Emicizumab in Type 3 von Willebrand Disease: Report of a Case with an Alloantibody and Literature Review.

Type 3 von Willebrand disease (VWD), the most severe form of VWD, is an inherited recessive bleeding disorder caused by the complete deficiency of von Willebrand factor (VWF). The reported prevalence is 1 per million but varies worldwide according to the frequency of consanguineous marriages. The clinical phenotype is characterized not only by mucocutaneous bleedings, but also by hemarthroses and muscle hematoma, as in patients with moderate hemophilia. Long-term prophylaxis with factor (F)VIII/VWF concentrates is recommended in patients with a history of severe and frequent bleeds. A rare complication of replacement therapy is the development of alloantibodies against VWF, with the consequences of an ineffective therapy and risk of anaphylactic reactions upon treatment. Emicizumab is the first bispecific monoclonal antibody that mimics FVIII coagulant activity and is approved for prophylaxis of bleeding in patients with inherited hemophilia A with or without inhibitors and recently also for acquired hemophilia. In this manuscript we report and discuss available data in the literature on the use of emicizumab in type 3 VWD and describe the case of a female patient with type 3 VWD with a history of alloantibodies against VWF and posttransfusion anaphylaxis, recently and successfully put on off-label prophylaxis with emicizumab.

Assessing joint health in haemophilia patients: The combined value of physical examination and ultrasound imaging.

INTRODUCTION: Early diagnosis of joint damage is pivotal in haemophilia to prevent the occurrence and progression of haemophilic arthropathy thus providing optimal personalised management. The haemophilia joint health score version 2.1 (HJHS) is based on a physical examination of the mainly affected joints. Musculoskeletal ultrasound has demonstrated the capability to detect early changes in terms of synovitis and osteochondral damage. The haemophilia early detection with ultrasound (HEAD-US) score has been proposed as a simple and reliable evaluation tool. AIM: This study aims to investigate the correlation between the HJHS and the HEAD-US scores performed by two independent operators (physical therapist and musculoskeletal ultrasound expert) for the evaluation of the joint health status of patients with haemophilia. METHODS: Consecutive adult patients independent of the severity degree were included. Elbows, knees and ankles were evaluated by a physical therapist by HJHS and by a musculoskeletal ultrasound expert following the HEAD-US protocol. RESULTS: We observed a good positive correlation between HJHS and HEAD-US (Spearman's rho 0.72). The main discrepancy in conceptually similar domains was found between the HJHS swelling and the HEAD-US synovitis (rho 0.17), as ultrasound was able to detect even mild synovitis when HJHS swelling was scored 0 in up to 40% of cases. CONCLUSIONS: The HJHS and HEAD-US correlate well even when performed by two independent operators. Musculoskeletal ultrasound is particularly useful for the early detection of synovitis. The routine assessment of both scores helps clinicians define the stage and extension of joint involvement and set up a personalised treatment.

Safety and efficacy of combined dual antiplatelet therapy and factor VIII prophylaxis in patients with haemophilia A after acute coronary syndrome.

INTRODUCTION: The increased life expectancy of patients with haemophilia A (HA) has led to a growing prevalence of cardiovascular risk factors and events. There is still scarce evidence on the safety and appropriate duration of dual antiplatelet therapy (DAPT) after acute coronary syndrome (ACS) in HA patients. AIM: We describe our experience on the clinical management of Italian HA patients after ACS. METHODS: Nine patients with congenital HA treated with DAPT after a revascularization procedure performed for ACS have been enrolled and followed at the Angelo Bianchi Bonomi Haemophilia and Thrombosis Center in Milan between 2005 and September 2022. The safety and efficacy of DAPT with or without FVIII prophylaxis were assessed. RESULTS: Ten ACS events occurred in the nine HA patients (four mild and five severe). All events were treated with percutaneous transluminal coronary angioplasty with deployment of 1 to 3 drug-eluting stents followed by DAPT for 1-12 months. All patients except one were treated with FVIII prophylaxis during DAPT aimed at achieving FVIII trough levels ≥20-30 IU/dL. DAPT was effective in all cases in preventing early ACS recurrence, with only a late recurrence. We observed two clinically relevant non-major bleeds (one in a patient without FVIII prophylaxis) and three minor bleeds. No venous thrombosis occurred. CONCLUSION: The long-term secondary antithrombotic prevention consisting of DAPT and FVIII prophylaxis achieving a trough level of 20-30 IU/dL can be effective and safe in HA patients.

Type 2M/2A von Willebrand disease: a shared phenotype between type 2M and 2A.

ABSTRACT: Four variants have been continuously subjected to debate and received different von Willebrand disease (VWD) classifications: p.R1315L, p.R1315C, p.R1374H, and p.R1374C. We chose to comprehensively investigate these variants with full set of VWD tests, protein-modeling predictions and applying structural biology. Patients with p.R1315L, p.R1315C, p.R1374H, and p.R1374C were included. A group with type 2A and 2M was included to better understand similarities and differences. Patients were investigated for phenotypic assays and underlying disease mechanisms. We applied deep protein modeling predictions and structural biology to elucidate the causative effects of variants. Forty-three patients with these variants and 70 with 2A (n = 35) or 2M (n = 35) were studied. Patients with p.R1315L, p.R1374H, or p.R1374C showed a common phenotype between 2M and 2A using von Willebrand factor (VWF):GPIbR/VWF:Ag and VWF:CB/VWF:Ag ratios and VWF multimeric profile, whereas p.R1315C represented a type 2M phenotype. There was an overall reduced VWF synthesis or secretion in 2M and cases with p.R1315L, p.R1374H, and p.R1374C, but not in 2A. Reduced VWF survival was observed in most 2A (77%), 2M (80%), and all 40 cases with p.R1315L, p.R1374H, and p.R1374C. These were the only variants that fall at the interface between the A1-A2 domains. p.R1315L/C mutants induce more compactness and internal mobility, whereas p.R1374H/C display a more extended overall geometry. We propose a new classification of type 2M/2A for p.R1315L, p.R1374H, and p.R1374C because they share a common phenotype with 2M and 2A. Our structural analysis shows the unique location of these variants on the A1-A2 domains and their distinctive effect on VWF.

Congenital fibrinogen disorders: a retrospective clinical and genetic analysis of the Prospective Rare Bleeding Disorders Database.

ABSTRACT: Congenital fibrinogen deficiency (CFD) is a rare bleeding disorder caused by mutations in FGA, FGB, and FGG. We sought to comprehensively characterize patients with CFD using PRO-RBDD (Prospective Rare Bleeding Disorders Database). Clinical phenotypes, laboratory, and genetic features were investigated using retrospective data from the PRO-RBDD. Patients were classified from asymptomatic to grade 3 based on their bleeding severity. In addition, FGA, FGB, and FGG were sequenced to find causative variants. A total of 166 CFD cases from 16 countries were included, of whom 123 (30 afibrinogenemia, 33 hypofibrinogenemia, 55 dysfibrinogenemia, and 5 hypodysfibrinogenemia) were well characterized. Considering the previously established factor activity and antigen level thresholds, bleeding severity was correctly identified in 58% of the cases. The rates of thrombotic events among afibrinogenemic and hypofibrinogenemic patients were relatively similar (11% and 10%, respectively) and surprisingly higher than in dysfibrinogenemic cases. The rate of spontaneous abortions among 68 pregnancies was 31%, including 86% in dysfibrinogenemic women and 14% with hypofibrinogenemia. Eighty-six patients received treatment (69 on-demand and/or 17 on prophylaxis), with fibrinogen concentrates being the most frequently used product. Genetic analysis was available for 91 cases and 41 distinct variants were identified. Hotspot variants (FGG, p.Arg301Cys/His and FGA, p.Arg35Cys/His) were present in 51% of dysfibrinogenemia. Obstetric complications were commonly observed in dysfibrinogenemia. This large multicenter study provided a comprehensive insight into the clinical, laboratory, and genetic history of patients with CFDs. We conclude that bleeding severity grades were in agreement with the established factor activity threshold in nearly half of the cases with quantitative defects.

Clinical and Laboratory Presentation and Underlying Mechanism in Patients with Low VWF.

BACKGROUND: Low von Willebrand factor (VWF) refers to subjects with plasma levels of 30 to 50 IU/dL. The mechanism of low VWF is poorly understood. We chose to determine the clinical presentation, laboratory phenotype, and underlying mechanisms of low VWF. MATERIAL AND METHODS: We included 250 patients characterized with low VWF. The International Society on Thrombosis and Haemostasis Bleeding Assessment Tool (ISTH-BAT) was used to assess clinical symptoms. To determine the underlying mechanisms of low VWF, we used as markers the VWF propeptide (VWFpp) assay and FVIII:C/VWF:Ag ratio for VWF synthesis and the VWFpp/VWF:Ag ratio for VWF clearance. Results were compared with those of 120 healthy controls. Cases with abnormal screening tests were further evaluated for coagulation factor levels and platelet disorders. RESULTS: The median age of the cohort was 35 years (range 3-85), 21% were children (n = 53), 34% were adult males (n = 85), and 45% (n = 112) were adult females. According to the ISTH-BAT, abnormal bleeding was found in 35% of children, 47% of males, and 49% of females. No association was found between VWF activity levels and ISTH-BAT. Patients showed an overall decreased VWF synthesis/secretion and an enhanced VWF clearance was identified in 33% of them. In 89 patients (36%), there were other hemostasis-related defects, but there was no difference in the ISTH-BAT between the two groups. CONCLUSION: Our findings indicate that reduced VWF synthesis/secretion and enhanced VWF clearance are major mechanisms of low VWF levels. Patients with low VWF have significant bleeding manifestations. While other hemostasis defects occurred together with low VWF, this combination did not exacerbate clinical symptoms.

Minimum factor VIII levels to prevent joint bleeding in mild hemophilia A.

The severity of the bleeding phenotype in patients with hemophilia A (HA) broadly correlates with the degree of coagulation factor VIII (FVIII) deficiency in plasma. However, the FVIII level necessary to achieve the goal of zero joint bleeds remains unclear. This study aimed to identify the minimum FVIII level necessary to prevent joint bleeds in patients with HA. In this retrospective study, patients with congenital mild HA treated on demand, aged ≥16 years, with no history of FVIII inhibitors, followed at the Angelo Bianchi Bonomi Hemophilia and Thrombosis Center in Milan, were enrolled. We investigated 270 male patients with a median age of 45 years (16-88) and median lifelong FVIII of 21 IU/dL. One hundred patients (37%) had a lifelong history of at least 1 joint bleed. The mean annualized joint bleeding rate (AJBR) and spontaneous AJBR were 0.016 (standard deviation [SD], 0.032) and 0.001 (SD, 0.010), respectively. After adjusting for age, for each IU/dL increase in FVIII, there was a 6% reduction in AJBR and an 11% reduction in spontaneous AJBR. The minimum FVIII levels needed to prevent lifelong any joint bleeds and spontaneous joint bleeds resulted to be 19.2 IU/dL and 17.7 IU/dL, respectively. In this large cohort of persons with mild HA, we identified the minimum FVIII levels needed to prevent total and spontaneous joint bleeds (19.2 IU/dL and 17.7 IU/dL, respectively). These findings could suggest important implications for the accurate design of prophylactic therapies for persons with moderate and severe HA, including gene therapy.

Characterization of Immunogenicity and Safety of COVID-19 mRNA-1273 in HIV-Positive Italian Patients with Hemophilia: A Prospective Single-Center Cohort Study.

To characterize the immunogenicity of mRNA-1273 (Moderna, Cambridge, MA, USA) vaccine in HIV-positive hemophilic patients during the third COVID-19 wave in Italy and to investigate biomarkers of coagulation and endothelial perturbation before and after complete vaccination schedule, twenty-three consecutive adult HIV-positive patients with hemophilia were included. Blood was collected before and two weeks after vaccination. We measured anti-SARS-CoV-2 spike protein antibodies to assess immunogenicity; circulating biomarkers of coagulation (protein C and D-dimer), endothelial perturbation (von Willebrand factor (VWF)) and anti-Platelet Factor 4 (PF4) antibodies were analyzed. Flow-based analysis of thrombus formation was performed in nine patients using a flow-chamber device. Two weeks after completing the vaccination schedule, all patients had anti-spike antibodies values consistent with an effective immunization. Mean (±standard deviation) basal values of protein C and VWF (106 ± 21% and 171 ± 45%, respectively) were not significantly different from data obtained two weeks after the second dose (103 ± 20%, 162 ± 43%, respectively). D-dimer median values (interquartile range) were not significantly different at baseline (442 (603-142) ng/mL) and after the second dose (477 (654-262) ng/mL). Anti-PF4 antibodies were detected in three patients with no associated clinical manifestations. No significant differences were found in flow-based analysis of thrombus formation. Our data demonstrate that in HIV-positive patients with hemophilia, SARS-CoV-2 vaccination is effective and safe, with no effects on coagulation and endothelial perturbation.

Residual burden of liver disease after HCV clearance in hemophilia: a word of caution in the era of gene therapy.

Ruling out advanced fibrosis/cirrhosis is mandatory for persons with hemophilia (PWH) who are candidates for gene therapy. However, clinical evaluation and noninvasive tests (NITs) may be inaccurate after hepatitis C virus (HCV) clearance. We conducted a prospective hepatological screening to detect advanced fibrosis/cirrhosis in PWH after HCV clearance. Any risk factor of chronic liver damage was registered by using biochemical data, liver stiffness measurement (LSM), and ultrasound (US). A pre/post-HCV clearance analysis was conducted prospectively in a subgroup of patients who underwent LSM, US, and NITs for fibrosis. We evaluated 119 patients (median age, 53 years; range, 36-87 years) with a previous HCV infection (hemophilia A, n = 108; hemophilia B, n = 11). Ninety-six (81%) presented at least 1 potential risk factor of chronic liver damage. Metabolic risk factors were the most prevalent, with 51 patients (44%) having US steatosis. In 21 patients (18%), clinical, biochemical, liver morphology, and/or LSM were suggestive of advanced fibrosis/cirrhosis. Furthermore, 10 patients (8%) had esophageal varices and 3 (3%) had hepatocellular carcinoma. In 57 patients included in the prospective analysis, LSM and NITs were reduced after HCV clearance (P < .05), but US signs specific of cirrhosis remained unchanged. Overall, 23 of 80 patients (29%) with LSM <10 KPa had at least 1 US sign suggestive of advanced fibrosis/cirrhosis. A similar proportion (18%) was observed for LSM <8 KPa. Overall, risk factors of chronic liver damage are frequent after HCV clearance, but changes in LSM and NITs after clearance may be inaccurate to rule out advanced fibrosis/cirrhosis. A specific diagnostic workup is warranted to evaluate liver health in PWH in the era of gene therapy.

Genetic determinants of enhanced von Willebrand factor clearance from plasma.

BACKGROUND: Enhanced von Willebrand factor (VWF) clearance from plasma is associated with von Willebrand disease (VWD). However, the genetic background of this disease mechanism is not well defined. OBJECTIVE: To determine VWF variants that are associated with reduced VWF survival. METHODS: Two hundred fifty-four patients with VWD (type 1 = 50 and type 2 = 204) were investigated, and the results were compared with 120 healthy controls. The patients were comprehensively characterized for phenotypic and genetic features. The ratio of VWF propeptide (VWFpp)/VWF antigen (VWFpp ratio) was used to establish in each patient the VWF clearance state. RESULTS: Out of 92 variants associated with type 1 (7 were novel) and type 2 VWD, 19 had a VWFpp ratio ranging from 1.7 to 2.2, 24 had a VWFpp ratio between 2.3 and 2.9, and 24 variants had a ratio of ≥3. The VWFpp median ratio in healthy controls was 0.98 (0.55-1.6) so that a cut-off value of >1.6 was considered an indicator of accelerated VWF clearance from plasma. An enhanced VWF clearance was observed in 34% of type 1 cases, 100% of type 1 Vicenza cases, 81% of 2A cases, 77% of 2B cases, 88% of 2M cases, and 36% of 2N cases. CONCLUSIONS: An accelerated VWF clearance was found in most patients with type 2A, 2B, and 2M VWD, with a lower proportion of type 1 and 2N. Sixty-seven different variants alone or in combination with other variants were associated with an increased VWFpp ratio. The variants with the highest VWFpp ratio were mostly located in the D3-A1 VWF domains.

Updates on Novel Non-Replacement Drugs for Hemophilia.

Over the last decade, the world of hemophilia has experienced an unprecedented therapeutic advance, thanks to the progress in bioengineering technologies, leading to the introduction of drugs with novel mechanisms of action based on restoring thrombin generation or coagulation factor VIII mimicking. Apart from the bispecific monoclonal antibody emicizumab, already approved for patients with severe hemophilia A with and without inhibitors, novel non-replacement drugs designed to reduce the treatment burden of patients with hemophilia A or B with or without inhibitors are undergoing evaluation in clinical trials. Thanks to their innovative mechanism of action and subcutaneous administration, these drugs promise to provide effective bleeding protection together with improved adherence and improve health-related quality of life for patients with hemophilia. On the other hand, rare thromboembolic events have been reported with some of these drugs and warrant continuous post-marketing surveillance and investigation of predisposing factors, although the overall safety profile of most of these drugs is good. Finally, new challenges need to be faced in the clinical and laboratory monitoring of the hemostatic status in patients treated with these innovative therapies. In this review, we provide an update on the available data on novel non-replacement drugs currently undergoing evaluation in clinical trials for patients with hemophilia.

Use of the von Willebrand factor concentrate with low factor VIII content to manage patients with inherited von Willebrand disease requiring surgical or secondary long-term prophylaxis: An expert opinion paper from an Italian panel.

OBJECTIVES: The present review aims to summarize the state-of-the-art von Willebrand disease (VWD) treatment focusing on specific clinical settings (obstetrics, surgery, long-term prophylaxis and comorbidities) as well as on the use of a Von Willebrand factor (VWF) concentrate with low FVIII content. METHODS: Literature research and case reports. RESULTS AND CONCLUSIONS: Considering that patients affected by VWD have an intact ability to synthesize FVIII, in order to avoid excessive levels of FVIII, a highly purified plasma VWF concentrate with low FVIII content could be particularly useful in those patients and clinical circumstances at high thrombotic risk as well as for long-term prophylaxis. When deciding the optimal therapeutic strategy, physicians should take into account both the patient's history and the differences among available concentrates according to the clinical situations requiring treatment.

Phenotypic and genetic characterizations of the Milan cohort of von Willebrand disease type 2.

von Willebrand disease (VWD) type 2 is caused by qualitative abnormalities of von Willebrand factor (VWF). This study aimed to determine the genotypic and phenotypic characterizations of a large VWD type 2 cohort from Milan. We included 321 patients (54% female) within 148 unrelated families from 1995 to 2021. Patients were fully characterized using laboratory phenotypic tests, and the genotypic diagnosis was confirmed by target genetic analysis using Sanger sequencing. Patients were diagnosed with type 2A (n = 98; 48 families), 2B (n = 85; 38 families), 2M (n = 112; 50 families), or 2N (n = 26; 12 families). Eighty-two unique VWF variants, including 8 novel variants, were found. The potential pathogenic effect of novel variants was assessed by in silico analysis. Most patients were heterozygous for a single variant (n = 259; 81%), whereas 37 cases (11%) had 2 variants (4 homozygous, 9 in trans, and 24 in cis). Twenty-five patients (8%) had ≥3 variants, mainly as a result of gene conversions. Among the 82 distinct variants identified, 5 different types, including missense (n = 64), gene conversion (n = 10), synonymous (n = 1), deletion (n = 4), and splice (n = 3), were observed. The results from this large cohort showed that VWD type 2 is invariably due to variants that do not prevent the synthesis of the protein, and a vast majority of patients (88%) had missense variants. Given the complexity of type 2 diagnosis and the necessity of performing several phenotypic tests, genetic analysis for patients suspected of having type 2 is beneficial to establish the correct diagnosis.

The dominant p.Thr274Pro mutation in the von Willebrand factor propeptide causes the von Willebrand disease type 1 phenotype in two unrelated patients.

BACKGROUND: von Willebrand factor propeptide (VWFpp) plays an important role in VWF multimerization and storage. VWFpp mutations have been previously associated with types 1, 3 and 2A/IIC von Willebrand disease (VWD). AIMS: To characterize the novel p.Thr274Pro variant identified in two unrelated type 1 VWD patients. METHODS: Phenotype tests were performed to evaluate patients' plasma and platelets following the current ISTH-SSC guidelines. Molecular analysis was performed using next-generation sequencing. The pcDNA3.1-VWF-WT and mutant pcDNA3.1-VWF-Thr274Pro expression vectors were transiently transfected into HEK293 cells to evaluate recombinant (r)VWF constitutive and regulated secretion. For the latter, the transfected cells were stimulated with phorbol-12-myristate-13-acetate. Immunofluorescence staining was performed to assess the localization of WT-rVWF and Thr274Pro-rVWF in endoplasmic reticulum, lysosomes, cis-/trans-Golgi and pseudo-Weibel Palade bodies. RESULTS: Biochemical characterization of patients' plasma samples indicated a type 1 VWD diagnosis. Both patients were heterozygous for the p.Thr274Pro variant. Hybrid Thr274Pro/WT-rVWF showed a secretion reduction of 36±4% according to patients' plasma VWF:Ag levels, whereas Thr274Pro-rVWF secretion was strongly impaired (21±2%). The amount of rVWF in cell lysates was nearly normal for both Thr274P (62±17%) and Thr274Pro/WT-rVWF (72±23%). The regulated secretion was impaired for Thr274Pro/WT-rVWF, whereas Thr274Pro-rVWF was not released at all. Immunofluorescence staining revealed no particular differences between WT and Thr274Pro-rVWF, although Thr274Pro-rVWF showed less pseudo-Weibel Palade bodies with a rounder shape than WT-rVWF. CONCLUSIONS: The novel p.Thr274Pro mutation has a dominant effect and it is responsible of patients' type 1 VWD phenotype through a combined mechanism of reduced synthesis, impaired secretion and multimerization.