Characterization of human platelet beta-adrenoceptors

THROMBOSIS RESEARCH 40; 757-767, 1985 0049-3848/85 $3.00 t .OO Printed in the USA. Copyright (c) 1985 Pergamon Press Ltd. All rights reserved.

CHARACTERIZATION

K.

Winther+,

'Coagulation '+Department

R.

of

OF

HUMAN

Klysner++,

PLATELET

A.

BETA-ADRENOCEPTORS

Geisler++,

Laboratory, The Municipal Pharmacology, University Copenhagen, Denmark

P.H.

Andersen+'

Hospital and of Copenhagen,

(Received (Received

27.12.1984; Accepted in revised form 2.7.1985 by Editor P. Olsson) in final form by the Executive Editorial Office 16.9.1985)

ABSTRACT The widespread use of beta-adrenoceptor antagonists againist hypertension, angina pectoris and migraine or as a after myocardial preventive treatment infarction has encouraged us to investigate the effects of these drugs on platelet function. The aim of this study was to examine whether beta-blocking drugs interfere with platelet betaadrenoceptors and whether this dependency is related to their selectivity for beta-adrenoceptor subtypes. Betaadrenoceptor human platelets with stimulation of isoprenaline increased cyclic AMP (CAMP), which is known to inhibit platelet aggregation. Furthermore, our studies showed that CAMP formation in vitro was stimulated by and non-selective beta -selective agonists, be ? a -agonist but not by the predominant prenalterol. Isoprenalinestimulated CAMP iormation was blocked by the nonselective beta-adrenoceptor antagonists propranolol, timolol, and alprenolol, while the beta -selective antagonists atenolol ana metoprolol had' no influence on an isoprenalineinduced CAMP formation. Receptor binding studies using (3H)-dihydroalprenolol revealed an IC for propranolol of 853 nM, while di;;l;ced the bound ( H)-dihydroalprenolol at far concentrations 20 uM). We conclude ( IC50, that beta-adrenoceptors the human platelet are mainly of the beta subtype and that betaadrenoceptor especially antagon P sts, the nonselective antagonists with interfere platelet function assessed as platelet CAMP formation.

Keywords:

platelets;

beta-adrenoceptors, 757

cyclic

AMP.

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INTRODUCTION The existence of two types of beta-adrenoceptors classified as beta,and beta receptors was first suggested by Lands et al. (1). The classi?ication was based on the order of potency of the natural agonists and some synthetic derivatives. The order of potency of the beta,-adrenocepior was isoprenaline > adrenaline - noradrenaline, and of the beta -adrenoceptor isoprenaline adrenaline > noradrenaline. Adregaline and noradrenaline can induce platelet aggregation (2, 3, 4, 51, which is totally blocked by phentolamine, showing that catecholamine-induced platelet aggregation is mediated by an alpha-adrenergic receptor (6, 7, 8). Beta-adrenoceptor stimulation of human platelets, e.g. by isoprenaline or adrenaline, in the presence of phentolamine, increases the cyclic 3',5'-AMP (CAMP) level. An elevation of this nucleotide has been shown to inhibit platelet aggregation (9, 10, 11). This suggests that the physiological function of beta-adrenoceptors on human platelets might be a modulation of the alpha-receptor-induced platelet aggregation. Beta-adrenoceptor antagonists are widely used, e.g. in the treatment of hypertension and as preventive treatment after myocardial infarction. We have previously shown that patients with treated proparnolol have enhanced platelet aggregatbility as compared with a place:: period (12). There is evidence that patients taking alprenolol also show enhanced platelet aggregation (13). We therefore found it of interest to characterize the subtype of beta-adrenoceptors present on the human platelet membrane.

METHODS Venous blood was drawn from a group of age with an equal sex distribution. haematological normal parameters disease.

of

volunteers, 25-35 years All were drug-free having chronic and no acute or

Cyclic AMP accumulation: Twenty ml of venous blood was collected in tubes containing 256 pl EDTA, 0.15 M. A suspension of intact obtained as described by Wang et al. platelets was (141, omitting the preincubation step with adenine. After the last centrifugation the platelets were resuspended in a buffer containing Tris-HCl 35 mM (pH 7.4, 37"C), NaHC03 3.3 mM, KC1 2.5 mM, Na HP0 0.4 mM, glucose 5.0 mM, EDTA 6.6 mM, and NaCl 90 mM. The figal jsmilarity was 280 mOsm/l. After preincubation for 10 minutes at 4"C, 100 pl of this incubated the same buffer suspension was with 100 pl of containing MgSO 10 mM and iheophylline 10 mM. Theophylline was added to inhibi .% CAMP break down during the incubation period. Beta-adrenoceptor performed in stimulatio always the was -!? presence of phentolamine 10 M. After incubation for 3 min at reaction 37°C the was terminated by boiling for 5 min. Thereafter the incubates were centrifuged for 20 min at 3000 x g, 4°C. aliquots of the supernatant were stored at -20°C until CAMP determination by a method (15). The protein bi ndi ng platelets were counted in a Coulter Counter.

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All preparatory steps were (3H)-dihydroalprenolol binding: oerformed at 4°C. Blood (120 ml) was collected in 1.5 ml EDTA After centrifugation at 200 x g for 15 min, the i3.15 M. rich plasma was centrifuged at 2500 x g for 10 min. plateletdecanted and the platelet pellet was The supernatant was washed twice in a buffer containing Tris-HCl 50mM (pH 7.5, NaCl 150 mM, and EDTA 20 mM. Then the platelets were 37"C), resuspenoed in a buffer composed of Tris-HCl 5.0 mM (pH 7.4, EDTA 4.75 mM and homogenized with a glass Teflon 37"Cj, min followed by homogenizator for 1 strokes) (10 centrifugation at 18,000 x g for 10 min. The platelet buffer containing membranes were finally resuspended in a Tris-HCl 50 mM (pH 7.4, 37°C) and EDTfi 4.75 mM. preparation was added to tubes The platelet membrane containing labelled and unlabel$ed ligands. The tota13volume ( Hl-dihydroalprenolol ( H-DHA) of each test tube was 200 ul. used in c 3 ncentrations of 0.25 9 nM. For was the displacement studies ( H-DHA) was used in a concentration of 3 which was displaced with different beta-adrenocepror nM, antagonists in concentrations 1 nM 10 mM. After the incubation for 10 min at 37"C, the incubate was poured through Uhatman GF/C filters at a vacuum of 10 mmHg. The filters were rapidly washed 3 times with 3 ml of ice-cold buffer containing Tris-HCl 50 mM (pH 7.4, 37°C) and EDTA 4.75 mM. Thereafter filters dried in the were scintillation vials, 2 ml of Instagel was added and the filters were counted in a scintillation counter. Displacement with beta-adrenoceptor agonists or antagonists was always made with addition of the phentolamige. Specific binding -#as defined as binding displaced by 10 M isoprenaline or 10 M propranolol. The protein concentration in each tube was approximataely 0.132 mg protein/ml, determined by the method of Lowry et al. (16). All d e t erminations are in triplicate and the data are expressed as means -+ SEM. The following drugs were used: l-(3H)-dihydroalprenflol 28 (mCi/mmol) was obtained from New Enoland Nuclear. ( HI-CAMP 27.5 (mCi/mmol) from The Radio Chemical Centre; Amersham, d-,lisoprenaline, l-adrenaline, and 1-noradrenaline from Sigma Chemicals Co. All other drugs were racemic mixtures. Phentolamine was a gift from Ciba-Geigy, propranolol and atenolol were from iC1 Ltd., alprenolol, metoprolol, and prenalterol from Hassle, timolol from Merck-Sharp & Dohme Ini., and terbutaline from Astra. RESULTS Cyclic AMP studies: Dose-response curves of different beta-aarenoceptor agonists on the CAMP accumulation in human platelets are shg#n in _F4ig. 1. Increasing concentrations of isoprenaline (10 - 10 M) increased5cAMP formation dose8 dependently (maximal stimulation at 10 M, IC with isoprenaline was Ml. The stimulation appr58xiamtatYelG 1000% above the unstimulated values (Fig. 1). In the presence of

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of phentolamine (10m5 Ml, adrenaline and noradrenaline induced CAMP accumulation dose-dependently in the order adrenaline > noradrenaline. Furthermore, the predominant beta -agonis.i ,terbutaline was able to induce CAMP formation, but witt? a lower than isoprenaline potency and adrenaline. Prenalterol was without agonistic property in this preparation.

pmoi oAMP/logplatelets 501

1

Basal

10-a

10-7

Figure

10-s

1o-5

lo-'M

1

Cyclic AMP accumulation in intact human platelets incubated isoprenaline (-o-J, adrenaline (-0-1, noradrenaline with terbutaling (- -i4 and prenalterol (- -1 in the (- -1, M, as described in Methods. The concentrations 10 to 10 data given are the means +SEM of an experiment using triplicate determinations. accumulation was blocked by Isoprenaline-stimulated CAMP beta-adrenoceptor antago$sts addition of the non-selective propranolol or alprenolol in the concentration 10 M. timolol, On the other hand, selective beta -antagonists such as atenolol without effect E oncentrations, and metoprolol were, in the same on the isoprenaline-stimulated CAMP accumulation (Fig. 2).

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pmol

cAMP/lOg

761

platelets

-L

T

D

BASAL

cl

ISOPRENALINE

T

N=3 SEM

20

II Ir

ADDIT

METOPROLO

TIMOLOL PROPRANOLOL

Figure

ALPRENOLOL

ATENOLOL

2

human platelets i_3cubated Cyclic AMP formation in intact in the presence or absence of isoprenaline, 10 M, as Various beta-adrenergic antago_r$ists described in Methods. of 10 M. were added simultaneously in concentrations using experiment SEM of an means + data are The triplicate determinations.

The binding of t3H)-DHA was rapidly reaching Binding studies: Specific incubated at 37°C. equilibrium within 3 min when and 37°C. binding was unchanged at temperatures between 20" the specific binding was constant between pH 6.0 Furthermore, - 8.0 but reduced by pH values below 6.0 and above 8.0. Magnesium chloride in concentrations up to 15 mM enhanced total binding and unspecific binding, leaving specific binding unaffected (data not shown).

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HUMAN PLATELET BETA-ADRENOCEPTORS

13H)-DHA non-selective of 1 nM to

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nM) was displaced by using (3 selective and beta-adrenoceptor antaggnists in concentrations 10 mM. Displacement of ( HI-DHA with propranolol value of 85 + 5 nM. Displacement with the rug metoproloT showed an IC50 value of 20 -t 4

Figure

3

Competition for (3H)-dihydroalprenolol t3H-DHA) binding sites in platelet lysates by unlabelled propranolol and Platelet lysates were incubated with 3 nM m~toprolol. ( HI-dihydroalprenolol varying concentrations of and either unlabelled propranolol (?? 1 or unlabelled Data are metoprolol (- 0 -1, as described in Methods. plotted as cent of specifically bound (3H)per dihydroalprenolol.

Scatchard analyses were performed by displacing t3H)-DHA (concentration range 0.25 nM to 9 nM) by 1 pM30f isoprenaline for ( HI-DHA of 2.0 t or 1 pM of propranolol, revealing a K 0.2 nM and a maximal binding capacit 9 (B,,,) of 14.1 + 1.2 fmol/mg protein (Fig. 4).

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lmol %f -DHA /mg

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BETA-ADRENOCEPTORS

Protein

15-

.

10.

. .

B/F . lci3 S

??

4 5. 2 . f mol W.DtfA/mg

vi\ 4

12 8 SCATCHARD ANALYSIS 2.5

10 nM3H-DHA

5

Figure

Protein

16

4

of (3H)-dihydroBinding of increasing concentrations lysates were incubated with varying alprenolol. Platele5 concentrations of ( H)-dihydroalprenolol in the presence or absence of 1 pM propranolol, as described in Methods. + SEM of triplicate Results represent means determinations. Insert: Scatchzrd analysis of the same data. DISCUSSION The present data characterize the type of beta-adrenoceptors present on human predominant beta the platelet. The adrenoceptor terbutaline agonist stimulated CA ?lP (17) formation, while prenalterol (181 had no effect on the cyclase activity (Fig. 1). Isoprenaline-stimulated CAMP formation was blocked by the nonselective beta-adrenoceptor antagonists propranolol, timolo concentration of 10 -4yM a~~i1ea'~~te~,9-'~l'ol \'ni' athtblolati17a 19, 20) were without any'effect on isoprenaline stimulation i; the same concentration (Fig. 2).

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The order of potency of the beta-adrenergic stimulation of activity in human adenylate platelets the cyclase was isoprenaline - adrenaline > noradrenaline. According to the classification of beta-adrenoceptors suggested by Lands et al. these indicate results the presence of a beta (11, adrenoceptor subtype. The present results, showing that no i;beta-adrenoceptor agonists were able to selective block formation, hormone-stimulated CAMP whereas beta'-receptor blockers left CAMP formation unaffected, support this assumpand indicate that in human platelets tion beta-receptors coupled to the adenylate cyclase system are mainly or exclusively of the beta2-adrenoceptor subtype. Several studies have shown that isoprenaline significantly increases the CAMP level in human platelets (9, 10, 11, 12). Jakobs et al. (10) further found that non-selective betaadrenoceptor antagonists enhanced the adrenaline-induced CAMP decrease, whereas beta,-selective antagonists were without this effect. This is in agreement with our studies indicating that only non-selective antagonists were able to inhibit betaadrenoceptor-induced CAMP formation. Our data also correlate very well with the results reported by Kerry and Scrutton (11) who found that isoprenaline blocked platelet aggregation, an effect which was antagonized by non-selective but not by beta -selective antagonists. Ix contrast to Jakobs et al. (10) we found that terbutaline exerted agonist properties (Fig. 11, which agrees with the report by Kerry and Scrutton (11) who showed that both salbutamol and inhibit terbutaline were able to platelet aggregation, but to a lower degree than isoprenaline. Scatchard analyses using ( H)-DHA indicated approximately 14.1 fmol beta-adrenoceptors per mg protein. This correlates very well with the observatio studied binding of ("I'~~~ybdyro~:~~~z,a,n,"inAdt,'e,'l "~r!~ ;Cn5 I)-cyanopindolol to human platelet membranes. They found and 18 14 fmol betaadrenoceptors protein, per mg respectively, which is close to the present data. Steer and Atlas (21) further concluded that the actual beta-adrenoceptor neither of the beta - nor . of the beta -subtype, ;a35 I)hydroxybenzylpindblol was displaced ?n the foll~:?~~ order: propranolol > isoprenaline > adrenaline > practolol > noradrenaline > phenylephrine. In our opinion, this might very well indicate a beta 2-jdrenoceptor subtype. Our displacement studies showing that ( HI-DHA is displaced in the following order: propranolol > metoprolol support these data, although a definite conclusion cannot be drawn from the present data. Displacement with metoprolol, however, results in a curve tending to be non-linear, suggesting the possible existence of beta - as well as beta -adrenoceptors (Fig. 3). Tt'ie binding studie 1 is human platelet membranes by Kerry and Scrutton (22) using ( HI-DHA in the concentration of 50 nM indicated no significant displacement when using isoprenaline or propranolol as displacing ligands in cone ntrations up to s 0.25 mM. Not only high concentrations of ( HI-DHA but also high concentrations of the displacing ligands were used. We have also performed studies using such high concentrations and

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HUMAN PLATELET BETA-ADRENOCEPTORS

of (3H)-DHA using proprafound no significant displacement nolo1 or isoprenaline as the unlabelled ligand. TherefoSe, the displacement of ( HI-DHA failure to observe any significant the not lead to concentrations should using high these conclusion that betaadrefoceptors are undetectable on human platelet membranes using ( HI-DHA as a ligand. The observation that alprenolol binds to platelet betaCAMP beta-adrenoceptor-induced inhibits adrenoceptors and formation might interfere with the beta-agonistic effect of Adrenaline and noradrenaline the circulating catecholaminer. betawell through alphaas their exert effects betaA blockade of the plateeit adrenoceptor stimulation. alpha-agonistic pure adrenoceptors would thus result in a This may explain why Jurgensen effect of these two hormones. et al. (13) found that paients with coronary heart disease showefl enhanced platelet receiving alprenolol, 200 mg b.i.d., compared with a placebo aggregability when tested at rest group. The present results are also in agreement with our earlier adrenawhich indicate a difference in published data (121, lineinduced platelet aggregation in patients treated with propranolol, 80 mg b.i.d., as compared with a period of or with placebo. treatment with metoprolol, 100 mg b.i.d., Contrary to this observation, Cambell et al. (23) reported a decrease in platelet aggregability in patients treated with high doses of propranolol (640 mg/day) compared with controls. affect However, propranolol in high doses is reported to thromboxane synthesis (231, an effect which may have a more powerful influence on platelet function than beta-adrenoceptor stimulation. On the basis of: the present findings, our earlier data (12) and the data of Jurgensen et al. (13) and Cambell et al. (23) a dose-dependent thrombotic risk should be considered when using non-selective beta-adrenoceptor antagonists. REFERENCES 1.

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