Essentials Patients with hemophilia A and inhibitors receiving emicizumab 
experience breakthrough bleeding. Safety concerns may exist when combining 
emicizumab with bypassing agents. Combined bypassing agent and bispecific 
antibody increased thrombin generation up to 17-fold. Thrombotic effects should 
be considered when combining emicizumab with plasma bypassing agent.
SUMMARY: Background Investigational non-factor products such as emicizumab offer 
a treatment option for patients with hemophilia and inhibitors. However, their 
mechanism of action raises questions regarding safety when they are combined 
with treatments for breakthrough bleeding. Objectives To evaluate in vitro 
thrombin generation (TG) and clot formation for combinations of activated 
prothrombin complex concentrate (aPCC), recombinant activated factor VII 
(rFVIIa), and a sequence-identical analog of emicizumab (SIA). Methods 
Therapeutic concentrations of SIA (20-600 nm) alone or with aPCC (0.05-1 U mL-1 
), isolated aPCC components or rFVIIa (0.88-5.25 μg mL-1 ) were tested for TG 
and compared with reference ranges for healthy donor plasma. Coagulation of 
FVIII-inhibited blood was determined with a widely established method, i.e. 
rotational thromboelastometry (ROTEM), and confirmed with the Total 
Thrombus-formation Analysis System. Results and conclusions SIA (600 nm) or aPCC 
(0.5 U mL-1 ) alone resulted in peak thrombin levels of 21.4 nm and 38.6 nm, 
respectively, both of which are lower than normal (83.7 ± 29.8 nm). SIA plus 
aPCC (0.5 U mL-1 ) increased the peak thrombin level 17-fold over SIA alone, 
exceeding the reference plasma value by 4.2-fold. This hypercoagulable effect 
occurred with 600 nmSIA combined with as little as 0.25 U mL-1 aPCC, confirmed 
by ROTEM. FIX was the main driver for enhanced TG. SIA plus rFVIIa (1.75 μg mL-1 
) induced a 1.8-fold increase in the peak thrombin level in platelet-rich 
plasma, but it did not reach the normal range. These in vitro experiments 
demonstrate excessive TG after administration of a combination of aPCC and SIA 
at clinically relevant doses. Careful judgement may be required when 
breakthrough bleeding is treated in patients receiving emicizumab.