PB0759 - MicroRNA-223-3p decreases platelet procoagulant activity through the regulation of TMEM16F

Background: Platelets are anucleate cells mostly involved in hemostasis through their aggregating properties and their ability to promote thrombin generation at their activated surface. Platelet reactivity is variable among individuals and microRNAs (miRNAs) may regulate platelet function. Among platelet-derived miRNAs, miR-223-3p is the most abundant. Furthermore, several studies have reported an association between miR-223-3p level and platelet reactivity or recurrence of cardiovascular events, but the impact of miR-223-3p on platelet function is poorly understood.

Aims: To investigate the implication of miR-223-3p in platelet reactivity in platelet-like structures (PLS) derived from human hematopoietic stem cells (CD34+).

Methods: MiR-223-3p upregulation and downregulation were carried out by transfecting CD34+-derived megakaryocytes with miR-223-3p mimic or CRISPR/Cas9 complexes, respectively. The quantification of procoagulant platelets (PP), defined as AnnexinV+ and CD62P+, was performed using flow cytometry. PLS-supported thrombin generation in human plasma was quantified with the Calibrated Automated Thrombogram using the velocity index. Finally, miR-223-3p’s target TMEM16F was validated using quantitative polymerase chain reaction and a reporter gene assay.

Results: Upregulation of miR-223-3p induced a 30±5% decrease in PP (n=7, p=0.001), a 10±3% decrease in PLS-supported thrombin generation (n=8, p=0.008), associated with a 47±10% decrease in TMEM16F mRNA expression (n=4, p=0.022), a phospholipid scramblase that has a key role in PP generation. Conversely, downregulation of miR-223-3p led to a 19±6% increase in PP (n=6, p=0.0092), and a 48±21% increase in TMEM16F mRNA expression (n=6, p=0.036). Co-transfection of the plasmid containing the 3’UTR_TMEM16F_WT and miR-223-3p in HT1080 cells resulted in a 24±3% reduction in luciferase activity (n=3 to 4, p=0.002), compared to control.

Conclusion(s): These data suggest that miR-223-3p modulates the generation of PP upon activation through the regulation of TMEM16F, a newly identified target of miR-223-3p.