Analysis of signal transduction system of Rho/Rac・cdc42 in the stimulation of proplatelet formation in megakaryocytes

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(1)Estimation of signal pathway of proplatelet formation (PPF) in megakaryocytes during the stimulation by PPF stimulating factor (TAT/HDL).Y27632, specific Rho kinase inhibitor, stimlated PPF of murine megakaryocytes. LY294002, specific PI3 kinase (PI3K) inhibitor, and/or PP1, specific Src family kinase (SrcFK) inhibitor, inhibited PPF of murine megakaryocytes. These data suggested that murine megakaryocytes' PPF might be stimulated through Rho/Rac・cdc42 pathways.(2)The purification of murine megakaryocytes.Murine sca-1(+) cells were purified from murine bone marrow cells. They were cultured with c-kit ligand and thrombopoietin in 10% FCS IMDM for 14 days. The purity of megakayocytes was determined by flow cytometric analysis using anti-CD61 antibody. Sca(+) cells (3.6x10^3) were obtained from murine bone marrow cells (9x10^7). Mature megakaryocytes (5x10^4) were produced from 3.6x10^3 sca-1(+) cells. It seemed to be difficult to use the purified mature megakaryocytes for the Western blot analysis.(3)Western blot analysis.Murine megakaryocytes were purified from murine bone marrow cells using the method described above. Murine megakaryocytes (5x10^6) were isolated from 20 mice. Activated Rac was detected at 60, 90, 120 and 150 min after the stimulation of murine megakaryocytes by PPF stimulating factor (HDL/TAT). Phospholylated cofolin was also detected with the similar manner.These data strongly suggested that HDL/TAT stimulate Rac/cdc42 activations, which induce the phospholylation of cofilin, followed by the inhibition of actin depolymerization, which induces the extension of actin.