D. Rataj, S. Werwitzke, B. Haarmeijer, A.Wünsch, A. Bähr, E. Wolf, N. Klymiuk, W. Ramackers, M. Winkler, A. Tiede
Background: An important cause of pig xenograft dysfunction is the initiation of coagulation. Acute vascular rejection arises, which originates within the porcine endothelium by binding of xenoreactive natural antibodies and complement proteins. We established a flow-chamber system consisting of porcine aortic endothelial cells (pAEC) perfused with human blood. This study aims to characterize the potential benefits of a triple-transgenic approach combining alpha-gal epitope (Galalpha1-3Galbeta1-(3)4GlcNAc-R) knockout, hCD46 based complement regulation and coagulation control via expression of human thrombomodulin.
Methods: Wild-type pAEC or αGALk.o./hCD46/hTBM pAEC (clone #1198) were grown to confluence in a Bioflux 200 (Fluxion Biosciences®) flow-chamber or 6-well culture dishes. Calcein-AM labeled platelet-rich plasma (PRP, 1U/ml Heparin) was used to study dysregulated coagulation by assessing thrombus formation (in % of thrombus coverage per viewing field, analyzed by ImageJ) over time (9 minutes) under perfusion (3 dyn/cm2). In addition, the activation of the complement system (C3a) - and coagulation (thrombin-antithrombin complex (TAT)), were assessed by ELISA upon co-incubation with platelet-free plasma (6-well culture dishes).
Results: Thrombus formation was observed when wild-type pAEC were perfused with human PRP (23.5 % ± 13.0 SD). In comparison, porcine PRP as well as citrate anti-coagulated human controls exhibited markedly reduced thrombus formation (4.1 % ± 5.1 SD and 1.4 % ± 1.2 SD, p<0.0001). Incubation of wild-type pAEC with human PFP revealed a time-dependent increase in TAT (mean 11339 ± 4514 SD μg/l, at 30 min) as well as C3a (mean 682.8 ± 136.9 SD ng/ml, at 30 min). αGALk.o./hCD46/hTBM pAEC resulted in significantly reduced thrombus formation (0.3 % ± 0.2, p<0.0001), TAT and C3a formation (mean 19.3 ± 1.4 SD μg/l at 30 min and 154.6 ng/ml ± 32.9 SD at 30 min).
Conclusion: In conclusion, we demonstrate that the donor modification αGALk.o./hCD46/hTBM exhibits a substantial improvement in complement- and coagulation control, as evaluated by platelet thrombi, TAT and C3a. In summary, our data indicate, that this donor modification might significantly help to extend the longevity of transplanted xenografts in vivo.