Thomas E. Starzl Transplantation Institute, University of Pittsburgh
Effect of pig genetic modification on human platelet aggregation in response to pig vascular endothelial cells
Hayato Iwase1, Burcin Ekser 1, Huidong Zhou1, Vikas Satyananda 1, Hidetaka Hara 1, Mohamed Ezzelarab1, Cassandra Long 1, Carol Phelps 2, David Ayares2, David K.C. Cooper 1.
1Surgery, Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, United States; 2Revivicor, Blacksburg, VA, United States
Background: Platelet aggregation plays a key role in dysregulation of coagulation (thrombotic microangiopathy [TM] and/or consumptive coagulopathy [CC]) in pig organ xenografts transplanted into baboon recipients. The present study was aimed at assessing in vitro the effect of pig genetic modification on human platelet aggregation induced by pig vascular endothelial cells.
Methods: (i) The expression of galactose-α1,3-galactose (Gal), N-glycolylneuraminic acid (NeuGc), human complement-regulatory proteins (CD46 and/or CD55), and human coagulation-regulatory proteins (thrombomodulin [TBM], endothelial protein C receptor [EPCR], CD39) was measured by flow cytometry on wild-type (WT) and genetically-modified pig (p) and human (h) aortic endothelial cells (AEC).
(ii) Using a two-sample four-channel Chrono-log Whole Blood Aggregometer, we evaluated human platelet aggregation after incubation of whole human blood with hAEC or pAEC using WT and various genetically-modified pigs.
Results: (i) pAEC from WT and α1,3-galactosyltransferase-gene knockout (GTKO) pigs transgenic for complement (CD46 or CD55)- and/or coagulation (TBM, EPCR, or CD39)-regulatory proteins expressed comparable or higher levels of these proteins than hAEC. No GTKO and NeuGcKO pAEC expressed Gal or NeuGc, respectively.
(ii) When incubated with human blood, hAEC induced minimal platelet aggregation (4%), while WT pAEC induced strong aggregation (54%). The transgenic expression of CD46 or CD55, or the absence of Gal expression (GTKO) was associated with a significant reduction in human platelet aggregation (CD46 38%, CD55 39%, GTKO 44%, all p<0.05 compared to WT pAEC). The expression of TBM or EPCR on GTKO/CD46 pAEC resulted in a further reduction in platelet aggregation (27% and 33%, both p<0.05 compared to GTKO/CD46 pAEC). Combined expression of TBM+EPCR (GTKO/CD46/CD55/TBM/EPCR) significantly reduced platelet aggregation (26%). Additional expression of CD39 (GTKO/CD46/CD55/TBM/EPCR/CD39) reduced aggregation a little further (23%). The additional deletion of NeuGc (GTKO/CD46/NeuGcKO) (39%) made little difference to platelet aggregation compared to GTKO/CD46 (41%).
Conclusions: The absence of Gal+/-NeuGc, and/or transgenic expression of human complement- and/or coagulation-regulatory proteins on pAEC are all associated with reduced human platelet aggregation in vitro. The GTKO/CD46/CD55/TBM/EPCR/CD39 pAEC is associated with the greatest reduction in aggregation, though this is still not comparable to the minimal aggregation induced by hAEC. These in vitro data suggest that the transplantation of an organ from a multi-transgenic pig is likely to be associated with reduced or delayed development of TM in the graft and/or CC in the recipient, which should result in prolongation of graft survival.
08:00 - 09:00
|Progress in Solid Organ Xenotransplantation||Progress in Kidney Xenotransplantation||Room 109|
11:00 - 12:30
|Inflammation & Coagulation||Effect of pig genetic modification on human platelet aggregation in response to pig vascular endothelial cells||Room 109|
15:30 - 17:00
|Solid Organ Xenotransplantation (1)||Factors contributing to pig kidney graft survival in a baboon for >4 months||Room 109|
17:30 - 18:30
|Joint Poster Session||Regulation of the inflammatory response by human thrombomodulin transgenic pigs in xenotransplantation.||Main Foyer 1 & 2|