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Published on: September 30, 2021
One amino acid in mouse activated factor VII defines its endothelial protein C receptor (EPCR) binding and modulates
G Pavani1, S M Zintner1, L Ivanciu1,2,3
1Department of Pediatrics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
Abstract:
Essentials The lack of factor (F) VIIa-endothelial protein C receptor (EPCR) binding in mice is unresolved. A single substitution of Leu4 to Phe in mouse FVIIa (mFVIIa) enables its interaction with EPCR. mFVIIa with a Phe4 shows EPCR binding-dependent enhanced hemostatic function in vivo vs. mFVIIa. Defining the FVIIa-EPCR interaction in mice allows for further investigating its biology in vivo.
Summary:
Background Human activated factor VII (hFVIIa), which is used in hemophilia treatment, binds to the endothelial protein C (PC) receptor (EPCR) with unclear hemostatic consequences. Interestingly, mice lack the activated FVII (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) by using three substitutions from mouse PC (mPC), i.e. 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 versus mFVIIa. These data implied 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 To determine the individual contributions of mPC Phe4, Met8 and Arg9 to 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 resulted in a 1.9-2.5-fold increased hemostatic capacity versus mFVIIa that was EPCR binding-dependent. This recapitulated previous observations made with triple-mutant mFVIIa-FMR. As Leu8 is crucial for hFVIIa-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.
Insights
A single amino acid change (Leu4 to Phe) in mouse factor VIIa (mFVIIa) enables binding to endothelial protein C receptor (EPCR). This mFVIIa-Phe4 variant shows enhanced hemostatic function in vivo, offering insights into factor VIIa-EPCR interactions.
Area of Science:
- Hemostasis and Thrombosis
- Molecular Biology
- Protein Engineering
Background:
- Human activated factor VII (hFVIIa) treats hemophilia but its interaction with endothelial protein C receptor (EPCR) has unclear hemostatic effects.
- Mice normally lack the activated FVIIa-EPCR interaction, hindering in vivo studies.
- Previous engineering of mouse FVIIa (mFVIIa) with three substitutions (mFVIIa-FMR) restored EPCR binding and enhanced hemostasis in hemophilic mice.
Purpose of the Study:
- To determine the specific contribution of individual amino acid substitutions (Phe4, Met8, Arg9) from mouse PC to mFVIIa-FMR's properties.
- To investigate the in vitro and in vivo effects of these individual substitutions on mFVIIa-EPCR interaction and hemostatic function.
Main Methods:
- Created single amino acid variants of mFVIIa or mPC at positions 4, 8, and 9.
- Assessed the mEPCR-binding properties of these variants.
- Evaluated the hemostatic capacity of mFVIIa variants in hemophilic mice.
Main Results:
- The substitution of Leucine 4 to Phenylalanine (Phe4) in mFVIIa or mPC was solely responsible for the interaction with mEPCR.
- Administration of mFVIIa with Phe4 in hemophilic mice resulted in a 1.9-2.5-fold increase in hemostatic capacity compared to wild-type mFVIIa.
- This enhanced hemostasis was dependent on EPCR binding, confirming the role of Phe4 and validating previous findings with the triple mutant.
Conclusions:
- Phenylalanine at position 4 is critical for the interaction between mouse FVIIa and mouse EPCR.
- The Leu4 to Phe substitution in mFVIIa significantly enhances hemostatic function in vivo in an EPCR-dependent manner.
- Understanding the sequence divergence in FVIIa-EPCR interactions between humans and mice allows for further in vivo characterization and potential development of improved FVIIa therapeutics.
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