Journal: International Journal of Molecular Sciences
Article Title: Characterization of Biomarkers of Thrombo-Inflammation in Patients with First-Diagnosed Atrial Fibrillation
doi: 10.3390/ijms25074109
Figure Lengend Snippet: Pro-inflammatory immune cell function in FDAF—Indications for PAR1 activation via thrombin. ( A ) Increased plasma levels of pro-inflammatory mediators, namely tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6), were seen in patients with FDAF. ( B ) Phenotyping via flow cytometry of peripheral blood mononuclear cells (PBMCs) revealed a higher percentage (% of total PBMCs) of circulating pro-inflammatory cells that possess thrombin-activated (cleaved) PAR1 (TNF-α + or IL-6 + cleaved PAR1 + ) in patients with FDAF. The antibody detects fragments of activated PAR1-cleaved-Ser42 protein. Cytokines TNF-α and IL-6 were visualized via intracellular staining of permeabilized cells. ( C – E ) Evaluation of different immune cell subsets expressing PAR1 via flow cytometry. Antigen-presenting cells (APCs; HLA-DR + ) ( C ), non-T cells (CD3 − ) ( D ), and lymphocytes ( E ). Left panel: percentage of a specified immune cell population expressing PAR1. Right panel: Increased mean fluorescence intensity (MFI) of PAR1. Extracellular (surface) PAR1 expression (=PAR1 that is accessible for activation via FIIa) was detected via staining of unpermeabilized PBMCs with a PAR1 antibody. Patients with FDAF ( n = 80) were compared to controls (patients with chronic cardiovascular diseases but without AF) ( n = 20). Results are expressed as single values, median, and p -values.
Article Snippet: The percentage of FIIa-activated PAR1-positive (the antibody detects fragments of activated PAR1-cleaved-Ser42 protein) (Sigma-Aldrich, St. Louis, MO, USA) (visualized with goat anti-rabbit IgG Alexa 405; Thermo Fisher Scientific, Waltham, MA, USA) circulating PBMCs was measured by using flow cytometry with FACSCaliburTM and CellQuest software 5.1 (both from Becton, Dickinson and Company, Franklin Lakes, NJ, USA).
Techniques: Cell Function Assay, Activation Assay, Flow Cytometry, Staining, Expressing, Fluorescence