A Modified Thrombin Generation Analysis to Measure the Plasma Coagulation Potency in the Presence of Anti-FIXa/FX Bispecific Antibody, Emicizumab

【Introduction】 Emicizumab is a humanized anti-factor (F)IXa/FX bispecific antibody with FVIIIa cofactor function. Since emicizumab, unlike FVIII, does not require activation by thrombin, its aPTT-shortening effect is much greater than that of FVIII. Thus, aPTT, a conventional assay to assess intrinsic coagulation potency, would have limited utility in emicizumab-administered hemophilia A-patients (HA-pts), because emicizumab would mask the effect of residual FVIII or a FVIII agent on aPTT. Also, aPTT cannot differentiate between different levels of emicizumab.

Recently we reported that clot waveform analysis (CWA) using a trigger reagent comprising a balanced mixture of ellagic acid (Elg) and tissue factor (TF) to reflect both intrinsic and extrinsic coagulation activities could provide a useful means of assessing plasma coagulation potential in HA-pts treated with emicizumab with enhanced activity neither masking nor being masked by FVIII or bypassing agents (BPAs)¹⁾. Thrombin generation assay (TGA) can provide multidimensional plasma coagulation potencies as well as CWA. We considered the possibility, therefore, that the TGA would also overcome the above issue on aPTT. In this study, we aimed to assess TGA using Elg/TF trigger and two reference triggers (FXIa, TF) to evaluate the coagulation potency of patients with HA receiving emicizumab.

【Method】 TGA was assayed using Elg/TF trigger consisted of TF (0.5 pM) and Elg (0.3 μM) with phospholipids vesicles (PL, 4 μM), FXIa trigger consisted of FXIa (0.47 nM) with PL (20 μM), and TF trigger (PPP-Reagent LOW^®). Various concentrations of emicizumab, FVIII agent, or BPAs (rFVIIa, aPCC) in clinical dosages were spiked into commercially available FVIII-deficient plasmas (George King) for testing. Then, FVIII or BPA was added to FVIII-deficient plasmas containing emicizumab.

【Result】 Emicizumab or FVIII showed dose-dependent increase in thrombin peak height in Elg/TF trigger as well as FXIa and TF triggers, but TF trigger had weak sensitivity to emicizumab and low range FVIII (1-10 IU/dl). Spiking both rFVIIa or aPCC showed dose dependent increase in thrombin peak height under Elg/TF trigger and TF trigger conditions, but the values were lower than the level of normal peak height. On the other hand, rFVIIa and aPCC resulted in little increase in peak height under FXIa trigger condition, suggesting that the FXIa trigger is not suitable for assessment of BPAs. These results indicated that Elg/TF trigger system was the most useful to evaluate the single-spiked effect of these agents.

Then, we examined the additional effects of FVIII or BPAs in the presence of emicizumab by Elg/TF trigger system. The combination of FVIII and emicizumab showed additive increase in peak thrombin height, but this effect was saturated at high dose of FVIII more than 100 IU/dl. rFVIIa and emicizumab showed additive effect on increased peak thrombin height, achieving normal level even at very low dose rFVIIa (0.67 μg/ml), but remains within normal ranges at high dose (6.0 μg/ml). aPCC and emicizumab showed increased peak thrombin height, but in this case, exceeding the normal level even at clinical dosage of aPCC (0.5-1.0 U/ml).

【Conclusion】 TGA with Elg/TF trigger could provide a useful monitoring tool of assessing global coagulation potential during emicizumab prophyaxis including concomitant therapy with FVIII or BPAs.

1) Nogami K, et al. Modified clot waveform analysis to measure plasma coagulation potential in the presence of the anti-factor IXa/factor X bispecific antibody emicizumab. J Thromb Haemost. 2018 Jun;16(6):1078-1088.