The “Feedback” Regulation of Thrombopoiesis: The Role of Serotonin on Proplatelet Formation and TPO Production

We previously reported that serotonin (5-HT) is a growth factor for hematopoietic stem cells and megakaryocytic cells (Yang & Ye et al, Stem Cells, 2007; 2014). This study further proposed a possible role of serotonin on platelet formation and TPO production. Results showed that there was a stimulating effect of serotonin on proplatelet formation in human bone marrow megakaryocytes. Human BM MK progenitors cultured in serum free medium with either 5-HT (200nM) or TPO (50 ng/ml) had more proplatelet bearing MKs than the control group (5-HT (11.33 % ± 4.93) vs. control (6 % ± 3.60), P=0.026; TPO (14.66 % ± 1.53) vs. control, P=0.043; n=3). The 5-HT treatment group showed more mature and more in the final stage MK cells as compared to TPO group. The effect of serotonin on proplatelet formation in Meg-01 cells were via 5-HT2 receptors.Meg-01 cells strongly expressed 5-HT 2A, 2B, 2C receptors by using western blot method. 5-HT also promoted proplatelet formation in these cells and this effect was reduced by 5-HT2 receptor inhibitor ketanserin (KE). Serotonin acted on cytoskeleton reorganization in human megakaryocytes via 5-HT2 receptors and ERK1/2 pathway. Using an immunofluorescence microscope with F-actin specific binder rhodamine-phalloidin staining, the polymerized actin level was lower in the control group (serum free) than the 5-HT group and actin distributed diffusely throughout the cytoplasm. In contrast, polymerization actin level was higher in 5-HT group. Adding ketanserin and ERK1/2 inhibitor PD98059 to 5-HT treatment, the fluorescence intensity was correspondingly reduced (5HT vs. Control, P=0.006; 5-HT vs.5-HT plus KE, P=0.014; n=6). Our data also demonstrated that ERK1/2 was activated in MK cells treated with 5-HT for 30 minutes (21.76 % ± 7.42). In a traumatic stress mice model, both of 5-HT and TPO were increased, but the increasing of TPO is posterior to 5-HT. After added LX1606, the synthesis inhibitor of 5-HT, the concentration of 5-HT was reduced markedly, as well as TPO. The expression of TPO mRNA and the production of TPO protein were increased as compared with the control. This study suggests that 5-HT promotes thrombopoiesis from two aspects: one is the direct effect on megakaryocytes. It can promote the cells proliferation and reduce the apoptosis, and accelerate the maturation of megakaryocytes. The second is the indirect effect by promoting the production of TPO from bone marrow stromal cells, which is a paracrine secretion to influence thrombopoiesis.