Mild Hyperhomocysteinemia and Fibrinolytic Factors in Patients with History of Venous Thromboembolism

Thrombosis Research 100 (2000) 271–278

ORIGINAL ARTICLE

Mild Hyperhomocysteinemia and Fibrinolytic Factors in Patients with History of Venous Thromboembolism Mojca Bozˇicˇ1, Mojca Stegnar1, Isabella Fermo2, Anka Ritonja3, Polona Peternel1, Janez Stare4 and Armando D’Angelo2 Department of Angiology, University Medical Centre, Ljubljana, Slovenia; 2H. San Rafaele Scientific Institute, Milan, Italy; 3Department of Biochemistry and Molecular Biology, J. Stefan Institute, Ljubljana, Slovenia; and 4Institute for Biomedical Informatics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia. 1

(Received 25 February 2000 by Editor H. Arnesen; revised/accepted 19 June 2000)

Abstract Mild hyperhomocysteinemia is recognized as a risk factor for venous thromboembolism (VTE), though its role in the thrombogenic processes is not understood. Its possible association with impaired fibrinolysis was investigated in 157 patients (61 women, 96 men) below the age of 60 years (43⫾11, mean⫾SD) with a history of objectively confirmed VTE. Patients had significantly higher fasting total plasma homocysteine (tHcy) levels than 138 apparently healthy subjects (8.0, 6.6–9.9 ␮mol/L vs. 7.2, 5.9–8.6 ␮mol/L, P⫽0.001; median, range between first and third quartile). In 17 of 157 patients (12%) hyperhomocysteinemia (tHcy⬎11.4 ␮mol/L for women and tHcy⬎12.6 ␮mol/L for men) was established. The adjusted odds ratio as an estimate of relative risk for VTE was 2.3 (0.8–7.0; 95% confidence interval). When patients with hyperhomocysteinemia were compared to patients without hyperhomocysteinemia, no significant differences in t-PA (antigen 9.2⫾5.5 ␮g/L and 9.7⫾4.7 ␮g/L, respectively; activity 1.3⫾0.5 IU/mL and 1.3⫾0.7 IU/mL, respectively) and PAI-1 (antigen 19.3⫾ 17.5 ␮g/L and 22.6⫾20.4 ␮g/L, respectively; activity Abbreviations: tHcy, total plasma homocysteine; t-PA, tissue plasminogen activator; PAI-1, plasminogen activator inhibitor type 1; VTE, venous thromboembolism. Corresponding author: Mojca Stegnar, University Medical Center, Department of Angiology, Riharjeva 24L, SI-1000 Ljubljana. Tel: ⫹386 (61) 333 500; Fax: ⫹386 (61) 333 155; E-mail: .

15.0⫾12.6 and 15.8⫾13.3 IU/mL, respectively) were observed. In conclusion, this study showed an association between mild hyperhomocysteinemia and VTE, but provided no evidence for an independent association between hyperhomocysteinemia and alterations in fibrinolytic proteins.  2000 Elsevier Science Ltd. All rights reserved. Key Words: Vascular disease; Venous thromboembolism; Hyperhomocysteinemia; Homocysteine; Fibrinolysis

M

ild hyperhomocysteinemia is recognized as an independent risk factor for arterial disease including coronary artery disease, cerebrovascular disease, and peripheral vascular disease [1]. Although venous thromboembolism (VTE) represents half of the complications in patients with severe hereditary forms of hyperhomocysteinemia (homocystinuria) [2], the association between mild hyperhomocysteinemia and VTE was not published until the 1990s [3–9]. Just recently a meta-analysis of ten case-control studies published from 1991 to 1997 supported fasting hyperhomocysteinemia as a risk factor for VTE, increasing the risk by 2.5-fold [10]. Moreover, two prospective studies showed that subjects with elevated tHcy are at an increased risk for future VTE, thus strengthening the assumption on causal relationship between hyperhomocysteinemia and VTE [8,11]. Mechanisms that lead to the atherogenic and thrombotic complications of hyperhomocysteine-

0049-3848/00 $–see front matter  2000 Elsevier Science Ltd. All rights reserved. PII S0049-3848(00)00324-8

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mia are not well understood. Studies in animals and in vitro studies suggest that hyperhomocysteinemia may predispose to atherosclerosis and thrombosis by injuring vascular endothelium, leading to endothelial dysfunction. It was proposed that endothelial damage is mediated through hydrogen peroxide production [12]. Homocysteine also alters normal antithrombotic phenotype of the endothelium by enhancing the activity of factor XII [13]. Homocysteine promotes factor V activity through the reduction of protein C activation [14] and blocks tissue plasminogen activator binding to human endothelial cells [15]. However, the relevance of the above studies on endothelial cell cultures has been questioned because of high homocysteine concentrations used, exceeding by several-fold concentrations of homocysteine observed in human plasma. In patients with hyperhomocysteinemia the observation of increased levels of endothelium-derived proteins such as von Willebrand factor, tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), thrombomodulin, and fibronectin would support the presumption on the noxious effect of homocysteine on the endothelial cells. Indeed, increased levels of von Willebrand factor and thrombomodulin but not of t-PA, PAI-1, and fibronectin were observed in hyperhomocysteinemic patients with peripheral arterial disease [16,17]. In patients with VTE, an increased level of PAI-1 is consistently found in up to 40% of cases [18–20]. In addition, an association between history of VTE and increased concentration of t-PA antigen is also observed occasionally [18,21]. Although elevated PAI-1 could partially be attributed to acute phase reaction associated with VTE [22], the underlying mechanism leading to increased PAI-1 and t-PA in VTE is mainly unresolved. The present study was undertaken in order to test the hypothesis that elevated fibrinolytic endothelial proteins (namely PAI-1 and t-PA) are associated with homocysteinemic status of VTE patients.

1. Subjects and Methods 1.1. Patients Patients with a history of venous VTE were recruited from consecutive patients treated at the

Department of Angiology in Ljubljana. One hundred and fifty-seven patients (61 women and 96 men) 19 to 60 (43⫾11) years old were included in the study at least three months (16⫾9 months, mean⫾SD) after objective confirmation of acute VTE. Diagnosis of VTE was established by at least one of the following methods: ultrasound vein imaging, impedance plethysmography, isotope, or contrast venography. Most patients (92%) had a history of leg deep venous thrombosis. A history of arm deep venous thrombosis was present in 8 patients, pulmonary embolism without signs of deep venous thrombosis (confirmed by perfusion/ ventilation lung scanning) in 3 patients, and thrombosis of the vena cava in 1 patient. Eighty-one percent of patients had a history of a single event. In 49% of patients no factors predisposing to VTE could be established. In the rest VTE was secondary to at least one predisposing factor such as surgery (n⫽29), trauma (n⫽21), immobilization (n⫽15), and bed rest (n⫽14). The most frequent predisposing factors in women were oral contraceptives (n⫽21), puerperium (n⫽6), pregnancy (n⫽4), and hormone replacement therapy (n⫽4). At the time of blood sampling 22% of patients were receiving oral anticoagulant treatment. None of the women included in the study were taking oral contraceptives or were on hormone replacement therapy. One hundred and thirty-eight apparently healthy subjects 19 to 60 (40⫾11, mean⫾SD) years of age (80 women and 58 men) were asked to participate as controls. This control group comprised mainly medical students, hospital staff, and their acquaintances. None had a history of VTE. All subjects participated in the study after they had given their full informed consent. The study was approved by the State Ethical Committee.

1.2. Blood Sampling Blood samples were obtained between 7 and 9 a.m. after an overnight fast and 20 minutes rest. Blood was collected from an antecubital vein, in most cases without the application of a tourniquet. For determination of tHcy and haemostatic parameters blood was collected in 10 mL Vacutainer威 tubes (Becton Dickinson, Heidelberg, Germany) with 0.13 mol/L trisodium citrate (1 volume of citrate to 9 volumes of blood). For determination of t-PA

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activity, blood was collected in 4.5 mL Stabilyte威 tubes (Biopool, Umea, Sweden) with acidic sodium citrate. Tubes were placed in ice water immediately after collection and centrifuged within 1 hour at 2000 g and 4⬚C for 30 minutes. Platelet poor plasma was transferred to small plastic vials, frozen in liquid nitrogen, and stored at⫺70⬚C until analysis.

1.3. Laboratory Methods In all the subjects tHcy in plasma was measured with high-performance liquid chromatography and fluorescent detection as previously described [7]. In the patient group, t-PA activity and PAI-1 activity were determined by amidolytic assays (Spectrolyse/fibrin, Biopool, Sweden) according to the instructions of the manufacturer. t-PA antigen and PAI-1 antigen were determined with commercial enzyme-linked immunosorbent kits (Imulyse tPA and Imulyse PAI-1, respectively, Biopool, Sweden). Antithrombin, protein C, and plasminogen were determined by amidolytic assays (Berichrom, Behring, Germany). Fibrinogen was determined by a clotting assay (Multifibren, Behring, Germany). In all subjects, total serum cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides were determined in fresh serum in biochemical analyzer (Ektachem 250, Kodak, Rochester, NY, USA). Low-density lipoprotein (LDL) cholesterol was calculated from total cholesterol, HDL cholesterol, and triglyceride concentrations [23]. Serum vitamin B12 and folate were determined in 54 patients (19 women and 35 men) and 61 healthy subjects (21 women and 40 men) with a radioassay (Dualcount SPNB Radioassay, Diagnostic Product Corporation, Los Angeles, CA, USA).

1.4. Statistical Methods For statistical analysis, Statistica 4.5 software (StatSoft Inc., Tulsa, OK, USA) and SPSS 9.0 for Windows (SPSS Inc., Chicago, IL, USA) were utilized. The distribution of variables was tested with the Kolmogorov-Smirnof test. The variables with normal distribution are shown as means and SDs, and statistical differences between groups were tested with Student’s t test. Variables with skewed distributions are shown as medians with ranges between the first and the third quartiles and statistical differences between groups tested with the Mann-Whit-

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ney U-test. Spearman’s correlation coefficient was used to evaluate association between variables. Odds ratios were calculated with a logistic regression model. Restricted cubic splines were utilized to present the association between the predictor (tHcy concentration) and the logarithm of odds. Patients with hyperhomocysteinemia and patients without hyperhomocysteinemia were compared using a general linear model, in which adjustment for total cholesterol and sex was performed.

2. Results Characteristics of the patient and control groups are shown in Table 1. Patients had significantly higher tHcy levels than healthy subjects (8.0, 6.6–9.9 ␮mol/L and 7.2, 5.9– 8.6 ␮mol/L, respectively, P⫽0.001). The difference between the two groups was predominantly attributed to a significantly higher tHcy level in male patients compared to healthy men (8.6, 7.1–10.9 ␮mol/L and 7.8, 6.7–9.0, respectively, P⫽0.03), since the difference between female cases and controls was not significant (7.1, 5.6–8.6 ␮mol/L and 6.5, 5.6–8.4 ␮mol/L, respectively, P⫽0.25). In patients with idiopathic VTE, tHcy levels were higher compared to patients with predisposing risk factors for VTE (8.4, 7.1–11.0 ␮mol/L and 7.6, 6.3–9.5 ␮mol/L, respectively, P⫽0.02). No statistically significant difference in tHcy levels in patients with their first episode compared to patients with a recurrent episode of VTE was observed, although patients with recurrent VTE showed slightly higher levels of tHcy (8.2, 7.3–10.2 vs. 7.2, 6.5–9.9, P⫽0.40). Oral anticoagulant therapy did not affect tHcy levels. The cut-off point for hyperhomocysteinemia was defined as the concentration of tHcy above the ninety-fifth percentile for control subjects (tHcy⬎ 11.4 ␮mol/L for women and tHcy⬎12.6 ␮mol/L for men). In this way hyperhomocysteinemia was established in 17 (10 male and 7 female) patients (12%). The odds ratio as an estimate of the relative risk for VTE was 2.7 (1.0–7.0). After adjustment for total serum cholesterol, the odds ratio was 2.3 (0.8–7.0). In all subjects together and in patients with VTE separately, tHcy positively correlated with age (␳⫽0.25, p⬍0.001 and ␳⫽0.27, p⬍0.001, respec-

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Table 1. Characteristics of patient and control groups. Variables with normal distribution are shown as means and standard deviations; variables with skewed distribution are given as medians with ranges between the first and the third quartile

Age Sex (female/male) Total cholesterol (mmol/L) High–density lipoprotein Cholesterol (mmol/L) Low–density lipoprotein Cholesterol (mmol/L) Triglycerides (mmol/L) Vitamin B12 (pmol/L) # Folate (nmol/L) #

Patients

Controls

p

43⫾11 61/96 6.1⫾1.2

40⫾11 82/63 5.2⫾1.2

⫽0.007 ⫽0.07 ⬍0.001

1.2 (1.0–1.4)

1.3 (1.0–1.5)

⫽0.27

4.0⫾1.0 1.5 (1.1–2.3) 219 (165–301) 22.1⫾6.6

3.3⫾1.1 1.2 (0.9–1.6) 183 (136–250) 19.4⫾7.1

⬍0.001 ⬍0.001 ⫽0.05 ⫽0.02

Serum concentrations of vitamin B12 and folate were determined in 54 patients (19 females/ 35 males) and 61 controls (21 females/ 40 males).

tively), total cholesterol (␳⫽0.25, p⬍0.001 and ␳⫽0.25, P⫽0.005, respectively), LDL cholesterol (␳⫽0.28, p⬍0.001 and ␳⫽0.25, P⫽0.007, respectively), and triglycerides (␳⫽0.16, P⫽0.01 and ␳⫽0.21, P⫽0.02, respectively). Negative correlation between tHcy and serum vitamin B12 (␳⫽ ⫺0.14, P⫽0.02 and ␳⫽⫺0.00, P⫽0.98, respectively) or folate (␳⫽⫺0.06, P⫽0.53 and ␳⫽-0.06, P⫽0.66, respectively) was not found. In patients tHcy correlated with t-PA antigen (␳⫽0.38, p⬍0.001), PAI-1 antigen (␳⫽0.18, P⫽0.03), and PAI-1 activity (␳⫽0.18, P⫽0.02). In a multiple regression model, only sex and total cholesterol were significantly associated with tHcy and explained 10% of tHcy variability. We found no threshold tHcy concentration. The risk for VTE rose proportionately to tHcy concentration (Figure 1). If all patients with hyperhomocysteinemia were compared with the patients without hyperhomocysteinemia in a generalized linear model, no statistically significant differences between the two

groups of patients were observed in any of the haemostatic variables determined (Table 2).

3. Discussion The results of this study provide further evidence on the relationship between mild hyperhomocysteinemia and history of VTE. It was observed that the group of patients with a history of VTE investigated had on average 11% higher tHcy than apparently healthy controls, which is in accordance with published data [5,7]. Twelve percent of the patients had tHcy above the ninety-fifth percentile of the control group (tHcy⬎11.4 ␮mol/L for women and tHcy⬎12.6 ␮mol/L for men). Other authors report similar frequencies of hyperhomocysteinemia (8– 18%) in patients with VTE [3–5,7,9]. In our study the odds ratio as an estimate of relative risk for VTE depended on the selected cut-off point. At the conventional ninety-fifth percentile as the cutoff point the adjusted odds ratio was 2.3. The linear

Fig. 1. Relationship between tHcy levels and relative risk for VTE expressed as the logarithm of odds in women (a) and in men (b). tHcy values were adjusted for total cholesterol.

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Table 2. Fasting total plasma homocysteine (tHcy) and haemostatic variables in patients with mild hyperhomocysteinemia and in patients with normal levels of tHcy. Data are presented as means⫾standard deviations

tHcy (␮mol/L) t–PA antigen (␮g/L) t–PA activity (IU/mL) PAI–1 antigen (␮g/L) PAI–1 activity (IU/mL) Plasminogen (%) Protein C*(%) Antithrombin (%) Fibrinogen (g/L)

Patients with hyperhomocysteinemia (n⫽17)

Patients without hyperhomocysteinemia (n⫽140)

p

18.7⫾11.3 9.2⫾5.5 1.3⫾0.5 19.3⫾17.5 15.0⫾12.6 107⫾17 112⫾14 110⫾18 2.56⫾0.45

8.1⫾2.0 9.7⫾4.7 1.3⫾0.7 22.6⫾20.4 15.8⫾13.3 110⫾14 102⫾17 106⫾15 2.66⫾0.62

⬍0.000 ⫽0.70 ⫽0.37 ⫽0.23 ⫽0.39 ⫽0.12 ⫽0.15 ⫽0.71 ⫽0.53

* Six patients on oral anticoagulant treatment and one patient with protein C deficiency were excluded.

relationship between the adjusted odds ratios and tHcy concentration implied that there was no threshold value for hyperhomocysteinemia. These results are in contrast to the results of den Heijer and co-workers [5] who established a substantial increase in the risk of thrombosis at the highest tHcy level, suggesting a threshold level above which tHcy exerts a thrombogenic effect. Hyperhomocysteinemia may be the result of a hereditary defect in the enzymes involved in methionine metabolism, or it might be acquired as a result of vitamin B6, B12, and folate deficiency. Levels of tHcy are also influenced by lifestyle factors such as smoking and coffee consumption [24]. In the present study the group of patients with VTE had increased levels of tHcy, though their levels of vitamins B12 and folate were not lower compared to the control group, suggesting hereditary causes of increased tHcy. In all subjects studied no negative correlation was observed between tHcy and serum vitamin B12 and folate. The absence of such a correlation was also reported by others [4,25]. However, if only subjects with folate concentration below the median value were considered, a statistically significant correlation between tHcy and folate was observed (data not shown). This association might support the previous observation that higher levels of folate counteract inadequate remethylation of tHcy in subjects with the inherited form of thermolabile methylenetetrahydrofolate reductase [26]. In our study only fasting tHcy was determined. It is believed that high fasting tHcy levels are more

associated with remethylation defects, while high tHcy levels observed after loading with methonine are more associated with transsulfuration defects. In patients with atherosclerotic vascular disease some authors observed higher frequency of mild hyperhomocysteinemia after methionine loading [27,28], while in patients with VTE similar prevalence of hyperhomocysteinemia was found regardless of measuring fasting or after methionine load [4]. The similarity of odds ratios for fasting and post-load tHcy calculated in the recent meta-analysis might imply that hyperhomocysteinemia in VTE is not more associated either with remethylation or transsulfuration defects [10]. The question of whether fasting tHcy is sufficient to detect hyperhomocysteinemia or methionine loading should be performed in VTE patients remains unanswered. The underlying mechanisms by which hyperhomocysteinemia could provoke thrombosis are unknown. There is evidence that tHcy contributes to endothelial damage in atherosclerosis [2], but it is not clear whether this mechanism is involved also in the pathogenesis of VTE. Increased levels of endothelium-derived proteins in association with increased tHcy would suggest such a role of tHcy. So far, association between t-PA and tHcy was observed in smaller groups of patients with arterial or venous thrombosis [29] and in patients with stroke [30]. In the VTE patients studied here, PAI-1 and t-PA antigens showed weak, though significant, association with tHcy. Associations were also observed between tHcy and age, sex, and serum lipids. Since the latter variables are also

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known to modify plasma levels of t-PA and PAI-1 [31–33], we compared patients with hyperhomocysteinemia to patients without hyperhomocysteinemia in order to evaluate the effect of hyperhomocysteinemia on t-PA and PAI-1 levels. The results showed no statistically significant differences between these two groups of VTE patients. Our observations, therefore, do not support the hypothesis that hyperhomocysteinemia in VTE patients facilitates alterations in endothelial cell function in such a way that it would be reflected in increased endothelial fibrinolytic proteins. Our study showed no significant differences in other haemostatic proteins (plasminogen and fibrinogen) and natural anticoagulants (antithrombin and protein C) between patients with and without mild hyperhomocysteinemia. In regard to natural anticoagulants, our results support those of Bienvenu and co-workers [34], who observed no reduction of antithrombin and protein C activity in patients with mild hyperhomocysteinemia in contrast to patients with severe forms of hyperhomocysteinemia in whom reduced antithrombin activity was established [35]. In conclusion, we found increased levels of tHcy in patients with a history of VTE and thus confirmed results of previous studies showing an association between hyperhomocysteinemia and VTE. Hyperhomocysteinemia was observed in 12% of patients and increased the risk for VTE by approximately two-fold. Since no evidence for independent association between levels of t-PA and PAI-1 and tHcy was apparent in our study, the results did not support the hypothesis that hyperhomocysteinemia causes disturbance of endothelial function, leading to increase in these proteins. The study was supported by the Slovenian Ministry of Science and Technology, grant No. L3-0368-0106.

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