Plasma-derived Von Willebrand Factor Research Articles

Nanobody activator improves sensitivity of the von Willebrand factor activity assay to multimer size

BackgroundThe activity of von Willebrand factor (VWF) in facilitating platelet adhesion and aggregation correlates with its multimer size. Traditional ristocetin-dependent functional assays lack sensitivity to multimer sizes. Recently, nanobodies targeting the autoinhibitory module and activating VWF were identified. ObjectivesTo develop an assay that can differentiate the platelet-binding activity of VWF multimers. MethodsA novel enzyme-linked immunosorbent assay (nanobody-triggered glycoprotein Ib binding assay [VWF:GPIbNab]) utilizing a VWF-activating nanobody was developed. Recombinant VWF, plasma-derived VWF (pdVWF), and selected gel-filtrated fractions of pdVWF were evaluated for VWF antigen and activity levels. A linear regression model was developed to estimate the specific activity of VWF multimers. ResultsOf the 3 activating nanobodies tested, 6C11 with the lowest activation effect exhibited the highest sensitivity for high-molecular-weight multimers (HMWMs) of VWF. VWF:GPIbNab utilizing 6C11 (VWF:GPIbNab6C11) produced significantly higher activity/antigen ratios for recombinant VWF (>2.0) and HMWM-enriched pdVWF fractions (>2.0) than for pdVWF (∼1.0) or fractions enriched with shorter multimers (<1.0). The differences were much larger than those produced by VWF:GPIbNab utilizing other nanobodies, VWF:GPIbM, VWF:GPIbR, or VWF:CB assays. Linear regression analysis of 5 pdVWF fractions of various multimer sizes produced an estimated specific activity of 2.7 for HMWMs. The analysis attributed >90% of the VWF activity measured by VWF:GPIbNab6C11 to that of HMWMs, which is significantly higher than all other activity assays tested. ConclusionThe VWF:GPIbNab6C11 assay exhibits higher sensitivity to HMWMs than ristocetin-based and collagen-binding assays. Future studies examining the application of this assay in clinical settings and any associated therapeutic benefit of doing so are warranted.

Cost-effectiveness of prophylaxis with recombinant vs plasma-derived VWF in severe von Willebrand disease in the United States

AbstractWe evaluated the cost-effectiveness of prophylaxis with recombinant von Willebrand factor (rVWF) vs with plasma-derived von Willebrand factor (pdVWF) for patients with severe Von Willebrand disease. We found that rVWF is a cost-saving factor replacement compared with pdVWF across all willingness-to-pay thresholds in the United States.

Use of Vonicog Alpha and Acquired von Willebrand Syndrome, a New Approach: A Case Report.

Therapeutic management of acquired von Willebrand syndrome (AVWS) can be challenging, particularly in cases of AVWS associated with monoclonal IgM such as Waldenström macroglobulinemia (WM) where several therapeutic options may be ineffective. Here, we describe the case of an 88-year-old patient who developed AVWS during follow-up for WM. The presence of a severe bleeding symptomatology not controlled by several therapies (plasma-derived von Willebrand factor, plasmapheresis) led us to introduce a supplementation with recombinant von Willebrand factor, vonicog α (Veyvondi, Takeda, Japan), starting at a dose of 50 IU/kg/d. This supplementation allowed clinical (no further bleeding) and biological (hemoglobin level, von Willebrand factor parameters) improvements. Because of the persistence of bleeding risk factors, the treatment was maintained at a prophylactic dose (20 UI/kg three times a week), without recurrence of bleeding events for a period of 9 months.

Optimal design of infusion tests for the identification of physiological models of acquired von Willebrand syndrome

Acquired von Willebrand syndrome (AVWS) is a bleeding disorder resembling inherited Von Willebrand disease (VWD) characterised by a qualitative and/or quantitative deficiency of von Willebrand factor (VWF) that occurs in patients with no personal or family history of bleeding as a result of underlying pathological conditions. To treat AVWS patients, desmopressin (DDAVP) and plasma-derived VWF concentrates are the primary therapies for spontaneous acute bleeding episodes and for preventing bleeding during invasive or surgical procedures. Pharmacokinetic (PK) models have been recently developed and applied to characterize VWD and AVWS, but these models cannot be calibrated from infusion tests data, and their calibration requires stressful 24-hours long tests to be carried out on subjects to achieve a satisfactory estimation of the individual haemostatic parameters. The objectives of this paper are: i) to present a new physiological model of VWD including exogenous infusion of plasma-derived VWF concentrates, suitable to describe AVWS; ii) to validate the newly proposed model from clinical data; iii) to quantify the information that can be obtained from clinical tests using different VWF concentrates by applying model-based design of experiments (MBDoE) techniques. Results show that the newly developed model calibrated from infusion data allows to estimate precisely the full set of haemostatic parameters for a subject affected by AVWS preserving the same level of information obtained from conventional tests. Most importantly, results demonstrate that the overall duration of infusion tests for the identification of key haemostatic parameters can significantly be reduced from 24 h to 2.5 h.

Cost-Effectiveness of Long-Term Prophylaxis with Plasma-Derived Vs. Recombinant Von Willebrand Factor in Severe Von Willebrand Disease

Introduction: von Willebrand disease (VWD) is the most common inherited bleeding disorder and exhibits significant variability in bleeding phenotype across patients. International guidelines conditionally recommend long-term prophylaxis to reduce bleeding events for patients with a severe phenotype: all patients with type 3 VWD and a subset of patients with severe phenotypes of types 1 and 2 VWD. At the time of this conditional recommendation, only 10% of patients with severe VWD are treated prophylactically, unlike patients with severe hemophilia (~45%). In the United States, there are currently two approved factor replacement therapies: plasma-derived von Willebrand factor (pdVWF) and recombinant von Willebrand factor (rVWF). The cost-effectiveness of prophylaxis options for patients with VWD in the United States is not known. We sought to fill this gap by determining the cost-effectiveness of pdVWF versus rVWF prophylaxis treatment options for patients with severe VWD. Methods: In this independent analysis free of industry influence, we constructed a Markov model of adult patients with severe VWD to compare the cost-effectiveness of long-term prophylaxis with pdVWF versus rVWF. Transition probabilities for patients' breakthrough bleeding events while on prophylaxis with each factor replacement product, and product utilization, were derived from an open-label, prospective, phase 3 clinical trial that employed a pdVWF versus rVWF switch design (NCT 02973087). Costs of prophylaxis and costs of VWD-specific breakthrough bleed health resource utilization in the United States (US) accounted for bleed category, severity, and bleed-specific mortality, and were sourced from previously published literature and the Centers for Medicare &amp; Medicaid Services. For effectiveness measured in quality-adjusted life-years (QALY), utilities for patients with severe VWD on prophylaxis were informed from EQ-5D-specific utility data for patients with severe hemophilia on prophylaxis. The primary outcome was the incremental cost-effectiveness ratio (ICER) or the incremental net monetary benefit (iNMB) if the intervention (rVWF) was found to be cost-saving, reported over a lifetime time-horizon across a range of accepted willingness-to-pay (WTP) thresholds in US dollars per QALY. The two secondary outcomes were threshold analyses for 1) the breakthrough bleed rate and 2) frequency of infusions necessary for the non-favored product (i.e., in the base-case) to become favored. In addition, we conducted a scenario analysis assuming the non-significant reduction in bleeding with rVWF (0.55 [95% confidence interval 0.086-3.523]) otherwise nullified in the base-case analysis, was significant. We proceeded with deterministic sensitivity analyses, varying all parameter estimates across the largest of their known 95% confidence intervals or +/-20% of absolute value. We concluded with a probabilistic sensitivity analysis, capturing uncertainty in all parameters simultaneously over 10,000 Monte Carlo simulations. Results: In the base-case, treatment with prophylactic pdVWF versus prophylactic rVWF yielded discounted lifetime costs of $21.2 million and $19.8 million, with both accruing 20.6 discounted QALYs. The average incremental net monetary benefit across accepted WTP thresholds of $50,000-$150,000/QALY was $1.38 million [95% credible interval $1.10-1.67 million], in favor of rVWF. Deterministic sensitivity analysis showed that the model was most sensitive to the frequency and cost of pdVWF infusions. In all three probabilistic sensitivity analyses across all WTP thresholds, rVWF was favored over pdVWF in 100% of 10,000 Monte Carlo iterations. Threshold analyses revealed that 1) the bleeding rate on pdVWF prophylaxis would have to reduce by at least 43% and, 2) the average frequency of pdVWF infusions would need to decrease from 3 to 2.75 infusions per week for pdVWF to be favored over rVWF prophylaxis. Scenario analysis revealed similar significant improvement in incremental net monetary benefit with rVWF. Conclusion: Twice-weekly rVWF versus thrice-weekly pdVWF demonstrated similar quality-adjusted life-expectancy, while rVWF was cost-saving due to its longer half-life requiring fewer lifetime infusions. At current pricing in the United States, prophylactic rVWF is a cost-saving option for patients averaging more than 2.75 weekly pdVWF infusions.

New and emerging therapies for women, girls, and people with the potential to menstruate with VWD

AbstractInnovation in therapies for patients with von Willebrand disease (VWD) has lagged far behind that for hemophilia, creating inequity in the bleeding disorder community. Although currently existing treatments of antifibrinolytics, desmopressin, and plasma-derived von Willebrand factor replacement are considered effective, multiple studies report poor quality of life in patients with VWD, especially those with heavy menstrual bleeding (HMB). This disconnect underscores the need for novel therapies that are safe and effective and that consider a patient’s specific contraceptive and reproductive needs. Recombinant von Willebrand factor is the most recent new therapy for VWD; the data specific to women are reviewed. We also present emerging data on emicizumab for the treatment of VWD, BT200 (rondoraptivon pegol), generalized hemostatic therapies (VGA039 and HMB-011), as well as treatments based on nanotechnology (platelet-inspired nanoparticles and KB-V13A12). We are optimistic as we move toward pivotal clinical trials for these elegant and innovative treatments.

In vitro field study and worldwide survey assessing how clinical haemostasis laboratories analyse recombinant and plasma-derived von Willebrand factor products.

Several well-established clinical laboratory methods are available to measure von Willebrand factor (VWF) in plasma samples, but few data are available on their use for analysing recombinant VWF (rVWF). To evaluate how clinical diagnostic laboratories analyse rVWF and plasma-derived VWF (pdVWF) spiked in vitro into VWF-deficient plasma using quantitative protein and functional assays of VWF. Human VWF-deficient plasma samples were spiked with rVWF (vonicog alfa; Takeda) or pdVWF/factor VIII (pdVWF/FVIII; antihemophilic factor/VWF complex [human], CSL Behring), each at final concentrations of 1.0, 0.6, 0.2, 0.1 IU/mL VWF:ristocetin cofactor activity (VWF:RCo) according to labelled VWF activity. The ISTH SSC secondary coagulation standard was used as a control. Participating laboratories received three sets of these blinded aliquots. Mean results per assay were compared with the expected potency based on the labelled VWF:RCo activity. Among 39 laboratories, the most commonly established assay was VWF:RCo; 22 laboratories reported data from 2214 tests. Despite a trend to lower values, VWF:RCo activities for rVWF were in agreement with target concentrations (71%-109%), whereas VWF:platelet glycoprotein Ib (VWF:GpIb) and VWF collagen-binding activity (VWF:CB) assays gave high recoveries (up to 132% and 127%, respectively). In contrast, pdVWF/FVIII was substantially underestimated by VWF:GpIb and VWF:CB assays (56%-86% recoveries), whereas the VWF:RCo assay gave recoveries of 47%-112% for pdVWF/FVIII. The results of VWF assays used in clinical laboratories differ between rVWF and pdVWF, particularly for VWF:GpIb and VWF:CB assays. These differences may arise from the higher multimeric structure of rVWF compared to pdVWF.

PB0840 Efficacy of Prophylaxis with a Plasma-Derived von Willebrand Factor/ Factor VIII Concentrate in Patients with von Willebrand Disease: Subgroup Analysis by Age and VWD Type

PB0840 Efficacy of Prophylaxis with a Plasma-Derived von Willebrand Factor/ Factor VIII Concentrate in Patients with von Willebrand Disease: Subgroup Analysis by Age and VWD Type

Desmopressin as a Treatment in Patients With Von Willebrand Disease: A Systematic Review.

Von Willebrand disease (VWD) and hemophilia A are the most common inherited bleeding disorders. Quantitative or qualitative von Willebrand factor (VWF) anomalies cause this disorder in men and women. VWF, a plasma glycoprotein, relies on platelets for primary hemostasis. It also carries and stabilizes factor VIII in the blood. VWD has several categories. Types 1 and 3 have partial or total VWF quantitative deficiencies. However, type 2 and its subtypes have VWF quality issues. The major treatment is desmopressin (DDAVP), which replaces endogenous VWF and factor VIII (FVIII). Plasma-derived VWF/FVIII products may also be substituted exogenously. Treatment with plasma-derived or recombinant VWF concentrates without FVIII is also possible.The purpose of this retrospective, single-center research was to evaluate DDAVP's efficacy in treating VWD based on many criteria established in the current literature. We looked at the results on Google Scholar, the Cochrane Library, and PubMed/Medline. There were a total of 10 papers found, evaluated, and accepted for inclusion in this study. A comprehensive analysis of DDVAP's role in VWD was compiled from the aforementioned papers. Various aspects of DDVAP were captured by including an analysis of complementary treatments used in surgical and clinical settings. We also describe the treatment's intended impact on the different variations of the disease. Given these results, further investigation is required to determine the most effective method for managing VWD so that it may be included in standard clinical practice.

An evaluation of von Willebrand factor (recombinant) therapy for adult patients living with severe type 3 von Willebrand disease

ABSTRACT Introduction Von Willebrand Factor (VWF) containing concentrates have been used for the treatment of von Willebrand Disease (VWD) for many years. Recently, however, a novel recombinant VWF (rVWF or vonicog alpha, VONVENDI [US], VEYVONDI [Europe]) has arrived to the market for the treatment of VWD. Initially, rVWF was approved by the U.S. Food and Drug Administration (FDA) for the on-demand treatment and control of bleeding episodes and for the perioperative management of bleeding for patients with VWD. More recently, however, the FDA has approved rVWF for routine prophylaxis to prevent bleeding episodes for those patients with severe type 3 VWD receiving on-demand therapy. Areas Covered This review will focus on recent phase III trial results from NCT02973087 regarding the use of long-term routine twice weekly prophylaxis with rVWF for the prevention of bleed events in patients with severe type 3 VWD. Expert Opinion A novel rVWF concentrate may have greater hemostatic potential over prior plasma-derived VWF concentrates and is now FDA approved for use in routine prophylaxis for patients with severe type 3 VWD in the United States. This greater hemostatic potential may be due to the presence of ultra-large VWF multimers and a more favorable high-molecular-weight multimer pattern compared to prior pdVWF concentrates.

The effect of recombinant versus plasma-derived von Willebrand factor on prolonged PFA closure times in ECMO patients with acquired von Willebrand syndrome – an observational study

BackgroundExtracorporeal membrane oxygenation (ECMO) is applied in patients with respiratory or cardiopulmonary failure, but bleeding is a frequent complication contributing to the high mortality rates in this patient collective. A major factor predisposing patients to bleeding events is an acquired von Willebrand syndrome (aVWS). So far, specific treatment options for this phenomenon are lacking. In hereditary von Willebrand disease (VWD), treatment with recombinant or plasma-derived von Willebrand factor (rVWF or pVWF) is common practice. Closure time measured by the Platelet Function Analyser-200 (PFA-200) is an established assay to detect defects in primary hemostasis and the method is useful to monitor the effect of hemostatic therapy. The aim of this study was to assess the effect of recombinant (rVWF) vs. plasma-derived von Willebrand factor (pVWF) on closure times measured by PFA in blood obtained from ECMO patients with aVWS.MethodsBlood was sampled from thirteen patients receiving extracorporeal membrane oxygenation and three patients with hereditary VWD. Diagnosis of aVWS was made by conventional coagulation parameters and by multimeric structure analysis. PFA analysis of blood spiked with rVWF or pVWF was performed.ResultsThirteen patients receiving ECMO were recruited. Ten patients survived and three patients suffered major bleeding complications. PFA closure times in ECMO patients with aVWS spiked with rVWF were significantly shorter at all concentrations than with pVWF (e.g., rVWF vs. pVWF: 1 U/ml: 150.4 ± 21.7 s vs. 263.8 ± 11.7 s; 4 U/ml: 97.8 ± 9.8 s vs. 195.8 ± 15.4 s, p<0.001). PFA closure times were also significantly shorter in three patients with hereditary VWD treated with rVWF compared to pVWF (e.g., 1 U/ml rVWF vs. pVWF: 73.7±1.33 s vs. 231.3±43.4 s, p<0.01)ConclusionIn summary, this study shows that rVWF compared to pVWF more effectively reduced PFA closures times in blood samples of ECMO patients with aVWS. Higher doses of VWF are needed to normalize PFA closure time in blood samples of patients with ECMO-induced aVWS compared to hereditary VWD. These data support the use of PFA-200 to monitor hemostatic effects in a future clinical trial involving ECMO patients with aVWS.

Efficacy and Safety of Prophylaxis with a Plasma-Derived Von Willebrand Factor/ Factor VIII Concentrate in Previously Treated Patients with Von Willebrand Disease

Introduction: Long-term prophylaxis with a von Willebrand factor (VWF) concentrate is recommended in von Willebrand disease (VWD) patients with a history of severe and frequent bleeds, regardless of age or VWD type. Data on the efficacy and safety of prophylaxis with a human plasma-derived VWF/factor VIII concentrate (pdVWF/FVIII; wilate®) that contains the two factors in a physiological 1:1 activity ratio have been collected previously from 19 VWD patients in four prospective, multicenter phase 2 and 3 trials. In these patients, bleeding rates were significantly reduced compared with previous treatment. Aims: To investigate the efficacy and safety of regular prophylaxis with a pdVWF/FVIII concentratein previously treated patients with VWD. Methods: WIL-31 (NCT04052698) was a prospective, non-controlled, international, multicenter phase 3 trial that enrolled patients of any gender with VWD type 1 (VWF:RCo <30 IU/dL), type 2 (except 2N) or type 3, who were at least 6 years old. Prior to entering the WIL-31 study, all patients had received on-demand treatment with pdVWF/FVIII during a 6-month, prospective, observational, run-in study (WIL-29); patients who experienced at least 6 bleeding episodes (BEs), excluding menstrual bleeds, with at least 2 of these BEs treated with a VWF-containing product, were eligible to enter WIL-31. Patients in WIL-31 received regular pdVWF/FVIIIprophylaxis, 2-3 times per week at a dose of 20-40 IU/kg, for 12 months. The primary endpoint of the study was to demonstrate that prophylaxis with pdVWF/FVIII lowered the mean total annualized bleeding rate (ABR) compared with that during 6 months of on-demand treatment by more than 50%. Secondary outcomes included spontaneous ABR and treatment-emergent adverse events. Results: Thirty-three patients were included in the modified full analysis set, with a median (range) age of 18 years (7-61). Fourteen (42.4%) patients were female, 9 (27.3%) patients were <12 years old. Six (18.2%) patients had severe Type 1 VWD, 7 (21.2%) had type 2, and 20 (60.6%) patients had type 3 VWD. Mean (median, range) total ABR in the on-demand run-in period and during pdVWF/FVIII prophylaxis was 33.4 (24.5, 11-115) and 5.2 (1.9, 0-35.8), respectively (Figure 1), meeting the primary endpoint of a ≥50% reduction in mean ABR. Mean (median, range) spontaneous ABR in the on-demand run-in period and during pdVWF/FVIII prophylaxis was 24.4 (18.7, 5-93) and 3.2 (1.0, 0-24.6), respectively. Of the 33 patients, 10 (30.3%) and 15 (45.5%) patients had zero total or spontaneous BEs during the study period, respectively. A total of 173 BEs occurred, the majority of which (146 BEs; 84.4%) were classified as minor. Of the 173 BEs, 121 (69.9%) were treated with pdVWF/FVIII. The overall efficacy of treatment was assessed as "excellent” or "good” for 120 (99.2%) BEs and "moderate” for 1 (0.8%) BE. Five serious adverse events were reported, none of which were assessed as related to the study treatment. Two patients had adverse events assessed as probably or possibly related to the study treatment by the investigator, and both patients discontinued from the study. One patient developed mild chest tightness (3 events), the other had hypersensitivity reactions of moderate severity (2 events). No thrombotic events occurred. Conclusions: Prophylaxis with a pdVWF/FVIII containing VWF and FVIII in a 1:1 activity ratio was effective in pediatric and adult patients with VWD of all types. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Empower Study - Design of a Randomized Clinical Trial to Assess the Efficacy and Safety of a Plasma-Derived Von Willebrand Factor / Factor VIII Concentrate for Heavy Menstrual Bleeding in Women with Von Willebrand Disease

Background: Although von Willebrand disease (VWD) is inherited by men and women equally, women are more likely to experience bleeding symptoms due to the challenges of menstruation and childbirth. Heavy menstrual bleeding (HMB) is the most common symptom observed in women and girls with VWD, and increases risk of iron deficiency anemia, blood transfusion, hospitalization and decreased health-related quality of life. While replacement therapy with von Willebrand factor (VWF) is recommended for prophylaxis in patients with VWD with severe and frequent bleeding, recommendations on its use for prophylaxis of HMB are limited due the lack of clinical trial data. Aims: The EMPOWER pilot trial will determine the feasibility of the trial design, as well as explore assay sensitivity to inform the determination of the primary outcome for the definitive randomized trial which will evaluate the effect of prophylaxis with plasma-derived VWF/FVIII (1:1; pdVWF:FVIII) concentrate compared with placebo on HMB in women with VWD. Methods: EMPOWER is a pilot, randomized, placebo-controlled, crossover, double-blind (patient and outcome assessor blinded), 2-year trial in female outpatients with VWD and HMB. We will evaluate the effect of 2-3 doses of prophylaxis with 40-60 IU VWF:RCo/kg pdVWF/FVIII concentrate when provided on the 2 heaviest days within the first 4 days of menstruation compared with placebo (normal saline). For the first treatment period, participants will be randomized to receive either pdVWF/FVIII concentrate or placebo for 4 cycles, crossing over to the comparator treatment during the second treatment period (see Figure 1). Non-pregnant female patients, ≥18 years of age with a diagnosis of inherited VWD (any type) with HMB defined as a modified pictorial blood assessment chart (mPBAC) score >100, on stable treatment for HMB and iron-deficiency anemia will be eligible. Patients with diagnosis of another bleeding disorder or the presence of >2 risk factors for venous thromboembolism will be excluded. The pilot trial viability outcomes include blinding index scores and participant drop-out rates. Feasibility outcomes include data completeness and rate of enrolment. The proposed primary clinical efficacy outcome is the mPBAC score and the key secondary efficacy outcome is the need for rescue therapy (i.e. >2 days of oral tranexamic acid use, additional treatment with pdVWF:FVIII concentrate, additional hormonal therapy for HMB, urgent/emergent gynecological surgery for HMB, treatment with intravenous iron, red blood cell transfusion, or hospital admission for HMB). Other secondary outcomes include major bleeding, clinically relevant non-major bleeding, red blood cell transfusion, fatigue and quality of life scores. Given the uncertainty in this area of research and the relative comparable clinical importance of proposed clinical efficacy outcomes, we will evaluate assay sensitivity to determine the outcome that has the greatest chance of detecting a difference between treatment periods. Assay sensitivity is defined as the ability of a scale to distinguish an effective from a less effective intervention or placebo. Assay sensitivity along with the proportion of missing data per clinical efficacy outcome and sample size considerations will influence the choice of primary outcome for the definitive trial. Safety will be evaluated by monitoring for hypersensitivity reactions, thromboembolic events and VWF inhibitors. The pilot trial will recruit 20 participants from Hemophilia Treatment Centres in Canada over 2 years. Results: The pilot trial is aiming to launch by the fall 2022. If the pilot trial is deemed feasible and viable, we will subsequently conduct a definitive international randomized controlled trial. Conclusion: Data from the EMPOWER trial will fill an important knowledge gap to facilitate evidence-based management of HMB in women with VWD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Efficacy and safety of von Willebrand factor concentrate almost devoid of factor VIII (Wilfactin®) in paediatric patients under 6 years of age with severe von Willebrand disease.

Plasma-derived von Willebrand factor (VWF) (Wilfactin®, LFB, France) was developed for prophylaxis and treatment of haemorrhages in both adults and adolescents with von Willebrand disease (VWD). Replacement therapy in paediatric patients is a key element of the clinical trial programme. Patients aged <6 years with severe VWD were enrolled in a multinational, open-label study to evaluate the in vivo recovery for Wilfactin®, and its efficacy in preventing and treating bleeding episodes and during surgery. Overall haemostatic efficacy based on a 4-point scale was assessed by investigators. The treatment period ≥18 months investigated the long-term safety. Nine patients, including 7 with type 3 VWD were exposed to treatment with Wilfactin® for up to 4.2 years. Recovery of VWF in 7 patients (n=5 type 3, n=1 type 2, n=1 type 1) was 1.8±0.4 IU/dL per IU/kg. Of the 62 bleeds, 89% were controlled with one (73%) or two (16%) infusions of Wilfactin®. The median dose per infusion was 54 IU/kg. A factor VIII dose was co-administered in 1.6% of bleeds. "Excellent"/"Good" haemostatic efficacy was achieved in 90.3% of episodes. Six patients underwent 11 minor surgical interventions. Treatment duration was 1 day (range: 1-6 days) with a dose administered 30-60 minutes before procedure of 56 IU/kg (range: 41-106 IU/kg). Haemostasis was rated as "Excellent" in all surgeries. During 4-year prophylactic treatment in one patient, breakthrough bleeds were reported in 2.2% of infusions. No VWF inhibitors, thromboembolic events or allergic/anaphylactic-type reactions were observed following a total exposure of 770 days. The results show that Wilfactin® provides a safe and effective treatment in patients <6 years of age with severe VWD.

Hydrogen-deuterium exchange of plasma-derived von Willebrand factor reveals similar dynamics to isolated A1 domain and autoinhibitory module

Von Willebrand factor (VWF) is a large multimeric, multidomain glycoprotein that circulates in the bloodstream and is essential for mediating platelet adhesion at sites of vascular injury through its A1 domain. Our lab has recently provided detailed characterizations of the autoinhibitory module (AIM), the discontinuous flanking regions that cooperatively shield the A1 domain and regulate its binding to cognate platelet receptor glycoprotein (GP) Ibα. While our recent analyses have described the mechanical and biochemical properties of AIM-A1 as an independent functional unit, the impact of the neighboring D’D3 and A2 domains on AIM dynamics remain unclear.

In Vitro Comparison of Recombinant and Plasma-Derived von Willebrand Factor Concentrate for Treatment of Acquired von Willebrand Syndrome in Adult Extracorporeal Membrane Oxygenation Patients

Coagulopathic bleeding is common during adult extracorporeal membrane oxygenation (ECMO), and acquired von Willebrand syndrome is a contributing factor. We compared ECMO patient blood samples that were treated in vitro with recombinant von Willebrand Factor concentrate and plasma-derived von Willebrand Factor concentrate. Our hypothesis was that recombinant von Willebrand Factor (vWF) would have greater efficacy in increasing vWF function. Secondarily, we hypothesized that recombinant vWF would have less impact on thrombin generation. Thirty ECMO patients and 10 cardiac surgical controls were enrolled in the study. ECMO patient blood samples were treated in vitro with low- and high-dose recombinant vWFs and low- and high-dose plasma-derived vWFs. Whole blood ristocetin-induced platelet aggregation (RIPA), plasma ristocetin cofactor activity (RCo), and thrombin generation were compared between ECMO patient blood samples and control blood samples and between vWF-treated ECMO patient blood samples and nontreated samples. ECMO patient blood samples had severely reduced median RIPA compared to control samples 2 ohms (1-12 [25th-75th percentile]) vs 20 ohms (11-42) (P < .001). Treatment of ECMO patient blood samples with high-dose recombinant vWF significantly increased median RIPA to 10 ohms (2-15) (P < .001), while low-dose recombinant vWF and low- and high-dose plasma-derived vWFs did not significantly increase RIPA; 6 ohms (3-14), 4 ohms (1-13), and 6 ohms (2-10), respectively (P = .25, >.99, and >.99). Treatment with high-dose recombinant vWF and low- and high-dose plasma-derived vWFs significantly increased median plasma RCo to 4.7 international units (IU)/mL (3.7-5.9), 3.3 IU/mL (2.7-4.8), and 3.9 IU/mL (3.4-5.3), respectively, compared to controls 1.8 IU/mL (1.5-2.3) (all P < .001). Treatment with low- and high-dose plasma-derived vWFs significantly increased mean endogenous thrombin potential (6270.2 ± 2038.7 and 6313.1 ± 1913.3) compared to nontreated samples (5856.7 ± 1924.6) (P = .04 and .006), whereas treatment with low- and high-dose recombinant vWFs had no significant effect on mean endogenous thrombin potential (5776.1 ± 2087.3 and 5856.2 ± 1946.4) (P > .99 for both comparisons). In vitro treatment of ECMO patient blood samples with high-dose recombinant vWF was superior to low-dose recombinant vWF and plasma-derived vWF in terms of improving RIPA. In addition, recombinant vWF treatment did not increase endogenous thrombin potential, which may reduce overall thrombotic risk if it used to treat acquired von Willebrand syndrome in ECMO patients.

Von Willebrand Factor Multimer Analysis by Low Resolution SDS-Agarose Gel Electrophoresis.

Von Willebrand factor (VWF) is a complex glycoprotein found in plasma, composed of disulfide-bond-linked multimers with apparent molecular weights between 500 kDa and 20,000 kDa. After release of VWF from storage granules, it is cleaved in flowing blood by the specific metalloproteinase ADAMTS13, resulting in a highly characteristic cleavage pattern and structure. As the structure of VWF multimers determines diagnosis of von Willebrand disease, which has different sub-types with different multimer- and cleavage patterns, VWF analysis is performed using low-resolution horizontal SDS-agarose gel electrophoresis. However, almost every laboratory uses a different protocol, and all experimental details are rarely, if at all, described. Therefore, the results from similar methods may be substantially different. Here, we present a detailed description of a validated VWF multimer method that we have developed. It has been successfully used for over more than 20 years in quality control of recombinant and plasma-derived VWF drug products, and in preclinical and clinical studies with VWF drug candidates. As most of the published methods, it enables visualization of VWF multimers separated by electrophoresis by immunostaining with a polyclonal anti-human VWF antibody followed by a secondary antibody coupled to alkaline phosphatase. VWF appears as a series of regularly spaced bands on the low and middle molecular weight range of the gel, with an unresolved zone in the high molecular weight (HMW) range, where ultra-large multimers are found. An example is shown below. This low-resolution agarose gel electrophoresis allows the determination of the number of VWF multimers with high reproducibility. Graphical abstract: Example of electrophoretic analysis of multimer structure of four batches of a recombinant VWF drug substance.

Exposure-Response Relationship between Von Willebrand Factor (VWF) Activity and Bleeding Events Following Prophylaxis with Recombinant VWF (rVWF, vonicog alfa) in Patients with Severe Von Willebrand Disease (VWD)

Exposure-Response Relationship between Von Willebrand Factor (VWF) Activity and Bleeding Events Following Prophylaxis with Recombinant VWF (rVWF, vonicog alfa) in Patients with Severe Von Willebrand Disease (VWD)

Mapping the Masked Region of the A1 Domain in Full-Length VWF

Von Willebrand factor (VWF) is the largest protein in plasma and promotes the early hemostatic response following vascular injury. The hemostatic response of VWF relies on the exposure of its A1 domain, which recognizes its receptor GPIbα on the platelet surface. To avoid binding and activating platelets in the absence of injury, the A1 domain is dormant in full-length VWF through a mechanism that is not completely understood. We recently characterized two recombinant A1 fragments (longA1, residues 1238-1472; and shortA1, residues 1261-1472) with differential GPIbα-binding affinities by hydrogen-deuterium exchange (HDX) mass spectrometry. We found that N- and C-terminal residues flanking the A1 domain (residues 1238-1271 and 1459-1472) cooperatively constitute an autoinhibitory module (AIM) that masks a region in A1 and impedes its binding to GPIbα[1]. To determine if the A1 domain in full-length VWF is “masked” in the same manner, we have characterized human plasma-derived VWF using the same HDX approach.On-column peptic fragmentation was performed following HDX from 10 to 10,000 sec. We obtained 946 sequenced peptides covering 96.4% of the VWF sequence with an average redundancy of 6 per residue. Among them, 113 peptides covered the A1 domain, allowing HDX analysis of A1 at high resolution. We found that residues around the 1272-1458 disulfide bond, helix α1, loops α1β2 and β3α2 in the A1 domain, which are protected by the AIM in the longA1 fragment (residues 1238-1472), are deeply protected in full-length VWF, suggesting that AIM in full-length VWF likely masks A1 in a similar pattern. Additionally, sheet β3, helix α3, and loops β4α3 in the A1 domain of full-length VWF reported lower HDX rates than their counterparts in the shortA1 fragment. These regions, along with those protected by AIM, form a contiguous surface that significantly overlaps with the GPIbα-binding interface (Fig. 1). Residues in loop β6α6, helix α6 and helix α1 also reported lower HDX rates, but they are separate from the regions protected by AIM and the GPIbα-binding interface.In summary, our results suggest that AIM and likely other domains in full-length VWF are in direct contact with the A1 domain and regulate the hemostatic properties of VWF.Figure 1: Difference in HDX between isolated VWF A1 domains and full length VWF.1 Deng W, Wang Y, Druzak SA, Healey JF, Syed AK, Lollar P, Li R. A discontinuous autoinhibitory module masks the A1 domain of von Willebrand factor. Journal of thrombosis and haemostasis : JTH . 2017. 10.1111/jth.13775. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Gastrointestinal Bleeding and Angiodysplasia in Von Willebrand Disease: Results from the Ongoing French HRQoL Study: Wish-Qol

Von Willebrand Disease (VWD), the most common inherited bleeding disorder, is related to a von Willebrand factor (VWF) defect and/or functional abnormality. VWD is primarily characterized by mucocutaneous bleedings as a result of primary haemostasis defect, excessive haemorrhage after invasive procedures, and, less commonly, by soft tissue haematomas and joint bleedings.Some patients with VWD may be subjected to recurrent overt or occult bleedings in the gastrointestinal (GI) tract associated with angiodysplasia which are challenging complications. Angiodysplasia occurs most commonly in middle-age or elderly patients in the cecum and ascending colon, but sometimes throughout the whole colon and stomach. These affected patients may need to be hospitalized for long periods of time and often require several weeks of therapy leading to high costs and poor quality of life. We report here the first results of GI bleedings impact on the Health-Related Quality of Life (HRQoL) of patients with VWD enrolled in the French WiSH-QoL study.As both VWD and its treatment affect patients' and, possibly, their families' everyday life, the French non-interventional study, WISH-QoL aims to assess HRQoL in VWD patients of any age and to evaluate associated costs related to VWD management.Clinical phenotype such as bleeding score (BS - Tosetto score), biological profile is documented. Patients are treated with the triple-secured plasma-derived VWF concentrate almost devoid of FVIII WILFACTIN. Patients fill in a booklet collecting information on socio-demographic and resources consumption. Clinical characteristics and therapeutic approach are recorded. HRQoL is assessed with the generic SF-36 (for adults) and the chronic-generic DISABKIDS Short Form (for children and adolescents) and the VWD-specific VWD-QoL (for adults, children and adolescents). In addition treatment satisfaction is evaluated. At least 350 patients will be followed during 2 years.Since October 2014, 327 patients have been included.At time of the current analysis, out of 312 patients with evaluable data, special attention is given to the specific clinical subgroup of 36 patients with recurrent bleeding from the gastrointestinal tract.These patients exhibited an age distribution ranging from 3.3 to 83.0 year-old with a 44.5 y.o median. Among them, 3 children were less than 6 years but elderly were more represented with 10 adults more than 65 years. Females were more represented than males 20 (55.5%) vs. 16 (45.5%), respectively.Among them, 5 had type 1 VWD (type 1Vincenza: 3; type 1: 2), 20 had type 2 (2A: 11; 2B: 6; 2M: 1 and 2 unspecified: 2), 10 had type 3 and 1 with a type undetermined (Table1).The median Tosetto bleeding score (BS) reported for 33 patients with GI bleeds was higher +12 ranging from +4 to +28 compared to +5 (-1 to +31) in patients without history of GI bleeds as described in Table1. It was also able to distinguish disease severity by VWD type; Type 3 VWD had the highest bleeding scores.At time of inclusion, 24 of these 36 patients were On-Demand treatment and 12 patients (4.7 - 70.4 years of age) were and are still requiring Long-Term secondary Prophylaxis (LTP) with this VWF because of joint and or gastro-intestinal bleeds. For 12 out of these 36 patients, the endoscopic procedure Argon plasma coagulation (APC) was used. For 5 of these patients, the procedure was associated with a secondary LTP and they were on regular prophylaxis with VWF concentrate at WiSH-QoL study entry.As frequently in VWD management, patients may need to stay on long-term iron therapy. This concomitant treatment has been recorded in 15 (45%) patients' file. Ten (28%) patients have also received anti-ulcer drugs.Thus, this study will provide an important contribution to knowledge in VWD patients with GI episodes that is challenging due to the severity and recurrence of bleeds. These findings may teach us what would be the best treatment such as LTP. With the HRQoL results especially the VWD-specific evaluation of HRQoL and treatment satisfaction, a deeper insight into these patients' real daily life and their specific health care needs will be gathered as well. [Display omitted] DisclosuresBorel-Derlon:LFB: Honoraria. Chatelanaz:LFB: Employment. Doriat-Robin:LFB: Employment. Goudemand:LFB: Honoraria. Von Mackensen:LFB: Honoraria.