Activated factor VII (FVIIa) plays a crucial role in the initiation of blood coagulation. This serine protease, derived from a vitamin K-dependent precursor, nevertheless exhibits only weak enzymatic activity in its free state: its active site remains immature, as the N-terminus of its protease domain fails to insert properly into the catalytic pocket. Binding to its cofactor, tissue factor (TF), stabilizes this insertion and markedly enhances FVIIa activity, both toward its protein substrates and toward small synthetic substrates. This dependence on TF raises a long-debated question: does the therapeutic effect of FVIIa, used in particular to restore hemostasis in the absence of factor VIII, operate through a TF-dependent or a TF-independent mechanism?
To address this question, the authors sought to generate a single-domain antibody (sdAb) capable of selectively recognizing free FVIIa rather than TF-bound FVIIa. Screening of a phage library derived from an immunized llama allowed the isolation of a candidate, designated KB-FVIIa-004. This antibody binds FVIIa but not its precursor FVII, nor homologous proteins such as prothrombin or factor X and their activated derivatives. It competitively inhibits the amidolytic activity of FVIIa (Ki of 28 to 45 nM) as well as FVIIa-mediated activation of factor X (Ki of 26 nM).
The antibody's behavior changes dramatically in the presence of TF. Indeed, the amidolytic activity of the FVIIa/TF complex and the FX activation it mediates remain unchanged, even at 50- to 1000-fold molar excesses of KB-FVIIa-004. Moreover, adding TF to previously inhibited FVIIa restores its activity, and prolongation of the prothrombin time appears only at very high excesses of the antibody. The authors propose that KB-FVIIa-004 recognizes a cryptic epitope near the active site, exposed only in the zymogen-like state of FVIIa: when the protease binds a substrate or TF, it adopts its active conformation and the epitope disappears, which would account for the mutual exclusion between TF and the antibody.
For in vivo testing, a KB-FVIIa-004/albumin fusion protein (004-HSA) was produced. In factor VIII-deficient mice, this molecule blocked the ability of FVIIa to correct bleeding: a ten-fold molar excess returned blood loss to the level of untreated animals. Because human FVIIa interacts weakly with murine TF, these results are consistent with a therapeutic effect of FVIIa exerted predominantly in the absence of TF. The authors thus describe the first antibody that specifically blocks the TF-independent activity of FVIIa, a valuable tool for studying the respective roles of free and TF-bound FVIIa, both in vitro and in vivo.