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Heterotopic cardiac transplant model is being used in xenotransplantation to investigate xenograft survival of multi-genie modified donor pig organs and optimizing immunosuppression (IS) regimen in non-human primate model. We have shown earlier that significant graft survival can be achieved in alpha 1,3 alpha[MM1] Galactosidase transferase enzyme knockout pigs (GTKO) also expressing human complement regulatory transgene CD46 (CD46Tg) and thrombomodulin (hTBM). In this study, we evaluated the necessity of hTBM expression in donor's hearts for the long-term graft survival using our costimulation blockade-based IS regimen.Heterotopic cardiac xenotransplantation were performed in specific pathogen-free baboons from genetically engineered (GE) two gene pig donors which do not express hTBM (i.e.GTKO.CD46 only) (n=3) and three gene pigs (GTKO.CD46.hTBM; (n=5). Recipient baboons were treated with a short course of anti CD20 antibody, cobra venom factor, anti-thymocyte globulin, and were maintained on anti CD40 antibody (clone 2C10R4), mycophenolate mofetil and tapering doses of steroids. All baboons received continuous intravenous heparin. The dose of heparin was adjusted based on ACT levels designed to maintain the ACT at twice the baseline level. Graft survival was monitored with continuous telemetry, periodic echocardiography, and manual palpation. Blood work (CBC, chemistry, troponin, and ACT) was performed at 1-2 week intervals.Cardiac xenograft survival in recipient baboon receiving xenograft from GE donor pig without hTBM expression was significantly less (Median 70 days) as compared to 3 genes expressing pig (Median 298 days). All the three xenografts from non-hTBM donor pigs failed on 9, 98 and 70 days respectively. Increased troponin release and poor contractility of the transplanted heart were noted at the time of rejection. Non-Gal IgG and IgM antibody levels in recipient baboons from non-TBM GE donor pigs were at the baseline level at the time of rejection. The animals receiving the grafts expressing TBM also maintained their coagulation profile (PTT, PT and Fibrinogen levels) better and had fewer episodes of bleeding. Also, there was considerably less inflammation observed in 3 gene grafts.Cardiac xenograft rejection is a complex phenomenon and a combination of manipulations are required to overcome this process. The study described above further supports this notion that hTBM gene expression on donor's hearts is required along with the anti-CD40 mAb-based IS regimen and this regimen alone is insufficient to achieve long-term graft survival. This study further reveals that each mechanism of xenograft rejection is needed to be addressed separately. These very critical findings will further help us advance our journey towards clinical xenotransplantation.