title: Mechanisms of xenogeneic baboon platelet aggregation and phagocytosis by porcine liver sinusoidal endothelial cells.
abstract: BACKGROUND
  Baboons receiving xenogeneic livers from wild type and transgenic pigs survive less than 10 days. One of the major issues is the early development of profound thrombocytopenia that results in fatal hemorrhage. Histological examination of xenotransplanted livers has shown baboon platelet activation, phagocytosis and sequestration within the sinusoids. In order to study the mechanisms of platelet consumption in liver xenotransplantation, we have developed an in vitro system to examine the interaction between pig endothelial cells with baboon platelets and to thereby identify molecular mechanisms and therapies.
  
  
  METHODS
  Fresh pig hepatocytes, liver sinusoidal and aortic endothelial cells were isolated by collagenase digestion of livers and processing of aortae from GTKO and Gal+ MGH-miniature swine. These primary cell cultures were then tested for the differential ability to induce baboon or pig platelet aggregation. Phagocytosis was evaluated by direct observation of CFSE labeled-platelets, which are incubated with endothelial cells under confocal light microscopy. Aurintricarboxylic acid (GpIb antagonist blocking interactions with von Willebrand factor/vWF), eptifibatide (Gp IIb/IIIa antagonist), and anti-Mac-1 Ab (anti-α(M)β(2) integrin Ab) were tested for the ability to inhibit phagocytosis.
  
  
  RESULTS
  None of the pig cells induced aggregation or phagocytosis of porcine platelets. However, pig hepatocytes, liver sinusoidal and aortic endothelial cells (GTKO and Gal+) all induced moderate aggregation of baboon platelets. Importantly, pig liver sinusoidal endothelial cells efficiently phagocytosed baboon platelets, while pig aortic endothelial cells and hepatocytes had minimal effects on platelet numbers. Anti-MAC-1 Ab, aurintricarboxylic acid or eptifibatide, significantly decreased baboon platelet phagocytosis by pig liver endothelial cells (P<0.01).
  
  
  CONCLUSIONS
  Although pig hepatocytes and aortic endothelial cells directly caused aggregation of baboon platelets, only pig liver endothelial cells efficiently phagocytosed baboon platelets. Blocking vWF and integrin adhesion pathways prevented both aggregation and phagocytosis.
authors.affiliation: Division of Transplant Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Peng
authors.forename: Heidi
authors.initials: H
authors.lastname: Yeh
authors.forename: Lingling
authors.initials: L
authors.lastname: Wei
authors.forename: Keiichi
authors.initials: K
authors.lastname: Enjyoj
authors.forename: Zurab
authors.initials: Z
authors.lastname: Machaidze
authors.forename: Eva
authors.initials: E
authors.lastname: Csizmad
authors.forename: Christian
authors.initials: C
authors.lastname: Schuetz
authors.forename: Kang Mi
authors.initials: KM
authors.lastname: Lee
authors.forename: Shaoping
authors.initials: S
authors.lastname: Deng
authors.forename: Simon C
authors.initials: SC
authors.lastname: Robson
authors.forename: James
authors.initials: J
authors.lastname: Markmann
authors.forename: Leo
authors.initials: L
authors.lastname: Buhler
mesh_terms: D000818:Animals; D001312:Aurintricarboxylic Acid / Q000008:administration & dosage; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D000077542:Eptifibatide; D022781:Hepatocytes / Q000276:immunology / Q000378:metabolism; D016031:Liver Transplantation / Q000009:adverse effects; D016177:Macrophage-1 Antigen / Q000235:genetics / Q000378:metabolism; D010215:Papio / Q000276:immunology / Q000502:physiology; D010455:Peptides / Q000008:administration & dosage; D010587:Phagocytosis / Q000235:genetics / Q000276:immunology; D010974:Platelet Aggregation / Q000235:genetics / Q000276:immunology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000037:antagonists & inhibitors; D013552:Swine / Q000235:genetics / Q000276:immunology; D014183:Transplantation, Heterologous / Q000009:adverse effects / Q000276:immunology; D014841:von Willebrand Factor / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D016177:Macrophage-1 Antigen; D010455:Peptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D014841:von Willebrand Factor; D001312:Aurintricarboxylic Acid; D000077542:Eptifibatide