27 Spence JD, Malinow MR, Barnett PA, Marian AJ, Freeman D,. Hegele RA. ... Belanger AJ, O'Leary DH, Wolf PA, Schaefer EJ, Rosenberg IH. Association ...
Journal of Thrombosis and Haemostasis, 2: 445–451
ORIGINAL ARTICLE
Homocysteine and markers of coagulation and endothelial cell activation V. E. A. GERDES,* H. A. KREMER HOVINGA,à H. TEN CATE,*§ M. R. MACGILLAVRY,* A. LEIJTE, P . H . R E I T S M A , § D . P . M . B R A N D J E S * and H . R . B U¨ L L E R à O N B E H A L F O F T H E A M S T E R D A M V A S C U L A R MEDICINE GROUP *Departments of Internal Medicine and Clinical Chemistry, Slotervaart Hospital, àDepartment of Vascular Medicine and §Laboratory for Experimental Internal Medicine, Academic Medical Center, Amsterdam, the Netherlands
To cite this article: Gerdes VEA, Kremer Hovinga HA, Ten Cate H, MacGillavry MR, Leijte A, Reitsma PH, Brandjes DPM, Bu¨ller HR on behalf of the Amsterdam Vascular Medicine Group. Homocysteine and markers of coagulation and endothelial cell activation. J Thromb Haemost 2004; 2: 445–51.
Introduction Summary. Objective: In vitro studies suggest an influence of hyperhomocysteinemia on the coagulation system, but the influence of mild hyperhomocysteinemia in vivo is unclear. Methods and results: We studied the relation between homocysteine and markers of coagulation activation and endothelial cell activation in 279 patients with established atherosclerotic disease. In addition, we performed an investigator-blinded placebo-controlled cross-over study to investigate the influence of acute hyperhomocysteinemia by oral methionine load on these markers in 20 healthy volunteers. In the atherosclerotic patients prothrombin fragment F1+2 and soluble thrombomodulin (sTM) were associated with homocysteine in univariate analyses (P ¼ 0.003 and P ¼ 0.001, respectively), but not in multivariate analyses. Age, creatinine and MTHFR C677T polymorphism were major determinants of homocysteine concentration. MTHFR C677T polymorphism status was not associated with F1+2 and sTM. Median homocysteine concentrations increased in the healthy volunteers after methione load. However, after methionine load or after placebo, we did not observe different plasma concentrations of F1+2 (0.9 nmol L)1 vs. 0.9 nmol L)1, P ¼ 0.39), d-dimer (153 lg L)1 vs. 151 lg L)1, P ¼ 0.63) and von Willebrand factor (103% vs. 107%, P ¼ 1.00). Conclusion: These results provide evidence against a major effect of mild hyperhomocysteinemia on activation of the coagulation system and endothelial cell activation in vivo. Keywords: atherosclerosis, coagulation, endothelium, homocysteine, methionine load.
Correspondence: V. E. A. Gerdes, Department of Vascular Medicine, Academic Medical Center, Meibergdreef 9, Postbus 22660, 1100 DD Amsterdam, the Netherlands. Tel.: +31 20 566 9111; fax: +31 20 566 9158; e-mail: v.e.gerdes@amc. uva.nl Received 22 October 2003, accepted 5 November 2003 2004 International Society on Thrombosis and Haemostasis
Mild hyperhomocysteinemia has been associated with arterial and venous vascular disease [1]. Patients with a myocardial infarction, ischemic stroke or peripheral arterial disease have higher homocysteine levels, fasting or after a methionine load, compared with healthy controls [2]. Higher homocysteine concentrations have been associated with a greater risk of future ischemic stroke and myocardial infarction, in asymptomatic individuals as well as patients with clinically manifest atherosclerosis [3–6]. The pathophysiological mechanism that explains the association between homocysteine and cardiovascular disease is unclear [1,5]. Since the coagulation system is an important factor in both arterial and venous vascular disease, the influence of homocysteine on the coagulation system may be one of the possible mechanisms. In vitro studies have shown different procoagulant effects of homocysteine on vascular endothelial cells, including activation of factor (F)V, inhibition of thrombomodulin-dependent protein C activation, impairment of von Willebrand factor (VWF) secretion and induction of tissue factor [7–10]. Tissue factor may be induced by homocysteine on monocytes as well [11]. There are only sparse data about the relationship between homocysteine and markers of coagulation in vivo. Acute hyperhomocysteinemia after oral methionine load induced increased concentrations of markers of coagulation and endothelial cell activation [12]. An association between markers of coagulation activation has been described in patients with premature arterial disease, in patients with venous thrombosis and in patients with acute coronary syndromes [13–15]. A weak association between hemostatic factors and homocysteine was described in individuals free of arterial disease [16]. The methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism has a clear influence on homocysteine concentrations. Homozygotes have approximately 25% higher homocysteine levels and the influence of folate deficiency on homocysteine levels is increased in these persons [17–19]. However, the role of this polymorphism in atherosclerosis is
446 V. E. A. Gerdes et al
controversial. The first reports suggested a higher prevalence of homozygotes among patients with a myocardial infarction [20–22]. This could not be confirmed by subsequent studies and a meta-analysis and is in contrast with the clear association between hyperhomocysteinemia and coronary artery disease [17,23–25]. A similar divergence in associations between homocysteine and arterial wall thickness, and between MTHFR and arterial wall thickness was reported [26,27]. This possible discrepancy between homocysteine and the MTHFR polymorphism, with a clear influence on homocysteine concentrations, is intriguing. It can be interpreted as an argument against a causal relationship between mildly elevated homocysteine and vascular disease. We investigated the relation between homocysteine and markers of coagulation and endothelial cell activation on the one hand, and between homocysteine and several parameters of atherosclerotic disease on the other hand in a cohort of patients with established atherosclerotic disease. In addition, we performed a placebo-controlled, investigator-blinded, randomized crossover trial in healthy individuals to test the influence of acute hyperhomocysteinemia after oral methionine load on markers of coagulation activation and endothelial cell activation. Methods Subjects
Atherosclerotic disease Consecutive patients with clinical manifestations of atherosclerosis were recruited in two teaching hospitals and classified according to the previous event: myocardial infarction, ischemic stroke or peripheral arterial disease. Only patients with a recent onset of myocardial infarction (£ 1 month) or ischemic stroke (£ 6 months) were included. Myocardial infarction was defined as typical chest pain for more than 20 min in combination with laboratory or ECG findings consistent with myocardial infarction. Ischemic stroke was defined as acute neurological deficit persisting at least 1 week. Intracerebral hemorrhage was ruled out by an early computed tomography scan. Peripheral arterial disease was defined as typical leg pain on walking and an ankle/arm blood pressure ratio lower than 0.85 in either leg at rest or a history of surgery for intermittent claudication. Smoking status, hypertension defined as a diastolic blood pressure of ‡ 95 mmHg or current treatment, and hypercholesterolemia defined as a total plasma cholesterol level of ‡ 7 mmol L)1 or current treatment were registered. Patients receiving anticoagulant treatment (coumarin or heparin) were not included. Healthy individuals (methionine load) Healthy volunteers were seen on two visits with an interval of 1 week. They were randomly assigned to oral methionine load (l-methionine 100 mg kg)1 in fruit juice) at the first or second visit. Blood samples were drawn after a 12-h overnight fast and 4 h after methionine load or placebo (fruit juice only). Participants were
allowed to drink only water during the experiment. Administration of methionine and placebo was done in a separate room. The research nurses who took the venous blood samples, laboratory personel and investigators were blinded during the experiment and the code was broken after laboratory investigations were completed. Both studies were approved by the Ethical Review Boards of the participating study centers and all patients gave their written informed consent to participate. Laboratory procedures
Homocysteine and coagulation markers were assessed in blood samples drawn 3 months after the acute event. Blood samples were collected after an overnight fast (> 10 h) by venepuncture from the antecubital vein and collected in tubes containing EDTA for homocysteine and citrate for coagulation assays. Homocysteine concentration was determined by high-performance liquid chromatography [28]. Commercial ELISAs for soluble thrombomodulin (sTM) and prothrombin fragment F1+2 (F1+2) were used (Diaclone, Besanc¸on, France and Dade Behring, Marburg, Germany, respectively). Lipids and creatinine were assessed by routine laboratory methods. Extraction of genomic DNA from peripheral leukocytes in citrated blood was performed using a QIAamp blood kit (Qiagen, Hilden, Germany). The primers and polymerase chain reaction (PCR) conditions used to determine MTHFR genotype were described previously [29]. In the healthy individuals homocysteine and prothrombin fragment F1+2 were measured with the same methods as in the patients with atherosclerotic disease. Commercial ELISAs for D-dimer (Bio Merieux Vidas, Marcy L’Etoile, France), VWF and thrombin–antithrombin complexes (Dade Behring) were used. B-mode ultrasound
The B-mode ultrasound intima-media thickness (IMT) measurement procedures have been described elsewhere [30]. B-mode ultrasound scans were performed by one sonographer. An ATL Ultramark IV (Advanced Technology Laboratories, Bothell, WA, USA) with a High Resolution Linear Array 7.5-MHz transducer was used. Subjects were scanned in the reclined position. Three right carotid, three left carotid, two right femoral and two left femoral artery wall segments were scanned. Images of each arterial wall segment were stored on S-VHS video tape. IMT of the posterior wall segments was measured off-line [31]. The average of the 10 segments was used for analysis. The sonographer and image analyst were blinded to the clinical status of the subjects. Statistical analysis
All analyses were performed with the Statistical Package for Social Science, version 10.0 (SPSS, Chicago, IL, USA). To explore the distribution of risk factors and laboratory variables 2004 International Society on Thrombosis and Haemostasis
Homocysteine and markers of coagulation and endothelial cell activation 447
the patients with atherosclerotic disease were divided into four subgroups defined by quartiles of homocysteine concentration. Frequencies were compared with v2 tests or Fisher’s exact tests when applicable. For comparison of variables with a normal distribution, Student’s t-test and analysis of variance tests were used, whereas the Mann–Whitney U-test and Kruskal–Wallis test were used for variables with a skewed distribution. To test for trend in the quartiles, tests for linearity were performed. We subsequently performed linear regression analyses with F1+2 and sTM as dependent variables. Variables with a skewed distribution were log-transformed and all variables with a P-value < 0.10 were selected for the multivariate model. A P-value of 0.05 was considered to be statistically significant. In the healthy volunteers study comparison of paired laboratory variables was performed using the non-parametric Wilcoxon signed ranks test. Results Homocysteine in patients with atherosclerotic disease
A total of 307 consecutive patients with manifest atherosclerotic disease were included. Homocysteine levels were determined in 279 patients from whom fasting plasma samples were available. Of these 279 patients, 95 had a recent ischemic stroke, 86 had a recent myocardial infarction and 98 had peripheral arterial disease. The mean age was 62.9 years. Median homocysteine concentration was 15.1 lmol L)1. We divided the group in quartiles according to homocysteine
concentration. Patient characteristics of the whole study group and the quartiles according to homocysteine concentration are shown in Table 1. Age, creatinine, F1+2, and sTM values were associated with homocysteine concentrations. Other patient characteristics were not different between the quartiles. The homocysteine concentrations were similar in patients with different types of arterial disease: myocardial infarction, ischemic stroke and peripheral arterial disease (medians 14.3 lmol L)1, 15.4 lmol L)1 and 15.2 lmol L)1, respectively; P ¼ 0.25, Kruskal–Wallis test). B-mode ultrasound IMT measurement was performed in 226 of the 279 patients, of whom homocysteine concentrations were determined. A slight increase of the median IMT values in the higher quartiles of homocysteine concentration was observed. However, this was not statistically significant, not even in the trend analysis (P for trend ¼ 0.14). This slight increase is probably attributable to difference in age among the quartiles. Homocysteine, F1 + 2 and sTM in patients with atherosclerotic disease
To test whether the relationship between homocysteine and F1+2 and sTM was independent, multivariate linear regression analyses with F1+2 and sTM as dependent variables were performed. In the analysis for F1+2 the univariate model selected homocysteine (B 0.18, SE 0.06, P ¼ 0.003), age, sTM, gender, IMT and type of arterial disease as explanatory variables of F1+2. In the multivariate model the association
Table 1 Characteristics: all patients and in quartiles according to homocysteine concentration
n Age (years) Male (%) Diabetes (%) Smoking (%) Hypertension (%) Creatinine (lmol L)1) Total cholesterol (mmol L)1) LDL (mmol L)1) HDL (mmol L)1) Triglycerides (mmol L)1) F1+2 (nmol L)1) sTM (ng mL)1) IMT (mm)
All patients
I
II
III
IV
279 63 63 11 85 41 101.7 88.4–113.2 6.1 5.4–6.9 4.0 3.5–4.7 1.11 0.96–1.35 1.7 1.2–2.3 1.33 1.04–1.73 8.9 7.0–11.5 0.94 0.77–1.11
68 58 54 15 78 38 96.0 85.9–106.1 6.0 5.4–6.8 3.9 3.4–4.7 1.11 0.95–1.32 1.7 1.2–2.3 1.09 0.94–1.41 7.8 6.5–10.2 0.89 0.75–1.08
71 62 61 4 87 39 98.1 87.5–112.3 6.3 5.7–7.0 4.0 3.3–4.6 1.12 0.96–1.43 1.8 1.2–2.4 1.34 1.10–1.67 8.5 6.6–11.5 0.92 0.72–1.09
69 64 77 19 86 46 106.0 92.8–121.6 6.1 5.4–6.9 4.1 3.5–5.0 1.10 0.94–1.29 1.7 1.4–2.4 1.25 1.01–1.88 9.3 7.7–12.3 1.01 0.78–1.18
71 68 62 6 87 46 106.1 89.3–125.5 6.2 5.4–6.8 4.1 3.5–4.7 1.16 0.98–1.32 1.6 1.3–2.3 1.47* 1.28–1.89 9.6* 7.2–12.8 0.97 0.81–1.14
sTM, Soluble thrombomodulin; IMT, intima-media thickness. For laboratory values and IMT medians and interquartile ranges are depicted. I: Homocysteine < 11.8 lmol L)1; II: 11.8–15.1 lmol L)1; III: 15.1–18.8 lmol L)1; IV: ‡ 18.8 lmol L)1. IMT measurement was performed in 226 patients. P < 0.001; *P < 0.01 (Kruskal–Wallis test). 2004 International Society on Thrombosis and Haemostasis
448 V. E. A. Gerdes et al
between homocysteine and F1+2 was not statistically significant any more, whereas age and type of arterial disease were the main determinants of F1+2 in these patients. In the linear regression analysis with sTM as dependent variable, univariate analysis selected age, diabetes, homocysteine (B 4.26, 95% confidence interval 1.77, 6.76, P ¼ 0.001), lipoprotein(a), F1+2, creatinine, IMT and type of arterial disease as variables associated with sTM. In the multivariate model, homocysteine was excluded and age, F1+2, lipoprotein(a) and peripheral arterial disease had an independent relationship with sTM. MTHFR polymorphism in patients with atherosclerotic disease
In 276 of the 279 patients MTHFR genotype could be determined in available DNA samples. We observed an allele frequency of 28%. The prevalence of homozygotes and heterozygotes of the MTHFR polymorphism was 9% and 39%, respectively. The allele frequency was similar in patients with different types of arterial disease. Median homocysteine levels were 14.3 lmol L)1, 16.1 lmol L)1 and 19.8 lmol L)1 for the CC, CT and TT genotype, respectively. This difference in homocysteine levels was significant for homozygotes compared with patients with the wild type (P ¼ 0.007, Mann– Whitney U-test), as well when we compared the three groups in Table 2 Homocysteine, markers of coagulation and endothelial cell activation and intima-media thickness according to MTHFR polymorphism status MTHFR )1
Hcy (lmol L ) F1+2 (nmol L)1) sTM (ng mL)1) IMT (mm)
CC (n ¼ 145)
TC (n ¼ 107)
TT (n ¼ 24)
14.3 11.7–17.5 1.33 1.01–1.71 9.4 7.1–12.2 0.96 0.74–1.14
16.1 11.7–19.1 1.32 1.06–1.69 8.2 6.9–11.1 0.91 0.79–1.09
19.8* 14.6–27.9 1.53 1.03–1.89 9.6 6.4–13.6 0.97 0.86–1.13
IMT, Intima media thickness; Hcy, homocysteine; sTM, soluble thrombomodulin. The medians and interquartile ranges are depicted. *P < 0.01 (Kruskal–Wallis test).
a Kruskal–Wallis test (P ¼ 0.008). In contrast, there was no association between MTHFR status and F1+2, sTM or IMT (Table 2). In none of the comparisons, either when we compared these variables between three groups, or when we compared these between homozygotes and carriers of the wild type, were statistically significant differences observed (all P-values > 0.18). Acute hyperhomocysteinemia in healthy volunteers
The group of healthy volunteers consisted of 10 male and 10 female individuals. The mean age was 33 (range 24–51) years. Median fasting homocysteine concentrations were 9.1 lmol L)1. After methionine load and placebo median homocysteine concentrations were 30.1 and 9.2 lmol L)1, respectively. Fasting homocysteine concentrations at the second visit were not influenced by oral methionine load at the first visit. There was no effect of acute hyperhomocysteinemia by the methionine load on VWF, F1+2, thrombin–antithrombin complexes or D-dimer concentrations (Table 3). Discussion Acute hyperhomocysteinemia by methionine load did not increase coagulation activation, or VWF concentration in the healthy volunteers. In our patients with established atherosclerotic disease we did not observe associations between homocysteine and markers of coagulation activation and endothelial cell activation in the multivariate analysis. These results provide evidence against a major direct effect of mildly elevated homocysteine on activation of the coagulation system and endothelial cell activation. In vitro studies suggested a procoagulant effect of homocysteine on endothelial cells [7–10]. However, most effects on endothelial cells were observed at higher homocysteine concentrations than those measured in patients with mild hyperhomocysteinemia. The in vitro studies may yield possible pathophysiological mechanisms for patients with severe hyperhomocysteinemia, but the relevance for patients with mild hyperhomocysteinemia is doubtful. A clear example are the
Table 3 Homocysteine and markers of coagulation in healthy volunteers, before and after methionine load or placebo Placebo
Hcy (lmol L)1) F1+2 (nmol L)1) TAT (ng mL)1) D-dimer
VWF (%)
(lg L)1)
Methionine
t¼0
t¼4
t¼0
t¼4
P
8.9 8.2–10.3 1.00 0.70–1.16 1.88 1.44–3.26 146 140–259 103 80–122
9.2 8.2–11.0 0.86 0.62–1.11 1.59 1.25–2.86 153 123–254 103 77–121
9.3 7.9–10.7 1.00 0.69–1.55 2.36 1.17–3.07 158 102–237 101 75–122
30.1 26.7–37.8 0.86 0.68–1.27 1.85 0.95–3.23 151 107–235 107 74–124
0.000 0.39 0.91 0.63 1.00
Hcy, Homocysteine; TAT, thrombin–antithrombin complexes; VWF, von Willebrand factor. Medians and interquartile ranges are depicted. P-value for comparison of difference (value at t ¼ 4 ) value at t ¼ 0) after methionine and placebo. 2004 International Society on Thrombosis and Haemostasis
Homocysteine and markers of coagulation and endothelial cell activation 449
studies on homocysteine and the protein C pathway. In vitro studies indicated that activation of protein C was suggested to be impaired in hyperhomocysteinemia [8,32]. Decreased thrombomodulin activity, which is essential for protein C activation, was observed in the aorta of hyperhomocysteinemic monkeys and mice [33,34]. However, human in vivo studies indicate that activation of protein C by thrombin and inactivation of plasma FVa by activated protein C are not impaired during moderate hyperhomocysteinemia [35,36]. Nappo and colleagues described increases of F1+2, D-dimer, soluble intracellular adhesion molecule-1 and soluble vascular cell adhesion molecule-1 after oral methionine load in healthy individuals [12], according to a protocol that was essentially identical to ours, except for an additional study arm with antioxidant vitamins in their investigation. We have no clear explanation for the discrepancy between their and our results, although the use of parametric testing by Nappa et al. on variables with a skewed distribution may have influenced the interpretation. Since others did not observe any influence of oral methionine loading on activated protein C resistance [35,36], and we could not confirm the results of Nappo et al, there is still no convincing evidence for a procoagulant effect of mild hyperhomocysteinemia by an oral methionine load in healthy individuals. In our patients with established atherosclerotic disease we observed, as shown previously, that homocysteine concentration was clearly associated with age, creatinine and MTHFR polymorphism. IMT values were slightly higher in patients with higher homocysteine concentrations in our cohort of patients with atherosclerotic disease. Since age was a major determinant of IMT in our study, the differences in age in the different quartiles of homocysteine concentration explain the observed differences in IMT. This is in agreement with a large population-based study in which an observed association between homocysteine and IMT disappeared after correction for other risk factors [37]. In a few other ultrasound studies an independent association between homocysteine and carotid artery changes was found, but these studies focused on different measures of atherosclerotic disease such as carotid plaque or stenosis, not IMT [27,38,39]. Although MTHFR homozygosity was associated with higher homocysteine concentrations in our patients, we did not detect any influence of MTHFR genotype on F1+2, sTM, or on IMT. These findings are compatible with other studies. McQuillan and Spence reported no influence of MTHFR on the carotid artery in their ultrasound studies [26,27]. In a number of studies and a meta-analysis a lack of association between MTHFR and cardiovascular disease was observed [17,23–25]. The independent associations of homocysteine and atherosclerotic disease found in cross-sectional and prospective trials do not prove a causal relationship. Homocysteine concentration is influenced by many factors. Mild hyperhomocysteinemia could be an epiphenomenon of atherosclerosis and subclinical atherosclerotic disease may explain the reported associations. Atherosclerosis is an inflammatory disease [40]. We suggest that cytokines, the mediators of inflammatory 2004 International Society on Thrombosis and Haemostasis
disease, may contribute to the reported association between homocysteine and atherosclerotic disease. When injected in healthy volunteers, lipopolysaccharide causes an increase in homocysteine levels [41], and homocysteine concentrations have been associated particularly with interleukin (IL)-6 and interleukin-8 concentrations in asymptomatic persons and patients with premature atherosclerosis [42,43]. IL-6 induces tissue factor, the main initiator of coagulation, in monocytes and endothelial cells, and induces tissue factor-dependent coagulation after endotoxin challenge in humans and primates [44,45]. Such a link between inflammation and coagulation may also explain the observed relation between homocysteine and F1+2 in the univariate analysis. Another possibility is the presence of yet undefined protective factors which counteract the direct influence of homocysteine on endothelial cells. These factors may be sufficient to regulate the effect of mild hyperhomocysteinemia as caused by the MTHFR polymorphism, and yield an alternative explanation for the lack of association of MTHFR with atherosclerotic disease. Further studies need to elucidate if mild hyperhomocysteinemia is an epiphenomenon or not. Acknowledgements We thank E. W. M. Vogels, Laboratory of Experimental Internal Medicine, who did the MTHFR PCR assay, and W. Mairuhu, who determined the homocysteine concentrations. H.t.C. is a Clinical Established Investigator from the Netherlands Heart Foundation. The Amsterdam Vascular Medicine Group
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