Introduction: Thrombomodulin (TBM) is a coagulation regulatory protein that has shown a protective role against coagulation dysregulation and prevents thrombotic microangiopathy and premature loss of cardiac xenografts. We have previously reported long-term cardiac xenograft survival with human TBM gene expression on genetically engineered (GE) pig hearts along with immunosuppression that includes anti-CD40 antibodies. In this study, we have investigated the correlation of hTBM expression level on GE donor pig hearts with xenograft survival.

Materials and Methods: Orthotopic cardiac xenotransplantation (XTx) was performed in SPF baboons from 3-gene (i.e. GTKO.CD46.hTBM; (n=3) and “multigene” xenografts with up to 10 gene modifications, which include overexpression of hEPCR, hCD47, hDAF, hHO1, and depletion of B4KO, CMAHKO, and GHRKO (n=7). Recipients were treated with anti-CD20 mAb, CVF, ATG, anti-CD40 mAb (clone 2C10R4), MMF, and a tapering dose of steroids. Recipients also received continuous intravenous heparin infusion. Xenograft survival was monitored with telemetry, echocardiography, and manual palpation. The level of hTBM gene expression on porcine endothelial cells (PAEC) and explanted heart samples were examined by quantitative real-time (RT) PCR.

Results and Conclusion: Life-supporting orthotopic cardiac XTx were performed without any difficulty and baboons were extubated immediately following surgery and were active, eating, and generally well soon thereafter. Orthotopic cardiac xenografts' survival ranged from few hours to 264 days. Quantitative RT PCR demonstrated variable hTBM expression (range 2 to 38 folds) on PAEC and explanted xenografts. The level of hTBM expression directly correlated with cardiac xenograft survival in 3-gene (GTKO.CD46.hTBM) porcine hearts  and  multigene expressing cardiac xenograft (R2 squared value 1 and 0.68 p=0.04). These results suggest the importance of hTBM expression with higher expression associated with prolonged cardiac xenograft survival.