Potential value of human thrombomodulin and DAF expression for coagulation control in pig-to-human xenotransplantation

Background: Problems of coagulation disorder remain to be resolved in pig-to-primate xenotransplantation. Molecular incompatibilities in the coagulation systems between pigs and humans, such as the thrombomodulin (TM)-protein C system or direct prothrombinase activity, have been suggested as possible causes. Coagulation and complement activation are closely related to each other. The purpose of this study was to elucidate the protective effects on the coagulation system of the expression of human TM and decay accelerating factor (hDAF) (for inhibition of complement activation) in pig endothelial cells. Methods: Human aortic endothelial cells (HAEC), porcine aortic endothelial cells (PAEC), hDAF-expressing PAEC (hDAF-PAEC), hDAF/Endo-beta-galactosidase C-expressing PAEC (hDAF/EndoGalC-PAEC), hTM-expressing PAEC (hTM-PAEC), hDAF/hTM expressing-PAEC (hDAF/hTM-PAEC), and hDAF/EndoGalC/hTM-expressing PAEC (hDAF/EndoGalC/hTM-PAEC) were used in this study. Coagulation activity was examined by clotting, activated protein C (APC), and thrombin generation assay. Results: A large difference was observed in clotting time of human plasma when exposed to PAEC (170 s) and HAEC (1020 s). hTM expression on PAEC was proven to produce a comparable level of APC to that produced by HAEC, which prolonged the clotting time, though not to the level of HAEC. Pretreatment with human sera considerably shortened the clotting time in PAEC (80 s). hDAF-PAEC significantly inhibited such a shortening of clotting time by reductions in tissue factor expression and thrombin generation. Thrombin generation through direct prothrombinase activity, which was detected only in PAEC, could be suppressed by hTM expression. Suppression of antibody binding and complement activation improved clotting time not in PAEC, but in PAEC expressing hTM. Conclusions: In addition to effective suppression of antibody-induced complement activation, hTM expression in PAEC may be essential for regulating procoagulant activity in xenotransplantation.