RNAseq of gingival fibroblasts exposed to activated platelets
Platelet-rich fibrin (PRF) contains not only platelets but also leucocytes and the coagulated plasmatic fraction of centrifuged blood. Conclusions about the contribution of platelets to the overall PRF activity are, therefore, indirect. To overcome this limitation, we have used washed leucocyte-depleted platelets in our bioassays. The platelets were activated with thrombin to release the contents of their granules into the serum-free culture medium. Gingival fibroblasts were exposed to this platelet-released supernatant (PRS), and transcriptional changes were identified by RNA sequencing. Under the strict criteria of a minimal 2.0-log2 fold-expression change and a significance level of 3.0-log10, we could identify 147 up-regulated and 39 down-regulated genes. Among the genes recently identified with PRF lysates were IL11 and CXCL8, and a series of genes that indicate the mitogenic activity of PRS such as centromere-associated proteins (CENPA, E, F, M, U, W), cell division cycle proteins (CDCA2, A3, A5, A8, 6, 20, 25A, 25C, 45), kinesin-like proteins (KIF11, 14, 18A, 18B, 20A, 23, 4A, C1), and shugoshin 1 and 2, as supported by gene ontology. Verification by RT-PCR and immunoassays further confirmed the significant increase of IL11 and CXCL8 when gingival fibroblasts were exposed to PRS. Consistently, PRS provokes TGF-ß but not NF-?B signaling, based on Smad2/3 and p65 translocation, respectively. In summary, our in vitro data provide support for the contribution of platelets in PRF, independent of leukocytes and plasma components, to elicit a robust response from gingival fibroblasts. Overall design: We used washed leucocyte-depleted platelets in our bioassays. The platelets were activated with thrombin to release the contents of their granules into the serum-free culture medium. Gingival fibroblasts were exposed to this platelet-released supernatant (PRS), and transcriptional changes were identified by RNA sequencing. Verification by RT-PCR and immunoassays further confirmed the significant increase of IL11 and CXCL8 when gingival fibroblasts were exposed to PRS.