PB0872 - MicroRNA-223-3p is a regulator of platelet procoagulant activity

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 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.

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

Methods: CD34+-derived megakaryocytes were transfected with a miR-223-3p mimic, or a scramble miRNA and PLS were timely collected for functional tests. PLS production was quantified using a Tali Image-Based cytometer. GPIIb/IIIa activation, P-selectin expression, phosphatidylserine (PS) exposure and calcium flux were 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 evaluate the impact of miR-223-3p on the expression of selected genes.

Results: Increased levels of miR-223-3p did not have a significant impact on PLS production, GPIIb/IIIa activation, P-Selectin expression and intracellular calcium concentration during the PLS activation. However, transfection with miR-223-3p mimic induced a 30±5% decrease of PS exposure after activation (n=7, p=0.001, Figure 1), and a 10±3% decrease in PLS-supported thrombin generation (n=8, p=0.008, Figure 2), compared to mock condition. These findings were associated with a 47±10% decrease in TMEM16F mRNA expression (n=4, p=0.022).

Conclusion(s): MiR-223-3p overexpression is associated with a decrease of PS exposure, PLS-supported thrombin generation, and TMEM16F mRNA expression, without impacting calcium flux. This observation suggests that miR-223-3p could regulate PLS procoagulant activity through the regulation of TMEM16F, a key protein involved in membrane phospholipid scrambling.