Xenogeneic serum induces the novel immune coagulant fgl2 in porcine aortic endothelial cells

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Abstract 090
Introduction Xenograft rejection is characterized by endothelial cell activation and thrombosis. fgl2 is a novel immune coagulant that acts as a direct prothrombinase. fgl2 has recently been shown by our laboratory to play a central role in the thrombosis associated with solid organ xenograft rejection in a rodent model. We hypothesized that fgl2 also plays a role in the thrombosis associated with pig-to-primate (discordant) xenotransplantation. We sought to investigate our hypothesis in vitro by exposing porcine aortic endothelial cells (PAEC) to primate serum and studying the expression of fgl2 as well as total cellular procoagulant activity (PCA).
Materials and methods Primary PAEC were isolated from wild-type pigs and hDAF (human decay accelerating factor) transgenic pigs. Cells were propagated as monolayers for four to five passages in media containing 10% ultra-low IgG fetal bovine serum (FBS). After serum starvation in 0.2% FBS, cells were incubated in media containing up to 50% heat-inactivated human or baboon serum; hDAF PAEC were also incubated in media containing non-heat-inactivated sera. Cells were harvested at 0, 8, 12, or 24 h. Total cellular PCA was assayed by incubating cell lysates with normal human plasma and factor VII-deficient human plasma (to minimize tissue factor-dependent coagulation); the length of time required for the formation of fibrin clot was measured in seconds. fgl2 expression was measured both by semiquantitative RT-PCR and by Northern analysis of mRNA extracted from harvested cells.
Results RT-PCR and Northern analysis demonstrated time and dose-dependent increases in the expression of fgl2 to an equivalent level in wild-type and hDAF PAEC in response to incubation with human or baboon sera. Furthermore, this observation was associated with a time and dose-dependent increase in total cellular PCA in a functional coagulation assay. The increase in PCA observed with normal plasma was not abrogated using factor VII-deficient plasma, suggesting that the clotting activity was not due to tissue factor.
Conclusions These observations demonstrate the importance of fgl2 in the thrombosis associated with pig-to-primate xenograft rejection. Cloning of the porcine fgl2 gene will enable further in vitro analysis of the transcriptional regulation of fgl2 as well as in vivo manipulation of this gene in models of discordant xenotransplantation.
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