Construction and in vitro characterization of rat FVIIa. (A) To generate rat FVIIa, we introduced a short amino acid sequence (RKRRKR) at the expected site for rat FVII cleavage between the heavy and light chains (R¹⁵² and I¹⁵³). The RKRRKR sequence is recognized and processed by the PACE/Furin intracellular protease, resulting in the secretion of 2-chain, activated rat FVII. A C-terminal HPC4 epitope tag was incorporated for immunoaffinity purification. (B) Polyacrylamide gel electrophoresis of recombinant rat FVIIa compared with rhFVIIa under reducing (R) and nonreducing (NR) conditions. The molecular size marker (M) is in kilodaltons. The heavy (∼35 kDa, H) and light (∼25 kDa, L) chains, as well as full-length (∼50 kDa, F), are indicated. (C) PT activity assay comparing activity of rat FVIIa (n = 7) relative to rhFVIIa (n = 7, 100%). Data are presented as mean ± standard deviation (SD). NS, nonsignificant difference (unpaired Student t test).