2011 - CTS-IXA


This page contains exclusive content for the member of the following sections: TTS, CTS, IXA. Log in to view.

Parallel Session 17- Coagulation and Thrombosis II (Xeno Track)

36.506 - Effect of alpha-Gal epitopes on the interaction between pig TFPI and human FXa on pig vascular endothelium

Presenter: Cristiana, Bulato, Padova, Italy
Authors: Cristiana Bulato1, Claudia Maria Radu1, Sabrina Gavasso1, Cesare Galli2, Andrea Perota2, Luca Spiezia1, Emanuele Cozzi3, Veronica Tisato3, Paolo Simioni1

506

Effect of alpha-Gal epitopes on the interaction between pig TFPI and human FXa on pig vascular endothelium

Cristiana Bulato1, Claudia Maria Radu1, Sabrina Gavasso1, Cesare Galli2, Andrea Perota2, Luca Spiezia1, Emanuele Cozzi3, Veronica Tisato3, Paolo Simioni1

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

 

Introduction: Tissue factor pathway inhibitor (TFPI), an endothelial protein with three Kunitz-type domains, is the primary inhibitor of tissue factor (TF)-induced coagulation. TFPI directly inhibits factor Xa (FXa) and blocks the procoagulant activity of the TF/factor VIIa (FVIIa) complex by forming a quaternary TF/FVIIa/FXa/TFPI complex. Pig organs transplanted into primates exhibit significant microvascular thrombosis leading to organ rejection. This coagulation disorders may, at least in part, be due to the failure of pig TFPI to bind human FXa efficiently and inhibit human FVII/TF activity. Recently, some authors have suggested that this interaction is prevented by the presence of alpha-Gal epitopes on pig vascular endothelium.

Aim of the study: To examine whether pig TFPI, expressed on porcine aortic endothelial cells (PAEC) and alpha1,3-galactosyltransferase gene knockout PAEC (GalT-KO-PAEC), are able to bind human FXa.

Methods: Human (HUVEC, HCAEC) and pig (PAEC and GalT-KO-PAEC) endothelial cells were incubated for 30 min at 37°C in 200ul of FXa-assay buffer containing 0.5 nM human FXa. The reaction was terminated by the addition of stop buffer (FXa-assay buffer containing EDTA instead of calcium) and residual amidolytic activity of FXa was measured using Spectrozyme FXa. The concentration of unbound FXa was determined by a standard curve of purified human FXa.

Results: We found a weak binding of human FXa by TFPI expressed on pig endothelium: the amount of unbound human FXa by PAEC and GalT-KO-PAEC was 0.21±0.05 nM and 0.22±0.01 nM, respectively, and the concentration of FXa in the absence of cells with and without recombinant human TFPI was 0.38±0.06 nM and 0.30±0.08 nM, respectively. We observed that TFPI associated with HUVEC ad HCAEC binds efficiently human FXa: the level of unbound FXa was 0.06±0.01 nM and 0.07±0.01 nM, respectively.

Conclusions: Pig endothelium fails to efficiently bind human FXa, suggesting that there is a molecular incompatibility between pig TFPI and human FXa. The amount of human FXa bound by PAEC and GalT-KO-PAEC is similar, this data indicate that the alpha-Gal epitopes, on pig vascular endothelium, do not interfere with the interaction between pig TFPI and human FXa.


Important Disclaimer

By viewing the material on this site you understand and accept that:

  1. The opinions and statements expressed on this site reflect the views of the author or authors and do not necessarily reflect those of The Transplantation Society and/or its Sections.
  2. The hosting of material on The Transplantation Society site does not signify endorsement of this material by The Transplantation Society and/or its Sections.
  3. The material is solely for educational purposes for qualified health care professionals.
  4. The Transplantation Society and/or its Sections are not liable for any decision made or action taken based on the information contained in the material on this site.
  5. The information cannot be used as a substitute for professional care.
  6. The information does not represent a standard of care.
  7. No physician-patient relationship is being established.

Social

Contact

Staff Directory
+1-514-874-1717
info@tts.org

Address

The Transplantation Society
International Headquarters
740 Notre-Dame Ouest
Suite 1245
Montréal, QC, H3C 3X6
Canada