|Budget Amount *help
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2002: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2001: ¥1,900,000 (Direct Cost: ¥1,900,000)
1. Purification of proplatelet formation (PPF) stimulatory factor.
We purified the PPF stimulatory factor from the normal human plasma and determined the amino acid sequence of the purified protein. It was antithrombin III, which in vitro activity expressed in association with high density lipoprotein (HDL).
2. Selection of human megakaryoblastic cell line, which respond to PPF stimulatory factor.
We established the quantitative assay system in which MEG cell line expressed PPF in response to AT III/HDL. AT III/HDL stimulated PPF of MEG cell line in a dose dependent manner (HDL 0 μg/ml 1.1%, HDL 50 μg/ml 30.8%, HDL 100 μg/ml 46.5%, HDL 200 μg/ml 57.8%).
3. Inhibition of PPF expression or not in the above assay system with the incubation of kinase inhibitors. PD98059(MAPK inhibitor), LY294002(PI3K inhibitor), SB203580(p38 MAPK inhibitor), KT5720(PKA inhibitor), BIM(PKC activator inhibitor) or Y27632(Rho kinase inhibitor) was added in the above assay system. PPF expression of MEG cell line was inhibited by the addition of BIM or Y27632 (BIM : 100 μM 70.7%, 500 nM 66.9%, 1 μM 54.0%, 10 μM 4.5%, 100 μM 5.6%, Y27632 :100 nM 97.6%, 1 μM 88.5%, 10 μM 61.3%).
Based on the results, PPF expression of megakaryocytes was deeply involved in the transduction systems of protein kinase C and Rho kinase.