The Roles of AGO2 in Megakaryopoiesis, Protein Expression, and Platelet Reactivity

Abstract

As the principal cellular agents initiating thrombus (clot) formation, the primary function of platelets is maintenance of blood hemostasis. These anucleate cell fragments lack nuclear DNA and so provide a unique model for investigating post-transcriptional regulation of gene expression via RNA interference (RNAi). Although platelets are not capable of transcribing nascent mRNA, they are able to maintain protein homeostasis throughout their ~10-day lifespan in circulation; how platelets are able to do so, considering they contain a functional proteasome, remain unclear. We hypothesized that platelet protein translation and homeostasis is driven by ongoing plasma-derived growth factor signaling balanced with degradation, which is regulated by Argonaute 2 (Ago2)-mediated RNAi. In nucleated cells, RNAi occurs via RNA-induced silencing complexes (RISC), in which a guide RNA such as a microRNA (miRNA) docked in an effector protein, typically Ago2, recruits a target mRNA via guide:target sequence complementarity and results in suppression of mRNA translation via multiple mechanisms. Though RNAi has not yet been fully explored in the context of anucleate cells, platelets contain the machinery and proteins necessary for RNAi as well as being highly enriched in a diverse array of miRNAs. Additionally, variations in platelet miRNAs in humans are associated with variable platelet reactivity and risk of thrombosis. We have found platelets to be capable of taking up exogenous double-stranded miRNA and have published on their subsequent ability to utilize the miRNA to modulate signal-activated translation and platelet function (Aim 1). In order to investigate the role of constitutive translation in circulating platelets (Aim 2) and the effect of Ago2 in regulation of platelet reactivity (Aim 3) we generated a novel platelet-specific Ago2 deleted (Ago2fl/fl/Pf4-Cre) mouse line and compared the proteome of knockout (KO) mice to wild-type (WT), by sex. Based on Ago2 roles in RNAi and miRNA enrichment in platelets, we predicted large-scale increases in platelet protein expression with megakaryocytic Ago2 deletion. However, only a small subset of genes was significantly upregulated. Among these was Ago1, which was significantly increased in both male and female Ago2 KO platelets. These results strongly suggest that Ago-mediated RNAi is essential for platelet development. Interestingly, deletion of platelet Ago2 resulted in decreased protein expression of a different subset of genes in platelet from female mice only; this sex-specific effect may be due to hormonal differences, namely estrogen, as many of these regulated proteins are encoded by estrogen-responsive genes. The gene set downregulated in Ago2 KO female platelets vs WT, but not males, corresponded to genes known to be coordinately regulated at the transcriptional level by ERβ in conjunction with Ago2 in breast cancer cells. These results point to a novel role for Ago2 in modulating the platelet transcriptome in megakaryocytes, affecting the mRNAome in the platelets they generate, in a hormone-dependent manner. Platelets from both male and female KO mice had significantly increased mean platelet volumes (MPV), not explained by changes in platelet production, lifespan, or protein content; however, KO megakaryocytes (MKs) had significantly increased size & ploidy, accounting for increased MPV, although femurs in the KO mice had fewer MKs. Furthermore, KO mice exhibited faster clot retraction and increased surface expression of integrin αIIbβ3. Platelets from male KO mice showed increased αIIbβ3 integrin activity and P-selectin expression following thrombin and U46619 stimulation, a thromboxane analogue, but these effects were not seen with collagen or ADP, pointing to agonist pathway-specific effects. Bioinformatics analysis revealed sex-specific changes in the platelet proteome of KOs to account for this change in reactivity; a cluster of mitochondrial enzymes modulated in male KOs only, involved in ROS metabolism and respiration, may be responsible for the observed difference in the thromboxane response. Taken together, our findings suggest Ago2-mediated RNAi to be an important regulator of megakaryopoeisis and platelet reactivity.