Thrombin-activatable fibrinolysis inhibitor (TAFI) levels and its polymorphism rs3742264 are associated with dyslipidemia in a cohort of Brazilian subjects

Clinica Chimica Acta 433 (2014) 76–83

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Thrombin-activatable fibrinolysis inhibitor (TAFI) levels and its polymorphism rs3742264 are associated with dyslipidemia in a cohort of Brazilian subjects Izabela R. Santos a, Ana P. Fernandes b, Maria G. Carvalho b, Marinez O. Sousa b, Cláudia N. Ferreira c, Karina B. Gomes a,b,⁎ a b c

Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Colégio Técnico, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

a r t i c l e

i n f o

Article history: Received 3 December 2013 Received in revised form 20 February 2014 Accepted 28 February 2014 Available online 11 March 2014 Keywords: Dyslipidemia Fibrinolysis Polymorphisms Risk factor TAFI

a b s t r a c t Background: Dyslipidemia, a metabolic alteration that affects lipoprotein levels, is considered a major risk factor for atherosclerosis and its complications. Dyslipidemia also affects the hemostatic system, especially impairing fibrinolysis, and increased levels of thrombin-activatable fibrinolysis inhibitor (TAFI) have been associated with cardiovascular events. Objectives and methods: This study evaluated the association of acquired risk factors (hypertension, body mass index — BMI, smoking, sedentary lifestyle, use or not of oral contraceptives and hormone replacement therapy, and post-menopause status), the polymorphisms Thr325Ile (rs1926447), Ala147Thr (rs3742264) and +1542C/G (rs940) in the TAFI gene, and TAFI plasma levels in 109 dyslipidemic and 105 normolipemic individuals. Biochemical analyses and TAFI levels were evaluated by colorimetric/turbidimetric assays and ELISA, respectively. Genotypic and allelic frequencies were determined by polymerase chain reaction (PCR). Results: Hypertension, increased BMI, and menopause were more common in dyslipidemic individuals, who had higher TAFI levels. The alleles 325Ile, Ala147, and C showed association with lower TAFI levels. The rs3742264 polymorphism was associated with dyslipidemia in males. Conclusions: The results suggest that TAFI levels are independently associated to dyslipidemia and that the polymorphism rs3742264 may be related to cardiovascular risk in male subjects. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Dyslipidemia, a metabolic complication characterized by high concentrations of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and triglycerides (TG), and by low levels of high-density lipoprotein cholesterol (HDL-C), is a major risk factor for cardiovascular diseases (CVD) [1]. CVD constitute a public health concern worldwide and their prevention is highly dependent on identification of risk factors. It has been estimated that, by 2030, CVD deaths may increase to 23.4 million accounting for 35% of all deaths [2]. Dyslipidemia triggers the formation of atherosclerotic plaque, facilitating the platelet deposition process and the action of clotting factors, which ultimately results in thrombus formation [3]. Therefore, the hemostatic system contributes significantly to the occurrence of CVD events. Indeed, it has been suggested that markers of endothelial ⁎ Corresponding author at: Faculdade de Farmácia, Universidade Federal de Minas Gerais, Avenida Antônio Carlos, 6627, Pampulha, Belo Horizonte, Minas Gerais 31270901, Brazil. Tel.: +55 31 3409 6895; fax: +55 31 3409 6985. E-mail address: [email protected] (K.B. Gomes).

http://dx.doi.org/10.1016/j.cca.2014.02.030 0009-8981/© 2014 Elsevier B.V. All rights reserved.

activation and hypofibrinolysis have important predictive value for assessing the risk of cardiovascular events [4]. The thrombin-activatable fibrinolysis inhibitor (TAFI) plays an important role in hemostasis, acting as a potent inhibitor of fibrinolysis in its activated form (TAFIa) [5,6]. Synthesized as a prepropeptide of 423 aminoacids, TAFI zymogen is activated by proteolytic cleavage mediated by thrombin [7], by the complex thrombin–thrombomodulin [8], or by plasmin [9]. TAFIa inhibits fibrinolysis by removing lysine residues from the carboxy-terminal fibrin molecule, during the process of fibrin clot lysis. Consequently, fibrin loses its cofactor activity, reducing the production of plasmin [10]. Thereby, TAFI establishes an important connection between the coagulation and the fibrinolytic systems [11]. The gene encoding TAFI (CPB2) locates on chromosome 13 (13q14.11), contains 11 exons, and spans 48 kb of genomic DNA [10]. The presence of the isoleucine aminoacid at 325 position, corresponding to the Thr325lle polymorphism or + 1040 C/T (rs1926447), increases the enzyme stability by two fold, and augments by 30–60% its antifibrinolytic effect as compared to threonine at this position [12–14]. The presence of alanine at the Ala147Thr polymorphism or +505 A/G (rs3742264) is associated to lower levels of TAFI [12,14,15]. Finally, the

I.R. Santos et al. / Clinica Chimica Acta 433 (2014) 76–83

exchange of a C to G at +1542 C/G (rs940) polymorphism in the 3′-UTR region affects the stability of the TAFI mRNA, leading to lower protein levels [16]. Association between elevated TAFI plasma levels and conventional risk factors for CVD [16], deep vein thrombosis (DVT) [17–19], coronary artery disease [20], stroke [13], angina pectoris [12], and acute myocardial infarction [14] has been demonstrated. Increased TAFI levels and activity have also been reported in hyperlipidemic patients, mainly those with hypercholesterolemia [6,21–24]. The role of TAFI levels in diseases which dyslipidemia is associated has been also analyzed, including patients on dialysis, with type 2 diabetes mellitus, metabolic syndrome and kidney transplant recipients, although not all individuals in these groups presented dyslipidemia [22,23,25,26]. A single study has reported TAFI levels in dyslipidemic individuals with no associated pathologies [21]. There is also a lack of studies on the possible association of known TAFI polymorphisms and dyslipidemia. Therefore, the relationship between TAFI levels and dyslipidemia, which precedes the development of associated pathologies and CVD and its genetic determinants remains unclear. Here, the levels of this enzyme in dyslipidemic and normolipemic individuals, characterized according to known cardiovascular risk factors, were compared and then the potential associations of TAFI gene polymorphisms Thr325lle (rs1926447), Ala147Thr (rs3742264), and + 1542 C/G (rs940) with this metabolic complication were explored. Moreover, the relationship of these polymorphisms and TAFI levels with gender was also investigated, in order to understand the role of genetic characteristics in dyslipidemic profile.

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Lipoprotein-a [LP(a)] was measured by immunoturbidimetric assay (Human®, Germany). Genomic DNA was extracted from peripheral blood leukocytes (Biopur Extraction Kit Mini Spin Plus — Biometrix ®). Genotyping of the rs1926447 polymorphism in the TAFI gene was performed by PCR-RFLP [12]. An allele-specific PCR was performed to identify the rs940 polymorphism, according to Henry et al. [15]. The rs3742264 polymorphism was genotyped by Taqman® (Applied Biosystems, USA, probe number: C_1872266_20). 2.3. Statistical analysis Statistical analysis was performed using the SPSS statistical package for Windows, version 13.0 (Statistical Package for Social Sciences, Inc., Chicago, Illinois, USA). The Shapiro–Wilk test was performed to assess data normality. The continuous variables were expressed as median and interquartile range if failing normality. The chi-square test was performed for the comparison of the categorical risk factors, genotypic and allelic frequencies. Kruskal Wallis and Mann–Whitney tests with Bonferroni correction were used to compare continuous variables between three or two groups, respectively. The Spearman rank correlation test was used for correlation between variables. The haplotype analysis was performed using the PHASE software, version 2.1.1 [30,31]. The Hardy–Weinberg equilibrium was evaluated by GENEPOP (http:// genepop.curtin.edu.au/). The multivariate logistic regression was performed primarily as a univariate analysis. We selected those with p b 0.2 to compose the multivariate regression analysis. The final model followed the adequacy, according to Hosmen and Lemeshow test. P-values b 0.05 were considered significant.

2. Materials and methods 3. Results 2.1. Subjects 3.1. Characterization of the studied groups Participants were subdivided into dyslipidemic and normolipemic groups, according to the National Cholesterol Education Program [27] criteria for lipid and lipoprotein levels. In the dyslipidemic group, 109 subjects (41 women and 68 men) were included. The control group was composed by 105 normolipidemic healthy volunteers (63 women and 44 men). Both groups aged 30 to 60 years, and were recruited at Socor Hospital or at Petrobrás — Gabriel Passos Refinery in Minas Gerais State, Brazil. Participants were diagnosed with dyslipidemia if they presented one or more of the following criteria: a plasma concentration of TC ≥ 6.24 mmol/L, TG ≥ 2.26 mmol/L; HDL-C b 1.04 mmol/L for men or b1.30 mmol/L for women; and LDL-C ≥ 4.14 mmol/L. Individuals with hyperglycemia or diabetes mellitus, hypothyroidism, renal/hepatic and auto-immune diseases, and subjects treated with lipid-lowering drugs, oral anticoagulants, and/or anti-inflammatory medications were excluded. The data for hypertension, BMI, smoking, sedentary lifestyle, use or not of oral contraceptives and hormone replacement therapy, and post-menopause status were obtained from medical records. Hypertension was defined as blood pressure ≥140/90 mm Hg, measured at 2 different occasions, and/or use of anti-hypertensive medication. Individuals who did not practice physical exercise regularly (for at least 40 min, three times a week) were considered sedentary [28]. Smokers were individuals with regular consumption of any quantity of cigarettes, for a period exceeding six months until the month prior to the completion of the clinical record, and alcoholism was considered heavy drinkers who consumed more than 30 mL of ethanol/day. The study was approved by the local Ethics Committee (COEPUFMG). Informed consent was obtained from all participants, after a full explanation of the study. 2.2. Methods Blood samples were collected after 12–14 h of fasting. Plasma TC, TG, and HDLc concentrations were measured enzymatically (Gold Analisa®, Brazil). LDLc was estimated by the Friedewald equation [29].

The individuals of the dyslipidemic group presented higher levels of TC, TG, VLDL, LDL-C, and non HDL-C, as compared to normolipemic individuals. As expected, the HDL-C levels were lower in the dyslipidemic group. No significant differences were observed for CRP and Lp(a) levels between the groups (Table 1). The characteristics of the dyslipidemic and normolipemic groups regarding demographic data and cardiovascular risk factors are shown in Table 2. The groups were similar with respect to age, sedentarism, smoking, and alcoholism. Regarding cardiovascular risk factors, there was a significant difference in the variables hypertension and BMI, which were more frequent in the dyslipidemic group. Significant differences were also observed regarding gender, with more women in the normolipemic group and more men in the dyslipidemic group. We also observed a higher number of post-menopausal dyslipidemic than normolipemic women. Thus, when the same analysis was presented according to gender, dyslipidemic men showed higher frequency of hypertension and BMI than men in the normolipemic group. Dyslipidemic women Table 1 Comparison of biochemical variables between dyslipidemic and normolipemic groups. Variables

Dyslipidemic (n = 109)

Normolipemic (n = 105)

p

TC TG HDL-C CRP VLDL-C LDL-C Lp(a) Non HDL-C

226 (59) 160 (136) 44 (15) 1.5 (2.3) 32 (27.2) 142.4 (53.4) 15.2 (25.7) 179 (49)

183 (32) 96 (58.5) 56 (16.5) 2.1 (3.15) 19.2 (11.7) 106.4 (26) 13.1 (21.65) 126

b0.001 b0.001 b0.001 ns b0.001 b0.001 ns b0.001

ns = not significant. Units for the total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), very low density lipoprotein (VLDL), lipoprotein (a) [Lp(a)], and non-HDL fraction: mg/dL. CRP unit: mg/L. P-value considered significant b0.05.

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Table 2 Comparison of demographic and clinical characteristics between dyslipidemic and normolipemic groups. Variables

DP (n = 109)

NDP (n = 105)

p

DP male (n = 68)

NDP male (n = 44)

p

DP female (n = 41)

NDP female (n = 61)

P

Age (years) Gender (F/M)

49 (9) F = 41 (37.6%) M = 68 (62.4%) 26.9 (5.3) 55 (50.5%) 20 (18.3%) 7 (6.4%) 30 (27.5%) 1 (2.4%) 22 (53.7%) 4 (9.8%)

47 (8) F = 61 (58.1%) M = 44 (41.9%) 24.8 (3.6) 51 (48.6%) 18 (17.1%) 2 (1.9%) 10 (9.5%) 7 (11.5%) 14 (23.0%) 6 (9.8%)

ns 0.004

48 (8) –

48 (8) –

ns –

51 (8) –

47 (8) –

0.008 –

b0.001 ns ns ns 0.001 ns 0.003 ns

26.8 (5.2) 34 (50.0%) 12 (17.6%) 7 (10.3%) 17 (25.0%) – – –

24.5 (3.38) 24 (54.5%) 12 (27.3%) 1 (2.3%) 4 (9.1%) – – –

b0.001 ns ns ns 0.047 – – –

27.6 (5.4) 21 (51.2%) 8 (19.5%) 0 13 (31.7%) 1 (2.4%) 22 (53.7%) 4 (9.8%)

24.8 (4.05) 27 (44.3%) 6 (9.8%) 1 (1.6%) 6 (9.8%) 7 (11.5%) 14 (23.0%) 7 (9.8%)

0.013 ns ns ns 0.009 ns 0.003 ns

BMI (kg/m2) Sedentarism Smoking Alcoholism Hypertension OCa Menopausea HRTa

ns = not significant; DP = dyslipidemic; NDP = normolipemic; F = female; M = male; BMI = body mass index; OC = oral contraceptives; HRT = hormone replacement therapy. P-value considered significant b0.05. a Frequency specific for women.

also presented higher age, BMI, and frequency of hypertension, in addition to postmenopausal status, compared to normolipemic women (Table 2). Biochemical variables were also evaluated by gender and similar differences were observed when comparing groups of dyslipidemic and normolipemic individuals (Tables 1 and S1). 3.2. Thrombin activatable fibrinolysis inhibitor (TAFI) levels There was a significant difference in TAFI levels between the dyslipidemic and normolipemic groups, with higher values observed in the dyslipidemic group (8.5–IQR 2.1 μg/mL) when compared to normolipidemic individuals (7.3–IQR 1.99 μg/mL; p b 0.001). Similar results were also found when gender was considered. Dyslipidemic men and women showed increased TAFI levels (8.20–IQR 1.4 and 9.50–IQR 1.92 μg/mL, respectively) in contrast to the gender normolipemic groups (7.550– IQR 2.06 μg/mL; p = 0.02 for men and 7.04–IQR 1.84 μg/mL; p b 0.001 for women). TAFI levels significantly correlated (all p b 0.05) with age (r2 = 0.025), BMI (r2 = 0.023), TC (r2 = 0.038), TG (r2 = 0.101), HDL-C (r2 = 0.095), VLDL-C (r2 = 0.077), LDL-C (r2 = 0.049), non HDL-C (r2 = 0.091), OC use (r2 = 0.038), hypertension (r2 = 0.041), and post-menopause status (r2 = 0.067). In contrast, gender, CRP, Lp(a), and HRT were not correlated with TAFI levels (p N 0.05). TAFI levels were then compared in the groups of women using OC or not, and post-menopausal or not. All women in both groups were considered because the number of contraceptive users was small. TAFI levels were lower in women on contraceptive use (6.90–IQR 2.08 μg/mL) when compared to non-users (8.22–2.30 μg/mL; p = 0.017). In addition, the post-menopausal group presented higher TAFI levels (8.98–IQR 2.05 μg/mL) than non-post-menopausal women (7.25–IQR 2.23 μg/mL; p b 0.001). 3.3. TAFI gene polymorphisms There was no significant difference in genic and genotypic frequencies for rs1926447 polymorphism, by comparing dyslipidemic and normolipemic groups (p N 0.05). However, a higher frequency of the T allele and the TT genotype in dyslipidemic than normolipemic men (p = 0.037 and 0.033, respectively) was observed. No significant difference was detected when dyslipidemic and normolipemic women were analyzed (Table 3). For the rs3742264 polymorphism, a higher frequency of GG genotype and a trend toward higher frequency of G allele were observed in the dyslipidemic group, when compared to normolipemic individuals (p = 0.012 for GG × AA and p = 0.073 G × A). However, this difference

was specific to the male dyslipidemic group (p = 0.016 for GG × AA and p = 0.003 for G × A) (Table 4). The allelic and genotypic frequencies of the rs940 polymorphism were similar between dyslipidemic and normolipemic groups, even when gender was considered in the analyses (p N 0.05, Table 5). A haplotype analysis was conducted for the three polymorphisms. The haplotypes CGC, CAC, TAC, and TAG were excluded, since their frequencies were less than 5% in any of the groups studied. No significant differences in haplotype frequencies were observed when dyslipidemic and normolipemic groups were compared (p N 0.05; Table S2). 3.4. Effect of TAFI gene polymorphisms on TAFI plasma levels in the dyslipidemic group Considering the rs1926447 polymorphism, TAFI levels were lower in TT than in CC and TC genotype carriers among dyslipidemic patients (TT × CC, p = 0.003; TT × TC, p b 0.001) and in men (TT × CC, p =0.004; TT × TC, p = 0.015). In dyslipidemic women, however, this difference was only observed between TT and CC genotypes (p = 0.039; Fig. 1A). For the rs3742264 polymorphism, TAFI levels were decreased in the presence of the GG, when compared to GA (p b 0.001) genotypes. This difference was also observed in male and female groups (p = 0.001 for both, Fig. 1B). Finally, the +1542CC genotype of the rs940 polymorphism was associated to lower TAFI levels when compared to GC and GG genotypes among dyslipidemic patients (p = 0.001 and 0.006, respectively). Men showed a difference in TAFI levels between the CC and GC/GG genotypes (p = 0.011 and 0.006, respectively), whereas among women no differences were detected for any of the genotypes analyzed (Fig. 1C). 3.5. Multivariate logistic regression A multivariate logistic regression was performed, regardless gender, and the final model is described in Table 6. This model was adequate, according to the Hosmen and Lemeshow test (p = 0.874). The presence of GA and GG genotypes (the latter being the reference test) of the rs3742264 polymorphism, plasma TAFI levels, male sex, and BMI were found to be independently associated with dyslipidemia. The groups were then classified by gender. The following final model was obtained, as described in Table 6, respectively for females and males (Hosmen and Lemeshow test p = 0.420 for females and p = 0.283 for males). In the female group, only TAFI levels were independently associated to dyslipidemia. Among males, TAFI levels, the presence of GA and GG genotypes of the rs3742264 polymorphism in the TAFI gene, and BMI were independently associated to the occurrence of dyslipidemia.

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Table 3 Genic and genotypic frequencies of rs1926447 polymorphism between dyslipidemic and normolipemic groups.

Genotypes TT TC CC Alleles T C

Dyslipidemic (n = 109)

Normolipemic (n = 104)

n

n

Frequencies (%)

Frequencies (%)

p

OR

IC

12 48 49

11.00 44.00 45.00

7 36 61

6.70 34.60 58.70

0.145 0.082 Reference

2.134 1.660 –

0.711–6.599 0.900–3.068 –

72 146

33.03 66.97

50 158

24.04 75.96

0.159

1.56

0.802–3.040

Frequencies (%)

p

OR

IC

Female dyslipidemic (n = 41)

Female normolipemic (n = 60)

N

n

Frequencies (%)

Genotypes TT TC CC

3 21 17

7.30 51.20 41.50

6 20 34

10.00 33.30 56.70

0.999 0.093 Reference

1.00 2.100 –

0.171–5.376 0.830–5.350 –

Alleles T C

27 55

32.93 67.07

32 88

26.67 73.33

0.355

1.332

0.695–2.557

Frequencies (%)

p

OR

IC

Male dyslipidemic (n = 68)

Male normolipemic (n = 44)

N

n

Frequencies (%)

Genotypes TT TC CC

9 27 32

13.20 39.70 47.10

1 16 27

2.30 36.40 61.40

0.033⁎ 0.422 Reference

7.594 1.427 –

0.875–170.284 0.592–3.443 –

Alleles T C

45 91

33.09 66.91

18 70

20.45 79.55

0.037⁎

1.923

0.982–3.792

⁎ Significant difference. For the rs1926447 polymorphism it was not possible to amplify a sample in the normolipemic group. P-value considered significant b0.05.

Table 4 Genic and genotypic frequencies of rs3742264 polymorphism between dyslipidemic and normolipemic groups.

Genotypes AA GA GG Alleles A G

Dyslipidemic (n = 109)

Normolipemic (n = 105)

n

n

Frequencies (%)

Frequencies (%)

p

OR

IC

7 46 56

6.40 42.20 51.40

16 51 38

15.20 48.60 36.20

0.012⁎ 0.092 Reference

0.297 0.612 –

0.099–0.863 0.331–1.130 –

60 158

27.52 72.48

83 127

39.52 60.48

0.073

0.583

0.309–1.099

Frequencies (%)

p

OR

IC

Female dyslipidemic (n = 41)

Female normolipemic (n = 61)

n

Frequencies (%)

n

Genotypes AA GA GG

2 19 20

4.90 46.30 48.80

8 26 27

13.10 42.60 44.30

0.287 0.974 Reference

0.338 0.987 –

0.044–2.034 0.397–2.451 –

Alleles A G

23 59

28.05 71.95

42 80

34.43 65.57

0.359

0.755

0.396–1.435

Frequencies (%)

p

OR

IC

Male dyslipidemic (n = 68)

Male normolipemic (n = 44)

n

Frequencies (%)

n

Genotypes AA GA GG

5 27 36

7.40 39.70 52.90

8 25 11

18.20 56.80 25.00

0.016⁎ 0.011⁎ Reference

0.191 0.330 –

0.042–0.828 0.126–0.852 –

Alleles A G

37 99

27.21 72.79

41 47

46.59 53.41

0.003⁎

0.417

0.221–0.785

⁎ Significant difference. P-value considered significant b0.05.

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Table 5 Genic and genotypic frequencies of rs940 polymorphism between dyslipidemic and normolipemic groups.

Genotypes CC CG GG Alleles C G

Dyslipidemic (n = 108)

Normolipemic (n = 103)

n

n

Frequencies (%)

Frequencies (%)

p

OR

IC

10 43 55

9.30 39.80 50.90

4 44 55

3.90 42.70 53.40

0.162 0.936 Reference

2.500 0.977

0.664–10.152 0.535–1.784 –

63 153

29.17 70.83

52 154

25.24 74.76

0.524

1.225

0.626–2.402

Frequencies (%)

p

OR

IC

Female dyslipidemic (n = 41)

Female normolipemic (n = 59)

n

n

Frequencies (%)

Genotypes CC CG GG

3 18 20

7.30 43.90 48.80

2 26 31

3.40 44.10 52.50

0.392 0.867 Reference

2.325 1.073 –

0.278–22.252 0.434–2.652 –

Alleles C G

24 58

29.27 70.73

30 88

25.42 74.58

0.524

1.225

0.626–2.402

Frequencies (%)

p

OR

IC

Male dyslipidemic (n = 67)

Male normolipemic (n = 44)

n

n

Frequencies (%)

Genotypes CC CG GG

7 25 35

10.40 37.20 52.20

2 18 24

4.50 40.90 54.50

0.465 0.905 Reference

2.400 0.952 –

0.399–18.418 0.397–2.286 –

Alleles C G

39 95

29.10 70.90

22 66

25.00 75.00

0.524

1.225

0.626–2.402

P-value considered significant b0.05. For the rs940 polymorphism it was not possible to amplify two samples in the normolipemic group.

4. Discussion This study investigated whether TAFI levels are associated with dyslipidemia through the evaluation of TAFI levels and TAFI gene polymorphisms in dyslipidemic and normolipemic subjects. As expected, hypertension and higher BMI were more prevalent among dyslipidemic individuals, since hypertension and obesity are commonly observed in dyslipidemic individuals and altogether these factors increase CVD risk [32–34]. Post-menopausal status was also more common among dyslipidemic women, who were also older in this group than in the control group. It is known that menopause is linked to decreased estrogen levels, increased TC levels, decreased HDL-C levels, and denser LDL-C particles [35], contributing to higher CVD risk. In fact, menopause status is, by itself, a risk factor for the development of dyslipidemia [36]. Our results indicated that TAFI levels are affected significantly in dyslipidemia, regardless gender. This finding agrees with the evidence that increases in plasma lipid levels are significant triggers for endothelial injury which exacerbates thrombin generation and, consequently TAFI activation and its levels [21]. Recently, TAFI has been implicated in anti-inflammatory process by removing C-terminal arginines in inflammatory mediators [37,38]. As inflammation plays an important role in the development of atherosclerosis in dyslipidemic subjects, it is plausible that TAFI levels are increased in dyslipidemic subjects due to low-grade inflammatory responses and therefore may contribute to decrease the inflammatory response [18]. It may be suggested that the increased release of TAFI in the dyslipidemic group results from increased thrombin generation, but also may participate on regulation of the thrombin's pro-inflammatory functions and the chronic inflammation commonly found in these individuals. The recognition of high activity and levels of TAFI in dyslipidemic patients with hypercholesterolemia [21,22,24,39] or type 2 diabetes mellitus [23] strengthens the need for prospective studies investigating

the association between TAFI levels and CVD. In one hand, high TAFI levels may facilitate the development of cardiovascular comorbidities, by shifting the hemostatic balance to a more hypofibrinolytic state, but in the other it may display a protective effect dampening the excessive inflammatory reaction. TAFI levels were positively correlated with the variables age, BMI, TC, TG, VLDL, LDL-C, non-HDL fraction, and the risk factor hypertension. There was an inverse correlation between TAFI and HDL-C levels. Similar correlations for TAFI levels and other variables in patients bearing pathological conditions were also reported [20]. Trégouët et al. [20] showed that TAFI levels correlated with age, BMI, TC, TG, and hypertension, while inversely correlated with HDL-C in patients undergoing coronary angiography. However, in contrast to the data presented here, these authors reported correlation with CRP. This discrepancy may be attributed to intrinsic differences of the patient groups analyzed in each study, including the presence of a pathological condition (myocardial infarction) in those studied by Trégouët et al. [20]. An association between the TT genotype and the T allele in the rs1926447 polymorphism with dyslipidemia in the male group was observed. The genotypic and allelic frequencies shown in this study are similar to those described by Tàssies et al. [14] in patients with acute coronarian syndrome and myocardial infarction. The distribution of genotypic frequencies found in this study is also similar those reported by the Prospective Epidemiological Study of Myocardial Infarction — PRIME [40], which evaluated the occurrence of CVD during 5 years in men aged 50–59 years, and is also similar to the Genetic risk factors for Arterial Thrombosis at a young age: the role of TAFI and other Coagulation factors — ATTAC study, which included patients with their firstever acute ischemic cardiac or focal cerebral event [41]. The GG genotype and the G allele in the rs3742264 polymorphism were more frequent in the dyslipidemic male group. Similar frequencies to those reported herein for this polymorphism in the dyslipidemic group were found by Trégouët et al. [20], in patients undergoing

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Fig. 1. TAFI levels (μg/mL) for the rs1926447, rs3742264, and rs940 polymorphisms in the TAFI gene.

coronary angiography, and by Brouwers et al. [12] in patients with unstable angina pectoris. We did not find differences between the groups for genotype and allele frequencies of the rs940 polymorphism, even when the groups were classified according to gender. Different frequencies were found by Morange et al. [40], in a sample from the general population of Northern Ireland, and by Henry et al. [15], in a Caucasian German population. Our contrasting results could be explained by the different population genetic background or the methodology used for genotyping this polymorphism in our study. Taken together our results suggest that the effect of the rs1926447 and rs3742264 polymorphisms on the TAFI protein could be regulated by gender-dependent factors. Further knowledge on this mechanism will require more investigation on hormonal interactions and control of TAFI gene expression. The analysis of haplotype frequencies showed no significant difference when the dyslipidemic and normolipemic groups were compared. These

results suggest that the rs1926447 and rs3742264 polymorphisms are associated with dyslipidemia independently of the haplotype. It is noteworthy that no other study has evaluated this same polymorphism and the same haplotype. In the dyslipidemic group, carriers of the TT genotype (Ile/Ile) of the rs1926447 polymorphism presented lower levels of the protein. When the groups were classified by gender, similar results were observed for this polymorphism. Curiously, Boffa and Koschinsky [10] showed that, while the T allele was associated with more stable TAFI protein, plasma levels were slightly lower, similarly to our results. Currently, genetic, non-modifiable factors are of particular interest since contradictory results have rose from studies that correlated plasmatic TAFI levels and the risk of CVD. A partial explanation for these discrepancies could be differences in the type of patients included, the time of sampling, or in the methods used to measure TAFI levels. Also, the effect of TAFI polymorphisms on TAFI antigen levels is unclear, since variable antibody reactivity to TAFI isoforms in some TAFI antigen ELISA tests underestimates real plasma TAFI levels, when an Ile is located at

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Table 6 Multivariate logistic regression among the whole group and according to gender. All groups together Variable analyzed

p

OR

IC

AA genotype (rs3742264 polymorphism) GA genotype (rs3742264 polymorphism) Male gender BMI TAFI (μg/mL)

b0.001 b0.001 b0.001 b0.001 b0.001

0.080 0.241 2.704 3.953 2.454

0.021–0.305 0.112–0.517 1.346–5.434 1.953–8.004 1.845–3.264

Variable analyzed

P

OR (B)

IC

TAFI (μg/mL)

b0.001

3.380

2.101–5.438

Variable analyzed

p

OR (B)

IC

AA genotype (rs3742264 polymorphism) GA genotype (rs3742264 polymorphism) BMI TAFI (μg/mL)

b0.001 0.002 0.001 b0.001

0.033 0.169 6.162 2.301

0.005–0.201 0.056–0.510 2.160–17.579 1.500–3.529

Female group

Male group

position 325. The methods used in the present study detect total TAFI (reflecting mostly the proenzyme) or the global capacity of TAFI to be activated, but not the actual activated TAFI in the circulation. Very recently, it has been suggested that activated/inhibited TAFI would have a better relationship with CVD than total TAFI [14]. Regarding the rs3742264 polymorphism, the TAFI levels were lower in the GG genotype (Ala/Ala) carriers when compared to the other genotypes. The same was observed when the groups were classified by gender. Tàssies et al. [14] and Martini et al. [17] also observed decrease in TAFI levels for the GG genotype. Due to the location of this polymorphism in the TAFI gene structure, it is possible that it may affect TAFI's protein synthesis, secretion, and half-life. However further studies will be necessary to establish these potential relationships. Regarding the rs940 polymorphism, we observed lower TAFI levels for the CC genotype among dyslipidemic individuals when compared to other genotypes. Similar results were found when gender analyses were performed. This suggests that the allele C, located in the 3′-UTR region, may affect TAFI mRNA stability. However, these data contrast to those reported elsewhere showing low levels of TAFI only when the G allele was present [10,15]. This inconsistency may result from the interaction with other polymorphisms that are characteristic of some specific populations or with other clinical variables of dyslipidemia. Finally, the multivariate analysis revealed that BMI and the rs3742264 polymorphism are independently associated to the dyslipidemic status in the male group. These analyses also showed that high TAFI levels are associated with dyslipidemia in both genders, and may compromise fibrinolysis in this situation. The small sample analyzed may be considered a limitation of the present study. Nevertheless, this is the first report indicating that high TAFI levels are associated to dyslipidemia, independently of other diseases. In particular, our study demonstrated for the first time the independent association of the polymorphism rs3742264 with dyslipidemia in male individuals, suggesting that this polymorphism may be further evaluated as risk factor for dyslipidemia. Future research following dyslipidemic patients to investigate the progression of TAFI levels and the association of TAFI polymorphisms with the occurrence of a cardiovascular event is warranted and may be largely informative. The authors state that they have no conflict of interest.

Acknowledgments The authors thank FAPEMIG, CNPq/Brazil, and PRPq/UFMG for the financial support.

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