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Journal of Thrombosis and Haemostasis, 3: 502–507

ORIGINAL ARTICLE

Thrombophilic risk factors in patients with severe carotid atherosclerosis R. MARCUCCI, F. SOFI, S. FEDI, B. LARI, I. SESTINI, A. P. CELLAI, R. PULLI*, G. PRATESI*, C . P R A T E S I * , G . F . G E N S I N I and R . A B B A T E Thrombosis Center, University of Florence, Dipartimento del Cuore e dei Vasi, Center for the Study at Molecular and Clinical Level of Chronic, Degenerative and Neoplastic Diseases to Develop Novel Therapies, Firenze, Italy; and *Unit of Vascular Surgery, University of Florence

To cite this article: Marcucci R, Sofi F, Fedi S, Lari B, Sestini I, Cellai AP, Pulli R, Pratesi G, Pratesi C, Gensini GF, Abbate R. Thrombophilic risk factors in patients with severe carotid atherosclerosis. J Thromb Haemost 2005; 3: 502–07.

Introduction Summary. Carotid stenosis and atrial fibrillation are the strongest risk factors for ischemic stroke. Ongoing prevention efforts include the identification of novel factors that increase the risk for carotid atherosclerosis. The aim of this study was to determine the thrombophilic risk profile of patients with severe carotid stenosis by evaluating a number of genetic and metabolic risk factors [factor (F)II G20210A, factor V Leiden, MTHFR C677T polymorphisms, anticardiolipin antibodies (aCL), lipoprotein(a) (Lp(a)), and homocysteine (Hcy)]. The study population consisted of 615 patients [(410 M/205 F; median age 73 (26–94) years] with severe (> 70%) carotid stenosis, and 615 apparently healthy subjects [(410 M/205 F; age 73 (31–92) years]. On multivariate analysis, independent risk factors were elevated Hcy [odds ratio (OR) 7.6, 95% confidence interval (CI) 4.8, 11.8] and Lp(a) levels (OR 2.9, 95% CI 2.1, 3.9), the presence of aCL (OR 5.7, 95% CI 3.1, 10.4) and heterozygosity for FII G20210A polymorphism (OR 2.8, 95% CI 1.3, 5.9). In the subgroup of women, independent risk factors for severe carotid atherosclerosis were: high levels of Hcy and Lp(a) and the presence of aCL, whereas hyperhomocysteinemia, elevated Lp(a) levels, aCL, FII G20210A and MTHFR 677TT polymorphisms remained independent risk factors in the subgroup of men. The results of the present study demonstrate that the prevalence of the thrombophilic risk factors is increased in patients with severe carotid atherosclerosis. Keywords: carotid artery, gene polymorphisms, hemostasis, homocysteine, lipoprotein(a). Correspondence: R. Marcucci, University of Florence, Dipartimento del Cuore e dei Vasi, Center for the Study at Molecular and Clinical Level of Chronic, Degenerative and Neoplastic Diseases to Develop Novel Therapies, Firenze, Italy. Tel.: +055 4279420; fax: +055 4279418; e-mail: r.marcucci@dac. unifi.it Received 14 September 2004, accepted 5 November 2004

Carotid stenosis and atrial fibrillation are the strongest risk factors for ischemic stroke. Approximately 7–10% of men and 5–7% of women older than 65 years have asymptomatic carotid stenosis of > 50%. Epidemiological studies suggest that the rate of unheralded stroke ipsilateral to stenosis is about 1–2% annually [1]. In addition to the treatment of well-established modifiable risk factors, ongoing prevention efforts include the identification and validation of novel biochemical and genetic factors that increase the risk for carotid atherosclerosis. Thrombophilic polymorphisms such as the factor (F)II gene mutation G20210A and the factor (F)V gene mutation G1691A, FV Leiden, are established risk factors for venous thromboembolism, but their role in atherosclerosis has been controversial. A meta-analysis failed to reveal a significant association between FV Leiden and ischemic stroke in a total number of 1504 patients, whereas a significant association was documented in the subgroup of 453 children [2]. An increased prevalence of FII G20210A polymorphism has been reported in patients with myocardial infarction and cerebral artery disease [3–8], but other studies have not been able to confirm these findings [9–11]. These conflicting results suggest that the importance of these polymorphisms as risk factors could be different in different patient groups and populations. Several retrospective studies document that anticardiolipin antibodies (aCL) are an independent risk factor for ischemic stroke, whereas two large prospective studies [12,13] did not document an independent association between the presence of aCL and the risk of future stroke. No data exist on the association between thrombophilic polymorphisms and carotid atherosclerosis. A number of studies have indicated that elevated lipoprotein(a) (Lp(a)) levels are a significant risk factor for both carotid atherosclerosis and stroke [14–20]. Finally, elevated plasma homocysteine (Hcy) levels have been consistently shown to be associated with carotid intimamedia thickness and carotid atherosclerotic plaques [21–26].  2005 International Society on Thrombosis and Haemostasis

Thrombophilia and carotid atherosclerosis 503

The aim of this study was to determine the thrombophilic risk profile of patients with severe carotid stenosis (> 70%) by evaluating in them a number of metabolic and genetic risk factors (Hcy, Lp(a), aCL antibodies, FV Leiden, FII G20210A and MTHFR C677T polymorphisms). Materials and methods Subjects examined

The study population consisted of 615 patients [(410 M/205 F; median age 73 (26–94) years] admitted to the Department of Vascular Surgery of the University of Florence from December 1999 to December 2003 to be evaluated for possible surgical intervention. Carotid stenosis was assessed by duplex scanning with color-coded echo flow imaging and confirmed by angiographic computed tomography, according to the NASCET criteria [27]. All patients had severe (defined by a stenosis > 70%) carotid stenosis. Preoperative neurological symptoms, defined as ipsilateral cerebrovascular events during 180 days within the intervention, were recorded in 218 patients; the remaining 397 were asymptomatic. All patients underwent a clinical cardiological evaluation, ECG, echocardiogram and peripheral artery echo color Doppler analysis. Exclusion criteria for patients were any history, symptom or sign of ischemic heart disease, peripheral artery disease or venous thrombosis. A group of 615 apparently healthy subjects, comparable for age and sex [(410 M/205 F; age 73(31–92) years], were recruited from the blood donors (n ¼ 65) of our hospital and from partners or friends (n ¼ 550) of the patients in the same time window as the patients. We used a structured questionnaire to identify symptom-free controls and to exclude subjects who were suspected of having any form of arterial or venous vascular disease. Consanguineous subjects were excluded; patients and controls were caucasian and drawn from the same area (Central Italy). Hypertension was defined as systolic pressure ‡ 140 mmHg and/or diastolic pressure ‡ 90 mmHg according to the Seventh report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure [28]; dyslipidemia was defined according to the Third Report of the National Cholesterol Education Program (NCEP) [29]; diabetes was defined in agreement with the American Diabetes Association [30]. Experimental procedure

Venous blood was collected from the basilic vein after an overnight fasting between 08.00 h and 08.30 h. Blood was drawn directly into plastic tubes containing sodium citrate 0.129 M (1/10, v/v) for determination of Lp(a) plasma levels. Plasma samples obtained after centrifugation were stored at ) 80 C except for Lp(a), which was assayed on fresh samples  2005 International Society on Thrombosis and Haemostasis

by an ELISA (IMUBIND Lp(a) Elisa Kit; American Diagnostic, Greenwich, CT, USA). Serum for testing aCL was obtained by centrifuging blood collected in evacuated tubes without anticoagulant at 1300 · g for 10 min and stored at ) 20 C. Stored serum was assayed within 15 days for the determination of all parameters. The aCL assay was performed by an ELISA (First Cardiolipin; Eurospital, Trieste, Italy) and aCL levels were reported in GPL units (for IgG) and in MPL units (for IgM). On the basis of the analysis of several hundred normal serum specimens performed in our laboratory in the past, and according to the literature, values > 20 U for both IgG and/or IgM were considered abnormal. To determine Hcy, whole venous blood was collected in tubes containing ethylenediaminotetracetate (EDTA) 0.17 mol L)1, immediately put in ice and centrifuged within 30 min at 4 C (1500 · g for 15 min). The supernatant was stored in aliquots at ) 80 C until assay. The plasma levels of total Hcy (free and protein bound) were determined by a Fluorimetric Polaryzed Immuno Assay (FPIA method) (IMX Abbott Laboratories, Oslo, Norway). For the detection of the C677T MTHFR polymorphism, the G1691A polymorphism of the FV gene and the G20210A polymorphism of the FII gene, genomic DNA was extracted from peripheral blood-cell lymphocytes, amplified by polymerase chain reaction and then digested with HinfI (C677T MTHFR), with MnlI (G1691A FV) and with HindIII (G20210A FII) restriction enzymes, respectively. Statistical analysis

Unless otherwise indicated, the results are given as median (range). The non-parametric Mann–Whitney test for unpaired data was used for comparisons between single groups. Univariate analysis was used to describe the association between carotid atherosclerosis and the presence of a hemostasis-related risk factor such as positivity of antiphospholipid antibodies, high levels of Lp(a) and Hcy, the presence of heterozygosity for FV Leiden and FII G20210A or the presence of homozygosity for MTHFR C677T. Lp(a) levels > 300 mg dL)1, a level associated with an increased risk of occlusive arterial disease [18], were considered over the normal range; high levels of Hcy were diagnosed when values exceeded the 95th percentiles of distribution in controls (Hcy, males 19 lmol L)1, females 15 lmol L)1). To perform the multivariate analysis, logistic regression was used with age, sex, smoking status (current smokers), hypertension, dyslipidemia, diabetes and each thrombophilic risk factor separately, as the independent variables. All odds ratios (OR) are given with their 95% confidence intervals (CI). All probability values are two-tailed, with values of < 0.05 considered statistically significant. Results Clinical characteristics of patients and controls are shown in Table 1.

504 R. Marcucci et al Table 1 Clinical characteristics of patients investigated Patients n ¼ 615

Controls n ¼ 615

410/205 73 (26–94) 241 (39.2%) 421 (68.5%) 81 (13.2%) 248 (40.3%)

410/205 73 (31–92) 124 (20.2%) 189 (30.7%) 24 (3.9%) 98 (15.9%)

Table 3 Genotype distribution of polymorphisms investigated according to sex Patients

M/F Age (years) Smoking habits Hypertension Diabetes Dyslipidemia

Data are expressed as n (%) or as median (range).

The prevalence of the classical cardiovascular risk factors—hypertension, smoking habit, dyslipidemia and diabetes—was significantly higher in patients with severe carotid atherosclerosis. We documented no classical risk factor in 66 (10.7%) patients, the presence of one risk factor in 219 (35.6%), two risk factors in 223 (36.2%), three risk factors in 100 (16.3%) and four risk factors in the remaining seven (1.2%) patients. Thrombophilic risk factors

FV Leiden, FII G20210A and MTHFR C677T genotype distributions in controls were compatible with the Hardy– Weinberg equilibrium. No significant difference was observed in the prevalence of heterozygosity for FV Leiden (3.3% vs. 3.9%), whereas the prevalence of heterozygosity for FII G20210A and homozygosity for MTHFR C677T was significantly higher in patients than in controls (Table 2). The genotype distributions of the three polymorphisms according to sex are reported in Table 3 (FII polymorphism, OR 2.8, 95% CI 1.2, 6.3; MTHFR, OR 1.8, 95% CI 1.3, 2.6). Heterozygosity for FII G20210A and homozygosity for MTHFR C677T were significantly more prevalent in male patients than in male controls, whereas no significant differences were found for females (Table 3). Hcy and Lp(a) levels were significantly higher in patients than in controls: 15.2 (3.6–72) lmol L)1 vs. 11 (6.0–70.6) lmol L)1 and 191 (1–2333) mg L)1 vs. 95 (9–695) mg L)1, respectively.

Table 2 Thrombophilic risk factors

Hyperhomocysteinemia Anticardiolipin antibodies positivity Lipoprotein(a) > 300 mg L)1 Heterozygosity for factor II G20210A Homozygosity for MTHFR C677T Heterozygosity for factor V Leiden

Patients N/615 (%)

Controls N/615 (%)

Unadjusted OR* (95% CI)

181 (29.4) 78 (12.7)

30 (4.9) 16 (2.6)

8.1 (5.4, 12.2) 5.4 (3.2, 9.4)

224 (36.4)

89 (14.5)

3.4 (2.6, 4.5)

12 (2)

2.5 (1.3, 4.9)

128 (20.8)

97 (15.8)

1.7 (1.2, 2.3)

20 (3.3)

24 (3.9)

0.8 (0.5, 1.5)

Males (n ¼ 410) FV Leiden +/– 14 (3.4%) – /– 396 (96.6%) Factor II G20210A +/– 22* (5.4%) – /– 388 (94.6%) MTHFR C677T +/+ 87* (21.2%) +/– 189 (46.1%) – /– 134 (32.7%)

Controls Females (n ¼ 205)

Males (n ¼ 410)

Females (n ¼ 205)

6 (2.9%) 199 (97.1%)

16 (3.9%) 394 (96.1%)

8 (3.9%) 197 (96.1%)

7 (3.4%) 198 (96.6%)

8 (2.0%) 402 (98.0%)

4 (2.0%) 201 (98.0%)

46 (22.5%) 97 (47.3%) 62 (30.2%)

62 (15.2%) 197 (48%) 151 (36.8%)

35 (17.0%) 89 (43.5%) 81 (39.5%)

*P < 0.005 vs. male controls.

Hyperhomocysteinemia (defined by Hcy levels above the 95th percentile of controls), Lp(a) levels > 300 mg L)1 and the aCL positivity were significantly more prevalent in patients than in controls (Table 2). The distribution of the thrombophilic risk factors in our patients showed that 409/615 (66.5%) patients had at least one thrombophilic risk factor. On multivariate analysis adjusted for age, sex and the classical cardiovascular risk factors, independent risk factors were elevated Hcy and Lp(a) levels, the presence of aCL and heterozygosity for FII G20210A polymorphism which determine a 1.6- to 7.6-fold increase in the odds of having severe carotid atherosclerosis (Table 4). No interactions between traditional and thrombophilic risk factors were detected. No differences were detected in the prevalence of thrombophilic risk factors between asymptomatic and symptomatic patients (Table 5). Thrombophilic risk factors in men and women

Hyperhomocysteinemia (OR 5.2, 95% CI 3.0, 8.8), elevated Lp(a) levels (OR 2.5, 95% CI 1.7, 3.7), aCL (OR 5.5, 95% CI 2.6, 11.5), FII G20210A (OR 2.9, 95% CI 1.2, 7.2) and MTHFR 677TT polymorphisms (OR 1.6, 95% CI 1.0, 2.4) were independent risk factors for severe atherosclerosis on multivariate analysis performed on the subgroup of men (410 patients vs. 410 controls), whereas FII G20210A (OR 2.3, 95% Table 4 Independent* risk factors for severe carotid atherosclerosis OR (95% CI)

29 (4.7)

*Odds ratio (OR) calculated by univariate logistic regression analysis.

Hyperhomocysteinemia Anticardiolipin antibodies Lipoprotein(a) > 300 mg L)1 Heterozygosity for factor II G20210A Homozygosity for MTHFR C677T

7.6 5.7 2.9 2.8 1.6

(4.8, (3.1, (2.1, (1.3, (1.1,

11.8) 10.4) 3.9) 5.9) 2.2)

OR (95% CI), Odds ratio (95% confidence intervals). *Multivariate analysis adjusted for age, sex, and traditional cardiovascular risk factors (hypertension, smoking, dyslipidemia and diabetes).  2005 International Society on Thrombosis and Haemostasis

Thrombophilia and carotid atherosclerosis 505 Table 5 Prevalence of thrombophilic risk factors in symptomatic and asymptomatic patients Symptomatic Asymptomatic patients patients (n ¼ 218) (n ¼ 397) Hyperhomocysteinemia Anticardiolipin antibody positivity Lipoprotein(a) > 300 mg L)1 Heterozygosity for factor II G20210A Homozygosity for MTHFR C677T Heterozygosity for factor V Leiden

66 29 76 12 45 6

(30.2) (13.3) (34.8) (5.5) (20.6) (2.7)

115 49 148 17 83 14

(28.9) (12.3) (37.2) (4.2) (20.9) (3.5)

Data are expressed as n (%).

CI 0.6, 8.8) and MTHFR C677T polymorphisms (OR 1.7, 95% CI 0.8, 2.9) did not remain independent risk factors in the subgroup of women (205 patients vs. 205 controls). Thrombophilic risk factors in patients with no classical risk factors

In 66 out of 615 patients and in 270 out of 615 controls no traditional risk factor for atherosclerosis was found. Forty-one out of 66 (62%) patients had at least one thrombophilic risk factor. We performed a multivariate analysis in this subgroup of subjects and we confirmed an independent role, as risk factors, of Hcy, Lp(a), the presence of aCL and FII G20210A polymorphism. As reported in Table 6, the ORs associated with the prothrombin polymorphism and hyperhomocysteinemia were higher in this subgroup than in the total population, whereas no differences with respect to the whole group were seen for the risk associated with elevated Lp(a) levels and aCL. Discussion In this study we have thoroughly evaluated the most relevant acquired and inherited thrombophilic risk factors in a large number of patients with severe carotid atherosclerosis. In particular, this is the first study in which FII polymorphism has been evaluated in patients with severe carotid atherosclerosis. In the whole group of our patients, the independent thrombophilic risk factors for severe carotid stenosis were: heterozygosity for FII G20210A, aCL, elevated levels of Lp(a) and hyperhomocysteinemia. In previous reports, a correlation between the G20210A polymorphism and levels of prothrombin, prothrombin Table 6 Independent* risk factors for severe carotid atherosclerosis in subjects with no classical risk factors (66 patients vs. 270 controls) OR (95% CI) Hyperhomocysteinemia Heterozygosity for factor II G20210A Anticardiolipin antibodies Lipoprotein(a) > 300 mg L)1

9.2 6.0 3.7 3.6

(3.5, (1.6, (1.2, (1.7,

24.4) 22.7) 11.9) 7.4)

OR (95% CI), Odds ratio (95% confidence intervals). *Multivariate analysis adjusted for age, sex and traditional cardiovascular risk factors (hypertension, smoking, dyslipidemia and diabetes).  2005 International Society on Thrombosis and Haemostasis

activation peptide F1+2 and thrombin–antithrombin complexes was described, suggesting that the mutation leads to increased rates of thrombin generation [5,31–33]. Thrombin is thought to be an important factor in arterial wall thickening [34–39] and therefore the G20210A polymorphism may contribute to the progression of atherosclerosis by causing more local thrombin generation. Regarding aCL, no patient had clinical signs of antiphospholipid syndrome. The prevalence of aCL found in our patients is probably overestimated as it is based on a single aCL determination which could have been influenced by a transient inflammatory state. Nevertheless, these data are consistent with those reported in a recent large study on antiphospholipid antibody-positive patients [40], even if for other authors aCL might be only an epiphenomenon of atherosclerosis [41]. In agreement with the results of our study, it has been recently demonstrated that IgG aCL titer independently predicts intima-media thickness [42] and it has been reported that warfarin treatment in aPL-positive patients significantly decreases the percentage of patients with a progression of atherosclerotic disease [43]. Lp(a) levels > 300 mg L)1 confer an increase, albeit moderate, of risk of severe carotid atherosclerosis. Lp(a) could determine a thrombogenic effect by its interference with intrinsic fibrinolysis and it has been associated with endothelial dysfunction [20]. Furthermore, Lp(a) activates monocytes, colocalizes with plaque macrophages, stimulates smooth-muscle cells and could induce inflammation [44]. Regarding Hcy, we confirm an increased prevalence of hyperHcy in patients with advanced atherosclerosis, independently of the classical risk factors. This association is maintained in both men and women. A moderate increase in the circulating levels of Hcy may be ascribed to genetic and/or nutritional factors. In particular, folic acid, vitamin B6 and vitamin B12 deficiencies are inversely associated with Hcy levels, and the polymorphism C677T of MTHFR is associated with higher Hcy levels. Furthermore, a recent meta-analysis [45] demonstrated a moderate increase in the risk of coronary artery disease associated with the homozygosity for MTHFR 677TT, independently of Hcy. Accordingly, our study confirms a similar OR associated with MTHFR 677TT also in patients with atherosclerosis confined to the carotid area. It should be emphasized that Hcy is the only parameter which can be easily corrected by an inexpensive therapy based on the supplementation of folic acid, vitamin B6 and B12. The importance of Hcy is documented by the clinical observations of the reduced rate of progression of plaque area or intima-media thickness in hyperhomocysteinemic patients treated with vitamin supplementation [46,47]. In this study no different prevalence of thrombophilic risk factors was detected between asymptomatic and symptomatic patients, suggesting that thrombophilic risk factors play a role primarily in atherogenesis. These results, even if in accordance with several papers, are not supported by studies which could not demonstrate any role

506 R. Marcucci et al

of decreased coagulability in atherogenesis, e.g. patients with hemophilia or von Willebrand’s disease are protected against fatal ischemic heart disease [48], but not against atherosclerosis assessed by intima-media thickness measurement [49]. There are sex-specific differences in the atherogenicity of G20210A prothrombin and C677T MTHFR polymorphisms within our group of patients. The explanation for this finding is unclear but, different from data in the literature [50], we cannot hypothesize that a higher background risk diluted the effects of these polymorphisms as females and males did not differ significantly in the age and in the number of risk factors. However, as phenotype related to MTHFR C677T polymorphism is modulated by sex hormones [51], it is conceivable that in women exposure to the risk factor determines an effect only after hormonal protection has ceased. The following limitations of this study should be taken into account: (i) some analytes—aCL, Hcy and Lp(a)—might have been influenced by transient states, i.e. inflammatory processes for aCL and Lp(a) levels or changes in dietary habits for Hcy. For this reason, data on these parameters should be treated with caution; (ii) the design of this study makes it unable to demonstrate if the altered hemostatic parameters are causally related to the presence and severity of carotid stenosis.; (iii) as our study design includes asymptomatic and symptomatic patients with severe carotid stenosis, and symptom-free controls, our results might be related to both the atherosclerosis status and the thromboembolic complications which make a plaque symptomatic. Neverthless, the fact that no differences were detected between symptomatic and asymptomatic patients suggests that hypercoagulability is associated with atherosclerosis per se. In conclusion, the results of the present study demonstrate that the Ôthrombophilia burdenÕ is increased in patients with severe carotid atherosclerosis, as more than half of patients (66.5%) had at least one thrombophilic risk factor. Contribution of authors Study design: G.F.G., R.A., R.M., C.P. Clinical evaluation of patients: R.P., G.P., C.P. Laboratory investigation: S.F., B.L., I.S., A.P.C. Statistical analysis: R.M., F.S. Writing up: R.M., R.A., G.F.G. References 1 Goldsteine LB, Adams R, Becker K. Primary prevention of ischemic stroke. Stroke 2001; 32: 280–99. 2 Juul K, Tybjaerg-Hansen A, Steffensen R, Kofoed S, Jensen G, Gronne Nordestgaard B. Factor V Leiden: The Copenhagen City Heart Study and 2 meta-analyses. Blood 2002; 100: 3–10. 3 Arruda VR, Annichino-Bizzacchi JM, Goncalves MS, Costa FF. Prevalence of the prothrombin gene variant (nt20210A) in venous thrombosis and arterial disease. Thromb Haemost 1997; 78: 1430–3. 4 Rosendaal FR, Siscovick DS, Schwartz SM, Psaty BM, Raghunathan TE, Vos HL. A common prothrombin variant (20210 G to A) increases the risk of myocardial infarction in young women. Blood 1997; 90: 1747–50.

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