Most patients with type 2 diabetes (T2D) die from complication of atherosclerosis  .
PAI-1 (plasminogen activator inhibitor-1) is a major regulator of fibrinolysis  , plasma PAI-1 Antigen (PAI-1Ag) level is increased in type 2 diabetes patients   and that may explain excess risk of cardiovascular disease. It also elevated in coronary artery disease patients  and its plasma level is determined by genetic  and environmental factors  .
The PAI-1 gene has been localized to q21.3-q22 of chromosome 7  . Several polymorphisms within the PAI-1 gene influence PAI-1 levels  . The most known polymorphism which influences PAI-1 level is −675 4G/5G insertion-deletion mutation-of PAI-1 promotor gene  and another single nucleotide polymorphism is −844 G/A    .
Environmental factors, like obesity and metabolic syndrome features also plays a role in Plasma PAI-1 variation in type 2 diabetes patients and in non diabetics   . The aim of this study was to examine the determinants of plasma PAI-1Ag level among adult patients with type 2 diabetes in Tunisia.
2. Patients and Methods
This was a cross sectional study involving 491 type 2 diabetic patients recruited from the outpatient’s endocrinology department at Farhat-Hachad hospital in Sousse-Tunisia during 2005-2006 period, written informed consent was obtained from participants, the study was approved by hospital ethic comity, inclusion criteria was: known type 2 diabetes, exclusion criteria were: cancer, coagulation disorders, pregnancy, end stage chronic kidney disease, all patients had clinical examination including (weight, high, BMI, Waist Circumference (WC)), laboratory investigations (Fasting blood glucose (FBG), Hb1Ac, cholesterol, triglyceride, HDL-cholesterol,) LDL was calculated by Fridewald formula(LDL (mmol/l) = total cholesterol -HDL-TG/2.26), after clear write consent plasma PAI-1 antigen level was done with ELISA, −675 4G/5G. PAI-1 gene promoter polymorphism genotyping was done by PCR-ASA(allele specific amplification) using common primer for 2 alleles in 5’P side and 2 specific primers for 2 alleles in 3’OH side and −844 G/A polymorphism genotyping was done by PCR-RFLP (restriction fragment length polymorphism) using 2 specific primers for 2 alleles and DNA was digested with restriction enzyme, allelic frequency was calculated with hardy-Weinberg law (p + q)2 = p2 + 2pq + q2 = 1, with p = n1 + n2/2n and q = n3 + n2/2n, n = number total of patients, n1 = 4G/4G carriers, n2 = 4G/5G, and n3 = 5G/5G, P = allele 4G frequency, q = 5G frequency. The same procedure was made with −844 G/A, and statistical analyses was performed using SPSS version 10.0 software.
The mean age of our T2D population was 58.3 ± 10.5 years, male/female-ratio = 0.92, mean PAI-1 level was 34.6 ± 21.3 ng/ml.
No significant difference found in PAI-1 Ag level between type 2 diabetes patients with hypertension and T2D without hypertension (Table 3).
In multivariate analysis, we found significant relationship between PAI-1 level and LDL-cholesterol (P = 0.05) (Figure 1).
T2D patients who have FBG > 11 mmol/l had PAI-1 Ag level higher than those who have FBG < 11 mmol/l (P = 0.034), but no difference found between T2D with high Hb1Ac > 8% and those with Hb1Ac < 8% (Table 4).
The Table 5 shows high correlation between PAI-1 Ag level and -675 4G/5G polymorphism genotypes, 4G/4Gcarriers had the highest PAI-1 level, 4G/5G had intermediary level and 5G/5G had the lowest level (P ˂ 0.001), No correlation was seen between PAI-1 Ag level and −844G/A polymorphism genotypes.
Using multiple variable linear regression analysis, the independent factor associated with plasma PAI-1 level was −675 4G/5G polymorphism (regression coefficient β = 4.6, P < 0.05).
Table 1. PAI-1 Ag level in diabetics in function of BMI (kg/m2).
SD: Standard Deviation; P: P-Value; NS: Non Significant.
Table 2. Mean PAI-1 Ag level in type 2 diabetes patients in function of WC (cm) and sex.
SD: Standard Deviation; P: P-Value; NS: Non Significant.
Figure 1. MeanPAI-1-Ag in relation to LDL in T2D patients.
Table 3. Mean PAI-1 Ag level in T2D patients in function of hypertension.
SD: Standard Deviation; P: P-Value; NS: Non Significant.
Table 4. Mean PAI-1 Ag in T2D in function of FBG (fast blood glucose) and Hb1Ac.
SD: Standard Deviation; P: P-Value; NS: Non Significant.
Table 5. Correlation between PAI-1 -Ag level and −675 4G/5G and -844G/A genotypes in T2D patients.
P: P-Value; NS: Non Significant.
PAI-1 level is increased in type 2 diabetic patients      in comparison with non diabetic.
In IRAS (insulin resistance atherosclerosis study)  high level of PAI-1 was a predictor of type 2 diabetes incidence, in multiple regression analyses, PAI-1 level still significantly linked to type 2 diabetes incidence. In the same study high PAI-1 level was linked to diabetes incidence.  , In Health, Aging and Body Composition Study  similar results were found.
In Framingham Offspring Study  , high PAI-1level was a risk factor of type 2 diabetes with relative risk (RR) of 1.4 for people who have PAI-1 level in upper normal range, this risk is independent of obesity and classical risk factors. In Strong Heart Study  , relationship between PAI-1 level and diabetes incidence was found but this relationship become non-significant after adjustment with other variables (age, sex, BMI, BP, triglyceride, CRP, fibrinogen and insulin), antidiabetic drug vildagliptin decrease PAI-1 level  .
A recent metanalysis  shows moderate association between PAI-1 and T2D independent of established diabetes risk factors.
In our study mean PAI-1 Ag level was 34.6 ± 21.4 ng/ml. we didn’t have control group due to financial limits (cost of dosage) and the comparison with other studies is difficult because measurements methods are different and non-standardized.
The PAI-1 level is correlated to insulin resistance markers (BMI, Waist circumference, glucose level and insulin)   .
In our study we didn’t find a positive correlation between BMI and PAI-1 but we found correlation between PAI-1 and WC which was most evident in men.
We had found correlation between PAI-1 and LDL cholesterol, LDL and VLDL cholesterol stimulate PAI-1 gene expression in vitro  , that may explain this correlation
The patients who have FBG > 11 mmol/l have PAI-1 level more than patients who have FBG < 11 mmol/l.
Glucose stimulate PAI-1 gene expression in vitro and that may explain relationship between PAI-1 and diabetes  , but this relationship is largely explained by metabolic syndrome.
Some studies found that PAI-1 level is linked to android fat distribution and endocrines and metabolic features of metabolic syndrome    .
People who have Metabolic syndrome with or without diabetes had elevated PAI-1 level   improvement of metabolic syndrome with weight loss decrease PAI-1 level  .
Some studies had found higher PAI-1 level in people with hypertension  .
In our study, we didn’t find significant difference between mean PAI-1 level of diabetic patients who have hypertension and diabetics without hypertension.
Pronounced elevations of PAI-1 antigen levels were seen in 4G carriers of −675 4G/5G polymorphism of T2D patients in a large number of studies,  as well as non-diabetic and in different ethnic populations like Tunisians    .
The most significant variation in PAI-1 expression resides in the PAI-1 4G/5G alleles. Unlike the 5G allele that binds a transcription repressor, resulting in low PAI-1 expression, the 4G allele does not bind a transcription repressor, thus conferring a “high PAI-1 expressor” nature to the allele I  .
Martinez-Calatrava  , had found that 4G allele is the principal determinant of PAI-1 level in study of 631 persons, independent of metabolic disorders.
These results are in agreements with our study who shown that −675 4G/5G polymorphism not metabolic disorders was the principal determinant of PAI-1 level. Another study show metabolic syndrome components explain only 12% of PAI-1variability in T2D patients  .
4G allele has been shown as a risk factor in cardio vascular disease in some studies  not others  , some studies show 4G as a risk factor of diabetes   , some studies show 4G allele association with obesity   and metabolic syndrome   .
About second polymorphism −844 G/A, we don’t found relationship between this polymorphism and PAI-1 level, this results is in agreement with the literature    .
A Mexican study revealed a relationship between −844 G/A and metabolic syndrome  . Another study revealed an association with cardio-vascular disease and dyslipidemia  .
The present study identifies −675 4G/5G not −844 G/A polymorphism of PAI gene as the principal determinant of plasma PAI-1 level in adult type 2 diabetes patients in Tunisia, and the android fat distribution, dyslipidemia and hyperglycemia play a modest role in this variation.
Conflicts of Interest
All authors declare no conflicts of interest.
All authors had participated actively in manuscript realization.
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