1.

Introduction
Cigarette smoke is a complex mixture of chemical compounds that are bound to
aerosol particles or are free in the gas phase. Chemical compounds in tobacco can
be distilled into smoke or can react to form other constituents that are then distilled
to smoke. Researchers have estimated that cigarette smoke has 7,357 chemical
compounds from many different classes (1) In assessing the nature of tobacco
smoke , scientists must consider chemical composition, concentrations of
components , particle size , and particle charge (2) . These characteristics vary with
the cigarette design and the chemical nature of the product. The chemicals in
tobacco smoke reach your lungs quickly when you inhale. What this new report
shows is that these same poisonous chemicals reach every organ in your body.
They go quickly from your lungs into your blood. Then the blood flows through
your arteries. It carries the chemicals to tissues in all parts of your body. Your
lungs, blood vessels, and other delicate tissues become inflamed and damaged
when you smoke (3) . Tobacco cigarette smoking is one of the major leading causes
of death and essential pubic health challenge in word over (4). It is the prime cause
of death word wide, resulting in millions of deaths annually, more than HIV\AIDS,
tuberculosis and malaria (5). Smoking change the pH in stomach that resulted in
peptic ulcers and gastric diseases. In Sudan, prevalence of cigarette smoking in the
population reached 12%\ Alternatively in the some developed countries, although
prevalence of cigarette smoking is almost double that of Sudan (6) Cigarette
smoking could cause alterations in the main hemostatic system, and coagulation
factors which in turn could affect their functions . Paucity of information on this
area, coupled with the increase in the number of chronic smokers necessitated the
present study which is aimed at evaluating the effect of smoking on the
coagulation markers PT and APTT . The results of the study will, therefore,
substantiate the predisposition or otherwise of smokers to coagulation disorders
and define the impact of long term smoking on coagulation.

1.1 Rational and Objectives:

1.1.1 Rationale:

The effect of tobacco cigarette smoking on human health are serious and in many
cases deadly

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1.1.2 Objectives:

1.1.2.1 General objective:

To assess the effect of cigarette smoking on the PT and PTT of male smokers in
Port Sudan city to add medical information to the local database table.

1.1.2.2 Specific objectives:

1- To estimate prothrombin time in smokers and non smokers.

2- To estimate partial thromboplastin time in smokers and non smokers.

3- To compare between the data obtained for smokers and non smokers.

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 2- Literature review
2.1 Component of normal hemostasis:
2.1.1 Blood vessel:
Blood vessel convey blood around the body and are classified in to three. main
types arteries, capillaries and vein . vessel wall is divided into three coats or
tunics. These coats are the tunica intimae ,tunica media and tunics adventitia the
tonics intimae from the smooth glistening. surface of endothelium that lines the
lumen (inner tubular cavity ) of all blood and lymphatic vessels and the heart . it
line with simple squamous epithelium referred to as endothelium the tunica intima
consists of single layer of endothelial cell thickened b a sub endothelial connective
tissue layer containing elastic fibers.
2.1.2 Platelets:
Platelets are made in the bone marrow Huge cells known as megakaryocytes.
(derived from hematopoietic stem cells) are the precursor to platelets , one
megkaryocyte can produce 2,000 platelets . Platelets bud off the edges of the
megakaryocyte which eventually. perishes by evaporating. It circulates in the
blood for 7-10 days. Its either . circulate freely or sequestered in the spleen .at any
given time one third of platelets are located in the spleen. Platelets are extremely
small and discoid, 3.0 x 0.5 micrometer in diameter with a mean volume 7-11
fl.thrombopietin is the major regulator of platelets production and constitutively
produced by the liver and kidneys.
2.1.3 Coagulation factors:
Bleeding from small blood vessel may be stopped vasoconstriction and the
formation of a platelet plug ,but the formation of (thrombus ) usually occurs as part
of the normal process of hemostasis. The soluble blood coagulation factor are
critical in the formation of a thrombus (table A) the coagulation factor have been
described as reacting in a cascading sequence . Modification of this sequence are
now know to occur as blood factor interact to form the final insoluble gelatinous
thrombus.

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Table 2-1: Coagulation proteins:
Factor Name Alternate terms
I Fibrinogen
II Prothrombin
V Proaccelertin Labile factor, Ac
globulin
VII Proconvertin Stabile factor, SPCA
VIII AHF AHG, anti-hemophilic
factor A
IX PTC Christmas factor, anti-
hemophilic factor B
X Stuart factor Stuart-Prower factor
XI Plasma thromboplastin PTA, anti-hemophilic
anteceden factor
XII Hageman factor Glass or contact factor
Other Prekllikrein Fletcher factor

HMW Kininoge HMW fitzgererald factor

Vwf Factor VIII–related

Fibronectin
Anti –thrombin III
Heparin cofactor II
Protein c
Protein s

2.1.4 Coagulation inhibitors:

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There are three separate inhibitors in this aspect, and they all have to do with
control of the production and function of thrombin. These factors are Circulating
antithrombin, protein C and thrombomodulin. More over, tissue factor pathway
inhibitor also act to inactivate thrombin. In addition, it has other anti –coagulant
action by inactivating XIIa, XIa, IXa, and Xa Thrombomodulin on the surface of
intact endothelial surfaces, binds both thrombin and protein C (and the binding to
protein C is strongly enhanced by the protein C receptor on the endothelium)
within this bound complex, thrombin loses its procoagulant properties and
becomes an anti coagulant, by the process of activating protein C. Activated
protein C on the surface of activated platelets (where the coagulation process is
going on), degrades Va and VIIIa, thus inhibiting further local coagulation.
Proteins C and S also require vitamin K-dependent post-translational carboxylation
for effect this is important when considering coagulation disorders in liver disease
and in instituting anti-coagulant therapy.
2.2 Primary homeostasis:
This is normally triggered by an injury to endothelium. This produces a gap in the
tissue and is immediately filled with blood from the damaged vessel. The blade
cuts through the tissue, through the sub endothelium, and then through the
endothelium producing a gap. The blade is withdrawn, and blood immediately fills
the gap.
2.2.1 Role of platelet in homeostasis:
2.2.1.1 Platelet Adhesion:
Platelet adhere to the sub endothelial collagen fibers ,spread psuedopods along the
surface and clump together (aggregate) when vascular injury exposes the end
endothelial surface and underlying collagen m, its adhesion to sub endothelial

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connective tissue ,especially collagen occur within 1 to 2 minutes after a break in
the endothelium .Epinephrine and serotonin promote Vasoconstriction.
2.2.1.2 Platelet aggregation:
It is the process in which adherent platelets become activated and release the
contents of storage granules, recruiting nearby platelet in circulation to from an
aggregate, the formation of the platelet aggregate or thrombus occurs via activation
of GPIIB-IIIa and binding of multivalent adhesive ligands, fibrinogen, or von Will
brand factor (VWF ) which crosslink the adjacent activated platelets.
2.2.1.3 Platelets activation and release reactions:
Platelets undergo aggregation and release the content of their dense granule and
alpha granule when exposed to agonist such as ADP epinephrine thrombin or
[13]
collagen ,ADP and serotonin released from the dense granules further enhance
the platelet activation processes .for example ,ADP released from the granules
interacts with receptors on platelets to enhance the activation process [14]
2.3 Secondary homeostasis:
Secondary homeostasis consist of the cascade of coagulation serine proteases that
cumulates in cleavage of soluble fibrinogen by thrombin, thrombin cleavage
generates insoluble fibrin that form a cross linked fibrin mesh at the site of an
injury. fibrin generation occur simultaneously to platelets aggregation [15]
2.3.1 Coagulation Pathways:
The plasmatic coagulation traditionally has been divided into twodifferent
pathways the intrinsic and extrinsic pathway. This understanding of coagulation
has been built on studies of clotting in arelatively cell-free plasma system in vitro.
However, such a division does not really occur in vivo because factor VIIa-TF
complex is a potent activator of factor IX as well as factor X. [16]

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2.3.2 Extrinsic Coagulation Pathway:
The extrinsic pathway is initiated by the entry of tissue thromboplastin into the
circulating blood. Tissue thromboplastin is derived from phospholipoproteins and
organelle membranes from disrupted tissue cells. These membrane lipoproteins,
termed tissue factors, are normally extrinsic to the circulation. Platelet phospholipids
are not necessary for activation of the extrinsic pathway because tissue factor supplies
its own phospholipids. Factor VII binds to these phospholipids in the tissue cell
membranes and is activated to factor VIIa, a potent enzyme capable of activating
factor X to Xa in the presence of ionized calcium. The activity of the tissue factor–
factor VII complex seems to be largely dependent on the concentration of tissue
thromboplastin. The photolytic cleavage of factor VIIa by factor Xa results in
inactivation of factor VIIa. Factor VII participates only in the extrinsic pathway.
Membranes that enter the circulation also provide a surface for the attachment and
activation off actors II and V. The final step is the conversion of fibrinogen to fibrin
by thrombin.
2.3.3 Intrinsic Coagulation Pathway:
This pathway is the longer pathway of secondary hemostasis . It begins with the
activation of factor XII (a zymogen, inactivated serine protease) which becomes
factor XIIa (activated serine protease) after exposure to endothelial collagen.
Endothelial collagen is only exposed when endothelial damage occurs. Factor XIIa
act as a catalyst to activate factor XI to factor XIa . Factor XIa then goes on to
activate factor IX to factor IXa goes on to serve as a catalyst for turning factor X
into factor Xa. This is known as a a cascade. When each factor is activated , it goes
on to activate many more factors in the next steps.
2.3.4 Common pathway:
This pathway begins at factor X which is activated to factor Xa. It goes on activate
factor II (prothrombin) into factor IIa (thrombin). Also , factor Xa requires factor V
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as a cofactor to cleave prothrombin into thrombin. Factor IIa (thrombin) goes on
to activate fibrinogen into fibrin. Thrombin also goes on to activate other factors in
the intrinsic pathway (factor XI) as well as cofactors V and VIII and factor XIII.
Fibrin subunits come together to form fibrin strands, and factor XIII act on fibrin
strands to form fibrin mesh. This mesh helps to stabilize the platelet plug.
2.4 Prothrombin time:
2.4.1 Principle:
The prothrombin time measures the clotting time of recalcified plasma in the
presence of an optimal concentration of tissue extract (thrombopolastin) and
indicates the overall efficiency of the extrinsic clotting system.
Although originally thought to measure prothrombin, the test also depends on
factors V, VII and X and on the fibrinogen concentration of the plasma.
2.4.2 Reagents:
Patient and control plasma samples.
Note that plasma stored at 4℃ have a shortened PT as a result of factor VII
activated in the cold.
2.4.2.1 Thromboplastin:
Thromboplastin were originally tissue extracts obtained from different species and
different organs containing tissue factor and phospholipid.
2.4.3 Reference values:
Normal values depend on the thromboplastin used, the exact technique and
whether visual or instrumental end point reading is used.
With most rabbit thromboplastin the normal range of the PT is between 11 and 16
sec. The common causes of a prolonged PT are as follows:
1. Administration of oral anticoagulant drugs (vitamin k antagonists).
2. The presence of a direct acting inhibitor of factor Xa.
3. Liver disease, particularly obstructive jaundice.
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 4. Vitamin K deficiency.
5. Disseminated intravascular coagulation.
6. Rarely , a previously undiagnosed factor VII, X, V, or prothrombin
deficiency or defect.
2.5 Activated partial thromboplastin time:
Specific variation of the APTT test are known as the partial thromboplastin time
with kaolin (PTTK) and thekaolincephalin clotting time (KCCT) reflecting the
methods used to perform the test.
2.5.1 Principle:
The test measures the ciotting time of plasma after the activation of contact factors
and the addition of phospholipid and CaC12 but without added tissue
thromboplastin and so indicates the overall efficiency of the intrinsic pathway The
plasma is first preincubated for a set period with a contact activator such as kaolin,
silica or ellagic acid During this phase of the test ,FXIIa is produced , which
cleaves FXI to FXIa , but coagulation does not proceed beyond this point in the
absence of calcium . After recalcification ,FXIa activates FIX and coagulation
follows. A standardized phospholipid is provided to allow the test to be performed
on PPP .the test depends not only on the contact factor and on factors VIII and IX
but also on the reactions with factor X,V,prothrombin and fibrinogen It is also
sensitive to the presence of circulating anticoagulants(inhibitors) and heparin.
2.5.2 Reagents:
Platelet poor plasma (PPP) from the patient and a control,

Kaolin. 5 g\L (laboratory grade) in barbitone buffered saline, PH 7.4. Add few
glass beads to aid resus pension .the suspension is stable at room temperature.
Other insoluble surface active substances such as silica, celite or ellagic acid can
also be used. Phospholipid .many reagents are available; these contain different
phospholipids. When choosing a reagent for the APTT, is important to establish
that the activator-phospholipid-incubation time combination is sensitive to
deficiencies of factor VIII, XI and XI at concentration of 0.35_0.4 iu\ml.
Combination that do not produce a prolonged clotting time at these levels are too
insensitive. The system should also be responsive to unfractionated heparin over
the therapeutic range of approximately 0.3_0.7 anti-Xaiu\ml in addition, some

9
laboratories will wish the system to the presence of lupus anticoagulants. Ca Cl 2.
0.025 mmol.

2.5.3 Reference ranges:

The normal range is typically 26 to 40s. the actual time depend on the reagent used
and the duration of the preincubation period which varies in manufacturers
recommendation for different reagents laboratories can choose appropriate
conditions to achieve the sensitivity they require. The common causes of a
prolonged APTT are as follows:
1. Disseminated intravascular coagulation.
2. Liver disease.
3. Massive transfusion with plasma-depleted red blood cell.
4. Administration of or contamination with heparin or other anticoagulants.
5 .A nonspecific circulating anticoagulant (such as an LAC).
6. The presence of a direct acting anticoagulant drug(e.g. anti all or anti-Xa agents)
7. Deficiency of a coagulation factor other than factor VII
The APTT is also moderately prolonged in patient taking oral anticoagulant drugs
and in the presence of vitamin K deficiency Occasionally, a patient with previously
undiagnosed haemophilia or another congenital coagulation disorder presents
With an isolated prolonged APTT. If the patientAPTT is abnormally long, mixing
tests , an inhibitor screen and factor assays should be considered.

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 3. Material and Methods
3.1 Study population and study area:
All adult smoker males in Port Sudan were subjected to be the study population of
this research.
3.2 Study sample:
A sample of 50 subjects, 25 smokers and 25non smokers.
3.2.1 Inclusion criteria:
- Subject 20-45 years old.
- Smoker who are smoking cigarette for a minimum period of 4-6 months.
- Individual who will affair that they have not smoked yet (or very rarely tried to
smoke) will be considered as non smokers.
3.2.2 Exclusion criteria:
- Non smokers, Subject who are on drugs which may affect coagulation marker,
and subject with severe illness.
3.3 Study design:
This is a cross sectional descriptive study.
3.4 Study duration:
The study was carried out during the period from August to October 2022.
3.5 Data analysis:
Data were analyzed by using a computer program SPSS software.
3.6 Procedures and techniques:
3.6.1 Collection of blood samples:
2.5 ml of blood was collected from each member of study group.
3.6.1. 1 Requirements:
- Disposable plastic 5 ml syringe with needle of (22Gx 1 1\4 AL-RJA).
- 2.5 ml trisodium citrate.
- Tourniquet.
- Adhesive dressing.
- 70% Alcohol.
3.6.1.2 Procedure:
- The skin of the collection was cleaned with 70% alcohol and was allowed to dry.
- The Tourniquet was applied.
- The needle was inserted into an antecubital vein and Tourniquet was loosed.

11
- The piston of the needle was then withdrawn slowly and 2.5 ml of blood was
obtained.
- The needle was removed and an adhesive dressing was placed over the puncture
site.
- The needle was detached and the blood was then carefully delivered from the
syringe into the trisodumcitrat blood container and is gently and thoroughly mixed
with the anticoagulant for at least 8 times.
- The blood container was finally labeled with donor’s number.

3.6.2. Tests procedure:

3.6.2.1 Prothrombin time:
1. Incubate PT reagent at 37℃ for at least 10 minutes.
2. Pipette 25 ul of sample into a test cuvette. Incubate at 37℃ for 1-2 minutes.
3. Add 50 ul of PT reagent and simultaneously start test.
4. Record the clotting time in seconds.
3.6.2.2 Activated partial thromboplastin time:
1- Pre-incubate the Calcium Chloride Reagent to 37c for at least 10 minutes.
Pipette 25 ul of test plasma into a test cuvette.
2- Incubate the plasma at 37c for 1-2 minutes.
3- Pipette 25 ul of the APTT reagent, into reagent cuvette containing the plasma.
Maintain the suspension of the APTT reagent by magnetic stirring or mixing by
inversion immediately prior to use.
4- Incubate at 37c for 3 minutes.
5- Add 25 ul preincubated Calcium Chloride solution and simultaneously start the
timer.
6- Record the clotting time in seconds.

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 4. Results
This is across sectional descriptive study conducted at Port Sudan ahlia collage in
the period from August to October 2022 and aimed at the determination of
prothrombin time and partial thrompoplastin time in smokers leaving in Port Sudan
Enrolled in this study are 25 adult smokers aged between 20 to 45 years old and
other 25 non smoker healthy appearns adult aged between 20 to 30 years old as a
control group.
The mean prothrombin time in smokers (13.2 second) differ significantly (P.value=
0.000) from the mean prhrombin time in non smokers (16.4 seconds). On the other
hand, the mean partial thromboplastin time in smokers (29.6 seconds) also differret
significantly (P. value = 0.001) from the mean partial thromboplastin time in non
smokers (34.0 seconds) see table 4-1
Table 4-1 coagulation resuit in smokers and non smokers:

Smokers Non smokers P value

Mean PT(second) 13.2 16.4 0.000
Mean PTT(second) 29.6 34.0 0.001

64%

32%

4%

5 -7. 8-10 11-13

Figure 4.1: Duration of cigarette smoking among test group.

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 52%

44%

4%

20-29 30-39 40-50

Figure 4.2: Age distribution among test group.

76%

16%
8%

5-10 11-15 16-20

Figure 4.3: Number of cigarette smoking among test group.

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 5. Discussion
Cigarette smoking is spread wildly in the population specially among middle age
group. It is well known that smoking has been shown to adversely affect
coagulation parameters resulting in haemostatic complications. This study was
design to evaluate the effect of smoking on PT and APTT results. Oke O.T et al
(2022)(7) studying the evaluation of changes on platelet, PT, and APTT of smokers
in Okehi and its environment in Nigeria found that the mean values of PT and
APTT of smokers were significantly lower compared to non smokers. Findings
that are absolutely consistent with our findings. Chizoba O.O and Eubka E.H(8)
studying the effect of cigarette smoking on some coagulation parameter of smokers
in Newi Metropolis, Nigeria recorded that the mean values of PT and APTT of
smokers was significantly prolonged compares to non smokers. Thesis finding are
different from ours results perhaps due to our smaller sample size and variation in
environmental factors. Ahmed M(2018)(9) focused the effects of cigarette smoking
on coagulation screening tests and platelet count in Sudanese male adult population
found that the mean PT was significantly lower in smokers while PTT had no
significant variation in smokers and non smokers. His findings about PT agreed
with what we have found but his findings about PTT is not agreed with our
findings perhaps due to environmental variation.

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 6. Conclusion and Recommendations
6.1 Conclusion:

Data obtained from this study Cleary indicated that cigarette smoking tent to
have lower PT and APTT results in smokers than non smokers. So that smoking
might be associated with bleeding disorders.

6.2 Recommendations:

1. It is advisable to establish governmental centers that work to stop the

tradition of smoking in the population.
2. Further studies are recommended that include larger sample size to
evaluate the effect of cigarette smoking on other coagulation parameters.
3. It is important to introduce lows that act to forbid and punch
individuals who smoke in popular area so as to minimize the effect of
passive smoking.

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 7. References
1 Rodgman A, perfetti TA. The chemical components of tobacco and tobacco
smoke. Boca Raton (FL): CRC Press, Taylor and Francis Group; 2009. {Ref list}

2 Dube MF, Green CR. Methods of collection of smoke for analytical purposes.
Recent Advances in Tobacco Science: Formation, Analysis, and Composition of
Tobacco Smoke. 1982;8:42_102.[Ref list].

3 A Report of the surgeon General; how Tobacco smoke causes disease.

4 KumeA,Kume T, Masude K, Shibuya F, Yamzaki Dose-dependent effect of

cigarette smoke on blood biomarkers in healthy volunteers: Observations from
smoking and non-smoking. Journal of Health Sciences 2009;55(2):259-264.

5 World Health Organization(WHO). Research for International Tobacco

Control. WHO report on the global tobacco epidemic, 2008:the MPOWER
package/ Geneva: World Health

WHO Organization.http://www.who.int/tobacco/mpower/mp Ower_

report_full_2008.

6 Oke OT, et al (2022). Evaluation of changes on Platelet, PT and PTT of

smokers in Okehi and its environment. International jernal of current resserch in
chemical and pharmaceutical science 9(1):1-6

7 Okeke OC, et al (2017). Effect of cigarette smoking on some coagulation

parameters of smokers in Nnewi metropolis. Article in journal of environmental
and Occupational Science 6(1): 12-16.

8 Ahmed M. Elkhalifa (2018). Effects of cigarette smoking on coagulation

screening tests and platelet counts in a sudanese male adults population.saudi med
journal: 36 (9): 897-901.

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 PERSONAL INFORMATION

Name: ………………………………………………………………………………

Age:

20-25 ( ) 25-35 ( ) 35-45 ( )

Number of cigarette/day:

5-10 ( ) 10-20 ( ) 20-25 ( )

Duration of smoking/years:

3-5 ( ) 5-7 ( ) 7-9 ( )

History:

Coagulation disorder( ) Diseases (

)

Any chronic disease: …………………………………………………..

Any Treatments: …………………………………………………………..

Laboratory investigation:

PT ………………………..

PTT …………………………. .

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