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. 2024 Aug;46(4):521-530.
doi: 10.1007/s10529-024-03505-z. Epub 2024 Jun 13.

Mutational analysis of pig tissue factor pathway inhibitor α to increase anti-coagulation activity in pig-to-human xenotransplantation

Chang-Hee Lee  1 , Hyeon Jeong Lee  1 , Si-Won Park  1 , Jiyoon Shin  1 , Seok-Jin Kang  1 , In-Byung Park  1 , Hyun Kyung Kim  2 , Taehoon Chun  3
Affiliations

Mutational analysis of pig tissue factor pathway inhibitor α to increase anti-coagulation activity in pig-to-human xenotransplantation

Chang-Hee Lee et al. Biotechnol Lett. 2024 Aug.

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 K36 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.

Keywords: Blood coagulation; Recombinant protein; Tissue factor; Tissue factor pathway inhibitor; Xenotransplantation.

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