ACTIVATED PROTEIN C CREATES STABLE BLOOD VESSELS BY A NOVEL MECHANISM OF DIRECT BINDING TO TIE2 — The Association Specialists
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ACTIVATED PROTEIN C CREATES STABLE BLOOD VESSELS BY A NOVEL MECHANISM OF DIRECT BINDING TO TIE2 (432)

Nikita Minhas 1 , Pradeep Naik 2 , Meilang Xue 1 , Chris Jackson 1
  1. Sutton Arthritis Research Laboratory, Institute for Bone and Joint Research, Kolling Institute of Medical Research, University of Sydney at Royal North Shore Hospital, St Leonards, NSW, Australia
  2. Biotechnology and Bioinformatics, Jaypee University of Information Technology, Solan, India

Angiopoietin(Ang)1 and Ang2 are the only known human ligands for the endothelial receptor, Tie2, which is crucial for vascular integrity and homeostasis. We have previously reported that Activated Protein C(APC) enhances endothelial barrier integrity via Tie2 by differentially regulating Ang1 and Ang2 (Faseb J, 2010). The aim of this study was to elucidate the exact mechanisms via which APC acts to exert its protective effect on endothelial cells. Using a mouse model of vascular leakage, we showed that APC reversed the effect of endotoxin-induced permeability, acting via Tie2 receptor at both 4 and 24hr timepoints. The vascular improvement at 24hr timepoint could be explained by APC increasing Ang1. However, these mechanisms do not explain the rapid protective effect of APC at 4hr. So we investigated how APC exerts these rapid effects. Computational analysis using molecular modelling indicated that APC binds to Tie2 in an antigen-antibody manner with high binding energy. Solid phase binding assays confirmed that APC does bind to Tie2 , remarkably, with ~10 times stronger binding affinity than its recognised ligand, Ang2. Strong binding between APC and Tie2 was entrenched by numerous techniques, including Biacore, cell-based immunoprecipitation and SDS-PAGE-based binding assays. When challenged with the native Ang ligands, APC competed for binding in a dose-dependent manner. We utilised functional assays to determine whether APC regulates endothelial barrier integrity after directly binding Tie2. Electiric cell impedeance and membrane insert permeability assays showed that APC rapidly (15mins) activates the Tie2 receptor and  reduced the leakiness of a human umbilical vein endothelial monolayer by >40%.

In summary, APC is a novel ligand for the endothelial receptor, Tie2 and activates the receptor to enhance endothelial barrier function. This novel mechanism helps explain how APC creates stable blood vesssels and is protective in vascular leakage related pathologies.