The interaction between the endothelial cell protein C receptor and protein C is dictated by the gamma-carboxyglutamic acid domain of protein C

The endothelial cell protein C receptor (EPCR) binds to both protein C and activated protein C (APC) with similar affinity. Removal of the Gla domain of protein C results in the loss of most of the binding affinity. This observation is compatible with at least two models: 1) the Gla domain of protein C interacts with phospholipid on cell surfaces to stabilize interaction with EPCR or 2) the Gla domain of protein C makes specific protein protein interactions with EPCR. The latter model predicts that chimeric proteins containing the protein C Gla domain should interact with EPCR, To test this, we constructed a prothrombin chimera in which the Gla domain and aromatic stack of prothrombin were replaced with the corresponding region of protein C. The I-125-labeled chimera (K-d = 176 nM) and I-125-APC (K-d = 65 nM) both bound specifically to 293 cells stably transfected with EPCR, but both bound poorly to sham-transfected cells. The chimera also blocked APC binding to EPCR-transfected cells in a dose-dependent fashion (K-i approximate to 139 nM) similarly to protein C (K-i approximate to 75 nM). Chimera binding to EPCR-transfected cells was blocked by soluble EPCR, demonstrating direct protein-protein interaction between the chimera and EPCR, Consistent with this conclusion, the isolated Gla domain of protein C blocked APC binding to EPCR-transfected cells (IC50 = 2 mu M). No inhibition was observed with the isolated prothrombin Gla domain, A protein C chimera with the prothrombin Gla domain and aromatic stack failed to bind to EPCR detectably. These data suggest that the Gla domain of protein C is responsible for much of the binding energy and specificity of the protein C-EPCR interaction.