Session: 321. Coagulation and Fibrinolysis: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Translational Research
Methods: We adapted the femoral vein thrombosis model (Gollomp et al., 2018; Welsh et al., 2019) to initiate coagulation with 2% FeCl3. Clot formation was directly visualized and platelet and fibrin accumulation were quantified by intravital confocal microscopy. Prior to injury, mice were administered fluorescently labeled non-inhibitory antibodies to platelets and fibrin with or without variable amounts of recombinant hFVIII protein in PBS. Then, topical application of 2% FeCl3 to the femoral vein was applied for 5 minutes. Thereafter, fluorescent images were collected over 30 minutes, and confocal time-lapsed images were analyzed with Slidebook 6 software. In vivo FVIII protein recovery was confirmed by measuring chromogenic FVIII activity from a blood draw collected within 5 minutes of injury; FVIII activity values were within 20% of the stated experimental group.
Results: HA mice demonstrated no platelet or fibrin accumulation. Consistent with model sensitivity to excess FVIII function, mice administered recombinant FVIII to achieve 300% FVIII activity had a 2-fold increase in platelet sum intensity compared to WT mice. To preliminarily support that the model was sensitive to disrupted FVIIIa regulation by activated protein C (APC), the assay was repeated on CRISPR/cas9 generated mice expressing a FVIII variant that cannot be cleaved by APC (FVIIIQQ mice). FVIIIQQ mice demonstrated a 3.3-fold increase in platelet accumulation compared to WT mice and a 1.4-fold increase compared to mice with 300% FVIII:Ag; these data are overall consistent with the established approximate 4-5-fold enhanced potency of FVIII-QQ over FVIII-WT (Wilhelm et al., 2021; Sternberg et al., 2024). Lastly, to confirm that observations may correlate with an established prothrombotic risk in mice and humans that similarly probes the APC pathway, we assayed homozygous FV-Leiden (FVL) mice. Homozygous FVL mice demonstrated a 4.5-fold increase in platelet accumulation compared to WT mice and a 1.8-fold increase compared to mice with 300% FVIII:Ag.
Conclusions: These data support that the 2% FeCl3 femoral vein injury model is sensitive to excess FVIII function and the impact of APC on FVIIIa regulation, and suggests this model may be used to study FVIII-associated prothrombotic risk in vivo. Long-term, we aim to employ this model to elucidate the mechanisms by which excess FVIII confers prothrombotic risk and to inform a therapeutic window of gain-of-function FVIII variants that bypass mechanisms of FVIIIa-regulation for second-generation hemophilia A gene therapy approaches.
Disclosures: George: CSL Behring: Consultancy; Tome Biosciences: Consultancy; Regeneron: Consultancy; Spark: Consultancy; Pfizer: Consultancy; Asklepios BioPharmaceutical: Patents & Royalties; Form Bio: Membership on an entity's Board of Directors or advisory committees.
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