Abstract
ObjectiveThe endothelial protein C-receptor (EPCR) is an endothelial transmembrane protein that binds protein C and activated protein C (APC) with equal affinity, thereby facilitating APC formation. APC has anticoagulant, antiapoptotic and antiinflammatory properties. Soluble EPCR, released by the endothelium, may bind activated neutrophils, thereby modulating cell adhesion. EPCR is therefore considered as a possible link between the anticoagulant properties of protein C and the inflammatory response of neutrophils. In the present study, we aimed to provide proof of concept for a direct binding of EPCR to the β2 –integrin Mac-1 on monocytic cells under static and physiological flow conditions.Measurements and Main ResultsUnder static conditions, human monocytes bind soluble EPCR in a concentration dependent manner, as demonstrated by flow cytometry. Binding can be inhibited by specific antibodies (anti-EPCR and anti-Mac-1). Specific binding was confirmed by a static adhesion assay, where a transfected Mac-1 expressing CHO cell line (Mac-1+ cells) bound significantly more recombinant EPCR compared to Mac-1+ cells blocked by anti-Mac-1-antibody and native CHO cells. Under physiological flow conditions, monocyte binding to the endothelium could be significantly blocked by both, anti-EPCR and anti-Mac-1 antibodies in a dynamic adhesion assay at physiological flow conditions. Pre-treatment of endothelial cells with APC (drotrecogin alfa) diminished monocyte adhesion significantly in a comparable extent to anti-EPCR.ConclusionsIn the present study, we demonstrate a direct binding of Mac-1 on monocytes to the endothelial protein C receptor under static and flow conditions. This binding suggests a link between the protein C anticoagulant pathway and inflammation at the endothelium side, such as in acute vascular inflammation or septicaemia.
Highlights
The endothelial protein C-receptor (EPCR) is an endothelial transmembrane type 1 molecule [1] that is expressed primarily on large blood vessels [2]
In the present study, we demonstrate a direct binding of Mac-1 on monocytes to the endothelial protein C receptor under static and flow conditions
Specific binding of a flag-tagged recombinant Soluble EPCR (sEPCR) to nonstimulated and Phorbol 12-myristate 13-acetate (PMA)-stimulated human monocytes was evaluated by flow cytometry in the presence or absence of anti–Mac-1 and anti-EPCR antibody
Summary
The endothelial protein C-receptor (EPCR) is an endothelial transmembrane type 1 molecule [1] that is expressed primarily on large blood vessels [2]. Protein C (PC) binding to EPCR facilitates formation of activated protein C (APC), but EPCR binds PC and APC with equal affinity [3]. The PC pathway plays a key role in the regulation of blood coagulation by inhibiting thrombin generation [4], and in limiting inflammatory response [3]. It is thought to decrease endothelial cell apoptosis in response to inflammatory cytokines and ischemia, thereby linking inflammation and endothelium [3,5]. A soluble form of EPCR that can be released by the endothelium into circulation retains full ligand-binding ability [6]. Soluble EPCR (sEPCR) binds to activated neutrophils [7], and increased levels of sEPCR were found in patients with sepsis or systemic lupus erythematosus [8]
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