In the previous ASH Meetings, we have reported that thrombin/antithrombin complex in association with high density lipoprotein (TAT/HDL) directly stimulated proplatelet formation (PPF) of human or murine megakaryocytes. In this study, we investigated the signal transduction pathways responsible for platelet production from megakaryocytes using human megakaryoblastic cell line (MEG).
TAT/HDL stimulated MEGs to induce the many platelet-like particles (PP) around the cells in the serum free culture during 12~24 hrs (Fig. 1). The frequency of MEGs with PP increased with the increase of TAT/HDL in a dose dependent manner (0 μg/ml: 1.8±0.5%, 50 μg/ml: 30.8±4.5%, 100 μg/ml: 46.5±7.8%, 200 μg/ml: 57.8±8.3%, mean±SD, n=4). The frequency of MEGs with PP, expressed as a pecentage of the control, was significantly decreased after the incubation with TAT/HDL(150 μg/ml) and Y27632, a specific inhibitor for Rho kinase(ROCK), (Y27632 10 μM: 61.3±7.9% of the control (p<0.01), 100 μM: 12.5±5.9% of the control (p<0.01), mean±SD, n=4). MEGs were incubated with TAT/HDL (150 μg/ml) in serum free condition for 5, 10, 15, 30, 60, 90, 120, 150 and 180 min. MEGs were havested, washed twice and lysed by the lysis buffer (50mM Tris/HCl, 150 mM NaCl. 0.5% Nonidet P-40 pH 6.8 with protease inhibitors’ cocktail) for the Western blot analysis. Activated Rho (GTP-bound state Rho), measured by the Western blot using Rhotekin-Rho binding domain (RBD) beads and anti-Rho antibody, increased at 90, 120 and 150 min after the incubation. ROCK was co-precipitated with activated Rho using Rhotekin-RBD beads and detected by the Western blot analysis using anti-ROCK antibody at 90, 120 and 150 min. LIM kinase was immunoprecipitated with ROCK/anti-ROCK antibody and protein G sepharose. Western blot analysis using anti-phosphothreonine antibody revealed that LIM-kinase was phospholylated at 90, 120 and 150 min after the incubation. Phospholylated cofilin (Ser3) expression was detected by the Western blot using anti-phosphocofilin antibody at 60 min, peaked at 120 min and tapered at 150 min after the incubation (Fig.2). The intensity of the phospholylated cofilin (Ser3) band decreased in the incubation of MEGs with TAT/HDL and Y27632 (100 μM) at each time. These results strongly suggested the platelet-like particles production from MEGs, stimulated by TAT/HDL, was involved in the pathway from Rho activation, ROCK activation, LIM kinase phospholylation and cofilin phospholylation, based on the evidence that phospholylated cofilin showed the inhibition of actin depolymerization and the stabilization of actin filaments.
Fig.1 (a) MEGs with no stimulation (b) MEGs with TAT/HDL (200 μg/ml) for 24h
Fig.2 Expression of phospholylated cofilin (Ser3).