Multicenter Evaluation of the Hemostatic Activity of Emicizumab in Patients with Severe Hemophilia a Using Thrombin Generation Assay
Authors: Josset Laurie , Janbain Maissaa , De Wreede Annaelle , Nougier Christophe , Desage Stephanie , Lienhart Anne , Delavenne Xavier , Dargaud Yesim G. 
Published: December 2023

Background: Emicizumab has been approved for the prophylaxis of patients with hemophilia A with or without inhibitors. However, the follow-up of these patients is poorly defined and makes difficult the management of those presenting breakthrough bleeds while on prophylaxis with emicizumab.

Objectives: The aim of this study was to evaluate the value of thrombin generation assay (TGA) as a surrogate marker of the hemostatic efficacy of emicizumab. The secondary objective was to study the correlation between TGA and the methods used to measure the blood concentration of emicizumab.

Methods: TGA was modified to enhance its sensitivity to emicizumab through the use of a reagent combining a low amount of tissue factor (TF) and activated factor XI (FXIa). Quantification of emicizumab was performed by three different methods, this modified one-step assay, and two methods based on liquid chromatography and mass spectrometry (LC-MS).

Results: Emicizumab procoagulant activity is of note highly dependent on FIX activity, while FIX itself is activated by FXIa, the latter depending on thrombin and platelets. Consistent with these findings, TGA measured in platelet rich plasma (PRP) showed a significantly higher coagulation capacity compared to platelet poor plasma (PPP) in patients receiving prophylaxis with emicizumab. These results emphasize the need for platelets to capture the full hemostatic effect of emicizumab. Of course, using fresh PRP is not classical in real life hospital settings. Although this appears as the best assessment measurement, this condition could be a major barrier to the deployment of PRP TGA in hospital laboratories to monitor emicizumab therapy and guide combined therapy with emicizumab and bypassing agents. In order to overcome this issue, we propose a modified TGA PPP assay using added traces of FXIa to the mixture of low TF and phospholipids. This provides a better activation of FIX, required for the procoagulant activity of emicizumab.

Using FXIa/TF-triggered TGA, a relationship was observed between the area under the TG curve (ETP) and the clinical response of patients to emicizumab. Patients who experienced breakthrough bleeds while they were on emicizumab, had significantly lower ETP than others without bleeding episodes. The ultrastructure of fibrin clots was consistent with TGA results and showed that the hemostatic activity of emicizumab is similar to Factor VIII activity between 20 and 30 IU/dL.

Finally, pharmacokinetic/pharmacodynamic analyses showed no correlation between ETP and LC-MS nor with modified one-stage activated partial thromboplastin time, but a statistically significant correlation was observed between the LC-MS methods and the time to peak results of TGA.

Conclusion: The modified TGA used here can segregate patients with good clinical response to emicizumab from those bleeding on emicizumab. This approach can be of valuable interest for the management of patients treated with emicizumab