Factor VIII antibody immune complexes modulate the humoral response to factor VIII in an epitope-dependent manner.

Introduction: Soluble antigens complexed with immunoglobulin G (IgG) antibodies can induce robust adaptive immune responses in vitro and in animal models of disease. Factor VIII immune complexes (FVIII-ICs) have been detected in individuals with hemophilia A and severe von Willebrand disease following FVIII infusions. Yet, it is unclear if and how FVIII-ICs affect antibody development over time. Methods: In this study, we analyzed internalization of FVIII complexed with epitope-mapped FVIII-specific IgG monoclonal antibodies (MAbs) by murine bone marrow-derived dendritic cells (BMDCs) in vitro and antibody development in hemophilia A (FVIII-/-) mice injected with FVIII-IC over time. Results: FVIII complexed with 2-116 (A1 domain MAb), 2-113 (A3 domain MAb), and I55 (C2 domain MAb) significantly increased FVIII uptake by BMDC but only FVIII/2-116 enhanced antibody titers in FVIII-/- mice compared to FVIII alone. FVIII/4A4 (A2 domain MAb) showed similar FVIII uptake by BMDC to that of isolated FVIII yet significantly increased antibody titers when injected in FVIII-/- mice. Enhanced antibody responses observed with FVIII/2-116 and FVIII/4A4 complexes in vivo were abrogated in the absence of the FVIII carrier protein von Willebrand factor. Conclusion: These findings suggest that a subset of FVIII-IC modulates the humoral response to FVIII in an epitope-dependent manner, which may provide insight into the antibody response observed in some patients with hemophilia A.

The impact of concurrent X chromosome anomalies on diagnosis and bleeding phenotype in children with hemophilia: A single-institution case series.

Hemophilia is an inherited X-linked bleeding disorder characterized by deficiencies of factors VIII or IX. Concomitant X chromosome disorders can impact bleeding phenotype, complicating timely diagnosis and disease management. Herein, we describe three cases of female and male pediatric patients with hemophilia A or B diagnosed between 6 days and 4 years old in the setting of skewed X chromosome inactivation, Turner syndrome, or Klinefelter syndrome. All of these cases had significant bleeding symptoms, and two patients required initiation of factor replacement therapy. One female patient developed a factor VIII inhibitor similar to that described in males with hemophilia A.

Building the foundation for a community-generated national research blueprint for inherited bleeding disorders: facilitating research through infrastructure, workforce, resources and funding.

BACKGROUND: The National Hemophilia Foundation (NHF) conducted extensive, inclusive community consultations to guide prioritization of research in coming decades in alignment with its mission to find cures and address and prevent complications enabling people and families with blood disorders to thrive. RESEARCH DESIGN AND METHODS: With the American Thrombosis and Hemostasis Network, NHF recruited multidisciplinary expert working groups (WG) to distill the community-identified priorities into concrete research questions and score their feasibility, impact, and risk. WG6 was charged with identifying the infrastructure, workforce development, and funding and resources to facilitate the prioritized research. Community input on conclusions was gathered at the NHF State of the Science Research Summit. RESULTS: WG6 detailed a minimal research capacity infrastructure threshold, and opportunities to enable its attainment, for bleeding disorders centers to participate in prospective, multicenter national registries. They identified challenges and opportunities to recruit, retain, and train the diverse multidisciplinary care and research workforce required into the future. Innovative collaborative approaches to trial design, resource networking, and funding to surmount obstacles facing research in rare disorders were elucidated. CONCLUSIONS: The innovations in infrastructure, workforce development, and resources and funding proposed herein may contribute to facilitating a National Research Blueprint for Inherited Bleeding Disorders.

Research is critical to advancing the diagnosis and care of people with inherited bleeding disorders (PWIBD). This research requires significant infrastructure, including people and resources. Hemophilia treatment centers (HTC) need many different skilled care professionals including doctors, nurses, and other providers; also statisticians, data managers, and other experts to process patients' clinical information into research. Attracting diverse qualified professionals to the clinical and research work requires long-term planning, recruiting individuals in training programs and retaining them as they become experts. Research infrastructure includes physical servers running database software, networks that link them, and the environment in which these components function. US Centers for Disease Control and Prevention (CDC) and American Thrombosis and Hemostasis Network (ATHN) coordinate and fund data collection at HTCs on the health and well-being of thousands of PWIBD into a registry used in research studies.National Hemophilia Foundation (NHF) and ATHN asked our group of health care professionals, technology experts, and lived experience experts (LEE) to identify the infrastructure, workforce, and resources needed to do the research most important to PWIBD. We identified the types of CDC/ATHN studies all HTCs should be able to perform, and the physical and human infrastructure this requires. We prioritized finding the best clinical trial designs to study inherited bleeding disorders, identifying ways to share personnel and tools between HTCs, and innovating how research is governed and funded. Involving LEEs in designing, managing, and carrying out research will be key in conducting research to improve the lives of PWIBD.

A review of the pharmacokinetics, efficacy and safety of high-purity factor X for the prophylactic treatment of hereditary factor X deficiency.

INTRODUCTION: Hereditary factor X (FX) deficiency (FXD) is a rare autosomal recessive bleeding disorder. Plasma-derived FX (pdFX) is a high-purity FX concentrate approved in the United States and Europe for the treatment and prophylaxis of bleeding episodes and for peri-operative management in patients with hereditary FXD (HFXD). AIM: To review pharmacokinetic dosing, efficacy, and safety data for pdFX as routine prophylaxis for HFXD. METHODS: Summary of the published pharmacokinetic and safety data from TEN01, TEN02, TEN05, and real-world publications of pdFX for prophylaxis. RESULTS: Pharmacokinetic modelling data from the phase 3 TEN01 study supported administration of pdFX 25 IU/kg twice weekly for routine prophylaxis in adolescents/adults (aged ≥12 years). Results from nine paediatric patients in the phase 3 TEN02 study and eight adolescents/adults (aged ≥12 years) in the retrospective data-collection TEN05 study, along with real-world evidence, showed that routine prophylaxis with pdFX ≈40 IU/kg twice weekly in patients aged <12 years and pdFX ≈25 IU/kg twice weekly in patients aged ≥12 years was effective in bleeding prevention. CONCLUSIONS: pdFX was well tolerated in clinical studies, with no new safety signals identified during routine prophylactic use. Based on current evidence, it is recommended that routine prophylaxis with pdFX be initiated at 25 IU/kg twice weekly in adults/adolescents ≥12 years of age, and at a dosage of 40 IU/kg twice weekly in children <12 years of age. Thereafter, FX levels should be closely monitored, and dosages should be adjusted according to clinical response and to maintain trough levels ≥5 IU/dl.

Iron deficiency anemia and bleeding management in pediatric patients with Bernard-Soulier syndrome and Glanzmann Thrombasthenia: A single-institution analysis.

INTRODUCTION: Frequent and severe bleeding events (SBE) in patients with inherited qualitative platelet disorders Bernard-Soulier Syndrome (BSS) and Glanzmann Thrombasthenia (GT) can lead to secondary iron deficiency anemia (IDA). SBE are primarily treated with platelet transfusions or recombinant activated factor VII (rFVIIa) infusions. The impact of IDA on bleeding management and disease outcomes is understudied. AIM: To evaluate bleeding management, outcomes, and any association with IDA in pediatric patients with BSS and GT. METHODS: Retrospective chart-review of pediatric patients with BSS or GT followed at a single hemophilia treatment center between 2007 and 2019. RESULTS: We identified 14 patients with BSS (n = 2) or GT (n = 12). Patients received rFVIIa (7%), platelet transfusions (7%), or a combination of both (57%) for SBE. Eleven patients (79%) had IDA requiring oral and/or intravenous iron replacement and 50% required red blood cell transfusions. Due to recurrent SBE and refractory IDA, three patients (21%) received rFVIIa prophylaxis at 90 µg/kilogram 2-3 times/week for ≥15 months. Patients initiated on rFVIIa prophylaxis had a median baseline hemoglobin of 9.8 g/dL (min-max: 8.0-10.7 g/dL) compared to 11.7 g/dL (8.4-13.8 g/dL) for patients treated on-demand. Following initiation of rFVIIa prophylaxis, median hemoglobin and ferritin increased by 1.3 g/dL (0.7-2.5 g/dL) and 14.6 ng/mL (0.2-42.9 ng/mL), respectively, and bleeding rates were reduced by 7-78%. CONCLUSION: IDA is a known complication of recurrent bleeding events in individuals with inherited bleeding disorders. Routine monitoring for IDA may help improve bleeding management and reduce bleed burden in BSS/GT.

Removal of single-site N-linked glycans on factor VIII alters binding of domain-specific monoclonal antibodies.

BACKGROUND: A portion of individuals with hemophilia A develop neutralizing antibodies called inhibitors to glycoprotein factor VIII (FVIII). There are multiple risk factors that contribute to the risk of inhibitor formation. However, knowledge of the role of FVIII asparagine (N)-linked glycosylation in FVIII immunity is limited. OBJECTIVE: To evaluate the effect of site-specific N-linked glycan removal on FVIII biochemical properties, endocytosis by murine bone marrow-derived dendritic cells (BMDCs), and antibody responses. METHODS: Four recombinant B domain-deleted (BDD) FVIII variants with single-site amino acid substitutions to remove N-linked glycans were produced for experimental assays. RESULTS: BDD FVIII-N41G, FVIII-N239A, FVIII-N1810A, and FVIII-N2118A with confirmed removal of N-linked glycans and similar glycosylation profiles to BDD FVIII were produced. There were no differences in thrombin activation or von Willebrand factor binding of FVIII variants compared with BDD FVIII; however, reduced FVIII expression, activity, and specific activity was observed with all variants. BDD FVIII-N41G and FVIII-N1810A had reduced uptake by BMDCs, but there were no differences in antibody development in immunized hemophilia A mice compared with BDD FVIII. Half of a repertoire of 12 domain-specific FVIII MAbs had significantly reduced binding to ≥1 FVIII variant with a 50% decrease in A1 domain MAb 2-116 binding to FVIII-N239A. CONCLUSIONS: Modifications of FVIII N-linked glycans reduced FVIII endocytosis by BMDCs and binding of domain-specific FVIII MAbs, but did not alter de novo antibody production in hemophilia A mice, suggesting that N-glycans do not significantly contribute to inhibitor formation.

Successful Perioperative Management of Orthotopic Cardiac Transplantation in a Pediatric Patient With Concurrent Congenital von Willebrand Disease and Acquired von Willebrand Syndrome Using Recombinant von Willebrand Factor.

Von Willebrand disease (VWD) is the most common bleeding disorder and reportedly affects 1:1,000 of the world's population. There are three subtypes of VWD characterized by a quantitative defect (types 1 and 3 VWD) or a qualitative defect (type 2 VWD). Type 1 VWD results in a partial deficiency of von Willebrand factor (VWF) and affects approximately 75% of individuals with VWD, whereas type 3 VWD results in a severe or complete deficiency of VWF. Individuals with type 2 VWD subtypes (types 2A, 2B, 2M, and 2N VWD) express a dysfunctional VWF protein that has impaired interactions with platelets or factor VIII. The majority of individuals with VWD have mild type 1 VWD and occasionally require bolus infusions of VWF for severe bleeding or major surgery. A subset of patients, especially those with type 2A or 3 VWD, may require more frequent VWF replacement or prophylaxis for refractory bleeding or bleeding prevention, respectively. Acquired von Willebrand syndrome (AVWS) is a rare bleeding disorder that primarily occurs as a result of an underlying disease or other pathologic mechanism. Cases of AVWS associated with heart valve defects, left ventricular assist devices, or congenital cardiac disease result from high shear stress in the circulation that induces VWF unfolding and subsequent proteolysis of high-molecular-weight multimers by ADAMTS-13. In rare instances, plasma-derived factor VIII-containing VWF concentrates have been administered to individuals with AVWS for persistent or challenging bleeding events. In this case report, the hemostatic challenges and the perioperative management of cardiac transplantation surgery using a novel recombinant VWF product in a pediatric patient diagnosed with AVWS concomitant with congenital type 1 VWD are described. Written informed consent was obtained from the patient's mother for this case report. The diagnosis of congenital VWD remains a challenge because of multiple potential modifiers that can alter VWF laboratory results. Concurrent conditions, such as congenital heart disease and the rare secondary condition of AVWS, in addition to congenital VWD, can further affect interpretation of coagulation studies. This can result in delays in diagnosis, increase severity of the bleeding phenotype, and complicate hemostatic management in individuals at risk for bleeding and thrombosis. A multidisciplinary approach, including anesthesiologists, cardiologists, cardiovascular surgeons, hematologists, and pharmacists, is critical to achieving optimal patient outcomes, as highlighted in this case report. As diagnostic capabilities and understanding of VWD broaden, future studies evaluating alternative treatment approaches for individuals with various types of VWD would be of great benefit to the medical community.

Reticular Dysgenesis: A Rare Immunodeficiency in a Neonate With Cytopenias and Bacterial Sepsis.

Severe combined immunodeficiency (SCID) consists of a group of disorders defined by abnormal B and T cell development that typically results in death within the first year of life if undiagnosed or untreated. Reticular dysgenesis (RD) is a rare but especially severe form of SCID that is caused by adenylate kinase 2 deficiency and is characterized not only by lymphopenia but also by profound neutropenia. RD predisposes patients to viral and fungal infections typical of SCID as well as serious bacterial infections atypical in the neonatal period in other SCID types. RD is also associated with sensorineural hearing loss not typically seen in other forms of SCID. Without rapid diagnosis and curative hematopoietic stem cell transplantation, RD is fatal within days to months due to overwhelming bacterial infection. The inclusion of the T cell receptor excision circle assay nationally in 2017 on the newborn screen has facilitated diagnosis of SCID in the neonatal period. This case reports on a male infant with RD who presented after preterm birth with severe cytopenias and a gastrointestinal anomaly and ultimately developed severe bacterial sepsis. Postmortem bone marrow evaluation and panel-based gene sequencing identifying 2 novel variants in the adenylate kinase 2 gene provided confirmation for a diagnosis of RD. This case emphasizes the importance of thorough diagnostic evaluation, including the newborn screen, in neonates and infants with persistent and unexplained cytopenias. Prompt hematology and/or immunology referral is advised for disease management and to facilitate hematopoietic stem cell transplantation to optimize long-term survival.

Rare Coagulation Factor Deficiencies (Factors VII, X, V, and II).

Although rare clotting factor deficiencies primarily referred to as rare bleeding disorders (RBD), including factors II, V, VII, and X, make up ∼5% of all inherited bleeding disorders worldwide, each of these clotting factors play a critical role in the coagulation cascade. Incomplete bleeding evaluation or misinterpretation of laboratory studies can result in delayed diagnoses that ultimately affect patient outcomes. Bleeding manifestations can range from mild to severe, but the most common are mucocutaneous bleeding. The ideal treatment in RBD is dedicated single-factor concentrates that can be used for acute bleeding events, surgical management, and prophylaxis.

Emicizumab in tolerized patients with hemophilia A with inhibitors: A single-institution pediatric cohort assessing inhibitor status.

BACKGROUND: The majority of patients with hemophilia A with inhibitors who undergo immune tolerance induction (ITI) achieve successful tolerance and transition to factor VIII (FVIII) prophylaxis. A portion of these patients have switched to emicizumab for bleeding prevention. However, the risk of inhibitor relapse on emicizumab is unclear. OBJECTIVE: To evaluate the inhibitor status of patients with hemophilia A and inhibitors who achieved successful/partial tolerance after ITI and transitioned from FVIII prophylaxis to emicizumab. METHODS: This is a single-institution, retrospective review of pediatric patients with severe hemophilia A who have completed ITI with FVIII and switched to emicizumab. RESULTS/CONCLUSIONS: Seven successfully tolerized and five partially tolerized patients were identified. Three patients continued intermittent FVIII infusions on emicizumab at 50-70 IU/kg twice weekly, once weekly, or every other week due to concerns for inhibitor relapse or loss of recent FVIII tolerance by the treating provider. Eleven of 12 patients (92%) maintained negative inhibitor titers at a mean follow-up of 14.2 ± 6.1 months. One individual had an inhibitor relapse with a peak titer of 2.5 BU/mL. Five of the 11 patients (45%) with negative inhibitor titers had detectable nonneutralizing anti-FVIII IgG4 antibodies, but none of the patients had detectable IgG1 antibodies. There were no inhibitor recurrences in a subset of six patients after FVIII re-exposure for bleeding events or surgery. Given that the presence of an inhibitor significantly impacts factor product choice for bleeding management, ongoing inhibitor monitoring in tolerized patients with hemophilia A who transition to emicizumab is strongly recommended.

Thrombocytosis with acquired von Willebrand disease in an adolescent with sickle cell disease.

Thrombocytosis is common in sickle cell disease and may contribute to vaso-occlusion. Hydroxyurea treats extreme thrombocytosis. Acquired von Willebrand disease should be considered prior to aspirin therapy.

Use of plasma-derived factor X concentrate in neonates and infants with congenital factor X deficiency.

BACKGROUND: Congenital factor X deficiency (FXD) is a rare bleeding disorder that often presents with severe bleeding in the neonatal period. Long-term prophylaxis with infusions of FX-containing products is recommended in patients with FXD and a personal or family history of severe bleeding. A plasma-derived FX concentrate (pdFX) is approved for on-demand and prophylactic therapy in adults and children with FXD. The safety and efficacy of pdFX has been demonstrated in patients <12 years of age, yet limited data exist regarding its use in infants. PATIENTS/METHODS: This retrospective case series details clinical experience using pdFX in four neonates with moderate and severe FXD across four institutions. RESULTS AND CONCLUSIONS: All four patients presented in the first week of life with severe bleeding. Following treatment of the acute bleed, prophylactic pdFX was initiated at an average of 29 days of life and a dose of 69 IU/kg every 48 hours. Incremental recovery (IR) in three infants averaged 1.42 IU/dL per IU/kg (min-max: 1.06-1.67 IU/dL per IU/kg). One patient experienced thrombotic complications in the setting of sepsis. After a median follow-up of 26.5 months, no patient has experienced breakthrough bleeding episodes. Our study supports the use of pdFX in neonates and infants and suggests that higher pdFX dosing of 70 to 80 IU/kg every 48 hours based on the smallest available vial size is feasible. Because of variability in IR, close monitoring of FX activity should be used to guide dosing in this age group.

Immune tolerance induction in paediatric patients with haemophilia A and inhibitors receiving emicizumab prophylaxis.

INTRODUCTION: The formation of neutralizing antifactor VIII (fVIII) antibodies, called inhibitors, is the most common complication in modern haemophilia A care. Novel non-factor replacement therapies, such as emicizumab, have sought to address the limitations of bypassing agents for bleeding management in patients with inhibitors. However, immune tolerance induction (ITI) remains the primary method for eradicating inhibitors and restoring the hemostatic response to fVIII. AIM: The aim of this study was to review a case series of paediatric patients with haemophilia A and inhibitors who have received ITI for inhibitor eradication concomitantly with emicizumab prophylaxis for bleeding prevention. METHODS: Single institution retrospective chart review of paediatric patients with severe haemophilia A receiving ITI and emicizumab. RESULTS: Seven patients were included in this cohort. Six patients used three different recombinant fVIII products at 100 IU/kg three times per week, and one patient used a plasma-derived fVIII product at an initial dose of 50 IU/kg three times per week. Three patients achieved a negative inhibitor titre <0.6 Chromogenic Bethesda Units per mL (CBU/mL), and two patients achieved a normal fVIII recovery ≥66%. There were nine bleeding events in four patients, but no thrombotic events. All patients remained on ITI and emicizumab. CONCLUSION: Immune tolerance induction while on emicizumab prophylaxis is a feasible approach in paediatric haemophilia A patients with inhibitors. This is the first report of the concomitant use of ITI in patients receiving emicizumab prophylaxis described as the 'Atlanta Protocol'.

Hemophilia and inhibitors: current treatment options and potential new therapeutic approaches.

The immune response to infused factor concentrates remains a major source of morbidity and mortality in the treatment of patients with hemophilia A and B. This review focuses on current treatment options and novel therapies currently in clinical trials. After a brief review of immune tolerance regimens, the focus of the discussion is on preventing bleeding in patients with hemophilia and inhibitors. Recombinant factor VIIa and activated prothrombin complex concentrates are the mainstays in treating bleeds in patients with inhibitors. Both agents have been shown to reduce bleeding episodes to a similar degree when infused prophylactically; however, individual patients may respond better to one agent over the other at any given time. The international immune tolerance trial revealed that a high-dose factor VIII regimen provided significantly better bleeding protection than the low-dose regimen. Given the high cost of treatment and the potential for a high-dose immune tolerance regimen to prevent bleeding in some patients, we discuss how we treat patients to maximize the prevention of bleeds while minimizing cost. Novel approaches to treatment of these patients are in development. These include agents that mimic factor VIII or augment thrombin generation by bypassing the inhibitor, as well as agents that inhibit the natural anticoagulants.

High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors.

Inhibitor formation in hemophilia A is the most feared treatment-related complication of factor VIII (fVIII) therapy. Most inhibitor patients with hemophilia A develop antibodies against the fVIII A2 and C2 domains. Recent evidence demonstrates that the C1 domain contributes to the inhibitor response. Inhibitory anti-C1 monoclonal antibodies (mAbs) have been identified that bind to putative phospholipid and von Willebrand factor (VWF) binding epitopes and block endocytosis of fVIII by antigen presenting cells. We now demonstrate by competitive enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry that 7 of 9 anti-human C1 mAbs tested recognize an epitope distinct from the C1 phospholipid binding site. These mAbs, designated group A, display high binding affinities for fVIII, weakly inhibit fVIII procoagulant activity, poorly inhibit fVIII binding to phospholipid, and exhibit heterogeneity with respect to blocking fVIII binding to VWF. Another mAb, designated group B, inhibits fVIII procoagulant activity, fVIII binding to VWF and phospholipid, fVIIIa incorporation into the intrinsic Xase complex, thrombin generation in plasma, and fVIII uptake by dendritic cells. Group A and B epitopes are distinct from the epitope recognized by the canonical, human-derived inhibitory anti-C1 mAb, KM33, whose epitope overlaps both groups A and B. Antibodies recognizing group A and B epitopes are present in inhibitor plasmas from patients with hemophilia A. Additionally, group A and B mAbs increase fVIII clearance and are pathogenic in a hemophilia A mouse tail snip bleeding model. Group A anti-C1 mAbs represent the first identification of pathogenic, weakly inhibitory antibodies that increase fVIII clearance.