Recombinant human antithrombin expressed in Chinese hamster ovary cells shows in vivo efficacy on rat DIC model similarly to plasma-derived antithrombin regardless of different N-glycosylation.
Plasma-derived human antithrombin (pAT) is used for the treatments of disseminated intravascular coagulation (DIC) and hereditary antithrombin deficiencies. We expressed recombinant human antithrombin (rAT) in Chinese hamster ovary (CHO) cells. The purified rAT is composed of 55% alpha-isoform and 45% beta-isoform. The structure of the N-linked oligosaccharides of rAT is the same biantennary complex type as previously found in pAT with less sialylated on the non-reducing ends. Most of the oligosaccharides of rAT are fucosylated at the reducing ends of N-acetylglucosamine, while those of pAT are not fucosylated. Despite of the difference in sialylation and fucosylation of the oligosaccharide units, rAT and pAT showed indistinguishable heparin cofactor and progressive activities, and they bound to thrombin in a one-to-one stoichiometric manner. In lipopolysaccharide (LPS)-induced and thromboplastin-induced DIC rat models, rAT reduced fibrinogen and platelet consumption to a similar extent with pAT. In LPS-induced DIC model, both ATs similarly restrained the increase of alanine aminotransferase and aspartate aminotransferase activities. Finally, pharmacokinetic analysis showed that both ATs had similar half-lives in the circulation of normal rats. Together, the present study demonstrated that rAT prepared in CHO cells has potential for a substitute of pAT in therapeutic use.
Hirose
Masaaki
M
Protein Research Laboratory, Pharmaceutical Research Division, Mitsubishi Pharma Corporation, 2-25-1, Shodai-Ohtani, Hirakata, Osaka, Japan. hirose.masaaki@mh.m-pharma.co.jp
Tsukada
Minoru
M
Hirayama
Fumihiro
F
Kubo
Yoshiji
Y
Kajii
Masahiko
M
Mochizuki
Shinobu
S
Hamato
Nobuaki
N
Ohi
Hideyuki
H
D000410:Alanine Transaminase / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D020152:Antithrombin III Deficiency / Q000188:drug therapy / Q000378:metabolism; D000991:Antithrombins / Q000096:biosynthesis / Q000737:chemistry / Q000627:therapeutic use*; D001219:Aspartate Aminotransferases / Q000187:drug effects / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004195:Disease Models, Animal; D004211:Disseminated Intravascular Coagulation / Q000188:drug therapy* / Q000378:metabolism; D005340:Fibrinogen / Q000187:drug effects; D006031:Glycosylation; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D020033:Protein Isoforms / Q000096:biosynthesis / Q000737:chemistry / Q000627:therapeutic use; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000627:therapeutic use*; D013237:Stereoisomerism; D013925:Thromboplastin / Q000494:pharmacology; D013997:Time Factors
D000991:Antithrombins; D008070:Lipopolysaccharides; D009844:Oligosaccharides; D020033:Protein Isoforms; D011994:Recombinant Proteins; D005340:Fibrinogen; D013925:Thromboplastin; D001219:Aspartate Aminotransferases; D000410:Alanine Transaminase