Effects of Serotonin on Proplatelet Formation and F-actin Reorganization in Human Megakaryocytes.

We previously reported that serotonin (5-HT) is a growth factor for hematopoietic stem cells and megakaryocytic progenitor (Yang et al, Stem Cells, 2007). We further proposed a possible role of serotonin on megakaryocyte differentiation and platelet formation. The effect of serotonin on proplatelet formation and F-actin reorganization in human megakaryocytes (MKs) was investigated in this study. Our 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); and

• 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). Our studies showed that serotonin had a stimulating effect on proplatelet formation and F-actin reorganization in human megakaryocytes and this effect involved the 5-HT2 receptors and the activation of ERK1/2 pathway.