Cigarette smoking and platelet function

THROMBOSIS RESEARCH 44; 85-94, 1986 0049-3848/86 $3.00 t .OO Printed in the USA. Copyright (c) 1986 Pergamon Journals Ltd. All rights reserved.

CIGARETTE SMOKING AND PLATELET FUNCTION

Bianca Marasini,, Maria Luisa Biondi*, Silvia Barbesti**, Giorgio Zatta** and Angelo Agostoni* *Clinica Medica V, +*Cattedra di Medicina Nucleare, Universita degli Studi di Milano, Ospedale S.Paolo, Milano, Italy

(Received 7.3.1986; Accepted in revised form 19.5.1986 by Editor P.M. Mannucci) (Received by Executive Editorial Office 10.7.1986)

ABSTRACT To

assess

influence

the

activation,

of

cigarette

studied

we

changes

platelet-released

the serotonin (5-HT)

platelet-associated

production

smoking in

and

on

plasma

of thromboxane

platelet

intraplatelet B2

levels

(TXB2 ),

and and

in 6 non

smokers and 6 habitual smokers, before and after acute exposure

to

smoke. Before smoking, habitual smokers showed slightly higher, albeit not significantly,

5-HT platelet concentrations

as well as lower TXB 2 platelet more after ADP stimulation 2.08+0.51

ng/108platelets

10 min after ADP, pcO.02).

production

and TXB2 plasma levels, after collagen

and even

(0.59+0.27 vs 1.35+0.46 and 0.99+0.47 vs for habitual smokers vs controls, 4 and No significant

differences

in platelet

5-HT release were observed. Acute smoking in either

did not induce any significant

5-HT or TXB2

TXB2 production

for controls,

from ADP-challenged

while

change

from

significantly

baseline reduced

platelets from habitual smokers

(0.30+0.15 vs 0.5920.27 ng/108 platelets, immediately after smoking

KEY WORDS: Platelet function, Serotonin, Thromboxane,

85

Cigarette smoking.

Vol. 44, No. 1

SMOKING AND PLATELETS

86

Ninety min after the completion of the vs baseline, p
for TXB2

platelet

production

(2.7621.78 vs 6.42k1.60, pcO.025 and 3.01+1.90 vs 6.44k2.26 ng/108 platelets, pcO.05, for habitual smokers vs controls, 4 and 10 min 0.30+0.15 vs 1.20+0.84 and after the addition of collagen; _ platelets, after ADP p co.05, 0.79+0.50 vs 1.7OkO.74 ng/108 stimulation). Differences were no longer significant 90 min after smoking. Our data indicate that cigarette smoking is associated of which seems due to impairment with platelet dysfunction, metabolic activation

platelet

capacity

than

rather

increased

platelet

in vivo.

INTRODUCTION Cigarette smoking is a major risk factor for development of cardiovascular disease (1). Carbon monoxide

(CO) and nicotine

are at present

considered

to

be the most likely mediators of vascular impairment from smoking and endothelial damage as well as platelet activation to be the putative targets of their actions confirmed

(5-7),

conflicting

(2-4). However, data

from

while endothelial

studies

dealing

with

damage has been largely platelet

function

are

(8-12).

The aim of the present study was to see whether or not changes in platelet release

of

vasoactive

metabolites

activation as a consequence

suggest

continuous

in

vivo

platelet

creatinine

sulphate

of smoking.

MATERIAL AND METHODS Reagents.

The following

monohydrate

(serotonin,

were used:

5-Hydroxytryptamine

5-HT) and 5-Hydroxymethyltryptamine

oxalate

(5-HMT)

(Janssen, indomethacin disodium Belgium); (Sigma Chem. Co, U.S.A.); ethylenediamine tetraacetic acid (EDTA) (Merck, W.Germany); collagen and adenosine (Eurochima,

diphosphate Italy);

(ADP)

HPLC

grade

(Mascia

Brunelli,

n-butanol

Italy);

and methanol

sodium

citrate

(Merck, W.Germany).

All other reagents were from C.Erba, Italy. Subjects: Twelve healthy volunteers, 6 non smokers and 6 habitual smokers (more than 20 cigarettes/day), who had not taken any drug for at least ten days prior to the experiment, entered the study. In the first group there were 4 men and 2 women, 30-44 years old (3556, mean +S.D.). The habitual smokers' group consisted of 4 men and 2 women, 24-40 years old (3157, mean +S.D.), who abstained from cigarette smoking overnight. The following measurements were made before smoking, immediately after smoking two medium-tar cigarettes in a 10 min period, and 90 min after the completion of the smoking:

Vol. 44, No. 1

1) 5-HT (intraplatelet concentration ADP-stimulated

87

SMOKING AND PLATELETS

and amounts released from collagen- and

platelets); (plasma levels and the amounts produced by platelets

2) thromboxane B2(TXB2)

after collagen and ADP stimulation); 3) platelet aggregation to collagen and ADP. All subjects were requested to inhale in a similar fashion. Procedure: into

tubes

drawn

Venous blood, containing

(platelet-rich

plasma,

PRP)

l/10 or

stasis,

was

volume

of

0.8/10

(EDTA)

ethylenediaminotetraacetate indomethacin

without

either

immediately 3.8%

volume

plus

0.5/10

of

collected

sodium 77

citrate

mM

volume

disodium

of

0.04

mM

(platelet-free plasma, PFP). PRP and PFP samples were prepared

as described previously (13). 5 PRP samples diluted with PFP to 1.1+0.2 x 10 platelets/p1 were used to measure intraplatelet 5-HT concentrations. Platelet aggregations and 5-HT and TXB liberation from stimulated platelets contain&g addition

3.OkO.5 of

x

lo5 platelets/pi.

collagen

or

ADP

were determined on PRP samples

Four

(4

and

and

ten

4

PM

minutes

after

respectively,

the final

pg/ml concentrations), reactions were stopped by adding 77 mM Na2EDTA and cooling and platelet metabolites assayed. 5-HT was extracted into butanol by the method of Shore and Olin into a high performance

(14) and the final aqueous extracts liquid chromatograph

Norwalk, CT, USA) equipped with an electrochemical Bioanalytical

Systems,

W.Lafayette,

with a thrombocounter-C. test platelet

aggregation.

A Born

IN, U.S.A.).

detector Platelet

type aggregometer

TXB2plasma

levels

were injected

(Series 2 pump,

Perkin-Elmer,

(L4 amperometer, counts

(Elvi 840)

and TXB2

were

was

made

used

to

platelet-associated

productions were assayed with the radioimmunoassay kit of Biodata-Serono Diagnostics. Statistics: Statistical evaluations were performed by using Student's t-test for paired data and determining

linear correlation coefficients.

RESULTS Platelets

from habitual

smokers

had

higher,

although

not

significantly,

basel'ne 5-HT contents than those of non smokers (116.75243.54 vs 82.1827.46 B platelets, mean+S.D.). _ Baseline 5-HT release from platelets from r&l0 smokers and non smokers after

addition

of collagen

or ADP

did not

differ

significantly (Table 1). The results in Table 2 show that baseline plasma TXB2 levels were somewhat, but not significantly, higher in smokers than in controls (0.15+0.03 vs 0.13+0.04 ng/ml, mean+S.D), while baseline TXB2 platelet habitual

production

after ADP stimulation was significantly lower in smokers 5-HT release production (p< 0.02). and TXB were significantly correlated (r=0.67 p< 0.01) A:Pfor but not for collagen-challenged platelets. Baseline platelet aggregations were similar in both groups (78.626.3 vs 83.8+16.0% after collagen stimulation and

75.7+8.0 vs 73.6+5.2% after ADP stimulation, for controls vs smokers). Intraplatelet 5-HT and plasma TXB levels did not significantly change after acute exposure to smoke (92.09&.90 vs 97.69544.38 and 78.1Ok18.67 vs

88

SMOKING AND PLATELETS

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SMOKING AND PLATELETS

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SMOKING AND PLATELETS

90

Vol. 44, No. 1

for 5-HT, immediately and 90 min after smoking 0.14+0.03 vs 0.15+0.05 and 0.14+0.03 vs after immediately and 90 min. levels plasma

109.43+48.99 *g/10* platelets for controls

vs habitual

smokers;

0.13+0.04 ng/ml for TXB2 controls vs habitual smoking,

5-HT

smokers).

release

from

stimulated

platelets after smoking was similar in the two groups (Table l), while TXB2 platelets immediately production from both collagen- and ADP-stimulated after smoking was significantly

lower in chronic

smokers

than

in controls

(Table 2). Differences were no longer significant 90 min after smoking. Immediately after acute exposure to smoke, there was significant correlation and between 5-HT and TXB2 production after both stimuli (r=0.79 pcO.01 r=0.91 p
in either tended

production

to

group have

even reduced

immediately after smoking long-term release. Immediately after smoking TXB2

ADP-stimulated

from

change from

though

platelets

from

habitual

smokers

was

significantly reduced in comparison with baseline (~~0.01). In contrast to controls, habitual smokers tended to have higher 5-HT release 10 min rather than 4 min after ADP challenge before as well as after smoking. A tendency toward a similar pattern of release following ADP challenge was observed in controls 90 min after acute exposure to smoke. Smoking

had

no effect

on

platelet

aggregation

to

collagen

though habitual smokers tended to have slightly higher values (73.4522.3 vs 82.6+9.4% with collagen vs 78.5+8.2% stimulation,

and 74.157.6

stimulation,

or

vs 78.6+_8.8% for controls

immediately

ADP,

vs smokers

and 90 min after smoking;

and

70.6+7.9 vs 79.1+17.4% for controls immediately and 90 min after smoking).

even

than controls

vs smokers

74.4211.8 with

ADP

DISCUSSION Even though platelets have been thought to play an important role in bringing about vascular impairment from cigarette smoking, there is little direct evidence

at present.

Augmented

platelet

activation

associated

with

smoking has been suggested,

but the data reported are inconsistent (8-12). The normal platelet aggregation following smoking found in this study, which agrees with most (9,10), but not all (12) previous rule

out

increased

platelet

activation.

investigations,

Measurement

of

does not

platelet-release

products However,

might provide more reliable indexes of platelet activity (15). studies dealing with measurements of several platelet-derived substances in plasma from smokers are conflicting (9, 16-19).

Although technical problems might interfere with the actual concentration of TXB2 in plasma, as recently pointed out (20), our data showing no significant changes for TXB2 plasma concentration, are not consistent with platelet activation in vivo. Platelet behaviour in response intriguing. After acute exposure

to exogenous stimuli in vitro is somewhat to smoke, a tendency toward decreased TXB

2

Vol.

44, No. 1

SMOKINGAND PLATELETS

91

production but not 5-HT release was observed in non smokers, while long-term smokers

liberated

depressed

5-HT

significantly

release

after

lesser amounts

of TXB2

ADP-stimulation.

and

Decreased

tended

5-HT

to have

(18,191,

but

normal TXB2 (21) liberation following smoking have been reported, but the data are difficult to compare because of technical differences. It has been suggested platelet

that

the depressed

TXA2

nicotine,

synthesis

impairs platelet

cytochromes

release

(19).

It

of

has

activation

5-HT

could

been

be

proposed

by binding

due that

to

decreased

CO,

to hemeproteins,

but not such as

interfering with TXA production 2 between TXB2 production and 5-HT (3,4,19). release from ADP- rather than from collagen-stimulated platelets of habitual smokers

and cyclooxygenase, thus The significant correlation

is in keeping

enzymatic

activities

with

an effect

leading

to

of

smoke

on cyclooxygenase

or other

TXA

synthesis, since ADP-induced 5-HT release is more dependent on TXA2 p?oduction. Moreover, the higher 5-HT release 10 min rather 4 min after ADP challenge we found in habitual smokers - in contrast with previous (22) and present observations dealing normal platelets in absence of smoke -, might be consistent with mechanism.

On

the

other

hand,

a

similar

platelet

behaviour

with such

following

exogenous stimulation in vitro, might reflect marked platelet activation in vivo. According to recent observations on platelets from patients suffering of

myocardial

consequence

of

infarction (231, depressed TXA2 liberation may be the limited substrate availability in "exhausted" platelets

following activation in vivo, rather than of biochemical enzymatic defects. However, the absence of significant changes in intraplatelet 5-HT would not support platelet activation in vivo (24,25). The smoking

behaviour

of platelet

might be due

5-HT

release

to a different

could be related to the hypothetical

we

observed

platelet

in

sensitivity

controls

after

to smoke,

which

altered sensitivity of chronic smokers'

platelets to the adrenergic effects of nicotine (26,29), even though different smoking technique of volunteers cannot be completely ruled out. In conclusion,

our data show that cigarette

of habitual smokers' platelets 1) a significant stimulation, stimulation,

reduced

to exogenous

TXB 2 platelet

toward 2) a tendency and 3) an abnormally

smoking affects

stimuli

formation

depressed

the response

in vitro, resulting after

5-HT

collagen

release

a

and

after

in: ADP ADP

slow release of 5-HT after ADP stimulation. These changes seem to reflect impairment of metabolic platelet capacity even though heightened platelet activation in vivo cannot be ruled out.

This work was supported by CNR "Gruppo Cardiorespiratorio", CT 84.00830.04.

Rome, Grant no.

Vol. 44, No. 1

SMOKING AND PLATELETS

92

REFERENCES of Smoking: Cardiovascular

1. The Health Consequences

the Surgeon General, publication

DMHS

Disease.

(PHS) 84-50204.

A Report of

U.S.Department

of

Health and Human Services, Public Health Services, Office on Smoking and Health. Rockville, MD, 1983. 2. BIERENBAUM,

M.L.,

FLEISCHMAN,

A.I.,

STIER,

A.,

SOMOL,

H.

and

WATSON,

P.B.: Effect of cigarette smoking upon in vivo platelet function in man. Thromb.Res. 12, 1051-1057, 3. MANSOURI, cigarette

A. and smoke

PERRY, and

1978. C.A.: Alteration of platelet aggregation by monoxide. Thromb.Haemostas. 48, 286-288,

carbon

1982. 4. WENNMALM,

A.:

cardiovascular

Interaction

of

nicotine

system. Prostaglandins

and

prostaglandins

in

the

23, 139-144, 1982.

5. ASMUSSEN, I. and KJELDSEN K.: Intimal ultrastructure

of human umbilical

arteries. Observations on arteries from newborn children non smoking mothers. Circ.Res. 36, 579-589, 1975.

of smoking

and

6. PITTILO, R.M., MACKIE, I.J., ROWLES, P.M., MACHIN, S.J. and WOOLF, N.: Effects of cigarette smoking on the ultrastructure of rat thoracic aorta and its ability to produce

prostacyclin.

Thromb.Haemostas.

48, 173-176,

1982. 7. DAVIS, J-W., I.S.:

SHELTON,

Effects

of

L.,

endothelium and platelets. 8. HAWKINS,

EIGENBERG,

tobacco

R.I.: Smoking,

and

D.A.,

HIGNITE,

non-tobacco

Clin.Pharmacol.

platelets

C.E.and

cigarette

Ther. 2,

and thrombosis.

WATANABE,

smoking

on

529-533, 1985. Nature

236,

450-452,

1972. 9. LASZLO, E., KALDI, N. and KCVACS, L.: Alteration in plasma proteins and platelet functions with aging and cigarette smoking in healthy men. Thromb.Haemostas.

49, 150, 1983.

10. RING, T., KRISTENSEN, H.

and

DYERBERG,

J.:

S.D., JENSEN, Cigarette

P.N., MOURITS-ANDERSEN,

smoking

shortens

the

T., MADSEN,

bleeding

time.

Thromb.Res. 32, 531-536, 1983. 11. RENAUD, S., BLACHE, D., DUMONT, E., THEVENON, C. and WISSENDANGER, Platelet function after cigarette smoking in relation to nicotine carbon monoxide. Clin.Pharrnac.Ther. 36, 389-395, 1984.

T.: and

12. MADSEN, H. and DYERBERG, J.: Cigarette smoking and its effects on the platelet-vessel wall interaction. Scand.J.Clin.Lab.Invest. 44, 203-206, 1984.

Vol. 44, No. 1

13. MARASINI,

93

SMOKING AND PLATELETS

BIONDI,

B.,

High-performance

PIETTA,

M.L.,

liquid

chromatographic

assay

A.:

AGOSTONI,

and

P.G. of

serotonin

and

chemical

in

human

plasma. La Ricerca Clin.Lab. 15, 63-69, 1985. 14. SHORE,

P.A.

and

norepinephrine

OLIN,

J.S.:

in brain

and

Identification

other

tissues.

assay

J.Pharmacol.exp.ther.

of 122,

295-300, 1958. 15. ZAHAVI, J. and KAKKA, U.V.: R-thromboglobulin

- a specific

vivo platelet release reaction. Thromb.Haemostas. 16. MEHTA, P. and MEHTA, J.: Effects thromboxane-prostacyclin balance

3,

of smoking on platelets in

man.

marker

on in

23-29, 1980. and on plasma

Prostaglandins,

Leukotrienes

and Medicine 9, 141-150, 1982. 17. SCHMIDT, K.G. and RASMUSSEN, J.W.: Acute platelet activation induced by smoking. In vivo and ex vivo studies in humans. Thromb.Haemostasis 51, 279-282, 1984. 18. SABA,

S.R.

and

MASON,

R.G.:

Some

effects

of

nicotine

on

platelets.

Thromb.Res. z, 819-824, 1975. 19. MANSOURI, A. and PERRY, C.A.: Inhibition of platelet ADP and serotonin release by carbon monoxide and in cigarette smokers. Experientia 40, 515-517, 1984. 20. PATRONO, C., CIABATTONI, FITZGERALD, circulation

G.A.:

G., PUGLIESE, F., PIERUCCI, A., BLAIR, I.A. and

Estimated

rate

of

thromboxane

secretion

into

the

of normal man. Clin.Res. 33, 287A, 1985.

21. STURM, H., BARDEN, A., BEILIN, L.J. and TAYLOR, R.R.: The measurement of thromboxane B2 plasma and the effect of smoking. Clin.Exp.Pharmac.Physiol. 11, 611-619, 1984. 22. MARASINI, B., BIONDI, M.L. and AGOSTONI, A.: Quantitative determination of serotonin released from human plasma. La Ricerca Clin.Lab. 15, 247-251, 1985. 23. MUELLER, H.S., RAO, P.S., GREENBERG

M.A., BUTTICK, P.M., SUSSMAN, I.I., V ., STRAIN, J.E. and SPAET,

LEVITE, H.A., GROSE, R.M., PEREZ-DAVILA, T.H.: Systemic and transcardiac platelet infarction

in

man:

resistence

to

activity

in acute

prostacyclin.

myocardial

Circulation

72,

1336-1345, 1983. 24. PARETI, F.I., CAPITANIO, A., MANNUCCI, L., PONTICELLI, C. and MANNUCCI, P.M.: Acquired dysfunction due to the circulation of "exhausted" platelets. Am. J. Med. 3,

235-240, 1980.

SMOKING AND PLATELETS

94

25. UMEGAKI,

K.,

platelets

INOVE,

at

hypertensive

the

Vol. 44, No. 1

I. and TOMITA, T.: The appearance time of smoke in stroke-prone

rats". Thromb. Haemostas.

on

the

serum

epinephrine

"exhausted"

spontaneously

54, 764-767, 1980.

26. HILL, P. and WYNDER, E.L.: Smoking and cardiovascular nicotine

of

and

disease. Effect of

corticoids.

Am.

Heart

J.

83,

491-496, 1974. 27. CRYER, P.E., Norepinephrine

HAYMOND, M.W., and epinephrine

smoking-associated

hemodynamic

SANTIAGO, J.K., and SHAH, S.D.: release and adrenergic mediation of and metabolic events. N.Eng.J.Med. 295,

573-577, 1976. 28. NADLER, J.L., VELASCO, prostacyclin

formation.

J.S. and HORTON, R.: Cigarette Lancet i, 1248-1250, 1983.

smoking

inhibits

29. BELCH, J.J.F., MCARDLE, B.M., BURNS, P., LOWE, G.D.O. and FORBES, The

effects

haemorheology

of acute

smoking

on platelet

behaviour,

in habitual smokers. Thromb.Haemostas.

fibrinolysis

51, 6-8, 1984.

C.D.: and