Background: Anucleate platelets are highly enriched in microRNAs (miRNAs), 19-22 nucleotide non-coding RNAs which function as translational suppressors by multiple mechanisms collectively referred to as RNA inhibition (RNAi). The ability to over-express or antagonize miRNAs ectopically in platelets is of substantial value for both research and clinical applications. Transfection of miRNAs in ex vivo platelets has been achieved using coupling reagents such as liposome carriers which are standard reagents for transfection of nucleated cells; however, transfection efficiencies with these approaches have generally been low as reported by us and others, limiting their utility.

Aims: We sought novel approaches to improve transfection efficiencies of microRNAs (miRNAs) in platelets, and to apply these approaches to investigate roles of miRNAs in regulating signal-activated protein translation and functional effects.

Methods: We found that ex vivo human platelets support gymnosis – internalization of ectopic miRNAs following co-incubation in the absence of conventional transfection reagents or schemes – and subsequently incorporate transfected miRNA into ARGONAUTE2 (AGO2)-based RNA-induced silencing complexes (RISC).

Results: Thrombin/fibrinogen stimulation activated translation of miR-223-3p target SEPTIN2, which was suppressed by miR-223-3p transfection in an AGO2/RISC-dependent manner. Thrombin/fibrinogen-induced exosome and microvesicle generation was inhibited by miR-223-3p transfection, and this effect was reversed with a RISC inhibitor. Platelet gymnosis of naked miRNAs was mediated in part by endocytic pathways including clathrin-dependent and fluid-phase endocytosis and caveolae.

Conclusions: These results demonstrate the ability of ex vivo platelets to internalize ectopic miRNAs by unassisted transfection, and utilize them to modulate signal-activated translation and platelet function. Our results identify new roles for miR-223-3p in extracellular vesicle generation in stimulated platelets. High efficiency gymnotic transfection of miRNAs in ex vivo platelets may be a broadly useful tool for exploring molecular genetic regulation of platelet function.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/high-efficiency-unassisted-transfection-of-platelets-with-naked-double-stranded-mirnas-modulates-signal-activated-translation-and-platelet-function/