Danazol Stimulates Proplatelet Formation of Megakaryocytes Via Rac Activation Pathway.

Abstract Danazol (C 22 H 27NO2; 17a-2,4-pregnadien-20-yno[2,3-d]isoxazol-17-ol) have been widely used for patients with endometriosis. Recently, many reports showed that danazol was effective especially in thrombopoietic action in patients with refractory chronic idiopathic thrombocytopenic purpura, apalstic anemia or myelodysplastic syndrome-refractory anemia (MDS/RA). The thrombopoietic mechanism of danazol has been unknown. In this study, we investigated the thrombopoietic effects of danazol on megakaryocytes, especially proplatelet formation (PPF). Murine megakaryocytes were isolated from bone marrow cells by Percoll®‚ density centrifugation and BSA gradient method. Isolated megakaryocytes (300/well) were cultured for 24 hrs in serum free condition with danazol and several kinase specific inhibitors including PPI (Src family kinase inhibitor), SU6695 (Src family kinase inhibitor), or LY294002 (PI3K inhibitor). PPF stimulation index (%) by danazol was calculated as (PPF with danazol)/(PPF without danazol). It was stimulated by danazol in a dose dependent manner(0μM: 100%, 0.2μM: 118±14.29%, 2μM: 136±10.3% (p<0.05), 12μM:176.7±17.4%(p<0.001), mean±SD, n=5). The inhibitiory index(%) by kinase specific inhibitor was calculated as (PPF with danazole and each kinase specific inhibitor)/(PPF without them). Src family kinase inhibitor including PPI or SU6656 did not affect the PPF of murine megakaryocytes at 0, 0.2, 2.4, and 12 μM concentrations of danazol. While, LY294002 inhibited PPF of murine megakaryocytes at 0μM of danazol (LY294002; 0μM: 100%, 10μM: 75.33±4.5% (p<0.01), 100μM: 61.3±9.4% (p<0.01) (n=4)), as shown in Figure 1. PPF with danazole 0.2μM/ LY294002 100μM was 86% of that with danazol 0.2μM/ LY294002 0μM, that with danazol 2.4μM/ LY294002 100μM was 78%(p<0.05) of PPF with danazol 2.4μM/ LY294002 0μM, and that with danazol 12μM/ LY294002 100μM was 60%(p<0.01) of PPF with danazol 12μM/ LY294002 0μM (Figure 1). As a result, the similar inhibitory effects of LY294002 on PPF of murine megakaryocytes at 0, 0.2, 2.4 and 12.0μM of danazol were also observed. These results strongly suggest that danazol is a powerful stimulating agent for PPF and that PI3K activation, followed by Rac activation might be the signal transduction pathway in PPF of megakaryocytes, based on that PI3K and Src family kinase stimulate Rac activation. Figure 1. %PPF by danazol and/ or LY294002(P13K inhibitor) Figure 1. %PPF by danazol and/ or LY294002(P13K inhibitor)