Quantification of the released polyP with JC-D8 polyP-specific fluorescent probe. and TF loading were assessed using fluorescence microscopy, circulation cytometry, myeloperoxidase(MPO)/DNA complex ELISA, and a Western blot. Ticagrelor interrupts plateletCneutrophil conversation by attenuating NETs induced by polyP. However, Ticagrelor does not impact polyP secretion from thrombin-activated platelets. Similarly, the intracellular production of TF in neutrophils brought on by IRA plasma is not hindered by Ticagrelor. Furthermore, DES induce NETs and synchronous activation with IRA plasma prospects to the formation of thrombogenic TF-bearing NETs. Ticagrelor inhibits stent-induced NET release. These findings suggest a novel immune-modulatory effect of Ticagrelor when it attenuates the formation of thrombogenic NETs. 0.05. All conditions were compared to untreated/control condition and statistical significance is usually indicated by the sign *. Any further statistical significance of other comparisons is usually indicated by the sign #. CHMFL-ABL/KIT-155 (d). Annexin V/Propidium Iodide circulation cytometry of control neutrophils in the presence or absence of Ticagrelor/Clopidogrel. One representative out of six impartial experiments is shown. Polymorphonuclear neutrophils (PMNs). In order to further strengthen our in vitro findings, we performed activation experiments in neutrophils obtained from coronary artery disease (CAD) patients receiving Ticagrelor or Clopidogrel and from healthy individuals (controls). The basal levels of NETs in CAD patients were low and comparable to that of controls (Physique 2e). Ticagrelor-treated CAD-patients-derived neutrophils were more resistant to NETotic activation from polyP when compared to control neutrophils under comparable polyP doses. This suggests that Ticagrelor exerts anti-thrombo-inflammatory effects by attenuating NETs (Physique 2a,b,d,e). On the other hand, Clopidogrel-treated CAD-patients-derived neutrophils do not have diminished NET release (Physique 2a,cCe). The formation of NETs was evaluated by Immunofluorescence, MPO/DNA ELISA. Open in a separate window Physique 2 Neutrophils from individuals receiving Ticagrelor were more resistant to NETotic activation from DNM1 polyP. (aCc). Fluorescence microscopy for cit-H3/NE staining in neutrophils isolated from a patient with a previous acute coronary syndrome and stent placement that receives Ticagrelor or Clopidogrel as a main antiplatelet treatment and neutrophils from a healthy individual, with or without synthetic polyP. One representative out of five impartial experiments is shown. Initial magnification: 600, Level bar: 5 m. Blue: DAPI, Green: NE, Red: cit-H3. (d). Percentage of NET-releasing neutrophils as assessed by immunofluorescence. (e). MPO-DNA complex levels in NET structures from these stimulations, as assessed by ELISA. Data from five impartial experiments offered as mean SD. Statistical significance * 0.05. All conditions were compared to untreated/control condition and statistical significance is usually indicated by the sign *. Polymorphonuclear neutrophils (PMNs). Since Ticagrelor inhibited the formation of NETs induced by polyP and considering that polyP is the major mediator of platelet-induced NETosis, we next investigated the role of Ticagrelor in polyP secretion from platelets. We found that Ticagrelor and Clopidogrel do not affect polyP release from thrombin-activated platelets, as assessed by circulation cytometry and fluorometry (Physique 3). Open in CHMFL-ABL/KIT-155 a separate window Physique 3 Ticagrelor does not inhibit polyP release from platelets. (a). Representative circulation cytometry analysis and (b). relative mean fluorescent intensity (MFI) of polyP on control platelets treated with thrombin, with or without pre-treatment with Ticagrelor or Clopidogrel. MFImean fluorescence intensity. (c). Quantification of the released polyP with JC-D8 polyP-specific fluorescent probe. Relative I integrated optical density OD was calculated compared to control platelets value. CHMFL-ABL/KIT-155 (a). One representative out of six impartial experiments is shown. (b,c) Data from six impartial experiments offered as mean SD. Statistical significance * 0.05. n.s.: non-significant. All conditions were compared to an untreated/control condition and statistical significance is usually indicated by the sign *. Any further nonstatistical significance of other comparisons is usually indicated by the sign n.s.. The results suggest that, beyond its antiplatelet effects, Ticagrelor exerts direct immune-regulatory properties on neutrophils without affecting polyP release from platelets. 2.2. Ticagrelor Effect on CHMFL-ABL/KIT-155 Neutrophils Does not Rely on P2Y12 Receptor and Autophagy We sought to investigate signaling pathways related to the action of Ticagrelor and NET formation, such as the P2Y12 receptor and the autophagy pathway, respectively. Based on the above.
Quantification of the released polyP with JC-D8 polyP-specific fluorescent probe