Background Blood clotting in humans is initiated by the binding of tissue factor to activated coagulation factor VII (FVIIa) in the plasma. Previous studies have reported that hepsin and factor VII (FVII)‐activating protease are responsible for generating FVIIa. Objectives We aimed to identify other proteases that may activate FVII using zebrafish as a model. Methods We screened 179 genes encoding serine protease domains using the piggyback knockdown method to identify genes involved in the activation of zebrafish Fvii. A prolonged kinetic prothrombin time (kPT) assay was used to detect gene knockdown effects. Results In the primary screen, 21 genes showed prolonged kPT. In the secondary screen, 14 of 21 genes showed positive results. In the tertiary screen, all 14 genes showed prolonged kPT. These 14 genes were knocked down again to estimate relative levels of zebrafish Fviia. Six genes, including known genes, such as f10 and novel prostasin and hepatocyte growth factor B ( hgfb ), showed lower Fviia levels. Fvii levels were affected only by the knockdown of f7 and not by the knockdown of the other five genes. Conclusions Prostasin and hgfb are involved in generating Fviia. We hypothesize that prostasin exerts serine protease activity directly or indirectly to activate Fvii. As Hgfb has a mutated serine protease domain, it may not cleave Fvii but may bind to Fvii to induce autoactivation. The approach developed here may be extended to design other large‐scale knockdown screens.
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