Analysis of Endothelial Protein C Receptor Functionality on Living Cells’



E Ducros1, S Mirshahi2, C Bermot1, M Mirshahi*, 1
1 Centre de Recherche des Cordeliers, Université Pierre et Marie Curie – Paris 6, UMRS 872, equipe 18, Paris
2 Stago R&D, Gennevilliers, France


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© Ducros et al.; Licensee Bentham Open.

open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

* Address correspondence to this author at the Centre de Recherche des Cordeliers, Université Pierre et Marie Curie – Paris 6, UMR S 872, Les Cordeliers, Paris, F-75006, France; Tel: 000 33 1 42 34 69 39; E-mail: massoud.mirshahi@upmc.fr


Abstract

Activated protein C (APC) is a major control system of blood coagulation. APC prevents coagulation pathway by degrading Va and VIIIa plasma’s coagulation factors. Protein C activation requires its binding to specific endothelial cell receptor (EPCR). APC binding to EPCR also activates a wide range of defense mechanisms (anti-inflammatory, antiapoptosis…). EPCR expression by cells can be detected by various methods, including immunoanalysis and molecular biology. However, no assays evaluate its functionality. A method, inspired of a standard fibrinoformation time assay, was developed to estimate EPCR ability to bind APC on living cell surface in vitro. Endothelial cells were incubated with APC and fibrinoformation on cells was followed by spectrophotometry (plasma absorbance increases with fibrin polymerization). Membrane-bound EPCR retain APC, thus prolonging fibrinoformation time in a dose-dependent manner. Control was realized with EPCR-negative cells. This new method can be used on any cell type to study the expression of other coagulation receptors.

Keywords: Thrombosis, activated partial thromboplastin time, endothelial protein C receptor, activated protein C, Endothelial protein C receptor functionality analysis.