Text Box: TSP-1 INDUCES APOPTOSIS IN MEGAKARYOCYTIC CELL LINES VIA CD36 AND CASPASE SIGNALING Yang M, Li K, Zhang XB, Su RJ, Yuen PMP, Li CK, Chik KW, Shing MK, Fok TF Department of Paediatrics, The Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong Objective: Megakaryocytopoiesis is positively regulated by TPO. It also seems to be regulated negatively by a feedback control mechanism that operates to maintain a constant level of platelets in peripheral circulation. Thrombospondin-1 (TSP1) is a major megakaryocyte and platelet alpha-granule glycoprotein. Our hypothesis is that TSP-1 may directly affect megakaryocytopoiesis by a feedback control mechanism through its binding to megakaryocytes via CD36 (TSP receptor), thus inducing a signal to cell apoptosis. Our previous results have showed that TSP1 significantly inhibited megakaryocyte growth in both murine and human CFU-MK assay. This inhibitory effect of TSP1 was reversed by the addition of anti-CD36 antibody (FA6-152) suggesting that the inhibition of TSP1 on megakaryocytopoiesis is probably mediated by TSP/CD36 interaction. Methods: The expression of CD36 on human platelets, megakaryocytes and the human megakaryoblastic cell lines: Meg-01, Dami, CHRF-288-11 and M-07e was investigated by RT-PCR, flow cytometry, ELISA and immunocytochemical staining. Results: The result showed that CD36 expression increases with megakaryocyte differentiation and maturation, suggesting that receptor for TSP1 may be upregulated during the cell differentiation. TSP1 (2.5 ug/ml) significantly inhibited Meg-01 (45.1%), Dami (43.3%) and CHRF-288-11 (31.2%) but not M-07e cell growth when compared with controls. This inhibitory effect was related to the levels of CD36 expression on the cells and the anti-CD36 antibodies abolished such effect. Furthermore, the apoptotic effect of TSP-1 was studied on megakaryocytic cell lines. Meg-01 and CHRF-288-11 cells were cultured for 2 days in IMDM with 2.5% FSC and TSP-1 (0 - 7.5 ug/ml). These cells were analyzed by total count, trypan blue exclusion assay, microscopy for apoptotic bodies, DNA ladder formation gel electrophoresis, annexin V and active caspase-3 expression using flow cytometry. Our results demonstrated that apoptosis, as indicated by all the studied parameters, was induced by TSP-1 at a dose dependent manner. At 2.5-5 ug/ml of TSP-1, we studied the effect of FA6-152 (10 ug/ml) and TPO (50 ng/ml) on the cultured cells. While FA6-152 alone did not exert any influence on the megakaryocytic cells, it significantly reversed the inhibition of TSP-1 (p< 0.05) in all the apoptotic parameters. The apoptotic effects of TSP-1 was also abolished by the addition of TPO (p < 0.05). Conclusions: Our results demonstrated that TSP-1 induced apoptosis in megakaryocytic cell lines via CD36 and caspase-3 signaling pathway. This mechanism could be regulated by growth factors such as TPO. These data provide supports to the possible existence of a negative feedback control on megakaryocytopoiesis exerted by the platelet granule protein TSP-1. 1987