plasma or washed suspensions, platelet aggregation, thromboxane (TX) B2 formation, arachidonic acid (AA) release, and cytosolic free calcium concentrations ([Ca2+]i) were measured in the presence or absence of thiopental (30-300 microM). Platelet activation was induced by adenosine diphosphate (ADP, 0.5-15 microM), epinephrine (0.1-20 microM) arachidonic acid (0.5-1.5 mM), or (+)-9,11-epithia-11,12-methano-TXA2 (STA2, 30-500 nM). Measurements of primary aggregation were performed in the presence of indomethacin (10 microM). Low concentrations of ADP and epinephrine, which did not induce secondary aggregation in a control study, induced strong secondary aggregation in the presence of thiopental (> or = 100 microM). Thiopental (> or = 100 microM) also increased the TXB2 formation induced by ADP and epinephrine. Thiopental (300 microM) increased ADP- and epinephrine-induced 3H-AA release. Thiopental (300 microM) also augmented the ADP- and epinephrine-induced increases in [Ca2+]i in the presence of indomethacin. Thiopental appears to enhance ADP- and epinephrine-induced secondary platelet aggregation by increasing AA release during primary aggregation, possibly by the activation of phospholipase A2.     

Second messengers in platelet aggregation evoked by serotonin and A23187, a calcium ionophore.

We investigated the combined effect of 5-hydroxytryptamine (5-HT, serotonin) and calcium ionophore (A23187) on human platelet aggregation. Aggregation, monitored at 37 degrees C using a Dual-channel Lumi-aggregometer, was recorded for 5 min after challenge by a change in light transmission as a function of time. 5-HT (2-200 microM) alone did not cause platelet aggregation, but markedly potentiated A23187 (low dose) induced aggregation. Inhibitory concentration (IC50) values for a number of compounds were calculated as means +/- SEM from dose-response determinations. Synergism between 5-HT (2-5 microM) and A23187 (0.5-2 microM) was inhibited by 5-HT receptor blockers, methysergide (IC50 = 18 microM) and cyproheptadine (IC50 = 20 microM), and calcium channel blockers (verapamil and diltiazem, IC50 = 20 microM and 40 microM respectively). Interpretation of the effects of these blockers is complicated by their lack of specificity. Similarly, U73122, an inhibitor of phospholipase C (PLC), blocked the synergistic effect at an IC50 value of 9.2 microM. Wortmannin, a phosphatidylinositide 3-kinase (PI 3-K) inhibitor, also blocked the response (IC50 = 2.6 microM). However, neither genistein, a tyrosine-specific protein kinase inhibitor, nor chelerythrine, a protein kinase C inhibitor, affected aggregation at concentrations up to 10 microM. We conclude that the synergistic interaction between 5-HT and ionophore may be mediated by activation of PLC/Ca2+ and PI 3-kinase signalling pathways, but definitive proof will require other enzyme inhibitors with greater specificity.     

Platelet dense granule release reaction monitored by high-performance liquid chromatography-fluorometric determination of endogenous serotonin.

The platelet dense granule release reaction was monitored by measuring the serotonin content of platelets collected by filtration following release. Sensitive HPLC-fluorometric analysis permitted release to be assessed using small (25-50 microliters) volumes of plasma and whole blood. Release could be examined for periods as short as 1 s due to the rapid stoppage effected by filtration. The half-life of thrombin-stimulated (1 U/ml) release was 2-3 s, the EC50 values observed for thrombin-stimulated release over a 60-s release period were 0.01-0.03 U/ml for diluted whole blood and plasma samples.     

Methodology of monitored release of a new preparation: buprenorphine.

The analgesic agent Temgesic (buprenorphine) was made available under monitored release regulations for one year, which yielded data from 1736 doctors on 9123 patients. An analysis of 17 120 administrations of the drug confirmed the results of the pre-licensing clinical studies encompassing data from nine British hospitals on 483 patients. No important new adverse effects attributable to the product were observed, and apart from giving reassurance, the usefulness of monitored release is questioned in the light of the relatively small amount of additional information arising from it.     

Correlated measurement of platelet release and aggregation in whole blood

We have used a technique for the simultaneous measurement of platelet activation and aggregation in whole blood using two-color immunofluorescence and flow cytometry to study the relationship between the release reaction and aggregation. A monoclonal antibody specific for the alpha granule membrane protein GMP-140 was used to measure the release reaction, and a monoclonal antibody specific for platelet membrane glycoprotein Ib (GPIb) was used to identify platelets and platelet aggregates. Aggregates were identified as particles expressing both levels of GPIb and size larger than that of resting single platelets. Anticoagulated whole blood was incubated with platelet agonists. At various times samples of the blood were removed and immediately fixed with paraformaldehyde. Blood that had been anticoagulated with ethylenediamine tetraacetic acid showed progressive release of platelets but little or no aggregation. However, blood anticoagulated with citrate or heparin showed correlated release and aggregation. The degree of aggregation was greater in heparin than in citrate. The expression of GPIb and GMP-140 increased in direct proportion to the size of the aggregates. Aggregates were observed varying in apparent diameter up to approximately 20 microns. During prolonged incubation there was progressive disaggregation of adenosine diphosphate (ADP)-induced aggregates. After disaggregation the proportion of GMP-140 negative single platelets increased, indicating that both released and nonreleased platelets participated in the aggregation. There was little or no disaggregation of phorbol myristate acetate (PMA)-induced aggregates. The relatively small size and reversibility of platelet aggregates that we have observed in whole blood may be relevant to phenomena occurring in vivo and in extracorporeal circulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endogenous serotonin release from the dopamine-deficient striatum of the weaver mutant mouse

In addition to an altered dopaminergic input, the striatum of the weaver mutant mouse (wv/wv) has increased serotonin tissue content and uptake compared to the wild-type mouse (+/+). To gain information regarding the functional status of serotonergic inputs to thewv/wv striatum, endogenous serotonin release fromwv/wv and +/+ striatum was measured under basal conditions as well as in the presence of fenfluramine or elevated concentrations of potassium (K⁺). Fractional basal release of serotonin from the +/+ striatum was significantly greater than that from thewv/wv striatum. In the presence of K⁺, evoked release (stimulated release minus basal release) was greater from the +/+ striatum than from thewv/wv striatum. In the presence of fenfluramine, evoked serotonin release was greater from thewv/wv striatum compared to the +/+ striatum. These data are consistent with the involvement of an additional transmitter(s) in modulating serotonin release to a greater extent in thewv/wv than the +/+ striatum. The data on fenfluramine-stimulated serotonin release suggest that the additional serotonin content found in thewv/wv striatum is in a releasable pool but that striatal serotonin release might be attenuated more inwv/wv than in +/+ mice.     

High concentrations of arachidonic acid induce platelet aggregation and serotonin release independent of prostagladin endoperoxides and thromboxane A2

We examined platelet aggregation and serotonin release, induced by less than 60 μM arachidonic acid, using washed platelet suspensions in the absense of albumin. The concentration of arachidonic acid use did not cause platelet lysis. Platelet responses induced by less than 20 μM arachidonic acid were inhibited by aspirin, whereas those induced by above 30 μM arachidonic acid were not inhibited, even by both aspirin and 5,8,11,14-eicosatetraynoic acid. Although phosphatidic acid and 1,2-diacylglcerol increased after the addition of arachidonic acid in aspirin-treated platelets, the amounts were not parallel to platelet aggregation. Oleic, linoleic and linolenic acids also induced platelet responses, while palmitic, stearic and arachidic acids did not. EDTA, dibutyryl cyclic AMP, apyrase and creatine phosphate / creatin phosphokinase brought about almost the same effects in platelet responses induced by the unsaturated fatty acids, other than arachodinic acid, as those induced by 40 μM arachodonic acid. These results suggest that the mechanism of the actions of more than 30 μM arachodinic acid on platelets is the same as that of the other unsaturated fatty acids and is independent of prostaglandin endoperoxides, thromboxane A₂ and, perhaps, phosphatidic acid and 1,2-diacylglycerol.     

The effect of halofenate and clofibrate on aggregation and release of serotonin by human platelets.

The hypolipidemic drugs halofenate and clofibrate inhibit the aggregation of human platelets induced by ADP, collagen and epinephrine. The effect of the drugs is primarily on the “second wave” of aggregation which is associated with the platelet release reaction. The drugs also inhibit the release of serotonin from platelets suggesting that the effect on aggregation is attributable to an inhibition of the release reaction. Halofenate is much more potent than clofibrate in inhibiting both the release of serotonin and the second wave of aggregation.     

Inhibition by sulfhydryl agents of arachidonic acid-induced platelet aggregation and release of potential inflammatory substances

Sulfhydryl agents (mercaptoethanol, thioglycerol, dithiotreitol, and sodium diethyldithiocarbamate) prevented aggregation of rabbit platelets and the accompanying generation of pharmacologically active substances due to arachidonic acid. Inhibition was also found after administration of the antagonists. This antagonism was suppressed if the inhibitors were removed from the platelet suspension by washing procedures, whereas inhibition by indomethacin was irreversible. Amino-thiol reagents either failed to antagonize the effects of AA or potentiated them. CuCl₂ increased the amounts of pharmacologically active substances generated in incubates of intact platelets with arachidonic acid, and reversed the inhibition due to thiol agents, but did not interfere with inhibition by indomethacin. Platelets suspended in Tyrode solution generated unstable pharmacologically active substances upon incubation with arachidonic acid ; stability of these substances could be maintained at 4°C. Generation of this temperature — sensitive material was inhibited by indomethacin and by thiol agents. Interference with a copper-containing component of PG synthetase or reduction of an intermediate lipoperoxide appear as two possible mechanisms of action of thiol agents.     

Human platelet electrorotation changed induced by activation: inducer specificity and correlation to serotonin release

Electrorotation of single platelets was compared with [¹⁴C]serotonin release, aggregation and electron microscopy. Activation of washed and degranulated platelets was induced by thrombin, arachidonic acid, collagen, adrenaline, platelet activation factor (PAF (PA), ADP and A23187. A strong correlation between electrorotation decrease and serotonin release was found. Electrorotation did not correlate with aggregation. It was concluded that an increase of the specific conductivity of the platelet membrane by three orders of magnitude (approx. 1.0–10⁻⁷ S · m⁻¹ to 1.0·10⁻⁴ S · m⁻¹) upon activation was responsible for the observed decrease of anti-field rotation and the shift of the first characteristic frequency towards higher values. Electrorotation allowed for time-dependent measurements of activation. Characteristic activation times in the order of minutes were found. There was the following sequence of activators classified by increasing activation time constants: A23187 was the fastest followed by thrombin, collagen, PAF, arachidonic acid, adrenaline, and ADP.     

Human platelet electrorotation change induced by activation: inducer specificity and correlation to serotonin release

Electrorotation of single platelets was compared with [14C]serotonin release, aggregation and electron microscopy. Activation of washed and degranulated platelets was induced by thrombin, arachidonic acid, collagen, adrenaline, platelet activation factor (PAF), ADP and A23187. A strong correlation between electrorotation decrease and serotonin release was found. Electrorotation did not correlate with aggregation. It was concluded that an increase of the specific conductivity of the platelet membrane by three orders of magnitude (approx. 1.0.10(-7) S.m-1 to 1.0.10(-4) S.m-1) upon activation was responsible for the observed decrease of anti-field rotation and the shift of the first characteristic frequency towards higher values. Electrorotation allowed for time-dependent measurements of activation. Characteristic activation times in the order of minutes were found. There was the following sequence of activators classified by increasing activation time constants: A23187 was the fastest followed by thrombin, collagen, PAF, arachidonic acid, adrenaline, and ADP.