Moving towards Normalization of haemostasis and health equity: Evolving treatment goals for haemophilia A.

BACKGROUND: Treatment options for people with haemophilia are evolving at a rapid pace and a range of prophylactic treatment options using various technologies are currently available, each with their own distinct safety and efficacy profile. TREATMENT GOALS: The access to replacement therapy and prophylaxis has driven a dramatic reduction in mortality and resultant increase in life expectancy. Beyond this, the abolition of bleeds and preservation of joint health represent the expected, but rarely attained, goals of haemophilia treatment and care. These outcomes also do not address the complexity of health-related quality of life impacted by haemophilia and its treatment. CONCLUSION: Capitalizing on the major potential of therapeutic innovations, 'Normalization' of haemostasis, as a concept, should include the aspiration of enabling individuals to live as normal a life as possible, free from haemophilia-imposed limitations. To achieve this-being supported by the data reviewed in this manuscript-the concept of haemostatic and life Normalization needs to be explored and debated within the wider multidisciplinary teams and haemophilia community.

Unravelling the Spectrum of von Willebrand Factor Variants in Quantitative Von Willebrand Disease: Results from a German Cohort Study.

INTRODUCTION: Von Willebrand disease (VWD), the most prevalent hereditary bleeding disorder, results from deficiency of von Willebrand factor (VWF). OBJECTIVES: This large cohort study aims to offer a comprehensive exploration of mutation spectra and laboratory features in quantitative VWF deficiencies, shedding light on genetic underpinnings and genotype-phenotype associations. PATIENTS/METHODS: Our cohort consisted of 221 Caucasian index patients with quantitative VWD, along with 47 individuals whose plasma VWF levels fell within the lower normal boundaries (50-70%). We conducted comprehensive VWF assays and genetic analyses, encompassing VWF gene sequencing, copy number variation investigations, and bioinformatic assessments. RESULTS: Following ISTH-SSC VWF guidelines, 77 index patients characterized as type 1 VWD (VWF:Ag < 30%), 111 as type 1 VWD (VWF:Ag 30-50%), and as 33 type 3 VWD. Mutation detection rates were 88%, 65%, and 92%, respectively. Notably, blood group O overrepresentation was evident in type 1 with VWF:Ag of 30-50%, particularly among mutation-negative patients, suggesting a potential causal role of blood group O. A total of 223 VWF variants, comprising 147 distinct variations, were identified in quantitative VWD patients, of which 57 were novel variants (39%). Additionally, approximately 70% of individuals with VWF levels within the lower normal boundaries (50-70%) displayed VWF variants. CONCLUSION: Our data advances our understanding of the molecular mechanisms underlying quantitative VWD, offering valuable insights for future research and clinical management. Distinct mutation patterns were observed among subgroups, particularly the contrast between type 1 VWD (VWF:Ag <30%) and type 1 (VWF:Ag 30-50%), an area with limited prior investigation.

Investigation of von Willebrand factor multimer abnormalities before and after aortic valve replacement using the Hydragel-5 assay.

BACKGROUND: Severe aortic stenosis (sAS) is associated with acquired von Willebrand syndrome (AVWS) by loss of high-molecular-weight multimers (HMWM) of von Willebrand factor (VWF), potentially resulting in perioperative bleeding. Analysis of VWF multimers remains challenging. Recently, the new, rapid Hydragel 5 assay has been developed, using electrophoretic protein separation for dividing VWF-multimers into low (LMWM), intermediate (IMWM), and HMWM, the hemostatically active part of VWF. Here, we evaluated its impact on predicting blood loss in presence of AVWS after surgical aortic valve replacement (SAVR). METHODS: We prospectively examined 52 patients (age: 68 ± 7 years; 54 % male) admitted to SAVR. They were divided in two groups (A: normal VWF, n = 28; B: abnormal VWF, n = 24, defined as VWF-activity/antigen (VWF:Ac/Ag)-ratio < 0.7 and/or HMWM loss). Blood samples and echocardiographic data were collected before, seven days and three months after SAVR. Blood loss and transfusions were recorded. RESULTS: Baseline characteristics and clinical data were similar in both groups. HMWM loss was present in 38.5 % of all patients. HMWM, the VWF:Ac/Ag- and HMWM/(IMWM+LMWM)-ratios were significantly decreased preoperatively in group B but normalized after SAVR. Bleeding, re-thoracotomy and transfusion rates were comparable. HMWM loss was inversely correlated with the peak aortic gradient (Pmax) and positively with the aortic valve area (AVA), while HMWM/(IMWM+LMWM)-ratio negatively correlated with the mean aortic gradient (Pmean). CONCLUSION: HMWM and HMWM/(IMWM+LMWM)-ratio inversely correlate with severity of AS and normalize after SAVR. The Hydragel-5 assay's might be valuable for routine diagnostics to assess bleeding risk and postoperative normalization of AS and VWF abnormalities in SAVR patients.

Quantitative analysis of heme and hemoglobin for the detection of intravascular hemolysis.

BACKGROUND: Intravascular hemolysis is associated with massive release of hemoglobin and consequently labile heme into the blood, resulting in prothrombotic and proinflammatory events in patients. Though heme is well-known to participate in these adverse effects, it is not monitored. Instead, haptoglobin and hemoglobin serve as clinical biomarkers. The quantification of labile heme together with hemoglobin, however, should be considered in clinical diagnosis as well, to obtain a complete picture of the hemolytic state in patients. So far, quantification techniques for labile heme were not yet systematically analyzed and compared for their clinical application potential, especially in the presence of hemoglobin. RESULTS: Two commercial assays (Heme Assay Kit®, Hemin Assay Kit®) and five common approaches (pyridine hemochromogen assay, apo-horseradish peroxidase-based assay, UV/Vis spectroscopy, HPLC, mass spectrometry) were analyzed concerning their linearity, accuracy, and precision, as well as their ability to distinguish between hemoglobin-bound heme and labile heme. Further, techniques for the quantification of hemoglobin (Harboe method, SLS method, Hemastix®) were included to study their selectivity for hemoglobin and potential interference by the presence of labile heme. Both, indirect and direct approaches were suitable for the determination of a wide concentration of heme (∼0.02-45 µM) and hemoglobin (∼0.002-17 µM). A clear distinction between hemoglobin-bound heme and labile heme with one method was not possible. Thus, a novel combined approach is presented and applied to human and porcine plasma samples for the determination of hemoglobin and labile heme. SIGNIFICANCE: Our results demonstrate the need to develop improved techniques to differentiate labile and protein-bound heme for early detection of intravascular hemolysis. Here, we present a novel strategy by combining two spectroscopic methods, which is most reliable as an easy-to-use tool for the determination of hemoglobin and heme levels in plasma samples for the diagnosis of intravascular hemolysis and in basic biomedical research.

High Prevalence of F2 20210G > A in Splanchnic Vein Thrombosis and Cerebral Venous Sinus Thrombosis: A Retrospective Cohort Study of Patients with Thrombosis in Atypical Sites.

INTRODUCTION: Atypical sites for thrombosis include deep vein thrombosis (DVT) of the upper extremity (UE-DVT), splanchnic vein thrombosis (SVT), and cerebral venous sinus thrombosis (CVST). In addition to specific pathogenic factors, their underlying mechanisms share similarities with typical venous thromboembolism (VTE), namely, DVT of the lower extremity and/or pulmonary embolism, but are less understood. METHODS: Records of unselected patients with a history of typical VTE (n = 2,011), UE-DVT (n = 117), SVT (n = 83), and CVST (n = 82), who were referred to the Institute in Bonn for ambulatory thrombophilia testing, were retrospectively analyzed. Acquired and hereditary thrombosis risk factors were comparatively assessed. RESULTS: UE-DVT was characterized by a high rate (50.4%) of site-specific acquired risk factors. Compared with typical VTE, SVT was more frequently associated with systemic inflammation, infection, or malignancy (2.2 vs. 12.0%, p = 3·10-8) and the JAK2 V617F mutation was present in 16.9%. In CVST compared with typical VTE, demographics and higher rates of oral contraception (43.2 vs. 57.6%, p = 0.011) and pregnancy (4.2 vs. 10.9%, p = 0.012) suggest a significant hormonal influence on etiology. While the prevalence of inhibitor deficiencies and factor V Leiden mutation did not differ between cohorts, the prevalence of F2 20210G > A was higher in SVT (15.7%, p = 0.003) and CVST (15.9%, p = 0.003) than in typical VTE (7.0%). CONCLUSION: The cohorts with thrombosis in atypical sites showed distinctive patterns of acquired risk factors. Further studies are warranted to provide additional mechanistic insight into the role of hormonal influence in CVST and the contribution of F2 20210G > A to the development of SVT and CVST.

Self-conducted sonographic monitoring of the knee in patients with haemophilia-A feasibility study.

INTRODUCTION/AIM: To evaluate whether patients with haemophilia (PwH) can be enabled to perform ultrasonography (US) of their knees without supervision according to the Haemophilia Early Arthropathy Detection with Ultrasound (HEAD-US) protocol and whether they would be able to recognize pathologies. METHODS: Five PwH (mean age 29.6 years, range 20-48 years) were taught the use of a portable US device and the HEAD-US protocol. Subsequently, the patients performed US unsupervised at home three times a week for a total of 6 weeks with a reteaching after 2 weeks. All images were checked for mapping of the landmarks defined in the HEAD-US protocol by a radiologist. In a final test after the completion of the self-sonography period, participants were asked to identify scanning plane and potential pathology from US images of other PwH. RESULTS: On the images of the self-performed scans, 82.7% of the possible anatomic landmarks could be identified and 67.5% of the requested images were unobjectionable, depicting 100% of the required landmarks. There was a highly significant improvement in image quality following reteaching after 2 weeks (74.80 ± 36.88% vs. 88.31 ± 19.87%, p < .001). In the final test, the participants identified the right scanning plane in 85.0% and they correctly identified pathology in 90.0% of images. CONCLUSION: Appropriately trained PwH can perform the HEAD-US protocol of their knee with high quality and are capable to identify pathologic findings on these standardized images. Asynchronous tele-sonography could enable early therapy adjustment and thereby possibly reduce costs.

Emicizumab versus immunosuppressive therapy for the management of acquired hemophilia A.

BACKGROUND: Acquired hemophilia A (AHA) is an autoimmune bleeding disorder caused by neutralizing antibodies against coagulation factor VIII (FVIII). Immunosuppressive therapy (IST) is standard of care to eradicate autoantibody production and protect from further bleeding but carries a risk of severe infection and mortality in frail patients with AHA. Recently, emicizumab has been studied for its potential to reduce the need for early and aggressive IST. OBJECTIVE: To compare outcomes of two studies that used either IST (GTH-AH 01/2010; n=101) or prophylaxis with emicizumab (GHT-AHA-EMI; n=47) early after diagnosis of AHA. METHODS: Baseline characteristics were balanced by propensity score matching. Primary endpoint was the rate of clinically relevant new bleeds during the first 12 weeks; secondary endpoints were adverse events and overall survival. RESULTS: The negative binominal model-based bleeding rate was 68% lower with emicizumab as compared with IST (incident rate ratio 0.325, 95% confidence interval (CI) 0.182-0.581). No difference was apparent in the overall frequency of infections (emicizumab 21%, IST 29%) during the first 12 weeks, but infections were less often fatal in emicizumab treated patients (0%) compared with IST treated patients (11%). Thromboembolic events occurred less often with emicizumab (2%) than IST (7%). Overall survival after 24 weeks was better with emicizumab (90% versus 76%, HR 0.44; 95% CI 0.24-0.81). CONCLUSION: Using emicizumab instead of IST in the early phase after initial diagnosis of AHA reduced bleeding and fatal infections, and improved overall survival.

Emicizumab is well tolerated and effective in people with congenital hemophilia A regardless of age, severity of disease, or inhibitor status: a scoping review.

Background: With the treatment landscape continually evolving, it is vital that the hemophilia community have an overview of all published data for approved therapies, such as emicizumab, to support shared decision making. Objectives: To bring together the clinical and real-world data for emicizumab use in people with congenital hemophilia A, regardless of age, disease severity, or factor VIII inhibitor status. Key focus areas were safety, efficacy, and quality of life (QoL). Methods: This scoping review used citation databases (PubMed, Embase, and the Cochrane Library) and manual searches of abstract books. Publications reporting original data for emicizumab in people with hemophilia A, published in English after December 2014, and reporting select endpoints were included. This narrative synthesis focused on zero bleeds, treated annualized bleeding rate (ABR), adverse events, and QoL measures. Results: Overall, 97 publications were included (cut-off: August 9, 2022). Treated ABR remained low (calculated mean and median treated ABRs ranged between 0.7-1.3 and 0.0-1.4, respectively), and the median percentage of people with zero treated bleeds was 66.7%. The proportion of people experiencing treatment-related adverse events ranged from 0.0% to 60.0%; most were injection-site reactions. Across 37 publications reporting on safety and enrolling >2300 individuals, 11 thrombotic events and 4 thrombotic microangiopathies were reported. Data from well-established tools show QoL benefits with emicizumab. Conclusion: This scoping review consolidates the global published experience for emicizumab in people with hemophilia A and supports the fact that emicizumab has an acceptable safety profile, is effective and efficacious in bleed prevention, and is associated with improvements in QoL.

Emicizumab is efficacious in people with hemophilia A with comorbidities aged ≥50 years: analysis of 4 phase III trials.

Background: The treatment of older people with hemophilia A (HA) can be complicated by comorbidities. Objectives: This post hoc analysis evaluates the efficacy and safety of emicizumab in people with HA aged ≥50 years with cardiovascular (CV) risk factors or HIV and/or hepatitis C virus (HCV) infection. Methods: The HAVEN 1 (NCT02622321), HAVEN 3 (NCT02847637), HAVEN 4 (NCT03020160), and STASEY (NCT03191799) studies enrolled adults/adolescents with severe HA. Participants were categorized as having a comorbidity if they had any CV risk factors (including history of CV disease, hypertension, diabetes, hyperlipidemia, prior stroke, or obesity), HIV, and/or HCV infection. Efficacy and safety outcomes were compared by age (<50 vs ≥50 years). Results: Of 504 participants at data cutoff, 408 were aged <50 years and 96 were aged ≥50 years. In people with HA aged <50 years, 26.7% had ≥1 CV risk factor and 29.4% had HIV and/or HCV infection. In people with HA aged ≥50 years, 72.9% had ≥1 CV risk factor and 74.0% had HIV and/or HCV infection. The mean (95% CI) annualized bleed rate for treated bleeds was 1.29 (0.07-6.06) for people with HA aged <50 years and 1.82 (0.19-6.93) for people with HA aged ≥50 years. No significant differences in annualized bleed rates were observed for those with comorbidities compared with those without. Safety outcomes were similar regardless of age. Conclusion: This pooled analysis suggests that emicizumab efficacy and safety in people with HA aged ≥50 years with CV and HIV/HCV comorbidities were consistent with those in people with HA aged <50 years enrolled in the HAVEN 1, 3, and 4 and STASEY studies.

Comparative Effectiveness of Valoctocogene Roxaparvovec and Prophylactic Factor VIII Replacement in Severe Hemophilia A.

INTRODUCTION: A prospective, non-interventional study (270-902) followed 294 adults with severe hemophilia A (SHA) receiving prophylactic factor VIII (FVIII). From these participants, 112 rolled over into a single-arm, multicenter, phase 3 trial (GENEr8-1; NCT03370913) that evaluated efficacy and safety of valoctocogene roxaparvovec, a gene therapy that provides endogenous FVIII in individuals with SHA. Participants from 270-902 who did not roll over provide an opportunity for a contemporaneous external control. Therefore, the comparative effectiveness of valoctocogene roxaparvovec vs FVIII prophylaxis was evaluated using propensity scoring (PS). METHODS: This post hoc analysis compared 112 participants from GENEr8-1 (treated cohort) to 73 participants in 270-902 who did not enroll in GENEr8-1 (control cohort). The primary analysis used standardized mortality ratio weighting to re-weight baseline characteristics of the control cohort to better match the treated cohort. Mean annualized bleeding rates (ABR) for treated and all bleeds were compared between cohorts along with the proportion of participants with zero bleeds (treated and all bleeds). Sensitivity and scenario analyses were also conducted. RESULTS: PS adjustments reduced differences in baseline characteristics between cohorts. Mean treated (4.40 vs 0.85; P < 0.001) and all (5.01 vs 1.54; P < 0.001) ABR were significantly lower, and the proportions of participants with zero treated bleeds (82.1% vs 32.9%; P < 0.001) and all bleeds (58.0% vs 28.5%; P < 0.001) were significantly higher in GENEr8-1. CONCLUSIONS: PS-adjusted analyses were consistent with prior intra-individual comparisons. Compared with participants receiving prophylactic FVIII, the participants receiving valoctocogene roxaparvovec experienced lower ABR, and a higher proportion had zero bleeds. TRAIL REGISTRATION: ClinicalTrials.gov identifier, NCT03370913.

Hemophilia A is a bleeding disorder where blood is unable to clot properly because of a missing protein called factor VIII (FVIII). Individuals with hemophilia A have an increased risk of prolonged bleeding episodes that can be deadly. To prevent bleeding, people with severe hemophilia A need to routinely inject treatment into the skin or vein (prophylaxis). While effective, some people find the time and effort needed to maintain frequent injections difficult, since some forms of the prophylaxis must be administered in a hospital setting. Valoctocogene roxaparvovec is a gene therapy where a single injection provides instructions to the liver of individuals with hemophilia A to make the missing protein (FVIII). Then, their own liver cells can produce FVIII protein and prevent bleeding episodes. The valoctocogene roxaparvovec clinical trial compared the number of treated bleeding episodes participants had prior to gene therapy, while using prophylaxis, with the number of treated bleeding episodes after gene therapy. On average, after gene therapy, participants had 4.1 fewer treated bleeding episodes per year. In this study, mathematical models were used to explore how differences in participant's physical characteristics, such as body weight or medical history, might influence the effectiveness of gene therapy. Even when considering differences in the participants' physical characteristics, the gene therapy reduced treated bleeding episodes by 3.6 events per year. This study confirms results originally presented from the valoctocogene roxaparvovec clinical trial and reinforces confidence in the ability of valoctocogene roxaparvovec to reduce bleeding outcomes for participants with hemophilia A.

Three-year outcomes of valoctocogene roxaparvovec gene therapy for hemophilia A.

BACKGROUND: Valoctocogene roxaparvovec transfers a human factor (F)VIII coding sequence into hepatocytes of people with severe hemophilia A to provide bleeding protection. OBJECTIVES: To present 3-year efficacy and safety in the multicenter, open-label, single-arm, phase 3 GENEr8-1 trial. METHODS: GENEr8-1 enrolled 134 adult males with severe hemophilia A who were receiving FVIII prophylaxis. Efficacy endpoints included annualized bleeding rate, annualized FVIII utilization, FVIII activity (chromogenic substrate assay; imputed as 1 IU/dL at baseline and 0 IU/dL after discontinuation), and the Haemophilia-Specific Quality of Life Questionnaire for Adults. Safety was assessed by adverse events (AEs). RESULTS: At week 156, 131 of 134 participants remained in the study; overall, 17 of 134 resumed prophylaxis. Mean annualized bleeding rate for treated bleeds decreased from 4.8 (SD, 6.5) bleeds/y at baseline to 0.8 (SD, 2.3; P < .0001) bleeds/y after prophylaxis (prophylaxis cessation to last follow-up) and 0.97 (SD, 3.48) bleeds/y during year 3. Annualized FVIII utilization decreased 96.8% from baseline after prophylaxis and 94.2% during year 3. At week 156, mean and median FVIII activity were 18.4 (SD, 30.8) and 8.3 IU/dL, respectively. FVIII activity decrease was lower between years 2 and 3 than between years 1 and 2. At the end of year 3, clinically meaningful improvements in the Haemophilia-Specific Quality of Life Questionnaire for Adults Total Score were observed (mean change from baseline, 6.6; 95% CI, 4.24-8.87; P < .0001). Mild alanine aminotransferase elevations remained the most common AE during year 3 (23.7% of participants). A serious AE of B-cell acute lymphoblastic leukemia was considered unrelated to treatment. CONCLUSION: Hemostatic efficacy was maintained, and safety remained unchanged from previous years.

In-depth structure-function profiling of the complex formation between clotting factor VIII and heme.

BACKGROUND AND AIMS: Blood disorders, such as sickle cell disease, and other clinical conditions are often accompanied by intravascular hemolytic events along with the development of severe coagulopathies. Hemolysis, in turn, leads to the accumulation of Fe(II/III)-protoporphyrin IX (heme) in the intravascular compartment, which can trigger a variety of proinflammatory and prothrombotic reactions. As such, heme binding to the blood coagulation proteins factor VIII (FVIII), fibrinogen, and activated protein C with functional consequences has been demonstrated earlier. METHODS: We herein present an in-depth characterization of the FVIII-heme interaction at the molecular level and its (patho-)physiological relevance through the application of biochemical, biophysical, structural biology, bioinformatic, and diagnostic tools. RESULTS: FVIII has a great heme-binding capacity with seven heme molecules associating with the protein. The respective binding sites were identified by investigating heme binding to FVIII-derived peptides in combination with molecular docking and dynamic simulation studies of the complex as well as cryo-electron microscopy, revealing three high-affinity and four moderate heme-binding motifs (HBMs). Furthermore, the relevance of the FVIII-heme complex formation was characterized in physiologically relevant assay systems, revealing a ~ 50 % inhibition of the FVIII cofactor activity even in the protein-rich environment of blood plasma. CONCLUSION: Our study provides not only novel molecular insights into the FVIII-heme interaction and its physiological relevance, but also strongly suggests the reduction of the intrinsic pathway and the accentuation of the final clotting step (by, for example, fibrinogen crosslinking) in hemolytic conditions as well as a future perspective in the context of FVIII substitution therapy of hemorrhagic events in hemophilia A patients.

Noninterventional study assessing joint health in persons with hemophilia A after switching to turoctocog alfa pegol: design of pathfinderReal.

Background: Joint damage affects the quality of life of persons with hemophilia A. The long-term safety and efficacy of turoctocog alfa pegol (N8-GP) prophylaxis in persons with hemophilia A has been investigated in pivotal phase 3 trials in children, adolescents, and adults (pathfinder program). However, there is a lack of data on joint health in adult persons with hemophilia A treated with N8-GP. Objectives: To describe the design of the ongoing pathfinderReal study investigating the joint health status in adult persons with hemophilia A after switching to N8-GP. Methods: pathfinderReal is a multicountry, noninterventional, single-arm study (NCT05621746) of joint health in adult (≥18 years) male persons with hemophilia A who have switched to N8-GP. Patients enrolled in other interventional studies and those who have previously terminated N8-GP treatment will be excluded. Approximately 124 adults with hemophilia A will be enrolled and followed up for a maximum of 24 months. Data from routine clinical assessments of patients' joint health will be collected. The primary endpoint is change in Hemophilia Joint Health Score (defined as a change in total score of ≤2) from initiation of N8-GP treatment until the end of the study. Secondary endpoints include number of bleeding episodes, number and resolution of target joints, patient-reported outcomes of problem joint score, pain score, and change in physical function levels. An exploratory endpoint is included to measure the number of patients achieving improved Hemophilia Joint Health Score from the initiation of N8-GP until the end of the study. Conclusion: The pathfinderReal study will provide insights regarding the impact of N8-GP on joint health in persons with hemophilia A in a real-world setting.

Comprehensive domain-specific analysis and immunoglobulin G profiling of anti-factor VIII antibodies using a bead-based multiplex immunoassay.

BACKGROUND: Antibodies against factor (F)VIII are a major complication in the treatment of patients with severe hemophilia A. The Nijmegen-Bethesda assay (NBA) is the gold standard for detection of neutralizing antibodies (inhibitors), whereas both inhibitors and nonneutralizing antibodies can be detected by immunoassays such as enzyme-linked immunosorbent assay (ELISA) and multiplex bead-based assays. OBJECTIVES: Evaluation of an in-house Luminex bead-based assay (LumiTope) compared with a commercially available ELISA and NBA. METHODS: The LumiTope method comprised full-length and B-domain-deleted FVIII as well as 9 purified FVIII single or multidomains. The respective proteins were coupled to magnetic beads to detect domain-specific immunoglobulin (IgG; IgG1-4) anti-FVIII antibodies in a large cohort of patients with hemophilia A with and without inhibitors. RESULTS: Overall, LumiTope assay had a high sensitivity (94.9%) and specificity (91.2%), particularly in patients with low-titer inhibitors compared with ELISA (sensitivity of 72.2% vs 27.7%). IgG4 was the most abundant IgG subclass in NBA-positive patients. NBA-positive and -negative patients showed different domain profiles. Patients with genetic variants in the heavy chain predominantly exhibited antibodies specific to this chain, while those with a light-chain variant showed a more diverse distribution of antibody specificities. Patients with an intron 22 inversion resembled those with a light-chain defect, with a majority of antibodies targeting the light chain. CONCLUSION: LumiTope assay provides a sensitive and specific method for not only detection but also domain specification of anti-FVIII-antibodies. Implementation of bead-based assays could improve antibody detection, profiling, and comparability of results and complement NBA.

Safety and Efficacy of Recombinant Fusion Protein Linking Coagulation Factor IX with Albumin (rIX-FP) in Previously Untreated Patients with Hemophilia B.

Introduction Recombinant fusion protein linking coagulation factor IX (FIX) with albumin (rIX-FP) has been shown to be an effective, well-tolerated treatment for patients with severe hemophilia B who had previously received factor replacement therapy. This study investigated the safety and efficacy of rIX-FP in previously untreated patients (PUPs). Methods Patients with moderately severe/severe hemophilia B (≤2% FIX) previously untreated with FIX replacement products received rIX-FP (25-75 IU/kg) prophylaxis weekly or on-demand treatment over ≥50 exposure days (EDs). Primary outcomes were the number of patients who developed FIX inhibitors and mean incremental recovery (IR) following a 50 IU/kg dose of rIX-FP. Secondary outcomes included incidence of adverse events (AEs) and annualized bleeding rates (ABRs). Results In total, 12 PUPs with a median age of 0 years (range, 0-11 years) were treated with rIX-FP for a median of 50 EDs (6/12 prophylaxis; 6/12 on-demand then prophylaxis). Overall, 11/12 patients did not develop FIX inhibitors; one 11-year-old patient developed an inhibitor against FIX after 8 EDs and was ultimately withdrawn. Mean (standard deviation) IR was 1.2 (0.4, n = 8) (IU/dL)/(IU/kg). Of the 137 treatment-emergent AEs recorded, five were attributed to rIX-FP. On the prophylaxis regimen, median ABR was 1.0 (range, 0-3.9, n = 12). No thromboembolic events or deaths occurred during the study. Conclusion This study provides data to support the safety and efficacy of rIX-FP in PUPs requiring on-demand or prophylactic treatment for moderately severe/severe hemophilia B, consistent with results in previously treated patients. Overall, 1/12 patients developed an inhibitor against FIX.

Fibrinolysis biomarker, thrombin, and activated protein C level alterations after coagulation activation depend on type of thrombophilia and clinical phenotype.

Background: Recently, we have shown alterations in the anticoagulant response to recombinant activated factor VII (rFVIIa)-induced coagulation activation in patients with thrombophilia. Objectives: This study aimed to extend this in vivo model to fibrinolysis biomarkers. Methods: This interventional in vivo study included 56 patients with thrombophilia and previous venous thromboembolism (VTE+), 38 without VTE (VTE-), and 35 healthy controls. Plasma levels of D-dimer, plasmin-α2-antiplasmin (PAP) complex, and plasminogen activator inhibitor-1 (PAI-1) were monitored for over 8 hours after rFVIIa infusion (15 µg/kg) along with thrombin markers and activated protein C (APC). Results: Throughout cohorts, median PAP increased by 40% to 52% (P < 3.9 × 10-10) and PAI-1 decreased by 59% to 79% (P < 3.5 × 10-8). In contrast to thrombin-antithrombin (TAT) complex, which also increased temporarily (44% to 115%, P < 3.6 × 10-6), changes in PAP and PAI-1 did not reverse during the observation period. The area under the measurement-time curves (AUCs) of PAP and TAT, which are measures of plasmin and thrombin formation, respectively, were each greater in the VTE+ cohort than in healthy controls (median PAP-AUC = 0.48 vs 0.27 ng·h/L [P = .003], TAT-AUC = 0.12 vs 0.03 nmol·h/L [P = 2.5 × 10-4]) and were correlated with one another (r = 0.554). As evidenced by the respective AUCs, asymptomatic factor (F)V Leiden carriers showed less PAP formation (0.22 vs 0.41 ng·h/L, P = 9 × 10-4), more pronounced PAI-1 decline (0.10 vs 0.18 ng·h/L, P = .01), and increased APC formation (28.7 vs 15.4 pmol·h/L, P = .02) than those within the VTE+ group (n = 19 each). Conclusion: rFVIIa-induced thrombin formation is associated with fibrinolysis parameter changes outlasting the concomitant anticoagulant response. Both correlate with thrombosis history in FV Leiden and might help explain its variable clinical expressivity.

The role of microRNAs in defining LSECs cellular identity and in regulating F8 gene expression.

Introduction: Coagulation Factor VIII (FVIII) plays a pivotal role in the coagulation cascade, and deficiencies in its levels, as seen in Hemophilia A, can lead to significant health implications. Liver sinusoidal endothelial cells (LSECs) are the main producers and contributors of FVIII in blood, a fact we have previously elucidated through mRNA expression profiling when comparing these cells to other endothelial cell types. Methods: Our current investigation focuses on small microRNAs, analyzing their distinct expression patterns across various endothelial cells and hepatocytes. Results: The outcome of this exploration underscores the discernible microRNAs expression differences that set LSECs apart from both hepatocytes (193 microRNAs at p < 0.05) and other endothelial cells (72 microRNAs at p < 0.05). Notably, the 134 and 35 overexpressed microRNAs in LSECs compared to hepatocytes and other endothelial cells, respectively, shed light on the unique functions of LSECs in the liver. Discussion: Our investigation identified a panel of 10 microRNAs (miR-429, miR-200b-3p, miR-200a-3p, miR-216b-5p, miR-1185-5p, miR-19b-3p, miR-192-5p, miR-122-5p, miR-30c-2-3p, and miR-30a-5p) that distinctly define LSEC identity. Furthermore, our scrutiny extended to microRNAs implicated in F8 regulation, revealing a subset (miR-122-5p, miR-214-3p, miR-204-3p, and miR-2682-5p) whose expression intricately correlates with F8 expression within LSECs. This microRNA cohort emerges as a crucial modulator of F8, both directly through suppression and indirect effects on established F8-related transcription factors. The above microRNAs emerged as potential targets for innovative therapies in Hemophilia A patients.

Classification of recombinant factor VIII products and implications for clinical practice: A systematic literature review.

INTRODUCTION: Consensus over the definition of recombinant factor VIII (rFVIII) product classification in haemophilia A is lacking. rFVIII products are often classified as standard half-life (SHL) or extended half-life (EHL); despite this, no universally accepted definition currently exists. One proposed definition includes half-life, area under the curve, and technology designed to extend half-life; however, the International Society on Thrombosis and Haemostasis defines activity over time as the most intuitive information for building treatment regimens and the World Federation of Hemophilia describes rFVIII product classification in terms of infusion frequency. AIM: To summarise published data on the clinical and pharmacokinetic criteria used to define rFVIII product classification. METHODS: PubMed and EMBASE database searches of English-language articles (2002-2022) were conducted using search strings to identify the relevant population, intervention, and outcomes (e.g., clinical and pharmacokinetic parameters). Articles then underwent title/abstract and full-text screens. RESULTS: Among 1147 identified articles, 62 were included. Half-life was the most widely reported outcome with no clear trends or product groupings observed. No clear groupings emerged among other outcomes, including infusion frequency, consumption, and efficacy. As activity over time was reported in few articles, further investigation of its relevance to rFVIII product classification is warranted. CONCLUSION: The findings of this systematic literature review suggest that parameters other than half-life might be important for the development of a comprehensive and clinically relevant rFVIII product classification definition. There seems to be an opportunity to consider parameters that are clinically meaningful and useful for shared decision-making in haemophilia A treatment.

Physical activity and factor VIII levels in patients with haemophilia: A real-world prospective observational study.

INTRODUCTION: Regular physical activity (PA) is recommended for patients with haemophilia (PwH). For PwH it is crucial to ensure a sufficient factor level to prevent PA-induced bleedings. However, there is a gap in the literature dealing with specific factor levels, which are needed when performing specific types of PA. AIM: To provide data on factor VIII (FVIII) levels at the start of PA performed by PwH. METHODS: In this prospective 12-month real-world observational study, 23 PwH recorded every PA they performed and the FVIII levels at the start of the PA using a pharmacokinetic application. PA types were clustered according to the collision and injury risk into three categories (Cat I = low, Cat II = medium, Cat III = high risk). Haemophilia Joint Health Scores (HJHS) were performed at baseline, after 6 and 12 months. RESULTS: 795 PA sessions of Cat I, 193 of Cat II, and 23 of Cat III were documented. FVIII levels at the start of PA were different between categories (Cat I: 29.8 ± 32.1%, Cat II: 38.3 ± 33.4%, Cat III: 86.6 ± 29.2%). Out of all PA sessions, 145 (14%) were performed at a factor level of ≤3%. Three PA-induced bleeding occurred. Baseline HJHS was 14.5 ± 13.6 points and did not change throughout the study. CONCLUSION: This study provides real-life data on FVIII levels at the start of 1011 PA sessions. PwH are mainly active in low-risk sports with higher FVIII levels observed in Cat II and III, respectively. Only three PA-induced bleeding occurred, even though several PA were started with low FVIII levels.

Genetic Landscape of Factor VII Deficiency: Insights from a Comprehensive Analysis of Pathogenic Variants and Their Impact on Coagulation Activity.

Congenital factor VII (FVII) deficiency is a rare genetic bleeding disorder characterized by deficient or reduced activity of coagulation FVII. It is caused by genetic variants in the F7 gene. We aimed to evaluate the rate of detection of pathogenic variants in the F7 gene in a large group of patients with FVII deficiency and investigate the correlations between the F7 genotype and FVII activity (FVII:C). Moreover, the influence of the common genetic variant rs6046: c.1238G>A; p.(Arg413Gln), designated as the M2 allele, on FVII:C was investigated. Genetic analysis of the F7 gene was performed on 704 index patients (IPs) using either direct Sanger- or next-generation sequencing. Genetic variants were detected in 390 IPs, yielding a variant detection rate (VDR) of 55%. Notably, the VDR exhibited a linear decline with increasing FVII:C levels. We identified 124 genetic variants, of which 48 were not previously reported. Overall, the frequency of the M2 allele was considerably higher in patients with mild deficiency (FVII:C > 20 IU/dl). Furthermore, IPs lacking an identified pathogenic variant exhibited a significantly higher prevalence of the M2 allele (69%) compared to IPs with a disease-causing variant (47%). These results strongly support the association of the M2 allele with decreased FVII:C levels. This study shows the utility of FVII:C as a predictive marker for identifying pathogenic variants in patients with FVII deficiency. The M2 allele contributes to the reduction of FVII:C levels, particularly in cases of mild deficiency.