Department of Cardiovascular Sciences, University of Leicester and Leicester NIHR Biomedical Research Centre, Glenfield Hospital, Leicester, UK.
Platelets. 2022 Oct 3;33(7):1031-1042. doi: 10.1080/09537104.2022.2027903. Epub 2022 Feb 8.
Extracellular vesicles (EVs) released from activated platelets contain microRNAs, the most abundant of which is hsa-miR-223-3p. Endogenous hsa-miR-223-3p suppresses the expression of tissue factor (TF), the initiator of the extrinsic coagulation pathway, in endothelial cells. Monocytes can be induced to express TF to enhance coagulation, but the role of hsa-miR-223-3p in regulating monocyte TF remains unknown. This study examined whether hsa-miR-223-3p from platelet-derived EVs (pdEVs) affects TF expression in monocytes. THP-1 cells, differentiated into a monocyte-like phenotype with 1α,25-dihydroxyvitaminD3, were transfected with hsa-miR-223-3p mimic or control microRNA. Alternatively, THP-1 cells were incubated with pdEVs from PAR1-agonist peptide activated-platelets, as platelet releasate, or pdEVs isolated by ultracentrifugation. Transfection with hsa-miR-223-3p mimic resulted in significant reductions in TF protein, determined by western blotting and flow cytometry and reduced procoagulant activity, measured by a TF-specific factor Xa generation assay, compared to cells transfected with control microRNA. This reduction was reversed by co-transfection with hsa-miR-223-3p inhibitor, AntagomiR-223. Incubation of THP-1 cells with pdEVs also decreased TF expression; however, this was not reversed by AntagomiR-223. Taken together, monocyte TF expression is downregulated by hsa-miR-223-3p, but when transferred via pdEVs the effect was not reversed with Antagomir-223, suggesting other pdEV components may contribute to TF regulation. Tissue factor (TF), Factor VII (FVII), activated Factor VII (FVIIa), Factor X (FX), activated Factor X (FXa), extracellular vesicles (EVs), microvesicles (MVs), platelet-derived extracellular vesicles (pdEVs), protease-activated receptor 1 agonist peptide (PAR1-AP), lipopolysaccharide (LPS), P-selectin glycoprotein ligand-1 (PSGL-1), Tris-Buffered Saline Tween (TBST), room temperature (RT)[Figure: see text].
从激活的血小板释放的细胞外囊泡 (EVs) 含有 microRNAs，其中最丰富的是 hsa-miR-223-3p。内源性 hsa-miR-223-3p 抑制内皮细胞中组织因子 (TF) 的表达，TF 是外源性凝血途径的启动子。单核细胞可以被诱导表达 TF 以增强凝血，但 hsa-miR-223-3p 在调节单核细胞 TF 中的作用尚不清楚。本研究探讨了血小板来源的 EVs (pdEVs) 中的 hsa-miR-223-3p 是否影响单核细胞中的 TF 表达。用 1α,25-二羟维生素 D3 将 THP-1 细胞分化为单核细胞样表型，然后用 hsa-miR-223-3p 模拟物或对照 microRNA 转染。或者，用 PAR1 激动肽激活血小板释放的血小板释放物或超速离心分离的 pdEVs 孵育 THP-1 细胞。与转染对照 microRNA 的细胞相比，hsa-miR-223-3p 模拟物转染导致 TF 蛋白的显著减少，这通过 Western blot 和流式细胞术测定，TF 特异性因子 Xa 生成测定法测量的促凝活性降低。用 hsa-miR-223-3p 抑制剂 AntagomiR-223 共转染可逆转这种减少。用 pdEVs 孵育 THP-1 细胞也降低了 TF 表达;然而，用 AntagomiR-223 并不能逆转这种情况。总之，单核细胞 TF 的表达被 hsa-miR-223-3p 下调，但当通过 pdEVs 转移时，用 Antagomir-223 并不能逆转这种作用，这表明 pdEV 的其他成分可能有助于 TF 的调节。组织因子 (TF)、因子 VII (FVII)、激活的因子 VII (FVIIa)、因子 X (FX)、激活的因子 X (FXa)、细胞外囊泡 (EVs)、微泡 (MVs)、血小板衍生的细胞外囊泡 (pdEVs)、蛋白酶激活受体 1 激动肽 (PAR1-AP)、脂多糖 (LPS)、P 选择素糖蛋白配体-1 (PSGL-1)、三羟甲基氨基甲烷缓冲盐水吐温 (TBST)、室温 (RT)[图:见正文]。
