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 t... More
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.
