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Background: The lethal thrombocytopenia that accompanies liver xenotransplantation is a barrier to clinical application. Human platelets are bound by the asialoglycoprotein receptor (ASGR) on pig sinusoidal endothelial cells and phagocytosed. Inactivation of the ASGR1 gene in donor pigs may prevent xenotransplantation-induced thrombocytopenia., Methods: Transcription activator-like effector nucleases (TALENs) were targeted to the ASGR1 gene in pig liver-derived cells. ASGR1 deficient pig cells were used for somatic cell nuclear transfer (SCNT). ASGR1 knock out (ASGR1-/-) fetal fibroblasts were used to produce healthy ASGR1 knock out piglets. Human platelet uptake was measured in ASGR1+/+ and ASGR1-/- livers., Results: Targeted disruption of the ASGR1 gene with TALENs eliminated expression of the receptor. ASGR1-/- livers phagocytosed fewer human platelets than domestic porcine livers during perfusion., Conclusions: The use of TALENs in liver-derived cells followed by SCNT enabled the production of healthy homozygous ASGR1 knock out pigs. Livers from ASGR1-/- pigs exhibit decreased human platelet uptake. Deletion of the ASGR1 gene is a viable strategy to diminish platelet destruction in pig-to-human xenotransplantation., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Hepatic failure has been treated successfully with clinical extracorporeal perfusions of porcine livers. However, dog-to-pig and pig-to-baboon liver xenotransplant models have resulted in severe bleeding secondary to liver xenograft-induced thrombocytopenia. Kupffer cells (KC) are abundant phagocytic cells in the liver. KC express the CD11b/CD18 receptor, which has been implicated in chilled platelet binding and phagocytosis through interaction with platelet surface proteins and carbohydrates. We sought to identify the role of KC CD18 in liver xenograft-induced thrombocytopenia., Methods: Primary pig KC were characterized by flow cytometry, immunoblots, and quantitative polymerase chain reaction. Pig KC were used in inhibition assays with fluorescently labeled human platelets. The CD18 receptor was targeted for siRNA knockdown., Results: Domestic and α1,3-galactosyltransferase double knockout porcine KC cultures were approximately 92% positive for CD18 as detected by quantitative polymerase chain reaction and flow cytometry. Use of CD18 blocking antibodies resulted in reduction of human platelet binding and phagocytosis. Additionally, asialofetuin, not fetuin, inhibited platelet phagocytosis suggesting the involvement of an oligosaccharide-binding site. Furthermore, reduced CD18 expression by siRNA resulted in decreased human platelet binding., Conclusions: Our data suggest that primary pig KC bind and phagocytose human platelets with involvement of CD18. Further understanding and modification of CD18 expression in pigs may result in a liver xenograft with reduced thrombocytopenic effects, which could be used as a bridge to allogeneic liver transplantation.

Background: Porcine liver xenografts represent a potential solution to the organ shortage, but thrombocytopenia occurs within minutes to hours after xenotransplantation, preventing clinical application. Recently, it was discovered that porcine liver sinusoidal endothelial cells (LSEC) bind and phagocytose human platelets. We examined the role of ASGR1 in binding and removing human platelets by the pig liver endothelium., Methods: Primary porcine enriched LSEC (eLSEC) were characterized by flow cytometry, immunoblot, quantitative PCR, and immunohistochemistry using confocal microscopy. Phagocytosis inhibition assays using anti-ASGR1 and an ASGR1 substrate were performed. ASGR1 was targeted for siRNA knockdown, and ASGR1-reduced cells were tested for human platelet binding and phagocytosis., Results: ASGR1 is expressed by eLSEC. Human platelet binding and phagocytosis by porcine eLSEC was inhibited by asialofetuin, but not fetuin, suggesting an interaction with galactose β1-4 N-acetyl glucosamine. Anti-ASGR1 antibodies inhibited human platelet binding in a dose-dependent manner. Knockdown experiments using siRNA reduced ASGR1 expression in asynchronous primary eLSEC by 40%-80%. There was a 20% reduction in translated protein significantly correlated with a 21% decrease in human platelet binding., Conclusions: ASGR1 on porcine eLSEC mediates phagocytosis of xenogeneic platelets., (© 2011 John Wiley & Sons A/S.)

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., (© 2010 John Wiley & Sons A/S.)