Research Assistant professor
Gastroenterological Surgery, Commissioned Research Project
Nagoya University Graduate School of Medicine
Study of proper expression level of human thrombomodulin in pig endothelial cell with effective anticoagulant activity and no bleeding tendency.
Yuko Miwa1, Kenta Iwasaki1,2, Syunichi Suzuki3, Masaki Iwamoto4, Akira Onishi3,5, Yasuhiro Kodera1, Takaaki Kobayashi1,6.
1Gastroenterological Surgery, Commissioned Research Project, Nagoya University Graduate School of Medicine, Nagoya, Japan; 2Kidney disease and Transplant Immunology, Aichi Medical University School of Medicine, Nagakute, Japan; 3Developmental Biology, National Institute of Agrobiological Sciences, Tsukuba, Japan; 4Prime Tech Ltd, Tsuchiura, Japan; 5Animal Science and Resources, Nihon University of College of Biosource Sciences, Fujisawa, Japan; 6Surgery (Renal Transplant Surgery), Aichi Medical University School of Medicine, Nagakute, Japan
Background: For successful xenotransplantation, in addition to a1,3-galactosyltransferase gene-knockout (GT-KO) and human complement regulatory protein (CD46, CD55, CD59) gene insertion, cloned pigs expressing human thrombomodulin (hTM) have been produced to solve the problem of molecular incompatibility in their coagulation system. Recently, it was reported that hearts expressing hTM have functioned for more than 1 year, but the effective expression level of hTM have not been investigated in detail yet. The purpose of this study was to clarify the proper expression level of human thrombomodulin in pig endothelial cell with effective anticoagulant activity and no bleeding tendency.
Methods: i) Activated partial thromboplastin time (APTT) and soluble hTM (S-hTM) in pig plasma were measured to check the bleeding tendency of hTM cloned pig. ii) Porcine aortic endothelial cell (PAEC), hTM-expressing PAEC (hTM-PAEC), and human umbilical vein endothelial cell (HUVEC) were used and compared the expression level of hTM and the production of activated protein C (APC). iii) Direct thrombin binding effect of hTM which lead to bleeding tendency, were measured using recombinant S-hTM (0.1,1,10 ug/ml) and hTM-PAEC.
Results: i) S-hTM concentrations in hTM-cloned pig plasma were 2 to 3 times higher than human normal concentrations, but APTT in hTM-cloned pig plasma were in the normal range and hTM-cloned pig have not tented bleeding. ii) hTM expression level(MFI) and APC production(ug/ml) between hTM-PAEC and HUVEC, (hTM-PAEC,HUVEC)=(MFI:177.6,160.0),(2.8,3.0 ug/ml), were same level. iii) Direct thrombin binding effect was not detected in hTM-PAEC, 10ug/ml of S-hTM extremely had direct thrombin binding effect.
Discussion: hTM have two potential anti coagulant functions which are inhibitory function of thrombin generation by APC and direct thrombin binding function, however clinical blood concentration of S-hTM for treatment of disseminated intravascular coagulation (DIC) sets the concentration (1.6ug/ml or less) which does not work direct thrombin binding effect with consideration for bleeding tendency. If a high level of expression hTM may cause risks of bleeding tendency in pigs and grafts, physiological expression level is recommended.
 Xenotransplantation 2012;19:82-91
 Transplantation 2015;99(4):702-709
11:00 - 12:30
|Inflammation & Coagulation||Study of proper expression level of human thrombomodulin in pig endothelial cell with effective anticoagulant activity and no bleeding tendency.||Room 109|