Abstract Number: PB0148
Meeting: ISTH 2020 Congress
Background: Localization of infused activated FVII (FVIIa) and its procoagulant reactions in vivo in hemophilia remain unclear. However, the prothrombinase complex was demonstrated to form on injured endothelium, at sites distinct from platelets, suggesting that activated endothelium can support fibrin-forming reactions. It is possible that FVIIa may also localize at sites distinct from platelets on the injured endothelium.
Aims: The aim of this study is to develop reagents and visualize FVIIa participation in clot formation in vivo under hemophilic conditions.
Methods: We generated biotinylated recombinant mouse FVIIa (mFVIIa) by adding a C-terminal, single biotin acceptor peptide tag (Avitag). Purified mFVIIa-Avitag was tested in vitro in a prothrombin time (PT) clotting assay and a thrombin generation assay (TGA) to determine protease activity. In vivo, mFVIIa-Avitag was visualized in a cremaster laser injury in hemophilia A (HA) mice. Prior to injury, HA mice were infused with fluorescently-labeled anti-CD41, anti-Fibrin, and streptavidin. Following injury, mice were infused with PBS or biotinylated mFVIIa-Avitag.
Results: We found that biotinylated mFVIIa-Avitag had 60% activity compared to mFVIIa in a PT assay, but reduction of activity was not observed in a TGA. Following infusion of mFVIIa-Avitag into HA mice that had undergone a laser injury, visualized mFVIIa-Avitag localized to the injury site. Three-dimensional reconstructions of confocal z-stack images revealed primarily extraluminal localization of mFVIIa-Avitag, and suggested additional binding to damaged endothelial cells adjacent to the injury site. The mFVIIa-Avitag did not substantially colocalize with platelets, it modestly increased platelet accumulation following injury, and had no effect on the extent of fibrin generation.
Conclusions: We generated site-specifically-labeled, biologically active mFVIIa. When infused in injured hemophilia A mice, it was localized at sites distinct from the growing platelet mass. These data suggest that, in this system, infused mFVIIa primarily localizes on the injured endothelium and extraluminally.
To cite this abstract in AMA style:Small JC, Stalker TJ, Dankner L, Zintner SM, Margaritis P. Real-time in vivo Visualization of Infused FVIIa in Hemophilia A Mice Following Injury [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/real-time-in-vivo-visualization-of-infused-fviia-in-hemophilia-a-mice-following-injury/. Accessed February 27, 2024.
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