Search

Background: Asialoglycoprotein receptor-1 (ASGR1) mediates capture and phagocytosis of platelets in pig-to-primate liver xenotransplantation. However, thrombocytopenia is also observed in xenotransplantation or xenoperfusion of other porcine organs than liver. We therefore assessed ASGR1 expression as well as ASGR1-mediated xenogeneic platelet phagocytosis in vitro and ex vivo on porcine aortic, femoral arterial, and liver sinusoidal endothelial cells (PAEC/PFAEC/PLSEC)., Methods: Porcine forelimbs were perfused with whole, heparinized human or autologous pig blood. Platelets were counted at regular intervals. Pig limb muscle and liver, as well as PAEC/PFAEC/PLSEC, were characterized for ASGR1 expression. In vitro, PAEC cultured on microcarrier beads and incubated with non-anticoagulated human blood were used to study binding of human platelets and platelet-white blood cell aggregation. Carboxyfluorescein diacetate succinimidyl ester-labeled human platelets were exposed to PAEC/PFAEC/PLSEC and analyzed for ASGR1-mediated phagocytosis., Results: Human platelet numbers decreased from 102 ± 33 at beginning to 13 ± 6 × 10/μL (P < 0.0001) after 10 minutes of perfusion, whereas no significant decrease of platelets was seen during autologous perfusions (171 ± 26 to 122 ± 95 × 10/μL). The PAEC, PFAEC, and PLSEC all showed similar ASGR1 expression. In vitro, no correlation was found between reduction in platelet count and platelet-white blood cell aggregation. Phagocytosis of human carboxyfluorescein diacetate succinimidyl ester-labeled platelets by PAEC/PFAEC/PLSEC peaked at 15 minutes and was inhibited (P < 0.05 to P < 0.0001) by rabbit anti-ASGR1 antibody and asialofetuin., Conclusions: The ASGR1 expressed on aortic and limb arterial pig vascular endothelium plays a role in binding and phagocytosis of human platelets. Therefore, ASGR1 may represent a novel therapeutic target to overcome thrombocytopenia associated with vascularized pig-to-primate xenotransplantation.

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: We have previously shown that recognition of human erythrocytes by porcine Kupffer cells is mediated by a carbohydrate-dependent mechanism. The present study explores the possible ligands existing on human glycophorin A and tests their ability to inhibit erythrocyte rosette formation., Methods: Human erythrocytes were tested for ABO and MN specificity and used as targets in a 51Chromium quantitative erythrocyte rosette assay. Monosaccharides present on human glycophorin A, neuraminyl lactoses, bovine and porcine submaxillary mucins (BSM and PSM), and hyaluronic acid as well as proteoglycan N-linked glycosidase F(PNGaseF)- and sialidase A-treated human erythrocyte glycoproteins (hEGP) and human erythrocytes were all tested for inhibitory potential in the rosetting assay., Results: Porcine Kupffer-cell recognition of human erythrocytes was insensitive to differences in blood groups A, B, O, or MN. At 30 mM, the monosaccharide, N-acetylneuraminic acid, and the trisaccharide mixture, neuraminyl lactoses, disrupted human erythrocyte recognition by 25% and 30%, respectively. A dilution of BSM but not PSM inhibited the rosetting assay by 17% (.2 mg/mL), 33% (1 mg/mL), and 53% (2 mg/mL). The same dilution of hyaluronic acid had no effect on rosetting. Removal of N-linked oligosaccharides from hEGP with PNGaseF did not impair its ability to inhibit the rosetting assay. In contrast, removal of sialic acid completely abrogated its inhibitory ability. Treatment of whole human erythrocytes with sialidase A likewise prevented recognition by porcine Kupffer cells., Conclusions: Terminal sialic acid on human erythrocytes is a target recognized by porcine Kupffer cells, suggesting a role for a sialic-acid receptor in innate cellular recognition of xenogeneic epitopes. Inasmuch as this work reveals a carbohydrate-recognition mechanism for cellular rejection, we shed light on a potential new boundary that will need to be overcome within xenotransplantation.

Background: We have previously shown that when porcine livers are perfused with human blood, porcine Kupffer cells extract up to 3 units of human erythrocytes over the course of a 72 hr perfusion. We have previously hypothesized that the recognition event responsible for this interaction involves a lectin receptor on the surface of the porcine Kupffer cell interacting with a carbohydrate epitope on the surface of the human erythrocytes., Methods: Treatments to disrupt the protein core of purified glycoproteins from the surface of human erythrocytes included: pronase, trypsin, beta-mercaptoethanol (2-ME), and heating to 90 degrees . Alternatively, we have removed the carbohydrate residues from purified human red blood cell (RBC) glycoproteins using glycosidases. Erythrocyte binding in the presence or absence of treated glycoproteins was quantified by Chromium-labeled RBC recognition by primary cultures of porcine Kupffer cells., Results: Human, but not porcine, erythrocytes were bound by in vitro primary cultures of porcine Kupffer cells. Binding was inhibited by preincubation of porcine Kupffer cells with purified human erythrocyte glycoproteins (hEGP) from human erythrocyte membranes. Pretreatment of human EGP with pronase, trypsin, 2-ME, or heating did not interfere with the ability of human EGP to inhibit the binding of human erythrocytes to porcine Kupffer cells. Deglycosylation of the purified hEGP completely disrupted hEGP ability to inhibit the binding of human erythrocytes to porcine Kupffer cells., Conclusion: We conclude that porcine Kupffer cells bind xenogeneic human RBC by recognition of a carbohydrate epitope on the surface of human erythrocytes. We hypothesize that this binding is mediated by a porcine Kupffer cell lectin receptor.