Search

Factor VIII (FVIII) functions as a cofactor of FIXa for FX activation in the intrinsic tenase complex. The 1811-1818 region in the FVIII A3 domain was observed to contribute to FIXa binding, and the K1813A/K1818A mutant increased the binding affinity for FIXa. The current study aims to identify mutated FVIII protein(s) that increase FVIIIa cofactor activity in the 1811-1818 region. FVIII mutants with K1813A, K1818A, and K1813A/K1818A were expressed in baby hamster kidney cells and were followed by assessments using purified and global coagulation assays for mouse models with hemophilia A (HA). A surface plasmon resonance–based assay revealed that the Kd value of FVIII-K1813A for FIXa interaction was lower than that of the wild-type (WT) (3.9±0.7/6.3±0.3 nM). However, the Km value of FVIII-K1813A for FIXa on tenase activity was comparable with that of the WT, whereas the kcat of this mutant was significantly greater than that of the WT. Thrombin-catalyzed FVIII-K1813A activation was ∼1.3-fold more enhanced than that of the WT, and the spontaneous decay of activated FVIII-K1813A was ∼2.5-fold slower than that of WT. The heat stability assay revealed that the decay rate of FVIII-K1813A was ∼2.5-fold slower than that of WT. Thrombin generation assay and rotational thromboelastometry using blood samples from patients with HA demonstrated that the addition of FVIII-K1813A (0.5 nM) exhibited a coagulation potential compatible with that of WT (1 nM). In the tail clip assay of HA mice, FVIII-K1813A showed a two- to fourfold higher hemostatic potential than that of the WT. FVIII-K1813A, with higher a FIXa binding affinity, enhances the global coagulation potential because of the stability of FVIII/FVIIIa molecules.

Background; Factor (F)V plays pivotal roles in both procoagulant and anticoagulant mechanisms. Genetic mutations, FV-W1920R (FVNara) and FV-A2086D (FVBesançon), in the C1 and C2 domains of FV light chain, respectively, appear to be associated with deep vein thrombosis. The detailed mechanism(s) between these mutations and thrombophilia remains to be fully explored, however. Aim; To clarify thrombotic mechanism(s) in the presence of these FV abnormalities. Methods and Results; Full-length wild-type (WT) and mutated FV were prepared using stable, human cell lines (HEK293T) with the piggyBac transposon system. The susceptibility of FVa-A2086D to activated protein C (APC) was reduced, resulting in significant inhibition of APC-catalyzed inactivation with little cleavage at Arg306 and delayed cleavage at Arg506. Furthermore, APC cofactor activity of FV-A2086D in APC-catalyzed inactivation of FVIIIa through cleavage at Arg336 was impaired. Surface plasmon resonance-based assays demonstrated that FV-A2086D bound to Glu-Gly-Arg-chloromethylketone active site-blocked APC and PS with similar affinities to FV-WT. However, deteriorated interaction between FVa-A2086D to phospholipid membranes was evident, by the prothrombinase assay. Moreover, the addition of FVa-A2086D to plasma failed to inhibit tissue factor (TF)-induced thrombin generation and prothrombin times. This inhibitory effect was independent from protein (P)C, PS, and antithrombin. The coagulant and anticoagulant characteristics of FV(a)-W1920R were similar to those of FV(a)-A2086D. Conclusions; FV-A2086D presented defects in the APC mechanisms associated with FVa inactivation and FV cofactor activity, similar to FV-W1920R. Moreover, both FV proteins that were mutated in the light chain impaired inhibition of TF-induced coagulation reactions. These defects were consistent with congenital thrombophilia.

Background: It is important to analyze the types of vaccines in travel clinics to determine the focus points in future practice. Methods We retrospectively reviewed the electronic medical records of all patients who visited the travel clinic of Nara Medical University between June 2013 and December 2019 to determine their background and the vaccines administered. The information regarding adverse events of the unapproved vaccines in Japan (Havrix®, Verorab®, Boostrix®, Priorix®, Typhim Vi®, and Mencevax®) was also collected. Results Of 645 patients, 58.6% were men and the median age was 31 years. Business was the most common travel purpose (34.9%), and Southeast Asia was the most common destination (40.2%). More than 80% of travelers to low- and middle-income countries were vaccinated against hepatitis A, while the rabies vaccination rate was approximately 50%. Typhoid vaccination coverage among travelers to South Asia was approximately 50%. The incidence of adverse events requiring medical consultation, telephonic consultation, or prolonged stay in the examination room was less than 5% for all unapproved vaccines in Japan. Conclusion More patient education is needed to increase the vaccination rate of rabies and typhoid vaccines. Adverse events to unapproved vaccines in Japan were not high and were well-tolerated.

Coagulation and fibrinolytic mechanisms are enhanced in patients with coronavirus (COVID-19), but disturbances in the balance of both functions in COVID-19 patients remain unclear. We assessed global coagulation and fibrinolysis in plasma from 167 COVID-19 patients (mild/moderate/severe: 62/88/17, respectively) on admission using clot-fibrinolysis waveform analysis (CFWA). Maximum coagulation velocity (|min1|) and maximum fibrinolysis velocity (|FL-min1|) were expressed as ratios relative to normal plasma. Ten patients (6.0%) developed thrombosis, 5 (3.0%) had bleeding tendency, and 13 (7.8%) died during admission. FDP levels increased with severity of COVID-19 symptoms (mild/moderate/severe; median 2.7/4.9/9.9 μg/mL, respectively). The |min1| ratios were elevated in all categories (1.27/1.61/1.58) in keeping with enhanced coagulation potential, with significant differences between mild cases and moderate to severe cases. The |FL-min1| ratios were also elevated in all groups (1.19/1.39/1.40), reflecting enhanced fibrinolytic potential. These data identified coagulation dominance in moderate to severe cases, but balanced coagulation and fibrinolysis in mild cases. There were significant differences in FDP and TAT, but no significant differences in |min1| or |FL-min1| ratios, between patients with and without thrombosis. CFWA monitoring of coagulation and fibrinolysis dynamics could provide valuable data for understanding hemostatic changes and disease status in COVID-19 patients.

Soft tissue infections with Mycobacterium mageritense are uncommon. We report the case of a 5-year-old girl who developed a subcutaneous abscess in her right ankle caused by M. mageritense. She had a history of acute encephalopathy and adrenal insufficiency and was hospitalized for acute pancreatitis. During hospitalization, the patient developed fever and tachycardia. Blood culture was positive for gram-positive bacilli. Although initial testing with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) reported a different organism, a repeat test identified M. mageritense. One month after the positive blood culture, she developed redness and swelling in the right ankle. The pus from the subcutaneous abscess after drainage grew M. mageritense, which was further confirmed by the sequencing of housekeeping genes. Based on sensitivity testing, the patient was treated with tosufloxacin and linezolid. The local inflammatory signs gradually improved on starting the treatment. The antibiotics were administered for 6 months, and she experienced no relapse during the 8 months of follow-up after the completion of therapy. This is the first case report of a pediatric M. mageritense infection, which also highlights an important potential pitfall of MALDI-TOF MS. Further, we observe that the choice of antimicrobials for the treatment of M. mageritense is more limited in children than in adults.

The human papillomavirus (HPV) vaccine is safe and effective for preventing HPV-related diseases. However, HPV vaccination rates in Japan are low because the "Ministry of Health, Labour and Welfare" had stopped recommending vaccination. We assessed healthcare workers' (HCWs) current recommendations regarding the HPV vaccine and how the provision of information about HPV vaccination affected their recommendations.A survey was conducted among nurses and physicians in Nara prefecture from March 2021 to July 2021. The questionnaire asked about their understanding, recommendations, and opinions regarding HPV vaccination. Before answering the last two questions (optional), the HCWs read evidence-based information quantifying the risks and benefits of HPV vaccination.A total of 441 HCWs completed the questionnaire. Only 19% of HCWs always recommended HPV vaccination for girls aged 12-16 years. The evidence-based information significantly improved the percentage of HCWs who would "always recommend" vaccination.This study showed that the proportion of HCWs who recommend HPV vaccination to adolescent girls remains low in Japan. However, we found that evidence-based information describing the causal relationship between adverse events and vaccination, quantifying the risks and benefits, noting the importance of HCW communications with families, and reporting the recommendations of national societies, might increase HCWs' recommendations for HPV vaccination.

Bypassing therapy is essential for the haemostatic management of patients with haemophilia A with inhibitor (PWHA-inh), but the therapeutic effects are inconsistent. We previously reported that activated prothrombin complex concentrates (aPCC) activated factor (F)VIIIin vitro, and was mediated mainly by the activated FVII (FVIIa) contained in aPCC. We have extended those studies to assess global coagulation in whole blood from 18 PWHA-inh in the co-presence of aPCC and FVIII using Ca2+ -triggered rotational thromboelastometry. The clot times (CTs) in the presence of both aPCC (0·05 iu/ml) and recombinant (r)FVIII (1 iu/ml) ex vivo were shortened compared to the aPCC alone (P

Introduction Acquired hemophilia A (AHA) is caused by autoantibodies against factor (F)VIII, and is characterized by severe, spontaneous bleeding, which can be life-threatening. Emicizumab, an anti-FIXa/FX bispecific antibody, significantly reduces bleeding events in congenital hemophilia A (HA) with and without inhibitors. The known pathophysiological mechanisms and current preclinical data in HA suggest that emicizumab could provide effective treatment for AHA, but the coagulation activities of emicizumab in these patients remain unknown. Aim To evaluate the coagulant effects of emicizumab in plasma from AHA patients. Methods and results Tissue factor-triggered thrombin generation assays using normal plasma preincubated with anti-FVIII monoclonal antibodies recognizing different epitopes demonstrated that 20 µg/mL emicizumab recovered the depressed peak levels of thrombin generation to 46% to 72%. Further studies were devised, therefore, to simulate the clinical course in AHA patients, including during the acute phase for severe bleeding requiring FVIII-bypassing therapy, and during the subacute/chronic phase with less bleeding. Various concentrations of emicizumab were used to represent the potential changes in plasma levels based on the half-life of the antibody (~30 days). The ex vivo addition of emicizumab to plasma samples from AHA patients (n = 16) increased peak thrombin in all cases, irrespective of the inhibitor epitope specificity. Thrombin generation at 20 and 100 µg/mL emicizumab was restored to (median) 43.9% and 92.2%, respectively. Differences were evident in some cases, however, and recovery rates appeared likely to be greater in patients with type 2 inhibitor than those with type 1. Conclusion Emicizumab improved ex vivo coagulation potential in plasma from AHA patients.

Background: Infantile pertussis is a major concern and causes a significant health burden worldwide. Maternal adult tetanus, diphtheria, and acellular pertussis (Tdap) vaccination is very effective way of preventing infantile pertussis. However, Tdap has not been approved by the Japanese government or been included in Japan’s national immunization program (NIP). We carried out a questionnaire survey to investigate whether Japanese women would accept the Tdap vaccination if they were provided with appropriate information. Methods: The questionnaire survey was administered to pregnant women who visited the Obstetrics and Gynecology Department and mothers who visited the Pediatrics Department of Nara Medical University, Kashihara City, Japan, between October 2018 and May 2019. The questionnaire included information about pertussis infection and maternal vaccination, followed by seven questions. Results: A total of 943 participants answered the questionnaire (481 pregnant women and 462 mothers). Before reading the information, just 4.6% of participants knew that infantile pertussis can be prevented by maternal vaccination. After reading the information, 93.0% and 92.6% of participants thought that the maternal Tdap vaccine should be approved by the Japanese government and be included in the NIP, respectively. Although only 67.6% of participants wished to have the maternal Tdap vaccine without government financial support after reading the information, 92.5% said they would have the vaccine with government support (P

Basic residues contained in the 39-, 60-, and 70–80-loops of activated protein C (APC) comprise an exosite that contributes to the binding and subsequent proteolytic inactivation of factor (F) VIIIa. Surface plasmon resonance (SPR) showed that WT APC bound to FVIII light chain (LC) and the FVIIIa A1/A3C1C2 dimer with equivalent affinity (Kd = 525 and 546 nM, respectively). These affinity values may reflect binding interactions to the acidic residue-rich a1 and a3 segments adjacent to A1 domain in the A1/A3C1C2 and a3 domain in LC, respectively. Results from SPR, using a panel of APC exosite variants where basic residues were mutated, in binding to immobilized FVIIIa A1/A3C1C2 or LC indicated ~4–10-fold increases in the Kd values relative to WT for several of the variants including Lys39Ala, Lys37-Lys38-Lys39/ Pro-Gln-Glu, and Arg67Ala. On the other hand, a number of APC variants including Lys38Ala, Lys62Ala, and Lys78Ala showed little if any change in binding affinity to the FVIII substrates. FXa generation assays and Western blotting, used to monitor rates of FVIIIa inactivation and proteolysis at the primary cleavage site in the cofactor (Arg336), respectively, showed marked rate reductions relative to WT for the Lys39Ala, Lys37-Lys38-Lys39/ Pro-Gln-Glu, Arg67Ala, and Arg74Ala variants. Furthermore, kinetic analysis monitoring FVIIIa inactivation by APC variants at varying FVIIIa substrate concentration showed ~2.6- to ~4.4-fold increases in Km values relative to WT. These results show a variable contribution of basic residues comprising the APC exosite, with significant contributions from Lys39, Arg67, and Arg74 to forming a FVIIIa-interactive site.

SummaryAlthough factor (F) VIIIa is inactivated by activated protein C (APC) through cleavages in the FVIII heavy chain-derived A1 (Arg336) and A2 subunits (Arg562), the FVIII light chain (LC) contributes to catalysis by binding the enzyme. ELISA-based binding assays showed that FVIII and FVIII LC bound to immobilised active site-modified APC (DEGRAPC) (apparent K d ~270 nM and 1.0 μM, respectively). Fluid-phase binding studies using fluorescence indicated an estimated K d of ~590 nM for acrylodan-labelled LC binding to DEGR-APC. Furthermore, FVIII LC effectively competed with FVIIIa in blocking APC-catalysed cleavage at Arg336 (K i = 709 nM). A binding site previously identified near the C-terminal end of the A3 domain (residues 2007–2016) of FVIII LC was subjected to Ala-scanning mutagenesis. FXa generation assays and western and dot blotting were employed to assess the contribution of these residues to FVIIIa interactions with APC. Virtually all variants tested showed reductions in the rates of APC-catalysed inactivation of the cofactor and cleavage at the primary inactivation site (Arg336), with maximal reductions in inactivation rates (~3-fold relative to WT) and cleavage rates (~3 to ~9-fold relative to WT) observed for the Met2010Ala, Ser2011Ala, and Leu2013Ala variants. Titration of FVIIIa substrate concentration monitoring cleavage by a dot blot assay indicated that these variants also showed ~3-fold increases relative to WT while a double mutant (Met2010Ala/Ser2011Ala) showed a >4-fold increase in K m. These results show a contribution of a number of residues within the 2007–2016 sequence, and in particular residues Met2010, Ser2011, and Leu2013 to an APC-interactive site.

Factor (F) VIII functions as a cofactor in FXase, markedly accelerating the rate of FIXa-catalyzed activation of FX. Earlier work identified a FX-binding site having μM affinity within the COOH-terminal region of the FVIIIa A1 subunit. In the present study, surface plasmon resonance (SPR), ELISA-based binding assays, and chemical cross-linking were employed to assess an interaction between FX and the FVIII light chain (A3C1C2 domains). SPR and ELISA-based assays showed that FVIII LC bound to immobilized FX (K(d) = 165 and 370 nM, respectively). Furthermore, active site-modified activated protein C (DEGR-APC) effectively competed with FX in binding FVIII LC (apparent K(i) = 82.7 nM). Western blotting revealed that the APC-catalyzed cleavage rate at Arg(336) was inhibited by FX in a concentration-dependent manner. A synthetic peptide comprising FVIII residues 2007-2016 representing a portion of an APC-binding site blocked the interaction of FX and FVIII LC (apparent K(i) = 152 μM) and directly bound to FX (K(d) = 7.7 μM) as judged by SPR and chemical cross-linking. Ala-scanning mutagenesis of this sequence revealed that the A3C1C2 subunit derived from FVIII variants Thr2012Ala and Phe2014Ala showed 1.5- and 1.8-fold increases in K(d) for FX, whereas this value using the A3C1C2 subunit from a Thr2012Ala/Leu2013Ala/Phe2014Ala triple mutant was increased >4-fold. FXase formed using this LC triple mutant demonstrated an ~4-fold increase in the K(m) for FX. These results identify a relatively high affinity and functional FX site within the FVIIIa A3C1C2 subunit and show a contribution of residues Thr2012 and Phe2014 to this interaction.

Factor (F) VIII functions as a cofactor in the tenase complex responsible for phospholipid surface-dependent conversion of FX to FXa by FIXa. FIXa binding sites were identified on A2, A3, and C2 domains of FVIII. Some earlier studies confirmed the FIXa-binding sites on residues 1811-1818 in FVIII A3 domain. According to the 3-D model of FVIIIa, the residues 1790-1798 in FVIII A3 domain form a loop and exist just close to residues 1811-1818 on the FVIIIa extended surface and might contribute to one of the FIXa binding regions. We, therefore, hypothesized that residues 1790-1798 might be one of the FIXa-binding components, and prepared synthetic peptides (1811-1818 and 1790-1798). The 1811-1818 peptide inhibited the FVIII light chain-EGR FIXa interaction (apparent Ki;10.5 ± 4.0 μM) on ELISA. The 1790-1798 peptide also inhibited these interaction (apparent Ki ;4.2± 1.2 μM), suggesting that the 1790-1798 region contributed to FIXa-interactive site. In general, the binding sites of serine protease in FVIII contain the acidic or basic rich residues. The residues 1790-1798, and 1811-1818 contain basic residues E1793, E1794, D1795, E1811, K1813, and K1818. To investigate the significance of these residues for FIXa-binding, the mutant forms of the A3 domain, converted to alanine, in BHK system and purified. Furthermore, because previous study demonstrated that F1816 might contribute to FIXa binding, we also prepared F1816A. Compared with wild type FVIII (Kd; 6.3± 0.3 nM), the binding affinity of F1816A and E1793A/E1794A/D1795A mutants for EGR FIXa were decreased by 1.4-fold (Kd; 9.1± 0.2 nM) and 1.3-fold (Kd; 8.4± 0.4 nM), respectively, on SPR-based assay, suggesting contribution of F1816, and E1793/E1794/D1795 to the binding interactions. On the other hand, the binding affinities of E1811A (Kd; 6.3± 2.1 nM) and K1818A (Kd; 5.1± 1.8 nM) mutants for FIXa were almost the same as that of wild type. Interestingly, the binding affinity of K1813A (Kd; 3.9± 0.7 nM) for FIXa was increased compared to wild type FVIII. For the functional evaluation of the association with FVIII mutants to FIXa, FVIII mutants were reacted with varying concentrations of FIXa in a FXa generation assay. F1816A and E1793A/E1794A/D1795A mutants decreased the FIXa affinity by 1.6-fold (Km; 10.6± 1.0 nM) and 2.8-fold (Km; 18.5± 4.3 nM), respectively, compared to wild type FVIII (Km; 6.6 ± 1.0 nM). These data suggested that not only F1816A but E1793A/E1794A/D1795A mutations contributed high Kmand low catalytic efficiency in FXase complex. E1811A (Km; 7.2 ± 0.7 nM), K1813A (Km; 4.5 ± 0.7 nM), and K1818A (Km; 4.5 ± 0.2 nM) mutants showed the similar FIXa affinities to wild type FVIII. Furthermore, to confirm that the F1816A and E1793A/E1794A/D1795A mutants were responsible for FIXa association, FIXa cleavages in these mutants were evaluated by SDS-PAGE and Western blot using anti-A1 monoclonal antibody. Compared with wild type FVIII, cleavages at Arg336 were slightly delayed in F1816A. On the other hand, those in E1793A/E1794A/D1795A were significantly delayed, indicative of contribution of these residues for FIXa bindings. After all, these results indicate that the 1790-1798 region in the FVIII A3 domain, and in particular a cluster of basic amino acids at residues 1793-1795, contributes to one of the FIXa-interactive sites. Disclosures 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. 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.

Summary The procoagulant activity of factors VIII and V depends on the presence of metal ion(s). We examined the effect of cation-exchange resins with different functional groups on both factors, of which only reaction with iminodiacetate resin resulted in the complete loss of their activity levels in plasma. However, the antigen level of factor VIII was preserved by >95%. This resin reduced divalent cations content present in factor VIII preparations, indicating that it inactivated this factor by direct deprivation of predominant Ca2+ (>Mn2+>>Cu2+), rather than adsorption of the factor itself. The antigen level of recombinant factor VIII alone was decreased by >95% by reaction with resin, whilst that complexed with von Willebrand factor was preserved by >95%. Iminodiacetate resin-treated plasma was evaluated by measuring factor VIII and V activity in plasma with various levels of either activity. These were significantly correlated to the values obtained using factor VIII- or V-deficient plasma prepared commercially by immunodepletion. We demonstrated that iminodiacetate resin-induced factors VIII and V inactivation is because of direct deprivation of metal ions, predominantly Ca2+, which is more essential for the functional structure of their molecules. Furthermore, iminodiacetate resin-treated plasma would be useful as a substrate for measuring the activity of these factors.

Essentials von Willebrand factor levels don't always reflect the severity of von Willebrand disease (VWD). Relationship between new flow system (T-TAS®) and bleeding score (BS) in type 1 VWD was studied. Patients with PL-T10 > 10 min had higher BS and particularly, PL-T10 > 8 min correlated best. T-TAS could be a useful tool for discriminating and predicting the BS in VWD type 1 patients. SummaryBackground The clinical phenotype of von Willebrand disease (VWD) is heterogeneous, and von Willebrand factor ristocetin cofactor activity (VWF:RCo) does not always reflect clinical severity, especially in VWD type 1. We have reported the potential of a microchip flow-chamber system (Total-Thrombus Formation Analysis System [T-TAS®]) for assessing physiologic hemostasis in VWD. Aim To evaluate the relationship between T-TAS, bleeding score (BS) and laboratory test results in type 1 VWD patients. Methods Microchips coated with collagen (platelet chip [PL-chip]) or collagen/thromboplastin (AR-chip) were used to assess platelet thrombus formation (PTF) at high shear rates or fibrin-rich PTF at low shear rates, respectively, in whole blood from 50 patients. The times needed for the flow pressure to increase by 10 kPa and 30 kPa (T10 and T30) from baseline were calculated from flow pressure curves. BS was determined by the use of a standardized questionnaire. Results PL-T10 values correlated with BS (R2 ~ 0.45) better than VWF:RCo (R2 ~ 0.36), irrespective of the flow rate, whereas AR-T10 showed only a weak correlation with BS (R2 ~ 0.18). Patients with PL-T10 > 10 min or AR-T10 > 30 min had lower VWF levels and higher BS than those with PL-T10 ≤ 10 min or AR-T10 ≤ 30 min, and the greatest differences were observed with PL-T10. Clinical severity appeared to correlate best with PL-T10 > 8 min. BS was significantly higher in patients with VWF:RCo of 8 min than in those with PL-T10 ≤ 8 min. Conclusion T-TAS could be a useful technique for discriminating and predicting BS in VWD type 1 patients.

Summary Background In mild hemophilia A (MHA) patients, the risk of inhibitor development is generally low, but some factor VIII (FVIII) gene missense mutations are associated with a higher inhibitor incidence. Objective To investigate the mechanism(s) of inhibitor development in MHA. Methods and results A patient, HA78, with MHA with a novel P1809L missense mutation in the A3 domain, exhibited significant residual FVIII activity (FVIII:C ~10 IU dL−1), despite the development of an inhibitor (5.6 BU mL−1). Purified HA78-IgG significantly depressed FVIII:C from normal plasma but not from patient's plasma without inhibitor, indicating that this IgG inhibited allogeneic but not autologous FVIII. The HA78-IgG blocked thrombin and FXa-catalyzed FVIII cleavage but had little effect on FVIII binding to von Willebrand factor and phospholipid. The IgG recognized a C2 epitope close or overlapping the previously described anti-C2 ESH8 epitope. Similarly, a recombinant FVIII-P1809L mutant was little inactivated by HA78-IgG. This mutant demonstrated ~3-fold lower binding affinities to von Willebrand factor and phospholipid compared with wild-type, while reactions with thrombin or FXa were not impaired. Reaction of FVIII-P1809L with the alternative anti-C2 ESH4 showed only an ~20% inhibition compared with wild-type FVIII but was similar to wild-type after incubation with ESH8. A surface plasmon resonance-based assay demonstrated that anti-C2 ESH4 bound to FVIII-P1809L with ~102-fold lower affinity compared with ESH8. Conclusion These results indicated that the P1809L mutation in A3 induced the conformational change in the FVIII molecule that hampered antigenic determinant(s) located in the C2 domain and might result in the inhibitor development.

Factor (F)VIII functions as a cofactor in the tenase complex responsible for phospholipid (PL) surface-dependent conversion of FX to FXa by FIXa. On the other hand, protein S (PS) functions as a cofactor of activated protein C that inactivates FVIII(a) and FV(a). We have reported a new regulatory mechanism on coagulation that PS directly impaired the FXase complex by competing the FIXa-FVIIIa interaction (Takeyama, Br J Haematol. 2008;143:409), and identified the PS-interactive site on the FVIII A2 domain (Takeyama, Thromb Haemost. 2009;102:645). However, the contribution of FVIII light chain (LC) to PS-binding has not been determined. In this study, several approaches were employed to assess a PS-FVIII LC interaction. The binding of FVIII LC to active site-modified FIXa (EGR-FIXa) was inhibited by PS dose-dependently (Ki; 4.6 nM) on ELISA. Because FVIII C2 domain has a binding site for FIXa, we examined whether FVIII C2 domain bound to PS. PS bound to FVIII C2 domain (Kdapp; 283 nM) by the ELISA. A SPR-based assay also revealed that FVIII C2 domain bound to PS (Kd; 62 nM). We have reported that a FIXa-interactive site exits on FVIII C2 domain (residues 2228-2240) (Soeda, J Biol Chem. 2009;284:3379). Therefore, we assessed the interaction of the synthetic peptide spanning the residues (2228-2240) with PS. ELISA showed that the peptide bound to PS (Kdapp; 104 µM). SPR-based assay also revealed that the peptide bound to PS (Kd; 31.4 µM), although scrambled peptide failed to bind to PS. Covalent cross-linking was observed between the biotynated 2228-2240 peptide and PS following reaction with EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) using SDS-PAGE. This cross-linking formation was blocked by the addition of the unlabeled peptide. Furthermore, N-terminal sequence analysis of the peptide-PS product showed that one basic residue (K2239) could not be detected, supporting that this residue participates in cross-link formation. These results indicate that the 2228-2240 residues in the FVIII C2 domain, in particular K2239, may contribute to a PS-interactive site. To inquire further into the interactive region of FVIII C2 domain to PS, we prepared a stably expressed recombinant B-domainless FVIII mutants (E2228A, D2233A, K2236A, and K2239A), which were polar amino acids in the 2228-2240 residues, subsequently compared interaction with PS of the mutants with B-domainless FVIII wild type. To evaluate the binding affinity to PS, we performed SPR-based assay with the wild type FVIII and the FVIII mutants. The assay showed K2239A bound to PS with ~1.9-fold higher Kd value (28.1 nM) than that of wild type (Kd; 14.7 nM), although E2228A, D2233A, or K2236A bound to PS (Kd; 12.7, 16.3, and 10.3 nM, respectively) with no significant difference compared with wild type. In addition, to evaluate the effect of mutants for FXa generation, we assessed FXa generation assay with wild type or K2239A in the presence or absence PS, because K2239 might contribute to a PS interaction of FVIII. After each FVIII (1 nM) was reacted with various concentrations of PS in the presence of PL (20 µM) for 30 minutes, FVIII was activated by thrombin (30 nM), followed by the reaction with FIXa (40 nM) and FX (300 nM) for 1 minute. Although generated FXa with both wild type and K2239A were reduced in the presence of PS dose-dependently, compared with its absence, the inhibition ratio of K2239A was less than that of wild type (18.2% and 31%, respectively in the presence of 500 nM PS). On the other hand, the Km value on FXa generation of K2239A for various concentrations of FIXa was ~1.8-fold higher than that of wild type (5.5±0.9 and 3.1±0.2 nM, respectively), suggesting that K2239 residue, involved in the FIXa-interactive site, might contribute to the inhibition of FVIII function by PS. In conclusion, FVIII C2 domain, in particular K2239, was possible to play an important role of the inhibitory mechanism to FVIII function by PS, due to the binding to PS. Disclosures Shima: Sysmex Corporation: Patents & Royalties, Research Funding; 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. Nogami:Sysmex Corporation: Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Chugai Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Factor (F)VIII functions as a cofactor in the tenase complex responsible for phospholipid (PL) surface-dependent conversion of FX to FXa by FIXa. On the other hand, protein S (PS) functions as a cofactor of activated protein C that inactivates FVIII(a) and FV(a). We have reported a new regulatory mechanism on coagulation that PS directly impaired the FXase complex by competing the FIXa-FVIIIa interaction (Takeyama, Br J Haematol. 2008;143:409), and identified the PS-interactive site on the FVIII A2 domain (Takeyama, Thromb Haemost. 2009;102:645). However, the contribution of FVIII light chain (LC) to PS-binding has not been determined. In this study, several approaches were employed to assess a PS-FVIII LC interaction. The binding of FVIII LC to active site-modified FIXa (EGR-FIXa) was inhibited by PS dose-dependently ( K i ; 4.6 nM) on ELISA. Because FVIII C2 domain has a binding site for FIXa, we examined whether FVIII C2 domain bound to PS. PS bound to FVIII C2 domain ( K d app ; 283 nM) by the ELISA. A SPR-based assay also revealed that FVIII C2 domain bound to PS ( K d ; 62 nM). We have reported that a FIXa-interactive site exits on FVIII C2 domain (residues 2228-2240) (Soeda, J Biol Chem. 2009;284:3379). Therefore, we assessed the interaction of the synthetic peptide spanning the residues (2228-2240) with PS. ELISA showed that the peptide bound to PS ( K d app ; 104 µM). SPR-based assay also revealed that the peptide bound to PS ( K d ; 31.4 µM), although scrambled peptide failed to bind to PS. Covalent cross-linking was observed between the biotynated 2228-2240 peptide and PS following reaction with EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) using SDS-PAGE. This cross-linking formation was blocked by the addition of the unlabeled peptide. Furthermore, N-terminal sequence analysis of the peptide-PS product showed that one basic residue (K2239) could not be detected, supporting that this residue participates in cross-link formation. These results indicate that the 2228-2240 residues in the FVIII C2 domain, in particular K2239, may contribute to a PS-interactive site. To inquire further into the interactive region of FVIII C2 domain to PS, we prepared a stably expressed recombinant B-domainless FVIII mutants (E2228A, D2233A, K2236A, and K2239A), which were polar amino acids in the 2228-2240 residues, subsequently compared interaction with PS of the mutants with B-domainless FVIII wild type. To evaluate the binding affinity to PS, we performed SPR-based assay with the wild type FVIII and the FVIII mutants. The assay showed K2239A bound to PS with ~1.9-fold higher K d value (28.1 nM) than that of wild type ( K d ; 14.7 nM), although E2228A, D2233A, or K2236A bound to PS ( K d ; 12.7, 16.3, and 10.3 nM, respectively) with no significant difference compared with wild type. In addition, to evaluate the effect of mutants for FXa generation, we assessed FXa generation assay with wild type or K2239A in the presence or absence PS, because K2239 might contribute to a PS interaction of FVIII. After each FVIII (1 nM) was reacted with various concentrations of PS in the presence of PL (20 µM) for 30 minutes, FVIII was activated by thrombin (30 nM), followed by the reaction with FIXa (40 nM) and FX (300 nM) for 1 minute. Although generated FXa with both wild type and K2239A were reduced in the presence of PS dose-dependently, compared with its absence, the inhibition ratio of K2239A was less than that of wild type (18.2% and 31%, respectively in the presence of 500 nM PS). On the other hand, the K m value on FXa generation of K2239A for various concentrations of FIXa was ~1.8-fold higher than that of wild type (5.5±0.9 and 3.1±0.2 nM, respectively), suggesting that K2239 residue, involved in the FIXa-interactive site, might contribute to the inhibition of FVIII function by PS. In conclusion, FVIII C2 domain, in particular K2239, was possible to play an important role of the inhibitory mechanism to FVIII function by PS, due to the binding to PS. Disclosures Shima: Sysmex Corporation: Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees; Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Patents & Royalties, Research Funding. Nogami: Sysmex Corporation: Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Chugai Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity9s Board of Directors or advisory committees, Patents & Royalties, Research Funding.

We have recently reported that cation-exchange iminodiacetate resin completely inactivated factor VIII (FVIII) by direct deprivation of metal ions, predominantly Ca2+, from its molecules, and that von Willebrand factor (VWF) protected FVIII antigen from resin-induced degradation. The present study was further developed to investigate this mechanism. Western blotting analysis and enzyme-linked immunosorbent assay showed that the antigenic structure of the FVIII light chain, especially the C2 domain, was completely impaired by the resin, whilst that of the heavy chain was little affected. However, the complex formation with VWF protected the C2 domain from the resin-induced degradation. The resin-treated C2 domain failed to interact with VWF and phospholipid. Furthermore, the addition of Ca2+ competitively blocked the resin-induced impairment of the C2 domain structure. These results demonstrate that VWF protects the Ca2+-dependent conformational structure of the FVIII light chain, especially the C2 domain, and may indicate that the C2 domain contains the Ca2+-binding site(s).

We have recently reported that plasmin likely associates with the factor VIII light chain to proteolyze at Lys36 within the A1 domain. In this study, we determined that the rate of plasmin-catalyzed inactivation on the forms of factor VIIIa containing A1-(1-336) and 1722A3C1C2, reflecting Lys36 cleavage, was reduced by approximately 60%, compared with those containing 1649A3C1C2 and 1690A3C1C2. SDS-PAGE analysis revealed that Lys36 cleavage of factor VIIIa with 1722A3C1C2 was markedly slower than those with 1649A3C1C2 and 1690A3C1C2. Surface plasmon resonance-based assays, using active site-modified anhydro-plasmin (Ah-plasmin) showed that 1722A3C1C2 bound to Ah-plasmin with an approximately 3-fold lower affinity than 1649A3C1C2 or 1690A3C1C2 (Kd, 176, 68.2, and 60.3 nM, respectively). Recombinant A3 bound to Ah-plasmin (Kd, 44.2 nM), whereas C2 failed to bind, confirming the presence of a plasmin-binding site within N terminus of A3. Furthermore, the Glu-Gly-Arg active site-modified factor IXa also blocked 1722A3C1C2 binding to Ah-plasmin by approximately 95%, supporting the presence of another plasmin-binding site overlapping the factor IXa-binding site in A3. In keeping with a major contribution of the lysine-binding sites in plasmin for interaction with the factor VIII light chain, analysis of the A3 sequence revealed two regions involving clustered lysine residues in 1690-1705 and 1804-1818. Two peptides based on these regions blocked 1649A3C1C2 binding to Ah-plasmin by approximately 60% and plasmin-catalyzed Lys36 cleavage of factor VIIIa with A1-(1-336) by approximately 80%. Our findings indicate that an extended surface, centered on residues 1690-1705 and 1804-1818 within the A3 domain, contributes to a unique plasmin-interactive site that promotes plasmin docking during cofactor inactivation by cleavage at Lys36.

Factor VIIIa functions as a cofactor for factor IXa in the phospholipid surface-dependent activation of factor X. Both the C2 domain of factor VIIIa and the Gla domain of factor IXa are involved in phospholipid binding and are required for the activation of factor X. In this study, we have examined the close relationship between these domains in the factor Xase complex. Enzyme-linked immunosorbent assay-based and surface plasmon resonance-based assays in the absence of phospholipid showed that Glu-Gly-Arg active site-modified factor IXa bound to immobilized recombinant C2 domain (rC2) dose-dependently (Kd = 108 nm). This binding ability was optimal under physiological conditions. A monoclonal antibody against the Gla domain of factor IXa inhibited binding by approximately 95%, and Gla domainless factor IXa failed to bind to rC2. The addition of monoclonal antibody or rC2 with factor VIIIa inhibited factor IXa-catalyzed factor X activation in the absence of phospholipid. Inhibition was not evident, however, in similar experiments in the absence of factor VIIIa, indicating that the C2 domain interacted with the Gla domain of factor IXa. A fragment designated C2-(2182-2259), derived from V8 protease-cleaved rC2, bound to Glu-Gly-Arg active site-modified factor IXa. Competitive assays, using overlapping synthetic peptides encompassing residues 2182-2259, demonstrated that peptide 2228-2240 significantly inhibited both this binding and factor Xa generation, independently of phospholipid. Our results indicated that residues 2228-2240 in the factor VIIIa C2 domain constitutes an interactive site for the Gla domain of factor IXa. The findings provide the first evidence for an essential role for this interaction in factor Xase assembly.

Protein S functions as an activated protein C (APC)-independent anticoagulant in the inhibition of intrinsic factor X activation, although the precise mechanisms remain to be fully investigated. In the present study, protein S diminished factor VIIIa/factor IXa-dependent factor X activation, independent of APC, in a functional Xa generation assay. The presence of protein S resulted in an c. 17-fold increase in K(m) for factor IXa with factor VIIIa in the factor Xase complex, but an c. twofold decrease in K(m) for factor X. Surface plasmon resonance-based assays showed that factor VIII, particularly the A2 and A3 domains, bound to immobilized protein S (K(d); c. 10 nmol/l). Competition binding assays using Glu-Gly-Arg-active-site modified factor IXa showed that factor IXa inhibited the reaction between protein S and both the A2 and A3 domains. Furthermore, Sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed that the cleavage rate of factor VIIIa at Arg(336) by factor IXa was c. 1.8-fold lower in the presence of protein S than in its absence. These data indicate that protein S not only down-regulates factor VIIIa activity as a cofactor of APC, but also directly impairs the assembly of the factor Xase complex, independent of APC, in a competitive interaction between factor IXa and factor VIIIa.

FVIII is activated by cleavage at Arg 372 , Arg 740 , and Arg 1689 by thrombin. This study showed that an anti-A2 monoclonal antibody, with a specific epitope for residues 484-509, and anti-FVIII inhibitor alloantibodies with similar A2 epitopes, inhibited thrombin-catalyzed FVIII activation. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis showed that cleavage at Arg 372 but not at Arg 740 occurred at approximately fourfold decreased rate in the presence of anti-A2 antibody. Peptide 484-509 also inhibited co-factor activation, consistent with inhibition of cleavage at Arg 372 . Direct binding studies using active-site modified thrombin showed that a 484-509 peptide as well as the anti-A2 antibodies blocked the A2-thrombin binding. Furthermore, covalent cross-linking was observed between the 484-509 peptide and thrombin following reaction with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. Mutant A2 molecules in which the clustered basic residues in this sequence were converted to alanine were used to assess the binding reactions in a surface plasmon resonance-based assay. Mutants R484A, R489A, R490A, H497A and K499A possessed two to fivefold lower affinity than wild-type A2. These findings demonstrate that clustered basic residues within the 484-509 region of the A2 domain play a part of key role in thrombin-binding, which is responsible for thrombin-catalyzed FVIII activation by cleavage at Arg 372 .

Acquired hemophilia A (AHA) is a rare bleeding disorder in which autoantibodies (autoAbs) against coagulation factor (F)VIII impair the coagulation system. Anti-FVIII autoAbs developed in AHA are polyclonal and the majority of these autoAbs bind to the A2, A3, and/or C2 domains in FVIII. Different inhibitory actions of anti-FVIII autoAbs, depending on their epitopes, are well-known. We have recently developed a humanized anti-FIXa/X bispecific antibody, ACE910, which mimics the cofactor function of FVIIIa even in the presence of anti-FVIII alloantibodies. In this study, we examined whether ACE910 improved the lower coagulation function in AHA patients using the comprehensive coagulation assays. Two representative experiments were performed as follows; 1) In vitro effects of ACE910 on normal pooled plasmas (PNPs) mixed with various anti-FVIII monoclonal Abs (mAbs) as the reconstituted AHA-models. For this experiments, several anti-FVIII mAbs; VIII-9222 (epitope: anti-a1), VIII-2236 (anti-A2), VIII-3776 (anti-A3C1), ESH4 (anti-C2) were prepared. 2) Ex vivo effects of ACE910 on plasmas obtained from nine patients with AHA, whose regions of Abs were A1, A2 and light chain for four patients, A2 and light chain for three patients, A2 for one patient and light chain for one patient. The coagulation functions in obtained samples were evaluated using the comprehensive coagulation assays, clot waveform analysis (CWA) and thrombin generation assays (TGA). In CWA, the minimum value of the first derivative (min1) and the second derivative (min2) were calculated as an index of the maximum acceleration of the reaction and the maximum velocity of coagulation, respectively. In TGA, calibrated automated thrombin generation assays derived the standard parameters; peak thrombin, lagtime, and time to peak. Total thrombin generation at intervals from the beginning to peak level was quantified. These values (nmol/l) were divided by the sample times (min, time to peak - lagtime), and represented the mean velocity of thrombin generation until the reach to peak level, expressed as mean velocity to peak thrombin (MV-peak thrombin). We defined the attenuation of inhibitory effect (% of AIE) represented that ACE910 attenuated the parameters inhibited by mAbs in artificial AHA or by allo-Abs in AHA patients, respectively. Figure 1 represents the % of AIE on thrombin peak in TGA. PNPs were incubated with various anti-FVIII mAbs (100-500 mcg/ml) at 37 degrees Celsius for 2 hrs, and FVIII activities in these samples were decreased to 2.0-43%. These reactant mixtures were reacted with ACE910 (0-20 mcg/ml) for a further 2 hr-incubation. ACE910 improved the parameters of CWA (|min1|; 40-62%, and |min2|; 49-74% of AIE) and of TGA (thrombin peak; 46-72%, and MV-peak thrombin; 51-70% of AIE, respectively) in all PNPs with mAbs. These data indicated that ACE910 improved the coagulant activity in AHA patients. Therefore, we determined the efficacy of ACE910 for nine AHA patients ex vivo. FVIII activities and FVIII inhibitors were Our results indicated that ACE910 could improve the coagulant activities in AHA patients independent of the regions of antibodies and might be useful for the treatment of AHA patients. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Takeyama: Chugai Pharmaceutical Co., Ltd.: Research Funding. Nogami:Bayer, NovoNordisk, Baxalta, Chugai, Kaketsuken, Pfizer, Biogen: Honoraria; Chugai: Membership on an entity's Board of Directors or advisory committees; Bayer, Novo Nordisk, Baxalta. Biogen: Research Funding. Matsumoto:Chugai Pharmaceutical Co., Ltd: Research Funding. Kitazawa:Chugai Pharmaceutical Co., Ltd: Employment, Equity Ownership, Patents & Royalties. Hattori:Chugai Pharmaceutical Co., Ltd: Employment, Equity Ownership, Patents & Royalties. Shima:Chugai Pharmaceutical Co., Ltd. and F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background: ACE910, a humanized bispecific antibody to factor (F) IXa and FX mimicking the functions of FVIII, exerts tenase activities without FVIII (a) (Kitazawa et al. Nature Medicine. 2012;18, 1570). In primate hemophilia A (HA) models, the hemostatic enhancing effect of ACE910 has been reported and the clinical study investigating the effect and safety of ACE910 for human HA patients is on-going. However, the hemostatic effect of ACE910 remains unquantified and difficult to be evaluated. Objectives: In this study, we evaluated the viscoelastmetric parameters in the whole blood obtained from HA patients under long-term treatment of ACE910 in phase 1 and its extension studies, utilizing rotation thromboelastometry (ROTEM), in order to investigate global hemostatic function of HA patients under treatment with ACE910. Methods: Ca2+-triggered hemostatic functions were assessed by ROTEM in the citrated whole blood samples obtained and stabilized at the indicated points from five severe HA cases with inhibitors (Case 1 : 39 BU/ml, 2 : 41 BU/ml, 3 : 17 BU/ml, 4 : 11 BU/ml and 5 : 111 BU/ml ) and two severe cases without inhibitors (Case 6 and 7) under subcutaneous administration of ACE910, with the different types of dosing regimen (group A with 0.3 mg/kg/week subcutaneous injection (loading dose of 1 mg/kg) for Case 1, 2, group B with 1 mg/kg/week (loading dose of 3 mg/kg) for Case 3, 4, group C with 3 mg/kg/week for Case 6 and group D changing from group A to group C for Case 5, 7) in a course of clinical study. The samples from the subjects prior to administration of ACE910 were spiked ex vivo with ACE910 and the hemostatic functions of them were also evaluated by ROTEM. The parameters of clot time (CT), clot formation time (CFT), maximum clot formation (MCF) and alpha angle were evaluated. The hemostatic potentials in the samples from twenty healthy volunteers and other ten HA patients with the various FVIII:C were evaluated by ROTEM as controls and the correlation between FVIII:C and CT was obtained. Annualized bleeding rates (ABR) of each case were calculated. The studies were approved by local ethics committee and the informed consent was obtained from each patient. Results: Addition of ACE910 (f.c. 10 or 30 μg/ml) into the sample from each case prior to administration of ACE910 shortened CT from 5,562 ± 374 sec (median 5,924 sec) to 1,475 ± 138 or 1,131 ± 82 sec, equivalent to FVIII:C 3.3 or 12.2 IU/dL, respectively, in an ACE910 concentration dependent manner. In group A, the plasma concentration of ACE910 got to 12 ± 5 μg/ml at 12 week. CT was shortened to 1,443 ± 24 sec, equivalent to FVIII:C 3.7 IU/dL maximally at 47 week consistent with the result of the spiked result. ABR decreased from 38.6 ± 25.8 to 1.1 ± 0.8, showing 97% decrease. As for Case 3 in group B (ACE910: 31 μg/ml at 12 week), CT was shortened to 1,164 sec, equivalent to FVIII:C 11.9 IU/dL maximally at 47 week consistent with the spiked data. ABR apparently decreased from 38.6 to 3.6. In Case 4 who dropped out the clinical study at 4 week, CT was shortened to 977 sec, equivalent to FVIII:C 22 IU/dL, maximally at 3 week, and continued to be shortened till 29 week (1,758 sec, equivalent to FVIII:C 1.1 IU/dL). In group C, the shortening of CT from 1,594 ± 103 sec, equivalent to FVIII:C 2.0 ± 1.0 (median 3.6) IU/dL (before administration of ACE910), to 1,226 ± 71 sec (8.5 ± 2.5 (median 10.4) IU/dL) (at steady state) was observed in consistent with the decrease of ABR from 8.1 to 1.1. In group D, CT was shortened from 3,405 ± 386 sec to 1,409 ± 85 sec, equivalent to FVIII:C 4.2 IU/dL after middle to high dose escalation in consistent with decrease of ABR from 22.3 ± 9.6 to 3.8 ± 2.6. Among all cases except Case 4, CT and ABR were of clinically relevant difference between before and after administration of ACE910. Conclusions: The comprehensive hemostatic function evaluated by ROTEM in hemophilia A patients irrespective of presence of inhibitors was improved after administration of ACE910 in a dose dependent fashion, resulting in the reduction of ABR, which suggested the hemostatic effectiveness of ACE910 for HA patients. Disclosures Yada: Chugai Pharmaceutical Co., Ltd: Research Funding. Nogami:Bayer, Novo Nordisk, Baxalta. Biogen: Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees; Bayer, NovoNordisk, Baxalta, Chugai, Kaketsuken, Pfizer, Biogen: Honoraria. Shida:Chugai Pharmacoceutical Co. Ltd.: Research Funding. Takeyama:Chugai Pharmaceutical Co., Ltd.: Research Funding. Kasai:Chugai Pharmaceutical Co., Ltd.: Employment. Shima:Chugai Pharmaceutical Co., Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Baxalta: Honoraria, Research Funding; Novo Nordisk: Honoraria, Research Funding; Kaketsuken: Honoraria; Bayer: Honoraria, Research Funding.

Acquired hemophilia A (AHA) is a rare hemorrhagic disease in which autoantibodies against coagulation factor (F) VIII impair the coagulation system. The inhibitors developed in AHA are polyclonal autoantibodies and the majority of FVIII inhibitors bind to the A2, A3, or C2 domains. Depending on the location of the epitope, different mechanisms of action for the anti-FVIII antibodies have been reported. Anti-A3 antibodies neutralize the procoagulant activity of FVIII by preventing its interaction with FIXa. Anti-C2 antibodies inhibit the binding of FVIII to phospholipid membrane and/or von Willebrand factor, whereas A2 and A3 inhibitors block the binding of FVIII to FIXa and FX, respectively, and obstruct the formation of the Xase complex. We have a case of AHA whose inhibitor recognizes only A2 domain and attempted several approaches to determine the mechanism of neutralizing FVIII. Thrombin and plasmin generation assay using patient’s plasma showed that the thrombin and plasmin generation in this AHA patient were decreased compared with that in congenital severe hemophilia A patient. Furthermore, FX generation (Coatest) in this AHA was also decreased compared with that in congenital severe hemophilia A patient (p Disclosures No relevant conflicts of interest to declare.

The development of inhibitory antibodies to factor (F)VIII in patients with mild hemophilia A is a rare, but significant event. We reported the mild hemophilia A associated with Pro(P)1809Leu(L) mutation in FVIII gene (FVIII-Tenri), developing the type II inhibitor (peak 5.6BU/ml), which inhibited allogeneic but not autologous FVIII recognizing the epitope(s) overlapping with that of anti-FVIII monoclonal antibody (mAb) ESH8 in the C2 domain remote from the mutated site in the A3 domain, at the 54th ASH meeting (#52283). However, the haemostatic characteristics of FVIII-Tenri remain unclear. In this study, to elucidate the characteristics of FVIII-Tenri, we prepared and stably expressed a recombinant, B-domainless FVIII mutant, P1809L. FVIII:C of the mutant reconstituted with FVIII-deficient plasma (0.3 nM) was 31.5 IU/dl, whilst that of the wild-type (WT) (0.3 nM) was 88.6 IU/dl, similar to the level of patient’s plasma. The polyclonal IgG immune-purified from this patient’s plasma decreased FVIII:C of WT by ∼60% at the maximum concentration of IgG, whilst any little affected in the mutant. The mutant was activated by thrombin and FXa, showing ∼16- and 7-fold increase in FVIII:C compared to baseline control, respectively, similar to WT. Furthermore, purified FXa generation assays showed that the mutant was any little different in Km for FIXa compared to WT (0.74 ± 0.07 and 0.72 ± 0.13 nM, respectively), supporting that this mutation little affected the association with FIXa. Interestingly, the mutant showed an ∼3-fold weak binding affinity (Kdapp 0.92 ± 0.23nM) to VWF compared to WT (Kdapp 0.33 ± 0.01nM), whilst any little difference was observed in binding affinity to phospholipid between the mutant and WT, in an ELISA. To further investigate the haemostatic mechanism(s) associated with less binding affinity to VWF, the residual FVIII:C of the mutant or WT (0.05-0.25nM) was measured in a one-stage clotting assay, after incubation with several anti-FVIII mAbs including anti-C2 mAbs ESH8 (residues 2248-2285) and ESH4 (2303-2332) with individual epitope overlapping the VWF-binding site(s) in the C2 domain. ESH8 conferred the ∼60% lower inhibition of FVIII:C in the mutant compared to that in WT, whilst ESH4 showed the similar inhibition of FVIII:C between in the mutant and WT. Anti-A2 mAb JR8 showed any little difference in the inhibition between in the mutant and WT. These results demonstrated that the mutation P1809L in the A3 domain did little affect upon activation by thrombin and FXa as well as association with FIXa and phospholipid, but attenuated the binding to VWF through the conformational change in the C2 domain. Taken together with the development of the allogeneic inhibitor associated with this mutation, these results strongly suggested that the conformational change occurred in the C2 domain remote from the mutated site in the A3 domain hampered the binding to VWF, resulting in the haemostatic impairment due to fragility of the mutant FVIII molecule as well as the change of the immunogenicity in the epitopes overlapping with the FVIII-VWF binding site (residue 2248-2285) presented on allogeneic FVIII. Disclosures: No relevant conflicts of interest to declare.

Abstract 3169 Poster Board III-110 Factor (F)VIIa with tissue factor (TF) is a primary trigger of blood coagulation. We have recently demonstrated that FVIIa/TF rapidly activated FVIII by proteolysis of the heavy chain (HCh), and served physiologically as a potent activator for up-regulation of FVIII activity in very early-timed phase (ASH #1036, 2008). FVIII inhibitors develop as alloantibodies in multi-transfused patients with hemophilia A and also arise as autoantibodies in normal individuals. FVIII inactivation by inhibitors is associated with impairment of FVIII(a) cofactor function through the binding to functional crucial epitopes in FVIII. Anti-C2 inhibitors prevent FVIII binding to phospholipid, von Willebrand factor, and FXa. Anti-A2 inhibitors prevent FVIII binding to FIXa and thrombin. However, effects of these inhibitors on FVIIa action for FVIII have remained to be studied. In this study, we prepared 13 of anti-FVIII inhibitor IgGs (2 of anti-A2, 7 of anti-C2 with type 1 behavior, and 4 of anti-C2 with type 2). We first examined FVIIa/TF-catalyzed FVIII activation in the presence of anti-FVIII inhibitors in one-stage clotting assay. The levels of FVIII activity (10 nM) elevated rapidly by ∼2.0-fold within 30 sec after adding of FVIIa/TF (1 nM), and subsequently decreased to the initial level within 20 min. The presence of anti-FVIII inhibitors did not significantly affect FVIIa/TF-catalyzed FVIII activation (by 1.7∼2.2-fold) compared to normal IgG. This action was independent of the difference of inhibitor epitopes. In addition, FVIIa-catalyzed FVIIIa inactivation with anti-A2 or anti-C2 with type 2 inhibitors was little affected, similar to that with normal IgG. However, of note, all of anti-C2 with type 1 significantly inhibited FVIIa-catalyzed inactivation of FVIIIa. Inactivation rates of FVIIa with anti-C2 with type 1 (k ∼0.15) was ∼40% less than that with control IgG (k ∼0.24), supporting that the presence of anti-C2 with type 1 might persist the activity of FVIIIa generated by FVIIa. To clarify this inhibitory mechanism of anti-C2 with type 1, we performed FVIIa-catalyzed FVIII cleavage in Western blotting. FVIIa/TF (1 nM) proteolyzed the HCh of FVIII (10 nM) rapidly by cleavages at Arg372 (and Arg740), whilst cleavage at Arg336 in the A1 domain was appeared at ∼2.5 min, supporting that cleavages at Arg372 and Arg336 by FVIIa contribute to the up- and down-regulation of FVIII(a) activity, respectively. All inhibitors, independent of recognizing epitopes, did not affect FVIIa-catalyzed cleavage at Arg372. However, the presence of anti-C2 type 1 delayed the cleavage at Arg336 in timed- and dose-dependent manners, whilst either anti-A2 or anti-C2 type 2 did not affect, consistent with the functional inactivation results. FVIIa binds to the A2, A3, and C2 domains in FVIII. Based on our findings, FVIIa-interactive sites on FVIII unlikely overlapped with anti-A2 and -C2 inhibitor epitopes, and inhibition of Arg336 cleavage may be due to conformational change caused by antibody binding. Furthermore, FVIIa indeed activates FVIII even in the presence of anti-FVIII inhibitors, different from thrombin, FXa, etc, and it would be important to predict the effect of FVIIa for FVIII to determine the characteristics of anti-FVIII inhibitors. Disclosures No relevant conflicts of interest to declare.

Abstract 2125 Poster Board II-103 Inflammation and coagulation are linked in a variety ways. Since both neutrophil elastase and cathepsin G bind to activated platelets, they can be localized on the platelet membrane providing negatively-charged phospholipid, that is essential for assembly of tenase complex. Although it has been reported that cathepsin G provides some procoagulant effect by activating factor (F)VIII, effect of elastase on FVIII is poorly understood. We now examine the effect of human neutrophil elastase (HNE) on FVIII(a). FVIII activity (used 100 nM) was rapidly decreased and was undetectable within 10 min after the addition of HNE (0.5 nM) in a one-stage clotting assay. This time-dependent inactivation was little or mildly affected (∼35% inhibition at 10 μg/ml) in the presence of phospholipid or von Willebrand factor, respectively. Rate constant of FVIII inactivation by HNE possessed ∼15-fold greater than that by APC/protein S (PS). SDS-PAGE analysis revealed that HNE proteolyzed the 90∼210-kDa heavy chain of FVIII to 47- and 37-kDa terminal products, via some proteolyses within the B domain. Western blot and N-terminal sequence analyses showed that these fragments derived from the heavy chain were identified as the A11-357 and A2375-708, respectively, by cleavages at Val357 and Val374 in A1 and Val708 in A2. The cleavages at Val374 and Val708 by HNE were faster than that at Val357. The 80-kDa light chain was proteolyzed to 75-kDa fragment by cleavage at Val1670, quite different with the pattern of FVIII cleavage by known serine proteases. Of note, HNE-catalyzed inactivation of FVIIIa was much slower (by ∼25-fold) than that of FVIII, supporting that HNE preferably attacked to FVIII. This discrepancy was attributed to much slower cleavage at Val708 in FVIIIa. Rate of inactivation of FVIIIa by HNE was ∼30-, ∼8-, and ∼3-fold lower than those by plasmin, APC/PS, and FXa, respectively. SDS-PAGE using FVIII(a) fragments revealed that each cleavage at Val357 and Val708 was regulated by the presence of the light chain and heavy chain (intact A1-A2), respectively. These results demonstrate that the importance of cleavage at Val708, constitutes a FIXa-interactive site in tenase complex, for the mechanism of HNE-catalyzed FVIII inactivation. Furthermore, this cleavage appears to be selectively modulated by the A1-A2 domain that might interact with HNE. Disclosures: No relevant conflicts of interest to declare.

Abstract 3177 Poster Board III-116 Activated factor (F)VII complex with tissue factor (FVIIa/TF) initiates the blood coagulation by generating FXa as extrinsic Xase complex (ex-Xase). Although FVIIa/TF also activates FIX, FIXa little functions without its cofactor, FVIIIa. A tiny amount of thrombin generated by FXa activates FV and FVIII, followed by forming of intrinsic Xase complex (in-Xase) and prothrombinase complex, respectively. These formations result in ‘thrombin burst’ and successful hemostasis. Although thrombin is thought to be a unique potent activator of FVIII in vivo, FXa and FVIIa/TF also activate FVIII in vitro. We have recently reported the detailed mechanism by which FVIIa/TF activated FVIII more rapidly in early timed-phase than thrombin (Blood Abst.1036, 2008). In this study, we further developed to examine whether TF affected FVIII(a) function. (1) FVIIa/TF rapidly increased FVIII activity by 4.7-fold of initial in the presence of Ca2+ and phospholipid (PL), following by inactivation, in one-stage clotting assay. However, since even in the presence of TF alone, FVIII activity elevated by 1.8-fold of initial, actual increase of FVIII activity by FVIIa/TF was 2.6-fold. A possibility that TF might bind to FVIIa contained in FVIII-deficient plasmas used, was negligible, since FVIIa-inhibitor used blocked an ex-Xase effect >95%. In the presence of FVIIa-inhibitor, residue FVIII activity with TF was ∼50%, thus TF alone affected FVIII cofactor activity independently of FVIIa. (2) Using SDS-PAGE, the addition of TF accelerated FVIII cleavage by FVIIa, whilst decelerated that by thrombin and FXa. (3) Surface plasmon resonance-based assays showed that FVIII(a) directly bound to TF with high affinity (Kd; ∼3 nM). (4) The effect of FVIIa/TF on in-Xase was evaluated in FXa generation assay. 0.1 nM FVIIa/TF, 1 nM FVIII, 90 nM FIX and 20 μM PL were reacted with 150 nM FX at various combinations. FVIIa/TF and FVIIa/TF/FVIII/FIX generated FXa with 3.9 and 10.4 nM/min, respectively. When FVIIa-inhibitor was added prior to addition of FX, FXa generated by FVIIa/TF and FVIIa/TF/FVIII/FIX were 5% and 46% (0.2 and 4.8 nM/min) of those without FVIIa-inhibitor, respectively. The latter was considered as FXa generated by in-Xase. Therefore, FXa derived from in-Xase was ∼40% of total FXa in this condition. (5) FVIIIa/FIXa (1 nM/2 nM)-dependent FXa generation in the presence of TF was evaluated. FXa generation in the presence of TF (0.02 and 0.3 nM) increased by ∼2 and ∼6-folds, respectively, of that in its absence. Furthermore, the functional affinity of FVIIIa for in-Xase complex in the presence of TF (0.1 nM), showed an ∼1.5-fold greater than that in its absence (Km; 4.9 ± 0.4 and 7.1 ± 0.9 nM, respectively). In conclusion, FVIIa/TF can generate FVIIIa in early timed-phase in vitro as well as FXa and FIXa, and possess potential of forming in-Xase. In addition, TF directly binds to FVIII(a), and functions in-Xase complex more efficiently by enhancing the affinity of FVIIIa for in-Xase. Although TF-dependent these reactions may be terminated rapidly via anticoagulant systems such as tissue factor pathway inhibitor, our data suggest that interactions of FVIII with TF might contribute to the acceleration of FXa generation in the initiation phase of blood coagulation. Disclosures Okuda: Sysmex Corporation: Employment.

Abstract 3483 Poster Board III-420 The development of factor (F)VIII autoantibody inhibitors results in severe hemorrhagic diathesis known as acquired hemophilia A (AHA), although many inhibitors with type 2 behavior incompletely inhibit FVIII activity at saturating concentrations. However, the mechanisms of AHA with type 2 behavior remain to be fully understood. In this study, we evaluated the detailed coagulation function and clarified inhibitory mechanisms for AHA with type 2. Plasma samples of patients were obtained from congenital hemophilia A with severe type {(FVIII:C Disclosures: No relevant conflicts of interest to declare.

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.

Plasmin (Plm), an active form of plasminogen (Plg), not only functions as a key enzyme in the fibrinolytic system, but also directly inactivates several coagulation factors. Especially, factor VIII is inactivated by Plm immediately after the activation by limited proteolytic cleavage at Lys36, Arg336, Arg372, and Arg740 in the heavy chain, and at Arg1689 and Arg1721 in the light chain (Nogami et al. J. Biol. Chem. 2007, 282, 5287). We recently have identified the plasmin-interactive sites on the A2 domain responsible for cleavages at Arg336 and Arg372, and on the light chain responsible for cleavage at Lys36 (Abst #1991/1709, BLOOD 102/108, 2005/2006). In the present study, we attempted to localize a factor VIII-interactive site on Plm (and Plg). Competitive binding assay using 6-aminohexanoic acid (6-AHA), a competitor of lysine-binding site (LBS) of Plm/Plg, showed that 6-AHA markedly inhibited (by >90%) the light chain binding to active-site modified Plm (anhydro-Plm), whilst inhibited weakly the A2 binding (by ∼30%). These results suggested that the light chain interaction with Plm was mainly dependent upon LBS, but the A2 interaction was independent. The addition of monoclonal antibody (mAb) against Plg kringle 5-catalytic domain (K5-CD) significantly inhibited Plm-catalyzed activation/inactivation of factor VIII or VIIIa with an ∼4-fold lower rate constant. On the other hand, anti-K1-3 and anti-K4 mAbs any little affected. SDS-PAGE analysis revealed that only anti-K5-CD mAb blocked Plm-catalyzed cleavages at Arg336 and Arg372 by ∼90% in dose-dependent manners (IC50: ∼20 nM). Surface plasmon resonance-based assays showed that the isolated K5-CD bound to factor VIII with an ∼50-fold higher affinity (Kd: 3 nM) compared to the K1-3 and K4, similar to the affinity obtained with anhydro-Plm (Kd: 4 nM). In particular, the K5-CD bound to the A2 domain with an ∼5-fold higher affinity (Kd: 42 nM) than those obtained with the K1-3 and K4. In contrast, both the K1-3 and K4 bound to the light chain predominantly (Kd: 43 and 87 nM, respectively), whilst the K5-CD failed to bind. Furthermore, the addition of a goat antibody against the CD (C-14; Santa Cruz Biotechnology) completely blocked the A2 and K5-CD interaction (by ∼95%). These findings suggest that the CD of Plm (and Plg) interacts with the factor VIII A2 domain through the LBS-independent mechanisms, whilst the K1-3 (and/or K4) interacts with the light chain through the LBS-dependent mechanisms. Furthermore, the CD and A2 interaction would regulate the activation/inactivation of factor VIII by proteolytic cleavages of Arg336 and Arg372.

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.

Factor VIII functions as a cofactor for factor IXa in the anionic phospholipid surface-dependent conversion of factor X to Xa. It is well-known that the A2 and A3 domains of factor VIII interact with the catalytic domain and EGF2 domain of factor IXa, respectively. Recently, Furie et al. have reported that the Gla domain of factor IXa (factor IXa-GD) interacts with the light chain of factor VIII. However, the factor IXa-GD-interactive site on the light chain remained to be investigated. In the current study, the recombinant C2 (rC2) domain of factor VIII was prepared using a yeast secretion system. ELISA-based assay in the absence of phospholipid showed the Glu-Gly-Arg-active site modified factor IXa (EGR-factor IXa) bound to the immobilized rC2 domain dose-dependently, and the binding ability was maximum under the condition of 150 mM NaCl/1 mM CaCl2. This binding was competitively inhibited by the addition of excess of factor VIII or rC2 domain, supporting the specificity of this interaction. Furthermore, the presence of high ionic strength and the metal-ion chelator EDTA blocked this binding by ∼95 and ∼75%, respectively. Surface plasmon resonance-based assay showed that the binding affinity (Kd) of rC2 domain for EGR-factor IXa was 108 ± 15.5 nM. GD less-factor IXa, deleting the GD completely, failed to bind to rC2 domain. A monoclonal antibody against factor IXa-GD specific for calcium-dependent conformation (mAbIXa-GD) also inhibited (∼ 95%) the rC2 domain binding to EGR-factor IXa in a dose-dependent manner (IC50; 758 nM), suggesting the authentic of the C2 domain and factor IXa-GD interaction. The addition of rC2 domain or mAbIXa-GD inhibited the factor IXa-catalyzed factor X activation with factor VIIIa in the absence of phospholipid (IC50; 15.7 μM or 43.2 nM, respectively), whilst both any little affected in the absence of factor VIIIa. In addition, the ∼8-kDa C2 fragment obtained by V8 protease digestion (residues 2182–2259) bound directly to EGR-factor IXa. Taken together, these results indicate that factor VIII C2 domain directly interacts with factor IXa-GD via both the electrostatic- and calcium-dependent interactions. Furthermore, our results provide the first evidence for an essential role of the C2 domain in the association between factor VIII and factor IXa in the factor Xase complex.

Factor VIII is inactivated by limited proteolytic cleavages at Arg336 and Lys36 by plasmin (Abst #1017 and 1018, BLOOD106, 2005). We recently identified a plasmin-interactive site in the A2 domain responsible for cleavage at Arg336, and also reported that the cleavage at Lys36 was selectively regulated by the light chain that might interact with its protease. In the current study, several approaches were employed to examine the localization of plasmin-interactive site(s) responsible for cleavage at Lys36. Rate constant of plasmin-catalyzed factor VIIIa inactivation by the addition of the 1649A3-C1-C2 (intact light chain) or 1690A3-C1-C2 fragment was reduced by ~2-folds in a dose-dependent manner using one-stage clotting assay, but not by the 1722A3-C1-C2. SDS-PAGE analysis revealed that Lys36 cleavage in A1/1722A3-C1-C2 dimer by plasmin was markedly slower than that in the A1/1649A3-C1-C2 or A1/1690A3-C1-C2 dimer. Surface plasmon resonance-based assay using anhydro-plasmin (Ah-plasmin), catalytically inactive derivative of plasmin in which the active-site serine was converted to dehydroalanine, showed that the 1722A3-C1-C2 bound to Ah-plasmin (Kd; 191 nM) with an ~3-fold lower affinity compared with 1649A3-C1-C2 or 1690A3-C1-C2 (68 or 83 nM, respectively). Recombinant A3 domain, expressed in E.Coli, also bound to Ah-plasmin with similar affinity (Kd; 44 nM), but the C2 failed to bind, suggesting the presence of a plasmin-binding site within N-terminus of the A3 domain. On the other hand, Glu-Gly-Arg-active-site modified factor IXa blocked the light chain binding to Ah-plasmin by ~60% (Ki: 10.5 nM), supporting that another plasmin-binding site juxtaposes or overlaps with a factor IXa-binding site (residues 1804–1818) in the A3 domain. The 6-aminohexanoic acid, a competitor of lysine-binding site of plasmin, blocked by >90% the light chain and Ah-plasmin interaction (Ki; 6.8 microM). Based on the major contribution of lysine-binding site(s) of plasmin for interaction with the light chain, evaluation of the A3 sequence revealed two regions involving clustered lysine residues of 1690–1705 and 1804–1818. Two synthetic peptides corresponding to these regions blocked the light chain binding to Ah-plasmin by ~50% (Ki: ~20 microM). We conclude that an extended surface centered the 1690–1705 and 1804–1818 regions within the A3 domain interacts with plasmin for the cleavage at Lys36 within the A1 domain.

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.

Factor VIII is activated by limited proteolytic cleavage at Arg372, Arg740, and Arg1689 by thrombin or factor Xa. Recently, it has been reported that the C2 domain and the acidic region comprising residues 389–394 in the A2 domain interacted with thrombin to facilitate cleavage at Arg1689 and Arg740, respectively. However, a thrombin-interactive site(s) responsible for cleavage at Arg372 remain to be determined. In the current study, several approaches were employed to examine the thrombin-interactive site(s). Thrombin-catalyzed factor VIII activation was inhibited by the addition of one anti-A2 monoclonal antibody 413 with an epitope of residues 484–509 in a dose-dependent manner using one-stage clotting assay. SDS-PAGE analysis revealed that cleavage rate at Arg372 in the heavy chain by thrombin selectively decreased by ~25% in the presence of the antibody. However, cleavage at Arg740 was not affected. Direct binding experiments by both surface plasmon resonance-based assay and ELISA using anhydro-thrombin (Ah-thrombin), catalytically inactive derivative of thrombin in which the active-site serine was converted to dehydroalanine, showed that anti-A2 antibody 413 inhibited the A2 subunit binding to Ah-thrombin. A synthetic peptide corresponding to the 484–509 residues also inhibited the A2/Ah-thrombin binding by ~40%. Mutant A2 molecules where the basic residues in the A2 domain were converted to alanine were evaluated for the binding of Ah-thrombin. Among the tested mutants, K466A mutant possessed ~330-fold lower affinity than the wild type A2 (Kd; 18400 nM, 55 nM, respectively), and the affinities of R471A, R484A, R489A, R490A, H497A, K499A, and K523A were decreased by 2–5-fold (Kd; 259, 212, 183, 163, 123, 255, and 165 nM, respectively). These findings demonstrate that clustered basic residues, in particular Lys466, encompassed the 484–509 region play a key role of thrombin-binding site within the A2 domain, responsible for thrombin-catalyzed factor VIII activation by cleavage at Arg372.

Factor VIII functions as a cofactor in the factor Xase complex responsible for phospholipid surface-dependent conversion of factor X to factor Xa by factor IXa. Factor VIIIa, activated form by thrombin and factor Xa, is down regulated by activated protein C (APC), and the reaction is enhanced by the presence of protein S, a cofactor for APC. It was previously reported that protein S inactivated directly factor Xa or factor Va, however, the direct regulation of factor VIII by protein S remains to be investigated. In the present study, surface plasmon resonance (SPR)-based assay showed that factor VIII bound directly to immobilized protein S (Kd; 70 nM). The isolated A2 and A3 domains also bound to protein S with similar modest affinity (Kd; 15 and 17 nM, respectively), whilst the isolated A1 and C2 domains failed to bind, suggesting the presence of protein S-binding sites within the A2 and A3 domain. Since it is known that factor IXa also interacts with the A2 and A3 domains in factor VIII, we examined the inhibitory effect of factor IXa on the factor VIII and protein S interaction in a SPR-based assay. Active-site modified (EGR−) factor IXa competitively inhibited the binding of protein S to both the A2 and A3-C1-C2 domains dose-dependently. Furthermore, Western blotting analysis using an anti-A1 monoclonal antibody revealed that Arg336 cleavage in factor VIII by factor IXa in the presence of protein S was slower with an ~1.8-fold lower cleavage rate than that in its absence, supporting that protein S competed the factor IXa interaction with factor VIII. Of interest, the reaction with protein S to factor VIII inhibited the generation of factor Xa dose-dependently in a factor Xa generation assay (IC50; 150 nM). The Km value for factor X obtained with factor Xase complex in the presence of physiological concentration of protein S was 19 nM, which was ~2-fold lower than that in its absence (45 nM). Whilst, the Km value for factor IXa in the presence of protein S was greater than 100 nM, which was ~5000-fold higher than that in its absence (21 pM). We demonstrate that protein S not only contributes to down-regulate factor VIIIa activity as a cofactor for APC, but also impairs the factor Xase complex by competing the binding of factor IXa to factor VIII.

Factor VIII (FVIII) is inactivated by limited proteolytic cleavage by plasmin immediately after the activation. However, the plasmin-interactive region(s) in FVIII remain to be determined. Recently, we reported that the A2 domain may interact with plasmin during FVIII inactivation by this protease (Abst #1991, BLOOD102, 2002). In the current study, several approaches were employed to examine the localization and role of plasmin-interactive region(s). Activation and inactivation rate constants of plasmin-catalyzed FVIII and FVIIIa by the addition of isolated A2 subunit were reduced by ~4 and ~13-folds, respectively, in dose-dependent manners using one-stage clotting assay. The addition of Glu-Gly-Arg active-site modified factor IXa, interacts with the A2 domain, also reduced the rate constant of FVIIIa inactivation by ~4-fold. SDS-PAGE analysis showed that an anti-A2 monoclonal antibody 413, recognizing residues 484–509 in factor IXa-interactive site, blocked the plasmin-catalyzed cleavages at Arg336, Arg372, and Arg740 in the heavy chain. Surface plasmon resonance-based assay using anhydro-plasmin, catalytically inactive derivative of plasmin in which the active-site serine was converted to dehydroalanine, showed that FVIII and isolated A2 subunit bound to anhydro-plasmin with Kd values of 4 and 21 nM, respectively. The binding assay using ELISA with immobilized anhydro-plasmin also showed the similar binding affinities. Monoclonal antibody 413 blocked the A2 subunit binding to anhydro-plasmin by ~80% (IC50: 151 nM). Furthermore, synthetic peptide with sequences 479–504 inhibited this binding by ~55% (Ki: 3 microM), however, peptide with sequences 489–514 had a very weak inhibition (by

Factor VIII (FVIII) functions as a cofactor for factor IXa in the intrinsic tenase complex. This tenase activity is down-regulated by activated protein C (APC) or factor Xa (FXa). Plasmin, the most potent fibrinolytic protease, inactivates FVIII as well as other clotting factors. However, the mechanism of FVIII inactivation by plasmin is poorly understood. FVIII activity reached to the peak value of ~2-fold increase at 3 min after the addition of plasmin in a one-stage clotting assay. Then, the activity was decreased rapidly and was undetectable within 30 min. This time-dependent reaction was not affected in the presence of von Willebrand factor and phospholipid. The activation of FVIII by plasmin was an ~50% level of that by FXa. The rate constant (min-1) of inactivation of FVIIIa by plasmin possessed ~11.3- and ~2.5-folds greater than those by FXa and APC in the presence of protein S, respectively. SDS-PAGE analysis showed that plasmin cleaved the 90~210-kDa heavy chain of FVIII to 50, 48,45, 40, and 38-kDa fragments via 90-kDa fragment. Using western blot and N-terminal sequence analyses, these fragments derived from the heavy chain were identified as A11-372, A1337-372-A2, A11-336, A2, and A137-336, respectively, by cleavages at Arg372, Arg740, Lys36 and Arg336 in the A1 domain. On the other hand, the 80-kDa light chain was cleaved to 67-kDa fragment via 70-kDa fragment by cleavages at Arg1721 and Arg1689, respectively, consistent with the pattern of cleavage by FXa. However, the cleavage at Arg336 by plasmin was much quicker than that at Arg372, contrast with that by FXa. Furthermore, this cleavage was faster than that by APC, consistent with rapid inactivation of FVIII. In addition, the cleavage at Arg336 of FVIIIa by plasmin was faster than that of isolated A1 or A1/A3-C1–C2 dimer, different with that by FXa. These results demonstrate the importance of cleavage at Arg336 for the mechanism of plasmin-catalyzed FVIII inactivation. Furthermore, this cleavage appears to be selectively modulated by the A2 domain that may interact with plasmin.

Factor VIII (FVIII) cofactor function is dependent on the presence of divalent cation, since its active form is metal-linked heterotrimer as well as factor V (FV). The activity of FVIII (FVIII:C) and FV in normal plasma were completely and selectively inactivated by the treatment with divalent cation exchange resin, imidinoacetate. However, FVIII antigen (FVIII:Ag) was preserved, suggesting that deprivation of FVIII:C by the resin was not due to absorption of FVIII but due to inactivation. Both FVIII:Ag and FVIII:C of recombinant FVIII were decreased by treatment with the resin. However, FVIII:Ag but not FVIII:C, was preserved in FVIII and von Willebrand factor complex (FVIII/VWF). Sandwich ELISA using anti-A3 and anti-A2 monoclonal antibodies revealed that association with heavy and the light chains were impaired in the resin-treated FVIII. These results suggested that FVIII was inactivated by deprivation of the metal ion by the resin and that VWF protected cation-dependent FVIII structure. Therefore, we tested if the resin deprives the metal ion such as Ca2+ from FVIII or FVIII/VWF. [Ca2+] in FVIII preparation was decreased from 1.30 to 0.07 mM by the resin. Furthermore, [Ca2+] was decreased from 0.39 to 0.01 mM in FVIII/VWF preparation. [Ca2+] was recovered completely by elution with 1 N HCl from the resins used in both preparations. FVIII:C of the resin-treated FVIII/VWF was partially recovered by addition of Ca2+, whilst FVIII:C of the resin-treated FVIII was not recovered. When FVIII was treated with the resin after addition of [Ca2+], the inactivation of FVIII was dose-dependently inhibited by ~20 % (at [Ca2+]: ~75 mM). On the other hand, the inactivation of FVIII was inhibited by ~60 % (at [Ca2+]: ~25 mM) in FVIII/VWF preparation. Present results demonstrated that FVIII was selectively inactivated by the cation exchange resin due to deprivation of Ca2+. Kinetic experiments by surface plasmon resonance using BIAcore demonstrated that the resin-treated FVIII as well as treated C2 didn’t interact with phospholipid and VWF. Furthermore, immunoblot analysis using the resin-treated FVIII revealed that anti-A2 monoclonal antibody reacted with the heavy chain, whilst anti-A3 and anti-C2 antibodies failed to react with the light chain. On the other hand, these antibodies reacted with the light chain in the experiment using FVIII/VWF, indicating that VWF protects antigenic conformation of the FVIII light chain. Present findings suggest another protection mechanism of VWF on FVIII through stabilization of Ca2+-dependent structure of the FVIII light chain.

Background: The replacement therapy with plasma-derived factor VIII (FVIII)/von Willebrand factor (VWF) concentrates is the first line treatment for the patients with type 3 von Willebrand disease (VWD). However, development of anti-VWF alloantibodies (inhibitor) is a major problem since the inhibitor neutralizes the VWF activity and may cause anaphylactic reactions. As an alternative treatment, the usage of FVIII concentrates has been reported but the mechanism of the hemostatic effects remains to be elucidated. Objectives: The purpose of this study is to address the role of FVIII in the hemostatic mechanism in the absence of VWF by in vitro and ex vivo analysis in the treatment for type 3 VWD with recombinant FVIII (rFVIII). Patient/Methods: The patient is a 55-year-old male with type 3 VWD. Blood samples were obtained before and 30 min after bolus administration. Rotating thromboelastometry (ROTEM) assay was performed to examine global interactions in hemostasis. To elucidate the effect on platelet activation, α-thrombin- and shear-induced platelet aggregation studies were performed. Further, α-thrombin-induced [Ca2+]i rise was assessed using fura2-AM loaded platelets. Results and Implications: The patient underwent two surgical procedures of multiple teeth extractions successfully with minimal bleeding by bolus administration of rFVIII (50 IU/kg) before procedure and followed by continuous infusion at rate of 10 IU/kg/h for 15 hours. FVIII:C was elevated from 1.0% to 20~30% 30 min after bolus infusion and maintained ~15% after 12 h-continuous infusion. ROTEM analysis showed that infusion of rFVIII shortened clotting time (preinfusion 2083.8±784.3 sec vs. post-infusion 1022.0±191.5 sec) and clot formation time (pre 1267.3±455.4 sec vs. post 705.8±261.8 sec) and increased α (pre 8.5±7.4 degree vs. post 23.5±4.4 degree). The α value and CFT indicate the rate of increase of elastic shear modulus. Addition of rFVIII to preinfusion blood in vitro corrected ROTEM parameters and thrombin-induced aggregation dose-dependently. Infusion of FVIII enhanced thrombin-induced platelet aggregation (% maximal aggregation: pre 26.3% vs. post 98.2%) as well as low shear-induced platelet aggregation (% maximal aggregation: pre 18% vs. post 52%). Furthermore, infusion of rFVIII meliorated thrombin-induced intracellular calcium flux of washed platelets (thrombin 10 nM, Ca flux: pre 414.0 nM vs. post 620.6 nM). Recently, the cell-based model of hemostasis provides a solid foundation for the relation between platelet and coagulation. Although coagulation initiation occurs normally via the extrinsic pathway, amplification mediated by the intrinsic pathway is seriously disturbed in type 3 VWD due to the marked decrease in FVIII. Therefore, correction of FVIII could result in the improvement of hemostasis. Our data demonstrated the effectiveness of FVIII in the surgical treatment for type 3 VWD and further suggested that FVIII molecules are incorporated into platelet phospholipids to facilitate platelet activation as well as act directly to intrinsic pathways to normalize coagulation. Conclusions: Our observations suggested that FVIII plays an essential role in hemostasis in the absence of VWF and provided the rationale for the usage of rFVIII in the hemostatic management of type 3 VWD.