Background Coagulation disorders remain barriers to successful pig-to-primate organ xenotransplantation. In vitro, we investigated the impact of pig genetic modifications on human platelet aggregation in response to pig aortic endothelial cells (pAEC). Methods In comparison to human (h)AEC and wild-type (WT) pAEC, the expression of human complement- (CD46, CD55) or coagulation (thrombomodulin [TBM], endothelial protein C receptor [EPCR]) -regulatory proteins on pAEC from WT or α1,3-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. Further, the inhibitory effect on aggregation of heparin, low molecular weight heparin, and hirudin was assessed. Results Heparin, low molecular weight heparin and hirudin almost completely prevented platelet aggregation induced by WT pAEC. The level of expression of human CD46, CD55, TBM and EPCR on pAEC was comparable to that on hAEC. Platelet aggregation induced by all genetically modified pAEC was significantly less (P < 0.05) than that by WT pAEC (which was 54%). GTKO/CD46/TBM pAEC induced the least platelet aggregation (27%) - a reduction of almost 50% - but this remained significantly greater (P < 0.01) than aggregation induced by hAEC (4%). There was significant positive correlation between reduction of aggregation and TBM or EPCR expression on pAEC (r = 0.89 and r = 0.86, respectively; P < 0.05). Platelet aggregation induced by GTKO/CD46/TBM pAEC in the presence of hirudin (1 IU/ml) was comparable to platelet aggregation induced by hAEC. Conclusions Genetic modification of pAEC is associated with significant reduction of human platelet aggregation in vitro. With concomitant thrombin inhibition, platelet aggregation was comparable to that stimulated by hAEC. © 2013 John Wiley & Sons A/S.