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Presenter: Claudia Maria, Radu, Padua, Italy
Authors: Claudia Maria Radu1, Cristiana Bulato1, Sabrina Gavasso1, Luca Spiezia1, Cesare Galli4, Paolo Simioni1, Emanuele Cozzi2,3
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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 Cozzi2,3
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.
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