Dogs with FVII-G96E mutation show reduced prothrombin time (PT) and minimal cFVII antigen by ELISA and western blot. (A) Using a PT clotting assay, hemostatically normal dogs (n = 5, WT) exhibited a clot time of 26 ± 7.2 seconds. In contrast, FVII-G96E dogs (n = 4) had a clot time of 106.4 ± 20 seconds (P < .05), corresponding to <1% cFVII activity. An ELISA was developed to detect cFVII antigen in canine plasma. Hemostatically normal dogs (n = 5, WT) were found to express 1040 ± 380 ng/mL, in contrast to FVII-G96E dogs (n = 4) that expressed ∼40 ng/mL. (B) Detection of cFVII-G96E or cFVII-WT in cell lysates by fluorescence-based western blot (shown in gray scale). HEK 293 cells were transiently transfected with a plasmid expressing cFVII-G96E (p-cFVII-G96E¹⁷ ). Untransfected control cell lysates without or with added recombinant cFVII-WT protein (1 µg) are shown. Black arrow indicates cFVII-WT or FVII-G96E. White arrow indicates the β-actin loading control. (C) Detection of endogenous canine WT FVII or FVII-G96E in canine plasma by fluorescence-based western blot (shown in gray scale). Lane 1, FVII-G96E plasma with 1 µg of recombinant cFVII-WT added (final sample diluted 1:16 in PBS); lanes 2 to 4, normal canine plasma diluted 1:16, 1:32, and 1:64 in PBS (respectively); lanes 5 to 7, FVII-G96E plasma diluted 1:16, 1:32, and 1:64 in PBS (respectively). Black arrow indicates canine WT FVII or FVII-G96E.