Large-scale lipidomics profiling reveals characteristic lipid signatures associated with an increased cardiovascular risk

Background and aims Patients with cardiovascular disease (CVD) are at high risk to develop adverse events. The distinct risk of developing adverse cardiovascular (CV) events is not solely explained by traditional risk factors. Platelets are essentially involved in progression of CVD including coronary artery disease (CAD) and platelet hyperreactivity leads to development of adverse CV events. Alterations in the platelet lipidome lead to platelet hyperresponsiveness and thus might alter the individual risk profile. In this study, we investigate the platelet lipidome of CAD patients by untargeted lipidomics and elucidate alterations in the lipid composition of patients with adverse CV events. Methods We characterized the platelet lipidome in a large consecutive CAD cohort (n = 1057) by an untargeted lipidomics approach using liquid chromatography coupled to mass spectrometry. Results The platelet lipidome in this study identified 767 lipids and characteristic changes occurred in patients with adverse CV events. The most prominent upregulated lipids in patients with cardiovascular events primarily belong to the class of phospholipids and fatty acyls. Further, upregulated platelet lipids are associated with an increased cardiovascular or bleeding risk and independently associated with adverse events. In addition, alterations of the platelet lipidome are associated with modulation of in vitro platelet functions. Conclusions Our results reveal that the composition of the platelet lipidome is altered in CVD patients with an increased cardiovascular risk and distinct platelet lipids may indicate adverse events. Results of this study may contribute to improved risk discrimination and classification for cardiovascular events in patients with CVD. Graphical abstract Main findings of this study and hypothetical impact of altered platelet lipid signatures in patients with adverse cardiovascular events on platelet function and clinical outcome. LPE lysophosphatidylethanolamines, CAR acylcarnitines, FA fatty acids.


Introduction
Platelets play a critical role in haemostasis and atherothrombosis [1].Platelet activation and hyperreactivity is associated with poor clinical outcome in patients with symptomatic coronary artery disease (CAD) [2][3][4].At site of a vulnerable plaque, platelet adhesion and aggregation trigger intracoronary thrombus formation, leading to myocardial ischaemia and acute coronary syndrome (ACS) [5].Beyond their critical role in thrombosis, platelets have been recognized to govern mechanisms of thrombo-inflammation and immuno-thrombosis, mechanisms involved in progression of CAD [6][7][8].A sustained hyperreactivity of circulating platelets is associated with adverse thrombo-ischaemic events in both chronic (CCS) and acute coronary syndrome (ACS) [9].
Dyslipoproteinaemia is a major cardiovascular risk factor that determines the clinical course of CAD [10,11].We and others found that enhanced levels of lipoproteins, such as low-density lipoprotein (LDL) promote platelet activation and thrombo-inflammation [12][13][14].Increased uptake of oxidized LDL enhances platelet activation and substantially modulates the platelet lipidome [12].Further, significant alterations of the platelet lipidome occur in patients with ACS, which may contribute to the pathophysiology of the disease [15,16].The assessment of distinct molecular lipid signatures in patients with CAD reflects therapeutic effects of lipid-lowering treatment and outperforms conventional measurements of lipoproteins [17].
In this study, we performed platelet lipidome profiling with mass spectrometry in a large-scale (n > 1000) prospective cohort study enrolling patients with symptomatic CAD and analysed the association of 767 distinct lipid species with platelet function and cardiovascular risk over 3 years after study enrolment.

Study population
One-thousand and fifty-seven (1057) patients were enrolled in this prospective, consecutive study (Table 1).All patients underwent catheter angiography within 24 h after hospital admission and were treated for symptomatic CAD.Blood sampling was performed according to a standardized protocol from peripheral venipuncture with patients fasting overnight for 12 h.Platelet isolation and preparation for mass spectrometry analysis was performed as previously described [15,16].
All patients completed a standardized questionnaire on smoking status, previous dietary factors and medication history.Further, medication on admission was assessed and

Platelet lipidomics
Preparation of platelets and analysis by mass spectrometry was performed as previously described [15] and summarized in the supplementary methods section.

Platelet function analysis
Analysis of platelet function was performed using whole blood impedance aggregometry (Multiplate) after stimulation with collagen, arachidonic acid, and ADP as previously described [9].

Statistical analysis
Patients baseline characteristics data and pre-processed lipidomics data were analysed using JMP ® Version 16.

Platelet lipidome profiling in CAD patients by untargeted liquid chromatography-mass spectrometry
Previously, we characterized changes of the platelet lipidome in patients with coronary artery disease (CAD) and showed that significant alterations occur in patients with acute coronary syndrome (ACS) when compared to chronic coronary syndrome (CCS) or healthy controls [12,15].In the present study, we prospectively analysed the platelet lipidome in a large cohort of patients with symptomatic CAD utilizing an untargeted UHPLC-ESI-QTOF-MS/MS approach to elucidate associations of distinct lipids with an increased cardiovascular risk.Baseline demographic, clinical and laboratory characteristics of the 1057 patients are shown in Table 1.Over a median follow-up period of 36 months of 1057 patients (2987 person-years), 72 individuals (6.8%) experienced a major cardiovascular (cardiac death, ischaemic stroke, myocardial infarction) or bleeding event corresponding to an incidence rate of 24.1 (95% CI 18.9-30.4)per 1000 person-years (Table 2).
To elucidate characteristic changes in the platelet lipidome associated with cardiovascular events, we compared the mean of relative concentrations of distinct lipids and their referring lipid subclasses between patients with and without adverse cardiovascular events during the followup period.We found that 18 lipids were significantly (p < 0.05, FDR < 5%) upregulated in patients with adverse cardiovascular events compared to patients without events (Fig. 2A).Significantly upregulated lipids mostly comprised lipids belonging to the category of glycerophos (Fig. 2A).Interestingly, LPE concentrations were significantly downregulated in patients with statin treatment in contrast to statin-naïve patients (Supplementary Fig. S7).
Metabolic pathway analysis of altered lipids revealed enrichment of fatty acyl and phospholipids metabolism in patients with adverse CV events when compared to patients without events during the follow-up (Supplementary Fig. S5).
To evaluate, whether critical changes in the total lipid composition of the platelet lipidome occur between patients with recurring adverse events and patients without CVD events, we performed principal component analysis (PCA) including all 767 detected lipids of this study.Clustering of patients enrolled into the 3-year follow-up showed a distinct separation of patients with adverse cardiovascular and bleeding events when compared to patients free from recurring adverse events (Fig. 3).Supervised orthogonal partial least square discriminant analysis (OPLS-DA) revealed a clear separation of patients with ischaemic events in contrast to bleeding events (Supplementary Fig. S1).Further, a distinct separation of patients with ischaemic or bleeding Fig. 2 Characteristic changes in the platelet lipidome occur in patients with adverse cardiovascular events.A Volcano plot of all identified platelet lipids in patients with CAD.The X-axis (fold change, FC) displays the base 2 logarithm of the concentration ratios between patients with cardiovascular events including cardiac death, myocardial infarction or stroke (ischaemic endpoint) to those without adverse events during the 3-year follow-up (ischaemic endpoint n = 51, no endpoint n = 1006).Values > 1 indicate upregulation in patients with cardiovascular events; < 1 downregulation compared to patients without incident CVD.Y-axis showing negative log10-transformed p values (BH p < 0.05).Significantly upregulated lipids with a > 1.5-fold change are labelled and mainly comprised lysophosphatidylethanolamines and lysophosphatidylserines. B Volcano plot of all identified platelet lipids comparing patients with major bleeding events (n = 21) to those without bleeding (n = 1036) during the follow-up period.Significantly (BH p < 0.05) upregulated lipids with a > 1.5-fold increase in patients with a major bleeding are displayed and exclusively belong to the category of fatty acyls events in contrast to patients with an event-free follow-up is depicted in Supplementary Figs.S2 and S3.This indicates that the platelet lipidome signature in patients with adverse ischaemic and bleeding events significantly differs from patients without cardiovascular events.Remarkably, concentrations of poly-unsaturated lysoglycerophospholipids (PUFA-LPLs) were increased in patients with recurrent adverse events, whereas glycerolipids and especially triglycerides were decreased by trend in contrast to patients without CVD events (Fig. 4A).Likewise, besides mono-and polyunsaturated fatty acyls, PUFA-PLs were increased in platelets of patients with major bleeding events.Interestingly, physiochemical attributes of distinct lipid classes significantly differed among patients with recurring cardiovascular events when compared to patients without any incident CVD during the follow-up (Fig. 4B).Side chain length (R = 0.760, p < 0.001) and degree of saturation (R = 0.873, p < 0.0001) of LPEs were significantly associated with a higher abundance in patients with recurrent cardiovascular adverse events when compared to patients without incidence of CVD events.Similar trends were observed for TG side chain length (R = 0.320, p = 0.002) and number of double bonds (R = 0.300, p = 0.003).In contrast, an inverse correlation of mean ratios between patients with recurrent cardiovascular events compared to the event-free group was shown for PC side chain length (R = − 0.391, p < 0.0001) and number of double bonds (R = − 0.392, p < 0.001) (Fig. 4C).Likewise, side chain length (R = 0.778, p < 0.0001) and degree of saturation (R = 0.807, p < 0.0001) of LPEs were associated with major bleeding events during the follow-up period.Similar results were achieved for TG side chain length (R = 0.312, p = 0.002) and double bonds (R = 0.292, p = 0.004).In contrast, FA side chain length (R = − 0.622, p = 0.041) and number of double bonds (R = − 0.635, p = 0.036) were inversely associated with major bleeding events (Fig. 4D).
In conclusion, it becomes evident that distinct lipid clusters including LPE and fatty acids might be suitable to predict the cardiovascular risk in patients with CAD.Fig. 4 Physicochemical attributes of platelet lipid species vary among patients with adverse cardiovascular events.A Dot plot of identified lipids in patients with symptomatic CAD.Each dot represents a unique lipid species.X-axis represents fatty acyl carbon chain number and y-axis the number of double bonds.Size is scaled by negative Log-transformed p-value of comparison between lipid levels in patients with and without cardiovascular events.Colour continuously represents fold change between patients subgroups.Upregulation of glycerophospholipids mainly with low fatty acyl carbon number (C < 25) and poly-unsaturated side chain (DB ≥ 2) is observed in patients with cardiovascular endpoints compared to those with-out incident CVD.B The right plot depicts upregulation of mainly fatty acyls and glycerophospholipids in patients with major bleeding in contrast to those without bleeding events.C Mean ratio of platelet lipid abundance between patients with cardiovascular events compared to patients without adverse events by fatty acid side chain length and degree of saturation in different lipid classes.Significant (p < 0.05) Spearman correlations (R s ) are shown.Ratio > 1 indicates a higher abundance in patients with adverse cardiovascular events.D Spearman correlations (R s ) of lipid abundance in patients with bleeding events and degree of saturation and side chain length

Extraction of platelet lipid signatures to predict CV risk in patients with CAD
In order to identify new biomarkers of the platelet lipidome in patients with CAD, being eligible to sufficiently determine the risk of adverse cardiovascular events, we applied a penalized model including all lipids.Thus, overfitting of lipids is minimized and thus variable selection and regularization is optimized leading to an enhanced prediction accuracy and interpretability in the setting of omics data and generation of new potential biomarkers.

Risk stratification in CAD patients integrating platelet lipid signatures
In this study, we performed a cross-validated approach of increasing the predictive value to discriminate the risk of adverse events in patients with CAD.Thus, we calculated concordance indices of conventional cardiovascular risk factors belonging to the Framingham risk score to discriminate the 3-year CV risk and added stepwise models including selected lipids of LASSO analysis (Table 3B).The addition of three lipids with highest ß-coefficients PC 34:2; O, LPE 20:3/0:0 and PI 16:0-20:4 to the baseline model (C index Fig. 5 Association of distinct platelet lipid signatures with an increased hazard of developing adverse cardiovascular events.A Forest plot of the estimated regression coefficient (95% CI) from Cox proportional hazard models on the association of significantly upregulated lipids in patients with ischaemic endpoints and the cardiovascular risk.All lipids were found to be independently associated with an increased cardiovascular risk and hazard ratios (HR) were calculated for a 1-SD unit higher lipid concentration and adjusted for age, gender and statin treatment.Thus, especially lysophosphatidylethanolamines might be independently suitable to predict the cardiovascular risk in patients with CVD.B Associations of significantly upregulated platelet lipids in patients with an increased bleeding risk.High levels of acylcarnitines and fatty acids FA18:1 and FA 18:2 might indicate an increased hazard to develop major bleeding events in patients with CVD 0.647, 95% CI 0.612-0.741)including conventional risk measures (including age, gender, diabetes mellitus, smoking status, arterial hypertension, total cholesterol, and HDL cholesterol) increased the C index by 0.006 (C index 0.650, 95% CI 0.623-0.754).However, an even stronger elevation of C index by 0.095 was achieved by adding six lipids including FA 18:2; 2O, TG 16:0-14:1-18:1 and PE O 16-1-18:2 (C index 0.739, 95% CI 0.703-0.816).Ultimately, the total cluster of nine lipids with addition of PI 18:1:20:4, FA18:1 and TG 14:0-16:0-18:1 only showed a marginal augmentation of C index by 0.001 (C index 0.740, 95% CI 0.710-0.820).To further elucidate the weight of adding new lipid biomarkers to established lipids risk measures, we replaced total cholesterol (TC) and high-density lipoprotein (HDL) with platelet lipid models.Here we found that replacing three lipids did not increase predictive value (C index 0.634, 95% CI 0.606-0.731),but six lipids were outperforming established measures by amplifying C index by 0.094 (C index 0.738, 95% CI 0.698-0.810).In addition, replacement of HDL and TC by all selected lipids caused the highest enhancement of C index by 0.097 (C index 0.741, 95% CI 0.695-0.812).Thus, addition of platelet lipid signatures to established cardiovascular risk factors might significantly augment the 3-year risk discrimination in patients with CAD.

Discussion
The major findings of the present study are: (1) changes of the platelet lipidome in patients with coronary artery disease are associated with adverse cardiovascular events; (2) a distinct platelet lipidome signature is associated with ischaemic and bleeding events; (3) changes of the platelet lipidome are associated with platelet function.Thus, it is tempting to speculate that assessment of distinct platelet lipids may improve evaluation of CAD patients at risk, improve the diagnostic value of conventional lipid measurement and provide new diagnostic and treatment perspectives.
Previously, substantial changes in the platelet lipidome of patients with CAD have been described [12,15].The platelet lipid signature is associated with disease severity and distinct platelet lipids are upregulated in ACS when compared to CCS or healthy controls [15].Changes in the platelet lipidome occur upon activation and promote enhanced ex vivo platelet functions [20][21][22].Beyond conventional cardiovascular risk factors, plasmatic lipoproteins such as low-density lipoprotein (LDL) are strongly associated with poor prognosis in CAD [23,24].Lowering LDL plasma levels has significantly improved the clinical outcome of CAD patients [25,26].
Recently, platelets have been recognized as a prominent compartment of lipid metabolism and both statins and PCSK9-inhibitors modulate the platelet lipidome signature [16,27].However, large-scale clinical studies that investigate the platelet lipidome in CAD and its association to adverse CV events are missing so far.
In this prospective study, we characterized the platelet lipidome in a consecutive cohort of patients with symptomatic CAD and further elucidated changes of distinct platelet lipids between patients with adverse CV events and patients without incident CV events.
We found that among 767 structurally annotated lipids, 18 lipids were significantly upregulated in patients with adverse CV events.Most of the lipids were upregulated in comparison to patients without recurrence of CV events.We found that distinct fingerprints of the platelet lipidome are associated with ischaemic and bleeding events.In patients with ischaemic events glycerophospholipids (lysophosphatidylethanolamine, lysophosphatidylserine and phosphatidylethanolamine) with mono-(MUFA) or poly-unsaturated fatty acyl (PUFA) side chains were significantly upregulated compared to individuals with an event-free follow-up.PUFA-phospholipids are important metabolites in thrombo-inflammatory cascades and regulate membrane integrity and thus, essentially modulate platelet function [28,29].Especially PUFA-PLs with eicosatetraenoic (20:4) and docosatetraenoic acid (22:4) are proinflammatory and proapoptotic [30][31][32].In our study, LPE 22:4/0:0 was significantly increased in patients with recurrence of CV events.Lysophosphatidylethanolamines promote platelet hyperreactivity [33].In the present study we found that ex vivo platelet aggregation is enhanced in patients with high LPE content.This indicates that LPEs play a role in favouring platelet hyperreactivity and may contribute to an enhanced risk for thromboischemic events in patients with CAD.Therefore, we hypothesize that especially patients with high platelet LPE concentrations and platelet hyperreactivity might benefit from effective antiplatelet therapy to prevent adverse thromboischemic events.Further, we depict that downregulation of LPE is accessible to high potency statin treatment as LPE concentrations were decreased in patients with statin treatment compared to statin-naïve patients in this study.Thus, beyond reduction of conventional lipid parameters, downregulation of platelet LPE through lipid-lowering therapy might serve as therapeutic target to attenuate the clinical course of CAD.Therefore, CAD patients with increased concentrations of platelet LPE might benefit from intensified patient management and a close clinical follow-up.
In patients with a major bleeding event during the follow-up period, 9 lipids were significantly increased in contrast to patients without bleeding and upregulated lipids mainly comprised fatty acyls including acylcarnitines and unsaturated fatty acids.Previously, plasma acylcarnitines were found to be elevated in patients with CAD and both lipid classes showed predictive potential for mortality or recurring myocardial infarction in patients with CAD [12,34].Unsaturated fatty acyls are inhibitors of platelet functions and have the potential to form conjugates with aspirin, and thus potentiate the antiplatelet therapy [35][36][37].
Acylcarnitines inhibit plasmatic coagulation and might act as anticoagulant lipids [38].In our study, an increase of platelet acylcarnitines was associated with a significant reduction of ex vivo aggregation response and high bleeding risk according to PREICSE-DAPT score.Thus, distinct platelet lipids were found to be associated with functional responsibility and an increased risk for adverse cardiovascular events.Therefore, it is tempting to speculate that CAD patients with high fatty acyl concentrations including platelet CAR might benefit from short-term and less aggressive dual antiplatelet treatment following PCI to prevent major bleeding events, and thus improve the long-term clinical outcome.
In our study, we highlighted that upregulated lipids were associated with an increased cardiovascular risk and especially lysophosphatidylethanolamines were significantly linked to an increased hazard of adverse CV events, whereas fatty acyls were associated with an increased risk of bleeding.To further assess the cardiovascular risk in patients with CAD with lipid measures beyond conventional parameters such as plasma lipoproteins, we selected important lipid parameters utilizing penalized LASSO algorithm.Here we included nine lipids mostly comprising glycerophospholipids.The addition of these lipids to conventional cardiovascular risk factors including traditional lipid measures improved prediction of recurrent adverse events in patients with CAD.Further, substitution of conventional lipid measures, i.e. total cholesterol and high-density lipoprotein by platelet lipids also enhanced risk discrimination of CV events in this study.Thus, we hypothesize that assessment of distinct platelet Fig. 6 Alterations of the platelet lipidome in patients with adverse cardiovascular events modulate platelet functions and thus, might influence disease progression and outcome of patients with incident CVD.A Patients were divided into quartiles (upper quartile = high/ lower quartile = low) according to the concentration of distinct lipids found to be significantly upregulated in patients with cardiovascular events when compared to those without adverse events.We found, that collagen-mediated platelet aggregation was significantly (Student's t test, p < 0.05) increased under high concentrations of LPE.In contrast, high concentrations of acylcarnitines were found to be significantly (p < 0.05) associated with a decrease of collagen, arachidonic acid and ADP-mediated platelet aggregation.B Correlation of lipids found to be significantly increased in patients with cardiovascular or bleeding events compared to patients without adverse events.Collagen-mediated platelet aggregation (AUC) significantly (p < 0.05) correlated with increased concentrations of LPE.In contrast, collagen-mediated platelet aggregation was inversely associated with enhanced concentrations of acylcarnitines ◂ Fig. 7 Platelet functions are altered in patients with distinct changes in the platelet lipidome.A The lipid class of lysophosphatidylethanolmines (LPE) was summarized by mean concentrations.All patients were divided into quartiles according to the LPE concentration (1st quartile < 25%, 2nd quartile < 50%, 3rd quartile < 75%, 4th quartile ≥ 75%).We found that collagen-mediated platelet aggregation was significantly (Student's t-test, p < 0.05) increased under high (≥ 75%) and intermediate (< 50%) concentrations of LPE in contrast to low (< 25%) concentrations.B In contrast, collagen-mediated platelet aggregation was significantly (Student's t test, p < 0.05) reduced under high (≥ 75%) concentrations of acylcarnitines (CAR) in contrast to intermediate (< 75%, < 50%) and low (< 25%) concentrations lipid biomarkers may improve prediction of adverse cardiovascular events in patients with CAD, and thus might significantly contribute to identify patients at high risk.
In conclusion, our present study implies a diagnostic value of platelet lipidomics which may be helpful to discriminate the individual risk of patients with CAD (graphical abstract and Supplementary Figure S6).

Table 1
Baseline characteristics of patient populationNormally distributed data were analysed using Student's t-test.Non-normally distributed data were compared using the Mann-Whitney U test.Mean values are presented as mean ± standard deviation (SD) and significant baseline characteristics (p < 0.05) are highlighted

Table 2
Clinical endpoints at 3-year follow-up