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Functional difference between membrane-bound and soluble human thrombomodulin

Yuko Miwa¹, Satoko Yazaki^2,3, Masaki Iwamoto^2,3, Kenta Iwasaki¹, Koji Yamamoto⁴, Masataka Haneda¹, Akira Onishi², Kazuharu Uchida⁵, Takaaki Kobayashi¹

¹Applied Immunology, Nagoya university School of Medicine, Nagoya; ²Developmental Biology, National Institute of Agrobiological Sciences, Tsukuba; ³Prime Tech.Ltd, Tsuchiura; ⁴Transfusion Medicine Medicine, Nagoya university School of Medicine, Nagoya; ⁵Transplant Surgery, Nagoya Daini Red Cross Hospital, Nagoya; Japan

Background: The production of genetically modified pigs has attracted considerable attention in xenotransplantation. In addition to deletion of α-Gal antigen (GT-KO) and insertion of human complement regulatory proteins (CD46, CD55, CD59), the resolution of coagulation dysregulation has become a next big challenge. Although we have successfully produced cloned pigs expressing human thrombomodulin (hTM) as a coagulation regulator, recombinant soluble thrombomodulin (S-hTM) has only recently been made commercially available. The purpose of this study to examine functional difference in endothelial cells between membrane-bound hTM (MB-hTM) and S-hTM and to elucidate the potential value of hTM-expressing cloned pigs.

Methods: The following points regarding coagulation control were compared between hTM-expressing endothelial cells (hTM-PAEC) derived from cloned pig and PAEC derived from non-transgenic cloned pigs in the presence of S-hTM (1ug/mL). (i) Activated protein C (APC) and (ii) thrombin production were assessed by specific chromogenic assay. (iii) Expression of tissue factor (TF) were analyzed by TF procoagulant activity assay in TNFa-activated cells. (iv) Anti-inflammatory effect was assessed by expression of pig E-selectin in TNFa-activated cells.

Results: (i) APC production: MB-hTM (1.1ug/mL) < S-hTM (2.9ug/ml), (ii) thrombin absorption: MB-hTM (23%) < S-hTM (65%), (iii) suppression of TF procoagulant activity: MB-hTM (47%) > S-hTM (13%), (iv) anti-inflammatory effect: MB-hTM (43%) > S-hTM (5%). As S-hTM had more potent capacity of APC production and thrombin absorption than MB-hTM, it would be effective under activated conditions. In contrast, MB-hTM appeared to exert a role as constant regulator under non-activated conditions.

Conclusion: Additional S-hTM treatment would be of assistance during high-risk periods for excessive thrombin formation (i.e, immediately after transplantation due to ischemia reperfusion injury). Considering the properties of MB-hTM exhibiting both protein C- activating cofactor activity and anti-inflammatory function, hTM-expressing cloned pigs might be indispensible to long-term stabilization of graft endothelial cells.