International Journal of Hematology

, Volume 107, Issue 5, pp 528–534 | Cite as

Protective effect of a newly developed fucose-deficient recombinant antithrombin against histone-induced endothelial damage

  • Toshiaki Iba
  • Tatsuhiko Hirota
  • Koichi Sato
  • Isao Nagaoka
Original Article


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.


Antithrombin Oligosaccharide Vascular endothelial cell Syndecan Histone 



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.

Compliance with ethical standards

Conflict of interest

The authors have no competing interests to declare.

Supplementary material

12185_2018_2402_MOESM1_ESM.pptx (94 kb)
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 (590 kb)
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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Copyright information

© The Japanese Society of Hematology 2018

Authors and Affiliations

  • Toshiaki Iba
    • 1
  • Tatsuhiko Hirota
    • 2
  • Koichi Sato
    • 3
  • Isao Nagaoka
    • 4
  1. 1.Department of Emergency and Disaster MedicineJuntendo University Graduate School of MedicineTokyoJapan
  2. 2.Department of Emergency and Disaster MedicineJuntendo University Graduate School of MedicineTokyoJapan
  3. 3.Department of Surgery, Juntendo Shizuoka HospitalJuntendo University Graduate School of MedicineTokyoJapan
  4. 4.Department of Host Defense and Biochemical ResearchJuntendo University Graduate School of MedicineTokyoJapan

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