Aim: Since RAP1B is critical for platelet functions, including hemostasis, this study was
conducted to identify RAP1B regulating microRNAs (miRNAs) in ex vivo stored platelets.
Background: Previous studies with platelets identified factors affecting RAP1B activity but
regulatory miRNAs that affect RAP1B protein expression have not been reported.
Objective: To understand the functional significance of miRNA mediated regulation of RAP1B in
stored platelets, using microRNA, miR-181a as an example.
Methods: A Tagged RNA Affinity approach (MS2-TRAP) was employed to identify miRNAs that
bound to the 3` untranslated region (3`UTR) of the RAP1B mRNA in HeLa cells as an assay system.
And subsequently, the mRNA 3’UTR:miRNA interactions were verified in platelets through the ectopic
expression of miR-181a mimic and appropriate controls. The interaction of such miRNAs with
RAP1B mRNA was also validated by qRT-PCR and Western analysis.
Results: Sixty-two miRNAs from MS2 assay were then compared with already known 171 platelet
abundant miRNAs to identify a common set of miRNAs. This analysis yielded six miRNAs (miR-
30e, miR-155, miR-181a, miR-206, miR-208a and miR-454), which are also predicted to target
RAP1B mRNA. From this pool, miR-181a was selected for further study since RAP1B harbors two
binding sites for miR-181a in its 3′UTR. Ectopic expression of miR-181a mimic in platelets resulted
in lowering the endogenous RAP1B at both mRNA and protein levels. Further, miR-181a ectopic
expression reduced the surface expression of the platelet activation marker, P-selectin.
Conclusion: MicroRNA-181a can target RAP1B and this interaction has the potential to regulate
platelet activation during storage.