Endothelins inhibit serotonin-induced platelet aggregation via a mechanism involving protein kinase C

European Journal of Pharmacology, 219 (1992) 289-293

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© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00

EJP 52613

Endothelins inhibit serotonin-induced platelet aggregation via a mechanism involving protein kinase C M i c h a l H. P i e t r a s z e k 1, Y u m i k o T a k a d a a n d A k i k a z u T a k a d a Department of Physiology. Hamamatsu University, School of Medicine, Handa-cho 3600, Hamamatsu-shi, Shizuoka-ken, Hamamatsu 431-31, Japan Received 12 March 1992, revised MS received 19 May 1992, accepted 9 June 1992

Endothelins are a family of three peptides that act as local hormones released by the endothelium. They were found to inhibit rabbit and dog platelet aggregation in vivo, but no effect was observed in vitro. In order to investigate the possible interaction between endothelins and human platelet serotonin receptors, their effects on platelet aggregation induced by serotonin was studied. Endothelin-1, -2 and -3 had a dual action, on platelet aggregation and calcium mobilization induced by serotonin. When added at the same time as serotonin, endothelin potentiated the response to the amine. On the contrary, preincubation of platelet suspension with endothelin resulted in a concentration-dependent inhibition of the serotonin-mediated platelet response. Moreover, endothelin-1 inhibited serotonergic amplification of epinephrine-induced aggregation of platelets. We hypothesize that endothelins can bind to the platelet membrane and interact with serotonin receptors. The diverse effect of endothelins on serotonin-induced aggregation and calcium mobilization may be due to stimulation of protein kinase C. Endothelin; 5-HT (5-hydroxytryptamine, serotonin); Platelet aggregation; Ca 2+ mobilization

1. Introduction

Endothelins are a family of three peptides consisting of 21 amino acids (Yanagisawa et al., 1988). The genes encoding for three isopeptides of the endothelin family (ET-1, ET-2, and ET-3) have been isolated (Inoue et al., 1989; Sakurai et al., 1990). The chemically and pharmacologically best characterized member of this family is endothelin-1. Its action as a vasoconstrictor and other effects suggest that it is designed to function as a local hormone released by the endothelial cells (Yanagisawa et al., 1989; D'Orleans-Juste et al, 1989). Circulating endothelin-1 was shown to release prostacyclin (Rae et al., 1989) and endothelium-derived relaxing factor (Warner et al., 1989), both of which possess very potent antiaggregatory properties. Endothelins were reported to inhibit rabbit and dog platelet aggregation in vivo (Thiemermann et al., 1988, 1990; Herman et al., 1989), whereas no effect was observed in vitro (Lidbury et al., 1990; Battistini et al., 1990; Edlund and Wennmalm, 1990). As shown by Matsumoto

Correspondence to: A. Takada, Department of Physiology, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 431-31, Japan. Tel. 81.53.435 2247, fax 81.53.435 2248. 1 On leave of absence from the Department of Pharmacodynamics, Medical Academy, Bialystok, Poland.

et al. (1990), endothelin-1 potentiates platelet activation induced by epinephrine. On the other hand, Yang et al. (1990) recently showed that threshold concentrations of endothelin-1 potentiated the vasoconstrictor effect of serotonin in the human internal mammary artery. Since blood platelets possess the same serotonin type 2 receptor, the purpose of our study was to investigate the possible influence of endothelins on platelet aggregation and calcium mobilization induced by serotonin in vitro.

2. Materials and methods

2.1. Materials Serotonin (5-hydroxytryptamine creatine sulfate complex) and phorbol 12,13-dibutyrate (PdBu) were purchased from Sigma (USA), f u r a - 2 / A M from Dojindo Lab (Japan) and H-7 from Seikagaku Kogyo Co. (Japan). ADP, epinephrine and collagen were purchased from Kyoto Daiichi Chemical Company (Japan) and dissolved in the buffer supplied by the manufacturer. Endothelin-1, -2, and -3 were obtained from the Peptide Institute, Inc. (Osaka, Japan) and were dissolved in distilled water, 0.1% aqueous acetic acid and sterilized water, respectively. Solutions were stored in aliquots at - 35°C.

290

2.2. Participants Thirty men, aged from 23 to 35 years (mean: 29) participated voluntarily in this study. None of them had taken any drug for at least 2 weeks prior to the study.

0.5; MgSO 4 1.0; CaCI 2 0.2; HEPES 10; and glucose 10, pH 7.4. The fluorescence of fura-2 was measured in a Hitachi F-2000 fluorescence spectrophotometer with excitation at 339 nm and emission at 500 nm. The platelets were prewarmed in a cuvette at 37°C with stirring and ligands were then added to the incubation medium.

2.3. Blood sampling 2.5. Statistical analysis Venous blood was obtained between 8 and 10 a.m. from the antecubital vein. Samples were collected into siliconized tubes, nine volumes of blood were anticoagulated with one volume of 3.8% trisodium citrate. Blood samples were immediately centrifuged at 200 × g for 10 min at room temperature to obtain platelet rich plasma (PRP). Platelet poor plasma (PPP) was prepared by additional centrifugation of the whole blood at 1200 x g for 20 min. PRP was diluted with autologous PPP and platelet counts were adjusted to 300 000-400 000 platelets//~l.

2.4. Assays Platelet aggregation was measured with an Elma (model PAT-2A) aggregometer using 200 ~1 of plasma in siliconized cuvettes stirred at 1100 rpm. Samples were prewarmed for 5 min at 37°C before the addition of aggregating agents. PRP was pre-incubated with endothelins at 37°C for 3 min prior to the addition of aggregating agonists. In another set of experiments, endothelin was added to the PRP at the same time as serotonin. The following concentrations of agonists were used: serotonin 1.0 or 0.1 /xM, ADP 4.0 /xM, collagen 2.0/~g/ml, and epinephrine 0.1/zM. The data are expressed (medium + S.D.) as the maximal increase in light transmission (LT) with PPP taken as the 100% level of LT. For intracellular Ca 2+ measurement the platelets were prepared from freshly drawn blood with ACD (11 mM citric acid monohydrate, 5.0 mM trisodium citrate dihydrate and 8.7 mM dextrose, final concentration). PRP was incubated with 3 / z M fura-2/AM for 30 min at 37°C. Thereafter fura-2/AM was removed from the platelets by Sepharose 2B chromatography in a Hepes buffer consisting of (mM): NaC1 145; KC1 5; NaEHPO 4

Statistical significance was set at P < 0.05 as determined by Student's paired t-test.

3. Results

3.1. The effect of endothelins on platelet aggregation induced by ADP and collagen In the first series of aggregation studies we confirmed the previous finding that endothelins (0.01-1.0 /zM) alone do not induce platelet aggregation. Furthermore, preincubation of platelets with endothelins does not modify the aggregatory response to ADP and collagen (data not shown).

3.2. The effect of endothelins on serotonin-induced aggregation of platelets Serotonin in the concentration of 1.0 /~M induced changes in light transmission by 35-45%. As seen in table 1 endothelins (0.1-1.0/~M), when incubated with platelet suspension for 3 min before then addition of serotonin, caused a concentration-dependent inhibition of platelet aggregation. Endothelin-1 appeared to be the most potent inhibitor. Ketanserin (0.1 jzM), an antagonist of 5-HT 2 receptors, completely abolished the platelet aggregatory response to serotonin. Figure 1 shows the profile of the antiaggregatory effect of endothelin-1 on serotonin-induced platelet aggregation. In another set of experiments, endothelins were added to PRP at the same time as serotonin. Surprisingly, all three endothelins produced a concentrationdependent potentiation of the serotonin-induced platelet aggregation (table 1). All members of endothe-

TABLE 1 The effect of endothelin-1, -2, and -3 on serotonin-induced human platelet aggregation. (A) platelet suspension was preincubated with endothelins 3 min prior to addition of serotonin. (B) endo.thelins were added to platelet suspension at the same time as serotonin. Results are expressed as means ± S.D., a p < 0.05, b p < 0.01. Endothelin-1

A B

Endothelin-2

Endothelin-3

0/zM

0.1 p.M

1.0 ~ M

0 ~M

0.1 ~ M

1.0 ttM

0/zM

0.1 / . L M

1.0/zM

38+7 39+6

27+5 a 44_+7 a

18+8 b 49+5 b

39+8 38-I-8

30+6 a 40+5

22+8 b 45+6 a

37+9 395:5

26+6 a 41+6

20+7 b 48+7 a

Ketanserin 0.1 ~ M 0 -

291 -,,~,~---.:--~ - - - ~ : - ~ , ~ - , ¢ ~

endothelin,-I

".~.,--'-,

"- - " ~ . ~ . . . ' , . . . , - . - ~ ' ~ " ~

e n dot

h e Iin - 1

O

O

e-

"~

5-HT

_

endothelin-1

5-HT

tel)

e-,

~

_ ~ / , , , ~ - ~

endothelin-1

+ 5-HT

+ 5-HT

1 min I

I

Fig. 1. T h e effect of preincubation of P R P with endothelin-1 (1.0 tzM) for 3 min on the aggregation of platelets induced by 5-HT (1.0 /.~M).

lins family had the same potency for this effect. Figure 2 shows the potentiating effect of endothelin-1 on aggregation of platelets by serotonin. We also investigated the effect of PdBu, an activator of protein kinase C, on the aggregation induced by serotonin. As seen in table 2, PdBu (10-20 nM), when preincubated for 3 min with PRP before the addition of serotonin, caused a marked inhibition of platelet aggregation. In contrast, addition of PdBu together with serotonin resulted in an increase of light transmission.

3.3. The effect of endothelin-1 on serotonergic amplification of the platelet response to epinephrine Since endothelin-1 appeared to be the most potent inhibitor of serotonin-induced platelet aggregation, we studied its effect on serotonergic amplification of the epinephrine-induced platelet response. As seen in fig. 3, epinephrine (0.1/zM) alone did not induce a platelet response. Serotonin (0.1 /zM) caused a modest, reversible aggregation, but when combined with an ineffective concentration of epinephrine, the aggregation proceeded to the complete response. The serotonergic amplification was inhibited by endothelin-1 (0.1 /zM) preincubated with PRP for 3 min prior to the addition of serotonin.

Fig. 2. T h e platelet aggregatory response to 5-HT (1.0 ~ M ) added at the same time as endothelin-1 (1.0 ~M).

3.4. The effect of endothelin-1 on serotonin-induced intracellular calcium mobilization and its modification by protein kinase C inhibitor, H-7 As shown in fig. 4, the mean resting intracellular Ca 2÷ concentration was 87.0 + 3.1 nM. Serotonin (10 ~M) caused a rapid mobilization of intracellular free Ca 2÷ in platelets to 202.7 + 7.4 nM (fig. 4A). Endothelin-1 (1.0 /zM) alone had no effect on intracellular Ca 2÷ levels (fig. 4B), but when incubated with platelets for 3 min before the addition of serotonin, the peptide caused inhibition of calcium release (120.5 + 5.6 nM, P < 0.05) (fig. 4C). Finally, when endothelin-1 was added to the platelet suspension at the same time as serotonin, the potentiation of serotonin-induced calcium mobilization was observed (242.3 + 8.1 nM, P < 0.05) (fig. 4D). We also investigated the effect of the protein kinase C inhibitor, H-7 (100 /zM), on 5-HTmediated Ca 2÷ release. H-7, which itself did not change intracellular Ca 2÷ levels, completely abolished the effect of endothelin-1 on serotonin-induced calcium mobilization. The preincubation of platelets with H-7 when endothelin-1 was added at the same time as serotonin, resulted in a calcium mobilization of 190.0 + 6.2 nM (fig. 4E), whereas when endothelin-1 was added 3 min before the addition of serotonin, the intracellular cal-

•-,,,,,

-,*-~,~-,---,0.,-'~--~ e p i n e p h r i n e

TABLE 2 T h e effect of phorbol 12,13-dibutyrate (PdBu) on platelet aggregation induced by serotonin. 5-HT + epinephrine

Results are expressed as m e a n s + S.D., a p < 0.05, b p < 0.01. 0 Three minutes preincubation prior to addition of serotonin PdBu added at the same time as serotonin

10 n M

20 nM l

38 ± 7 37 ± 9

I

31±6 46±8 b

e n d o t h e l i n - I + 5-HT + epinephrine

rain I

22±8 a 49±9 b

Fig. 3. The effect of endothelin-1 (1.0 ~ M ) on the serotonergic amplification of epinephrine-induced platelet aggregation. T h e concentration of 5-HT was 0.1 ~ M and epinephrine 0.1 ~ M .

292 5HT 3001"

~

5HT

ET-L

ET-I

0 300r

ET~1+SHT

0 i

ET-I+SHT H-7•

ET-1 5HT H-7

3 i

i

i

Time (rain) Fig. 4. T h e effects of e n d o t h e l i n - 1 on s e r o t o n i n - m e d i a t e d intracellu-

lar calcium mobilization in human platelets. Each trace represents at least three experiments. Serotonin at the concentration of 10 ~M was added at the arrows. (A) Control, (B) endothelin (1 ~M), (C) endothelin was added 3 min before 5-HT stimulation, (D) endothelin was added at the same time as 5-HT, (E) H-7 was added 1 min before endothelin and serotonin, (F) H-7 at the concentration of 100 ~M was added 1 min before the addition of endothelin, which was followed by 5-HT stimulation after 3 min. cium concentration increased to 192.5 + 5.8 (fig. 4F). It should be pointed out that this effect was observed only in 20 cases out of 30 (60%).

4. Discussion The present study demonstrated for the first time that endothelin-1, -2 and -3 have a dual effect on platelet aggregation and intracellular calcium mobilization induced by serotonin. Platelets (when preincubated with endothelins for 3 min) showed a decreased response to serotonin, whereas their addition at the same time as serotonin resulted in potentiation of both aggregation and calcium release. The pattern of inhibition of aggregation was the same as that obtained with PdBu, a classic activator of protein kinase C, but different from that of ketanserin, an antagonist of 5-HT 2 receptors. Furthermore, endothelin-1, a most potent inhibitor of 5-HT-induced platelet aggregation, partially abolished the serotonergic amplification of the platelet response to subthreshold concentrations of epinephrine. Finally, the inhibitor of protein kinase C (H-7) suppressed the effect of endothelin-1 on platelet Ca 2÷ mobilization. Endothelins had been shown to inhibit human platelet function in vivo (Herman et al., 1989; Thiemermann et al., 1989, 1990; Lidbury et al., 1990), whereas they fail to induce platelet aggregation under in vitro conditions (Battistini et al., 1990; Edlund and Wennmaim, 1990; Lidbury et al., 1990). In the first part of our study we confirmed the observation that endothelins have no effect on aggregation of platelet induced by A D P and collagen in vitro (data not shown).

The inhibitory action of endothelins on platelet aggregation in vivo had been related to the release of prostaglandin (PG) 12 from the endothelium (Thiemermann et al., 1988, 1989, 1990; Herman et al., 1989; Lidbury et al., 1990). Endothelin-1 has also been shown to induce an elevation of cAMP levels which would suppress aggregation (Thiemermann et al., 1989). The inhibitory effect of endothelins on platelet function in vivo was blocked by pretreatment with indomethacin, suggesting clearly that the rise in cAMP levels was due to the release of prostacyclin (Thiemermann et al., 1989). We have now shown that endothelins, when preincubated with platelet suspension, inhibited the response of platelets to serotonin. The mechanisms underlying these findings seem to be complicated. One hypothesis could be that endothelin activates protein kinase C. This has been documented by Takuwa et al. (1989) in an experiment with fibroblasts, in which endothelin was found to be an activator of phospholipase C with concomitant production of two second messengers, inositol trisphosphate and 1,2-diacylglycerol, leading to Ca 2÷ mobilization and activation of protein kinase C. Induction of platelet aggregation by serotonin operates through 5-HT 2 receptor, the stimulation of which enhances the turnover of polyphosphoinositides, resulting in activation of protein kinase C and an increase in cytoplasmic levels of Ca 2÷ (De Clerck and Vanhoutte, 1982). It was shown that protein kinase C has diverse effects on platelet activation, depending on whether it is activated before or during platelet activation (Siess, 1991). If protein kinase C is activated prior to the addition of an agonist, it suppresses signal transduction between receptor and phospholipase C and inhibits platelet aggregation. If activated during the stimulation of platelet activation it produces a maximal platelet response and inhibits by negative feedback the coupling between receptor activation and phospholipase C. The present experiments indicate that endothelins inhibit serotonin-induced aggregation and calcium mobilization by stimulation of protein kinase C. This possibility is supported by our observation that a classic activator of protein kinase C, PdBu, at low concentrations led to the same inhibitory effects as endothelins and H-7, an inhibitor of protein kinase C, reversed the action of endothelin on 5-HT-mediated calcium release. The most striking observation was that H-7 did not suppress the effect of endothelin on serotonin-induced calcium translocation in all cases (60%), while serotonin-promoted aggregation of platelets was modified by endothelin in all subjects. Thus, the other plausible hypothesis is that endothelin may interact with the platelet 5-HT z receptor. In favour of such an explanation are the findings of Consigny (1990), who showed that endothelin-1 increases arterial sensitivity to serotonin. In our experiment, the endothelins-medi-

293

ated inhibition of serotonin-induced platelet aggregation, according to its kinetics, seemed to be non-competitive (data not shown). It is also possible that endothelins modify the platelet response to serotonin, involving both mechanisms, non-competitive interaction with serotonin receptors and stimulation of protein kinase C. Such an interrelationship was proposed by Wang and Friedman (1990) and Kagaya et al. (1990), who showed that the stimulation of the 5-HT 2 receptor increases protein kinase C activity and that this activation could be inhibited by the protein kinase C-mediated negative feedback system of 5-HT 2 receptors. Nevertheless, a radioligand binding study is needed to clarify the mechanism by which endothelins inhibit the aggregation of platelets promoted by 5-HT. In conclusion, the present study demonstrated that endothelins inhibit serotonin-induced human platelet aggregation and intracellular calcium mobilization. Based on our results, we hypothesize that endothelin may stimulate protein kinase C activity a n d / o r bind to the platelet membrane interacting with 5-HT z receptors.

Acknowledgement This study was supported by a Sasakawa Scientific Research Grant, Tokyo, Japan.

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