The endothelial protein C receptor (EPCR) has been demonstrated to bind activated FVII (FVIIa) through the Gla domain with equal affinity to Protein C (PC). Mouse studies suggest that EPCR is involved in the extravasation of infused human FVIIa, leading to an extended extravascular tissue persistence, longer than expected based on its circulating half-life. This provides a plausible explanation for the long-term benefits of hemophilic patients on human FVIIa prophylaxis. Collectively, these data suggest that EPCR sequesters administered FVIIa in tissues where it may have a hemostatic effect. However, the role of the endogenous FVII-EPCR interaction in normal conditions is largely unknown. For this, we have developed a mouse model to better understand this interaction in vivo. Endogenous mouse FVII and FVIIa (mFVII/FVIIa) do not bind mouse EPCR. However, our laboratory has demonstrated that L4F, L8M, and T9R substitutions in the Gla domain of mFVIIa enable its interaction with mouse EPCR while retaining full enzymatic activity in vitro. Based on that data, we utilized CRISPR/Cas9 technology to knock-in L4F, L8M, and T9R into the mFVII Gla domain in the mouse F7 locus (F7FMR), thereby developing mice with a chimeric endogenous FVII capable of binding EPCR. Founder animals were generated and capable of producing offspring, indicating that the gain-of-function in mFVII was compatible with life. Animals were subsequently backcrossed to wildtype C57BL/6 mice in order to remove potential off-target effects of the CRISPR/Cas9. Resultant heterozygous animals (F7FMR/WT) from the final cross were bred to generate F7FMR/FMR, F7FMR/WT, and F7WT/WTlittermates. We generated 59 male and 52 female animals and a binomial distribution test demonstrated that sex is equally distributed in the population. Moreover, the genotypes expected from the heterozygous crosses were inherited in a 1:2:1 ratio, further indicating that the gain-of-function in FVII is not lethal during development. As additional metrics of health, we measured weight longitudinally during weeks 1-10 of life and found no differences between the three genotypes for either gender. Complete blood counts (CBCs) revealed no differences between the F7FMR/FMR, F7FMR/WT, and F7WT/WTgenotypes, with the exception of a mild elevation in F7FMR/WTanimals compared to animals with wildtype FVII. Collectively, we found that the gain-of-function in EPCR binding by endogenous FVII is not detrimental to the overall health of the mice. Subsequently, we determined the mFVII levels in the F7FMR/FMR, F7FMR/WT, and F7WT/WTanimals using an in-house ELISA. We observed that plasmatic mFVII levels were dependent on the EPCR-binding capacity of the endogenous mFVII. Specifically, F7WT/WTmice, whose mFVII does not bind EPCR, had a plasmatic mFVII concentration of ~690 ng/ml. In contrast, F7FMR/FMRhomozygote mice had ~350 ng/ml of mouse FVII, approximately half the plasma levels of the F7WT/WT. Heterozygote animals F7FMR/WThad an intermediate plasmatic mFVII level (~550 ng/ml), suggesting that EPCR may regulate plasmatic FVII levels in vivo. Lastly, we determined the hemostatic response to injury in the F7FMR/FMR, F7FMR/WT, and F7WT/WTanimals. We did this in two ways, by measuring blood loss following tail clip assay and by determining time to vessel occlusion following ferric chloride injury of the carotid artery. We observed no differences between the three genotypes in response to either injury model. In conclusion, we have generated and characterized a novel mouse model in which endogenous FVII is capable of binding EPCR. Using this model, we demonstrated that EPCR can modulate plasmatic FVII levels in vivo but does not appear to affect hemostasis. Since this model mimics the FVII-EPCR interaction in humans, it can now be used to further investigate how this interaction participates in other normal or pathologic states that depend on FVII and/or EPCR.
Disclosures
Margaritis: Bayer Hemophilia Awards: Research Funding; Bristol-Myers Squibb: Other: Salary (spouse); CSL Behring: Other: Salary (spouse); NovoNordisk A/S: Research Funding.
Blocking endothelial protein C receptor (EPCR) accelerates thrombus development in vivo