Safety of recombinant activated factor VII for treatment of breakthrough bleeds in patients with congenital haemophilia A and inhibitors receiving emicizumab prophylaxis: Review of the real-world evidence.

BACKGROUND: Emicizumab is used as a subcutaneous prophylaxis for prevention of bleeding episodes in patients with haemophilia A (HA) with and without inhibitors. While low bleeding rates were observed in clinical trials, patients still experience breakthrough bleeds (BTBs) with emicizumab in the real-world. Current guidelines recommend use of recombinant activated factor VII (rFVIIa) for treatment of BTBs in patients with inhibitors. Due to thrombotic events observed in the HAVEN 1 study, activated prothrombin complex concentrate (aPCC) should be used with caution. OBJECTIVES: The objective of this review is to identify and discuss real-world data on the frequency of BTBs and the safety of concomitant rFVIIa use in patients with inhibitors on emicizumab prophylaxis. METHODS: A search of the following databases was conducted on 15 July 2022: BIOSIS Previews® , Current Contents Search® , Embase® , MEDLINE® . Search terms included 'real world', 'haemophilia A', and 'emicizumab'. RESULTS AND CONCLUSIONS: Eleven relevant publications were identified (seven original research articles and four congress abstracts). The frequency of BTBs specifically for HA patients with inhibitors was described in three publications with 5%-56% patients on emicizumab reporting ≥1 bleeding episode. Treatment of these BTBs appeared to be managed according to relevant guidelines. Importantly, no thrombotic complications occurred during concomitant rFVIIa use. Due to the nature of real-world studies, direct comparison of the results between studies is limited. However, real-world data show that BTBs in inhibitor patients during emicizumab prophylaxis can be safely treated with rFVIIa.

Befovacimab, an anti-tissue factor pathway inhibitor antibody: Early termination of the multiple-dose, dose-escalating Phase 2 study due to thrombosis.

INTRODUCTION: Befovacimab (formerly BAY 1093884) is a fully human monoclonal antibody able to bind to tissue factor pathway inhibitor (TFPI) and developed as a non-replacement therapy for individuals with haemophilia A/B, with or without inhibitors. AIM: To assess the safety of multiple escalating doses of befovacimab in individuals with severe haemophilia A/B with or without inhibitors. METHODS: In this non-randomised, open-label Phase 2 study (NCT03597022), adult males with <1% factor VIII or <2% factor IX and ≥4 bleeds in the previous six months were enrolled in three dose cohorts (100/225/400 mg). Participants received befovacimab subcutaneously once weekly. The primary endpoint was safety; secondary endpoints included annualised bleeding rate (ABR) and pharmacokinetics/pharmacodynamics (PK/PD) of befovacimab. RESULTS: A total of 24 participants (n = 8 in each dose cohort) were treated for 2-47 weeks. Patients treated with 100 mg and 225 mg doses of befovacimab demonstrated improved bleeding control compared with pre-study bleeding rates, with a dose-dependent effect. Dosing was suspended and the study prematurely terminated following three drug-related thrombotic serious adverse events (SAEs): two at the 225 mg dose and one at the 400 mg dose. These occurred in the absence of bleeding episodes or concomitant use of replacement/bypass therapies. No laboratory abnormalities were observed, and PK/PD data did not show correlation between SAE occurrence and levels of circulating befovacimab or free TFPI. CONCLUSION: Despite favourable initial results from preclinical and clinical studies, a positive safety profile of befovacimab was not confirmed. The lack of SAE-related laboratory abnormalities or differentiating PK/PD characteristics in participants experiencing SAEs raises concerns about the predictability of thrombosis following befovacimab treatment and emphasises the need for further investigation into the therapeutic window of anti-TFPI treatment.

Prophylaxis Using a Mixture of Plasma-Derived Activated Factor VII and Factor X (pdFVIIa/FX) in a Patient with Hemophilia B Complicated by Inhibitors and Allergy to Factor IX Concentrates: A Case Report.

Treating patients with hemophilia and inhibitors is often problematic. The presence of inhibitors negatively impacts the effectiveness of treatment to achieve hemostasis especially in patients with hemophilia B, owing mainly to allergic reactions to factor IX (FIX) concentrates and the low success rate of immune tolerance therapy. A 9-month-old boy had intracranial hemorrhage and was diagnosed with hemophilia B. After replacement therapy, he developed inhibitors and an allergic reaction to FIX. Prophylactic therapy was initiated with recombinant activated factor VII (rFVIIa) and later switched to pdFVIIa/factor X (FX; 120 µg/kg as the FVII dose, every other day) because of a recurrence of intracranial hemorrhage. Since then, he remained well without life-threatening bleeding for more than 2 years. Our case suggests that pdFVIIa/FX may be useful for prophylactic therapy in hemophilia B complicated by inhibitors and allergic reaction to FIX concentrates.

Prediction of the haemostatic effects of bypassing therapy using comprehensive coagulation assays in emicizumab prophylaxis-treated haemophilia A patients with inhibitors.

In emicizumab prophylaxis, the concomitant therapy using bypassing agents (BPAs) is required for breakthrough bleeding and invasive procedures with attention to thrombotic complications. To predict coagulant effects of BPAs in emicizumab-treated patients with haemophilia A (PwHA) with inhibitor (PwHAwI), blood samples from emicizumab-treated PwHAwI (n = 8) and PwHA without inhibitor (n = 2) in phase 1/2 and HAVEN 1 study, spiked with activated prothrombin complex concentrates (aPCC) or recombinant factor VIIa (rFVIIa) ex vivo, and blood samples from emicizumab-treated PwHAwI-receiving BPAs were analysed by Ca2+ -triggered rotational thromboelastometry (ROTEM) and ellagic acid/tissue factor-triggered clot waveform analysis (CWA). Spiked aPCC, corresponded to 10-100 U/kg, markedly shortened ROTEM parameters beyond the normal range, while spiked rFVIIa, corresponded to 90-270 µg/kg, shortened them within near-normal range. Each of the spiked BPA-improved adjusted maximum coagulation velocity of CWA to within or near the normal range. In blood samples at post-infusion of aPCC (44-73 U/kg) or rFVIIa (79-93 µg/kg), the parameters of both assays improved to approximately the normal range. Taken together, ex vivo results of spiking tests in ROTEM and CWA, except aPCC spiking test in ROTEM, were relatively consistent with in vivo ones, and could usefully predict the coagulant effects of concomitant bypassing therapy for emicizumab-treated PwHAwI.

Outcomes in children with hemophilia A with inhibitors: Results from a noninterventional study.

BACKGROUND: Data regarding management of pediatric persons with hemophilia A (PwHA) with factor VIII (FVIII) inhibitors are limited. This prospective noninterventional study (NCT02476942) evaluated annualized bleeding rates (ABRs), safety, and health-related quality of life (HRQoL) in pediatric PwHA with FVIII inhibitors. PROCEDURE: PwHA aged <12 years with current FVIII inhibitors and high-titer inhibitor history were enrolled. Participants remained on usual treatment; no interventions were applied. Outcomes included ABR, safety, and HRQoL. RESULTS: Twenty-four PwHA aged 2-11 years (median 7.5) were enrolled and monitored for 8.7-44.1 weeks (median 23.4). In the episodic (n = 10) and prophylactic (n = 14) groups, respectively, 121 of 185 (65.4%) and 101 of 186 (54.3%) bleeds were treated using activated prothrombin complex concentrate (aPCC) and/or recombinant activated FVII (rFVIIa). ABRs (95% confidence interval) were 19.4 (13.2-28.4) and 18.5 (14.2-24.0) for treated bleeds, and 32.7 (20.5-52.2) and 33.1 (22.4-48.9) for all bleeds, respectively. Most prophylactic group participants (92.9%) were prescribed aPCC; 50% adhered to their prescribed treatment regimen. Adherence to prophylactic rFVIIa was not assessed. Serious adverse events included hemarthrosis (12.5%) and mouth hemorrhage (12.5%); the most common nonserious adverse event was viral upper respiratory tract infection (12.5%). HRQoL showed functional impairment at baseline; scores remained stable throughout, with little intergroup variation. CONCLUSIONS: ABRs remained high in pediatric PwHA with inhibitors receiving standard treatment. This study demonstrates the need for more effective treatments, with reduced treatment burden, to prevent bleeds, increase prophylaxis adherence, and improve patient outcomes.

Leveraging Human Genetics to Estimate Clinical Risk Reductions Achievable by Inhibiting Factor XI.

Background and Purpose- Coagulation factor XI (FXI) is a novel target for antithrombotic therapy addressed by various therapeutic modalities currently in clinical development. The expected magnitude of thrombotic event reduction mediated by targeting FXI is unclear. Methods- We analyzed the association of 2 common genetic variants, which alter levels of FXI, with a range of human phenotypes. We combined variants into a genetic score standardized to a 30% increase in relative activated partial thromboplastin time, equivalent to what can be achieved with pharmacological FXI reduction. Using data from 371 695 participants in the United Kingdom Biobank and 2 large-scale genome-wide association studies, we examined the effect of this FXI score on thrombotic and bleeding end points. Results- Genetic disposition to lower FXI levels was associated with reduced risks of venous thrombosis (odds ratio, 95% CI; P value; odds ratio=0.1, 0.07-0.14; P=3×10-43) and ischemic stroke (odds ratio=0.47, 0.36-0.61; P=2×10-8) but not with major bleeding (odds ratio=0.7, 0.45-1.04; P=0.0739). The observed relative risk reductions were consistent within a range of subgroups that were at high risk for thrombosis. Consistently, we observed higher absolute risk reductions conferred by genetically lower FXI levels in high-risk subgroups, such as patients with atrial fibrillation. Conclusions- Human genetic data suggest that pharmacological inhibition of FXI may achieve considerable reductions in ischemic stroke risk without clear evidence for an associated risk of major bleeding. The quantitative framework developed can be used to support the estimation of achievable risk reductions with pharmacological modulation of FXI.

Bleeding and safety outcomes in persons with haemophilia A without inhibitors: Results from a prospective non-interventional study in a real-world setting.

INTRODUCTION: Prospectively collected real-world data on bleeds, haemophilia treatment and safety in persons with haemophilia A (PwHA) without factor VIII (FVIII) inhibitors are limited. A global, non-interventional study (NIS; NCT02476942) prospectively collected real-world data in PwHA who were treated per local routine clinical practice. AIM: Assess annualized bleeding rate (ABR), haemophilia treatment practices and adverse events (AEs) in adult/adolescent PwHA without inhibitors. METHODS: Eligible participants aged ≥12 years with severe HA without history of inhibitors prospectively collected bleeding and treatment information. RESULTS: Ninety-four participants were enrolled (median [range] age, 34 [12-76] years) and monitored for a median (range) of 29.8 (12.4-47.7) weeks. In the episodic (n = 45) and prophylactic (n = 49) treatment groups, respectively, 872/1066 (81.8%) and 151/189 (79.9%), bleeds were treated; ABRs (95% confidence interval) were 36.1 (30.8-42.3) and 5.0 (3.3-7.5), respectively, for treated bleeds and 43.1 (36.5-50.9) and 6.2 (4.2-9.2), respectively, for all bleeds, and median (interquartile range) ABRs were 31.1 (19.8-51.6) and 1.9 (0.0-8.2), respectively, for treated bleeds and 35.3 (21.7-62.9) and 2.7 (0.0-9.4), respectively, for all bleeds. Half of the participants on FVIII prophylaxis had relatively high adherence to treatment, using 2.9 and 2.1 median doses/wk of standard and extended half-life FVIII, respectively. Serious AEs included gastrointestinal polyp haemorrhage and haemarthrosis; the most common AE was viral upper respiratory tract infection. CONCLUSION: PwHA without inhibitors continue to bleed on prophylaxis, consistent with the literature, and require treatment for breakthrough bleeds. This prospective NIS demonstrates the need for more efficacious haemostatic approaches.

Recombinant factor VIII products and inhibitor development in previously untreated patients with severe haemophilia A: Combined analysis of three studies.

INTRODUCTION: Standard treatment of congenital haemophilia A is based on replacement therapy with coagulation factor VIII (FVIII) products. A major complication of FVIII therapy is the occurrence of IgG alloantibodies (inhibitors) that neutralize FVIII activity. AIM: The aim of the analysis was estimating the risk of high-titre inhibitor associated with the second-generation full-length product compared to third-generation full-length product and other recombinant FVIII (rFVIII). METHODS: We conducted a combined analysis of individual patient data from three large studies in previously untreated patients (PUPs) with severe haemophilia A. RESULTS: A total of 1109 PUPs were treated from 1993 to 2013 including 787 PUPs treated from 2004 onwards (primary analysis cohort). A total of 322 patients (29.0%) developed an inhibitor, of which 192 (17.3%) a high-titre inhibitor. In the primary analysis set, 29.9% of patients developed an inhibitor and 17.2% a high-titre inhibitor. The combined analysis indicated a lower risk of high-titre inhibitor development for the third-generation rFVIII product compared to the second-generation rFVIII product (primary analysis: adjusted hazard ratio (HR) = 0.72, 95% CI: 0.49 to 1.06). Adjusted HR for all inhibitor development was significantly lower for the third-generation product compared to the second-generation product. CONCLUSION: The trend of an increased risk of inhibitor development in PUPs for one recombinant product illustrates that extrapolation from one recombinant factor VIII product to other products might not be justified.

Bleeding events and safety outcomes in persons with haemophilia A with inhibitors: A prospective, multi-centre, non-interventional study.

INTRODUCTION: Prospectively collected, real-world data on bleeds, haemophilic treatment and safety outcomes in persons with haemophilia A (PwHA) with factor VIII (FVIII) inhibitors are limited. A prospective, global, multi-centre, non-interventional study (NIS; NCT02476942) collected detailed real-world data in PwHA treated per local routine clinical practice. AIM: To characterize bleeding rates, haemophilic treatment practices, prophylaxis adherence and adverse events (AEs) in adult/adolescent PwHA with inhibitors in the NIS. METHODS: Participants aged ≥12 years with congenital haemophilia A/documented high-titre FVIII inhibitor history were enrolled. Participants remained on their usual treatment; no interventions were applied. RESULTS: Overall, 103 PwHA with inhibitors enrolled, (median [range] age 31 [12-75] years) and were monitored for median (range) 26.0 (4.1-69.6) weeks. In the episodic (n = 75) and prophylactic (n = 28) treatment groups, respectively, 1244 and 325 bleeds were reported, and 528 (42.4%) and 104 (32.0%) were not treated; annualized bleeding rates (ABRs; 95% confidence interval) were 18.6 (15.2-22.8) and 14.9 (10.5-21.2) for treated bleeds, and 32.7 (27.3-39.1) and 25.0 (18.4-34.0) for all bleeds. Coagulation products used included activated prothrombin complex concentrate (aPCC) and/or recombinant activated FVII. Among participants prescribed aPCC prophylaxis, 35.0% adhered to both prescribed frequency of aPCC administration and prescribed dose. Serious AEs of haemarthrosis and muscle haemorrhage were reported; most common AEs were arthralgia, viral upper respiratory tract infection and pyrexia. CONCLUSIONS: ABRs (treated bleeds and all bleeds) remain high on standard treatment; this prospective NIS demonstrates the need for more effective treatments for PwHA with inhibitors to reduce/prevent bleeds, with potential to improve prophylaxis adherence and further improve outcomes.

In silico and in vitro immunogenicity assessment of B-domain-modified recombinant factor VIII molecules.

INTRODUCTION: B-domain modification can improve production of recombinant factor VIII (rFVIII) proteins. However, the engineered junction results in non-native peptide sequences with the potential to elicit immune responses via major histocompatibility complex class-II (MHC-II)-binding and CD4+ T cell activation. AIM: Assess the immunogenic potential of B-domain junction peptides of turoctocog alfa and other B-domain-modified rFVIII proteins using in silico and in vitro immunogenicity assessment techniques. METHODS: Peptides with amino acid sequences identical to the B-domain junction of turoctocog alfa, simoctocog alfa and moroctocog alfa were evaluated by in silico peptide-MHC-II binding prediction, in vitro peptide-MHC-II-binding measurement and in vitro T cell-activation assays. Moreover, turoctocog alfa was assessed for peptide presentation on dendritic cells (DCs) using MHC-associated peptide proteomics. RESULTS: In silico analysis predicted virtually no neo-epitopes in the B-domain junction for turoctocog alfa, whereas some were predicted for simoctocog alfa and moroctocog alfa. Turoctocog alfa and moroctocog alfa peptides showed minimal capacity to bind high-frequency MHC-II molecules in vitro, whereas simoctocog alfa peptide demonstrated some degree of binding to approximately half of the MHC-II molecules tested. In line with this, no B-domain peptides from turoctocog alfa were found to be presented on MHC-II complexes on DCs. B-domain junction peptides from all 3 compounds induced T cell responses in only a few percentages of donors. CONCLUSION: All 3 junction peptides were found to have a low immunogenicity potential, suggesting that modification of the B-domain does not constitute an increased immunogenicity risk for any of the products examined.

Identification of mutations in the F8 and F9 gene in families with haemophilia using targeted high-throughput sequencing.

INTRODUCTION: At present, many methods are available for the genetic diagnosis of haemophilia, including indirect linkage analysis, direct sequencing. However, these methods are time-consuming, labourious, and limited in their application. Therefore, the development of new, more effective techniques is necessary. AIM: To detect the F8 and F9 gene mutations in patients with haemophilia and their female relatives in 29 haemophilia A (HA) and 11 haemophilia B (HB) families. FVIII: C and FIX:C were analyzed using one-stage method, and factor VIII and factor IX inhibitors were tested using the Bethesda method. Intron 22 and one inversions were identified using long-distance polymerase chain reaction (PCR) and standard PCR. Non-inversion mutations of the F8 and F9 gene were identified by targeted high-throughput sequencing. All mutations were verified by Sanger sequencing. RESULTS: Intron 22 inversion was detected in eight HA families and intron one inversion was detected in one HA family. Apart from the inversion mutations, 20 mutations were identified in HA families, including 17 previously reported and three novel mutations: c.5724G>A (p.Trp1908*), c.6116-1_6120delGAGTGTinsTCC (p.Lys2039Ilefs*13), and c.5220-2A>C. We found a complex rearrangement in HA: intron one inversion concomitant with exon one deletion. In HB, eight recurrent mutations were detected, including six missense mutations and two nonsense mutations. CONCLUSION: Targeted high-throughput sequencing is an effective technique to detect the F8 and F9 gene mutations, especially for the discovery of novel mutations. The method is highly accurate, time-saving and shows great advantage in uncovering large deletion mutations and also in distinguishing the wild-type genotype and heterozygous large deletions.

FEIBA in the treatment of acquired haemophilia A: results from the prospective multicentre French 'FEIBA dans l'hémophilie A acquise' (FEIBHAC) registry.

Factor VIII inhibitor bypass activity (FEIBA) is a recommended first-line bypassing agent for bleeding episodes in patients with acquired haemophilia A (AHA). Due to the low incidence of AHA, available clinical data on FEIBA treatment are limited. The study aim was to delineate practice patterns in FEIBA treatment of AHA patients, the haemostatic efficacy of FEIBA, including criteria for its assessment, and safety. A prospective registry was established of AHA patients receiving FEIBA for bleeding episodes or prophylaxis at the time of invasive procedures. Data were collected at 16 participating centres in France. Patients were followed up for 3 months. Haemostatic efficacy, FEIBA regimen and FEIBA-related adverse events were documented. Thirty-four patients averaging 81.8 years old with standard deviation (SD) 8.1 years were included in the study: 33 for acute bleeding and one for haematoma evacuation. The mean initial dose of FEIBA for acute bleeding was 75.4 U kg(-1) (SD, 7.7 U kg(-1) ), most often administered twice daily, and the median duration of FEIBA treatment was 4.0 days (interquartile range, 2.2-8.0 days). FEIBA was effective in managing 88.0% of bleeding episodes (95% confidence interval, 75.8-94.5%). No baseline variables influencing treatment response could be identified. The sensitivity and specificity of an objective haemostatic efficacy scale in predicting sequential investigator assessments of haemostatic efficacy were 45.3% and 84.1% respectively. Four patients experienced a total of six serious adverse events possibly related to FEIBA. In the first prospective study specifically focused on FEIBA treatment of patients with AHA, 88.0% of bleeding episodes were effectively managed.

Safety of recombinant activated factor VII (rFVIIa) in patients with congenital haemophilia with inhibitors: overall rFVIIa exposure and intervals following high (>240 µg kg⁻¹) rFVIIa doses across clinical trials and registries.

Recombinant activated factor VII (rFVIIa) is indicated for treatment of bleeding in congenital haemophilia with inhibitors (CHwI) using 90 µg kg(-1) every 2-3 h (EU and US) or a single 270 µg kg(-1) dose (EU only) with ~90% efficacy reported for both regimens. Dosing of rFVIIa varies, and home treatment makes assessment of frequency of doses >90 µg kg(-1), the intervals before additional treatment, and the risk for thromboembolic events (TEs) more difficult. This post hoc analysis assessed the safety and distribution of rFVIIa dosing in CHwI and the impact of >240 µg kg(-1) dosing on subsequent bypassing agent (BPA) dosing interval and frequency. Data regarding on-demand or prophylactic rFVIIa dosing, TE incidence and subsequent BPA dosing after high rFVIIa doses were compiled from multiple sources incorporating safety surveillance. A total of 61 734 rFVIIa doses were reported in 481 patients treated for 3947 bleeds and for 43 135 prophylaxis days. Over half (52%) exceeded 120 µg kg(-1), 37% exceeded 160 µg kg(-1) and 15% exceeded 240 µg kg(-1). Subsequent doses of BPA(s) were administered after 38% of initial and 49% of any rFVIIa dose >240 µg kg(-1), and were most frequently administered ≥24 h after initial (40%) or any (53%) doses >240 µg kg(-1). No TEs were reported. The findings of this analysis show that rFVIIa doses >90 µg kg(-1) are utilized for 'real-world' treatment of children and adults. When additional BPA was administered following an rFVIIa dose >240 µg kg(-1), reported intervals were prolonged, often ≥24 h. No safety issues were identified in the 61,734 doses analysed.

Novel family of insect salivary inhibitors blocks contact pathway activation by binding to polyphosphate, heparin, and dextran sulfate.

OBJECTIVE: Polyphosphate and heparin are anionic polymers released by activated mast cells and platelets that are known to stimulate the contact pathway of coagulation. These polymers promote both the autoactivation of factor XII and the assembly of complexes containing factor XI, prekallikrein, and high-molecular-weight kininogen. We are searching for salivary proteins from blood-feeding insects that counteract the effect of procoagulant and proinflammatory factors in the host, including elements of the contact pathway. APPROACH AND RESULTS: Here, we evaluate the ability of the sand fly salivary proteins, PdSP15a and PdSP15b, to inhibit the contact pathway by disrupting binding of its components to anionic polymers. We attempt to demonstrate binding of the proteins to polyphosphate, heparin, and dextran sulfate. We also evaluate the effect of this binding on contact pathway reactions. We also set out to determine the x-ray crystal structure of PdSP15b and examine the determinants of relevant molecular interactions. Both proteins bind polyphosphate, heparin, and dextran sulfate with high affinity. Through this mechanism they inhibit the autoactivation of factor XII and factor XI, the reciprocal activation of factor XII and prekallikrein, the activation of factor XI by thrombin and factor XIIa, the cleavage of high-molecular-weight kininogen in plasma, and plasma extravasation induced by polyphosphate. The crystal structure of PdSP15b contains an amphipathic helix studded with basic side chains that forms the likely interaction surface. CONCLUSIONS: The results of these studies indicate that the binding of anionic polymers by salivary proteins is used by blood feeders as an antihemostatic/anti-inflammatory mechanism.

Development of a novel fully functional coagulation factor VIII with reduced immunogenicity utilizing an in silico prediction and deimmunization approach.

BACKGROUND: Up to 30% of hemophilia A patients develop inhibitory antibodies against the infused factor VIII (FVIII). The development of a deimmunized FVIII is an unmet high medical need. Although improved recombinant FVIII (rFVIII) products evolved within the last years, the immunogenicity has not been solved. A deimmunized FVIII could reduce the probability of inhibitor development, providing safer therapy. OBJECTIVE: To develop a deimmunized FVIII molecule by modifying major histocompatibility complex (MHC) class II presentation, leading to a functional but less immunogenic molecule. METHODS: We performed (1) in silico prediction of potentially immunogenic T cell epitopes and their modification by amino acid substitutions in the FVIII sequence, (2) evaluation of functional and structural similarity of the modified rFVIII to unmodified FVIII and registered products, and (3) confirmation of the reduced immunogenicity by in vitro testing. RESULTS: A partially deimmunized fully functional FVIII molecule incorporating 19 amino acid substitutions was generated. The substitutions led to a reduction of the immunogenicity score, indicating a reduced immunogenicity based on in silico calculations. This was confirmed in an in vitro dendritic cell (DC)--T cell assay. Using this assay, cells from healthy donors proved the significantly reduced immunogenicity of the modified FVIII variant by revealing less proliferation of T helper cells to this variant than to the unmodified FVIII. CONCLUSION: In silico predictions resulted in a partially deimmunized FVIII. This FVIII is fully functional and was demonstrated to be less immunogenic in in vitro testing. This approach may result in a reduction of the inhibitor risk for patients with hemophilia A.

Clot waveform analysis for perioperative hemostatic monitoring of arthroscopic synovectomy in a pediatric patient with hemophilia A and inhibitor receiving emicizumab prophylaxis.

Emicizumab reduces bleeding in patients with hemophilia A and inhibitors (PwHA-I). Coagulation potential during the perioperative period in emicizumab-treated PwHA-I undergoing surgery remains to be evaluated. We describe a 14-year-old boy with HA-I receiving emicizumab prophylaxis who experienced arthroscopic synovectomy. He was treated with a bolus infusion of recombinant factor VIIa (rFVIIa; 80 µg/kg) immediately before surgery, and treatment continued at the same dose every 3 h on day 1, every 4 h on day 2, and every 6 h on day 3. Treatment with rFVIIa was discontinued on day 4. No perioperative bleeding or thrombotic events were observed. Coagulation potential throughout the perioperative period was retrospectively assessed with an easy-to-use clot waveform analysis (CWA). Measurements from CWA returned to within or near the normal range, suggesting successful hemostatic management. Coagulation potentials assessed by CWA showed a significant correspondence with those from a thrombin generation assay (TGA) that is already in use. CWA and TGA could both provide useful data for assessing coagulation potential in the perioperative hemostatic management of emicizumab-treated PwHA-I.

Non-additive effect on thrombin generation when a plasma-derived factor VIII/von Willebrand factor (FVIII/VWF) is combined with emicizumab in vitro.

BACKGROUND: Emicizumab is an alternative non-factor approach for treating patients with hemophilia A. However, there is a potential risk of thrombotic events when emicizumab is concomitantly administered with pro-hemostatic therapies. OBJECTIVES: To assess the hemostatic effect in vitro when a plasma-derived factor VIII concentrate containing von Willebrand factor (pdFVIII/VWF) was added to hemophilia A plasma (HAp) in combination with emicizumab. METHODS: HAp and HAp with FVIII inhibitors (HAp-i) samples with different concentrations of emicizumab (50 and 100 µg/mL) were combined with activated prothrombin complex concentrate at 0.5 to 1 U/mL, recombinant activated factor VII (rFVIIa) at 0.5 to 7 µg/mL, and pdFVIII/VWF at 0.1 to 4.5 IU/mL. Thrombin generation (TG; thrombin peak and endogenous thrombin potential) was determined using a Calibrated Automated Thrombogram assay. RESULTS: When activated prothrombin complex concentrate was added to HAp and HAp-i with emicizumab, TG dramatically increased (multiplier effect > 4.5×). Addition of rFVIIa to HAp or HAp-i with emicizumab moderately increased TG in a concentration-related manner compared with rFVIIa alone. Addition of pdFVIII/VWF to HAp or HAp-i with emicizumab induced a TG response equivalent to those samples without emicizumab. In an in vitro model of immune tolerance induction with bleeds (HAp-i 15 Bethesda units), combination of pdFVIII/VWF, emicizumab, and rFVIIa did not trigger a multiplying effect on TG. CONCLUSIONS: pdFVIII/VWF showed a non-additive effect on TG when combined in vitro with emicizumab. This finding suggests that emicizumab has limited ability to promote factor X activation in the presence of pdFVIII/VWF, thus reducing the risk of thrombotic events.

Factors Involved in the Development of Inhibitory Antibodies in Patients with Hemophilia in Colombia: A Case-Control Study.

BACKGROUND: The appearance of inhibitory antibodies against antihemophilic factors is one of the most serious complications related to hemophilia. OBJECTIVE: The objective of this study was to identify variables and factors related to the development of inhibitory antibodies in a group of patients undergoing antihemophilic therapy in Colombia. METHODS: A case-control study in patients with hemophilia treated in Specialized Healthcare Provider Institutions (IPS-E) in 21 cities of Colombia of any age and with a diagnosis of inhibitory antibodies against factor VIII or IX during 2016. Four controls per case paired by age and type of hemophilia were used. Sociodemographic, clinical, and pharmacological variables were identified and analyzed. RESULTS: Seventeen patients with inhibitory antibodies and 68 controls with hemophilia were identified. The mean age was 28.3 ± 17.8 years. A total of 94.1% had hemophilia A, and 88.2% of the cases and 50.0% of the controls had severe hemophilia; 47.1% of the cases and 54.4% of the controls were receiving prophylaxis with coagulation factors. Multivariate analysis showed that having severe hemophilia (OR:17.0, 95%CI:1.32-219.60) and lack of knowledge of the coagulation factor with which the patient was treated before entering the care program in the IPS-E (OR:8.9, 95%CI:1.82-43.75) were significantly associated with a higher probability of developing inhibitory antibodies. CONCLUSION AND RELEVANCE: Coagulation factors associated with the development of inhibitory antibodies were severe hemophilia and lack of knowledge of the type of factor used prior to entering the follow-up cohort.

Quantitative HLA-class-II/factor VIII (FVIII) peptidomic variation in dendritic cells correlates with the immunogenic potential of therapeutic FVIII proteins in hemophilia A.

BACKGROUND: Plasma-derived (pd) or recombinant (r) therapeutic factor VIII proteins (FVIIIs) are infused to arrest/prevent bleeding in patients with hemophilia A (PWHA). However, FVIIIs are neutralized if anti-FVIII-antibodies (inhibitors) develop. Accumulating evidence suggests that pdFVIIIs with von Willebrand factor (VWF) are less immunogenic than rFVIIIs and that distinct rFVIIIs are differentially immunogenic. Since inhibitor development is T-helper-cell-dependent, human leukocyte antigen (HLA)-class-II (HLAcII) molecules constitute an important early determinant. OBJECTIVES: Use dendritic cell (DC)-protein processing/presentation assays with mass-spectrometric and peptide-proteomic analyses to quantify the DP-bound, DQ-bound, and DR-bound FVIII-derived peptides in individual HLAcII repertoires and compare the immunogenic potential of six distinct FVIIIs based on their measured peptide counts. PATIENTS/METHODS: Monocyte-derived DCs from normal donors and/or PWHA were cultured with either: Mix-rFVIII, a VWF-free equimolar mixture of a full-length (FL)-rFVIII [Advate® (Takeda)] and four distinct B-domain-deleted (BDD)-rFVIIIs [Xyntha® (Pfizer), NovoEight® (Novo-Nordisk), Nuwiq® (Octapharma), and Afstyla® (CSL Behring GmBH)]; a pdFVIII + pdVWF [Beriate® (CSL Behring GmBH)]; Advate ± pdVWF; Afstyla ± pdVWF; and Xyntha + pdVWF. RESULTS: We showed that (i) Beriate had a significantly lower immunogenic potential than Advate ± pdVWF, Afstyla - pdVWF, and Mix-rFVIII; (ii) distinct FVIIIs differed significantly in their immunogenic potential in that, in addition to (i), Afstyla + pdVWF had a significantly lower immunogenic potential than Beriate, while the immunogenic potential of Beriate was not significantly different from that of Xyntha + pdVWF; and (iii) rFVIIIs with pdVWF had significantly lower immunogenic potentials than the same rFVIIIs without pdVWF. CONCLUSIONS: Our results provide HLAcII peptidomic level explanations for several important clinical observations/issues including the differential immunogenicity of distinct FVIIIs and the role of HLAcII genetics in inhibitor development.

Clinical Care of Bone Health in Patients on the Immune Tolerance Induction's Protocols With an Immunosuppressive Agent for Inhibitor Eradication in Hemophilia.

Nowadays, the development of factor VIII and IX inhibitors in patients with hemophilia is considered as the most challenging in the treatment of hemophilia. Immune tolerance induction (ITI) therapy is an approach for eradication of inhibitors. Some ITI protocols are routinely in use for the eradication of inhibitors in patients with hemophilia. Moreover, such a therapeutic regimen may facilitate the tendency to reduced bone density in patients with inhibitor. This study scheduled to investigate whether that predisposing role of ITI protocols with an immunosuppressive agent has considered or not. By a literature review, published ITI protocols in hemophilia with inhibitors were evaluated. Among them, 51 papers found and studied thoroughly. None of them had performed the bone mineral examination in patients with hemophilia and inhibitor under treatment. Since there are 2 coexisting facilitating factors in these protocols, considering the bone mineral density study for patients with inhibitor who are undergoing ITI protocols with an immunosuppressive agent is recommended.