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CC BY-NC-ND 4.0 · TH Open 2017; 01(01): e3-e10
DOI: 10.1055/s-0037-1603926
Original Article
Georg Thieme Verlag KG Stuttgart · New York

The Comparison of the Protective Effects of α- and β-Antithrombin against Vascular Endothelial Cell Damage Induced by Histone in Vitro

Toshiaki Iba
1   Department of Emergency and Disaster Medicine, Juntendo University Graduate School of Medicine, Tokyo, Japan
,
Tetsuya Sasaki
2   Nihon Pharmaceutical Co. Ltd., Research Laboratory, Narita, Japan
,
Kazutoshi Ohshima
2   Nihon Pharmaceutical Co. Ltd., Research Laboratory, Narita, Japan
,
Koichi Sato
3   Department of Surgery, Juntendo Shizuoka Hospital, Juntendo University Graduate School of Medicine, Tokyo, Japan
,
Isao Nagaoka
4   Department of Host Defense and Biochemical Research, Juntendo University Graduate School of Medicine, Tokyo, Japan
,
Jecko Thachil
5   Department of Haematology, Manchester Royal Infirmary, Manchester, United Kingdom
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Publikationsverlauf

Publikationsdatum:
28. Juni 2017 (online)

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Abstract

Antithrombin is a promising option for the treatment of sepsis, and vascular endothelium is an important target for this fatal condition. Here, we aimed to evaluate the protective effects of different glycoforms of antithrombin on histone-induced endothelial cell damage and explore the responsible mechanisms in an experimental model in vitro. Endothelial cells were treated in vitro using histone H4 to induce cellular damage. Various doses of either α- or β-antithrombin were used as treatment interventions, and both cell viability and the levels of lactate dehydrogenase (LDH) in the medium were assessed. Endothelial cell damage was also assessed using microscopic examination and immunofluorescent staining with anti-syndecan-4 and anti-antithrombin antibodies. As a result, both glycoforms of antithrombin significantly improved cell viability when administered at a physiological dose (150 μg/mL). Cellular injury as evaluated using the LDH level was significantly suppressed by β-antithrombin at a supranormal dose (600 μg/mL). Microscopic observation suggested that β-antithrombin suppressed the endothelial cell damage more efficiently than α-antithrombin. β-Antithrombin suppressed the intensity of syndecan-4 staining which became evident after treatment with histone H4, more prominently than α-antithrombin. The distribution of antithrombin was identical to that of syndecan-4. In conclusion, both α- and β-antithrombin can protect vascular endothelial cells from histone H4-induced damage, although the effect was stronger for β-antithrombin. The responsible mechanisms might involve the binding of antithrombin to the glycocalyx on the endothelial surface. These results provide a theoretical basis for the application of antithrombin to the prevention and treatment of sepsis-related endothelial damage.

Keywords

antithrombin - glycoform - vascular endothelial cell - glycocalyx - syndecan-4

Contributions of Authors

The study design was performed by T.I. and J.T. I.N. and K.S. were involved in the study conduct. Data collection was performed by T.S. and K.O. Data analysis was performed by T.I. and K.S., and I.N., T.I., and J.T. were involved in drafting and revising of the manuscript. All authors read and approved the final manuscript.


Supplementary Material

 

  • References

  • 1 Bucur SZ, Levy JH, Despotis GJ, Spiess BD, Hillyer CD. Uses of antithrombin III concentrate in congenital and acquired deficiency states. Transfusion 1998; 38 (05) 481-498
    CrossrefPubMedGoogle Scholar
  • 2 Quinsey NS, Greedy AL, Bottomley SP, Whisstock JC, Pike RN. Antithrombin: in control of coagulation. Int J Biochem Cell Biol 2004; 36 (03) 386-389
    CrossrefPubMedGoogle Scholar
  • 3 Levy JH, Sniecinski RM, Welsby IJ, Levi M. Antithrombin: anti-inflammatory properties and clinical applications. Thromb Haemost 2016; 115 (04) 712-728
    Artikel in Thieme ConnectPubMedGoogle Scholar
  • 4 Iba T, Thachil J. Present and future of anticoagulant therapy using antithrombin and thrombomodulin for sepsis-associated disseminated intravascular coagulation: a perspective from Japan. Int J Hematol 2016; 103 (03) 253-261
    CrossrefPubMedGoogle Scholar
  • 5 Peterson CB, Blackburn MN. Isolation and characterization of an antithrombin III variant with reduced carbohydrate content and enhanced heparin binding. J Biol Chem 1985; 260 (01) 610-615
    PubMedGoogle Scholar
  • 6 McCoy AJ, Pei XY, Skinner R, Abrahams JP, Carrell RW. Structure of beta-antithrombin and the effect of glycosylation on antithrombin's heparin affinity and activity. J Mol Biol 2003; 326 (03) 823-833
    CrossrefPubMedGoogle Scholar
  • 7 Karlaftis V, Sritharan G, Attard C, Corral J, Monagle P, Ignjatovic V. Beta (β)-antithrombin activity in children and adults: implications for heparin therapy in infants and children. J Thromb Haemost 2014; 12 (07) 1141-1144
    CrossrefPubMedGoogle Scholar
  • 8 de la Morena-Barrio ME, García A, Martínez-Martínez I. , et al. A new method to quantify β-antithrombin glycoform in plasma reveals increased levels during the acute stroke event. Thromb Res 2015; 136 (03) 634-641
    CrossrefPubMedGoogle Scholar
  • 9 Iba T, Saitoh D. Efficacy of antithrombin in preclinical and clinical applications for sepsis-associated disseminated intravascular coagulation. J Intensive Care 2014; 2 (01) 66
    CrossrefPubMedGoogle Scholar
  • 10 Warren BL, Eid A, Singer P. , et al; KyberSept Trial Study Group. Caring for the critically ill patient. High-dose antithrombin III in severe sepsis: a randomized controlled trial. JAMA 2001; 286 (15) 1869-1878
    CrossrefPubMedGoogle Scholar
  • 11 Kienast J, Juers M, Wiedermann CJ. , et al; KyberSept investigators. Treatment effects of high-dose antithrombin without concomitant heparin in patients with severe sepsis with or without disseminated intravascular coagulation. J Thromb Haemost 2006; 4 (01) 90-97
    PubMedGoogle Scholar
  • 12 Gauthier VJ, Tyler LN, Mannik M. Blood clearance kinetics and liver uptake of mononucleosomes in mice. J Immunol 1996; 156 (03) 1151-1156
    PubMedGoogle Scholar
  • 13 Xu J, Zhang X, Pelayo R. , et al. Extracellular histones are major mediators of death in sepsis. Nat Med 2009; 15 (11) 1318-1321
    CrossrefPubMedGoogle Scholar
  • 14 Ishiyama M, Tominaga H, Shiga M, Sasamoto K, Ohkura Y, Ueno K. A combined assay of cell viability and in vitro cytotoxicity with a highly water-soluble tetrazolium salt, neutral red and crystal violet. Biol Pharm Bull 1996; 19 (11) 1518-1520
    CrossrefPubMedGoogle Scholar
  • 15 Wildhagen KC, Wiewel MA, Schultz MJ. , et al. Extracellular histone H3 levels are inversely correlated with antithrombin levels and platelet counts and are associated with mortality in sepsis patients. Thromb Res 2015; 136 (03) 542-547
    CrossrefPubMedGoogle Scholar
  • 16 Ekaney ML, Otto GP, Sossdorf M. , et al. Impact of plasma histones in human sepsis and their contribution to cellular injury and inflammation. Crit Care 2014; 18 (05) 543
    CrossrefPubMedGoogle Scholar
  • 17 Alhamdi Y, Abrams ST, Cheng Z. , et al. Circulating histones are major mediators of cardiac injury in patients with sepsis. Crit Care Med 2015; 43 (10) 2094-2103
    CrossrefPubMedGoogle Scholar
  • 18 Brinkmann V, Reichard U, Goosmann C. , et al. Neutrophil extracellular traps kill bacteria. Science 2004; 303 (5663): 1532-1535
    CrossrefPubMedGoogle Scholar
  • 19 Chaaban H, Keshari RS, Silasi-Mansat R. , et al. Inter-α inhibitor protein and its associated glycosaminoglycans protect against histone-induced injury. Blood 2015; 125 (14) 2286-2296
    CrossrefPubMedGoogle Scholar
  • 20 Weinbaum S, Tarbell JM, Damiano ER. The structure and function of the endothelial glycocalyx layer. Annu Rev Biomed Eng 2007; 9: 121-167
    CrossrefPubMedGoogle Scholar
  • 21 Reitsma S, Slaaf DW, Vink H, van Zandvoort MA, oude Egbrink MG. The endothelial glycocalyx: composition, functions, and visualization. Pflugers Arch 2007; 454 (03) 345-359
    CrossrefPubMedGoogle Scholar
  • 22 Pries AR, Kuebler WM. Normal endothelium. Handb Exp Pharmacol 2006; 176 (176, Pt 1): 1-40
    PubMedGoogle Scholar
  • 23 Schmidt EP, Yang Y, Janssen WJ. , et al. The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis. Nat Med 2012; 18 (08) 1217-1223
    CrossrefPubMedGoogle Scholar
  • 24 Johansson PI, Stensballe J, Rasmussen LS, Ostrowski SR. A high admission syndecan-1 level, a marker of endothelial glycocalyx degradation, is associated with inflammation, protein C depletion, fibrinolysis, and increased mortality in trauma patients. Ann Surg 2011; 254 (02) 194-200
    CrossrefPubMedGoogle Scholar
  • 25 Nelson A, Berkestedt I, Schmidtchen A, Ljunggren L, Bodelsson M. Increased levels of glycosaminoglycans during septic shock: relation to mortality and the antibacterial actions of plasma. Shock 2008; 30 (06) 623-627
    CrossrefPubMedGoogle Scholar
  • 26 Chappell D, Hofmann-Kiefer K, Jacob M. , et al. TNF-alpha induced shedding of the endothelial glycocalyx is prevented by hydrocortisone and antithrombin. Basic Res Cardiol 2009; 104 (01) 78-89
    CrossrefPubMedGoogle Scholar
  • 27 Branzk N, Lubojemska A, Hardison SE. , et al. Neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens. Nat Immunol 2014; 15 (11) 1017-1025
    CrossrefPubMedGoogle Scholar
  • 28 Couchman JR. Transmembrane signaling proteoglycans. Annu Rev Cell Dev Biol 2010; 26: 89-114
    CrossrefPubMedGoogle Scholar
  • 29 Iba T. Glycocalyx regulates the intravascular hemostasis. Juntendo Medical J 2016; 62: 444-449
    PubMedGoogle Scholar
  • 30 De Jong MC, Walstra CM. Immunofluorescent localization of antithrombin III in human skin. Br J Dermatol 1982; 106 (03) 281-285
    CrossrefPubMedGoogle Scholar
  • 31 Chappell D, Jacob M, Hofmann-Kiefer K. , et al. Antithrombin reduces shedding of the endothelial glycocalyx following ischaemia/reperfusion. Cardiovasc Res 2009; 83 (02) 388-396
    CrossrefPubMedGoogle Scholar
  • 32 Becker BF, Chappell D, Bruegger D, Annecke T, Jacob M. Therapeutic strategies targeting the endothelial glycocalyx: acute deficits, but great potential. Cardiovasc Res 2010; 87 (02) 300-310
    CrossrefPubMedGoogle Scholar
  • 33 Horie S, Ishii H, Kazama M. Heparin-like glycosaminoglycan is a receptor for antithrombin III-dependent but not for thrombin-dependent prostacyclin production in human endothelial cells. Thromb Res 1990; 59 (06) 895-904
    CrossrefPubMedGoogle Scholar
  • 34 Kaneider NC, Förster E, Mosheimer B, Sturn DH, Wiedermann CJ. Syndecan-4-dependent signaling in the inhibition of endotoxin-induced endothelial adherence of neutrophils by antithrombin. Thromb Haemost 2003; 90 (06) 1150-1157
    Artikel in Thieme ConnectPubMedGoogle Scholar
  • 35 Pemberton AD, Brown JK, Inglis NF. Proteomic identification of interactions between histones and plasma proteins: implications for cytoprotection. Proteomics 2010; 10 (07) 1484-1493
    CrossrefPubMedGoogle Scholar
  • 36 Iba T, Hamakubo T, Nagaoka I, Sato K, Thachil J. Physiological levels of pentraxin 3 and albumin attenuate vascular endothelial cell damage induced by histone H3 in vitro. Microcirculation 2016; 23 (03) 240-247
    CrossrefPubMedGoogle Scholar
  • 37 Dickneite G, Leithäuser B. Influence of antithrombin III on coagulation and inflammation in porcine septic shock. Arterioscler Thromb Vasc Biol 1999; 19 (06) 1566-1572
    CrossrefPubMedGoogle Scholar
  • 38 Becker BF, Jacob M, Leipert S, Salmon AH, Chappell D. Degradation of the endothelial glycocalyx in clinical settings: searching for the sheddases. Br J Clin Pharmacol 2015; 80 (03) 389-402
    CrossrefPubMedGoogle Scholar
  • 39 Nordling S, Hong J, Fromell K. , et al. Vascular repair utilising immobilised heparin conjugate for protection against early activation of inflammation and coagulation. Thromb Haemost 2015; 113 (06) 1312-1322
    Artikel in Thieme ConnectPubMedGoogle Scholar