Properties of a recombinant bovine tissue factor expressed by Silkworm pupae and its performance as an Owren-type prothrombin time reagent for warfarin monitoring

• Published in: Thrombosis research 2012-09, Vol.130 (3), p.520-527
• Main Authors: Okuda, Masahiro, Taniguchi, Tomokuni, Takamiya, Osamu
• Format: Article
• Language: English
• Subjects: Animals; baculovirus; Biological and medical sciences; Blood and lymphatic vessels; Blood coagulation; Bombyx - genetics; Bombyx - metabolism; bovine brain; Cardiology. Vascular system; Cattle; Cross Reactions; Diseases of the peripheral vessels. Diseases of the vena cava. Miscellaneous; Hematology, Oncology and Palliative Medicine; Humans; Indicators and Reagents - chemistry; Indicators and Reagents - metabolism; INR; Investigative techniques, diagnostic techniques (general aspects); ISI; Medical sciences; Owren PT; Prothrombin Time - methods; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Reproducibility of Results; Sensitivity and Specificity; Silkworm pupae; Thromboplastin - metabolism; Warfarin - blood
• ISSN: 0049-3848; 1879-2472
• DOI: 10.1016/j.thromres.2012.05.012

Abstract Tissue factor (TF), or thromboplastin, is a glycoprotein that triggers the extrinsic coagulation pathway. In blood coagulation testing, TF has been used as a natural source for determining Quick prothrombin time (PT) or the Owren PT (OBT). Currently, natural sources are being replaced with recombinant proteins because of their uniform characteristics and the possibility of stable mass production of PT reagents. Because bovine spongiform encephalopathy (BSE)-infected cows are widespread in Japan, we prepared a recombinant bovine TF (rbTF) with a baculovirus expression system using silkworms. To overcome the limitations of natural TF, especially in bovine brain, we expressed a full-length rbTF protein in Silkworm pupae with a baculovirus expression system. Baculovirus inactivation and the presence of DNA fragments in the rbTF fraction were confirmed using Reed-Muench and polymerase chain reaction methods after inactivation with a detergent. The rbTF fraction prepared by an immobilized anti- Silkworm pupae fluid protein Sepharose 4B column was identified as a visible band on western blots with a polyclonal antibody against human TF with cross-reactivity with TFs. The inhibition of the polyclonal antibody against human TF by the clotting assay for PT was identified, and amidolytic biological activity through activated factor VII on S-2288 substrate was observed. In conclusion, the rbTF expressed by the baculovirus system using Silkworm pupae was uniformly specific for bovine TF. The OBT reagent incorporated by this rbTF was similar to those of commercial reagents. It also showed a suitable International Sensitivity Index and reproducibility precision, thereby allowing for diagnostic use.