1. Mutational analysis of pig tissue factor pathway inhibitor α to increase anti-coagulation activity in pig-to-human xenotransplantation.
- Author
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Lee CH, Lee HJ, Park SW, Shin J, Kang SJ, Park IB, Kim HK, and Chun T
- Subjects
- Animals, Humans, Swine, CHO Cells, Cricetulus, Thromboplastin genetics, Thromboplastin metabolism, Mutagenesis, Site-Directed, DNA Mutational Analysis, Transplantation, Heterologous, Lipoproteins genetics, Lipoproteins metabolism, Blood Coagulation genetics
- Abstract
Blood coagulation mediated by pig tissue factor (TF), which is expressed in pig tissues, causes an instant blood-mediated inflammatory reaction during pig-to-human xenotransplantation. Previously, we generated a soluble pig tissue factor pathway inhibitor α fusion immunoglobulin (TFPI-Ig) which inhibits pig TF activity more efficiently than human TFPI-Ig in human plasma. In this study, we generated several pig TFPI-Ig mutants and tested the efficacy of these mutants in preventing pig-to-human xenogeneic blood coagulation. Structurally important amino acid residues of pig TFPI-Ig were changed into different residues by site-directed mutagenesis. Subsequently, a retroviral vector encoding each cDNA of several pig TFPI-Ig mutants was cloned and transduced into CHO-K1 cells. After establishing stable cell lines expressing each of the pig TFPI-Ig mutants, soluble proteins were produced and purified for evaluating their inhibitory effects on pig TF-mediated blood coagulation in human plasma. The replacement of K
36 and K257 with R36 and H257 , respectively, in pig TFPI-Ig more efficiently blocked pig TF activity in human plasma when compared with the wild-type pig TFPI-Ig. These results may provide additional information to understand the structure of pig TFPIα, and an improved pig TFPI-Ig variant that more efficiently blocks pig TF-mediated blood coagulation during pig-to-human xenotransplantation., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)- Published
- 2024
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