von Willebrand disease (VWD) is a genetic bleeding disease due to a defect of von Willebrand factor (VWF), a glycoprotein crucial for platelet adhesion to the subendothelium after vascular injury. ...VWD include quantitative defects of VWF, either partial (type 1 with VWF levels <50 IU/dL) or virtually total (type 3 with undetectable VWF levels) and also qualitative defects of VWF (type 2 variants with discrepant antigenic and functional VWF levels). The most bleeding forms of VWD usually do not concern type 1 patients with the mildest VWF defects (VWF levels between 30 and 50 IU/dL). The French reference center for VWD performed a laboratory phenotypic and genotypic analysis in 1167 VWD patients (670 families) selected by their basic biologic phenotype: type 3, type 2, and type 1 with VWF levels <30 IU/dL. In these patients indeed, to achieve an accurate diagnosis of VWD type and subtype is crucial for the management (treatment and genetic counseling). A phenotype/genotype correlation was present in 99.3% of cases; 323 distinct VWF sequence variations (58% of novel) were identified (missense 67% versus truncating 33%). The distribution of VWD types was: 25% of type 1, 8% of type 3, 66% of type 2 (2A: 18%, 2B: 17%, 2M: 19%, 2N: 12%), and 1% of undetermined type. Type 1 VWD was related either to a defective synthesis/secretion or to an accelerated clearance of VWF. In type 3 VWD, bi-allelic mutations of VWF were found in almost all patients. In type 2A, the most frequent mechanism was a hyper-proteolysis of VWF. Type 2B showed 85% of patients with deleterious mutations (distinct from type 2B New York). Type 2M was linked to a defective binding of VWF to platelet glycoprotein Ib or to collagen. Type 2N VWD included almost half type 2N/3. This biologic study emphasizes the complex mechanisms for both quantitative and qualitative VWF defects in VWD. In addition, this study provides a new epidemiologic picture of the most bleeding forms of VWD in which qualitative defects are predominant.
Abstract von Willebrand factor (VWF) is a multimeric protein, the size of which is regulated via ADAMTS13-mediated proteolysis within the A2 domain. We aimed to isolate nanobodies distinguishing ...between proteolyzed and non-proteolyzed VWF, leading to the identification of a nanobody (designated KB-VWF-D3.1) targeting the A3 domain, the epitope of which overlaps the collagen-binding site. Although KB-VWF-D3.1 binds with similar efficiency to dimeric and multimeric derivatives of VWF, binding to VWF was lost upon proteolysis by ADAMTS13, suggesting that proteolysis in the A2 domain modulates exposure of its epitope in the A3 domain. We therefore used KB-VWF-D3.1 to monitor VWF degradation in plasma samples. Spiking experiments showed that a loss of 10% intact VWF could be detected using this nanobody. By comparing plasma from volunteers to that from congenital von Willebrand disease (VWD) patients, intact-VWF levels were significantly reduced for all VWD types, and most severely in VWD type 2A–group 2, in which mutations promote ADAMTS13-mediated proteolysis. Unexpectedly, we also observed increased proteolysis in some patients with VWD type 1 and VWD type 2M. A significant correlation (r = 0.51, P < .0001) between the relative amount of high–molecular weight multimers and levels of intact VWF was observed. Reduced levels of intact VWF were further found in plasmas from patients with severe aortic stenosis and patients receiving mechanical circulatory support. KB-VWF-D3.1 is thus a nanobody that detects changes in the exposure of its epitope within the collagen-binding site of the A3 domain. In view of its unique characteristics, it has the potential to be used as a diagnostic tool to investigate whether a loss of larger multimers is due to ADAMTS13-mediated proteolysis.
•Nanobody KB-VWF-D3.1 binds to the collagen-binding site in the VWF A3 domain, and it loses its binding upon proteolysis of VWF by ADAMTS13.•KB-VWF-D3.1 identified VWF degradation in patients with ...VWD, which correlated with a loss of larger VWF multimers.
Display omitted
von Willebrand factor (VWF) is a multimeric protein, the size of which is regulated via ADAMTS13-mediated proteolysis within the A2 domain. We aimed to isolate nanobodies distinguishing between proteolyzed and non-proteolyzed VWF, leading to the identification of a nanobody (designated KB-VWF-D3.1) targeting the A3 domain, the epitope of which overlaps the collagen-binding site. Although KB-VWF-D3.1 binds with similar efficiency to dimeric and multimeric derivatives of VWF, binding to VWF was lost upon proteolysis by ADAMTS13, suggesting that proteolysis in the A2 domain modulates exposure of its epitope in the A3 domain. We therefore used KB-VWF-D3.1 to monitor VWF degradation in plasma samples. Spiking experiments showed that a loss of 10% intact VWF could be detected using this nanobody. By comparing plasma from volunteers to that from congenital von Willebrand disease (VWD) patients, intact-VWF levels were significantly reduced for all VWD types, and most severely in VWD type 2A–group 2, in which mutations promote ADAMTS13-mediated proteolysis. Unexpectedly, we also observed increased proteolysis in some patients with VWD type 1 and VWD type 2M. A significant correlation (r = 0.51, P < .0001) between the relative amount of high–molecular weight multimers and levels of intact VWF was observed. Reduced levels of intact VWF were further found in plasmas from patients with severe aortic stenosis and patients receiving mechanical circulatory support. KB-VWF-D3.1 is thus a nanobody that detects changes in the exposure of its epitope within the collagen-binding site of the A3 domain. In view of its unique characteristics, it has the potential to be used as a diagnostic tool to investigate whether a loss of larger multimers is due to ADAMTS13-mediated proteolysis.
Introduction
With current molecular diagnosis, about 1 to 5% of haemophilia A (HA) patients remain genetically unresolved. In these cases, deep intronic variation or structural variation disrupting ...the F8 gene could be causal.
Aim
To identify the causal variation in four genetically unresolved mild‐to‐severe HA patients using an F8 mRNA analysis approach.
Methods
Ectopic F8 mRNA analysis was performed in four unrelated HA patients. An in vitro minigene assay was performed in order to confirm the deleterious splicing impact of each variation identified.
Results
In all probands, mRNA analysis revealed an aberrant splicing pattern, and sequencing of the corresponding intronic region found a deep intronic substitution. Two of these were new variations: c.2113+601G>A and c.1443+602A>G, while the c.143+1567A>G, found in two patients, has previously been reported. The c.1443+602A>G and the c.143+1567A>G variants both led to the creation of a de novo acceptor or donor splice site, respectively. Moreover, the c.143+1567A>G was found in 3/6 patients with genetically unresolved moderate HA registered in our laboratory. Haplotype analysis performed in all patients carrying the c.143+1567A>G variation suggests that this variation could be a recurrent variation. The c.2113+601G>A led to the exonization of a 122‐bp antisense AluY element by increasing the strength of a pre‐existing cryptic 5’ splice site. For each point variation, in vitro splicing analysis confirmed its deleterious impact on splicing of the F8 transcript.
Conclusion
We identified three deep intronic variations, leading to an aberrant mRNA splicing process as HA causing variation.
von Willebrand disease (VWD) is a genetic bleeding disease due to a defect of von Willebrand factor (VWF), a glycoprotein crucial for platelet adhesion to the subendothelium after vascular injury. ...VWD include quantitative defects of VWF, either partial (type 1 with VWF levels <50 IU/dL) or virtually total (type 3 with undetectable VWF levels) and also qualitative defects of VWF (type 2 variants with discrepant antigenic and functional VWF levels). The most bleeding forms of VWD usually do not concern type 1 patients with the mildest VWF defects (VWF levels between 30 and 50 IU/dL). The French reference center for VWD performed a laboratory phenotypic and genotypic analysis in 1167 VWD patients (670 families) selected by their basic biologic phenotype: type 3, type 2, and type 1 with VWF levels <30 IU/dL. In these patients indeed, to achieve an accurate diagnosis of VWD type and subtype is crucial for the management (treatment and genetic counseling). A phenotype/genotype correlation was present in 99.3% of cases; 323 distinct VWF sequence variations (58% of novel) were identified (missense 67% versus truncating 33%). The distribution of VWD types was: 25% of type 1, 8% of type 3, 66% of type 2 (2A: 18%, 2B: 17%, 2M: 19%, 2N: 12%), and 1% of undetermined type. Type 1 VWD was related either to a defective synthesis/secretion or to an accelerated clearance of VWF. In type 3 VWD, bi-allelic mutations of VWF were found in almost all patients. In type 2A, the most frequent mechanism was a hyper-proteolysis of VWF. Type 2B showed 85% of patients with deleterious mutations (distinct from type 2B New York). Type 2M was linked to a defective binding of VWF to platelet glycoprotein Ib or to collagen. Type 2N VWD included almost half type 2N/3. This biologic study emphasizes the complex mechanisms for both quantitative and qualitative VWF defects in VWD. In addition, this study provides a new epidemiologic picture of the most bleeding forms of VWD in which qualitative defects are predominant.
A rapid lateral flow immunoassay (LFIA) (STic Expert® HIT), recently developed for the diagnosis of heparin‐induced thrombocytopenia (HIT), was evaluated in a prospective multicentre cohort of 334 ...consecutive patients. The risk of HIT was estimated by the 4Ts score as low, intermediate and high in 28·7%, 61·7% and 9·6% of patients, respectively. Definite HIT was diagnosed in 40 patients (12·0%) with positive results on both enzyme‐linked immunosorbent assay (Asserachrom® HPIA IgG) and serotonin release assay. The inter‐reader reproducibility of results obtained was excellent (kappa ratio > 0·9). The negative predictive value of LFIA with plasma samples was 99·6% with a negative likelihood ratio (LR) of 0·03, and was comparable to those of the particle gel immunoassay (H/PF4‐PaGIA®) performed in 124 cases. Positive predictive value and positive LR were 44·4% and 5·87, respectively, and the results were similar for serum samples. The probability of HIT in intermediate risk patients decreased from 11·2% to 0·4% when the LFIA result was negative and increased to 42·5% when it was positive. In conclusion, the STic Expert® HIT combined with the 4Ts score is a reliable tool to rule out the diagnosis of HIT.
Background: The use of extending half-life (EHL) FVIII or FIX products is today a current strategy in Hemophilia A (HA) patients for improving prophylaxis and reducing the number of IV injections. Fc ...fusion technology is based on the use of the neonatal Fc receptor and endogenous Fc recycling pathway, thereby prolonging the half-life (T1/2) of rFVIII-Fc. A single dose phase 1/2 pharmacokinetic (PK) study performed in 16 severe HA patients demonstrated a prolonged T ½ of rFVIII-Fc equal to 18.8 hours (mean) compared to 12.2 hours with one conventional rFVIII (Malhangu et al. Blood 2014).
The aim of the present study was to analyze PK data collected with Elocta® in “real life” i.e. in a large cohort of patients treated in 13 different French hemophilia care centers, and results were compared to those obtained with conventional FVIII, when available. Importantly, this study was performed without any involvement of Sobi, the pharmaceutical company that provides Elocta® in France.
Patients and methods: 113 severe Hemophilia A (HA) patients with the following characteristics were included: mean age 30 years (range 3 - 70); weight 65 Kg (17-125); total FVIII-Fc dose injected 2650 IU (500-5750); FVIII-Fc IU/Kg: 41 (25 - 59); VWF Ag 98% (41-279). The FVIII recovery (R) was calculated as follows: (body weight (Kg) x observed increased in FVIII (%))/administered dose (IU/Kg). The T1/2 was calculated with the following formula: Ln2/((Ln FVIII% T1 - Ln FVIII%T2)/T2 - T1)), with T1 ≥ 4 hours and T2 ≥ 24 hours.
Results were compared to those performed with conventional FVIII (non EHL-FVIII) in 48 patients (Advate® n = 14, Refacto® n = 2, Helixate®/Kogenate®/Kovaltry® n = 29, Factane® n = 3)
Results: rFVIII-Fc activity measured by one stage clotting assay (OSA) was 20% lower than those obtained with chromogenic assay (CSA) in samples with FVIII levels higher than 20%, but this difference was lower than 10% when FVIII levels < 20%. Therefore, rFVIII-Fc recovery (R) always appeared lower when measured with OSA (Mean 2.38, range 1.33 - 5.7) than with CSA (mean 2.82, ranges 1.35 - 5.5) (p < 0.0001). No correlation was found between this recovery and age, weight, injected doses or VWF Ag levels. Mean T1/2 measured with rFVIII-Fc equaled 15 hours whatever the measurement method used (OSA or CSA), and was strongly correlated with vWFAg levels (R2 = 0.57). Using OSA, significantly lower recovery (1.86 vs. 2.49, p = 0.0002) and T1/2 values (11.75 vs. 15.13 hours, p = 0.0004) were measured in children (< 10 years, n = 19) compared to adults. Similar differences were evidenced with data obtained by CSA (recovery : 2.26 vs. 2.93, p = 0.0009 and T1/2 : 11.4 vs. 15.6 h, p = 0.004, n = 14 children < 10 years).
PK parameters of FVIII-Fc were compared to those obtained with non EHL-FVIII (rFVIII or pdFVIII) in 47 patients (mean T1/2 equal to 10.0 hours; range 5.3 - 21.2), and half-lives of these two categories of products were well correlated (r2 = 0.57). However, the apparent benefit provided by FVIII-Fc was variable from one patient to another, with a mean T1/2 rFVIII-Fc / T1/2 FVIII ratio ranging from 0.6 to 2.4 (mean 1.4). Interestingly, the increase in T1/2 with FVIII-Fc was lower than 20% only in patients previously treated with BHK-derived rFVIII i.e. Helixate®/Kogenate®/ Kowaltry® (n=10). Whatever the FVIII injected (FVIII-Fc or other non EHL-FVIII), the T1/2 measured was also strongly correlated to vWF levels, which were significantly lower in patients for whom the mean T1/2 rFVIII Fc / T1/2 FVIII ratio was > 1.3 (mean 79% vs 116% in the others, p=0.017).
Conclusion: This study is the first to report PK data obtained with rFVIII-Fc (Elocta®) in a large group of HA patients. Our results confirm the benefit of rFVIII-Fc in most HA patients, adults or children, but also emphasize the impact of vWF on half-life of rFVIII-Fc or conventional non EHL-FVIII. Indeed, the benefit of rFVIII-Fc clearly appears higher in patients with lower vWF levels, with a more significant prolongation of T1/2.
No relevant conflicts of interest to declare.