Construction and in vitro characterization of canine FVII and canine FVIIa. (A) The canine FVII and FVIIa constructs contained a C-terminal epitope tag (HPC4) for immunoaffinity purification. To generate canine FVIIa, we introduced a short amino acid sequence (RKRRKR) at the normal site of cleavage of canine FVII (Arg¹⁵²-Ile¹⁵³, indicated), which is recognized by an intracellular protease of the PACE/furin type. Cleavage at this sequence (indicated by ) results in the secretion of a 2-chain, activated molecule. (B) Polyacrylamide gel electrophoresis of zymogen (Z) cFVII and cFVIIa under reducing conditions. The molecular size marker (M) bands in kDa are indicated as well as the heavy (∼35 kDa, H) and light (∼20 kDa, L) chains. (C) TF-dependent activity of cFVII and cFVIIa in a PT-based clotting assay using canine FVII-deficient plasma relative to rhFVIIa (100%). *P < .05 versus rhFVIIa. (D) TF-independent activity of cFVII and cFVIIa in an aPTT-based clotting assay using either canine hemophilia A (HA) or B (HB) plasma, relative to rhFVIIa (100%). ND indicates nondetectable activity.