ALBERT

Description: Factor VIIa (FVIIa), complexed with tissue factor (TF), is a trigger of blood coagulation. Analog of recombinant FVIIa (rFVIIa), NN1731 (V158D/E296V/M298Q) possesses a greater hemostatic effect than rFVIIa and has been expected in clinical application. Factor X activation rate of NN1731 compared to rFVIIa was 1.2-fold in the presence of TF (TF(+)), and was 30-fold on activated platelets in its absence (TF(−))(Allen, Arterioscler Thromb Vasc Biol.2007; 27: 683). This TF-independent mechanism likely attributes to excellent effects by NN1731. More recently, we reported the physiological role of FVIIa/TF-dependent FVIII activation in the early phase of blood coagulation. Therefore, we were tempted to investigate the action of NN1731 in FVIII activation. Time-dependent change in FVIII activity after the addition of rFVIIa/NN1731 was examined by one-stage clotting assay under the presence of phospholipids (PS:PC:PE=1:6:3), CaCl2 and TF(+)/TF(−). NN1731 raised FVIII activity up to peak level rapidly within 30 sec (TF(+)), following by inactivation. Peak level of FVIII activity by NN1731 in TF(−) reached to the same peak level of that in TF(+) within 5 min, and this peak level persisted for ~30 min. Whilst, peak FVIII level by rFVIIa in TF(−) showed only ~35% of that in TF(+) even at 30 min. FVIII activating rate of NN1731 was observed to be 1.2-fold (TF(+)) and 3.8-fold (TF(−)) of rFVIIa-catalyzed activation. Kinetics by the Xa generation assay showed the Km values of NN1731 in FVIII activation were ~1.5-fold lower than those of rFVIIa (NN1731/rFVIIa; TF(+) 27.3/49.2 nM and TF(−) 50.5/68.1 nM). Vmax values of NN1731 in FVIII activation, however, showed the obvious difference between TF(+) (2.3-fold; NN1731/rFVIIa 70.0/30.4 nM•min−1) and TF(−) (7.9-fold; 92.5/11.7 nM•min−1), compared to rFVIIa. Inactivation of FVIIIa by NN1731 was somewhat faster than that by rFVIIa. FVIII cleavages by NN1731 were analyzed using SDS-PAGE/Western blotting. The heavy chain of FVIII was proteolyzed at Arg740 (A2-B junction), Arg372 (A1-A2 junction) and Arg336 (within the A1), faster by NN1731 than by rFVIIa. These predominant cleavages by NN1731 were more evident in TF(−). However, little cleavage of the light chain of FVIII was observed by both proteases. FVIII cleavages were correlated with the observations of FVIII activation and/or inactivation. To further localize the binding region for NN1731, we evaluated the interactions between FVIII and Glu-Gly-Arg-active site modified (EGR-) NN1731, lacking enzymatic activity, in a surface plasmon resonance-based assay. The Kd value of EGR-NN1731 with FVIII was similar to that of EGR-rFVIIa (6.3 and 7.8 nM, respectively). Binding was particularly evident with the A2, A3, and C2 domains, whilst the A1 domain failed to bind, similar to the results obtained by rFVIIa. We demonstrated that NN1731 possesses higher potential as an activator for up-regulation of FVIII activity than rFVIIa. Furthermore, catalytic activity of NN1731 in TF(−), rather than binding affinity, likely attributes to this potential of its analog. We concluded that the analog has another novel mechanism in its potent hemostatic effect through FVIII activation in TF-independent manner.

Description: Protein S functions as a cofactor of activated protein C that inactivates factor VIII(a) and factor V(a). We recently have reported a new regulatory mechanism that protein S interacted with both the A2 and A3 domains in factor VIII, and consequently this cofactor directly impaired the factor Xase complex by competing the interaction of factor IXa to factor VIIIa (Blood2006; 108, 487a). Since factor IXa blocked the binding of A2 subunit to protein S, we attempted several approaches to localize the protein S-interactive site(s) on the factor VIII A2 domain. An anti-A2 monoclonal antibody (mAb413) with the 484–509 epitope, recognizing a factor IXa-interactive site on the A2, inhibited the A2 binding to immobilized protein S up to approximately 90% in a dose-dependent manner in a surface plasmon resonance-based assay. Furthermore, ELISA-based assay showed that a synthetic peptide corresponding to residues 484–509 directly bound to protein S dose-dependently. Covalent cross-linking was observed between the 484–509 peptide and protein S following reaction with EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) using SDS-PAGE. The cross-linked product formed with EDC was consistent with 1:1 stoichiometry of reactants, suggesting specificity in the 484–509 peptide and protein S interaction. This cross-linking formation was blocked by the addition of the 484–497 peptide, whilst not by the 498–509 peptide, supporting the presence of protein S-interactive site within residues 484–497. Furthermore, N-terminal sequence analysis of the 484–509 peptide-protein S product showed that three sequential basic residues (S488, R489 and R490) could not be detected, supporting that three residues participate in cross-link formation. To confirm the significance of these residues in A2 domain for protein S-binding, the mutant forms of the A2 domain, converted to alanine, were expressed in baculovirus system and purified. Compared with wild type A2 (Kd: ∼9 nM), each binding affinity of S488A, R489A, or R490A A2 mutant for protein S was decreased by 4∼5-fold (32, 40 and 40 nM, respectively). These results indicate that the 484–509 region in the factor VIII A2 domain, and in particular a cluster of basic amino acids at residues 488–490, contributes to a unique protein S-interactive site.

Description: Abstract 1126 Exogenous factor VIII (FVIII) is used to reduce bleeding complications in patients with severe hemophilia A. However, there are two drawbacks of current routine prophylaxis by FVIII. One is the requirement of frequent intravenous administration due to its short half-life and low subcutaneous bioavailability of FVIII. Second is the development of anti-FVIII antibodies (inhibitors) in approximately 30% of the severe patients which deprives the patients from routine prophylaxis by FVIII. To overcome these drawbacks, bispecific IgG antibody against activated factor IX (FIXa) and factor X (FX), which mimics the cofactor function of FVIII by placing these two factors into spatially appropriate positions, was screened from approximately 40,000 bispecific antibodies recognizing FIXa by the one arm and FX by the other arm. The therapeutic potential of the bispecific antibody identified from the screening was marginal due to insufficient FVIII-mimetic activity and poor pharmacokinetics, and moreover, large scale purification of recombinant bispecific IgG antibody was challenging. Therefore, the lead bispecific antibody, after humanization, was subjected to multidimensional optimization process in order to improve both the therapeutic potential and the manufacturability of the bispecific antibody. FVIII-mimetic activity was improved by modifying its binding properties to FIXa and FX, and the pharmacokinetics was improved by engineering the charge properties of the variable region. Difficulty of manufacturing bispecific antibody was overcome by identifying common light chain for anti-FIXa and FX heavy chain through framework/complementarity determining region shuffling, and by isoelectric point engineering of the two heavy chain variable regions to facilitate ion exchange chromatography purification of the bispecific antibody. Engineering to overcome low solubility and deamidation was also performed to enable stable high concentration liquid formulation for clinical use. ACE910, multidimensionally optimized bispecific antibody, exhibited potent FVIII-mimetic activity in human FVIII deficient plasma (more than 10% of FVIII activity at 300 nM in thrombin generation assay), and half-life of approximately 3 weeks with high subcutaneous bioavailability in cynomolgus monkey, enabling effective prophylaxis by subcutaneous administration with long dosing interval. In silico immunogenicity prediction analysis suggested that ACE910 was minimally immunogenic in human, in contrast to high immunogenicity of FVIII in human. Importantly, the activity of ACE910 was not affected by the presence of inhibitors, while polyclonal anti-ACE910 antibody did not inhibit FVIII activity, allowing the use of ACE910 without considering the development or presence of inhibitors. Furthermore, ACE910 could be purified in a large scale manufacturing, and formulated into patient-friendly subcutaneously injectable liquid formulation for clinical use. We believe that ACE910, with its multidimensionally optimized profile, would significantly improve the quality of life of hemophilia A patients by reducing not only bleeding but also the burden on the patients themselves, their parents, and all medical staff. Disclosures: Igawa: Chugai Pharmaceutical Co.,Ltd: Employment. Sampei:Chugai Pharmaceutical Co., Ltd.: Employment. Soeda:Chugai Pharmaceutical Co.,Ltd: Employment. Okuyama-Nishida:Chugai Pharmaceutical Co., Ltd.: Employment. Moriyama:Chugai Pharmaceutical Co.,Ltd: Employment. Wakabayashi:Chugai Pharmaceutical Co.,Ltd: Employment. Tanaka:Chugai Pharmaceutical Co.,Ltd: Employment. Muto:Chugai Pharmaceutical Co., Ltd.: Employment. Kojima:Chugai Pharmaceutical Co.,Ltd: Employment. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Employment. Yoshihashi:Chugai Pharmaceutical Co.,Ltd: Employment. Harada:Chugai Pharmaceutical Co.,Ltd: Employment. Funaki:Chugai Pharmaceutical Co.,Ltd: Employment. Haraya:Chugai Pharmaceutical Co.,Ltd: Employment. Tatsuhiko:Chugai Pharmaceutical Co.,Ltd: Employment. Suzuki:Chugai Pharmaceutical Co.,Ltd: Employment. Esaki:Chugai Pharmaceutical Co.,Ltd: Employment. Nabuchi:Chugai Pharmaceutical Co.,Ltd: Employment. Hattori:Chugai Pharmaceutical Co., Ltd.: Employment.

Description: Key Points A long-term acquired hemophilia A model expressing spontaneous joint bleeds and other bleeds was newly established in nonhuman primates. Weekly SC dose of the anti-FIXa/X bispecific antibody ACE910 prevented joint bleeds and other bleeds in the primate hemophilia A model.

Description: 【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. 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. Matsumoto:Shire Japan Co. Ltd: Research Funding. Noguchi-Sasaki:Chugai: Employment. Soeda:Chugai: Employment, Patents & Royalties: Patents related to emicizumab. Matsumoto:Chugai Pharmaceutical Co., Ltd: Employment, Equity Ownership. Hirata:Chugai Pharmaceutical Co., Ltd: Employment, Equity Ownership. Shima: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; F. Hoffmann-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 42 Background: Hemophilia A is treated by intravenous replacement therapy with factor VIII (FVIII), either on demand to resolve bleeding or as a prophylactic to prevent bleeding. Recently, routine prophylactic treatment is recommended to effectively prevent bleeding and to reduce bleeding-related chronic joint damage. However, the need for frequent intravenous injections of FVIII negatively affects patients' quality of life and their adherence to the routine prophylactic regimen. More importantly, approximately 30% of severe hemophilia A patients develop inhibitory antibodies toward the injected FVIII, rendering the replacement therapy ineffective. To overcome these drawbacks, we generated a bispecific antibody (termed ACE910) against activated factor IX (FIXa) and factor X (FX), which mimics the cofactor function of FVIII. Objectives: The aims of the present study were to examine the FVIII-mimetic cofactor activity of ACE910 in vitro and its hemostatic activity in vivo. Methods: The FVIII-mimetic cofactor activity of ACE910 was evaluated by a thrombin generation assay in human FVIII-deficient plasma as well as by an enzymatic assay using purified coagulation factors. For in vivo studies, an acquired hemophilia A model was established in cynomolgus monkeys by a single intravenous injection of mouse monoclonal anti-FVIII neutralizing antibody, which was cross-reactive to cynomolgus monkey FVIII but not to porcine FVIII. After artificial bleeding had been induced, ACE910 or porcine FVIII was intravenously administered in a single dose or in twice-daily repeated doses, respectively. Bleeding symptoms, including anemia and skin bruising, were monitored for three days. A pharmacokinetic study of ACE910 was also performed with a single intravenous or subcutaneous administration to cynomolgus monkeys. Results: ACE910 concentration-dependently showed FVIII-mimetic cofactor activity in the enzymatic assay and improved thrombin generation parameters in human FVIII-deficient plasma. Intravenous administration of ACE910 (a single dose of 3 mg/kg) significantly reduced the bleeding symptoms in the acquired hemophilia A model of a non-human primate. This hemostatic effect was comparable to twice-daily intravenous administration of porcine FVIII (repeated doses of 10 U/kg). The half-life of ACE910 was approximately three weeks for both single intravenous and subcutaneous administrations. The subcutaneous bioavailability of ACE910 was nearly 100%. Conclusion: The bispecific antibody against FIXa and FX, ACE910, exerted FVIII-mimetic cofactor activity in vitro. Furthermore, a single dose of ACE910 demonstrated hemostatic activity comparable to twice-daily repeated doses of 10 U/kg porcine FVIII in vivo. Moreover, ACE910 exhibited high subcutaneous bioavailability and approximately three-week half-life in a non-human primate. Our bispecific antibody against FIXa and FX is a subcutaneously injectable, long-acting agent that removes the need to consider the induction or presence of FVIII inhibitors and may establish a novel principle for the prophylactic treatment of hemophilia A patients. Disclosures: Muto: Chugai Pharmaceutical Co., Ltd.: Employment. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Employment. Yoshihasi:Chugai Pharmaceutical Co., Ltd.: Employment. Takeda:Chugai Pharmaceutical Co., Ltd.: Employment. Soeda:Chugai Pharmaceutical Co., Ltd.: Employment. Igawa:Chugai Pharmaceutical Co., Ltd.: Employment. Sampei:Chugai Pharmaceutical Co., Ltd.: Employment. Sakamoto:Chugai Pharmaceutical Co., Ltd.: Employment. Okuyama-Nishida:Chugai Pharmaceutical Co., Ltd.: Employment. Saito:Chugai Pharmaceutical Co., Ltd.: Employment. Kawabe:Chugai Pharmaceutical Co., Ltd.: Employment. Shima:Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Research Funding. Hattori:Chugai Pharmaceutical Co., Ltd.: Employment.

Description: Emicizumab (also termed ACE910) is a humanized anti-factor (F)IXa/FX bispecific antibody with FVIIIa cofactor function. A clinical phase 3 study was initiated in 2015 for hemophilia A patients (HA-pts) with FVIII inhibitors. 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 whole coagulation potency, would have limited utility in emicizumab-administered HA-pts, because emicizumab would mask the effect of residual FVIII or a FVIII agent on APTT. Clot waveform analysis (CWA) can provide multidimensional coagulation potencies by monitoring the process of plasma clot formation with an automated coagulation analyzer. We considered the possibility, therefore, that this assay system would overcome the above issue on APTT. In this study, we aimed to optimize concentrations of tissue factor (TF) and ellagic acid (Elg) in a trigger reagent for CWA as well as CWA parameters to provide precise evaluation of coagulation potency even in the presence of emicizumab with neither masking nor being masked by FVIII or bypassing agents. First, we determined an optimal concentration of TF/Elg trigger reagent. Various concentrations (10, 30, 100, and 300 μg/mL) of emicizumab were spiked into commercially available FVIII-deficient plasmas (George King) for testing. Recombinant (r)FVIII (Kogenate FS; Bayer)-spiked samples were also tested as a reference. PT reagent (under development; Sysmex) and APTT reagent (Thrombocheck APTT-SLA; Sysmex), used as a source of TF and Elg, respectively, were mixed in various ratios. The optimized mixture ratio (PT:APTT:buffer=1:15:135) was chosen to ensure that the maximum coagulation velocity (|min1|) in the presence of emicizumab would be in agreement with the animal study-based estimated conversion rate "0.2-0.4 IU/dL of equivalent FVIII per 1 μg/mL of emicizumab" (Muto. J Thromb Haemost. 2014). When evaluating several lots of FVIII-deficient plasmas, however, we observed large variations in transmittance depending on fibrinogen concentration of each plasma, which resulted in large variations of |min1| between donor plasmas. To decrease the bias due to fibrinogen concentration, % transmittance of clot waveform (CW) was adjusted to 100% and 0% at the pre- and post-coagulation phase, respectively. By using |min1| from the adjusted CW (adjusted-|min1|), we successfully reduced the inter-donor variations and chose it as a main parameter. Next, we evaluated adjusted-|min1| using plasmas from HA-pts without inhibitors (severe; n=2, moderate; n=2) and HA-pts with inhibitors (10 BU/mL; n=2) by adding emicizumab (30, 100, and 300 μg/mL) in vitro. After the addition of emicizumab, concentration-dependent increases in adjusted-|min1| were observed in all plasmas with rather small individual variations. Finally, we examined whether adjusted-|min1| reflected the effects of FVIII or bypassing agent that was added to plasmas containing emicizumab. Additive effects of a plasma-derived FVIII agent (CROSS EIGHT M; Japan Blood Products Organization), a rFVIII agent (ADVATE; Baxalta) and activated prothrombin complex concentrate (FEIBA; Baxalta) were confirmed by the increase of adjusted-|min1|. As for rFVIIa agent (NovoSeven; Novo Nordisk), its additive effect on adjusted-|min1| was not clear enough in this assay condition, but its additive effects were confirmed by the clot time. In conclusion, we established the Elg/TF-triggered CWA assay condition and parameters for measuring coagulation potency in plasmas from HA-pts even in the presence of emicizumab and a FVIII/bypassing agent without masking each other. Disclosures Nogami: Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees. Matsumoto:Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties, Research Funding. Tabuchi:Sysmex Corporation: Employment, Patents & Royalties; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties. Soeda:Sysmex Corporation: Patents & Royalties; Chugai Pharmaceutical Co., Ltd.: Employment, Patents & Royalties. Arai:Sysmex Corporation: Employment. Kitazawa:Chugai Pharmaceutical Co.: Employment, Equity Ownership, Patents & Royalties; Sysmex Corporation: Patents & Royalties. Takaoka:Sysmex Corporation: Employment. Hattori:Chugai Pharmaceutical Co.: Employment, Equity Ownership, Patents & Royalties. Shima:F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Sysmex Corporation: Patents & Royalties, Research Funding.

Description: Factor VIIIa functions as a cofactor for the factor IXa in the membrane-dependent conversion of factor X to factor Xa. We recently have reported the C2 fragment (residues 2182–2259) of factor VIIIa directly interacts with the Gla domain of factor IXa via both the electrostatic- and calcium-dependent interactions and this association plays a significant role in the factor Xase complex (Blood2007; 110, 2687). In this study, we further localized a factor IXa-interactive site within the 2182–2259 region of C2 domain. The competitive binding assays in ELISA using several overlapping synthetic peptides encompassing residues 2182–2259 demonstrated that one peptide 2228–2240 (EWLQVDFQKTMKV; C22228–2240), supposed to be exposed on the molecular surface according to the crystal structure of FVIII, significantly inhibited the binding of active-site modified EGR-factor IXa to the recombinant C2 domain (residues 2169-2332) by ~80% (IC50; ~400 μM), whilst a control peptide, comprising the 2228-2240 residues in a random sequence, failed to inhibit. This peptide did not affect both bindings of factor VIII and factor IXa to phospholipid. The addition of C22228–2240 inhibited the factor VIIIa/factor IXa-mediated factor X activation in the presence of phospholipid dose-dependently (IC50; ~10 μM), suggesting that residues 2228–2240 of the C2 domain significantly contribute to the interaction with the Gla domain of factor IXa. The amino acid sequences were well-conserved, independently of species. Of note, this inhibitory effect was much greater than those obtained by the 484–509 peptide and 1804–1818 peptide, corresponding to other factor IXa-interactive sites (IC50; ~60 and ~180 μM, respectively). Furthermore, we studied the inhibitory effect of C22228–2240 on the blood coagulation quantitatively using both the clot-waveform analyses by a photo-optical automated coagulation analyzer and thromboelastography. This peptide (500 μM) slightly prolonged the activated partial thromboplastin time (APTT) by ~1.3-fold. The maximum coagulation velocity (min1) and maximum coagulation acceleration (min2) were significantly reduced (by ~95%) by the addition of peptide, whilst the prothrombin time (PT) was little affected. A thromboelastography revealed that the C22228–2240 prolonged the value of clot formation time and decreased α angel and maximal clot firmness in dose-dependent manners. These inhibitory effects obtained by both assays were equivalent to those of representative anticoagulants, low weight molecular heparin (at ~0.3 IU/ml) and fondaparinux (at ~0.5 IU/ml). Of interest, this peptide showed little effect on the precoagulation phase (clotting time) in both assays. These data indicated that C22228–2240 possessed an anticoagulant effect for the process of clot formation. In conclusion, we indentified a factor IXa-interactive site within a 2228–2240 region of the C2 domain of factor VIIIa. Furthermore, these interactions on the factor Xase assembly likely play an essential role for propagation of intrinsic coagulation.

Description: Epitopes of anti-factor VIII (FVIII) neutralizing antibodies distribute all of the FVIII domains. They inhibit FVIII for instance by blocking the FVIII interaction with factor IXa, factor X/factor Xa (FX/FXa), phospholipid, von Willebrand factor, or thrombin. Therefore, localization of such FVIII inhibitory effect gives us useful information for understanding of FVIII structure and function. We expect FVIII enhancing antibody also may be a useful tool. In this study, we found an anti-FVIII monoclonal antibody (named by moAb216) that increased the FVIII cofactor activity. The addition of moAb216 increased FVIII activity by ~1.6-fold dose-dependently in one-stage clotting assay. The increase of FVIII activity in the presence of moAb216 correlated with that of generated thrombin or factor Xa in thrombin or FXa generation based-assay. Blotting analysis revealed that this antibody reacted with only intact FVIII molecule, whilst failed to react with either SDS-treated FVIII, FVIIIa (active-form), or isolated each A1, A2, and A3-C1-C2 subunit. Individual monoclonal antibody, with an epitope of the A1 or A3 acidic region in FVIII, competitively inhibited FVIII binding to moAb216 as well as the increase of FVIII activity by its antibody. However, anti-A2 or anti-C2 monoclonal antibody did not affect. These results supported that this unique antibody, with a discontinuous epitope spanning both acidic regions in the A1 and A3 domains, recognized the native conformation of FVIII. To examine the mechanism(s) of increasing effect by moAb216, we focused on the activation and/or inactivation of FVIII. Rate constants on thrombin- and FXa-catalyzed activation of FVIII in the presence of moAb216 were ~2 and 3-fold greater, respectively, in dose-dependent manners compared with that of FVIII in its absence. On the other hand, the antibody inhibited the activated protein C (APC)-catalyzed FVIII inactivation with ~10-fold lower of inactivation rate constant. SDS-PAGE analysis revealed that moAb216 accelerated the cleavage at Arg372 in the A1-A2 junction by thrombin and FXa, whilst decelerated the cleavage at Arg336 within the A1 domain by APC. In addition, FVIII activity in the presence of moAb216 was more stable following heat denaturation analysis than that in its absence. We demonstrated that the increasing effect of FVIII activity by moAb216 was attributed to the change of cleavage at Arg372 and/or Arg336 in the heavy chain caused by interaction with the A1/A3 domains. Furthermore, moAb216 would be useful as a new replacement therapy for hemophilia A patients, since this increases and stabilizes FVIII activity, and can function even in the presence of anti-FVIII (A2 and C2) inhibitors.