Active Endothelial protein C receptor (EPCR)

Endothelial protein C receptor (EPCR) also known as CD201, is a transmembrane glycoprotein expressed on vascular endothelial cells and functions as a negative regulator of thrombosis. It is expressed most strongly in the endothelial cells of arteries and veins in heart and lung. Mature human EPCR consists of a 193 amino acid (aa) extracellular domain (ECD), a 21 aa transmembrane segment, and a 7 aa cytoplasmic tail. Within the ECD, human EPCR shares 63% and 66% aa sequence identity with mouse and rat EPCR, respectively. EPCR inhibits thrombosis through its interactions with Protein C, activated Protein C (APC), and Coagulation Factors VII, and VIIa. Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human EPCR and recombinant rat Coagulation Factor VII (F7). Briefly, EPCR was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to F7-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-EPCR pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody for 1h at 37℃, wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50 µL stop solution to the wells and read at 450/630nm immediately. The binding activity of recombinant human EPCR and recombinant rat F7 was shown in Figure 1, the EC50 for this effect is 0.06 ug/mL.