Abstract 212: Thrombin Stimulation Induces de novo SDF-1a Synthesis and Alters MicroRNA Profile in Human Platelets
Platelets are anucleated cells, but still retain certain capacity of protein synthesis. Platelets are also known to contain microRNAs (miRNAs), which can negatively regulate mRNA translation/protein synthesis. We asked if platelet activation can initiate de novo protein synthesis of angiogenic regulators and if platelet activation induces changes of platelet miRNA expression. Platelet rich plasma was prepared from citrated blood of 12 healthy subjects. Platelets were purified by a second centrifugation and using leukocyte-depleting CD45-Dynabeads, and then treated without or with thrombin (0.1 U/ml, 30 min, 37°C). Platelet lysates were prepared immediately after treatments or after 16 h-culture for Western blottings of stromal cell derived factor-1a (SDF1a) and angiostatin. Total platelet RNA was isolated with a mirVana miRNA isolation kit, and platelet miRNA expression profiling was performed using an Agilent’s miRNA microarray system. Thrombin stimulation reduced platelet immunoblotting intensities/protein contents of SDF-1a and angiostatin as compared to unstimulated platelets, suggesting release of the angiogenic regulators upon platelet activation. SDF-1a, but not angiostatin, content was partially recovered after 16 h culture, indicating de novo protein synthesis. Using mature mRNA-specific primers, SDF-1a, but not angiostatin, mRNA was detected in thrombin-activated but not in unstimulated platelets. Furthermore, miRNA array analysis demonstrated that thrombin stimulation down-regulated a panel of platelet miRNA expression (e.g., miR-23a, miR-23b, miR-24, miR-106b, and miR-107) but also up-regulated a group of miRNA expression (e.g., miR-96, miR-212, miR-449a, and miR-629). In conclusion, thrombin stimulation induces mRNA splicing and protein synthesis of SDF-1a in platelets. Thrombin activation also alters platelet miRNA profile that may have a major impact on de novo protein synthesis in activated platelets.
- © 2012 by American Heart Association, Inc.