G is associated with myocardial infarction

Atherosclerosis 177 (2004) 189–191

Tissue factor promotor polymorphism −603 A/G is associated with myocardial infarction Ilka Ott∗ , Werner Koch, Nicolas von Beckerath, Rene de Waha, Anna Malawaniec, Julinda Mehilli, Albert Sch¨omig, Adnan Kastrati Deutsches Herzzentrum und 1, Medizinische Klinik der Technischen Universit¨at, Lazarettstr. 36, 80636 M¨unchen, Germany Received 26 January 2004; received in revised form 14 June 2004; accepted 2 July 2004 Available online 25 August 2004

Abstract Tissue factor (TF), the main initiator of the extrinsic coagulation cascade is expressed in atherosclerotic lesions and contributes to coronary thrombus formation in myocardial infarction (MI). Circulating TF reflects intravascular TF activation but also adds to prothrombotic activation. Because the G allele of the TF promotor polymorphism −603 A/G is associated with monocytic mRNA expression we evaluated its association with myocardial infarction, based on a recessive deleterious effect assumption. Patients with MI (MI; n = 793) and age and sex matched control subjects without coronary artery disease (C; n = 340) undergoing coronary angiography were included. In patients with MI, the −603 G (MI: 76%, C: 70%) allele was prevalent compared to the control group (P = 0.04). Multivariate analysis revealed an odds ratio of 1.44 (confidence interval 1.07–1.93). Carriage of the −603 G allele is associated with an increased risk for myocardial infarction. Because higher plasma TF concentrations are found in −603 G carriers enhanced TF expression may be the mechanism underlying this association. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Myocardial infarction; Coagulation; Tissue factor

1. Introduction Tissue factor (TF) is a 47 kDa transmembrane glycoprotein, that after binding of factor VIIa (FVIIa) activates the coagulation factor FX to FXa and, thereby, initiates the extrinsic coagulation cascade. TF is normally not expressed in circulating leukocytes and endothelial cells, however, TF transcription can be induced by proinflammatory cytokines, growth factors, shear forces and balloon injury of the vessel wall [1]. In atherosclerotic lesions, TF is expressed in macrophages and smooth muscle cells [2]. Plaque rupture in acute myocardial infarction (MI) exposes TF in the vessel wall with circulating coagulation factors and contributes to local thrombosis [3]. Recent studies have shown that increased circulating TF may also contribute to procoagulant ∗

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0021-9150/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2004.07.006

responses [4,5]. In addition to circulating microparticles a spliced variant of TF that lacks the transmembrane domain, exists in a soluble form in blood and may contribute to the circulating procoagulant activity [6]. Plasma concentrations of TF are increased in patients with diabetes mellitus, hypercholesterolemia, smokers [7] and in acute myocardial infarction [8]. Because monocytic TF mRNA expression was found to be significantly higher in carriers of the G allele we analyzed the association between myocardial infarction and G allele carriage [18].

2. Methods Cases were represented by a consecutive series of 793 patients with MI. The controls were recruited during the same time period as the patients with myocardial infarction and were matched with those for age and sex. In these subjects

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I. Ott et al. / Atherosclerosis 177 (2004) 189–191

coronary angiography was performed to exclude the presence of coronary artery disease. Both groups were of Caucasian origin and underwent coronary angiography. Acute MI, present in 369 patients, was defined on the basis of prolonged chest pain, significant ST-segment elevation and rise of creatine kinase [10]. The diagnosis of old MI, established for 424 patients, was based on the presence of unequivocal electrocardiographic changes. Of the patients with old MI, 35.8% had angiographically occluded infarct-related arteries and 90.3% showed regional wall motion abnormalities corresponding to the electrocardiographic infarct localization. Control subjects (n = 340) had no evidence of myocardial infarction, angina pectoris, stroke, peripheral vascular disease or malignancy or angiographically coronary stenosis of more than 10%. All subjects enrolled in this study gave written informed consent. The Ethics Committee of the Technical University approved this study. Risk factors were defined as described previously [10]. For genotyping genomic DNA was extracted [10] and genotyping was performed using allele-specific fluorogenic probes [11]. We used the forward primer 5 -GCATTCCCACCGCCTTTC-3 , reverse primer 5 -AGCCACGGTGGCT TCTTCTAC-3 , allele A603 probe 5 -AGGTCAAGAATACTTGGCCTGCCCA-3 , allele G603 probe 5 -AGGTCAAGAATACCTGGCCTGCCC-3 . Allele A603 probe was labeled with 6-carboxy-fluorescein (FAM) and allele G603 probe with 2,7-dimethoxy-4,5-dichloro6-carboxy-fluorescein (VIC) at their 5 ends and with the quencer 6-carboxy-tetramethyl-rhodamine (TAMRA) at their 3 ends. The PCR included 40 cycles of denaturation at 95 ◦ C for 10 s and annealing/extension at 60 ◦ C for 60 s. Statistical analysis consisted of comparing separately genotype distributions between MI patients and controls. Discrete variables are expressed as counts (%) and compared by chi-square or Fisher’s exact test. Continuous variables are expressed as mean ± S.D. and compared by the means of the unpaired, two-sided t-test. The independent association between MI and the presence of the −603 G allele was assessed after adjusting for potential confounding factors using multiple logistic regression analysis and calculating the adjusted odds ratios and their 95% confidence intervals (CI). All statistical analyses were performed using S-Plus software (Mathsoft, Inc., Seatle, WA). Statistical significance was accepted for P-values of <0.05.

3. Results Genotype distribution in the control subjects was in Hardy–Weinberg equilibrium. The characteristics of the patients and age- and sex-matched controls are shown in Table 1. Among the coronary risk factors significant more patients with MI were smokers and had diabetes. For patients with MI the frequency for the −603 A/A genotype was 24%, for the −603 A/G genotype 51% and for the −603 G/G genotype 25%. In the control group the frequency for the −603 A/A genotype was 30%, for the −603 A/G 44%

Table 1 Baseline characteristics of control subjects and MI patients

Gender (M/F) Age (years) (mean ± S.D.) Active smokers (%) Hypercholesterolemia (%) Hypertension (%) Diabetes mellitus (%)

Control subjects (n = 340)

MI patients (n = 793)

P

226/84 63.4 ± 10.3 18.8 40.6 60.6 10.3

614/179 62.6 ± 11.6 24.3 37.8 62.7 21.6

NS NS <0.001 NS NS <0.001

Table 2 The −603 G allele of the −603 A/G tissue factor promoter polymorphism and risk factors: correlation with myocardial infarction −603 G allele Gender Age Active smoking Hypercholesterolemia Hypertension Diabetes mellitus

Odds ratio 1.44 0.97 0.91 2.33 0.82 1.1 2.72

P 0.01 0.85 0.33 0.00 0.15 0.49 0.00

and for the −603 G/G genotype 26%. The proportion of −603 G allele carriers was significantly higher in the patients with MI than in the control subjects: 76% versus 70% (P = 0.04). After adjusting for other baseline characteristics (age, sex, hypercholesterolemia, hypertension, diabetes and smoking) carriage of the G allele was associated with an odds ratio of 1.44 (Table 2, 95% CI 1.07–1.93, P = 0.01). The multivariate model did not show a significant interaction between −603 G allele carriage and other cardiovascular risk factors (including diabetes and smoking) regarding the risk of myocardial infarction.

4. Discussion The present study provides the first evidence of the association between a common polymorphism of the TF promoter and MI. We found that the −603 G allele in the TF gene was significantly more frequent in MI patients than in control subjects which suggests that patients with the −603 G allele are prone to myocardial infarction. Tissue factor is a cellular receptor that initiates blood coagulation [12]. The TF gene is constitutively expressed in some extravascular cell types and expression can be stimulated in several vascular cell types, including monocytes and vascular cells [1]. Induction of TF occurs at a transcriptional level by cell type specific promoters. Activation of AP-1 and NF-kappa B sites induce TF in endothelial and monocytic cells, whereas activation of Egr-1 and Sp1 sites stimulate TF in epithelial, smooth muscle cells and monocytes [1]. Different TF promoter polymorphisms have been described that were frequent and completely concordant (−1812 C/T, −1322 C/T, −1208 D/I and −603 A/G). An association of the −1208 D polymorphisms with TF plasma concentrations

I. Ott et al. / Atherosclerosis 177 (2004) 189–191

has been described, where −1208 D homozygotes present with lower circulating TF levels 9. Furthermore, the −603 G phenotype was associated with an increased TF mRNA expression in monocytes compared with the AA genotypes [18]. The four TF promoter polymorphisms are in linkage disequilibrium where one genotype has −1812 C, −1322 C, −1208 D and −603 A whereas the other genotype has −1812 T, −1322 T, −1208 I and −603 G. Therefore, lower plasma TF levels or decreased monocyte TF expression in −1208 D/−603 A phenotypes may be protective for individuals bearing two −603 A/−1208 D alleles, whereas individuals carrying the −603 G allele may favor thrombosis. Although the molecular mechanisms of these observations have not been identified they support the clinical observations made in our study. Recent evidence suggests the existence of a blood-borne pool of TF that may play a role in the propagation of thrombosis [4]. As high plasma levels of TF antigen have been reported in patients with acute coronary syndromes versus patients with stable angina [13] this may contribute to subsequent thrombotic complications. Circulating TF is not homogeneous: it consists of soluble forms of TF and procoagulant microparticles that contribute to thrombus formation to an ill defined extent [6,14]. Another source of TF within the blood represent activated monocytes [15,16]. Increased monocytic TF expression and activity may depict an additional mechanism for a procoagulant state in MI. Finally, higher TF content at the site of the plaque in patients with myocardial infarction is associated with a local increase in thrombin generation, and suggests a link of TF expression with the in vivo thrombogenicity of the plaque [17]. An increase in circulating TF as well as an enhanced response of activated monocytes and vascular cells in MI in carriers of the −603 G allele might contribute an enhanced thrombotic predisposition. The present population has been the basis of other association studies published previously [10]. This may increase the risk of type 1 error and should be acknowledged as a limitation. In contrast to our results, a previous case-control study could not detect an association of the TF promoter polymorphism with MI 9. The phenotypic characterization of the controls and cases in the latter study was based on clinical and electrocardiographic criteria (MONICA criteria). In addition to these criteria we used coronary and left ventricular angiography to differentiate between controls and cases in the present study. An additional reason for our discordant results may be the age- and sex-related restrictions in the inclusion criteria applied in the study by Arnaud [9]. In conclusion, our study suggest that the −603 A/G TF promoter polymorphism associated with MI. Although this might be due to an increased basal TF expression [18] the underlying molecular mechanism for the increase in TF plasma levels and their contribution to coronary thrombosis remain to be elucidated. A longitudinal prospective study could be of interest to confirm these results.

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Acknowledgments The study was supported in part by grants from the Deutsche Forschungsgemeinschaft (Ot 158/4-1) and the Bayerische Wissenschafts-ministerium (Bayerischer Habilitationsf¨orderpreis I. Ott). We thank W. Latz, M. Eichinger and A. Ehrenhaft for invaluable technical assistance. References [1] Mackman N. Regulation of the tissue factor gene. FASEB J 1995;9:883–9. [2] Toschi V, Gallo R, Lettino M, et al. Tissue factor modulates the thrombogenicity of human atherosclerotic plaques. Circulation 1997;95:594–9. [3] Ardissino D, Merlini PA, Ariens R, et al. Tissue-factor antigen and activity in human coronary atherosclerotic plaques. Lancet 1997;349:769–71. [4] Giesen PL, Rauch U, Bohrmann B, et al. Blood-borne tissue factor: another view of thrombosis. Proc Natl Acad Sci USA 1999;96:2311–5. [5] Rauch U, Nemerson Y. Circulating tissue factor and thrombosis. Curr Opin Hematol 2000;7:273–7. [6] Bogdanov V, Balasubramanian V, Hathcock J, et al. Alternatively spliced human tissue factor: a circulating, soluble, thrombogenic protein. Nat Med 2003;9:458–62. [7] Sambola A, Osende J, Hathcock J, et al. Role of risk factors in the modulation of tissue factor activity and blood thrombogenicity. Circulation 2003;107:973–7. [8] Soejima H, Ogawa H, Yasue H, et al. Angiotensin-converting enzyme inhibition reduces monocyte chemoattractant protein-1 and tissue factor levels in patients with myocardial infarction. J Am Coll Cardiol 1999;34:983–8. [9] Arnaud E, Barbalat V, Nicaud V, et al. Polymorphisms in the 5’ regulatory region of the tissue factor gene and the risk of myocardial infarction and venous thromboembolism: the ECTIM and PATHROS studies. Etude Cas-Temoins de l’Infarctus du Myocarde. Paris Thrombosis case-control Study. Arterioscler Thromb Vasc Biol 2000;20:892–8. [10] Koch W, Kastrati A, Bottiger C, et al. Interleukin-10 and tumor necrosis factor gene polymorphisms and risk of coronary artery disease and myocardial infarction. Atherosclerosis 2001;159:137–44. [11] Livak K. Allelic discrimination using fluorogenic probes and the 5’ nuclease assay. Genet Anal 1999;14:143–9. [12] Edgington TS, Dickinson CD, Ruf W. The structural basis of function of the TF. VIIa complex in the cellular initiation of coagulation. Thromb Haemost 1997;78:401–5. [13] Soejima H, Ogawa H, Yasue H, et al. Heightened tissue factor associated with tissue factor pathway inhibitor and prognosis in patients with unstable angina. Circulation 1999;99:2908–13. [14] Mallat Z, Benamer H, Hugel B, et al. Elevated levels of shed membrane microparticles with procoagulant potential in the peripheral circulating blood of patients with acute coronary syndromes. Circulation 2000;101:841–3. [15] Ott I, Neumann FJ, Kenngott S, et al. Procoagulant inflammatory responses of monocytes after direct balloon angioplasty in acute myocardial infarction. Am J Cardiol 1998;82:938–42. [16] Ott I, Andrassy M, Zieglgansberger D, et al. Regulation of monocyte procoagulant activity in acute myocardial infarction: role of tissue factor and tissue factor pathway inhibitor-1. Blood 2001;97:3721–6. [17] Ardissino D, Merlini PA, Bauer KA, et al. Thrombogenic potential of human coronary atherosclerotic plaques. Blood 2001;98:2726–9. [18] Reny JL, Laurendeau I, Fontana P, et al. Tissue factor −603 A–G promotor polymorphism is associated with human monocyte constitutive but not with LPS induced gene expression. Thromb Haemost 2004;91:248–54.