The fate of human platelets perfused through the pig liver: implications for xenotransplantation

Background: Pig liver xenotransplantation could offset the shortage of livers available for orthotopic liver transplantation. Studies in pig and baboon liver xenografts revealed the main obstacle to be a lethal thrombocytopenia that occurred within minutes to hours of transplantation. Methods: We have created a model of xenotransplantation-induced thrombocytopenia using ex vivo pig liver perfusion with human platelets. Thrombocytopenia was examined using fluorescently labeled platelets during the ex vivo perfusion and coculture with primary liver sinusoidal endothelial cells (LSEC). Results: Ex vivo liver perfusion revealed that 93% of human platelets were removed from circulation after 15 min. Endothelial cells and platelets were not activated based on tissue factor release into the perfusate. Biopsies from the ex vivo perfusion at 15 and 30 min and in vitro analysis indicated that human platelets are phagocytosed by pig LSEC and degraded in phagosomes. Sixty to 120 min after the addition of platelets to the ex vivo perfusion system, we observed platelet fragments and degraded platelets in hepatocytes. Platelet phagocytosis was not mediated by opsonization as Fc blocking had no effect on platelet phagocytosis. In vitro uptake of human platelets by primary LSEC cultures peaked at 15 min followed by a greater than 55% decrease in platelet fluorescence after 3 h. Primary pig LSEC phagosomes containing human platelets were colocalized with lysosomes positive for lysosome-associated membrane protein-1 (LAMP1), indicating the formation of mature phagosomes within pig LSEC. Conclusions: Our observation of pig LSEC phagocytosis of human platelets describes a novel mechanism of large-particle uptake in the liver. The creation of a model system to study xenotransplantation-induced thrombocytopenia makes possible the investigation into mechanisms that mediate platelet loss.