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 highly expressed. 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. The objective of the project is to investigate the role of miR-223-3p on platelet reactivity in platelet-like structures (PLS) derived from human hematopoietic stem cells (CD34+).

MiR-223-3p upregulation and downregulation were carried out by transfecting CD34+-derived megakaryocytes with miR-223-3p mimic or CRISPR/Cas9 complexes, respectively. PLS were then timely collected for functional tests. Phosphatidylserine (PS) exposure was performed using flow cytometry. PLS-supported thrombin generation in human plasma was quantified with the Calibrated Automated Thrombogram using the velocity index. Finally, quantitative polymerase chain reaction was used to quantify the expression of miRNAs and selected mRNAs.

Increased levels of miR-223-3p induced a 30±5% decrease in PS exposure after activation (n=7, p=0.001), and a 10±3% decrease in PLS-supported thrombin generation (n=8, p=0.008), compared to mock condition. These findings were associated with a 47±10% decrease in TMEM16F mRNA expression (n=4, p=0.022), a major contributor in PS exposure. Transfection with CRISPR/Cas9 complexes led to a 56±5% decrease in the expression of miR-223-3p (n=7, p<0.0001) and induced a 19±6% increase in PS exposure after activation (n=6, p=0.0092), compared to the negative control.

MiR-223-3p upregulation is associated with a decrease of PS exposure, PLS-supported thrombin generation, and TMEM16F mRNA expression. This observation suggests that miR-223-3p could regulate PLS procoagulant activity through the regulation of TMEM16F. CRISPR/Cas9 editing tool allowed to significantly decrease miR-223-3p and confirmed the involvement of miR-223-3p in PS exposure regulation.