Thrombin-induced Serotonin Release Research Articles

Anti‐platelet Activity of Erythro‐(7S,8R)‐7‐acetoxy‐3,4,3′,5′‐tetramethoxy‐8‐O‐4′‐neolignan from Myristica fragrans

Platelets play a critical role in pathogenesis of cardiovascular disorders and strokes. The inhibition of platelet function is beneficial for the treatment and prevention of these diseases. In this study, we investigated the anti-platelet activity of erythro-(7S,8R)-7-acetoxy-3,4,3',5'-tetramethoxy-8-O-4'-neolignan (EATN), a neolignan isolated from Myristica fragrans, using human platelets. EATN preferentially inhibited thrombin- and platelet-activating factor (PAF)-induced platelet aggregation without affecting platelet damage in a concentration-dependent manner with IC50 values of 3.2 ± 0.4 and 3.4 ± 0.3 μM, respectively. However, much higher concentrations of EATN were required to inhibit platelet aggregation induced by arachidonic acid. EATN also inhibited thrombin-induced serotonin and ATP release, and thromboxane B2 formation in human platelets. Moreover, EATN caused an increase in cyclic AMP (cAMP) levels and attenuated intracellular Ca(2+) mobilization in thrombin-activated human platelets. Therefore, we conclude that the inhibitory mechanism of EATN on platelet aggregation may increase cAMP levels and subsequently inhibit intracellular Ca(2+) mobilization by interfering with a common signaling pathway rather than by directly inhibiting the binding of thrombin or PAF to their receptors. This is the first report of the anti-platelet activity of EATN isolated from M. fragrans.

The effect of Sildenafil on human platelet secretory function is controlled by a complex interplay between phosphodiesterases 2, 3 and 5

Human platelets contain the cyclic nucleotide-hydrolyzing phosphodiesterases (PDEs) 2, 3 and 5. The cGMP-PDE5 inhibitors Sildenafil and Zaprinast have been demonstrated to potentiate the anti-platelet aggregatory effect of NO donors like sodium nitroprusside (SNP) in vitro but the mechanisms of Sildenafil's action on the secretory function of human platelets have not been analysed in detail. In the present paper, we show (1) that both compounds potentiate the SNP-induced increase in cGMP in human platelets concentration-dependently. (2) However, whereas Sildenafil plus SNP treatment only partially inhibits thrombin-induced release of serotonin, the less selective Zaprinast plus SNP cause a complete inhibition. (3) The inhibition mediated by Sildenafil plus SNP is limited to low compound concentrations at which cAMP levels are increased, probably due to cGMP-mediated inhibition of PDE3. (4) High concentrations of Sildenafil (plus SNP) neither affect cAMP levels, likely due to the activation of PDE2, nor inhibits the release of serotonin. Thus, increases in both cyclic nucleotides seem to control platelet function. (5) Accordingly, treatment with increasing concentrations of Sildenafil plus SNP and a selective PDE2 inhibitor, which by its own has no effect, induced a concentration-dependent increase in cAMP and complete inhibition of platelet activation. In summary, our data indicate that Sildenafil inhibits secretory function of human platelets at least in part due to the cGMP-mediated effects on intracellular cAMP and that entire inhibition of serotonin release from thrombin-activated platelets is controlled by both cyclic nucleotides.

Expression of scinderin in megakaryoblastic leukemia cells induces differentiation, maturation, and apoptosis with release of plateletlike particles and inhibits proliferation and tumorigenesis

Rapid proliferation of atypical megakaryoblasts is a characteristic of megakaryoblastic leukemia. Cells from patients with this disorder and cell lines established from this type of leukemia showed the presence of gelsolin but the absence of scinderin expression, 2 filamentous actin-severing proteins present in normal megakaryocytes and platelets. Vector-mediated expression of scinderin in the megakaryoblastic cell line MEG-01 induced a decrease in both F-actin and gelsolin. This was accompanied by increased Rac2 expression and by activation of the PAK/MEKK.SEK/JNK/c-jun, c-fos transduction pathway. The Raf/MEK/ERK pathway was also activated in these cells. Transduction pathway activation was followed by cell differentiation, polyploidization, maturation, and apoptosis with release of platelet-like particles. Particles expressed surface CD41a antigen (glycoprotein IIb/IIIa or fibrinogen receptor), had dense bodies, high-affinity serotonin transport, and circular array of microtubules. Treatment of particles with thrombin induced serotonin release and aggregation that was blocked by CD41a antibodies. PAC-1 antibodies also blocked aggregation. Exposure of cells to PD98059, a blocker of MEK, inhibited antigen CD41a expression, increases in cell volume, and number of protoplasmic extensions. Cell proliferation and cell ability to form tumors in nude mice were also inhibited by the expression of scinderin. MEG-01 cells expressing scinderin had the same fate in vivo as in culture. Thus, when injected into nude mice, they entered apoptosis and released platelet-like particles. The lack of scinderin expression in megakaryoblastic leukemia cells seems to be responsible for their inability to enter into differentiation and maturation pathways characteristic of their normal counterparts.

Myristoylated alanine-rich C kinase substrate phosphorylation is involved in thrombin-induced serotonin release from platelets.

Stimulation of platelets by thrombin induces protein kinase C (PKC) activation, phosphorylation of pleckstrin, aggregation and serotonin release. Here, we demonstrate that, in human platelets, thrombin stimulation also induced phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) and serotonin release in intact and digitonin-permeabilized platelets. MARCKS is known to bind actin and cross-link actin filaments, and this is inhibited by PKC-evoked MARCKS phosphorylation. MARCKS phosphorylation and serotonin release in response to increasing concentrations of thrombin have a similar EC50 and time course and, in permeabilized platelets, peptide MPSD, with an amino acid sequence corresponding to the phosphorylation site domain of MARCKS, blocked both responses. However, pleckstrin and myosin light chain phosphorylations were not modified. Ala-MPSD, in which the four serine residues of MPSD were substituted by alanines was ineffective. The results suggest a role for MARCKS in platelet secretion. The fact that pleckstrin phosphorylation has a different time course and was not modified in the presence of MPSD when MARCKS phosphorylation and serotonin release were inhibited would suggest either that pleckstrin phosphorylation is unrelated to secretion or that it might only be involved upstream in the events leading to secretion.

Recombinant fragment of von Willebrand factor AR545C inhibits platelet binding to thrombin and platelet adhesion to thrombin-treated endothelial cells.

Activated, but not resting, platelets are capable of adhering to intact endothelial cells (ECs). We evaluated the effect of a recombinant von Willebrand factor (VWF) fragment AR545C, which inhibits glycoprotein Ib (GPIb)/VWF binding, on platelet adhesion to human ECs under static or flow conditions. Incubation of resting platelets with intact endothelium under flow conditions (350/s) resulted in minimal platelet adhesion. The adhesion was enhanced two- to threefold after either platelet activation by thrombin receptor agonist peptide (TRAP) or EC pretreatment with thrombin. The enhancing effect of thrombin was abolished by addition of either hirudin (10 u/ml) or PGE1 (1 microg/ml). Preincubation of resting platelets with increasing concentrations of AR545C under static or flow conditions resulted in a dose-dependent inhibition of thrombin-induced enhanced adhesion to ECs. AR545C (0.3 microM) completely abolished the effect of thrombin, reducing platelet adhesion to the control level observed with non-treated ECs. In contrast, the same concentration of AR545C had no effect on the adhesion of TRAP-activated platelets to ECs. AR545C also inhibited thrombin-induced platelet aggregation and binding in a dose-dependent manner. In addition, 0.3 microM of AR545C reduced thrombin-induced serotonin release by 57%, whereas monoclonal antibody AN51, which inhibits ristocetin-induced platelet aggregation, had no effect on either thrombin-induced platelet aggregation or binding or on serotonin release. Similarly, AR545C had no effect on TRAP-induced serotonin release. These findings suggest that (i) AR545C inhibits platelet activation mediated by thrombin and this inhibition occurs through blocking the high-affinity thrombin binding sites on the GPIb/IX complex and (ii) AR545C has no effect on the moderate affinity thrombin receptor (seven transmembrane domain thrombin receptor; STDR).

Different adhesion types and active sensitivity of platelet subpopulations

Two different adhesion patterns of blood platelets with different sensitivity to adenosine were demonstrated by means of reflection contrast microscopy with consecutive image analysis and cell affinity chromatography. High-performance liquid chromatography revealed that thrombin-induced serotonin release of adenosine-sensitive platelets was lower than that of adenosine-resistant cells. Our results indicate platelet heterogeneity and suggest that the platelets with lower adenosine sensitivity may be actively involved in the early interaction between platelets and injured endothelium. © 1996 Wiley-Liss, Inc.

Different adhesion types and active sensitivity of platelet subpopulations

Two different adhesion patterns of blood platelets with different sensitivity to adenosine were demonstrated by means of reflection contrast microscopy with consecutive image analysis and cell affinity chromatography. High-performance liquid chromatography revealed that thrombin-induced serotonin release of adenosine-sensitive platelets was lower than that of adenosine-resistant cells. Our results indicate platelet heterogeneity and suggest that the platelets with lower adenosine sensitivity may be actively involved in the early interaction between platelets and injured endothelium.

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.

Beta 2-glycoprotein I is a requirement for anticardiolipin antibodies binding to activated platelets: differences with lupus anticoagulants

Antiphospholipid (aPL) antibodies are of major interest not only because the lupus anticoagulant (LA) causes an inhibition of in vitro blood coagulation, but also because the presence of aPL antibodies confers a risk of thrombosis. The inhibition of in vitro phospholipid- dependent coagulation (LA) is thought to be caused by the binding of LA to procoagulant phospholipid surfaces, thus impeding the clotting process. Another class of aPL antibodies are those originally described to be directed against negatively charged phospholipids, in particular cardiolipin (ACA). ACA are usually directed against a complex antigen consisting of negatively charged phospholipid and a plasma protein, beta 2-glycoprotein I (beta 2-GPI). Further, there is antibody heterogeneity even within individual patients so that ACA and LA are separable using physicochemical techniques such as ion exchange chromatography and chromatofocusing. Using such techniques we have enriched Ig fractions for LA and ACA from two patient plasmas. The majority of Ig with LA activity had a pI of 7.2 to 7.3 whereas ACA had a pI of 5.0 to 5.2. Using these enriched fractions labeled with [125I]- iodine we have shown that LA binds to platelets in a specific and saturable manner. Binding is dependent on thrombin activation. [125I]- ACA behaves differently. Like LA, binding is specific and dependent on thrombin activation but in this case requires the presence of beta 2- GPI. ACA, in the presence of beta 2-GPI, competes for binding with LA suggesting the same or contiguous site. There is no cross-reactivity of these antibodies with GPIIb/IIIa and the most likely binding site is phospholipid. In neither case does LA nor ACA have an effect on thrombin-induced release of serotonin or beta-thromboglobulin nor do they affect platelet aggregation induced by a number of agonists. This antibody binding may play an etiological role in thrombocytopenia associated with aPL, but does not explain thrombosis on the basis of hyperaggregability or increased platelet release.

β2-Glycoprotein I Is a Requirement for Anticardiolipin Antibodies Binding To Activated Platelets: Differences With Lupus Anticoagulants

Antiphospholipid (aPL) antibodies are of major interest not only because the lupus anticoagulant (LA) causes an inhibition of in vitro blood coagulation, but also because the presence of aPL antibodies confers a risk of thrombosis. The inhibition of in vitro phospholipid-dependent coagulation (LA) is thought to be caused by the binding of LA to procoagulant phospholipid surfaces, thus impeding the clotting process. Another class of aPL antibodies are those originally described to be directed against negatively charged phospholipids, in particular cardiolipin (ACA). ACA are usually directed against a complex antigen consisting of negatively charged phospholipid and a plasma protein, beta 2-glycoprotein I (beta 2-GPI). Further, there is antibody heterogeneity even within individual patients so that ACA and LA are separable using physicochemical techniques such as ion exchange chromatography and chromatofocusing. Using such techniques we have enriched Ig fractions for LA and ACA from two patient plasmas. The majority of Ig with LA activity had a pI of 7.2 to 7.3 whereas ACA had a pI of 5.0 to 5.2. Using these enriched fractions labeled with [125I]-iodine we have shown that LA binds to platelets in a specific and saturable manner. Binding is dependent on thrombin activation. [125I]-ACA behaves differently. Like LA, binding is specific and dependent on thrombin activation but in this case requires the presence of beta 2-GPI. ACA, in the presence of beta 2-GPI, competes for binding with LA suggesting the same or contiguous site. There is no cross-reactivity of these antibodies with GPIIb/IIIa and the most likely binding site is phospholipid. In neither case does LA nor ACA have an effect on thrombin-induced release of serotonin or beta-thromboglobulin nor do they affect platelet aggregation induced by a number of agonists. This antibody binding may play an etiological role in thrombocytopenia associated with aPL, but does not explain thrombosis on the basis of hyperaggregability or increased platelet release.

Indapamide Inhibits Human Platelet Aggregation in vitro

Among antihypertensive drugs with diuretic properties, indapamide was shown to inhibit platelet growth factors production in diabetic hypertensive patients, suggesting an antiplatelet activity. The present study aimed to demonstrate the antiaggregating properties of indapamide. The effect of indapamide on platelet function was compared in vitro to that of hydrochlorothiazide. Indapamide (100 microM) inhibited the second wave of adenosine diphosphate-induced aggregation and inhibited collagen-induced aggregation of platelet rich plasma by 50%. Using isolated platelets, indapamide also inhibited aggregation induced by low doses of thrombin (70% inhibition with 0.035 U/ml). This inhibition was dose-dependent and was still observed in presence of high thrombin concentrations, although the inhibition was moderate. Inhibitory effect of indapamide was more pronounced on the release reaction. Indapamide inhibited the thrombin-induced release of serotonin from dense granules by up to 80%. Hydrochlorothiazide at the same concentrations had no effect on platelet aggregation, and the inhibitory effect on the secretion was inconsistent and never exceeded 30%. By contrast, when the aggregation inducer was arachidonic acid, indapamide had no effect either on aggregation or on thromboxane formation, indicating that it was not acting on arachidonic catabolism. Calcium mobilization evoked by thrombin stimulation and measured with the fluorescent dye Indo 1 was also reduced in presence of indapamide by 30%. Myosin light chain and pleckstrin phosphorylation induced by thrombin were also reduced. These results demonstrate that indapamide inhibits platelet responses by inhibiting calcium mobilization. The anti-aggregating properties of indapamide could contribute to normalize the hyperresponsiveness of platelets from hypertensive patients.

Thrombin-induced effects are selectively inhibited following treatment of intact human platelets with okadaic acid.

The involvement of protein phosphatases in regulating platelet activation was studied. The major portion of the phosphorylase phosphatase activity found in platelet lysates appears to be of the type 1 variety. The identification of this enzyme was based on the finding that greater than 80% of protein phosphatase activity was inhibited by the heat-stable inhibitor protein inhibitor 2 and, while only 20% of the phosphorylase phosphatase activity in platelet extracts was inhibited by 2 nM okadaic acid, greater than 95% of the activity was inhibited in the presence of 1 microM okadaic acid. Increases in protein phosphorylations occurred and thrombin-induced release of serotonin was prevented as a result of artificially inhibiting the enzyme with okadaic acid in intact platelets. This implies either that the regulation of okadaic acid sensitive protein phosphatases is necessary for some agonist-induced effects or that okadaic acid sensitive phosphatases are required for maintaining platelets in a responsive state.

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.

Platelet phospholipase C activity in salt-dependent hypertension.

In spontaneously hypertensive rats (SHR), enhanced responsiveness of phospholipase C has been reported in various cells and tissues. In SHR and in some patients with essential hypertension particularly, the increased phospholipase C responsiveness of platelets has been described as involved in the hyperreactivity to thrombin. To determine the relation between such an enzymic abnormality and hypertension, the platelet phospholipase C activity was investigated in various models of experimental hypertension (i.e., in the Dahl salt-resistant and salt-sensitive strains inbred by John Rapp at Toledo, Ohio, SR/Jr and SS/Jr, respectively) fed either on a low or a high NaCl-containing diet, and in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. In phosphorus-32-prelabeled platelets, phospholipase C was determined by measurement of the thrombin-induced [32P]phosphatidic acid formation; the labeling of the P47 protein with 32P was also measured. In parallel experiments, the platelet reactivity was assessed by measurement of the thrombin-induced serotonin release. Under thrombin (0.05-0.5 units/ml) stimulation, phospholipase C activity, [32P]P47 labeling, and serotonin release were significantly increased in SS/Jr rats fed a high NaCl diet compared with SS/Jr rats fed a low NaCl diet. NaCl-rich diet did not modify phospholipase C in SR/Jr rats. Platelet reactivity and phospholipase C responsiveness were also normal in DOCA-salt hypertensive rats compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet activating factor and sheep platelets: A sensitive new bioassay

The in vitro response of sheep platelets to platelet activating factor (PAF) was investigated. Sheep platelet-rich plasma aggregated in response to PAF with an EC 50 of 10 nM. Platelets isolated via arabinogalactan density gradient centrifugation displayed an EC 50 of 50 nM with a threshold response at 0.1 pM. PAF-induced release of 14C-serotonin from isolated sheep platelets was comparable with an EC 50 of 50 pM and threshold release at 10 fM. PAF-induced aggregation was specific in that it could be blocked by the competitive receptor antagonists Alprazolam (Upjohn, IC 50 = 40 μM), L-652, 731 (MSD, IC 50 = 6 μM), and WEB 2086 (Boehringer Ingelheim, IC 50 = 0.8 μM). At micromolar concentrations, WEB 2086 did not inhibit ADP- or thrombin-induced aggregation nor thrombin-induced serotonin release. However, at higher concentrations of WEB 2086 some inhibition of thrombin-induced platelet aggregation and release was observed. Subsequent experiments demonstrated that high concentrations of WEB 2086 can inhibit thrombin-induced clotting (K i = 866 μM) and cleavage of the chromogenic substrate Spectrozyme-TH (K i = 842 μM). In summary, the response of isolated sheep platelets to PAF was specifically inhibitable and was 10 to 100 times more sensitive than washed rabbit platelets, the most popular bioassay currently in use.

Thrombin-induced serotonin release as an in vitro indicator of the functional integrity of stored platelets

Measurements of endogenous serotonin and the thrombin-induced serotonin release reaction in platelet concentrates were compared with other methods of quality control: particle counting and pH and gas analyses. A discrepancy between the serotonin release reaction and the other data was observed after seven days of storage. The decline in the release reaction was not predictable from pH measurements or the number of platelets or leukocytes in the concentrate. There was a significant positive correlation between endogenous serotonin content and the thrombin-induced serotonin secretion after seven days of storage. We conclude that the current methods for routine control of platelet concentrates may not ensure the quality of the product after it has been stored for seven days. The thrombin-induced serotonin release may provide a valuable addition to the battery of tests available.

Thrombin-stimulated calcium uptake is altered in platelets from spontaneously hypertensive rats. Effect of external magnesium.

Thrombin-induced calcium uptake and serotonin release were measured on platelets from spontaneously hypertensive rats (SHR) and normotensive control animals (WKY). Both 45Ca uptake and serotonin release induced by thrombin are enhanced in platelets from SHR in the presence of various Ca2+ concentrations. In the presence of [Ca2+] 10(-6)M a correlation exists between thrombin-induced calcium uptake and serotonin release; platelets from SHR and WKY do not differ in this respect, which demonstrates that the anomaly observed in platelets from SHR concerns an early reaction in the stimulation-response coupling. The inhibitory effect of magnesium on thrombin-induced serotonin release, less important in platelets from SHR than in control ones, seems to pass through two phenomena: an inhibition of calcium influx occurring at high concentrations and less effective on SHR platelets and a stimulation of calcium influx, probably corresponding to intracellular calcium sequestering, observed in the presence of low magnesium concentrations and enhanced in platelets from SHR. This study therefore evidence a relationship between the increases observed in both serotonin secretion and calcium influx on thrombin-stimulated SHR platelets and suggests cellular mechanisms involved in the inhibitory action of magnesium impaired in these SHR platelets.

A preferential inhibition by Zn 2+ on platelet activating factor- and thrombin-induced serotonin secretion from washed rabbit platelets

Zinc ions at micromolar levels exhibited a significant inhibitory activity toward platelet activating factor (AGEPC)- and thrombin-induced serotonin release from washed rabbit platelets. In the ranges from 25 to 30 μ m and 10 to 50 μ m, respectively, zinc essentially prevented any serotonin release from 1.25 × 10 8 cells/ μl by 1 × 10 −10 m AGEPC and by 0.2 unit thrombin/ml. This inhibition by zinc ions, in micromolar range, occurred in the presence of 1.0 m m Ca 2+. The amount of zinc needed for inhibition was inversely proportional to the amount of AGEPC present and further zinc must be added prior to or at the same time as the AGEPC to be effective. Introduction of zinc ions after the AGEPC essentially abolished the inhibitory properties of this divalent cation. Other cations such as Cu 2+, La 3+, Cd 2+, and Mg 2+ were ineffective as inhibitors at concentrations where zinc showed its maximal effects. Under conditions similar to those noted above, aggregation induced by AGEPC was blocked only to the extent of 25% of a control. No inhibitory action by zinc on thrombin-induced aggregation was noted. It is apparent that zinc ions influence a site(s) on the rabbit platelet of considerable importance to the activation (or signaling) process by AGEPC and thrombin in these cells, as expressed by serotonin release. Zinc should provide a suitable probe to explore the mechanism of action of these agonists in their interaction wtih sensitive cells and to define in more specific biochemical terms the putative receptor for these molecules.

Determination of Serotonin Release from Platelets by Using A Serotonin-Selective Membrane Electrode

Abstract A serotonin-selective membrane electrode was constructed for the first time by using sodium 2,4,6-trinitrobenzenesulfonate as an ion-exchanger and tris(2-ethylhexyl) phosphate as a membrane solvent, respectively, and was applied for the detection of thrombin-induced serotonin release from rabbit platelets. This method made it possible to monitor continuously the endogeneous serotonin secretion process from platelets in situ

The effect of calcium channel blockers on blood platelet function, especially calcium uptake

The effects of organic and inorganic calcium antagonists on washed platelets from rat and human been studied. Platelet aggregation was assessed by turbidimetry. Endogenous serotonin release was measured on the same sample by means of electrochemically treated carbon fiber electrodes. The organic calcium antagonist, nitrendipine, and the inorganic calcium channel blockers (Co 2+, Mn 2+, Cd 2+, La 3+) drastically inhibited rat and human platelet aggregation induced by thrombin, ADP or adrenaline in teh presence of 0.32 mM Ca 2+. In our conditions, the thrombin-induced release of endogenous serotonin was found to be external Ca 2+-dependent and completely inhibited by 20 μM nitrendipine or 1 mM Cd 2+. In addition, Ba 2+ or Sr 2+ ions can be substituted for Ca 2+ to bring about platelet aggregation as well as endogenous serotonin secretion. In Ba 2+ or Sr 2+-containing media, rat platelet aggregation and/or serotonin secretion can be inhibited by either nitrendipine or Cd 2+. Finally, we have also studied the thrombin- and external Ca 2+-dependence of radiolabeled calcium uptake by rat platelets. We found that the thrombin-induced 45Ca uptake was inhibited by either 18 μM nitrendipine or 1 mM Cd 2+. These results provide strong evidence for the existence of an influx of divalent cations (Ca 2+, Sr 2+, Ba 2+) triggering platelet function. They also suggest, although they do not prove, that the translocation of these cations occurs through an agonist-operated channel as proposed by Hallam and Rink (FEBS Lett. 186 (1986) 175–179).