Pig endothelial protein C receptor is functionally compatible with the human protein C pathway.
coagulation
endothelial protein C receptor
protein C pathway
thrombomodulin
xenotransplantation
Journal
Xenotransplantation
ISSN: 1399-3089
Titre abrégé: Xenotransplantation
Pays: Denmark
ID NLM: 9438793
Informations de publication
Date de publication:
03 2020
03 2020
Historique:
received:
03
05
2019
revised:
12
08
2019
accepted:
11
09
2019
pubmed:
27
9
2019
medline:
9
6
2021
entrez:
27
9
2019
Statut:
ppublish
Résumé
Endothelial protein C receptor (EPCR) plays an anticoagulant and anti-inflammatory role by promoting the activation of protein C by thrombin bound to thrombomodulin (TBM). Incompatibility between pig TBM and human/primate thrombin is thought to contribute to dysregulated coagulation in pig-to-primate organ xenografts, and expression of human TBM (hTBM) in pigs has shown benefit in preclinical models. However, it is not known whether there are incompatibilities-or molecular barriers-between endogenous pig EPCR (pEPCR) and transgenically expressed human TBM. To clone and express pEPCR, and determine its function in the human protein C pathway in vitro. Pig endothelial protein C receptor cDNA was generated from pig lung RNA by RT-PCR. Primate COS-7 transfectants expressing various combinations of human and pig TBM and EPCR were incubated with human thrombin and human protein C, and tested for TBM cofactor activity. The predicted protein sequence of pEPCR shared 72.3% amino acid sequence identity with hEPCR, and residues critical for protein C binding were conserved. COS-7 cells transfected with hEPCR, pEPCR or vector showed minimal TBM cofactor activity (0.13 ± 0.04, 0.13 ± 0.02 and 0.14 ± 0.06 U, respectively). The cofactor activity of hTBM-transfected cells (1.18 ± 0.29 U) was 8-fold higher than vector-transfected cells (P = .004) and further increased 4-fold and 3-fold by co-transfection with hEPCR (5.01 ± 1.12 U, P = .004) or pEPCR (3.73 ± 0.65 U, P = .003), respectively. Our data show that pEPCR is largely compatible with the human TBM/thrombin complex, when expressed on COS-7 cells in vitro, promoting the activation of human protein C. These findings suggest that endogenous pEPCR will enhance the activity of transgenic hTBM in the xenograft setting.
Sections du résumé
BACKGROUND
Endothelial protein C receptor (EPCR) plays an anticoagulant and anti-inflammatory role by promoting the activation of protein C by thrombin bound to thrombomodulin (TBM). Incompatibility between pig TBM and human/primate thrombin is thought to contribute to dysregulated coagulation in pig-to-primate organ xenografts, and expression of human TBM (hTBM) in pigs has shown benefit in preclinical models. However, it is not known whether there are incompatibilities-or molecular barriers-between endogenous pig EPCR (pEPCR) and transgenically expressed human TBM.
AIM
To clone and express pEPCR, and determine its function in the human protein C pathway in vitro.
METHODS
Pig endothelial protein C receptor cDNA was generated from pig lung RNA by RT-PCR. Primate COS-7 transfectants expressing various combinations of human and pig TBM and EPCR were incubated with human thrombin and human protein C, and tested for TBM cofactor activity.
RESULTS
The predicted protein sequence of pEPCR shared 72.3% amino acid sequence identity with hEPCR, and residues critical for protein C binding were conserved. COS-7 cells transfected with hEPCR, pEPCR or vector showed minimal TBM cofactor activity (0.13 ± 0.04, 0.13 ± 0.02 and 0.14 ± 0.06 U, respectively). The cofactor activity of hTBM-transfected cells (1.18 ± 0.29 U) was 8-fold higher than vector-transfected cells (P = .004) and further increased 4-fold and 3-fold by co-transfection with hEPCR (5.01 ± 1.12 U, P = .004) or pEPCR (3.73 ± 0.65 U, P = .003), respectively.
CONCLUSIONS
Our data show that pEPCR is largely compatible with the human TBM/thrombin complex, when expressed on COS-7 cells in vitro, promoting the activation of human protein C. These findings suggest that endogenous pEPCR will enhance the activity of transgenic hTBM in the xenograft setting.
Substances chimiques
Endothelial Protein C Receptor
0
Protein C
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e12557Subventions
Organisme : NIAID NIH HHS
ID : P01 AI045897
Pays : United States
Informations de copyright
© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Références
Cooper DK, Gollackner B, Sachs DH. Will the pig solve the transplantation backlog? Annu Rev Med. 2002;53:133-147.
Cowan PJ, Tector AJ. The resurgence of xenotransplantation. Am J Transplant. 2017;17(10):2531-2536.
Cowan PJ, Robson SC, d'Apice AJ. Controlling coagulation dysregulation in xenotransplantation. Curr Opin Organ Transplant. 2011;16(2):214-221.
Siegel JB, Grey ST, Lesnikoski BA, et al. Xenogeneic endothelial cells activate human prothrombin. Transplantation. 1997;64(6):888-896.
Kopp CW, Grey ST, Siegel JB, et al. Expression of human thrombomodulin cofactor activity in porcine endothelial cells. Transplantation. 1998;66(2):244-251.
Roussel JC, Moran CJ, Salvaris EJ, Nandurkar HH, d'Apice AJ, Cowan PJ. Pig thrombomodulin binds human thrombin but is a poor cofactor for activation of human protein C and TAFI. Am J Transplant. 2008;8(6):1101-1112.
Esmon CT. The protein C pathway. Chest. 2003;124(3 Suppl):26S-32S.
Stearns-Kurosawa DJ, Kurosawa S, Mollica JS, Ferrell GL, Esmon CT. The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex. Proc Natl Acad Sci USA. 1996;93(19):10212-10216.
Jackson C, Whitmont K, Tritton S, March L, Sambrook P, Xue M. New therapeutic applications for the anticoagulant, activated protein C. Expert Opin Biol Ther. 2008;8(8):1109-1122.
Bae JS, Yang L, Rezaie AR. Lipid raft localization regulates the cleavage specificity of protease activated receptor 1 in endothelial cells. J Thromb Haemost. 2008;6(6):954-961.
Crikis S, Zhang XM, Dezfouli S, et al. Anti-inflammatory and anticoagulant effects of transgenic expression of human thrombomodulin in mice. Am J Transplant. 2010;10(2):242-250.
Wuensch A, Baehr A, Bongoni AK, et al. Regulatory sequences of the porcine THBD gene facilitate endothelial-specific expression of bioactive human thrombomodulin in single- and multitransgenic pigs. Transplantation. 2014;97(2):138-147.
Mohiuddin MM, Singh AK, Corcoran PC, et al. Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft. Nat Commun. 2016;7:11138.
Iwase H, Ekser B, Satyananda V, et al. Pig-to-baboon heterotopic heart transplantation - exploratory preliminary experience with pigs transgenic for human thrombomodulin and comparison of three costimulation blockade-based regimens. Xenotransplantation. 2015;22(3):211-220.
Langin M, Mayr T, Reichart B, et al. Consistent success in life-supporting porcine cardiac xenotransplantation. Nature. 2018;564(7736):430-433.
Godwin JW, d'Apice AJ, Cowan PJ. Characterization of pig intercellular adhesion molecule-2 and its interaction with human LFA-1. Am J Transplant. 2004;4(4):515-525.
Petersen TN, Brunak S, von Heijne G, Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods. 2011;8(10):785-786.
Liaw PC, Mather T, Oganesyan N, Ferrell GL, Esmon CT. Identification of the protein C/activated protein C binding sites on the endothelial cell protein C receptor. Implications for a novel mode of ligand recognition by a major histocompatibility complex class 1-type receptor. J Biol Chem. 2001;276(11):8364-8370.
Simmonds RE, Lane DA. Structural and functional implications of the intron/exon organization of the human endothelial cell protein C/activated protein C receptor (EPCR) gene: comparison with the structure of CD1/major histocompatibility complex alpha1 and alpha2 domains. Blood. 1999;94(2):632-641.
Hayashi T, Nakamura H, Okada A, et al. Organization and chromosomal localization of the human endothelial protein C receptor gene. Gene. 1999;238(2):367-373.
Blackhart BD, Ruslim-Litrus L, Lu CC, et al. Extracellular mutations of protease-activated receptor-1 result in differential activation by thrombin and thrombin receptor agonist peptide. Mol Pharmacol. 2000;58(6):1178-1187.
Wang L, Cooper D, Burdorf L, Wang Y, Iwase H. Overcoming coagulation dysregulation in pig solid organ transplantation in nonhuman primates: recent progress. Transplantation. 2018;102(7):1050-1058.
Laszik Z, Mitro A, Taylor FB Jr, Ferrell G, Esmon CT. Human protein C receptor is present primarily on endothelium of large blood vessels: implications for the control of the protein C pathway. Circulation. 1997;96(10):3633-3640.
Regan LM, Mollica JS, Rezaie AR, Esmon CT. The interaction between the endothelial cell protein C receptor and protein C is dictated by the gamma-carboxyglutamic acid domain of protein C. J Biol Chem. 1997;272(42):26279-26284.
Taylor FB Jr, Peer GT, Lockhart MS, Ferrell G, Esmon CT. Endothelial cell protein C receptor plays an important role in protein C activation in vivo. Blood. 2001;97(6):1685-1688.
Byrne GW, Stalboerger PG, Du Z, Davis TR, McGregor CG. Identification of new carbohydrate and membrane protein antigens in cardiac xenotransplantation. Transplantation. 2011;91(3):287-292.