Mediterranean Diet improves thrombosis biomarkers in high cardiovascular risk individuals: a randomized controlled trial

Following a Traditional Mediterranean diet (TMD) decreases cardiovascular risk. However, its anti-thrombotic effects are unexplored. We aimed to assess whether this diet improved platelet, coagulation, and fibrinolysis-related biomarkers in cardiovascular risk subjects. In 358 random PREDIMED Study volunteers (Prevencion con Dieta Mediterranea), we assessed: 1) 1-year effects on thrombosis of TMD, enriched in virgin olive oil (TMD-VOO; N=120) or nuts (TMD-Nuts; N=119), versus a low-fat diet (N=119), and 2) associations between 1-year biomarker changes and increments in Mediterranean Diet adherence and key food group intakes. TMD-Nuts attenuated pro-thrombotic status (P=0.029), whilst TMD-VOO did it in a marginally significant way (P=0.076). TMD-VOO decreased HDL-bound alpha1-antitrypsin levels (HDL pro-inflammatory/pro-thrombotic properties, P=0.034) and increased HDL platelet activating factor-acetylhydrolase activity (P=0.024). TMD-Nuts diminished non-esterified fatty acid levels (P=0.034). Only the low-fat diet was associated with increased platelet factor-4 concentrations (P=0.012) and thrombin activation (higher prothrombin factor1+2 levels, P=0.003) versus baseline. 2-point TMD adherence increments were linked to 10.2 mg/dL fibrinogen decrements (P=0.011). 1-serving increments in vegetable intake were associated with platelet factor-4 concentration decreases (-9.21%, P=0.031). 1-portion increments in fruit and fatty fish consumption were linked to thrombin generation decrements (-7.72% -P=0.007- and -13.2% -P=0.046-, respectively). Increases in antithrombin levels were related to 1-serving increments in nut intake (+2.34%, P=0.033) and reductions in processed meat consumption (+2.00%, P=0.018). Finally, 1-portion decreases in processed meat intake were associated with reduced plasminogen activator inhibitor-1 levels (-10.4%, P=0.008). Summarizing, TMD and its typical food items were associated with improved thrombosis biomarkers in high cardiovascular risk individuals.

Finally, 1-portion decreases in processed meat intake were associated with reduced plasminogen activator inhibitor-1 levels (-10.4%, P=0.008). Summarizing, TMD and its typical food items were associated with improved thrombosis biomarkers in high cardiovascular risk individuals. All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/19001909 doi: medRxiv preprint

INTRODUCTION
Following a traditional Mediterranean Diet (TMD) prevents the development of cardiovascular outcomes 1,2 . The PREDIMED Study (Prevención con Dieta Mediterránea) was the largest intervention study performed so far to assess the effects of this dietary pattern on the primary prevention of major cardiovascular outcomes 1 . In its context, our group and others have reported that TMD effects on cardiovascular protection may be mediated by improvements in risk factors related to blood pressure, glucose metabolism, oxidative stress, and low-grade inflammation 3 . However, very scarce evidence related to TMD effects on other mechanisms such as thrombosis is available.
Thrombosis consists of the formation of a clot to avoid blood loss in case of a vessel disruption, whose excessive activation is also linked to an increased risk of atherosclerotic outcomes 4 . Thrombus generation is based on the occurrence of four phenomena (apparition of an endothelial lesion, platelet activation and aggregation, activation of the coagulation cascade, and fibrinolysis) which, in turn, are constituted by a chain of intertwined biochemical responses 4 . Excessive levels of biomarkers related to these responses (or defective, in the case of fibrinolysis) are linked to an increased risk of suffering atherosclerotic events. This association has been reported for plasma biomarkers of endothelial damage (von Willebrand factor 5 ), platelet aggregation (P-selectin 6 , platelet factor-4 7 ), activation of coagulation (fibrinogen 8 , thrombin formation -directly proportional to the levels of prothrombin fragment 1+2-9 , antithrombin 10 ), and fibrinolysis (plasminogen activator inhibitor-1 -PAI1-11 , D-dimer 12 ). Other substances (such as non-esterified fatty acids and nitrite/nitrate levels) have also been shown to be involved in the development of cardiovascular diseases through their capacity to modulate endothelial stress and platelet activation 13,14 . Finally, novel functional biomarkers such as the antithrombotic capacity of high-density lipoproteins (HDLs) (based on the interrelation between low-grade All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/19001909 doi: medRxiv preprint 5 inflammation -in which HDLs are involved-and thrombosis 8 and the activity of HDL-bound enzymes to hydrolyze pro-thrombotic signals such as the platelet activating factor 15 -the platelet activating factor acetylhydrolase, PAF-AH-) could also contribute to protecting against cardiovascular disease.
Although their link with atherosclerotic diseases is consistent, very few interventions with healthy dietary patterns have been associated with changes in thrombosis biomarkers.
Following a TMD has only been associated with a decrease in P-selectin levels in high cardiovascular risk subjects in the context of the PREDIMED Trial 16 . Beyond our study, a cross-sectional study reported an inverse association between adherence to a Mediterranean-like diet and platelet levels 17 . Other small-scale prospective analysis with 21 young, healthy male volunteers assessed the impact of a TMD-like diet for 90 days on this process, and reported that this dietary pattern was associated with lower fibrinogen levels and a moderation of the coagulation response 18 . However, no extensive analysis of the long-term TMD effects on thrombosis mechanisms has been performed to date in a large-scale intervention trial.
Our main aim was to assess whether a 1-year intervention with TMD improved a comprehensive set of endothelial stress, platelet, coagulation, and fibrinolysis-related biomarkers in high cardiovascular risk subjects. Our secondary aims were to determine whether there were associations between 1-year changes in thrombosis biomarkers and: 1) increments in Mediterranean Diet adherence score values (dose-response compliance analysis), and 2) differences in the consumption of key food group whose intake was modified in the TMD intervention (virgin olive oil, nuts, fruits, vegetables, legumes, fish, processed meat, and wine).
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Study population
Study subjects belonged to the PREDIMED Study (Prevención con Dieta Mediterránea). It was a large-scale, parallel, multicenter, randomized controlled trial assessing the longterm effects of following a TMD on the prevention of primary cardiovascular disease in a population at high cardiovascular risk 1 . Volunteers were randomly assigned to follow one of the three following intervention groups (in a 1:1:1 ratio): 1) a TMD supplemented with virgin olive oil (TMD-VOO); 2) a TMD supplemented with mixed nuts (TMD-Nuts); and 3) a low-fat control diet. Further details of inclusion and exclusion criteria and the dietary intervention are available in Supplemental Methods.
We selected a random subsample of 358 subjects (4.8% of the total PREDIMED population, belonging to Hospital Clinic and IMIM study sites, with biological samples at baseline and after 1 year of dietary intervention) for the present analyses. 120 volunteers were allocated to the TMD-VOO intervention group, 119 to the TMD-Nuts one, and 119 to the low-fat diet. In these individuals, age, sex and educational level data were gathered at baseline. At baseline and after 1 year of intervention, trained clinical personnel collected the following information: 1) body mass index, waist circumference, blood pressure, smoking, and medication use; 2) physical activity levels (using a validated Minnesota Leisure-Time Physical Activity Questionnaire; 3) adherence to a Mediterranean diet (the 14-item Mediterranean Diet adherence score 19 , used as a continuous variable); and 4) intake of 137 food items in the previous year (using a validated food frequency questionnaire). From the information in the food frequency questionnaires, we calculated the intakes of key TMD food groups (Supplemental Methods). In addition, trained nurses collected plasma and serum samples, and stored at -80ºC upon use and to quantify the All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/19001909 doi: medRxiv preprint 7 levels of fasting glucose, total and HDL cholesterol, and triglycerides by automatized methods 1,20 .
All participants provided written informed consent before joining the study. The trial protocol was approved by local institutional ethic committees, registered under the International Standard Randomized Controlled Trial Number ISRCTN35739639 (http://www.isrctn.com/ISRCTN35739639), and thoroughly described in previous publications 1,20 .

Thrombosis biomarkers
We determined thrombosis biomarkers in biological samples collected at baseline and after 1 year of intervention. We used ELISA kits to quantify the levels of: fibrinogen author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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Sample size
Since the only thrombosis-related parameter assessed in the PREDIMED Study was Pselectin, we assumed that the variability of this parameter would be similar to that in our variables. Thus, a sample size of 119 individuals per group (total sample size: N=357) allowed ≥80% power to detect a 10% change relative to the mean baseline value of Pselectin levels (7.65 ng/mL) between post-and pre-intervention values, and a 14% change relative to the mean baseline value (10.8 ng/mL) in the differences among the three diets, considering a 2-sided type I error of 0.05, a loss rate of 5%, and the standard deviation of the differences in P-selectin values in this intervention (SD=28.9 ng/mL) 16 .

Statistical analyses
We first checked the distribution of continuous variables in normality plots and by the Shapiro-Wilk test. We assessed whether there were differences in baseline values among the three intervention groups with one-way ANOVA tests for normally distributed continuous variables, Kruskal-Wallis tests for non-normally distributed continuous ones, and chi-squared tests (or exact Fisher tests when expected frequencies were <5) for categorical parameters.
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author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/19001909 doi: medRxiv preprint We first calculated a continuous score reflecting the general activation of the thrombosis mechanisms at baseline and after 1-year of intervention. In biomarkers where high concentrations were associated with a more pro-thrombotic state (von Willebrand factor, platelet factor-4, fibrinogen, prothrombin fragment 1+2, D-dimer, PAI-1, non-esterified fatty acids, and α1-antitrypsin in HDL), we assigned values from 1 to 5 to the volunteers located in increasing quintiles. On the contrary, in biomarkers where high levels were linked to a less pro-thrombotic state (antithrombin, nitrites+nitrates, and PAF-AH activity in HDL), we assigned values from 5 to 1 to the volunteers located in increasing quintiles. By summing these scores, we obtained a general pro-thrombotic score ranging from 11 (all biomarkers located in the least pro-thrombotic quintile) to 55 (all biomarkers located in the most prothrombotic quintile), in which higher scores indicated a higher pro-thrombotic state.
We analyzed differences between pre-and post-intervention values in every intervention by paired t tests in normally distributed variables and Wilcoxon signed rank tests in nonnormally distributed parameters. We checked whether there were differences in the 1-year changes in thrombosis biomarkers (calculated by subtracting baseline to post-intervention values) among the three intervention groups by multivariate linear regression analyses.
We assessed whether there were relationships between 1-year changes by Pearson's correlation tests. Finally, we studied whether there were associations between 1-year changes in the Mediterranean diet adherence score or the intake of key food groups (virgin olive oil; mixed nuts; fruits; vegetables; legumes; fish; processed meat; eggs; and wine) and the 1-year changes in thrombosis biomarkers by additional multivariate linear regression analyses. Associations with 1-year increments in the intake of food servings were performed with percentage changes, calculated as follows: (1-year valuebaseline value)/baseline value x100). All regression models were adjusted for: age, sex, study site, educational level, baseline value of the variable, fasting glucose levels, use of antidiabetic drugs (yes/no), cholesterol concentrations, use of cholesterol-lowering drugs (yes/no), All rights reserved. No reuse allowed without permission.
author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/19001909 doi: medRxiv preprint triglyceride values, use of triglyceride-lowering drugs (yes/no), systolic blood pressure, use of antihypertensive drugs (yes/no), use of antithrombotic drugs (yes/no), tobacco use (never/former/actual smoker), body mass index, waist circumference, leisure-time physical activity, and two propensity scores to correct for the theoretical deviations in the randomization process (calculated from 30 baseline variables) 1 . Models were plotted using the "lme" package in R Software 22 .
We considered any two-sided P-value<0.05 as significant and performed statistical analyses with R software version 3.5.0 23 .

Data sharing statement
Considering that we do not have the explicit written consent of the study volunteers to yield their deidentified data at the conclusion of the study, individual participant data cannot be shared. The study protocol is available in the main publication of the study 1 and in the study website (http://www.predimed.es/uploads/8/0/5/1/8051451/_1estr_protocol_olf.pdf), and a summary of the dietary intervention is also available in Supplemental Methods.

Study population
We found no significant differences in clinical variables at baseline among the individuals assigned to the three intervention groups ( Table 1). Regarding the total PREDIMED Study population, there were 5.9% more women and 7% more antithrombotic drug users among our volunteers and they presented higher glucose (6 mg/dL), total cholesterol (4 mg/dL), and leisure-time physical activity levels (22 METs·min/d) (P<0.05) (Supplemental Table   1). Physical activity did not change throughout the study (P>0.05), and volunteers' All rights reserved. No reuse allowed without permission.
author/funder, who has granted medRxiv a license to display the preprint in perpetuity. year of intervention in the TMD-VOO and TMD-Nuts interventions, respectively; P<0.001 relative to baseline and the control intervention). TMD adherence increased in the TMD-VOO group due to increments in the intake of virgin olive oil, vegetables, legumes, total and fatty fish, white meat, and wine, and decrements in the consumption of red and processed meat (virgin olive oil, vegetable, legume, total and fatty fish intake changes were significant relative to the low-fat diet, P<0.05). In the TMD-Nuts group, TMD adherence increased because of increments in the consumption of nuts, virgin olive oil, fruits, legumes, and fatty fish and decreases in the intake of red and processed meats (nut, fatty fish, and processed meat consumption changes were significant relative to the control diet, P<0.05). Finally, low-fat diet compliance was observed as a reduction in the consumption of fats, based on a decrease of the intake of mixed nuts and the substitution of red for white meat (red meat consumption decreased while meat did not) and the substitution of fatty for non-fatty fish (total fish intake increased while fatty fish did not) (P<0.05) (Supplemental Table 2).

TMD interventions and thrombosis biomarkers
Overall pro-thrombotic status (measured as the thrombosis score) was attenuated by the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
Changes in HDL thrombosis-related properties were correlated as well, since increments All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

DISCUSSION
Our results show that 1 year of intervention with TMD improves pro-thrombotic state in high cardiovascular risk individuals. To the best of our knowledge, this trial is the largest (N=358) and more comprehensive study on the effects of a healthy dietary pattern on the molecular mechanisms related to thrombosis.
Thrombosis, and particularly platelet and coagulation activation, seems to be deeply affected by inflammation and oxidative stress 24,25 . TMD is a lifestyle intervention known to improve both risk factors 3 , and our data indicate that following this dietary pattern was also associated with an improvement in the overall pro-thrombotic response of high cardiovascular risk subjects. Several data from our study and others reinforce the hypothesis that TMD effects of thrombosis could be due to its capacity to moderate oxidative stress and low-grade inflammation. First, TMD adherence increments were particularly associated with fibrinogen decreases in our results. Fibrinogen is a pivotal molecule connecting hemostasis and inflammatory responses 26 and this fact emphasizes the possibility of an interlinked anti-inflammatory/antithrombotic TMD capacity. Second, TMD is a dietary pattern based in high intakes of food groups and nutrients able to decrease oxidative stress and low-grade inflammation responses (antioxidants 27 , and polyunsaturated 28 and fiber-derived short-chain fatty acids 29 ). Our results indicate that All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/19001909 doi: medRxiv preprint 14 increments in the consumption of some of the previous food groups (fruits, vegetables, nuts, and fatty fish) were linked to better levels of platelet factor-4, thrombin activation, and antithrombin (reflecting a lower activation of platelet function and the coagulation cascade). Finally, we have also observed that the decreases in inflammation-related biomarkers were also related to decrements in pro-thrombotic responses in our data.
HDLs have been shown to be able to induce beneficial effects on some hemostatic mechanisms 30 . In this regard, the TMD-VOO intervention led to a particular decrease in α1antitrypsin levels and an increase in PAF-AH activity in HDL particles, two potential HDL antithrombotic properties. It is not the first time that we report that the TMD-VOO intervention promotes HDL functionality in the PREDIMED Study 21 . However, our data extend this protection to anti-inflammatory and anti-thrombotic HDL functions (also interconnected in our results). Antioxidant protection on HDL components of typical TMD food items could contribute to explaining an improved activity of the lipoprotein protective enzymes (due to a greater preservation of their non-oxidized structure) 31 . In addition, the capacity of this dietary pattern to decrease the levels of inflammation biomarkers in circulation may be related to a decrement in the concentrations of pro-inflammatory molecules bound to HDL particles 32 .
We observed a decrease in non-esterified fatty acid levels after 1 year of TMD-Nuts intervention. Non-esterified fatty acids promote endothelial stress and platelet aggregation and, therefore, are considered an emergent pro-thrombotic risk factor in the cardiovascular risk states in which their levels are high (e.g., obesity, hypertriglyceridemia) 13 . The capacity of polyunsaturated fatty acids (whose intake was particularly increased in this group) to bind to GPR120 receptors in adipose tissue in order to decrease lipolysis 33,34 could be an explanation for this effect. In addition, TMD is a fiber-rich dietary pattern, whose intestinal metabolism leads to blood peaks of short-chain fatty acids (known to be able to bind other GPR family receptors with a similar anti-lipolytic effect 35 ). Thus, a synergistic effect All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/19001909 doi: medRxiv preprint 15 between these two nutrients could also be expected in order to explain this particular decrease in the levels of these fatty acids in the TMD-Nuts intervention group.
Increments in processed meat consumption have been associated in our data with several deteriorations in the coagulation cascade (decreased antithrombin levels), fibrinolysis (PAI-1 concentrations), and other thrombosis biomarkers (nitrite + nitrate levels, PAF-AH activity in HDLs). These changes are plausible in the context of the cardiovascular risk associated with their intake. Consumption of processed meats been consistently associated with augmented incidences of atherosclerotic 36 and thromboembolic outcomes 37 . The pro-inflammatory state induced by processed meat components could contribute to explaining a possible pro-thrombotic response. Some of them (e.g., smoke flavors) may promote pro-inflammatory responses through the activation of aryl hydrocarbon receptor-mediated pathways 38 . In parallel, other substrates originated due to their digestion (hydrogen sulfide, other sulfur compounds, nitrosamines, ammonia, amino acid metabolism residues, etc.) may compromise intestinal integrity, increasing the colon permeability to pro-inflammatory bacterial endotoxins such as lipopolysaccharide 39 .
Our study has several strengths. First, it presents a large sample size (N=358). Second, it is based on a randomized design with an active comparator (low-fat control diet) and a long-term duration (1 year). Last, it is the first analysis of the effects of a dietary pattern on a comprehensive, hypothesis-driven set of thrombosis-related biomarkers, performed after a strict laboratory quality control. However, it also presents limitations. First, our volunteers were elderly people at high cardiovascular risk and, therefore, the extrapolation of our conclusions to other populations is hindered. In addition, we report modest changes after the interventions. However, the PREDIMED trial is based on modest real-life dietary modifications and the control diet is already a well-known healthy dietary pattern (rich in fruits, vegetables, and legumes, and poor in fresh and processed meats). All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/19001909 doi: medRxiv preprint In conclusion, 1 year of intervention with TMD improves pro-thrombotic state in high cardiovascular risk individuals. The TMD-VOO intervention improved HDL antithrombotic properties, the TMD-Nuts one enhanced the levels of non-esterified fatty acids, both TMD groups were not related to increments in the levels of platelet factor-4 and prothrombin activation (whilst the low-fat diet was), and increases in TMD adherences were dosedependently related to decreases in fibrinogen levels. These effects seemed to be particularly based on increments in the intake of fruits, vegetables, nuts, and fatty fish, and decreases in processed meat consumption. As far as we know, this is the largest, most comprehensive, hypothesis-based analysis of the effects of a healthy dietary pattern on thrombosis mechanisms in high cardiovascular risk subjects. Our results support the improvement of thrombosis status after following a TMD. Further studies are needed to confirm whether these improvements mediate the reported cardioprotective benefits of this dietary pattern.
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author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. author/funder, who has granted medRxiv a license to display the preprint in perpetuity. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/19001909 doi: medRxiv preprint  Figure 1. TMD effects on thrombosis biomarkers.
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author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/19001909 doi: medRxiv preprint Figure 2. Associations between changes in food group intakes (1 serving) and 1-year changes in thrombosis biomarkers