Once-weekly subcutaneous administration of emicizumab markedly decreased the bleeding rate in patients who had hemophilia A with or without factor VIII inhibitors. (Funded by Chugai Pharmaceutical; JapicCTI number, 121934.).

Introduction Emicizumab is a recombinant humanized bispecific monoclonal antibody mimicking the cofactor function of activated factor VIII. Aim In this multicentre, open‐label study (HOHOEMI), we evaluated the efficacy, safety and pharmacokinetics of emicizumab in Japanese paediatric patients aged <12 years with severe haemophilia A without factor VIII (FVIII) inhibitors. Methods Emicizumab was administered subcutaneously, with four loading doses of 3 mg/kg every week followed by maintenance doses of 3 mg/kg every 2 weeks (Q2W) or 6 mg/kg every 4 weeks (Q4W) in 6 and 7 patients, respectively. Results All patients completed at least 24 weeks of treatment. Baseline ages ranged from 4 months to 10 years, and all patients had been treated with FVIII prophylaxis prior to enrolment except a 4‐month‐old patient untreated with FVIII previously. In the respective Q2W and Q4W cohorts, 2/6 and 5/7 patients experienced no treated bleeding events, and annualized bleeding rates for treated bleeding events were 1.3 (95% confidence interval [CI], 0.6‐2.9) and 0.7 (95% CI, 0.2‐2.6). All caregivers preferred emicizumab to the patient's previous treatment. Only one related adverse event (injection site reaction) was observed. There were no thromboembolic events or thrombotic microangiopathy. Individual trough plasma concentrations of emicizumab were within the variability observed in preceding adult/adolescent studies. All patients tested negative for anti‐emicizumab antibodies. Conclusions Emicizumab administered Q2W or Q4W was efficacious and safe in paediatric patients with severe haemophilia A without inhibitors. This study was registered at http://www.clinicaltrials.jp (JapicCTI‐173710).

Summary 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 ( K D = 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 K D -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 K D -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. Institution where the work was carried out: Research Division, Chugai Pharmaceutical Co., Ltd.Supplementary Material to this article is available online at www.thrombosis-online.com.

Key Points Long-term, once-weekly emicizumab was well tolerated with no thromboembolic adverse events in patients with hemophilia A. Long-term, once-weekly emicizumab shows encouraging efficacy irrespective of the presence of FVIII inhibitors in patients with hemophilia A.

Background Emicizumab is an anti-activated factor IX/FX bispecific antibody that mimics activated FVIII cofactor function. Emicizumab does not require activation by thrombin, and its effect on shortening the activated partial thromboplastin time (APTT) is much greater than that of FVIII. Therefore, the APTT has limited utility in hemophilia A (HA) patients treated with emicizumab. Aim To evaluate the global coagulation potential of emicizumab. Methods Clot waveform analysis (CWA) with prothrombin time (PT)/APTT mixed reagents was used to define hemostatic monitoring protocols in HA patients. A modified parameter, adjusted-|min1| (Ad|min1|), was developed. Maximum and minimum percentage transmittance were defined as 100% and 0% in the precoagulation and postcoagulation phases, respectively. Ad|min1| was calculated as an index of the maximum velocity of the coagulation process. Results Ad|min1| obtained with mixed-trigger reagent (PT/APTT/buffer, 1 : 15 : 135) in the presence of emicizumab optimally corresponded to the conversion rate estimated in animals; 0.2-0.4 IU dL equivalent FVIII per 1 μg mL emicizumab). Ex vivo addition of emicizumab to HA plasma with or without inhibitors resulted in concentration-dependent increases in Ad|min1|, with some individual variations. The addition of various concentrations of FVIII to HA plasma mixed with emicizumab resulted in dose-dependent increases in Ad|min1|. Similarly, mixtures of activated prothrombin complex concentrate and emicizumab added to HA plasma resulted in dose-dependent increases in Ad|min1|. In contrast, enhanced coagulation potential appeared to be better defined by the clot time than by Ad|min1| in experiments using recombinant activated FVII. Conclusion The PT/APTT reagent-triggered adjusted CWA could provide a useful means of assessing global coagulation potential in emicizumab-treated HA patients, with enhanced activity neither masking nor being masked by FVIII or bypassing agents.

Introduction:The efficacy and safety of recombinant factor VIII Fc fusion protein (rFVIIIFc) as an extended half-life treatment for severe haemophilia A were demonstrated in the Phase 3 A-LONG and Kids A-LONG studies. Eligible subjects who completed A-LONG and Kids A-LONG could enrol in ASPIRE (NCT01454739), an open-label extension study.This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Thromboelastography (TEG) was first described by Hartert in 1948, and was designed to monitor viscoelastic clot strength in whole blood in real time. The current TEG method and Rotational Thromboelastometry (ROTEM) were subsequently developed from the original principles. Both of the modern methods provide data by measuring changes in the viscoelastic strength of a small sample of clotting blood in response to a constant rotational force. The important advantage of these techniques is to visually observe and quantify blood coagulation including the propagation, stabilization and dissolution phases of clot formation under low shear conditions. Analysis of the results provides detailed kinetic data on fibrin generation, clot strength and fibrinolysis. These TEG/ROTEM analyses therefore enable evaluation of global clotting function and the monitoring of haemostatic treatment in various clinical situations, not only in patients with genetic bleeding disorders, such as haemophilia, but also in patients undergoing cardiac surgery, liver transplantation or suffering from traumatic injury. Some evidence suggests that haemostatic management using TEG/ROTEM leads to a reduction in total transfusions of whole blood or clotting factors. Wider clinical application of this technology seems likely.

Factor VIIIa consists of subunits designated A1, A2, and A3-C1-C2. The limited cofactor activity observed with the isolated A2 subunit is markedly enhanced by the A1 subunit. A truncated A1 (A1 336 ) was previously shown to possess similar affinity for A2 and retain ϳ60% of its A2 stimulatory activity. We now identify a second site in A1 at Lys 36 that is cleaved by factor Xa. A1 truncated at both cleavage sites (A1 37-336 ) showed little if any affinity for A2 (K d >2 M), whereas factor VIIIa reconstituted with A2 plus A1 37-336 /A3-C1-C2 dimer demonstrated significant cofactor activity (ϳ30% that of factor VIIIa reconstituted with native A1) in a factor Xa generation assay. These affinity values were consistent with values obtained by fluorescence energy transfer using acrylodan-labeled A2 and fluorescein-labeled A1. In contrast, factor VIIIa reconstituted with A1 37-336 showed little activity in a one-stage clotting assay. This resulted in part from a 5-fold increase in K m for factor X when A1 was cleaved at Arg 336 . These findings suggest that both A1 termini are necessary for functional interaction of A1 with A2. Furthermore, the C terminus of A1 contributes to the K m for factor X binding to factor Xase, and this parameter is critical for activity assessed in plasmabased assays.Factor VIII, a plasma protein that participates in the blood coagulation cascade, is deficient or defective in individuals with hemophilia A. Factor VIII functions as a cofactor for the serine protease, factor IXa, in the anionic phospholipid surface-dependent conversion of factor X to Xa. Factor VIII is synthesized as a multi-domain, single chain molecule (A1-A2-B-A3-C1-C2) (1) with a molecular mass of ϳ300 kDa (2, 3). Factor VIII is processed to a series of divalent metal ion-linked heterodimers by cleavage at the B-A3 junction, generating a heavy chain consisting of the A1-A2-B domains and a light chain consisting of the A3-C1-C2 domains. This procofactor is activated by cleavage at Arg 372 , Arg 740 , and Arg 1689 by thrombin and factor Xa, converting the dimer into the factor VIIIa trimer composed of the A1, A2, and A3-C1-C2 subunits (4, 5). The resulting factor VIIIa heterotrimer retains the metal ion-dependent linkage between the A1 and A3-C1-C2 subunits, whereas A2 is associated with a weak affinity by electrostatic interactions (5, 6). Factor VIIIa is unstable, and loss of activity is due to the dissociation of the A2 subunit from the A1/A3-C1-C2 dimer (5-7). Under physiological conditions, the K d for this interaction is ϳ260 nM (8, 9); however, at slightly acidic pH and low ionic strength, this interaction is facilitated by an ϳ10-fold increase in the affinity (K d ϭ ϳ30 nM) (8).The role of factor VIIIa in the intrinsic factor Xase is to bind factor IXa, which increases the k cat for factor Xa formation by several orders of magnitude compared with factor IXa alone (10). Interactive sites for factor IXa are localized to A2 and A3 domains (11-13). Recent studies have shown that modulation of factor IXa by the isolated A2...