Low Factor VIII Levels Research Articles

Clinical, Laboratory, and Molecular Aspects of Factor VII Deficiency.

Congenital factor VII (FVII) deficiency, the most frequent among the recessively inherited disorders of blood coagulation, is characterized by a wide range of symptoms, from mild mucosal bleeds to life-threatening intracranial hemorrhage. Complete FVII deficiency may cause perinatal lethality. Clinically relevant thresholds of plasma levels are still uncertain, and modest differences in low FVII levels are associated with large differences in clinical phenotypes. Activated FVII (FVIIa) expresses its physiological protease activity only in a complex with tissue factor (TF), which triggers clotting at a very low concentration. Knowledge of the FVIIa-TF complex helps to interpret the clinical findings associated with low FVII activity as compared with other rare bleeding disorders and permits effective management, including prophylaxis, with recombinant FVIIa, which, however, displays a short half-life. Newly devised substitutive and nonsubstitutive treatments, characterized by extended half-life properties, may further improve the quality of life of patients. Genetic diagnosis has been performed in thousands of patients with FVII deficiency, and among the heterogeneous F7 mutations, mostly missense changes, several recurrent variants show geographical distribution and identity by descent. In the general population, common F7 polymorphisms explain a large proportion of FVII level variance in plasma through FVII-lowering effects. Their combination with pathogenic variants may impact on the frequent detection of FVII coagulant levels lower than normal, as well as on mild bleeding conditions. In the twenties of this century, 70 years after the first report of FVII deficiency, more than 200 studies/reports about FVII/FVII deficiency have been published, with thousands of FVII-deficient patients characterized all over the world.

The Role of Clot Waveform Analysis and Related Parameters in the Diagnosis and Treatment of Hemophilia A.

Hemophilia A (HA) is an inherited bleeding disorder caused by a deficiency or defect in factor VIII (FVIII). We investigated the role of clot waveform analysis (CWA) of activated partial thromboplastin time in the diagnosis and therapeutic monitoring of HA. The changes in CWA parameters the maximum clotting velocity (|Min1|), maximum clotting acceleration (|Min2|), and maximum clotting deceleration (|Max2|) were detected among mild, moderate, and severe HA groups. As the severity of HA subtypes increased, the levels of |Min1|, |Min2|, and |Max2| progressively decreased (p < 0.05). Receiver operating characteristic curve analysis showed that |Max2| and |Min2| were more effective than |Min1| in distinguishing different types of HA patients, with higher diagnostic efficacy. The standard curves based on Actin FSL reagent for normal and low levels of FVIII:C-|Max2| were established, with R2 values of 0.98 and 0.99, respectively. These curves can be utilized for monitoring during replacement therapies involving full-length recombinant FVIII and B-domain-deleted FVIII. Thirty cases of HA patients utilized the FVIII-|Max2| standard curve to obtain individual pharmacokinetics characteristic parameters. The clearance, half-life (t1/2), time to FVIII:C of 1% above baseline (tt1%), and predicted dosage showed no statistically significant differences compared with one-stage assay (p > 0.05). CWA is an economical and practical tool, and its related parameters are associated with the severity of HA. It has promising clinical prospects in predicting FVIII:C levels and individualized treatment when HA patients undergo replacement therapy.

Key Drivers of Coagulation Factor Use in Von Willebrand Disease During Hospitalization: An Overview of the French BERHLINGO Cohort.

Von Willebrand disease (VWD) is the most common inherited bleeding disorder. However, studies of hospitalisation patterns with replacement treatment are scarce. The aim of this study was to investigate the current therapeutic management of VWD and determine the key drivers of coagulation factor uses in patients during hospitalisation. Hopscotch-WILL was a multi-centric retrospective study conducted over a 48-month period in any patients with VWD. The data were collected from the BERHLINGO Research Database and the French Hospital database. A total of 988 patients were included; 153 patients (15%) were hospitalised during 293 stays requiring treatment with von Willebrand factor (VWF) concentrates-pure or in association with Factor VIII (FVIII). Their median basal concentrations of VWF and FVIII were significantly lower than in untreated patients: VWF antigen < 30 IU/dL, VWF activity < 20 IU/dL and FVIII:C < 40 IU/dL. The median (interquartile range) concentrate consumption was similar between highly purified VWF or VWF combined with FVIII (72 [110] vs 57 [89] IU/kg/stay, p = 0.154). The use of VWF was highly heterogeneous by VWD type; type 3 had a particularly high impact on VWF consumption in non-surgical situations. The main admissions were for ear/nose/throat, hepato-gastroenterology, and trauma/orthopaedic conditions, besides gynaecological-obstetric causes in women. The use of VWF concentrates is mostly influenced by low basal levels of VWF and FVIII, but also by VWD type or the cause for hospitalisation. These results could inform future studies of newly released recombinant VWF.

Functional Assessment of VWD Type 2N Variants Using Mammalian Cell Displayed Surface-Tethered Von Willebrand Factor

Background von Willebrand factor (VWF) is a large plasma protein critical in hemostasis and thrombosis. Defects in VWF cause the inherited autosomal bleeding disorder von Willebrand disease (VWD). One critical function of VWF is to bind circulating coagulation factor VIII (FVIII) and protect it from rapid clearance. Type 2N VWD is a recessive form of VWD characterized by low FVIII levels due to impairment in VWF-FVIII binding. Type 2N VWD clinically is similar to mild-to-moderate hemophilia A, an X-linked bleeding disorder caused by deficiency in FVIII. It is critical to distinguish type 2N VWD from hemophilia A because the treatments are different. VWF genotyping can inform accurate diagnoses, treatment, and testing in relatives. With the rapid expansion of DNA sequencing, a growing number of DNA variants are being identified in the VWF gene. However, the interpretation of significance of these variants is often challenged by insufficient evidence to inform confident classification regarding pathogenicity. In vitro functional assays provide an avenue to develop the evidence needed to accurately assign functional significance to DNA variants. We adapted multiplexed surface tethering of extracellular proteins (MultiSTEP), our mammalian cell surface display system for secreted proteins, to display VWF and test the functional impact of VWF variants on VWF-FVIII binding. Methods We constructed a VWF cDNA of common VWF coding sequences by mutating rare variants to the more common nucleotides. The regions encoding the VWF propeptide were deleted (∆Pro-VWF). ∆Pro-VWF was cloned into our MultiSTEP cassette that provides a C-terminal flexible linker, Strep II tag, and CD28 transmembrane domain. Three VWF missense variants implicated in type 2N VWD (Table 1) that had varying levels of evidence to support classification of pathogenicity were chosen for study - VWF c.2561 G&amp;gt;A (p.R854Q), c.2572 T&amp;gt;A (p.C858S), and c.2635 G&amp;gt;A (p.D879N). Variants were subcloned into our ∆Pro-VWF cassette and then recombined into the genomically integrated MultiSTEP landing pad in HEK293 Freestyle cells. Following induction of expression, VWF-displaying cells were incubated with recombinant FVIII. VWF expression was measured using a polyclonal anti-VWF antibody, and VWF-FVIII binding was detected with two different monoclonal antibodies to FVIII. Cells were incubated with fluorescently-conjugated secondary antibodies and cell surface VWF and FVIII abundance determined by flow cytometry. Results We show that ∆Pro-VWF robustly displays in our MultiSTEP system and binds FVIII. All three VWF missense variants express VWF at levels similar to ∆Pro-VWF. Relative to ∆Pro-VWF all three variants show marked impairment in VWF-FVIII binding (Figure 1), providing strong in vitro evidence for pathogenicity in type 2N VWD useful in variant reinterpretation (bold column in Table 1). Conclusions We have developed an in vitro mammalian cell surface display system for the interrogation of VWF variants and adapted it to study VWF-FVIII binding. The Pathogenic variant p.R854Q shows decreased VWF-FVIII binding in this assay, as expected. The Likely Pathogenic variant p.D879N and the Variant of Uncertain Significance p.C858S both exhibit a profound loss of VWF-FVIII binding in this assay. Our results provide evidence for reclassification of p.C858S to Likely Pathogenic for type 2N VWD. We expect our system can be used at scale to similarly interrogate the functional significance of a multiplexed library of VWF variants and improve variant interpretation in VWD.

Sensitive Measurement of Clinically Relevant Factor VIII Levels in Thrombin Generation Assays Requires Presence of Factor XIa.

Hemophilia A (HA) is characterized by decreased or absent factor VIII (FVIII) activity. Current FVIII assays are based on clotting time and thus only provide information about the initiation of coagulation. In contrast, thrombin generation assays (TGAs) can be used to measure the full coagulation spectrum of initiation, propagation, and termination that provide information on the whole course of thrombin generation and inhibition. However, the commercially available TG kits lack sensitivity for measurements of hemophilia plasma within lower FVIII ranges, which is essential for explaining differences in bleeding phenotypes in hemophiliacs at clinically low levels of FVIII. Optimization of the TGA for measurements of low FVIII levels in severe HA patients. TGA measurements were performed in severe HA pooled plasma (n = 10). Investigations of several preanalytical and analytical variables of the assay were performed in a stepwise process and adjusted based on sensitivity toward intrinsic coagulation activation. TGA initiated by tissue factor (TF) alone at varying concentrations was unable to significantly differentiate between FVIII levels below 20%. In contrast, TGA activation with low concentrations of TF in presence of FXIa appeared to be highly sensitive for FVIII changes both in high and low ranges. In addition, a representative TGA curve at trough levels could only be produced using the dual TF/FXIa TGA. We propose a critical optimization for the setup of the TGA for measurements in severe HA plasma. The dual TF/FXIa TGA shows increased sensitivity, especially in lower FVIII ranges, which allows for better individual characterization at baseline, prediction of interventions, and follow-up.

A novel F8 variant in a Chinese hemophilia A family and involvement of X-chromosome inactivation: A case report.

Hemophilia A (HA) is an X-linked recessive bleeding disorder, which shows factor VIII (FVIII) deficiency caused by genetic variant in F8 gene. Males with F8 variants are affected, whereas female carriers with a wide range of FVIII levels are usually asymptomatic, it is possible that different X-chromosome inactivation (XCI) may effect the FVIII activity. We identified a novel variant F8: c.6193T > G in a Chinese HA proband, it was inherited from the mother and grandmother with different FVIII levels. We performed Androgen receptor gene (AR) assays and RT-PCR. AR assays revealed that the X chromosome with the F8 variant was severely skewed inactivated in the grandmother with higher FVIII levels, but not in the mother with lower FVIII levels. Further, RT-PCR of mRNA confirmed that only the wild allele of F8 was expressed in the grandmother, with lower expression in the wild allele of the mother. Our findings suggest that F8: c.6193T > G could be the cause of HA and that XCI affected the FVIII plasma levels in female carriers.

In vitro validation of chromogenic substrate assay for evaluation of surrogate FVIII-activity of emicizumab

[Introduction] Emicizumab, a bispecific antibody mimicking activated factor VIII (FVIII), is increasingly used in prophylaxis against bleeding in hemophilia A. Human factor-based chromogenic substrate assay (hCSA) shows concentration-dependency between emicizumab and reported FVIII activity. However, the assay measurement settings have not been optimized for emicizumab, and the reported FVIII activity cannot be directly referred as surrogate FVIII activity.[Materials and Methods] For in vitro validation of hCSA-reported surrogate FVIII activity, we compared the equation curves for emicizumab concentration with surrogate FVIII activity using spiked plasma in the thrombin generation assay (TGA), hCSA, and clot waveform analysis (CWA). Then, we generated conversion equations for hCSA-reported surrogate FVIII value to that of TGA. We also assessed the additive effect of rFVIII onto 340 nM (i.e., 50 μg/mL) emicizumab using the same assays.[Results] With 1:20 diluted plasma, halving hCSA-reported surrogate FVIII activity can be approximated to that in TGA triggered by the extrinsic pathway reagent (27.3 IU/dL vs. 13.9 IU/dL) under therapeutic emicizumab concentration. Both in TGA and hCSA, the additive effect of added FVIII on therapeutic emicizumab concentration (340 nM) was maintained at low levels of FVIII but gradually decreased at higher levels.[Conclusions] Surrogate FVIII activity can be estimated simply by halving hCSA-reported FVIII value, and the additive effect of FVIII on emicizumab diminishes at high concentrations. Based on our in vitro study, a clinical study is currently being conducted to compare individual variation of surrogate FVIII activity in hCSA and TGA.

Polyphosphate Activates von Willebrand Factor Interaction with Glycoprotein Ib in the Absence of Factor VIII In Vitro.

Polyphosphate (polyP), a phosphate polymer released by activated platelets, may modulate various stages of hemostasis by binding to blood proteins. In this context, we previously reported that polyP binds to the von Willebrand factor (VWF). One of the most significant functions of VWF is to bind to and protect the blood circulating Factor VIII (FVIII). Therefore, here, we study the role of polyP in the VWF-FVIII complex in vitro and suggest its biological significance. Surface plasmon resonance and electrophoretic mobility assays indicated that polyP binds dynamically to VWF only in the absence of FVIII. Using the VWF Ristocetin Cofactor assay, the most accepted method for studying VWF in platelet adhesion, we found that polyP activates this role of VWF only at low levels of FVIII, such as in plasmas with chemically depleted FVIII and plasmas from severe hemophilia A patients. Moreover, we demonstrated that FVIII competes with polyP in the activation of VWF. Finally, polyP also increases the binding of VWF to platelets in samples from patients with type 2 and type 3 von Willebrand disease. We propose that polyP may be used in designing new therapies to activate VWF when FVIII cannot be used.

Bleeding Phenotype Does Not Appear to Correlate with Factor VIII Levels or Genetic Mutation in an Exclusive Pediatric Cohort of Hemophilia a Carriers

Introduction: Inherited Hemophilia A is defined as Factor VIII (FVIII) deficiency, with levels <0.40 IU/ml. Female carriers exhibit varying degrees of X chromosome inactivation, with resultant variable FVIII activity levels. Bleeding phenotype in Hemophilia A carriers has been thought to be due to low FVIII levels; however, adult studies have shown lack of correlation of FVIII levels and bleeding phenotype in some patients. Our study aimed to correlate FVIII levels with bleeding phenotype and genetic mutations in pediatric hemophilia A carriers. Methods: Retrospective medical record review of Hemophilia A carriers <18 years of age was undertaken at our center. Inclusion criteria were family history of Hemophilia A and FVIII mutation or FVIII level <0.40 IU/ml in patients without genetic testing done. Patients with other co-existing bleeding disorders were excluded. Statistical analysis was done by Chi Square/Exact tests. Results: Twenty-three pediatric Hemophilia A carriers were eligible, with median age of 12 years (range1-16). Fifteen were adolescents >10 years. Fourteen (60.9%) had FVIII level >0.40 IU/ml with 8 (57.1%) categorized as asymptomatic, and 6 (42.9%) symptomatic carriers in accordance with the new ISTH nomenclature. Nine (39.1%) patients had FVIII <0.40 IU/ml, with 8 (88.8%) categorized as mild (FVIII 0.05-0.40 IU/ml), 1 (11.1%) as moderate (FVIII 0.01-0.05 IU/ml), and none as severe (<0.01 IU/ml) Hemophilia A, as per the new nomenclature. Bleeding Scores (BS) were obtained in all patients using the ISTH Bleeding Assessment Tool (ISTH-BAT) with a median BS of 1 (range 0-7). A significant BS of >2 was present in 4/9 (44.4%) patients with FVIII <0.40 IU/ml, and 4/14 (28.6%) with FVIII >0.40 IU/ml, the difference was not statistically significant (p=0.40) (Table 1). Two symptomatic carriers with FVIII >0.4 IU/ml, had BS of 1 and 2. 5/15 adolescents (33.3%) had BS>2 (BS of 4 in 3; BS of 7 in 2). FVIII was measured with one stage assay (OSA) (N=17; 73.9%), chromogenic substrate assay (CSA) (N= 2; 8.7%) or both (N=4; 17.4%), with 1 patient's (4.3%) CSA discrepantly lower than OSA by 0.62 IU/ml and none with inverse discrepancy. Genetic testing was done in 20/23 (86.9%) patients, with FVIII missense mutations in 14 (70%), intron inversions in 4 (20%), and deletions in 2 (10%), their association with BS did not show statistically significant difference (p=0.6) (Table 2). Conclusion: Our study was done exclusively in pediatric Hemophilia A carriers, as it is important to identify children at risk of bleeding and institute preventive measures early. Unlike in adult women, higher number (one-third) of our patients experienced mucosal bleeding, and none reported joint or muscle bleeding. Our study showed lack of statistically significant association of FVIII levels with bleeding phenotype. According to the new ISTH nomenclature, 39.1% of our study patients have the diagnosis of Hemophilia A, of whom 55.6% did not have a significant BS. Of the 60.9% categorized as carriers, 28.6% had a significant BS. In addition, we found lack of statistically significant correlation of genetic mutation type with the bleeding phenotype, with none, 5% and 25% of the patients with deletion, intron inversion and missense mutation having a significant BS respectively. Follow-up/BS reassessment in our cohort is planned to monitor BS increase with age. Our study limitations include small cohort and retrospective analysis. Future pediatric studies are required to identify coagulation/genetic parameters that correlate better with bleeding phenotype, which may then help with early recognition and prompt institution of preventive therapy to pediatric Hemophilia A carriers at risk of bleeding, in order to avoid bleeding complications during adulthood. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Novel cysteine substitution p.(Cys1084Tyr) causes variable expressivity of qualitative and quantitative VWF defects

Abstractvon Willebrand factor (VWF) is an extremely cysteine-rich multimeric protein that is essential for maintaining normal hemostasis. The cysteine residues of VWF monomers form intra- and intermolecular disulfide bonds that regulate its structural conformation, multimer distribution, and ultimately its hemostatic activity. In this study, we investigated and characterized the molecular and pathogenic mechanisms through which a novel cysteine variant p.(Cys1084Tyr) causes an unusual, mixed phenotype form of von Willebrand disease (VWD). Phenotypic data including bleeding scores, laboratory values, VWF multimer distribution, and desmopressin response kinetics were investigated in 5 members (2 parents and 3 daughters) of a consanguineous family. VWF synthesis and secretion were also assessed in a heterologous expression system and in a transient transgenic mouse model. Heterozygosity for p.(Cys1084Tyr) was associated with variable expressivity of qualitative VWF defects. Heterozygous individuals had reduced VWF:GPIbM (<0.40 IU/mL) and VWF:CB (<0.35 IU/mL), as well as relative reductions in high-molecular-weight multimers, consistent with type 2A VWD. In addition to these qualitative defects, homozygous individuals also displayed reduced factor VIII (FVIII):C/VWF:Ag, leading to very low FVIII levels (0.03-0.1 IU/mL) and reduced VWF:Ag (<0.40 IU/mL) and VWF:GPIbM (<0.30 IU/mL). Accelerated VWF clearance and impaired VWF secretion contributed to the fully expressed homozygous phenotype with impaired secretion arising because of disordered disulfide connectivity.

Relationship between Endogenous, Transgene FVIII Expression and Bleeding Events Following Valoctocogene Roxaparvovec Gene Transfer for Severe Hemophilia A: A Post-Hoc Analysis of the GENEr8-1 Phase 3 Trial

Relationship between Endogenous, Transgene FVIII Expression and Bleeding Events Following Valoctocogene Roxaparvovec Gene Transfer for Severe Hemophilia A: A Post-Hoc Analysis of the GENEr8-1 Phase 3 Trial

Single Centre Retrospective Analysis of Patients Who Underwent Emergency Reversal of Warfarin Anticoagulation

IntroductionWarfarin is used in the treatment and prevention of thromboembolic disorders, with the risk of bleeding ranging from 1% to 3% per year resulting in hospitalisation or death. Emergency reversal of warfarin is indicated when warfarin therapy is complicated by bleeding, prior to emergency procedures / surgeries, and in patients who have supra-therapeutic INR with high risk of bleeding. Launceston General Hospital is a 330-bed hospital in regional Australia and we practise warfarin reversal in accordance with the national guidelines. Prothrombinex-VF (PTx-VF) is a 3-factor prothrombin complex concentrate (PCC) used in Australia, which contains factors II, IX, and X mainly. Due to low factor VII levels in PTx-VF, addition of 1 unit of FFP with PTx-VF is generally recommended, however there is lack of supportive evidence. There are a few reports of successful use of 3-factor PCC without the use of FFP. Hence in our hospital, the addition of FFP to PTx-VF for warfarin reversal is based on clinician's preference.Aim of studyTo study the clinical, laboratory, therapeutic and outcome data on patients who underwent emergency reversal of warfarin anticoagulation, and to compare the warfarin reversal (INR values) after administration of PTx-VF vs PTx-VF + FFP.MethodsWe performed a retrospective study of patients who underwent emergency reversal of warfarin in our hospital between January 2011 and December 2013. All the patients who underwent emergency reversal of warfarin with PTx-VF +/- FFP were captured from the blood bank database. Patients who received PTx-VF for indications other than emergency warfarin reversal and those with insufficient data were excluded from the study. The clinical, laboratory and transfusion data of these patients and the warfarin reversal strategies and clinical outcomes across different clinical settings were analysed. Non-parametric statistical analysis was used to compare the differences in post-treatment INR values in the PTx-VF vs PTx-VF + FFP groups and statistical significance was assumed at a P value of less than 0.05.ResultsA total number of 125 patients who underwent emergency warfarin reversal were included in the study. The median age was 77 years (range: 28 - 95) with 78 males (62%) and 48 females (38%). Table-1 presents clinical characteristics of the patients. Three clinical subgroups were identified - major bleeding in 61 patients (49%), emergency surgery in 54 patients (43%) and supra-therapeutic INR with high risk of bleeding in 10 patients (8%). Table-2 compares the clinical, laboratory, management and outcome characteristics in these subgroups. According to clinicians' preference, 56% of patients received PTx-VF alone, while 44% received PTx-VF + FFP. Statistically there were no differences in median INR values performed within 6 - 12 hours after administration of PTx-VF vs PTx-VF + FFP in all patients (P = 0.10), bleeding subgroup (P = 0.08) and emergency surgery subgroup (P = 0.24); and the number of patients in the supra-therapeutic INR subgroup was very small for evaluation. Overall 4 patients died due to warfarin-related complications, all of them in the bleeding subgroup.ConclusionIn our study majority (64%) of patients were above 75 years and the most common (69%) reason for warfarin anticoagulation was atrial fibrillation, which were similar in other studies. We observed that GI bleeding was the most common cause for warfarin reversal. Rapid INR correction with good clinical haemostasis occurred in most of the patients after administration of PTx-VF +/- FFP. The mortality rate in our cohort was 3% (4 of 125), and intracranial bleeding accounted for 75% (3 of 4) deaths. Among 14 patients who experienced intracranial bleeding, either spontaneous or induced by trauma, three (21%) died. There were no significant differences in the post-treatment INR values with PTx-VF alone when compared to PTx-VF + FFP, overall and also in the clinical subgroups. The major advantage of warfarin over DOACs is the rapid reversibility in emergency settings and we demonstrated that our 3-factor PCC (PTx-VF) was effective in emergency reversal of warfarin, without the need for FFP. There are a few limitations in this study since it is retrospective and observational. Hence prospective studies are required for better evaluation of the clinical characteristics, and the efficacy of 3-factor PCC without FFP in emergency reversal of warfarin anticoagulation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pediatric Bleeding Questionnaire as a predictor of perioperative bleeding in patients with low factor VII levels

IntroductionFactor VII is an essential blood coagulation factor, yet the minimum level of Factor VII activity (FVII:C) for hemostasis is not well defined. Therefore, patients with seemingly low FVII:C levels diagnosed on presurgical screening are often exposed to potentially unnecessary treatment to mitigate bleeding risk that may not exist. The Pediatric Bleeding Questionnaire (PBQ) a bleeding risk assessment tool has not been validated in patients with low FVII:C. ObjectiveTo assess the utility of the PBQ as a bleeding risk predictor in patients with low FVII:C level undergoing surgical procedures. MethodsPatients with low FVII:C levels diagnosed in the pre-surgical setting with a completed PBQ were eligible. Patient’s FVII:C, PBQ score, surgery type, perioperative hemostatic measures and bleeding events were recorded. ResultsFifty-Two patients (mean age 10.6 years) with mean FVII:C of 39.7% were evaluated. All but 3 patients had a PBQ score ≤2. No perioperative bleeding prophylaxis was administered to 41 (78.8%) patients. None of the patients reported prolonged perioperative bleeding. ConclusionIndividuals with low FVII:C detected on presurgical screening are not at high risk for bleeding in the absence of a personal bleeding history and the PBQ did not predict bleeding risk in this setting.

X Chromosome inactivation: a modifier of factor VIII and IX plasma levels and bleeding phenotype in Haemophilia carriers.

Haemophilia A and B are X-linked hemorrhagic disorders caused by gene variants in the F8 and F9 genes. Due to recessive inheritance, males are affected, while female carriers are usually asymptomatic with a wide range of factor VIII (FVIII) or IX (FIX) levels. Bleeding tendency in female carriers is extremely variable and may be associated with low clotting factor levels. This could be explained by F8 or F9 genetic variations, numerical or structural X chromosomal anomalies, or epigenetic variations such as irregular X chromosome inactivation (XCI). The aim of the study was to determine whether low FVIII or FIX coagulant activity in haemophilia carriers could be related to XCI and bleeding symptoms. HUMARA assay was performed on 73 symptomatic carriers with low clotting activity ≤50 IU/dL. Bleeding Assessment Tool (BAT) from the International Society on Thrombosis and Haemostasis (ISTH) was used to describe symptoms in the cohort of carriers. In 97% of haemophilia carriers, a specific gene variant in heterozygous state was found, which alone could not justify their low FVIII or FIX levels (≤50 IU/dL). A statistical association between XCI pattern and FVIII and FIX levels was observed. Moreover, female carriers with low coagulant activity (≤20 IU/dL) and high degree of XCI ( ≥ 80:20) had a higher ISTH-BAT score than the carriers with the opposite conditions (>20 IU/dL and <80:20). In our cohort of haemophilia carriers, XCI was significantly skewed, which may contribute to the low expression of clotting factor levels and bleeding symptoms.

Defining the Optimal FVIII Transgene for Placental Cell-Based Gene Therapy to Treat Hemophilia A

The delivery of factor VIII (FVIII) through gene and/or cellular platforms has emerged as a promising hemophilia A treatment. Herein, we investigated the suitability of human placental cells (PLCs) as delivery vehicles for FVIII and determined an optimal FVIII transgene to produce/secrete therapeutic FVIII levels from these cells. Using three PLC cell banks we demonstrated that PLCs constitutively secreted low levels of FVIII, suggesting their suitability as a transgenic FVIII production platform. Furthermore, PLCs significantly increased FVIII secretion after transduction with a lentiviral vector (LV) encoding a myeloid codon-optimized bioengineered FVIII containing high-expression elements from porcine FVIII. Importantly, transduced PLCs did not upregulate cellular stress or innate immunity molecules, demonstrating that after transduction and FVIII production/secretion, PLCs retained low immunogenicity and cell stress. When LV encoding five different bioengineered FVIII transgenes were compared for transduction efficiency, FVIII production, and secretion, data showed that PLCs transduced with LV encoding hybrid human/porcine FVIII transgenes secreted substantially higher levels of FVIII than did LV encoding B domain-deleted human FVIII. In addition, data showed that in PLCs, myeloid codon optimization is needed to increase FVIII secretion to therapeutic levels. These studies have identified an optimal combination of FVIII transgene and cell source to achieve clinically meaningful levels of secreted FVIII.

Discovery of Type 3 von Willebrand Disease in a Cohort of Patients with Suspected Hemophilia A in Côte d'Ivoire.

BackgroundType 3 von Willebrand disease (VWD) is the most severe form of VWD, characterized by a near-total absence of von Willebrand factor (vWF), leading to a massive deficiency in plasmatic factor VIII (FVIII). VWD may be confused with hemophilia A, sometimes leading to misdiagnosis. The purpose of this work was to finalize the biological diagnosis of patients with FVIII activity deficiency in Abidjan in order to guide the best type of management.MethodsWe conducted a cross-sectional descriptive study from June 2018 to April 2019. Forty-nine patients, all of whom had lower FVIII levels or had been referred for a bleeding disorder, were monitored in the clinical hematology service. The pro-coagulant activity of coagulation factors was performed in Abidjan. The assays for von Willebrand antigen and activity were performed at Nîmes University Hospital in France.ResultsThe mean age of patients was 13.8 years (1 – 65) and 86% were Ivorian. FVIII deficiency was discovered during a biological checkup, circumcision or post-traumatic bleeding, in 33%, 31% and 29% respectively. The FVIII deficiency of patients was classified as severe (89.8%), moderate (8.2%) and mild (2%). Only one patient had a quantitative deficiency of von Willebrand factor (vWF: Ag <3%) with undetectable von Willebrand factor activity (vWF: Ac) and an FVIII level <1%.ConclusionsNot all of the congenital deficiency of FVIII are represented by hemophilia A. It was crucial to assess the Willebrand factor of these patients followed in Côte d’Ivoire for whom hemophilia A had been suspected.

The Discordance between Offsite to Onsite Testing for Von Willebrand Disease in Post-Menarchal Females: A Multi-Center Study

The Discordance between Offsite to Onsite Testing for Von Willebrand Disease in Post-Menarchal Females: A Multi-Center Study

VWF and FVIII Levels after Desmopressin Are Associated with the Bleeding Phenotype in Type 1 VWD

Introduction The bleeding phenotype of type 1 von Willebrand disease (VWD) patients is very heterogeneous. We hypothesized that part of this heterogeneity is explained by variability in response of von Willebrand factor (VWF) and factor VIII (FVIII) levels to stress during hemostatic challenges. Patients who have a strong increase in VWF and FVIII levels during hemostatic challenges, may have less frequent or less severe bleeding episodes compared to patients who have a smaller increase in VWF and FVIII levels. Therefore, we investigated whether VWF and FVIII levels after desmopressin, which mimic in vivo hemostatic response during hemostatic challenges, are associated with the bleeding score, and therefore may explain part of the heterogeneity in bleeding phenotype of type 1 VWD patients. Methods In this retrospective cohort study, we included type 1 VWD patients of all ages who were diagnosed and treated in the Erasmus University Medical Center. All patients had a hemorrhagic diathesis or family history of VWD and historically lowest VWF antigen (VWF:Ag) and/or VWF Ristocetin Cofactor activity (VWF:RCo) ≤ 0.30 IU/mL with VWF activity/ VWF antigen ratio above 0.60. All patients participated in the Willebrand in the Netherlands (WiN) study, in which a self-administered Tosetto bleeding score was assessed. Results We identified 173 eligible type 1 VWD patients in our treatment center who participated in the WiN study. After exclusion of 50 patients who did not receive a desmopressin test dose, and 1 patient who did not have a bleeding score assessment, we included 122 type 1 VWD patients. Table 1 shows the patient characteristics. Higher FVIII activity during the complete time course (i.e. one, three and 5-6 hours) after desmopressin, and higher VWF and FVIII levels combined at three hours after desmopressin, were associated with a lower bleeding score, respectively β=-0.9 (95% CI: -1.7; -0.1) and β=-1.2 (-1.9; -0.5), both adjusted for age, sex, BMI and comorbidities. FVIII:C at three hours after desmopressin was significantly associated with a lower bleeding score: β=-1.5 (-3.0; -0.1), whereas VWF levels were not associated (Figure 1). Moreover, patients with VWF and FVIII levels in the highest quartile three hours after desmopressin administration had a lower bleeding score compared to other patients (Figure 2). The largest difference was found for FVIII:C: β=-5.1 (-8.4; -1.8), adjusted for age, sex, BMI, comorbidities and baseline FVIII:C. These patients also less often had an abnormal bleeding score: OR=0.1 (0.0-0.5), adjusted for age, sex, BMI, comorbidities and baseline FVIII:C. Lastly, patients with an increased clearance of VWF, defined as VWF propeptide/VWF:Ag ratio &gt;2.2, had lower VWF:Ag (β=-0.3, -0.6; -0.1), VWF:Act (β=-0.5, -0.7; -0.2), VWF:CB (β=-0.5, -1.0; -0.1) and FVIII:C (β=-0.7, -1.0; -0.4) at three hours after desmopressin administration compared to other type 1 VWD patients, all adjusted for age, sex, BMI and comorbidities. An increased clearance of VWF was also associated with a higher bleeding score: β=3.2 (0.2; 6.2), adjusted for age, sex, BMI, comorbidities and baseline VWF and FVIII levels. Discussion In type 1 VWD patients, the desmopressin induced increase of VWF and FVIII levels is associated with a lower bleeding score. Especially higher VWF and FVIII levels at three hours after desmopressin were associated with a milder bleeding phenotype, suggesting that sustained VWF and FVIII levels after a hemostatic challenge may be important to prevent bleeding. This was strengthened by an association between increased clearance of VWF and a higher bleeding score. Furthermore, patients with VWF and FVIII levels in the highest quartile three hours after desmopressin had 5 points lower bleeding score, and patients with FVIII:C in the highest quartile had 10 times less often an abnormal bleeding score. These results indicate that a good hemostatic response may have important and clinically relevant consequences on the bleeding phenotype, and may compensate for the low baseline VWF and FVIII levels in type 1 VWD patients. In conclusion, VWF and FVIII levels after desmopressin administration, which mimic in vivo hemostatic response to hemostatic challenges, are associated with the bleeding phenotype in type 1 VWD patients. This may partly explain the well-known variability in bleeding phenotype of type 1 VWD patients. Disclosures Atiq: Sobi: Other: Travel grant (ISTH 2019); Professor Heimburger Award 2018 (CSL Behring): Research Funding. Schütte:CSL Behring: Other: Travel grant. Boender:Sobi: Employment; Professor Heimburger Award 2016 (CSL Behring): Research Funding. Kruip:Bayer: Research Funding; Pfizer: Research Funding; Daiichy Sankyo: Research Funding; Boehringer Ingelheim: Research Funding. Leebeek:Shire/Takeda: Consultancy; UniQure: Consultancy; Novo Nordisk: Consultancy; Shire/Takeda: Research Funding; Sobi: Other: Travel grant; CSL Behring: Research Funding.

Accurate measurement of extended half-life and unmodified factor VIII low levels with one-stage FVIII assays is dependent on the matrix of calibration curves.

The monitoring of factor VIII (FVIII) replacement therapy relies on the accurate measurement of FVIII activity over a large concentration range. However, unexplained overestimation of low FVIII levels has recently been reported with extended half-life recombinant FVIIIs. The objective of this study was to confirm previous publications indicating that the reagents used to generate the calibration curves determine the accuracy of the measurement of low FVIII levels. We generated FVIII calibration curves with FVIII-deficient plasmas or a commercial diluent buffer. We then measured FVIII levels in FVIII-deficient plasma spiked with plasma FVIII, a full-length recombinant FVIII (Advate® , Shire, Brussels, Belgium) and two extended half-life recombinant FVIIIs (Elocta® , Swedish Orphan Biovitrum, Woluwe Saint-Lambert, Belgium and Afstyla® , CSL Behring, Mechelen, Belgium). FVIII levels were also analyzed in spiked samples prediluted two- and fourfold, either in diluent buffer or in FVIII-deficient plasma to evaluate parallelism. Coagulation times of calibration curves generated with diluent buffer were longer than those with FVIII-deficient plasmas. This resulted in an overestimation of FVIII levels lower than 25IU/dL in spiked samples and in detection of FVIII activity (≥1IU/dL) in FVIII-deficient plasma. Predilution of samples with diluent buffer rather than with FVIII-deficient plasma also led to discordant results. Our data confirm that the generation of calibration curves by dilution in FVIII-deficient plasma is crucial for the accurate measurement of low FVIII levels. When serial dilutions of samples are analyzed, predilution in FVIII-deficient plasma is required to respect the parallelism criteria. These methods should be more generally implemented for coagulation instruments.

Long-Term Expression of Von Willebrand Factor Via Piggybac-Mediated Gene Transfer

von Willebrand disease (vWD), caused by a deficiency in von Willebrand factor (vWF), is the most common inherited bleeding disorder, affecting up to 1% of the population; those affected have a varied bleeding phenotype. von Willebrand factor plays two important roles in hemostasis including platelet adhesion and stability of factor VIII (FVIII). As such, patients with von Willebrand disease type 3 have no measurable vWF protein and demonstrate low levels of FVIII. To date, persistent long-term expression of full-length vWF using gene transfer strategies has not been demonstrated, largely due to the immense size of vWF cDNA (8.4 kb). Non-viral vector systems, such as piggyBac, are used increasingly in gene targeting technologies and as tools for gene transfer applications. Nonviral DNA transposons are genetic elements consisting of inverted terminal DNA repeats which in their naturally occurring configuration flank a transposase coding sequence. The transposase follows a "cut and paste" mechanism to excise the transposon from its original genomic location and insert it into a new locus. The insect derived piggyBac (PB) can be engineered to carry a therapeutic transgene between the inverted terminal repeats. Advantages of this nonviral vector system include a large transgene cassette capacity, ease of production and purification, and potential for site-specific integration. We hypothesize PB-mediated vWF gene transfer will confer long-term expression and improve the bleeding phenotype in an animal model of vWD. We engineered PB transposon to carry a human von Willebrand factor cDNA (PB vWF). When transfected with PB-vWF and a hyperactive transposase, iPB7, the hepatocarcinoma cell line, HepG2, demonstrated secretion of vWF into supernatants compared to mock transfected controls (p < 0.01). Next, we evaluated the in vivo gene transfer efficiency in von Willebrand deficient mice by hydrodynamic tail-vein injection using PB-vWF driven by the murine albumin enhancer/human alpha anti-trypsin promoter. vWF null mice received 25 or 50 micrograms each of the PB-vWF transposon and iPB7 to determine long-term expression and phenotypic correction. vWF levels were measured prior to injection and then every 4 weeks for up to 24 weeks. Results revealed therapeutic levels (on average 50% normal mice) of vWF post gene transfer with stable expression for 24 weeks in most mice. These data indicate the PB transposon vector system may provide a long-term gene transfer strategy for vWD. To evaluate phenotypic correction, a tail clip assay was performed at the end of the study. PB-vWF gene transfer resulted in at least partial bleeding phenotype correction via tail clip. Additionally, treated mice demonstrated a rescue of the FVIII activity. These data show that the PB vector can be used to deliver this large transgene expression cassette to the liver and achieve long-term expression and phenotypic correction in vivo. DisclosuresStaber:Emergent BioSolutions: Honoraria; Baxalta: Honoraria.