thromboplastin and Antithrombin-III-Deficiency

Topics: alpha 1-Antitrypsin; alpha-Macroglobulins; Antithrombin III; Antithrombin III Deficiency; Blood Transfusion; Complement C1 Inactivator Proteins; Fibrinolysis; Heparin; Humans; Sphingomyelins; Thromboplastin

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.

Topics: Alanine Transaminase; Animals; Antithrombin III Deficiency; Antithrombins; Aspartate Aminotransferases; CHO Cells; Cricetinae; Cricetulus; Disease Models, Animal; Disseminated Intravascular Coagulation; Fibrinogen; Glycosylation; Humans; Lipopolysaccharides; Male; Oligosaccharides; Protein Isoforms; Rats; Rats, Wistar; Recombinant Proteins; Stereoisomerism; Thromboplastin; Time Factors

We reported that recombinant human soluble thrombomodulin (rhs-TM) is effective for disseminated intravascular coagulation (DIC) in vivo, in mice and rats. In the present work, we investigated the effects of decreased plasma antithrombin III (ATIII) levels on anticoagulant effects of rhs-TM, as compared to findings with heparin, of which effect is lowered by the decreased plasma ATIII levels in patients with DIC. Rat plasma ATIII levels decreased when we mixed plasma with anti-rat ATIII antibody and the potential of heparin to prolong APTT or PT was markedly diminished. The potential of rhs-TM to prolong APTT and PT was not affected. In rats injected with anti-rat ATIII antibody, plasma ATIII levels decreased immediately. When the rats were infused with tissue factor (TF), DIC was induced. At doses of rhs-TM and heparin which were equally effective at inhibiting the decrease in platelet count and fibrinogen level in control rats treated with TF, only rhs-TM remained effective in preventing DIC in rats with reduced ATIII levels. Heparin was not effective when administered to these rats with reduced ATIII levels. Therefore, rhs-TM effectively inhibits coagulation independent of ATIII levels, in contrast to heparin, which depends on the ATIII level.

Topics: Animals; Anticoagulants; Antithrombin III; Antithrombin III Deficiency; Disseminated Intravascular Coagulation; Heparin; Humans; Immune Sera; Male; Partial Thromboplastin Time; Prothrombin Time; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Thrombomodulin; Thromboplastin