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Trimethylamine N-Oxide and Adenosine Diphosphate–Induced Platelet Reactivity Are Independent Risk Factors for Cardiovascular and All-Cause Mortality

2020; Lippincott Williams & Wilkins; Volume: 126; Issue: 5 Linguagem: Inglês

10.1161/circresaha.119.316214

1524-4571

Martin Berger, Marcus E. Kleber, Graciela E. Delgado, W. März, Andreas Meinitzer, Peter Hellstern, Nikolaus Marx, Katharina Schuett,

Heart Failure Treatment and Management

HomeCirculation ResearchVol. 126, No. 5Trimethylamine N-Oxide and Adenosine Diphosphate–Induced Platelet Reactivity Are Independent Risk Factors for Cardiovascular and All-Cause Mortality Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBTrimethylamine N-Oxide and Adenosine Diphosphate–Induced Platelet Reactivity Are Independent Risk Factors for Cardiovascular and All-Cause Mortality Martin Berger, Marcus E. Kleber, Graciela E. Delgado, Winfred März, Meinitzer Andreas, Peter Hellstern, Nikolaus Marx and Katharina A. Schuett Martin BergerMartin Berger From the Department of Internal Medicine I, University Hospital Aachen, Germany (M.B., N.M, K.A.S.) , Marcus E. KleberMarcus E. Kleber Vth Department of Medicine (Nephrology, Hypertensiology, Rheumatology, Endocrinology, Diabetology), Medical Faculty Mannheim, University of Heidelberg, Germany (M.E.K., G.E.D., W.M.) , Graciela E. DelgadoGraciela E. Delgado Vth Department of Medicine (Nephrology, Hypertensiology, Rheumatology, Endocrinology, Diabetology), Medical Faculty Mannheim, University of Heidelberg, Germany (M.E.K., G.E.D., W.M.) , Winfred MärzWinfred März Vth Department of Medicine (Nephrology, Hypertensiology, Rheumatology, Endocrinology, Diabetology), Medical Faculty Mannheim, University of Heidelberg, Germany (M.E.K., G.E.D., W.M.) Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University Graz, Austria (W.M., M.A.) SYNLAB Academy, SYNLAB Holding Deutschland GmbH, Mannheim and Augsburg, Germany (W.M.) , Meinitzer AndreasMeinitzer Andreas Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University Graz, Austria (W.M., M.A.) , Peter HellsternPeter Hellstern Center of Hemostasis and Thrombosis Zurich, Switzerland (P.H.). , Nikolaus MarxNikolaus Marx From the Department of Internal Medicine I, University Hospital Aachen, Germany (M.B., N.M, K.A.S.) and Katharina A. SchuettKatharina A. Schuett https://orcid.org/0000-0002-0162-5219 From the Department of Internal Medicine I, University Hospital Aachen, Germany (M.B., N.M, K.A.S.) Originally published20 Jan 2020https://doi.org/10.1161/CIRCRESAHA.119.316214Circulation Research. 2020;126:660–662Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: January 20, 2020: Ahead of Print Trimethylamine N-oxide (TMAO) is an intestinal microbiota-derived metabolite shown to be associated with major adverse cardiovascular events and all-cause mortality.1,2 Recently, TMAO-induced platelet hyperreactivity received increased attention by the observation that TMAO predicts atherothrombotic events, enhances platelet activation through altered Ca2+ signaling, and increases in vivo thrombosis when injected intraperitoneally in mice.1 However, despite clear evidence from animal studies, the role of TMAO exposure on platelet reactivity (PR) in humans remains conflicting.3 Here, we investigate the role of TMAO on PR and subsequent all-cause and cardiovascular mortality in the LURIC study (Ludwigshafen Risk and Cardiovascular Health).TMAO and PR were measured in patients who underwent coronary angiography between 1997 and 2000 and participated in the LURIC study.4 TMAO was measured by high-performance liquid chromatography as previously described.2 On the day of study inclusion, PR was measured on a Coulter FC500 flow cytometer (Coulter Krefeld, Germany) by adenosine diphosphate (ADP; 10 µM, ADP) and thrombin receptor agonist peptide (TRAP; 10 µM) induced P-selectin expression (CD62P) relative to basal. Data regarding TMAO and PR were available in 1627 patients. Statistical analysis was performed using SPSS v25.0.In line with previous studies, ADP-induced PR was an excellent mortality predictor in the LURIC study (Figure [Aii]).5 Both TMAO and PR were stratified into tertiles for cox regression models (ie, low, medium, and high; Figure [Ai, Aii]). Among the 1627 patients analyzed, 468 deaths (295 cardiovascular [CV]) occurred within a mean follow-up of 9.7 years. Compared with patients in the lowest tertile, patients in the highest tertile of TMAO and PR had significantly higher age- and sex adjusted hazard ratios (HR) for death (Figure Ai and Aii, Model 1). Further adjustment of TMAO and PR for coronary artery disease, hsCRP and glomerular filtration rate did not affect its association with all-cause mortality (TMAO: HR, 1.5 [95% CI, 1.2–2.0; 1.6 for CV death]; PR: HR, 1.3 [95% CI, 1.0–1.6; 1.3 for CV death]). Adjustment for additional CV risk factors did not lead to a significant different model (Figure Ai and Aii, Model 4). Critically, neither adjustment of TMAO for PR nor adjustment of PR for TMAO did affect the significant association with mortality in both models (Figure Ai and Aii, Model 2). To analyze the association between TMAO plasma levels and platelet activation, we calculated correlation coefficients for ADP- and TRAP induced CD62P expression (Figure Bi and Bii). ADP-induced platelet CD62P expression correlated weakly with TMAO plasma levels and explained 140 mmHg), body mass index (BMI), LDL-C (low-density lipoprotein cholesterol), HDL-C (high-density lipoprotein cholesterol), triglycerides, cholesterol, and HbA1c. Inclusion criteria for the LURIC study were: German ancestry, clinical stability except for acute coronary syndromes, and the availability of a coronary angiogram. TMAO was measured with the use of a stable-isotope-dilution assay and high-performance liquid chromatography with electrospray ionization tandem mass spectrometry on a Voyager TSQ Quantum triple quadrupole instrument equipped with an Ultimate 3000 chromatography system. Platelet reactivity was measured on the day of study inclusion. Citrated whole blood was diluted 1:10 with 0.5% albumin-supplemented Tyrode buffer and incubated for 5 min with 10 µM TRAP or 10 µM ADP in parallel tubes followed by parallel anti-CD41-FITC and anti-CD62P-FITC (Becton Dickinson, Heidelberg, Germany) staining for 15 min. The reaction was quenched with 1 mL of ice-cold Tyrode buffer. Ten thousand platelets were counted according to forward- and sideward scatter characteristics and CD41 expression. Antibody staining was measured on a Coulter FC 500 flow cytometer (Coulter Krefeld, Germany) and expressed as median fluorescence intensity (MFI) relative to basal (B) correlation between TMAO plasma levels (µmol) and ADP (Bi) and TRAP-induced CD62P expression (MFI, relative to basal; Bii). Correlation was tested by Spearman correlation. N=1627 patients. C, Hazard ratios for all-cause mortality stratified by combined tertiles of TMAO and ADP-induced platelet reactivity. Hazard ratios were calculated by cox proportional hazards model. Low-TMAO and low platelet-reactivity served as reference group. The model was adjusted for age, sex, coronary artery disease, eGFR (CKD-EPI) and hsCRP, arterial hypertension, BMI, cholesterol, LDL-C, HDL-C, triglycerides, and HbA1c. N=1622 patients; 5 patients not included due to missing values for multivariable adjustment. Numbers on bars denote patients/group (D). Cumulated survival functions calculated by cox proportional hazards model. Risk groups were stratified in 4 groups according to the first (low) and third (high) tertile of TMAO and ADP-induced platelet reactivity, respectively. Low-TMAO and low platelet-reactivity served as reference group. N=706 patients with 233 events. Model adjusted as in C.In summary, the present study is the largest observational study to date that evaluates the association between TMAO and PR and extends the current understanding with respect to relevant clinical outcomes. Here, we demonstrate that TMAO and ADP-induced PR represent both significant but independent predictors of all-cause and cardiovascular mortality in the LURIC study. Therefore, in a real-life patient scenario, alternative mechanisms that may explain the pathological effects of TMAO and PR deserve further attention.Nonstandard Abbreviations and AcronymsHRhazard ratioLURICLudwigshafen Risk and Cardiovascular HealthPRplatelet reactivityTMAOtrimethylamine N-oxideTRAPthrombin-receptor agonist peptideAcknowledgmentsWe thank the participants of the LURIC study (Ludwigshafen Risk and Cardiovascular Health); without their collaboration this article would not have been written. The authors also thank the LURIC study team members who were either temporarily or permanently involved in patient recruitment and sample and data handling; the laboratory staff at the Ludwigshafen General Hospital; and the Universities of Freiburg, Ulm, and Graz.Sources of FundingThis work received funding from the European Union's Horizon 2020 Research and Innovation Programme under the ERA-Net Cofund action N° 727565 (OCTOPUS project) and the German Ministry of Education and Research (grant number 01EA1801A).DisclosuresK.A. Schuett and N. Marx received funding from the Deutsche Forschungsgemeinschaft (grant number SFB/TRR219 C-07, M-05, M-03). K.A. Schuett and N. Marx have received funding from the Corona-Stiftung, Germany. W. März reports grants and personal fees from Abbott Diagnostics, Aegerion Pharmaceuticals, Akcea Therapeutics, Alexion Pharmaceuticals, AMGEN, BASF, Berlin-Chemie, Numares AG, Sanofi, and grants from Astrazeneca, Bayer Vital GmbH, bestbion dx GmbH, Boehringer Ingelheim Pharma GmbH Co KG, Immundiagnostik GmbH, Merck Chemicals GmbH, MSD Sharp and Dohme GmbH, Novartis Pharma GmbH, Olink Proteomics, Siemens Healthineers, all outside the submitted work. W. März is employed with SYNLAB Holding Deutschland GmbH. The other authors report no conflicts.FootnotesFor Sources of Funding and Disclosures, see page 661.References1. Zhu W, Gregory JC, Org E, Buffa JA, Gupta N, Wang Z, Li L, Fu X, Wu Y, Mehrabian M, et al. Gut microbial metabolite TMAO enhances platelet hyperreactivity and thrombosis risk.Cell. 2016; 165:111–124. doi: 10.1016/j.cell.2016.02.011CrossrefMedlineGoogle Scholar2. Schuett K, Kleber ME, Scharnagl H, Lorkowski S, März W, Niessner A, Marx N, Meinitzer A. Trimethylamine-N-oxide and heart failure with reduced versus preserved ejection fraction.J Am Coll Cardiol. 2017; 70:3202–3204. doi: 10.1016/j.jacc.2017.10.064CrossrefMedlineGoogle Scholar3. Haissman JM, Haugaard AK, Ostrowski SR, Berge RK, Hov JR, Trøseid M, Nielsen SD. Microbiota-dependent metabolite and cardiovascular disease marker trimethylamine-N-oxide (TMAO) is associated with monocyte activation but not platelet function in untreated HIV infection.BMC Infect Dis. 2017; 17:445. doi: 10.1186/s12879-017-2547-xCrossrefMedlineGoogle Scholar4. Winkelmann BR, März W, Boehm BO, Zotz R, Hager J, Hellstern P, Senges J; LURIC Study Group (LUdwigshafen RIsk and Cardiovascular Health). Rationale and design of the LURIC study–a resource for functional genomics, pharmacogenomics and long-term prognosis of cardiovascular disease.Pharmacogenomics. 2001; 2:S1–73. doi: 10.1517/14622416.2.1.S1CrossrefMedlineGoogle Scholar5. Puurunen MK, Hwang S, Larson MG, Vasan RS, O'Donnell CJ, Tofler G, Johnson AD. ADP platelet hyperreactivity predicts cardiovascular disease in the FHS (Framingham Heart Study).J Am Heart Assoc. 2018; 7:1–9. doi: 10.1161/JAHA.118.008522LinkGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited By Canyelles M, Plaza M, Rotllan N, Llobet D, Julve J, Mojal S, Diaz-Ricart M, Soria J, Escolà-Gil J, Tondo M, Blanco-Vaca F and Souto J (2022) TMAO and Gut Microbial-Derived Metabolites TML and γBB Are Not Associated with Thrombotic Risk in Patients with Venous Thromboembolism, Journal of Clinical Medicine, 10.3390/jcm11051425, 11:5, (1425) Li D, Lu Y, Yuan S, Cai X, He Y, Chen J, Wu Q, He D, Fang A, Bo Y, Song P, Bogaert D, Tsilidis K, Larsson S, Yu H, Zhu H, Theodoratou E, Zhu Y and Li X (2022) Gut microbiota–derived metabolite trimethylamine-N-oxide and multiple health outcomes: an umbrella review and updated meta-analysis, The American Journal of Clinical Nutrition, 10.1093/ajcn/nqac074 Wei S, Ma W, Zhang B and Li W (2021) NLRP3 Inflammasome: A Promising Therapeutic Target for Drug-Induced Toxicity, Frontiers in Cell and Developmental Biology, 10.3389/fcell.2021.634607, 9 February 28, 2020Vol 126, Issue 5 Advertisement Article InformationMetrics © 2020 American Heart Association, Inc.https://doi.org/10.1161/CIRCRESAHA.119.316214PMID: 31958034 Originally publishedJanuary 20, 2020 Keywordsplatelet activationcoronary angiographyattentionthrombinmortalityPDF download Advertisement SubjectsBiomarkersMortality/SurvivalPlateletsThrombosis