Introduction: To overcome various features of xeno lung rejection, multiple genetic modifications and drug treatments have been proposed. Here, we evaluate whether genetic expression of human tissue factor pathway inhibitor (TFPI, extrinsic coagulation pathway inhibitor) and human CD47 ("don’t eat me" immunoregulator), alone or with combined PSGL-1 (selectin) and GMI (integrin) inhibitors of platelet and neutrophil rolling and adhesion processes (“adhesion inhibition”), influences GalTKO.hCD46 pig lung injury during perfusion with human fresh blood.

Methods: Expression of TFPI and CD47 was confirmed by immunochemistry. TFPI function on pAECs to inhibit thrombin formation in human plasma was assessed using a thrombin generation assay. In a well-established paired ex vivo lung perfusion model, GalTKO.hCD46.hTFPI.hCD47 transgenic pig lungs (n=7) were compared to reference GalTKO.hCD46 lungs (n=5). All lungs were treated with a thromboxane synthase inhibitor (1-BIA), anti-histamine (Famotidine), and anti-GPIb integrin-blocking Fab, and donor pigs were pre-treated with Desmopressin to reduce vWF expression. In both genotypes, one lung of each pair was additionally treated with adhesion inhibitors (n=6 hTFPI.hCD47, n=3 reference). Lungs were perfused for up to 8 hours or until lung failure.

Results: In vitro, GalTKO.CD46.TFPI.CD47 pAEC triggered less thrombin formation compared to GalTKO (normalized AUC: 93 ± 2 vs. 100 ± 0.4, p<0.001). Ex vivo, all except for two GalTKO.hCD46 lungs (183, 440 min) survived until elective termination at 8 hours. Additional adhesion inhibition moderately lowered PVRs in lungs expressing hTFPI.hCD47 during the first 2 hours. Neutrophil sequestration was significantly delayed during the first hour (P<0.05) in association with hTFPI.hCD47 expression, but not with adhesion inhibition (Fig.A). Lungs expressing hTFPI.hCD47 significantly attenuated terminal platelet activation measured by CD62P expression compared to GalTKO.CD46 (P<0.0001) (Fig.B). Additional adhesion inhibitors didn't further suppress platelet activation in GalTKO.hCD46 or GalTKO.hCD46.hTFPI.hCD47-expressing lungs.

Conclusion: Expression of hTFPI.hCD47 on pig lung transiently delays, but does not prevent, neutrophil and platelet sequestration or modulate xenograft injury in ex vivo perfusion model. While additional adhesion inhibition transiently reduced PVR elevation associated with hTFPI.hCD47 expression, it didn’t significantly attenuate neutrophil or platelet sequestration by these lung genotypes. We conclude that non-canonical adhesive mechanisms mediate the adhesion of human formed blood elements to pig endothelium in the context of these multiple genetic modifications and drug treatments designed to attenuate lung xenograft injury by these pathways.