Dicer1-mediated miRNA processing shapes the mRNA profile and function of murine platelets

Human platelets contain microRNAs (miRNAs) and miRNA processing machinery, but their contribution to platelet function remains incompletely understood. Here, we show that murine megakaryocyte (MK)-specific knockdown of Dicer1, the ribonuclease that cleaves miRNA precursors into mature miRNAs, reduces the level of the majority of miRNAs in platelets. This leads to altered platelet messenger RNA (mRNA) expression profiles and mild thrombocytopenia. Fibrinogen receptor subunits Itga2b (alpha(IIb)) and Itgb3 (beta(3)) mRNAs were among the differentially expressed transcripts that are increased in platelets lacking Dicer1. Argonaute 2 (Ago2), a member of the miRNA silencing complex, co-immunoprecipitated with alpha(IIb) and beta(3) mRNAs in wild-type platelets. Furthermore, co-immunoprecipitation experiments suggested reduced alpha(IIb)/beta(3)/Ago2 complexes in miRNA-deficient platelets. These results suggested that miRNAs regulate both integrin subunits. Subsequent 39 untranslated region luciferase reporter assays confirmed that the translation of both alpha(IIb) and beta(3) mRNAs can be regulated by miRNAs miR-326, miR-128, miR-331, and miR-500. Consistent with these molecular changes, the deletion of Dicer1 resulted in increased surface expression of integrins alpha(IIb) and beta(3), and enhanced platelet binding to fibrinogen in vivo and in vitro. Heightened platelet reactivity, shortened tail-bleeding time, and reduced survival following collagen/epinephrine-induced pulmonary embolism were also observed in Dicer1-deficient animals. Combined Pf4-cre-mediated deletion of Drosha and Dicer1 did not significantly exacerbate phenotypes observed in single Dicer1 knockout mice. In summary, these findings indicate that Dicer1-dependent generation of mature miRNAs in late-stageMKs and platelets modulates the expression of target mRNAs important for the hemostatic and thrombotic function of platelets.