Presence Of Factor VIII Inhibitors Research Articles

Association between interleukin-2 (rs2069762) gene polymorphism and FVIII inhibitor development in Indonesian patients with severe hemophilia A

BACKGROUND Factor VIII (FVIII) inhibitors in hemophilia A (HA) patients render FVIII replacement therapy ineffective. Although its development cause is unclear, it has been classified into therapeutic and genetic-related etiologies. Single nucleotide polymorphisms (SNPs) in several cytokine genes, including interleukin (IL)-2, could increase the risk of FVIII inhibitor development. This study aimed to evaluate the association between IL-2 (rs2069762) gene SNP and FVIII inhibitor development in Indonesian patients with severe HA.
 METHODS The IL-2 (rs2069762) gene SNP was examined in 119 HA patients. The presence of FVIII inhibitors was quantified using an enzyme-linked immunosorbent assay, with a titer of <0.28 ng/ml considered negative. Patients were divided into two groups: 59 with FVIII inhibitors (positive group) and 60 without inhibitors (negative group). The genotype of the subjects was determined using peripheral blood mononuclear cells and tetra-primer amplification refractory mutation system-polymerase chain reaction.
 RESULTS There was no association between IL-2 (rs2069762) gene polymorphism and FVIII inhibitor development on genotypes (p = 0.138) and allele frequencies (p = 0.780).
 CONCLUSIONS IL-2 (rs2069762) gene polymorphism is not a risk factor in the development of FVIII inhibitors in Indonesian patients with severe HA. Thus, further polymorphism studies in other cytokine genes are required to gain a comprehensive understanding of the FVIII inhibitor development.

Recombinant porcine factor VIII corrects thrombin generation in vitro in plasma from patients with congenital hemophilia A and inhibitors

BackgroundNeutralizing factor VIII (FVIII) antibodies are a major complication in hemophilia A. Antihemophilic factor VIII (recombinant), porcine sequence (rpFVIII; susoctocog alfa; Baxalta US Inc., a Takeda company) has low cross‐reactivity to anti‐human FVIII antibodies and can provide functional FVIII activity in the presence of FVIII inhibitors. ObjectivesEvaluate in vitro thrombin generation and clot formation responses to rpFVIII in blood from patients with congenital hemophilia A. MethodsIn this multicenter study, blood was obtained for in vitro analyses that included human and porcine FVIII inhibitors, low <5 Bethesda units (BU)/ml or high ≥5 BU/ml titer (Nijmegen‐modified Bethesda assay); thrombin generation assay (TGA), clot viscoelasticity (thromboelastography), fibrin clot structure analysis (scanning electron microscopy), and epitope mapping. ResultsBlood samples were from 20 patients with congenital hemophilia A (FVIII activity <1%, mean [range] inhibitor titers: anti‐human FVIII, 14 [1–427] BU/ml [n = 13 high, n = 6 low, n = 1 data unavailable]); anti‐porcine FVIII, 12 (0–886) BU/ml (n = 11 high, n = 8 low, n = 1 data unavailable). Porcine inhibitor titer and TGA response measured by endogenous thrombin potential showed an inverse correlation (2.7–10.8 U/ml rpFVIII Spearman correlation coefficient: −0.594 to −0.773; p< 0.01). Clot structures in low anti‐porcine inhibitor titer plasmas were similar to those in noninhibitor plasma. ConclusionsRecombinant porcine factor VIII demonstrated a dose‐dependent correction of thrombin generation and clot formation in vitro, dependent on the anti‐porcine FVIII inhibitor titer. Procoagulant responses to rpFVIII occurred in plasma containing FVIII inhibitors.

In vitro effects of emicizumab on activated clotting time in blood samples from cardiac surgical patients.

Heparin management in hemophilia A (HA) patients with a factor VIII (FVIII) inhibitor can be challenging due to severe activated clotting time (ACT) prolongations. It is important to better understand the impact of emicizumab, a FVIII mimetic on ACT, and tissue factor (TF)-based coagulation assays. Whole blood from 18 patients undergoing cardiopulmonary bypass (CPB) were mixed in vitro with pooled normal plasma, FVIII-deficient or FVIII-inhibitor plasma to affect functional FVIII levels. ACTs and heparin concentration by protamine titration were measured in whole blood mixture with/without emicizumab (50-100μg/ml). Thrombin generation and plasmin generation were measured in the patient's plasma mixed with normal plasma or FVIII-inhibitor plasma to assess the impact of emicizumab under low TF activation. FVIII inhibitors prolonged ACTs by 2.2-fold compared to those in normal plasma mixture at baseline. During CPB, ACTs in normal plasma mixture, and FVIII-deficient mixture were in 400s, but ACTs reached 900s in FVIII-inhibitor mixture. Emicizumab shortened ACTs by up to 100s in normal plasma mixture, and FVIII-deficient mixtures. ACTs remained over 600s in FVIII-inhibitor mixture, despite adding emicizumab at 100μg/ml. Heparin concentration measured by TF-based protamine titration was unaffected. Emicizumab enhanced thrombin peak in the presence of FVIII inhibitors, whereas plasmin generation was mainly affected by thrombin generation, and systemic use of ɛ-aminocaproic acid. FVIII inhibitors extensively prolong ACTs in heparinized whole blood, and clinical levels of emicizumab partially reverse ACT values. Protamine titration should be considered for optimal heparin monitoring in emicizumab-treated patients with FVIII inhibitors.

Congenital hemophilia A with low activity of factor XII: a case report and literature review

BackgroundCongenital hemophilia A is a recessive inherited hemorrhagic disorder. According to the activity of functional coagulation factors, the severity of hemophilia A is divided into three levels: mild, moderate and severe. The first bleeding episode in severe and moderate congenital hemophilia A occurs mostly in early childhood and mainly involves soft tissue and joint bleeds. At present, there are limited reports on severe congenital hemophilia A with low factor XII (FXII) activity during the neonatal period.Case presentationA 13-day-old neonate was admitted to the hospital with hematoma near the joints of both upper arms. Coagulation tests showed he had low activity of factor VIII (FVIII) and FXII. He was diagnosed with congenital hemophilia A and treated with human coagulation factor VIII (recombinant FVIII). Although the hematoma became smaller, FVIII activity was only increased to a certain extent and FXII activity decreased gradually. Unfortunately, the child responded poorly to recombinant human coagulation factor VIII and his guardian rejected prophylactic inhibitors and genetic testing and refused further treatment. Three months later, the child developed intracranial hemorrhage (ICH) due to low FVIII activity.ConclusionsIn hemophilia A, the presence of FVIII inhibitors, drug concentration and testing are three important aspects that must be considered when FVIII activity does not reach the desired level. Early positive disease treatment and prophylaxis can decrease the frequency of bleeding and improve quality of life. We recommend that pregnant women with a family history of hemophilia A undergo early prenatal and neonatal genetic testing.

Anti-FVIII Antibody and Inhibitor Development Patterns in Hemophilia A Mouse Models: A Systematic Review

Background: Management of hemophilia A with exogenous factor VIII (FVIII) infusions can result in the development of alloantibodies that neutralize FVIII activity (inhibitors). The presence of FVIII inhibitors is associated with high morbidity and a decreased quality of life. Hemophilia A mouse models are a valuable tool for studying inhibitor development and hemophilia A. These models have been shown to develop inhibitor upon treatment with human FVIII. However, there are numerous hemophilia A mouse models, each with potentially varying patterns of inhibitor development. Objective: To identify existing hemophilia A mouse models and characterize anti-FVIII antibody and inhibitor development patterns. Methods: A systematic literature review was conducted using Ovid MEDLINE and EMBASE. The Databases were searched from inception through May 2019 for full-length publications, published abstracts, and conference proceedings that reported use of mouse models of hemophilia A to investigate total anti-FVIII antibody levels and/or inhibitor titers. The search strategy used the categorical search terms: "hemophilia A" AND "mouse" AND "animal model" AND "inhibitor" (Figure 1). Results: Our review consisted of a total of 88 studies which reported the use of 11 different hemophilia A mouse models. Out of the 88 studies, 19 studies only reported total anti-FVIII antibody data without reporting inhibitor data. In 30% of the studies that reported both total anti-FVIII antibody and inhibitor data, not all mice that developed anti-FVIII antibodies had positive inhibitor titres. Our systematic review identified the E16 - C57BL/6 model as the most frequently used model, as 27 studies (31%) reported using this model. The E16 - C57BL/6, E16 - Balb/c, E16 - C57BL6/129, E17 - C57BL/6, E17 - C57BL6/129, and FVIII total knockout (TKO) models demonstrate high immunogenicity to exogenous FVIII and were used in 65% of the studies. These mice all consistently develop anti-FVIII antibodies and inhibitors at &gt;95% incidence following treatment with at least 4 doses of full-length recombinant human FVIII (rhFVIII). The E16 - R593C model and the E17 - HLA-DRB1*1501 model both display anti-FVIII antibody and inhibitor development patterns that are representative of the human clinical scenario (~30% inhibitor development incidence). The only model completely immunotolerant to rhFVIII is the E17 - subline E model generated by van Helden et al. (2011) which is a transgenic mouse line on the hemophilic E17 background that expresses human full-length F8 cDNA. Treatment with 8 weekly doses of native human FVIII resulted in no anti-FVIII antibody or inhibitor development in this model. Conclusions: This systematic review demonstrated that mice of the most frequently used models almost always develop inhibitors when treated with full-length rhFVIII. Humanized models may be ideal for use in studies that aim to replicate human inhibitor development patterns. In addition, immunotolerant models may be best used for in vivo studies in hemophilia A that aim to avoid both antibody and inhibitor development. Lastly, this systematic review emphasises that it is important to report both anti-FVIII antibodies and neutralizing activity in Bethesda Units since in 30% of the studies that reported both values, the incidence of inhibitor development was less than the incidence of anti-FVIII antibody development in mice. Disclosures Matino: Bioviiix: Honoraria; Roche: Research Funding; Sanofi: Honoraria; Sobi: Honoraria, Research Funding; Bayer: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding.

Evaluation of a Blood Coagulation Factor IX Variant That Functions Independently of Factor VIII As an Alternative Treatment for Hemophilia A

The serine protease factor IXa (FIXa) serves an important role in coagulation by catalyzing the proteolytic activation of factor X (FX) together with its cofactor VIIIa (FVIIIa). Being a critical protease in coagulation, the FIXa structure has evolved to be subjected to strict regulatory mechanisms. While FIXa displays considerable structural homology with other coagulation serine proteases, its active site is uniquely controlled by the 99-loop that blocks access to the active site pocket. Cofactor-mediated interaction of FIXa with its substrate FX induces a conformational change that allows for active site engagement and substrate catalysis. Previously, the molecular constraints of the 99-loop were lifted due to specific modifications in both the 99-loop (K265A), the S1 active site subpocket (V181I, I383V), and the L6F substitution, thereby generating FIX-FIAV [Quade-Lyssy et al. J. Thromb. Haemost. 2014]. As a result, this variant is capable of functioning independently of factor VIII (FVIII). Moreover, FIX-FIAV was demonstrated to ameliorate the hemophilia A phenotype both in vitro and in vivo. To further evaluate its therapeutic potential, FIX-FIAV was stably expressed in HEK293 cells and purified by ion-exchange and hydrophobic interaction chromatography. Evaluation of the kinetics of tissue factor-factor VIIa (TF-FVIIa) activation of FIX-FIAV revealed kinetic parameters similar to those of human wild-type FIX(-WT). Analysis of FIX activation intermediates that are formed upon proteolysis by TF-FVIIa or factor XIa demonstrated prolonged formation of FIX-FIAVα, while no FIXa-WTα could be observed. This is consistent with delayed cleavage at position 180, likely resulting from the V181I substitution in FIX-FIAV. Given that the activation mechanism of FIX-FIAV is unperturbed, we next assessed the specific FVIII clotting activity and demonstrated that FIX-FIAV exhibited significant FVIII-like clotting activity (56 ± 4 U/mg) as opposed to FIX-WT (&lt;13 U/mg). These values correlate with up to 28% of FVIII-independent activity for FIX-FIAV at FIX plasma levels (5 ug/mL), confirming that FIX-FIAV has the potential to enhance thrombin generation in FVIII deficiency. To validate this, tissue factor-initiated (0.5 or 1.0 pM) thrombin generation was assessed in FVIII-immunodepleted plasma, leading to a severely reduced thrombin peak (88% or 81% reduction, respectively) relative to conditions with 100% FVIII. Addition of FIX-FIAV (5 ug/mL) partially restored thrombin generation, demonstrated by an up to ~30% increase in both thrombin peak and endogenous thrombin potential. Evaluation of the FVIII-independent activity of FIX-FIAV in severe hemophilia A patient plasma with or without an inhibitor resulted in an up to 18% or 32% FVIII-like activity, respectively, demonstrating efficacy of FIX-FIAV in the presence of FVIII inhibitors. Although unlikely, it remains to be determined whether specific FVIII-inhibitors may impact FIX-FIAV function. Adding 100% FVIII or low- to mid-range therapeutic concentrations of the bispecific antibody emicizumab to FVIII-deficient plasma incubations with FIX-FIAV resulted in a synergistic enhancement of thrombin generation, demonstrated by a 9-fold increase in thrombin peak. This is consistent with the previously demonstrated hyperactivity of FIX-FIAV in a cofactor-dependent system. In contrast, no synergistic effect on thrombin generation was observed when combining FIX-FIAV with physiologically relevant concentrations of FEIBA or NovoSeven. Summarizing, FIX-FIAV is characterized by a preserved mechanism of activation in addition to being capable of sustaining therapeutic levels of coagulation activity in FVIII deficiency. This provides support for the use of FIX-FIAV as an alternative treatment for hemophilia A. Disclosures Strijbis: uniQure Biopharma B.V.: Research Funding. Konstantinova:uniQure Biopharma B.V.: Employment. Liu:uniQure Biopharma B.V.: Employment. van Deventer:uniQure Biopharma B.V.: Employment. Bos:uniQure Biopharma B.V.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Evaluation of Global Hemostatic Assays in Response to Factor VIII Inhibitors.

Global hemostatic assays including thromboelastography (TEG), Innovance ETP (endogenous thrombin potential), and Thrombinoscope could measure thrombin generation potential and be useful to guide management of patients with factor VIII (FVIII) inhibitors. However, the performance characteristics of these global assays in the presence of FVIII inhibitors are incompletely characterized. In this study, the normal range of thrombin generation potential was measured in 20 healthy individuals by all 3 assays. In 5 commercial and 7 clinical samples with FVIII inhibitors, it was shown that PPP-reagent thrombinoscope shows a dose-dependent response to different levels of FVIII inhibitors from the same patients, while Innovance ETP shows virtually no response to FVIII inhibitors. The TEG is more sensitive to FVIII inhibitors than thrombinoscope. Importantly, we show the same levels of FVIII inhibitor from different patients results in different levels of inhibition for thrombin generation potential by thrombinoscope, which potentially explains the phenotypic heterogeneity of patients with FVIII inhibitors. Global assays such as thrombinoscope, but not Innovance ETP, show appropriate sensitivity to FVIII inhibitors that could offer an objective and clinically relevant marker to guide patient management.

Effects of pre-analytical heat treatment in factor VIII (FVIII) inhibitor assays on FVIII antibody levels.

The use of pre-analytical heat treatment (PHT) with the Nijmegen-Bethesda assay (NBA) for inhibitors to factor VIII (FVIII) can remove/destroy infused or endogenous FVIII from patient plasma samples, allowing testing of recently infused patients with haemophilia. Two PHT methods have been described as follows: heating to 56°C for 30minutes and heating to 58°C for 90minutes. Data examining the effects of PHT on anti-FVIII IgG4 , the antibodies known to correlate most closely with the presence of FVIII inhibitors, are limited. To assess the effect of PHT on the levels of detectable anti-FVIII IgG4 . Nijmegen-Bethesda assay-positive specimens were incubated at 56, 58 or 60°C for 90minutes, and anti-FVIII IgG4 was measured by fluorescence immunoassay (FLI) at 30-minute intervals. The effects of PHT on the ability of recombinant FVIII (rFVIII) to inhibit detection of patient antibodies by FLI was also examined to assess the stability of rFVIII under the various PHT conditions tested. Levels of anti-FVIII IgG4 showed little change following incubations at 56°C (mean 101% of original value at 30minutes and 100% at 60minutes) but decreased upon exposure to 58°C (mean 85% at 30minutes and 66% at 60minutes). In addition, heating to 56°C effectively decreased the ability of rFVIII to block antibody binding compared to unheated rFVIII. The optimal temperature for PHT in the FVIII NBA is 56°C. Higher temperatures may lead to loss of inhibitory antibodies.

Neutralisation of factor VIII inhibitors by anti-idiotypes isolated from phage-displayed libraries.

Following replacement therapy with coagulation factor VIII (FVIII), up to 30 % of haemophilia A patients develop FVIII-specific inhibitory antibodies (FVIII inhibitors). Immune tolerance induction (ITI) is not always successful, resulting in a need for alternative treatments for FVIII inhibitor-positive patients. As tolerance induction in the course of ITI appears to involve the formation of anti-idiotypes specific for anti-FVIII antibodies, such anti-idiotypes might be used to restore haemostasis in haemophilia A patients with FVIII inhibitors. We isolated anti-idiotypic antibody fragments (scFvs) binding to murine FVIII inhibitors 2-76 and 2-77 from phage-displayed libraries. FVIII inhibitor/anti-idiotype interactions were very specific as no cross-reactivity with other FVIII inhibitors or isotype controls was observed. ScFvs blocked binding of FVIII inhibitors to FVIII and neutralised their cognate inhibitors in vitro and a monoclonal mouse model. In addition, scFv JkH5 specific for FVIII inhibitor 2-76 stained 2-76-producing hybridoma cells. JkH5 residues R52 and Y226, located in complementary determining regions, were identified as crucial for the JkH5/2-76 interaction using JkH5 alanine mutants. SPR spectroscopy revealed that JkH5 interacts with FVIII inhibitor 2-76 with nanomolar affinity. Thus, FVIII inhibitor-specific, high-affinity anti-idiotypes can be isolated from phage-displayed libraries and neutralise their respective inhibitors. Furthermore, we show that anti-idiotypic scFvs might be utilised to specifically target inhibitor-specific B cells. Hence, a pool of anti-idiotypes could enable the reestablishment of haemostasis in the presence of FVIII inhibitors in patients or even allow the depletion of inhibitors by targeting inhibitor-specific B cell populations.

A Shot in the Bone Corrects a Genetic Disease

A Shot in the Bone Corrects a Genetic Disease

Is some better than none: are TEG and TGA profiles different in severe FVIII-deficient patients with inhibitors?

Severe factor VIII (FVIII)-deficient patients with and without FVIII inhibitors cannot be distinguished using FVIII levels. The FVIII assay is sensitive to detect factor levels below 1%. While severe FVIII-deficient, non-inhibitor patients have FVIII<1%, they may retain unmeasurable residual factor activity. In contrast, inhibitor patients have a FVIII antibody that presumably fully eliminates FVIII activity. It is unknown whether thromboelastography (TEG) and thrombin generation assay (TGA) can differentiate between patients with FVIII<1% with and without the presence of FVIII inhibitors. The primary objective was to discern whether TEG and TGA could differentiate between severe FVIII-deficient patients with and without the presence of FVIII inhibitors. A secondary objective was to correlate TEG and TGA to annualized bleeding rates. This observational study performed TEG and TGA in healthy volunteers (N=15), severe FVIII-deficient (N=15) and severe FVIII-deficient patients with inhibitors (N=15). Kaolin-activated TEG was better at differentiating reaction time (31.3 vs. 120min respectively, P=0.004) and kinetics time (6.1 vs. 23.1min respectively, P=0.028) between the non-inhibitor and inhibitor patients. TEG activated by tissue factor in plasma-containing corn trypsin inhibitor failed to differentiate groups. The TGA failed to differentiate peak thrombin, endogenous thrombin potential and lag time between groups. There was no correlation between TEG and TGA with annualized bleeding rates. Kaolin-activated TEG, but not TGA, differentiated between severe FVIII-deficient patients with and without inhibitors. These assays did not find a correlation to annualized bleeding rate.

Genetic changes in severe haemophilia A

Haemophilia A is an X-linked bleeding disorder caused by reduced or absent clotting factor VIII (FVIII) activity, determined by heterogeneous mutations in the F8 gene. Identification of these pathogenic mutations is important for genetic counseling and the assessment of clinical manifestations. Although more than 700 mutations of the F8 gene have been reported as responsible for severe haemophilia (FVIII: C<1%), the corresponding data is currently insufficient for southern Brazilian populations, and world reviews concerning these changes are scarce. Thirty-six unrelated severe haemophilia A patients who showed negative results for introns 22 and 1 inversions were studied for gross exon deletions and mutations there and in adjacent regions. Missense mutations were examined using molecular structural methods. The presence of FVIII inhibitors was also investigated. The results were compared with the information available from respectively 2878 and 1952 patients from all over the world. Twenty-nine different genetic changes were found, 16 of them novel. Seventeen of the carriers developed FVIII inhibitors, and molecular analysis suggested that Asp542Gly and Ser109Pro may interfere with calcium binding, whereas Leu2297Arg clearly affects the molecule's electrostatic surface. The main aetiological factor in the severe form of haemophilia seems to be missense mutations. Of all genetic changes occurring in these patients, large deletions are the most important in inhibitors formation.

Identification and Multidimensional Optimization of an Asymmetric Bispecific IgG Antibody Mimicking the Function of Factor VIII Cofactor Activity

In hemophilia A, routine prophylaxis with exogenous factor VIII (FVIII) requires frequent intravenous injections and can lead to the development of anti-FVIII alloantibodies (FVIII inhibitors). To overcome these drawbacks, we screened asymmetric bispecific IgG antibodies to factor IXa (FIXa) and factor X (FX), mimicking the FVIII cofactor function. Since the therapeutic potential of the lead bispecific antibody was marginal, FVIII-mimetic activity was improved by modifying its binding properties to FIXa and FX, and the pharmacokinetics was improved by engineering the charge properties of the variable region. Difficulties in manufacturing the bispecific antibody were overcome by identifying a common light chain for the anti-FIXa and anti-FX heavy chains through framework/complementarity determining region shuffling, and by pI engineering of the two heavy chains to facilitate ion exchange chromatographic purification of the bispecific antibody from the mixture of byproducts. Engineering to overcome low solubility and deamidation was also performed. The multidimensionally optimized bispecific antibody hBS910 exhibited potent FVIII-mimetic activity in human FVIII-deficient plasma, and had a half-life of 3 weeks and high subcutaneous bioavailability in cynomolgus monkeys. Importantly, the activity of hBS910 was not affected by FVIII inhibitors, while anti-hBS910 antibodies did not inhibit FVIII activity, allowing the use of hBS910 without considering the development or presence of FVIII inhibitors. Furthermore, hBS910 could be purified on a large manufacturing scale and formulated into a subcutaneously injectable liquid formulation for clinical use. These features of hBS910 enable routine prophylaxis by subcutaneous delivery at a long dosing interval without considering the development or presence of FVIII inhibitors. We expect that hBS910 (investigational drug name: ACE910) will provide significant benefit for severe hemophilia A patients.

Acquired hemophilia complicated by cardiorenal syndrome type 3

Development of autoantibodies against coagulation factor VIII (FVIII) leads to a rare condition defined as acquired hemophilia (AH). If not diagnosed and treated early, AH may be associated with high mortality and morbidity. A 65-year-old woman presented with history of macrohematuria, acute renal failure, cardiogenic shock, and acute respiratory failure. Blood investigation revealed azotemia, prolonged activated partial thromboplastin time (aPTT), coagulation FVIII level of <1%, and presence of FVIII inhibitor. Echocardiography showed global hypokinesia and ultrasonography and computed tomography (CT) revealed bilateral hydroureteronephrosis. The final diagnosis was acquired hemophilia A, complicated by acute obstructive renal failure and cardiorenal syndrome (CRS) type 3. Patient was managed with mechanical ventilation, heparin-free hemodialysis, negative fluid balance, recombinant activated factor VII, and prednisolone. Hematuria was relieved, renal function improved, and cardiac function showed improvement on repeat echocardiography. Patient was discharged on prednisolone with subsequent follow ups.

Hemostatic Effect of a Novel Bispecific Antibody (ACE910) Against Activated Factor IX and Factor X in an Acquired Hemophilia A Model

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

Cerebellar hemorrhage as a first presentation of acquired Hemophilia A

Acquired hemophilia A (AHA) is an uncommon coagulation disorder caused by the development of autoantibodies against coagulation factor VIII (FVIII). While intracranial hemorrhage is a known complication of AHA, intracranial hemorrhage as the presenting manifestation of AHA has only been described in three previous case reports. We report a case of an 86-year-old woman with no previously reported history of coagulopathy presenting with an acute intraparenchymal cerebellar hemorrhage and laboratory studies demonstrating an isolated prolonged activated partial thromboplastin time (aPTT). We discuss an approach to the prolonged aPTT, and review the literature concerning the diagnosis and treatment of AHA. Occipital decompressive craniectomy with evacuation of the hemorrhage was performed. Eight hours following the procedure, the patient's status acutely declined with demonstration of a reoccurrence of the cerebellar hemorrhage and new right frontal lobe hemorrhage. After discussion with the patient's family, life-sustaining support measures were withdrawn. Postmortem analysis revealed a low FVIII activity level and the presence of FVIII inhibitor. The presentation of intracranial hemorrhage with an isolated prolonged aPTT is concerning for an acquired hemophilia with FVIII deficiency. Other causes of isolated prolonged aPTT such as a lupus anticoagulant must also be considered. Preoperative identification and work-up of the coagulation abnormality is essential to guide initial treatment.

Analysis of cytokine genes polymorphism as markers for inhibitor development in haemophilia A

Antibodies that block factor VIII (FVIII) activity appear in some haemophilia A patients treated with FVIII replacement therapy and severely impaired treatment. To date, the mechanisms that lead to this immune response are unknown. In this work, haplotypes of cytokine interleukin 10 (IL-10) gene have been associated with the presence of FVIII inhibitors in a group of Brazilian haemophilia A patients. The coexistence of a haplotype defining high IL-10 synthesis and one defining an intermediate production of cytokines is found to be associated with the group of patients who have a history of inhibitor development. Additionally, the coexistence of haplotypes defining high and low IL-10 syntheses is strongly associated with the group of negative inhibitors. These results have shown that the simple association considering only the presence or the absence of a haplotype and the development of inhibitors in haemophilia A is not sufficient. Data obtained in this work sustain the idea that the genetic studies may partly explain why only approximately 25% of haemophilia A patients develop FVIII inhibitors. Other genetic issues and details of the protein replacement therapy should be considered to measure the chances of a patient to develop anti-FVIII antibodies.

Platelet-Derived Factor VIII (FVIII) Corrects the Murine Hemophilia A Phenotype Even in the Presence of FVIII Inhibitors.

Inhibitory antibody development is not only the most severe and important complication of FVIII infusion in hemophilia A patients, but also a major potential concern in patients following hemophilia A gene therapy. We have developed a transgenic model in which human B-domain deleted FVIII (hFVIII) expression is controlled by the platelet-specific αIIb promoter (2bF8) and results in FVIII storage together with VWF in platelets. The transgenic mice were then bred into FVIII knockout mice (FVIIInull) so that the only FVIII in peripheral blood is derived in platelets. The bleeding phenotype was corrected in the 2bF8/FVIIInull transgenic mice, even though FVIII was not detectable in plasma. We further hypothesize that platelet-derived FVIII in a complex with VWF will 1) protect FVIII from inactivation by circulating FVIII-specific antibodies and 2) release FVIII in sufficiently high local concentration at sites of vessel damage so as to overcome immediate inactivation by FVIII inhibitors. To address our hypothesis, we used three strategies -1) an acute model infusing plasma from highly immunized FVIIInull mice, 2) immunization of 2bF8 transgenic mice with human FVIII, and 3) a model using transplantation of spleen cells from highly immunized mice into sub-lethally irradiated 2bF8 transgenic mice. Thirteen mice were infused with plasma containing 48-64 BU and 12 survived following tail clipping using a 1.59 mm template. FVIII-immunized transgenic mice (given adjuvant) generated anti-FVIII inhibitory antibodies as high as 350 BU after a single immunization. The level of platelet-FVIII and the platelet number in these mice were not significantly different from pre-immunization levels and 13 of 15 survived tail clipping. In the 3rd group, spleen cell transplantation resulted it titers of >5000 BU and 8 of 12 transgenic mice survived tail clip. In contrast, only 2 of 7 wild type mice survived tail clip with inhibitor titers >5000 BU after spleen cell transplantation. None of 9 untransplanted FVIIInull mice survived. These results demonstrate that platelet-derived FVIII is protected from inhibitor inactivation and correct the phenotype of murine hemophilia A even in the presence of a high-titer anti-FVIII antibody. This approach may be useful not only for preventing inhibitor formation in hemophilia A gene therapy but also for gene therapy in the case of pre-existing inhibitors.

Restricted BV gene usage by factor VIII-reactive CD4+ T cells in inhibitor-positive patients with severe hemophilia A.

In the present study, we have analyzed the T cell receptor (TCR) repertoires of CD4+ T cells isolated from peripheral blood of 10 inhibitor-positive patients with severe hemophilia A. The distribution of complementarity determining region (CDR3) lengths of the beta chain of the TCRs was analyzed by spectratyping prior to and following in vitro stimulation of the cells with human factor VIII (FVIII). The repertoires of CD4+ T cells of patients were perturbed when compared to those of healthy blood donors. The perturbations of T cell repertoires were heterogeneous among patients with respect to the number and the nature of V-beta (BV) families that exhibited expansion following incubation with FVIII. Some patients showed alterations in one or two BV families, others exhibited more perturbed repertoires affecting 5 to 8 of the 14 BV families tested. Alterations of BV2, BV5 and/or BV9 were consistently found after incubation of CD4+ T cells in the presence of FVIII in 80% of the patients. These findings indicate that the presence of FVIII inhibitors in patients with severe hemophilia A is associated with measurable perturbations of the CD4+ T cell repertoire that results from oligoclonal expansion of FVIII-specific cells and may be relevant for the design of strategies aimed at modulating the anti-FVIII immune responses by T cell-targeted therapy

Acquired von Willebrand factor deficiency during high-dose infusion of recombinant factor VIII.

Constant infusion of factor VIII (FVIII) into patients with haemophilia A after major surgery has been recommended as optimal treatment to avoid peaks and valleys in the circulating levels of FVIII and to allow the use of much lower doses of FVIII than are historically required. One of our young patients with severe (< 0.01 U/ml FVIII) haemophilia suffered a subdural haematoma for which he received treatment with 815190 recombinant FVIII (rFVIII) units over a period of 52d. 2 weeks after admission, because of low FVIII levels and the presence of FVIII inhibitors, the infusion rate was increased to > 100 U/kg/h for 14d. During this time the FVIII level fluctuated between 0.6 and 4.2 U/ml. For some period it was not possible to detect ristocetin co-factor activity in this patient's plasma and the von Willebrand factor (VWF) level and VWF multimer pattern resembled those of a patient with von Willebrand's disease. Subsequently, when the rFVIII dose was increased 2-fold, this was not reflected by the plasma level of FVIII although antibodies were not detected. The data suggest that the prolonged infusion of very high levels of rFVIII which is deficient in von Willebrand factor can result in depletion of VWF from existing stores, producing a laboratory picture which is consistent with the diagnosis of von Willebrand's disease. Further, in the absence of complexing with VWF, FVIII appears to be cleared from the circulation at an increased rate. This is expensive and potentially compromising. Therefore, when administering very high doses of FVIII concentrates devoid of VWF for prolonged periods of time, ristocetin cofactor and VWF levels should be monitored.