Abstracts
Antithrombin is expected to modulate both prothrombotic and proinflammatory reactions in sepsis; vascular endothelium is the primary target. In the present study, we sought to evaluate the protective effects of a newly developed fucose-deficient recombinant antithrombin. Endothelial cells were treated in vitro with histone H4 to induce cellular damage. Low to high doses of either plasma-derived antithrombin or recombinant thrombomodulin were used as treatment interventions. Morphological change, apoptotic rate, cell viability, cell injury, and syndecan-4 level in the medium were evaluated. Immunofluorescent staining with anti-syndecan-4 was also performed. Both types of antithrombin reduced cellular damage and apoptotic cell death. Both plasma-derived and recombinant antithrombin improved cell viability and reduced cellular injury when administered at a physiological concentration or higher. Syndecan-4 staining became evident after treatment with histone H4, and both antithrombins suppressed the staining intensity at similar levels. The syndecan-4 level in the medium was significantly decreased by both antithrombins. None of the indicators showed a significant difference between plasma-derived and recombinant antithrombin. In conclusion, both recombinant and plasma-derived antithrombin can protect vascular endothelial cells. Recombinant antithrombin may represent a useful new therapeutic agent for sepsis-associated vascular damage.
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This work was supported by the fund from Ministry of Education, Culture, Sports, Science and Technology-Supported Program for the Strategic Research Foundation at Private Universities 2016. All the authors have read and approved the final manuscript.
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12185_2018_2402_MOESM1_ESM.pptx
Supplement 1. Time-course and dose–response of cell viability after treatment with histone H4. Vascular endothelial cell viability was measured based on the CCK-8 level in the culture medium. Cell viability started to decrease 6 h after the treatment with histone H4, and the level decreased in a dose-dependent manner. Cell viability was decreased nearly half of the initial level when the dose was 50 μg/mL. Data are expressed as the mean ± standard error. CCK-8: cell counting kit-8 (PPTX 94 kb)
12185_2018_2402_MOESM2_ESM.ppt
Supplement 2. SDS-PAGE for various types of antithrombin. The cathode (-) is located at the top of the gel (pH4), while the anode (+) is at the bottom (pH6.5). Fifteen micrograms of protein was loaded in each lane of either 20% or 7.5% (w/v) acrylamide gel. Each protein band was stained with Coomassie Brilliant Blue. Both α- and β-antithrombin were recognized as blue bands located near 58 kDa. In contrast, AT-γ was recognized as wider bands. SDS-PAGE: Sodium dodecyl sulfate–polyacrylamide gel electrophoresis, α-AT: α-antithrombin, β-AT: β-antithrombin, Pd-AT: plasma-derived antithrombin, AT-γ: antithrombin-γ (PPT 589 kb)
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Iba, T., Hirota, T., Sato, K. et al. Protective effect of a newly developed fucose-deficient recombinant antithrombin against histone-induced endothelial damage. Int J Hematol 107, 528–534 (2018). https://doi.org/10.1007/s12185-018-2402-x
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DOI: https://doi.org/10.1007/s12185-018-2402-x