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 G la domain of protein C results in the loss of most of the binding affi nity. This observation is compatible with at least two models: 1) the Gla domain of protein C interacts with phospholipid on cell surfaces t o stabilize interaction with EPCR or 2) the Gla domain of protein C ma kes specific protein protein interactions with EPCR. The latter model predicts that chimeric proteins containing the protein C Gla domain sh ould interact with EPCR, To test this, we constructed a prothrombin ch imera in which the Gla domain and aromatic stack of prothrombin were r eplaced 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 specific ally to 293 cells stably transfected with EPCR, but both bound poorly to sham-transfected cells. The chimera also blocked APC binding to EPC R-transfected cells in a dose-dependent fashion (K-i approximate to 13 9 nM) similarly to protein C (K-i approximate to 75 nM). Chimera bindi ng to EPCR-transfected cells was blocked by soluble EPCR, demonstratin g direct protein-protein interaction between the chimera and EPCR, Con sistent with this conclusion, the isolated Gla domain of protein C blo cked APC binding to EPCR-transfected cells (IC50 = 2 mu M). No inhibit ion 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 pro tein C is responsible for much of the binding energy and specificity o f the protein C-EPCR interaction.