Emicizumab, a humanised bispecific antibody recognising factors (F) IX/IXa and 
X/Xa, can accelerate FIXa-catalysed FX activation by bridging FIXa and FX in a 
manner similar to FVIIIa. However, details of the emicizumab-antigen 
interactions have not been reported so far. In this study, we first showed by 
surface plasmon resonance analysis that emicizumab bound FIX, FIXa, FX, and FXa 
with moderate affinities (KD = 1.58, 1.52, 1.85, and 0.978 µM, respectively). We 
next showed by immunoblotting analysis that emicizumab recognised the antigens' 
epidermal growth factor (EGF)-like domains. We then performed KD-based 
simulation of equilibrium states in plasma for quantitatively predicting the 
ways that emicizumab would interact with the antigens. The simulation predicted 
that only a small part of plasma FIX, FX, and emicizumab would form 
antigen-bridging FIX-emicizumab-FX ternary complex, of which concentration would 
form a bell-shaped relationship with emicizumab concentration. The bell-shaped 
concentration dependency was reproduced by plasma thrombin generation assays, 
suggesting that the plasma concentration of the ternary complex would correlate 
with emicizumab's cofactor activity. The simulation also predicted that at 
10.0-100 µg/ml of emicizumab-levels shown in a previous study to be clinically 
effective-the majority of plasma FIX, FX, and emicizumab would exist as 
monomers. In conclusion, emicizumab binds FIX/FIXa and FX/FXa with micromolar 
affinities at their EGF-like domains. The KD-based simulation predicted that the 
antigen-bridging ternary complex formed in circulating plasma would correlate 
with emicizumab's cofactor activity, and the majority of FIX and FX would be 
free and available for other coagulation reactions.