Abstract 1934: Platelet Activation Regulates Levels of MicroRNA
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. Platelets are without genomic DNA but are known to retain a small amount of megakaryocyte-derived messenger RNA (mRNA) that can regulate protein expression. We have found that platelets have miRNAs by Real-Time PCR and in-situ hybridization and their regulatory proteins, Dicer and Ago2 by western blot, immuno-staining, and confocal microscopy. We assessed the hypothesis that miRNAs may play an important role in platelet functions via protein regulation. We performed miRNA profiling using miRNA microarray analysis after activating human platelets with thrombin or immune stimulation (PAM3CSK4) vs. resting platelets. Isolated and magnetically purified human platelets (pooled from 9 different subjects) were activated with 0.5 u/ml thrombin or 10 μg/ml PAM3CSK4 for two hours at room temperature. After lyses, total RNA including the small RNAs were isolated. Isolated RNAs underwent miRNA profiling using miRCURY LNA microRNA Profiling (Exiqon, Denmark). There was 1.6 – 4.4 fold increase in the specific levels of miRNAs; hsa-miR-107, hsa-miR-126*, hsa-miR-148b, hsa-miR-19a, hsa-miR-19b, hsa-miR-27a, hsa-miR-29a, hsa-miR-30b, hsa-miR-30c, hsa-miR-107, and hsa-miR-600. There was a 1.38 and 2.04 fold decrease in hsa-miR-26b using PAM3CSK4 or thrombin activation, respectively, vs. resting platelet. In summary, these data demonstrate that platelet miRNA profile is affected by thrombin and PAM3CSK4 activation. These findings also suggest a potentially new mechanism for platelet control of function via microRNA expression.