One amino acid in mouse factor VIIa defines its endothelial protein C receptor binding and modulates its EPCR‐dependent hemostatic activity in vivo

Background Human activated Factor VII (hFVIIa), used in hemophilia treatment, binds to the endothelial Protein C (PC) receptor (EPCR) with unclear hemostatic consequences. Interestingly, mice lack the Factor VIIa (FVIIa)-EPCR interaction. Therefore, to investigate the hemostatic consequences of this interaction in hemophilia, we previously engineered a mouse FVIIa (mFVIIa) molecule that bound mouse EPCR (mEPCR) using 3 substitutions from mouse Protein C (mPC, Leu4→Phe, Leu8→Met and Trp9→Arg). The resulting molecule, mFVIIa-FMR, modeled the EPCR-binding properties of hFVIIa and showed enhanced hemostatic capacity in hemophilic mice vs. mFVIIa. These data inferred a role of EPCR in the action of hFVIIa in hemophilia treatment. However, the substitutions in mFVIIa-FMR only broadly defined the sequence determinants for its mEPCR interaction and enhanced function in vivo. Objectives Determine the individual contribution of the mPC Phe4, Met8 or Arg9 in the in vitro/in vivo properties of mFVIIa-FMR. Methods The mEPCR binding properties of single amino acid variants of mFVIIa or mPC at position 4, 8 or 9 were investigated. Results and Conclusions Phe4 in mFVIIa or mPC was solely critical for interaction with mEPCR. In hemophilic mice, administration of mFVIIa harboring a Phe4 showed a 1.9-2.5 fold increased hemostatic capacity vs. mFVIIa that was EPCR binding-dependent. This recapitulated previous observations with triple mutant mFVIIa-FMR. Since Leu8 is crucial for the human FVIIa-EPCR binding, we describe the sequence divergence of this interaction in mice, now allowing its further characterization in vivo. We also illustrate that modulation of the EPCR-FVIIa interaction may lead to improved FVIIa therapeutics. This article is protected by copyright. All rights reserved.