Introduction

Platelet sequestration and inappropriate coagulation cascade activation are associated with poor outcomes in multiple liver xenotransplant models. Here we evaluate a cassette of additional genetic modifications, primarily targeting coagulation and complement pathway regulation, and donor pharmacologic treatments on these phenomena.

Methods

Livers from α1,3-galactosyl transferase knockout (GalTKO), and human membrane cofactor (hCD46) pigs (Group 1, n=3) and GalTKO.hCD46 pigs also transgenic for human endothelial protein C receptor (hEPCR), thrombomodulin (hTBM), integrin associated protein (hCD47), and heme oxygenase 1 (HO-1) treated with DDAVP and clodronate liposomes (Group 2, n=4) were perfused ex vivo with whole human blood. Heparin was titrated to maintain an ACT> 400 seconds. Complete blood counts were measured at timed intervals by hemocytometer. Macrophage and platelet activation was evaluated by thromboxane (TXB2) levels, and thrombin production, as measured by prothrombin fragment F1+2 release, was assayed via ELISA. Perfusions were terminated when vascular flows declined due to high resistance or there were uncorrectable metabolic derangements.

Results

Mean survival in the GalTKO.hCD46 group was 305 minutes (SEM 148 min, range 115-597 min) and 856 minutes (SEM 61 min, range 720-960 min) in Group 2 (p=0.012). Average heparin required was 8837 U/hr in Group 1 and 1354 U/hr in Group 2 (p=0.048). Platelet counts tended to remain higher in Group 2 over the first hour (p = 0.158). TXB2 levels were lower in Group 2 over the first hour (p = 0.044). Similarly, F1+2 levels tended to be higher in Group 1 though decreased survival times limit analysis (p=0.058). AST and ALT values were not significantly different between the two groups.

Conclusions

Transgenic expression of the hEPCR.hTBM.hCD47.HO-1 cassette, along with donor pig DDAVP and clodronate liposome pretreatment, was associated with improved liver xenograft survival, reduced coagulation pathway perturbations (reduced heparin requirements, platelet consumption and F1+2 release), and decreased TXB2 elaboration versus GalTKO.hCD46 alone.

CITATION INFORMATION: Cimeno A., Burdorf L., Ayares D., Azimzadeh A., Pierson III R., Barth R., LaMattina J. hEPCR.hTBM.hCD47.HO-1 Contributes to Increased Survival, Reduced Platelet Sequestration, and Modulation of Coagulation Activation in GalTKO.hCD46 Porcine Livers Perfused with Human Blood Am J Transplant. 2017;17 (suppl 3).

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