PMID- 26179123 OWN - NLM STAT- MEDLINE DCOM- 20160506 LR - 20150728 IS - 1399-3089 (Electronic) IS - 0908-665X (Linking) VI - 22 IP - 4 DP - 2015 Jul-Aug TI - Human thrombomodulin regulates complement activation as well as the coagulation cascade in xeno-immune response. PG - 260-72 LID - 10.1111/xen.12173 [doi] AB - BACKGROUND: With the introduction of the alpha1, 3-galactosyltransferase gene-knockout (GT-KO) pig and its pivotal role in preventing hyperacute rejection (HAR), coagulation remains a considerable obstacle yet to be overcome in order to provide long-term xenograft survival. Thrombomodulin (TBM) plays a critical anticoagulant and anti-inflammatory role in its part of the protein C pathway. Many studies have demonstrated the strong anticoagulant effects of TBM in xenotransplantation, but its complement regulatory effects have not been appropriately examined. Here, we investigate whether TBM can regulate complement activation as well as coagulation in response to xenogeneic stimuli. METHODS: We transfected porcine endothelial cells (MPN-3) with adenovirus vectors containing the human TBM gene (ad-hTBM), or a control gene containing GFP (ad-GFP). The expression level of ad-hTBM was measured by flow cytometry. To confirm the anticoagulant effect of TBM, coagulation time was measured after treatment with recalcified human plasma in ad-hTBM-transfected MPN-3, and a thrombin activity assay was performed after treatment with 50% human serum in ad-hTBM-infected MPN-3. RESULTS: Thrombin generation was significantly decreased in a dose-dependent manner in ad-TBM group, and coagulation time was increased in the ad-hTBM group when compared to the ad-GFP group. Complement-dependent serum toxicity assays were performed after treatment with 20% human serum or heat-inactivated human serum by LDH assay. Complement-dependent toxicity was significantly attenuated in the ad-hTBM group, but complement-independent toxicity was not attenuated in the ad-hTBM group. These results suggest that human thrombomodulin (hTBM) has complement regulatory effects as well as anticoagulant effects. To further investigate the mechanisms of complement regulation by hTBM, we deleted the EGF5, 6 domains that are involved in thrombin generation or the lectin-like domain involved in inflammation of TBM and functional tests were performed using these modified forms. We showed that the EGF5, 6 domain of TBM principally inhibits complement activation rather than the lectin domain. CONCLUSION: The EGF5, 6 domains of TBM appear to be the major domains for down-regulating the complement system rather than the lectin-like domain during xenogenic stimuli. The role of EGF5, 6 domains of hTBM may be due to inhibition of thrombin as thrombin can cleave C3a and C5a directly and hTBM may also be involved in complement regulation. Clearly then human TBM has complement regulatory effects as well as anticoagulant effects in xeno-immune response, and it is a promising target for attenuating xenograft rejection. CI - (c) 2015 The Authors Xenotransplantation Published by John Wiley & Sons Ltd. FAU - Kim, Hwajung AU - Kim H AUID- ORCID: 0000-0001-8470-1514 AD - Transplantation Research Institute, Seoul National University, Seoul, Korea. FAU - Hawthorne, Wayne J AU - Hawthorne WJ AD - Centre for Transplant and Renal Research, Westmead Millennium Institute, The University of Sydney at Westmead Hospital, Westmead, NSW, Australia. FAU - Kang, Hee Jung AU - Kang HJ AD - Department of Laboratory Medicine, Hallym University College of Medicine, Anyang, Korea. FAU - Lee, Yoo Jin AU - Lee YJ AD - Transplantation Research Institute, Seoul National University, Seoul, Korea. FAU - Hwang, Jong-Ik AU - Hwang JI AD - Graduate School of Medicine, Korea University, Seoul, Korea. FAU - Hurh, Sunghoon AU - Hurh S AD - Transplantation Research Institute, Seoul National University, Seoul, Korea. FAU - Ro, Han AU - Ro H AD - Gachon University Gil Medical Center, Inchon, Korea. FAU - Jeong, Jong Cheol AU - Jeong JC AD - Transplantation Research Institute, Seoul National University, Seoul, Korea. AD - Transplantation Center, Seoul National University Hospital, Seoul, Korea. FAU - Cho, Bumrae AU - Cho B AD - Designed Animal & Transplantation Research Institute, Institute of Green Bio Science & Technology, Seoul National University, Pyeongchang, Gangwon-do, Korea. FAU - Yang, Jaeseok AU - Yang J AD - Transplantation Research Institute, Seoul National University, Seoul, Korea. AD - Transplantation Center, Seoul National University Hospital, Seoul, Korea. FAU - Ahn, Curie AU - Ahn C AD - Transplantation Research Institute, Seoul National University, Seoul, Korea. AD - Transplantation Center, Seoul National University Hospital, Seoul, Korea. AD - Designed Animal & Transplantation Research Institute, Institute of Green Bio Science & Technology, Seoul National University, Pyeongchang, Gangwon-do, Korea. AD - Division of Nephrology, Seoul National University College of Medicine, Seoul, Korea. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20150714 PL - Denmark TA - Xenotransplantation JT - Xenotransplantation JID - 9438793 RN - 0 (Recombinant Proteins) RN - 0 (THBD protein, human) RN - 0 (Thrombomodulin) RN - EC 3.4.21.5 (Thrombin) SB - IM MH - Animals MH - Animals, Genetically Modified MH - Blood Coagulation/*immunology MH - Cell Line MH - Complement Activation/*immunology MH - Endothelial Cells/immunology/metabolism MH - Graft Rejection/immunology/prevention & control MH - Heterografts/*immunology MH - Humans MH - Protein Structure, Tertiary MH - Recombinant Proteins/chemistry/genetics/immunology MH - Swine MH - Swine, Miniature MH - Thrombin/metabolism MH - Thrombomodulin/chemistry/genetics/*immunology MH - Transfection MH - Transplantation, Heterologous/adverse effects OTO - NOTNLM OT - acute humoral xenograft rejection OT - coagulation cascade OT - complement cascade OT - thrombomodulin OT - xenotransplantation EDAT- 2015/07/17 06:00 MHDA- 2016/05/07 06:00 CRDT- 2015/07/17 06:00 PHST- 2014/11/07 00:00 [received] PHST- 2015/05/22 00:00 [accepted] PHST- 2015/07/17 06:00 [entrez] PHST- 2015/07/17 06:00 [pubmed] PHST- 2016/05/07 06:00 [medline] AID - 10.1111/xen.12173 [doi] PST - ppublish SO - Xenotransplantation. 2015 Jul-Aug;22(4):260-72. doi: 10.1111/xen.12173. Epub 2015 Jul 14.