Deletion of Cytidine Monophospho-N-Acetylneuraminic Acid Hydroxylase in Pigs Decreases Human Xenogeneic Platelet Uptake.

Abstract

Xenotransplantation of porcine livers maintains to be the closest and most likely candidate to solve the human donor liver shortage problem. Cells from pigs with deletion of both alpha(1,3)galactosyl transferase (GGTA1) and cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) genes have decreased human antibody binding. Livers from these pigs have yet to be tested for xenogeneic human platelet uptake, which is the major issue preventing liver xenotransplantation. This work examines human platelet uptake by livers from GGTA1-/-CMAH-/- (DKO) pigs. GGTA1-/- or DKO pig livers were continuously perfused with platelets ex vivo; platelets were counted at 5, 10, 15, 20, 30, 45, 60, 90, and 120 minutes. Initial platelet loss was also examined using a one-pass perfusion system in which human platelets were perfused through either domestic or DKO porcine livers once and did not cycle back through liver. The main cell types responsible for xenogeneic platelet phagocytosis are kupffer cells (KC) and liver sinusoidal endothelial cells (LSEC). Human and autologous platelet phagocytosis was examined using LSEC and KC isolated from DKO pig livers. Over 2 hours there is a greater loss of human platelets perfused through a GGTA1-/- pig liver than through a DKO pig liver (Figure). The initial loss of human platelets/g liver is significantly greater in domestic pig livers than DKO pig livers. KC, but not LSEC from DKO pig livers phagocytose xenogeneic human platelets. These results indicate that deletion of the CMAH gene not only reduced human antibody binding, but also led to a reduction in xenogeneic human platelet loss by porcine livers and in particular LSEC.Figure 1: GGTA1-/-CMAH-/- livers have reduced human platelet loss over 2 hours. Human platelets were perfused through GGTA1-/-/CMAH-/- (n= 3), GGTA1-/- (n=2) livers, or system without a liver (n=1)(No liver). Perfusate samples were taken at times indicated and platelets counted.