Mechanisms of Xenogeneic Baboon Platelet Aggregation and Phagocytosis by Porcine Liver Sinusoidal Endothelial Cells

Qiang Peng,Shaoping Deng,Keiichi Enjyoj,Zurab Machaidze,Simon C Robson,Leo Buhler,Christian Schuetz,James Markmann,Kang Mi Lee,Eva Csizmad,Lingling Wei,Heidi Yeh,Peter J Lenting

doi:10.1371/journal.pone.0047273

Abstract

BackgroundBaboons 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.MethodsFresh 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-αMβ2 integrin Ab) were tested for the ability to inhibit phagocytosis.ResultsNone 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).ConclusionsAlthough 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.

Highlights

An increasing shortage of suitable donor livers has led to high rates of mortality of those patients awaiting liver allotransplantation
Isolation of fresh of aortic or liver sinusoidal endothelial cells and hepatocytes Aortic endothelial cells became confluent after 14 days and were CD31+ (Figure 1a and d)
Hepatocytes became adherent and were identified after 7 days and were OCH1E5+ (Figure 1b and e).Liver sinusoidal endothelial cells became confluent after 10 days and were identified by Dil-LDL uptake (Figure 1c and f)

Summary

Introduction

An increasing shortage of suitable donor livers has led to high rates of mortality of those patients awaiting liver allotransplantation. Baboon recipients of GTKO pig livers survive for less than 10 days, with profound thrombocytopenia accompanied by fatal bleeding being major limitations [6]. The endothelial lining of the liver sinusoids differ from the heart and kidney in lacking a well developed basement membrane. Baboons receiving xenogeneic livers from wild type and transgenic pigs survive less than 10 days. 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

Results

Discussion

Conclusion