Geneva Platelet Group, Faculty of Medicine, University of Geneva, Geneva, Switzerland.
Unité Mixte de Recherche S1255, INSERM, Strasbourg, France.
Thromb Haemost. 2021 Sep;121(9):1206-1219. doi: 10.1055/a-1497-9649. Epub 2021 Jun 18.
Several platelet-derived microRNAs are associated with platelet reactivity (PR) and clinical outcome in cardiovascular patients. We previously showed an association between miR-204-5p and PR in stable cardiovascular patients, but data on functional mechanisms are lacking.
To validate miR-204-5p as a regulator of PR in platelet-like structures (PLS) derived from human megakaryocytes and to address mechanistic issues.
Human hematopoietic stem cells were differentiated into megakaryocytes, enabling the transfection of miR-204-5p and the recovery of subsequent PLS. The morphology of transfected megakaryocytes and PLS was characterized using flow cytometry and microscopy. The functional impact of miR-204-5p was assessed using a flow assay, the quantification of the activated form of the GPIIbIIIa receptor, and a fibrinogen-binding assay. Quantitative polymerase chain reaction and western blot were used to evaluate the impact of miR-204-5p on a validated target, CDC42. The impact of CDC42 modulation was investigated using a silencing strategy.
miR-204-5p transfection induced cytoskeletal changes in megakaryocytes associated with the retracted protrusion of proPLS, but it had no impact on the number of PLS released. Functional assays showed that the PLS produced by megakaryocytes transfected with miR-204-5p were more reactive than controls. This phenotype is mediated by the regulation of GPIIbIIIa expression, a key contributor in platelet-fibrinogen interaction. Similar results were obtained after CDC42 silencing, suggesting that miR-204-5p regulates PR, at least in part, via CDC42 downregulation.
We functionally validated miR-204-5p as a regulator of the PR that occurs through CDC42 downregulation and regulation of fibrinogen receptor expression.
几种血小板衍生的 microRNA 与心血管疾病患者的血小板反应性 (PR) 和临床结局相关。我们之前曾显示 miR-204-5p 与稳定型心血管患者的 PR 相关，但缺乏关于功能机制的研究数据。
验证 miR-204-5p 作为人巨核细胞衍生的血小板样结构 (PLS) 中 PR 的调节剂，并解决相关机制问题。
用人造血干细胞分化为巨核细胞，从而实现 miR-204-5p 的转染和随后 PLS 的回收。使用流式细胞术和显微镜对转染巨核细胞和 PLS 的形态进行特征描述。通过流式分析、GPIIbIIIa 受体激活形式的定量、纤维蛋白原结合分析评估 miR-204-5p 的功能影响。使用定量聚合酶链反应和 Western blot 评估 miR-204-5p 对已验证靶标 CDC42 的影响。使用沉默策略研究 CDC42 调节的影响。
miR-204-5p 转染诱导巨核细胞骨架发生变化，与 proPLS 的回缩突起相关，但对释放的 PLS 数量没有影响。功能分析表明，转染 miR-204-5p 的巨核细胞产生的 PLS 比对照更具反应性。这种表型是通过 GPIIbIIIa 表达的调节介导的，GPIIbIIIa 是血小板-纤维蛋白原相互作用的关键贡献者。在 CDC42 沉默后也获得了类似的结果，这表明 miR-204-5p 通过下调 CDC42 至少部分调节 PR。
我们从功能上验证了 miR-204-5p 作为 PR 的调节剂，其通过下调 CDC42 和调节纤维蛋白原受体表达而起作用。
