The Function And Mechanism Of Thrombin And Coagulation Factor XA In MEG-01 Cells Differentiation

Thrombin and coagulation factor Xa (FXa) are the important blood coagulation factors, the function and mechanism of thrombin and FXa on the differentiation of megakaryocyte was studied.Thrombin is one kind of mitogen, which can induce many kinds of cell function. Thrombin was used as the agonist firstly, the function and mechanism of thrombin in Meg-01 cells differentiation were investigated. TPO was chosen as positive control. Meg-01 cells proliferation was detected by CCK-8 assay. The viability of platelet-like particles was analyzed by AlamaBlue kit. The mRNA expression levels of GATA-I, Bcl-2, PAR1 and PAR4 were quantified by real-time PCR. MAPK/ERK pathway, PI3K/AKT pathway and the expression of PTEN were assayed by Western blot. The expression of CD41b that is the marker of megakaryocyte differentiation was analyzed by Western blot and Flow Cytometry. The expression of CD61, cell cycle and apoptosis were detected by Flow Cytometry. After treated with thrombin, the aggregation of the platelet-like particles released from Meg-01 cells was stimulated with ADP. The results showed that the expression of CD61 in Meg-01 cells was up to 92.4%. Meg-01 cells developed pseudopodia after exposure to thrombin. Thrombin suppressed Meg-01 cells proliferation and induced apoptosis. Cell cycle was arrest at GO/GI after thrombin treatment. Further more, thrombin up-regulateed the expression of CD41b. which is one of the most important megakaryocyte markers. GATA-1, an important transcriptional regulator, controls megakaryocytes development and maturation. The expression of GATA-1 was also up-regulated by thrombin in Meg-01. The expression of PAR1 and PAR4 were also up-regulated by thrombin. The expression of Bcl-2, which is an apoptosis-inhibitory protein, was down-regulated. What is more, thrombin down-regulated the expression of PTEN and up-regulated ERK and AKT phosphorylation The platelet-like particles released from Meg-01 cells after thrombin treatment were viable. After treated with ADP, the platelet-like particles became aggregated. In conclusion, all these data indicated that thrombin may play an important role in megakaryocytes differentiation.Thrombin was used as agonist which can induce many cell functions. Firstly, thrombin was used as an agonist, the function and mechanism of thrombin in Meg-01 cells differentiation was investigated. TPO was used as positive control. Meg-01 cells proliferation was detected by CCK-8 assay. The viability of platelet-like particles was analyzed by AlamaBlue kit. MAPK/ERK pathway and PI3K/AKT pathway were assayed by Western blot. GATA-1, Bcl-2, PAR1 and PAR4 mRNA expression levels were quantified by real-time PCR. The expression of CD41b was analyzed by Western blot and Flow Cytometry. Cell cycle and apoptosis were detected by Flow Cytometry, Then ADP was added to the platelets-like particls, the state of aggregation was observed under microscope.The results show that the expression of CD61 was up to 92.4%. Meg-01 cells produced pseudopodias and platelets-like particles after exposure to thrombin. Thrombin can suppress Meg-01 cells proliferation and induce apoptosis. Cell cycle was arrest at G0/G1 stage by thrombin. Thrombin up-regulates the Expression of GATA-1, PAR1 and PAR4 and down-regulates the expression of Bcl-2. Further more, thrombin up-regulated the expression of CD41b, which is one of the most important megakaryocyte markers. Thrombin activated the MAPK/ERK pathway and PI3/AKT pathway. The platelets-like particles were viable.The expression of CD61 in particles treated with thrombin was similar to particles treated with TPO. In conclusion, thrombin maybe display some function on the differentiation of megakaryocytes into platelets.FXa is one of the key blood coagulation factors, which is similar to thrombin. FXa is a kind of serine proteinase. Since thrombin can display some function on the differentiation of megakaryocytes into platelets, FXa may also play a role inthe differentiation of megakaryocytes. Then the function and mechanism of FXa in Meg-01 cells differentiation was investigated. Meg-01 cells proliferation was detected by CCK-8 assay. The viability of platelet-like particles was analyzed by AlamaBlue kit. The expression of Skp2 which is related to cell cycle, MAPK/ERK pathway and PI3K/AKT pathway were assayed by Western blot. The expression of CD41b was analyzed by Western blot and Flow Cytometry. The results showed that FXa inhibited cell viability, induced apoptosis. FXa triggered cell arrest at the G2/M stage and down-regulated the expression of Skp2. After Meg-01 cells were stimulated by FXa, the expression of CD41b was up-regulated and ERK and AKT phosphorylation were up-regulated. Meg-01 cells induced by FXa produced viable platelet-like particles. These results indicated that FXa maybe display some function in the differentiation of megakaryocytes into platelets.In summary, both thrombin and FXa can change Meg-01 cells shape.They promoted the appearance of pseudopodias and platelet-like particles. Both thrombin and FXa suppressed Meg-01 cells proliferation and induced apoptosis. So, Both of them may induce megakaryocytes differentiation.