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Activated protein C levels generated by GalT-KO PAEC expressing human endothelial protein C receptor and thrombomodulin in the early stage of interaction with human protein C and thrombin

Cristiana Bulato¹, Claudia Maria Radu¹, Sabrina Gavasso¹, Cesare Galli², Andrea Perota², Federica Besenzon³, Emanuele Cozzi³, Paolo Simioni¹

¹Department of Cardiologic, Thoracic and Vascular Sciences, University of Padua, Padua; ²Laboratorio di Tecnologie della Riproduzione, Avantea, Cremona; ³Consortium for Research on Organ Transplantation, CORIT, Padua; Italy

Introduction: Kidneys from GalT-KO pigs are rejected when transplanted into primates, due to the formation of thrombi in the microvasculature. Thrombosis could be prevented by the co-expression on vascular endothelium of these transgenic pigs of two human anticoagulant proteins, endothelial protein C receptor (EPCR) and thrombomodulin (TM), which are involved in the activation of protein C (PC).

Aim of the study: We have already demonstrated that GalT-KO PAEC, expressing both human EPCR and TM, activate human PC more efficiently than PAEC expressing only one receptor. This result is observed after incubation of cells with human PC and thrombin for long periods (1 to 4 hours). Aim of the research is to determine the levels of human activated PC (APC) produced at the beginning of the reaction.

Methods: The activation PC assay was performed in the following cell lines: Human Coronary Artery Endothelial Cells (HCAEC), GalT-KO PAEC expressing hEPCR or hTM and GalT-KO PAEC expressing both human receptors. Human PC and thrombin were added to confluent cells and APC formed was measured by a chromogenic substrate after 10, 20 and 30 min of incubation.

Results: hEPCR-GalT-KO PAEC did not activate human PC. The APC generated by hTM-GalT-KO PAEC, after exposure to human PC and thrombin for 10, 20 and 30 min, was 0.57±0.01 ug/ml, 1.47±0.01 ug/ml and 3.40±0.02 ug/ml, respectively. The APC level detected in hEPCR-hTM-GalT-KO PAEC after 10, 20 and 30 min was 6.06±0.01 ug/ml, 10.49±0.07 ug/ml and 13.36±0.17 ug/ml, respectively. Finally, the APC produced by our human cell line at 10, 20 and 30 min was 0.69±0.02 ug/ml, 1.45±0.01 ug/ml and 2.53±0.02 ug/ml, respectively.

Conclusions: The co-expression of both human EPCR and TM on GalT-KO PAEC is required to obtain the optimal activation of human PC, even during the early stage of interaction of pig cell surface with human PC and thrombin.