PF806 SEPSIS INDUCED MRNA AND MIRNA LEVELS IN MEGAKARYOCYTES AND PLATELETS GENERATE ADVERSE PLATELET REACTIVITY

Abstract

Background:In sepsis, platelet activation by lipopolysaccharide (LPS) via TLR4 can result in microvascular thrombosis. However, megakaryocytes (MKs) also express TLRs, thus severe infection can modulate thrombopoiesis. Both MKs and platelets are rich in microRNAs (miRNAs) to regulate messenger RNA (mRNA) function and different cellular events. Murine MKs were previously described to produce platelets with altered mRNA profile in septic mice.Aims:We characterized sepsis‐induced expression of mRNAs with their regulatory miRNAs in ex vivo and in vitro stimulated platelets as well as in MK cell cultures. The contribution of Dicer function to abnormal miRNA levels was also investigated.Methods:Leukocyte‐depleted platelets of 22 septic and 26 healthy individuals were analyzed for P2RY12 and SELP (P‐selectin) mRNAs. TaqMan Open Array was performed for miRNAs, and RT‐qPCR were used for verification. The effects of sepsis were also studied in isolated platelets and MEG‐01 cells in vitro after treatment with recombinant TNF‐α (100 ng/mL) or LPS (O55:B5, 100 ng/mL) with lipoprotein binding protein (100 ng/mL) and soluble CD14 (150 ng/mL) for 1–24 h. Dicer level was observed by western blotting and fluorescence microscope, while its function was tested by calpain inhibitor (calpeptin, 10 μmol/L) in septic MKs and silencing by Dicer siRNA (40 pmol) in MEG‐01 cells in comparison to control samples with NEG‐01 siRNA for P2RY12 and SELP expression. To prove the effect of LPS, TLR4 expression was observed on platelets and MKs by flow cytometry. Induced inflammatory conditions were evaluated by miR‐155 expression and IL‐1β/IL‐6 mRNA levels by RT‐qPCR and via nuclear translocation of p65 subunit of NF‐κB pathway by fluorescence microscope.Results:There was augmented platelet activation as surface and soluble P‐selectin were increased in septic patients (P < 0.001). Platelets and MKs upregulated P2RY12 and SELP gene expression (P < 0.01) accompanied with elevated inflammation‐specific miR‐155 and decreased miR‐223/26b. Additionally, 64 other platelet miRNAs (e.g. miR‐150, let‐7e) indicated ≥2‐fold decrease, while 37 (e.g. miR‐191) were increased at the same degree in sepsis vs. controls. Septic conditions resulted in substantial p65 translocation into nuclei causing at least 2‐fold elevated miR‐155 and IL‐1β or IL‐6 mRNA levels (P < 0.05) in platelets and MKs in the ex vivo and in vitro experiments via TLR4 receptors that could be detected on the surface of platelets (3–7%) and MEG‐01 cells (8–12%). After a transient induction of Dicer and miRNAs at 1 h, reduced Dicer mRNA and protein levels were seen in LPS/TNF‐α treated platelets/MKs after 4–24 h causing attenuated miR‐223/26b. Calpain inhibition restored miRNA levels, while downregulation of Dicer generated higher P2RY12 and SELP levels in MEG‐01.Summary/Conclusion:Alteration in Dicer‐dependent miRNAs contribute to elevated mRNA levels in MKs and reactive platelets during sepsis.This study is supported by the GINOP‐2.3.2–15–2016–00043‐IRONHEART project.