Exploring nonreplacement therapies' impact on hemophilia and other rare bleeding disorders.

The management of hemophilia, von Willebrand disease (VWD), and rare coagulation disorders traditionally relied on replacement therapies, such as factor concentrates, to address clotting factor deficiencies. However, in recent years, the emergence of nonreplacement therapies has shown promise as an adjunctive approach, especially in hemophilia, and also for patients with VWD and rare bleeding disorders. This review article offers an overview of nonreplacement therapies, such as FVIII-mimicking agents and drugs aimed at rebalancing hemostasis by inhibiting natural anticoagulants, particularly in the management of hemophilia. The utilization of nonreplacement therapies in VWD and rare bleeding disorders has recently attracted attention, as evidenced by presentations at the International Society on Thrombosis and Haemostasis 2023 Congress. Nonreplacement therapies provide alternative methods for preventing bleeding episodes and enhancing patients' quality of life, as many of them are administered subcutaneously and allow longer infusion intervals, resulting in improved quality of life and comfort for patients.

Global Prevalence of Hereditary Thrombotic Thrombocytopenic Purpura Determined by Genetic Analysis.

Hereditary thrombotic thrombocytopenic purpura (hTTP) is a rare autosomal recessive, life-threatening disorder caused by a severe deficiency of the plasma enzyme, ADAMTS13. The current estimated prevalence of hTTP in different regions of the world, 0.5-2.0 patients/million, is determined by the frequency of diagnosed patients. To evaluate more accurately the worldwide prevalence of hTTP, and also the prevalence within distinct ethnic groups, we used data available in exome and genome sequencing of 807,162 (730,947 exomes, 76,215 genomes) subjects reported recently by the Genome Aggregation Database (gnomAD-v4.1). Among 1,614,324 analyzed alleles in the gnomAD population we identified 6,321 distinct ADAMTS13 variants. Of these 6,321 variants, 758 were defined as pathogenic; 140 (18%) variants had been previously reported and 618 (82%) were novel (predicted as pathogenic). 10,154 alleles (0.6%) were carrying the reported or predicted pathogenic variants; 7,759 (77%) with previously reported variants. Considering all 758 pathogenic variants and also only the 140 previously reported variants, we estimated a global hTTP prevalence of 40 and 23 cases/106, respectively. Considering only the 140 previously reported variants, the highest estimated prevalence was in East Asians (42/106). The estimated prevalences of other populations were: Finnish, 32/106; non-Finnish Europeans, 28/106; Admixed Americans, 19/106; Africans/African Americans, 6/106; and South Asians, 4/106. The lowest prevalences were Middle Eastern, 1/106 and Ashkenazi Jews, 0.7/106. This population-based genetic epidemiology study reports that hTTP prevalence is substantially higher than the currently estimated prevalence based on diagnosed patients. Many hTTP patients may not be diagnosed or may have died during the neonatal period.

Application of genetic testing for the diagnosis of von Willebrand disease.

von Willebrand disease (VWD) is the most frequent inherited bleeding disorder, with an estimated symptomatic prevalence of 1 per 1000 in the general population. VWD is characterized by defects in the quantity, quality, or multimeric structure of von Willebrand factor (VWF), a glycoprotein being hemostatically essential in circulation. VWD is classified into 3 principal types: low VWF/type 1 with partial quantitative deficiency of VWF, type 3 with virtual absence of VWF, and type 2 with functional abnormalities of VWF, being classified as 2A, 2B, 2M, and 2N. A new VWD type has been officially recognized by the ISTH SSC on von Willebrand factor which has also been discussed by the joint ASH/ISTH/NHF/WFH 2021 guidelines (ie, type 1C), indicating patients with quantitative deficiency due to an enhanced VWF clearance. With the advent of next-generation sequencing technologies, the process of genetic diagnosis has substantially changed and improved accuracy. Therefore, nowadays, patients with type 3 and severe type 1 VWD can benefit from genetic testing as much as type 2 VWD. Specifically, genetic testing can be used to confirm or differentiate a VWD diagnosis, as well as to provide genetic counseling. The focus of this manuscript is to discuss the current knowledge on VWD molecular pathophysiology and the application of genetic testing for VWD diagnosis.

Immune gene polymorphisms associated with poor response to platelet transfusion and recombinant factor VII administration in Glanzmann thrombasthenia.

INTRODUCTION: Poor response to platelet and recombinant factor VII administration is a major problem in patients with Glanzmann Thrombasthenia (GT). The risk factors associated with poor response to treatment in these patients are unknown. Some genetic variations of cytokines may contribute to therapy resistance. AIMS: We evaluated, for the first time, whether genetic polymorphisms on cytokine genes are related to poor treatment response in GT patients. METHODS: We enrolled 30 patients with GT (15 resistant and 15 non-resistant) and 100 healthy controls. Gene polymorphisms of IL-10 and TNF-α were analysed using TaqMan Realtime PCR, and IL-1, IL-1R1 and IL-1RN were investigated with the RFLP method. In-silico analyses were performed to predict the potential impact of these polymorphisms. RESULTS: In the resistant group, all patients had a variant of the IL-10 gene at the -1082 position (rs1800896), with a GG genotype that was significantly more frequent than the non-resistant group. Analysis between healthy controls and GT patients revealed a probable correlation between rs3783550, rs3783553, rs3917356 and rs2234463 and GT. The In-silico study indicated that TNF-α rs1800629 and IL-10 rs1800896 polymorphisms result in different allelic expressions which may contribute to poor response to therapy. CONCLUSIONS: These findings suggest that polymorphisms in the IL-10 and IL-1 receptor antagonist genes may play a role in poor therapy response in GT patients. In addition, some polymorphisms in IL-1α, IL1-ß, IL-1R1 and IL-R antagonists might be involved in the GT progression.

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.

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.

Population-based prevalence and mutational landscape of von Willebrand disease using large-scale genetic databases.

Von Willebrand disease (VWD) is a common bleeding disorder caused by mutations in the von Willebrand factor gene (VWF). The true global prevalence of VWD has not been accurately established. We estimated the worldwide and within-population prevalence of inherited VWD by analyzing exome and genome data of 141,456 individuals gathered by the genome Aggregation Database (gnomAD). We also extended our data deepening by mining the main databases containing VWF variants i.e., the Leiden Open Variation Database (LOVD) and the Human Gene Mutation Database (HGMD) with the goal to explore the global mutational spectrum of VWD. A total of 4,313 VWF variants were identified in the gnomAD population, of which 505 were predicted to be pathogenic or already reported to be associated with VWD. Among the 282,912 alleles analyzed, 31,785 were affected by the aforementioned variants. The global prevalence of dominant VWD in 1000 individuals was established to be 74 for type 1, 3 for 2A, 3 for 2B and 6 for 2M. The global prevalences for recessive VWD forms (type 2N and type 3) were 0.31 and 0.7 in 1000 individuals, respectively. This comprehensive analysis provided a global mutational landscape of VWF by means of 927 already reported variants in the HGMD and LOVD datasets and 287 novel pathogenic variants identified in the gnomAD. Our results reveal that there is a considerably higher than expected prevalence of putative disease alleles and variants associated with VWD and suggest that a large number of VWD patients are undiagnosed.

Laboratory Testing for von Willebrand Factor Activity by a Glycoprotein Ib-Binding Assay (VWF:GPIbR): HemosIL von Willebrand Factor Ristocetin Cofactor Activity on ACL TOP®.

von Willebrand disease (VWD) is a lifelong and common inherited bleeding disorder caused by a quantitative deficiency and/or qualitative defect of von Willebrand factor (VWF). In order to establish the correct diagnosis of VWD, various tests must be conducted, including evaluation of factor VIII activity (FVIII:C), VWF antigen (VWF:Ag), and VWF functional activity. The platelet-dependent VWF activity is measured in different ways, with the historical ristocetin cofactor assay (VWF:RCo) using platelet aggregometry now replaced with newer assays that offer better precision, lower limits of detection, low coefficient of variation, and are fully automated. The VWF activity by glycoprotein Ib-binding assays (VWF:GPIbR) measured on the ACL TOP® platform represents an automated assay that instead of using platelets employs latex beads coated with recombinant wild-type GPIb. VWF in the test sample agglutinates the polystyrene beads coated with GPIb in the presence of ristocetin. The reduction of turbidity as beads agglutinate represents a linear relationship with VWF:GPIbR activity. Using a ratio of VWF:GPIbR/VWF:Ag, the VWF:GPIbR assay also provides good sensitivity and specificity for distinguishing type 1 VWD from type 2. The following chapter describes a detailed protocol for the VWF:GPIbR assay.

Diagnostic utility of bleeding assessment tools in congenital fibrinogen deficiencies.

BACKGROUND: The assessment of clinical history is crucial before referring a patient for further laboratory testing. Bleeding assessment tools (BAT) are developed to standardize clinical evaluation. A small number of patients with congenital fibrinogen deficiencies (CFDs) have been evaluated with these tools without definitive results. AIMS: We compared the adequacy of the ISTH-BAT and the European network of rare bleeding disorders bleeding score system (EN-RBD-BSS) to identify patients with CFDs. The correlation between the two BATs and fibrinogen levels and patient clinical grade severity was further analyzed. METHODS: We included 100 Iranian patients with CFDs. Routine coagulation and fibrinogen-specific tests (fibrinogen antigen [Fg:Ag] and activity [Fg:C]) were performed. The ISTH-BAT and EN-RBD-BSS were used to assess the bleeding score (BS) of all patients. RESULTS: The ISTH-BAT and EN-RBD-BSS median (range) were 4 (0-16) and 2.21 (-1.49 to 6.71), with a statistically significant moderate correlation between the two systems (r = .597, P < .001). In patients with quantitative deficiencies (afibrinogenemia and hypofibrinogenemia), the correlation between Fg:C and the ISTH-BAT was moderately negative (r = -.4, P < .001), while the correlation between Fg:C and the EN-RBD-BSS was weakly negative (r = -.38, P < .001). Overall, 70% and 72% of patients with fibrinogen deficiencies were correctly identified by both the ISTH-BAT and EN-RBD-BSS, respectively. CONCLUSION: These results suggest that in addition to the ISTH-BAT, the EN-RBD-BSS may also be useful in identifying CFD patients. We found a significant level of sensitivity for detecting fibrinogen deficiency in the two BATs, and bleeding severity classification correctly identified severity grades in almost two-thirds of patients.

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.

Prevalence of hemorrhagic ovarian cysts in patients with rare inherited bleeding disorders.

BACKGROUND: In comparison with the general population, women with bleeding disorders are more prone to develop obstetrical and gynecological problems. However, no comprehensive evaluation has investigated the prevalence of hemorrhagic ovarian cysts (HOCs) in rare bleeding disorders (RBDs). In this study, we sought to determine the prevalence of HOCs in a large cohort of Iranian patients with RBDs. METHODS: A total of 210 symptomatic patients suspected of HOCs with RBD were included. The median age of the study population was 24 years. Patients were diagnosed with fibrinogen disorders (n = 7, 3%), factor (F) II (n = 4, 2%), FV (n = 28, 13%), FVII (n = 4, 2%), FX (n = 6, 3%), FXIII (n = 122, 58%), combined FV and FVIII (n = 8, 4%), Glanzmann's thrombasthenia (n = 10, 5%), and von Willebrand disease (VWD) type 3 (n = 21, 10%). RESULTS: Following further clinical and ultrasound examinations of these 210 patients, 68 (32.4%) were confirmed with a diagnosis of HOCs. Of which, FXIII deficiency with 46 cases (67.6%), followed by VWD type 3 (6 cases, 8.8%) showed the highest number. Other coagulation defects associated with HOCs were including fibrinogen deficiency (n = 2, 3%), FII (n = 2, 3%), FV (n = 4, 6%), FVII (n = 2, 3%), FX (n = 1, 1.5%), combined FV and FVIII (n = 2, 3%), and Glanzmann's thrombasthenia (n = 3, 4.5%). CONCLUSION: This study found a high prevalence of HOCs in patients with RBDs, indicating the importance of early diagnosis and optimal management of obstetric and gynecological complications in these patients.

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.

Clinical and molecular characterization of Iranian patients with congenital fibrinogen disorders.

INTRODUCTION: Congenital fibrinogen disorders (CFDs) are caused by mutations in the FGA, FGB and FGG genes and are classified as quantitative and qualitative fibrinogen defects. This study sought to determine the genetic background of CFDs in Iran and to examine the genotype-phenotype correlation. METHODS: Fourteen patients with a CFD diagnosis were included. Fibrinogen antigen and activity were measured by the immunoturbidimetric and Clauss methods respectively. Gene sequencing was performed following a polymerase chain reaction amplification of fibrinogen's genes. The ISTH Bleeding Assessment Tool was also evaluated for all cases. RESULTS: Patients were diagnosed with dysfibrinogenemia (n = 10), hypodysfibrinogenemia (n = 2) and afibrinogenemia (n = 2). Seven different mutations located on FGA exon 2 (57 %), exon 4 (7%), exon 5 (7%) and FGG exon 8 (29 %) were identified. In patients with qualitative deficiencies, mutations were including p.Arg38Thr, p.Arg35His, p.Arg35Cys, p.Val145Asp, and p.Arg301Cys and were including p.Gly316GlufsX105 and p.Trp52stop in afibrinogenemic patients. In dysfibrinogenemia, two hotspot mutations, FGA Arg35 and FGG Arg301 were identified in 60 % of patients and the remaining (40 %) had p.Arg38Thr mutation. The p.Val145Asp and two hotspot mutations, p.Arg35His, p.Arg35Cys, were identified for the first time in Iran. The overall median (range) bleeding score (BS) was 4 (0-6) in all patients and it was 3.5 (0-5) in dysfibrinogenemia. Cutaneous bleeding and menorrhagia were the most common bleeding manifestations. CONCLUSION: There was a weak genotype-phenotype correlation in CFDs and patients with dysfibrinogenemia were more symptomatic than in previous studies. Despite ethnic's differences, the prevalence of hotspot mutations in dysfibrinogenemia was similar to the other studies.

Von Willebrand disease combined with coagulation defects in Iran.

BACKGROUND: Although Von Willebrand disease (VWD) is the most common inherited bleeding disorder, few cases of VWD combined with coagulation defects have been reported. This study sought to determine the clinical and laboratory features of VWD combined with other coagulation defects and to evaluate the prevalence of this combination in Iran. MATERIAL AND METHODS: A total of 3,120 cases were evaluated to confirm a suspected diagnosis of VWD. Clinical and laboratory phenotypes, including bleeding scores (BS), were also obtained. RESULTS: A diagnosis of VWD was established for 130 patients. Following their further characterisation, a subgroup of 25 patients with a dual or triple combination of VWD with coagulation defects (FXII, FXI, FIX, FVII, FV, and lupus anticoagulant) was identified. Their laboratory and clinical data were compared with those of healthy controls (n=25) and VWD-only patients (n=25). No differences were observed for VWF-related laboratory measurements between the combined deficient cases and those with VWD only, results being expectedly lower than in healthy controls. The median BS of combined patients was 4, higher than for VWD-only and control groups (median BS 3 and 1; p=0.55 and p<0.001, respectively). DISCUSSION: The prevalence of combined coagulation defects was 19.2% among all the VWD cases. The co-occurrence of VWD with clotting factor deficiencies may lead to more severe clinical presentations. To ensure adequate treatment, combined defects should be considered in VWD patients presenting with a more severe bleeding phenotype than expected or with a poor response to treatment.

Von Willebrand disease type 2N: An update.

Quantitative or qualitative defects of von Willebrand factor (VWF) are responsible for the most common inherited bleeding disorder, von Willebrand disease (VWD). Type 2N VWD is an uncommon recessive disorder that results from gene mutations located in the region coding for the binding site of VWF for factor VIII (FVIII). This narrative review describes the pathophysiology, diagnostic procedures and treatment as well as the molecular biology of type 2N VWD. Although other VWF-dependent functions like binding to platelets and collagen are preserved, FVIII plasma levels are low due to the rapid clearance of this moiety in the absence or reduction of its binding to VWF. The diagnosis of type 2N should be considered in patients with low FVIII coagulant activity (FVIII:C) and disproportionally higher VWF antigen, especially when they present with an autosomal recessive pattern of inheritance. Because an accurate diagnosis is essential for genetic counseling and optimal treatment, type 2N must be distinguished from mild/moderate hemophilia A and its carrier state. This differential diagnosis can be obtained by using the laboratory assay of the FVIII binding capacity of VWF (VWF:FVIIIB) or analysis of the FVIII binding site on the VWF gene.

Prophylactic management of patients with von Willebrand disease.

Von Willebrand disease, the most common inherited bleeding disorder that affects both males and females, is due to quantitative or qualitative defects of the multimeric glycoprotein von Willebrand factor, which cause mucous membrane bleeding but also soft tissue bleeding owing to the secondary deficiency of factor VIII. The aim of treatment is to correct this dual defect of hemostasis. In addition to the episodic management of bleeding episodes, therapy includes their short- or long-term prevention. Short-term prophylaxis is mainly warranted in order to provide effective hemostatic coverage to patients undergoing surgery or invasive procedures and to affected women at the time of delivery or during menstruations associated with excessive bleeding. The aim of long-term prophylaxis is to prevent bleeding in particular categories of patients at increased risk of frequent and spontaneous bleeding in the joints, nose, and gastrointestinal tract.