Expression of Human Thrombomodulin by Porcine Endothelium Attenuates Thrombus Formation and Platelet Activation in a...

Long term xenografts often fail in association with evidence of coagulation pathway dysregulation, which may be caused in part by the inefficient binding of inactivated protein C (PC) to porcine thrombomodulin (TBM). This study evaluates the effect of human TBM (hTBM) expression by genetically modified pig endothelium on human platelet activation, adhesion, and aggregation within a dynamic in vitro xenoperfusion model. Porcine aortic endothelial cells (PAECs) with a variety of genetic modifications and immortalized human coronary endothelial cells (HCECs) (n = 9) were cultured to confluence in microfluidic channels and perfused for one hour with human whole blood stained with 0.5 mM Calcein to visualize thrombosis formation. After one hour of perfusion, platelet adhesion (as percent surface area coverage) and aggregation (fluorescence intensity) were significantly reduced in association with human APC (hAPC) pretreatment of GTKO, and with DKO.hCD55.hTBM or 10-GE ECs relative to GT/DKO ± hCD55 ECs. Fold change in frequency of activated (CD62P+) platelets in channel effluent was significantly decreased in association with GTKO+hAPC (1.5 ± 0.03 p = 0.01, n = 6), and DKO.hCD55.hTBM (1.6 ± 0.03 p = 0.02, n = 12) or 10-GE ECs (1.3 ± 0.04 p < 0.0001, n = 9) relative to GT/DKO ± hCD55 ECs (1.8 ± 0.07, n = 8). No significant difference was observed between DKO.hCD55.hTBM and GTKO+hAPC. We conclude that, under conditions mimicking microcirculatory flow, hTBM expression confers anti-thrombotic effects comparable to those with hAPC pretreatment of GTKO ECs. Consequently, hTBM expression is a promising method to inhibit platelet dysregulation in xenotransplant models.