Abstract 278: Platelet MicroRNA is Altered by Thrombin-Induced Aggregation
MicroRNAs (miRNAs) are recently discovered small RNAs that play an important role in the negative regulation of gene expression. MicroRNAs are approximately 22 nucleotides in length and derived from larger hairpin-forming precursors. Pre-miRNAs are cleaved by a protein (Dicer) to release the mature miRNA which binds to Argonaute 2 (Ago2) and mediates gene silencing. Platelets are without genomic DNA but are known to retain a small amount of megakaryocyte-derived messenger RNA (mRNA) that can regulate protein expression. It is not known if platelets have miRNA; therefore, we isolated human platelet RNA and proteins to determine if this system is present. Total RNA, including small RNAs, were isolated from washed platelets and studied by real-time PCR (qRT-PCR). 34 mature miRNA were found to be highly expressed in platelets. Specific miRNA (hsa-miR-142–3p) was confirmed to be present in platelets and megakaryocytes by in situ hybridization and confocal microscopy. The regulatory proteins, Dicer and Ago2 were present in platelets as seen by western blot, immunostaining, and confocal microscopy. Dicer, Ago2, and hsa-miR-142–3p were found in the same platelet using a combination of in situ hybridization and immunostaining with confocal microscopy. Dicer and Ago2 mRNA was also present in platelets as measured by qRT-PCR. When platelets were thrombin activated, expression of the specific miRNAs; hsa-let7a, hsa-let7d, hsa-miR-26a, hsa-miR-106a, hsa-miR-125b, hsa-miR-126#, hsa-miR-142–3p, hsa-miR-222, and hsa-miR-335 were down regulated after 2 hours between 0.76 fold to 0.53 fold (P<0.05). In summary, these data demonstrate for the first time that platelets have miRNA essential proteins and select mature miRNAs. In addition, platelet activation can regulate miRNA expression. These findings suggest a potentially new mechanism for platelet control of function via protein expression.