PB0394 - Argonaute2 (Ago2) modulates megakaryocyte development and platelet reactivity in a sex-specific manner via suppression of mitochondrial genes; evidence for diverse RNA granule species in platelets.

Background: Platelets are enriched in miRNAs and harbor Ago2 as the principal RNA silencing Argonaute. However, roles in thrombopoiesis and platelet function remain poorly understood.

Aims: Determine gene regulatory and functional roles of Ago2 in megakaryocytes and platelets.

Methods: We generated megakaryocyte/platelet-specific Ago2-deleted mice and assessed proteomic and functional effects.

Results: We predicted platelet hyperreactivity with Ago2 deletion due to large-scale upregulated protein expression. Platelet counts were normal. Mean volumes were increased, associated with larger, though fewer megakaryocytes. Cremaster muscle arteriolar clot dynamics after laser injury were normal, and no effects on hemostasis were observed by tail tip amputation. Ex vivo platelets from male mice showed hyperreactivity to thromboxane but not other agonists, whereas female-derived platelets showed no changes in reactivity. We compared expressed proteomes of resting Ago2-deleted and WT platelets by LC/MS/MS-based label-free quantitation. Again surprisingly, proteomes were mostly similar, although multiple mitochondrial proteins were increased. However, Ago1 - undetectable in WT platelets - was upregulated in Ago2-deleted platelets; immunoblotting confirmed Ago1 induction. Female Ago2-deleted platelets selectively showed downregulation of a protein cohort, established in breast cancer cells to be transcriptionally regulated by Ago2 coupled to estrogen receptor-beta. Interestingly, male Ago2-deleted platelets showed increased mitochondrial membrane potential. Immunolabeling of unstimulated human platelets revealed AGO2 condensation in punctate structures distinct from early/late endosomes and alpha/dense granules, and in mitochondria. MitoRNAs co-precipitated with AGO2 from human platelets. In addition, immunolabeling with selected markers revealed distinct Ago2-negative punctate structures suggestive of P-bodies (p54/DDX6) and GW-bodies (GW182) – non-membranous ribonucleoprotein granules involved in mRNA storage and translational regulation, previously unreported in platelets.

Conclusion(s): Ago2-mediated RNA silencing is important for platelet development and function, putatively rescued by Ago1. Ago2-specific modulation of mitochondrial genes regulates platelet reactivity via control of mitochondrial activity. Sex-specific platelet reactivity controlled by Ago2 may reflect sex-specific regulation, putatively at the transcriptional level in megakaryocytes.