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Role of the expression of human endothelial protein C receptor and thrombomodulin on GalT-KO PAEC in PC activation

Claudia Maria Radu¹, Cristiana Bulato¹, Sabrina Gavasso¹, Luca Spiezia¹, Cesare Galli⁴, Paolo Simioni¹, Emanuele Cozzi^2,3

¹Cardiologic, Thoracic and Vascular Sciences, University of Padua, Padua; ²Surgical and Gastrointestinal Sciences, University of Padua, Padua; ³Consortium for Research in Organ Transplantation (CORIT), Padua; ⁴Istituto Sperimentale Italiano Lazzaro L. Spallanzani, AVANTEA, Cremona; Italy

Introduction: The protein C (PC) anticoagulant pathway plays a key role in the control of haemostasis. PC activation is catalyzed on endothelium by a complex composed by thrombin, thrombomodulin (TM) and endothelial PC receptor (EPCR). Microvascular thrombosis observed in pig organs transplanted into primates is probably associated to the inability of EPCR and TM, expressing on pig vascular endothelium, to activate efficiently primate circulating PC.

Aim of the study: To understand the importance of expression of both human receptors (EPCR and TM) on GalT-KO PAEC surface reproducing in vitro the PC pathway.

Materials and methods: Confluent cells were pre-incubated with saturating concentrations of anti-human EPCR (5 ug/ml) or anti-human TM (20 ug/ml) or both antibodies. At the end of incubation, human PC and thrombin were added and after 120 min at 37°C the activated PC (APC) generated was measured using a chromogenic substrate.

Results: The pre-incubation of hEPCR-hTM-GalT-KO PAEC with anti-human EPCR or anti-human TM results in a reduction of PC activation of 32% and 80%, respectively. In the presence of both antibodies, the generation of APC is reduced by 97%.

Conclusions: These result confirm that TM is an essential protein for PC activation in the presence or absence of EPCR, while the function of EPCR is to amplify the catalytic reaction mediated by the thrombin-TM complex. The generation of genetically modified pigs that express inhibitors of coagulation could overcome the develop of microvasculat thrombosis in transplanted organs.