Vol.2, No.5, 454-457 (201
doi:10.4236/health.2010.25068
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Association of the plasminogen activator
inhibitor-1(PAI-1) gene 4G/5G promoter polymorphism
in Buerger’s disease (Tromboangiitis obliterans)
Sinasi Manduz1, Nurkay Katrancioglu1, Oguz Karahan1, Oztürk Ozdemir2*
1Department of Cardiovascular Surgery, Faculty of Medicine, Cumhuriyet University, Sivas, Turkey
2Department of Medical Genetics, Faculty of Medicine, Cumhuriyet University, Sivas and University of COMU, Faculty of Medicine,
Canakkale, Turkey; *Corresponding Author:
Received 3 January 2010; revised 26 January 2010; accepted 27 January 2010.
ABSTRACT
Thromboangiitis obliterans (TAO) is an unusual
tobacco-associated vasculopathy that is a non-
atherosclerotic inflammatory disorder of unkn-
own etiology that affects small and medium-
sized vessels of the extremities. The single
guanosine nucleotide deletion/insertion polym-
orphism (4G/5G) at -675 bp in promoter region
of the PAI-1 gene is the major genetic determi-
nant of PAI-1 expression. Plasma PAI-1 level is
higher in people with the homozygous 4G gen-
otype than in those with the 5G/5G genotype
and renders higher transcription activity. The
aim of this study was to determine the status
and the role of PAI-1 gene 4G/5G promoter pol-
ymorphism in patients with Buerger’s disease
(Thromboangiitis obliterans—TAO). The current
case-control study included 30 consecutive pat-
ients with Buerger’s disease (mean age 42.9 ±
14.3 years, 28 men and 2 women), and 30 heal-
thy volunteers (mean age 40.9 ± 4.79 years, 27
men and 3 women) between January 2006 and
September 2009. Patients and control cases were
genotyped for the 4G/5G polymorphism using
the multiplex PCR based stripassay reverse
hybridisation technique. It was found that hete-
rozygote PAI-1 gene polymorphisms (p < 0.05)
was significantly more frequent in patients with
TAO in the current results. There was a signif-
icant difference in genotype distribution between
the two groups (P < 0.001). The 4G allele occur-
red more frequently in the patient group of
heavy smokers (P = 0.05). The current study sh-
ows the higher prevalence of of 4G allele in
TAO patients in Sivas population means gene
may predispose to TAO.
Keywords: Thromboangiitis Obliterans; Genetic
Polymorphisms; PAI-1
1. INTRODUCTION
In 1908 Buerger suggested the name thromboangiitis
obliterans (TAO) for a disease of the peripheral arteries
which he had studied in eleven lower limbs amputated for
“presenile spontaneous gangrene” [1]. TAO (Buerger’s
syndrome) is an unusual tobacco-associated vasculopa-
thy that is a nonatherosclerotic inflammatory disorder of
unknown etiology that affects small and medium-sized
vessels of the extremities [1-3]. Etiopathogenesis of dis-
ease could not be evenly revealed. Researchers think that
tobacco is rolled for starting and progressing of disease
and by the way genetic factors, hipercoagulability, en-
dothelium function and immune mechanisms etc. are
suspicious factors that enrolled such as tobacco [1,2,4,5].
For example HLA-9 and HLA-B5 was excessively doc-
umented in patients. This may sign that genetic factors
likely play a role [5].
Highly cellular thrombus occludes much of the lumen
and lymphocytes, giant cells, fibrous hyperplasia, inti-
mae thickness have seen around vaso-vasorums in his-
tological investigation of vessel that affected by TAO [5].
A highly cellular thrombus occludes much of the lumen.
A multinucleate giant cell and microabscess are present
within the thrombus [5].
Both genetic and thrombotic events, reminds that
prothrombotic gene deletions might act a part in this
disorder. Prothrombotic genes detected significantly in
some studies about TAO [6]. Plasminogen is one of me-
diators of fibrinolysis that synthesized in the liver and
then circulates in plasma and other extracellular fluids.
This respect presents in all tissues [7]. Urokinase-type
plasminogen activator (uPA) and the tissue-type PA (tPA)
are two regulative enzymes that directed the modulation
of plasminogen to plasmin. The enzymatic activity of
uPA can be inhibited plasminogen activator inhibitor

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(PAI) -1 and PAI-2 [8]. PAI-1 is a major inhibitor of the
fibrinolytic system. The PAI-1 gene polymorphism is
known as a deletion/insertion of G base (5G/4G) in
codon -675 and the major genetic determinant of PAI-1
expression. The 4G allele renders higher transcription
activity than the PAI-1 promoter with the 5G allele in
stimulated MCs [9]. The polymorphic PAI-1 gene is a
member of plasminogen cascade with an inhibitory role
in plasmin activation by response to cytokines, hor-
mones and many growth factors. Isordila et al., claimed
that the 4G allele is an independent risk factor for acute
myocardial infarction in young patients, as are smoking,
hypertension and a family history of inherited cardio-
vascular disease [9]. It was aimed to find out the possi-
ble role of polymorphic 5G/4G alleles of PAI-1 gene in
TAO in the current results.
2. MATERIAL AND METHOD
Thirty patients diagnosed with TAO at 1998 to 2009 in
our clinic were included the study. Total genomic DNA
from peripheral blood samples were used for each patient
and control for 4G/5G polymorphism in PAI-1 gene.
Control group was composed of healthy individuals who
had no vascular complaints.
Clinical diagnosis of TAO was made according to cri-
teria of Shionoya and defined as fallows; a history of
smoking, disease on set before the age of 50 years, in-
frapopliteal arterial occlusions, upperlimb involvement
or phlebitis migrans, and the absence of atherosclerotic
risk factors other than smoking. In Doppler ultrasound of
cases, the decreased arterial flow pattern was also re-
vealed. Because of this arteriography was applied all
cases. In arteriography, arterial occlusion and corkscrew
collaterals revealed typically.
Mutation Analysis: Two ml of peripheral blood-EDTA
samples were used for total genomic DNA isolation (In-
vitek, Berlin, Germany). Multiplex PCR-based reverse
hybridisation stripAssay technique (Vienna Lab, Labor-
diagnostika GmbH, and Vienna, Austria) was used for
mutation analysis (ProfiBlot T48, Tecan, Switzerland).
Genotyping of PAI-1 gene was made and compared to
the control group individuals.
Statistical Analysis: The SPSS software (ver; 14.0)
was used for evaluation of data, χ²-test (chi-square) was
used to compare the significance of the differences be-
tween groups. The test of importance between the mean of
two groups was used for accordance of two groups and
margin of error was approved to as 0.05. Furthermore
appropriateness of groups was evaluated with importance
test of difference of between two groups.
3. RESULTS
The informed consents of the patients were also obta-
ined. 30 TAO patients (28 males, 2 females and mean
ages was 42.9 ± 14.3 years) and 30 healthy controls (27
males, 3 females and mean ages was 40.9 ± 4.79 years)
were included in the current study. Two groups were
similar regarding age and sex distribution [(t = 0.69); P =
0.487; p > 0.05]. Twenty nine (97%) cases had a history
of smoking in TAO group and 22 (73%) cases had a his-
tory of smoking in control group. Detection of etiopa-
thogenesis is important for meticulous care of these pa-
tients. Homozygous 4G/4G profile was 7% for TAO and
4% for control individuals. The homozygous wide type
5G/5G genotype was 10 % in patients with TAO and
53% in healthy controls (Table 1). Heterozygote muta-
tion of 4G/5G profile was detected in 25 (83.3%) pa-
tients with TAO and 13 (43.3%) control subjects. The
current results indicate that the 4G/5G gene polymor-
phism of PAI-1 is significantly associated with TAO
while 4G/4G type may probably be an important heredi-
tary risk factor as well. We found that heterozygote PAI
gene polymorphisms (p < 0.05) were significantly more
frequent in patients with TAO. We detected PAI-1 5G/
4G polymorphism was significantly higher in TAO
group (p = 0.001) (Table 1 and Figure 1). Most possibly
the 4G/5G gene polymorphism for PAI-1 may be an ac-
ceptable risk factor of TAO.
Table 1. The percantages of polymorphic alleles (5G/4G) for
PAI-1 gene in the current TAO and control groups.
GROUP ALTERNATIVE GENOTYPES FOR PAI-1
GENE
4G/4G 4G/5G 5G/5G Total
(n-%)
TAO
(n-%) 2–7 25–83 3–10 30–100
Control
(n-%) 1–4 13–43 16–53
*P 0.05 0.001 0.001
30–100
*p < 0.05 is significant
Figure 1. Shows the barr diagram of alternative 5G/4G alleles
of PAI-1 gene in TAO and control groups.

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4. DISCUSSIONS
Some association has been suggested between Buerger’s
disease (TAO) and other syndromes but there is still no
consensus about diagnostic criteria. TAO is a nonathero-
sclerotic inflammatory disorder of unknown etiology
that affects small and medium-sized vessels of the ex-
tremities [1-3]. It is usually present at less than 40 years
old in male sex. However, last studies show that this
disease can be appears in smoking females [5]. In our
study, there were 28 (93%) males, 2 (7%) females’ cases
in TAO group and the mean age was 42.9 ± 14.3 years.
On the other hand, control group was consisted of 27
(90%) males, 3 (10%) females. Additionally 29 (97%)
subjects had smoking history in TAO group and 22 (73%)
in control group. TAO is diagnosed and treated with dif-
ficulty because of elusive etiology (10]. There is no suf-
ficient knowledge about the other risk factors and pre-
disposing conditions in the literature [11,12]. It was
documented that there was a strongly relationship be-
tween TAO and HLA-9 and HLA-B5. This situation
remind to genetical predisposition can be rolled in back-
ground of this disorder [5]. The effected vessel was
filled with the thrombus formation in microscopical
scanning [5]. Prothrombotic risk factors were observed
in this disease because of genetical suspicions and
thrombotic findings [6]. The 4G/5G alleles in PAI-1 and
MTHFR C677T gene polymorphisms may help to iden-
tify the couples at risk for recurrent pregnancy loss [13].
Abboud et al. showed that the risk of myocardial infarc-
tion (MI) was notably high in 4G and -844A carriers
with elevated plasma PAI-1 and were associated with
reduced tPA levels [14]. PAI-1 concentration increases
because of the 4G deletion of 675th promoter region of
PAI gene. Therefore, fibrinolytic activity is reduces and
predisposing thrombotic events are increases [15]. It was
demonstrated that all alone or combined effect of this
gene deletion was impacted deep venous thrombosis
(DVT), in studies [16]. Choudhury and friends detected
that the free PAI-1 levels can be significant venous oc-
clusion in TAO patients [17]. This gene mutation ac-
cused in Turkish DVT patients [18]. A meta-analyze
signs to 4G/4G genotype was 20% folds increased of
myocardial infarction risk that have included 9 studies
[19]. Another meta-analyze points to each 4G allele was
slightly increased of myocardial infarction risk that have
included 37 studies [20]. Recently another meta-analyze
that included 17 studies points that there was average
relation ship between 4G allele and venous thrombo-
emboli in subjects which have no genetical and acquired
prothrombotic risk factors [21]. In our study we detected
PAI-1 4G/5G deletion in 25 (83%) subjects in TAO
group (Figure 1). Although there were 13 (43%) sub-
jects detected with PAI-1 4G/5G deletion in control
group. Evaluation of the difference between two groups
was statistically significant (p = 0.001) (Table 1). Anal-
ysis of etiopathogenesis of TAO disease has importance
for loss of extremity and labor force by reason of this
disease and these results should be verified by larger
population studies. In view of previous and current res-
ults, there is a clear need to redefine the diagnostic algor-
ithm and the criteria for diagnosing TAO (Buerger’s
disease).
In conclusion, our study revealed the presence of sus-
picious relationship between TAO and PAI-1 gene
polymorphism. However this is a pilot study including
limited number of subjects and it must be supported with
large cohort studies. In addition, it is believed that the
current results could be helpful for analysis of etiopa-
thogenesis of TAO in the future studies. Additionally,
analyses of etiopathogenesis of the TAO disease have
importance for prevention of potential losses of extrem-
ity and labor force caused by this disease.
REFERENCES
[1] Buerger, L. (1952) Thrombo-angiitis obliterans: A study
of the vascular lesions leading to presenile spontaneous
gangrene. American Journal of Medicine, 13(5), 526-
532.
[2] Brodmann, M., Renner, W., Stark, G., Winkler, M., Pabst,
E., Hofmann, C. and Pilger, E. (2000) Prothrombotic risk
factors in patients with thrombangitis obliterans. Throm-
bosis Research, 99(5), 483-486.
[3] Cooper, L.T., Tse, T.S., Mikhail, M.A., Mcbane, R.D.,
Stanson, A.W. and Ballman, K.V. (2004) Long-term sur-
vival and amputation risk in thromboangiitis obliterans
(Buerger’s disease). Journal of the American College of
Cardiology, 44(12), 2410-2411.
[4] Taşoğlu, I., Hanedan, O. and Ulus, A.T. (2008) Throm-
boangiitis obliterans (buerger hastalığı). Turkiye Klinik-
leri Journal of General Surgery Special Topics, 1(3), 15-
25.
[5] Szuba, A. and Cooke, J.P. (1998) Thromboangiitis oblit-
erans. An update on Buerger’s disease. Western Journal
of Medicine, 168(4), 255-260.
[6] Özen, F., Manduz, Ş., Katrancıoğlu, N., Karahan, O.,
Köksal, B. and Özdemir, Ö. (2009) Tromboanjiitis oblit-
erans hastalarında protrombotik gen polimorfizminin rolü
turkiye klinikleri. Journal of Cardiovascular Science,
21(2), 160-164.
[7] Isserhoff, R.R. and Rifkin, D.B. (1983) Plasminogen is
present in the basal layer of the epidermis. Journal of
Investigative Dermatology, 80, 297-299.
[8] Herouy, Y., Trefzer, D., Hellstern, M.O., Stark, G.B.,
Vanscheıdt, W., Schoè, P.F.E. and Norgauer J. (2000)
Plasminogen activation in venous leg ulcers. British
Journal of Dermatology, 143(5), 930-936.
[9] Isordia-Salas, I., Leaños-Miranda, A., Sainz, I.M., Reyes-
Maldonado, E. and Borrayo-Sánchez, G. (2009) Associa-
tion of the plasminogen activator inhibitor-1 gene 4G/5G
polymorphism with ST elevation acute myocardial in-
farction in young patients. Revista Española de Cardi-
ología, 62(4), 365-372.

S. Manduz et al. / HEALTH 2 (2010) 454-457
Copyright © 2010 SciRes. http://www.scirp.org/journalT / Openly accessible at /HEALH
457
457
[10] Foley, S. and Gibbs, H. (2008) Muir painful digital in-
farction in a male smoker treated as Buerger’s disease.
Australasian Journal of Dermatology, 49(2), 94-97.
[11] Stammler, F., Diehm, C., Hsu, E., Stockinger, K. and Ame-
ndt, K. (1996) The prevalence of hyperhomocysteinemia
in thrombangitis obliterans. Does homocsteine play a role
pathogenetically? Deutsche medizinische Wochenschrift,
121(46), 1417-1423.
[12] Chenl, Z., Takahashi, M., Naruse, T., Nakajima, T., Chen,
Y.W., Inoue, Y., Ishikawa, I., Iwail, T. and Kimura, A.
(2007) Synergistic contribution of CD14 and HLA loci in
the susceptibility to Buerger disease. Human Genetics,
122(3-4), 367-372.
[13] Yenicesu, G.I., Cetin, M., Ozdemir, O., Cetin, A., Ozen,
F., Yenicesu, C., Yildiz, C. and Kocak, N. (2009) A pro-
spective case-control study analyzes 12 thrombophilic
gene mutations in turkish couples with recurrent preg-
nancy loss. American Journal of Reproductive Immuno-
logy, 63(2), 126-136.
[14] Abboud, N., Ghazouani, L., Saidi, S., Ben-Hadj-Khalifa,
S., Addad, F., Almawi, W.Y. and Mahjoub, T. (2010) As-
sociation of PAI-1 4G/5G and -844G/A gene polymor-
phisms and changes in PAI-1/tissue plasminogen activa-
tor levels in myocardial infarction: A case-control study.
Genetic Testing and Molecular Biomarkers, 14(1), 23-
27.
[15] Francis, C.W. (2002) Plasminogen activator inhibitor-1 le-
vels and polymorphisms. Archives of Pathology & Labo-
ratory Medicine, 126(11), 1401-1404.
[16] Akar, N., et al. (2000) Effect of plasminogen activator
inhibitor-1 4G/5G polymorphism in turkish deep vein
thrombotic patients with and without FV1691 G-A.
Thrombosis Research, 97, 227-230.
[17] Choudhury, N.A., Pietraszek, M.H., Hachiya, T., Baba, S.,
Sakaguchi, S., Takada, Y. and Takada, A. (1992) Plasmi-
nogen activators and plasminogen activator inhibitor 1
before and after venous occlusion of the upper limb in
thromboangiitis obliterans (Buerger’s disease). Thrombo-
sis Research, 66(4), 321-329.
[18] Eroglu, A., Ulu, A. and Akar, N. (2006) Plasminogen
activator inhibitor-1 gene 4G/5G polymorphism in cancer
patients with and without thrombosis. Journal of Throm-
bosis and Thrombolysis, 22(2), 111-112.
[19] Boekholdt, S.M., Bijsterveld, N.R., Moons, A.H., Levi,
M., Büller, H.R. and Peters, R.J. (2001) Genetic variation
in coagulation and fibrinolytic proteins and their relation
with acute myocardial infarction: A systematic review.
Circulation, 104(25), 3063-3068.
[20] Ye, Z., Liu, E.H., Higgins, J.P., Keavney, B.D., Lowe, G.D.,
Collins, R. and Danesh J. (2006) Seven haemostatic gene
polymorphisms in coronary disease: Meta-analysis of 66,
155 cases and 91,307 controls. Lancet, 367(9511), 651-
658.
[21] Tsantes, A.E., Nikolopoulos, G.K., Bagos, P.G., Rapti, E.,
Mantzios, G., Kapsimali, V. and Travlou, A. (2007)
Association between the plasminogen activator inhibi-
tor-1 4G/5G polymorphism and venous thrombosis. A
meta-analysis. Journal of Thrombosis and Haemostasis,
97(6), 907-913.