BACKGROUND:Coagulation factor XI (FXI) influences both thrombotic risk and myocardial function, making its relationship with mortality crucial for guiding therapies, especially in coronary artery disease (CAD). METHODS:We analyzed data from 3,170 participants who underwent coronary angiography; 67% were diagnosed with CAD. Participants were followed for a median of 14.5 years. Mortality risk was assessed using Cox proportional hazards models with restricted cubic splines and Wald statistics. Models were adjusted for age, sex, BMI, and further cardiovascular risk factors. Interactions between FXI activity, N-terminal pro-B-type natriuretic peptide (NT-proBNP), and CAD were explored. FINDINGS:A U-shaped association between FXI activity and mortality was observed (p = 0.027), with the lowest risk at an FXI activity of 115.6%. Among patients without CAD, this U-shaped relationship persisted. In contrast, patients with CAD demonstrated a linear relationship, where higher FXI activity correlated with increased mortality (p interaction < 0.0001). NT-proBNP levels significantly modified these associations, particularly in patients with CAD. CONCLUSIONS:These findings emphasize the dual role of FXI activity in hemostasis, which could have profound implications for pharmacological interventions. The variable effects of FXI activity based on underlying cardiovascular conditions suggest that a personalized approach to treatment is necessary. Consequently, future studies on FXI inhibitors should carefully examine these modulating factors to optimize therapeutic strategies. FUNDING:The LURIC study was supported by the Ludwigshafen Heart Centre and academic collaborators, including the universities of Freiburg, Ulm, and Düsseldorf and the Centre Nationale de Genotypage in France, through internal institutional resources.

Einleitung & Ziel: Der Gerinnungsfaktor XI (FXI) ist wichtig für die Blutgerinnung und an der Entstehung von Thrombosen beteiligt. Neue Daten weisen auf kardioprotektive Effekte besonders bei diastolischer Dysfunktion hin, einem Merkmal der diabetischen Kardiomyopathie. Wir analysierten den Zusammenhang von FXI und Mortalität, und die Modifikation dieses Effektes durch Diabetes und vorhandene Herzinsuffizienz in der LURIC-Kohorte.

Abstract Background Metabolic clusters can stratify subgroups of individuals at risk for type 2 diabetes mellitus and related complications. Since obesity and insulin resistance are closely linked to alterations in hemostasis, we investigated the association between plasmatic coagulation and metabolic clusters including the impact on survival. Methods Utilizing data from the Ludwigshafen Risk and Cardiovascular Health (LURIC) study, we assigned 917 participants without diabetes to prediabetes clusters, using oGTT-derived glucose and insulin, high-density lipoprotein cholesterol, triglycerides, and anthropometric data. We performed a comprehensive analysis of plasmatic coagulation parameters and analyzed their associations with mortality using proportional hazards models. Mediation analysis was performed to assess the effect of coagulation factors on all-cause mortality in prediabetes clusters. Results Prediabetes clusters were assigned using published tools, and grouped into low-risk (clusters 1,2,4; n = 643) and high-risk (clusters 3,5,6; n = 274) clusters. Individuals in the high-risk clusters had a significantly increased risk of death (HR = 1.30; CI: 1.01 to 1.67) and showed significantly elevated levels of procoagulant factors (fibrinogen, FVII/VIII/IX), D-dimers, von-Willebrand factor, and PAI-1, compared to individuals in the low-risk clusters. In proportional hazards models adjusted for relevant confounders, elevated levels of fibrinogen, D-dimers, FVIII, and vWF were found to be associated with an increased risk of death. Multiple mediation analysis indicated that vWF significantly mediates the cluster-specific risk of death. Conclusions High-risk prediabetes clusters are associated with prothrombotic changes in the coagulation system that likely contribute to the increased mortality in those individuals at cardiometabolic risk. The hypercoagulable state observed in the high-risk clusters indicates an increased risk for cardiovascular and thrombotic diseases that should be considered in future risk stratification and therapeutic strategies. Graphical Abstract

Introduction & Objective: We investigated the effect of FXI on mortality in a cohort at increased risk for cardiac death, with a specific focus on risk-modification in case of type 2 diabetes (T2D) or current heart failure. Methods: The prospective LURIC study recruited 3063 persons undergoing coronary diagnostics. Mortality risk was assessed using Cox proportional hazards models employing restricted cubic splines adjusted for age, sex, BMI and coronary artery disease, with interaction terms between FXI activity and T2D status as well as between FXI activity and brain natriuretic peptide (NTproBNP). Results: In the unadjusted model, FXI activity demonstrated a U-shaped association with mortality risk (p=0.025): It was protective between 89-139%, but the risk increased at levels outside this range. A significant interaction of FXI activity with T2D (p=0.036) revealed that higher FXI activity was linked to reduced mortality in people without diabetes. However, this relation was inverted in participants with T2D (Fig. 1). NTproBNP was a further significant modifier of the relationship between FXI activity and mortality (p=0.0085). Conclusion: T2D and NTproBNP significantly alter FXI activity's effect on mortality, underscoring a multifaceted interplay. Upcoming FXI-targeted therapies should be carefully evaluated in persons with diabetes with regards to their efficacy and safety. Disclosure K. Prystupa: None. M. Heni: Research Support; Boehringer-Ingelheim. Advisory Panel; Amryt Pharma Plc. Speaker's Bureau; Amryt Pharma Plc. Advisory Panel; Boehringer-Ingelheim, Boehringer-Ingelheim. Speaker's Bureau; Lilly Diabetes, Novartis AG, Novo Nordisk, Sanofi. S. Hörber: None. A. Peter: None. P. Hellstern: None. M. Kelm: Research Support; Edwards Lifesciences Coroporation, Microvision Medical, B.Braun, IPP Med GmbH - Institut für Pharmakologie u. präventive Medizin GmbH, Mars Scientific Advisory Council (MSAC). Other Relationship; Bayer Inc., Abiomed. Board Member; ESC-European Society of Cardiology, DGIM-Deutsche Geselleschaft für Innere Medizin, DSHF-Deutsche Stiftung für Herzforschung, DGK-Deutsche Gesellschaft für Kardiologie. Other Relationship; Kel Con GmbH, diaplan. H. Yamazaki: Other Relationship; AstraZeneca, Janssen Pharmaceuticals, Inc., Mitsubishi Tanabe Pharma Corporation, Kowa Company, Ltd., Kyorin Pharmaceutical Co. Ltd, Takeda Pharmaceutical Company Limited, Takeda Pharmaceutical Company Limited, Magmitt Pharmaceutical Co. M. Roden: Advisory Panel; Eli Lilly and Company. Research Support; Boehringer-Ingelheim. Advisory Panel; Novo Nordisk. Research Support; Novo Nordisk. Advisory Panel; TARGET PharmaSolutions, Inc. Speaker's Bureau; AstraZeneca. R. Wagner: Speaker's Bureau; Sanofi. Advisory Panel; Lilly Diabetes. Speaker's Bureau; Boehringer-Ingelheim, Novo Nordisk.

BACKGROUND:Direct oral anticoagulants (DOAC) such as Xa inhibitors rivaroxaban (riva) and apixaban (apix) are increasingly replacing Vitamin K antagonists in prophylaxis and treatment of venous thromboembolism (VTE). Measurements of DOAC plasma levels may be necessary in certain clinical conditions to determine the further dosage. Making decisions is made more difficult by the fact that the peak and trough plasma levels are subject to strong inter-individual fluctuations with overlapping reference ranges. We wanted to find out whether the peak and trough levels can be narrowed if they are determined based on age and gender.METHODS:Therefore, we collected data on peak and trough anti-Xa concentrations in patients treated with either rivaroxaban (n = 93) or apixaban (n = 51) in one center. After exclusion of blood samples of uncertain oral intake, 83 samples for rivaroxaban and 49 samples for apixaban remained for further analysis. Differences between male (riva n = 42, apix n = 28) and female (riva n = 41 and apix n = 21) as well as young (≤ 60 years, riva n = 44, apix n = 23) and elder (> 60 years) patients (riva n = 39 and apix n = 26) were analyzed by Student`s t-test and retrospective regression.RESULTS:We found no differences in age and gender for the apix peak levels. But women had significantly higher riva peak concentrations than men (308.8 ± 178.1 ng/mL versus 206.4 ± 80 ng/mL, p = 0.013). Patients older than 60 years had significantly higher riva peak levels than those younger than 60 (293.7 ± 126.7 ng/mL versus 211.7 ± 158.4 ng/mL, p = 1.29 x 10-8).CONCLUSIONS:In search of narrowing standard peak and trough levels in patients' sera we found significant differ-ences between patients below and above sixty years of age. Gender-associated differences were found in rivaroxa-ban levels possibly explaining DOAC associated hypermenorrhea. In conclusion, gender and age should be included in the determination of peak blood concentration references.

BACKGROUND:Thrombophilia testing is controversial, not least because of its high cost. Because comprehensive valid testing requires standardized blood collection close by the specialized laboratory, and interpretation of findings together with clinical data, often only part of the necessary laboratory analyses can be performed in remote central laboratories. Restrictive indications for testing, as have been recommended by previous reviews on the topic, have been based on incomplete analytics, studies with small case numbers, or short observation periods, and on an inappropriate, simple risk stratification for venous thromboembolism (VTE), further subdivided into provoked and unprovoked events.METHODS:The authors reviewed four electronic databases for all peer-reviewed and in-press articles about thrombophilia, VTE, obstetric complications, and arterial thrombosis. After confirmation for relevance to the topic, 201 articles were accepted for inclusion in this article. This review summarizes the studies relevant to the evaluation of thrombophilic conditions, and their combination with each other and with clinical risk factors, to stratify individual risk for thromboembolism and obstetric complications.RESULTS:Thrombophilia testing requires highly skilled personnel for laboratory analysis and interpretation. Clinical conditions that influence the results as well as special preanalytical, analytical, and postanalytical aspects must be considered if valid results are to be obtained. Tests involved include the natural anticoagulants antithrombin, protein C, and protein S; the procoagulants fibrinogen (dysfibrinogen), prothrombin (mutation G20210A), factor V (Leiden mutation), factor VIII/von Willebrand factor/blood group ABO, factor IX, and factor XI; the anti-phospholipid antibodies to detect an antiphospholipid syndrome and potentially additional uncertain thrombophilic conditions. The risks of thrombophilic conditions and clinical risk factors for VTE are cumulative or even supra-additive. Scores from thrombophilic conditions and other genetic and nongenetic risk factors permit estimation of risk for first and recurrent VTE. Therapeutic strategies can be derived from this risk stratification.CONCLUSIONS:Thrombophilia testing is indicated when the results have potential to influence the type and duration of treatment. Indications include certain patients after VTE; or patients without previous VTE but with positive family history regarding VTE or thrombophilia before major surgery, pregnancy, combined oral contraceptives, or hormone replacement therapy. Whether or not thrombophilia is present should help determine anticoagulation, hormonal contraception, or hormone replacement.

Background: The clinical and prognostic implications of platelet reactivity (PR) testing in a P2Y12-inhibitor naïve population are poorly understood. Objectives: This explorative study aims to assess the role of PR and explore factors that may modify elevated mortality risk in patients with altered PR. Methods: Platelet ADP-induced CD62P and CD63 expression were measured by flow-cytometry in 1520 patients who were referred for coronary angiography in the Ludwigshafen Risk and Cardiovascular Health Study (LURIC). Results: High- and Low-platelet reactivity to ADP were strong predictors of cardiovascular and all-cause mortality and risk equivalent to the presence of coronary artery disease. (High platelet reactivity 1.4 [95% CI 1.1–1.9]; Low platelet reactivity: 1.4 [95% CI 1.0–2.0]). Relative weight analysis indicated glucose control (HbA1c), renal function ([eGFR]), inflammation (high-sensitive C-reactive protein [hsCRP]) and antiplatelet therapy by Aspirin as consistent mortality risk modifiers in patients with Low- and High-platelet reactivity. Pre-specified stratification of patients by risk modifiers HbA1c (<7.0%), eGFR (>60 mL/min/1.73 m2) and CRP (<3 mg/L) was associated with a lower mortality risk, however irrespective of platelet reactivity. Aspirin treatment was associated with reduced mortality in patients with high platelet reactivity only (p for interaction: 0.02 for CV-death [<0.01 for all-cause mortality]. Conclusions: Cardiovascular mortality risk in patients with High- and Low platelet reactivity is equivalent to the presence of coronary artery disease. Targeted glucose control, improved kidney function and lower inflammation are associated with reduced mortality risk, however independent of platelet reactivity. In contrast, only in patients with High-platelet reactivity was Aspirin treatment associated with lower mortality.

BACKGROUND:Surgical and technological advances have resulted in the widespread adoption of microsurgical breast reconstruction. Many comorbidities that potentially might impair vasculature and wound healing are no longer considered contraindications for these procedures. However, some uncertainty still prevails regarding the perioperative management of patients with disorders of hemostasis.METHODS:The authors combined a literature review with a retrospective chart review of patients with disorders of hemostasis who had undergone microsurgical breast reconstruction at the senior author's (J.F.) center between 2015 to 2020. Several disorders associated with thrombotic and/or hemorrhagic complications were identified, and a standardized risk assessment and management strategy was developed in cooperation with a hematologist.RESULTS:Overall, 10 studies were identified comprising 29 patients who had a defined disorder of hemostasis and underwent microsurgical breast reconstruction. Seventeen microsurgical breast reconstructions were performed on 11 patients at the senior author's (J.F.) center. High factor VIII levels, heterozygous factor V Leiden, and heterozygous prothrombin mutation G20210A were the most common genetic or mixed genetic/acquired thrombophilic conditions. As expected, hereditary antithrombin, protein C, or protein S deficiencies were rare. Among hemorrhagic disorders, thrombocytopenia, platelet dysfunction, and von Willebrand disease or low von Willebrand factor levels were those factors most frequently associated with increased perioperative bleeding.CONCLUSIONS:Patients should be screened for elevated risk of thrombosis or bleeding before undergoing microsurgical breast reconstruction, and positive screening should prompt a complete hematologic evaluation. Interdisciplinary management of these disorders with a hematologist is essential to minimize risks and to obtain optimal reconstructive results.CLINICAL QUESTION/LEVEL OF EVIDENCE:Risk, IV.

Introduction: Elevated leukocyte counts are associated with cardiovascular disease. Smoking induces inflammation and alters levels of leukocyte subtypes. Aims and Methods: Our aim was to investigate the effect of smoking on circulating immune cells and their association with mortality. Lymphocyte subtypes were identified by flow cytometry of fluorescent-labeled cells. We analyzed the association of leukocytes with mortality using Cox regression and assessed their effect on risk prediction based on principle components (PCs) using area under the receiver operating characteristic curve and net-reclassification in 2173 participants from the Ludwigshafen Risk and Cardiovascular Health Study, a prospective case-control study in patients who underwent coronary angiography. Results: The numbers of T cells, monocytes, and neutrophils were higher and natural killer cells were lower in smokers compared with never-smokers. In never-smokers, lymphocyte counts were inversely associated with mortality while a positive association was observed for neutrophils.The neutrophil-to-lymphocyte ratio (NLR) had the strongest association in never-smokers with a hazard ratio (95% confidence interval) of 1.43 (1.26-1.61). No associations were found in smokers. Adding the first five PCs or the NLR to a risk prediction model based on conventional risk factors did not improve risk prediction in smokers, but significantly increased the area under the curve from 0.777 to 0.801 and 0.791, respectively, in never-smokers. Conclusions: Lymphocyte counts were inversely associated with mortality in never-smokers but not in active smokers. Markers of innate immunity, namely total neutrophils and CD11b+/CD18+ and CD31+/CD40- granulocytes, were directly associated with mortality. Adding markers of immune function like PCs or the NLR to basic risk models improved risk prediction in never-smokers only.

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 <1% of the variance in expression of the activation markers (CD62P R2=0.004; P=0.015). Platelet stimulation by TRAP showed no significant correlation (CD62P R2=0.000009; P=0.885). To further dissect the role of TMAO and PR on all-cause and cardiovascular mortality, we stratified patients in 9 groups according to PR and TMAO tertiles and calculated HRs using a multivariable-adjusted cox-regression model (Figure C). TMAO remained a significant predictor of all-cause mortality even in patients with low PR with a HR of 1.9 (95% CI, 1.2–3.0; P=0.007 [1.8 for CV death], highest versus lowest TMAO tertile). Accordingly, in patients with low TMAO levels, PR significantly predicted mortality with a HR of 1.9 ([95% CI, 1.2–3.0]; P=0.009 [1.6 for CV death], highest versus lowest PR tertile). The weak correlation of TMAO with markers of platelet activation and the unrelated effects of both TMAO and PR on mortality suggested that these markers represent independent risk factors. To test this hypothesis, we calculated survival curves for all-cause mortality and CV death after stratifying patients into 4 risk-groups according to the first (low) and third (high) tertile of TMAO and PR, respectively. The multivariable-adjusted HR were as follows: low TMAO—low PR, 1.0 (reference category), low TMAO—high PR, 1.9 ([95% CI, 1.2–3.0]; 1.7 for CV-death), high TMAO—low PR, 2.0 ([95% CI, 1.2–3.2]; 2.0 for CV death), and high TMAO—high PR, 2.5 ([95% CI, 1.6–4.0]; 3.0 for CV death; Figure D). Additional interaction analyses revealed no significant interaction between both markers for all-cause and CV mortality and suggested an independent relationship (P value for interaction: 0.22 [all-cause mortality] 0.42 [CV death]; data not shown). Accordingly, combined assessment of TMAO and PR only marginally increased the discriminatory power for all-cause and cardiovascular mortality (C statistic 0.639; [0.630 for CV death]) compared with TMAO alone (C statistic, 0.636; P=0.16; [0.634 for CV death; P=0.65]).Download figureDownload PowerPointFigure. Trimethylamine N-oxide (TMAO) and adenosine diphosphate (ADP) are independent risk factors for cardiovascular and all-cause mortality. A, Crude and risk-factor adjusted hazard ratios for all-cause mortality in participants of the LURIC study (Ludwigshafen Risk and Cardiovascular Health) according to TMAO plasma levels (Ai) and ADP-induced CD62P expression (Aii), respectively. All markers were stratified in tertiles, and hazard ratios were calculated by cox proportional hazards model using the lowest tertile of each marker as reference group. Crude hazard ratios were stepwise adjusted for age, sex, self-reported antiplatelet therapy, angiographically verified coronary artery disease, high sensitive CRP (C-reactive protein), eGFR (estimated glomerular filtration rate; chronic kidney disease epidemiology collaboration [CKD-EPI] formula), arterial hypertension (systolic blood pressure >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

Platelet quality in different platelet concentrates (PCs) has been the subject of several studies. Nonetheless, there is a lack of robust data on the correlation and agreement among platelet function tests as a prerequisite for the association of PC functionality in vitro with platelet function in vivo post PC transfusion. The purpose of our study was to correlate a larger panel of platelet function assays in PCs and to assess whether the methods agree sufficiently and can be used interchangeably. Twelve apheresis platelet concentrates in plasma (APC), 16 pooled platelet concentrates in plasma (PPC), and 12 PPC in T-sol (PPCA) were examined on days 1 and 4 after production. PCs were tested for platelet count, light transmission aggregation (LTA) induced by ADP, collagen, or TRAP; platelet ATP release induced by collagen; and spontaneous and ADP and TRAP-induced increase in CD62P and PAC1 expression measured by flow cytometry. All tests were performed in undiluted platelet-rich plasma, recalcified and mixed with an inhibitor of factor Xa and thrombin. Most platelet function parameters correlated significantly with each other, but agreement among methods was insufficient. A proper inverse correlation was observed between ADP-induced LTA and spontaneous platelet activation assessed by CD62P expression (r=-0.61, p<0.0001). Spontaneous CD62P correlated also significantly with spontaneous PAC1 (r=0.69, p<0.0001) and inversely with TRAP-induced CD62P expression (r=-0.86, p<0.0001). We found significant correlations among all flow cytometric assays measuring platelet CD62P and PAC1 expression induced by ADP or TRAP. Subsequent Bland Altman analysis revealed insufficient agreement between methods. With one exception (collagen-induced LTA compared with TRAP-induced LTA, percentage error=16%) the limits of agreement expressed as percentage error exceeded the chosen acceptable difference of 30%.In APC, platelet count was 41% and 44% higher, respectively, than in PPC and PPCA (p<0.0001). Spontaneous CD62P and PAC1 expression were significantly greater, and ADP-induced aggregation and agonist-induced increase in CD62P and PAC1 were significantly lower in PPCA compared to APC and PPC on day 4 of storage. ADP and TRAP-induced CD62P and PAC1 activatability fell significantly during storage between day 1 and day 4 in APC and PPCA, but not in PPC. In conclusion, different platelet function tests capture different aspects of platelet function and do not correlate and agree sufficiently to be used interchangeably.

To investigate whether adenosine diphosphate (ADP)-induced platelet hyperaggregability is associated with nonarteritic anterior ischemic optic neuropathy (NAION) or retinal vein occlusion (RVO). We retrospectively reviewed thrombophilia screening data of patients with NAION or RVO without a history of arterial hypertension, diabetes mellitus, hyperlipidemia, obesity, and cigarette abuse. Patients with a positive family history for thromboembolism were not excluded. Platelet aggregation (area under the curve, AUC) after induction of 0.5, 1.0, and 2.0 µmol of ADP was estimated in 25 NAION and RVO patients and compared with 25 healthy controls. We observed significantly greater platelet aggregation post 0.5 (P = 0.002) and 1.0 (P = 0.008) µmol of ADP among NAION and RVO patients compared with healthy controls. Platelet hyperaggregability was significantly more prevalent in patients than in controls (56% vs. 8%; P = 0.0006). Our results suggest that in NAION and RVO patients without a history of arterial hypertension, diabetes mellitus, hyperlipidemia, obesity, and cigarette abuse, platelets are significantly hyperreactive after induction of very low concentrations of ADP when compared with healthy individuals. This hyperreactivity is particularly evident in patients with a family history of thromboembolism.

BACKGROUND:Thus far, no data has become available to evaluate systematically the prevalences of prothrombin polymorphism A19911G (PT A19911G), factor V HR2 haplotype A4070G (FV A4070G), or plasminogen activator-inhibitor-1 polymorphism 4G/5G (PAI-1 4G/5G) in patients who develop retinal vein occlusion (RVO) without cardiovascular risk factors.MATERIALS AND METHODS:We retrospectively evaluated comprehensive thrombophilia data from 42 preselected RVO patients without cardiovascular risk factors. The prevalences of different gene mutations and polymorphisms including factor V Leiden mutation G1691A (FVL), FV A4070G, prothrombin mutation G20210A, PT A19911G, and PAI-1 4G/5G were compared with 241 healthy controls matched for age and sex.RESULTS:A total of 20 patients (47.7%) were found to carry thrombophilic gene polymorphisms including FVL, FV A4070G, and homozygous PT A19911G compared with 72 of 241 controls (29.9%; p = 0.03). Subgroup analysis of patients with a significant personal or family history of thromboembolism revealed a high prevalence of FVL, FV A4070G, and homozygous PT A19911G (p = 0.005). FV A4070G was found to be significantly associated with at least two other heterozygous or one homozygous gene polymorphisms (p = 0.02). Multivariate analysis revealed the presence of FVL (p = 0.0017) and homozygous PT A19911G (p = 0.03) polymorphism as independent risk factors for the development of RVO.CONCLUSIONS:Our results indicate that in selected RVO patients screening for thrombophilic gene polymorphisms including FVL, FV A4070G and homozygous PT G19911A may be helpful in a high percentage of cases. Our findings suggest that hereditary thrombophilia associated with RVO is more likely to be multigenic than caused by any single risk factor.

Platelet function assays are commonly used diagnostic tools in patients with suspected inherited or acquired hemostatic disorders, and to monitor the efficacy of anti-platelet medication.[1] [2] Beside different newer point-of-care assays, such as the platelet function analyzer (PFA) or the whole blood aggregometry (WBA),[3] [4] the classical Born's light transmission platelet aggregometry (LTA) is an indispensable diagnostic tool for hemostaseologists and therefore still frequently performed in specially equipped laboratories.[5] For routine testing of platelet function by the LTA assay, the patient's platelet rich plasma (PRP) is mixed with different platelet activating agonists, such as ristocetin, ADP, collagen, and arachidonic acid.[5] In few patients, testing their PRP by the LTA assay leads to an isolated missing response in the collagen-induced aggregation, while the platelets respond normally to all the other agonists.[6] [7] [8] Regarding the collagen-induced aggregation, the platelet membrane glycoprotein VI (GP6) plays a central role.[9] [10] It is expressed as a complex with the Fc receptor γ-chain (FcRγ) and has been identified as the main physiological platelet receptor for collagen.[11] When collagen binds to GP6, an activation cascade is initiated by tyrosine phosphorylation inducing the formation of a complex of signal-transducing proteins.[11] The collagen/GP6 interaction finally leads to a physiologic activation of platelets, which can be measured in vitro by the formation of platelet aggregates from PRP. On the other hand, in patients with bleeding tendency displaying a negative collagen reaction in the LTA assay but positive results for all other agonists including arachidonic acid, an acquired or inherited functional defect of GP6 could be the possible cause of the bleeding disorder.[12] [13] [14] [15] [16]

Purpose: To investigate the prevalence of various thrombophilic disorders among young patients with retinal artery occlusion (RAO). Procedures: We retrospectively reviewed thrombophilia screening data of young patients ≤60 years of age with RAO and healthy controls matched for gender and age. Results: Thrombophilia screening data of 25 young patients and 62 healthy controls were analyzed. Mean patient age by the time of the RAO was 43.3 ± 10.8 years. Overall, thrombophilic defects were found to be present in 17 patients (68%) compared with 11 of 62 controls (17.7%; p < 0.0001). Multivariate logistic regression analysis confirmed a statistically significant association between the development of RAO and increased levels of lipoprotein(a) (odds ratio: 9.48; p = 0.001) and factor VIII (odds ratio: 6.41; p = 0.024). There was a strong association between the presence of thrombophilic disorders and a personal or family history of thromboembolism (p = 0.01). Conclusions: Our results indicate that screening for thrombophilic disorders among selected young patients with RAO yields positive results in a high percentage of cases.

Pathogen reduction (PR) systems for platelets, based on chemically induced cross-linking and inactivation of nucleic acids, potentially prevent transfusion transmission of infectious agents, but can increase clinically significant bleeding in some clinical studies. Here, we documented the effects of PR systems on microRNA and mRNA levels of platelets stored in the blood bank, and assessed their impact on platelet activation and function. Unlike platelets subjected to gamma irradiation or stored in additive solution, platelets treated with Intercept (amotosalen+ ultraviolet-A [UVA] light) exhibited significantly reduced levels of 6 of the 11 microRNAs, and 2 of the 3 anti-apoptotic mRNAs (Bcl-xl and Clusterin) that we monitored, compared with platelets stored in plasma. Mirasol (riboflavin+ UVB light) treatment of platelets did not produce these effects. PR neither affected platelet microRNA synthesis or function nor induced cross-linking of microRNA-sized endogenous platelet RNA species. However, the reduction in the platelet microRNA levels induced by Intercept correlated with the platelet activation (p < 0.05) and an impaired platelet aggregation response to ADP (p < 0.05). These results suggest that Intercept treatment may induce platelet activation, resulting in the release of microRNAs and mRNAs from platelets. The clinical implications of this reduction in platelet nucleic acids secondary to Intercept remain to be established.

BackgroundThe quality of whole blood (WB)‐derived plasma preparations has been the subject of several studies, but there has been a lack of robust, comparative data for the different methods of processing and freezing.Study Design and MethodsSix WB‐derived plasma units were pooled and split (n = 16) and frozen within either 8 or 24 hours after WB collection, stored at 4°C or at room temperature (RT), and then frozen either slowly at −20°C or rapidly to below −30°C. Plasma units were tested for fibrinogen, Factor (F)V, FVII, FVIII, FXI, and von Willebrand factor (VWF), protein C (PC), protein S (PS) activity and free PS, prothrombin time, and partial thromboplastin time.ResultsFVIII was reduced by 9% to 19% after having been stored for 24 hours irrespective of storage temperature. Slow freezing (SF) reduced FVIII by 17% to 25% compared to rapid freezing (RF) to below −30°C. Storage temperature, but not 24‐hour storage, decreased PS activity by 20% to 28%. PS activity was 8% to 17% lower in plasma units frozen slowly compared to RF. Storage and freezing had no influence on free PS. SF caused small losses of FVII and FXI activity.ConclusionTwenty‐four‐hour hold at RT and SF both reduce FVIII levels below 70 U/dL in many plasma units. PS activity is affected substantially by storage temperature and SF, but free PS is not. With regard to plasma quality, freezing to below −30°C within 1 hour is superior to SF at −20°C.