214: Interpreting troponin elevation in relation to symptom onset in intermediate-risk pulmonary embolism

BackgroundTroponin elevation in the setting of acute pulmonary embolism (PE) is of small magnitude and short duration and can go unnoticed in pts referred late after symptom onset.MethodsProspective, single-center registry of pts with confirmed intermediate-risk PE, defined as at least 1 echocardiographic finding of right ventricular (RV) dysfunction (endo-diastolic (EDRV)/left ventricular (EDLV) end-diastolic diameter ratio >=1 in the 4-chamber view, paradoxical septal systolic motion or pulmonary hypertension defined as RV/atrial gradient >30mmHg), or positive troponin test. Combined in-hospital endpoint was defined as death, non-fatal recurrent PE, or residual pulmonary vascular obstruction (RPVO) ≥35%.Results282 pts were included, age 66±14 years, 59% women, 174 (62%) referred ≤5 days after symptom onset, 108 (38%) after >5 days. Troponin elevation was observed in 126 (72%) treated within ≤5 days, in 60 (56%) treated after >5 days (p=0.004). A significant interaction was observed between time since symptom onset and both troponin elevation and persistence of EDRV/EDLV diameter ratio>1 at 48h. The negative predictive value of troponin elevation was 85% in patients treated within 5 days of symptoms, but fell to 70% in those admitted >5 days after symptom onset (p=0.002). Positive troponin was an independent predictor of adverse outcome (OR=1.43 [1.08-5.56]). ROC curves show that prognostic value of positive troponin test was higher in pts referred ≤5 days than in pts referred >5 days after symptom onset (p=0.01).ConclusionThere is a significant relation between troponin elevation and time since symptom onset in patients with intermediate-risk PE. Negative predictive value of troponin elevation is adequate in pts treated early (≤5 days) but is suboptimal in pts treated >5 days after symptom onset.TableResults<=5 days since symptom onset>5 days since symptom onsetpSensitivity72% (61.3-82.7)51% (42.4-59.6)0.005Specificity42% (44.5-49.5)47% (39.1-54.9)0.33PPV26% (18.4-33.6)30% (22.2-37.8)0.81NPV85% (78.4-91.6)70% (63-77)0.00

HISTAMINE ET ARYTHMIES DE REPERFUSION (UNE ETUDE PAR MICRODIALYSE SUR COEURS ISOLES DE RAT (DES BIOL. MED.))

BESANCON-BU Médecine pharmacie (250562102) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Les anévrismes de l'aorte thoracique

BESANCON-BU Médecine pharmacie (250562102) / SudocSudocFranceF

L'expression des récepteurs AT2 de l'angiotensine II dans le syndrome coronarien aigu et impact des statines

BESANCON-BU Médecine pharmacie (250562102) / SudocSudocFranceF

Protection myocardique (de l'approche pharmacologique à la thérapie cellulaire)

BESANCON-BU Médecine pharmacie (250562102) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Comparisons of detergents for purification of platelet membrane lipid rafts: a lipidomics and proteomics analysis focusing on P2XR localization

Introduction: Platelet membrane contains cholesterol and sphingomyelin-rich microdomains also known as lipid rafts (LR). LRs have been considered as pharmacological target of anti-aggregation drugs as they suggested providing a platform for physiological activity of target proteins such as purinergic receptors (P2XR, P2YR) activated by nucleotides (ATP and ADP respectively). Localization of P2XR in these microdomains remains mostly unknown. The aim of this study was to compare the effectivity of different conventional detergents for lipid rafts isolation and investigation on the lipidomics and proteomics features of these microdomains focusing their potentiality of containing P2XR. Obviously, given the lack of well-established methods for platelet membrane studies, we also aimed at comparing the effectivity of different conventional detergents studies of plasma membrane studies. Material and methods: Platelets were obtained from healthy donors from Etablissement Franc_ais du Sang. Platelets were lysed with three different detergents (Brij 35, lubrol and TritonX100) in low and high doses (0.05% and 1%). After density separation of platelets lysate using ultracentrifugation with sucrose gradient (5, 35 and 45%), 12 equal sucrose fractions were collected and concentration of cholesterol (Ch), sphingomyelin (SM) and phosphatidyl-choline (PC) were quantified. Based on these lipidomics results, fractions containing raft and nonraft domains were subjected to proteomics analysis. Results: LRs mainly were observed in fractions 1–4 in all detergents except Triton 0.05% that was not being able to extract lipid rafts. Ch and SM enrichment analysis were performed by Ch/PC and SM/PC ratio in each fraction. It showed that Lubrol 1% and Triton 1% were more suitable detergents as they were able to isolate raft fractions enriched in Ch and SM about 1.5 times more than other detergents (Lubrol 1%: Ch/PC = 1.52 and SM/PC= 1.44; Triton 1%: Ch/PC = 1.77 and SM/PC= 1.48). By proteomics analysis; overall 822 proteins were identified in platelet membrane. Each detergent revealed a different profile of protein’s set. P2XR was found only in fractions contain lipid rafts by Brij 1% and Lubrol 0.05%. Discussion / Conclusion: Investigating on P2XR on the membrane of platelets, Brij 1% and Lubrol 0.05% would be suitable detergents. Our results suggest that depending on the focal objective of study, adopting a compatible detergent has a great importance

Impact of ticagrelor on P2Y1 and P2Y12 localization and on cholesterol levels in platelet plasma membrane

Ticagrelor is an antiplatelet agent that inhibits platelet activation via P2Y12 antagonism. There are several studies showing that P2Y12 needs lipid rafts to be activated, but there are few data about how ticagrelor impacts lipid raft organization. Therefore, we aimed to investigate how ticagrelor could impact the distribution of cholesterol and consequently alter the organization of lipid rafts on platelet plasma membranes. We identified cholesterol-enriched raft fractions in platelet membranes by quantification of their cholesterol levels. Modifications in cholesterol and protein profiles (Flotillin 1, Flotillin 2, CD36, P2Y1, and P2Y12) were studied in platelets stimulated by ADP, treated by ticagrelor, or both. In ADP-stimulated and ticagrelor-treated groups, we found a decreased level of cholesterol in raft fractions of platelet plasma membrane compared to the control group. In addition, the peak of cholesterol in different experimental groups changed its localization on membrane fractions. In the control group, it was situated on fraction 2, while in ADP-stimulated platelets, it was located in fractions 3 to 5, and in fraction 4 in ticagrelor-treated group. The proteins studied also showed changes in their level of expression and localization in fractions of plasma membrane. Cholesterol levels of plasma membranes have a direct role in the organization of platelet membranes and could be modified by stimulation or drug treatment. Since ticagrelor and ADP both changed lipid composition and protein profile, investigating the lipid and protein composition of platelet membranes is of considerable importance as a focus for further research in anti-platelet management

Membrane Environment Modulates Ligand-Binding Propensity of P2Y12 Receptor

International audienceThe binding of natural ligands and synthetic drugs to the P2Y12 receptor is of great interest because of its crucial role in platelets activation and the therapy of arterial thrombosis. Up to now, all computational studies of P2Y12 concentrated on the available crystal structures, while the role of intrinsic protein dynamics and the membrane environment in the functioning of P2Y12 was not clear. In this work, we performed all-atom molecular dynamics simulations of the full-length P2Y12 receptor in three different membrane environments and in two possible conformations derived from available crystal structures. The binding of ticagrelor, its two major metabolites, adenosine diphosphate (ADP) and 2-Methylthioadenosine diphosphate (2MeS-ADP) as agonist, and ethyl 6-[4-(benzylsulfonylcarbamoyl)piperidin-1-yl]-5-cyano-2-methylpyridine-3-carboxylate (AZD1283)as antagonist were assessed systematically by means of ensemble docking. It is shown that the binding of all ligands becomes systematically stronger with the increase of the membrane rigidity. Binding of all ligands to the agonist-bound-like conformations is systematically stronger in comparison to antagonist-bound-likes ones. This is dramatically opposite to the results obtained for static crystal structures. Our results show that accounting for internal protein dynamics, strongly modulated by its lipid environment, is crucial for correct assessment of the ligand binding to P2Y12

Validation of an HPLC–MS/MS Method for the Determination of Plasma Ticagrelor and Its Active Metabolite Useful for Research and Clinical Practice

Ticagrelor is an antiplatelet agent which is extensively metabolized in an active metabolite: AR-C124910XX. Ticagrelor antagonizes P2Y12 receptors, but recently, this effect on the central nervous system has been linked to the development of dyspnea. Ticagrelor-related dyspnea has been linked to persistently high plasma concentrations of ticagrelor. Therefore, there is a need to develop a simple, rapid, and sensitive method for simultaneous determination of ticagrelor and its active metabolite in human plasma to further investigate the link between concentrations of ticagrelor, its active metabolite, and side effects in routine practice. We present here a new method of quantifying both molecules, suitable for routine practice, validated according to the latest Food and Drug Administration (FDA) guidelines, with a good accuracy and precision (&lt;15% respectively), except for the lower limit of quantification (&lt;20%). We further describe its successful application to plasma samples for a population pharmacokinetics study. The simplicity and rapidity, the wide range of the calibration curve (2&ndash;5000 &micro;g/L for ticagrelor and its metabolite), and high throughput make a broad spectrum of applications possible for our method, which can easily be implemented for research, or in daily routine practice such as therapeutic drug monitoring to prevent overdosage and occurrence of adverse events in patients

Comparative lipidomics and proteomics analysis of platelet lipid rafts using different detergents

Lipid rafts play a pivotal role in physiological functions of platelets. Their isolation using nonionic mild detergents is considered as the gold standard method, but there is no consensual detergent for lipid raft studies. We aimed to investigate which detergent is the most suitable for lipid raft isolation from platelet membrane, based on lipidomics and proteomics analysis. Platelets were obtained from healthy donors. Twelve sucrose fractions were extracted by three different detergents, namely Brij 35, Lubrol WX, and Triton X100, at 0.05% and 1%. After lipidomics analysis and determination of fractions enriched in cholesterol (Ch) and sphingomyelin (SM), proteomics analysis was performed. Lipid rafts were mainly observed in 1-4 fractions, and non-rafts were distributed on 5-12 fractions. Considering the concentration of Ch and SM, Lubrol WX 1% and Triton X100 1% were more suitable detergents as they were able to isolate lipid raft fractions that were more enriched than non-raft fractions. By proteomics analysis, overall, 822 proteins were identified in platelet membrane. Lipid raft fractions isolated with Lubrol WX 0.05% and Triton X100 1% contained mainly plasma membrane proteins. However, only Lubrol WX 0.05 and 1% and Triton X100 1% were able to extract non-denaturing proteins with more than 10 transmembrane domains. Our results suggest that Triton X100 1% is the most suitable detergent for global lipid and protein studies on platelet plasma membrane. However, the detergent should be adapted if investigation of an association between specific proteins and lipid rafts is planned