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Biochemical, Molecular, and Genetic Mechanisms

Plasma Triacylglycerol and Coagulation Factor Concentrations Predict the Anticoagulant Effect of Dietary Fish Oil in Overweight Subjects¹

² Departments of Human Biology and ³ Biochemistry, Nutrition and Toxicology Research and Cardiovascular Research Institutes of Maastricht, Maastricht University, 6200 MD Maastricht, The Netherlands; ⁴ Department of Hematology, Erasmus Medical Centre, Rotterdam, The Netherlands; and ⁵ Department for Thrombosis Research, University of Southern Denmark, Esbjerg, Denmark

^* To whom correspondence should be addressed. E-mail: k.vanschoonbeek{at}hb.unimaas.nl.

	ABSTRACT

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
Fish oil, containing (n-3) PUFA, is associated with a moderate reduction in cardiovascular disease through a multifactorial mechanism involving a decrease in plasma lipids and anticoagulant activity. Two intervention studies on subjects at risk were performed to determine the relation of these 2 fish-oil effects. In study 1, 54 overweight subjects consumed 3.1 g (n-3) PUFA daily. In study 2, which involved 42 overweight patients with type 2 diabetes, 20 subjects consumed (n-3) PUFA, whereas 22 others ingested a preparation rich in (n-6) PUFA. Tissue factor-induced thrombin generation (thrombin potential) was determined as an integrated measure of plasma coagulant activity. In both studies, multivariate analysis indicated a strong clustering of fasting concentrations of triacylglycerols, prothrombin, factor V, factor VII, and factor X with one another at baseline. This cluster of factors determined partly the interindividual variation in thrombin generation, of which prothrombin and triacylglycerol concentrations were the main determinants. In both healthy subjects and diabetes patients, high triacylglycerol concentrations (>1.69 mmol/L) at baseline were closely linked to a strong fish oil–induced lowering of triacylglycerol and coagulation factor V, VII, and X concentrations, and thrombin generation. We conclude that high fasting triacylglycerol concentrations predict high procoagulant activity and a lowering of thrombin potential with dietary fish oil.

	Introduction

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

Fish oil also has antiplatelet and anticoagulant activities, although these 2 effects vary considerably among published studies (9). In earlier work, we reported considerable interindividual variation in the anticoagulant effect of fish oil (10). This variation became apparent using a sensitive coagulation assay that measures in an integrated way the kinetics and extent of tissue factor–induced thrombin generation in full plasma (11,12). Part of the intersubject variability could be explained by coagulation factor polymorphisms (10), but it is likely that other variables contribute to this as well.

The literature provides indications that the metabolism of coagulation factors and that of lipids in plasma are interrelated; for instance, concentrations of the vitamin K-dependent factors, prothrombin, factor VII, and factor X correlate with plasma lipid concentrations (13). Further, hypertriglyceridemia is a risk factor for thrombosis, and patients with high triacylglycerol concentrations also have high concentrations of factor VII (14). In the same patient group, postprandial hyperlipemia is accompanied by factor VII activation and increased coagulant activity (15). In contrast, there is evidence that triacylglycerols do not directly influence factor VIIa activity or coagulation (16). Thus, whereas the plasma concentration of triacylglycerol seems to be related to coagulant activity, exactly how is unclear.

In this study, we investigated whether subjects' plasma triacylglycerol concentrations and hemostatic variables determine the lipid-lowering and hypocoagulant effects of dietary fish oil. For this purpose, we performed 2 (n-3) PUFA intervention studies with 2 groups of overweight subjects: healthy volunteers and patients with type 2 diabetes and a tendency for hypertriglyceridemia.

	Subjects and Methods

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
    Subjects and study design. Two intervention studies investigated the anticoagulant effect of fish oil in overweight subjects with increased thrombotic risk. Study protocols were in agreement with the Helsinki declaration and approved by the Medical Ethical Committee of the Academic Hospital Maastricht. Subjects were only included after they provided written informed consent.

Study 1 was performed with 57 overweight but healthy Caucasian men. Inclusion criteria were BMI >25 kg/m² and the absence of lipid-lowering, antiplatelet, or anticoagulant medication. The subjects did not suffer from clinical cardiovascular diseases or hypertension and had normal fasting blood glucose concentrations, indicating the absence of (type 2) diabetes. Nine of the subjects were smokers (Table 1). Blood samples were collected before the intervention (baseline) and after 4 wk of fish oil intake. This intervention period was based on an earlier intervention study (10). During the intervention period, all subjects took 9 soft gelatin capsules (Pharma Nord) per day in addition to consuming their normal diet. Each capsule contained 500 mg fish oil (90% free fatty acids with 45% EPA and 25% DHA; Table 2) with a total (n-3) PUFA intake of 3.1 g/d. Data from 3 of 57 subjects were excluded from the analysis of intervention effects because the subjects became ill during the intervention (2 had influenza and 1 had periodontal disease), which was also apparent from high plasma concentrations of C-reactive protein (>10 mg/L).

The fatty acid composition of the capsules was as presented (Table 2). For both studies, the subjects were asked to maintain their normal dietary, smoking, and alcohol consumption pattern, but not to take other fish or marine products 4 wk before or during the intervention period. Subjects completed a short dietary questionnaire, in which this was verified. At 2 d prior to baseline blood sampling, dietary food intake records were obtained. These were used to standardize dietary intake prior to the postintervention blood sampling. Food intake was recorded and details on energy intake and macronutrient composition of the diet before both blood samples were compared.

    Hematological measurements and plasma coagulation factor concentrations. After an overnight fast, blood was collected into open tubes with 129 mmol/L trisodium citrate (10% v) or with potassium EDTA. The citrate blood was immediately used to prepare platelet-free plasma (PFP) by centrifugation at 870 x g; 10 min, followed by 2 centrifugation steps at 18,000 x g; 10 min. PFP was snap-frozen in liquid nitrogen, and stored at –80°C for coagulation measurements.

The EDTA-anticoagulated blood was used to determine standard hematological variables. Remaining plasma samples, centrifuged twice at 870 x g; 10 min, were stored at –80°C and later used to perform lipid analysis. Triacylglycerol in PFP was measured as total glycerol content (GPO-trinder 337B; Sigma), from which the free glycerol content (148270; Roche Diagnostics) was subtracted. Total cholesterol and HDL cholesterol were determined with reagents from ABX Diagnostics. Plasma LDL cholesterol was not directly measured, but was calculated by LDL cholesterol = total cholesterol – HDL cholesterol – triacylglycerol/2.2 (all in mmol/L).

In citrate PFP, plasma concentrations of prothrombin and factors V, VII, and X were measured at 2 dilutions using factor-deficient plasmas. Test kits were from Dade Behring and BioMérieux. Concentrations of antithrombin were determined with a test kit from Chromogenix (Mölndal). Fibrinogen activity in plasma (Claus method) and C-reactive protein were determined with reagents from Roche Diagnostics. Factor concentrations were measured using factor-deficient plasma and expressed as percentages of activity compared with a normal plasma pool from 40 healthy subjects (males and females).

    Thrombin generation measurements. Thrombin generation was measured in PFP using the automated thrombogram method (12,17). Briefly, triplicate samples of 80 µL freshly isolated PFP were pipetted into a 96-well plate, each well containing 20 µL recombinant human tissue factor (30 pmol/L; Dade) and 4 µmol/L phospholipids (phosphatidylserine:phosphatidylcholine:phosphatidylethanolamine, 1:3:1) in buffer A (20 mmol/L Hepes, 140 mmol/L NaCl, and 5 g/L bovine serum albumin, pH 7.35). This gave optimal final concentrations of procoagulant phospholipids and tissue factor. Polystyrene well plates (Immulon 2HB, Dynex Technologies) were used with minimal contact activation. The plates were inserted into a Fluoroskan Ascent well-plate reader (Thermo Labsystems), and preheated to 37°C. Coagulation was started by automated addition of 20 µL fluorescent thrombin substrate Z-GGR-AMC (2.5 mmol/L, benzyloxycarbonyl Gly-Gly-Arg 7-amido-4-methyl coumarin; Bachem), suspended in buffer B (20 mmol/L Hepes, 0.1 mol/L CaCl₂ and 60 g/L bovine serum albumin, pH 7.35) by shaking. Fluorescence accumulation was measured, in time, at excitation and emission wavelengths of 390 and 460 nm, respectively (37°C). First-derivative curves showing the accumulation of fluorescence intensity were converted to curves of thrombin nanomolars using a human thrombin calibrator and thrombinoscope software (Synapse) to correct for nonlinearity of the fluorescence with AMC concentration, depletion of fluorescent substrate, and fluorescence accumulation due to noncoagulant ₂-macroglobulin-bound thrombin (17).

Reference samples contained the same volume of plasma, to which a fixed amount of stable thrombin calibrator was added. Thrombograms were run in triplicate and analyzed with respect to the time to thrombin peak (equivalent to the clotting time), the thrombin peak height (indicative of the maximal rate of thrombin formation), and the area under the curve or endogenous thrombin potential (ETP, representing total thrombin activity or the thrombin potential of plasma) (17). The assay variability of the thrombogram parameters was <3% (18).

    Determination of genetic polymorphisms. Leukocyte DNA was isolated from blood buffy coats using the high-pure kit from Roche Diagnostics, according to instructions of the manufacturer. Genetic polymorphisms were determined using PCR primers (Eurogentec) suitable for restriction fragment length polymorphism (RFLP) analysis as described: the T312A polymorphism of fibrinogen- (19), the G20210A (20), and A19911G (21) variants of prothrombin, and the A4070G (R₂ factor V) (22) and G1691A (factor V_Leiden) (23) variants of factor V. PCR was for 30–35 cycles (temperature conditions according to characteristics of the primers) with 50 ng isolated DNA, dNTPs (2 mmol/L of each), 1.5 mmol/L MgCl₂, forward and reverse primers (10 µmol/L), Taq polymerase (5 U/µL) and suitable PCR buffer. Taq DNA polymerase, buffer, and MgCl₂ were obtained from Amersham Pharmacia Biotech. Restriction enzymes with buffers were from the following sources: Mnl I from New England Biolabs; Rsa I from Promega; Hind III and dNTPs from Fermentas. Generated PCR fragments and endonuclease digestion products were controlled for purity and fragment size by electrophoresis on ethidium bromide-stained agarose gels.

    Statistics. Data are expressed as means ± SEM, except when indicated otherwise. Principal component analysis was performed to determine the relation between plasma lipids and coagulation factors. The contribution of individual (coagulation) factors to the process of thrombin generation was evaluated with multivariate regression analysis. Within groups, effects of fish or corn oil on plasma variables were compared with baseline values with the paired t test or, if parameters were not normally distributed, the nonparametric Wilcoxon Signed Rank test. Baseline values of different groups were compared with one another with the Mann-Whitney U test. The Pearson test was used to determine whether baseline concentrations and fish oil effects were correlated for individual variables. Statistical significance was set at P < 0.05. All calculations were performed using the statistical package for the social sciences 12.0 (SPSS).

	Results

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
    Relation between plasma concentrations of (anti)coagulant factors and lipids and thrombin generation at baseline. In study 1, body weight was 92.1 ± 10.9 kg and BMI was 28.7 ± 2.6 kg/m² (mean ± SD). Plasma concentrations of triacylglycerol, lipoproteins, and coagulation factors were all in the normal range (24,25) (Table 1). Principle component analysis was performed for all 57 subjects' baseline plasma samples to determine the relation between lipids and coagulation factors. This analysis showed a strong association among the concentrations of triacylglycerol, total cholesterol, prothrombin, factor VII, and factor X (Fig. 1A, component 1, 29.6%). Variables that clustered differently were fibrinogen/factor V (component 2, 18.7%) and antithrombin/HDL (component 3, 16.0%). Subsequent correlation analysis indicated that concentrations of triacylglycerol correlated with those of prothrombin (r = 0.33, P = 0.011) and factor X (r = 0.42, P = 0.001) and tended to correlate with factor VII (r = 0.25, P = 0.066). Calculated concentrations of LDL cholesterol correlated only with fibrinogen.

View larger version (18K): [in this window] [in a new window]   Figure 1  Principal component analysis of relations between baseline concentrations of lipids and coagulation factors in plasma from 57 overweight healthy men (study 1) (A) and 42 overweight type 2 diabetes patients (study 2) (B). In the component matrix, variables with values closer to 1.0 are contributing more to the clustered component. AT, antithrombin; F, factor; Fbg, fibrinogen; HDL, HDL cholesterol; TC, total cholesterol; TG, triacylglycerol. LDL cholesterol was not included in the analysis, because this concentration was calculated from other variables.

Tissue factor–triggered thrombin generation was used to measure the integrated coagulant activity of the plasmas. Plasma samples from all overweight subjects and diabetic patients had normal thrombin generation curves (Table 1). In both studies, multiple regression analysis indicated that plasma concentrations of triacylglycerol, total cholesterol, fibrinogen, prothrombin, factor V, factor VII, and factor X determined the thrombin peak and/or ETP values, whereas the anticoagulant factor antithrombin correlated inversely with the ETP. The variables of component 1 (triacylglycerol, total cholesterol, prothrombin, factor VII, and factor X) predicted 32.9 and 42.7% of the ETP variation in overweight subjects and diabetic patients, respectively. Prothrombin and antithrombin were the main determinants of the ETP in the overweight subjects (r = 0.53, P < 0.001) and diabetics (r = 0.68, P < 0.001). In combination, the concentrations of triacylglycerol and total cholesterol correlated with the ETP in overweight (r = 0.40, P = 0.009) and diabetic (r = 0.42, P = 0.025) subjects. Only triacylglycerol contributed to the variation in study 1 (P = 0.019). Together, these results indicate that in both studies, plasma triacylglycerol and total cholesterol concentrations are associated with total thrombin generation, i.e., the integrated coagulant activity of plasma.

    Intervention studies with fish oil. Dietary questionnaires showed that none of the subjects had consumed fish (oil) during 4 wk prior to the start of the intervention. Accordingly, fish oil carry over effects were minimal. Furthermore, the habitual consumption pattern did not change prior to and during the intervention periods. Energy intake and macronutrient composition of the regular diet was similar before both blood samples. The recorded mean energy intake was 120.4 ± 4.5 kJ/kg body mass, with 35.7 ± 0.8% of energy as carbohydrates, 43.9 ± 1.0% of energy as fat, 17.5 ± 0.4% of energy as protein, and 2.4 ± 0.4% of energy as alcohol.

    Effects of fish oil on thrombin generation. In study 1, plasma concentrations of triacylglycerol were moderately reduced, whereas coagulation factors were not affected (Table 3). Overall, the fish oil intervention resulted in a prolonged time to thrombin peak. For the whole population of subjects (for subpopulations, see below), these results point to a slightly weaker anticoagulant effect than in an earlier fish oil intervention study (10).

    Increased anticoagulant effect of fish oil in subjects with high plasma triacylglycerol concentrations. For the overweight subjects of study 1, correlation analysis showed that the fish oil effect on plasma triacylglycerol strongly correlated with the baseline triacylglycerol concentration (r = –0.45, P = 0.001) as well as with the fish oil effect on factor VII (r = 0.28, P = 0.042) and factor X (r = 0.25, P = 0.070). Interestingly, such a relation was not found for the type 2 diabetes patients of study 2, whom all had high triacylglycerol at baseline (>1.50 mmol/L) and a consistent lipid-lowering and anticoagulant response, as described above.

Because recent risk assessments indicate that fasting triacylglycerol concentrations of >150 mg/dL (>1.69 mmol/L) are a matter of health concern (26), we used this as a cut-off point to distinguish between subjects with low and high triacylglycerol concentrations. For the overweight subjects of study 1, this resulted in 2 subgroups, one consisting of 39 subjects with low concentrations of triacylglycerol (0.99 ± 0.05 mmol/L) and one consisting of 15 subjects with moderately increased concentrations of triacylglycerol (2.68 ± 0.32 mmol/L). The BMI of the subgroups were 28.6 ± 0.6 and 28.4 ± 0.4 kg/m², respectively. At baseline, plasma concentrations of total and HDL cholesterol did not differ between the subgroups (Fig. 2A). However, the high-triacylglycerol subgroup had also higher concentrations of prothrombin (P = 0.024) and factor X (P = 0.003), and tended to have a higher concentration of factor VII (P = 0.079), but not of factor V and antithrombin (Fig. 2B). Thrombin generation did not differ between the subgroups.

View larger version (22K): [in this window] [in a new window]   Figure 2  Effects of fish oil on plasma lipids, coagulation factors and thrombin generation in 54 overweight healthy subjects with low and high baseline triacylglycerol concentrations (study 1). Fasting triacylglycerol concentrations at baseline were considered to be high at >1.69 mmol/L. Values are means ± SEM. Data presented from subjects with low (n = 39) and subjects with high (n = 15) triacylglycerol concentrations. Baseline concentrations, *P < 0.05 (Mann-Whitney U test) (A, B). Effects of fish oil intervention compared with baseline, *P < 0.05 (paired t test or Wilcoxon signed rank-test) (C, D). Abbreviations: AT, antithrombin; F, factor; Fbg, fibrinogen; HDL, HDL cholesterol; TC, total cholesterol; TG, triacylglycerol; TTP, time-to-peak.

    Increased anticoagulant effect of fish oil in subjects with high thrombin generation or fibrinogen. We also evaluated whether high baseline concentrations of plasma components other than triacylglycerol could determine the fish oil effect on those components. This was the case for the thrombogram (thrombin generation curve) and for the fibrinogen concentration. In the overweight subjects of study 1, thrombin peak height (r = –0.35, P = 0.010) and ETP (r = –0.36, P = 0.007) negatively correlated with the fish oil effect on these variables. Further, in studies 1 and 2 baseline fibrinogen concentrations negatively correlated with the fish oil effect on fibrinogen (r = –0.42, P = 0.001 and r = –0.47, P = 0.037, respectively). In contrast, such a relation between baseline concentration and effect was not present for other coagulation factors (prothrombin, factor V, factor VII, factor X, and antithrombin). This is of interest because it suggests that the anticoagulant effect of fish oil is typically highest in individuals who not only have high triacylglycerol or fibrinogen concentrations but also high coagulant activity.

    Genetic variation and effects of fish oil. We have found that healthy carriers of the fibrinogen- chain 312Ala variant had relatively high fibrinogen concentrations and a strong reduction in thrombin generation in response to fish oil (10). For the overweight subjects of study 1, we determined a number of coagulation factor polymorphisms that have been associated with increased thrombotic risk, i.e., the fibrinogen -chain T312A polymorphism; the G20210A and A19911G polymorphisms of prothrombin; and the A4070G (HR2 haplotype) and G1691A (factor V_Leiden) polymorphisms of factor V. At baseline, data analysis confirmed the known linkage of G20210A carriers with a tendency to increased prothrombin concentrations (n = 3, P = 0.138) and a linkage of A4070G carriers with decreased factor V concentrations (n = 8, P = 0.019) (Table 4). Differences in fish oil effects between carriers and noncarriers were only observed for the fibrinogen- 312Ala carriers, which had a borderline decrease in thrombin peak concentration (P = 0.082) and for the factor V A4070G carriers, which tended to decrease factor V concentrations (P = 0.058). However, correlation analysis indicated that the genetic variation was only a minor determinant of the subject-dependent fish-oil effects compared with the variation in triacylglycerol concentrations.

	Discussion

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

In the second study, almost all diabetic patients had high triacylglycerol concentrations (>1.50 mmol/L) at baseline. Intake of fish oil, but not of corn oil [containing (n-6) PUFA], reduced plasma triacylglycerol concentration, accompanied by a decrease in concentrations of prothrombin, factor V, factor VII, and factor X, as well as by a potent reduction in thrombin generation. These findings likely are of clinical relevance because type 2 diabetes patients usually have an atherogenic lipoprotein profile with high triacylglycerol and LDL cholesterol and show an increased risk of thrombosis (28,29). It has been established that diabetic patients respond to fish oil by a partial correction of dyslipidemia (30,31). We now provide evidence that the triacylglycerol-lowering effect in these patients is associated with an anticoagulant effect.

It was proposed that dietary fish oil may interfere with the production of vitamin K-dependent coagulation factors (32). Earlier, this hypothesis was challenged in animal studies, indicating that fish oil also lowers the vitamin K-independent factor V and, further, that the anticoagulant effect of fish oil is not sensitive to vitamin K depletion (33,34). In the present human intervention studies, we again found a reduction in factor V by fish oil, particularly in subjects with high triacylglycerol concentrations. Despite this, factor V was not associated with the concentrations of vitamin K-dependent factors (prothrombin, factor VII, and factor X) at baseline, suggesting that its steady-state concentration in plasma is regulated separately. The physiological relevance of this becomes apparent from the clear correlation between the extent of thrombin generation (peak height and ETP) at baseline and the fish oil–provoked reduction in thrombin generation.

Other variables also appear to contribute to the subject-dependent response to fish oil. As we and others have observed before (10,35,36), baseline fibrinogen concentrations were correlated with the effect of fish oil on this plasma variable. Compared with these likely determinants, statistical analysis indicates that genetic polymorphisms of prothrombin or factor V do not play an important role in the fish oil response, whereas the fibrinogen- T312A polymorphism is of secondary importance. Together, this suggests that subjects with high concentrations of triacylglycerol, coagulation factors, coagulation activity, and fibrinogen, and thus with increased risk of thrombosis, would benefit most from fish oil intake.

Hyperlipidemic patients have elevated concentrations of factor VII (13,15,37), and there is also evidence that these patients have elevated concentrations of prothrombin and factor X (38). This compares well with the present key observation that subjects with high triacylglycerol concentrations, both healthy subjects and patients, responded better to fish oil by a greater decrease in triacylglycerol, factor VII, and thrombin generation than those with lower triacylglycerol concentrations. Small changes in prothrombin are likely to contribute to this effect, as multivariate analysis showed that triacylglycerol, together with prothrombin (and antithrombin), contributes to the variation in thrombin generation. Taken together, we conclude that the plasma triacylglycerol concentration is predictive of the (pro)coagulant state of plasma, likely by reflecting the overall activity of several coagulation factors. In fact, the present data suggest that measurement of overall thrombin generation (thrombin potential), or the triacylglycerol concentration as predictive value, gives a better indication of the (pro)coagulant state of plasma than measurement of individual hemostatic factors. High triacylglycerol concentrations also seem to predict the effect of dietary fish oil on plasma (pro)coagulant activity. Although these conclusions come from data obtained with 96 subjects, we note that larger epidemiological analyses are needed for confirmation. The intervention studies were carried out with relatively large amounts of (n-3) PUFA (3.1 g/d), which is equivalent to a daily intake of 100–200 g fatty fish.

The present results indicate that the coagulation-lowering effect of fish oil is part of a broader mechanism that is not confined to coagulation factors alone, but also involves regulation of plasma lipids. One of the first reports to suggest a link between hypertriglyceridemia and hypercoagulability is the Northwick Park Heart Study, showing an overall correlation between plasma triglycerides and factor VII activity (39). In the present study of 99 subjects, the principal component analysis indicated that fasting triacylglycerol clustered with the steady-state concentrations of a number of coagulation factors, namely, prothrombin, factor VII, and factor X. Covariance analysis confirmed that triacylglycerol correlated with these coagulation factors. This is likely to point to a common regulation by the liver, insofar as the majority of triacylglycerol in fasting plasma comes from VLDL particles produced by the liver, which organ is also the source of most coagulation factors. The hepatic regulation of triacylglycerol in VLDL by fish oil is still a matter of debate. Mechanisms proposed include downregulation of gene expression in the liver by the (n-3) PUFA delivered via chylomicron remnants (40); suppression of VLDL apo B production (41,42), or stimulation of apo B degradation (43); increased clearance of chylomicrons (44,45); and higher conversion of VLDL into LDL in the peripheral tissues (46,47). The present data argue for a common hepatic effect of (n-3) PUFAs, e.g., jointly controlling the production and/or secretion of VLDL and vitamin K (in)dependent coagulation factors.

	ACKNOWLEDGMENTS

 
We acknowledge Dr. S. Loman (Pharma Nord, Vejle, Denmark) for the kind supply of capsules for intervention studies. We thank Dr. P. Marckmann (Esbjerg, Denmark) for his help in the intervention studies and collection of patient data and samples.

	FOOTNOTES

 
¹ This work was supported by the Netherlands Organization for Scientific Research (NWO 980-10-018).

⁶ Abbreviations used: DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; ETP, endogenous thrombin potential; PFP, platelet-free plasma.

Manuscript received 26 June 2006. Initial review completed 30 July 2006. Revision accepted 16 October 2006.

	LITERATURE CITED

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 

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