Investigating the interconnection between complement and coagulation in xenotransplantation
R. Sfriso1, N. Klymiuk2, E. Wolf2, J. Seebach3, R. Rieben1 (1Bern, Switzerland, 2Munich, Germany, 3Geneva, Switzerland)
Vascular wall biology and disorders
Time: 17:15 - 18:15
Objective: Acute vascular rejection is one of the main issues that hamper xenotransplantation to be clinically feasible. It is characterized by the activation of complement and coagulation systems converging into the onset of thrombotic microangiopathy which leads to the xenograft failure. In this study porcine endothelial cells (pEC), both wild type (wt) and lacking the major xenoantigen Gal-alpha-1,3-Gal (GTKO) were analysed for the activation of complement and coagulation in a xenotransplantation setting. Furthermore, genetically modified pEC overexpressing the human complement regulatory protein CD46 (hCD46) and human Thrombomodulin (hTM) were tested in a whole blood clotting assay.
Methods: Cell ELISA was carried out to analyse immunoglobulin and complement deposition after incubation of pEC with normal human serum (NHS). Activation of pEC was evaluated by immunofluorescence staining of Tissue Factor (TF) and E-Selectin after treatment with TNF or NHS. The anticoagulant properties were analysed by performing a whole blood clotting assay which involves the cultivation of different types of pEC on collagen coated microcarrier beads to increase the endothelial surface-to-blood volume ratio mimicking the in vivo situation in small vessels. After confluency, microcarrier beads were incubated with freshly drawn non anticoagulated human blood and the clotting time was measured. The values obtained with the different types of cells were graphed and compared. Beads were retrieved during the experiment and stained for von Willebrand Factor (vWF) and TF.
Results: A lower deposition of IgM, IgG, C3b/c and C4b/c was observed on GTKO pEC as compared with wildtype, which is due to the GalKO phenotype. NHS incubation led to the activation of pEC as showed by the expression of E-selectin and TF which was lower in GalKO pEC. GalKO/hCD46/hTM transgenic pEC delayed the clotting time of non-anticoagulated human blood in the microcarrier bead assay in a higher extent compared to the other cell lines. At the end of the experiment vWF and TF were strongly upregulated.
Conclusion: Data showed that NHS treatment leads to activation of pEC and genetically modified cells are able to prolong the clotting time of non-anticoagulated human blood much longer than wild type cells. This result suggests that the overexpression of hCD46 and hTM has a beneficial effect on the regulation of coagulation in xenotransplantation.