The Endothelial Protein C Receptor Supports TF-VIIa-Xa Ternary Complex Signaling through Protease-Activated Receptors.

Abstract 1144

Protease-activated receptor (PAR) signaling is closely linked to the cellular activation of the pro- and anticoagulant pathways. In contrast to thrombin that directly binds to and activates PAR1, other coagulation factors are dependent on co-receptors for efficient PAR cleavage. The endothelial protein C receptor (EPCR) is crucial for protein C (PC) activation by thrombomodulin-bound thrombin and supports signaling of activated PC through PAR1. EPCR may play additional roles by interacting with procoagulant proteases. EPCR binds the Gla-domain of human coagulation factor VII and conflicting reports exist about the role of EPCR as a receptor for FX. We studied the interaction of murine soluble EPCR extracellular domain (sEPCR) under physiological concentrations of divalent cations Ca²⁺/Mg²⁺. BIAcore measurements showed that murine sEPCR bound both human and mouse complexes of soluble tissue factor (TF) with FVIIa, as well as human FX. In a lipid free system measuring only protein-protein interactions, amidolytic activity of soluble TF-FVIIa was not changed by sEPCR. However, sEPCR dose dependently inhibited FX activation by both human and mouse soluble TF-FVIIa, indicating that sEPCR interacted with the TF-FVIIa-FX extrinsic activation complex. On human cells, TF forms two signaling complexes, the non-coagulant TF-FVIIa binary complex that activates PAR2 and the ternary TF-FVIIa-FXa complex that signals through PAR1 or PAR2. Overexpression of PAR2 in HUVECs resulted in PAR2 activation by activated PC, and in these transduced cells TF-FVIIa-FXa ternary complex signaling was inhibited by antibody blockade of EPCR. Human HaCaT keratinocytes constitutively synthesize TF and represent a well characterized model of TF binary and ternary complex signaling through PAR2. FACS analysis showed that EPCR was expressed on the cell surface and antibody blockade of EPCR prevented TF-FVIIa-Xa ternary, but not TF-FVIIa binary complex signaling. Mutation of FVIIa Gla residues had no effect on ternary complex signaling, indicating a primary interaction of EPCR with FX/FXa. To expand these studies to murine cells that constitutively express EPCR and TF, we isolated lung smooth muscle cells (SMC) from wild-type, PAR1-/-, PAR2-/-, and EPCR^low mice. Stimulation of SMC with thrombin, high concentrations of FXa, or FVIIa/FX, but not FVIIa alone induced PAR1-dependent signaling. While thrombin signaling was unaltered, EPCR^low SMC showed no response to the ternary complex measured by ERK1/2 phosphorylation. In order to exclude indirect effects on SMC phenotypes due to prolonged EPCR-deficiency in vivo, we further deleted EPCR in vitro by adenoviral transduction with cre recombinase from EPCR^floxflox SMC or blocked EPCR function with antibodies to murine EPCR. Both approaches inhibited TF ternary complex, but not thrombin signaling. These results show that EPCR interacts with the TF coagulation initiation complex to enable specifically ternary complex signaling and suggest that EPCR may play a role in regulating the biology of TF expressing extravascular and vessel wall cells that are exposed to limited concentrations of FVIIa and FX provided by ectopic synthesis or vascular leakage.