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
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