Regulation Of Human Platelet Aggregation By Genetically Modified Pig Endothelial Cells And Thrombin Inhibition

Background: Coagulation disorders remain barriers to successful pig-to-primate organ xenotransplantation. In vitro, we investigated the impact of pig genetic modifications and thrombin inhibition on human platelet aggregation in response to pig aortic endothelial cells(p AEC). Methods: ①In comparison to human(h AEC) and wild-type(WT) p AEC, the expression of human complement-(CD46, CD55) or coagulation(thrombomodulin [TBM], endothelial protein C receptor [EPCR])-regulatory proteins on p AEC from WT or a1, 3-galactosyltransferase gene-knockout(GTKO) pigs was studied by flow cytometry. Using platelet-aggregometry, human whole blood platelet aggregation was evaluated after co-incubation with various AEC. ②Further, the inhibitory effect on aggregation of heparin, low molecular weight heparin, and hirudin was assessed.③ Last but not least, adding thrombin inhibition in various AEC the inhibitory effect on aggregation was assessed. Results: ① Platelet aggregation induced by all genetically modified p AEC was significantly less(P < 0.05) than that by WT p AEC(which was 54%). GTKO/CD46/TBM p AEC induced the least platelet aggregation(27%) — a reduction of almost 50% — but this remained significantly greater(P < 0.01) than aggregation induced by h AEC(4%). ②Heparin, low molecular weight heparin and hirudin almost completely prevented platelet aggregation induced by WT p AEC. ③ Platelet aggregation induced by GTKO/CD46/TBM p AEC in the presence of hirudin(1 IU/ml) was comparable to platelet aggregation induced by h AEC. Conclusions: ① Genetic modification of p AEC is associated with significant reduction of human platelet aggregation in vitro. ② Thrombin inhibition can reduce human platelet aggregation in vitro. ③With concomitant thrombin inhibition, platelet aggregation was comparable to that stimulated by h AEC.