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In te rn at io na l Journa l of Basic Me di cal Science - December 20 12, Vol : 3, Issue : 5 125 Original Article Mrunal R. Shenwai , N.V. Aundhakar 1 2 STUDY OF EFFECT OF CIGARETTE SMOKING ON PLATELET COUNT AND PLATELET AGGREGABILITY IN YOUNG MALE SMOKERS STUDY OF EFFECT OF CIGARETTE SMOKING ON PLATELET COUNT AND PLATELET AGGREGABILITY IN YOUNG MALE SMOKERS INTRODUCTION MA TERIAL & METHODS Ci garette smoking which was an uncommon behav iour ear lier in the 19 centu ry has incr eased tremendously over a period of years, despite of the fac t tha t it is pos itivel y associated wit h many diseases. Growing incidence of smoking in young population of both sexes is still a matter of serious concern for health professionals. Majority of people who succumb to the habit are well educat ed. Coronary heart disease is one of the important causes of mortality in human beings and cigarette smoking is considered as an independent risk factor for it. Components of smoke like nicotine, carbon monoxide and tar exert direct as well as indirect effects on many systems of the body especially cardiovascular system. Heavy smoking is the commonest cause of ischemic heart disease and death in 30-40 yrs of age group who are likely to be free from other myocardial risk factors. Hyper thro mbic state in smokers may be due to increase in the platelet aggregability and increased pla tel et act ivi ty whi ch ini tia tes clot for mat ion leading to occlusi ve vascular di sease . Ma ny previous studies on this have reported conflicting results. According to some researchers, platelet aggr egabi lity is not affe cted by smok ing; rather sometimes it may even decrease . Similarly effect of smoking on platelet count is a matter of debate. Hence the present work is undertak en to study the eff ect of cig arette smoki ng on pla tel et count & platelet aggra gability. The focus of the study was on young well educated male smokers with an aim of creating awareness amongst them regarding their healt h & soci al resp onsib ility . Sel ection of sub jects: The pre sent study was conducted between two groups i.e. smokers and non-smokers. All were app arentl y health y male subjects between the age group of 26-40 yrs. Total th 1 2,3 4,5 ABSTRACT Back groun d & Obje ctive : Cigar ette smok ing has become a major avoidable cause of diseases like stroke, Ischemic heart disease and occlusive vas cular disease. Smoki ng increases mortalit y nearly five times between age group of 30-40 years who are likely to be free from other myocardial risk factors. Hyperthrombic state in smokers may be attributed to enhanced platelet activity which may be reflected in terms of platelet count and platelet aggragabilty. Previous studies have reported many conflicting results on these parameters.The present work was undertaken to study the effect of cigarette smoking on platelet count and platelet aggregability in young & well educated male smokers andcomparetheresultswithnon-smokers. Methods: T otal sample size was 70 which in cluded a mi xed pop ula tio n of male doc tors & eng ine ers [smokers (n=35) & non-smokers (n=35)] between the age group 26-40 yrs. They were selected on the basis of smoking of filtered cigarettes minimum 5 per day for durat ion 10 years . Plate let count was done on Sysmex K- 4500 Auto analy ser. Platelet aggr agbil ity was estimated by O'Br ien's method using ADP. Observations: Our results showed a statistically significant increase in the platelet aggragbility (P< 0.01 & Z >2). The change in the platelet count was statistically insignificant. Concl usion : As per our stud y incr eased platele t adhesiveness is evidenced even in young smokers with duration of smoking less than 10 years. Such young population which is otherwise free from the pred ispo sing fact ors lik e obesi ty , hype rtens ion, diabetes etc. can be encouraged to adopt healthier lifestyles and quit smoking so that future health rel ate d con seq uencescan be avo ide d. Key words: young smokers,Platelet count Platelet aggregability

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International Journal of Basic Medical Science - December 2012, Vol : 3, Issue : 5 125

Original Article

Mrunal R. Shenwai , N.V. Aundhakar1 2

STUDY OF EFFECT OF CIGARETTE SMOKING ON PLATELET COUNT AND

PLATELET AGGREGABILITY IN YOUNG MALE SMOKERS

STUDY OF EFFECT OF CIGARETTE SMOKING ON PLATELET COUNT AND

PLATELET AGGREGABILITY IN YOUNG MALE SMOKERS

INTRODUCTION

MATERIAL& METHODS

Cigarette smoking which was an uncommon

behaviour earlier in the 19 century has increased

tremendously over a period of years, despite of the

fact that it is positively associated with many

diseases. Growing incidence of smoking in young

population of both sexes is still a matter of serious

concern for health professionals. Majority of people

who succumb to the habit are well educated.Coronary heart disease is one of the important

causes of mortality in human beings and cigarette

smoking is considered as an independent risk factor

for it. Components of smoke like nicotine, carbon

monoxide and tar exert direct as well as indirect

effects on many systems of the body especially

cardiovascular system. Heavy smoking is the

commonest cause of ischemic heart disease and

death in 30-40 yrs of age group who are likely to be

free from other myocardial risk factors.

Hyperthrombic state in smokers may be due to

increase in the platelet aggregability and increased

platelet activity which initiates clot formation

leading to occlusive vascular disease . Many

previous studies on this have reported conflicting

results. According to some researchers, platelet

aggregability is not affected by smoking; rather

sometimes it may even decrease . Similarly effect

of smoking on platelet count is a matter of debate.

Hence the present work is undertaken to study the

effect of cigarette smoking on platelet count & platelet aggragability. The focus of the study was on

young well educated male smokers with an aim of

creating awareness amongst them regarding their

health & social responsibility.

Selection of subjects: The present study was

conducted between two groups i.e. smokers and

non-smokers. All were apparently healthy male

subjects between the age group of 26-40 yrs. Total

th

1

2,3

4,5

ABSTRACT

Background & Objective: Cigarette smoking has

become a major avoidable cause of diseases like

stroke, Ischemic heart disease and occlusive

vascular disease. Smoking increases mortality

nearly five times between age group of 30-40 years

who are likely to be free from other myocardial risk

factors. Hyperthrombic state in smokers may be

attributed to enhanced platelet activity which maybe reflected in terms of platelet count and platelet

aggragabilty. Previous studies have reported many

conflicting results on these parameters.The

present work was undertaken to study the effect of

cigarette smoking on platelet count and platelet

aggregability in young & well educated male smokers

andcompare theresults with non-smokers.

Methods: Total sample size was 70 which included a

mixed population of male doctors & engineers

[smokers (n=35) & non-smokers (n=35)] betweenthe age group 26-40 yrs. They were selected on the

basis of smoking of filtered cigarettes minimum 5

per day for duration 10 years. Platelet count was

done on Sysmex K-4500 Autoanalyser. Platelet

aggragbility was estimated by O'Brien's method

using ADP.

Observations: Our results showed a statistically

significant increase in the platelet aggragbility (P<

0.01 & Z >2). The change in the platelet count was

statistically insignificant.

Conclusion: As per our study increased platelet

adhesiveness is evidenced even in young smokers

with duration of smoking less than 10 years. Such

young population which is otherwise free from the

predisposing factors like obesity, hypertension,

diabetes etc. can be encouraged to adopt healthier

lifestyles and quit smoking so that future health

related consequencescanbeavoided.

Key words: young smokers,Platelet count Platelet

aggregability

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International Journal of Basic Medical Science - December 2012, Vol : 3, Issue : 526

www.ijbms.com Mrunai R. Shenwai et al, Effect of Cigarette Smoking

sample size was 70, out of which study group

included smokers (n=35) and control group included

non-smokers (n=35). The study group included male

smokers who have been smoking filtered cigarettes

minimum 5 (maximum 10) per day for duration 10

yrs. Out of 35, 18 subjects were engineers

belonging to a Pune based Software Company & 17

were doctors from BJMC & Sassoon General

Hospital. The control group included 18 subjects

(non-smokers) from the same software company & 

17 doctors (non-smokers) from BJMC & Sassoon

General Hospital. Doctors & engineers were chosen

as subjects because they are considered as role

models of the society. The socio-economic status,

age, height, weight, daily activity and levels of stresswere comparable between study group and the

control group. Subjects suffering from coagulation

disorders, diabetes, hypertension or any infection

and those who are on any medication like aspirin or

non-steroidal anti-inflammatory drugs (NSAIDs)

were excluded from the study. All subjects were free

from other habits like tobacco chewing and alcohol

intake.

 The study was conducted at B.J.

Medical College, Pune. Study protocol was approvedby the ethical committee of the B.J. Medical College,

Pune.

For Platelet Aggregability, O'Brien's method of ADP

(Adenine DiPhosphate) induced platelet aggregation

was used. PRINCIPLE Addition of ADP to platelet

rich plasma (PRP) causes change in the absorbance.

 The change in the absorbance reading is expressed

as platelet aggregation time. “Absorbance of

platelet rich plasma is directly proportional to the

concentration of platelets in it”. More the decrease

in the absorbance more is the platelet aggregability.

. Platelet count was estimated by Sysemex K-

4500 Autoanalyser. It is an 18-parameter, 3 part

differential analyzer which works on the principle of

Aperture Impedance.

Written informed consent was taken from all the

subjects before the procedure. By a clean

venepuncture of anticubital vein, 7ml of blood was

collected into a plastic syringe with all aseptic

precautions out of which 2ml was immediately

Study Protocol:

:

6,7

transferred to an EDTA (anticoagulant) bulb and

automated counting was done using sysmex

autoanalyser. The remaining 5ml was collected in a

plastic centrifuge test tube containing 0.5ml of

3.8% trisodium citrate as an anticoagulant. The

sample was centrifuged at 1300 rpm for 15 min to

obtain platelet rich plasma (PRP). Colorimeter was

adjusted for the operative wavelength of 550nm in

such a way that the absorbance for dark was infinity

and distilled water was zero. It was kept constant

for each test. 2ml of PRP was taken in a colorimeter

tube and absorbance reading (optical density) was

noted. Then 0.1ml of ADP solution was added to the

sample and mixed by stirring. ADP induces platelet

aggregation and platelet clumps are formed whichsettle down at the bottom of the test tubes. Since

the aggregates tend to disperse after 20 seconds

absorbance readings were noted at the end of 20

seconds. Change in the optical density is taken as a

measure of platelet aggregability. ADP solution used

(200g/ml) was prepared by dissolving weighed

amounts of ADP powder (Sigma Pharmaceuticals)

(200 mg/L) in glassdistilledwater.

Sample collection time was kept constant between

9.30- 11.30A.M. to avoid the effect of diurnalvariationonplatelet aggregation.

 The results are presented as mean ± S.D. All the

results were statistically analysed by applying 'Z'

test, as the sample size was more than 30. 'Z' value

i.e. relative deviate and 'P' value were found out.

'Z'value of> 2 and 'P' value of < 0.001 has beentaken

as statisticallysignificant.

Our results showed that platelet aggregability

increases significantly in smokers as compared to

non-smokers (P< 0.01 & Z >2). [Table I]. No

significant difference was observed in the platelet

count of smokersandnon-smokers. [Table II]

STATISTICAL ANALYSIS OF THE DATA

RESULTS

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International Journal of Basic Medical Science - December 2012, Vol : 3, Issue : 5 127

Mrunai R. Shenwai et al, Effect of Cigarette Smoking www.ijbms.com

n=35 Mean ± SD Z value P Value

Smokers 0.066±0.021

7.32 *P<0.01Non smokers 0.035±0.012

TABLE I : EFFECT OF SMOKING ON PLATELET AGGREGABILITY

*P < 0.01 = statistically highly significant

Parameter

n= 35

Smokers

Mean± S.D.

Non-Smokers

Mean± S.D.Z value P value

Platelet count

(cells/cu.mm.)

225085.7 ±

46660.1

222657.1 ±

47589.90.27 N.S.

TABLE II : EFFECT OF SMOKING ON PLATELET COUNT

N.S. = statistically not significant

DISCUSSION

Increased platelet aggregabitity in smokers as

observed in the present study goes in accordance

with the findings reported by Davis, Rival J et al,

Chiang V.L.The increase in platelet aggregability can

be explained on the basis of raised levels of several

platelet aggregating agents and injury to the

endothelium ordue tothe direct effect of smoking on

platelets. The most important p latelet

aggregating agent elevated in smokers is

epinephrine and also nor-epinephrine. This is due to

thenicotine induced stimulation of adrenal medulla & 

sympathetic ganglia Epinephrine binds with

specif ic receptors on platelet membrane,

stimulates prostaglandin synthesis from the

phospholipids leading to formation of thromboxane

A , reduces cAMP levels and increases intracellular

Ca concentration. Increased intracellular Ca

stimulates therelease of ADP andother substancesfrom platelet granules leading to further activation & 

aggregation of platelets. Thus thromboxane A & 

Ca act as positive feedback mechanisms in the

promotion of platelet aggregation. Smoking also

stimulates thromboxane A synthesis by the

endothelial cells which also contributes to increased

platelet aggregation in smokers. Epinephrine also

induces exposure of f ibrinogen receptors

(Glycophorin IIb – IIIa) on the platelets and favours

binding of fibrinogen with these receptors. This

8-11.

12,13

2+ 14 2+

2+

15

16,17

2

2

2

helps in the formation of interplatelet bridges

causing 1 or reversible platelet aggregation.

Increase in the levels of long chain fatty acids & 

cortisol in smokers also contributes to increased

fibrinogen synthesis. Nicotine in tobacco smoke

also causes endothelial cell injury which exposes

subendothelial collagen and activates platelets.

 This leads to decreased nitric oxide production in

smokers which is a known vasodilator thus leading to

vasoconstriction and activation of platelets.

Increased platelet aggregability may be responsible

for the hyperthrombotic state and increased risk of

cardiovascular disease in smokers.The result

obtained in case of platelet count were statistically

not significant. Some researchers have reportedthat an increase in the epinephrine levels due to

nicotine causes contraction of spleen and release of

platelets in circulation leading to increased platelet

count. But many other workers found that there

is no significant change in the platelet counts in

smokers and it is only the increased reactivity and

adhesiveness of platelets which is responsible for

the hyperthrombic state. It appears that the

formation of platelet and fibrin thrombi in the

coronary arteries during an acute coronary event is

promoted by the adhesion of platelets to endothelial

cells, platelet aggregation and increased

procoagulant activity of platelets. Some of the

recent studies have observed that Smoking

apparently promotes the procoagulant activity of

platelets and enhances the prothrombotic state.

 The sharp exacerbation of the prothrombotic state

following smoking is also assumed to be related

closely pathophysiologically to theonsetof theacute

coronary syndrome. Our study corroborates with

all these findings. The younger age group and limitednumberof subjectsare someof the limitations of our

study. In future we would like to extend our study in

smokers of higher age group with duration of

smokingmore than 15years.

As per our study smoking causes significant

increase in theplatelet aggregability. These changes

are evidenced even in younger age group who are

smoking for a period between 6-10 years. Increased

0

18

8

19

20,21

9,22,23

25

CONCLUSION

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International Journal of Basic Medical Science - December 2012, Vol : 3, Issue : 5 129

Mrunai R. Shenwai et al, Effect of Cigarette Smoking www.ijbms.com

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1.Dept. of Physiology,

Smt. Kashibai Navale Medical College [SKNMC],

Narhe – Ambegaon, Pune - 41.

Mrunal R. Shenwai 2.Professor & Head,

Dept. of Physiology,

R.C.S.M. Govt. Medical College,

Kolhapur.

N.V. Aundhakar