Development of coagulation disorders remains a major challenge in pig-to-primate organ xenotransplantation. Our previous studies demonstrated that porcine aortic endothelial cells (pAEC) activate human platelets to express tissue factor (TF). In this study, we investigated the molecular interaction between human platelets and pAEC to identify possible targets for further genetic modification and/or systemic therapy. Human platelets were incubated with pAEC from wild-type (WT), α1,3-galactosyltransferase gene-knockout (GTKO), and GTKO pigs expressing human CD46, after which the platelets were analyzed for TF expression, TF mRNA level and TF function. pAEC were analyzed for von Willebrand factor (vWF) expression and mRNA level as well. Neutralizing antibodies for P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) were used to block the molecular interaction between platelets and pAEC. GTKO and GTKO/CD46 pAEC-activated human platelets to induce human TF activity equivalently to WT pAEC. Simultaneously, after incubation with pAEC, platelets co-expressed TF and P-selectin. TF expression was blocked when pAEC and platelets were pre-incubated with anti-human P-selectin or anti-human PSGL-1 antibodies, but not by anti-porcine P-selectin antibody. Activated pAEC up-regulated TF on platelets through the interaction of porcine vWF with the human GPIb receptor. Up-regulation of TF on human platelets by GTKO and GTKO/CD46 pAEC was comparable to that by WT pAEC, which is associated with concomitant expression of P-selectin and PSGL-1, forming an auto-augmented loop of pAEC and platelet activation. Blocking of P-selectin and PSGL-1 interaction may be required to prevent up-regulation of recipient TF in vivo after organ xenotransplantation. © 2013 John Wiley & Sons A/S.