Assessment of Emicizumab-Driven Clot Stability in Hemophilia a Model
Authors: Ogiwara Kenichi , Horiuchi Hiroki , Nogami Keiji , Kitazawa Takehisa , Shima Midori 
Published: October 2019

Abstract
Clot stability is an important hemostatic aspect in treating patients with hemophilia A (HA). Effects of factor VIII (FVIII), recombinant activated FVII (rFVIIa), FXIII and tranexamic acid on clot stability in hemophilia had been reported1). Emicizumab (Emi), FVIIIa-mimicking bispecific antibody, is a recently approved novel therapeutics for HA with inhibitor. Although hemostatic activity of Emi was confirmed by preclinical and clinical studies, whether Emi-driven clot stability is similar to that of FVIII or not remains unknown. Furthermore, in clinical trial of Emi, thrombotic events were reported when high doses of activated prothrombin complex concentrates (aPCC) were used concurrently with Emi. Effect of aPCC on clot stability and its possible association with the thrombotic events should be also clarified. In this study, we investigated the effect of Emi on clot stability with a thromboelastometry (ROTEM) -based assay using tissue plasminogen activator (tPA) as a fibrinolysis trigger, and compared it with that of FVIII or bypassing agents (BPAs). Furthermore, we assessed the effect of BPAs on Emi-driven clot stability.
Normal blood from healthy volunteer (n=22) was incubated with anti-FVIII antibody (FVIII:C <1 IU/dL), which was regarded as HA model blood. HA model blood was spiked with Emi (50 μg/ml), rFVIIa (22 nM), and aPCC (1 U/ml). ROTEM was performed with/without tPA (2 nM). We used ellagic acid (Elg, x300 INTEM®) as a trigger in addition to tissue factor (TF, 0.5 pM) since TF-trigger was not sensitive to Emi. Residual area under the curve in 30 min (%AR30) was used as a clot stability parameter. Median values (25-75%tile) of %AR30 were compared among the groups with Dunn's multiple comparison test [*p<0.05, **p<0.01, ***p<0.001].
In Elg-trigger ROTEM, %AR30 of HA model blood had no values since coagulation did not occur within test time. However, coagulation was improved in HA model spiked with Emi and its clot stability showed slightly weak value with no statistical significance [50% (30-76), p>0.05] compared to normal blood [75% (56-90)]. Whilst, HA model + rFVIIa [13%*** (7-22)] and HA model + aPCC [41%*** (20-71)] showed significantly weak values, indicating that effect of Emi on clot stability was comparable to normal blood and better than BPAs in Elg trigger. Concomitant effect of Emi and BPAs were evaluated. Compared to HA model + Emi [50% (30-76)], clot stability increased in the co-presence of Emi and aPCC [99%*** (96-101)] but not in that of Emi and rFVIIa [53% (29-78)].
TF trigger, which was sensitive to BPAs but not to Emi, was also used in order to evaluate concomitant effect of Emi and BPAs. In TF trigger, clot stability of HA model blood + Emi [31% (19-47)] was similar to that of HA mode blood [27% (20-44)] or normal blood [32% (22-55)], but increased in HA model + rFVIIa [43%** (30-62)] and HA model + aPCC [41%** (29-68)]. rFVIIa increased clot stability of HA model blood in the presence of Emi [56%** (28-65)] more than its absence. Effect of the aPCC in the presence of Emi [87%*** (78-91)] was much more remarkable than that of rFVIIa.
In conclusion, effect of Emi on clot stability in HA model blood was comparable to that of normal blood, indicating that clot formation under Emi had no qualitative difference with FVIII. Clot stability remarkably increased in Emi in the presence of aPCC, suggesting that hyper clot stability might be one of the reasons of thrombotic events in the concomitant use of Emi and aPCC.
1) Tran HT, et al. Tranexamic acid as adjunct therapy to bypassing agents in haemophilia A patients with inhibitors. Haemophilia. 2014 May;20(3):369-75.

Disclosures
Ogiwara: CSL Behring: Research Funding. Nogami:Chugai Pharmaceutical Co., Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Anti-FIXa/X bispecific antibodies , Research Funding, Speakers Bureau. Kitazawa:Chugai Pharmaceutical Co., Ltd: Employment, Equity Ownership, Patents & Royalties: Patents related to emicizumab. Shima:F. Hoffmann-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees; Chugai Pharmaceutical Co., Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Anti-FIXa/X bispecific antibodies , Research Funding, Speakers Bureau.