A single‐domain antibody that blocks factor VII a activity in the absence but not presence of tissue factor

Background Activated factor VII ( FVII a) is pertinent to the initiation of blood coagulation. Proteolytic and amidolytic activity of FVII a are greatly enhanced by its cofactor, tissue factor ( TF ). Objective We aimed to generate a single‐domain antibody (sdAb) that recognizes free FVII a rather than TF ‐bound FVII a. Methods A llama‐derived phage library was used to screen for anti‐ FVII a sdAbs. Results One sdAb, KB ‐ FVII a‐004, bound to FVII a, but not to its precursor FVII or to homologous proteins (prothrombin, factor X, or their activated derivatives). FVII a amidolytic activity was inhibited by KB ‐ FVII a‐004 ( K i  = 28‐45 nM ) in a competitive manner. KB ‐ FVII a‐004 also inhibited FVII a‐mediated FX activation ( K i  = 26 nM ). In contrast, KB ‐ FVII a‐004 was inefficient in prolonging the clotting time of the prothrombin time‐test, which was prolonged by a maximum of 10 s at high sdAb concentrations (10 μM). Furthermore, FVII a/ TF amidolytic activity or FVII a/ TF ‐mediated FX activation remained unaffected up to a 50‐fold to 1000‐fold molar excess of KB ‐ FVII a‐004. These data suggest that KB ‐ FVII a‐004 loses its inhibitory activity in the presence of TF . A KB ‐ FVII a‐004/albumin fusion‐protein (004‐ HSA ) was generated for in vivo testing. By using 004‐ HSA , we observed that this sdAb blocked the therapeutic capacity of FVII a to correct bleeding in FVIII ‐deficient mice. Discussion This observation is compatible with the view that FVII a functions independently of TF under these conditions. In conclusion, we have generated a sdAb that specifically blocks TF ‐independent activity of FVII a. This antibody can be used to gain insight into the roles of TF ‐bound and TF ‐free FVII a.