ALBERT

Description: Abstract 2237 Introduction: von Willebrand disease (VWD) has clinically heterogeneous phenotype. Routine measurements of von Willebrand factor (VWF) antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo) and FVIII activity (FVIII:C) do not always reflect clinical severity, especially in type 1 VWD. These assays evaluate VWF function under non-physiological static condition - lacking blood flow. We had reported that a new microchip flow chamber system, total-thrombus-formation analysis system (T-TAS®, Fujimori Kogyo, Tokyo) would be a clinically useful flow assay for VWD (ASH 2011). In this study, we extended this study for application to evaluation and hemostatic monitoring for type 1 VWD. Methods: Citrated or hirudin-added blood from 15 patients with type 1 VWD was utilized. Re-calcified citrated blood added corn trypsin inhibitor was injected to a microchip in T-TAS at a constant flow rate (240 s−1), which flow surface was coated by collagen and tissue factor (AR chip). Hirudin-added blood was injected to a microchip in T-TAS at higher shear rate (1,000 s−1) which surface was coated by collagen (PL chip). Flow pressure curve was visualized and time until reach to 10 kPa (T10) was evaluated. AR chip promoted thrombus formation by both platelet aggregation and fibrin generation, whilst PL chip promoted thrombus formation by platelet alone. Standard laboratory tests for VWD were also performed. Clinical severities of VWD patients were evaluated by using a quantitative bleeding score (BS, from −3 without any symptoms to +45 with all major symptoms) previously reported by Tosetto (JTH, 2006). Results: Fifteen patients with type 1 VWD showed low levels of VWF:Ag [median 14% (range 1.3–51%)], VWF:RCo [8% (1.6–32%)], and FVIII:C [31% (3.0–68%)]. T10 in AR chip or PL chip was 17.7 min (11-〉30) or 7.1 min (3.3-〉10) [normal control (n=20); 12.2 min (8.6–16.6) or 3.5 min (2.4–6.6), respectively], showing delayed thrombus formation in type 1 VWD. Correlations between VWF:Ag and VWF:RCo (r2=0.80, p

Description: Introduction: Emicizumab (Emi) is an anti-factor (F)IXa/FX bispecific antibody that mimics FVIIIa cofactor function. The clinical trials on Emi prophylaxis for hemophilia A patients with inhibitor (HA-Inh) demonstrated marked reduction in bleeding rates. However, Emi at clinically relevant plasma concentrations (~50 mg/ml) corresponding to ~15% of FVIII equivalent activity may not provide enough hemostasis at severe bleeding or major surgical intervention. In these circumstances, additional infusions of bypassing agents (BPAs) may be needed for hemostatic management. In the HAVEN 1 study, thrombotic events/ thrombotic microangiopathy occurred in Emi-treated HA-Inh receiving consecutive infusions of aPCC in higher doses. After these reports, guidance was provided to avoid the use of aPCC as first choice during Emi prophylaxis. If aPCC is the only available BPA, use of the lowest dose expected to achieve hemostasis with the initial dose ≦50 U/kg was recommended. To further support these recommendations, more investigation is required for the use of BPAs under Emi prophylaxis. Objective: We examined the coagulation effects by spiking BPAs to whole blood or plasma samples from Emi-treated HA. Methods: Samples from eight Emi-treated HA-Inh and two hemophilia A patients without inhibitors (HA-Inh(-)) in phase 1/2 and HAVEN 1 were tested. The Emi doses were 0.3-3.0 mg/kg/w or 1.5 mg/kg/w, respectively. Either aPCC or rFVIIa was spiked to patients' whole bloods or plasmas, and assessed by rotational thromboelastometry (ROTEM; Ca2+ trigger) and clot waveform analysis (CWA; ellagic acid/tissue factor-mixed trigger; Nogami, JTH2018). Furthermore, samples in Emi-treated HA-Inh of phase 1/2 (n=1) or HAVEN 2 (n=2) receiving BPAs infusion for hemostatic management were assessed by both assays. Results and Discussion: The baseline levels, before spiking BPAs, in ROTEM parameters, such as clot time (CT) and clot formation time (CFT), of Emi-treated HA were 1,380/521sec(s) (reference range 762-1,127/207-511s), respectively. Spiking aPCC markedly shortened CT/CFT to 167/81.5s and 148/78.0s at 0.65 and 1.3 U/mL corresponding to 50 and 100 U/kg infusion, respectively. Even at 0.13 U/mL (10 U/kg), CT/CFT shortened to 290/92.6s, which was shorter than that of reference range. Its effect was dose-dependent. Spiking rFVIIa also improved CT/CFT to 726/179s, 551/141s at 32.3 and 112 μg/mL (90 and 270 μg/kg). In CWA, the baseline levels of adjusted-|min1| (ad|min1|), indicative of maximum coagulation velocity, in Emi-treated HA were 5.53 (normal control 7.98±0.24). Ad|min1| was improved to 6.48 and 8.04 by spiking aPCC (10 and 100 U/kg), or 7.11 and 7.12 by spiking rFVIIa (90 and 270 μg/kg), respectively. For rFVIIa its effect was not dose-dependent. According to the investigation for in vivo effect by BPAs infusion in a total of 9 treatment events of Emi-treated HA-Inh, CT/CFT (1,812/553s) was improved to 1,151/346s, of which was within normal range. Ad|min1| (5.7) was improved to 6.6 30 min(m) after aPCC infusion (44-74 U/kg). By rFVIIa infusion (90-119 μg/kg), CT/CFT (1,962/758s) improved to 912/207s, and ad|min1| (4.9) also improved to 6.5 30m after infusion, of which improvement remained within normal range. The hemostasis effects were clinically satisfying and no thrombosis occurred in all cases. The ROTEM parameters by spiking aPCC were apparently hypercoagulant relative to those by infusion at same dose, supporting our previous report (Furukawa JTH2015). The shortening effect on the ROTEM parameters by spiking aPCC 100 U/kg in Emi-untreated HA seemed to be similar to that by spiking aPCC 10 U/kg in Emi-treated HA. In addition, ad|min1| by spiking aPCC 10 U/kg with Emi also improved as well as ~100 U/kg without Emi in our previous report (Nogami JTH2018). Taken together, it is suggested that the hemostatic effect of aPCC infusion (10 U/kg) to Emi-treated HA could be comparable to that of 100 U/kg to Emi-untreated HA-Inh, though there is a limitation to predicting the hemostatic effect of aPCC in a clinical setting from these ex vivo results. Conclusion: Spiking tests in Emi-treated HA-Inh by ROTEM and CWA are useful to evaluate clinical coagulation potentials and dose finding of BPAs in Emi-treated HA receiving BPAs. Additionally, under Emi prophylaxis, a lower dose of aPCC infusion might be a possible option to treat breakthrough bleed in Emi-treated HA-Inh. Disclosures Furukawa: CSL Behring: Research Funding. Nogami:Bayer: Consultancy, Honoraria, Research Funding; Shire: Consultancy, Honoraria, Research Funding; Novo Nordisk: Consultancy, Honoraria, Research Funding; Sysmex: Consultancy, Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Anti-FIXa/X bispecific antibodies, Research Funding, Speakers Bureau. Matsumoto:Shire Japan Co. Ltd: Research Funding. Kasai:Chugai Pharmaceutical Co., Ltd: Employment. Shima:F. Hoffmann-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees; Chugai Pharmaceutical Co., Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Anti-FIXa/X bispecific antibodies , Research Funding, Speakers Bureau.

Description: We have experienced a case of acquired hemophilia A with inhibitor recognizing only a factor (F) VIII A2 epitope, and reported the inhibitory mechanism for disappearing FVIII activity (Blood, 124, 4226, 2014). In summary, the patient's inhibitor IgG bound to FVIII A2N (residue 372-562) fragment and inhibited Arg372 cleavage in FVIII by FXa, suggesting that FX(a) bound to FVIII A2 domain. ELISA-based assay showed that FVIII A2 fragment bound to FX (Kd; 338 nM). We hypothesized that FVIII A2 residues 400-429 might be FX binding site according to the 3-D model of FVIII molecule, and prepared synthetic peptides (400-409, 409-419, and 420-429). The 400-409 peptide inhibited the FVIII A2-FX interaction, suggesting that the 400-409 region contributed to FX-interactive site. In this current study, we further performed the localization of a FX-interactive site on the 400-409 region in the A2 domain. A purified FXa generation assay demonstrated the 400-409 peptide decreased the generation of FXa in a dose-dependent manner up to 38% of 100 μM (Ki; 23 ± 9 nM). In comparison, scrambled peptide of 400-409 decreased up to 10% of 100 μM. These data demonstrated that the 400-409 peptide inhibited the generated FXa, suggesting the 400-409 region contributed to regulate the coagulation function. Covalent cross-linking was observed between the biotinylated 400-409 peptide and FX following reaction with EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) using SDS-PAGE. This cross-linking formation was blocked by the addition of unlabeled 400-409 peptide. N-terminal sequence analysis of the peptide-FX product demonstrated that two sequential residues (Lys408 and Ser409) could not be detected, supporting that two residues participate in cross-link formation. To confirm the significance of these residues in A2 domain for FX-binding, the mutant forms of the A2 domain, converted to alanine, were expressed in BHK system and purified. Compared with wild type FVIII (Kd; 10 ± 3 nM), the binding affinity of Ser409Ala FVIII mutant for FX was no significant difference (Kd; 14 ± 1 nM) on SPR-based assay. Lys408Ala or Lys408Ala/Ser409Ala double FVIII mutant, however, decreased the binding affinity by 3.6~4.3-fold (Kd; 36 ± 7 or 43 ± 2 nM, respectively), suggesting contribution of Lys408Ala to the binding interaction. For the functional evaluation of the association with FVIII mutants to FX, a FXa generation assay was repeated. Lys408Ala, Ser409Ala, or Lys408Ala/Ser409Ala FVIII mutant reacted with varying concentrations of FX decreased by 1.2~1.6-fold (Km; 53 ± 12, 69 ± 15, or 65 ± 15 nM, respectively) compared to wild type FVIII (Km; 43 ± 9 nM), supporting a contribution of these mutants to Km and overall catalytic efficiency. Vmax values were largely unaffected by the mutations with most values within approximately 30% of the wild-type value. On the other hand, Kcat/Km value of Lys408Ala, Ser409Ala, or Lys408Ala/Ser409Ala FVIII mutant were decreased by 0.5~0.7-fold (Kcat/Km; 1.0, 1.3, or 0.9 nM-1min-1, respectively) compared to wild type FVIII (Kcat/Km; 1.8 nM-1min-1), suggesting low catalytic efficacy of Lys408Ala and Ser409Ala. These results indicate that the 400-409 region in the FVIII A2 domain, and in particular Lys408 and Ser409, may contribute to a unique FX-interactive site. Disclosures Nogami: Chugai Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sysmex Corporation: Patents & Royalties, Research Funding. Shima:Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.